αA-Crystallin Interacts with TBK1 to Modulate NF-κB Pathway in Mediating Stress-Induced Changes of the Immune Microenvironment and Inflammatory Responses in the Ocular Lens

Abstract

Purpose : The immune microenvironment plays an important role in maintaining tissue homeostasis and function. Recent studies have shown that disturbance of the immune microenvironment is also implicated in cataractogenesis. αA-Crystallin is known to prevent uveitis of the ocular retinas. Whether it plays a role in regulating the immune microenvironment of the ocular lens remains to be investigated

Methods : CRISPR/Cas9 technology and shRNA-mediated silence were used to knockdown alphaA-crystallin, p65 of NF-κB, and TBK1 in mouse lens epithelial cell line, αTN4-1. QRT-PCR was used to analyze the mRNA expression of the inflammatory cytokines. Western blot was used to analyze expression levels of NF-κB pathway components. Co-IP assays were used to verify the interactions between alphaA and TBK1 in both mouse lens epithelial cell line, αTN4-1 and human lens epithelial cell line, FHL-124.

Results : Knockdown of αA-crystallin in αTN4-1 cells or in αA(-/-) mice leads to enhanced phosphorylation of NF-κB and increased expression levels of inflammatory cytokines. Conversely, overexpression of αA inhibits phosphorylation of NF-κB and decreases expression levels of inflammatory cytokines. Mechanistically,αA-crystallin directly interacts with the upstream kinase, TBK1 to modulate the phosphorylation status of NF-κB and the expression levels of inflammatory cytokines.

Conclusions : Our results demonstrate that αA-crystallin plays a fundamental role in regulating lens inflammatory response. Through interactions with TBK1 and p65 of NF-κB, αA-crystallin regulates the immune microenvironment of the ocular lens. [Supported by National Natural Science Foundation of China (Grants 82271071, 81970787, 81770910) and the Joint Key Project from the Natural Science Foundation of Guangdong Province and Guangzhou City (2019B1515120014)，and the Fundamental Funds of the State Key Laboratory of Ophthalmology of Zhongshan Ophthalmic Center (3030901010111)].

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.