Download Bacterial and viral infection systems

ARVO 2015 Annual Meeting Abstracts
467 Bacterial and viral infection systems
Wednesday, May 06, 2015 3:45 PM–5:30 PM
702/704/706 Paper Session
Program #/Board # Range: 4841–4847
Organizing Section: Immunology/Microbiology
Program Number: 4841
Presentation Time: 3:45 PM–4:00 PM
MyD88 Adaptor Protein is Required for Adenovirus Keratitis
Mirja Ramke, Xiaohong Zhou, Ashish Chintakuntlawar, Jaya Rajaiya,
James Chodosh. Ophthalmology, Massachusetts Eye and Ear
Infirmary, Harvard Medical School, Boston, MA.
Purpose: The innate immune response is robust in the development
of epidemic keratoconjunctivitis (EKC). EKC is a hyperacute and
highly contagious ocular surface infection by human adenovirus
species D (HAdV-D) types 8, 37, 53, 54, 56, and 64. Toll-like
receptors (TLRs) are important contributors to innate immunity, and
recruit intracellular adaptor protein myeloid differentiation factor
88 (MyD88) to form active signaling complexes. Initiation of TLR
and MyD88 downstream signaling cascades leads to activation of
transcription factors and secretion of proinflammatory and antiviral
cytokines. The role of TLR-dependent pathways in adenovirus
keratitis is unknown.
Methods: Stromal keratitis was induced by intracorneal injection
of 105 infectious units of HAdV-D37 or dialysis buffer control in
C57Bl/6j (WT), TLR2-/-, TLR9-/-, MyD88-/-, and TLR2/9-/- mice.
Flow cytometry, ELISA, real-time RT-PCR, confocal microscopy,
and Western blot analysis were performed on the cornea 4 days post
infection (dpi). Cultured human corneal fibroblasts (HCF) at 2nd
passage were infected with HAdV-D37 or dialysis buffer control, and
treated with MyD88 inhibitory peptide prior to Western blot analysis
and real-time RT-PCR.
Results: Clinically evident keratitis developed in TLR2-/-, and
TLR9-/-mice within 1 dpi, whereas keratitis was not evident or was
significantly reduced in MyD88-/- and TLR2/9-/- mice, respectively.
Flow cytometric analysis showed reduction in CD45+ leukocytes
in infected MyD88-/- corneas as compared to WT. ELISA showed
significantly higher CXCL1, IL-6, CCL2, and CXCL2 expression in
infected WT corneas than in MyD88-/- or TLR2/9-/-. By confocal
microscopy using Cy3-labeled HAdV-D37, viral entry appeared
similar between WT and MyD88-/-, but E1A gene expression was
reduced in MyD88-/-corneas. Src kinase phosphorylation and
CXCL1 induction were also decreased in infected MyD88-/- corneas
as compared to WT. Pretreatment with MyD88 inhibitory peptide
in HCF in vitro reduced the phosphorylation of Src kinase and
decreased CXCL8 and CCL2 gene expression.
Conclusions: Cytokine expression in adenovirus keratitis is MyD88
and Src dependent, and relies on the synergistic activation of TLR2
and 9. The innate immune response is critical to the development of
stromal keratitis in EKC.
Commercial Relationships: Mirja Ramke, None; Xiaohong Zhou,
None; Ashish Chintakuntlawar, None; Jaya Rajaiya, None; James
Chodosh, None
Support: NIH grants EY013124, EY021558, and EY014104, and
Research to Prevent Blindness.
Program Number: 4842
Presentation Time: 4:00 PM–4:15 PM
Interferon regulator factor 8 (IRF-8) limits ocular pathology
during HSV-1 infection by restricting the activation and
expansion of CD8 T cell
Lin Sun, Anthony St Leger, Chengrong Yu, Chang He, Rashid M.
Mahdi, Chi-Chao Chan, CHRALES E EGWUAGU. Immunology,
NEI, Bethesda, MD.
Purpose: Interferon Regulatory Factor 8 (IRF-8) plays an important
role in myeloid and B cell differentiation. Although expression of
IRF-8 is induced in T cells in response to antigen stimulation, its role
in T cell mediated regulation of immune responses or host immunity
is still unknown. In this study, we have used an ocular herpes simplex
virus 1 (HSV-1) infection model to investigate the function of IRF-8
in CD8 T cell mediated control of viral infection.
Methods: We generated mice with targeted deletion of IRF-8 in the
CD4 compartment (CD4-IRF-8KO) by breeding IRF-8fl/fl mouse
(kindly provided by Dr. Herbert Morse, NIH) with mice expressing
Cre recombinase under the CD4 promoter (CD4/Cre mice). WT
and CD4-IRF8KO mice were infected with HSV-1 by corneal
scarification and HSV-specific CD8 T cell responses were examined
and characterized using the virus-specific gB-Tetramer reagent, flow
cytometry and intracellular cytokine staining assays. Viral clearance
was determined by plaque assay and ocular pathology was assessed
by histological analysis.
Results: Compared to WT, the CD4-IRF8KO mice generated
robust immune responses characterized by increased numbers of
virus-specific CD8 T cells and markedly elevated levels of proinflammatory cytokines, resulting in excessive ocular inflammation.
CD4-IRF8KO CD8 T cells expressed high levels of chemokine
receptors and up-regulation of the chemotactic molecules facilitated
the infiltration of protective CD8 T cells into the trigeminal ganglia
(TG). In addition, viral clearance in the eyes of CD4-IRF8KO mice
was found to correlate with a dramatic increase of HSV-specific CD8
T cells in the TG.
Conclusions: The data presented here suggest that an important
function of IRF-8 in T cells may be to restrain lymphocyte activation,
proliferation and to regulate the expression of molecules that mediate
lymphocyte chemotaxis. Specifically, loss of IRF-8 led to exuberant
inflammatory response, resulting in exacerbated immune-mediated
ocular pathology in HSV-1 infected mice. Our data showing that
IRF-8 suppresses T cell expansion and effector functions suggest
that IRF-8 is a potential therapeutic target that can be exploited to
mitigate pathological autoimmunity.
Commercial Relationships: Lin Sun, None; Anthony St Leger,
None; Chengrong Yu, None; Chang He, None; Rashid M. Mahdi,
None; Chi-Chao Chan, None; CHRALES E EGWUAGU, None
Program Number: 4843
Presentation Time: 4:15 PM–4:30 PM
Proteotyping as a tool to study the evolution of human
adenoviruses associated with epidemic keratoconjunctivitis
James Chodosh1, Gurdeep Singh1, Christopher Robinson1, Jeong
Yoon Lee1, Jaya Rajaiya1, Morris Jones2, David Dyer3, Donald Seto2.
1
Ophthalmology, Mass Eye & Ear Infirmary/HMS, Boston, MA;
2
George Mason University, Manassas, VA; 3University of Oklahoma
Health Sciences Center, Boston, OK.
Purpose: Human adenoviruses of species D (HAdV-D) cause
infections of mucosal epithelia, including the ocular surface.
HAdV-D types 8, 37, 53, 54, 56, and 64 are the known etiologic
agents of epidemic keratoconjunctivitis, a severe and highly
contagious ocular surface infection associated with prolonged
corneal inflammation. HAdV-D is also the largest and fastest growing
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
HAdV species, populated by 45 of 70 typed HAdVs in GenBank,
and 13 of the 18 HAdVs most recently typed. Because the viruses
within HAdV-D appear to evolve by multiple rounds of homologous
recombination, the degree of relatedness across numerous types
at specific genetic loci can be difficult to discern by traditional
bioinformatics tools. Proteotyping is a novel bioinformatics approach
that provides a unique overview of homologous recombination events
in genome evolution.
Methods: In proteotyping, maximum likelihood trees are used to
align amino acid sequences of hypervariable, frequently recombined
proteins. The clade-guided consensus sequence is determined, and
each amino acid is assigned a unique, arbitrary color. Consensus
residues are colored white, and gaps in the alignment are colored
black. A threshold of <10% sequence divergence is used to
distinguish unique proteotypes. Proteotyping analysis was applied to
open reading frames across 38 whole genome sequenced HAdV-Ds,
including those associated with epidemic keratoconjunctivitis.
Results: Among hypervariable regions, the hexon protein was found
to be the most variable (least recombined) with 28 proteotypes, while
the putative E3 CR1α protein had only 6 proteotypes for 38 viruses,
suggesting significant prior recombination. The E3 CR1β and CR1γ,
and fiber proteins had 14, 15, and 22 proteotypes, respectively. The
fiber proteotype most closely predicted corneal tropism. Highly
conserved genes such as DNA polymerase had only one proteotype.
HAdV-D proteotyping results correlated well with the results of
traditional recombination analyses, including Bootscan and SimPlot.
Conclusions: Proteotyping identifies unique amino acid
signatures across a virus species, providing both an overview
of recombination patterns as well as detailed information about
conserved and variant amino acid positions that phylogenetic trees
cannot offer. Proteotyping represents a valuable tool to discern
the evolution of viruses within HAdV-D associated with epidemic
keratoconjunctivitis.
Commercial Relationships: James Chodosh, None; Gurdeep
Singh, None; Christopher Robinson, None; Jeong Yoon Lee, None;
Jaya Rajaiya, None; Morris Jones, None; David Dyer, None;
Donald Seto, None
Support: NIH grants EY013124, EY021558, and P30EY014104, a
Senior Scientific Investigator Award (to JC) from Research to Prevent
Blindness, the Massachusetts Lions Eye Research Fund, and the Falk
Foundation
Program Number: 4844
Presentation Time: 4:30 PM–4:45 PM
Identification and characterization of a novel microbial
virulence-associated transcription factor and its role in ocular
infection
Robert M. Shanks, Nicholas A. Stella, Kimberly M. Brothers,
Kristin M. Hunt, Xinu Liu, Eric G. Romanowski, Regis P. Kowalski.
Chemistry, University of Pittsburgh, Pittsburgh, PA.
Purpose: To identify bacterial regulators necessary for microbial
keratitis. Bacterial keratitis progresses rapidly and can cause vision
loss even with effective antibiotics. An unknown gene was isolated
in genetic screens. Its role in controlling virulence factor production,
cytotoxicity to human corneal cells, corneal wound healing, and
keratitis was determined.
Methods: Molecular genetic, biochemical, RNA-Seq, proteomic, and
metabolomic approaches were used to characterize an eepR mutant
in a keratitis isolate of the bacteria Serratia marcescens. HCLE cells
were used in cytotoxicity assays and cell migration assays. Pig eyes
were used for corneal organ culture, and NZW rabbits were used for
keratitis studies.
Results: Transposon mutations were isolated in an uncharacterized
gene in a screen for genes involved in secretion of protease and
hemolysin activity using S. marcescens. Targeted deletion of eepR
in clinical and laboratory strains and complementation analysis
confirmed the importance of EepR in transcription of metalloprotease
and hemolysin genes. EMSA analysis confirmed a direct binding
of EepR to the hemolysin gene promoter, but not the protease gene
promoter. Proteomic analysis supported the importance of EepR in
protease secretion. Mass-spec and metabolomic analysis confirmed
loss of hemolysin production in the eepR mutant. Bacteria lacking
the eepR gene lost cytotoxicity to HCLE cells. This defect could
be restored by expression of the metalloprotease gene in the eepR
mutant. The eepR mutant was unable to inhibit corneal cell migration
and wound healing. In a rabbit keratitis model, EepR was necessary
for proliferation of the bacteria with a significant reduction in CFU
recovered from corneas infected with the eepR mutant compared to
the parental strain.
Conclusions: These results indicate EepR is a novel bacterial
transcription factor required for expression of secreted protease and
hemolysin activity from ocular clinical bacterial isolates. The EepR
protein regulates cytotoxicity through protease regulation and hostpathogen interactions through a yet unknown mechanism. EepR is
required for bacterial proliferation in mammalian corneas. Further
studies are required to determine the mechanism behind EepR’s role
in proliferation and corneal wound healing inhibition.
Commercial Relationships: Robert M. Shanks, None; Nicholas
A. Stella, None; Kimberly M. Brothers, None; Kristin M. Hunt,
None; Xinu Liu, None; Eric G. Romanowski, None; Regis P.
Kowalski, None
Support: NIH Grant AI085570, NIH Grant EY08098, Research to
Prevent Blindness
Program Number: 4845
Presentation Time: 4:45 PM–5:00 PM
Impact of microbiome on ocular immunity
Mihaela G. Gadjeva1, Abirami Kugadas1, Laura Ruiz2, Sharmila
Masli2. 1Medicine, Brigham and Womens Hospital, Boston, MA;
2
Department of Ophthalmology, Boston University, Boston, MA.
Purpose: How microbiome affects the ocular immunity is at its
early stage of accumulating experimental evidence. Here, we carried
out experiments to evaluate the repertoire of ocular commensals
in healthy mice, and autoimmune-prone mice. To determine the
significance of microbiome in promoting health, we compared the
susceptibility to infection of germ-free (GF) mice, conventional mice,
and GF mice reconstituted with murine or human microbiota.
Methods: To characterize the ocular commensals in healthy mice and
mice that develop Sjögren syndrome-like disease, conjunctival swabs
were collected from the conventionally bred wild type (C57BL6)
mice and thrombospondin-1 (Tsp-1 -/-) deficient mice and plated
on selective agar media. To evaluate the ocular immune status of
the different groups of mice, quantitative LC-MS/MS analysis of
ocular surface proteomes were carried out. To evaluate the impact of
microbiome on sensitivity to infection, mice were challenged with P.
aeruginosa 6294, bacterial presence in the cornea and the degree of
developed ocular pathology during keratitis were quantified.
Results: We found that the repertoire of the ocular microbiota in mice
was limited to Staphylococcus aureus, Staphylococcus coagulase
negative sp, and Streptococcus sp. The Tsp-1-/- mice showed
significant increase in Staphylococcus aureus, Staphylococcus
coagulase negative sp ocular commensals, suggestive of a defect
in the clearance mechanisms. Quantitative LC-MS/MS analysis
of ocular surface proteomes demonstrated that in the absence of
commensals, the tear-film components were altered. For example,
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
complement pathway components and iron-scavenging proteins
were significantly reduced in the GF animals. In agreement with this
finding, the GF mice were more sensitive to ocular P. aeruginosa–
induced keratitis than the conventional mice. This was exemplified
by increased bacterial presence and elevated corneal pathology. Upon
reconstitution of GF mice with either mouse or human microbiota,
the resistance to infection and the levels of ocular innate immune
mediators were recovered.
Conclusions: Our data suggest that tonic signals from local
commensal flora continuously induce increased synthesis of ocular
innate immune effectors to limit microbial presence at the ocular
surfaces. Furthermore, when the host fails to limit commensal
outgrowth, autoimmune disease states could occur.
Commercial Relationships: Mihaela G. Gadjeva, None; Abirami
Kugadas, None; Laura Ruiz, None; Sharmila Masli, None
Support: NIH/NEI R01 EY022054
Program Number: 4846
Presentation Time: 5:00 PM–5:15 PM
Toxigenic Staphylococcus Aureus bacteria activate conjunctival
goblet cell NLRP3 inflammasomes using TLR 2, TLR1, and α
toxin
Darlene A. Dartt1, 2, Dayu Li1, 2, Robin R. Hodges1, 2, Michael
Gilmore3, 2, Meredith S. Gregory-Ksander1, 2. 1Schepens Eye Research
Institute/MEEI, Boston, MA; 2Ophthalmology, Harvard Medical
School, Boston, MA; 3Massachusetts Eye and Ear Infirmary, Boston,
MA.
Purpose: To determine the Toll-like receptor (TLR) and pore forming
signals used by toxigenic Staphylococcus aureus (S. aureus) bacteria
in conjunctival goblet cells to activate the NLRP3 inflammasome to
produce IL-1β.
Methods: Cultured human goblet cells were incubated for 72 hrs
with siRNA for NLRP3, TLR 1, 2, and 6; scrambled siRNA; or
vehicle. Cells were then stimulated with no additions or S. aureus
RN6390. NLRP3 activation was determined by measuring proIL1β expression by western blotting and mature IL-1β secretion by
ELISA. The amount of TLR-1 and -2 expression in cultured cells
was measured by Q-PCR. Cultured goblet cells were stimulated by
no additions, α toxin, toxigenic S. aureus RN6390, and S. aureus
ALC837 that only lacked α toxin. Caspase-1 activity was measured
by FLICA along with pro-IL-1 β expression and mature IL-1β
secretion.
Results: NLRP3 siRNA completely decreased NLRP3 expression,
pro-IL-1β expression, and mature IL-1β secretion stimulated by S.
aureus RN6390. Use of non-target siRNA had no effect. When cells
were stimulated with S. aureus RN6390, TLR2 siRNA completely
blocked pro-IL-1β expression and mature IL-1β secretion. Nontarget siRNA had no effect. Stimulation of pro-IL-1β expression
and mature IL-1β secretion by S. aureus RN6390 in the presence
of TLR1 siRNA was significantly, but not completely decreased. In
contrast in the presence of TLR6 siRNA, S. aureus RN6390 did not
significantly alter pro-IL-1β expression and mature IL-1β secretion.
Values were the same as in the presence of non-target siRNA. TLR1
was expressed at much lower levels than TLR2 in cultured goblet
cells. S. aureus RN6390, but not α toxin nor S. aureus ALC837,
stimulated pro-IL-1β expression and mature IL-1β secretion. The α
toxin was active as it stimulated caspase-1 activity to the same level
as S. aureus RN6390. Simultaneous addition of α toxin and S. aureus
ALC837 restored pro-IL-1β expression and mature IL-1β secretion to
the same level as produced by S. aureus RN6390.
Conclusions: We conclude that in conjunctival goblet cells toxigenic
S. aureus stimulates the NLRP3 inflammasome to secrete mature IL-
1β by activating TLR2, to a lesser extent TLR1, but not TLR 6 and
by the pore forming action of its α toxin.
Commercial Relationships: Darlene A. Dartt, None; Dayu Li,
None; Robin R. Hodges, None; Michael Gilmore, None; Meredith
S. Gregory-Ksander, None
Support: NIH grant EY017381
Program Number: 4847
Presentation Time: 5:15 PM–5:30 PM
Pseudomonas aeruginosa isolates with lasR mutations were
associated with worse visual outcomes in the Steroids For
Corneal Ulcer Trial (SCUT)
Michael E. Zegans1, Kathryn Ray2, Wesley Hebert1, Amanda Naimie1,
John H. Hammond1, Lalitha Prajna3, Deborah Hogan1, Antonio
DiGiandomenico4, Thomas M. Lietman2, Muthiah Srinivasan3.
1
Microbiology and Immunology, Geisel School of Medicine at
Dartmouth, Lebanon, NH; 2Ophthalmology, F.I Proctor Foundation,
UCSF, San Francisco, CA; 3Ophthalmology, Aravind Eye Hospital,
Maduri, India; 4MedImmune, Gaithersburg, MD.
Purpose: Pseudomonas aeruginosa (PA) uses quorum sensing
to measure cell density and regulate production of virulence
factors. In PA, quorum sensing is mediated through the activity
of 3 hierarchically arranged signaling cascades, with the LasR
transcription factor positioned as the master regulator. While lasR up
regulates known virulence factors such as proteases, lasR mutants are
frequently isolated from infections, suggesting that lasR is selected
against in certain clinical contexts. We investigated 101 PA keratitis
isolates from the SCUT study for evidence of lasR mutations and the
effect of these mutations on visual outcomes. This is the first large
scale study of lasR among PA keratitis strains.
Methods: 101 PA SCUT isolates were screened for the lasR mutant
sheen colony phenotype by streaking them on a lysogeny broth agar
plate. Colonies with an iridescent sheen were considered possible
lasR mutants. The lasR gene including the promoter was sequenced
in all candidate isolates. Normal LasR function is associated with
production of proteases, surfactant and homoserine lactones (HSL).
Our candidate lasR mutants were assessed for these phenotypes. As
a control, all of the above assays were performed with randomly
selected SCUT PA isolates without the iridescent sheen phenotype.
Finally, we compared vision at presentation and at 3 months in
patients infected with las-intact vs. las- deficient Pseudomonas.
Results: 22 of 101 PA isolates from the SCUT study were noted
to have colony morphology with a sheen phenotype characteristic
of lasR mutations. Mutations in the lasR gene or promoter were
identified among the candidate lasR mutants, but not in the other
SCUT PA isolates. We verified the loss of LasR signaling by
demonstrating decreased protease activity (p<0.01), decreased
surfactant production (p<0.01), and a lack of production of HSL.
Finally, SCUT patients infected with strains with lasR mutations had
worse vision at presentation and at 3 months.
Conclusions: Mutations of lasR were common (22/101) among
PA keratitis isolates from the SCUT study and associated with
worse vision at presentation and at 3 months compared to patients
infected with las-intact strains. These data suggest that conventional
assumptions about the relationship between quorum sensing and
virulence may need to be re-evaluated.
Commercial Relationships: Michael E. Zegans, None; Kathryn
Ray, None; Wesley Hebert, None; Amanda Naimie, None; John H.
Hammond, None; Lalitha Prajna, None; Deborah Hogan, None;
Antonio DiGiandomenico, MedImmune (E); Thomas M. Lietman,
None; Muthiah Srinivasan, None
Support: NEI U10 EY015114
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Clinical Trial: NCT00324168.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].