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University of South Florida Scholar Commons 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] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the Biology Commons, Immunology and Infectious Disease Commons, and the Virology Commons Scholar Commons Citation Wong, Terianne Maiko, "Innate Immune Responses to Respiratory Syncytial Virus: Age-associated Changes" (2013). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/4854 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. 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, 57 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 58 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 59 (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 60 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, 61 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. 62 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 63 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 64 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 65 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). 66 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 67 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 68 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 69 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. 70 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. 72 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). 75 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 76 used to construct individual gene expression graphs. Young (dotted) and aged (solid) mice lines ± SEM, n=3/group. 77 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. 78 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 79 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 80 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 81 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 82 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) 83 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 84 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 85 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 86 (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 87 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 88 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 89 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 90 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 91 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 92 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 93 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. 94 RGB Original 400X 200X 40X RGB-Blue Channel 95 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 96 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. 97 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. 98 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 99 percentage of RSV-positive cells from the total number of cells. The threshold was optimized by comparing mock-infected sections. 100 101 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. 102 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 103 individual mice (n=4-6/group) and graph represents the mean ±SEM from triplicate experiments. 104 105 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. 106 107 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. 108 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 109 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. 110 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 111 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 112 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 113 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 114 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. 115 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 116 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. 118 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 119 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 120 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 121 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 122 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, 123 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 124 (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. 125 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 126 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). 127 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 128 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. 129 130 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. 131 132 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) 133 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. 134 135 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. 136 137 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. 138 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 139 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 140 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. 141 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. 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Lucin, Egfr expression is linked to osteopontin and nf-kappab signaling in clear cell renal cell carcinoma. Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico 15, 65 (Jan, 2013). 167 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).