Mutual regulation between butyrate and hypoxia‐inducible factor‐1α in epithelial cell promotes expression of tight junction proteins

Inflammatory bowel disease is a kind of multi‐aetiological chronic disease that is driven by multidimensional factors. Hypoxia‐inducible factor‐1α (HIF‐1α) plays an important role in anti‐inflammatory and cellular responses to hypoxia. Previous studies have found that B or T‐cell‐specific HIF‐1α knock out mice exhibit severe colonic inflammation. However, we know very little about other functions of HIF‐1α in intestinal epithelial cells (IECs). In our study, HIF‐1α^ΔIEC mice were used to study the function of HIF‐1α in IECs. HIF‐1α was knocked down in Caco‐2 cells by transfection with a small interfering (si) RNA. Immunohistochemical staining and western blotting were used to detect the expression of zonula occluden‐1 (ZO‐1) and Occludin. The content of colon was harvested for high‐performance liquid chromatography analysis to examine the levels of butyrate in the gut. Our research found that HIF‐1α played a protective role in dextran sulphate sodium‐induced colitis, which was partly due to its regulation of tight junction (TJ) protein expression. Further study revealed that HIF‐1α mediated TJ proteins levels by moderating the content of butyrate. Moreover, we found that butyrate regulated TJ protein expression, which is dependent on HIF‐1α. These results indicated that there is a mutual regulatory mechanism between butyrate and HIF‐1α, which has an important role in the maintenance of barrier function of the gastrointestinal tract.     

Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression

Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-kappaB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.     

Indomethacin-induced translocation of bacteria across enteric epithelia is reactive oxygen species-dependent and reduced by vitamin C

The enteric epithelium must absorb nutrients and water and act as a barrier to the entry of luminal material into the body; this barrier function is a key component of innate immunity. Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy occurs via inhibition of prostaglandin synthesis and perturbed epithelial mitochondrial activity. Here, the direct effect of NSAIDs [indomethacin, piroxicam (cyclooxygenase 1 and 2 inhibitors), and SC-560 (a cyclooxygenase 1 inhibitor)] on the barrier function of human T84 epithelial cell line monolayers was assessed by transepithelial electrical resistance (TER) and internalization and translocation of a commensal Escherichia coli. Exposure to E. coli in the presence and absence of drugs for 16 h reduced TER; however, monolayers cotreated with E. coli and indomethacin, but not piroxicam or SC-560, displayed significant increases in internalization and translocation of the bacteria. This was accompanied by increased reactive oxygen species (ROS) production, which was also increased in epithelia treated with E. coli only. Colocalization revealed upregulation of superoxide synthesis by mitochondria in epithelia treated with E. coli + indomethacin. Addition of antioxidants (vitamin C or a green tea polyphenol, epigallocathechin gallate) quenched the ROS and prevented the increase in E. coli internalization and translocation evoked by indomethacin, but not the drop in TER. Evidence of increased apoptosis was not observed in this model. The data implicate epithelial-derived ROS in indomethacin-induced barrier dysfunction and show that a portion of the bacteria likely cross the epithelium via a transcellular pathway. We speculate that addition of antioxidants as dietary supplements to NSAID treatment regimens would reduce the magnitude of decreased barrier function, specifically the transepithelial passage of bacteria.     

Bacterial translocation from the intestines

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal (GI) tract through the mucosal epithelium to other sites, such as the mesenteric lymph nodes, spleen, liver and blood. This paper reviews results from animal models utilized to obtain information concerning the defense mechanisms operating in the healthy host to confine bacteria to the GI tract. Gnotobiotic and antibiotic-decontaminated mice colonized with particular bacteria demonstrated that the indigenous GI flora maintains an ecologic equilibrium to prevent intestinal bacterial overgrowth and translocation from the GI tract. Studies with athymic (nu/nu) mice, thymus-grafted (nu/nu) mice, neonatally thymectomized mice, and mice injected with immunosuppressive agents demonstrated that the host immune system is another defense mechanism inhibiting bacterial translocation from the GI tract. Ricinoleic acid given orally to mice disrupted the intestinal epithelial barrier allowing indigenous bacteria to translocate from the GI tract. Thus, bacterial translocation from the GI tract of healthy adult mice is inhibited by: (a) an intact intestinal epithelial barrier, (b) the host immune defense system, and (c) an indigenous GI flora maintaining ecological equilibrium to prevent bacterial overgrowth. Deficiencies in host defense mechanisms act synergistically to promote bacterial translocation from the GI tract as demonstrated by animal models with multiple alterations in host defenses. Bacterial translocation occurred to a greater degree in mice with streptozotocin-induced diabetes, mice receiving nonlethal thermal injury, and mice receiving the combination of an immunosuppressive agent plus an oral antibiotic than in mice with only a primary alteration in host defenses. The study of bacterial translocation in these complex models suggests that opportunistic infections from the GI tract occur in discrete stages. In the healthy adult animal, bacterial translocation from the GI tract either does not occur or occurs at a very low level and the host immune defenses eliminate the translocating bacteria. Bacterial translocation does take place if one of the host defense mechanisms is compromised, such as a deficiency in the immune response, bacterial overgrowth in the intestines, or an increase in the permeability of the intestinal barrier. In this first stage, the bacteria usually translocate in low numbers to the mesenteric lymph node, and sometimes spleen or liver, but do not multiply and spread systemically.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epithelial permeability induced by neutrophil transmigration is potentiated by hypoxia: Role of intracellular cAMP

Mucosal tissues, such as the lung and intestine, are primary targets for ischemic damage. Under these conditions, neutrophil (polymorphonuclear leukocyte; PMN) infiltration into the protective epithelium has been implicated as a pathophysiologic mediator. Because PMN transepithelial migration results in increased paracellular permeability, and because our previous data revealed that epithelial hypoxia enhances PMN transmigration, we hypothesized that macromolecular permeability may be altered in epithelium exposed to hypoxia and reoxygenation (H/R) in the presence of PMNs. Human intestinal epithelia (T84) were grown on permeable supports, exposed to cellular hypoxia (pO₂ 20 torr) for 0–72 hr, and examined for increases in PMN-evoked permeability by using standard flux assays. Increasing epithelial hypoxia potentiated PMN-induced permeability of labeled paracellular tracers (size range 3–500 kD). Such increases were blocked by monoclonal antibody (mAb) to the PMN integrin CD11b (82 ± 1% decreased compared with control mAb) and were partially blocked by anti-CD47 mAb(51 ± 1%). Assessment of barrier recovery revealed that monolayers exposed to H/R were significantly diminished in their ability to reseal following PMN transmigration (recovery of 36 ± 6% in H/R vs. 94 ± 2% in normoxic controls). Because intracellular cyclic AMP (cAMP) has been demonstrated to regulate epithelial permeability, and because PMN-derived compound(s), (i.e., 5′-adenosine monophosphate; AMP) elevate epithelial cAMP, we examined the impact of hypoxia on epithelial cAMP responses. These experiments revealed that hypoxic epithelia were diminished in their ability to generate cAMP, and pharmacologic elevation (8-bromo-cAMP) of intracellular cAMP in hypoxic cells normalized both PMN-induced permeability changes and restoration of barrier function. These results support a role for PMN in increased intestinal permeability associated with reperfusion injury and imply a substantial role for cAMP signaling in maintenance of permeability during PMN transmigration. J. Cell. Physiol. 176:76–84, 1998. © 1998 Wiley-Liss, Inc.     

Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro

Previous experimental studies in a standard Transwell culture system have shown Streptococcus suis ability to compromise barrier function of porcine choroid plexus epithelial cells (PCPEC). The development of an 'inverted' Transwell filter system of PCPEC enables us now for the first time to investigate bacterial invasion and translocation from the physiologically relevant basolateral (blood) to the apical (cerobrospinal fluid) side. Most importantly, we observed specific invasion and translocation of S. suis across the PCPEC exclusively from the basolateral side. During this process, bacterial viability and the presence of a capsule as well as cytoskeletal regulation of PCPEC seemed to play an important role. No loss of barrier function was observed. Bacterial translocation could be significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002, but not by its inactive analogue   Ly303511 or dexamethasone. Apotome imaging as well as electron microscopy revealed intracellular bacteria often in cell vacuoles. Thus, possibly regulated by the presence of a capsule, S. suis induces signals that depend on the lipid kinase phosphatidylinositol 3-kinase pathway, which paves the way for cellular uptake during the bacterial transcellular translocation process. Taken together, our data underline the relevance of the blood–cerebrospinal fluid barrier as a gate for bacterial entry into the central nervous system.     

A metalloproteinase secreted by Streptococcus pneumoniae removes membrane mucin MUC16 from the epithelial glycocalyx barrier

The majority of bacterial infections occur across wet-surfaced mucosal epithelia, including those that cover the eye, respiratory tract, gastrointestinal tract and genitourinary tract. The apical surface of all these mucosal epithelia is covered by a heavily glycosylated glycocalyx, a major component of which are membrane-associated mucins (MAMs). MAMs form a barrier that serves as one of the first lines of defense against invading bacteria. While opportunistic bacteria rely on pre-existing defects or wounds to gain entry to epithelia, non opportunistic bacteria, especially the epidemic disease-causing ones, gain access to epithelial cells without evidence of predisposing injury. The molecular mechanisms employed by these non opportunistic pathogens to breach the MAM barrier remain unknown. To test the hypothesis that disease-causing non opportunistic bacteria gain access to the epithelium by removal of MAMs, corneal, conjunctival, and tracheobronchial epithelial cells, cultured to differentiate to express the MAMs, MUCs 1, 4, and 16, were exposed to a non encapsulated, non typeable strain of Streptococcus pneumoniae (SP168), which causes epidemic conjunctivitis. The ability of strain SP168 to induce MAM ectodomain release from epithelia was compared to that of other strains of S. pneumoniae, as well as the opportunistic pathogen Staphylococcus aureus. The experiments reported herein demonstrate that the epidemic disease-causing S. pneumoniae species secretes a metalloproteinase, ZmpC, which selectively induces ectodomain shedding of the MAM MUC16. Furthermore, ZmpC-induced removal of MUC16 from the epithelium leads to loss of the glycocalyx barrier function and enhanced internalization of the bacterium. These data suggest that removal of MAMs by bacterial enzymes may be an important virulence mechanism employed by disease-causing non opportunistic bacteria to gain access to epithelial cells to cause infection.     

Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcgammaRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.     

Effect of Wild-Type Shigella Species and Attenuated Shigella Vaccine Candidates on Small Intestinal Barrier Function,Antigen Trafficking,and Cytokine Release

Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria''s ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to the large intestine where they invade colonocytes inducing a strong inflammatory response.     

PHD3 Stabilizes the Tight Junction Protein Occludin and Protects Intestinal Epithelial Barrier Function

Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.     

Good bug, bad bug: in the case of enteric inflammatory disease does the epithelium decide?

Many studies demonstrate that intestinal inflammation is either initiated or exaggerated by a component of the normal microbiota, most likely commensal bacteria or products derived from these organisms. We review the nature of human inflammatory bowel disease, the evidence for the involvement of the normal bacterial flora in these disorders and the relevance of maintaining the integrity of the epithelial barrier. Moreover, we, and others, have shown abnormal mitochondria structure in tissue resections from patients with inflammatory bowel disease and tissues from rodents that demonstrated psychological stress-induced increases in epithelial permeability. Thus, we also consider the possibility that a defect in epithelial mitochondrial function would predispose an individual to respond to their commensal bacteria flora--no longer considering them as a beneficial passive inhabitant, but rather perceiving them as a threatening and pro-inflammatory stimulus. In support of this postulate, we discuss our recent findings from an in vitro model showing that the human colon-derived T84 cell line exposed to the metabolic stressor, dinitrophenol, and the non-pathogenic, non-invasive, Escherichia coli (strain HB101) display a loss of barrier function, increased signal transduction and increased production of the chemokine, interleukin 8.     

Hypoxia and inflammatory bowel disease

Inflammatory bowel disease (IBD) is a general term to describe inflammatory diseases of the gastrointestinal tract such as Crohn's disease and ulcerative colitis. IBD affects approximately 1 in 200 individuals and exerts a significant health and quality of life burden on patients. Surgical intervention can be curative in ulcerative colitis but there is currently no cure for Crohn's disease. Since this is the case, and the fact that patients are often diagnosed at a young age, IBD exerts a significant financial burden on the health care system, and society as a whole.The underlying pathology of IBD is complex and involves a combination of genetic, environmental and microbial factors. Regardless of the underlying causes of the condition, this disease is universally characterized by disruption to the protective epithelial barrier separating the intestinal lumen above from the mucosal immune system below. Once this barrier becomes compromised a sequence of events ensues, that can occur in repetitive cycles to ensure long-term and serious damage to the gut.The role of hypoxia and hypoxia-dependent signalling pathways are increasingly appreciated to play a role in the physiology and pathophysiology of the intestine. The intestinal epithelium normally exists in a state of physiological hypoxia, with additional tissue hypoxia a feature of active inflammatory disease. Furthermore, recent pre-clinical animal studies have clearly supported the rationale for pharmacologically manipulating the oxygen-sensitive hypoxia-inducible factor (HIF) pathway in models of IBD. Thus, this review will discuss the contribution of hypoxia sensitive pathways in the pathology of IBD. Finally we will discuss the emerging evidence for manipulation of hypoxia-sensitive pathways in the treatment of IBD.     

烧伤后免疫抑制引起肠道细菌易位和脓毒症的病理学研究

近年来,创伤后体内细菌易位造成的内源性感染逐渐受到重视,为了探讨细菌易位的条件和途径等机理。本文利用烧伤后注射大剂量地塞米松的大鼠动物模型,模拟烧伤后应激状态下体内肾上腺皮质激素分泌增多,免疫机能下降的条件,观察肠道和其它脏器的病理改变。观察结果证实,烧伤与大剂量皮质激素确有引起肠道细菌易位和内源性感染,造成脓毒症的叠加作用。其基本条件包括肠道菌群微生态平衡的改变,宿主免疫机能下降和肠粘膜屏障的破坏。易位的肠道细菌主要是通过肠粘膜的坏死灶,上皮细胞坏变缺损处进入肠粘膜淋巴管和小静脉的。超微结构改变包括肠上皮细胞表面的微绒毛倒伏、变形、脱落、上皮细胞顶端崩解破坏,细胞间隙泡状增宽。脓肿中多数细菌微细结构完好,粒细胞和吞噬细胞胞浆中有多量糖元颗粒聚集,提示糖皮质激素可能影响吞噬细胞的糖代谢而降低其吞噬功能,使易位的细菌得以定植形成脓肿感染。