Dataset Information

Autophagy Regulates Muller Glial Cell Inflammatory Activation.

ABSTRACT:

Purpose

We tested whether Müller cells utilize autophagy to support immune privilege in the eye.

Methods

The essential autophagy gene Atg5 was deleted in retinal Müller cells. Inflammation was induced by intravitreal injection of lipopolysaccharide (LPS) that was monitored by hematoxylin and eosin (H&E) staining, immunofluorescent confocal microscopy, and flow cytometry. Single-cell RNA sequencing was performed on retinal Müller cells isolated from control and Atg5-deficient mice. Markers of Müller cell gliosis were assessed, and cytokine production in the eye was measured. Small interfering RNA knockdown techniques were used to examine LPS-induced inflammatory pathways in culture.

Results

We observed increased and prolonged intraocular inflammation when Müller cells were autophagy (Atg5) deficient. Müller cell gliosis was significantly increased, and the retinae contained increased inflammatory mediators. Gene expression analysis revealed a heterogeneous response to LPS in Müller cells, revealing two states of activation. The normal retinae contained both basal and activated Müller cells, whereas the autophagy-deficient retinae contained only activated cells. Analysis of the gliosis markers glial fibrillary acidic protein (Gfap) and lipocalin-2 (Lcn2) confirmed this heterogeneity, as in control eyes basal and activated (gliotic) Müller glia were observed; however, with autophagy deficiency, all Müller cells were gliotic. Activated cells were largely indistinguishable between autophagy-sufficient and -deficient Müller cells. In cultured Müller cells, knockdown of Atg5 resulted in heightened mechanistic target of rapamycin (mTOR) activation, increased Gfap expression, and upregulated cytokine/chemokine production in response to LPS.

Conclusions

Autophagy regulates the activation state of Müller cells in response to LPS. Thus, autophagy restrains cellular activation and inflammation, supporting immune privilege by preventing excessive and potentially destructive immune responses.

SUBMITTER: Doggett TA

PROVIDER: S-EPMC12400976 | biostudies-literature | 2025 Aug

REPOSITORIES: biostudies-literature

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Publications

Autophagy Regulates Müller Glial Cell Inflammatory Activation.

Doggett Teresa A TA Zhou Zhenqing Z Rebba Sohini S Unsinger Jacqueline J Ruzycki Philip A PA Ferguson Thomas A TA

Investigative ophthalmology & visual science 20250801 11

<h4>Purpose</h4>We tested whether Müller cells utilize autophagy to support immune privilege in the eye.<h4>Methods</h4>The essential autophagy gene Atg5 was deleted in retinal Müller cells. Inflammation was induced by intravitreal injection of lipopolysaccharide (LPS) that was monitored by hematoxylin and eosin (H&E) staining, immunofluorescent confocal microscopy, and flow cytometry. Single-cell RNA sequencing was performed on retinal Müller cells isolated from control and Atg5-deficient mice. ...[more]

PMID: 40879296

Similar Datasets

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.