Glutathione Trisulfide Prevents Lipopolysaccharide-induced Retinal Inflammation via Inhibition of Proinflammatory Cytokine Production in Glial Cells

Glutathione Trisulfide Prevents Lipopolysaccharide-induced Retinal Inflammation via Inhibition of Proinflammatory Cytokine Production in Glial Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Glutathione Trisulfide Prevents Lipopolysaccharide-induced Retinal Inflammation via Inhibition of Proinflammatory Cytokine Production in Glial Cells Hiroshi Tawarayama, Kota Umeki, Maki Inoue-Yanagimachi, Noriko Himori, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2425068/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jul, 2023 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract We aimed to investigate the impact of glutathione trisulfide (GSSSG) on lipopolysaccharide (LPS)-induced inflammation in retinal glia. Inflammatory responses in mouse-derived glial cells and Wistar rat retinas were stimulated with intravitreal LPS injection. Cell survival and proinflammatory cytokine production were examined using the Calcein-AM assay, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Retinal microglia were visualized with immunohistochemistry for Iba1. Administration of LPS (10 µg/mL) or GSSSG (less than 100 µM) did not affect survival of cultured primary Müller cells and established microglial cells. RT-qPCR and ELISA indicated that GSSSG inhibited LPS-induced gene upregulation and protein secretion of proinflammatory cytokines in these glial cells and rat retinas. Finally, immunohistochemical studies indicated that GSSSG inhibited LPS-induced accumulation of Iba1-immunopositive microglia in rat retinas. GSSSG has the potential to prevent pathogenesis of inflammation-associated ocular diseases by inhibiting proinflammatory cytokine expression in retinal glial cells. Biological sciences/Neuroscience/Glial biology/Microglia Biological sciences/Neuroscience/Visual system/Retina Biological sciences/Immunology/Cytokines/Interleukins Biological sciences/Immunology/Inflammation/Acute inflammation glutathione trisulfide glia retina cytokine inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Many cells respond to injury by secreting proinflammatory cytokines and chemokines, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and C-C motif chemokine ligand 2 (Ccl2). 1 – 6 TNF-α and IL-1β are produced in the early stage of inflammation and upregulate expression of themselves and other cytokines including IL-6. 7 – 12 IL-1β and IL-6 work synergistically to activate macrophages and B- and T-cells. 13 – 17 Ccl2 recruits monocytes and lymphocytes to inflammation sites via chemotaxis. 18 , 19 These molecular and cellular events lead to a severe inflammatory state in a broad range of tissues, causing irreversible damage. Retinal glial cells, including Müller cells and microglia, play crucial roles in maintaining retinal structure, homeostasis, and nutrition. 20 , 21 Müller cells are prominent glia that interact with the majority of retinal cell types and have the ability to produce inflammatory mediators. 22 – 26 Microglia are resident macrophage-like cells that release inflammatory mediators and reactive oxygen species (ROS) in response to harmful stimuli. 27 Past studies indicated that dysregulation of these mediators in glial cells contributed to the pathogenesis of retinal degeneration diseases in human patients and retinal damage in experimental animal models. 20 , 28 , 29 Thus, glial cells are a potential therapeutic target to prevent inflammation-related retinal degeneration diseases and to attenuate the symptoms. Glutathione trisulfide (GSSSG) belongs to a group of reactive sulfane sulfur species and is found endogenously in animal- and human-derived samples. 30 – 32 Glutathione polysulfides, mainly consisting of GSSSG, effectively quench ROS in the presence of glutathione disulfide reductase, which converts the oxidized form of glutathione into the reduced form. 31 GSSSG displays stronger inhibitory effects on oxidative stress-induced cell death in vitro compared to its relative, GSSG. 30 , 31 Furthermore, a recent in vitro study revealed a novel function of GSSSG, i.e., inhibition of stimulant-induced proinflammatory gene upregulation in a retinal pigment epithelial cell line established from human eyes. 33 However, it remains elusive whether GSSSG exerts similar inhibitory effects on other retinal cell types including glial cells, which are major sources of proinflammatory cytokines in pathological conditions. Attenuation of proinflammatory gene expression reduces the risk of retinal degeneration. In the present study, we examined the impacts of the reactive sulfur species GSSSG on LPS-induced proinflammatory responses in retinal glial cells in vitro and in vivo . Methods Animals C57BL/6J mice and Wistar rats (8–10 weeks old) were purchased from Japan SLC (Shizuoka, Japan) and maintained at animal facilities in Tohoku University Graduate School of Medicine (Sendai, Japan) under a 12-h light/dark cycle. Male and female mice were mated to obtain pups. All animal experiments were approved by the Committee on Animal Research at Tohoku University, and performed in agreement with the the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research and ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines. Cell culture Mouse-derived Müller cells were obtained as described previously. 34 – 36 Briefly, eyes dissected from postnatal day (P)5 to P8 pups were incubated in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific, Waltham, MA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific) at room temperature overnight. Retinas were isolated from eyes using sharp forceps 15 min after treatment with 0.25% trypsin-EDTA solution at 37°C for 15 min and dissociated into small pieces by pipetting several times. The small retinal explants were cultured in DMEM containing 10% FBS in a 5% CO 2 incubator at 37°C. The mouse brain-derived microglial cell line BV-2 was substituted for primary retinal microglia in this study since the number of retina-derived primary microglia was expected to be small. Past studies indicated that both primary microglia and BV-2 cells possess common properties and produce the same kinds of proinflammatory cytokines. 37 – 39 Cell viability assay Cell viability was determined using Calcein-AM (Dojindo, Kumamoto, Japan). Müller (0.5 × 10 4 cells/well) and BV-2 cells (5 × 10 4 cells/well) were cultured in 96-well cell culture plates in a medium containing various concentrations of GSSSG (Kyowa Hakko Bio, Tokyo, Japan) or LPS (Sigma-Aldrich, St. Louis, MO, USA) for 6 h. After washing with Dulbecco’s phosphate-buffered saline (DPBS), cells were incubated in DPBS containing 2 µM Calcein-AM for 30 min at 37°C. Cells were lysed with DPBS solution containing 5% Triton X-100 (FUJIFILM Wako pure chemical, Osaka, Japan), and then fluorescence intensity was measured at 515 nm (excitation: 490 nm) using a SpectraMax M2e microplate reader (Molecular Devices, San Jose, CA). In vitro treatment with GSSSG and LPS Müller and BV-2 cells were maintained and expanded in 10-cm dishes containing DMEM (10% FBS) in a 5% CO 2 incubator at 37°C. One day before the experiments, Müller (0.5 × 10 4 cells/well) and BV-2 cells (5 × 10 4 cells/well) were seeded in each well of 96-well culture plates. The next day, cells were pretreated with various concentrations of GSSSG for 1 h. Subsequently, 10 µg/mL of LPS (Sigma-Aldrich) was added to the cultures, and the cells were incubated for 6 more hours to induce the expression of proinflammatory genes. Enzyme-linked immunosorbent assay (ELISA) Concentrations of inflammation-related proteins in cell culture media were quantified using Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, glial cells were treated with GSSSG and LPS as described above. The supernatants were collected 6 h and 24 h after LPS treatment and used for ELISA-based quantification of IL-6 (6 h), and TNF-α, IK-1β, and Ccl2 (24 h). In vivo administration of glutathiones and LPS Rats were anesthetized using intraperitoneal injection of 8 mg/kg xylazine (Bayer Yakuhin, Osaka, Japan) and 80 mg/kg ketamine (Daiichi Sankyo, Tokyo Japan). Rats were intravitreally administered with 2 µL of a mixture of LPS (250 ng/µL) and GSSSG or GSSG (7.5 or 30 nmol/µL) in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS; Nacalei Tesque, Kyoto, Japan) using a micro-syringe with a 32G needle (Ito, Shizuoka, Japan) and then sacrificed at 10 h and 48 h post-administration for quantification of proinflammatory gene expression and microglial activation, respectively. The appropriate LPS concentration to stimulate inflammatory responses was determined in our preliminary experiments. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) For in vitro experiments, cell lysis and cDNA synthesis were performed using the SuperPrepII cell lysis & RT kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. For in vivo experiments, total RNA was extracted from the rat retina using the miRNeasy mini kit (QIAGEN, Hilden, Germany) and then reverse-transcribed into cDNA using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific, Waltham, MA, USA). RT-qPCR was performed in a 7500 fast real-time PCR system (Thermo Fisher Scientific) using TaqMan fast universal PCR master mix (Thermo Fisher Scientific) and a mixture of predesigned TaqMan primers and probes (Thermo Fisher Scientific or Integrated DNA Technologies, Coralville, IA, USA) (see Table 1 ). Table 1 List of the primer and probe mixtures used for quantitative RT-PCR. Species Genes Supplier Assay ID mouse Gapdh Integrated DNA Technologies Mm.PT.39a.1 mouse Il6 Thermo Fisher Scientific Mm.00446190_m1 mouse Il1ß Thermo Fisher Scientific Mm.00434228_m1 mouse Ccl2 Thermo Fisher Scientific Mm.00441242_m1 mouse Tnf-α Integrated DNA Technologies Mm.PT.58.12575861 rat Gapdh Thermo Fisher Scientific Rn.01775763_g1 rat Il6 Thermo Fisher Scientific Rn.01410330_m1 rat Il1ß Thermo Fisher Scientific Mm.00434228_m1 rat Ccl2 Thermo Fisher Scientific Rn.00580555_m1 Immunohistochemistry Immunostaining on whole retinas was performed following as previously described. 40 Briefly, retinas were dissected from eyes fixed with 4% paraformaldehyde in PBS for 1 h at room temperature and then postfixed with the same fixative solution overnight at 4°C. After treating with 10% normal donkey serum in PBS containing 0.1% tween 80, retinas were incubated with antibodies for Iba1 (FUJIFILM Wako pure chemical; 019-19741; 1:500 dilutions) for 3 days, washed, and incubated with Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch; 711-165-152; 1:500 dilution). Image acquisition and quantification of Iba1-immunopositive microglia Four fluorescent images per retina were captured 1 mm from the edge of the optic nerve head using a BZ-9000 fluorescence microscope with a 10× objective lens (Keyence, Osaka, Japan). Contrast and brightness adjustment and photo trimming were performed in Adobe Photoshop Elements (Adobe Systems, San Jose, CA, USA). The number of Iba1-immunopositive microglia was counted with ImageJ software (NIH, Bethesda, MD, USA), and cell density is expressed per mm 2 . The average was calculated based on data obtained from four retinas per each experimental group. Statistical analyses Quantitative data were analyzed using Welch’s t -test for two experimental groups, and analysis of variance, followed by Tukey–Kramer and Dunnett’s post-hoc tests, was performed for more than two experimental groups. Analyses were performed using JMP Pro 14 software (SAS Institute, Cary, NC, USA), and P < 0.05 was considered significant. Availability of Data and Materials The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Results Cell viability of Müller and BV-2 cells treated with GSSSG or LPS We first investigated the toxicity of GSSSG and LPS on mouse-derived Müller and BV-2 microglial cells. Cells were treated with various concentrations of GSSSG (25–200 µM) or LPS (10 µg/mL) for 6 h, and then living cells were detected using Calcein-AM. GSSSG had no effect on the Calcein-AM signal in Müller or BV-2 cells at concentrations less than 100 µM (Fig. 1A and B). However, a higher concentration (200 µM) of GSSSG decreased the viability of Müller but not BV-2 cells (Fig. 1A and B). LPS treatment did not affect the viability of both Müller and BV-2 cells in the assay period examined (Fig. 1A and B). GSSSG inhibited LPS-induced upregulation of proinflammatory genes and secretion of proteins We then examined the effects of GSSSG pretreatment on LPS-induced expression of mRNAs and proteins from proinflammatory genes in the culture media of Müller and BV-2 cells using RT-qPCR and ELISA, respectively. LPS stimulated expression of IL-6 and Ccl2 mRNAs (Fig. 2A and B) and secretion of their respective proteins (Fig. 2C and D) in Müller cells. However, pretreatment of Müller cells with GSSSG resulted in inhibition of LPS-induced increases in mRNA expression and protein secretion (Fig. 2A-D). Upregulated expression of proinflammatory genes was also detected in LPS-stimulated BV-2 cells. In addition to Ccl2 and IL-6, the expression levels of inflammatory mediators TNF-α and IL-1β were significantly increased in the presence of LPS (Fig. 3A-D). However, GSSSG pretreatment attenuated LPS-induced upregulation of these inflammatory genes (Fig. 3A-D). ELISA-based protein quantification indicated that LPS stimulated secretion of proinflammatory proteins including TNF-α, Ccl2, and IL-6 from BV-2 cells (Fig. 3E-G). However, GSSSG administration attenuated the increased secretion of these proteins (Fig. 3E-G). The concentration of IL-1β protein was below the limit of detection (data not shown). GSSSG inhibited proinflammatory gene expression in LPS-challenged rat retinas We then examined whether GSSSG exerted similar anti-inflammatory activity in animal retinas as that observed in vitro . To this end, GSSSG or GSSG was intravitreally administered to rat eyes together with LPS, and changes in the expression of proinflammatory genes were analyzed at 10 h post-administration by RT-qPCR. Intravitreal injection of LPS (500 ng) upregulated IL-6 , IL-1β , and Ccl2 expression in the retina (Fig. 4A and B), whereas concomitant administration of GSSSG (60 nmol) decreases IL-6 expression, although not significantly (P = 0.08) (Fig. 4A). However, GSSSG administration with a higher amount (60 nmol) significantly attenuated IL-6 , IL-1β , and Ccl2 upregulation in LPS-challenged retinas, whereas GSSG administration led to no significant reduction in their expression (Fig. 4B). GSSSG attenuates microglial accumulation in LPS-challenged rat retinas We then examined the impact of GSSSG on microglial accumulation in LPS-challenged rat retinas. Rats were intravitreally administered with LPS alone or in combination with GSSSG and sacrificed 48 h later, and then dissected retinas were immunostained for the microglial marker Iba1 (Fig. 5A). The immunohistochemical analysis of flat-mounted retinas indicated that LPS administration resulted in vigorous extension of microglial processes and a significant increase in the number of Iba1-immunopositive microglia (Fig. 5B and C). However, concomitant administration of GSSSG attenuated LPS-induced accumulation of microglia (Fig. 5B and C). Discussion Glial cells, including Müller cells and microglia, as well as retinal pigment epithelial cells are major sources of proinflammatory cytokines in retinas. 41–43 Excessive expression of these proteins exacerbates inflammation. Thus, it is important to properly control the expression level of proinflammatory cytokines upon exposure to harmful stimulants. In this study, we examined the impact of a glutathione polysulfide GSSSG on retinal inflammation and proinflammatory cytokine production in glial cells using LPS-induced in vitro and in vivo inflammation models. Our previous in vitro study indicated that GSSSG exerted an inhibitory effect on proinflammatory gene expression in retinal pigment epithelial cells stimulated with LPS. 33 Consistently, the present study revealed that GSSSG could attenuate LPS-induced increase of proinflammatory cytokines at the mRNA and protein levels in glial cells, including Müller cells and microglia, which are key players responsible for regulation of the inflammatory state in the retina under pathological conditions. 42,44 Past studies indicated that attenuation of pathogen-associated molecular pattern (PAMP)-induced proinflammatory responses is coincident with inactivation of the NF-κB signaling pathway and hyperactivation of extracellular signal-regulated kinase (ERK) 1/2 following administration of N-acetyl-L-cysteine polysulfides or GSSSG in various kinds of established cells, including mouse macrophage-like and human retinal pigment epithelial cells. 33,45 Furthermore, ERK1/2 hyperactivation inhibited LPS-induced upregulation of proinflammatory genes without GSSSG. 33 Thus, it is possible that GSSSG exerts anti-inflammatory effects in glial cells via the same intracellular mechanisms. This study is the first to demonstrate that GSSSG has the potential to attenuate LPS-induced inflammatory responses using in vivo experiments. GSSSG, but not its related molecule GSSG, known as a principal endogenous antioxidant, significantly inhibited proinflammatory gene upregulation in LPS-challenged rats. LPS administration leads to oxidative stress induced by reactive oxygen species (ROS). Furthermore, oxidative stress activates the NF-кB pathway to stimulate proinflammatory cytokine production. 46,47 GSSSG and GSSG both quench ROS through the redox reaction of thiol groups, although GSSSG is more effective. 31 However, elimination of oxidative stress seems not to be the mechanism underlying the GSSSG-mediated inhibition of LPS-induced proinflammatory gene upregulation. In a previous study, GSSG did not inhibit LPS-induced proinflammatory gene upregulation but rather stimulated it. 33 This is also supported with the finding that deficiency of nuclear factor-erythroid 2-related factor 2 (Nrf2), a master regulator of antioxidant defense responses including glutathione synthesis, had no effect on GSSSG-mediated inhibition of LPS-induced proinflammatory gene upregulation. 33 Thus, GSSSG would exert its anti-inflammatory activity by NF-кB inactivation and ERK hyperactivation through unidentified mechanisms other than antioxidation. We found that intravitreal administration of GSSSG inhibited not only proinflammatory gene upregulation but also microglial accumulation in the retina of LPS-challenged rats. Resident microglia change the morphology from a ramified to larger and round shape upon activation with stimulants, as shown in Fig. 5B. 48 Activated microglia increase expression of proinflammatory cytokines including IL-1, IL-6, TNF-α, and Ccl2, which stimulate microglial proliferation. 49–53 Treatment with LPS led to activation of the Toll-like receptor 4-mediated NF-кB signaling pathway, implicated in proinflammatory cytokine upregulation, in microglia. 54,55 Our previous study indicated that GSSSG attenuates LPS-induced activation of the NF-кB signaling pathway through inhibition of p65 phosphorylation. 33 Taken together, a negative feedback effect of GSSSG on LPS-induced activation of the NF-кB signaling pathway and cytokine production may explain the mechanism underlying the GSSSG-mediated inhibition of microglial proliferation; GSSSG deactivates the NF-кB signaling pathway, which results in downregulation of proinflammatory gene expression in retinal cells including microglia. Consequently, decreased cytokine production leads to attenuation of LPS-induced microglial proliferation. Conclusion In the present study, we examined the effects of a reactive sulfur species GSSSG on proinflammatory cytokine expression in glial cells in vitro and on retinal inflammation in rodents using LPS-induced inflammation models. Consequently, we found that GSSSG inhibits upregulation of proinflammatory cytokine expression in Müller cells and microglia and secretion of these cytokines and on microglial accumulation in retinas. Glial cells are major sources of proinflammatory cytokines in retinas and are implicated in inflammatory eye diseases. Thus, GSSSG-mediated regulation of glial inflammation would be effective to prevent the pathogenesis of such diseases. Declarations Acknowledgments We thank Junko Sato and Mayumi Suda and the Biomedical Research Unit of Tohoku University Hospital for technical assistance with the experiments. This research was supported by AMED (Grant Numbers JP18lm0203046, JP19lm0203046, JP20lm0203046 and JP21ym0126032) and JSPS KAKENHI (Grant Number JP21K09714). Author contributions Hiroshi Tawarayama: Conceptualization, Formal analysis, Funding acquisition, Investigation, Validation, Visualization, Writing – original draft, Writing – review & editing. Kota Umeki: Investigation, Validation. Maki Unoue-Yanagimachi: Investigation. Naoki Takahashi: Investigation. Noriko Himori: Supervision. Satoru Tsuda: Supervision. Hiroshi Kunikata: Supervision. Takaaki Akaike: Supervision. Toru Nakazawa: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing. Competing financial interests References Medzhitov R. Inflammation 2010: New Adventures of an Old Flame. Cell . 2010;140(6):771-776. doi:10.1016/j.cell.2010.03.006 Le J, Vilcek J. Interleukin 6: A multifunctional cytokine regulating immune reactions and the acute phase protein response. 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Microglia and Inflammatory Responses in Diabetic Retinopathy. Front Immunol . Published online 2020. doi:10.3389/fimmu.2020.564077 Zhang T, Ono K, Tsutsuki H, et al. Enhanced Cellular Polysulfides Negatively Regulate TLR4 Signaling and Mitigate Lethal Endotoxin Shock. Cell Chem Biol . 2019;26(5):686-698.e4. doi:10.1016/j.chembiol.2019.02.003 Takada Y, Mukhopadhyay A, Kundu GC, Mahabeleshwar GH, Singh S, Aggarwal BB. Hydrogen peroxide activates NF-κB through tyrosine phosphorylation of IκBα and serine phosphorylation of p65. Evidence for the involvement of IκBα kinase and Syk protein-tyrosine kinase. J Biol Chem . Published online 2003. doi:10.1074/jbc.M212389200 Canty TG, Boyle EM, Farr A, Morgan EN, Verrier ED, Pohlman TH. Oxidative stress induces NF-κB nuclear translocation without degradation of IκBα. Circulation . Published online 1999. doi:10.1161/circ.100.suppl_2.ii-361 Yang P, De Vos AF, Kijlstra A. Macrophages in the retina of normal Lewis rats and their dynamics after injection of lipopolysaccharide. Investig Ophthalmol Vis Sci . Published online 1996. Janabi N, Hau I, Tardieu M. Negative feedback between prostaglandin and α- and β-chemokine synthesis in human microglial cells and astrocytes. J Immunol . Published online 1999. Zielasek J, Hartung HP. Molecular mechanisms of microglial activation. Adv Neuroimmunol . Published online 1996. doi:10.1016/0960-5428(96)00017-4 Klein MA, Möller JC, Jones LL, Bluethmann H, Kreutzberg GW, Raivich G. Impaired neuroglial activation in interleukin-6 deficient mice. Glia . Published online 1997. doi:10.1002/(SICI)1098-1136(199703)19:33.0.CO;2-W Hopkins SJ, Rothwell NJ. Cytokines and the nervous system I: expression and recognition. Trends Neurosci . Published online 1995. doi:10.1016/0166-2236(95)80029-2 Mander PK, Jekabsone A, Brown GC. Microglia Proliferation Is Regulated by Hydrogen Peroxide from NADPH Oxidase. J Immunol . Published online 2006. doi:10.4049/jimmunol.176.2.1046 He Y, Taylor N, Yao X, Bhattacharya A. Mouse primary microglia respond differently to LPS and poly(I:C) in vitro. Sci Rep . Published online 2021. doi:10.1038/s41598-021-89777-1 Das A, Chai JC, Kim SH, et al. Transcriptome sequencing of microglial cells stimulated with TLR3 and TLR4 ligands. BMC Genomics . Published online 2015. doi:10.1186/s12864-015-1728-5 Datta S, Cano M, Ebrahimi K, Wang L, Handa JT. The impact of oxidative stress and inflammation on RPE degeneration in non-neovascular AMD. Prog Retin Eye Res . 2017;60:201-218. doi:10.1016/j.preteyeres.2017.03.002 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 17 Jul, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 02 Feb, 2023 Reviews received at journal 24 Jan, 2023 Reviewers agreed at journal 13 Jan, 2023 Reviewers invited by journal 02 Jan, 2023 Editor assigned by journal 02 Jan, 2023 Editor invited by journal 02 Jan, 2023 Submission checks completed at journal 02 Jan, 2023 First submitted to journal 29 Dec, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2425068","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":164245235,"identity":"b89663d8-2463-4ce0-a767-bb5d9e53623b","order_by":0,"name":"Hiroshi Tawarayama","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Tawarayama","suffix":""},{"id":164245237,"identity":"c71d65f6-33e0-429f-bdf1-ba132a7e4ef8","order_by":1,"name":"Kota Umeki","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kota","middleName":"","lastName":"Umeki","suffix":""},{"id":164245238,"identity":"4bb4cf83-2569-47eb-a64d-0b20c60d7286","order_by":2,"name":"Maki Inoue-Yanagimachi","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Maki","middleName":"","lastName":"Inoue-Yanagimachi","suffix":""},{"id":164245239,"identity":"6cec8b12-e97d-4f49-bfe5-32333796808d","order_by":3,"name":"Noriko Himori","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Noriko","middleName":"","lastName":"Himori","suffix":""},{"id":164245240,"identity":"b2cbac15-77c6-4aa3-8c3d-ee5c8291d318","order_by":4,"name":"Naoki Takahashi","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Takahashi","suffix":""},{"id":164245244,"identity":"a6b53fe0-45c5-4363-997c-cd82c4cef6a1","order_by":5,"name":"Satoru Tsuda","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Satoru","middleName":"","lastName":"Tsuda","suffix":""},{"id":164245248,"identity":"a1b79e97-26b6-4b5b-a071-4dc9281bd400","order_by":6,"name":"Hiroshi Kunikata","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Kunikata","suffix":""},{"id":164245253,"identity":"60cb8279-6182-4d24-a3ee-cf35b84c7845","order_by":7,"name":"Takaaki Akaike","email":"","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Takaaki","middleName":"","lastName":"Akaike","suffix":""},{"id":164245256,"identity":"692fc53f-0958-4c80-b2ee-de4168e23315","order_by":8,"name":"Toru Nakazawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYNCCCgYGAxDNwwBisEEEGRtwqAbLn4FrMSBSC2MbNi24gLl887HPvPNs8swZuBMfvGH4I2fO3pYmwVBjx8A8G7s1lm1sybN5t6UVWzbwbjacw2BgbNlz7JgEw7FkBsY5B7BqMTjGY8zMu+1w4oYDvNukgQ5L3HAjvU2Cge0AA+OMBDxa5oC1bP+N0PKPkJYGiC3MEC1pxyQY2/BpSUtmnHMsrdjgMO9myTkGxsYGZ44lWyT2JfPg9Mvhw4cZ3tTY5Bkc79344U2FnJzB8TbDGx++2ckZ4ggxGEhgYAabAOcy8BjOwKuDAYuz5SXwaxkFo2AUjIIRAwDIjVkYCZkwRQAAAABJRU5ErkJggg==","orcid":"","institution":"Tohoku University Graduate School of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Toru","middleName":"","lastName":"Nakazawa","suffix":""}],"badges":[],"createdAt":"2022-12-29 11:29:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2425068/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2425068/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-38696-4","type":"published","date":"2023-07-17T21:40:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":31173223,"identity":"6fe34d9d-be2a-49a1-b8f3-6c705abb06b3","added_by":"auto","created_at":"2023-01-05 16:24:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":663263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell survival of Müller and BV-2 cells treated with GSSSG or LPS. \u003c/strong\u003eCell viability of Müller (\u003cstrong\u003eA\u003c/strong\u003e) and BV-2 cells (\u003cstrong\u003eB\u003c/strong\u003e) treated with various concentrations of GSSSG or LPS for 6 h. Living cells were detected using the Calcein-AM assay, and viability is shown as a percentage of LPS(−)GSSSG(−) controls.\u003csup\u003e **\u003c/sup\u003eP \u0026lt; 0.01 vs. LPS(−)GSSSG(−) (Dunnett’s test; \u003cem\u003en\u003c/em\u003e = 4).\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2425068/v1/d7c7d8661989aca8f410bb9b.jpg"},{"id":31173224,"identity":"3b4101b1-5fdb-427e-850e-213b31149269","added_by":"auto","created_at":"2023-01-05 16:24:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1110919,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibitory effects of GSSSG on LPS-induced expression of proinflammatory cytokines in Müller cells. \u003c/strong\u003eChanges of IL-6 and Ccl2 mRNA expression\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003eA \u003c/strong\u003eand\u003cstrong\u003e B\u003c/strong\u003e) and protein secretion (\u003cstrong\u003eC \u003c/strong\u003eand\u003cstrong\u003e D\u003c/strong\u003e) in mouse primary Müller cells treated with GSSSG and/or LPS. Error bars indicate standard error of the mean. \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01 vs. LPS(−)GSSSG(−) controls (Welch’s \u003cem\u003et\u003c/em\u003e test; \u003cem\u003en\u003c/em\u003e = 4); \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01 vs. LPS(+)GSSSG(−) (Dunnett’s test; \u003cem\u003en\u003c/em\u003e = 4).\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2425068/v1/b40231d1fc57500fe3970256.jpg"},{"id":31173222,"identity":"b7fda850-8f7b-4115-ac6c-88be94446e78","added_by":"auto","created_at":"2023-01-05 16:24:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1850827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibitory effects of GSSSG on LPS-induced expression of proinflammatory cytokines in BV-2 cells. \u003c/strong\u003eChanges of\u003cstrong\u003e \u003c/strong\u003eTNF-α, Ccl2, IL-6 and IL-1β mRNA expression\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003eA-D\u003c/strong\u003e) and protein secretion (\u003cstrong\u003eE-G\u003c/strong\u003e) in mouse-derived BV-2 cells treated with GSSSG and/or LPS. Error bars indicate standard error of the mean. \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01 vs. LPS(−)GSSSG(−) controls (Welch’s \u003cem\u003et\u003c/em\u003e test; \u003cem\u003en\u003c/em\u003e = 4); \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01 vs. LPS(+)GSSSG(−) (Dunnett’s test; \u003cem\u003en\u003c/em\u003e = 4).\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2425068/v1/e537911920e3e1d5b6fca366.jpg"},{"id":31173225,"identity":"b2b0ce9c-c9d3-4b2c-85d0-0a3299867710","added_by":"auto","created_at":"2023-01-05 16:24:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":896404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e effects of GSSSG on LPS-induced upregulation of proinflammatory cytokine genes in rat retinas. \u003c/strong\u003eChanges in \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eIL-1β\u003c/em\u003e, and \u003cem\u003eCcl2\u003c/em\u003e mRNA levels in retinas dissected from rat eyes, which were intravitreally administered LPS (500 ng) and GSSSG at approximate final concentrations of 300 µM (\u003cstrong\u003eA\u003c/strong\u003e) and 1,200 µM (\u003cstrong\u003eB\u003c/strong\u003e) in the vitreous humor. Error bars indicate standard error of the mean.\u003csup\u003e ##\u003c/sup\u003eP \u0026lt; 0.01 vs. LPS(−)GSSSG(−) controls; \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01 vs. LPS(+) controls (Tukey–Kramer test; \u003cem\u003en\u003c/em\u003e = 6−12).\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2425068/v1/18318794fd6939766a4600d0.jpg"},{"id":31173565,"identity":"a265134d-7d67-41f0-8b81-c562db64d176","added_by":"auto","created_at":"2023-01-05 16:32:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2221934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibitory effects of GSSSG on LPS-induced accumulation of microglia in rat retinas.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Experimental schema. (\u003cstrong\u003eB\u003c/strong\u003e) Iba1 immunostaining of flat-mounted retinas prepared from LPS- and GSSSG-challenged rats. Scale bars: 100 µm. (\u003cstrong\u003eC\u003c/strong\u003e) Quantification of Iba1-immunopositive microglia in retinas treated with LPS and GSSSG. \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01 (Tukey–Kramer test; \u003cem\u003en\u003c/em\u003e = 4).\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2425068/v1/3dfab954d55bf923ebb635cf.jpg"},{"id":44734212,"identity":"41195b20-a24f-44d3-80f0-169b3e0c029a","added_by":"auto","created_at":"2023-10-16 22:15:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":851883,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2425068/v1/cd629919-89e5-447e-882a-88388a910dcb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Glutathione Trisulfide Prevents Lipopolysaccharide-induced Retinal Inflammation via Inhibition of Proinflammatory Cytokine Production in Glial Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMany cells respond to injury by secreting proinflammatory cytokines and chemokines, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and C-C motif chemokine ligand 2 (Ccl2).\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e TNF-α and IL-1β are produced in the early stage of inflammation and upregulate expression of themselves and other cytokines including IL-6.\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e IL-1β and IL-6 work synergistically to activate macrophages and B- and T-cells.\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Ccl2 recruits monocytes and lymphocytes to inflammation sites via chemotaxis.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e These molecular and cellular events lead to a severe inflammatory state in a broad range of tissues, causing irreversible damage.\u003c/p\u003e \u003cp\u003eRetinal glial cells, including M\u0026uuml;ller cells and microglia, play crucial roles in maintaining retinal structure, homeostasis, and nutrition.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e M\u0026uuml;ller cells are prominent glia that interact with the majority of retinal cell types and have the ability to produce inflammatory mediators.\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Microglia are resident macrophage-like cells that release inflammatory mediators and reactive oxygen species (ROS) in response to harmful stimuli.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Past studies indicated that dysregulation of these mediators in glial cells contributed to the pathogenesis of retinal degeneration diseases in human patients and retinal damage in experimental animal models.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Thus, glial cells are a potential therapeutic target to prevent inflammation-related retinal degeneration diseases and to attenuate the symptoms.\u003c/p\u003e \u003cp\u003eGlutathione trisulfide (GSSSG) belongs to a group of reactive sulfane sulfur species and is found endogenously in animal- and human-derived samples.\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Glutathione polysulfides, mainly consisting of GSSSG, effectively quench ROS in the presence of glutathione disulfide reductase, which converts the oxidized form of glutathione into the reduced form.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e GSSSG displays stronger inhibitory effects on oxidative stress-induced cell death \u003cem\u003ein vitro\u003c/em\u003e compared to its relative, GSSG.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Furthermore, a recent \u003cem\u003ein vitro\u003c/em\u003e study revealed a novel function of GSSSG, i.e., inhibition of stimulant-induced proinflammatory gene upregulation in a retinal pigment epithelial cell line established from human eyes.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e However, it remains elusive whether GSSSG exerts similar inhibitory effects on other retinal cell types including glial cells, which are major sources of proinflammatory cytokines in pathological conditions.\u003c/p\u003e \u003cp\u003eAttenuation of proinflammatory gene expression reduces the risk of retinal degeneration. In the present study, we examined the impacts of the reactive sulfur species GSSSG on LPS-induced proinflammatory responses in retinal glial cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eC57BL/6J mice and Wistar rats (8\u0026ndash;10 weeks old) were purchased from Japan SLC (Shizuoka, Japan) and maintained at animal facilities in Tohoku University Graduate School of Medicine (Sendai, Japan) under a 12-h light/dark cycle. Male and female mice were mated to obtain pups. All animal experiments were approved by the Committee on Animal Research at Tohoku University, and performed in agreement with the the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research and ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eMouse-derived M\u0026uuml;ller cells were obtained as described previously.\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Briefly, eyes dissected from postnatal day (P)5 to P8 pups were incubated in Dulbecco\u0026rsquo;s modified Eagle medium (DMEM; Thermo Fisher Scientific, Waltham, MA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific) at room temperature overnight. Retinas were isolated from eyes using sharp forceps 15 min after treatment with 0.25% trypsin-EDTA solution at 37\u0026deg;C for 15 min and dissociated into small pieces by pipetting several times. The small retinal explants were cultured in DMEM containing 10% FBS in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C. The mouse brain-derived microglial cell line BV-2 was substituted for primary retinal microglia in this study since the number of retina-derived primary microglia was expected to be small. Past studies indicated that both primary microglia and BV-2 cells possess common properties and produce the same kinds of proinflammatory cytokines.\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eCell viability was determined using Calcein-AM (Dojindo, Kumamoto, Japan). M\u0026uuml;ller (0.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) and BV-2 cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were cultured in 96-well cell culture plates in a medium containing various concentrations of GSSSG (Kyowa Hakko Bio, Tokyo, Japan) or LPS (Sigma-Aldrich, St. Louis, MO, USA) for 6 h. After washing with Dulbecco\u0026rsquo;s phosphate-buffered saline (DPBS), cells were incubated in DPBS containing 2 \u0026micro;M Calcein-AM for 30 min at 37\u0026deg;C. Cells were lysed with DPBS solution containing 5% Triton X-100 (FUJIFILM Wako pure chemical, Osaka, Japan), and then fluorescence intensity was measured at 515 nm (excitation: 490 nm) using a SpectraMax M2e microplate reader (Molecular Devices, San Jose, CA).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vitro\u003c/span\u003e \u003cb\u003etreatment with GSSSG and LPS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eM\u0026uuml;ller and BV-2 cells were maintained and expanded in 10-cm dishes containing DMEM (10% FBS) in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C. One day before the experiments, M\u0026uuml;ller (0.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) and BV-2 cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded in each well of 96-well culture plates. The next day, cells were pretreated with various concentrations of GSSSG for 1 h. Subsequently, 10 \u0026micro;g/mL of LPS (Sigma-Aldrich) was added to the cultures, and the cells were incubated for 6 more hours to induce the expression of proinflammatory genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eConcentrations of inflammation-related proteins in cell culture media were quantified using Quantikine ELISA Kit (R\u0026amp;D Systems, Minneapolis, MN, USA) according to the manufacturer\u0026rsquo;s instructions. Briefly, glial cells were treated with GSSSG and LPS as described above. The supernatants were collected 6 h and 24 h after LPS treatment and used for ELISA-based quantification of IL-6 (6 h), and TNF-α, IK-1β, and Ccl2 (24 h).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vivo\u003c/span\u003e \u003cb\u003eadministration of glutathiones and LPS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRats were anesthetized using intraperitoneal injection of 8 mg/kg xylazine (Bayer Yakuhin, Osaka, Japan) and 80 mg/kg ketamine (Daiichi Sankyo, Tokyo Japan). Rats were intravitreally administered with 2 \u0026micro;L of a mixture of LPS (250 ng/\u0026micro;L) and GSSSG or GSSG (7.5 or 30 nmol/\u0026micro;L) in Ca\u003csup\u003e2+\u003c/sup\u003e- and Mg\u003csup\u003e2+\u003c/sup\u003e-free phosphate-buffered saline (PBS; Nacalei Tesque, Kyoto, Japan) using a micro-syringe with a 32G needle (Ito, Shizuoka, Japan) and then sacrificed at 10 h and 48 h post-administration for quantification of proinflammatory gene expression and microglial activation, respectively. The appropriate LPS concentration to stimulate inflammatory responses was determined in our preliminary experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcription-polymerase chain reaction (RT-qPCR)\u003c/h2\u003e \u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e experiments, cell lysis and cDNA synthesis were performed using the SuperPrepII cell lysis \u0026amp; RT kit (Toyobo, Osaka, Japan) according to the manufacturer\u0026rsquo;s instructions. For \u003cem\u003ein vivo\u003c/em\u003e experiments, total RNA was extracted from the rat retina using the miRNeasy mini kit (QIAGEN, Hilden, Germany) and then reverse-transcribed into cDNA using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific, Waltham, MA, USA). RT-qPCR was performed in a 7500 fast real-time PCR system (Thermo Fisher Scientific) using TaqMan fast universal PCR master mix (Thermo Fisher Scientific) and a mixture of predesigned TaqMan primers and probes (Thermo Fisher Scientific or Integrated DNA Technologies, Coralville, IA, USA) (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of the primer and probe mixtures used for quantitative RT-PCR.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSupplier\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAssay ID\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGapdh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIntegrated DNA Technologies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMm.PT.39a.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIl6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMm.00446190_m1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIl1\u0026szlig;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMm.00434228_m1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCcl2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMm.00441242_m1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTnf-α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIntegrated DNA Technologies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMm.PT.58.12575861\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003erat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGapdh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRn.01775763_g1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003erat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIl6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRn.01410330_m1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003erat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIl1\u0026szlig;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMm.00434228_m1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003erat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCcl2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRn.00580555_m1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eImmunostaining on whole retinas was performed following as previously described.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Briefly, retinas were dissected from eyes fixed with 4% paraformaldehyde in PBS for 1 h at room temperature and then postfixed with the same fixative solution overnight at 4\u0026deg;C. After treating with 10% normal donkey serum in PBS containing 0.1% tween 80, retinas were incubated with antibodies for Iba1 (FUJIFILM Wako pure chemical; 019-19741; 1:500 dilutions) for 3 days, washed, and incubated with Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch; 711-165-152; 1:500 dilution).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImage acquisition and quantification of Iba1-immunopositive microglia\u003c/h2\u003e \u003cp\u003eFour fluorescent images per retina were captured 1 mm from the edge of the optic nerve head using a BZ-9000 fluorescence microscope with a 10\u0026times; objective lens (Keyence, Osaka, Japan). Contrast and brightness adjustment and photo trimming were performed in Adobe Photoshop Elements (Adobe Systems, San Jose, CA, USA). The number of Iba1-immunopositive microglia was counted with ImageJ software (NIH, Bethesda, MD, USA), and cell density is expressed per mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The average was calculated based on data obtained from four retinas per each experimental group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eQuantitative data were analyzed using Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test for two experimental groups, and analysis of variance, followed by Tukey\u0026ndash;Kramer and Dunnett\u0026rsquo;s post-hoc tests, was performed for more than two experimental groups. Analyses were performed using JMP Pro 14 software (SAS Institute, Cary, NC, USA), and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eAvailability of Data and Materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCell viability of M\u0026uuml;ller and BV-2 cells treated with GSSSG or LPS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first investigated the toxicity of GSSSG and LPS on mouse-derived M\u0026uuml;ller and BV-2 microglial cells. Cells were treated with various concentrations of GSSSG (25\u0026ndash;200 \u0026micro;M) or LPS (10 \u0026micro;g/mL) for 6 h, and then living cells were detected using Calcein-AM. GSSSG had no effect on the Calcein-AM signal in M\u0026uuml;ller or BV-2 cells at concentrations less than 100 \u0026micro;M (Fig. 1A and B). However, a higher concentration (200 \u0026micro;M) of GSSSG decreased the viability of M\u0026uuml;ller but not BV-2 cells (Fig. 1A and B). LPS treatment did not affect the viability of both M\u0026uuml;ller and BV-2 cells in the assay period examined (Fig. 1A and B). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGSSSG inhibited LPS-induced upregulation of proinflammatory genes and secretion of proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then examined the effects of GSSSG pretreatment on LPS-induced expression of mRNAs and proteins from proinflammatory genes in the culture media of M\u0026uuml;ller and BV-2 cells using RT-qPCR and ELISA, respectively. LPS stimulated expression of \u003cem\u003eIL-6\u003c/em\u003e and \u003cem\u003eCcl2\u003c/em\u003e mRNAs (Fig. 2A and B) and secretion of their respective proteins (Fig. 2C and D) in M\u0026uuml;ller cells. However, pretreatment of M\u0026uuml;ller cells with GSSSG resulted in inhibition of LPS-induced increases in mRNA expression and protein secretion (Fig. 2A-D).\u003c/p\u003e\n\u003cp\u003eUpregulated expression of proinflammatory genes was also detected in LPS-stimulated BV-2 cells. In addition to Ccl2 and IL-6, the expression levels of inflammatory mediators TNF-\u0026alpha; and IL-1\u0026beta; were significantly increased in the presence of LPS (Fig. 3A-D). However, GSSSG pretreatment attenuated LPS-induced upregulation of these inflammatory genes (Fig. 3A-D). ELISA-based protein quantification indicated that LPS stimulated secretion of proinflammatory proteins including TNF-\u0026alpha;, Ccl2, and IL-6 from BV-2 cells (Fig. 3E-G). However, GSSSG administration attenuated the increased secretion of these proteins (Fig. 3E-G). The concentration of IL-1\u0026beta; protein was below the limit of detection (data not shown). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGSSSG inhibited proinflammatory gene expression in LPS-challenged rat retinas\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then examined whether GSSSG exerted similar anti-inflammatory activity in animal retinas as that observed \u003cem\u003ein vitro\u003c/em\u003e. To this end, GSSSG or GSSG was intravitreally administered to rat eyes together with LPS, and changes in the expression of proinflammatory genes were analyzed at 10 h post-administration by RT-qPCR. Intravitreal injection of LPS (500 ng) upregulated \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eIL-1\u0026beta;\u003c/em\u003e, and \u003cem\u003eCcl2\u003c/em\u003e expression in the retina (Fig. 4A and B), whereas concomitant administration of GSSSG (60 nmol) decreases \u003cem\u003eIL-6 \u003c/em\u003eexpression, although not significantly (P = 0.08) (Fig. 4A). However, GSSSG administration with a higher amount (60 nmol) significantly attenuated \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eIL-1\u0026beta;\u003c/em\u003e, and \u003cem\u003eCcl2\u003c/em\u003e upregulation in LPS-challenged retinas, whereas GSSG administration led to no significant reduction in their expression (Fig. 4B). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGSSSG attenuates microglial accumulation in LPS-challenged rat retinas\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then examined the impact of GSSSG on microglial accumulation in LPS-challenged rat retinas. Rats were intravitreally administered with LPS alone or in combination with GSSSG and sacrificed 48 h later, and then dissected retinas were immunostained for the microglial marker Iba1 (Fig. 5A). The immunohistochemical analysis of flat-mounted retinas indicated that LPS administration resulted in vigorous extension of microglial processes and a significant increase in the number of Iba1-immunopositive microglia (Fig. 5B and C). However, concomitant administration of GSSSG attenuated LPS-induced accumulation of microglia (Fig. 5B and C).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGlial cells, including M\u0026uuml;ller cells and microglia, as well as retinal pigment epithelial cells are major sources of proinflammatory cytokines in retinas.\u003csup\u003e41\u0026ndash;43\u003c/sup\u003e Excessive expression of these proteins exacerbates inflammation. Thus, it is important to properly control the expression level of proinflammatory cytokines upon exposure to harmful stimulants. In this study, we examined the impact of a glutathione polysulfide GSSSG on retinal inflammation and proinflammatory cytokine production in glial cells using LPS-induced \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e inflammation models.\u003c/p\u003e\n\u003cp\u003eOur previous \u003cem\u003ein vitro\u003c/em\u003e study indicated that GSSSG exerted an inhibitory effect on proinflammatory gene expression in retinal pigment epithelial cells stimulated with LPS.\u003csup\u003e33\u003c/sup\u003e Consistently, the present study revealed that GSSSG could attenuate LPS-induced increase of proinflammatory cytokines at the mRNA and protein levels in glial cells, including M\u0026uuml;ller cells and microglia, which are key players responsible for regulation of the inflammatory state in the retina under pathological conditions.\u003csup\u003e42,44\u003c/sup\u003e Past studies indicated that attenuation of pathogen-associated molecular pattern (PAMP)-induced proinflammatory responses is coincident with inactivation of the NF-\u0026kappa;B signaling pathway and hyperactivation of extracellular signal-regulated kinase (ERK) 1/2 following administration of N-acetyl-L-cysteine polysulfides or GSSSG in various kinds of established cells, including mouse macrophage-like and human retinal pigment epithelial cells.\u003csup\u003e33,45\u003c/sup\u003e Furthermore, ERK1/2 hyperactivation inhibited LPS-induced upregulation of proinflammatory genes without GSSSG.\u003csup\u003e33\u003c/sup\u003e Thus, it is possible that GSSSG exerts anti-inflammatory effects in glial cells via the same intracellular mechanisms.\u003c/p\u003e\n\u003cp\u003eThis study is the first to demonstrate that GSSSG has the potential to attenuate LPS-induced inflammatory responses using \u003cem\u003ein vivo\u003c/em\u003e experiments. GSSSG, but not its related molecule GSSG, known as a principal endogenous antioxidant, significantly inhibited proinflammatory gene upregulation in LPS-challenged rats. LPS administration leads to oxidative stress induced by reactive oxygen species (ROS). Furthermore, oxidative stress activates the NF-кB pathway to stimulate proinflammatory cytokine production.\u003csup\u003e46,47\u003c/sup\u003e GSSSG and GSSG both quench ROS through the redox reaction of thiol groups, although GSSSG is more effective.\u003csup\u003e31\u003c/sup\u003e However, elimination of oxidative stress seems not to be the mechanism underlying the GSSSG-mediated inhibition of LPS-induced proinflammatory gene upregulation. In a previous study, GSSG did not inhibit LPS-induced proinflammatory gene upregulation but rather stimulated it.\u003csup\u003e33\u003c/sup\u003e This is also supported with the finding that deficiency of nuclear factor-erythroid 2-related factor 2 (Nrf2), a master regulator of antioxidant defense responses including glutathione synthesis, had no effect on GSSSG-mediated inhibition of LPS-induced proinflammatory gene upregulation.\u003csup\u003e33\u003c/sup\u003e Thus, GSSSG would exert its anti-inflammatory activity by NF-кB inactivation and ERK hyperactivation through unidentified mechanisms other than antioxidation.\u003c/p\u003e\n\u003cp\u003eWe found that intravitreal administration of GSSSG inhibited not only proinflammatory gene upregulation but also microglial accumulation in the retina of LPS-challenged rats. Resident microglia change the morphology from a ramified to larger and round shape upon activation with stimulants, as shown in Fig. 5B.\u003csup\u003e48\u003c/sup\u003e Activated microglia increase expression of proinflammatory cytokines including IL-1, IL-6, TNF-\u0026alpha;, and Ccl2, which stimulate microglial proliferation.\u003csup\u003e49\u0026ndash;53\u003c/sup\u003e Treatment with LPS led to activation of the Toll-like receptor 4-mediated NF-кB signaling pathway, implicated in proinflammatory cytokine upregulation, in microglia.\u003csup\u003e54,55\u003c/sup\u003e Our previous study indicated that GSSSG attenuates LPS-induced activation of the NF-кB signaling pathway through inhibition of p65 phosphorylation.\u003csup\u003e33\u003c/sup\u003e Taken together, a negative feedback effect of GSSSG on LPS-induced activation of the NF-кB signaling pathway and cytokine production may explain the mechanism underlying the GSSSG-mediated inhibition of microglial proliferation; GSSSG deactivates the NF-кB signaling pathway, which results in downregulation of proinflammatory gene expression in retinal cells including microglia. Consequently, decreased cytokine production leads to attenuation of LPS-induced microglial proliferation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, we examined the effects of\u0026nbsp;a reactive sulfur species\u0026nbsp;GSSSG on proinflammatory cytokine expression in glial cells \u003cem\u003ein vitro\u003c/em\u003e and on retinal inflammation in rodents using LPS-induced inflammation models. Consequently, we found that GSSSG inhibits upregulation of proinflammatory cytokine expression in M\u0026uuml;ller cells and microglia and secretion of these cytokines and on microglial accumulation in retinas. Glial cells are major sources of proinflammatory cytokines in retinas and are implicated in inflammatory eye diseases. Thus, GSSSG-mediated regulation of glial inflammation would be effective to prevent the pathogenesis of such diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Junko Sato and Mayumi Suda and the Biomedical Research Unit of Tohoku University Hospital for technical assistance with the experiments. This research was supported by AMED (Grant Numbers JP18lm0203046, JP19lm0203046, JP20lm0203046 and JP21ym0126032) and JSPS KAKENHI (Grant Number JP21K09714).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHiroshi Tawarayama:\u003c/strong\u003e Conceptualization, Formal analysis, Funding acquisition, Investigation, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eKota Umeki:\u003c/strong\u003e Investigation, Validation. \u003cstrong\u003eMaki Unoue-Yanagimachi:\u003c/strong\u003e Investigation. \u003cstrong\u003eNaoki Takahashi:\u003c/strong\u003e Investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNoriko Himori:\u003c/strong\u003e Supervision. \u003cstrong\u003eSatoru Tsuda:\u003c/strong\u003e Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHiroshi Kunikata:\u003c/strong\u003e Supervision. \u003cstrong\u003eTakaaki Akaike:\u003c/strong\u003e Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eToru Nakazawa:\u0026nbsp;\u003c/strong\u003eConceptualization,\u0026nbsp;Funding acquisition, Project administration, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting financial interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMedzhitov R. 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Published online 2015. doi:10.1186/s12864-015-1728-5\u003c/li\u003e\n\u003cli\u003eDatta S, Cano M, Ebrahimi K, Wang L, Handa JT. The impact of oxidative stress and inflammation on RPE degeneration in non-neovascular AMD. \u003cem\u003eProg Retin Eye Res\u003c/em\u003e. 2017;60:201-218. doi:10.1016/j.preteyeres.2017.03.002\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"glutathione trisulfide, glia, retina, cytokine, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-2425068/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2425068/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe aimed to investigate the impact of glutathione trisulfide (GSSSG) on lipopolysaccharide (LPS)-induced inflammation in retinal glia.\u003cstrong\u003e \u003c/strong\u003eInflammatory responses in mouse-derived glial cells and Wistar rat retinas were stimulated with intravitreal LPS injection. Cell survival and proinflammatory cytokine production were examined using the Calcein-AM assay, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Retinal microglia were visualized with immunohistochemistry for Iba1.\u003cstrong\u003e \u003c/strong\u003eAdministration of LPS (10 µg/mL) or GSSSG (less than 100 µM) did not affect survival of cultured primary Müller cells and established microglial cells. RT-qPCR and ELISA indicated that GSSSG inhibited LPS-induced gene upregulation and protein secretion of proinflammatory cytokines in these glial cells and rat retinas. Finally, immunohistochemical studies indicated that GSSSG inhibited LPS-induced accumulation of Iba1-immunopositive microglia in rat retinas. GSSSG has the potential to prevent pathogenesis of inflammation-associated ocular diseases by inhibiting proinflammatory cytokine expression in retinal glial cells.\u003c/p\u003e","manuscriptTitle":"Glutathione Trisulfide Prevents Lipopolysaccharide-induced Retinal Inflammation via Inhibition of Proinflammatory Cytokine Production in Glial Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-05 16:24:50","doi":"10.21203/rs.3.rs-2425068/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-02-02T07:57:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-01-25T02:07:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1cd85885-979a-4d03-b70d-bca7854e4c93","date":"2023-01-14T03:33:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-01-02T13:06:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-01-02T13:03:44+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-01-02T08:32:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-01-02T08:25:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-12-29T11:19:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"153ab6e0-ea8f-4ba3-ab39-2bbca9d43e89","owner":[],"postedDate":"January 5th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":18072404,"name":"Biological sciences/Neuroscience/Glial biology/Microglia"},{"id":18072405,"name":"Biological sciences/Neuroscience/Visual system/Retina"},{"id":18072406,"name":"Biological sciences/Immunology/Cytokines/Interleukins"},{"id":18072407,"name":"Biological sciences/Immunology/Inflammation/Acute inflammation"}],"tags":[],"updatedAt":"2023-10-16T21:58:12+00:00","versionOfRecord":{"articleIdentity":"rs-2425068","link":"https://doi.org/10.1038/s41598-023-38696-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-07-17 21:40:27","publishedOnDateReadable":"July 17th, 2023"},"versionCreatedAt":"2023-01-05 16:24:50","video":"","vorDoi":"10.1038/s41598-023-38696-4","vorDoiUrl":"https://doi.org/10.1038/s41598-023-38696-4","workflowStages":[]},"version":"v1","identity":"rs-2425068","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2425068","identity":"rs-2425068","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}