Goblet Cell-associated Antigen Passages Research Articles

The right educational environment: Oral tolerance in early life.

Oral tolerance promotes the suppression of immune responses to innocuous antigen and is primarily mediated by regulatory T cell (Tregs). The development of oral tolerance begins in early life during a "window of tolerance," which occurs around weaning and is mediated by components in breastmilk. Herein, we review the factors dictating this window and how Tregs are uniquely educated in early life. In early life, the translocation of luminal antigen for Treg induction is primarily dictated by goblet cell-associated antigen passages (GAPs). GAPs in the colon are negatively regulated by maternally-derived epidermal growth factor and the microbiota, restricting GAP formation to the "periweaning" period (postnatal day 11-21 in mice, 4-6 months in humans). The induction of solid food also promotes the diversification of the bacteria such that bacterially-derived metabolites known to promote Tregs-short-chain fatty acids, tryptophan metabolites, and bile acids-peak during the periweaning phase. Further, breastmilk immunoglobulins-IgA and IgG-regulate both microbial diversity and the interaction of microbes with the epithelium, further controlling which antigens are presented to T cells. Overall, these elements work in conjunction to induce a long-lived population of Tregs, around weaning, that are crucial for maintaining homeostasis in adults.

Intergenerational protective anti-gut commensal immunoglobulin G originates in early life

Maternal immunoglobulins of the class G (IgGs) protect offspring from enteric infection, but when, where, and how these antibodies are physiologically generated and confer protection remains enigmatic. We found that circulating IgGs in adult mice preferentially bind early-life gut commensal bacteria over their own adult gut commensal bacteria. IgG-secreting plasma cells specific for early-life gut bacteria appear in the intestine soon after weaning, where they remain into adulthood. Manipulating exposure to gut bacteria or plasma cell development before, but not after, weaning reduced IgG-secreting plasma cells targeting early-life gut bacteria throughout life. Further, the development of this anti-gut commensal IgG response coincides with the early-life interval in which goblet cell-associated antigen passages (GAPs) are present in the colon. Offspring of dams "perturbed" by B cell ablation or reduced bacterial exposure in early life were more susceptible to enteric pathogen challenge. In contrast to current concepts, protective maternal IgGs targeted translocating gut commensals in the offspring, not the enteric pathogen. These early-life events affecting anti-commensal IgG production have intergenerational effects for protection of the offspring.

A nerve-goblet cell association promotes allergic conjunctivitis through the rapid antigen passage.

The penetration of allergens through the epithelial layer is the initial step in the development of allergic conjunctivitis. Although the pollinosis patients manifest symptoms in minutes after pollen exposure, the mechanisms of the rapid allergen transport remain unclear. In the present study, we found that the instillation of pollen shells rapidly induces a large number of goblet cell-associated antigen passages (GAPs) in the conjunctiva. Antigen acquisition by the stromal cells including macrophages and CD11b+ dendritic cells correlated with the surface GAP formation. Furthermore, a substantial amount of antigen was transported to the stroma during the first 10 minutes of the pollen exposure, which was sufficient for the full induction of an allergic conjunctivitis mouse model. This inducible rapid GAP formation and antigen acquisition was suppressed by topical lidocaine or trigeminal ablation, indicating that the sensory nervous system plays an essential role. Interestingly, pollen shell-stimulated GAP formation was not suppressed by topical atropine, suggesting that the conjunctival GAPs and intestinal GAPs are differentially regulated. These results identify pollen shell-induced GAP as a novel therapeutic target for allergic conjunctivitis.

Maternal Antibiotic Treatment Dysregulates Goblet Cell Associated Antigen Passages in Neonatal Mice

Abstract Maternal antibiotic treatment (MAT) disrupts the development of the neonatal microbiome, intestinal immune development and increases susceptibility to sepsis in mouse models. Mechanisms by which MAT disrupts neonatal intestinal immune function will facilitate the development of novel strategies to reduce neonatal sepsis risk. Intestinal goblet cell associated antigen passages (GAPs) are portals for physiologic exposure of the immune system to commensal gut bacteria and luminal antigens. GAPs are inhibited by epidermal growth factor receptor (EGFR) signaling, colonic GAPs are closed prior to postnatal day (P) 10 in mice when luminal EGF levels from breast milk are high, then open after P10 when breast milk EGF levels decline. When colonic GAPs are opened inappropriately due to low levels of EGF prior to P10 or dysbiosis of the gut microbiota after weaning, they can facilitate pathological bacterial translocation. Pups of dams treated with ampicillin and neomycin in their drinking water from pup P1–8 had significantly more open GAPs in the small intestine (SI) compared to controls. These data suggest that MAT creates a permissive environment for SI GAPs in the neonatal period which could allow pathologic bacterial translocation. MAT pups weighed significantly less and were significantly hypoglycemic compared to pups from control dams, after weaning MAT pups recover to a normal weight. We hypothesize that MAT pups have reduced intestinal EGFR signaling, leading to stunted growth and irregular opening of GAPs in the SI and colon. These data suggest maternal antibiotic treatment may be a risk factor for sepsis and growth failure in premature neonates.

Premature Treg induction in early life results in FoxP3 instability post-weaning

Abstract Peripherally-derived RORγt+ regulatory T cells (Tregs) induced in early life are crucial for maintaining oral tolerance later in life, and the loss of these long-lived cells is associated with increased allergic and inflammatory phenotypes in several animal models. We have identified a predominant pathway of luminal antigen delivery that is associated with Treg development. Around weaning, microbial and dietary proteins are delivered to the colonic lamina propria via goblet cell-associated antigen passages (GAPs) and taken up by antigen presenting cells. GAP formation is inhibited by breastmilk-derived epidermal growth factor (EGF) which is highest in maternal milk at parturition and is diminished by weaning. We have previously shown that disruption of this process – via EGF receptor inhibition (EGFRi) day of life (DOL)4–8 or by conditional deletion of EGFR from goblet cells – results in premature antigen delivery. Consequently, this results in an early initial increase in FoxP3 expression in the colon that is unstable and, ultimately, lost by Tregs primed pre-weaning; this suggests that earlier education of T cells is incompatible with regulatory T cell differentiation in early life. EGFRi-treated FoxP3eGFP-Cre-ERT2ROSAlsl-YFPmice, whose FoxP3+ cells were YFP-labeled at DOL21, had a larger number of YFP+ T cells that lost FoxP3 expression than that of control mice, by DOL28. Further, compared to controls, wild-type mice treated with EGFRi DOL4–8 had increased IFNγ, TNFα, and IL-17a-expressing CD4 T cells when challenged with DSS colitis later in life. This suggests that early antigen delivery-associated FoxP3 instability may contribute to an increased propensity toward an inflammatory phenotype later in life.

Protective colonic iNKT cells are expanded by Goblet cell-associated antigen passages in CD1d-dependent manner in adults

Abstract Invariant natural killer T (iNKT) cells are unique innate immune cell which secrete cytokines and immune mediators. These cells recognize self or microbial glycolipids presented in the context of CD1d. iNKT cells can contribute to host protection or pathogenesis during intestinal inflammation. Colonic iNKT cells are established under the influence of the microbiota in early life and the current understanding is that colonic iNKT cells are fixed and not manipulable in later life. Goblet cells (GCs) or/and goblet cell associated passages (GAPs) deliver luminal antigens and maintain peripheral T regulatory cells in the steady state. However, GAPs in the colon are absent in adult mice due to GCs microbial sensing. We found that the glycolipids can be delivered through GAPs and that inducing GAPs in the colon in adult mice resulted in significant iNKT cell expansion. Deletion of GCs or deletion of CD1d on GCs prevented iNKT cell expansion suggesting a role for colonic GCs in presenting glycolipids to iNKT cells. Single cell RNA sequencing of colonic-iNKT cells showed significantly increased iNKT2 and iNKT1 subsets after colonic GAP induction. Proteomic analysis of these iNKT cells further confirmed the characteristics of expanded iNKT subsets. Moreover, iNKT cells expanding after colonic GAP induction persisted and were protective in DSS-induced colitis. Adoptive transfer of the expanded colonic iNKT cells protected in the DSS-induced colitis model, indicating that expanded colonic iNKT cells were sufficient for protection in this model. These findings indicate that colonic iNKT cells are not fixed during early life but can be expanded in a GAP dependent and GC CD1d dependent manner and can be protective in some models of colitis. Crohn's and Colitis Foundation

Goblet cell-associated antigen passage: A gatekeeper of the intestinal immune system.

Effective delivery of luminal antigens to the underlying immune system is the initial step in generating antigen-specific responses in the gut. However, a large body of information regarding the immune response activation process remains unknown. Recently, goblet cells (GCs) have been reported to form goblet cell-associated antigen passages (GAPs). Luminal antigens can be transported inside GAPs and reach subepithelial immune cells to induce antigen-specific immune responses, contributing largely to gut homeostasis and the prevention of some intestinal diseases like allergic enteritis and bacterial translocation. In this article, we summarized recent observations on the formation of intestinal GAPs and their roles in mucosal immunity. We hope that this review can offer a fresh perspective and valuable insights for clinicians and researchers interested in studying the intestinal immune system.

Autofluorescence imaging permits label free cell type assignment in unmodified airway and reveals the dynamic formation of airway secretory cell associated antigen passages (SAPs).

The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a physiologic response. Modern approaches for identifying and dissecting novel physiologic mechanisms would benefit from an ability to identify specific cell types in live tissues that could then be imaged in real-time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the simultaneous study of 3 or 4 cell types at a time. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in mouse tracheal explant in real-time. Furthermore, we find that this methodology for direct cell type specific identification avoids pitfalls associated with the use of ostensibly cell type-specific markers that are, in fact, altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology and identify dynamic secretory cell associated antigen passages (SAPs) that form in response to cholinergic stimulus. The identical process has been well documented in the intestine where the dynamic formation of secretory and goblet cell associated antigen passages (SAPs and GAPs) enable luminal antigen sampling. Given that airway secretory cells can be stimulated to make mucous within hours, we suspect that both SAPs and GAPs are also used for luminal antigen sampling in the airway. This hypothesis is supported by our observation that secretory cells with airway SAPs are frequently juxtaposed to antigen presenting cells.

An optimized method to visualize the goblet cell-associated antigen passages and identify goblet cells in the intestine, conjunctiva, and airway

The goblet cell-associated antigen passages (GAPs), formed in the intestine and conjunctiva, contribute to immune responses by delivering antigens to immune cells. It has been proved that dysfunctions of GAPs may contribute to intestinal inflammation and dry eye disease. GAPs were first discovered in the intestine with the help of intravital two-photon microscopy. The imaging of GAPs was subsequently captured in a frozen section and whole-mount immunofluorescent staining. Both protocols have their features. This study provides a new protocol for visualizing GAPs with better mucus preservation by combining 4% PFA and methanol-Carnoy’s solution (PFA-MC). In addition, embedding samples in paraffin provides an opportunity for antigen retrieval for immunohistochemistry analysis. Using this protocol, we have successfully visualized GAPs in the intestine, intestinal organoids, conjunctiva, and trachea. It is worth mentioning that the visualization of GAPs in intestinal organoids and airways has rarely been discussed before, which may assist researchers in their studies. Moreover, we also discovered the active uptake of dextran of club cells in terminal bronchioles, which may provide a new direction for studying the function of club cells. Overall, we developed a new method to visualize GAP formation and identify GCs simultaneously in the intestine, organoid, conjunctiva, and airway.

Maternally derived epidermal growth factor mediates protection in neonates from opportunistic intestinal pathogens.

Abstract The intestinal epithelial barrier is vital in preventing translocating bacteria and subsequent systemic infections. This protection is provided by host factors, by the intestinal microbiota, and, during early life, by breast milk. While the beneficial functions of breast milk are numerous to the nursing infant, it has been observed that proteins such as antibodies and growth factors are secreted in higher concentrations early during lactation and decrease as the milk matures. One such growth factor, epidermal growth factor (EGF), regulates the development of colonic goblet cell-associated antigen passages (GAPs). Decrease of EGF through disruption of synchronous breastfeeding or deletion of the epidermal growth factor receptor from goblet cells alters the kinetics of GAP formation, which can serve as a passage for pathogenic bacteria. In a murine model of early GAP formation utilizing an inhibitor of EGF receptor, we observed a variety of gut-resident pathogens were able to utilize GAPs to disseminate systemically and cause disease, including Escherichia coli, Enterococcus faecalis, Enterobacter cloacae, and Pseudomonas aeruginosa. In human studies, we found a strong correlation between concentrations of EGF in the diet and stool of very low birth weight infants. Notably, formula-fed infants contained significantly less EGF in both diet and stool. Future work will assess EGF in donor milk, which we find has highly variable EGF concentration. This work has strong implications for how diet influences risk of developing infections from gut-resident pathogens and how maternal EGF present in breastmilk is synchronized to the newborn infant to provide appropriate protection in early life. Supported by grants from NIH (AI144721, DK122187)

Intestinal goblet cells sample and deliver lumenal antigens by regulated endocytic uptake and transcytosis.

Intestinal goblet cells maintain the protective epithelial barrier through mucus secretion and yet sample lumenal substances for immune processing through formation of goblet cell associated antigen passages (GAPs). The cellular biology of GAPs and how these divergent processes are balanced and regulated by goblet cells remains unknown. Using high-resolution light and electron microscopy, we found that in mice, GAPs were formed by an acetylcholine (ACh)-dependent endocytic event remarkable for delivery of fluid-phase cargo retrograde into the trans-golgi network and across the cell by transcytosis - in addition to the expected transport of fluid-phase cargo by endosomes to multi-vesicular bodies and lysosomes. While ACh also induced goblet cells to secrete mucins, ACh-induced GAP formation and mucin secretion were functionally independent and mediated by different receptors and signaling pathways, enabling goblet cells to differentially regulate these processes to accommodate the dynamically changing demands of the mucosal environment for barrier maintenance and sampling of lumenal substances.

367: Goblet cell-associated antigen passages and tolerogenic dendritic cells are increased in the intestinal-specific CFTR KO mouse intestine

367: Goblet cell-associated antigen passages and tolerogenic dendritic cells are increased in the intestinal-specific CFTR KO mouse intestine

EVALUATING THE ROLE OF GOBLET CELL ASSOCIATED ANTIGEN PASSAGES (GAPS) IN THE DEVELOPMENT OF MUCOSAL IMMUNE TOLERANCE IN THE CFTR KO INTESTINE

Greater than 70,000 individuals worldwide are living with the monogenetic disease cystic fibrosis (CF). The development of chronic intestinal inflammation, with clinical signs resembling inflammatory bowel disease-like conditions, is a common yet poorly understood occurrence in CF patients. This inflammation is typically neutrophilic in human and animal models with a heightened basal pro-inflammatory cytokine release. Prior research utilizing intestinal organoids (enteroids) cultured from Cftr knockout mice has shown that goblet cells in the CF mouse intestine demonstrate defective clearance of mucin granules and abnormal mucus retention. Goblet cell-associated antigen passages (GAPs), located in the small intestine and colon, deliver intraluminal antigens to antigen-presenting dendritic cells in the submucosa. This mechanism serves as an important step in the development and maintenance of tolerogenic dendritic cell populations expressing receptors to luminal antigens with involvement of regulatory T cell activation and release of IL-10. We hypothesized that mucus plugging of goblet cells in the CF intestine leads to defective GAP formation and a consequent decrease in the expansion of tolerogenic dendritic cells. To test this hypothesis, Cftrtm1Unc (Cftr KO) and wild type (WT) sex-matched littermate pairs (n=2) maintained on a commercially available liquid diet (Peptamen®) were anesthetized with ketamine/xylazine for a laparotomy to inject a luminal fluorescent 10kD dextran dye into the mid-jejunum. After 30 min, the mice were euthanized with CO2, and the intestine was collected for immunofluorescent staining to evaluate GAP formation. In the WT intestine, the dextran dye was observed within goblet cells outlined by CK18 immunofluorescence, a goblet cell marker. Punctate dextran dye was observed in the submucosa, suggestive of dendritic cell uptake. In contrast, the Cftr KO mice demonstrated defective GAP formation, i.e., without dye penetration of goblet cells, and the lack of punctate dextran fluorescence in the submucosa. To evaluate the population of tolerogenic dendritic cells, small intestinal segments from Cftr KO-WT sex-matched littermate pairs (3-female and 2-male pairs) were collected for FACS sorting of submucosal CD103+ (tolerogenic) and CD103- (pro-inflammatory) dendritic cells. The WT mice had a significantly higher population of CD103+ tolerogenic dendritic cells compared to the CF mice (WT: 20.5+/-2, CF: 9.2+/-3, P < 0.006). A trend towards an increase in CD103- dendritic cells was seen in the CF intestine. In summary, the CF mice were found to have defective intraluminal antigen transfer through the GAP pathway and a significant decrease in tolerogenic dendritic cells in the intestine.

EVALUATING THE ROLE OF GOBLET CELL ASSOCIATED ANTIGEN PASSAGES (GAPS) IN THE DEVELOPMENT OF MUCOSAL IMMUNE TOLERANCE IN THE CFTR KO INTESTINE

Abstract Greater than 70,000 individuals worldwide are living with the monogenetic disease cystic fibrosis (CF). The development of chronic intestinal inflammation, with clinical signs resembling inflammatory bowel disease-like conditions, is a common yet poorly understood occurrence in CF patients. This inflammation is typically neutrophilic in human and animal models with a heightened basal pro-inflammatory cytokine release. Prior research utilizing intestinal organoids (enteroids) cultured from Cftr knockout mice has shown that goblet cells in the CF mouse intestine demonstrate defective clearance of mucin granules and abnormal mucus retention. Goblet cell-associated antigen passages (GAPs), located in the small intestine and colon, deliver intraluminal antigens to antigen-presenting dendritic cells in the submucosa. This mechanism serves as an important step in the development and maintenance of tolerogenic dendritic cell populations expressing receptors to luminal antigens with involvement of regulatory T cell activation and release of IL-10. We hypothesized that mucus plugging of goblet cells in the CF intestine leads to defective GAP formation and a consequent decrease in the expansion of tolerogenic dendritic cells. To test this hypothesis, Cftrtm1Unc (Cftr KO) and wild type (WT) sex-matched littermate pairs (n=2) maintained on a commercially available liquid diet (Peptamen®) were anesthetized with ketamine/xylazine for a laparotomy to inject a luminal fluorescent 10kD dextran dye into the mid-jejunum. After 30 min, the mice were euthanized with CO2, and the intestine was collected for immunofluorescent staining to evaluate GAP formation. In the WT intestine, the dextran dye was observed within goblet cells outlined by CK18 immunofluorescence, a goblet cell marker. Punctate dextran dye was observed in the submucosa, suggestive of dendritic cell uptake. In contrast, the Cftr KO mice demonstrated defective GAP formation, i.e., without dye penetration of goblet cells, and the lack of punctate dextran fluorescence in the submucosa. To evaluate the population of tolerogenic dendritic cells, small intestinal segments from Cftr KO-WT sex-matched littermate pairs (3-female and 2-male pairs) were collected for FACS sorting of submucosal CD103+ (tolerogenic) and CD103- (pro-inflammatory) dendritic cells. The WT mice had a significantly higher population of CD103+ tolerogenic dendritic cells compared to the CF mice (WT: 20.5+/-2, CF: 9.2+/-3, P &amp;lt; 0.006). A trend towards an increase in CD103- dendritic cells was seen in the CF intestine. In summary, the CF mice were found to have defective intraluminal antigen transfer through the GAP pathway and a significant decrease in tolerogenic dendritic cells in the intestine.

The relationship between intestinal goblet cells and the immune response.

Goblet cells (GCs) are single-cell glands that produce and secrete mucin. Mucin forms a mucus layer, which can separate the materials in cavities from the intestinal epithelium and prevent the invasion of pathogenic microorganisms in various ways. GCs can also participate in the immune response through nonspecific endocytosis and goblet cell-associated antigen passages (GAPs). GCs endocytose soluble substances from the lumen and transmit antigens to the underlying antigen-presenting cells (APCs). A variety of immuno-regulatory factors can promote the differentiation, maturation of GCs, and the secretion of mucin. The mucin secreted by GCs forms a mucus layer, which plays an important role in resisting the invasion of foreign bacteria and intestinal inherent microorganisms, regulating the immune performance of the body. Therefore, the present study mainly reviews the barrier function of the mucus layer, the mucus secreted by goblet cells, the protective effect against pathogenic bacteria, the delivery of luminal substances through GAPs and the relationship between GCs and the immune response.

Small Bowel Resection Increases Paracellular Gut Barrier Permeability via Alterations of Tight Junction Complexes Mediated by Intestinal TLR4

BackgroundShort bowel syndrome resulting from small bowel resection (SBR) is associated with significant morbidity and mortality. Many adverse sequelae including steatohepatitis and bacterial overgrowth are thought to be related to increased bacterial translocation, suggesting alterations in gut permeability. We hypothesized that after intestinal resection, the intestinal barrier is altered via toll-like receptor 4 (TLR4) signaling at the intestinal level. MethodsB6 and intestinal-specific TLR4 knockout (iTLR4 KO) mice underwent 50% SBR or sham operation. Transcellular permeability was evaluated by measuring goblet cell associated antigen passages via two-photon microscopy. Fluorimetry and electron microscopy evaluation of tight junctions (TJ) were used to assess paracellular permeability. In parallel experiments, single-cell RNA sequencing measured expression of intestinal integral TJ proteins. Western blot and immunohistochemistry confirmed the results of the single-cell RNA sequencing. ResultsThere were similar number of goblet cell associated antigen passages after both SBR and sham operation (4.5 versus 5.0, P > 0.05). Fluorescein isothiocyanate–dextran uptake into the serum after massive SBR was significantly increased compared with sham mice (2.13 ± 0.39 ng/μL versus 1.62 ± 0.23 ng/μL, P < 0.001). SBR mice demonstrated obscured TJ complexes on electron microscopy. Single-cell RNA sequencing revealed a decrease in TJ protein occludin (21%) after SBR (P < 0.05), confirmed with immunostaining and western blot analysis. The KO of iTLR4 mitigated the alterations in permeability after SBR. ConclusionsPermeability after SBR is increased via changes at the paracellular level. However, these alterations were prevented in iTLR4 mice. These findings suggest potential protein targets for restoring the intestinal barrier and obviating the adverse sequelae of short bowel syndrome.

Maternal activation of the EGFR prevents translocation of gut-residing pathogenic Escherichia coli in a model of late-onset neonatal sepsis

Late-onset sepsis (LOS) is a highly consequential complication of preterm birth and is defined by a positive blood culture obtained after 72 h of age. The causative bacteria can be found in patients' intestinal tracts days before dissemination, and cohort studies suggest reduced LOS risk in breastfed preterm infants through unknown mechanisms. Reduced concentrations of epidermal growth factor (EGF) of maternal origin within the intestinal tract of mice correlated to the translocation of a gut-resident human pathogen Escherichia coli, which spreads systemically and caused a rapid, fatal disease in pups. Translocation of Escherichia coli was associated with the formation of colonic goblet cell-associated antigen passages (GAPs), which translocate enteric bacteria across the intestinal epithelium. Thus, maternally derived EGF, and potentially other EGFR ligands, prevents dissemination of a gut-resident pathogen by inhibiting goblet cell-mediated bacterial translocation. Through manipulation of maternally derived EGF and alteration of the earliest gut defenses, we have developed an animal model of pathogen dissemination which recapitulates gut-origin neonatal LOS.

In vivo labeling of epithelial cell-associated antigen passages in the murine intestine.

The intestinal immune system samples luminal contents to induce adaptive immune responses that include tolerance in the steady state and protective immunity during infection. How luminal substances are delivered to the immune system has not been fully investigated. Goblet cells have an important role in this process by delivering luminal substances to the immune system through the formation of goblet cell-associated antigen passages (GAPs). Soluble antigens in the intestinal lumen are transported across the epithelium transcellularly through GAPs and delivered to dendritic cells for presentation to T cells and induction of immune responses. GAPs can be identified and quantified by using the ability of GAP-forming goblet cells to take up fluorescently labeled dextran. Here, we describe a method to visualize GAPs and other cells that have the capacity to take up luminal substances by intraluminal injection of fluorescent dextran in mice under anesthesia, tissue sectioning for slide preparation and imaging with fluorescence microscopy. In contrast to in vivo two-photon imaging previously used to identify GAPs, this technique is not limited by anatomical constraints and can be used to visualize GAP formation throughout the length of the intestine. In addition, this method can be combined with common immunohistochemistry protocols to visualize other cell types. This approach can be used to compare GAP formation following different treatments or changes to the luminal environment and to uncover how sampling of luminal substances is altered in pathophysiological conditions. This protocol requires 8 working hours over 2-3 d to be completed.

Goblet cell associated antigen passages support the induction and maintenance of oral tolerance.

Tolerance to innocuous antigens from the diet and the commensal microbiota is a fundamental process essential to health. Why tolerance is efficiently induced to substances arising from the hostile environment of the gut lumen is incompletely understood but may be related to how these antigens are encountered by the immune system. We observed that goblet cell associated antigen passages (GAPs), but not other pathways of luminal antigen capture, correlated with the acquisition of luminal substances by lamina propria (LP) antigen presenting cells (APCs) and with the sites of tolerance induction to luminal antigens. Strikingly this role extended beyond antigen delivery. The GAP function of goblet cells facilitated maintenance of pre-existing LP T regulatory cells (Tregs), imprinting LP-dendritic cells with tolerogenic properties, and facilitating LP macrophages to produce the immunomodulatory cytokine IL-10. Moreover, tolerance to dietary antigen was impaired in the absence of GAPs. Thus, by delivering luminal antigens, maintaining pre-existing LP Tregs, and imprinting tolerogenic properties on LP-APCs GAPs support tolerance to substances encountered in the hostile environment of the gut lumen.

Intestinal Goblet cells and Goblet Cell Associated Antigen Passages regulate the balance between Tregs and Th17

Abstract Tolerance towards dietary antigens and luminal microbes is essential to maintain homeostasis, the breakdown of which results in development of inflammatory bowel disease (IBD). T-helper 17 (Th17) and T-regulatory (Treg) cells are abundant in the intestinal mucosa, where they function to protect the host from pathogens and to restrain excessive effector T-cell responses, respectively. How the balance and switch between Treg and Th17 responses is maintained in the steady state to promote tolerance and how this balance becomes altered to favor Th17 responses in the setting of IBD is not known. We have identified that intestinal goblet cell (GC) associated antigen passages (GAPs), are a major pathway for steady-state luminal antigen transfer to lamina propria - dendritic cells (LP-DCs). In addition, GAPs are necessary for the imprinting of LP-DCs with tolerogenic properties, for the de novo induction of Tregs specific for dietary antigens, and the maintenance of pre-existing LP Tregs. Moreover, GAP inhibition results in the expansion of Th17 cells in the small intestine LP. Consistent with this, single cell profiling of intestinal lymphocytes revealed enhanced Th17 pathway gene signatures in mice lacking GCs. These observations strongly support that GCs/GAPs have a previously unappreciated role in maintaining the functional balance between Treg and Th17 cell.