Fig. 2: Myeloid EGFR deficiency led to increased resident macrophage subclusters but relative decreases in infiltrating macrophages.

A Both male EGFR^f/f (WT) and CD11b-Cre: EGFR^f/f (myeloid EGFR^−/−) mice underwent ischemic injury. The animals were sacrificed at days 1 and 3, and kidney leukocytes were enriched with CD45 microbeads for single cell RNA-seq (scRNA-seq). B scRNA-seq analysis identified clusters of kidney cells, and canonical markers of kidney cell populations were used to identify major cell types in the kidney: macrophages (Cd68, C1qb), neutrophils (S100a9, S100a8), B lymph cells (Cd79b), T lymph cells (Cd3e), NK cells (Nkg7), cDC1(Cd68, C1qb, Xcr1), cDC2 (Cd68, C1qb, Cd209a), and pDC (Siglech). C Egfr mRNA was mainly detected in myeloid neutrophils and macrophages in WT mice. D Eleven subclusters were identified corresponding to macrophages: Mac1-11. E Violin plot of representative genes in each subcluster. C1qc^High HP^low Mac: Mac1, 2, 4, 6, 8, 9, and 10, which tended to be resident macrophages; C1qc^low HP^high Mac: Mac3, 5, 7, and 11, which tended to be infiltration macrophages; proliferating macrophages (Mac 6), proinflammatory macrophages (Mac7 and Mac11) and pro-fibrotic macrophages (Mac 5, 7, and 11). F Gene Ontology (GO) analysis suggested that Mac1 and 2 clusters were associated with myeloid cell differentiation and small GTPase mediated signal transduction, Mac10 cluster with energy metabolism and Mac 9 cluster with regulation of apoptotic signaling pathways. G, H The percentage of cells in each cluster. Compared to WT mice, myeloid EGFR^−/− mice had significantly increased Mac1, 2, 6, 8, 9 and 10 but not Mac 3, 5, 7, and 11 subclusters. I Flow cytometry confirmed that 3 days after ischemic injury myeloid EGFR^−/− mouse kidneys had increased C1q^highHp^low resident macrophages with less apoptosis, while the C1q^lowHp^high infiltrating macrophages were decreased and had increased apoptosis compared to WT mice (n = 9 and 10). Data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, analyzed using 2 tailed Student’s t test.