Fig. 4.

Microbiota affects tissue specific iNKT cell accumulation by genetic modifications of Cxcl16. (A) The CXCL16 concentration in the serum of each group was measured with ELISA in SPF, GF, GF/a, and GF/n as described above (n = 4 mice per group). (B) Colon and lung tissue samples were harvested from age-matched mice from each group and analyzed for Cxl16 expression (n = 4 mice per group). (C) Colon, ileum, and lung tissue samples were analyzed from age-matched SPF and GF mice at different time points for Cxcl16 expression (n = 5 mice per group). (D) SPF (open circles) and GF (closed circles) newborn mice were treated three times a week intraperitoneal with 25 μg of a neutralizing CXCL16 antibody (αCXCL16) or its isotype control IgG2a antibody and analyzed at the age of 2 weeks for iNKT cell percentages by means of flow cytometry. Each circle represents a mouse. (E) Analysis of DNA methylation of five CpG sites of Cxcl16 by bisulfite pyrosequencing. For each group, the mean of DNA methylation of 5 CpG sites is shown as a percentage according to the methylated control (Methyl. control) (n ≥ 3 mice per group). (F) Cxcl16 qPCR analysis of colonic DNA after performing a 5-hydroxymethylated DNA immunoprecipitation (5-hmC IP DNA) (n ≥ 3 mice per group). All data were obtained from at least two independent experiments with similar results. Error bars indicate the SD. *P ≤ 0.05, **P < 0.01, unpaired t test.