{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:01:00Z","timestamp":1785427260126,"version":"3.56.0"},"posted":{"date-parts":[[2025,9,19]]},"group-title":"Molecular Biology","reference-count":50,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T00:00:00Z","timestamp":1758240000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["5T32EY007043-45"],"award-info":[{"award-number":["5T32EY007043-45"]}],"id":[{"id":"https:\/\/ror.org\/03wkg3b53","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000053","name":"National Eye Institute","doi-asserted-by":"crossref","award":["R01EY030218"],"award-info":[{"award-number":["R01EY030218"]}],"id":[{"id":"10.13039\/100000053","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000053","name":"National Eye Institute","doi-asserted-by":"crossref","award":["P30EY003176"],"award-info":[{"award-number":["P30EY003176"]}],"id":[{"id":"10.13039\/100000053","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,19]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Purpose<\/jats:title>\n                  <jats:p>\n                    M\u00fcller glia play dual roles in glaucoma, contributing to both retinal homeostasis and neuroinflammation; their activation by elevated intraocular pressure through the mechanosensitive channel TRPV4 promotes a reactive state that drives retinal ganglion cell (RGC) loss. Lipoxin B\n                    <jats:sub>4<\/jats:sub>\n                    (LXB\n                    <jats:sub>4<\/jats:sub>\n                    ), an endogenous lipid mediator produced by retinal astrocytes, has been shown to suppress glial reactivity and directly protect RGCs. This study investigated whether LXB\n                    <jats:sub>4<\/jats:sub>\n                    modulates TRPV4-driven M\u00fcller glial activation and inflammation and whether M\u00fcller glia themselves contribute to this retinal lipoxin pathway.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>\n                    Ocular hypertension (OHT) was induced in mice via a silicone oil model, and reactive M\u00fcller glia were isolated via magnetic sorting for transcriptomic analysis.\n                    <jats:italic>In vitro<\/jats:italic>\n                    , primary and immortalized M\u00fcller glia were treated with a TRPV4 agonist with or without LXB\n                    <jats:sub>4<\/jats:sub>\n                    . Glial reactivity was assessed by flow cytometry, immunostaining, qPCR, and western blotting. LC\u2013MS\/MS-based lipidomics was used to quantify lipoxin pathway metabolites, and single-cell RNA-seq was used to examine transcriptional responses to LXB\n                    <jats:sub>4<\/jats:sub>\n                    treatment. GFAP and TRPV4 expression was evaluated via immunohistochemistry in retinal sections.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                    RNA bulk-sequencing analysis and qPCR revealed that M\u00fcller glia express both 5- and 15-lipoxygenase. Lipidomic analysis confirmed that the lipoxin pathway is functional and that M\u00fcller glia endogenously produce LXB\n                    <jats:sub>4<\/jats:sub>\n                    , establishing this essential cell type as a source of anti-inflammatory and neuroprotective LXB\n                    <jats:sub>4<\/jats:sub>\n                    in the retina. TRPV4 activation induced a reactive glial phenotype characterized by increased GFAP and IL6 expression, increased STAT3 phosphorylation, and increased production of lipoxins, suggesting that biomechanical stress simultaneously triggers both gliosis and protective lipid signaling. Treatment with LXB\n                    <jats:sub>4<\/jats:sub>\n                    suppressed TRPV4-induced gliosis\n                    <jats:italic>in vitro<\/jats:italic>\n                    by downregulating IL6 and inhibiting STAT3 activation, and\n                    <jats:italic>in vivo<\/jats:italic>\n                    by reducing the expression of\n                    <jats:italic>Stat3, Il6<\/jats:italic>\n                    , and\n                    <jats:italic>TNF-<\/jats:italic>\n                    \u03b1 during OHT while attenuating TRPV4 upregulation in M\u00fcller glia.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion<\/jats:title>\n                  <jats:p>\n                    M\u00fcller glia are a significant source of LXB\n                    <jats:sub>4<\/jats:sub>\n                    in the retina. This neuroprotective M\u00fcller glia pathway is amplified during chronic TRPV4 activation to counter-regulate gliosis. The findings support targeting of the TRPV4\u2013lipoxin pathway as a potential approach to protect against OHT-induced neurodegeneration in glaucoma.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.09.17.673801","type":"posted-content","created":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T15:10:20Z","timestamp":1758294620000},"source":"Crossref","is-referenced-by-count":0,"title":["Lipoxin B\n                  <sub>4<\/sub>\n                  Mitigates TRPV4-Activated M\u00fcller Cell Gliosis During Ocular Hypertension"],"prefix":"10.64898","author":[{"given":"Matangi","family":"Kumar","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shruthi","family":"Karnam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shubham","family":"Maurya","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rama","family":"Nagireddy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John G.","family":"Flanagan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karsten","family":"Gronert","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025092303401093000_2025.09.17.673801v1.1","unstructured":"David Kri\u017eaj . 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