Fig 2: IL-22 signaling deficiency does not increase NEC susceptibility(A) Representative histology of the small intestine of dam-fed (DF) pups (left panel) or pups that have been subjected to a NEC protocol (NEC, right panel). Scale bar, 200 μm.(B) Il22 and Il22ra1 expression in the terminal ileum of DF mice and pups subjected to experimental NEC.(C) Representative histology of non-NEC control and NEC human infant small intestine. Scale bar, 200 μm.(D) Expression levels of IL22 (left panel) or IL22RA1 (right panel) in the ileum of infants with NEC and non-NEC controls.(E) Effect of intestinal epithelial-specific IL22ra1 deficiency on Il1b expression in the intestine of DF pups and mice subjected to experimental NEC. Data were combined from independent experiments.(F) Representative H&E staining of terminal ileum sections from each genotype. Scale bar, 200 μm.(G) Histological score of the intestinal lesions in pups subjected to NEC (n = 11–15 per group). Histological scores are displayed as mean ± SEM as the percentage of lesion extent from the total length of the entire tissue section.(H) Effect of IL-22 deficiency on Il1b expression in the intestine of DF pups and neonatal mice subjected to NEC. Data were combined from at least three independent experiments.(I) Representative H&E staining of terminal ileum sections from each genotype. Scale bar, 200 μm.(J) Histological score of the intestinal lesions in the experimental NEC pups (n = 7–8 per group).All data are presented as mean ± SEM percentage of lesion extent from the total length of the tissue section. All gene expression data are displayed as mean ± SEM and were analyzed using a Mann-Whitney test. ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 3: IL-22 treatment induces an antimicrobial transcriptional program in the small intestine of neonates(A) Heatmap analysis showing the differential gene expression of adult mouse small intestinal enteroids cultured in the presence of rIL-22 (100 ng/mL) or an equivalent volume of PBS for 24 h (left column) as well as the small intestine of adult of IL22ra1fl/fl Vil cre+ and IL22ra1fl/fl Vil cre− mice (right column).(B) qPCR quantification of Retnlb, Reg3g, and Fut2 expression in neonatal mouse intestinal enteroids cultured in the presence of rIL-22 (2 ng/mL) or an equivalent volume of PBS for 24 h. Data are representative of three independent experiments.(C) Impact of IL-22 treatment on ileal expression of Retnlb, Reg3g, and Fut2 in DF pups and pups subjected to NEC. All gene expression data are displayed as mean ± SEM and were analyzed using Mann-Whitney test. Data were combined from at least three independent experiments.(D) Gene expression of RETNLB and REG3G in human neonatal intestinal enteroids treated for 24 h with PBS, 1, 10, or 100 ng/mL of human rIL-22. Unpaired Student’s t test was used for this analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig 4: Neonatal intestinal immune cells display low levels of IL-22 production(A and B) Il22 (A) and Il22ra1 (B) gene expression was quantified in the terminal ileum of embryonic day 15 (e15) and e17, as well as postnatal days 1, 4, 7, 14, 21, 28, and 49, mice (n = 3 embryonic mice, n = 10 postnatal mice as biological replicates per group).(C) IL-22 intracellular staining of LP cells isolated from the small intestine of 1- and 8-week-old mice. Data are representative of two independent experiments.(D) Percentage of CD45+IL22+ cells in the small intestine.Data are displayed as mean ± SEM. Results were analyzed using a Mann-Whitney test. ∗∗p < 0.01.

Fig 5: IL-22 treatment attenuates experimental NEC by enhancing epithelial cell regeneration(A) Four-day-old pups were either subjected to the NEC protocol or kept with the dams as controls for 3 days. The pups received a daily i.p. injection of recombinant IL-22 (rIL-22, 100 μg/kg) or an equivalent volume of PBS. Expression levels of Il1b, Cxcl1, and Cxcl2 were measured in the terminal ileum of the pups after 3 days of PBS or IL-22 treatment. Data were combined from at least three independent experiments.(B) Representative H&E staining of terminal ileum sections from each experimental group. Scale bar, 200 μm.(C) Histological score of the intestinal lesions in pups subjected to NEC and either PBS or rIL-22 injections (n = 6–9 per group). Data are presented as mean ± SEM percentage of lesion extent from the total length of the tissue section.(D) Representative confocal images of ileal samples of pups with NEC and indicated treatments stained with antibodies against proliferating cell nuclear antigen (PCNA, green) and Olfactomedin 4 (OLFM4, red). The nuclei were stained with Hoechst (blue). Scale bar, 50 μm.(E) Quantification of epithelial cell proliferation in the terminal ilea of pups with NEC treated with rIL-22 or PBS.All gene expression data are displayed as mean ± SEM and were analyzed using Mann-Whitney test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.