Fig 2: 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 3: 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 4: 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.

Fig 5: Antimicrobial response initiated by IL-22 in the small intestine does not impact the composition of the intestinal microbiome(A) DF pups and pups subjected to NEC received a daily i.p. injection of recombinant Reg3g (rReg3g, 2 ng/pup) or an equivalent volume of PBS. Expression levels are shown of Il1b in the terminal ileum of either DF or pups subjected to NEC plus PBS or rReg3g.(B) Representative H&E staining of terminal ileal sections from each experimental group. Scale bar, 200 μm.(C) Histological score of the intestinal lesions in pups subjected to NEC and treated with PBS or IL-22 (n = 8–11 per group).(D) Microbial community composition of colon contents collected from pups with NEC injected with PBS or rIL-22 (NEC + rIL-22).(E) Shannon, Chao1, and Simpson indices demonstrating overall bacterial diversity in the microbiome of a subset of pups with NEC injected with either PBS or rIL-22 (NEC + rIL-22).(F) Boxplots of alpha diversity indices (Shannon, Chao1, and Simpson, respectively) reflecting the abundance and the diversity of the operational taxonomic units (OTUs) in the colon contents of pups subjected to NEC and injected with either PBS or rIL-22. Data are displayed as mean ± SEM and were analyzed using Mann-Whitney test. ∗p < 0.05, ∗∗p < 0.01.