Fig. 6. R848 signals via TLR-7–expressing CD11c⁺ dendritic cells to induce IL-22, which is necessary to reestablish colonization resistance against VRE.

(A and B) C57BL/6, Tlr7^−/−/B6, and Tlr7^−/−/CD11c.DTR mixed BMC mice were treated with ampicillin for 3 days, treated with diphtheria toxin, and administered PBS or R848 (50 µg) daily orally. Frequency (A) and number (B) of IL-22⁺ ILCs in the ileum of R848-treated C57BL/6, Tlr7^−/−/B6, and Tlr7^−/−/CD11c.DTR mixed BMC mice after ex vivo incubation in medium in the presence of BFA for 3 hours. FACS plots gated on live, CD45⁺, non-T non-B, Gr-1^neg CD90⁺ CD127⁺ cells. Data are representative of two independent experiments (*P ≤ 0.05, **P ≤ 0.01, Mann-Whitney test; n = 3). (C) Il22 gene expression in the ileum of Tlr7^−/−/B6 and Tlr7^−/−/CD11c.DTR mixed BMC mice as quantified by qRT-PCR and displayed as fold increase over PBS-treated B6 BMC mice and normalized to Hprt. Data are representative of two independent experiments (*P ≤ 0.05, **P ≤ 0.01, Mann-Whitney test; n = 3). Data are means ± SEM. (D) ABX-treated or untreated C57BL/6 or Il22^−/− BMC mice were administered PBS or R848 (50 µg) daily orally. Protein extracts from the ileum were analyzed by Western blotting using the Reg3γ-specific antiserum. Data are representative of two independent experiments. Each lane is for a representative mouse from the indicated group. (E) C57BL/6 and Il22^−/− were treated with ampicillin for 3 days and administered PBS or R848 (50 µg) daily orally. Mice were inoculated with VRE, and CFUs in the ileal content were assessed 24 hours after infection. Data are sum of three independent experiments (**P ≤ 0.01, Mann-Whitney test; n = 10 to 14).