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.

The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells.

Intestinal microfold cells (M cells) are an enigmatic lineage of intestinal epithelial cells that initiate mucosal immune responses through the uptake and transcytosis of luminal antigens. The mechanisms of M-cell differentiation are poorly understood, as the rarity of these cells has hampered analysis. Exogenous administration of the cytokine RANKL can synchronously activate M-cell differentiation in mice. Here we show the Ets transcription factor Spi-B was induced early during M-cell differentiation. Absence of Spi-B silenced the expression of various M-cell markers and prevented the differentiation of M cells in mice. The activation of T cells via an oral route was substantially impaired in the intestine of Spi-B-deficient (Spib(-/-)) mice. Our study demonstrates that commitment to the intestinal M-cell lineage requires Spi-B as a candidate master regulator.

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.

Regulation of oral antigen delivery early in life: Implications for oral tolerance and food allergy.

The increasing incidence of food allergy remains a significant public health concern. Food allergy is partially due to a lack, or loss of tolerance to food allergens. Clinical outcomes surrounding early life practices, such as breastfeeding, antibiotic use and food allergen exposure, indicate the first year of life in children represents a unique time for shaping the immune system to reduce allergic outcomes. Animal models have identified distinctive aspects of when and where dietary antigens are delivered within the intestinal tract to promote oral tolerance prior to weaning. Additionally, animal models have identified contributions from maternal proteins from breast milk and bacterial products from the gut microbiota in regulating dietary antigen exposure and promoting oral tolerance, thus connecting decades of clinical observations on the benefits of breastfeeding, early food allergen introduction and antibiotic avoidance in the first year of life in reducing allergic outcomes. Here, we discuss how exposure to gut luminal antigens, including food allergens, is regulated in early life to generate protective tolerance and the implications of this process for preventing and treating food allergies.

IL-13-induced intestinal secretory epithelial cell antigen passages are required for IgE-mediated food-induced anaphylaxis.

BACKGROUND: Food-induced anaphylaxis (FIA) is an IgE-dependent immune response that can affect multiple organs and lead to life-threatening complications. The processes by which food allergens cross the mucosal surface and are delivered to the subepithelial immune compartment to promote the clinical manifestations associated with food-triggered anaphylaxis are largely unexplored. OBJECTIVE: We sought to define the processes involved in the translocation of food allergens across the mucosal epithelial surface to the subepithelial immune compartment in FIA. METHODS: Two-photon confocal and immunofluorescence microscopy was used to visualize and trace food allergen passage in a murine model of FIA. A human colon cancer cell line, RNA silencing, and pharmacologic approaches were used to identify the molecular regulation of intestinal epithelial allergen uptake and translocation. Human intestinal organoid transplants were used to demonstrate the conservation of these molecular processes in human tissues. RESULTS: Food allergens are sampled by using small intestine (SI) epithelial secretory cells (termed secretory antigen passages [SAPs]) that are localized to the SI villous and crypt region. SAPs channel food allergens to lamina propria mucosal mast cells through an IL-13-CD38-cyclic adenosine diphosphate ribose (cADPR)-dependent process. Blockade of IL-13-induced CD38/cADPR-dependent SAP antigen passaging in mice inhibited induction of clinical manifestations of FIA. IL-13-CD38-cADPR-dependent SAP sampling of food allergens was conserved in human intestinal organoids. CONCLUSION: We identify that SAPs are a mechanism by which food allergens are channeled across the SI epithelium mediated by the IL-13/CD38/cADPR pathway, regulate the onset of FIA reactions, and are conserved in human intestine.

Goblet cells deliver luminal antigen to CD103+ dendritic cells in the small intestine.

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 cells. 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(+) CX(3)CR1(-) DCs, which promote IgA production, imprint gut homing on lymphocytes and induce the development of regulatory T cells, and CD103(-) CX(3)CR1(+) DCs (with features of macrophages), which promote tumour necrosis factor-α (TNF-α) production, colitis, and the development of T(H)17 T cells. 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.

CCR6 promotes steady-state mononuclear phagocyte association with the intestinal epithelium, imprinting and immune surveillance.

The intestinal lamina propria (LP) contains antigen-presenting cells with features of dendritic cells and macrophages, collectively referred to as mononuclear phagocytes (MNPs). Association of MNPs with the epithelium is thought to play an important role in multiple facets of intestinal immunity including imprinting MNPs with the ability to induce IgA production, inducing the expression of gut homing molecules on T cells, facilitating the capture of luminal antigens and microbes, and subsequent immune responses in the mesenteric lymph node (MLN). However, the factors promoting this process in the steady state are largely unknown, and in vivo models to test and confirm the importance of LP-MNP association with the epithelium for these outcomes are unexplored. Evaluation of epithelial expression of chemoattractants in mice where MNP-epithelial associations were impaired suggested CCL20 as a candidate promoting epithelial association. Expression of CCR6, the only known receptor for CCL20, was required for MNPs to associate with the epithelium. LP-MNPs from CCR6-/- mice did not display defects in acquiring antigen and stimulating T-cell responses in ex vivo assays or in responses to antigen administered systemically. However, LP-MNPs from CCR6-deficient mice were impaired at acquiring luminal and epithelial antigens, inducing IgA production in B cells, inducing immune responses in the MLN, and capturing and trafficking luminal commensal bacteria to the MLN. These findings identify a crucial role for CCR6 in promoting LP-MNPs to associate with the intestinal epithelium in the steady state to perform multiple functions promoting gut immune homeostasis.

Antibiotics promote inflammation through the translocation of native commensal colonic bacteria.

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 whether 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 characterise 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 goblet cells (GCs), was associated with the formation of colonic GC-associated antigen passages, was abolished when GCs were depleted and required CX3CR1(+) dendritic cells. 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.

Streptococcus agalactiae and Escherichia coli Induce Distinct Effector γδ T Cell Responses During Neonatal Sepsis.

Neonates born prematurely are highly vulnerable to life-threatening conditions such as bacterial sepsis. Streptococcus agalactiae, also known as group B Streptococcus (GBS) and Escherichia coli are frequent causative pathogens of neonatal sepsis, however, it remains unclear if distinct sepsis pathogens induce differential adaptive immune responses. In the present study, we find that γδ T cells in neonatal mice rapidly respond to single-organism GBS and E. coli bloodstream infections and that these pathogens induce distinct activation and cytokine production from IFN-γ and IL-17 producing γδ T cells, respectively. We also report differential reliance on γδTCR signaling to elicit effector cytokine responses during neonatal sepsis, with IL-17 production during E. coli infection being driven by γδTCR signaling, and IFN-γ production during GBS infection occurring independently of γδTCR signaling. Furthermore, we report that the divergent effector responses of γδ T cells during GBS and E. coli infections impart distinctive neuroinflammatory phenotypes on the neonatal brain. The present study reveals that the neonatal adaptive immune system differentially responds to distinct bacterial stimuli, resulting in unique neuroinflammatory phenotypes.

Streptococcus agalactiae and Escherichia coli induce distinct effector γδ T cell responses during neonatal sepsis.

Neonates born prematurely are vulnerable to life-threatening conditions such as bacterial sepsis. Streptococcus agalactiae (GBS) and Escherichia coli are frequent causative pathogens of neonatal sepsis, however, it remains unclear if these pathogens induce differential immune responses. We find that γδ T cells rapidly respond to single-organism GBS and E. coli bloodstream infections in neonatal mice. Furthermore, GBS and E. coli induce distinct cytokine production from IFN-γ and IL-17 producing γδ T cells, respectively. We also find that IL-17 production during E. coli infection is driven by γδTCR signaling, whereas IFN-γ production during GBS infection occurs independently of γδTCR signaling. The divergent effector responses of γδ T cells during GBS and E. coli infections impart distinctive neuroinflammatory phenotypes on the neonatal brain. Thus, the neonatal adaptive immune system differentially responds to distinct bacterial stimuli, resulting in unique neuroinflammatory phenotypes.

Neonatal intestinal colonization of Streptococcus agalactiae and the multiple modes of protection limiting translocation.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a predominant pathogen of neonatal sepsis, commonly associated with early-onset neonatal sepsis. GBS has also been associated with cases of late-onset sepsis potentially originating from the intestine. Previous findings have shown GBS can colonize the infant intestinal tract as part of the neonatal microbiota. To better understand GBS colonization dynamics in the neonatal intestine, we collected stool and milk samples from prematurely born neonates for identification of potential pathogens in the neonatal intestinal microbiota. GBS was present in approximately 10% of the cohort, and this colonization was not associated with maternal GBS status, delivery route, or gestational weight. Interestingly, we observed the relative abundance of GBS in the infant stool negatively correlated with maternal IgA concentration in matched maternal milk samples. Using a preclinical murine model of GBS infection, we report that both vertical transmission and direct oral introduction resulted in intestinal colonization of GBS; however, translocation beyond the intestine was limited. Finally, vaccination of dams prior to breeding induced strong immunoglobulin responses, including IgA responses, which were associated with reduced mortality and GBS intestinal colonization. Taken together, we show that maternal IgA may contribute to infant immunity by limiting the colonization of GBS in the intestine.

Transepithelial antigen delivery in the small intestine: different paths, different outcomes.

PURPOSE OF REVIEW: The intestinal epithelium is a dynamic barrier protecting the body from the multitudes of luminal micro-organisms present in the gut. However, this barrier is not impermeable and mechanisms exist that allow small amounts of antigen to traverse the epithelium in controlled manner to maintain tolerance and to mount immune responses. This review will summarize our current understanding of how luminal antigens traverse the small intestine epithelium without disrupting the epithelial barrier and how these antigen delivery pathways might influence the resulting immune responses. RECENT FINDINGS: Recent findings have revealed four pathways for transepithelial antigen delivery in the absence of barrier disruption. We propose that during homeostasis, antigen introduced through microfold cells induces immunoglobulin A responses, antigen delivered by goblet cell-associated antigen passages contributes to peripheral tolerance, and antigen delivered by paracellular leak initiates immune responses in the mesenteric lymph node. In contrast, dendritic cell transepithelial dendrites may play an important role in host protection during pathogen infection, but do not appear to play a role in antigen capture by lamina propria dendritic cells in the steady state. SUMMARY: These observations indicate that the route by which antigen crosses the epithelium directs the outcome of the subsequent immune response.

Distinct developmental requirements for isolated lymphoid follicle formation in the small and large intestine: RANKL is essential only in the small intestine.

Cryptopatches (CPs) and isolated lymphoid follicles (ILFs) are organized intestinal lymphoid tissues that develop postnatally in mice and include stromal cells expressing the receptor activator of nuclear factor kappa-B ligand (RANKL). We investigated how stromal RANKL influences the development and differentiation of CPs and ILFs by analyzing the development of these lymphoid structures in knockout mice lacking RANKL. We found that RANKL(-/-) mice had a fourfold reduction in the overall density of CPs in the small intestine compared to control mice, with the largest decrease in the proximal small intestine. No B cells were present in CPs from the small intestine of RANKL(-/-) mice and ILF formation was completely blocked. In sharp contrast, colonic ILFs containing B cells were present in RANKL(-/-) mice. Stromal cells within CPs in the small intestine of RANKL(-/-) mice did not express CXCL13 (originally called B lymphocyte chemoattractant) and often lacked other normally expressed stromal cell antigens, whereas colonic lymphoid aggregates in RANKL(-/-) mice retained stromal CXCL13 expression. The CXCL13-dependent maturation of precursor CPs into ILFs is differentially regulated in the small intestine and colon, with an absolute requirement for RANKL only in the small intestine.

CCR6hiCD11c(int) B cells promote M-cell differentiation in Peyer's patch.

M cells are responsible for uptake of mucosal antigens in Peyer's patches (PPs). Differentiation of M cells is thought to be induced by interactions between follicle-associated epithelium and PP cells; however, it remains elusive what types of immune cells function as M-cell inducers. Here, we attempted to identify the cells that serve as an M-cell inducer in PP. We found that a unique B-cell subset characterized by CCR6(hi)CD11c(int) resided in the subepithelial dome (SED) in mouse PP. CCR6(hi)CD11c(int) B cells showed chemotactic migration in response to CCL20. Furthermore, this unique B-cell subset substantially decreased in PP of CCR6-deficient mice, indicating that the SED localization of CCR6(hi)CD11c(int) B cells is most likely regulated by the CCL20-CCR6 system. Concomitantly, CCR6 deficiency caused remarkable decrement of M cells. Moreover, adoptive transfer of CCR6(hi)CD11c(int) B cells from wild-type mice restored the M-cell decrement in CCR6-deficient mice. Collectively, the spatial regulation of CCR6(hi)CD11c(int) B cells via the CCL20-CCR6 system may play a vital role in M-cell differentiation in mice.

RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium.

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-kappaB 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.

Sepsis, Cytokine Storms, and Immunopathology: The Divide between Neonates and Adults.

Sepsis can result from a variety of pathogens, originating from a range of sources. A vast range of presenting symptoms is included in the catch-all term of "bacteremia," making diagnosis and prognosis particularly troublesome. One underexplored factor contributing to disparate outcomes is the age of the patient. Neonatal sepsis in very-low-birth-weight infants can result in vastly different immunological outcomes unique from sepsis in adults. It is also becoming increasingly clear, both from preclinical experimental models and clinical observations, that the age and history of previous microbial exposures can significantly influence the course of infection from sepsis and cytokine storms to immunopathology. In this study, we will explore key differences between neonatal and adult sepsis, experimental models used to study sepsis, and how responses to the surrounding microbial universe shape development of the immune system and impact, positively or negatively, the course of disease.

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.

Cells in the gut need to be protected against the many harmful microbes which inhabit this environment. Yet the immune system also needs to 'keep an eye' on intestinal contents to maintain tolerance to innocuous substances, such as those from the diet. The 'goblet cells' that are part of the gut lining do both: they create a mucus barrier that stops germs from invading the body, but they also can pass on molecules from the intestine to immune cells deep in the tissue to promote tolerance. This is achieved through a 'GAP' mechanism. A chemical messenger called acetylcholine can trigger both mucus release and the GAP process in goblet cells. Gustafsson et al. investigated how the cells could take on these two seemingly opposing roles in response to the same signal. A fluorescent molecule was introduced into the intestines of mice, and monitored as it pass through the goblet cells. This revealed how the GAP process took place: the cells were able to capture molecules from the intestines, wrap them in internal sack-like vesicles and then transport them across the entire cell. To explore the role of acetylcholine, Gustafsson et al. blocked the receptors that detect the messenger at the surface of goblet cells. Different receptors and therefore different cascades of molecular events were found to control mucus secretion and GAP formation; this explains how the two processes can be performed in parallel and independently from each other. Understanding how cells relay molecules to the immune system is relevant to other tissues in contact with the environment, such as the eyes, the airways, or the inside of the genital and urinary tracts. Understanding, and then ultimately harnessing this mechanism could help design of new ways to deliver drugs to the immune system and alter immune outcomes.

Understanding the Elements of Maternal Protection from Systemic Bacterial Infections during Early Life.

Late-onset sepsis (LOS) and other systemic bloodstream infections are notable causes of neonatal mortality, particularly in prematurely born very low birth weight infants. Breastfeeding in early life has numerous health benefits, impacting the health of the newborn in both the short-term and in the long-term. Though the known benefits of an exclusive mother's own milk diet in early life have been well recognized and described, it is less understood how breastfed infants enjoy a potential reduction in risk of LOS and other systemic infections. Here we review how gut residing pathogens within the intestinal microbiota of infants can cause a subset of sepsis cases and the components of breastmilk that may prevent the dissemination of pathogens from the intestine.

Regulatory T Cells Developing Peri-Weaning Are Continually Required to Restrain Th2 Systemic Responses Later in Life.

Atopic disorders including allergic rhinitis, asthma, food allergy, and dermatitis, are increasingly prevalent in Western societies. These disorders are largely characterized by T helper type 2 (Th2) immune responses to environmental triggers, particularly inhaled and dietary allergens. Exposure to such stimuli during early childhood reduces the frequency of allergies in at-risk children. These allergic responses can be restrained by regulatory T cells (Tregs), particularly Tregs arising in the gut. The unique attributes of how early life exposure to diet and microbes shape the intestinal Treg population is a topic of significant interest. While imprinting during early life promotes the development of a balanced immune system and protects against immunopathology, it remains unclear if Tregs that develop in early life continue to restrain systemic inflammatory responses throughout adulthood. Here, an inducible deletion strategy was used to label Tregs at specified time points with a targeted mechanism to be deleted later. Deletion of the Tregs labeled peri-weaning at day of life 24, but not before weaning at day of life 14, resulted in increased circulating IgE and IL-13, and abrogated induction of tolerance towards new antigens. Thus, Tregs developing peri-weaning, but not before day of life 14 are continually required to restrain allergic responses into adulthood.

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.