Schmalen, A. | Search bioRxiv

bioRxiv2026

Interactive downstream proteomics analysis with MiraProt using Mueller cell proteomes from equine recurrent uveitis

Mass spectrometry-based proteomics requires downstream analysis of processed protein abundance data, including data inspection, filtering, statistical testing, functional enrichment, protein set comparison, network analysis, and visualization. MiraProt was developed as a modular, metadata-aware R Shiny platform that integrates these steps in a single interactive workflow for processed protein-level proteomics data. Its metadata-aware design enables identifiers, sample information, experimental conditions, transformations, and derived data columns to be defined during data preparation and reused consistently across downstream analyses. To demonstrate its use, we reanalyzed a previously published label-free proteomic dataset of primary retinal Mueller cells from healthy horses and horses with equine recurrent uveitis (ERU). ERU is a naturally occurring autoimmune eye disease of horses characterized by recurrent intraocular inflammation triggered by autoreactive T-cells. Mueller cells are specialized retinal macroglia with various functions such as maintaining retinal ion homeostasis and supporting retinal neuron metabolism. Of 193 proteins with an adjusted p-value [≤] 0.05, 187 also showed at least a twofold abundance difference between ERU-derived and control Mueller cells. Functional enrichment highlighted nuclear RNA processing, chromatin-associated structures, DNA and RNA binding, interferon responses, and cell-cycle-associated programs. Gene set enrichment analysis identified positive enrichment of Interferon Alpha Response, Interferon Gamma Response, and MYC-, E2F-, and G2M-associated gene sets. Network analysis of shared proteins further linked this signature to DNA replication, mitotic checkpoint control, and RNA processing. ERU-derived Mueller cells also showed increased abundance of MHC class II-associated proteins. Together, these findings identified an interferon-responsive, cell-cycle-associated, and MHC class II-associated Mueller cell protein signature in ERU and generated experimentally testable hypotheses for further mechanistic studies. MiraProt provides an accessible, metadata-aware framework for reproducible downstream exploration of processed proteomic datasets and prioritization of candidate proteins and pathways for experimental follow-up.

bioinformatics↗

bioRxiv2021

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

Muller 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 Muller 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 Muller cells in chronic retinal inflammation remains to be elucidated. In order to assess the signaling capacity of Muller cells and their role in retinal inflammation, we performed in-depth proteomic analysis of Muller cell proteomes and secretomes after stimulation with INF{gamma}, TNF, IL-4, IL-6, IL-10, VEGF, TGF{beta}1, TGF{beta}2 and TGF{beta}3. We used both, primary porcine Muller cells and the human Muller cell line MIO-M1 for our hypothesis generating approach. Our results point towards an intense signaling capacity of Muller cells, which reacted in a highly discriminating manner upon treatment with different cytokines. Stimulation of Muller cells resulted 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 Muller 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 Muller cell signaling in the inflamed retina, indicating an active role in chronic retinal inflammation underlying the pathogenesis of diabetic retinopathy.

immunology↗