Lung monocytes express GPR183 and migrate toward 7α,25-dihydroxycholesterol. (A) Gpr183-GFP expression by the indicated mouse lung myeloid cells. Green and grey histograms show Gpr183-GFP expression of lung cells from Gpr183^GFP/+ and Gpr183^+/+ mice, respectively. Gating strategy for monocytes, neutrophils, and eosinophils is shown in Fig. S1 A. Dendritic cells were gated as live CD45⁺ lineage (CD3/CD19/NK1.1)⁻CD64⁻Ly6G^-CD11c⁺MHCII⁺ single cells. Data are representative of greater than or equal to three independent experiments with a total of n ≥ 6 mice per genotype. (B)Gpr183-GFP expression by lung lymphocytes from Gpr183^GFP/+ (green histograms) and Gpr183^+/+ mice (grey histograms). After gating on live CD45⁺F4/80⁻ single cells, B cells were gated as B220⁺CD3⁻ cells, CD4 T cells as B220^-CD3⁺TCRβ⁺CD4⁺ cells, and CD8 T cells as B220⁻CD3⁺TCRβ⁺CD8α⁺ cells. Data are representative of two independent experiments with a total of n = 6 mice per genotype. (C) Chemotaxis of mouse bone marrow (BM) monocytes toward 7α,25-dihydroxycholesterol (7α,25-OHC). Left panel shows the transwell migration of bone marrow monocytes from B6 mice to the indicated concentrations of 7α,25-dihydroxycholesterol. Right panel shows the transwell migration of bone marrow monocytes from Gpr183^+/+ and Gpr183^−/− mice to 7α,25-dihydroxycholesterol. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA with Tukey’s multiple comparison post hoc test. Data are pooled from two (left panel) or three (right panel) independent experiments. Each experiment was performed with cells from one mouse per genotype and with two technical replicates per condition (total n = 4–6).