Lipoxin B4 Mitigates TRPV4-Activated Muller Cell Gliosis During Ocular Hypertension
Purpose: Müller 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 B4 (LXB4), an endogenous lipid mediator produced by retinal astrocytes, has been shown to suppress glial reactivity and directly protect RGCs. This study investigated whether LXB4 modulates TRPV4-driven Müller glial activation and inflammation and whether Müller glia themselves contribute to this retinal lipoxin pathway.  Methods: Ocular hypertension (OHT) was induced in mice via a silicone oil model, and reactive Müller glia were isolated via magnetic sorting for transcriptomic analysis. In vitro, primary and immortalized Müller glia were treated with a TRPV4 agonist with or without LXB4. Glial reactivity was assessed by flow cytometry, immunostaining, qPCR, and western blotting. LC–MS/MS-based lipidomics was used to quantify lipoxin pathway metabolites, and single-cell RNA-seq was used to examine transcriptional responses to LXB4 treatment. GFAP and TRPV4 expression was evaluated via immunohistochemistry in retinal sections.  Results: RNA bulk-sequencing analysis and qPCR revealed that Müller glia express both 5- and 15-lipoxygenase. Lipidomic analysis confirmed that the lipoxin pathway is functional and that Müller glia endogenously produce LXB4, establishing this essential cell type as a source of anti-inflammatory and neuroprotective LXB4 in the retina. TRPV4 activation induced a reactive glial phenotype characterized by increased GFAP and IL6 expression, increased STAT3 phosphorylation, and increased production of lipoxin pathway metabolites, suggesting that biomechanical stress simultaneously triggers both gliosis and lipid signaling. Exogenous LXB4 suppressed TRPV4-induced gliosis in vitro by downregulating IL6 and inhibiting STAT3 activation, and in vivo treatment during OHT reduced the expression of Stat3, Il6, and TNF-a while attenuating TRPV4 upregulation in Müller glia.  Conclusion: These findings identify a self-regulating lipid circuit in Müller glia and support the targeting of the TRPV4–lipoxin pathway to inhibit gliosis and protect against neurodegeneration in glaucoma. Overall design: RNA seq analysis for magnetically sorted Müller glia from Control (n=3) and Ocular hypertension (n=3) and untreated primary Müller glia (n=3)