Figure 4

MHCII⁺ monocytes and macrophages are the major producers of interleukin-23 (IL-23) during Helicobacter hepaticus (Hh)-induced colitis. CD11c^IL-23+ mice (Cre-GFP⁺) were infected with Hh combined with anti-IL-10R monoclonal antibody (mAb) treatment and analyzed 4 days after infection alongside uninfected controls (Ctrl). (a) Quantitative PCR (qPCR) analysis of Il23a mRNA expression in colonic tissue collected from five individual mice at the indicated time points. (b) Total number of lamina propria cells per mouse at day 4. (c) Representative fluorescence-activated cell sorting (FACS) plots and (d,e) frequencies of the indicated myeloid subsets (as defined in Supplementary Figure S2) among colonic CD45⁺ leukocytes. (f,g) Representative FACS plots of CD11c-Cre-associated green fluorescent protein (GFP) expression vs. (f) CD11c and (g) MHCII. (h) qPCR analysis of Il23a mRNA expression by Cre-GFP⁺ and Cre-GFP^- cells FACS-sorted from colonic leukocytes. (i) Frequency of CD11c-Cre-associated GFP expression among the indicated colonic myeloid subsets. Rectangle indicates Cre-positive subsets that were selected for further analysis in j. (j) qPCR analysis of Il23a mRNA expression by myeloid subsets FACS-sorted from CD11c^IL-23+ and CD11c^IL-23- mice. Statistics comparing cells from CD11c^IL-23+ and CD11c^IL-23- mice are shown. (k) qPCR analysis of Il23a mRNA expression by FACS-sorted MHCII^- and MHCII⁺ monocytes. Data points represent individual mice, bars indicate medians. All bar charts of FACS-sorted cells represent the mean±s.e.m. of 10–15 pooled mice, sorted in two biological replicates. Data are representative of two independent experiments. *P<0.05, **P<0.01, ***P<0.001, as determined by (a, j) one-way analysis of variance (ANOVA) with Bonferroni’s post-test or (b–h, k) Mann–Whitney U-test. DC, dendritic cell; MHCII, major histocompatibility complex class II.

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