Shuttling of information between the mucosal and luminal environment drives intestinal homeostasis

The gastrointestinal tract is a passageway for dietary nutrients, microorganisms and xenobiotics. The gut is home to diverse bacterial communities forming the microbiota. While bacteria and their metabolites maintain gut homeostasis, the host uses innate and adaptive immune mechanisms to cope with the microbiota and luminal environment. In recent years, multiple bi-directional instructive mechanisms between microbiota, luminal content and mucosal immune systems have been uncovered. Indeed, epithelial and immune cell-derived mucosal signals shape microbiota composition, while microbiota and their by-products shape the mucosal immune system. Genetic and environmental perturbations alter gut mucosal responses which impact on microbial ecology structures. On the other hand, changes in microbiota alter intestinal mucosal responses. In this review, we discuss how intestinal epithelial Paneth and goblet cells interact with the microbiota, how environmental and genetic disorders are sensed by endoplasmic reticulum stress and autophagy responses, how specific bacteria, bacterial- and diet-derived products determine the function and activation of the mucosal immune system. We will also discuss the critical role of HDAC activity as a regulator of immune and epithelial cell homeostatic responses.     

Activation of Toll-like receptor 2 on human tracheobronchial epithelial cells induces the antimicrobial peptide human beta defensin-2

As pattern recognition receptors capable of eliciting responses to a diverse array of microbial products, Toll-like receptors (TLRs) participate in the activation of host defense mechanisms that protect against infectious pathogens. Given that epithelial cells lie at the interface between the host and its environment, we designed experiments to determine whether human airway epithelial cells express TLRs and respond to TLR agonists. Immunohistochemical labeling of TLR2 in normal human airways revealed TLR2 expression throughout the epithelium, with an apparently higher level of expression on noncolumnar basal epithelial cells. Two-color immunofluorescent labeling of TLR2 and cytokeratins 8 and 15 revealed that TLR2 is coexpressed with the epithelial cell markers. In addition, airway epithelial cells grown at air-liquid interface responded to bacterial lipopeptide in a TLR2-dependent manner with induction of mRNA and protein of the antimicrobial peptide human beta defensin-2. Stimulation of epithelial cell cultures with lipopeptide resulted in a small and variable reduction of bacteria on the apical surface. Together, these data suggest that TLRs monitor epithelial surfaces to enhance host defense by inducing the production of an antimicrobial peptide.     

Microbial responses to microgravity and other low-shear environments.

Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.     

The immunomodulatory properties of probiotic microorganisms beyond their viability (ghost probiotics: proposal of paraprobiotic concept)

The probiotic approach represents a potentially effective and mild alternative strategy for the prevention and treatment of either inflammatory or allergic diseases. Several studies have shown that different bacterial strains can exert their probiotic abilities by influencing the host's immune system, thereby modulating immune responses. However, the emerging concern regarding safety problems arising from the extensive use of live microbial cells is enhancing the interest in non-viable microorganisms or microbial cell extracts, as they could eliminate shelf-life problems and reduce the risks of microbial translocation and infection. The purpose of this review is to provide an overview of the scientific literature concerning studies in which dead microbial cells or crude microbial cell fractions have been used as health-promoting agents. Particular attention will be given to the modulation of host immune responses. Possible mechanisms determining the effect on the immune system will also be discussed. Finally, in the light of the FAO/WHO definition of probiotics, indicating that the word 'probiotic' should be restricted to products that contain live microorganisms, and considering the scientific evidence indicating that inactivated microbes can positively affect human health, we propose the new term 'paraprobiotic' to indicate the use of inactivated microbial cells or cell fractions to confer a health benefit to the consumer.     

Events at the host-microbial interface of the gastrointestinal tract. II. Role of the intestinal epithelium in pathogen-induced inflammation

An immense number of bacteria reside within the intestinal lumen. The task of appropriately identifying and responding to microbial threats lies primarily with the single layer of cells that line the intestinal tract. Intestinal epithelial cells have developed a number of strategies aimed at identifying microorganisms and eliciting the appropriate inflammatory response. The pathogen recognition mechanisms and the signaling and inflammatory events that ensue within the intestine are the focus of this review.     

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.     

Immunophysiology of epithelial cells and pattern-recognition receptors

Many types of pattern-recognition receptors, including the group of signaling Toll-like receptors, have been found in epithelial cells. They serve for recognizing microorganisms, which induces the activation of epithelial cells followed by production of chemokines and antimicrobial substances. On the one hand, this attracts macrophages, neutrophils, and other cells of innate immunity eliminating microorganisms to the epithelial locus. The same pattern-recognition receptors are involved in cell activation resulting in the production of cytokines that trigger the controlled activation of adaptive-immunity cells. This leads to the formation of anti-bodies or cytotoxic T lymphocytes. On the other hand, activated epithelial cells produce an array of antimicrobial substances suppressing enteropathogenic microflora inhabiting external epithelial spaces. Conversely, local tolerance is developed with respect to predominant microflora of epithelial tracts. This tolerance encompasses both innate and adaptive immunities of subepithelial spaces. Although much remains to be understood regarding the mechanisms of its formation and maintenance, it is obvious that epithelial cells play an important role in these processes. Thus, epithelial cells “conduct” immune responses to both pathogenic microorganisms penetrating the host body and microorganisms inhabiting epithelial cellular spaces. The maintenance of the optimal composition of the microbial biocenosis of the human body is a function of its immune system.     

Toll-like receptors and innate immunity

Toll-like receptors have a crucial role in the detection of microbial infection in mammals and insects. In mammals, these receptors have evolved to recognize conserved products unique to microbial metabolism. This specificity allows the Toll proteins to detect the presence of infection and to induce activation of inflammatory and antimicrobial innate immune responses. Recognition of microbial products by Toll-like receptors expressed on dendritic cells triggers functional maturation of dendritic cells and leads to initiation of antigen-specific adaptive immune responses.     

Damaged Intestinal Epithelial Integrity Linked to Microbial Translocation in Pathogenic Simian Immunodeficiency Virus Infections

The chronic phase of HIV infection is marked by pathological activation of the immune system, the extent of which better predicts disease progression than either plasma viral load or CD4⁺ T cell count. Recently, translocation of microbial products from the gastrointestinal tract has been proposed as an underlying cause of this immune activation, based on indirect evidence including the detection of microbial products and specific immune responses in the plasma of chronically HIV-infected humans or SIV-infected Asian macaques. We analyzed tissues from SIV-infected rhesus macaques (RMs) to provide direct in situ evidence for translocation of microbial constituents from the lumen of the intestine into the lamina propria and to draining and peripheral lymph nodes and liver, accompanied by local immune responses in affected tissues. In chronically SIV-infected RMs this translocation is associated with breakdown of the integrity of the epithelial barrier of the gastrointestinal (GI) tract and apparent inability of lamina propria macrophages to effectively phagocytose translocated microbial constituents. By contrast, in the chronic phase of SIV infection in sooty mangabeys, we found no evidence of epithelial barrier breakdown, no increased microbial translocation and no pathological immune activation. Because immune activation is characteristic of the chronic phase of progressive HIV/SIV infections, these findings suggest that increased microbial translocation from the GI tract, in excess of capacity to clear the translocated microbial constituents, helps drive pathological immune activation. Novel therapeutic approaches to inhibit microbial translocation and/or attenuate chronic immune activation in HIV-infected individuals may complement treatments aimed at direct suppression of viral replication.     

Microbial Responses to Microgravity and Other Low-Shear Environments

Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.     

Modulation of neonatal microbial recognition: TLR-mediated innate immune responses are specifically and differentially modulated by human milk

The mechanisms controlling innate microbial recognition in the neonatal gut are still to be fully understood. We have sought specific regulatory mechanisms operating in human breast milk relating to TLR-mediated microbial recognition. In this study, we report a specific and differential modulatory effect of early samples (days 1-5) of breast milk on ligand-induced cell stimulation via TLRs. Although a negative modulation was exerted on TLR2 and TLR3-mediated responses, those via TLR4 and TLR5 were enhanced. This effect was observed in human adult and fetal intestinal epithelial cell lines, monocytes, dendritic cells, and PBMC as well as neonatal blood. In the latter case, milk compensated for the low capacity of neonatal plasma to support responses to LPS. Cell stimulation via the IL-1R or TNFR was not modulated by milk. This, together with the differential effect on TLR activation, suggested that the primary effect of milk is exerted upstream of signaling proximal to TLR ligand recognition. The analysis of TLR4-mediated gene expression, used as a model system, showed that milk modulated TLR-related genes differently, including those coding for signal intermediates and regulators. A proteinaceous milk component of > or =80 kDa was found to be responsible for the effect on TLR4. Notably, infant milk formulations did not reproduce the modulatory activity of breast milk. Together, these findings reveal an unrecognized function of human milk, namely, its capacity to influence neonatal microbial recognition by modulating TLR-mediated responses specifically and differentially. This in turn suggests the existence of novel mechanisms regulating TLR activation.     

Salmonella Typhimurium Type III Secretion Effectors Stimulate Innate Immune Responses in Cultured Epithelial Cells

Recognition of conserved bacterial products by innate immune receptors leads to inflammatory responses that control pathogen spread but that can also result in pathology. Intestinal epithelial cells are exposed to bacterial products and therefore must prevent signaling through innate immune receptors to avoid pathology. However, enteric pathogens are able to stimulate intestinal inflammation. We show here that the enteric pathogen Salmonella Typhimurium can stimulate innate immune responses in cultured epithelial cells by mechanisms that do not involve receptors of the innate immune system. Instead, S. Typhimurium stimulates these responses by delivering through its type III secretion system the bacterial effector proteins SopE, SopE2, and SopB, which in a redundant fashion stimulate Rho-family GTPases leading to the activation of mitogen-activated protein (MAP) kinase and NF-κB signaling. These observations have implications for the understanding of the mechanisms by which Salmonella Typhimurium induces intestinal inflammation as well as other intestinal inflammatory pathologies.     

Exploring host-pathogen interactions at the epithelial surface: application of transcriptomics in lung biology

The epithelial surface of the airways is the largest barrier-forming interface between the human body and the outside world. It is now well recognized that, at this strategic position, airway epithelial cells play an eminent role in host defense by recognizing and responding to microbial exposure. Conversely, inhaled microorganisms also respond to contact with epithelial cells. Our understanding of this cross talk is limited, requiring sophisticated experimental approaches to analyze these complex interactions. High-throughput technologies, such as DNA microarray analysis and serial analysis of gene expression (SAGE), have been developed to screen for gene expression levels at large scale within single experiments. Since their introduction, these hypothesis-generating technologies have been widely used in diverse areas such as oncology and brain research. Successful application of these genomics-based technologies has also revealed novel insights in host-pathogen interactions in both the host and pathogen. This review aims to provide an overview of the SAGE and microarray technology illustrated by their application in the analysis of host-pathogen interactions. In particular, the interactions between epithelial cells in the human lungs and clinically relevant microorganisms are the central focus of this review.     

Adrenomedullin expression in pathogen-challenged oral epithelial cells

Adrenomedullin, a multifunctional peptide, is expressed by many surface epithelial cells and, previously, we have demonstrated that adrenomedullin has antimicrobial activity. The oral cavity contains an epithelium that is permanently colonized by microflora, yet infections in a host are rare. We exposed oral keratinocytes to whole, live cells from four microorganisms commonly isolated from the oral cavity, Porphyromonas gingivalis, Streptococcus mutans, Candida albicans and Eikenella corrodens. There was upregulation of protein and gene expression in these cells in response to bacterial suspensions, but not with the yeast, Candida albicans. We propose there is a potential role for microbial products in enhancing mucosal defense mechanisms and that adrenomedullin participates in the prevention of local infection, thus contributing to host defense mechanisms.     

Type 1 fimbriae deliver an LPS- and TLR4-dependent activation signal to CD14-negative cells

Fimbriae target bacteria to different mucosal surfaces and enhance the inflammatory response at these sites. Inflammation may be triggered by the fimbriae themselves or by fimbriae-dependent delivery of other host activating molecules such as lipopolysaccharide (LPS). Although LPS activates systemic inflammation through the CD14 and Toll-like receptor 4 (TLR4) pathways, mechanisms of epithelial cell activation by LPS are not well understood. These cells lack CD14 receptors and are unresponsive to pure LPS, but fimbriated Escherichia coli overcome this refractoriness and trigger epithelial cytokine responses. We now show that type 1 fimbriae can present an LPS- and TLR4-dependent signal to the CD14-negative epithelial cells. Human uroepithelial cells were shown to express TLR4, and type 1 fimbriated E. coli strains triggered an LPS-dependent response in those cells. A similar LPS- and fimbriae-dependent response was observed in the urinary tract of TLR4-proficient mice, but not in TLR4-defective mice. The moderate inflammatory response in the TLR4-defective mice was fimbriae dependent but LPS independent. The results demonstrate that type 1 fimbriae present LPS to CD14-negative cells and that the TLR4 genotype determines this response despite the absence of CD14 on the target cells. The results illustrate how the host "sees" LPS and other microbial products not as purified molecules but as complexes, and that fimbriae determine the molecular context in which LPS is presented to host cells.     

Escherichia coli P fimbriae utilize the Toll-like receptor 4 pathway for cell activation

Fimbriae mediate bacterial attachment to host cells and provide a mechanism for tissue attack. They activate a host response by delivery of microbial products such as lipopolysaccharide (LPS) or through direct fimbriae-dependent signalling mechanisms. By coupling to glycosphingolipid (GSL) receptors, P fimbriae trigger cytokine responses in CD14 negative host cells. Here we show that P fimbriae utilize the Toll-like receptor 4 (TLR4)-dependent pathway to trigger mucosal inflammation. Escherichia coli strains expressing P fimbriae as their only virulence factor stimulated chemokine and neutrophil responses in the urinary tract of TLR4 proficient mice, but TLR4 defective mice failed to respond to infection. Mucosal cells were CD14 negative but expressed several TLR species including TLR4, and TLR4 protein was detected. Infection with P fimbriated bacteria stimulated an increase in TLR4 mRNA levels. The activation signal did not involve the LPS-CD14 pathway and was independent of lipid A myristoylation, as shown by mutational inactivation of the msbB gene. Co-staining experiments revealed that TLR4 and the GSL receptors for P fimbriae co-localized in the cell membrane. The results demonstrate that P fimbriae activate epithelial cells by means of a TLR4-dependent signalling pathway, and suggest that GSL receptors for P fimbriae can recruit TLR4 as co-receptors.