NF-κB signaling in Cx3cr1+ cells is required for neonatal intestinal homeostasis and for NEC-induced monocyte differentiation

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Abstract Necrotizing enterocolitis (NEC) is a life-threatening intestinal disease affecting premature infants. Intestinal macrophages (Mφ) express Cx3cr1 and have been reported to mediate the gut immune response at this age. At birth in mice, embryonic (e) Mφ (Ly6C−CD64+MHCII+Tim4+) are the primary intestinal myeloid cell with few Ly6C+ monocytes. At 3 weeks, differentiated monocyte-derived-Mφ replace intestinal eMφ. In a NEC model, we showed decreased intestinal Ly6C+ cells due to monocyte activation, recruitment, and differentiation. NF-κB inhibition in monocytes decreased their recruitment and differentiation, improved NEC, but did not reduce IL1β. Whether eMφ contribute to inflammation in the neonatal intestine remains unknown. When IKKβ was deleted in Cx3cr1+ cells, we unexpectedly found intestinal IL1β mRNA to be increased in pups exposed to experimental NEC (8.2 ± 0.8; p≤0.05) but also in dam fed (DF) pups (12.6 ± 2.5; p≤0.01). When small intestinal lamina propria Cx3cr1+ cells were traced (using Cre-reporter mice) by flow cytometry, both eMφ (CD11b+Ly6C−CD64+Tim4+) and differentiating monocytes (CD11b+Ly6C+CD64+) expressed Cx3cr1. In mice lacking IKKβ in Cx3cr1+ cells (Cx3cr1cre+/−IKKβf/f but not in Cre- IKKβf/f controls, experimental NEC caused a 2.4 (±0.4)-fold increase in Ly6C+ cells compared to DF, and 30.4% (±8.3) of the Ly6C+ cells expressed CD64 compared to 6.7 (±0.26) in the DF (p≤0.05). CD64+ cells had a 2.4 (± 0.64; p≤0.05) and 8.3 (± 2.9; p≤0.05)-fold decrease in Tim4 and MHCII MFI respectively. Therefore NF-κB signaling in eMφ is required for neonatal intestinal homeostasis and for completing monocyte Ly6C down-regulation and MHCII up-regulation upon gaining CD64 during NEC-induced monocyte differentiation.