Abstract
Purpose :
To compare gene expression profiles, transcriptional trajectories and transcription factor accessibility in immune cell populations in the non-stressed mouse conjunctiva to those after desiccating stress (DS) induced dry eye using single cell RNA sequencing (scRNA-seq) and ATAC-seq.
Methods :
CD45+ cells were sorted from conjunctival suspensions prepared from nonstressed (NS) naïve mice and those subjected to DS for 1-10 days were sorted. scRNA-seq and scATAC-seq were performed with the 10x Genomics platform. Pseudotime trajectory and RNA velocity were calculated with CellRank and Palantir. ArchR was used to identify changes in transcription factor motifs in open chromatin.
Results :
Six cell types comprised 69% of conjunctival immune cells in the NS conjunctiva [macrophage (MΦ), three distinct monocyte (Mo) populations, conventional DC2 (cDC2), and gamma delta (gd) T cells] with myeloid cells accounting for the majority (70%) of these (Fig1A). Myeloid cells included Ccl12hi MΦ (the largest population that doubled in size during DS), Acehi Mo, Ly6c2hi Mo, and Fn1hi Mo (Fig1A,B). The expression profile of Ccl12 hi MΦs is similar to tumor associated macrophages (TAM) with high expression of phagocytic genes (Apoe, Selenop, C1qa-c) and Il10. Fn1hi Mo have the highest expression of inflammatory genes, including H2-a2, Il1 (a and b) and Cxcl10, all increasing over 10 days. Lineage trajectory from Ly6c2hi Mo to Acehi Mo and Mmp14hi MΦ to Ccl12hi MΦ was observed (Fig1C) and pseudotrajectory analysis (deterministic model) predicted 2 terminal states (Ly6c2/Ace Mo and Ccl12 MΦ, Fig1D). Significant increases in certain transcription factor motifs, including Nkb1, Jun, Rela, Relb, RXRα, were found in these clusters.
Conclusions :
The greatest DS induced changes are found in myeloid cells of the innate immune system consisting of distinct populations expressing anti-inflammatory, phagocytic, and inflammatory genes. The functional significance of these findings is being investigated.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.