Cryptic activation of an Irf8 enhancer governs cDC1 fate specification.

Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification.

Commensal Cryptosporidium colonization elicits a cDC1-dependent Th1 response that promotes intestinal homeostasis and limits other infections.

Cryptosporidium can cause severe diarrhea and morbidity, but many infections are asymptomatic. Here, we studied the immune response to a commensal strain of Cryptosporidium tyzzeri (Ct-STL) serendipitously discovered when conventional type 1 dendritic cell (cDC1)-deficient mice developed cryptosporidiosis. Ct-STL was vertically transmitted without negative health effects in wild-type mice. Yet, Ct-STL provoked profound changes in the intestinal immune system, including induction of an IFN-γ-producing Th1 response. TCR sequencing coupled with in vitro and in vivo analysis of common Th1 TCRs revealed that Ct-STL elicited a dominant antigen-specific Th1 response. In contrast, deficiency in cDC1s skewed the Ct-STL CD4 T cell response toward Th17 and regulatory T cells. Although Ct-STL predominantly colonized the small intestine, colon Th1 responses were enhanced and associated with protection against Citrobacter rodentium infection and exacerbation of dextran sodium sulfate and anti-IL10R-triggered colitis. Thus, Ct-STL represents a commensal pathobiont that elicits Th1-mediated intestinal homeostasis that may reflect asymptomatic human Cryptosporidium infection.

High-fat diet prevents the development of autoimmune diabetes in NOD mice.

AIMS: Type 1 diabetes (T1D) has a strong genetic predisposition and requires an environmental trigger to initiate the beta-cell autoimmune destruction. The rate of childhood obesity has risen in parallel to the proportion of T1D, suggesting high-fat diet (HFD)/obesity as potential environmental triggers for autoimmune diabetes. To explore this, non-obese diabetic (NOD) mice were subjected to HFD and monitored for the development of diabetes, insulitis and beta-cell stress. MATERIALS AND METHODS: Four-week-old female NOD mice were placed on HFD (HFD-NOD) or standard chow-diet. Blood glucose was monitored weekly up to 40 weeks of age, and glucose- and insulin-tolerance tests performed at 4, 10 and 15 weeks. Pancreata and islets were analysed for insulin secretion, beta-cell mass, inflammation, insulitis and endoplasmic reticulum stress markers. Immune cell levels were measured in islets and spleens. Stool microbiome was analysed at age 4, 8 and 25 weeks. RESULTS: At early ages, HFD-NOD mice showed a significant increase in body weight, glucose intolerance and insulin resistance; but paradoxically, they were protected from developing diabetes. This was accompanied by increased insulin secretion and beta-cell mass, decreased insulitis, increased splenic T-regulatory cells and altered stool microbiome. CONCLUSIONS: This study shows that HFD protects NOD mice from autoimmune diabetes and preserves beta-cell mass and function through alterations in gut microbiome, increased T-regulatory cells and decreased insulitis. Further studies into the exact mechanism of HFD-mediated prevention of diabetes in NOD mice could potentially lead to interventions to prevent or delay T1D development in humans.

Yersinia pseudotuberculosis supports Th17 differentiation and limits de novo regulatory T cell induction by directly interfering with T cell receptor signaling.

Adaptive immunity critically contributes to control acute infection with enteropathogenic Yersinia pseudotuberculosis; however, the role of CD4+ T cell subsets in establishing infection and allowing pathogen persistence remains elusive. Here, we assessed the modulatory capacity of Y. pseudotuberculosis on CD4+ T cell differentiation. Using in vivo assays, we report that infection with Y. pseudotuberculosis resulted in enhanced priming of IL-17-producing T cells (Th17 cells), whereas induction of Foxp3+ regulatory T cells (Tregs) was severely disrupted in gut-draining mesenteric lymph nodes (mLNs), in line with altered frequencies of tolerogenic and proinflammatory dendritic cell (DC) subsets within mLNs. Additionally, by using a DC-free in vitro system, we could demonstrate that Y. pseudotuberculosis can directly modulate T cell receptor (TCR) downstream signaling within naïve CD4+ T cells and Tregs via injection of effector molecules through the type III secretion system, thereby affecting their functional properties. Importantly, modulation of naïve CD4+ T cells by Y. pseudotuberculosis resulted in an enhanced Th17 differentiation and decreased induction of Foxp3+ Tregs in vitro. These findings shed light to the adjustment of the Th17-Treg axis in response to acute Y. pseudotuberculosis infection and highlight the direct modulation of CD4+ T cell subsets by altering their TCR downstream signaling.

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.

The cytotoxic necrotizing factor of Yersinia pseudotuberculosis (CNFY) enhances inflammation and Yop delivery during infection by activation of Rho GTPases.

Some isolates of Yersinia pseudotuberculosis produce the cytotoxic necrotizing factor (CNFY), but the functional consequences of this toxin for host-pathogen interactions during the infection are unknown. In the present study we show that CNFY has a strong influence on virulence. We demonstrate that the CNFY toxin is thermo-regulated and highly expressed in all colonized lymphatic tissues and organs of orally infected mice. Most strikingly, we found that a cnfY knock-out variant of a naturally toxin-expressing Y. pseudotuberculosis isolate is strongly impaired in its ability to disseminate into the mesenteric lymph nodes, liver and spleen, and has fully lost its lethality. The CNFY toxin contributes significantly to the induction of acute inflammatory responses and to the formation of necrotic areas in infected tissues. The analysis of the host immune response demonstrated that presence of CNFY leads to a strong reduction of professional phagocytes and natural killer cells in particular in the spleen, whereas loss of the toxin allows efficient tissue infiltration of these immune cells and rapid killing of the pathogen. Addition of purified CNFY triggers formation of actin-rich membrane ruffles and filopodia, which correlates with the activation of the Rho GTPases, RhoA, Rac1 and Cdc42. The analysis of type III effector delivery into epithelial and immune cells in vitro and during the course of the infection further demonstrated that CNFY enhances the Yop translocation process and supports a role for the toxin in the suppression of the antibacterial host response. In summary, we highlight the importance of CNFY for pathogenicity by showing that this toxin modulates inflammatory responses, protects the bacteria from attacks of innate immune effectors and enhances the severity of a Yersinia infection.

Local complement activation and modulation in mucosal immunity.

The complement system is an evolutionarily conserved arm of innate immunity, which forms one of the first lines of host response to pathogens and assists in the clearance of debris. A deficiency in key activators/amplifiers of the cascade results in recurrent infection, whereas a deficiency in regulating the cascade predisposes to accelerated organ failure, as observed in colitis and transplant rejection. Given that there are over 60 proteins in this system, it has become an attractive target for immunotherapeutics, many of which are United States Food and Drug Administration-approved or in multiple phase 2/3 clinical trials. Moreover, there have been key advances in the last few years in the understanding of how the complement system operates locally in tissues, independent of its activities in circulation. In this review, we will put into perspective the abovementioned discoveries to optimally modulate the spatiotemporal nature of complement activation and regulation at mucosal surfaces.

Small Intestinal Goblet Cells Control Humoral Immune Responses and Mobilization During Enteric Infection.

Humoral immune responses within the gut play diverse roles including pathogen clearance during enteric infections, maintaining tolerance, and facilitating the assemblage and stability of the gut microbiota. How these humoral immune responses are initiated and contribute to these processes are well studied. However, the signals promoting the expansion of these responses and their rapid mobilization to the gut mucosa are less well understood. Intestinal goblet cells form goblet cell-associated antigen passages (GAPs) to deliver luminal antigens to the underlying immune system and facilitate tolerance. GAPs are rapidly inhibited during enteric infection to prevent inflammatory responses to innocuous luminal antigens. Here we interrogate GAP inhibition as a key physiological response required for effective humoral immunity. Independent of infection, GAP inhibition resulted in enrichment of transcripts representing B cell recruitment, expansion, and differentiation into plasma cells in the small intestine (SI), which were confirmed by flow cytometry and ELISpot assays. Further we observed an expansion of isolated lymphoid follicles within the SI, as well as expansion of plasma cells in the bone marrow upon GAP inhibition. S1PR1-induced blockade of leukocyte trafficking during GAP inhibition resulted in a blunting of SI plasma cell expansion, suggesting that mobilization of plasma cells from the bone marrow contributes to their expansion in the gut. However, luminal IgA secretion was only observed in the presence of S. typhimurium infection, suggesting that although GAP inhibition mobilizes a mucosal humoral immune response, a second signal is required for full effector function. Overriding GAP inhibition during enteric infection abrogated the expansion of laminar propria IgA+ plasma cells. We conclude that GAP inhibition is a required physiological response for efficiently mobilizing mucosal humoral immunity in response to enteric infection.

Vancomycin-induced gut microbial dysbiosis alters enteric neuron-macrophage interactions during a critical period of postnatal development.

Vancomycin is a broad-spectrum antibiotic widely used in cases of suspected sepsis in premature neonates. While appropriate and potentially lifesaving in this setting, early-life antibiotic exposure alters the developing microbiome and is associated with an increased risk of deadly complications, including late-onset sepsis (LOS) and necrotizing enterocolitis (NEC). Recent studies show that neonatal vancomycin treatment disrupts postnatal enteric nervous system (ENS) development in mouse pups, which is in part dependent upon neuroimmune interactions. This suggests that early-life antibiotic exposure could disrupt these interactions in the neonatal gut. Notably, a subset of tissue-resident intestinal macrophages, muscularis macrophages, has been identified as important contributors to the development of postnatal ENS. We hypothesized that vancomycin-induced neonatal dysbiosis impacts postnatal ENS development through its effects on macrophages. Using a mouse model, we found that exposure to vancomycin in the first 10 days of life, but not in adult mice, resulted in an expansion of pro-inflammatory colonic macrophages by increasing the recruitment of bone-marrow-derived macrophages. Single-cell RNA sequencing of neonatal colonic macrophages revealed that early-life vancomycin exposure was associated with an increase in immature and inflammatory macrophages, consistent with an influx of circulating monocytes differentiating into macrophages. Lineage tracing confirmed that vancomycin significantly increased the non-yolk-sac-derived macrophage population. Consistent with these results, early-life vancomycin exposure did not expand the colonic macrophage population nor decrease enteric neuron density in CCR2-deficient mice. Collectively, these findings demonstrate that early-life vancomycin exposure alters macrophage number and phenotypes in distinct ways compared with vancomycin exposure in adult mice and results in altered ENS development.

Lung epithelial cell-derived C3 protects against pneumonia-induced lung injury.

The complement component C3 is a fundamental plasma protein for host defense, produced largely by the liver. However, recent work has demonstrated the critical importance of tissue-specific C3 expression in cell survival. Here, we analyzed the effects of local versus peripheral sources of C3 expression in a model of acute bacterial pneumonia induced by Pseudomonas aeruginosa. Whereas mice with global C3 deficiency had severe pneumonia-induced lung injury, those deficient only in liver-derived C3 remained protected, comparable to wild-type mice. Human lung transcriptome analysis showed that secretory epithelial cells, such as club cells, express high levels of C3 mRNA. Mice with tamoxifen-induced C3 gene ablation from club cells in the lung had worse pulmonary injury compared with similarly treated controls, despite maintaining normal circulating C3 levels. Last, in both the mouse pneumonia model and cultured primary human airway epithelial cells, we showed that stress-induced death associated with C3 deficiency parallels that seen in Factor B deficiency rather than C3a receptor deficiency. Moreover, C3-mediated reduction in epithelial cell death requires alternative pathway component Factor B. Thus, our findings suggest that a pathway reliant on locally derived C3 and Factor B protects the lung mucosal barrier.

Gut microbiota induces weight gain and inflammation in the gut and adipose tissue independent of manipulations in diet, genetics, and immune development.

Obesity and the metabolic syndrome are complex disorders resulting from multiple factors including genetics, diet, activity, inflammation, and gut microbes. Animal studies have identified roles for each of these, however the contribution(s) specifically attributed to the gut microbiota remain unclear, as studies have used combinations of genetically altered mice, high fat diet, and/or colonization of germ-free mice, which have an underdeveloped immune system. We investigated the role(s) of the gut microbiota driving obesity and inflammation independent of manipulations in diet and genetics in mice with fully developed immune systems. We demonstrate that the human obese gut microbiota alone was sufficient to drive weight gain, systemic, adipose tissue, and intestinal inflammation, but did not promote intestinal barrier leak. The obese microbiota induced gene expression promoting caloric uptake/harvest but was less effective at inducing genes associated with mucosal immune responses. Thus, the obese gut microbiota is sufficient to induce weight gain and inflammation.

Emerging roles of the complement system in host-pathogen interactions.

The complement system has historically been entertained as a fluid-phase, hepatically derived system which protects the intravascular space from encapsulated bacteria. However, there has been an increasing appreciation for its role in protection against non-encapsulated pathogens. Specifically, we have an improved understanding of how pathogens are recognized by specific complement proteins, as well as how they trigger and evade them. Additionally, we have an improved understanding of locally derived complement proteins, many of which promote host defense. Moreover, intracellular complement proteins have been identified that facilitate local protection and barrier function despite pathogen invasion. Our review aims to summarize these advances in the field as well as provide an insight into the pathophysiological changes occurring when the system is dysregulated in infection.

Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens.

Trophoblast organoids derived from placental villi provide a 3D model system of human placental development, but access to first-trimester tissues is limited. Here, we report that trophoblast stem cells isolated from naive human pluripotent stem cells (hPSCs) can efficiently self-organize into 3D stem-cell-derived trophoblast organoids (SC-TOs) with a villous architecture similar to primary trophoblast organoids. Single-cell transcriptome analysis reveals the presence of distinct cytotrophoblast and syncytiotrophoblast clusters and a small cluster of extravillous trophoblasts, which closely correspond to trophoblast identities in the post-implantation embryo. These organoid cultures display clonal X chromosome inactivation patterns previously described in the human placenta. We further demonstrate that SC-TOs exhibit selective vulnerability to emerging pathogens (SARS-CoV-2 and Zika virus), which correlates with expression levels of their respective entry factors. The generation of trophoblast organoids from naive hPSCs provides an accessible 3D model system of the developing placenta and its susceptibility to emerging pathogens.

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.

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.

Synchronization of mothers and offspring promotes tolerance and limits allergy.

Allergic disorders, characterized by Th2 immune responses to environmental substances, are increasingly common in children in Western societies. Multiple studies indicate that breastfeeding, early complementary introduction of food allergens, and antibiotic avoidance in the first year of life reduces allergic outcomes in at-risk children. Why the benefit of these practices is restricted to early life is largely unknown. We identified a preweaning interval during which dietary antigens are assimilated by the colonic immune system. This interval is under maternal control via temporal changes in breast milk, coincides with an influx of naive T cells into the colon, and is followed by the development of a long-lived population of colonic peripherally derived Tregs (pTregs) that can be specific for dietary antigens encountered during this interval. Desynchronization of mothers and offspring produced durable deficits in these pTregs, impaired tolerance to dietary antigens introduced during and after this preweaning interval, and resulted in spontaneous Th2 responses. These effects could be rescued by pTregs from the periweaning colon or by Tregs generated in vitro using periweaning colonic antigen-presenting cells. These findings demonstrate that mothers and their offspring are synchronized for the development of a balanced immune system.

Intestinal Macromolecular Transport Supporting Adaptive Immunity.

The gastrointestinal tract performs opposing functions of nutrient absorption, barrier maintenance, and the delivery of luminal substances for the appropriate induction of tolerogenic or protective adaptive immunity. The single-layer epithelium lining the gastrointestinal tract is central to each of these functions by facilitating the uptake and processing of nutrients, providing a physical and chemical barrier to potential pathogens, and delivering macromolecular substances to the immune system to initiate adaptive immune responses. Specific transport mechanisms allow nutrient uptake and the delivery of macromolecules to the immune system while maintaining the epithelial barrier. This review examines historical observations supporting macromolecular transport by the intestinal epithelium, recent insights into the transport of luminal macromolecules to promote adaptive immunity, and how this process is regulated to promote appropriate immune responses. Understanding how luminal macromolecules are delivered to the immune system and how this is regulated may provide insight into the pathophysiology of inflammatory diseases of the gastrointestinal tract and potential preventative or therapeutic strategies.