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.

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.

The delivery of antigens across the intestinal epithelium is an early and critical event in homeostatic responses to innocuous antigens and inflammatory responses to potential pathogens. The factors and mechanisms regulating antigen delivery to the lamina propria (LP) immune system are largely unknown. We recently demonstrated when small intestinal goblet cells (GCs) secrete, they form goblet cell-associated antigen passages (GAPs) and deliver luminal antigens to LP dendritic cells (DCs). Here we report in the basal state, GAP formation is driven by acetylcholine (Ach) signaling via the muscarinic ACh receptor 4 (mAChR4) expressed by GCs. In contrast to conventionally housed mice, GAPs were present in the colon of mice with a reduced microbiota or altered microbial sensing. Formation of colonic GAPs in these mice was dependent upon mAChR4 signaling. Moreover, in mice with a reduced microbial load, luminal microbial stimuli inhibited GAP formation within minutes. In vitro studies revealed GC intrinsic sensing of the microbiota rapidly inhibited ACh induced GC secretion, and conditional deletion of Myd88 in GCs in conventionally housed mice resulted in the spontaneous formation of colonic GAPs, and the delivery of luminal antigen to colonic LP DCs. These findings identify a role for microbial sensing by colonic GCs to rapidly regulate antigen delivery to the LP immune system, and preventing delivery of luminal antigens when the luminal microbial load is abundant and complex.