Inflammatory bowel diseases are associated with dysregulated immune responses in the intestinal tissue. Four molecularly identified macrophage subsets control immune homeostasis in healthy gut. However, the specific roles and transcriptomic profiles of the phenotypically heterogeneous CD14 + macrophage-like population in inflamed gut remain to be investigated in Crohn's disease (CD). Here we identified two phenotypically, morphologically and functionally distinct colonic HLADR + SIRPα + CD14 + subpopulations that were further characterized using single-cell RNA-sequencing (scRNAseq) in CD. Frequencies of CD64 hi CD163 −/dim cells selectively augmented in inflamed colon and correlated with endoscopic score of disease severity. IL-1β and IL-23-producing CD64 hi CD163 −/dim cells predominated over TNF-α-producing CD64 hi CD163 hi cells in lesions. Purified "inflammatory monocyte-like" CD163 − , but not "macrophage-like" CD163 hi cells, through IL-1β, promoted Th17/Th1 but not Th1 responses in tissue memory CD4 + T cells. Unsupervised scRNAseq analysis that captures the entire HLADR + SIRPα + population revealed six clusters, two of which were enriched in either CD163 − or CD163 hi cells, and best defined by TREM1/FCAR/FCN1/IL1RN or CD209/MERTK/MRCI/CD163L1 genes, respectively. Selected newly identified discriminating markers were used beyond CD163 to isolate cells that shared pro-Th17/Th1 function with CD163 − cells. In conclusion, a molecularly distinct pro-inflammatory CD14 + subpopulation accumulates in inflamed colon, drives intestinal inflammatory T-cell responses, and thus, might contribute to CD disease severity.

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In the original version of this article, the data availability section was missing. The PDF and HTML versions of this article have been updated to include the following details: DATA AVAILABILITY The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus, and are accessible through GEO Series accession number GSE140123 (https://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE140123).