Bacterial Sphingolipids Exacerbate Colitis by Inhibiting ILC3-derived IL-22 Production

Abstract

Background & aims: Gut bacterial sphingolipids, primarily produced by Bacteroidetes, have dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells. Patients with inflammatory bowel disease display altered sphingolipids profiles in fecal samples. However, how bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remains unclear.

Methods: We used dextran sodium sulfate (DSS)-induced colitis in mice monocolonized with Bacteroides fragilis strains expressing or lacking sphingolipids to assess the influence of bacterial sphingolipids on intestinal inflammation using transcriptional, protein, and cellular analyses. Colonic explant and organoid were used to study the function of bacterial sphingolipids. Host mucosal immune cells and cytokines were profiled and characterized using flow cytometry, enzyme-linked immunosorbent assay, and Western blot, and cytokine function in vivo was investigated by monoclonal antibody injection.

Results: B fragilis sphingolipids exacerbated intestinal inflammation. Mice monocolonized with B fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin (IL)-22 by ILC3. Mice colonized with B fragilis lacking sphingolipids following DSS treatment showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production. Protection against DSS colitis associated with B fragilis lacking sphingolipids was reversed on IL22 blockade. Furthermore, bacterial sphingolipids restricted epithelial IL18 production following DSS treatment and interfered with IL22 production by a subset of ILC3 cells expressing both IL18R and major histocompatibility complex class II.

Conclusions: B fragilis-derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL18 expression and concomitantly suppressing the production of IL22 by ILC3 cells.

Keywords: Colitis; IL22; ILC3 Cells; Microbiota; Sphingolipids.

Graphical abstract

Figure 1

B fragilis-derived sphingolipids do not disrupt host homeostasis but exacerbate DSS-induced colitis. (A) Body weight gaining postweaning of monocolonized mice (BFWT mice: n = 12; BFΔSPT: n = 10). (B) CFU of B fragilis in fecal samples from monocolonized mice (BFWT mice: n = 6; BFΔSPT: n = 6). (C) Colon length, (D) spleen weight, and (E) mesenteric lymph node (mLN) weight of 12-week-old monocolonized mice (BFWT mice: n = 5; BFΔSPT: n = 5). Monocolonized mice were exposed to 1.5% DSS in their drinking water for 14 days, and the following parameters were monitored: (F) DSS water intake, (G) body weight, (H) diarrhea scores, (I) rectal bleeding scores, and (J) overall survival rate. Additionally, (K) spleen weight and (L and M) colons length were assessed on Day 14. Subsequently, (N) hematoxylin & eosin (H&E)-stained colon sections were examined and (O) histopathology scores of the colon were assessed. Initially, both groups started with 12 mice each; however, because of the loss of 3 mice in the WT group, the final counts were BFWT mice: n = 9; BFΔSPT: n = 12. In a separated DSS experiment, colon samples were collected on Day 14 for immunofluorescence staining and flow cytometry analysis. (P) Representative immunofluorescence sections of the colon showing Ki67 (pink) and DAPI (blue) staining were analyzed. (Q) Quantification of Ki67⁺ cells number per crypt. (BFWT mice: n = 9; BFΔSPT: n = 12). (R) Representative flow cytometry plots and (S) quantification of colonic Ki67⁺ epithelial cells based on the flow cytometry results. IECs were gated on singlet/live/CD45-/EpCAM⁺ cells. (BFWT mice: n = 9; BFΔSPT: n = 12). The data presented are from 1 experiment but represent findings consistent across 2 independent experiments. Results are shown in box and whisker plots with individual data points. Statistical significance was determined using the nonparametric Mann-Whitney test (GraphPad Prism version 10.2.2), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.

Figure 2

B fragilis–derived sphingolipids suppress STAT3 in colonic epithelial cells. (A) Representative images of organoids and (B) quantification of the number of spheres formed, and (C) average sphere area of organoids treated with total lipids or purified sphingolipids derived from B fragilis. (D) Quantification of the percentage of Ki67⁺ cells in cells extracted from organoids treated with total lipids or purified sphingolipids from B fragilis. N = 4 in each treatment. The data presented are from 1 experiment but represent findings consistent across 3 independent experiments. (E) Volcano plot showing differentially expressed genes in colonic epithelial cells from monocolonized mice treated with 1.5% DSS in drinking water for 14 days. (F) Quantitative reverse transcription PCR (qRT-PCR) analysis of Reg3b, Reg3g, and Mmp13 expression in colonic epithelial cells from monocolonized mice treated with 1.5% DSS for 14 days. (n = 5 mice per group). (G) Immunoblot analysis and (H) corresponding quantification of phosphorylated and total STAT3 in colonic epithelial cells from monocolonized mice treated with 1.5% DSS in drinking water for 14 days (n = 6 mice per group). (I) Immunoblot analysis and (J) corresponding quantification of phosphorylated and total STAT3 in colonic epithelial cells from monocolonized mice treated with 1.5% DSS in drinking water for 14 days (n = 3 mice per group). Two groups of mice were orally gavaged with atovaquone (a STAT3 inhibitor) or PBS every other day. (K) Weight loss, (L) hematochezia scores, and (M) diarrhea scores were monitored during the 8-day DSS treatment in monocolonized mice that received atovaquone (n = 5 mice per group) or PBS gavage (n = 4 mice per group). The data presented are from 1 experiment but represent findings consistent across 2 independent experiments. Results are shown in box and whisker plots with individual data points. Statistical significance was determined using the nonparametric Mann-Whitney test (GraphPad Prism version 10.2.2), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.

Figure 3

B fragilis–derived sphingolipids suppress STAT3 by constraining the IL22 signal. (A) Representative Immunoblot phosphorylated and total STAT3 in mouse colonic organoids treated with total lipids extracted from WT or mutant B fragilis or purified B fragilis sphingolipids. (B and C) Concentrations of IL22, IL10, and IL6 were measured by ELISA in the medium of colon explant cultures from monocolonized mice challenged with 1.5% DSS in drinking water for 14 days. Protein extraction was performed from the colon explants after culture, and cytokine concentrations were normalized to protein weight (n = 8 individual mice per group). (D-J) Monocolonized mice were treated with 1.5% DSS in drinking water for 14 days and injected with either anti-IL22 or Isotype IgG. (D) Immunoblot analysis and (E) quantification of phosphorylated and total STAT3 in colonic epithelial cells from monocolonized mice treated with 1.5% DSS in drinking water for 14 days (n = 3 mice per group). (F) Weight loss, (G) diarrhea scores, and (H) hematochezia scores were recorded during the DSS treatment. (I) Colon length and (J) spleen weight were assessed on Day 14 (n = 3-4 individual mice per group). The data presented are from 1 experiment but represent findings consistent across 2 independent experiments. Results are shown in box and whisker plots with individual data points. Statistical significance was determined using the nonparametric Mann-Whitney test (GraphPad Prism version 10.2.2), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.

Figure 4

B fragilis–derived sphingolipids suppress colonic IL22 and IL18 activity by interacting with ILC3s and epithelial cells. Monocolonized mice were challenged with 1.5% DSS in drinking water for 7 days. (A) Representative flow cytometry plots and quantification of IL22⁺ ILC3 cells in the colon (n = 10 mice per group). (B) Representative flow cytometry plots and quantification of IL22⁺ NK cells in the colon (n = 5 mice per group). (C) Representative flow cytometry plots and quantification of IL22⁺ γδT cells in the colon (n = 5 mice per group). (D) Representative flow cytometry plots and quantification of IL22⁺ CD4+ T cells in the colon (n = 5 mice per group). (E) Concentrations of IL23, IL18, and IL1β were measured in the medium of colon explant cultures. Protein extraction was performed from the colon explants after culture, and cytokine concentrations were normalized to protein weight (n = 8 individual mice per group). (G-F) Immunoblot analysis and quantification of IL18 and inflammasome proteins in colonic epithelial cells (n = 6 mice per group). (H) Representative Immunoblot analysis of IL18 and inflammasome proteins in mouse colonic organoids treated with total lipids extracted from WT or mutant B fragilis or purified B fragilis sphingolipids. The data presented are from 1 experiment but represent findings consistent across 2 independent experiments. Results are shown in box and whisker plots with individual data points. Statistical significance was determined using the nonparametric Mann-Whitney test (GraphPad Prism version 10.2.2), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.

Figure 5

B fragilis–derived sphingolipids modulate ILC3-derived IL22 by targeting IL18R⁺MHCII⁺ILC3 cells. Monocolonized mice were challenged with 1.5% DSS in drinking water for 7 days. (A) Representative flow cytometry plots of IL18R⁺/IL18R^- ILC3 cells, and IL22⁺ cells in IL18R⁺/IL18R^- ILC3 cells in the colon. (B) Representative flow cytometry plots and quantification of IL18R⁺ cells in ILC3 cells in the colon. (C) Representative flow cytometry plots and quantification of IL22⁺ cells in IL18R⁺ ILC3 cells in the colon. (D) Representative flow cytometry plots of MHCII ⁺/MHCII^- ILC3 cells. (E) Representative flow cytometry plots and quantification of MHCII⁺ ILC3 cells. (F) Representative flow cytometry plots and quantification of IL22⁺ cells in MHCII⁺ ILC3 cells. (G) Representative flow cytometry plots of IL18R and MHC II expression in IL-22+ / IL-22- cells in ILC3 cells. (H) Representative flow cytometry plots and quantification of IL18R+ MHCII⁺ cells in ILC3 cells. (I) Representative flow cytometry plots and quantification of IL22⁺ cells in IL18R⁺MHCII⁺ ILC3 cells. ILC3 cells are gated on single/live/CD45⁺/Lin-/Thy1.2⁺/IL7R⁺/RORγt⁺ cells. n = 4–5 individual mice per group. The data presented are from 1 experiment but represent findings consistent across 2 independent experiments. Results are shown in box and whisker plots with individual data points. Statistical significance was determined using the nonparametric Mann-Whitney test (GraphPad Prism version 10.2.2), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.

Figure 6

B fragilis–derived sphingolipids suppress IL22 production in ILC3 cells. (A) Representative flow cytometry plots and quantification of IL22⁺ ILC3 cells after purified B fragilis sphingolipids treatment and IL23 and IL18 stimulation. (B) Representative flow cytometry plots and quantification of IFNγ⁺ ILC3 cells after purified B fragilis sphingolipids treatment and IL23 and IL18 stimulation. (C) Representative flow cytometry plots and quantification of IL17A⁺ ILC3 cells after purified B fragilis sphingolipids treatment and IL23 and IL18 stimulation (D) Representative flow cytometry plots and (E) MFI (Mean Fluorescence Intensity) quantification of phosphorylated p38 MAPK (Thr180, Tyr182), phosphorylated AKT-1 (Ser473), phosphorylated ERK1/2 (Thr202, Tyr204), and phosphorylated STAT3 (Tyr705) in ILC3 cells after purified B fragilis sphingolipids treatment and IL23 and IL18 stimulation. ILC3 cells sorted from the colon and mLN of germ-free mice were treated with purified sphingolipids of WT B fragilis overnight. Cells were then stimulated by IL23 and IL18 to induce IL22 production. ILC3 cells were sorted based on single/live/CD45⁺/Lin^-/Thy1.2⁺/NK1.1^-/KLRG1^- cells. n = 6 individual wells of 24-well-plate per group. The data presented are from 1 experiment but represent findings consistent across 3 independent experiments. Results are shown in box and whisker plots with individual data points. Statistical significance was determined using the nonparametric Mann-Whitney test (GraphPad Prism version 10.2.2), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.

Figure 7

B fragilis–derived sphingolipids aggravate C rodentium infection. (A) Schematic representation of the experimental procedures. (B) Weight loss and (C) fecal C rodentium CFU were examined postinfection. (D) Spleen weight was assessed on Day 9 (n = 5 individual mice per group). (E) Representative flow cytometry plots and (F) quantification of IL22+ ILC3 cells in the colon of monocolonized mice 9 days post–C rodentium infection. ILC3 cells were gated on single/live/CD45⁺/Lin^-/Thy1.2⁺/IL7R⁺/RORgt⁺ cells. (G) Immunoblot analysis and (H) corresponding quantification of phosphorylated and total STAT3 in colonic epithelial cells from mono-colonized mice 9 days post–C rodentium infection (n = 5 mice per group). (I) Schematic representation of the experimental procedures with anti-IL22 application. (J) Weight loss and (K) fecal C rodentium CFU were examined postinfection. (L) Spleen weight and (M and N) colon length were assessed on Day 9. (n = 4 individual mice per group). The data presented are from 1 experiment but represent findings consistent across 2 independent experiments. Results are shown in box and whisker plots with individual data points. Statistical significance was determined using the nonparametric Mann-Whitney test (GraphPad Prism version 10.2.2), ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.