Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells

Abstract

Coevolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Here, we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host's endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood, and hosts are protected against experimental iNKT cell-mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids-exemplified by an isolated peak (MW = 717.6) called GSL-Bf717-reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive.

Figure 1

B. fragilis sphingolipids modulate homeostasis of colonic LP iNKT cells. Representative FACS plots of iNKT cell gating are shown in (A). Total numbers of colonic LP iNKT cells (B) and their percentages in CD3⁺ populations (C) were higher in GF and BFΔSPT than in SPF and BFWT mice. B. fragilis mono-colonized mice had iNKT cell counts similar to those of GF mice in lung (D), liver (E) and small intestine (F). Data in panels (B) and (C) (days 21,42 and 63) were confirmed to have normal distribution by the KS normality test with α=0.05, analyzed by the Student’s t test and are presented as mean ± SEM (standard error of the mean); n≥3 for each group. Data in panels (D)–(F) were analyzed by the Mann Whitney test. See also Fig. S1.

Figure 2

B. fragilis sphingolipids modulate host oxazolone-induced iNKT cell–mediated colitis phenotype. Upon oxazolone colitis challenge, BFΔSPT mice had more severe weight loss (A; n≥6; P values compare BFWT Oxa and BFΔSPT Oxa at days 3 and 4, respectively) and higher cumulative histopathology scores (B) than did BFWT mice. In tissue explant cultures, levels of IL-13 (C) and IL-4 (D) production in colonic tissue after oxazolone challenge were higher in BFΔSPT mice than in BFWT mice although IL-1β production was not (E). Data in (A) were confirmed to have normal distribution by the KS normality test with α=0.05, analyzed by the Student’s t test and are presented as mean ± SEM; Data in (B)–(E) were analyzed by the Mann Whitney test; n≥3 for each group; representative of 3 experiments. See also Fig. S2.

Figure 3

B. fragilis sphingolipids inhibit colonic LP iNKT cell proliferation during neonatal development and restrict the accumulation of these cells in adult mice. Proliferation rates (A and B; n≥3) were higher in GF and BFΔSPT mice than in SPF and BFWT mice at early ages. Colonic LP iNKT cell counts were not restored in GF-WT(adu) mice, but GF-WT(neo) mice had levels of colonic LP iNKT cell proliferation and total cell numbers similar to those in BFWT mice (C and D). After oxazolone challenge, only GF-WT(adu) mice had severe weight loss and high cumulative histopathology scores (E and F; n≥10; P value in panel E compares BFWT and BF-WT(adu) mice at day 4). Data in (A) (days 8 and 12) and (E) were confirmed to have normal distribution by the KS normality test with α=0.05, analyzed by the Student’s t test and are presented as mean ± SEM. MFI, mean fluorescence intensity. Data in (B)–(D) and (F) were analyzed by the Mann Whitney test. See also Fig. S3.

Figure 4

Chemical analysis of B. fragilis glycosphingolipid peak GSL-Bf717. Three distinct sphingolipid clusters were identified in B. fragilis (A). None of these clusters activated iNKT cells (B). GL-Cers was inhibitory to iNKT cell activation by KRN7000 in vitro (C). Data in panel (B) are representative of 2 experiments, each with triplicates, and presented as median ± range. Data in (C) complied three experiments (n≥6) and were confirmed to have normal distribution by the KS normality test with α=0.05, analyzed by the Student’s t test and are presented as mean ± SEM. P values compare KRN7000 + GSL-Bf717 (at ratios of 1:10 and 1:30) with KRN7000 alone (ratio 1:0). (D) GL-Cers had 5 chain-length variants, C32–C36, and the C34 with M.W.=717.6 (m/z 716) was predominant. A peak from this variant, GSL-Bf717, was characterized by MS/MS (E), HPAEC (F) and ¹H-NMR (G) and was found to have an α-galactosyl linkage. In (F), the hydrolyzed hexose head group co-eluted with the spiked galactose standard (blue) at 12.6 min. In (H), the proposed molecular configuration of GSL-Bf717 is shown for comparison with that of the prototypical agonist KRN7000.

Figure 5

B. fragilis GSL-Bf717 inhibits iNKT cell activation. GSL-Bf717 did not activate iNKT cells (A and C; data are representative of 2 independent experiments with each in triplicates) but did inhibit iNKT cell activation by agonist KRN7000 in both hybridomas 24.7 and DN32.D3 (B and D, data are representative of 2 independent experiments with each in triplicates; P value compares KRN7000 + GSL-Bf717 at a 1:30 ratio with KRN7000 alone, which is at ratio 1:0). GSL-Bf717 also inhibited activation by agonist β-GlcCer (right columns in B; P values compare β-GlcCer + GSL-Bf717 at ratios 40:1 and 20:1 with β-GlcCer alone, which is at ratio 1:0). The loading efficiency of PBS-44 to empty CD1d tetramer (phycoerythrin-labeled) was decreased significantly in presence of GSL-Bf717 as evidenced by the lowered MFI of CD1d tetramer-stained iNKT cells (E); n=4. GSL-Bf717 reduced serum levels of IFN-γ (F) and IL-4 (G) released by KRN7000-stimulated iNKT cells in vivo. 100% production was defined as the level of cytokines produced in KRN7000 only-treated mice; n≥4. Data in (A)–(F) are presented as median ± range. Data in (B), (D)–(F) were analyzed by the Mann Whitney test. Data in (G) were confirmed to have normal distribution by the KS normality test with α=0.05, analyzed by the Student’s t test and are presented as mean ± SEM. See also Fig. S4.

Figure 6

GSL-Bf717 treatment of neonatal BFΔSPT mice restores colonic iNKT cell homeostasis and protects mice from colitis in adulthood. GSL-Bf717-treated mice had lower Ki-67 expression at 8-day (A; n≥4) and lower total counts of colonic LP iNKT cells at 8 weeks (B; n≥6). These mice were protected against the oxazolone challenge, with less weight loss (C; P values compare the two groups at days 1 and 4) and lower cumulative histopathology scores (D). Data are representative of 2 independent experiments; n=6 for each group. Data in (A), (B) and (D) were analyzed by the Mann Whitney test. Data in (C) were confirmed to have normal distribution by the KS normality test with α=0.05, analyzed by the Student’s t test and are presented as mean ± SEM.