Investigation of inflammatory cytokines on Muller cell expression of gliosis associated proteins.

Abstract

Purpose : Previous proteomic studies of gliotic retina show increased upregulation of intermediate filament proteins such as GFAP and vimentin, as well as other proteins such as galectin and tenascin C. Since upregulation of these proteins is associated with retinal damage, it is important to identify mechanisms that may control their expression and release in order to limit retinal damage and perhaps promote repair. This study therefore investigated the effect of various cytokines upregulated during gliosis on the expression of these proteins

Methods : Human Muller cells (MIO-M1) were cultured with the recombinant cytokines TNFα, IL-6 or TGFβ1, which are known to be upregulated during retinal injury. After incubation with these inflammatory factors, RNA was extracted and mRNA levels of tenascin C (TNC), vimentin, GFAP and galectin-1 were assessed. In addition, protein levels of GFAP and involvement of the JAK/STAT signalling pathway were investigated

Results : Addition of recombinant TGFβ1 to cell culture medium significantly reduced mRNA expression of TNC (P=0.0059) and GFAP (p=0.0070). Incubation of Müller cells with TNFα resulted in a significant decrease in GFAP mRNA(p=0.0348), whereas IL-6 increased mRNA levels of this intermediate filament (p=0.0049). No differences in the expression of mRNA coding for vimentin or galectin 1 were observed after incubation with either of the three cytokines. Further analyses showed that TGFβ1 and TNFα significantly reduced protein expression of GFAP (p<0.05). Interestingly, TNFα caused a significant reduction in the phosphorylation of STAT3 (P=0.0491).

Conclusions : Our results showed that although TGFβ1 and IL-6 modify the expression of GFAP, TNFα causes a pronounced reduction in the expression of this intermediate filament protein. Although TGFβ1 also downregulated TNC, neither galectin or vimentin was modified by any of the cytokines investigated. We also showed that TNFα activation of Müller glia causes a decrease in STAT3 (Y705) phosphorylation, suggesting that the JAK/STAT signalling pathway is involved in the regulation of GFAP expression. Further studies will explore these mechanisms that may aid in the design of new treatments to prevent and control scarring of the human retina.

This is a 2020 ARVO Annual Meeting abstract.