Mucus enhances gut homeostasis and oral tolerance by delivering immunoregulatory signals

Abstract

A dense mucus layer in the large intestine prevents inflammation by shielding the underlying epithelium from luminal bacteria and food antigens. This mucus barrier is organized around the hyperglycosylated mucin MUC2. Here we show that the small intestine has a porous mucus layer, which permitted the uptake of MUC2 by antigen-sampling dendritic cells (DCs). Glycans associated with MUC2 imprinted DCs with anti-inflammatory properties by assembling a galectin-3-Dectin-1-FcγRIIB receptor complex that activated β-catenin. This transcription factor interfered with DC expression of inflammatory but not tolerogenic cytokines by inhibiting gene transcription through nuclear factor κB. MUC2 induced additional conditioning signals in intestinal epithelial cells. Thus, mucus does not merely form a nonspecific physical barrier, but also constrains the immunogenicity of gut antigens by delivering tolerogenic signals.

Fig. 1. MUC2 imprints DCs with tolerogenic properties

(A) Confocal microscopy of human SI-LP stained for CD11c, MUC2, CD103 and DNA-capturing 4′-6- diamidino-2-phenylindole (DAPI). Boxes and arrowheads: MUC2⁺CD103⁺ DCs. Original magnification, ×63. (B) Enzyme-linked immunosorbent assay (ELISA) of IL-12p70, IL-10 and TGF-β1 and flow cytometry (FC) of active RALDH in human DCs cultured for 2 days with or without LPS and/or MUC2. (C–E) FC of IFN-γ, Foxp3, CD4 and CFSE in human naïve CD4⁺ T cells cultured for 4 days with allogeneic DCs stimulated with or without LPS and/or MUC2 for 2 or 5 days in the absence or presence of control (ctr) IgG Ab, ctr vehicle, neutralizing Abs to TGF-β1 or IL-10 or LE540. (F) FC of CD103 and CX3CR1 on DCs cultured for 2 days with or without LPS and/or MUC2. Data summarize 3 experiments (error bars, s.d.; unpaired t test, *P <0.05) or show one of 4 experiments with similar results.

Fig. 2. MUC2 delivers anti-inflammatory signals to gut DCs

(A) ELISA of TNF and IL-10 from mouse SI-LP DCs cultured for 2 days with or without flagellin and/or MUC2. (B) Light microscopy of Alcian blue-stained mucin and fluorescence in situ hybridization (FISH) of bacterial 16S ribosomal RNA in DAPI-stained SI-LP from WT and Muc2⁻/⁻ mice. Original magnification, ×10. (C–E) Quantitative real-time polymerase chain reaction (qRT-PCR) of mRNAs for TNF, IL-12p35 (Il12a), IL-12p40 (Il12b), IL-10, RALDH1 (Aldh1a1) and TGF-β1 in SI-LP DCs and FC of Foxp3 and CD4 on SI-LP T cells from WT and Muc2⁻/⁻ mice before and after oral antibiotics. RE, relative expression compared to Gapdh encoding glyceraldehyde 3-phosphate dehydrogenase. (F) ELISA of proliferation-induced bromodeoxyuridine (BrdU) and IFN-γ from OT-II cells activated for 5 days by OVA-pulsed SI-LP DCs from WT and Muc2⁻/⁻ mice with or without MUC2. Data summarize 2 experiments with ≥3 mice/group (error bars, s.d.; unpaired Student’s t test, *P <0.05) or show one of 4 experiments with similar results.

Fig. 3. MUC2 enhances gut homeostasis and oral tolerance

(A) FC of CD103 and qRT-PCR of Il12a, Il12b, Il10, Aldh1a1 and Tgfb1 in SI-LP CD103⁺ DCs from WT or Muc2⁻/⁻ mice gavaged for 5 days with phosphate buffer solution (PBS) or MUC2. RE, relative expression compared to Gapdh. (B) FC of Foxp3, IFN-γ, IL-17 and CD4 in SI-LP T cells from WT or Muc2⁻/⁻ mice treated as in (A). (C) FC of Foxp3 and CD4 in naïve OT-II cells cultured for 5 days with SI-LP CD103⁺ DCs from WT or Muc2⁻/⁻ mice treated as in (A) and intragastrically immunized with OVA. CD4⁺CD25⁺ OT-II cells from these cultures were incubated for 5 days with CFSE-labeled naïve OT-II cells and Abs to CD3 and CD28; divided CFSElow cells were quantified by FC. (D) ELISA of fecal OVA-specific IgG from WT and Muc2⁻/⁻ mice tolerized with PBS, OVA or OVA plus MUC2 for 5 days and immunized as in (C). (E) ELISA of IFN-γ from OT-II cells incubated for 5 days with MLN CD103⁺ DCs from WT or Muc2⁻/⁻ mice tolerized and immunized as in (D). (F) OVA-induced DTH in WT or Muc2⁻/⁻ mice tolerized as in (C) and subcutaneously immunized with OVA. (G) ELISA of proliferation-induced BrdU from SPL CD4⁺ T cells activated for 5 days with OVA-pulsed SPL DCs from WT or Muc2⁻/⁻ mice tolerized and immunized as in (F). (H–J) DTH, OVA-specific serum IgG and IgE, and SPL CD4⁺ T cell proliferation and IFN-γ secretion in WT or Muc2⁻/⁻ mice immunized and tolerized as in (F) after oral antibiotics. Data summarize 2 experiments with ≥4 mice/group (error bars, s.d.; unpaired Student’s t test, *P <0.05) or show one of 4 experiments with similar results.

Fig. 4. MUC2 binds galectin-3, dectin-1 and FcγRIIB on DCs

(A) FC of CFSE-MUC2 on human DCs pre-incubated with unlabeled native MUC2, deglycosylated (dgl) MUC2, MUC2 peptide, mannan or lactose. % of MUC2 binding compared to medium alone. (B) ELISA of native or dgl MUC2 interaction with galectins. (C) CFSE-MUC2 or CFSE-dgl MUC2 binding to DCs pre-incubated with PBS, human serum albumin (HSA) or galectin-3. (D) CFSE-MUC2 binding to DCs before and after pre-incubation with a fluorescent Ab to galectin-3. (E) IFA of CD11c, galectin-3, Muc2 and DAPI in mouse PP sections. Original magnification ×5 (upper panel) and °—63 (bottom panel). (F) qRT-PCR of mRNA for galectin-3 in DC subsets from mouse PPs, SI-LP and SPL. RE, relative expression compared to Gapdh. (G) Immunoprecipitation (IP) with control or anti-galectin-3 Abs of proteins from human DCs treated without (ctr) or with MUC2 for 30 min, followed by immunoblotting of FcγRIIB, Dectin-1 and galectin-3. (H) ELISA of IL-12p70 from human DCs exposed to scrambled (ctr) or LGALS3 (galectin-3), FCGR2B (FcγRIIB) or CLEC7A (Dectin-1) small interfering RNAs (siRNAs) and cultured with or without LPS and/or MUC2 for 2 days. (I) Binding of CFSE-MUC2 to SI-LP DCs from WT, Lgals3⁻/⁻, Clec7A⁻/⁻ or Fcgr2b⁻/⁻ mice. Data summarize experiments with 3 donors or 3 mice from each strain (error bars, s.d.; unpaired t test, *P <0.05) or show one of 3 experiments with similar results.

Fig. 5. Galectin-3, Dectin-1 and FcγRIIB enhance gut homeostasis and oral tolerance

(A) FC of galectin-3, FcγRIIB and Dectin-1 on SI-LP DCs from WT mice. MFI, mean fluorescence intensity. (B) Quantification of MUC2⁺ SI-LP DCs from WT, Lgals3⁻/⁻, Clec7A⁻/⁻ or Fcgr2b⁻/⁻ mice by IFA of 10–12 SI-LP sections/group. (C) qRT-PCR of Muc2 in IECs and SI-LP DCs from WT mice. RE, relative expression compared to Gapdh. (D and E) qRT-PCR of Il12a, Il12b, Aldh1a1 and Tgfb1 in SI-LP DCs and FC of Foxp3 and IFN-γ in SI-LP CD4⁺ T cells from WT, Lgals3⁻/⁻, Clec7A⁻/⁻ or Fcgr2b⁻/⁻ mice. RE, relative expression compared to Gapdh. (F) DTH and ELISA of OVA-specific IgE in WT, Lgals3⁻/⁻, Clec7A⁻/⁻ or Fcgr2b⁻/⁻ mice intragastrically tolerized with PBS or OVA for 5 days and subcutaneously immunized with OVA. (G) ELISA of proliferation-induced BrdU from SPL CD4⁺ T cells activated for 5 days by OVA-pulsed SPL DCs from WT, Lgals3⁻/⁻, Clec7A⁻/⁻ or Fcgr2b⁻/⁻ mice tolerized and immunized as in (F). Data summarize 2 experiments with ≥4 mice/group (error bars, s.d.; unpaired Student’s t test, *P <0.05) or show one of 4 experiments with similar results.

Fig. 6. MUC2 impairs NF-κB-driven inflammatory signals via β-catenin

(A) WB of cytoplasmic or nuclear phospho (p)-AKT, AKT, pGSK-3β, GSK-3β, β-catenin (β-cat), dephospho (dp)-β-cat, actin and octamer-1 (Oct-1) from human DCs cultured with or without LPS and/or MUC2 for 10 min. (B) Electrophoretic mobility gel shift assay of IL12A-bound NF-κB p65-p50 and consensus DNA-bound Oct-1 in DCs cultured as in (A) for 3 hours. (C) Chromatin IP (ChIP) of IL12A-bound NF-κB p65 in DCs cultured as in (A) for 3 hours. RDQ, relative DNA quantity. (D) IP with control (ctr) IgG Ab or anti-dp-β-cat Ab of nuclear proteins from DCs cultured with or without LPS and/or MUC2 for 10 min, followed by WB of NF-κB p65 and dp-β-cat. (E) IL12A transcription in DCs cultured as in (A) for 2 days. (F and G) NF-κB-mediated transcription and ELISA of IL-12p70 and IL-10 in DCs cultured as in (E) in the presence of scrambled (ctr) or CTNNB1 (β-cat) siRNAs. (H) IFA of pAKT, pGSK-3β, β-cat, galectin-3, CD11c and DAPI in mouse PPs. Original magnification, ×5. Data show one of 3 experiments yielding similar results or summarize 3 experiments (error bars, s.d.; unpaired t test, *P <0.05).