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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].