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Lipoxin B4 Mitigates TRPV4-Activated Muller Cell Gliosis During Ocular Hypertension

ABSTRACT: 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-α 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE306785 | GEO | 2026/02/11

REPOSITORIES: GEO

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Project description:Purpose. Immune privilege in the eye is a compilation of anti-inflammatory mechanisms that protect vision from the damaging sequelae of intense immune responses. Although the breakdown of privilege can lead to ocular disease, little is known about how these mechanisms are regulated. Since retinal Müller glial cells and autophagy also have anti-inflammatory properties, we tested the idea that Müller cells utilize autophagy to support immune privilege. Methods. The essential autophagy regulator Atg5 was deleted in retinal Muller cells using a tamoxifen inducible GlastCre ERT X Atg5f/f strain. Intraocular inflammation was induced by intravitreal injection of LPS and monitored by H&E staining, immunofluorescent confocal microscopy, and flow cytometry. scRNA-seq was performed on retinal Müller cells isolated from control and Atg5-deficient mice. Additionally, markers of Müller cell gliosis and function were assessed by western blotting and cytokines were detected by Luminex cytokine/chemokine arrays. In cultured Müller cells siRNA knockdown techniques were used to examine the role of autophagy in regulation of LPS-induced inflammatory pathways. Results. We observed increased and prolonged intraocular inflammation when Müller cells were autophagy (Atg5) deficient. Müller cell gliosis (as measured by Gfap expression) was significantly increased and inflammation was sustained over 5 days post-LPS injection compared to control. The retinae with Atg5-deficient Müller cells also contained increased inflammatory mediators and neuroprotective molecules. Gene expression analysis revealed a heterogeneous response to LPS in Müller glial cell population that revealed 2 states of activation. The normal retinae contain Müller cells in both a basal and an activated state, while autophagy deficient retinae (Atg5iΔMüller) contained only activated Müller cells. Analysis of gliosis markers Gfap and Lcn2 confirmed this heterogeneity showing that in control eyes basal and activated (gliotic) Müller glia were observed; however, with autophagy deficiency nearly all Müller cells were gliotic. Interestingly, activated cells were largely indistinguishable between autophagy sufficient and deficient Müller cells. In cultured Müller cells, siRNA knockdown of autophagy genes resulted in heightened mTOR activation, increased Gfap expression, and upregulated cytokine/chemokine production. Conclusions. Autophagy deficiency in Müller cells leads to enhanced intraocular inflammation though activation of the mTOR pathway. Our data suggests that autophagy defines a novel perspective on Müller glial heterogeneity based on their activation state. Thus, autophagy restrains cellular activation and inflammation, supporting immune privilege by preventing excessive and potentially destructive immune responses.

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Lipoxin B4 Mitigates TRPV4-Activated Muller Cell Gliosis During Ocular Hypertension

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