Figure 2. ILCs are a major source of GM-CSF.
(A) Representative flow cytometric analysis of ILC populations at day 3 following anti-CD40 treatment. Surface marker and GM-CSF expression is shown following 3 hr stimulation with PMA, ionomycin, monensin, and brefeldin A (n=6). Cells are gated on a live cell gate with doublets excluded. GM-CSF expression gating is based on an FMO control. Red marks ILCs and black marks NK cells. Lower left panel shows IL-7Rα staining in live CD45+ cells compared with FMO control. (B) Representative flow cytometric analysis showing GM-CSF production for ILC populations in untreated or anti-CD40 treated mice. The solid grey histogram represents the isotype control. Following doublet exclusion, cells are gated on a live cell gate, then gated on lineage-, CD45+ Thy1.2+ IL-7Rα+ cells. (C) Percent and median fluorescence intensity of GM-CSF expression in CD4- NKp46- ILCs isolated from mice 7d following anti-CD40 injection. Total cLP cells were stimulated over night with IL-12, IL-23 or in medium alone, followed by 3 hr stimulation with PMA, ionomycin, monensin and brefeldin A (n=7). Results are representative of 2–3 independent experiments. (D) mRNA expression of Il23a and Csf2 in proximal colonic lamina propria at various time points following anti-CD40 injection. Results are shown as fold change in target gene relative to hprt compared with day 0 uninjected control mice. Data are shown as means and SEM. Results are pooled from 2 independent experiments with n=4–6 mice per group per experiment. *, p<0.05, **, p<0.01, one-way ANOVA with Bonferroni’s post test. (Figure 2—figure supplement 1) shows analysis of lack of NK cell contribution to colitis.
Figure 2—figure supplement 1. NK cells are not required for anti-CD40 induced colitis.
