Figure 6. Systemic Reg3b Substitution Rescues Il23R^ΔIEC Mice by Increasing Local IL-22 Production.

DSS colitis was induced in Il23R^ΔIEC + Reg3b (n = 5; 3 m, 2 f) and Il23R^ΔIEC + (n = 9; 5 m, 4 f) mice as described above. Mice received 2 μg rec. Reg3b or an equivalent volume of PBS i.p. on day 1 and every second day.

(A) Weight loss was monitored from day 3 every day until day 10.

(B) Colon Swiss rolls from Il23R^ΔIEC and Il23R^ΔIEC + Reg3b mice were immunostained with antibodies for BrdU. Representative pictures are shown, with red lines indicating the BrdU-negative area.

(C) Representative pictures of colonic sections stained with anti-BrdU⁺ antibody.

(D and E) Representative H&E pictures of colon showing increased neutrophil influx in Reg3b-treated mice (D) and statistical evaluation of counted neutrophils per HPF (E).

(F) IL-22 ELISA of colon explants from Il23R^ΔIEC+Reg3b versus Il23R^ΔIEC mice.

(G) Principle coordinate analysis (Bray Curtis distance matrices) of fecal flora obtained by 16S rDNA V4 sequencing from Il23R^ΔIEC+Reg3b versus Il23R^ΔIEC animals after DSS treatment (see the Supplemental Experimental Procedures for details).

(H and I) Abundances of dominant bacterial groups (genus level) after DSS treatment in the feces of Il23R^ΔIEC+ Reg3b versus Il23R^ΔIEC mice according to the flagellatin status (flagellated, non-flagellated, or unknown) in response to DSS (H), IL-22 ELISA from WT or Reg3b KO explants after stimulation with 1 μg ultrapure flagellin for 2 hr i.p. (I).

(J–Q) Peritoneal exudates of five wild-type mice were harvested 6 hr after intraperitoneal injection of either sterile saline solution (K) or 100 μg/kg of purified, endotoxin-free Reg3b (N), and relative numbers of neutrophils or macrophages were assessed by cytospin (J and M), or Gr-1 and F4/80 staining was quantified by FACS analysis (K, L, N, and O). Representative flow cytometric analyses (K and N) of peritoneal cell populations and statistical analysis (L and O) show that Reg3b triggers the influx of neutrophils. Also shown is an ELISA on the indicated cytokines on peritoneal fluid (P) and sera (Q) after Reg3b stimulation as described above. Significance was determined using two-tailed Student’s t test and is expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.