Abstract
Purpose :
To identify distinct populations expressing homeostatic and regulatory genes and specific identity markers based on gene expression profiles.
Methods :
Female C57BL/6J (B6) mice aged 6–8 weeks were allowed to house in 50–75% relative humidity before the experiments. The CD45+ cells were sorted from the conjunctiva of B6 mice using the Aria-II cell sorter. Single-cell gene expression libraries was prepared using the Chromium Single Cell Gene Expression 3v3.1 kit and sequenced on the NovaSeq 6000 Sequencing System using the S2 v1.0 Flowcell. Raw sequence reads in the FASTQ format were aligned to the mouse reference genome using Cell Ranger Count v6.0.1 and analyzed with Seurat v3.1. Cell-cell interactions were analyzed by Cell Chat and Gene pathway analysis using Qiagen IPA. Immunofluorescence staining in whole-mount conjunctival tissue samples was visualized using laser scanning confocal microscope.
Results :
Sixteen distinct clusters were identified, including myeloid cells (neutrophils, monocytes, macrophages), dendritic cells (DC), and lymphoid cells (B, T, gdT, ILC2, and NK) lineages. Novel neutrophil [lipocalin (Lcn2) high and low), MHCIIlow macrophage (MP), and Retnla DC2 clusters were identified. Approximately half of the cells map to myeloid and dendritic cell populations with differential expression profiles that include genes with homeostatic and regulatory functions. Serpinb2 (MHCIIlo macrophage), Apoe (monocyte), Cd209a (macrophage), Cst3 (cDC1), and IL4i1 in migratory DC (mDC). Suppressed inflammatory and activated anti-inflammatory/regulatory pathways were observed in certain myeloid and DC populations. Confocal immunolocalization of identity markers showed cDC2 and mDC located on or within the conjunctival epithelium. Monocyte, macrophage, cDC1 and IL-13/IL-5+ ILC2 were located below the conjunctival epithelium and goblet cells.
Conclusions :
This study provides valuable information that can be used for more specific cell identification and cell-specific gene expression profiles that can be compared with those in ocular surface inflammatory diseases, such as dry eye where there is recruitment and activation of immune cells. It provides biomarkers that can be used to determine factors, such as diet and microbiome, that maintain the production of the homeostatic and regulatory factors by the conjunctival immune cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.