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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Immunology/Microbiology ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Immunology/Microbiology 106 Posterior Segment Infection/AIDS-Related Ocular Disease Methods: Immunosuppressed female Balb/c mice were injected via Sunday, May 05, 2013 8:30 AM-10:15 AM the supraciliary route with m38.5 and m41.1 mutant murine Exhibit Hall Poster Session cytomegaloviruses (MCMV) and K181 parent MCMV virus. Eyes Program #/Board # Range: 126-139/C0131-C0144 were collected at days 4 and 7 post infection (p.i.) and sectioned for Organizing Section: Immunology/Microbiology immunohistochemistry or homogenized for plaque assay. Double staining for MCMV Early Antigen (EA) and TUNEL were Program Number: 126 Poster Board Number: C0131 performed. Virus titers were performed by plaque assay on Presentation Time: 8:30 AM - 10:15 AM monolayers of mouse embryo fibroblast (MEF) cells and in-vitro Infiltrating granulocytes and resident Muller cells are major studies were performed using an organotypic retinal culture model. sources for suppressor of cytokine signaling (SOCS)1 and SOCS3 Results: Staining for MCMV EA showed more cells to be positive in production during murine cytomegalovirus (MCMV) retinitis in the m38.5 and m41.1 mutant viruses than in the K181 parent virus. mice with retrovirus-induced immunosuppression (MAIDS) Late stage apoptosis activity was observed by TUNEL staining and in Richard D. Dix1, 2, Christine I. Alston1, Emily L. Blalock1, Jessica both m38.5 and m41.1 mutant viruses more DNA fragmentation was Fleming1, Hsin Chien1. 1Department of Biology, Georgia State seen than in the K181 virus. Virus titers were lower in the injected University, Atlanta, GA; 2Ophthalmology, Emory University School eyes of the m38.5 and m41.1 mutants compared to the K181 virus of Medicine, Atlanta, GA. after day 7 p.i.. Likewise, in retinal cultures at both days 4 and 7 p.i., Purpose: SOCS family proteins govern the regulation of immune less virus was recovered from cultures infected with mutant viruses responses to pathogen invasion by a negative feedback regulatory than from cultures infected with the parent virus. mechanism to prevent cytokine over-expression. SOCS1 is an Conclusions: Our results indicate that MCMV lacking m38.5 or inhibitor of interferon signaling, whereas SOCS3 regulates the m41.1 has a reduced capacity to prevent viral infected cells, of divergent actions of IL-6 and IL-10. We have shown previously that immunosuppressed mice, from undergoing BAX and BAK mediated SOCS1 and SOCS3 mRNAs and proteins are upregulated in MCMV- apoptosis. In altering the apoptotic process of the viral infected cells, infected eyes with retinitis during MAIDS. These findings prompted further spread of the virus to the uninfected cells may be reduced and us to define the cell types that serve as sources for SOCS1 and further retinal damage potentially eliminated. SOCS3 production. Commercial Relationships: Jason Covar, None; Juan Mo, None; Methods: Groups of C57BL/6 mice with MAIDS were injected Brendan Marshall, None; Sally S. Atherton, None; Ming Zhang, subretinally with MCMV or mock injected (control). At 6 and 10 None days postinfection, whole eyes were collected, cryostat sectioned, and Support: NIH RO1 EY009169 subjected to immunostaining for detection and localization of SOCS1 or SOCS3 production to specific cells types within the retina. These Program Number: 128 Poster Board Number: C0133 included infiltrating macrophages (F4/80), infiltrating granulocytes Presentation Time: 8:30 AM - 10:15 AM (neutrophils) (Ly-6G), resident Muller cells (GFAP), and resident Suppressor of cytokine signaling (SOCS)1 and SOCS3 expression microglial cells (Iba-1). is upregulated following intraocular, but not systemic, murine Results: When compared with mock-infected eyes, MCMV-infected cytomegalovirus (MCMV) infection of mice with retrovirus- eyes of MAIDS mice that showed retinitis exhibited expression of induced immunosuppression (MAIDS) SOCS1 and SOCS3 in infiltrating macrophages, infiltrating Christine I. Alston1, Hsin Chien1, Moon K. Han1, Richard D. Dix1, 2. granulocytes, resident Muller cells, and resident microglia cells of the 1Biology, Viral Immunol Ctr, Georgia State Univ, Atlanta, GA; retina at 10 days postinfection. Quantification of SOCS1 and SOCS3 2Ophthalmology, Emory University School of Medicine, Atlanta, production by these cells suggested infiltrating granulocytes ≈ Muller GA. cells > microglial cells > infiltrating macrophages. Purpose: SOCS family proteins govern the regulation of immune Conclusions: The sources of SOCS1 or SOCS3 production in the responses to pathogen invasion by a negative feedback regulatory retina during development of MAIDS-related MCMV retinitis mechanism to prevent cytokine over-expression. SOCS1 is an includes infiltrating granulocytes and infiltrating macrophages as inhibitor of interferon (IFN) signaling, whereas SOCS3 regulates the well as resident Muller cells and resident microglial cells. Of these divergent actions of IL-6 and IL-10. We have shown previously that cell types, however, major sources for SOCS1 and SOCS3 SOCS1 and SOCS3 mRNA and protein are robustly upregulated in production are infiltrating granulocytes and resident Muller cells. MCMV-infected eyes with retinitis during MAIDS. We therefore Commercial Relationships: Richard D. Dix, None; Christine I. sought to determine the patterns of SOCS1 and SOCS3 expression as Alston, None; Emily L. Blalock, None; Jessica Fleming, None; well as SOCS-regulated cytokines during systemic MCMV infection Hsin Chien, None of mice with MAIDS that fail to develop retinitis. Support: NIH Grant EY010568, NIH/NEI Core Grant Methods: Groups of C57BL/6 mice with MAIDS or healthy mice P30/EY006360, Research to Prevent Blindness, and Fight for Sight were inoculated systemically with MCMV or mock infected (control). At 2, 4, 7, and 10 days postinfection, eyes and spleens were Program Number: 127 Poster Board Number: C0132 collected from all groups and quantified for SOCS1 and SOCS3 Presentation Time: 8:30 AM - 10:15 AM mRNA expression as well as mRNAs for IFNs and IL-6 by real-time Murine Cytomegalovirus (MCMV) Lacking m38.5 or m41.1 RT-PCR assay. Increases Apoptosis of Viral Infected Retinal Cells Results: Systemic MCMV infection of MAIDS mice resulted in no Jason Covar, Juan Mo, Brendan Marshall, Sally S. Atherton, Ming statistically significant upregulation of SOCS1, SOCS3, Type I IFNs, Zhang. Georgia Health Sciences University, Augusta, GA. or IL-6 mRNAs in whole eyes or whole splenic cells when compared Purpose: Previous results in our laboratory showed that during with controls. In comparison, Type II IFN mRNA expression was MCMV retinitis, uninfected retinal cells become apoptotic while upregulated significantly in whole eyes, but not in whole splenic infected cells do not. The purpose of this study was to determine if cells, of MAIDS mice following systemic MCMV infection. MCMV lacking m38.5 or m41.1 prevent viral infected cells from Conclusions: Whereas direct intraocular MCMV infection and BAX and BAK mediated apoptosis. development of retinitis during MAIDS resulted in robust ©2013, 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 2013 Annual Meeting Abstracts by Scientific Section/Group – Immunology/Microbiology upregulation of SOCS1 and SOCS3 mRNA and protein within the River (C), Nordic Biotech (C), PanOptica (C), Pfizer (C), Salutaris ocular compartment, systemic MCMV infection without retinitis Medical Devices (C), Sanofi-Fovea (C), Valeant Ophthalmics (C), during MAIDS did not lead to significant upregulation of SOCS1, Imagen Biotech (I); Richard D. Dix, None SOCS3, Type I IFNs, or IL-6 mRNAs within the ocular compartment Support: NIH Grant EY010568, NIH/NEI Core Grant or spleen, although there was a surprising upregulation of Type II P30/EY006360, Research to Prevent Blindness, Fight for Sight IFN in the ocular compartment without retinitis. We conclude that upregulation of SOCS1 and SOCS3 expression takes place within the Program Number: 130 Poster Board Number: C0135 ocular compartment only during MCMV retinitis development in Presentation Time: 8:30 AM - 10:15 AM mice with MAIDS. The precise role in SOCS1 and SOCS3 in the Autophagy Is Anti-apoptotic during Murine Cytomegalovirus pathogenesis of MAIDS-related MCMV retinitis requires further (MCMV) Infection of the Retina or RPE Cells investigation. Juan Mo, Ming Zhang, Brendan Marshall, Jason Covar, Sally S. Commercial Relationships: Christine I. Alston, None; Hsin Atherton. Cellular Biology & Anatomy, Georgia Health Sciences Chien, None; Moon K. Han, None; Richard D. Dix, None University, Augusta, GA. Support: NIH Grant EY010568, NIH/NEI Core Grant Purpose: The contribution of apoptosis and autophagy to the P30/EY006360, Research to Prevent Blindness, Fight for Sight pathogenesis of cytomegalovirus infection has not been explored. The purpose of this study was to determine if MCMV infection Program Number: 129 Poster Board Number: C0134 affects apoptosis and autophagy during MCMV retinitis, and if so, Presentation Time: 8:30 AM - 10:15 AM how these processes influence the pathogenesis of MCMV infection. The M33 gene of murine cytomegalovirus (MCMV) is involved in Methods: In vitro, RPE cells were mock-infected or infected with the stimulation of VEGF-A production by mouse macrophages MCMV at an MOI of 1 PFU/cell and treated with
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