Microbial sensing by goblet cells controls immune surveillance of luminal antigens in the colon

Abstract

The delivery of luminal substances across the intestinal epithelium to the immune system is a critical event in immune surveillance, resulting in tolerance to dietary antigens and immunity to pathogens. How this process is regulated is largely unknown. Recently goblet cell-associated antigen passages (GAPs) were identified as a pathway delivering luminal antigens to underlying lamina propria (LP) dendritic cells in the steady state. Here, we demonstrate that goblet cells (GCs) form GAPs in response to acetylcholine (ACh) acting on muscarinic ACh receptor 4. GAP formation in the small intestine was regulated at the level of ACh production, as GCs rapidly formed GAPs in response to ACh analogs. In contrast, colonic GAP formation was regulated at the level of GC responsiveness to ACh. Myd88-dependent microbial sensing by colonic GCs inhibited the ability of colonic GCs to respond to Ach to form GAPs and deliver luminal antigens to colonic LP-antigen-presenting cells (APCs). Disruption of GC microbial sensing in the setting of an intact gut microbiota opened colonic GAPs, and resulted in recruitment of neutrophils and APCs and production of inflammatory cytokines. Thus GC intrinsic sensing of the microbiota has a critical role regulating the exposure of the colonic immune system to luminal substances.

Figure 1. GAP formation is driven by mAChR₄ signaling in the SI at the basal state and in the colon following treatment with antibiotics

a) Intravital two-photon (2P) images of the SI following the luminal administration of dextran (green) and DAPI (blue) and b) number of GAPs per villus from mice treated with carbamylcholine (CCh; a pan-nicotinic pan-muscarinic receptor agonist), atropine (Atr; a pan-muscarinic receptor antagonist), tropicamide (Trop; a mAChR₄ antagonist), piperidine (Pip; a mAChR₃ antagonist), or cholera toxin (CT; a GC secretagogue, in b only). c) Quantitative real time RNA for the genes encoding mAChR₃ and mAChR₄ (Chrm3 and Chrm4 respectively) from FACS sorted SI and colonic goblet cells. d) 2P images of the SI and colon following the administration of luminal dextran (red) and DAPI (blue) in SPF-housed mice untreated or treated with antibiotics (Abx). e) 2P images of the colon of Math1^iΔvil mice or littermate controls treated with tamoxifen and antibiotics following the administration of luminal dextran (green) and DAPI (blue). f) 2P and fluorescent IHC images of the colon following the administration of luminal dextran (green) and DAPI (blue) in dtom^iMath1 mice treated with RU486 and antibiotics demonstrating dextran (green) colocalizing with Math1 expressing epithelial cells with GC morphology (red). g and h) 2P images of the colon and i) number of GAPs per crypt from untreated SPF housed mice and antibiotic treated SPF housed mice given vehicle, cholinergic agonists, non-specific or specific mAChR antagonists, or cholera toxin. White arrows denote GAPs, yellow arrows denote the colonic crypt lumen. Data is presented as the mean +/− SEM, * = p<0.05, ND = not detected, ns = not significant, n = 3 or more mice for each condition in panels a–d, and g–i, n = 2 in panels e and f, scale bar = 50µm, unless otherwise noted.

Figure 2. GC intrinsic Myd88-dependent microbial sensing inhibits GAPs in the colon, but not the SI

a–c) Number of GAPs per colonic crypt or SI villus from SPF housed mice, mice treated with antibiotics, germfree (GF) mice, MyD88^−/− mice, and Myd88^iΔMath1 mice treated with RU486 given a) tropicamide (Trop), b) carbamylcholine (CCh), or c) luminal heat killed cecal contents from an SPF housed mouse (CC). d) Quantitative real time PCR for the mRNA expression of TLRs, Myd88, and the epidermal growth factor receptor (EGFR) from FACS sorted SI or colonic goblet cells isolated from SPF housed mice. e) 2P images from Myd88^iΔMath1 mice or Myd88^fl/fl littermates treated with RU486. Data is presented as the mean +/− SEM, * = p<0.05, ND = not detected, ns = not significant, n = 4 or more mice for each condition in panels a–d, panel e is representative of one of three mice from each condition, scale bar = 50µm.

Figure 3. GC intrinsic microbial sensing activates the EGFR, p42/p44 mitogen activated protein kinase (MAPK), and suppresses mAChR₄ dependent GAP formation

a) Immunofluorescence staining for phosphorylated EGFR (pEGFR; red), phosphorylated p42/p44 MAPK (pMAPK; red), and cytokeratin 18 (cyt18; green) in the SI and colon of SPF housed mice. White arrow denotes GCs enlarged in inserts with individual channels. b) ELISAs for pMAPK and pEGFR on isolated SI and colonic epithelium from untreated mice (UT), mice treated with an EGFR inhibitor (EGFRi), Myd88^−/− mice, and GF mice. c) Immunofluorescence for cyt18, pEGFR and pMAPK in the colon of Myd88^iΔMath1 mice and littermate controls treated with RU486. White arrow indicates the location of a cyt18+ cell in each panel. d) Number of colonic GAPs per crypt in mice treated with EGFRi or MAPKi and antagonists for mAChR₃ and mAChR₄. e) Number of colonic GAPs per crypt from SPF housed mice with or without antibiotic treatment, GF mice, MyD88^−/− mice, and Myd88^iΔMath1 mice treated with RU486 and given vehicle or luminal EGF. f) Number of SI GAPs per villus in SPF housed mice treated with EGF and MAPKi. g) Immunofluorescence for cyt18 (green), pEGFR (red), and pMAPK (red) in the colon of GF mice following intraluminal vehicle (left panel), LPS (middle panel), or LPS and i.p. EGFRi treatment (right panel). White arrows denotes GCs enlarged in insets with individual channels. h) Number of GAPs per crypt from the colon of GF mice treated with LPS with and without EGFRi treatment. i) Number of GAPs per crypt from the colon of antibiotic treated mice given EGF with and without MAPKi treatment. Data is presented as the mean +/−SEM, * = p<0.05, ND = not detected, ns = not significant, n = 3 or more mice for each condition in panels a, b, and d–i, panel c representative of one of two mice for each condition, scale bar = 20µm in panels a and g; scale bar = 5 µm in panel c.

Figure 4. Antigen Presenting Cells (APCs) are recruited to the colonic epithelium and acquire luminal antigen in a mAChR₄ dependent manner when GAPs are present

a) 2P images of the colon 20µm below the surface following the administration of luminal dextran (red) and DAPI (blue) in SPF housed CD11c^YFP reporter (green) mice treated 24 hours earlier with vehicle (left panel), MAPKi (middle panel) or EGFRi (right panel) b) 3D reconstruction of Z-stacks from the treatment groups with red channel removed to better identify CD11c^YFP+ cells (major ticks = 20µm). c) The number of CD11c^YFP+ cells in each group in a volume of 250µm(x) × 220µm(y) × 20µm(z) obtained from the colonic epithelial surface extending into the LP in the z-plane. d) Number of CD11c⁺MHCII⁺ LP cells and e) CD103+ and CD103- CD11c⁺MHCII⁺ LP cells in the colon and SI 24 hours following treatment with vehicle or EGFRi as assessed by flow cytometry. f and g) Luminal antigen acquisition and presentation capacity of colonic CD103⁺ and CD103⁻ LP-APCs isolated from f) vehicle treated, MAPKi treated, or EGFRi treated SPF housed mice or EGFRi treated GC depleted Math1^iΔvil mice or isolated from g) SPF housed mice treated with EGFRi and mAChR₄ antagonists following the administration of luminal PBS or Ova. Luminal antigen acquisition and presentation capacity was assessed by the expansion of Ova specific OTI T cells following 72 hours of culture. Data is presented as the mean +/− SEM, * = p<0.05, ND = not detected, ns = not significant, n = 3 or more mice for each condition in panels a–e, data in panels f and g are representative of one of three replicates generated by pooling LP-APCs from two mice in each group, scale bar = 50µm.

Figure 5. Overriding GC intrinsic microbial sensing and opening colonic GAPs results in an influx of leukocytes and inflammatory cytokine production in response to a non-pathogenic commensal microbiota

a) Representative flow plots of CD45+CD11c+MHCII+ cells in Myd88^iΔMath1 mice and Myd88^fl/fl littermates treated with RU486. b and c) Number of CD45+CD11c+MHCII+ LP cells as assessed by flow cytometry in b) Myd88^−/− mice, and Myd88^iΔMath1 mice and Myd88^fl/fl littermates treated with RU486 or c) SPF housed C57BL/6 mice treated with or without antibiotics and MAPKi or EGFRi. d) Luminal antigen acquisition and presentation capacity of colonic CD103⁺ and CD103⁻ LP-APCs isolated from Myd88^iΔMath1 mice and Myd88^fl/fl littermates treated with RU486 following luminal PBS or Ova as assessed by the expansion of Ova specific OTI T cells following 72 hours of culture. e) IL-6 (left panel) and IL-17 (right panel) levels in the serum and culture supernatants from mesenteric lymph node (MLN) or colonic LP cells and f) CXCL1 levels in SI or colon tissue homogenates from Myd88^iΔMath1 mice and Myd88^fl/fl littermates treated with RU486 for four days. g) Representative flow plots, h) number of CD11b⁺Ly6G⁺ cells in colon, and i) immunofluorescence images Ly6G+ cells in the colon of Myd88^iΔMath1 mice and Myd88^fl/fl littermates treated with RU486 for four days. Data is presented as the mean +/− SEM, * = p<0.05, ND = not detected, ns = not significant, n = 3 or more mice for each condition. scale bar = 50µm.