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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 Retinal Cell Biology Neuroretinal-Derived Caveolin-1 Promotes Endotoxin-Induced Inflammation in the Murine Retina Jami M. Gurley,1 Grzegorz B. Gmyrek,1 Mark E. McClellan,1 Elizabeth A. Hargis,1 Stefanie M. Hauck,2 Mikhail G. Dozmorov,3 Jonathan D. Wren,4 Daniel J. J. Carr,1,5 and Michael H. Elliott1 1Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, Oklahoma, United States 2Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Munich, Germany 3Department of Biostatistics, Virginia Commonwealth University (VCU), Richmond, Virginia, United States 4Arthritis and Clinical Immunology Research Program, Division of Genomics and Data Sciences, Oklahoma Medical Research Foundation (OMRF), Oklahoma City, Oklahoma, United States 5Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, Oklahoma, United States Correspondence: Michael H. Elliott: PURPOSE. The immune-privileged environment and complex organization of retinal tissue Department of support the retina’s essential role in visual function, yet confound inquiries into cell- Ophthalmology/Dean McGee Eye specific inflammatory effects that lead to dysfunction and degeneration. Caveolin-1 (Cav1) Institute, University of Oklahoma is an integral membrane protein expressed in several retinal cell types and is impli- Health Sciences Center, Oklahoma cated in immune regulation. However, whether Cav1 promotes or inhibits inflammatory City, OK, USA; [email protected]. processes in the retina (as well as in other tissues) remains unclear. Previously, we showed that global-Cav1 depletion resulted in reduced retinal inflammatory cytokine production Received: April 24, 2020 but paradoxically elevated retinal immune cell infiltration. We hypothesized that these Accepted: October 6, 2020 disparate responses are the result of differential cell-specific Cav1 functions in the retina. Published: October 20, 2020 METHODS. We used Cre/lox technology to deplete Cav1 specifically in the neural reti- Citation: Gurley JM, Gmyrek GB, McClellan ME, et al. nal (NR) compartment to clarify the role NR-specific Cav1 (NR-Cav1) in the retinal Neuroretinal-derived caveolin-1 immune response to intravitreal inflammatory challenge induced by activation of Toll- promotes endotoxin-induced like receptor-4 (TLR4). We used multiplex protein suspension array and flow cytometry inflammation in the murine retina. to evaluate innate immune activation. Additionally, we used bioinformatics assessment Invest Ophthalmol Vis of differentially expressed membrane-associated proteins to infer relationships between Sci. 2020;61(12):19. NR-Cav1 and immune response pathways. https://doi.org/10.1167/iovs.61.12.19 RESULTS. NR-Cav1 depletion, which primarily affects Müller glia Cav1 expression, signif- icantly altered immune response pathway regulators, decreased retinal inflammatory cytokine production, and reduced retinal immune cell infiltration in response to LPS-stimulated inflammatory induction. CONCLUSIONS. Cav1 expression in the NR compartment promotes the innate TLR4-mediated retinal tissue immune response. Additionally, we have identified novel potential immune modulators differentially expressed with NR-Cav1 depletion. This study further clarifies the role of NR-Cav1 in retinal inflammation. Keywords: caveolin-1, caveolin, caveolae, inflammation, neural retina, retina, retinal degeneration, immune response cular inflammation promotes the development and by passive and active mechanisms intended to reduce O progression of retinal degenerative diseases that inflammatory responses that lead to collateral damage.3,4 lead to vision loss. Many of these retinal degenerative Uncontrolled inflammatory activation or dysregulation of diseases, such as age-related macular degeneration, diabetic ocular immune suppression contributes to development and retinopathy, and glaucoma, are major causes of blind- progression of retinal degeneration, retinal disease, and ness and are strongly linked to dysregulated immune vision loss.1 Unfortunately, the leading treatment option for responses that damage retinal tissue and decrease visual dysregulated retinal inflammation is localized corticosteroid function.1 administration, which increases the risk for ocular condi- The retina is a complex and delicate tissue that is highly tions that exacerbate vision loss (e.g., cataracts, glaucoma). susceptible to inflammation-mediated damage.1,2 While Therefore, understanding how to control chronic inflamma- the immune-privileged retina elicits immune responses tion in the context of the immune-privileged retinal envi- when necessary, these responses are relatively suppressed ronment is important for developing novel therapeutics that Copyright 2020 The Authors iovs.arvojournals.org | ISSN: 1552-5783 1 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 10/02/2021 Neuroretinal-Cav1 Promotes Retinal Inflammation IOVS |October2020|Vol.61|No.12|Article19|2 preserve retinal and visual function while minimizing off- Chx10-promoter-driven Cre recombinase (stock no. 005105; target effects. The Jackson Laboratory, Bar Harbor, ME, USA) were bred Several studies have implicated caveolin-1 (Cav1) as with animals carrying the Cav1 gene flanked by Lox P sites an established immune regulator in lung, brain, and located upstream and downstream of exon 2.26 Endothelial- myeloid-derived immune cells.5–7 In most cell types, Cav1 specific Cav1 knock-out mice (Tie2-Cre;flox/flox Cav1 )were is integrated into specialized plasma membrane domains generated similarly, using endothelial cell–specific recombi- called caveolae, which are involved in a variety of cellu- nation (B6.Cg-Tg (Tek-cre) 1Ywa/J; stock no. 008863; The lar processes including clathrin-independent endocytosis, Jackson Laboratory).27 Cre-negative littermates were used endothelial transcytosis, lipid transport, vascular tone regu- as controls. Mice were screened for rd1 and rd8 mutations lation, fibrosis, cell proliferation, monocyte/macrophage before this study. All procedures were approved by the differentiation, and signaling pathway regulation.6,8–15 Addi- University of Oklahoma Health Sciences Center Institutional tionally, Cav1 and caveolar domains play complex roles in Animal Care and Use Committee and comply with the Asso- immune regulation with studies suggesting that Cav1 can ciation for Research in Vision and Ophthalmology Statement either promote or inhibit inflammatory processes. For exam- for the Use of Animals in Ophthalmic and Visual Research. ple, in the lung, Cav1 has been shown to promote inflam- mation by inhibiting endothelial nitric oxide synthase– Whole Retinal Tissue Lysis dependent downregulation of proinflammatory cytokine production6; whereas, in macrophages, Cav1 inhibits TLR4 Total retinal protein was obtained via whole retinal tissue (Toll-like receptor 4)-mediated inflammatory stimulation via lysis by brief sonication in ×2 lysis buffer (120 mM caveolae-dependent sequestration, which prevents recep- octyl glucoside, 2% Triton X-100, 20 mM Tris-HCl, pH 7.4, tor complex formation and downstream signaling.10 Impor- 200 mM NaCl, 1 mM EDTA) containing a protease inhibitor tantly, this suggests that such differences are due to distinct cocktail (Roche). Lysates were cleared by centrifugation at cell-specific Cav1-dependent functions. 13,000 rpm, at 4°C. Protein concentration was determined In the retina, Cav1 is involved in important visual via BCA assay (ThermoSci, Cat no. 23225) and samples were processes such as phagolysosomal digestion of photore- diluted in ×6 Laemmli buffer. ceptor outer segments by the retinal pigment epithe- 16 lium (RPE), maintenance of retinal vascular blood-retinal- Western Blotting barrier integrity,17–20 and retinal neovascularization.21 We and others have also implicated Cav1 function in reti- Retinas were homogenized in buffer containing 100 mM nal inflammation and immune response modulation.22–24 sodium carbonate and 1 mM EDTA using a plastic pellet However, the precise immunomodulatory role for Cav1 pestle and brief pulse sonication on ice. Membranes in retinal tissue is as perplexing as it appears to be in were isolated by centrifugation at 16,000g in a bench- extraocular tissues, and has only begun to be investigated.22 top microcentrifuge and membrane pellets were lysed Previously, we showed that global-Cav1 deletion resulted in 1X lysis buffer. Protein content of lysates was deter- in suppression of endotoxin-mediated retinal inflammatory mined by the BCA assay with bovine serum albumin cytokine production suggesting that Cav1 promotes retinal as the standard. Protein separation was achieved via inflammatory activation.22 Surprisingly, global Cav1 dele- SDS-PAGE and proteins were transferred to nitrocellulose tion paradoxically enhanced retinal immune cell infiltra- for immunoblotting with the following primary antibody tion implying that Cav1 function suppresses the immune dilutions: rabbit monoclonal anti–Caveolin-1 (1:1000; Cell response. Because Cav1 is expressed in several retinal cell Signaling no. 3267), mouse monoclonal anti–Actin (1:1000; populations, we reasoned that these paradoxical phenotypes no. MA1-744; ThermoFisher, St. Louis, MO, USA), rabbit could be explained by differential cell-specific Cav1 func- polyclonal anti-TRAF3 (TNF Receptor-Associated
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