Microbiota Sensing by Mincle-Syk Axis in Dendritic Cells Regulates Interleukin-17 and -22 Production and Promotes Intestinal Barrier Integrity

Abstract

Production of interleukin-17 (IL-17) and IL-22 by T helper 17 (Th17) cells and group 3 innate lymphoid cells (ILC3s) in response to the gut microbiota ensures maintenance of intestinal barrier function. Here, we examined the mechanisms whereby the immune system detects microbiota in the steady state. A Syk-kinase-coupled signaling pathway in dendritic cells (DCs) was critical for commensal-dependent production of IL-17 and IL-22 by CD4⁺ T cells. The Syk-coupled C-type lectin receptor Mincle detected mucosal-resident commensals in the Peyer's patches (PPs), triggered IL-6 and IL-23p19 expression, and thereby regulated function of intestinal Th17- and IL-17-secreting ILCs. Mice deficient in Mincle or with selective depletion of Syk in CD11c⁺ cells had impaired production of intestinal RegIIIγ and IgA and increased systemic translocation of gut microbiota. Consequently, Mincle deficiency led to liver inflammation and deregulated lipid metabolism. Thus, sensing of commensals by Mincle and Syk signaling in CD11c⁺ cells reinforces intestinal immune barrier and promotes host-microbiota mutualism, preventing systemic inflammation.

Keywords: IL-17; IL-22; Mincle; Syk kinase; T lymphocyte; antimicrobial defense; dendritic cell; gut microbiota translocation; innate lymphoid cells; intestinal barrier; lipid metabolism; liver inflammation.

Graphical abstract

Figure 1

Mincle and Syk Signaling in DCs Control Microbiota-Driven Th17 Differentiation (A–C) Naive OT-II T cells were co-cultured with GM-BMs (1:2 ratio) from: (A) WT mice or mice lacking MyD88 (Myd88^−/−) or Syk in the CD11c⁺ compartment (CD11cΔSyk); (B) WT mice or mice lacking Clec7a or mice lacking Fcer1g; (C) WT mice or mice lacking Clec4n or Clec4e, the indicated genotypes loaded with OVA peptide in the presence or absence of microbiota (10:1 GM-BM ratio), and IL-17 was measured by ELISA in the supernatant 3 days later. (D–G) Naive OT-II T cells were co-cultured with GM-Macs or GM-DCs (1:1 ratio) loaded with OVA peptide in the presence or absence of microbiota (10:1 DC ratio) , and IL-17 was measured by ELISA in the supernatant 3 days later (D). IL-17 (E), IL-22 (F), and IFN-γ (G) production after re-stimulation was measured by intracellular staining and flow cytometry in OT-II T cells from the co-cultures. To the left is a representative plot, and to the right quantification is shown. Individual data represent data generated from independent GM-BM cultures (biological replicates) in pools of at least two independent experiments. Individual data and arithmetic mean are shown. ^∗∗∗p < 0.001 (one-way ANOVA and Bonferroni post hoc test). See also Figure S1.

Figure 2

Mucosa-Associated Commensals Are Sensed by PP DCs Expressing Mincle (A) Representative plots (left) and graph depicting the frequency of SPF microbiota stained with control-hFc or Mincle-hFc. Shown is the arithmetic mean + SEM of a pool of three replicates from two independent experiments. (B) Analysis by stimulated emission depletion super-resolution microscopy of SPF-mouse mucosa-associated commensals labeled with control-hFc or Mincle-hFc. Scale bar, 2 μm. (C) Frequency of SPF-mouse luminal and mucosal microbiota stained with control-hFc or Mincle-hFc by flow cytometry. (D) Luminal microbiota was stained as in (A), sorted into Mincle-hFc-enriched (Mincle-hFc⁺), Mincle-hFc-depleted (Mincle-hFc⁻), and control-hFc-enriched (control-hFc⁺) fractions, and analyzed by 16S sequencing. Shown on the left is the relative abundance of each genus from two independent experiments. To the right are the enrichment index and specificity index, calculated as explained in the STAR Methods. (E) Mucosa-associated commensals from WT SPF mice gavaged with Celltrace-violet-labeled Lactobacillus plantarum (L. plantarum) or in-vitro-grown L. plantarum were stained with control-hFc or Mincle-hFc and analyzed by flow cytometry. Shown on the left is representative staining. Shown on the right is the frequency of bacteria positive for the indicated staining and pre-gated on cell-violet-positive bacteria. (F) Mincle expression in PPs from Mincle-deficient (Clec4e^−/−) mice and WT littermates analyzed by qPCR and normalized to Gapdh. (G) Mincle expression in the indicated genotypes by flow cytometry in PPs. On the left are representative plots. On the right, mean fluorescence intensity (MFI) is shown. (H) Mincle expression by flow cytometry in the myeloid populations from WT mice PPs. On the left is a representative plot. On the right, the frequency of Mincle⁺ cells in the depicted subset is shown. (I) Frequency of Mincle⁺ cells in the myeloid subsets from WT small intestine LP. (J) Mincle expression in LP mononuclear cells from three colonic and three duodenal samples from healthy individuals by flow cytometry. On the left, the gating strategy is shown. In the middle, Mincle versus fluorescence minus one (FMO) expression in the CD11c⁻HLA-DR⁻ and CD11c⁺HLA-DR⁺ populations is shown. Shown on the right is the MFI of Mincle expression or the FMO in six independent samples. Data represent one representative experiment of two performed (B, C, E, F, and J) or were pooled from at least two independent experiments (G, H, and I). ^∗p < 0.05, ^∗∗p < 0.01 (unpaired two-tailed Student’s t test). See also Figure S2.

Figure 3

DCs from PPs Instruct Mincle- and Syk-Dependent Th17 Differentiation (A–C) Naive OT-II T cells were co-cultured for 3 days with dome CD11b⁺ DCs, CD8α⁺ DCs, LysoMacs, or LysoDCs from the indicated genotypes, sorted from PPs (1:1 ratio), and loaded with OVA_323–339 peptide. (A) IL-17 secretion by ELISA. Each dot represents an independent co-culture where myeloid cells were from different mice from two independent experiments. (B and C) Representative FACS plots of IL-17 (B) and IL-22 (C) intracellular staining after OTII re-stimulation. (D) Il6, Il23a, Tgfb, and Il12b transcripts in PPs of the indicated genotypes by qPCR; they were normalized to Gapdh. (E and F) Analysis of IL-6 (E) and IL-12p40 (F) intracellular staining in CD11c⁺MHC-II⁺CD19⁻ cells from PPs in the indicated genotypes. Shown on the left are representative histograms. On the right is MFI of staining. (G) ELISA of IL-6 and IL-23 production by sorted GM-DCs from WT and Mincle-deficient (Clec4e^−/−) mice untreated (medium) or stimulated with gut microbiota (10:1 DC ratio) for 12 h. Data represent two independent pooled experiments (A) or one representative experiment of at least two performed (B–G). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (A: one-way ANOVA and Bonferroni post hoc test; D, E, and G: unpaired two-tailed Student’s t test). See also Figure S3.

Figure 4

Mincle and Syk in DCs Are Needed for Intestinal IL-17 and IL-22 Steady-State Production (A–E) Representative plots and summary graph of IL-17 and IL-22 (A and B), IFN-γ (C and D), and IL-17, IFN-γ, and IL-10 (E) production by intracellular staining after phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation in CD4⁺ T cells from PPs in the indicated genotypes. (F and G) Frequency of CCR6⁺ ILCs from PPs producing IL-22 and IL-17 in the steady state by intracellular staining in the indicated genotypes. At least two independent experiments were pooled. Each symbol represents an individual mouse. The arithmetic mean for each group is indicated. ^∗∗p < 0.01, ^∗∗∗p < 0.001 (unpaired two-tailed Student’s t test). See also Figure S4.

Figure 5

Commensal Bacteria Are Required for Mincle-Dependent Th17 Generation in PPs (A and B) Frequency of IL-17 production by intracellular staining in re-stimulated CD4⁺ T cells from PPs of the indicated genotypes after treatment with an antibiotic cocktail (ABX) containing ampicillin, neomycin, metronidazole, and vancomycin (A) or vancomycin only (B) in the drinking water over 4 weeks. (C) Frequency of IL-17 and IL-22 production by intracellular staining of re-stimulated OT-II T cells co-cultured with sorted WT and Mincle-deficient (Clec4e^−/−) GM-DCs loaded with OVA_323–339 peptide, as in Figures 1D and 1E; the cells were pulsed or not (medium) with mucosal-associated commensals from WT mice treated with ABX during gestation and lactation and gavaged with L. plantarum at weaning as indicated (L. plantarum-enriched mucus; 10:1 DC ratio). (D) ELISA of IL-6 and IL-23 from the co-culture of GM-DC in (C). (E and F) WT littermates and Mincle-deficient (Clec4e^−/−) mice were treated with ABX during gestation and lactation and were gavaged with L. plantarum (1 × 10⁶) at weaning (+L. plantarum gavage) or not (WT), as indicated in the figure. The frequency of IL-17- and IL-22-producing re-stimulated CD4⁺ T cells (E) or CCR6⁺ ILCs (F) from PPs by intracellular staining in the indicated genotypes and conditions is shown. Data represent one representative experiment of two performed (A and F) or were pooled from at least two independent experiments (B–E). Each symbol represents an individual mouse. The arithmetic mean for each group is indicated. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (A–E: one-way ANOVA and Bonferroni post hoc test; F: unpaired two-tailed Student’s t test). See also Figure S5.

Figure 6

The Mincle-Syk Axis Contributes to Intestinal Barrier Function (A and B) Reg3g transcripts were measured by qPCR and normalized to Gapdh in the jejunum of the indicated genotypes during the steady state (A) or after L. plantarum gavage, as described in the scheme (B). (C) Quantification of total IgA measured by ELISA in the intestinal lumen of the indicated genotypes. (D) Frequency of IgA⁺ bacteria by flow cytometry in the indicated genotypes. (E and F) Representative plots and summary graphs of CD45⁺B220⁻IgA⁺ (E) or IgG⁺ (F) plasmatic cells from the small intestine LP of Mincle-deficient (Clec4e^−/−) mice and WT littermate controls. (G) Representative plots and summary graph of CD4⁺PD-1^high T cells from PPs of the indicated genotypes. Data were pooled from at least two independent experiments. Individual mice and the arithmetic mean of each group are indicated. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (A and B: one-way ANOVA and Bonferroni post hoc test; C–H: unpaired two-tailed Student’s t test). See also Figure S6.

Figure 7

Mincle-Syk Pathway Promotes Commensal Bacteria Containment (A) Bacterial translocation into the liver of Mincle-deficient (Clec4e^−/−) mice and WT littermates. On the left is the bacterial load as colony-forming units (CFUs) per organ, indicating the limit of detection (LOD). On the right, the frequencies of the mice of each genotype show more than 40 CFUs per organ (2× LOD). (B) 16S rRNA sequencing analysis of LB-grown commensals from the liver (left) or bacterial DNA directly extracted from the liver (right) in the indicated genotypes. The graph shows the percentage of total reads corresponding to each phylum. Each bar represents four pooled LB plates per mouse and four mice per sample of each genotype (LB-grown commensals) or six pooled mice per genotype (commensals from the liver). (C) ELISA of serum IgG against intestinal bacteria in the indicated genotypes. (D) Total numbers of CD45⁺CD11b^high cells infiltrated in the liver of the indicated genotypes in the steady state or after administration of an antibiotic cocktail (ABX). (E) Total (left) or direct (right) bilirubin in the indicated genotypes. (F) Acc, Fas, Scd1, Srebpc, G6pase, Pepck, Cpt1a, and Ppara transcripts from the liver of 15 mice of the indicated genotypes were analyzed by qPCR and normalized to Gapdh in three independent experiments; the graph shows fold induction compared with the WT. (G and H) Individual lipid species of diacylglycerides (DAGs) (G) and the margaric (C17:0) and linoleic (C18:2) acid classes of free fatty acids (H) measured by a liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) metabolomics-based profiling approach. Data are presented as the metabolites’ abundance. Data were pooled from at least two independent experiments. Individual mice and the arithmetic mean of each group are shown (A, C–E, G, and H). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (A–C and E–H [margaric acid]: unpaired two-tailed Student’s t test; H [linoleic acid]: Mann-Whitney U test; D: one-way ANOVA and Bonferroni post hoc test). See also Figure S7.