Single-cell mRNA analysis of colon phagocyte heterogeneity identifies two major macrophage developmental pathways

Tissue macrophages (MPs) develop distinctive functions due to unique transcriptional programs driven by local environmental ques. In the intestine, it is known that MPs are heterogeneous cells that differentiate from blood monocytes and embryonic precursors, and are essential for homeostasis with the vast microbiome through their ability to kill invading microbes, clear apoptotic cells, and produce regulatory cytokines. However, the full extent of MP heterogeneity, their developmental relationships, and how the intestinal microbiota affects MP generation is not known. Here, we performed single cell RNA sequencing of colonic myeloid cells from specific pathogen free (SPF) and germ free (GF) C57BL/6 mice and found extensive heterogeneity of colon MP, with at least six different identifiable populations, with less heterogeneity of dendritic cells (DCs). Unsupervised modeling of developmental pathways combined with inference of gene regulatory networks suggested two major trajectories from common CCR2+ precursors resulting in CD11c+CD9+MMRintCD121b+ and CD11c-CD9intMMRhiCD121b- MP populations with unique transcription factors activities to coordinate downstream target molecules. The generation of both mature populations of colon MPs was impaired in germ-free mice, which coincided with the appearance of a dominant unique population of MPs, whereas the generation of DC populations in GF mice were unaffected or increased. Furthermore, the two major MP populations were clearly distinct from other populations regarding their gene expression profile, localization within the lamina propria, and ability to phagocytose macromolecules from the blood. These data uncover the diversity of intestinal myeloid cell populations, highlight the importance of microbiota on the unique developmental as well as anatomical and functional fates of colon MPs. We anticipate that these findings will form the basis for future studies on how tissue myeloid cell development is influenced either directly or indirectly by signals from endogenous microbiota in the intestine, as well as other tissues. To understand how commensal microbiome affect the heterogeneity of colon macrophages and dendritic cells, colon macrophages and dendric cells were isolated from either SPF and GF mice and profiled using 3’-droplet-based (dropseq) single-cell RNAseq. Cell surface markers of macrophages clusters were carefully selected by downstream analysis of scRNAseq data. To confirm the selected markers are well to identify subsets of colon macrophages and get more reliable dataset with deeper sequencing depth, 8 different subsets of colon macrophages were further FACs-sorted and their transcriptomic profiles were examined by bulk RNAseq.