Microbial exposure during early life has persistent effects on natural killer T cell function

Abstract

Exposure to microbes during early childhood is associated with protection from immune-mediated diseases such as inflammatory bowel disease (IBD) and asthma. Here, we show that in germ-free (GF) mice, invariant natural killer T (iNKT) cells accumulate in the colonic lamina propria and lung, resulting in increased morbidity in models of IBD and allergic asthma as compared with that of specific pathogen-free mice. This was associated with increased intestinal and pulmonary expression of the chemokine ligand CXCL16, which was associated with increased mucosal iNKT cells. Colonization of neonatal-but not adult-GF mice with a conventional microbiota protected the animals from mucosal iNKT accumulation and related pathology. These results indicate that age-sensitive contact with commensal microbes is critical for establishing mucosal iNKT cell tolerance to later environmental exposures.

Fig. 1

Intestinal bacteria-dependent accumulation of colonic iNKT cells in GF mice leads to high mortality in oxazolone-induced colitis. (A to C) The percentage of CD1d tetramer–positive cells (iNKT cells) within the live lymphocytes from the lamina propria (LP) of gender-matched GF and SPF SW mice (7 to 8 weeks old) was analyzed by means of flow cytometry (A). Representative dot-plots are shown in (B), and the absolute number of iNKT cells is shown in (C). Each circle in (A) represents an individual mouse. (D) Percentages of colonic iNKT cells of live LP lymphocytes in gender-matched SPF and GF mice were analyzed at different ages by means of flow cytometry (n = 4 mice per group). (E to H) Eight-week-old GF and SPF mice were monitored and scored after rectal challenge with 1% oxazolone (ox) or 50% ethanol for survival and body weight loss for 5 days. On day 5, the colons were collected and dissected for histological analysis (n = 5 mice per group). (G) Scale bar, 50 μm. (I) The concentration of IL-4, IL-13, and IL-1β in the supernatant of 24 hours–colon organ explant cultures determined by means of enzyme-linked immunosorbent assay (ELISA) on day five (n = 5 mice per group). All data were obtained from three independent experiments with similar results. In all panels, error bars represent the SD. *P < 0.05, **P < 0.01, unpaired t test and *P < 0.05, log-rank test in (H).

Fig. 2

Microbial colonization during early life prevents the CD1d/iNKT cell-dependent high mortality in oxazolone colitis in GF mice. (A to C) Eight-week-old GF and SPF mice were treated once with 1 mg of 19G11 or isotype control IgG2b antibody before presensitization and rectal oxazolone challenge. (A) Survival after oxazalone challenge is shown. (B) On day 5, the colons were collected for histological analysis, and (C) the cytokine concentration in the supernatant of 24 hours-colon organ explant cultures was measured with ELISA (n ≥ 4 mice per group). (D) Colonic LP lymphocytes were analyzed for iNKT cells by flow cytometry. (GF/a) mice were GF mice that were exposed to SPF environmental conditions at the age of 5 weeks and maintained for 4 more weeks. (GF/n) mice were pups exposed to SPF conditions on their first day of life and maintained under SPF environmental conditions for 8 to 9 weeks. Each circle represents a mouse. (E to F) Colonized mice were treated as above with oxazolone (n = 5 mice per group). All data were obtained from more than two independent experiments with similar results. Error bars indicate SD. *P < 0.05, **P < 0.01, unpaired t test and *P < 0.05, log-rank test in (A) and (E).

Fig. 3

The increased CD1d/iNKT cell-mediated allergic response sensitivity of GF mice is dependent on age of colonization. (A to C) Lymphocytes from lungs of 8-week-old mice in each group were analyzed for iNKT cells by means of flow cytometry (A). Representative dot-plot is shown in (B), and the absolute number of iNKT cells is shown in (C) (n = 6 mice per group). (D to F) Age-matched mice from each group were treated once before OVA presensitization with 1 mg of 19G11 or IgG2b isotype control antibody and with 0.5 mg of 19G11 or IgG2b antibody before the first and the third serial OVA exposure. Twenty-four hours after the last challenge with 5% aerosolized OVA, the mice were analyzed for (D) airway resistance (Rn), (E) percentage of eosinophils of total BALF cells, and (F) IgE (n = 4 mice per group). (G) Lung lymphocytes were analyzed for iNKT cells by means of flow cytometry of age- and gender-matched GF/n and GF/a mice. (H to J) Mice were presensitized with OVA and analyzed ater the last serial OVA challenge as described above (n ≥ 4 mice per group). Each circle in (A), (D), (G), and (H) represents an individual mouse. All data were obtained from more than two independent experiments with similar results. Error bars indicate the SD. *P ≤ 0.05, **P < 0.01, unpaired t test.

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.