Compensatory expression of Crystallin γS in retinal microglia neutralizes endotoxin-induced neuroinflammation

Abstract

Purpose : To explore the origin of Crygs expression in the retina and assess its role against neuroinflammation

Methods : Retinal CD45⁺ CD11b⁺ cells from C57BL/6 mice were sequenced using single-cell RNAseq after inflammatory insult. Crygs expression was assessed using CX3CR1^+/EGFP mice with immunohistochemistry and flow-cytometry. The function of Crygs was evaluated in Crygs^-/- or wild-type mice that received recombinant murine Crygs (rCrygs) injection in the eye following acute retinal inflammation induced by ocular injection of LPS (10ng/mL) or ocular surface injury with NaOH. qPCR was used to quantify retinal inflammatory cytokines. The neutralizing effect of exogenous Crygs in LPS-mediated retinal inflammation was evaluated ex vivo in CX3CR1^GFP/+ mice using flow cytometry and immunofluorescent microscopy. Retinal lysates from AMD patients and healthy control were also analyzed by Western blot for Crygs expression

Results : Single cell RNAseq identified a subset of CD45⁺ CD11b⁺ retinal immune cells with compensatory expression of Crygs in response to inflammation. Studies in CX3CR1^GFP/+ mice showed that activated microglia express Crygs in response to injury. Administration of rCrygs in LPS injected mouse eyes led to significant suppression of TNF-α (65% suppression), IL-1β (60% suppression), and Crygs (95% suppression) within 24 hours, and reduced MHC-II expression in microglia from 51.6% to 12.27%. rCrygs was shown to mediate neuroprotection by physically binding to LPS, thereby disrupting its biologic activity. In a separate model of ocular alkali burn, deletion of Crygs gene exacerbated retinal TNF-α and IL-1β expression after injury while restoration of the gene in CX3CR1⁺ cells by adoptive transfer suppressed inflammation in injured Crygs^-/-mice. Lastly, we were able to detect expression of Crygs by Western blots in retinal extracts from patients with AMD, a condition known to have activated microglia, but not in normal controls.

Conclusions : Here we show for the first time that Crygs is expressed in retinal microglia during retinal inflammation or pathology. Its expression is compensatory in nature, leading to suppression of inflammation and associated retinal neurodegeneration. Further studies are required to assess the functional and therapeutic roles of Crygs in other retinal and CNS pathologies

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.