The intestinal immune system is exposed to a mixture of foreign antigens from diet, commensal flora, and potential pathogens. Understanding how pathogen-specific immunity is elicited while avoiding inappropriate responses to the background of innocuous antigens is essential for understanding and treating intestinal infections and inflammatory diseases. The ingestion of protein antigen can induce oral tolerance, which is mediated in part by a subset of intestinal dendritic cells (DCs) that promote the development of regulatory T cells1. The lamina propria (LP) underlies the expansive single cell absorptive villous epithelium and contains a large population of DCs (CD11c+ CD11b+ MHCII+ cells) comprised of two predominant subsets; CD103+ CX3CR1− DCs, which promote IgA production, imprint gut homing on lymphocytes, and induce the development of regulatory T cells2–9, and CD103− CX3CR1+ DCs (with features of macrophages), which promote TNFα production, colitis, and the development of Th17 T cells5–7,10. However the mechanisms by which different intestinal LP-DC subsets capture luminal antigens in vivo remains largely unexplored. Using a minimally disruptive in vivo imaging approach we show that in the steady-state, small intestine goblet cells (GCs) function as passages delivering low molecular weight soluble antigens from the intestinal lumen to underlying CD103+ LP-DCs. The preferential delivery of antigens to DCs with tolerogenic properties implies a key role for this GC function in intestinal immune homeostasis.

Microfold cells (M cells) are specialized epithelial cells situated over Peyer’s patches (PP) and other organized mucosal lymphoid tissues that transport commensal bacteria and other particulate Ags into intraepithelial pockets accessed by APCs. The TNF superfamily member receptor activator of NF-κB ligand (RANKL) is selectively expressed by subepithelial stromal cells in PP domes. We found that RANKL null mice have <2% of wild-type levels of PP M cells and markedly diminished uptake of 200 nm diameter fluorescent beads. Ab-mediated neutralization of RANKL in adult wild-type mice also eliminated most PP M cells. The M cell deficit in RANKL null mice was corrected by systemic administration of exogenous RANKL. Treatment with RANKL also induced the differentiation of villous M cells on all small intestinal villi with the capacity for avid uptake of Salmonella and Yersinia organisms and fluorescent beads. The RANK receptor for RANKL is expressed by epithelial cells throughout the small intestine. We conclude that availability of RANKL is the critical factor controlling the differentiation of M cells from RANK-expressing intestinal epithelial precursor cells.

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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 passages (GAPs) were identified as a pathway delivering luminal antigens to underlying lamina propria (LP) dendritic cells (DCs) in the steady state. Here we demonstrate that goblet cells (GCs) form GAPs in response to acetycholine (ACh) acting on muscarinic acetylcholine receptor (mAChR) 4. GAP formation in the small intestine (SI) was regulated at the level of ACh production, as GCs rapidly formed GAPs in response to ACh analogues. 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 opened colonic GAPs and resulted in recruitment of neutrophils and APCs and production of inflammatory cytokines. Thus GC intrinsic sensing of the microbiota plays a critical role regulating the exposure of the colonic immune system to luminal substances.

Objective Antibiotic use is associated with an increased risk of developing multiple inflammatory disorders, which in turn are linked to alterations in the intestinal microbiota. How these alterations in the intestinal microbiota translate into an increased risk for inflammatory responses is largely unknown. Here we investigated if and how antibiotics promote inflammation via the translocation of live native gut commensal bacteria. Design Oral antibiotics were given to wildtype and induced mutant mouse strains and the effects on bacterial translocation, inflammatory responses, and the susceptibility to colitis were evaluated. The sources of the bacteria and the pathways required for bacterial translocation were evaluated using induced mutant mouse strains, 16s rRNA sequencing to characterize the microbial communities, and in vivo and ex vivo imaging techniques. Results Oral antibiotics induced the translocation of live native commensal bacteria across the colonic epithelium, promoting inflammatory responses, and predisposing to increased disease in response to coincident injury. Bacterial translocation resulted from decreased microbial signals delivered to colonic goblets cells (GCs), was associated with the formation of colonic goblet cell associated antigen passages (GAPs), was abolished when GCs were depleted, and required CX3CR1+ dendritic cells (DCs). Bacterial translocation occurred following a single dose of most antibiotics tested, and the predisposition for increased inflammation was only associated with antibiotics inducing bacterial translocation. Conclusions These findings reveal an unexpected outcome of antibiotic therapy and suggest that bacterial translocation as a result of alterations in the intestinal microflora may provide a link between increasing antibiotic use and the increased incidence of inflammatory disorders.

We have a mutually beneficial relationship with the trillions of microorganisms inhabiting our gastrointestinal tract. However, maintaining this relationship requires recognizing these organisms as affable and restraining inflammatory responses to these organisms when encountered in hostile settings. How and when the immune system develops tolerance to our gut microbial members is not well understood. Here we identify a specific pre-weaning interval in which gut microbial antigens are encountered by the immune system to induce antigen specific tolerance to gut bacteria. Intriguingly for some bacterial taxa, physiologic encounters with the immune system are restricted to this interval, despite abundance of these taxa in the gut lumen at later times outside this interval. Antigen specific tolerance to gut bacteria induced during this pre-weaning interval is stable and maintained even if these taxa are encountered later in life in an inflammatory setting. However, inhibiting microbial antigen encounter during this interval or extending these encounters beyond the normal interval, results in a failure to induce tolerance and robust antigen specific effector responses to gut bacteria upon reencounter in an inflammatory setting. Thus, we have identified a defined pre-weaning interval critical for developing tolerance to gut bacteria and maintaining the mutually beneficial relationship with our gut microbiota.

Goblet cells (GCs) are specialized epithelial cells that line multiple mucosal surfaces and have a well-appreciated role in barrier maintenance through the secretion of mucus. Moreover, GCs secrete anti-microbial proteins, chemokines, and cytokines demonstrating functions in innate immunity beyond barrier maintenance. Recently it was appreciated that GCs can form goblet cell-associated antigen passages (GAPs) and deliver luminal substances to underlying lamina propria (LP) antigen-presenting cells (APCs) in a manner capable of inducing adaptive immune responses. GCs at other mucosal surfaces share characteristics with the GAP forming intestinal GCs, suggesting that GAP formation may not be restricted to the gut, and that GCs may perform this gatekeeper function at other mucosal surfaces. Here we review observations of how GCs contribute to immunity at mucosal surfaces through barrier maintenance, the delivery of luminal substances to APCs, interactions with APCs, and secretion of factors modulating immune responses.

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