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Necrotizing enterocolitis (NEC) is a severe gastrointestinal complication of prematurity. Using suspension and imaging mass cytometry coupled with single-cell RNA sequencing, we demonstrate severe inflammation in patients with NEC. NEC mucosa could be subtyped by an influx of three distinct neutrophil phenotypes (immature, newly emigrated, and aged). Furthermore, CD16+CD163+ monocytes/Mϕ, correlated with newly emigrated neutrophils, were specifically enriched in NEC mucosa, found adjacent to the blood vessels, and increased in circulation of infants with surgical NEC, suggesting trafficking from the periphery to areas of inflammation. NEC-specific monocytes/Mϕ transcribed inflammatory genes, including TREM1, IL1A, IL1B, and calprotectin, and neutrophil recruitment genes IL8, CXCL1, CXCL2, CXCL5 and had enrichment of gene sets in pathways involved in chemotaxis, migration, phagocytosis, and reactive oxygen species generation. In summary, we identify a novel subtype of inflammatory monocytes/Mϕ associated with NEC that should be further evaluated as a potential biomarker of surgical NEC and a target for the development of NEC-specific therapeutics.

CD16+CD163+ monocytes traffic to sites of inflammation during necrotizing enterocolitis in premature infants