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.     

Hypothesis: inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis.

Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity in preterm infants. We hypothesize that the intestinal injury in this disease is a consequence of synergy among three of the major risk factors for NEC: prematurity, enteral feeding, and bacterial colonization. Together these factors result in an exaggerated inflammatory response, leading to ischemic bowel necrosis. Human milk may decrease the incidence of NEC by decreasing pathogenic bacterial colonization, promoting growth of nonpathogenic flora, promoting maturation of the intestinal barrier, and ameliorating the proinflammatory response.     

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.     

Host Responses to Intestinal Microbial Antigens in Gluten-Sensitive Mice

Background and Aims

Excessive uptake of commensal bacterial antigens through a permeable intestinal barrier may influence host responses to specific antigen in a genetically predisposed host. The aim of this study was to investigate whether intestinal barrier dysfunction induced by indomethacin treatment affects the host response to intestinal microbiota in gluten-sensitized HLA-DQ8/HCD4 mice.

Methodology/Principal Findings

HLA-DQ8/HCD4 mice were sensitized with gluten, and gavaged with indomethacin plus gluten. Intestinal permeability was assessed by Ussing chamber; epithelial cell (EC) ultra-structure by electron microscopy; RNA expression of genes coding for junctional proteins by Q-real-time PCR; immune response by in-vitro antigen-specific T-cell proliferation and cytokine analysis by cytometric bead array; intestinal microbiota by fluorescence in situ hybridization and analysis of systemic antibodies against intestinal microbiota by surface staining of live bacteria with serum followed by FACS analysis. Indomethacin led to a more pronounced increase in intestinal permeability in gluten-sensitized mice. These changes were accompanied by severe EC damage, decreased E-cadherin RNA level, elevated IFN-γ in splenocyte culture supernatant, and production of significant IgM antibody against intestinal microbiota.

Conclusion

Indomethacin potentiates barrier dysfunction and EC injury induced by gluten, affects systemic IFN-γ production and the host response to intestinal microbiota antigens in HLA-DQ8/HCD4 mice. The results suggest that environmental factors that alter the intestinal barrier may predispose individuals to an increased susceptibility to gluten through a bystander immune activation to intestinal microbiota.     

Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: a potential role in neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. NEC is believed to occur when intestinal bacteria invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Mucins are produced and secreted by epithelial goblet cells as a key component of the innate immune system and barrier function of the intestinal tract that help protect against bacterial invasion. To better understand the role of mucins in NEC, we quantified the number of mucus-containing small intestinal goblet cells present in infants with NEC and found they had significantly fewer goblet cells and Paneth cells compared with controls. To test whether inflammation has a developmentally dependent effect on intestinal goblet cells, TNF-α was injected into mice at various stages of intestinal development. TNF-α caused a loss of mucus-containing goblet cells only in immature mice and induced Muc2 and Muc3 mRNA upregulation only in mature ileum. Only minimal changes were seen in apoptosis and in expression of markers of goblet cell differentiation. TNF-α increased small intestinal mucus secretion and goblet cell hypersensitivity to prostaglandin E2 (PGE(2)), a known mucus secretagogue produced by macrophages. These TNF-α-induced changes in mucus mRNA levels required TNF receptor 2 (TNFR2), whereas TNF-α-induced loss of mucus-positive goblet cells required TNFR1. Our findings of developmentally dependent TNF-α-induced alterations on intestinal mucus may help explain why NEC is predominantly found in premature infants, and TNF-α-induced alterations of the intestinal innate immune system and barrier functions may play a role in the pathogenesis of NEC itself.     

Neutrophil Elastase Alters the Murine Gut Microbiota Resulting in Enhanced Salmonella Colonization

The intestinal microbiota has been found to play a central role in the colonization of Salmonella enterica serovar Typhimurium in the gastrointestinal tract. In this study, we present a novel process through which Salmonella benefit from inflammatory induced changes in the microbiota in order to facilitate disease. We show that Salmonella infection in mice causes recruitment of neutrophils to the gut lumen, resulting in significant changes in the composition of the intestinal microbiota. This occurs through the production of the enzyme elastase by neutrophils. Administration of recombinant neutrophil elastase to infected animals under conditions that do not elicit neutrophil recruitment caused shifts in microbiota composition that favored Salmonella colonization, while inhibition of neutrophil elastase reduced colonization. This study reveals a new relationship between the microbiota and the host during infection.     

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.     

The Yin and Yang of Bacterial Resilience in the Human Gut Microbiota

The human gut is home to trillions of microbes that form a symbiotic relationship with the human host. During health, the intestinal microbiota provides many benefits to the host and is generally resistant to colonization by new species; however, disruption of this complex community can lead to pathogen invasion, inflammation, and disease. Restoration and maintenance of a healthy gut microbiota composition requires effective therapies to reduce and prevent colonization of harmful bacteria (pathogens) while simultaneously promoting growth of beneficial bacteria (probiotics). Here we review the mechanisms by which the host modulates the gut community composition during health and disease, and we discuss prospects for antibiotic and probiotic therapy for restoration of a healthy intestinal community following disruption.     

Strain competition restricts colonization of an enteric pathogen and prevents colitis

The microbiota is a major source of protection against intestinal pathogens; however, the specific bacteria and underlying mechanisms involved are not well understood. As a model of this interaction, we sought to determine whether colonization of the murine host with symbiotic non‐toxigenic Bacteroides fragilis could limit acquisition of pathogenic enterotoxigenic B. fragilis. We observed strain‐specific competition with toxigenic B. fragilis, dependent upon type VI secretion, identifying an effector–immunity pair that confers pathogen exclusion. Resistance against host acquisition of a second non‐toxigenic strain was also uncovered, revealing a broader function of type VI secretion systems in determining microbiota composition. The competitive exclusion of enterotoxigenic B. fragilis by a non‐toxigenic strain limited toxin exposure and protected the host against intestinal inflammatory disease. Our studies demonstrate a novel role of type VI secretion systems in colonization resistance against a pathogen. This understanding of bacterial competition may be utilized to define a molecularly targeted probiotic strategy.     

Transcriptional Modulation of Intestinal Innate Defense/Inflammation Genes by Preterm Infant Microbiota in a Humanized Gnotobiotic Mouse Model

Background and Aims

It is known that postnatal functional maturation of the small intestine is facilitated by microbial colonization of the gut. Preterm infants exhibit defects in gut maturation, weak innate immunity against intestinal infection and increased susceptibility to inflammatory disorders, all of which may be related to the inappropriate microbial colonization of their immature intestines. The earliest microbes to colonize the preterm infant gut encounter a naïve, immature intestine. Thus this earliest microbiota potentially has the greatest opportunity to fundamentally influence intestinal development and immune function. The aim of this study was to characterize the effect of early microbial colonization on global gene expression in the distal small intestine during postnatal gut development.

Methods

Gnotobiotic mouse models with experimental colonization by early (prior to two weeks of life) intestinal microbiota from preterm human infants were utilized. Microarray analysis was used to assess global gene expression in the intestinal epithelium.

Results and Conclusion

Multiple intestinal genes involved in metabolism, cell cycle regulation, cell-cell or cell-extracellular matrix communication, and immune function are developmental- and intestinal microbiota- regulated. Using a humanized gnotobiotic mouse model, we demonstrate that certain early preterm infant microbiota from prior to 2 weeks of life specifically induce increased NF-κB activation and a phenotype of increased inflammation whereas other preterm microbiota specifically induce decreased NF-κB activation. These fundamental differences correlate with altered clinical outcomes and suggest the existence of optimal early microbial communities to improve health outcomes.     

Longitudinal Analysis of the Premature Infant Intestinal Microbiome Prior to Necrotizing Enterocolitis: A Case-Control Study

Necrotizing enterocolitis (NEC) is an inflammatory disease of the newborn bowel, primarily affecting premature infants. Early intestinal colonization has been implicated in the pathogenesis of NEC. The objective of this prospective case-control study was to evaluate differences in the intestinal microbiota between infants who developed NEC and unaffected controls prior to disease onset. We conducted longitudinal analysis of the 16S rRNA genes of 312 samples obtained from 12 NEC cases and 26 age-matched controls with a median frequency of 7 samples per subject and median sampling interval of 3 days. We found that the microbiome undergoes dynamic development during the first two months of life with day of life being the major factor contributing to the colonization process. Depending on when the infant was diagnosed with NEC (i.e. early vs. late onset), the pattern of microbial progression was different for cases and controls. The difference in the microbiota was most overt in early onset NEC cases and controls. In proximity to NEC onset, the abundances of Clostridium sensu stricto from Clostridia class were significantly higher in early onset NEC subjects comparing to controls. In late onset NEC, Escherichia/Shigella among Gammaproteobacteria, showed an increasing pattern prior to disease onset, and was significantly higher in cases than controls six days before NEC onset. Cronobacter from Gammaproteobacteria was also significantly higher in late onset NEC cases than controls 1-3 days prior to NEC onset. Thus, the specific infectious agent associated with NEC may vary by the age of infant at disease onset. We found that intravenously administered antibiotics may have an impact on the microbial diversity present in fecal material. Longitudinal analysis at multiple time points was an important strategy utilized in this study, allowing us to appreciate the dynamics of the premature infant intestinal microbiome while approaching NEC at various points.     

肠道微生物组与宿主的表观遗传修饰

高通量测序技术已经增加了人们对肠道微生物组和表观遗传学修饰的理解,将肠道微生物组和宿主表观遗传学修饰紧密联系起来,阐明了很多疾病的发生过程如免疫、代谢、心血管疾病甚至是癌症。肠道微生物组与宿主具有相互作用,与人体密不可分,相辅相成。肠道微生物组的生态失调可能诱导疾病的发生并能调控宿主表观遗传学修饰。宿主表观遗传学调控和肠道微生物组(或其代谢产物)变化的相互关系在很多疾病中都有报道。因此,肠道微生物组可作为某些疾病的诊断标记,健康肠道微生物组的移植会逆转这种微生态失调,可作为一种有效的治疗策略。本文主要探讨了肠道微生物组直接调控宿主表观修饰和通过小分子生物活性物质和其他酶辅因子间接影响表观修饰,以及基于肠道微生物组调控宿主表观修饰的诊断和治疗应用等。     

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.     

The roles of bacteria and TLR4 in rat and murine models of necrotizing enterocolitis

Bacteria are thought to contribute to the pathogenesis of necrotizing enterocolitis (NEC), but it is unknown whether their interaction with the epithelium can participate in the initiation of mucosal injury or they can act only following translocation across a damaged intestinal barrier. Our aims were to determine whether bacteria and intestinal epithelial TLR4 play roles in a well-established neonatal rat model and a novel neonatal murine model of NEC. Neonatal rats, C57BL/6J, C3HeB/FeJ (TLR4 wild type), and C3H/HeJ (TLR4 mutant) mice were delivered by Cesarean section and were subjected to formula feeding and cold asphyxia stress or were delivered naturally and were mother-fed. NEC incidence was evaluated by histological scoring, and gene expression was quantified using quantitative real-time PCR from cDNA generated from intestinal total RNA or from RNA obtained by laser capture microdissection. Spontaneous feeding catheter colonization or supplementation of cultured bacterial isolates to formula increased the incidence of experimental NEC. During the first 72 h of life, i.e., the time frame of NEC development in this model, intestinal TLR4 mRNA gradually decreases in mother-fed but increases in formula feeding and cold asphyxia stress, correlating with induced inducible NO synthase. TLR4, inducible NO synthase, and inflammatory cytokine induction occurred in the intestinal epithelium but not in the submucosa. NEC incidence was diminished in C3H/HeJ mice, compared with C3HeB/FeJ mice. In summary, bacteria and TLR4 play significant roles in experimental NEC, likely via an interaction of intraluminal bacteria and aberrantly overexpressed TLR4 in enterocytes.     

The murine caecal microRNA signature depends on the presence of the endogenous microbiota

The intestinal messenger RNA expression signature is affected by the presence and composition of the endogenous microbiota, with effects on host physiology. The intestine is also characterized by a distinctive micronome. However, it is not known if microbes also impact intestinal gene expression epigenetically. We investigated if the murine caecal microRNA expression signature depends on the presence of the microbiota, and the potential implications of this interaction on intestinal barrier function. Three hundred and thirty four microRNAs were detectable in the caecum of germ-free and conventional male mice and 16 were differentially expressed, with samples from the two groups clustering separately based on their expression patterns. Through a combination of computational and gene expression analyses, including the use of our curated list of 527 genes involved in intestinal barrier regulation, 2,755 putative targets of modulated microRNAs were identified, including 34 intestinal barrier-related genes encoding for junctional and mucus layer proteins and involved in immune regulation. This study shows that the endogenous microbiota influences the caecal microRNA expression signature, suggesting that microRNA modulation is another mechanism through which commensal bacteria impact the regulation of the barrier function and intestinal homeostasis. Through microRNAs, the gut microbiota may impinge a much larger number of genes than expected, particularly in diseases where its composition is altered. In this perspective, abnormally expressed microRNAs could be considered as novel therapeutic targets.     

Mucosal gene expression profiles following the colonization of immunocompetent defined-flora C3H mice with Helicobacter bilis: a prelude to typhlocolitis

An aberrant immune response to the commensal microbiota is widely hypothesized to contribute to the development of inflammatory bowel disease. Helicobacter bilis colonization of defined-flora mice has been shown to trigger host immune responses to the commensal flora. However, the magnitude of the effects on mucosal homeostasis following colonization with H. bilis has not been determined. Using microarray analysis, differential gene expression within the cecal mucosa was assessed at 15, 30, or 45 days following H. bilis colonization using Affymetrix Genechips. H. bilis colonization induced marked upregulation of genes associated with protein metabolism, immune responses, and downregulation of genes associated with fatty acid metabolism and detoxification which peaked at 15 days postinfection. A set of genes associated with glycoprotein synthesis and detoxification including Fut2, B3galt5, Ceacam12, Cyp4b1, and Ugt8a were uniquely identified and found to be similarly expressed following the induction of typhlocolitis by dextran sodium sulfate or Brachyspira hyodysenteriae. This study provides preliminary evidence as to the types of factors or changes in the intestinal mucosa that potentially predispose the host to the development of typhlocolitis.     

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.     

Lactobacillus reuteri strains reduce incidence and severity of experimental necrotizing enterocolitis via modulation of TLR4 and NF-κB signaling in the intestine

Necrotizing enterocolitis (NEC) is the leading gastrointestinal cause of mortality and morbidity in the premature infant. Premature infants have a delay in intestinal colonization by commensal bacteria and colonization with potentially pathogenic organisms. Lactobacillus reuteri is a probiotic that inhibits enteric infections, modulates the immune system, and may be beneficial to prevent NEC. In previous studies, L. reuteri strains DSM 17938 and ATCC PTA 4659 differentially modulated inflammation in vitro; however, the strains had equivalent anti-inflammatory responses in LPS feeding-induced ileitis in neonatal rats in vivo. The impact of these two strains in the prevention of NEC has not been previously investigated. NEC was induced in newborn rats by orogastric formula feeding and exposure to hypoxia. L. reuteri was added to the formula to prevent NEC. NEC score, Toll-like receptor (TLR)-signaling genes, phospho-IκB activity, and cytokine levels in the intestine were examined. Both strains significantly increased survival rate and decreased the incidence and severity of NEC, with optimal effects from DSM 17938. In response to probiotic, mRNA expression of IL-6, TNF-α, TLR4, and NF-κB was significantly downregulated, while mRNA levels of anti-inflammatory cytokine IL-10 were significantly upregulated. In parallel, L. reuteri treatment led to decrease intestinal protein levels of TLR4 and cytokine levels of TNF-α and IL-1β in newborn rats with NEC. Both strains significantly inhibited not only intestinal LPS-induced phospho-IκB activity in an ex vivo study but also decreased the levels of phospho-IκB in the intestines of NEC rat model. Cow milk formula feeding produced a similar but milder proinflammatory profile in the intestine that was also ameliorated by 17938. Our studies demonstrate that each of the two L. reuteri strains has potential therapeutic value in our NEC model and in enteritis associated with cow milk feeding. These results support the concept that L. reuteri may represent a valuable treatment to prevent NEC.