Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis

Toll-like receptors (TLRs) play a crucial role in host defense against microbial infection. The microbial ligands recognized by TLRs are not unique to pathogens, however, and are produced by both pathogenic and commensal microorganisms. It is thought that an inflammatory response to commensal bacteria is avoided due to sequestration of microflora by surface epithelia. Here, we show that commensal bacteria are recognized by TLRs under normal steady-state conditions, and this interaction plays a crucial role in the maintenance of intestinal epithelial homeostasis. Furthermore, we find that activation of TLRs by commensal microflora is critical for the protection against gut injury and associated mortality. These findings reveal a novel function of TLRs-control of intestinal epithelial homeostasis and protection from injury-and provide a new perspective on the evolution of host-microbial interactions.     

Leaky guts and lipid rafts

The intestinal epithelium functions as a physical barrier separating luminal microorganisms from the underlying immune system. There is compelling evidence that several intestinal diseases are associated with the translocation of commensal bacteria across the epithelial barrier. Recent work has identified a novel mechanism by which normally non-invasive enteric bacteria breach the intestinal epithelium during periods of inflammation.     

Molecular interactions between bacteria, the epithelium, and the mucosal immune system in the intestinal tract: implications for chronic inflammation

In the last few years, advances in immunology, metabolomics and microbial ecology have shown that the contribution of the intestinal microbiota to the overall health status of the host has been so far underestimated. In this context, intestinal epithelial cells play a crucial role in the maintenance of intestinal homoeostasis. Indeed, at the interface between the luminal content and host tissues, the intestinal epithelium must integrate pro- and anti-inflammatory signals to regulate innate and adaptative immune responses, i.e. to control inflammation. However, under the influence of environmental factors, disturbance of the dialog between enteric bacteria and epithelial cells contributes to the development of chronic inflammation in genetically susceptible hosts. The present review covers the state of knowledge of the host response, especially in intestinal epithelial cells, to enteric bacteria, including colitogenic and probiotic bacteria. It also seeks to give an overview of potential regulatory mechanisms involved in the maintenance of intestinal homeostasis, and discusses the clinical implications for inflammatory bowel diseases.     

Epithelial cells and their neighbors. IV. Bacterial contributions to intestinal epithelial barrier integrity

Mammalian intestinal surfaces are in constant and intimate contact with a vast consortium of indigenous commensal bacteria. As a result, gut epithelia have evolved an array of strategies for limiting bacterial invasion into deeper tissues, helping to preserve the mutually beneficial nature of intestinal host-microbial relationships. In this review, we discuss a growing body of evidence indicating that commensal bacteria are actively involved in shaping the very barriers that confine them to the gut lumen. By modulating epithelial inflammatory responses, antimicrobial protein expression, and tissue repair functions, indigenous microbial populations are essential for the maintenance of healthy mucosal surfaces.     

Stress at the intestinal surface: catecholamines and mucosa–bacteria interactions

Psychological stress has profound effects on gastrointestinal function, and investigations over the past few decades have examined the mechanisms by which neural and hormonal stress mediators act to modulate gut motility, epithelial barrier function and inflammatory states. With its cellular diversity and large commensal bacterial population, the intestinal mucosa and its overlying mucous environment constitute a highly interactive environment for eukaryotic host cells and prokaryotic bacteria. The elaboration of stress mediators, particularly norepinephrine, at this interface influences host cells engaged in mucosal protection and the bacteria which populate the mucosal surface and gut lumen. This review will address growing evidence that norepinephrine and, in some cases, other mediators of the adaptation to stress modulate mucosal interactions with enteric bacteria. Stress-mediated changes in this delicate interplay may shift the microbial colonization patterns on the mucosal surface and alter the susceptibility of the host to infection. Moreover, changes in host-microbe interactions in the digestive tract may also influence ongoing neural activity in stress-responsive brain areas.     

Identification of genes involved in mucosal defense and inflammation associated with normal enteric bacteria

Normal luminal bacteria and their products play a role in experimental colitis and inflammatory bowel disease. However, what molecules from what cells are responsible for mounting and maintaining the mucosal defense against luminal flora is still uncertain. The aim of this study was to identify epithelial gene products involved in mucosal defense and inflammation associated with ubiquitous enteric bacteria. Germ-free ICR mice were given an oral bacterial suspension prepared from conventional components (bacterial reconstitution). Small intestinal and colonic epithelial cells were isolated from bacteria-reconstituted, germ-free, and specific pathogen-free mice. Differential gene expression was investigated by differential display, Northern blot, and sequence analysis. Bacterial reconstitution resulted in acute but self-limited colitis. In epithelial cells, we observed the induction of small intestine-specific genes of the cryptdin family and colon-specific expression of serum amyloid A1 gene. This novel approach allows the identification of known and novel gene products involved in mucosal defense against luminal microorganisms and the associated inflammatory response.     

The role of microbiota in infectious disease

The intestine harbors an ecosystem composed of the intestinal mucosa and the commensal microbiota. The microbiota fosters development, aids digestion and protects host cells from pathogens - a function referred to as colonization resistance. Little is known about the molecular basis of colonization resistance and how it can be overcome by enteropathogenic bacteria. Recently, studies on inflammatory bowel diseases and on animal models for enteric infection have provided new insights into colonization resistance. Gut inflammation changes microbiota composition, disrupts colonization resistance and enhances pathogen growth. Thus, some pathogens can benefit from inflammatory defenses. This new paradigm will enable the study of host factors enhancing or inhibiting bacterial growth in health and disease.     

The intestinal epithelium as guardian of gut barrier integrity

A single layer of epithelial cells separates the intestinal lumen from the underlying sterile tissue. It is exposed to a multitude of nutrients and a large number of commensal bacteria. Although the presence of commensal bacteria significantly contributes to nutrient digestion, vitamin synthesis and tissue maturation, their high number represents a permanent challenge to the integrity of the epithelial surface keeping the local immune system constantly on alert. In addition, the intestinal mucosa is challenged by a variety of enteropathogenic microorganisms. In both circumstances, the epithelium actively contributes to maintaining host–microbial homeostasis and antimicrobial host defence. It deploys a variety of mechanisms to restrict the presence of commensal bacteria to the intestinal lumen and to prevent translocation of commensal and pathogenic microorganisms to the underlying tissue. Enteropathogenic microorganisms in turn have learnt to evade the host's immune system and circumvent the antimicrobial host response. In the present article, we review recent advances that illustrate the intense and intimate host‐microbial interaction at the epithelial level and improve our understanding of the mechanisms that maintain the integrity of the intestinal epithelial barrier.     

Pathophysiology of enteric infections with Giardia duodenalius

Giardia is the most prevalent human intestinal parasitic protist in the world, and one of the most common parasite of companion animals and young livestock. Giardia is a major cause of diarrhea in children and in travelers. The host-microbial interactions that govern the outcome of infection remain incompletely understood. Findings available to date indicate that the infection causes diarrhea via a combination of intestinal malabsorption and hypersecretion. Malabsorption and maldigestion mainly result from a diffuse shortening of epithelial microvilli. This enterocytic injury is mediated by activated host T lymphocytes. Pathophysiological activation of lymphocytes is secondary to Giardia-induced disruption of epithelial tight junctions, which in turn increases intestinal permeability. Loss of epithelial barrier function is a result of Giardia-induced enterocyte apoptosis. Recent findings suggest that these effects may facilitate the development of chronic enteric disorders, including inflammatory bowel disease, irritable bowel syndrome, and allergies, via mechanisms that remain poorly understood. A newly discovered SGLT-1 glucose uptake-mediated host cytoprotective mechanism may represent an effective modulator of the epithelial apoptosis induced by this parasite, and, possibly, by other enteropathogens. A better understanding of the pathogenesis of giardiasis will shed light on new potential therapeutic targets.     

Commensal microbial regulation of natural killer T cells at the frontiers of the mucosal immune system

The commensal microbiota co-exists in a mutualistic relationship with its human host. Commensal microbes play critical roles in the regulation of host metabolism and immunity, while microbial colonization, conversely, is under control of host immunity and metabolic pathways. These interactions are of central importance to the maintenance of homeostasis at mucosal surfaces and their perturbation can provide the basis for atopic and chronic inflammatory diseases such as asthma and inflammatory bowel disease (IBD). Recent evidence has revealed that natural killer T (NKT) cells, a subgroup of T cells which recognizes self and microbial lipid antigens presented by CD1d, are key mediators of host-microbial interactions. Mucosal and systemic NKT cell development is under control of the commensal microbiota, while CD1d regulates microbial colonization and influences the composition of the intestinal microbiota. Here, we outline the mechanisms of bidirectional cross-talk between the microbiota and CD1d-restricted NKT cells and discuss how a perturbation of these processes can contribute to the pathogenesis of immune-mediated disorders at mucosal surfaces.     

TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.     

TLR signaling in the gut in health and disease

The human intestine has evolved in the presence of diverse enteric microflora. TLRs convert the recognition of pathogen-associated molecules in the gut into signals for anti-microbial peptide expression, barrier fortification, and proliferation of epithelial cells. Healing of injured intestinal epithelium and clearance of intramucosal bacteria require the presence of intact TLR signaling. Nucleotide oligomerization domain (Nod)1 and Nod2 are additional pattern recognition receptors that are required for defense against invasive enteric pathogens. Through spatial and functional localization of TLR and Nod molecules, the normal gut maintains a state of controlled inflammation. By contrast, patients with inflammatory bowel disease demonstrate inflammation in response to the normal flora. A subset of these patients carry polymorphisms in TLR and CARD15/NOD2 genes. A better understanding of the delicate regulation of TLR and Nod molecules in the gut may lead to improved treatment for enteric infections and idiopathic inflammatory bowel diseases.     

A New Zebrafish Model of Oro-Intestinal Pathogen Colonization Reveals a Key Role for Adhesion in Protection by Probiotic Bacteria

The beneficial contribution of commensal bacteria to host health and homeostasis led to the concept that exogenous non-pathogenic bacteria called probiotics could be used to limit disease caused by pathogens. However, despite recent progress using gnotobiotic mammal and invertebrate models, mechanisms underlying protection afforded by commensal and probiotic bacteria against pathogens remain poorly understood. Here we developed a zebrafish model of controlled co-infection in which germ-free zebrafish raised on axenic living protozoa enabled the study of interactions between host and commensal and pathogenic bacteria. We screened enteric fish pathogens and identified Edwardsiella ictaluri as a virulent strain inducing a strong inflammatory response and rapid mortality in zebrafish larvae infected by the natural oro-intestinal route. Using mortality induced by infection as a phenotypic read-out, we pre-colonized zebrafish larvae with 37 potential probiotic bacterial strains and screened for survival upon E. ictaluri infection. We identified 3 robustly protective strains, including Vibrio parahaemolyticus and 2 Escherichia coli strains. We showed that the observed protective effect of E. coli was not correlated with a reduced host inflammatory response, nor with the release of biocidal molecules by protective bacteria, but rather with the presence of specific adhesion factors such as F pili that promote the emergence of probiotic bacteria in zebrafish larvae. Our study therefore provides new insights into the molecular events underlying the probiotic effect and constitutes a potentially high-throughput in vivo approach to the study of the molecular basis of pathogen exclusion in a relevant model of vertebrate oro-intestinal infection.     

Luminal host-defense mechanisms against invasive amebiasis

Most humans infected with the virulent protozoan parasite Entamoeba histolytica do not develop invasive disease. Available evidence indicates that beneficial bacteria and the mucus gel layer in the colon lumen protect the host mucosa. Glycosidases produced by some normal colonic bacteria and luminal proteases degrade the key adherence lectin on E. histolytica trophozoites and decrease their adherence to epithelial cells. The mucus gel layer prevents those trophozoites that escape the hydrolases from reaching the epithelial cells. Trophozoite mucosal invasion is triggered only when both protective mechanisms are lost, as might occur during an unrelated pathogenic enteric bacterial infection. A newly developed gnotobiotic model of intestinal amebiasis should enable testing of this hypothesis and provide clues to help design practical studies in humans.     

A role for IL-22 in the relationship between intestinal helminths,gut microbiota and mucosal immunity

The intestinal tract is home to nematodes as well as commensal bacteria (microbiota), which have coevolved with the mammalian host. The mucosal immune system must balance between an appropriate response to dangerous pathogens and an inappropriate response to commensal microbiota that may breach the epithelial barrier, in order to maintain intestinal homeostasis. IL-22 has been shown to play a critical role in maintaining barrier homeostasis against intestinal pathogens and commensal bacteria. Here we review the advances in our understanding of the role of IL-22 in helminth infections, as well as in response to commensal and pathogenic bacteria of the intestinal tract. We then consider the relationship between intestinal helminths and gut microbiota and hypothesize that this relationship may explain how helminths may improve symptoms of inflammatory bowel diseases. We propose that by inducing an immune response that includes IL-22, intestinal helminths may enhance the mucosal barrier function of the intestinal epithelium. This may restore the mucosal microbiota populations from dysbiosis associated with colitis and improve intestinal homeostasis.     

The protective roles of NLRP6 in intestinal epithelial cells

The evolution of chronic inflammatory diseases is thought to be due to a combination of host genetic variations and environmental factors that include the alteration of intestinal flora, termed “dysbiosis.” The intestinal mucosal barrier includes a chemical barrier and physical barrier that have important roles in protecting the intestine against inflammatory injury. The chemical barrier includes antimicrobial peptides (AMPs), and the physical barrier includes a mucous layer, a monolayer of intestinal epithelial cells and cell junctions. The intestinal mucosal barrier is not a static barrier, but rather, it strongly interacts with the gut microbiome and cells of the immune system. Correct expression of AMPs, together with mucus and balanced epithelial cell proliferation, prevents the occurrence of disease. NLRP6, a member of the nucleotide‐binding domain, leucine‐rich repeat‐containing (NLR) innate immune receptor family, participates in the progression of intestinal inflammation and enteric pathogen infections. It has become apparent in recent years that NLRP6 is important in disease pathogenesis, as it responds to internal ligands that lead to the release of AMPs and mucus, thus regulating the regeneration of intestinal epithelial cells. This review summarizes the activation of NLRP6 and its protective role in the intestinal epithelial cell.     

Carcinoembryonic antigen (CEACAM) family members and Inflammatory Bowel Disease

Inflammatory bowel disease (IBD), encompassing Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic intestinal inflammatory condition with increasing incidence worldwide and whose pathogenesis remains largely unknown. The collected evidence indicates that genetic, environmental and microbial factors and a dysregulated immune response are responsible for the disease. IBD has an early onset and long term sufferers present a higher risk of developing colitis associated cancer (CAC). The carcinoembryonic antigen-related adhesion molecules (CEACAM) are a subgroup of the CEA family, found in a range of different cell types and organs including epithelial cells in the intestine. They can act as intercellular adhesions molecules for e.g. bacteria and soluble antigens. CEACAMs are involved in a number of different processes including cell adhesion, proliferation, differentiation and tumour suppression. Some CEACAMs such as CEACAM1, CEACAM5 and CEACAM6 are highly associated with cancer and are even recognised as valid clinical markers for certain cancer forms. However, their role in IBD pathogenesis is less understood. The purpose of this review is to provide a comprehensive summary of published literature on CEACAMs and intestinal inflammation (IBD). The interactions between CEACAMs and bacteria adhesion in relation to IBD pathophysiology will be addressed and potential new therapeutic and diagnostic opportunities will be identified.