Abstract
Purpose :
Macrophages (MF) and dendritic cells (DC) are categorized as two separate cell types with distinct roles in innate immunity, but also can have overlapping functions given their highly complex and plastic nature. In the cornea, current classifications for these two population of mononuclear phagocytes are still limited to only several markers or genes. Thus, a better accounting of the full range and dynamics of their cell states is needed to more completely understand the distinct roles of these cells and how they fulfill their functions. We addressed this knowledge gap here at the transcriptome level, by applying single cell RNA-seq of mononuclear phagocytes from the cornea and their precursors from the blood in normal adult mice.
Methods :
Corneas from n=25 male and n=25 female eight-week-old C57BL/6 mice were harvested, prepared as single cell suspensions, and FACS sorted for live CD45+ populations. Additionally, the peripheral blood of n=5 male and n=5 female mice were FACS sorted for live CD45+, CD11b+, CD115+, Ly6G- cells to enrich for circulating monocytes and precursor DCs. FACS sorted cornea and blood were pooled by sex before sequencing.
Results :
Our dataset included n=3,237 viable cells and n=15,831 genes. Unbiased cluster analysis at a resolution of 1.5 via Seurat resulted in n=25 clusters. To understand the potential relationships between these clusters we conducted a pseudotime analysis. Any clusters with lymphoid or granulocyte signatures were excluded. Langerhan’s cells were also excluded given their fetal origins. Our results revealed two main trajectory branches. One branch transitioned through a DC-like state, while the other did not. Interestingly, both branches converged into several closely related clusters dominated primarily by a MF gene signature, with genes related to DC function.
Conclusions :
Collectively, our results suggest that corneal mononuclear phagocytes derived from adult blood precursors represent MFs of which had either transitioned through a DC-like state or did not, yet retained DC features. Hence, rather than a two-cell type model for DCs and MFs, our findings suggest that they exist in a continuum of cell states and expressed gene programs in the cornea. These implications may better explain how mononuclear phagocytes maintain such a diverse functional repertoire with efficient on-demand responses involved in corneal homeostasis and disease.
This is a 2021 ARVO Annual Meeting abstract.