Figure 2.

The microbiota promotes MyD88-mediated signaling for IL-1β induction in resident intestinal macrophages. (A) Bone marrow chimeras were generated by reconstituting lethally irradiating SPF wild-type recipients with 10⁶ donor bone marrow cells isolated from the indicated mice. Intestinal sT_H17 response was assessed at 10 wk after reconstitution. Production of IL-17, IL-22, and IFN-γ by (10⁶ cells/ml) total LP cells was assessed in the indicated chimera mice after ex vivo stimulation with anti-CD3. The results are shown from one of two independent experiments with five chimera mice per group per experiment. (B, left) Representative FACS dot plot analysis of total intestine LP cells based on CD11b and CD11c expression. (B, middle) R1, R2, and R3 populations were FACS sorted from SPF wild-type mice, and IL-1β levels in total cell lysates from the indicated populations were determined by ELISA and normalized to total protein concentration. (B, right) R1, R2, and R3 subsets were sorted from the LP of the indicated mice, and IL-1β levels were determined. (C) IL-1β mRNA levels measured by real-time RT-PCR in sorted R1 population from indicated mice. (D) Protein extracts form sorted intestinal R1 population from indicated mice were immunoblotted with anti–IL-1β or anti–β-actin (loading control). (E) Total LP cells were isolated from either SPF (n = 20) or GF (n = 9) wild-type mice and the level of spontaneous IL-6 production in overnight cultured supernatant was determined by ELISA. Representative results are shown from one of at least two to three independent experiments. Experiments in panels C and D are representative of two experiments using pooled cells from n = 2–3 mice.