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Graduate Theses and Dissertations
Graduate School
January 2013
Innate Immune Responses to Respiratory Syncytial
Virus: Age-associated Changes
Terianne Maiko Wong
University of South Florida, [email protected]
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Wong, Terianne Maiko, "Innate Immune Responses to Respiratory Syncytial Virus: Age-associated Changes" (2013). Graduate Theses
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Innate Immune Responses to Respiratory Syncytial Virus: Age-associated Changes
by
Terianne Maiko Wong
A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
in Medical Sciences
Department of Molecular Medicine
Morsani College of Medicine
University of South Florida
Major Professor: Shyam Mohapatra, Ph.D.
Michael Teng, Ph.D.
Alberto Luis van Olphen, D.V.M, Ph.D.
Andreas Seyfang, Ph.D.
Subhra Mohapatra, Ph.D.
Shyam Biswal, Ph.D.
Date of Approval:
August 8, 2013
Keywords: Innate Immunity, Antiviral Responses, Viral infections, Respiratory
Infections, Aging
Copyright © 2013, Terianne Maiko Wong
Dedication
I would like to dedicate this first and foremost to my parents, Dr. Stephen Wong
and Merle Wong, who gave me everything I would need as a budding scientist. Their
love and enthusiasm for the sciences have helped me get to a career that I will be
passionate about for the rest of my life. I also thank them for giving me the best role
model I could ask for, my older sister and best friend, Stephanie Wong-Nguyen. Without
her guidance and care, I am certain I could not be where I am now. I am also grateful for
a loving brother-in-law who has taught me so much in life beyond my academic career.
Words cannot express how privileged I was to have such a supportive, thoughtful,
and generous lab environment while under my major professor, Dr. Shyam Mohapatra.
Dr. Gary Bentley has probably spent days, if not weeks, editing and refining all of my
writing to make me a better scientist and author. Dr. Sandhya Boyapalle has been
instrumental to my development as a scientist and her training provided a foundation for
everything else I have learned. Dr. Subhra Mohapatra was not only on my committee,
but also served a comforting maternal role, with valuable suggestions, academic and not.
To my closest lab-mates, Viviana Sampayo, Rimi Bedi, Huy Nguyen, Michael
Cheung, and Hetty Hong—they each have helped me so much the past chaotic year or
more; without their help and encouragement, I would likely not be completing in this
timeframe. Moreover, I am appreciative of all the fun times together intended to release
stress and anxiety—our entire Nanomedicine Research Center group is practically a
family, with all of our holiday potlucks and birthday parties. I thank everyone for being
unforgettable members of this wonderfully close-knit lab and I will miss them terribly
when I leave. I also would like to thank Kim Teng, Dr. Xiaoyuan Kong, and Dr.
Weidong Zhang for providing valuable training in RSV and molecular biology.
Outside of lab, I must thank my closest friends Lisa Le and Samantha Phouthone
for always being there for me during my long academic career. Their love and support
kept me from giving up no matter how exhausted I became. Lastly, my love and
gratitude is extended to my other best friend in life and companion for my decade-long
academic career, my husband Khoa, for supporting my dreams and accepting the
challenges we have had to endure together so I could achieve my own goals.
Acknowledgments
First, I must acknowledge my major professor, Dr. Shyam Mohapatra, for his
years of support and guidance. His dedication to help me, with each and every
fellowship application, manuscript, and presentation, has been unforgettable. He has
made my graduate training an extremely rewarding experience and I am immensely
grateful for all the opportunities he has provided for me and earnestly supported. Next, I
would like to extend my appreciation and acknowledgement to my dissertation
committee members, Dr. Michael Teng, Dr. Alberto van Olphen, Dr. Andreas Seyfang,
and Dr. Subhra Mohapatra. Each of them has dedicated their valuable time to see me
progress over the years and provide scientific guidance that has been instrumental to my
graduate career. I would like to thank the entire USF Graduate Program in Integrative
Biomedical Sciences, including the Molecular Medicine (MM) department Chair Dr.
Robert Deschenes and Dr. Burt Anderson for their continued support and improvements
to the MM department. Special thanks for the all of the administrative staff: Dana
Pettaway, Kim Davis, Lady Washington, Andrew Conniff, Helen Chen-Duncan, Melanie
Martin-Lewis, Michael Ramsamooj, and Kathy Zahn, who have all helped simplify the
confusion as a Molecular Medicine graduate student.
Table of Contents
List of Figures .................................................................................................................... iv
Abstract .............................................................................................................................. vi
Chapter 1: Introduction and Background .............................................................................1
1.1 Epidemiology and significance ..........................................................................3
1.2 Innate immunity .................................................................................................6
1.3 Immunobiology of RSV...................................................................................11
1.4 Therapeutic options for RSV infections ..........................................................13
1.5 Inflammatory responses to RSV infection .......................................................13
1.6 Concluding remarks .........................................................................................15
Chapter 2: General Methods ..............................................................................................17
2.1 Cells and viruses ..............................................................................................18
2.2 Mice .................................................................................................................20
2.3 Protein analysis ................................................................................................21
2.4 Nucleic acid analysis........................................................................................23
2.5 Immunocytochemistry and immunohistochemistry .........................................25
Chapter 3: RSV-mediated Evasion of Host Innate Immunity ...........................................27
Note to reader .........................................................................................................27
3.1 Introduction ......................................................................................................27
3.2 Materials and methods .....................................................................................32
3.2.1 Vector construction and establishment of the stable cell line,
Flp-In 3XFLAG-NS1 ...........................................................................32
3.2.2 Protein analysis .................................................................................33
3.2.3 Tetracycline induction ......................................................................34
3.2.4 Live-cell imaging of His6-tagged NS1 in RSV-infected cells ..........34
3.3 Results ..............................................................................................................36
3.3.1 RSV NS1 localization to the mitochondria in vitro ..........................36
3.3.2 RSV NS1 binds to mitochondrially-associated MAVS and
prevents interaction with RIG-I ...........................................................38
3.3.3 RSV NS1 expression kinetics are parallel with IFN-regulator
LGP2 ....................................................................................................39
3.3.4 Induction of NS1 expression restores RSV replication and
transcription of viral RNA ...................................................................40
i
3.4 Discussion ........................................................................................................41
Chapter 4: Differential RSV-induced Gene Expression in Young and Aged mice ...........54
4.1 Introduction ......................................................................................................54
4.2 Materials and methods .....................................................................................59
4.2.1 Mice and animal studies....................................................................59
4.2.2 Gene profiler RNA expression analysis ............................................60
4.2.3 Minitab design of experiment (DOE) analysis .................................61
4.2.4 Isolation of alveolar macrophages and stimulation of TLR7/8 ........62
4.3 Results ..............................................................................................................63
4.3.1 Age-associated alterations in antiviral gene expression and
kinetics .................................................................................................63
4.3.2 Statistical analysis with DOE identifies genes that are altered
by age and infection .............................................................................65
4.3.3 TLR7/8 signaling is impaired in aged alveolar macrophages ...........66
4.4 Discussion ........................................................................................................67
Chapter 5: RSV-induced Pathology in Young and Aged Mice .........................................79
5.1 Introduction ......................................................................................................79
5.2 Materials and methods .....................................................................................83
5.2.1 Tissue collection and histological processing ...................................83
5.2.2 Immunohistochemical staining and image analysis with
ImageJ software ...................................................................................84
5.3 Results ..............................................................................................................87
5.3.1 Aging is associated with delayed cellular clearance of RSV
rA2-L19F .............................................................................................87
5.3.2 Mucus production is altered in aged mice upon rA2-L19F
infection ...............................................................................................88
5.3.3 Infection with RSV rA2-L19F is enhanced and prolonged in
aged BALB/c mice ...............................................................................89
5.4 Discussion ........................................................................................................90
Chapter 6: Effects of Aging on RSV-infection Induced Expression of IL-1β and
OPN......................................................................................................................109
6.1 Introduction ....................................................................................................109
6.2 Materials and methods ...................................................................................115
6.2.1 Mice ................................................................................................115
6.2.2 Tissue collection and histological processing .................................116
6.2.3 Enzyme-linked immunosorbent assay ............................................116
6.2.4 Immunohistochemical staining and image analysis with
ImageJ ................................................................................................117
6.3 Results ............................................................................................................118
6.3.1 Aging results in altered IL-1β induction upon RSV infection ........118
ii
6.3.2 OPN induction is delayed but prolonged in aged mice in
response to RSV .................................................................................120
6.3.3 Mucus secretion is enhanced in OPN KO mice after infection
with rA2-L19F ...................................................................................121
6.3.4 OPN KO mice have increased resistance to rA2-L19F ..................122
6.4 Discussion ......................................................................................................123
Chapter 7: Conclusions and Future Directions ................................................................139
7.1 Subversion of antiviral interferon responses by RSV NS1 ............................139
7.2 Aging leads to impairment of antiviral gene expression ...............................140
7.3 Age-associated changes in lung microenvironment affect RSV
outcomes ........................................................................................................143
7.4 Age-associated changes to cytokine induction in RSV infections.................144
7.4 Final remarks .................................................................................................146
References ........................................................................................................................148
About the Author ....................................................................................................End page
iii
List of Figures
Figure 1. Construction of FLAG-tagged human codon-optimized vectors for
transient transfection and inducible cell line construction. ....................................45
Figure 2. Procedure for live-cell tracking of polyhistidine-tagged RSV infection. ...........46
Figure 3. Subcellular localization of RSV NS1 in transiently transfected A549...............47
Figure 4. Tetracycline-inducible model for controlled expression of RSV NS1. ..............48
Figure 5. Cell-permeable fluorophore implemented to track subcellular
localization of polyhisitidine-tagged RSV NS1 in live, RSV-infected cells. ........49
Figure 6. RSV NS1 associates with MAVS protein in pNS1-transfected A549
and RSV-infected A549. ........................................................................................50
Figure 7. Kinetics of RSV NS1 expression in A549 and induction of IFNregulator protein LGP2. .........................................................................................51
Figure 8. Induction of NS1 restores infectivity and viral replication. ...............................52
Figure 9. Proposed virulence activities of RSV NS1 in disruption of host innate
immunity. ...............................................................................................................53
Figure 10. Baseline antiviral gene expression in young and aged mice. ...........................72
Figure 11. Differential antiviral gene expression upon RSV-infection in aged
mice. .......................................................................................................................74
Figure 12. Five antiviral genes altered by age and infection. ............................................76
Figure 13. TLR 7/8 activation in aged alveolar macrophages is impaired. .......................78
Figure 14. Quantification of cell infiltration density in hematoxylin-stained lung
sections. ..................................................................................................................96
iv
Figure 15. PAS-quantification with color deconvolution and threshold area
measurement. .........................................................................................................98
Figure 16. Method for quantifying RSV-positive fluorescence in tissue sections. ...........99
Figure 17. Aged mice have enhanced pathology upon infection with mucogenic
RSV ......................................................................................................................102
Figure 18. Mucus secretion increases after rA2-L19F infection in young but not
aged mice. ............................................................................................................103
Figure 19. Infection with rA2-L19F is enhanced in aged mice. ......................................106
Figure 20. RSV clearance is delayed in aged mice upon rA2-L19F ..............................108
Figure 21. Age differential expression of IL-1β upon RSV infection .............................131
Figure 22. Aging results in diminished OPN production in response to 2-20 RSV. .......133
Figure 23. Mucus secretion is enhanced in OPN KO mice upon rA2-L19F
infection. ..............................................................................................................136
Figure 24. OPN KO mice are resistant to rA2-L19F infection compared to WT. ...........138
Figure 25. Age-differential innate immune responses to RSV. ......................................147
v
Abstract
Respiratory syncytial virus (RSV) infection causes ~64 million cases of
respiratory disease and 200,000 deaths annually worldwide, yet there is no broadly
effective prophylactic or treatment regimen. RSV can produce acute respiratory illness in
patients of all ages but strikes the age extremes, infants and the elderly, with highest
frequency presumably due to innate immune deficiencies. A higher morbidity and
mortality has been reported for the elderly above 65 years of age, which has been
attributed to immune senescence. Efforts to generate an effective vaccine have thus far
been unsuccessful.
The innate immune system provides the first line of defense against viral
pathogens with a repertoire of anatomical barriers, phagocytic immune cells, pattern
recognition receptors (PRRs) and antiviral cytokines like interferons (IFNs). The precise
mechanism of subversion of innate immunity in young and aged is poorly understood. A
better understanding of innate immune pathways is expected to aid in the development of
appropriate vaccines or prophylactics for these high-risk groups.
Previously, the RSV nonstructural protein 1 (NS1) was shown to antagonize IFN
responses by disrupting components of the innate immune system, although the
mechanism is not well defined. We hypothesized that NS1 targets constituents of the
vi
PRR pathways to evade innate immunity and thus ensure viral survival. Using
microscopy and co-immunoprecipitation assays, we found that NS1 localizes to the
mitochondria and binds to the mitochondrially associated adaptor protein MAVS, thus
preventing MAVS interaction with the RNA helicase, RIG-I. Expression of NS1 was
also correlated with upstream IFN-response regulator, LGP2, and its expression was
inducible in the absence of a viral infection. Tetracycline-inducible expression of
recombinant NS1 in a cell model also promoted viral replication and emphasizes the key
contribution of NS1 to RSV survival. Through this study, we demonstrated a mechanism
for RSV NS1 in the disruption of early innate responses through mitochondrial
localization and alteration of the RLH signaling.
Whereas the above studies showed the importance RSV-induced innate immune
pathways, whether the expression and signaling of innate immune pathways were
adversely affected upon RSV infection in the high-risk groups remains unknown. Since
elderly individuals are at an increased risk for severe bronchiolitis and RSV-induced
pneumonia, often resulting in hospitalization and medical intervention and adaptive
immune cell functionality and responsiveness reportedly decline with age, we
hypothesized a similar age-related deterioration of the innate antiviral system. In this
investigation, we used an aged mouse model to correlate age-associated changes in innate
immune gene expression with RSV pathology. Of 84 antiviral genes examined, five
genes including RIG-I, IFNAR1, TLR8, IL-1β, and osteopontin (OPN) were associated
with both age and infection. In response to RSV infection, aged mice had delayed
induction of antiviral genes and diminished ability to secrete IL-6 in response to TLR7/8
vii
agonist in primary alveolar macrophages. Lungs from aged, RSV-infected mice had
increased cellular infiltration and prolonged infection as compared to young mice. In
summary, age-related decline in expression and functionality of antiviral defenses were
correlated with enhance RSV-induced lung disease in aged mice.
In the absence of infection, aged mice chronically overproduced IL-1β and OPN
relative to young mice. Upon infection, aged mice had impaired ability to secrete higher
levels of IL-1β and mucus. In contrast, OPN secretion remained high and prolonged in
aged mice throughout infection. The age-related decline in host antiviral gene induction
and delayed cytokine production correlated with enhanced disease pathology. Using a
transgenic strain of mice deficient in OPN (OPN-KO), we observed greater resistance to
RSV and enhanced secretion of mucus, but unaltered cellular infiltration into the lungs.
Therefore, OPN overproduction and defective mucus production likely contribute to
pathology in aged mice.
These findings demonstrate that RSV targets the innate virus recognition and
antiviral cytokine activation pathways but also that the antiviral defense system is
significantly affected by age. Consequently, efforts to generate vaccines or develop
therapies that stimulate IFN induction may prove unsuccessful in the elderly given that
RSV virulence factors and age weaken these responses. This study contributes to our
understanding of how aging relates to the RSV subversion of the host antiviral response
and should help with the development of better antiviral therapies suited to the growing
elderly population.
viii
Chapter 1: Introduction and Background
Respiratory syncytial virus (RSV) is a leading cause of respiratory and pulmonary
illness resulting in significant worldwide morbidity and mortality. In healthy individuals,
the innate immune system provides defense against RSV and enables prompt immune
responses to limit the spread and propagation of the microorganism; however, in
premature, immunocompromised, or elderly individuals, responses to pathogens is
impaired, leading to more severe disease. The molecular mechanisms that RSV uses to
subvert the host immune response are a growing concern for vaccine development;
further, examining age differential responses to RSV may provide clues as to whether
certain high-risk populations are qualified candidates for therapy.
To contribute to the field of innate immunology in the context of RSV-induced
lung disease, RSV was examined both in vitro and in vivo in the following studies. The
overall hypothesis was the following: regulated innate immune responses are critical for
preventing severe RSV disease. To test this, the following areas were examined: RSVmediated evasion of pattern-recognition receptor (PRR) immune surveillance; ageassociated immunodeficiencies in the innate system; effects of age on lung pathology
induced by mucogenic strains of RSV; and the influence of proinflammatory cytokines in
antiviral defenses. I examine multiple areas of innate immunity, including viral
1
recognition through PRR, cytokine induction, viral clearance, and changes in the lung
microenvironment, particularly mucus secretion.
In Chapter 2, I will introduce the general methods and techniques used and
present the experimental designs. The goal of Chapters 3-6 is to address four areas of
innate immunity that are influenced by RSV infections: 1) RSV-mediated mechanisms
for evading immune surveillance; 2) the effects of aging on innate gene expression in an
aged mouse model of RSV; 3) the effects of aging on pathology; and 4) the correlation of
innate cytokine profiles with RSV-induced lung disease. Each chapter will begin with
relevant background information and the specific methods for the experiments. Standard
experimental methods were used, including viral titration, immunoassays, and
histological staining, and are described in Chapter 2. In addition, several novel methods
are introduced in these investigations, including a strategy for monitoring polyhistidinetagged viral proteins in live cells for in vivo tracking. I also customized macro scripts
and outlined methods for analyzing histological sections using the ImageJ Java platform.
Through these four aims and experimental design, evidence will support the hypothesis
that innate immune responses are necessary for RSV antiviral defenses and prevent
severe RSV-lung disease.
The findings from these investigations are particularly important to the largest atrisk population for RSV infections. With no effective vaccine available to seniors, the
elderly remain susceptible to disease. Moreover, the current antiviral strategies that rely
on fully functional innate and adaptive immunity will likely lose efficacy in populations
with diminished immune statuses. By examining aging antiviral responses and
2
pulmonary health, I hope to contribute to the development of therapy options that are
specifically adapted to elderly persons and age-related needs.
1.1 Epidemiology and significance
RSV is a major etiological agent for serious lower respiratory infections
worldwide. As a member of the Paramyxoviridae family, which includes the viruses that
cause mumps and measles, RSV afflicts nearly all children by the age of 3 years and
reinfection is common since natural infection of RSV fails to adequately stimulate
production of protective antibodies (1). No vaccine is available and prophylaxis is
restricted to high risk infants because of the expense and low cost-effectiveness among
healthy infants and adults (2, 3). Infections are generally mild in healthy adults but
disease can progress to bronchiolitis and pneumonia in susceptible immunocompromised
populations such as infants, transplant patients, and the elderly (4-8).
In a ten-year study conducted in the United States, RSV was associated with an
estimated 24% of nearly 5.5 million LRTI-associated hospitalizations of children <5
years old or approximately 132,000-172,000 hospitalizations annually (9). Preterm
infants with or without co-morbidities are particularly susceptible to severe viral and
bacterial infections since their immune systems are incompletely developed (10-12);
further, neonates rely on maternal antibodies for immune defense, although decay is
rapid, lasting only through the first 6 months of life (13-15). This makes neonates and
preterm infants particularly susceptible to nosocomial infections. In a multicenter study
conducted in France, 40-60% of infants born prematurely and hospitalized for LRTI
3
infections had illnesses attributed to RSV; further, administration of the RSV
prophylactic antibody, palivizumab, significantly reduced the incidence of RSVassociated LRTI hospitalizations (16).
Premature infants who develop severe RSV-
related LRTI have worse lung function and increased lung resistance at the time of
neonatal discharge (36 weeks gestational age)(17). Preexisting conditions substantially
increase morbidity associated with RSV infections. Neonates born with congenital heart
disease (CHD) have significantly higher mortality rates from RSV infection than infants
without CHD, 37% vs. 1.5 %, respectively (8); the duration of hospital stay and incidence
of nosocomial RSV infections are significantly higher in CHD infants (10, 18). Children
<5 yr. old infected with human immunodeficiency virus (HIV) are at increased
susceptibility for RSV-induced pneumonia as compared to HIV-uninfected infants, with
incidence rates of 92.3% vs. 68.8%, (p<0.002), respectively (19). RSV bronchiolitis is
the most frequent reason for hospitalization and severe illness may require mechanical
ventilation, extended hospitalization stays, and other medical interventions to prevent
respiratory or pulmonary failure and death (20-22). From 1997-2000, 311,077 infants
(<1 yr. old) were reported to be hospitalized for RSV bronchiolitis, resulting in an
estimated $3 billion in medical care expenses (23). The impact of RSV on infant and
childhood morbidity and mortality rates make it a particularly important respiratory
pathogen in the fields of pediatric respiratory and pulmonary medicine.
In persons over the age of 65, RSV accounts for an estimated 30% of respiratoryrelated healthcare visits (4) with a mortality rate of 8% annually (24). Nosocomial RSV
infections are commonly acquired by seniors residing in community centers or long-term
4
care facilities (25, 26). Between 20 and 40% of community-acquired pneumonia cases in
adults require in-patient treatment or hospitalization (27); furthermore, several
investigations suggest RSV is responsible for annual hospitalization rates of 8.2-25.4 per
10,000 for patients >50 years of age (28). Chronic pulmonary disease significantly
increases the risk of hospitalization for RSV in patients ≥65 years of age; additionally, an
inverse relationship was found between low neutralizing serum anti-RSV antibodies and
duration of hospital stay (29). Despite its reputation for being a pediatric respiratory
disease, the annual number of deaths attributed to RSV and underlying
respiratory/circulatory disease is substantially higher in adults ≥65 years old, with
mortality rates more than twice that of children younger than 5 years of age (24). The
molecular mechanisms which increase susceptibility are largely unknown; however,
deficits in both the adaptive and innate immune systems would suggest that aging leads to
diminished defenses against ongoing and new infections.
Currently, measures to prevent RSV disease are limited to fusion-inhibitor
prophylaxis prescribed to select high-risk infants (21). Palivizumab, a humanized murine
monoclonal antibody targeting the fusion glycoprotein of RSV (F), is a clinically
effective method for preventing severe RSV-induced lung disease among high-risk
neonates, children with CHD or chronic lung disease (CLD), and children <2 years born
prematurely(3, 12, 21, 30). Given through intramuscular injection, palivizumab was
approved in 1998 and decreased rates of RSV-associated hospitalizations by 19-45% in
infants born with CHD (31, 32). Despite the efficacy, analyses worldwide suggest the
5
expense of palivizumab at 5 monthly doses of 15 mg/kg does not make it cost-effective to
administer to healthy children, adults, or seniors (2, 3, 33).
RSV infections occur seasonally and individuals are prone to reinfection each
year. For example, in the United States during the 2011-2012 season, RSV infections
began in late October, continued into January and ended between March and May, with
the exception of Florida which has an earlier onset and longer season than other states
(34). Unlike influenza, which is capable of genome segment reassortment resulting in
antigenic shifts each season, variations in RSV antigenicity do not appear to be the major
reason for annual reinfections (35). Two major antigenic groups have been described
worldwide, A and B (36-38); group A tends to be more frequent during epidemics (39),
although incidence of RSV B genotype has risen in recent studies. Of the ten viral
proteins encoded by the RSV genome, RSV attachment glycoprotein (G) varies the
greatest among the two major antigenic groups A or B; importantly, the pathogenicity of
RSV strains within antigenic groups also appears to involve attachment proteins, F and
G, as both proteins have significant roles in disrupting the antiviral immune response to
RSV (40-43). Understanding the molecular mechanisms which determine strain virulence
and disease pathology may help provide solutions for treating and preventing RSVinduced lung disease.
1.2 Innate immunity
Adequate innate immunity is well recognized as an essential defense against
microbial infections; furthermore, deficiencies in either adaptive or innate immunity are
6
predictive of clinical outcomes during microbial pathogenesis (44). Upon binding of
pathogen-associated molecular patterns (PAMPs) (45) or danger-associated molecular
patterns (DAMPs) (46) to pattern recognition receptors (PRRs), innate immune responses
are activated, leading to release of proinflammatory mediators that serve in clearance of
pathogens and also contribute to tissue injury and disease pathology. The activation of
innate immune responses are often examined in the context of microbial infections (45,
47-49), innate autoimmunity, cancer, and with the recent discovery of innate lymphoid
cells, other inflammatory diseases such as allergic asthma (50-52). Furthermore,
bacterial and viral respiratory pathogens are reported to contribute to, if not exacerbate,
the symptoms and recurrence of asthma, suggesting that microbial pathogens have both
acute and chronic consequences on respiratory health (53-56).
PRRs are stimulated by pathogen-associated proteins, nucleic acid sequences, or
evolutionarily conserved molecules that the host immune system recognizes as nonself.
The airway system is comprised of a heterogeneous network of epithelial, endothelial,
and immune cells, many of which serve in rapid innate responses to clear respiratory
pathogens. Anatomical and physiochemical barriers serve to prevent invasion of
pathogens and aide in clearance. Mucociliary clearance is particularly important to
respiratory health and decline in function leads to airway obstruction, inflammation, and
possibly death (57-60). Extracellular PRRs such as Toll-like receptor 4 (TLR4)
recognize foreign microorganisms or viral glycoproteins upon attachment. Activation of
TLR4 with the prototypical bacterial PAMP lipopolysaccharide (LPS) initiates
downstream signals, cytokine production, immune cell recruitment, and inflammatory
7
responses (61). TLRs are transmembrane-spanning proteins that are homologous to
interleukin-1 (IL-1) receptors (62); ligand binding to hetero- or homo-dimers of TLRs
initiates recruitment of adaptor proteins, such as myeloid differentiation factor 88
(MyD88), Toll/IL-1R (TIR)-domain-containing adaptor protein-inducing IFN-β (TRIF),
TRIF-related adaptor molecule (TRAM), and TIR-associated protein (TIRAP)(63).
Following adaptor protein recruitment, a series of protein complexes are formed to
initiate nuclear factor-kappa B (NF-κB) nuclear translocation for induction of proinflammatory cytokines and mitogens (64). In airway epithelial cells, TLR activation
leads to synthesis and release of pro-T helper type 2 (Th2) cytokines and activation of
neighboring dendritic cells (if they have not been activated by their own PRRs already).
In addition to PAMPs, some TLRs can recognize danger signals DAMPs released by
neighboring tissues after injury which then initiates sterile inflammation (65). Aberrant
TLR activation has been associated with inflammatory autoimmune diseases, cancer cell
survival, and evasion of immune surveillance (66); overexpression of TLR on cancer
cells promotes tumor cell proliferation/growth while sustaining proinflammatory
cytokine-rich microenvironments (67). Regulated TLR activity is essential for effective
defense against a wide variety of microbial pathogens, including fungi (68), bacteria (69),
and viruses (70); moreover, deficiencies in TLR3 and TLR7 lead to more mucin
production in the mouse model of RSV infection (71, 72), and human TLR
polymorphisms increase disease severity in human individuals (73, 74). Consequently,
TLR function and regulation is particularly important in understanding RSV
pathogenesis.
8
During viral genome replication, double-stranded RNA (dsRNA) is recognized by
a group of PRRs known as retinoic-inducible gene I (RIG-I like) helicases (RLHs), and
initiates the cascade of signals towards type-I IFN production (75). Upon preferential
binding to dsRNA, RNA sensors like melanoma-differentiation-associated gene 5
(MDA5) or RIG-I associate with mitochondrial antiviral-signaling (MAVS) protein and
activate the RLH-MAVS pathway through interaction of their homotypic caspase
activation and recruitment domains (CARDs). RIG-I is stimulated by the uncapped 5’triphosphate end of viral RNA in Paramyxoviruses, Orthomyxoviruses, and
Rhabdoviruses (75), while MDA5 is reported to preferentially recognize Picornaviruses;
however, expression of RIG-I, MDA5, and MAVS are all upregulated upon RSV
infection (76). Further, MAVS and MDA-5 have been found to colocalize with RSVinduced inclusion bodies, enigmatic cytoplasmic structures that contain components or
RNA from RSV after infection in vitro (77). Lung fibroblasts, bone-marrow derived
macrophages, and conventional dendritic cells from MAVS-deficient mice were unable
to produce type-1 IFN upon infection with RSV A2 (78). Although its function in RSV
infections is unknown, a third RLH lacking the CARD region known as LGP2 acts
upstream of MAVS and is suspected to play a regulatory role in RIG-I-mediated
responses without requiring binding with MAVS (79-82).
Endosomal TLRs like TLR7 and TLR8 recognize single-stranded RNA and
guanosine-enriched oligonucleotides, while TLR9 is activated by bacterial or viral DNA
(83). Once activated, endosomal TLR7/8/9 utilize MyD88 for production of type I IFNs;
TLR3 recruits TRIF and mediates NF-κB transcription factor activity for induction of
9
IFN and TNFα. Alternatively, NF-κB and mitogen-activated protein kinase (MAPK)
signaling can be activated through a recently described cytosolic family of PRRs known
as Nod-like receptors (NLRs) (84). Several NLRs known as NOD receptors recognize
peptidoglycan-related molecules (84, 85) while a subset of NLRs known as Nucleotidebinding oligomerization domain, Leucine-rich Repeat and Pyrin domain containing
(NLRP) proteins are capable of sensing danger signals, viral DNA, bacterial toxins,
lysosomal damage, and reactive oxygen species released upon infection (86, 87).
Activation of NLR adaptor protein initiates inflammasome activation for caspase-1mediated cleavage of pro-IL-1β; subsequent release of bioactive IL-1β promotes cell
proliferation, inflammation, mucus secretion, and other anti-pathogen activities. The
inflammasome multiprotein complex tightly regulates caspase-1 activity and processing
of pro-IL-1β (87, 88), thus inflammasome activity may play a greater role in immune
responses than previously understood.
Together, the three main PRR pathways described, TLRs, RLRs, and NLRs,
collectively function in antimicrobial defense and activation of inflammatory responses.
Furthermore, PRR signaling is often required for the regulation of adaptive responses.
CD8+ T-cell expansion is dependent on TLR-MyD88 signaling in acute lymphocytic
choriomeningitis virus (89), RSV (90), and vaccinia virus infection (91). Deficiency of
MAVS in a mouse model of West Nile virus infection led to aberrant CD8+ T-cell
activity, accompanied by uncontrolled inflammation, enhanced immunopathology and
increased mortality (92). Lastly, NLRP3-/- and Casp-1-/- mice are more susceptible to
severe influenza virus infections than wild-type (WT) mice (93). Neonates that were
10
RSV-positive had elevated transcript levels of MDA5, RIG-I, TLR7, and TLR8,
Unfortunately for the host, microorganisms have developed unique strategies to
circumvent steps within PRR pathways and thus evade immune responses (94-99).
These are additional obstacles to the research and development of antimicrobial
treatments for against pathogens.
1.3 Immunobiology of RSV
The RSV single-stranded RNA genome encodes eleven proteins, including
structural components that make up the nucleocapsid, envelope and matrix. The most
promoter-proximal genes in sequence are nonstructural proteins 1 and 2 (NS1/2), which
are absent in the RSV virion but are detectable in infected cells (100, 101). Similar to
other pathogenic viruses, RSV has acquired several strategies for disrupting host antiviral
immune responses. RSV NS2 and other viral nonstructural proteins, such as hepatitis C
Virus NS3/4 and influenza NS1, have been previously demonstrated to have proteolytic
capabilities, subcellular localization signaling sequences, RNA synthesis regulation, and
interference with JAK-STAT and interferon-related protein production (102-106). The
NS1 and NS2 genes are more abundantly transcribed than promoter-distal genes due to
polar transcription (107). NS1 protein is detectable by immunoblot of lysates from RSVinfected cells in culture with polyclonal anti-RSV antibodies 6-8 hours post-infection
(p.i.), yet NS1 expression in vitro is not maximal until 12-16 h p.i (100, 108, 109). NS1
has been found to alter STAT1-mediated signaling, disrupt TRAF3 and IKKε signaling,
and suppress types I, II, and III IFNs (100, 110-112); RSV NS2 inhibits IFN-β through
11
direct interaction with RIG-I and promotes ubiquitin proteaosomal degradation of
STAT2, likely in conjunction with NS1 (96, 105, 113). Although not critical for
replication, NS1 seems to negatively regulate viral genome synthesis in early infection,
limiting stimulation of an immune response (109). Despite the multifunctional roles of
NS1, the physiological expression and subcellular localization of NS1 throughout an
infection is unknown.
Aside from the nonstructural proteins, RSV has alternative strategies for host
immune evasion. RSV interferes with assembly of immunological synapses between
dendritic cells and lymphocytes through unclear mechanisms and subsequently prevents
T-cell activation (114). Other RSV proteins appear to have cooperative roles in diverting
host immune responses; co-expression of RSV phosphoprotein (P) and nucleoprotein (N)
is required for the formation of inclusion bodies and subsequent decrease in type I IFN
(77). RSV glycoproteins G and F serve in viral attachment to the host cell and fusion to
neighboring cells but also stimulate secretion of pro-inflammatory cytokines (115). RSV
G can competitively interact with the chemokine CX3CR1 to inhibit fractalkine-mediated
responses and alter cellular recruitment and function (116); further, G induces
suppressors of cytokine signaling 1 and 3 (SOCS 1/3) to negatively regulate IFN
responses (97). RSV infection upregulates TLR4 (117) as well as the surface expression
of intercellular adhesion molecular-1 (ICAM-1), with gene expression paralleling that of
RSV F (117, 118). Given the various tactics for modulating the host response to RSV
infection, RSV proves to be evolutionarily well-adapted for persistent and recurrent
12
infection in humans; importantly, better understanding of these viral proteins may help
with the development of antiviral therapies or vaccines.
1.4 Therapeutic options for RSV infections
RSV vaccine attempts have been hindered because natural RSV infection poorly
induces a long-lasting, protective antibody production. Previous attempts to use
formalin-inactivated RSV failed in human trials. In contrast to the successful Salk
poliovirus vaccine, formalin-inactivation of RSV lead to unforeseen negative
consequences, leading to the death of two toddlers during the clinical study (14). It was
later determined that formalin-inactivation was immunogenic but failed to stimulate
innate PRR signals for antibody affinity maturation (119); as a result, immunopathology
mediated by CD4+ T-cells was exacerbated, accompanied with pulmonary eosinophilia,
deposit of low-affinity, non-protective antibodies in tissue, and pneumonia (14). Further,
RSV is a poor inducer of IFN because of the aforementioned viral strategies to escape the
antiviral immune response, therefore antiviral control of an ongoing infection is fairly
limited. This exemplifies how critical PRR-mediated viral recognition is for activation of
innate as well as adaptive immune responses to invading viral pathogens.
1.5 Inflammatory responses to RSV infection
Activation of proinflammatory responses leads to cytokine production and
release, resulting in fever, increased blood circulation and inflammation. Infiltration of
leukocytes into airways leads to tissue remodeling, airway hyperresponsiveness (AHR),
13
mucus hyper-secretion, and airway obstruction (120-122). Several well-described
cytokines, including interleukin-6 (IL-6), IL-1β, TNFα, RANTES (CXCL5), macrophage
inflammatory protein 1α (MIP-1α), IL-8, and others, increase in expression upon RSV
infection in airway epithelia (121, 123-126). Pro-Th2 cytokines, such as GM-CSF, IL-1α,
IL-25, and IL-33 are upregulated upon PRR stimulation through microbial and danger
triggers (127). Changes in cytokine profiles in the airway result in mucus hypersecretion
and structural changes in the airway walls.
Alterations in pulmonary microenvironment by viral and bacterial pathogens has
also been correlated with asthma exacerbations in young children. More than 300 million
individuals of all ages and ethnic groups are afflicted by asthma, a leading chronic
inflammatory respiratory disease (128). Over 7 million children under the age of 18 have
been diagnosed with asthma in the US alone and deaths associated with asthma are an
estimated 250,000 worldwide. Growing evidence suggests the asthmatic phenotype is
associated with increased susceptibility to virus-induced lower respiratory tract infections
(LRTI), particularly human rhinovirus (HRV) and respiratory syncytial virus (RSV)
(129). Inflammatory mediators, such as CCL5 (RANTES), IL-6, IL-1β, CCL3 (MIP-α),
and many others are induced upon pathogen exposure; pulmonary inflammation is
exacerbated by virus-induced recruitment of immune cells, particularly eosinophils,
macrophages, and neutrophils (121). The causal relationship between viral and bacterial
infections and asthma remains debatable (130), but the high frequency of co-infections
during asthma exacerbations strongly suggests respiratory pathogens contribute to
cellular and cytokine inflammatory responses observed in severe asthma. Of particular
14
importance, prophylactic administration of palivizumab during their first year of life
prevent recurrent wheeze by 80% in nonatopic children between ages 2 to 5; however, no
effect was observed among infants from families with medical history of atopy (131).
Thus, RSV infections contribute but are not a sole factor to the development of asthma
exacerbations.
1.6 Concluding remarks
The failure of the RSV vaccination trial exemplifies the importance of
understanding the molecular mechanism of the innate immune response to RSV and the
interplay of innate PRR signaling with adaptive immunity. With the absence of vaccine
and cost-effective prophylaxis, RSV infections recur seasonally and remain a constant
risk for various populations, spanning different age groups, predisposed conditions, and
immune statuses. In the following chapters, the properties of RSV will be defined and
the sequences of innate immune response to RSV will be explained. Chapters 4, 5, and 6
will present the findings on RSV nonstructural protein 1, the aged mouse model of RSV,
and inflammatory signaling in response to RSV infections respectively.
The purpose of the following studies was to examine how innate responses to
RSV infections are affected by viral protein NS1, age, and cytokine profiles. In
agreement with others in the field, our lab has repeatedly linked RSV NS1 with viral
subversion of the host-immune response but the mechanisms remained unclear (100, 108,
132). Determining the subcellular localization of the RSV nonstructural proteins could
help understand their role in host-immune disruption (100, 133-135). The decline in
15
adaptive and innate responses can be attributed to age, therefore an aged mouse model of
RSV was examined to study gene expression, pathology, and disease severity. Cytokines
upregulated upon RSV infection were examined in both young and aged mice to identify
age-specific cytokine production upon RSV infection.
16
Chapter 2: General Methods
Two experimental models were used in the following three aims: mammalian
epithelial cell culture and intranasally infected mice. These are well-established models
for studying RSV replication, protein interactions, and pathogenesis associated with RSV
infections (136-139). Cell culture investigations were performed in a biosafety-level 2
(BSL-2)-certified laboratory, with all cell culture, virus propagation, and viral titration
techniques performed in BSL2, HEPA-filtered cell culture hoods. The animal studies
were carried out at the USF Morsani College of Medicine, an AALAC accredited
institution with Animal Welfare Assurance number A-4100-01. The animals were
housed in polycarbonate cages covered with a HEPA-filtered lid with food and water
available ad libitum. All animals were housed in a temperature-controlled room
(22ºC±3ºC) and a 12 h light-dark cycle in a BSL-2-validated room. Intranasal infections,
euthanasia, and tissue collection were all performed in a BSL2, HEPA-filtered, ventilated
hood. The animals were checked daily to ensure they had adequate food and water and
that the rooms and cages were clean. Protective clothing, face masks, and gloves were
worn upon entry into the BSL-2 animal rooms and discarded upon exit. All equipment
and resources that had contact with RSV were sterilized with 70% ethanol, sporicidin,
10% bleach, or by autoclaving where applicable.
17
Standard molecular biology procedures for protein isolation, immunoblotting,
enzyme-linked immunostaining assay (ELISA), immunohistochemistry, and subcloning
were performed with aseptic technique following optimized protocols for the outlined
purposes. All nucleic acid experiments were performed under RNase- and DNAse-free
conditions, using diethyl pyrocarbonate-treated water where applicable. Cell pellets,
tissue, serum, and nucleic acid samples were maintained on ice and stores in non-frostfree freezers (freeze-thaw cycles were avoided). Control samples were included with
each experiment to confirm and ensure minimal variation between repetitions of a
procedure.
2.1 Cells and viruses
Human epithelial HeLa contaminant (HEp-2, CCL-23), African green monkey
kidney (Vero, CCL-81), and human alveolar epithelial (A549, CCL-185) were all
purchased from American Type Culture Collection (Rockville, MD) and grown in
standard Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetalbovine serum, L-glutamine, 100 IU/ml penicillin, and 100µg/ml streptomycin. The FlpIn™ T-Rex-293™ cell line (Cat. No. R780-07) was purchased from Invitrogen and
maintained in DMEM containing 10% tetracycline-free FBS (Clontech); as needed,
growth media was supplemented with antibiotics for clonal selection. Cell lines were
routinely tested for mycoplasma using the LookOut Mycoplasma PCR Detection Kit
(Sigma). Opti-MEM, supplemented with 2% FBS was used to maintain HEp-2 cell
cultures during virus propagation and semi-clarification. A master stock of RSV A2, an
18
A subtype, was obtained from the ATCC (Cat. No. VR1302) and working stocks of RSV
were propagated by infecting semiconfluent monolayers of mycoplasma-free HEp-2
cells. After substantial cytopathic effects were visible in approximately 80% of the
monolayer, supernatant media and cell debris were pooled, subjected to a single round of
freeze-thaw cycles, and semi-clarified by centrifugation (20 min at 1000 x g at 4°C);
aliquots were made, snap-frozen with dry ice, and stored at -80°C. Recombinant RSV
strains, rA2, rA2∆NS1, rA2∆NS2, rA2-His6NS1 were gifts of Dr. Michael Teng (USF)
and recombinant green-fluorescent protein-expressing RSV was provided by Dr. Mark E.
Peeples (Nationwide Children’s Hospital). Master stocks of recombinant RSV A2
expressing fusion protein from Line 19 (rA2-L19F) and clinical RSV isolate 2-20 were
generous gifts from Dr. Martin L. Moore (Emory University) and working stocks were
propagated in the same way as RSV A2 (41). Recombinant strains rA2∆NS1 and
rA2∆NS2 strains were propagated in Vero cells due to reduced infectivity in HEp-2 cells
(100, 140, 141). Mock-supernatant was derived using the same culture conditions as for
RSV propagation, but without infection.
Viral titers were determined using the methylcellulose-immunostaining method
previously described (111). Cell culture and homogenized tissue supernatants were
centrifuged at 4°C for 10 min at 300 x g and immediately serially diluted with FBS-free
media for infection in monolayer cells. HEp-2 or Vero cells were plated in 24-well cell
culture plates and grown to 80% confluency; supernatant dilutions were used to infect
cells and after an hour of infection at 37°C with frequent rocking, the inoculum was
replaced with 5% DMEM containing 0.8% methylcellulose, L-glutamine, and
19
penicillin/streptomycin/amphotericin B. After four to five days of growth, the overlay
was discarded and plaques were fixed with 80% methanol overnight at 4°C. Fixative was
discarded and plaques were blocked with 5% non-fat dry milk in phosphate-buffered
saline (PBS) at room-temperature for ½ h. A primary monoclonal antibody solution
containing 1:2000 dilution of mouse anti-RSV F (AbDSerotec, MCA490) was added to
plaques and incubated for 2 h at room temperature. After three washes of 5 min each, a
solution of horseradish peroxidase-conjugated anti-mouse secondary antibody diluted to
1:2000 was added to plaques for an additional one hour. Plaques were visualized using
4CN substrate (Kirkegaard and Perry Laboratories) and counted to calculate total number
of plaques per mL of supernatant, with appropriate correction for dilution.
2.2 Mice
Old (19-21 months) and young (2-3 mos) BALB/c mice were purchased from
Charles River Laboratories (Wilmington, MA) under a contractual agreement with the
National Institute of Aging. All animal work was approved by and performed in
accordance with the policies of the University of South Florida Institutional Animal Use
and Care Committee. Animals received a minimum of a week to acclimate to new
housing facilities upon arrival at the vivarium. Mice were observed for pathological
abnormalities, indications of masses, or dermatitis. For RSV infection, mice were lightly
anaesthetized with inhaled isoflurane and a single volume of 60 µl was given intranasally
dropwise at 106 plaque forming units (pfu) per mouse RSV A2, a dose previously shown
to yield plaque titers from lung homogenates (142, 143). For mucin studies, 105 pfu per
20
mouse of RSV 2-20 or rA2-L19F were given intranasally; doses were based on
references that characterized mucin production in BALB/c after infection with either 2-20
or rA2-L19F (40-42). Mice were euthanized by carbon dioxide inhalation on days 3-8.
For collection of bronchoalveolar lavage fluid (BALF), 1 ml of room-temperature PBS
containing 5 mM EDTA was intratracheally injected and recovered with two repetitions.
BALF was pooled, kept on ice, centrifuged at 300 x g for 10 min, and supernatant was
reserved in aliquots while cells were resuspended in pre-warmed (37°C) RPMI culture
medium containing 10% FBS for alveolar macrophage studies.
For histological and
protein/RNA analysis, the right bronchus was clamped with a hemostat and the left lung
was gently perfused with 4% paraformaldehyde (PFA) in PBS through the trachea. Right
lobes were quickly removed after perfusion, cut into 1 mm pieces, portioned for
RNA/protein, and snap-frozen on dry ice; the left lobe was immediately immersed in 4%
PFA for histological analysis. The sample size used in these experiments were calculated
based on pilot studies and were intended to minimize animal use by dividing the tissues
for multiple methods of measure.
2.3 Protein analysis
Lung tissue was weighed and homogenized with glass Dounce homogenizers in 5
volumes (wt./vol) of pre-chilled FBS-free DMEM; whole mammalian cell pellets were
collected by centrifugation and resuspended in homogenization lysis buffer containing
10mM Tris-HCl, 150 mM NaCl, 1% NP-40, 10% glycerol, 5 mM EDTA, and protease
inhibitor cocktail. Lysed tissues or cell supernatants were aliquoted, frozen immediately
21
on dry ice, and subjected to a single freeze-thaw cycle. Upon thawing on ice, protein
concentrations were measured using the bicinchoninic acid (BCA) protein assay (Thermo
Scientific Pierce) and calculated for either immunoprecipitation, western blot, or ELISA
using modified protocols recommended by AbCam.
Whole-cell lysates (500 µg) were pre-cleared with agarose beads (Thermo
Scientific Pierce) and incubated overnight with primary immunoprecipitating antibody.
Agarose beads were added to samples and antibody-bead complexes were mixed by
agitation for an additional 4 h at 4°C. Whole-cell lysates or immunoprecipitated proteins
were heated in 2X protein denaturing Laemmli buffer (4% SDS, 20% glycerol, 10% 2mercaptoethanol, 0.004% bromophenol blue, 0.125 M Tris HCl) for 5 min at 95 °C.
Lysates were run on 4-20% pre-cast sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) gels (Mini-PROTEAN® TGX™, BioRad) and transferred
to nitrocellulose membranes for immunoblotting. Blots were blocked for ½ h with 5%
nonfat dry milk and 0.1% Tween-20 Tris-buffered saline at room temperature. All
primary antibodies were diluted with blocking solution and incubated with blots for
overnight at 4°C on a rocker. The following day, blots were washed thoroughly, probed
for an additional 2 h at room temperature with secondary antibodies diluted in blocking
solution, washed again, then visualized with ECL (ThermoFisher Scientific, SuperSignal
West Femto).
Protein quantification via ELISA was conducted per the manufacturer’s
instructions. In brief, 96-well ELISA plates were coated with capture antibody overnight,
washed thoroughly, blocked with supplied assay diluent, and then diluted samples of
22
lysates, plasma, or BALF were added to plate. Plates were washed after each step before
detection antibody, Avidin-HRP, and substrate solution were added to wells; 2N H2SO4
stop solution was added and plates were immediately read at 450 nm. Standard curves
were derived from dilutions of the cytokine standards provided by the kits. Samples were
run in triplicate and dilutions were optimized for sample types.
2.4 Nucleic acid analysis
Large quantities of endotoxin-free plasmid DNA was isolated using PureLink®
HiPure Filter Plasmid Kits (Invitrogen) and resuspended in Tris-EDTA (TE) buffer.
DNA was quantified using a NanoDrop® ND-1000 spectrophotometer and stored in
aliquots at -20 °C. Subcloning methods were performed on circularized plasmid DNA
using restriction enzymes (New England Biolabs, MA) and customized primers designed
with restriction enzyme sequences. PCR inserts and plasmid constructs were confirmed
with gel electrophoresis and sequences were analyzed at the Moffitt Cancer Center
Molecular Genomics Core (Tampa, FL). Transfection with Lipofectamine 2000
(Invitrogen) of plasmid DNA was performed per manufacturer’s instructions.
Transfection reagent or plasmid DNA was diluted separately in serum-free Opti-Mem for
5 min before combining to make DNA-liposome complexes. After 20 min at room
temperature, complexes were added to 80-90% confluent cell cultures and incubated at
37 °C for 16 h. For transient transfection, cells were washed with cold PBS and
harvested for protein or RNA extraction. For stable cell-line selection, cells were
maintained at 30-50% confluency in complete growth medium containing selective
23
antibiotics for 2-3 weeks, replacing the medium every two days. Protein expression was
confirmed by western blot and clones were propagated and stored at -80°C after freezing
in DMEM containing 30% FBS and 10% DMSO.
RNA was isolated from cell or lung tissue using TRIzol® Reagent (Invitrogen)
and chloroform/isopropanol precipitation method. After resuspension of precipitated
RNA pellets with TE, RNA was treated with DNase I (Roche, Germany) for 20 min; to
remove excess enzyme and buffer, samples were processed with RNeasy Mini Kit Clean
Up (Qiagen) columns. 1 µg of total RNA was converted into cDNA using reverse
transcriptase enzyme (Fermentas Maxima First Strand cDNA synthesis for qRT-PCR,
Thermo Fisher Scientific) and cDNA template was diluted 1:5 with DNase-free water,
then further diluted with 4X Yellow Sample Buffer for use with DyNAmo Flash SYBR
Green qPCR reagent (Thermo Fisher). A master PCR mix was prepared using 2.5 µl of
2X Master mix, 0.15 µl of appropriate forward or reverse primers (stock concentration 10
µM), and 1.2 µl of DNase-free PCR water. 1 µl of diluted cDNA template was added to 4
µl of master PCR mix in individual wells of a 384-well clear PCR reaction plate (Thermo
Fisher). Samples, including no reverse transcriptase (NRT) and no template (water)
controls, were run in four replicates using BioRad CFX284™ Real-time PCR detection
system. Data was analyzed using delta cycle threshold (∆Ct) and delta-delta Ct (∆∆Ct)
derivations by comparing target gene expression to relative expression of endogenous
housekeeping genes, as previously described (144).
24
2.5 Immunocytochemistry and immunohistochemistry
A549 cells were seeded in chambered coverglass slides (Nunc® Lab-Tek® II,
Thermo Fisher) at 60% confluency and 24 hours later infected with rA2, rA2∆NS1, or
rA2∆NS2 RSV strains at a multiplicity of infection (MOI) of 0.1 and 12-24 h later, cells
were stained for mitochondria (MitoTracker® Red CMXRos, Molecular Probes
Invitrogen), fixed with 4% paraformaldehyde, permeabilized with cold 0.2% Triton X100 in PBS (PBST) for 10 min, washed thoroughly, then stained with primary antibodies
diluted in PBST containing 2.5% bovine serum albumin (BSA). Zenon® Alexa® Fluor
conjugation was conducted per the manufacturer’s instructions (Invitrogen); Alexa 488
was conjugated to anti-His6NS1 antibody and anti-MAVS antibody was conjugated to
Alexa 647. Cells were viewed by Leica TCS SP2 laser scanning confocal microscopy. A
minimum of ten images was collected per experimental group and experiments were
performed in triplicate.
Histological analyses were performed using modified IHC protocols from
AbCam, Inc. After euthanasia, lungs were gently perfused, removed, and immersed in
4% PFA for 6 h at 4°C. An equal volume of 30% sucrose in PBS was added to the vials
and fixation continued for 16 h at 4°C. After fixing, the solution was replaced with 30%
sucrose in PBS and tissues were kept at 4°C for an additional 24 h before gently blotting
for OCT medium embedding (Tissue-Tek®) and snap-freezing on dry ice. A minimum of
5 µm longitudinal sections were mounted on glass slides (SuperFrost™ Microscope)
using a cryomicrotome; sections were made throughout different depths of the tissue to
span the dorsoventral axis. Sections were dried on slides in a humidified chamber,
25
washed with PBS, then either heated at 95 °C in unmasking solution for 20 min for
antigen retrieval for IHC or directly stained with periodic-acid Schiff (PAS) or
hemaxotoxylin-eosin (HE).
26
Chapter 3: RSV-mediated Evasion of Host Innate Immunity
Note to reader
Portions of these results have been previously published (Boyapalle et. al, 2012)
and are utilized with permission of the publisher.
3.1 Introduction
Respiratory syncytial virus fails to stimulate a protective antiviral interferon (IFN)
response and consequently, re-infections with RSV are common among individuals,
particularly the elderly (5). The Mohapatra lab has previously observed upregulation of
the IFN-inducible genes STAT1, IRF1, IRF3, IRF7, and IFN-β after silencing RNA
(siRNA) was used to downregulate the virulence factor, nonstructural protein 1 (NS1) in
A549 cells infected with WT RSV (145). Prophylactic delivery of siRNA against NS1
regenerated the early antiviral immune response both in vitro and in vivo, and
significantly attenuated RSV infection. This suggests that RSV NS1 plays a significant
role in viral propagation during the early infection; however, few studies have examined
the individual role of RSV NS1 in the absence of an active viral infection or study the
subcellular localization of NS1 in culture. Herein, we demonstrate subcellular
27
localization of RSV NS1 serves function in the disruption of host antiviral immune in
response to RSV promotes RSV survival.
During viral genome replication, double-stranded RNA (dsRNA) is recognized by
PRRs such as endosome-associated Toll-like receptors (TLRs) and cytoplasmic receptors,
and initiates the cascade of signals towards type-I IFN production (75). Upon
preferential binding to dsRNA, retinoic-inducible gene-1 (RIG-I)-like receptor helicases
(RLHs) like melanoma-differentiation-associated gene 5 (MDA5) or RIG-I interact with
mitochondrially-associated adaptor protein MAVS and homodimerization of caspase
activation and recruitment domains (CARDs) leads to activation of downstream
molecules such as IκB kinases (IKKs) and TANK-binding kinase 1 (TBK1). This
mediates phosphorylation and degradation of IκB, thereby permitting nuclear
translocation of transcription factors NFκB, IRF3, or IRF7 for the induction of type-I
IFNs and proinflammatory cytokines (146, 147). Signaling cascades leading to cytokine
production are often redundant, involving crosstalk among components of the TLR and
RLR families; recent evidence also suggests RIG-I activity is upstream in the activation
of NOD-like receptors (NLRs), inflammasomes, and antiviral IL-1β production (148).
Importantly, RLH signaling is tightly regulated by host IFN-inducible ubitiquitin
ligase (149) and components of the autophagosome (150) to prevent aberrant production
of cytokines. Viruses have evolved virulence factors to evade host antiviral defenses,
through competitive interaction with antiviral accessory proteins or posttranslational
modification sites (95, 151), proteolytic cleavage of receptors or functional domains
(152), inhibition of NFκB or IRF3 activation (153), and manipulation of host-RLH
28
regulatory proteins. Influenza A virus utilizes its nonstructural protein 1, which has no
sequence homology to the RSV NS1, to target RIG-I to modulate IFN and IL-1β
production in lung epithelial cells(154). Other viruses target components of the RLRsystem to evade recognition by the antiviral immune response, including hepatitis C virus
nonstructural protein 3/4 A (NS3/4A), which directs proteolytic cleavage of MAVS to
prevent MAVS oligomerization and downstream signaling (152).
Importantly, RSV has more than one strategy for interfering with antiviral
immunity. The RSV G protein has cysteine-rich repeats that serve to dampen TLRmediated inflammatory cytokine and IFN production during early RSV infection(155);
G-protein directed modulation of suppressors of cytokine signaling (SOCS) proteins
SOCS1 and SOCS3 also inhibits type-I IFN production (97, 156). Although dispensable
for RSV replication, RSV NS1 and NS2 aid in viral persistence by disrupting host IFN
production. NS2 inhibits IFN-β through direct interaction with RIG-I (105) and
decreases TRAF and STAT2. RSV NS1 and NS2 genes are located in the 3’ region of
the negative-sense RNA viral genome and are more abundantly transcribed than
promoter-distal genes due to polar transcription (107). NS1 protein is detectable by
immunoblot with polyclonal anti-RSV antibodies in RSV-infected lysates 6-8 hours postinfection, yet protein expression is not maximal until 12-16 hours p.i (100, 108, 109).
RSV NS1 reportedly inhibits nuclear translocation of STAT1, IRF3 and NFκB (100,
141). Recent supporting evidence with two-dimensional differential gel electrophoresis
found RSV NS1 targets STAT1, but not STAT2 (112), and both genes are effective in
decreasing levels of the signal transducer, TNF receptor-associated factor 3 (TRAF3);
29
although NS1 more so than NS2 (157). Although not critical for viral replication, NS1
seems to negatively regulate synthesis of the viral genome in early infection to limit
stimulation of an antiviral response (19). The translocation of RSV nonstructural
proteins to the nucleus suggests that they may regulate transcription, similar to the
transcriptional suppressors that disrupt IFN or Jak-STAT activation as reported in other
RNA viruses (13, 20-22). Despite years of research on the multifunctional roles of NS1
and its potential to translocate into the nucleus, the temporal expression and cellular route
of NS1 throughout an infection are still unknown. In our investigations, we questioned
whether subcellular localization of NS1 could serve in immune subversion and whether
NS1 functioned within the cytoplasmic region like RSV NS2.
MAVS-deficient mice are reportedly unable to produce type-1 IFN upon infection
with WT RSV (78), indicating the importance of MAVS to antiviral immunity to RSV.
Since RSV NS1 was not found to directly interact with RIG-I, RSV NS1 could negatively
regulate alternate cytosolic antiviral signaling receptors such as MAVS or the recently
discovered cytoplasmic RNA helicase, LGP2. LGP2 is reported to play important roles
in modifying and sequestering viral RNA upstream of MDA5 and RIG-I (158, 159).
While the role of LGP2 in RSV infection is not fully clear, LGP2 is known to lack the
CARD and therefore acts upstream of MAVS without directly interacting with it.
Although its regulatory function remains controversial, LGP2 appears to modulate the
efficiency of viral RNA recognition by RIG-I and MDA5, thereby regulating downstream
IFN induction (82). LGP2 expression is stimulated by dsRNA, including the synthetic
dsRNA mimic, polyinosinic: polycytidylic acid (poly I: C), and the LGP2 gene contains
30
promoter motifs that are highly responsive to IRF and STAT molecules, particularly
IRF3 in human LGP2 (160).
We hypothesized that RSV NS1 regulates a number of antiviral genes in the host
and is a promising target for prophylaxis against RSV pneumonia in highly susceptible
populations, such as infants, the immunocompromised, and elderly individuals. We
hypothesized that RSV NS1 is a potent inhibitor of MAVS and disrupts the binding of
RIG-I to MAVS to prevent development of an antiviral response. To demonstrate this,
we used antibodies to immunoprecipitate and track in vitro the localization of NS1 in
epithelial cells during transfection and RSV infection. We also employed a novel cellpermeable fluorophore that utilized polyhistidine-nickel chelating properties for live, in
vivo subcellular tracking of recombinant histidine-tagged RSV NS1 in infected
mammalian cells. Since transient transfections activate the IFN response (161) and other
viral proteins could alter the cellular localization of NS1 (162), we wanted to examine the
role of inducible NS1 in a human cell model of RSV infection. For inducible expression
of NS1, we used Invitrogen’s Flp-In T-REx HEK 293 cell line, which stably expresses
the TetR repressor and a FRT-Zeocin-resistance target for recombinase-mediated site
integration. This cell line is a derivative of HEK 293, which is susceptible to RSV
infection (163) and highly-transfectable. Site-directed integration of the FLAG-tagged
human codon-optimized RSV NS1 (3XFLAG-NS1) sequence into the host cell genome
yielded a homogeneous, tet-inducible NS1-producing, hygromycin-resistant population
of cells and eliminated the need for individual clone selection. Constitutive expression of
the stably integrated TetR gene suppresses the expression of the transgene to basal levels
31
in the absence of Tet; subsequently, the inducible-cell line model provided a strategy to
study NS1 in the absence of RSV infection for temporal and controlled expression.
Collectively, we have used several approaches to study the subcellular localization of
RSV NS1, in artificial transfection and live, active RSV infections of mammalian cells.
3.2 Materials and methods
The following methods are specific to Chapter 3 for investigating the early innate
immune responses to RSV infections in mammalian cell culture. These methods were in
addition to the general methods previously described in Chapter 2.
3.2.1 Vector construction and establishment of the stable cell line, Flp-In
3XFLAG-NS1
The human codon-optimized sequence of RSV NS1 (145) was amplified by PCR
with customized primers containing 5’-HindIII and 3’-EcoRV restriction enzyme (RE)
sequences. The parental expression vector p3XFLAG-CMV™-10 (pFLAG, Sigma) was
linearized with HindIII and EcoRV and the NS1 sequence was inserted using the Quick
Ligation kit (New England Biolabs). The final plasmid vector product was denoted as
p3XFLAG-NS1 (pNS1). A secondary set of primers containing 5’-BamHI and 3’-XhoI
RE sites were designed to span the length from 5’-end of the 3XFLAG to the 3’-end of
the RSV NS1 sequence; the double RE digest product of p3XFLAG-NS1 (Figure 1A)
was then PCR-amplified and inserted into the mammalian tetracycline-inducible
expression vector, pcDNA™5/FRT/TO (Invitrogen), thus yielding pcDNA5-3XFLAG32
NS1. Flp-In™ T-Rex-293™ (Flp-In HEK 293) cells, maintained in 10% DMEM with
100 µg/ml Zeocin and 15 µg/ml blasticidin, were co-transfected with pcDNA5-3XFLAGNS1 and Flp-recombinase containing vector, pOG44 (Invitrogen). The Flp-In HEK 293
are a derivative of human embryonic kidney cell line that were adenovirus transformed
and frequently referred to as HEK 293. Transfectants that retained the antibiotic
resistance genes were selected with a combination of hygromycin B (100 µg/ml) and
blasticidin (15 µg/ml) over a period of 2-3 weeks and the final cell line was named as
Flp-In 3XFLAG-NS1 cells (Figure 1B). The expression of 3XFLAG-NS1 was confirmed
by qRT-PCR and western blot analysis as described below.
3.2.2 Protein analysis
20 x 106 A549 cells were transfected with either p3XFLAG-NS1 (pNS1) or
pFLAG vector alone and the mitochondrial fraction was obtained using a kit
(ThermoFisher Scientific). Alternatively, A549 cells were infected with rA2 His6NS1
RSV at MOI of 1 or mock infected then lysed with lysis buffer for collection of wholecell lysates. Protein in mitochondrial fractions or whole-cell lysates was measured using
the BCA protein assay. Whole-cell lysates were subjected to immunoprecipitation with
anti-MAVS (3 µg/500µg of total protein, Santa Cruz) or anti-FLAG (3 µg/500µg of total
protein, Sigma) then analyzed with western blot. Blots were probed overnight at 4°C
with the following antibody solutions in blocking solution for the detection of target
genes: rabbit anti-FLAG antibody (1:2000, Sigma), mouse anti-MAVS (1:2000, Santa
Cruz), anti-COX-IV (1:1000, Cell Signaling), anti-RIG-I antibody (1:1000, Cell
33
Signaling) or polyclonal rabbit antiserum against recombinant His6-NS1 (1:1000) were
detected by ECL (ThermoFisher Scientific, SuperSignal West Pico).
3.2.3 Tetracycline induction
To induce NS1 expression, stably integrated pcDNA5-3XFLAG-NS1 Flp-In™ TRex-293™ cells (Flp-In 3XFLAG-NS1 cells) were incubated with increasing
concentrations (10 ng/ml -10 µg/ml) of tetracycline and then harvested at 24 h posttreatment. Alternatively, cells were incubated with 1 µg/ml of tetracycline and cell
pellets were harvested at different time points for western blot and detection with mouse
anti-FLAG (Sigma) antibody; portion of cell pellets were reserved for RNA isolation and
qt-RT-PCR analysis for NS1 expression. Alternatively, induction of the gene of interest
was confirmed by immunocytochemistry. Paraformaldehyde-fixed cells were
permeabilized with 0.1% Triton X-100 containing PBS prior to 2h incubations with
mouse anti-FLAG primary antibody, followed by Alexa Fluor 488-conjugated anti-mouse
secondary (Invitrogen). As experimental controls, tetracycline treatments, cell harvests,
and immunocytochemistry were performed on Flp-In HEK 293 (Flp-In 293) cells that
were not stably transfected with pcDNA5-3XFLAG-NS1.
3.2.4 Live-cell imaging of His6-tagged NS1 in RSV-infected cells
A nickel-conjugated fluorophore composed of two cys-nitrilotriacetic acid
moieties and dibromobimane (Ni2+-NTA2-BM) (164, 165) was used to label rA2-His6NS1-infected live HEp-2 cells. HEp-2 cells were seeded in 35 mm glass-bottom confocal
34
culture dishes and infected with rA2-His6-NS1 at an MOI of 2 for 10 h in complete
growth DMEM media (cDMEM). To prepare the fluorophore, 50 μM NTA2-BM was
complexed with a 10-fold molar excess of NiSO4. The complex was added to
ammonium bicarbonate buffer-washed RSV-infected cells that were stained
simultaneously for mitochondria and nuclei (MitoTracker Red and DRAQ5, Molecular
Probes Invitrogen). Figure 2 illustrates the detailed steps for rA2-His6NS1 RSV
infection, fluorophore preparation, and staining. Laser confocal fluorescence microscopy
was used to visualize fluorophore-tagged NS1 in cells at wavelengths of 405, 563 and
633 nm. Differential interference contrast (DIC) images of the cells were also obtained.
A series of 0.5 μm Z-stack images was collected of each specimen and experiments were
performed in triplicate; semi-quantitative analysis was performed using the JACoP
ImageJ plugin which provides Pearson's and Mander's overlap coefficients
(http://rsb.info.nih.gov/ij/plugins/track/jacop.html).
35
3.3 Results
3.3.1 RSV NS1 localization to the mitochondria in vitro
MAVS-deficient mice reportedly fail to produce IFN during WT RSV infection
(78), suggesting the MAVS-related signaling is necessary in innate immunity but may
also be a target for the virus to disrupt host antiviral responses. Using a vector containing
the 3XFLAG sequence fused to the amino-terminus of RSV NS1 (pNS1), the NS1 was
detectable in mitochondrial fractions as shown in Figure 3A. Moreover, NS1 was present
in both nuclear and cytosolic fractions (Figure 3B & C). To examine how the inducible
cell line Flp-In 3XFLAG-NS1 is regulated with tetracycline, increasing doses were given
to cells for single time point harvest at 24 h or cells were collected at various time points
after treatment with a single dose of 1μg tetracycline/ml. Induction of 3XFLAG-NS1
was found to be both time- and dose-dependent (Figure 4A). We found that tetracycline
specifically induced NS1 as compared to Flp-In 293 that lack the RSV NS1 sequence,
confirming NS1 expression was unaltered by the inducible subcloning or antibiotic
selection (Figure 4B). We observed widespread distribution of tetracycline induced-RSV
NS1 in mammalian cells using immunocytochemistry with a FLAG-specific antibody and
Alexa Fluor-488-conjugated secondary antibody (Figure 3C). Green-stained particles
were found throughout the cell, including the DAPI-stained nucleus (blue).
We further examined RSV NS1 localization to the mitochondria using NS1
antibody-mediated confocal microscopy analysis and gold-antibody nanoparticles
analyzed with electron microscopy (data not shown; published in Boyapalle et al. 2011).
To determine whether transfection and activation of IFN-signaling from liposome
36
treatment (161, 166) could alter the subcellular localization of RSV NS1, we used a cellpermeable fluorophore to track subcellular localization of a hexahistidine-tagged NS1 in
live, RSV-infected cells. HEp-2 cells were infected with recombinant RSV containing a
hexahistidine-tagged NS1 (rA2 His6-NS1) and at 10 h p.i., a metal-chelating fluorophore,
Ni2+-NTA2-BM was added (Figure 5A). Mitochondrial and nuclear dyes were added to
the cells prior to laser fluorescence confocal microscopy analysis. Cells were maintained
in cell culture growth conditions and 0.5 μm Z-stack images were collected to accurately
assess whether His6-NS1 was found on the surface or inside cellular compartments. To
assess specificity of the metal-chelating fluorophore, recombinant rA2 RSV lacking
polyhistidine-tagged NS1 was used to infect HEp-2 cells. Representative images at 120X
magnification show NTA2-BM was only present in rA2 His6-NS1 RSV-infected cells,
and not rA2 (Figure 5B). The merged image illustrates regions of the cell where
mitochondrial stain and NTA2-BM overlapped, producing yellow-to-orange pixels. The
extent of green and red colocalization was analyzed using Renyi Entropy AutoThreshold
ImageJ plugin (Landini) and is based on RGB channel separation, binary image
conversion, and threshold particle quantification. An average of ten individual cells per
field of view (total of 10 images collected at 120X) were used to calculate the Pearson's
correlation and Mander's co-occurrence coefficients using the JACoP plugin (Bolte &
Cordelieres). Pearson’s coefficient assesses how well particles relate to each other by
correlation in positive or negative values and such that 0 indicate no relationship exists.
Mander’s co-occurrence coefficients measure intensity of an area of colors thus M1
signifies how much the mitochondria overlapping His6-NS1, while M2 indicates the
37
overlap of His6-NS1 to mitochondria(167). These results show that His6-NS1 is localized
with mitochondria in rA2 His6-NS1-infected cells. At 10 h p.i., approximately 70% of the
NTA2-BM-stained His6-NS1 was found to overlap the mitochondria, according to the M2
(Figure 5C) and further supports our hypothesis that RSV NS1 is partially localized on
the mitochondria.
3.3.2 RSV NS1 binds to mitochondrially-associated MAVS and prevents
interaction with RIG-I
Cross-reactivity with a second nonstructural protein, NS2, and poor antigenicity
of NS1 has previously hindered the production of an NS1-specific antibody for use in
protein-protein interaction studies. To contend with this obstacle, we used FLAG-tagged
NS1-transfected cells and the recombinant rA2 His6-NS1 RSV strain and His6-NS1
antibody for specific detection of NS1 (140). A549 were transfected with pNS1
expressing 3XFLAG-NS1 or control vector, pFLAG expressing 3XFLAG, and 24 h posttransfection, cell lysates were immunoprecipitated with anti-MAVS antibody and
analyzed by western blot.
3XFLAG-tagged NS1, but not 3XFLAG alone, was found to
precipitate with MAVS (Figure 6A); in contrast, co-immunoprecipitation of RIG-I
decreases at 12 h and 24 h with expression of 3XFLAG-NS1 in pNS1 transfected lysates
(Figure 6B). To examine RIG-I and MAVS binding in RSV infection, HEp-2 cells were
infected with mock-supernatant or with RSV strains rA2ΔNS1 or rA2 His6-NS1, and
lysates were examined by western blot for relative protein level of RIG-I, MAVS, and
NS1 (Figure 6C). RIG-I expression was found to be substantially reduced in mock- and
38
rA2ΔNS1-infected cells as compared to cells from rA2-infection. The same lysates were
then used for immunoprecipitation with RIG-I-antibody. RIG-I was found to precipitate
MAVS as well as NS1 in rA2 RSV infected cells but not mock-infected or cells infected
with ∆NS1 RSV. Notably, the amount of RIG-I precipitated using anti-RIG-I was
substantially lower in mock- and rA2ΔNS1 infected cells, which was similar to the
immunoblot of whole-cell lysates prior to immunoprecipitation. Densitometry was
performed on the western blot images and the ratio of MAVS to RIG-I (MAVS/RIG-I)
was calculated (Figure 6D). More MAVS was precipitated in rA2ΔNS1-infected cells as
compared to rA2, which had the lowest ratio of MAVS/RIG-I.
3.3.3 RSV NS1 expression kinetics are parallel with IFN-regulator LGP2
The kinetics and temporal expression of RSV NS1 were first examined with qRTPCR and western blot analysis and compared to mock or ΔNS1-infection (Figure 7A &
B). Expression was normalized to mock-infected cells and NS1was absent in mock and
rA2ΔNS1 infections while NS1 expression gradually increased with time from 16 h to 40
h with rA2. Expression of the IFN-regulator protein, LGP2, was also assessed with qRTPCR and LGP2 expression paralleled that of RSV NS1 in rA2 RSV infected cells (Figure
7C). Expression was below detection on western blot and could not be compared (data
not shown). Alternatively, Flp-In 3XFLAG-NS1 cells were incubated with various
concentrations of tetracycline (tet) and harvested for western blot analysis 20 h posttreatment. Lysates were probed for LGP2, FLAG, and β-actin. LGP2 was detectable at
all tet concentrations, but highest at 10 μg/ml when 3XFLAG-NS1 was the highest
39
(Figure 7D). Optical density was quantified and background and β-actin quantities were
substracted to yield values for comparing normalized expression of LGP2 and NS1, as
shown in D. In a dose-dependent manner, LGP2 expression increased with tetracyclineinduced 3XFLAG-NS1 expression in the Flp-In 3XFLAG-NS1 cell line.
3.3.4 Induction of NS1 expression restores RSV replication and transcription
of viral RNA
Flp-In 3XFLAG-NS1 cells were incubated with 10 μg tetracycline /ml for 4 h
prior to infection with rA2ΔNS1 at a MOI of 1 and infection proceeded for an additional
20 h in culture medium containing tetracycline (10 μg/ml). Control samples received an
equal volume of vehicle, 1% ethanol. Cells were harvested for isolation of RNA and
examined for gene expression of NS1, LGP2, and RSV N at 24 hours post-infection
(hpi). NS1 expression increased only when tetracycline was added (Figure 8A); in a
similar manner, LGP2 expression was maximal in the tetracycline-treated rA2ΔNS1infected cells as compared to mock-infected or vehicle-treated rA2ΔNS1-infected cells
(Figure 8B). RSV N expression was significantly higher in tetracycline-treated
rA2ΔNS1-infected cells than in cells given vehicle instead of tetracycline (Figure 8C).
Plaque assays were attempted but plaques were not detectable due to low infectivity and
viral titers from rA2ΔNS1 24 hpi (data not shown).
40
3.4 Discussion
Innate antiviral signaling is an essential defense mechanism against invading
pathogens; consequently, viruses have evolved various strategies to evade host immunity
and inhibit production of antiviral cytokines. RSV NS1 is recognized as a key virulence
factor in RSV infectivity although the mechanism of host antiviral disruption remains
incompletely understood. In these studies, we found that mitochondrial localization of
NS1 could disrupt the central adaptor protein involved in RLH-signaling, MAVS. In
multiple instances using transient transfection, a stably-integrated inducible cell line, and
live imaging of RSV infection, we observed localization of NS1 to the mitochondria;
therefore, the likelihood NS1 served antagonist roles against IFN responses while near
the mitochondria was not beyond possibility. Upstream interaction with RIG-I was found
to be impaired when RSV NS1 was transiently expressed in uninfected A549 cells. The
degree of MAVS/RIG-I interaction was substantially enhanced during rA2ΔNS1
infections compared to rA2 infections, suggesting that the presence of NS1 hinders the
interaction of RIG-I and MAVS. Disruption of RIG-I/MAVS complexes through
competitive inhibition of MAVS binding would be an effective strategy for disrupting
RLH-signaling in RSV infections; however, the precise domains permitting binding
between NS1 and MAVS have yet to be determined. Lastly, we observed upregulation of
a lesser-known RLH, LGP2, when NS1 expression is abundant. Recognized as an
upstream modulator of dsRNA recognition, LGP2’s role in RSV infection has not been
characterized. Our investigations suggest that RSV NS1 induces LGP2 expression, both
in RSV-infected cells and in the NS1-inducible Flp-In 3XFLAG cell line without active
41
RSV infection. Further, we found upregulation of LGP2 in rA2ΔNS1 RSV infected cells
when 3XFLAG-NS1 was induced with tetracycline; as an indicator of RSV replication
efficiency, RSV N gene expression was also examined and found to be highest when
RSV NS1 was induced prior to rA2ΔNS1 infection. This is further evidence to suggest
that RSV NS1 promotes RSV survival and therefore, may be a promising target for
antiviral strategies.
Through these studies, we have identified possible mechanisms for RSV NS1mediated disruption of MAVS interactions and downstream signaling. The localization
of proteins often reveals their function and we hypothesized that mitochondrial
localization of NS1 disrupted the mitochondrially-associated protein MAVS since
MAVS-deficient mice failed to produce IFN upon RSV infection (78); we sought to
understand why we frequently observed NS1 on the mitochondria and what role it could
have prior to inhibition nuclear translocation of transcription factor such as STAT1 or
IRFs. RIG-I disruption is frequently a viral strategy for disrupting host immunity and we
found MAVS-RIG-I interactions to be diminished in the presence of NS1. In contrast to
RSV NS2 which was found to directly interact with RIG-I, we predict NS1 binds to
MAVS and serves to compete with RIG-I. Alternatively, LGP2 sequesters dsRNA so
that it cannot interact with MDA5 and RIG-I (168). Dysregulated LGP2 activity could
potentially alter the efficiency of dsRNA recognition and may hinder RLH-signaling
upstream of RIG-I, similar to what was observed with other Paramyxoviruses,
particularly Parainfluenza accessory V protein which binds directly to LGP2 to prevent
RIG-I signaling (169); whether these specific protein-protein interactions occurs in RSV
42
remains to be investigated. Although beyond the scope of this investigation, the nuclear
localization of NS1 remains to be examined further and will likely provide a better
understanding of RSV NS1’s function as a potential transcription regulator of IFNinducible genes.
The multifunctional activity of RSV NS1 promotes RSV survival and
pathogenesis; a summary of the three NS1-mediated strategies for host immune
disruption are illustrated in Figure 9. As a virulence factor, RSV NS1 is a good target for
antiviral therapies and as proof-of-concept, silencing of NS1 attenuated disease and
infection in previous studies (145, 170, 171). Alternatively, live-attenuated, deletion
mutant strains of RSV demonstrate promise as vaccine candidates and may provide
preventive therapy high-risk populations. Together, these results demonstrate that RSV
NS1 is important as an antagonist of innate immune responses and provide further
evidence that the host innate immune system is fragile, susceptible to virus-mediated
disruption, and needs further investigation to better understand viral pathogenesis.
43
44
Figure 1. Construction of FLAG-tagged human codon-optimized vectors for
transient transfection and inducible cell line construction.
Vectors were digested with the indicated restriction enzymes and PCR-amplified
products were inserted using the Quick Ligation kit. Expression was confirmed by PCR
and sequence analysis, and tested by immunoblot after transfection in mammalian cells.
(A) pNS1 was constructed by inserting the RSV NS1 sequence on the carboxyl terminus
of the 3XFLAG sequence in the parental vector, pFLAG. (B) pCDNA5-3XFLAG-NS1
was the coexpression vector used for stably integrating the 3XFLAG-NS1 sequence in
Flp-In 293. The resulting cell line, Flp-In 3XFLAG-NS1, was selected with antibiotics
and proved to be free of mycoplasma using a test kit.
45
Figure 2. Procedure for live-cell tracking of polyhistidine-tagged RSV infection.
The protocol outline is modified from Krishnan et al. 2007 and altered to accommodate
fluorescent subcellular differential stains for identifying mitochondria (MitoTracker Red)
and nucleus (DRAQ5). The dibromobimane fluoresces after excitation at 405 nm. Laser
scanning at 563 and 633 nm and differential interference contrast (DIC) were also used to
collect a series of 0.5 µm Z-stack images for each specimen and experiments were
performed in triplicate.
46
Figure 3. Subcellular localization of RSV NS1 in transiently transfected A549.
(A-C) A549 cells were transfected with pNS1 or control vector pFLAG and (A)
mitochondrial, (B) nuclear, and (C) cytoplasmic fractions were obtained using
commercial kits prior to western blot analysis, yielding a band with molecular weight
~18kDa.
47
Figure 4. Tetracycline-inducible model for controlled expression of RSV NS1.
(A) Flp-In 3XFLAG-NS1 cells given various doses of tetracycline and harvested at 24 h
or given a single dose of 1 μg/ml and harvested at timepoints indicated and analyzed by
western blot. (B) Flp-In 3XFLAG-NS1 or control Flp-In 293 cells were treated with 1
μg/ml tetracycline for 24 h and analyzed with qRT-PCR for expression of RSV NS1. C)
Flp-In 3XFLAG-NS1 cells were incubated with tetracycline or vehicle (1% ethanol) for
24 h before immunocytochemical staining for FLAG. Shown are DAPI (blue) and FLAG
(green) merged images at 200X magnification
48
Figure 5. Cell-permeable fluorophore implemented to track subcellular localization
of polyhisitidine-tagged RSV NS1 in live, RSV-infected cells.
(A) Diagram of the chelating interaction between hexahistidine sequences on rA2His6NS1 RSV and Ni2+-NTA2-BM that permits tracking of histidine-tagged proteins
without permeabilization or fixation. (B) A solution of Ni2+-NTA2-BM in ammonium
bicarbonate buffer was added to RSV-infected HEp-2 10h p.i. in confocal culture dishes.
Ni2+-NTA2-BM (green) stained cells were counterstained with mitochondrial stain (red)
and nuclear dye (blue) before confocal microscopy analysis at 240X. Cells were
maintained at 37°C in an atmosphere of 5% CO2 during image collection on an Olympus
FV1000 MPE multiphoton laser scanning microscope and a series of 0.5 µm Z-stack
images for each specimen were collected. (C) Images were used to calculate the
Pearson’s correlation and Mander’s co-occurrence coefficients. Experiments were
performed in triplicate and representative images are shown.
49
Figure 6. RSV NS1 associates with MAVS protein in pNS1-transfected A549 and
RSV-infected A549.
(A) A549 were transfected with pNS1 or pFLAG for 24h and immunoprecipitated with
anti-MAVS antibody and analyzed by western blot analysis with anti-FLAG and antiMAVS antibodies. (B) Upon 12 h or 24 h post-transfection with pFLAG or pNS1,
MAVS-immunoprecipitated lysates were probed for RIG-I and MAVS. (C) A549 were
mock-infected or infected with rA2ΔNS1 or rA2 for 24 h and analyzed with western blot
for detection of MAVS, RIG-I and NS1. The same lysates were immunopreciptated with
anti-RIG-I antibody. (D) Densitometry was performed on the western blot and the ratio
of MAVS/RIG-I is shown in a bar graph, comparing MAVS-RIG-I binding when NS1 is
absent or present.
50
Figure 7. Kinetics of RSV NS1 expression in A549 and induction of IFN-regulator
protein LGP2.
A549 cells were mock-infected or infected with either rA2ΔNS1 RSV or rA2 RSV. (A)
Gene expression was examined with qRT-PCR and normalized to mock-infected or 0
hours post-infection (hpi) and in reference to mouse HPRT. (B) Western blot analysis
was performed on lysates from RSV-infected A549 compared with rA2ΔNS1 RSV for
expression of NS1 using NS1-antibody. (C) Gene expression of LGP2 was analyzed with
qRT-PCR with the same samples from (A) and in reference to mouse HPRT. (D) Flp-In
3XFLAG-NS1 cells were incubated with various concentrations of tetracycline in the
absence of RSV infection and immunoblotted for LGP2, FLAG, and loading control βactin. Optical density (OD) of each band was quantified with densitometer and LGP2
and NS1 were normalized to β-actin (OD-Bkgd) and illustrated on a bar graph.
51
Figure 8. Induction of NS1 restores infectivity and viral replication.
Flp-In 3XFLAG-NS1 cells were treated with 10 μg/ml tetracycline or vehicle for 4 h to
initiate induction. Media was replaced with ΔNS1 RSV inoculum with MOI 1 and
volume was supplemented with 10 μg/ml tetracycline or vehicle. After 1 ½ h of infection,
media was replaced with fresh media containing 10 μg/ml tetracycline or vehicle. (A-C)
Cells were harvested for isolation of RNA and analysis with qRT-PCR for expression of
(A) NS1, (B) LGP2 and (C) RSV N and normalized to HPRT. Experiment was
performed in duplicate; representative graphs shown are from a single experiment and are
similar to the findings in the replicate experiment.
52
Figure 9. Proposed virulence activities of RSV NS1 in disruption of host innate
immunity.
Upon RSV attachment and infection, viral RNA is replicated by viral RNA polymerase.
Double-stranded RNA (dsRNA) is recognized by receptors of the RLH-signaling
pathway, including LGP2. LGP2, which contains a RNA-helicase domain (shown in
darken orange) modifies the efficiency of dsRNA recognition by RIG-I or MDA-5, which
leads to homodimerization of CARD domains on the RIG-I and MAVS, resulting in
activation of IRF3/7 and NFκB for the synthesis of antiviral mediators.
We examined two proposed roles RSV NS1 may serve in the disruption of the host innate
immunity. (1) RSV NS1 expression is temporal and increases with kinetics of RSV
replication; we also induced expression in the inducible cell line. NS1 induction parallels
with LGP2, which may regulate downstream IFN signaling. (2) RSV NS1 localizes on
the mitochondria and binds with MAVS, thus preventing MAVS-RIG-I interaction. (3)
RSV NS1 may alter nuclear translocation of transcription factors and prevent activation
of type I IFN synthesis or cytokine production. In our study, we examined (1) and (2)
using microscopy, immunoprecipitation, and gene expression analysis.
53
Chapter 4: Differential RSV-induced Gene Expression in
Young and Aged mice
4.1 Introduction
The innate immune system serves as the first line of defense against invading
microbial pathogens. As discussed in the previous chapter, pattern recognition receptors
and downstream signaling components are frequently targeted by microbial pathogens to
disrupt activation of host immunity and aide in microbial survival. Moreover, genetic
deficiencies of PRR-associated signaling pathways, particularly Toll-IL-1R (TIR) and
NFκB, increase morbidity and mortality associated with infectious diseases (62).
Because of their diminished immune status, premature infants, immunocompromised
patients, and the elderly remain at increased susceptibility to nosocomial and communityacquired infections, particularly RSV, often resulting in severe disease or even death. A
goal in this chapter is to better understand the age-related molecular mechanisms that
influence innate immune responses to RSV and subsequently, how those changes affect
pulmonary health.
As of 2011, there was an estimated 41.4 million elderly Americans over the age of
65, contributing an estimated 12% to the nation’s total population (172). According to
the National Center for Health Statistics, only 68% of the elderly received influenza
54
vaccinations within the year of 2011. The average life expectancy continues to grow
over previous decades, with a current estimated additional 19.2 years expected after the
age of 65, thus the elderly population continues to grow; by 2040, seniors are expected to
make up 21% of the population. Approximately 3.6% of the senior population lives in
institutional settings, such as nursing or palliative care, and the proportion that requires
supportive medical assistance increases with age. Hospitalization rates associated with
respiratory viruses like influenza, RSV and human metapneumovirus are estimated to be
6.5%, 6.1%, and 4.5%, respectively, although surveillance for viral infections is often
restricted to particular seasons and geographical regions (28); consequently,
hospitalization rates are highly variable and dependent on study design. Long-term care
facilities (LTCFs) are often sites of RSV infection outbreaks and rates range widely from
1-89% based on 1975-2005 data (5, 173, 174). The mortality rate associated with RSV
infections is estimated to be 7.2% and the number of total RSV-associated deaths are
substantially higher in the elderly in comparison to young children despite RSV’s
reputation as a pediatric disease (24).
The effects aging on RSV pathogenesis in adult humans remains incompletely
understood since the majority of epidemiological studies investigate pediatric populations
and do not study cytokine responses, viral loads, or cellular immunity in senior adults;
recent investigations led by Walsh and Falsey attempt to address these areas (175, 176).
Older subjects tend to have increased nasal titers, elevated serum and nasal antibody
responses to RSV, and shed RSV longer in nasal secretions than younger counterparts.
Interestingly, underlying medical conditions, not older age alone, were statistically
55
correlated with RSV-disease severity. Still, the aforementioned study was quite small
(111 RSV-infected adults ≥ 21 years of age) with a heterogeneous subject population.
The older subjects were recruited disproportionally from the hospitalized population and
frequently suffered underlying medical conditions, such as chronic obstructive pulmonary
disease (COPD), congestive heart failure, and other comorbidities. As one of the first
investigations to examine inflammatory cytokines associated with RSV-induced lung
disease, further investigations are still needed to confirm these groundwork findings.
Aging results in a significant decline in humoral and cellular adaptive immunities
in the airway, leading to the term ‘immunosenescence’ (177). The predominant research
on age-related changes in the immune system examined the efficacy of vaccines or
therapies on animal models or human clinical studies that; dramatic changes in antigenpresentation (178, 179), disproportional memory T- and B-cell populations (180-182),
reduced immunoglobulin class switching (183), and decreased antibody diversity and
specificity (184, 185) are all credited with contributing to poor vaccine responsiveness
and subsequent reinfection. The particular challenge in vaccine development remains
how to adequately stimulate adaptive responses among the elderly when cellular
immunity such as T-cell responsiveness (182, 186, 187) and dendritic cell (DC) function
decline with age. Importantly, diminished DC functionality is associated with defects in
innate immunity, such as impaired TLR signaling and expression; declining TLR
function in DCs from elderly persons correlated well with poor influenza vaccine
responsiveness (188). Increased age, but not body mass or gender, is also found to
correlate with declining functional plasmacytoid DC populations and TLR9 expression
56
(189) and may help explain the reduced secretion of antiviral mediators, such as IFN-α
(179). Moreover, altered cytokine production by lymphocytes also may imbalance the
Th1 and Th2 responses specifically associated with RSV, potentially reducing the ability
to defend against reinfections (180) or exacerbating underlying diseases.
Unfortunately, adequate information regarding senior populations and respiratory
viral infections is currently lacking. The few published studies conducted among senior
populations are often outdated or lack the most recent viral diagnostic methods, such as
rapid real-time PCR tests (24). Additionally, it is particularly difficult to define when the
first day of exposure occurs, therefore duration of illness varies greatly. Consequently,
the role of aging on innate immunity is poorly understood despite its relevance to vaccine
development research against RSV. Results from a variety of animal models of aging
and respiratory infections suggest that declining adaptive as well as innate immunity
contributes to enhanced disease in the elderly. The aged mouse model has provided
substantial information regarding the effect of aging on TLR function against bacterial,
yeast, and viral infections, particular within macrophages (190-192). The absolute
number of bone marrow macrophages reportedly increases with age (193), but cytokine
production induced by TLR1/ 2, 2/6,TLR3, TLR4, TLR5, and TLR9 are reportedly
diminished in peritoneal macrophages (190, 191, 194). Additionally, TLR signaling and
expression of the TLR -adaptor protein, MyD88, was reduced in the macrophages of
LPS-stimulated aged mice (195). TLRs 4 and 5 were reportedly elevated in PBMCs from
aged macaques while expression of RIG-I and MDA5 was substantially lower (196).
Human myeloid-derived dendritic cells also demonstrated declining IFN-signaling,
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particularly RIG-I and TLR expression, as compared to young DCs in in vitro infection
with West Nile Virus (197). Our understanding of TLR signaling often is derived from
bone marrow-derived macrophages, dendritic cells, or PBMCs, and functionality is
assessed by their ability to secrete proinflammatory mediators, such as IL-6 (190), TNFα, or IL-1β; expression of TLR8 on primary monocytes isolated from RSV-infected
infant patients was directly correlated with disease severity and impaired TNF-α
production (74). Respiratory pathogens like RSV that predominantly infect airway
epithelium encounter alveolar macrophages, which are phenotypically unique from bone
marrow-derived macrophages (198). Consequently, the PRR signaling cascades induced
by respiratory pathogens in the context of alveolar or pulmonary antigen-presenting cells
still are largely undefined.
Overall, the significance of age on antiviral PRR signaling in RSV infections
remains largely unknown despite the epidemiological importance to elderly populations.
Two prior studies have previously assessed the effects of aging on RSV infections using
laboratory strain A2, which fails to induce substantial lung pathology in the BALB/c
mouse; consequently, histopathology was previously not examined. Instead, older age
was associated with reduced cytotoxic lymphocyte activity by CD8+ T-cells (143) and a
reduction was observed in vaccine immunogenicity to adjuvanted RSV subunit vaccine
(199). Importantly, both studies were primarily interested in age-related changes in
adaptive, not innate responses. Here, we examined differences in RSV-mediated
induction of innate antiviral immunity using an aged BALB/c mouse model. We found
PRR and cytokine activation to be impaired with older age, despite elevated induction of
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proinflammatory genes in the absence of infection. The findings from this study will
help predict the age-associated efficacy of RSV antivirals or adjuvants that rely on the
PRR stimulation.
4.2 Materials and methods
The following methods are in addition to the general methods described in
Chapter 2 and were specific to examining antiviral gene expression and function in young
and aged mice.
4.2.1 Mice and animal studies
Young and aged BALB/c mice were lightly anaesthetized with inhaled isoflurane
and intranasally inoculated with 106 pfu RSV A2 per mouse or with 105 pfu of RSV 2-20
or rA2-L19F per mouse. Blood was collected via cardiac puncture and allowed to clot
for 30 min at RT before centrifugation at 1000 x g to separate serum; serum was snapfrozen on dry ice and stored until analyzed by ELISA. For bronchoalveolar lavage
(BAL), 1 ml of room-temperature PBS containing 5 mM EDTA was intratracheally
injected and recovered. The BAL was repeated twice for a total of 3 ml; BAL fluid was
pooled among mice in a group and reserved in aliquots while BAL cells were
resuspended in pre-warmed (37°C) 10% FBS-containing RPMI culture medium for
alveolar macrophage studies. A separate set of mock- or RSV-infected animals was used
for histological and protein/RNA analysis. The right bronchus was clamped with a
hemostat and the left lung was gently perfused intratracheally with 4% paraformaldehyde
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(PFA) in PBS. The right lobes were minced into 1 mm pieces, portioned for
RNA/protein, and snap-frozen on dry ice; the left lobe was immersed in 4% PFA for
histological analysis.
4.2.2 Gene profiler RNA expression analysis
RNA was isolated with TRIzol from right lung tissue from RSV A2-infected
young and aged mice at 1, 3 and 5 dpi. RNA was treated with DNAse I and further
purified with RNeasy mini columns. 1 μg of total RNA was converted into cDNA using
the SABiosciences RT2 First Strand kit and combined with RT2 qPCR Master Mix as per
the manufacturer’s instructions. Antiviral gene expression analysis was performed using
RT2PCR Profiler Array PAMM-122 on a BioRad CFX96 real-time PCR system using the
manufacturer’s recommended thermocycler conditions. The same cycle threshold (Ct)
value was applied to all PCR runs and samples were performed in triplicate. Baseline
gene expression scatter-plots and network maps were generated from the SABiosciences
RT2PCR Profiler Data PCR array analysis software and all data quality checks were
performed. Experimental gene expression was defined as the Delta-Delta cycle threshold
(ΔΔCt) and calculations were in reference to the arithmetic mean of five endogenous
housekeeping genes (Gusb, Hprt, Hsp90ab1, Gapdh, and Actb); relative gene expression
(no normalization) was used for Minitab statistical analysis while normalization to mocktreated, age-matched controls was specific for heatmap and pathway map analysis.
GENE-E software (http://www.broadinstitute.org/cancer/software/GENE-E/) was
used to generate a heat map of the mean age-matched, normalized gene expression values
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of all 84 antiviral genes for young and aged mice over the 1, 3, or 5 day infection with
A2. Data was sorted by one minus the Pearson’s correlation hierarchical clustering;
columns indicate the timepoint while rows indicate the gene examined. Gradients within
each row ranges shade from blue to red to indicate minimum to maximum expression of
the indicated genes.
Gene expression changes >2-fold higher than age-matched mock-treated mice
were separately analyzed using GeneMania predictive interaction pathway maps (200) to
identify biologically relevant networks. Linkages between genes is indicated by coexpression (purple), colocalization (light blue), predicted interaction (orange), shared
protein domains (light yellow), or other undefined relationships (gray). Miniaturized
gene expression charts were generated using the fold-regulation values for each timepoint
and age group and illustrate relative kinetics. Individual mouse RNA array analysis was
performed in a single experiment with three different time points (n=3 mice/group) and
gene expression data are displayed as mean values +/- SEM; statistical significance was
assessed using 2-way ANOVA.
4.2.3 Minitab design of experiment (DOE) analysis
The difference between the individual gene of interest Ct value (Ct GOI) and the
arithmetic mean of five endogenous housekeeping Ct values (Ct HSKG) was designated as
the first Delta Ct (ΔCt) value. ΔCt was assigned as the response variable and two
processing conditions, age or RSV exposure, were defined in a two-level factorial DOE
analysis with alpha = 0.05. Factorial fit values were generated with the terms age,
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infection, or age*infection. Significance between terms was assumed when p<0.05 and
estimated effects and coefficients were tabulated for all 84 antiviral genes.
4.2.4 Isolation of alveolar macrophages and stimulation of TLR7/8
Bronchoalveolar lavage fluid was pooled from mice of the same group (n = 4) and
centrifuged at 300 x g to collect BAL cells. Cells were resuspended in 10% FBS-RPMI
and seeded at a density of 8 x 105 cells per well in a 24-well tissue culture plate and
incubated for 2 h at 37°C to isolate macrophages by adherence (198). Cells were washed
twice with 37 °C PBS to remove lymphocytes. Equal numbers of adherent macrophages
were incubated with 2.5 μg/ml of the TLR7/8 ligand, R848. After 20 h, cell culture
supernatants were collected and analyzed for the presence of the IL-6 by ELISA using
manufacturer’s instructions. Experiments were performed in triplicate and data are
represented as mean ± SEM; significance was determined by 2-way ANOVA, p<0.05.
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4.3 Results
4.3.1 Age-associated alterations in antiviral gene expression and kinetics
We were interested in identifying antiviral genes that are significantly altered by
age and infection with the well-characterized RSV A2 strain; RT2PCR Profiler array
analysis was performed on total lung RNA from mice at 1, 3, and 5 dpi, while A2 was
still present in the lungs. Baseline gene expression of 84 antiviral genes was compared
between mock-infected young and aged mice. In the absence of infection, 14 genes
showed expression >2-fold higher in aged mice compared to young (Figure 10A) and
statistical significance with asterisks (p<0.05) or p-value is indicated. The 14 genes were
used to construct a network map, which demonstrates that the majority of genes
upregulated are involved in TLR-signaling (Figure 10B) or are proinflammatory
cytokines or chemokines.
Assigning the age-matched mock-infected mice as controls for comparison, ΔΔCt
values were derived for samples obtained on 1, 3 or 5 dpi. Mean gene expression values
were tabulated and a heatmap was constructed using GENE-E software (Figure 11A).
Within each row, shades of blue to red represent the mean normalized gene expression
values, then compare age groups within the same row. The heatmap was divided into
two large columns to adjust for large size. Upon examination of the heatmap, differences
were observed in the induction of gene clusters. The first column contains a variety of
chemokines (CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11) and inflammatory
cytokines (IL-6, TNF-α), which were induced at 1 dpi in young but not aged mice.
Interestingly, most of these genes were upregulated in aged mice in the absence of
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infection; consequently, the change in gene expression may be reduced in aged mice 1
dpi as a result of high baseline expression. In the second column of the heatmap,
hierarchical clustering grouped several MAPK-associated genes and TLR or RLH
adaptor molecules that had similar gene induction between young and aged. The lower
half of the second column contains a cluster of genes, many of which are involved in
NLR signaling like IL-1β, NLRP3, Casp 1, Nod2, and Mefv, that are all found to have
age-altered gene kinetics. This minor cluster tended to have early and sustained
induction in young mice but induction was more variable in aged mice, often with
downregulation of genes at 3 dpi. The induction of antiviral genes is generally delayed or
diminished in aged mice in comparison to young, suggesting that stimulation or
activation of antiviral responses is impaired.
Genes upregulated >2-fold in young mice at 1 dpi (left column) were further
examined in the context of a biologically relevant pathways using Gene-Mania. 27 genes
were upregulated and served as inputs to the analysis. Of the original 27 genes provided
as input, 11 genes were predicted to be involved in cytokine signaling pathways with a
low false discovery rate (FDR) or 8.69 e-24; coverage spanned 16 of 88 genes in the
databases associated with cytokine signaling (200). Normalized gene expression of the
11 genes is shown above or below the gene symbols and demonstrates the difference in
magnitude and induction of genes involved in cytokine signaling. Gene expression of
young mice (dotted lines) was often induced earlier and with greater magnitude than
expression from aged mice (solid line) (Figure 11B). The unusual exception was aged
IFNβ1 transcripts which were elevated above young gene expression over the time course
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of RSV A2 infection; further investigations will determine if induction leads to functional
secretion IFNβ1 and activation of IFN-inducible signaling, particularly since IFNβ1
production reportedly declines in pDCs(188) and monocytes (201) with age.
4.3.2 Statistical analysis with DOE identifies genes that are altered by age and
infection
Representation of gene expression changes through fold-regulation has limitations
in that normalization allows for comparison of gene kinetics among the same age group
and not multiple ages over multiple days. To better understand the interactions, statistical
analysis was performed to determine if age and/or infection were significantly
contributing to changes in the ΔCt on 1, 3 or 5 dpi. Age alone was a statistically
significant factor for 15 genes and these are shown on the left circle in the Venn diagram
(Figure 12A); 73 genes were statistically altered as a result of RSV infection on any of
the days examined. A minor group of genes was found to be unchanged in ΔCt on any of
the days post infection regardless of age; included in this group were several genes that
were expected to be significantly altered by the presence of RSV, including MyD88, IL18, CD40 and CD80, although several studies have also reported minimal changes in
endogenous gene expression (78, 90, 202).
Five antiviral genes were found to have significantly altered expression kinetics,
including RIG-I, IFNAR1, IL-1β, OPN and TLR8. These genes are shown in line graphs
of non-normalized relative expression comparing induction from baseline (mock) to
when significance was observed at 3 dpi with RSV A2 (Figure 12B). Change in relative
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expression was evident in both magnitude and direction; compared to baseline
expression, most genes decline in expression at 3 dpi or expression remains unaltered. It
should be noted that the relative expression of RIG-I, IL-1β, OPN and TLR8 appears
marginal at 3 dpi in young mice, but as shown in the heatmap (Figure 11A), expression
was rapidly induced at 1 dpi and expression levels were reduced by 3 and 5 dpi. The
exception was IFNAR1 whose expression initially dropped at 1 dpi then increased after 3
dpi. IL-1β and OPN are examined more extensively because of their role as
proinflammatory cytokines in Chapter 6.
4.3.3 TLR7/8 signaling is impaired in aged alveolar macrophages
Since TLR8 function was correlated with disease severity in RSV-infected infants
(74) and was influenced by age and RSV infection in our array study, we examined TLR8
signaling ex vivo using alveolar macrophages collected from young and aged BALB/c
mice (Figure 13). Stimulation of alveolar macrophages with TLR7/8 agonist R848
resulted in secretion of the proinflammatory cytokine IL-6 in cells from young but not
aged mice; in the absence of stimulation, IL-6 secretion was below detection. The
significant reduction in IL-6 suggests impairment of TLR7/8 and provides evidence to
support our PCR array findings that TLR8 expression and function decline with age;
secretion of IL-1β was also assessed by ELISA but was below the detectable levels (data
not shown).
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4.4 Discussion
Current knowledge about innate responses and aging is still limited despite its
impact on geriatric health and vaccine research. Human aging studies are often
confounded by underlying diseases and environmental factors, making it more difficult to
study age-specific immune responses. Studying innate immunity is particularly
challenging because responses involve heterogeneous populations of epithelial,
endothelial, mucosal, and leukocytic cells interacting through receptors and adaptor
proteins during acute stimulation (177); subsequent inflammation associated with innate
immunity serve to protect but also contribute to disease. In this series of studies, we used
pathogen-free aged and young BALB/c to model RSV-induced activation of innate,
antiviral responses.
We found that several PRR components were diminished in expression and
function upon infection in aged mice. Importantly, several genes associated with TLRs
(TLR7-9) and inflammasome activation (Card9, Casp1, Nod2) were found significantly
altered by age alone. Antiviral genes RIG-I, IFNAR1, IL-1β, OPN, and TLR8 had
statistically significant decline or delay in expression at 3 dpi in aged mice. The observed
decline in RIG-I is particularly intriguing because of its role in recognizing viral RNA
during early stages of infection. TLR7/8 function was impaired in primary alveolar
macrophages stimulated ex vivo as less IL-6 was produced upon stimulation. We
attempted to measure IL-1β, another indicator of TLR functionality but were unable to
detect levels with our methods. IL-1β and OPN will be separately discussed in the
following chapters. Several genes such as IL-18, TLR7-adaptor protein MyD88, and
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costimulatory molecules CD40 and CD80 were unaltered in expression upon RSV
infection despite expected roles in antiviral immunity; in agreement with our results,
other studies found that CD40/80 expression was unchanged after RSV infection in
mDCs and pDCs (202) and therefore, their function may not be specific to RSV (203).
MyD88 was previously found dispensable for type I IFN production and CD8+
lymphocyte-mediated RSV clearance (78), and Ifih1 was also not altered by age or
infection but its expression is generally associated with recognition of Picornaviruses
(204). Jun is potentially a cis-regulatory factor for the induction of CCL5, but other
genes such as p38 also play similar regulator roles to CCL5 induction (205). In
summary, the majority of our PCR aging data was validated by evidence reported by
other studies. Surprisingly, NFκB1 not found associated with RSV or age, although
NFκB activation was reported in the nuclear extracts of lungs from RSV-infected mice at
5 and 7 dpi (206) by detection with electrophoretic mobility shift assay (EMSA).
Noteworthy, the aforementioned study did not specifically examine NFκB1 or p50 after
24 h therefore alternate NFκB-associated factors could contribute to NFκB activation.
Alternatively, the authors identified a challenge using mock-supernatant which causes
significant early induction of NFκB1 and therefore, induction of NFκB1 specifically from
RSV may not appear statistically significant in our investigation. Further investigations
may be needed on nuclear lung extracts to specifically monitor induction of transcription
regulators, such as NFκB1.
Similar to what was observed for gene expression in PBMCs from human and
macaques (196), we also saw decreased expression of several antiviral PRRs, particularly
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RIG-I, TLR8, and IFNAR1. Age-related changes in the TLR-family of protein are better
characterized in the context of bacterial infection. RLHs are recognized as essential
mediators that help initiate adaptive immune responses to viral infections but the effects
of aging have not be examined thoroughly; the NLR family, regardless of age, remains
the least understood of the PRRs. New evidence suggests activation of the NLRP3
inflammasome complex formation is an essential innate defense strategy against
Influenza A virus (154) and also declines in function with older age (207).
Inflammasome activation is mediated by RIG-I and TLR3 and positively controlled by
the binding of type I IFNs to IFNAR1, which was significantly downregulated upon
infection in our PCR array. The proposed model of inflammasome activation sheds new
light on crosstalk between TLRs, RLRs, and NLRs. However, the redundancy in the
PRR pathways also complicates our abilities to correlate antiviral gene function directly
with disease pathology or antimicrobial defense. The following chapters will attempt to
correlate age-related decline of PRR and cytokine gene induction with RSV-induced lung
pathology.
Further investigation is still needed to assess if RIG-I, TLR8 or IFNAR1 function
are substantially reduced by age and whether stimulation of these receptors could also be
assayed for NFκB activation or type I IFNs. Although RSV is a poor inducer of IFN due
to the virulence factors described in Chapter 3, further examination of IFNAR1 signaling
may reveal age-related differences in activation of the inflammasome complex and
subsequent release of IL-1β. We identified particular age-associated alterations in PRR
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signaling and our data suggest that therapeutics aimed at senior populations may require
strategies for reactivating or bypassing PRRs that have age-related decline in function.
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71
Figure 10. Baseline antiviral gene expression in young and aged mice.
(A) Young and aged mock-infected mice (n = 3) were compared for differences in
baseline gene expression using a RT2PCR Profiler array analysis. Genes with asterisks
were found statistically upregulated in mock-infected aged mice as compared to mockinfected young. Genes upregulated in aged mice, but not statistically significant (p<0.05)
are in parentheses with calculated p-value. (B) Genes elevated in aged mice were then
used as input genes to construct a network map using the SABioscience Gene Network
Generator Pro. Genes were categorized based on association with PRR pathways
suggested by the SABiosciences network maps.
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73
Figure 11. Differential antiviral gene expression upon RSV-infection in aged mice.
Antiviral gene expression was examined over 1, 3, and 5 dpi with RSV A2 using RT2PCR Profiler array analysis. (A) Mean, normalized gene expressions of 84 genes were
used to generate a heatmap using GENE-E software. Rows were sorted in ascending
gene expression order and processed for hierarchical clustering using one minus
Pearson’s correlation. Within each row, gradients from blue to red indicate minimum to
maximum expression of the indicated gene, respectively. Columns indicate Days 1, 3, or
5 post-infection in either aged or young mice. The heatmap was split into two: the top74
half shows clustered genes upregulated in young mice 1 dpi and the second-half is a
cluster of genes that are commonly upregulated in young and aged groups. The lower
half of the second column illustrates a cluster of genes that remain upregulated on 5 dpi
in young mice. Individual mouse RNA array analysis was performed in a single
experiment with three different time points (n=3 mice/group). (B) 27 genes found
upregulated >2-fold on 1 dpi in young mice vs. aged mice were used to construct a
cytokine signaling network map using GeneMania (11 genes are involved from the 27
genes initially entered as input). Genes share predicted linkages through co-expression
(purple), colocalization (light blue), predicted interaction (orange), shared protein
domains (light yellow), or other known relationship (gray). Relative gene expression
charts of each of the 11 gene is displayed (above or below) of aged (solid) or young
(dotted) over the time course of RSV A2 infection. Fold-regulation changes were derived
by ∆∆Ct calculation and in reference to age-matched mock-infected control group (n = 3
per group).
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Figure 12. Five antiviral genes altered by age and infection.
Age and/or infection were found to statistically alter expression of antiviral genes on 1
dpi, 3 dpi, 5 dpi, or combination of all days (p<0.05). (A) A Venn diagram illustrates
relationships from the 84 genes screened and identifies genes significantly associated
only with age, RSV A2 infection, a combination of both factors, and neither on 1 dpi, 3
dpi, or 5 dpi. (B) Genes found significantly influenced by age and infection at 3 dpi were
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used to construct individual gene expression graphs. Young (dotted) and aged (solid)
mice lines ± SEM, n=3/group.
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Figure 13. TLR 7/8 activation in aged alveolar macrophages is impaired.
TLR7/8 activation was examined in alveolar macrophages collected from uninfected
young and aged BALB/c (n = 4 mice/group). Primary alveolar macrophages harvested
from lavage were incubated with 2.5 μg/ml of the TLR7/8 ligand, R848, for 20 h. As an
indicator of TLR7/8 function, culture supernatant was examined for IL-6 using ELISA.
Significance was determined with ANOVA 2-way analysis and experiment was
performed in triplicate.
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Chapter 5: RSV-induced Pathology in Young and Aged Mice
5.1 Introduction
Studying age-associated changes to antiviral innate immunity in humans remains
a challenge limited availability of rapid methods for diagnosing RSV; further antiviral
gene induction occurs rapidly after exposure. Unfortunately, it is particularly difficult to
identify the first day after RSV exposure (175) and clinical examination is often
conducted after symptomatic physiological changes and viral shedding has already
occurred. Moreover, the frequency of comorbidities or confounding underlying medical
conditions in elderly patients increase subject variation and make it difficult to derive
age-specific conclusions. Consequently, pathological studies are often performed in
controlled settings, such as animal models, which can provide important information
regarding cellular changes immediately and soon after RSV exposure. Unfortunately,
some limitations to the current mouse model of RSV A2 complicates our understanding
of PRR signaling in studying RSV pathogenesis and antiviral immunity. Despite general
similarities to the human immune system, BALB/c mice are only semi-permissive to
RSV replication, thus lung viral loads often remain low despite intranasal instillation of
large inoculums of A2 (106 pfu/mouse) (208, 209); typically lung pathology was
negligible in previous studies. However, recent studies have identified RSV strains that
induce greater lung pathology and mucus hypersecretion, which mimic RSV
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pathogenesis in humans more accurately (40, 41). RSV-induced lung pathology involves
complex changes to airway microenvironments, including mucin production, perivascular
edema, and cellular infiltration (40, 71, 72) and these pathological events are aggravated
when TLR signaling is impaired. Mice deficient in TLR3 or TLR7 have significant lung
pathology, even with the non-mucogenic RSV strain A2; notably, absence of either TLR
did not affect RSV replication; thus these receptors influence airway pathology but not
necessarily viral clearance (71, 72).
The impact of pathogen-induced mucus hypersecretion in pulmonary health
continues to be a controversial subject. Rhinovirus, influenza, RSV, and many other
respiratory pathogens induce mucus hypersecretion in humans and animal models;
similar to what occurs in allergen-induced asthma (50, 210-212), pathogen-induced
mucus hypersecretion can obstruct airways and increase airway hyperresponsiveness.
Postmortem studies suggest airway obstruction is a frequent cause of death in COPD,
asthma, and severe RSV infection (213). Conversely, regulated production of mucus is
critical for effective mucociliary-mediated clearance of microorganisms and deficiency or
inactivation of mucogenesis increases host susceptibility to infections with Streptococcus
pneumoniae (214) and Pseudomonas aeruginosa (215), which activate mucin production
through TLR5 (216). Production of mucins, MUC5AC and MUC5B, declines in cystic
fibrosis patients and possibly increases susceptibility to respiratory pathogens (217). The
specific role of MUC5AC is still unclear: mucus is often cited with enhanced pathology
and virus-induced inflammation (40, 71, 218), but is still a fairly recent subject in RSV
research. Moreover, overexpression of mucin genes has not been explored with RSV
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infection, although overexpression of mucin-associated gene IL-13 led to reduced RSV
illness (219).
Nasomucociliary clearance declines with underlying medical conditions (diabetes
and hypertension) and with older age (220). In humans, subjects over the age of 40 have
a reduction in mechanical ciliary beats (221), which may contribute to obstruction of the
airways and may explain the increased frequency and severity of respiratory illness
among elderly persons. Moreover, the responses to environmental and allergen triggers
appears significantly altered in aged mice. Ovalbumin-sensitized aged mice have
enhanced mucus and goblet cell metaplasia, although airway hyperresponsiveness and
eotaxin levels diminish with age in the mouse model (177). The population of
fibroblasts, the predominant source of eotaxin, decreases with age, possibly due to
skewed differentiation of lung fibroblasts into myofibroblasts, which are less regenerative
and have potential to contribute to the development of fibrosis (222). Age-related
changes in allergen-induced pulmonary cytokines, including increased IFN-γ and IL-5,
and diminished Th2-responses, demonstrates significant changes in lung
microenvironments as a result of age. Similar changes are expected in responses to
respiratory pathogens, although research investigating these specific age-related changes
to pulmonary health are certainly lacking.
Histological analysis of lung sections helps assess airway inflammation but is
largely dependent on a blinded pathologist who can provide clinical scores. Generally,
pathological scores include thickness of alveolar walls, the size or diameter of lesions
(214), the frequency of immune cells surrounding the site of injury, and intensity or area
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of staining (40). The length of time required to interpret each image varies depending on
pathologist’s experience. Advances in technology now offer alternatives to man-made
pathological scoring, which may reduce person-to-person variability as well as conserve
time. ImageJ is a freely available, Java-based platform for image processing and
analysis. With over 25,000 downloads per month, ImageJ has become an indispensable
tool for biologists (223). Capable of color separation, pixel measurements, densitometry,
multipoint selection, and many other features, ImageJ utilizes application macros, or
sequences of computer instructions, to execute small commands that can be combined to
construct larger programs or activate plugins. The use of macro scripts and Java make
ImageJ simple to use and easily customizable. Readily-accessible plugins and macro
script forums provide support for developing custom macros. Digital, quantitative
computer analysis has been shown to reproduce results from pathological scoring system
and may provide a more rapid, cost-effective method for better turnaround times (224).
We hypothesized the altered antiviral gene expression observed in Chapter 4
resulted in impaired or delayed defenses against RSV infections. To test this, we
performed histological analysis, viral titration, and immunohistochemistry to monitor the
progression of RSV-induced lung disease caused by three RSV strains, recombinant A2
expressing Line 19 fusion protein (rA2-L19F), 2-20 and non-mucogenic A2. The two
mucogenic strains were previously characterized in the BALB/c mouse to induce greater
pathology and mucus production than the laboratory strain A2. Although no ageassociated difference was previously reported with A2, we were interested in testing
more virulent strains of RSV to see whether age has a significant influence on disease
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severity. Periodic-acid Schiff (PAS) and hematoxylin-stained tissues were first analyzed
with ImageJ using particle analysis often referred to ‘Find Maxima.’ Portions of the
macro scripts were previously constructed elsewhere but have been combined for rapid
use with lung pathology sections. Additionally, we quantified cellular infiltration and
area of mucin-staining, then enumerated RSV-infected cells from the total cell
population. Multiple methods of analysis should strengthen our findings and serve as a
foundation for future histopathological studies.
5.2 Materials and methods
The following methods are in addition to the general methods described in
Chapter 2 and are specific to the characterization of RSV-induced lung disease in young
and aged mice.
5.2.1 Tissue collection and histological processing
Young and aged BALB/c mice were lightly anaesthetized with inhaled isoflurane
and intranasally inoculated with 106 pfu RSV A2 per mouse or with 105 pfu of RSV 2-20
or rA2-L19F per mouse. Blood was collected via cardiac puncture and allowed to clot
for 30 min at RT before centrifugation at 1000 x g to separate serum; serum was snapfrozen on dry ice and stored until analyzed by ELISA. For BAL, 1 ml of roomtemperature PBS containing 5 mM EDTA was intratracheally injected and recovered.
The BAL was repeated twice for a total of 3 ml; BAL fluid was pooled among mice in a
group and reserved in aliquots while BAL cells were resuspended in pre-warmed (37°C)
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10% FBS RPMI culture medium for alveolar macrophage studies.
A separate set of
mock- or RSV-infected animals was used for histological and protein/RNA analysis. The
right bronchus was clamped with a hemostat and the left lung was gently perfused
intratracheally with 4% PFA in PBS. The right lobes were minced into 1 mm pieces,
portioned for RNA/protein, and snap-frozen on dry ice; the left lobe was immersed in 4%
PFA for histological analysis.
5.2.2 Immunohistochemical staining and image analysis with ImageJ software
Paraformaldehyde-fixed left lungs were cryopreserved in increasing
concentrations of sucrose and PBS, then embedded with OCT-freezing media. Lungs
were frozen over dry-ice then sectioned at 5 µm with a microtome; a minimum of 4
lengthwise sections were placed on a glass slide and heated at 37°C for ½ h. Slides were
rinsed in PBS then stained with PAS reagent per manufacturer’s instructions (RichardAllan Scientific) and counterstained with hematoxylin QS (Vector Labs). PAS-stained
slides were dehydrated in alcohol gradients and cleared with several changes of xylene
immediately before being mounted with a glass coverslip. Slides were allowed to dry
completely overnight prior to image collection; a minimum of 10 images per mouse
(n=4-6 mice/group) were collected at 40X and 200X. Images were collected at constant
exposure, brightness, and white or black background correction was applied for
brightfield or fluorescence, respectively.
For indirect immunofluorescence and immunohistochemical staining, slides were
steamed at 95°C for 20 min in pH 9.0 (Vector Labs) unmasking solution. Tissues were
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permeabilized with methanol and nonspecific sites were blocked for ½ h with 5% bovine
serum albumin (BSA) and 0.1%Tween-20 in Tris-buffered saline (TBST). Primary
polyclonal goat anti-RSV (Millipore, Ab1128) was diluted in 2.5% BSA/TBST and
added to sections for overnight incubation at 4°C. Dilutions were optimized and
antibody specificity was confirmed by comparing with sections stained with control goat
IgG. After washes, slides stained for RSV antigens were incubated with Alexa Fluor
555-conjugated donkey anti-goat secondary antibody for 2 h, followed by counterstaining
with DAPI-containing antifade media for coverslip mounting; slides were kept at -20°C
until imaging was done.
5.2.2.1 Quantification of cellular infiltration in hematoxylin-stained lung
To quantify the number of cells within an area, particularly in regions surrounding
bronchioles, alveolar sacs, and blood vessels, areas were manually annotated for selection
using ImageJ. Entire RGB images were split into three channels at 8-bit and the ‘blue’
channel was selected to avoid bleed-through of pink coloration. Selected regions were
analyzed with the macro Count and Mark cells using the Find Maxima Java script and
specified threshold. Figure 14 contains screenshots of how images appear during
processing and white dots indicate where maxima of dark peak values were found within
the threshold range. The size of the selected area was measured in microns using area
calibration. Various settings in the Java script were modified, including scale, threshold,
and size of marker to enumerate cells with minimal detection of PAS or nonspecific dark
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matter; the displayed macro script was used for all images at 40X after bronchiole regions
were selected.
5.2.2.2 Quantification of PAS-stained areas using color deconvolution analysis
Post-hoc Rolling background subtraction was applied to all PAS-stained images at
200X magnification. To utilize the ImageJ Color Deconvolution plugin, singly-stained
sections were analyzed to find individual stain vector settings; the Color Deconvolution
Java script was modified for customized stain settings. Set Measurements was adjusted
to include Area and the Set Scale setting was changed to account for area, converting
distance of pixels into microns. To rapidly analyze images, all PAS-stained images at
200X magnification were compiled into a single folder and execution of the Batch macro
performed all sequential steps outlined in Figure 15 on all of the sections.
5.2.2.3 Quantification of RSV-positive cells in immunofluorescently-stained lung
sections
Left lung 5 µm sections were probed with goat polyclonal antibody for detection
of RSV antigens and Alexa Fluor 555-conjugated secondary anti-goat antibody was used
for indirect immunofluorescence. A minimum of 10 images per mouse at 200X with
excitation at 350 nm and 596 nm were taken using a DP72 digital camera on an IX710
Olympus fluorescence microscope; RGB images collected at 350 nm were split into three
8-bit channels and the ‘blue’ channel was selected, while RGB images obtained at 596
nm were split and the ‘red’ channel was reserved for analysis. Selected images were
inverted and individually analyzed with the Image-based Tool for Counting Nuclei
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(ITCN; http://www.bioimage.ucsb.edu/automatic-nuclei-counter-plug-in-for-imagej). To
approximate the number of red cells and blue nuclei, the ITCN plugin settings were
adjusted such that cell widths were set to 22 pixels, while DAPI-stained nuclei were
analyzed at a width of 18 pixels. Minimum distance was set as half of width values and
threshold was set to 0.3. Analyses were set to find dark maxima peaks, which were
indicated with white dots. As presented in Figure 16, the number of cells were then
divided by the total number of nuclei, yielding a percentage of RSV-positive cells.
5.3 Results
5.3.1 Aging is associated with delayed cellular clearance of RSV rA2-L19F
To assess the effect of aging on RSV-induced lung disease, young and aged
BALB/c mice were intranasally infected with three different RSV strains: rA2-L19F, 220, and A2. Lung tissue was collected at the peak of infection (4 or 5 dpi, depending on
the strain) and 8 dpi. Mock-infection in either age group did not result in observable
infiltration or differences between ages (Figure 17A, top row). Infection with rA2-L19F
resulted in the greatest pathology, particularly in aged mice. On both 5 and 8 dpi with
rA2-L19F, leukocytes and other nucleated-cells were easily visible around the bronchus
and bronchioles. Cellular infiltration was visible in young rA2-L19F-infected mice, but
pathology was reduced compared to aged mice. RSV 2-20 infections also resulted in
dense aggregation of nucleated cells along the periphery of bronchioles and blood
vessels; in comparison, the lungs of young 2-20-infected mice appear to have
perivascular edema and slight infiltration of nucleated cells, but still comparatively less
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than in aged mice. RSV A2 infections failed to induce significant pathology at 5 and 8
dpi in either age group. Infrequently, regions of the lungs of aged mice had minor sites
of infiltration.
Cellular infiltration densities surrounding bronchioles, alveolar sacs, and blood
vessels were calculated with ImageJ software (Figure 17B). Graphs represent the mean
infiltration density derived from 3-5 bronchioles per image, with a minimum of 8 images
collected per mouse (n=4-6 mice/group). Cellular infiltration was increased in both
young and aged mice upon 2-20 infection and remained significantly higher than mock at
8 dpi; it is noteworthy that the cellular infiltration of rA2-L19F and 2-20 at the peak of
infection, 4 or 5 dpi, respectively, are significantly different in young but not in aged
mice. By 8 dpi, cellular infiltration is reduced and no longer significant in young mice; in
contrast, aged mice still have substantial infiltration 8 dpi in comparison to mock-infected
animals. The difference in cellular clearance is statistically significant between young
and aged mice at 8 dpi with rA2-L19F. A2 did not result in changes in cellular
infiltration in either age group. These data suggest RSV strain virulence plays an
important role in cellular clearance.
5.3.2 Mucus production is altered in aged mice upon rA2-L19F infection
Infection with rA2-L19F reportedly induces mucus hypersecretion and more
severe pathology than the laboratory strain A2, therefore we examined PAS-staining in
lung sections from young and aged mice infected with either rA2-L19F or A2. After
stain-separation analysis by color deconvolution, the PAS-stained areas were compared
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to the complete tissue stained within a single field of view. This ratio is defined as
PAS/Hematoxylin Area and values >1 suggest that secretion of mucin exceeds the area of
the alveolar epithelial network, which acquires a faint blue coloration during hematoxylin
staining (Figure 18A). Mock-infection did not elicit mucus secretion, and significant
mucus was quantifiable in young but not aged mice at 8 dpi; A2 infection did not elicit
significant or measurable mucus (Figure 18B). In contrast, aged mice had earlier
induction of mucus at 5 dpi but the mucus level was not significantly higher than mock.
This age-related alteration in mucus production was further examined with qRT-PCR
analysis for expression of MUC5AC (Figure 18C). Unlike young mice, relative gene
expression of MUC5AC was not statistically changed in aged mice upon rA2-L19F 8 dpi.
Mucus was elevated at 5 dpi among aged mice and was statistically different from young
mice at 5 dpi. In agreement with previous studies, A2 did not result in a significantly
elevated PAS/hematoxylin ratio and MUC5AC was not induced on 8 dpi.
5.3.3 Infection with RSV rA2-L19F is enhanced and prolonged in aged
BALB/c mice
Since cellular clearance was delayed in aged mice upon RSV rA2-L19F infection,
we examined age-specific viral infectivity with the three strains of RSV (Figure 19A, left
column). RSV N expression was found to be significantly higher in aged mice upon rA2L19F infection 8 dpi; infection with 2-20 was significantly higher in aged mice on both 4
and 8 dpi. Expression of the RSV nucleocapsid protein N was comparable between age
groups after RSV A2 infection. Plaque assays performed on homogenized right lung
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tissue indicated that rA2-L19F but not 2-20 or A2, have significantly higher plaque titers
in aged mice on the day of peak infection (5 dpi) (Figure 19B, right column). No plaques
could be obtained from lung tissue at 8 dpi from mice infected with any of the virus
strains because of the relative insensitivity of the assay.
Because of the limit of detection in plaque assay, immunofluorescent staining of
lung sections for RSV antigens was performed on 8 dpi to assess whether viral clearance
was delayed in aged mice (Figure 20A). Infections with either RSV 2-20 or A2 were
barely visible or absent 8 dpi; in contrast, RSV antigens from rA2-L19F virus were still
visible throughout the left lung 8 dpi. The percentage of RSV-positive cells was obtained
by ITCN ImageJ analysis and percentages averaged from >10 images per mouse were
used to generate the individual box plots (Figure 20B). Significantly more RSV-positive
cells were counted in the lungs of aged and young mice with rA2-L19F as compared to 220 or A2; among the mice infected with rA2-L19F, aged mice had significantly higher
percentages than young. Infection with 2-20 resulted in a significant percentage of RSVpositive cells in aged but not young mice, although staining was more variable among
this set of mice.
5.4 Discussion
The elderly are more susceptible to severe RSV infections, as characterized by
prolonged hospitalization and greater viral loads from nasal washes. Senior populations
have greater risk for community-acquired diseases while in long-term assistive living
homes or when frequenting hospitals. RSV leads to significant morbidity among the
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elderly populations and frequent confounding comorbidities skew age-related
investigations. Predominant knowledge regarding RSV-induced lung pathology comes
from histopathological examination of lungs from infants, not elderly adults, with severe
RSV and lethal obstruction of airways (213). Open lung biopsies have been collected
from adults, but often when subjects were immunocompromised or lung transplant
recipients (225). These individuals demonstrate characteristics of RSV pneumonitis,
including alveolar damage and perivascular exudate. Radiological examination often
reveal unilateral infiltrates and small pleura effusion, but correlating chest radiograph
diagnosis with symptoms of pneumonia remains to be a challenge. Consequently, little
remains known regarding RSV-induced lung pathology and age-related changes to viral
clearance in the lung.
Interestingly, the earlier study of aging immunity and RSV A2 infections
conducted in 2001 did not find differences in viral loads between age groups but
identified a decline in function of CD8+ T-cell CTL activity (143). In agreement, we
found the histopathology was not significant in the RSV A2 infected aged mice.
However, these findings did not appear clinically relevant or plausible since elderly
humans have prolonged viral shedding and disease symptoms (175). Therefore, we chose
to use more virulent and mucogenic strains of RSV to compare disease severity in our
mouse model. In our study, we found that RSV-induced lung pathology was enhanced in
aged mice upon infection with virulent RSV strain, rA2-L19F, and infection was
prolonged when assessed by viral RNA, viral load, and levels of viral antigens. Cellular
infiltration, measured by enumeration of hematoxylin-stained cells with ImageJ, was
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highest in aged rA2-L19F and 2-20 infected mice. Infection with isolate 2-20 resulted in
greater viral RNA at 4 and 8 dpi in aged mice, but plaques and IHC demonstrated similar
pathology between age groups. Perivascular edema was present but not quantified.
Reduced cellular clearance at 8 dpi suggests delayed viral clearance, which was evident
by the presence of viral antigens in the lung sections on day 8; our application of antiRSV antibodies on lung sections permitted comparison at later stages of infection,
beyond the sensitivity of the standard plaque assay. Alternatively, enhanced cellular
infiltration could indicate reduced ability to effectively remove apoptotic leukocytes after
they were recruited to the site of inflammation. Phagocytosis and clearance of apoptotic
bodies reportedly decline with older age (226). Since viral shedding and prolonged
hospitalization are common in elderly patients, our aged mouse model better resembles
the age-specific pathology observed in humans than the previous A2 aged mouse studies
(143, 199) Reexamination of CTL activity may be necessary in the future using
mucogenic RSV strains to test whether there are additional age-related adaptive immune
changes not previously observed. Further, changes in the mucosal response to rA2-L19F
and 2-20 may also indicate virus specific antiviral gene induction. We screened antiviral
genes associated with low virulence RSV A2 as the virus is fully sequenced and
characterized in cell-culture and animal models and these genes were found altered in
function in aged mice. A2 was previously shown to induce Type-I interferon better than
rA2-L19F and 2-20, therefore antiviral gene induction by rA2-L19F and 2-20 may be less
than A2 in either age group. Our investigation did not examine age related differences in
all of the 84 antiviral genes after infection with mucogenic RSV strains because mucosal
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responses to mucogenic strains of RSV is still incompletely understood. Difficulty arises
when attempting to correlate mucosal disease severity and antiviral gene induction
associated with virulent strains: does enhanced viral loads induce greater immune
responses and enhances secretion of mucus or has tissue injury from virulent RSV strains
cause physiological changes to the airways, altering airway surface liquid balance? To
correspond with the age-specific pathology observed in this chapter, we anticipate
antiviral gene induction would still be diminished in aged mice with either mucogenic
strain; we also expect induction and kinetics to be altered from the infections induced by
A2 as rA2-L19F and 2-20 are more virulent. Alternatively, cytokines and
proinflammatory genes associated with tissue injury and cellular infiltration are expected
to be induced to a greater extent during infections with mucogenic RSV strains. We have
attempted to partially address subject by examining antiviral gene induction of IL-1β and
OPN in the following chapter to correlate mucogenesis and disease severity.
In a published study that examined an aged mouse model of influenza, older mice
had increased granulocyte infiltration, delayed recruitment of adaptive cells, and
prolonged disease (227); possibly the infiltration observed in our study is composed of
similar immune cells. Further examination for anti-RSV antibody titers and flow
cytometric analysis of lung suspensions could provide more information regarding which
cell types are present at sites of inflammation. The implications of TLR dysfunction on
RSV-induced lung pathology are still unclear, particularly since mucus hypersecretion
occurs independently of lung viral loads or replication. Regulated mucus secretion likely
provides undervalued antiviral benefits, as overexpression of MUC5AC in mouse lungs
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was found to protect against infection with H1N1 influenza A strain PR8 (228).
Determining which features of PRR signaling, mucus hypersecretion, or activation of
cytokines are impaired due to older age remains a challenging task. In the following
chapter, we examine how cytokine induction from Chapter 4 and age-associated
pathological changes may be correlated with production of two cytokines found
significantly associated with RSV and aging.
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RGB Original
400X
200X
40X
RGB-Blue Channel
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Figure 14. Quantification of cell infiltration density in hematoxylin-stained lung
sections.
Lungs were processed for OCT-embedding and 5 μm sections were stained with PAS and
hematoxylin. Images were collected at 40X, 200X and 400X magnification with white
background correction and area calibration. Bronchiole regions were manually annotated
to minimize empty space during area calculations. Original images were converted into
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8-bit by splitting RGB channels using ImageJ; the blue channel was inverted and the
threshold was optimized specific for magnification. The ImageJ macro Count and Mark
Cells was executed to find and quantify maxima of dark peaks. The area was measured
and results were compiled from a minimum of six images per mouse at 40X. After
bronchioles were designated on tissues, the following macro script was executed and
performed all aforementioned commands.
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Figure 15. PAS-quantification with color deconvolution and threshold area
measurement.
Lung sections were stained with PAS and hematoxylin and images were collected with
white background and identical exposure/conditions. Post-hoc background subtraction
was performed using Rolling Ball Subtraction (229). Singly-stained tissues (no stain,
hematoxylin, or PAS-alone) were used to calibrate the ImageJ Color Deconvolution
plugin and obtain customized PAS vector values. Auto-threshold was performed on
stain-separated images and area was calculated. The resulting ratio of areas between PAS
(Color 1): Hematoxylin (Color 3) were used to compare relative PAS-staining in tissues.
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Figure 16. Method for quantifying RSV-positive fluorescence in tissue sections.
Lungs were prepared for histology and sectioned at 5 μm thickness. Sections were
stained for RSV antigens and followed with Alexa Fluor-555 secondary antibody and
mounting with DAPI-containing antifade media. At 200X magnification, a minimum of
10 images were obtained per mouse (n =4-6/group). Single-channel RGB images were
split, inverted, followed by analysis with ITCN ImageJ plugin to estimate the number of
nuclei (18 pixels width) and number of cells (22 pixels width); results yielded the
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percentage of RSV-positive cells from the total number of cells. The threshold was
optimized by comparing mock-infected sections.
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101
Figure 17. Aged mice have enhanced pathology upon infection with mucogenic RSV
Young and aged mice were intranasally infected with a dose of 105 pfu/mouse of either
RSV 2-20 or rA2-L19F, or 106 pfu/mouse of A2 for comparison. Lungs were harvested
for histology on the days indicated, sectioned at 5μm, stained with PAS and
counterstained with hematoxylin. (A) Representative images of either aged or young
mice are shown in two columns; rows indicate infection with either rA2-L19F, 2-20 or
A2; scale bars indicate 200 μm (first column, 40X) or 40μm (second column, 200X),
respectively. At 40X magnification, hematoxylin stained nuclei were quantified with
ImageJ. (B) Five bronchioles were quantified per field of view to calculate the mean
infiltration density (#cells/mm2) and a minimum of 8 images were collected from a single
mouse (n=4-6 mice/group). (***) indicates significance between aged and young.
Significance was obtained through ANOVA, p<0.05. Histological analysis shown is
representative of total mean infiltration densities within a group from three separate
experiments.
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Figure 18. Mucus secretion increases after rA2-L19F infection in young but not
aged mice.
(A-B) Lung sections were stained with PAS and hematoxylin, and relative
PAS/Hematoxylin was estimated by ImageJ color deconvolution analysis. The ratio of
PAS to hematoxylin indicates how much area the PAS exceeds the area simultaneously
stained for hematoxylin and provides a relative measure of PAS abundance between
RSV-groups and age-groups. (A) rA2-19F or (B) A2 strains of RSV. Bars indicate the
mean ±SEM the total PAS/hematoxylin ratios collected from a minimum of 8 images per
mouse (n=4-6 mice/group), * = p<0.05 comparing aged from young, from two separate
experiments. (C) Gene expression of MUC5AC was examined with qRT-PCR and is
shown in reference to endogenous HPRT expression. Expression was obtained from
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individual mice (n=4-6/group) and graph represents the mean ±SEM from triplicate
experiments.
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105
Figure 19. Infection with rA2-L19F is enhanced in aged mice.
Young and aged BALB/c mice (n=4-6/group) were intranasally infected with 106
pfu/mouse of A2 or 105 pfu/mouse of rA2-L19F or 2-20 and total lung RNA was isolated
on days indicated in columns. (A) RNA expression of RSV N was analyzed with qRTPCR and represented as a ratio of target gene expression to endogenous mouse HPRT
with arbitrary units. Significance was determined with ANOVA, p<0.05 (B) Right lung
tissues from RSV-infected mice were gently homogenized in five equivalent volumes of
medium (wt/vol) immediately prior to serial dilution for infection in semi-confluent HEp2 cells for plaque assay using methylcellulose and immunostaining. Dotted horizontal
lines at 5 x103 indicates the limit of detection and graphs show the mean plaque titers
obtained from mice (n=4-6 mice per group) and experiments were repeated in triplicate.
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Figure 20. RSV clearance is delayed in aged mice upon rA2-L19F
(A) Left-lung sections were stained for RSV antigens and a minimum of 10 images per
mouse were taken at 200X magnification. Representative merged images display DAPI(blue) and RSV-positive (red) cells at 8 dpi with either RSV rA2-L19F, 2-20, or A2 in
young and aged mice. (B) The number of RSV-positive cells was enumerated using
ITCN ImageJ analysis. Single channels were quantified separately with width of 18 for
blue-nuclear counts and 22 for red-cell counts; the percentage of RSV-positive cells was
calculated by dividing the number of red-positive cells by the total number of blue-nuclei
present in a field of view. Individual dot plots were constructed with Minitab and
statistical significance was determined using ANOVA, p<0.05.
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Chapter 6: Effects of Aging on RSV-infection Induced
Expression of IL-1β and OPN
6.1 Introduction
Respiratory syncytial virus infections in the BALB/c mouse induce secretion of
cytokines and chemokines including TNF-α, IL-6, IFN-γ, CCL3, CCL5 and CXCL1
(209). For the purposes of studying RSV pathogenesis, the BALB/c mouse model has
some limitations, but it provides insight in areas that are otherwise unobtainable in cell
culture, such as aging and genetic deficiencies. Lung histology, lavage fluid, serum, and
tissue homogenates can be simultaneously harvested to provide better insight into what
occurs under physiological conditions with a heterogeneous population of cell types.
Dysregulated cytokine secretion and inflammation can be better examined over an
infection time course that resembles human infection and viral shedding (175). Chapter 3
described how RSV NS1 antagonized host immunity through binding to the adaptor
protein MAVS. Antiviral gene expression was then compared with disease severity in an
aged mouse model of RSV. Several antiviral proteins and genes were significantly
altered as a result of natural aging. A goal in this chapter is to correlate RSV-induced
pathology with the production of two inflammatory cytokines that are significantly
altered by RSV infection and older age. We studied the effects of the absence of this
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gene in the mouse and demonstrated how innate immune responses to RSV activate viral
recognition receptors, cytokine signaling, and physiochemical changes to the airways.
Respiratory infections are a leading cause of morbidity and mortality for
individuals over the age of 65, and in a 3-yr study in Tennessee, an estimated 25.4 per
10,000 persons over 65 years old were hospitalized for RSV-related illnesses (28).
Disease symptoms are exacerbated by preexisting medical conditions, such as COPD,
asthma, cardiac disease, and diabetes; epidemiological studies on aging are often
confounded by the disproportionate number of elderly participants who are hospitalized
for other underlying medical conditions (175). A recent study examined RSV pathology
in 111 RSV-infected individuals and compared persons <65 or ≥65 years old. Among
outpatients who demonstrated fewer disease symptoms (<65 years (n=37) versus ≥65
years (n=24)), elderly persons ≥65 years old had higher nasal viral titers and antibody
titers, although secretion of cytokines was insignificant, with the exception of IL-8.
When cytokines were measured early during infection (1-4 days of illness), CCL3 (or
MIP-1α) was significantly higher in older subjects than young. When severely ill
subjects were included in the analysis, nasal IL-6 and CCL3 levels were significantly
higher than levels from less ill individuals. It is important to note, however, that this
study was small and one of the first to assess cytokine levels from nasal samples;
furthermore, the study could not determine the first day of infection but instead relied on
first day of symptom onset for assessing disease length. These reported findings are
difficult to interpret because underlying conditions could contribute to cytokine levels.
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As a result, the study excluded a substantial number of subjects; further investigations are
needed before conclusions can be made about human aging and RSV infections.
The aged mouse model can provide clues regarding cytokine signaling and
physiochemical changes as a result of cytokine secretion. Recent use of clinical isolates
Line 19 and 2-20 have provided new information on RSV pathogenesis and host
responses to respiratory infections. RSV A2 infection in semi-permissive BALB/c mice
fails to induce substantial lung pathology at moderate to high doses (106 pfu/mouse);
obstruction of airways and mucin hypersecretion are often absent even though they are
histopathological hallmarks of severe RSV disease in human subjects (14, 230). The
recombinant A2 strain that expresses the F protein from Line 19 (rA2-L19F) grows more
efficiently in mice and induces greater pathology. As compared to the A2 or Long
strains, lung levels of IFN-α levels are reduced 24 h p.i. in rA2-L19F infected mice and
IL-13 are increased (41). IL-13 shares similarities to the Th2-associated cytokine, IL-4,
and is associated with airway hyperreactivity in animal models of asthma and RSV. IL13 also mediates airway inflammation and regulates mucus hypersecretion (50, 127, 231)
through STAT6 signaling, although the relationship between mucogenesis and IL-13 is
quite complex. Neutralization of IL-13 during RSV 2-20 infection alleviated goblet cell
metaplasia and pulmonary inflammation and reduced viral antibody titers although viral
loads were unchanged(232). In contrast, IL-13 overexpression was associated with
reduced RSV A2 illness and viral loads while deficiency of IL-13 increased lung viral
loads (219). To further complicate the story, IL-13 potentially negatively regulates
another mucin-associated cytokine, IL-17A, through upregulation of IL-10 in A2
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infections in STAT1-deficient mice (233) which suggests that drugs targeting IL-13 may
have unforeseen consequences in dysregulation of IL-17A. The conclusions from each
study may be specific to the RSV subtype A strains used but essentially all studies
implicate IL-13 in the regulation of mucin hypersecretion and alterations to the airway
microenvironment. Whether these mucosal changes are beneficial or pathogenic appears
dependent on strain virulence and degree of inflammation induced; further investigation
is needed with other mucogenic RSV strains.
Previously in Chapter 4, we identified two cytokines that were significantly
associated with age and RSV A2 infection, IL-1β and OPN. IL-1β, an acute
inflammatory cytokine that mediates mucus production through transcription regulation
(234, 235), is found elevated in the sputum of COPD and asthma patients during
exacerbations (60). Other cytokines found elevated in sputum are IL-6, TNF-α, and IL-8,
which are also associated with induction of mucus hypersecretion and inflammation (58).
Importantly, IL-1β is also found upregulated in influenza A virus in vitro and in vivo and
serves protective roles by mediating communication between epithelial and immune cells
during early stages of inflammation (154). The activation cascade for IL-1β is mediated
by a complex of proteins. It is also known as inflammasome activation and involves
multiple PRR components including RIG-I, TLR3, NLRP3, and caspase-1 for cleavage of
pro-IL-1β into the bioactive form. Defective signaling caused by a loss of any of these
components resulted in diminished IL-1β, which is correlated with enhanced influenza
disease (93, 148, 207). Similar to the mucin genes, IL-6, TNF-α, CCL5, and IL-1β are
elevated during disease, but whether they exacerbate disease or are byproducts of
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continuing damage and repair mechanisms remains debatable (127, 195, 212, 213, 236239).
These examples of cytokine pleiotropy and redundancy can lead to changes in
physiopathology in unexpected ways. Osteopontin (OPN) is associated with functions in
tissue remodeling (240), leukocyte recruitment (240, 241), activation of Th1 responses
(242, 243), and release of IL-12. OPN is critical for mucosal protection, particularly for
maintenance of epithelial barriers in gastrointestinal and respiratory systems. OPN was
recently credited with a role in antimicrobial defense against Klebsiella pneumoniae
(244) and Mycobacterium tuberculosis (245), although OPN was dispensable for viral
immunity against influenza (246, 247). Despite significantly increased levels of OPN in
the lung after influenza infection in wild-type mice, OPN knockout (OPN KO) studies
using low inoculum of influenza (50 pfu/mouse) did not see significant differences in
viral loads, virus-specific CD8+ T-cells, or lung pathology (246, 247). Instead,
deficiency of OPN had no effect on primary infection and led to enhanced differentiation
of memory CD8+ T-cells that resulted in lower viral loads during secondary exposure to
influenza (247). The authors suggested that OPN regulated IL-12 production by DCs
during the acute phase of influenza infection which then preferentially produced terminal
effector CD8+ T-cells. Consequently, a deficiency of OPN leads to rapid viral clearance
during secondary infections because of an abundance of memory effector CD8+ cells but
also enhances lung pathology. In contrast, infection with lethal doses of influenza (103
pfu/mouse) led to significant increase in disease severity and a reduction in natural killer
cells was observed (248). The rationale for conflicting results was not addressed in these
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papers, but may suggest OPN has beneficial and pathogenic roles in antiviral immunity.
Of interest, OPN deficiency was found to also protect against airway remodeling after
allergen-induced asthma (249, 250). In OPN KO mice, ovalbumin-induced asthma
symptoms including mucus production and airway hyperresponsiveness were
significantly reduced (249, 251) and disease was alleviated. Importantly, OPN mediates
neutrophil recruitment in a mouse model of ozone inhalation (252) and experimental
colitis (253). Migration of immune cells by OPN is partially mediated by OPN-receptor,
CD44, and is more important for macrophages and less for neutrophils. Since neutrophils
have significant roles in inflammation, development of airway hyperresponsiveness,
tissue injury, and mucus regulation, OPN likely regulates early phases of inflammatory
responses (251, 253-255). Cumulatively, OPN can act both beneficially and
detrimentally in lung disease.
Cytokine production is usually tightly regulated in healthy individuals but aging is
often characterized by chronic overproduction of proinflammatory mediators, particularly
C-reactive protein (CRP), IL-6, and TNF-α (256). Recent evidence suggests other
cytokines are altered by age, including IL-1β (257, 258) and OPN (259). Since both
genes were found upregulated in our earlier studies in Chapter 4, we hypothesized the
altered expression of IL-1β and OPN contributed to prolonged disease or enhanced
pathology that was observed in Chapter 5. We also examined RSV-lung disease in
transgenic mice that are deficient in OPN and compared the pathology with wild-type
C57BL/6 mice. Previous studies found C57BL/6 to be more resistant to RSV A2
therefore a higher virus inoculum was used to increase infectivity (260). This series of
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experiments were intended to provide insight into RSV-induced lung pathology in the
aged mouse and an understanding of how cytokine dysregulation influences viral
infectivity and pathogenesis.
6.2 Materials and methods
The following methods are in addition to the general methods described in
Chapter 2 and are specific to studying the role of IL-1β and OPN in RSV-induced lung
disease in young and aged mice. Moreover, transgenic mouse model used similar
histopathological methods described in Chapter 5.
6.2.1 Mice
Old (19-21 months) and young (2-3 mos) BALB/c mice were purchased from
Charles River Laboratories (Wilmington, MA) under a contractual agreement with the
National Institute of Aging. OPN KO (B6.129S6 (Cg)-Spp1tm1Blh/J) with C57BL/6
genetic background (4-6 weeks) were purchased from JAX laboratories as a single
homozygous breeding pair and bred for 3 generations. For comparison, age-matched
wild-type (WT) C57BL/6 (4-6 weeks) were purchased from JAX laboratories. All
animal work was approved by and performed in accordance with the policies of the
University of South Florida Institutional Animal Use and Care Committee.
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6.2.2 Tissue collection and histological processing
Mice were lightly anaesthetized with inhaled isoflurane and infected with 106 pfu
RSV A2 per mouse or 105 pfu RSV 2-20 or rA2-L19F per mouse. Because C57BL/6 are
reportedly more resistance to RSV infections than BALB/c, a higher inoculum (106
pfu/mouse) was used in OPN KO studies. Blood was collected via cardiac puncture and
allowed to clot for 30 min at RT before the serum was collected by centrifugation at 1000
x g. Serum was aliquoted and stored at -80 °C until analysis with ELISA. For
histological and protein/RNA analysis, the right bronchus was clamped with a hemostat
and the left lung was gently perfused intratracheally with 4% PFA in PBS. The right
lobes were minced into 1 mm pieces and apportioned for RNA/protein, and snap-frozen
on dry ice; the left lobe was immediately immersed in 4% PFA and processed for
histological analysis. A separate group of mice was used to study cytokines in BALF: 1
ml of room-temperature PBS containing 5 mM EDTA was intratracheally injected and
repeated twice for a total of 3 ml lavage fluid; BALF was pooled among mice in a group
and reserved in aliquots for future analysis by ELISA.
6.2.3 Enzyme-linked immunosorbent assay
Lung tissue was weighed and homogenized with glass Dounce homogenizers in 5
volumes (wt/vol) of pre-chilled FBS-free DMEM, and centrifuged at 1200 x g for 15 min
at 4 °C. Supernatants were aliquoted, frozen immediately on dry ice, and subjected to a
single freeze-thaw cycle. Upon thawing on ice, protein concentrations were measured
using the bicinchoninic acid (BCA) protein assay (Thermo Scientific Pierce). Serum was
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diluted 1:3 and BALF was analyzed neat (undiluted). Protein quantification via ELISA
was conducted per the manufacturer’s instructions. In brief, 96-well ELISA plates were
coated with capture antibody overnight, washed thoroughly, blocked with supplied assay
diluent, and then diluted samples of lysates, plasma, or BALF were added to the plate.
Plates were washed after each step before detection antibody, avidin-HRP, and substrate
solution were added to wells; 2N H2SO4 stop solution was added and plates were
immediately read at 450 nm. Standard curves were derived from dilutions of the
cytokine standards provided by the kits. Samples were run in triplicate.
6.2.4 Immunohistochemical staining and image analysis with ImageJ
Paraformaldehyde-fixed left lungs were cryopreserved in increasing
concentrations of sucrose and PBS, then embedded with OCT-freezing media. Lungs
were frozen over dry-ice then sectioned at 5 µm with a microtome; a minimum of four
lengthwise sections were placed on a glass slide and heated at 37 °C for ½ h. Slides were
steamed at 95 °C for 20 min in pH 9.0 unmasking solution. Tissues were then
permeabilized with methanol containing 3% hydrogen peroxide and nonspecific sites
were blocked for ½ h using 5% bovine serum albumin (BSA) Tris-buffered saline (TBS)
and 0.1%Tween-20 (TBST) buffer. Diluted primary polyclonal antibody goat anti-mouse
OPN (R &D AF808) was diluted 1:40 in 2.5% BSA/TBST and added to sections for
overnight incubation at 4 °C. Dilutions were optimized and antibody specificity was
confirmed by comparing staining with control goat IgG. After primary antibody
incubation, sections were processed with ABC Vectastain and nickel-3, 3’117
diaminobenzidine tetrahydrochloride (DAB) kits (Vector Labs). Nickel was added to
DAB reagent for greater contrast. Sections were counterstained with hematoxylin and
eosin before dehydration, clearing and mounting of coverslips.
6.2.4.1 Quantification of DAB staining
To obtain the percentage of DAB-positive cells from the total number of
hematoxylin-stained cells, lung sections were analyzed with ImmunoRatio ImageJ plugin
(261). Blank-field image correction and threshold were adjusted before the macro was
executed on groups of images, yielding a montage of >6 images from a single mouse
(n>4 mice/group). Background and nonspecific nickel-DAB staining was subtracted
through comparison with control sections stained with normal goat IgG. Individual
percentages of DAB to hematoxylin were tabulated to generate box plots and analyzed
with Minitab software with 2-way ANOVA to determine statistical significance.
6.3 Results
6.3.1 Aging results in altered IL-1β induction upon RSV infection
We intranasally infected young and aged BALB/c mice with three different RSV
strains, rA2-L19F, 2-20, and A2 and collected blood, tissue, or BALF at the peak of
infection (1, 4 or 5 dpi, depending on the strain) and 8 dpi. RNA obtained from a portion
of the right lung was analyzed by qRT-PCR for expression of IL-1β in respect to
endogenous gene HPRT (Figure 21A). At baseline, uninfected aged mice had higher
expression of IL-1β than young mock-infected mice. Expression was rapidly upregulated
in young mice after rA2-L19F and 2-20 1 dpi, but aged mice had downregulation.
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Kinetics of IL-1β differed between rA2-L19F and 2-20 in young mice at the peak of
infection (5 dpi and 4 dpi, respectively) and by 8 dpi with rA2-L19F, aged and young
mice had the same expression level; in contrast, 2-20 infection resulted in upregulation of
gene expression of IL-1β in aged mice by 4 dpi and continued to rise above baseline by 8
dpi. A similar slow, upwards trend was observed in RSV A2 infection in aged, but the
induction was less pronounced. Upregulation was observed 1 dpi with A2 in young mice,
but induction was mild. By 8 dpi with A2, young mice IL-1β were below that at
baseline. Overall, induction of IL-1β was delayed compared to young counterparts.
Homogenized lung tissues from young and aged mice infected by either rA2L19F or 2-20 were analyzed for IL-1β by ELISA (levels of IL-1β in young A2 infected
mice were below detection and are not shown). Infection in young mice resulted in a
peak production of IL-1β at 5 dpi and 4 dpi for rA2-L19F and 2-20, respectively (Figure
21B). Levels decreased slightly by 8 dpi but remained significantly higher than baseline.
In contrast, levels of IL-1β remained unchanged until 8 dpi with either RSV strain in aged
mice. For rA2-L19F infections, young mice had higher levels of IL-1β on 5 and 8 dpi.
Serum and BALF was analyzed for levels of IL-1β to examine changes in
systemic IL-1β or local IL-1β, respectively (Figure 21C). At baseline, aged mice produce
more serum IL-1β than young. Levels of IL-1β in serum were inversely related between
RSV strains in young mice: for rA2-L19F, IL-1β was produced late, while IL-1β was
circulating earlier after 2-20 infection. No significant change was observed for serum IL1β in aged mice regardless of RSV strain. Detection of IL-1β was attempted for BALF
but levels were frequently below detection limits. A significant increase in BALF IL-1β
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was observed in rA2-L19F-infected young and aged mice at 5 but not 8 dpi. Surprisingly,
IL-1β levels peaked in BALF on 8 dpi upon 2-20 in aged mice but were undetectable at 4
dpi.
6.3.2 OPN induction is delayed but prolonged in aged mice in response to
RSV
OPN was found to be significantly altered by age as well as RSV infection,
therefore we compared expression and secretion of OPN in BALB/c mice using the three
RSV strains. RNA from portions of the right lungs of individual mice was analyzed for
OPN expression using qRT-PCR. Expression of OPN was elevated in aged mockinfected mice and expression kinetics of OPN were age-and RSV strain-specific (Figure
22A). The greatest change in OPN expression occurred in rA2-L19F infections with
both age groups; however, as with all three strains, young mice had induction of OPN at
4 or 5 dpi but levels decreased by 8 dpi. In contrast, aged mice had the greatest induction
at 8 dpi with rA2-L19F and 2-20.
To assess whether OPN induction changed locally in airways upon infection,
immunohistochemistry was performed for detection of OPN in rA2-L19F and 2-20
(Figure 22B). In the absence of infection, aged mice had greater staining for OPN than
young. For rA2-L19F, OPN was induced at 5 dpi for young but diminished by 8 dpi;
conversely, aged mice had little change in OPN at 5 dpi, and at 8 dpi the airways were
still infiltrated and OPN staining was still prominent. 2-20 infections resulted in a similar
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age-specific trend: young mice had temporary induction of OPN by 4 dpi, but levels were
less by 8 dpi; aged mice had very little change in IHC.
The percentage of OPN-positive cells was derived by quantifying DAB and
hematoxylin staining in ImageJ. The bar graphs illustrate what was shown in the
representative images (Figure 22C): aged mice had reduced induction of OPN in
comparison to young but induction was prolonged at 8 dpi. OPN-staining in mockinfected aged mice was nearly twice that of mock-infected young (44.1% vs. 21.0%,
respectively ±SEM). Infection with 2-20 showed no significant change in OPN
production in aged mice, which contrasts with the increases in OPN seen in young 2-20
infected mice.
Since OPN induction was diminished in aged mice, we examined a receptor for
CD44 with qRT-PCR (Figure 22D). Expression was significantly different at baseline
and at 1 dpi, suggesting early induction of OPN may be altered due to age and may
partially explain the observed delayed induction of OPN in aged mice.
6.3.3 Mucus secretion is enhanced in OPN KO mice after infection with rA2L19F
To examine the significance of OPN in the context of mucogenic RSV infections,
we intranasally infected young (4-6 weeks old) OPN KO and WT C57BL/6 for
comparison (n=3) with rA2-L19F at 106 pfu/mouse and experiments were performed
twice; a higher dose of virus was used because C57BL/6 mice have increased resistance
to RSV (260). No observable differences were visible in OPN KO or WT mice after
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infection and body weight did not significantly change with infection (data not shown).
Perfused left lung and right lung tissues were collected 5 and 8 dpi and stained with PAS
and hematoxylin (Figure 23A). Mock-infected OPN KO and WT were compared for
lung integrity and were not visibly different. Lungs were stained for PAS and compared
at 5 and 8 dpi. In contrast to WT mice, OPN KO had more visible magenta PAS staining
along the bronchioles but cellular infiltration was not altered between mice strains (data
not shown). PAS staining was quantified with ImageJ and constructed into a graph
(Figure 23B). At 200X magnification, more PAS-stained areas were detectable in OPN
KO than in WT mice at 5 and 8 dpi, suggesting mucin production is enhanced in OPN
KO mice upon infection. We examined two genes associated with mucin production, IL13 and MUC5AC with qRT-PCR analysis and found increased expression of both genes
in OPN KO mice after rA2-L19F infection. No observable differences were detected in
IL-13 or MUC5AC expression in the absence of infection.
6.3.4 OPN KO mice have increased resistance to rA2-L19F
Viral RSV N gene expression and plaques were examined in OPN KO and WT
mice after rA2-L19F infection to assess whether OPN is required for viral clearance.
RSV N expression was found reduced in OPN KO, although large variation was seen in
WT mice at 8 dpi (Figure 24A). Lung homogenates were then examined for viral load by
plaque assay. Similar to qRT-PCR results, WT mice had greater lung viral loads than
OPN KOs on 5 and 8 dpi (Figure 24B). Usually in young BALB/c mice, rA2-L19F is
usually cleared by 8 dpi and we observed viral plaques present on 8 dpi in young WT
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C57BL/6 mice; this is likely from higher viral dose initially given (106 pfu versus 105
pfu, C57BL/6 and BALB/c respectively). Immunofluorescence was performed on lung
sections from OPN KO and WT mice and prolonged rA2-L19F infection was visible in
the airways of WT but not OPN KO mice (Figure 24C). Images were quantified for the
percentage of RSV-positive cells (Figure 24D). At 5 dpi, the number of RSV-positive
cells was not statistically different between WT and OPN KO; however, at 8 dpi, virus
was cleared from the lungs of OPN KO but could still be detected in the lungs of WT
mice. The number of cells surrounding bronchiole regions was also quantified with
ImageJ at 40X magnification as previously described in Chapter 5. No significant
difference was observed between WT and OPN KO at 8 dpi (data not shown). These
data suggest deficiency of OPN promotes viral clearance without affecting cellular
infiltration or inflammation.
6.4 Discussion
Our results demonstrate age-specific cytokine responses to mucogenic RSV
strains. Comparative analysis was performed on the lung sections and tissues from rA2L19F, 2-20 and A2 infected mice to determine if aging contributes to more severe
pathology and whether antiviral responses are impaired. Gene expression of IL-1β was
compared and found to be different depending on the RSV strain. Aged mice had an
elevated baseline of IL-1β in the absence of infections, which is in agreement with other
reports on aging and chronic inflammation (257). Infection with rA2-L19F in young
mice led to substantial increases in expression and production of IL-1β in lung tissue,
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serum, and lavage fluid, particularly at 5 dpi when infection peaked in young mice. In
aged mice, IL-1β production induced by the rA2-L19F strain was delayed and
significantly less than in the young in local tissue and BALF at 5 dpi; circulating serum
levels remained elevated in the aged and did not change upon infection with rA2-L19F.
Infection with rA2-L19F was prolonged in aged mice, with viral RNA peaking at 8 dpi
instead of 5 dpi. As seen with the young mice, local production of IL-1β is maximal at
the peak of infection (5 dpi). 2-20 infection also increased IL-1β gene expression in lung
tissue in young and aged mice; RNA and protein levels were more comparable, if not
higher, than levels observed in young mice. The induction of IL-1β correlates well with
infection, regardless of strain in young mice, but is dysregulated in aged counterparts;
interestingly, the induction of IL-1β in aged mice lacks consistency between strains,
further supporting the three strains demonstrate unique pathogenesis in the mouse
model(40). Importantly, infections with 2-20 yielded fewer plaques and RSV-positive
cells in IHC at 8 dpi than with rA2-L19F (Chapter 5, Figure 20) suggesting that 2-20 is
cleared by 8 dpi in either age group. Further examination at earlier time points may
indicate whether IL-1β levels are altered during early 2-20 infection.
OPN was next examined because of possible roles in leukocyte recruitment, tissue
remodeling, or activation of Th1 responses. Aged mice expressed more OPN than young
at baseline and regardless of RSV strain; however the degree of change in expression
from baseline to 5 dpi was substantially diminished in older mice. In contrast to the
young, RSV 2-20 did not induce any significant change in OPN in aged mice. In healthy
individuals, OPN is usually tightly regulated and is only expressed upon tissue injury
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(241, 262). As infiltration was substantially greater in aged mice, tissue damage could
also be greater. Alternatively, the prolonged and excessive production of OPN may
suggest age-related loss in OPN regulation or signaling. As such, we observed irregular
expression of CD44, a known receptor for OPN that is predominantly used for
macrophage but not neutrophil recruitment (253), in aged mice. This may indicate a
delay in recruitment of macrophages and instead, prolonged neutrophil migration;
however further examination is still needed. Additionally, it was reported that aging
resulted in delayed CD44 expression on mitogen activated CD8+ T-cells (178, 242, 262)
therefore age may impact upstream OPN signaling; further investigation is needed to
assess whether downstream OPN signaling is impaired with age. Alternatively, OPN
undergoes numerous post-translational modifications, including glycosylations,
phosphorylations, and sialylations, which could substantially alter ligand-receptor
interactions (263). Additionally, three known variants of osteopontin, A, B, and C, occur
in nature but only isoform A is recognized by commercial antibodies; consequently, our
methods were limited to detection of osteopontin A although isoforms may exist during
lung injury. Additionally, the intracellular and extracellular osteopontin variants
contribute to unique immunes responses (91). Our investigation did not explicitly
examine a particular variant, as little remains known about the functionality of the
osteopontin secreted in the airways. Further examination, including proteomic analysis
of airway osteopontin, is still needed to determine the predominant isoform expressed
during RSV infection and whether the secretion contributes or suppresses antiviral
responses to RSV.
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Age-related changes in expression and abundance of IL-1β and OPN were
correlated with age-associated RSV disease pathology as described in Chapter 5. To test
whether OPN could be contributing to prolonged pathology or disease, we used OPN KO
mice and infected them with mucogenic rA2-L19F. Our findings suggest that OPN
attenuates mucus production in the airways as we saw quantitatively fewer PAS-positive
areas in the lungs of rA2-L19F-infected WT than OPN KO. Further confirming our
visual observations, mucin-associated genes were upregulated when OPN expression was
absent. Since OPN may be involved in viral clearance, we examined viral loads in
infected OPN KO and WT mice. OPN KO mice demonstrated resistance to rA2-L19F
infections and had lower viral RNA expression, viral loads from the lung, and fewer
RSV-positive cells. Infiltration was also analyzed but found unaltered by the absence of
OPN.
In our investigations, we observed differences in mucus production at the peak of
infection (5 dpi). Whether the observed correlation of OPN and mucus hypersecretion is
the predominant explanation for reduced RSV infection in OPN KO mice still requires
further investigation. Currently, the knowledge regarding osteopontin induction during
viral infections remains poorly understood and literature suggest infectivity, strain, and
duration of illness can contribute to altered osteopontin expression. While we cannot
conclude osteopontin contributes directly to tissue injury or infiltration, as we have not
studied the inflammatory cell types or early production of osteopontin, we associated
delayed but prolonged induction of osteopontin with enhanced disease. Additionally, the
observed changes in mucosal responses and increased resistance to RSV in OPN KO
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mice suggest excessive osteopontin could alter airway surface liquid homeostasis,
whether directly or indirectly. Further examination with small-interfering RNA targeting
OPN may assist with conditional suppression of OPN in the airways and better clarify the
role of OPN in lung disease. Suppression of aberrant OPN in aged mice is predicted to
attenuate RSV disease in aged mice and restore mucogenesis. To further demonstrate the
potentially negative role of OPN on airway health, aerosolized OPN in young mice is
expected to exacerbate lung disease and further restrict mucogenic responses to RSV;
these experiments remain to be conducted in future studies. Ultimately, our goal was to
study aging and innate immunity and we observed age-associated, dysregulated
overexpression of IL-1β and OPN during mucogenic RSV infections. This suggests viral
pathogenesis is significantly altered by age and therefore efforts to treat disease may need
to be modified to adjust for chronic cytokine hyperproduction.
The role of OPN in antiviral immunity is an ongoing investigation, not only with
RSV, but also for infections with mouse-adapted, mucogenic strain of influenza virus
PR8 (A/PR/8/34 H1N1). Similar to our findings, recruitment of inflammatory cells was
unchanged in OPN KO upon low-dose(50 pfu/mouse) infection with influenza PR8 at
days 7, 10, and 13 post-infection (246, 247). Upon secondary infection, OPN acted in
preferential generation of memory CD8+ T-cells (264). Interestingly, when given as low
inoculum (50 pfu), the investigators did not find statistical differences in influenza viral
load but in a third study involving OPN KO mice, high, sublethal doses of PR8 (103 pfu)
resulted in a substantial increase in disease severity early in infection (5 dpi);
interestingly, mucus production was not assessed in any of the investigations (246).
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Conflicting results suggest the role of OPN remains uncertain and may emphasize the
differences in mucogenic responses induced by influenza and RSV.
Of interest, a recent transgenic mouse model overexpressing mucin MUC5AC
(MUC5AC OE) was found more resistant to PR8 infection at a high inoculum (300 pfu)
and resulted in reduced neutrophil recruitment (228). This may suggest that
physiochemical changes, such as mucus, can affect the disease outcome. The
investigation with MUC5AC OE mice showed that mucus hypersecretion alone does not
cause inflammation or obstruction of the airways--quite the opposite: secretion of mucus
captured virion particles and reduced infectivity (228). Differences between the OPN
KO and MUC5AC OE studies may be related to different viral inoculums used in the two
studies (50 pfu versus 700 pfu, respectively) as well as days post-infection when mice
were euthanized for plaque titers (days 7 and 11 versus days 2 and 4). Consequently,
provided the evidence from the studies in Chapter 5 and 6, we would anticipate future
studies with OPN KO and early PR8 infections may have more similar results to that of
the MUC5AC OE.
Importantly, RSV and influenza differ greatly at a structural and genetic level, and
RSV lacks viral hemagglutinin-neuraminidase (HN) activity during entry and exit from
target cells. Neuraminidase cleaves sialic acid groups on glycoprotein to mediate release
from host cell, evade mucin defense mechanisms, and to prevent virion aggregation
(265). This was previously demonstrated by treating RSV particles with neuraminidase
which enhanced infectivity and promoted cell-to-cell fusion. Although RSV does not
utilize sialic acid residues for entry like influenza (226), evidence that neuraminidase
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treatment improves RSV infectivity in vitro suggests RSV encounters difficulties when
invading and exiting target cells that are mucin-enriched. Since mucogenic strains were
only recently described, little is known regarding binding of mucins to RSV virions or
how mucus affects RSV infectivity. Instead of HN, the RSV fusion protein F and
glycoprotein G mediate cell-to-cell fusion and stimulate mucin-production and neutrophil
recruitment (225). The authors replaced wild-type fusion protein with fusion proteins
from clinical isolate Line 19 and 2-20 and found the mucogenic F protein also induced
neutrophil recruitment; neutrophil depletion reduced mucin production, therefore the
authors have made the first correlation between RSV F, neutrophilia, and mucogenesis;
further investigation is still needed to understand the mechanisms. Since cellular
infiltration was not significantly different between the OPN KO and WT RSV-infected
mice, we predict alternate mechanisms led to increased mucus production in OPN KO,
however this remains to be investigated. Lastly, little remains known regarding agerelated changes in airway surface liquid homeostasis, particularly whether ion channel
transport or mucociliary clearance is impaired due to age or hyperinflammatory cytokine
responses; our investigations have only partially examined age-related changes in lung
physiology.
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Figure 21. Age differential expression of IL-1β upon RSV infection
Young and aged BALB/c mice (n=4-6/group) were intranasally infected with a dose of
106 pfu/mouse of A2 or 105 pfu/mouse of rA2-L19F or 2-20 and lavage fluid, serum, and
lung tissue were collected on the days indicated. (A) RNA transcripts of IL-1β were
analyzed with qRT-PCR and represented as a relative ratio of target gene expression to
endogenous mouse HPRT. (B) Lung tissue homogenates were analyzed for levels of IL1β by ELISA; tissues from rA2-L19F and 2-20 infected young and aged mice were
weighed and levels are given in respect to pg of IL-1β/g of tissue. (C) Levels of IL-1β in
serum and BALF from individual mice were analyzed using standard ELISA (n=4-6 per
group). The horizontal line indicates the limit of detection of the ELISA method. Data
are represented as means ±SEM. Experiments were performed in triplicate.
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Figure 22. Aging results in diminished OPN production in response to 2-20 RSV.
Young and aged BALB/c mice (n=4-6/group) were intranasally infected with 106
pfu/mouse of A2 or 105 pfu/mouse of rA2-L19F or 2-20 and left lungs were
formaldehyde-fixed for histology and right lungs were used for RNA isolation. (A) RNA
transcripts of OPN were analyzed with qRT-PCR and represented as a relative ratio of
target gene expression to endogenous mouse HPRT. (B) 5 μm lung sections from RSVinfected mice were collected on days indicated. Sections were immunostained for antimouse OPN and enhanced with nickel-DAB reagent before counterstaining with
hematoxylin and eosin. Representative images shown are at 100X magnification with
400X inset with dark brown/black nickel-DAB staining; hematoxylin is light blue nuclear
stain. Representative images are shown with scale bar indicating 100 μm. (C)
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Enumeration of OPN-positive cells was performed with ImmunoRatio ImageJ analysis on
200X magnified lung sections from 8 dpi and values are shown as a percentage of total
hematoxylin-stained cells in an individual box plot with mean interval bars. A minimum
of 8 images were collected per mouse (n=4-6/group) and individual dot plots are shown
of aged or young mice with interval bars ± SEM. (D) qRT-PCR was performed on total
right lung RNA from young and aged 2-20 RSV-infected mice for mRNA expression of
OPN receptor CD44. Statistical significance was determine with ANOVA 2-way analysis
with p<0.05. All experiments were performed in triplicate.
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Figure 23. Mucus secretion is enhanced in OPN KO mice upon rA2-L19F infection.
Lung sections from young (4-6 weeks) OPN KO and WT C57BL/6 mice were stained
with PAS and a minimum of 10 images per mouse at 40X and 200X were collected (n=3
mouse/group). Experiments were performed twice. (A) Mucin staining with PAS in OPN
KO and WT C57BL/6 is shown at 200X magnification on 8 dpi with rA2-L19F. (B) PAS
staining quantified with ImageJ color deconvolution analysis; the measured areas of PAS
positivity were divided by the total stained tissue area (hematoxylin). (C) Gene
expression of IL-13 was analyzed by qRT-PCR from right lung RNA. (D) Gene
expression of mucin-associated gene MUC5AC was analyzed by qRT-PCR. Significance
could not be calculated due to the small number of mice.
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Figure 24. OPN KO mice are resistant to rA2-L19F infection compared to WT.
OPN KO and C57BL/6 mice were intranasally infected with 106 pfu/mouse with RSV
rA2-L19F and on 5 dpi and 8 dpi, left lungs were formaldehyde-fixed and right lungs
were reserved for RNA isolation and plaque titration. (A) Lung RNA was analyzed with
qRT-PCR for RSV N expression and is shown with relative gene expression when
normalized to HPRT. (B) Lung tissue was homogenized in five equivalent volumes of
culture media and serially diluted for infection in HEp-2 cells. Plaques from individual
mice were obtained by methylcellulose overlay and immunostaining. The graph shows
the mean plaque titers (n=3 mice/group) and experiments were performed twice. (C)
Lung sections were stained with polyclonal antibody against RSV antigens and enhanced
with Alexa Fluor 555-secondary antibody. Images with RSV-positive (red) cells and
DAPI (blue) nuclear staining were collected, with a minimum of 10 images per mouse
(n=3 mice/group). (D) The average percentage of RSV-positive cells is shown ±SEM in a
graph.
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Chapter 7: Conclusions and Future Directions
7.1 Subversion of antiviral interferon responses by RSV NS1
The mammalian innate immune system has several strategies for recognizing
pathogens such as through RNA-helicases that respond to double-stranded RNA and by
activating adaptor proteins that lead to synthesis of one of the earliest antiviral cytokines,
type I IFNs. To promote their survival, viruses have evolved tactics to antagonize the
host’s early innate immunity. RSV is a particularly poor-inducer of IFN, most likely in
part because of the two nonstructural proteins, NS1 and NS2, although their roles as
virulence factors remain incompletely understood. We found localization of NS1 to the
mitochondria to cause disruption of the MAVS/RIG-I binding that occurs upstream of
IFN signaling. We also observed temporal expression of NS1 paralleling that of the IFN
regulator LGP2; restoration of NS1 in recombinant RSV that lacked NS1 also helped
restore infectivity.
The findings emphasize the importance of MAVS/RIG-I and other PRR
components in the context of early innate immune responses to RSV infections.
Silencing RSV NS1 has previously been demonstrated to have therapeutic benefits in
attenuating RSV infection; our data suggests a possible mechanism for RSV NS1mediated disruption of the antiviral interferon response and viral recognition pathways.
This investigation also emphasizes how critical MAVS is to the RSV-induced immune
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response. MAVS dysfunction in humans is predicted to result in enhanced RSV
exacerbations. Genetic polymorphisms, underlying medical illnesses that disrupt innate
immune responses, and other environmental factors that could contribute to MAVS/RIGI dysfunction may serve as predictors of RSV disease severity.
Although incompletely understood, we also observed that the upstream RLH
molecule, LGP2, was upregulated by NS1, even in the absence of a viral infection. This
could implicate NS1 as a possible transcription regulator or other role for inducing more
LGP2. Alternatively, NS1’s activity could have an effect on RLH signaling that causes
feedback induction of LGP2. Whether these proposed pathways are correct remains to be
investigated. Further examination is expected to shed light on the role of LGP2, whose
role as a positive or negative regulator in viral recognition remains under debate.
Nonetheless, our studies conclude that NS1 serves important roles in host innate immune
disruption and viral survival and therefore is a good target for the development of RSV
antivirals.
7.2 Aging leads to impairment of antiviral gene expression
Our understanding of aging and pulmonary health is surprisingly limited despite a
growing senior population and demand for age-specific therapeutics. RSV is a leading
cause of respiratory disease in persons over the age of 65, but few studies have identified
why elderly are more susceptible for severe disease. To better understand changes in the
innate immune responsiveness to RSV infection, we use PCR arrays on A2 infected
young and aged mice. Over 27 genes were upregulated more than 2-fold in young mice
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on 1 dpi, but fewer genes were changed in aged mice. Similar to how NS1-mediated
disruption of RLH signaling increases viral survival, diminished induction and activation
of antiviral genes due to older age and may partially explain the mechanism of agerelated enhanced disease in humans. We also identified genes that are affected by age
and RSV infection as well as other genes that are unaffected by RSV infections in either
age group. Upon further investigation, we found RIG-I to be downregulated in aged mice
upon RSV infection. As an essential receptor for RLH and IFN signaling, defective RIGI activity may have severe consequences in respiratory health. Additional functions have
been associated with RIG-I activity, particularly the RIG-I/TLR3/NLRP3 inflammasome
activation during influenza A virus infections (154). NLRP3 inflammasome activation
and bioactive IL-1β are credited with antiviral roles in the defense against influenza .
Although beyond of the scope of this particular study, we anticipate declining RIG-I
function due to age contributes to poor inflammasome activation and reduction of
subsequent antiviral responses; declining NLRP3 inflammasome associated with age was
previously reported in an influenza mouse model(207)
. Indeed, we found IL-1β, a byproduct of caspase 1-directed cleavage, had
delayed induction and secretion in aged mice compared to young mice upon mucogenic
RSV infections. Moreover, NLRP3 inflammasome activation declines with older age in
the mouse model of influenza but can be restored with nigericin, a purinoceptor
potassium ionophore that restimulates inflammasome activity (207). A similar
protective role against RSV is predicted but has yet to be studied.
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Ex vivo stimulation of alveolar macrophages demonstrated age-related impairment
in TLR7/8 and reduced ability to secrete IL-6 upon stimulation. IL-6 is a
proinflammatory cytokine associated with injury, acute phase responses, and
inflammation. IL-6 is also credited with protective roles in antimicrobial defense and
immune cell differentiation (266-269). Thus, local, controlled production of IL-6 is
important for antiviral responses; unfortunately, IL-6 and other proinflammatory
cytokines may be chronically over-produced in elderly subjects. Whether signaling
cascades leading to cytokine production are defective or a maximum threshold of
cytokine production was reach resulting in feedback inhibition occurs in elderly remains
to be determined. Through this investigation, we demonstrate that, in addition to having
chronic proinflammatory cytokine profiles at baseline, elderly mice demonstrate
defective antiviral responses that lead to impaired or delayed cytokine induction upon
new infections, which may subsequently increase susceptibility for future infections.
Further studies could examine age-related changes to transcription factors NFκB and
IRFs because of their canonical roles in antiviral immunity.
These data provide evidence that age is a significant contributing factor to
respiratory disease. Diminished ability to induce antiviral gene expression has
noteworthy clinical significance: vaccine candidates often rely on functional PRR activity
and downstream activation. TLR expression and function serve as helpful indicators of
influenza vaccine responsiveness (188); possibly a similar correlation can be made with
RSV. Our studies suggest not only expression, but also innate immune function, is
impaired with age. Consequently, therapeutic strategies may need to be adapted to
142
restore diminished host immunity and focus on areas of human immunity that are not
negatively altered by age.
7.3 Age-associated changes in lung microenvironment affect RSV
outcomes
In Chapter 5, we implemented an aged mouse model of RSV and included two
mucogenic RSV strains to characterize RSV pathogenesis that resembles disease in
humans. Virulence was greatest among mucogenic strains and was particularly enhanced
in aged mice. These results contradict previous studies with non-mucogenic RSV A2 and
suggest that age is a significant contributing factor in disease severity. Cellular
infiltration was considerably enhanced while mucus secretion and viral clearance were
delayed in aged mice. The differences observed between A2 and mucogenic rA2-L19F
and 2-20 suggest A2 has substantial limitations in the mouse model and may previously
not provide an adequate model for RSV-induced lung disease in the mouse. The
presence of pathology and viral loads on 8 dpi with rA2-L19F indicates the virulence is
substantially higher and may serve as a better model for severe disease. Further
examination is needed to see how mucus production is altered in aged mice, although
nasal mucociliary clearance reportedly declines with age in humans (220, 221). Ageassociated changes in the lung microenvironment, including cellular composition, airway
surface liquid integrity, and mechanical function of cilia remain to be examined in future
studies.
143
7.4 Age-associated changes to cytokine induction in RSV infections
Once histopathology was characterized in our aged mouse models of mucogenic
RSV strains, we sought to correlate disease severity with age-related changes in antiviral
responses. Five antiviral genes including IL-1β and OPN were altered by both age and
RSV A2 infection. Induction of IL-1β and OPN was highly dependent on RSV strain and
virulence; peak viral titer and secretion of IL-1β were correlated in rA2-L19F and 2-20
infections; importantly, IL-1β failed to be induced in local tissues of aged mice. A
similar loss of NLRP3 inflammasome activation and IL-1β production was observed in
aged mice infected with influenza (207). In contrast, OPN expression was elevated at
baseline, but showed a reduced slope of induction upon RSV infection in aged compared
to young mice. This change in gene expression kinetics was apparent at the protein level,
where OPN was abundant and its expression was prolonged in older mice. A receptor of
OPN that is used for macrophage, but not neutrophil, migration was found diminished at
baseline and 1 dpi in elderly mice but restored by later stages of infection.
To better understand the role of OPN in RSV-induced lung disease, OPN KO
mice were infected and compared to WT C57BL/6. No pathological differences were
apparent between mouse strains upon mock-infection. In a similar manner as BALB/c,
WT C57BL/6 developed infiltration and mild mucus production with rA2-L19F;
C57BL/6 mice are more resistant to RSV than BALB/c and infection was expected to be
mild. Instead, infection with rA2-L19F resulted in infection of the airways with plaques
reaching ~4 x104 pfu/g of lung tissue and sustained infection up to 8 dpi. Conversely,
144
OPN KO had reduced viral loads and infection, and increased mucus and expression of
mucin-associated genes, IL-13 and MUC5AC.
OPN-dependent changes in mucin response were not anticipated, although OPN is
involved in tissue remodeling, modulation of extracellular matrix, and fibrosis (240, 254,
262). Evidence suggest the absence of OPN may alter airway microenvironments that
favor mucus secretion. One possible mechanism may involve epidermal growth factor
receptor (EGFR) which is upstream of MUC5AC and mediates mucus metaplasia (270).
Influenza induces mucus and MUC5AC expression through a transcriptional signaling
cascade that involves EGFR (271). OPN recently was found to have protein levels that
are inversely proportional to EGFR and is frequently identified with EGFR as a
biomarker of disease severity (272); however, the precise relationship between OPN and
EGFR remains unknown as these relationships were only recently described in 2012.
Alternatively, OPN may alter the recruitment of neutrophils, which are credited to mucus
production in young mice(225). Although total cellular infiltration was not altered in
OPN KO mice, we did not specifically stain for presence of macrophages or neutrophils,
which may likely be reduced in the OPN KO mice. We anticipate a decline in infiltrating
neutrophils would consequently involve alternate means for mucus secretion yet this
remains to be investigated. The effect of OPN on leukocyte migration has been
previously established, yet little remains known in the context of RSV infections.
145
7.5 Final remarks
The studies performed and described in this dissertation are expected to contribute
to pulmonary research and reveal the undervalued importance of innate immunity in RSV
infections. The changes in PRR signaling lead to exacerbated disease; unfortunately,
with natural age, there appears to be a decline in function and expression of antiviral
mediators. To further the damage, excessive proinflammatory cytokine production and
dysregulated IL-1β and OPN production may exacerbate pathology. These studies found
RSV strain virulence plays an important role in age-related studies, although several
genes were commonly affected by age and infection. We also observed a loss of mucus
secretion in aged mice and that would coincide with our findings with OPN KO mice.
Overproduction of OPN in the elderly may be just one example of many age-associated
dysfunctions in the innate immune response to RSV. We summarize the findings of this
investigation in the following illustration, Fig. 25. Our findings contribute to the
understanding of innate antiviral immunity and conclude RSV-induced antiviral
responses are diminished or altered with older age. Ultimately, the future direction of
this research will be to investigate age-specific therapies designed to alleviate aberrant
inflammation in the airways of elderly subjects and prevent enhanced disease.
146
Figure 25. Age-differential innate immune responses to RSV.
A comparison of innate immune responses to RSV infections is illustrated in the
flowcharts and predicts age-related outcomes to viral infections. Early innate immune
responses to RSV and other respiratory viruses were found impaired in aged mice and
was associated with enhanced lung pathology and increased viral loads. Additionally,
innate immune responses can be altered by RSV NS1, which mediates disruption of IFN
activation. Aberrant changes to antiviral responses by either the viral pathogen or by age
negatively affect pulmonary health.
147
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About the Author
Terianne Maiko Wong received her Bachelor of Science from the University of
South Florida with a major in Microbiology in 2007. After participating in
Undergraduate Research in a water ecology/quality research lab in the College of Arts
and Sciences, she spent a year employed as a hematology technical assistant at the
clinical laboratory company, LabCorp of America. In August of 2008, she returned to
the University of South Florida, Morsani College of Medicine to pursue a Ph.D. in
Medical Sciences. In fall of 2011, she was awarded the USF Signature Research
Doctoral Fellowship and simultaneously earned her Master of Science in Medical
Sciences. In addition to active participation in the USF Health Research Days, USF
Graduate Student and Post-doctoral Annual Symposiums, and Veterans Affairs Research
Days, she served as lead in Sponsorship and Awards Committee when helping organize
the USF NanoFlorida Symposium 2013. She received multiple travel grants for national
conferences, Association of Medical Science Graduate Students (AMSGS) Travel Grant
(2011), USF Signature Interdisciplinary Program of Allergy, Immunology, & Infectious
Disease Travel Award (SIPAIID) Travel award (2011), Graduate School Conference
Presentation Grant Program (2012 & 2013) and Fellow In-Training Travel Award from
the American Academy of Allergy, Asthma and Immunology (2011, 2012, & 2013).