Alpha A-Crystallin Modulates Neuroinflammation In Retina Via Stress Specific Inflammatory Pathways

Abstract Purpose We have previously demonstrated that αA-crystallin, a molecular chaperone, plays an important intrinsic neuroprotective role during diabetes, by its phosphorylation on residue 148. We also reported that αA-crystallin is highly expressed by glial cells. There is a growing interest in the potential causative role of low-grade inflammation in diabetic retinopathy pathophysiology and retinal Müller glial cells (MGCs) participation in the inflammatory response. MGCs indeed play a central role in retinal homeostasis via secreting various cytokines and other mediators. Hence, this study was carried out to delineate and understand the regulatory function of αA-crystallin in the inflammatory response associated with metabolic stresses. Methods Primary MGCs were isolated from Ko-αA-crystallin mice. These primary cells were then transfected with plasmids encoding either wild-type (WT), phosphomimetic (T148D), or non-phosphorylatable mutants (T148A) of αA-crystallin. The cells were exposed to multiple metabolic stress including serum starvation (SS) or high glucose with TNF-alpha (HG + T) before being further evaluated for the expression of inflammatory markers by qPCR. The total protein expression along with subcellular localization of NF-kB & NLRP3 component was assessed by western blot. Results Elevated levels of IL-6, IL-1β, MCP-1, and IL-18 in SS were significantly diminished in MGCs overexpressing WT and further in T148D as compared to EV. The HG + T-induced increase in these inflammatory markers was also dampened by WT and even more significantly by T148D overexpression, whereas T148A was ineffective in either stress. Further analysis revealed that overexpression of WT or the T148D, also lead to a significant reduction of Nlrp3, Asc, and caspase-1 transcript expression in serum-deprived MGCs and nearly abolished the NF-kB induction in HG + T diabetes-like stress. This mechanistic effect was further evaluated at the protein level and confirmed the stress-dependent regulation of NLRP3 and NF-kB by αA-crystallin. Conclusion The data gathered in this study demonstrate the central regulatory role of αA-crystallin and its modulation by phosphorylation on T148 in retinal MGCs. For the first time, this study demonstrates that αA-crystallin can dampen the stress-induced expression of pro-inflammatory cytokines through modulation of multiple key inflammatory pathways, therefore, suggesting its potential as a therapeutic target for modulation of chronic neuroinflammation.