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. Author manuscript; available in PMC: 2015 Sep 14.
Published in final edited form as: Nat Commun. 2015 Aug 13;6:8010. doi: 10.1038/ncomms9010

Figure 4. Activation of RORγt+ ILCs requires IL-1β produced by monocyte-derived intestinal macrophages during Citrobacter rodentium infection.

Figure 4

(a) CD3−RORγt+ ILCs from uninfected RORγtGFP/+ reporter mice and MP1 cells from C. rodentium-infected CD115gfp were isolated. ILCs and MP1 cells (1 × 106 cells ml−1) were cultured alone or co-cultured with or without stimulation with heat-killed C. rodentium (MOI=10) for 24 hrs. Neutralizing antibodies for IL-23 (10 μg ml−1), IL-1β (10 μg ml−1), or isotype controls were used to block cytokines. Data are given as mean ± SD (n=4). *p<0.05; N.S., not significant by Dunnett’s test (compared to isotype control). (b) Schematic illustrating experimental protocol of mixed bone-marrow chimera for IL-1β monocyte/macrophage-conditional depletion. Lethally irradiated C57BL/6 recipient mice were reconstituted with mixed bone-marrows from Ccr2WT or Ccr2DTR and Il1b−/− mice (1:1 ratio) for 8 weeks. After 8 weeks, mice were infected with C. rodentium, and CCR2+ monocytes and monocyte-derived MP1 cells were depleted by DT injection (10 ng g−1 body weight) on days 5, 7, 9, 11 post-infection. (c) Survival of chimeric mice infected with C. rodentium (n=7). ** p<0.01 by Log-rank test. Results are pooled data of 2 independent experiments with 3–4 mice each. (d) CCR2WT/Il1b−/− control chimera (IL-1βWT) and CCR2DTR/Il1b−/− chimera (IL-1βΔMo/MP) were infected with C. rodentium and DT was injected (10 ng g−1 body weight) on days 5, 7. LPMCs were isolated on day 8 post-infection. 2 × 106 cells ml−1 LPMCs were cultured with or without stimulation with heat-killed C. rodentium (MOI=10) for 24 hrs. Cytokines in the culture supernatant were analyzed by ELISA. Data are given as mean ± s.d. (n=4, representative of 2 independent experiments). * p<0.05; ** p<0.01; *** p<0.001 by Student’s t test. (e) Isolated LPMCs in (d) were cultured in the presence of heat-killed C. rodentium (MOI=10) for 16 hrs. IL-22 production in CD4− ILCs (Lin−Thy-1+CD3−CD4−) and CD4+ ILCs (Lin−Thy-1+CD3−CD4+) was assessed by flow cytometry. Data are representative of 4 individual mice.