Lipoxin B₄ Regulates Disease-Associated Microglia in the Myelinated Optic Nerve During Ocular Hypertension

Abstract

Purpose : To investigate the homeostatic and neuroprotective action of Lipoxin B₄ (LXB₄) and its specific targets on microglial function in the retina and optic nerve. LXB₄ is produced under homeostatic conditions by astrocytes in the retina and directly acts on retinal ganglion cells (RGCs). Retinal insult, both excitotoxic and ocular hypertension (OHT) induced neuropathy, has been shown to impair the resident astrocytes-RGC lipoxin circuit. LXB₄ is the most potent neuroprotective lipoxin in the retina.

Methods : We used single-cell RNA sequencing to understand the direct action of LXB₄ and the regulation of different cell types in the retina. Silicone oil-induced OHT was used to model neurodegeneration in mice. To understand the microglial phenotype in the retina and optic nerve, we used MorphOMICs and pseudo-time trajectory analysis for morphological characterization. RNA sequencing of optic nerves followed by STRING network and KEGG pathway enrichment analysis was performed to identify novel action and targets for LXB₄. Immunostaining was used to validate the findings of RNAseq.

Results : LXB₄ treatment significantly downregulated the microglia ‘sensome’ transcriptomics in the retina (log2FC< -1, p<0.05). OHT-induced distinct and temporally defined microglial functional phenotypes during sustained hypertension in both the retina and distal myelinated optic nerve (Fig. 1). Microglial expression of CD74, a marker of disease-associated microglia, was only induced in the optic nerve (3-fold, p=0.0017) but not in retinal microglia. LXB₄ treatment during OHT shifted optic nerve microglia toward a homeostatic morphology (Fig. 2) and downregulated expression of CD74 (1.1-fold, p=0.0054). Furthermore, we identified a correlation between CD74 and phospho(p)-PI3K expression levels in the optic nerve during OHT(r= 0.74, p=0.004) and OHT+LXB₄ (r=0.91, p=0) conditions. Interestingly, the expression of p-PI3K was reduced by LXB₄ treatment following OHT (0.43-fold, p=0.0072).

Conclusions : We established dynamic and early functional change in the microglia phenotype in both the retina and distal myelinated optic nerve during OHT. LXB₄ restored the optic nerve homeostatic microglia phenotype and inhibited the disease-associated phenotype. These findings identify LXB₄ microglia regulation as a neuroprotective mechanism and optic nerve microglia responses as an early event in OHT pathogenesis.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.