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

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.

米勒胶质细胞（Müller cells）是视网膜的主要大胶质细胞，具备多种功能以维持视网膜稳态。当视网膜发生病理改变时，它们会发生胶质增生，兼具保护与有害双重效应。越来越多的研究证据表明，米勒胶质细胞在糖尿病视网膜病变（diabetic retinopathy, DR）的发病机制中发挥关键作用。尽管作为视网膜固有免疫细胞的小胶质细胞被认为是糖尿病视网膜病变相关炎症过程的主要参与者，但活化的米勒胶质细胞在慢性视网膜炎症中的作用仍有待阐明。为评估米勒胶质细胞的信号传导能力及其在视网膜炎症中的作用，本研究采用INFγ、TNFα、IL-4、IL-6、IL-10、TGFβ1、TGFβ2及TGFβ3对细胞进行刺激后，对米勒胶质细胞的蛋白质组与分泌组开展了深度蛋白质组学分析。本研究同时使用原代猪源米勒胶质细胞与人类米勒胶质细胞系MIO-M1进行假说生成类实验。研究结果显示，米勒胶质细胞具备强大的信号传导能力，可对不同细胞因子刺激产生高度特异性的应答反应。米勒胶质细胞受刺激后，主要呈现促炎表型，可分泌细胞因子与补体系统相关组分。此外，本研究观察到线粒体功能障碍的相关证据，提示经多种细胞因子处理后可引发氧化应激。最后，MIO-M1细胞与原代猪源米勒胶质细胞均表现出非典型抗原呈递细胞的若干特征：它们可诱导表达主要组织相容性复合体（major histocompatibility complex, MHC）I类与II类分子，以及共刺激分子。与此一致，这些细胞可表达与吞噬体形成及成熟相关的蛋白质。综上，本研究结果凸显了米勒胶质细胞信号传导在炎症视网膜中的重要性，表明其在驱动糖尿病视网膜病变发病机制的慢性视网膜炎症中发挥主动作用。