Abstract
Purpose :
Interleukin-6 (IL-6) plays a prominent role in retinal inflammation in diabetic retinopathy (DR). IL-6 trans-signaling using soluble IL-6 receptor (IL-6R) is pro-inflammatory, whereas classical signaling via the membrane-bound receptor is needed for the regenerative or anti-inflammatory activities of IL-6. Müller glial cells (MGCs) express the membrane bound IL-6R and therefore are capable of both classical and trans-signaling. Hypoxia is well known to cause ischemia and thus is often used to mimic DR pathology. The purpose of this study was to investigate the hypoxia-mediated effects on MGCs in the presence and absence of IL-6R using our MGC-specific IL-6R knockout (KO) mouse.
Methods :
MGCs were isolated from C57BL/6J (WT) and KO mouse pups at P7 and cultured in vitro. WT and KO cells were exposed to 1% O2 in a hypoxia chamber for 24 hours. Control cultures were incubated under standard normoxic conditions for the same duration. The effects of hypoxia on genes involved in glutamate metabolism, endoplasmic reticulum (ER) stress, mitochondrial function, and apoptosis were studied using RT-PCR and Western blot analyses.
Results :
Exposure of WT MGCs to hypoxia induced a significant upregulation of vascular endothelial growth factor (Vegf), coronin 1C (Coro1c), and retinol binding protein 1 (Rbp1) genes, whereas the expression of glutamate aspartate transporter 1 (Glast), heat shock protein A5 (Hspa5), Hsp40 member C3 (Dnajc3), thymopoietin (Tmpo), myeloid cell leukemia-1(Mcl1), and BCL2 antagonist/killer 1 (Bak1) were significantly downregulated. Both Glast and GS are known to mediate glutamate regulation. Similarly, Hspa5 and Dnajc3 regulate ER homeostasis, thrombospondin 1 (Thsb1) and Vegf are involved in angiogenesis and inflammation, and Mcl1, Bak1 and Bcl2 are key regulators of apoptosis. Glast, GS, Vat1, and Mcl1 levels were significantly downregulated, and expression of Thsb1 and Tmpo was upregulated in MGCs isolated from KO mice compared to WT. In KO MGCs, hypoxia caused a reduction in the expression of Glast, Thsb1, Hspa5, and Tmpo, whereas the levels of Vegf, Coro1c, and Rbp1 were increased.
Conclusions :
Loss of IL-6R causes a significant change in the gene expression profile of MGCs, and hypoxia mediated effects are stronger in KO cells as compared to WT. In the future, these KO MGCs will be helpful in evaluating the effects of IL-6 signaling on structural and metabolic functions of Müller glia.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.