Dataset Information

Proteomic phenotyping of stimulated Müller cells uncovers profound pro-inflammatory signaling and antigen-presenting capacity

ABSTRACT: Müller cells are the main macroglial cells of the retina exerting a wealth of functions to maintain retinal homoeostasis. Upon pathological changes in the retina, they become gliotic with both protective and detrimental consequences. Accumulating data also provide evidence for a pivotal role of Müller cells in the pathogenesis of diabetic retinopathy (DR). While microglial cells, the resident immune cells of the retina are considered as main players in inflammatory processes associated with DR, the implication of activated Müller cells in chronic retinal inflammation remains to be elucidated. In order to assess the signaling capacity of Müller cells and their role in retinal inflammation, we performed in-depth proteomic analysis of Müller cell proteomes and secretomes after stimulation with INFγ, TNFα, IL-4, IL-6, IL-10, TGFβ1, TGFβ2 and TGFβ3. We used both, primary porcine Müller cells and the human Müller cell line MIO-M1 for our hypothesis generating approach. Our results point towards an intense signaling capacity of Müller cells, which reacted in a highly discriminating manner upon treatment with different cytokines. Stimulation of Müller cells results in a primarily pro-inflammatory phenotype with secretion of cytokines and components of the complement system. Furthermore, we observed evidence for mitochondrial dysfunction, implying oxidative stress after treatment with the various cytokines. Finally, both MIO-M1 cells and primary porcine Müller cells showed several characteristics of atypical antigen-presenting cells, as they are capable of inducing MHC class I and MHC class II with co-stimulatory molecules. In line with this, they express proteins associated with formation and maturation of phagosomes. Thus, our findings underline the importance of Müller cell signaling in the inflamed retina, indicating an active role in chronic retinal inflammation underlying the pathogenesis of diabetic retinopathy.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Sus Scrofa Domesticus (domestic Pig)

SUBMITTER: Stefanie Hauck

LAB HEAD: Stefanie M. Hauck

PROVIDER: PXD028390 | Pride | 2022-02-16

REPOSITORIES: Pride

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Publications

Proteomic Phenotyping of Stimulated Müller Cells Uncovers Profound Pro-Inflammatory Signaling and Antigen-Presenting Capacity.

Schmalen Adrian A Lorenz Lea L Grosche Antje A Pauly Diana D Deeg Cornelia A CA Hauck Stefanie M SM

Frontiers in pharmacology 20211029

Müller cells are the main macroglial cells of the retina exerting a wealth of functions to maintain retinal homoeostasis. Upon pathological changes in the retina, they become gliotic with both protective and detrimental consequences. Accumulating data also provide evidence for a pivotal role of Müller cells in the pathogenesis of diabetic retinopathy (DR). While microglial cells, the resident immune cells of the retina are considered as main players in inflammatory processes associated with DR, ...[more]

PMID: 34776983

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Project description:Diabetic retinopathy (DR), the leading cause of blindness in working-age adults, is thought to be primarily a microvascular complication of diabetes. Müller cells, the major macroglia of the retina, are important for maintaining a healthy and functional retina and play a critical role in several pathological events during DR disease progression as they are a central element of the retinal neurovascular unit. Here, we aim to improve our understanding of Müller cell-specific signaling pathways that are altered during disease progression in order to develop novel gene therapy strategies targeting Müller cells in DR. To achieve this, we used a multi-omics approach on purified Müller cells from 6-month-old control and diabetic db/db mice, including (i) RNA-seq followed by oPOSSUM-3 transcription factor (TF) binding site cluster analysis, (ii) glial chromatin landscape analysis by ATAC-seq, and (iii) Müller cell proteomics by MS/MS mass spectrometry. This led to the identification of the glucocorticoid receptor (GR, gene ID: Nr3c1) as the most promising candidate. Its transcripts and protein were most highly expressed in Müller cells and significantly reduced in cells from diabetic mice, its target gene cluster was downregulated in Müller cells from diabetic animals, and importantly, it was identified as a potential master regulator not only by oPOSSUM analysis based on differentially expressed mRNA in Müller cells, but also validated by the ATAC-seq approach. Next, we investigated the effect of GR modulation in an in vitro cortisol-stimulated retinal explant model. Cortisol not only increased GR phosphorylation levels, but also induced changes in the expression of known downstream GR target genes. Finally, we evaluated whether AAV-mediated GR overexpression in Müller cells improves retinal functionality and found moderate functional improvements in electroretinogram recordings. Although synthetic and topical glucocorticoids are widely used in ophthalmology with undeniable beneficial effects, our study provides valuable new insights into the role of GR signaling and glial alterations in the diabetic retina and thus supports the therapeutic concept of locally enhancing the GR signaling axis. However, our finding of reduced GR levels in Müller cells of the diabetic retina suggests that therapeutic approaches should aim at increasing the expression of the receptor rather than adding more ligands. This may result in optimized local efficacy with fewer systemic unwarranted side effects.

Dataset Information

Proteomic phenotyping of stimulated Müller cells uncovers profound pro-inflammatory signaling and antigen-presenting capacity

Publications

Proteomic Phenotyping of Stimulated Müller Cells Uncovers Profound Pro-Inflammatory Signaling and Antigen-Presenting Capacity.

Similar Datasets

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