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Neuroretinal-Derived Caveolin-1 Promotes Endotoxin-Induced Inflammation in the Murine Retina

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Neuroretinal-Derived Caveolin-1 Promotes Endotoxin-Induced Inflammation in the Murine Retina bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899377; this version posted January 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Neuroretinal‐Cav1 promotes retinal inflammation Neuroretinal-derived caveolin-1 promotes endotoxin-induced inflammation in the murine retina Jami M. Gurley1, Grzegorz Gmyrek1, Mark E. McClellan1, Stefanie M. Hauck2, Mikhail G. Dozmorov3, Jonathan D. Wren4, Daniel J. J. Carr1,5, and Michael H. Elliott1* 1 Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center (OUHSC) 2 Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH) 3 Department of Biostatistics, Virginia Commonwealth University (VCU) 4 Arthritis and Clinical Immunology Research Program, Division of Genomics and Data Sciences, Oklahoma Medical Research Foundation (OMRF) 5 Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center (OUHSC) Running title: Neuroretinal-Cav1 promotes retinal inflammation *To whom correspondence should be addressed: Michael H. Elliott: Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City OK 73104; [email protected] ; Tel.(405) 271-8001, ext.30024; Fax.(405) 271-8128. Keywords: caveolin-1; caveolin; caveolae; inflammation; neural retina; retina; retinal degeneration; immune response ABSTRACT model to deplete Cav1 specifically in the neural Chronic ocular inflammation is associated retinal (NR) compartment in order to clarify the with many retinal degenerative diseases that result role of neural retinal-specific Cav1 (NR-Cav1) in in vision loss. The immune-privileged the retinal immune response to intravitreal LPS environment and complex organization of retinal (lipopolysaccharide) challenge. Our data support tissue allow for the retina’s essential role in visual that neural retinal-derived Cav1 promotes retinal function processes, yet confound inquiries into tissue inflammation as Chx10-mediated Cav1 cell-specific inflammatory effects that lead to depletion was sufficient to suppress both retinal retinal dysfunction and degeneration. Caveolin-1 cytokine production and immune cell infiltration (Cav1) is an integral membrane protein expressed following inflammatory stimulation. Additionally, in many retinal cell populations and has been we identify Traf3 (tumor necrosis factor (TNF) implicated in retinal immune regulation. However, receptor-associated factor 3) as a highly expressed the direction (i.e., promotion or inhibition) in potential immune modulator in retinal tissue that is which Cav1 regulates inflammatory processes in upregulated with NR-Cav1 depletion. the retina (as well as in other tissues) remains Furthermore, this study highlights the importance unclear. Previously, we showed that global-Cav1 for understanding the role of Cav1 (and other depletion in the retina paradoxically resulted in proteins) in cell-specific contexts. reduced retinal inflammatory cytokine production with concurrent elevated retinal immune cell INTRODUCTION infiltration. We hypothesized that our previous Chronic ocular inflammation promotes the results could be explained by cell-specific Cav1 development and progression of retinal functions in the retina. Here, we utilized our degenerative diseases that lead to vision loss. Chx10 (visual system homeobox 2)-Cre knockout Many of these retinal degenerative diseases, such 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899377; this version posted January 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Neuroretinal‐Cav1 promotes retinal inflammation as age-related macular degeneration, diabetic inflammatory stimulation via caveolar retinopathy, and glaucoma, are major causes of sequestration, which prevents receptor complex blindness and are strongly linked to dysregulated formation and downstream signaling (10). immune responses that damage retinal tissue and In the retina, Cav1 affects important visual decrease visual function (1). processes such as phagolysosomal digestion of The retina, which is located in the back of photoreceptor outer segments by the retinal the eye and is responsible for processing light in pigment epithelium (RPE) (16), maintenance of order to facilitate visual function, is a complex and retinal vascular blood-retinal-barrier integrity (17- delicate tissue that is highly susceptible to 19), and retinal neovascularization (20). We and inflammation-mediated damage (1,2). As it is an others have also implicated Cav1 function immune-privileged tissue, the retina can elicit an specifically in retinal inflammation and immune immune response when necessary, but can also response modulation (21-23). However, the engage immune-suppressive mechanisms in order specific role for Cav1 in retinal immunity is as to control inflammatory responses that could lead perplexing as it has proven to be in extraocular to collateral damage (3,4). Under retinal tissue immune responses, and has only begun to be degenerative disease conditions, uncontrolled investigated (21). Previously, we showed that inflammatory activation and/or dysregulation of global-Cav1 deletion resulted in suppression of ocular immune suppression are key contributors endotoxin-mediated retinal inflammatory cytokine toward the development and progression of retinal production, which suggested that Cav1 promotes disease and vision loss (1). Unfortunately, the retinal inflammatory activation (21). Surprisingly, leading treatment option for dysregulated retinal Cav1 deletion enhanced retinal immune cell inflammation is localized corticosteroid infiltration, which paradoxically implied that Cav1 administration, which increases the risk for ocular function suppresses the immune response. conditions that exacerbate vision loss (e.g., Because Cav1 is expressed throughout retinal cataracts, glaucoma). Thus, understanding how to tissue in several retinal cell populations, we control inflammatory responses in the context of reasoned that these paradoxical phenotypes could the immune-privileged retinal environment is be explained by differential cell-specific Cav1 important for developing novel therapeutic functions in the retina. interventions that will preserve retinal and visual The aim of this study was to identify the function. Cav1-expressing retinal compartment responsible Several studies have implicated Cav1 as for promotion of endotoxin-induced inflammation. an immune regulator in lung, brain, and myeloid- Here, we show that neural retinal-derived Cav1 derived immune cells (5-7). In most cell types, depletion is sufficient to suppress inflammatory Cav1 is integrated into specialized plasma processes in the retina, which highlights the membrane domains called caveolae, which are importance of the neural retinal compartment in involved in a variety of cellular processes immune activation. This study (in conjunction including clathrin-independent endocytosis, with ours and other previous work) contributes to endothelial transcytosis, lipid transport, vascular our understanding of complex cell-specific Cav1 tone and contraction, fibrosis, cell proliferation, functions. Additionally, we identify Traf3 as a monocyte/macrophage differentiation, and potential retinal immune modulator that deserves signaling pathway regulation (6,8-15). The role of future study in regards to our search for novel Cav1 and caveolae in immune regulation is therapeutic interventions aimed at suppressing the complex. Collectively, existing studies suggest retinal immune response under conditions of that Cav1-dependent inflammatory promotion or chronic ocular inflammation. inhibition can occur via various mechanisms that depend on cell-specific contexts. For example, in Results the lung, Cav1 has been shown to promote Neural retinal depletion of CAV1 protein in inflammation by inhibiting eNOS-dependent retinal tissue downregulation of proinflammatory cytokine Our previously published results presented production (6); whereas, in macrophages, Cav1 seemingly contradictory data where global-Cav1 inhibits TLR4 (Toll-like receptor 4)-mediated depletion resulted in suppression of retinal 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.899377; this version posted January 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Neuroretinal‐Cav1 promotes retinal inflammation inflammatory cytokine production with a Müller glia have a unique morphology where their concurrent elevation in retinal immune cell cellular processes span the retina from inner infiltration (21). Because we hypothesized that this limiting membrane at the edge of the RGC layer to paradoxical result was due to divergent cell- the outer limiting membrane at the edge of the specific Cav1 functions in different retinal cell outer nuclear layer (ONL); whereas, the retinal populations, we wished to separate potential cell- vasculature appears as discrete vessel
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