Functional characterization and Toll-like receptor (TLR) responsiveness of colonic CX3CR1-defined myeloid cells in healthy and inflamed intestine. (a) Colonic LP cells isolated from resting CX3CR1+/gfp mice, or from mice receiving 2% dextran sodium sulfate (DSS) for 4 days, were cultured for 4.5 h in medium (top panels) or with 1 μg ml−1 lipopolysaccharide (LPS) (lower panels). Interleukin 10 (IL10) and tumor necrosis factor α (TNFα) production was assessed by intracellular cytokine staining. Results shown are mean proportions of IL10+, TNFα+, and IL10+TNFα+ cells within P1, P2, P3, and P4 cells in each condition + 1 s.d. for 3–4 mice per group. (b) Proportions of total cytokine-producing cells within P1 to P4 that produce IL10 alone, TNFα alone, or both IL10 and TNFα in resting (top panels) or inflamed colon (lower panels) ± lipopolysaccharide (LPS). Data representative of two individual experiments. (*P<0.05, **P<0.01, ***P<0.001 and ¶P<0.05, ¶¶P<0.01, ¶¶¶P<0.001 versus P1–P4 subsets from resting mice cultured in medium alone; †P<0.05, ††P<0.01, †††P<0.001 versus P1–P4 subsets from colitic mice cultured in medium alone.) (c) Quantitation of messenger RNA (mRNA) for IL10 and TNFα by fluorescence-activated cell sorted subsets P1–P4 from resting colon. (d) Quantitation of mRNA for pro-inflammatory cytokines and TLR by P1 subset of colonic myeloid cells from resting and colitic mice. Results are shown as mean expression relative to cyclophilin A (CPA) using the 2−ΔC(t) and are from three independent experiments using pooled cells from 12 mice per experiment (resting mice) or one experiment using pooled cells from 10 mice fed DSS for 4 days.