Enteric bacterial pathogens such as enterohemorrhagic E. coli (EHEC) and Salmonella Typhimurium target the intestinal epithelial cells (IEC) lining the mammalian gastrointestinal tract. Despite expressing innate Toll-like receptors (TLRs), IEC are innately hypo-responsive to most bacterial products. This is thought to prevent maladaptive inflammatory responses against commensal bacteria, but it also limits antimicrobial responses by IEC to invading bacterial pathogens, potentially increasing host susceptibility to infection. One reason for the innate hypo-responsiveness of IEC is their expression of Single Ig IL-1 Related Receptor (SIGIRR), a negative regulator of interleukin (IL)-1 and TLR signaling. To address whether SIGIRR expression and the innate hypo-responsiveness of IEC impacts on enteric host defense, Sigirr deficient (−/−) mice were infected with the EHEC related pathogen Citrobacter rodentium. Sigirr −/− mice responded with accelerated IEC proliferation and strong pro-inflammatory and antimicrobial responses but surprisingly, Sigirr −/− mice proved dramatically more susceptible to infection than wildtype mice. Through haematopoietic transplantation studies, it was determined that SIGIRR expression by non-haematopoietic cells (putative IEC) regulated these responses. Moreover, the exaggerated responses were found to be primarily dependent on IL-1R signaling. Whilst exploring the basis for their susceptibility, Sigirr −/− mice were found to be unusually susceptible to intestinal Salmonella Typhimurium colonization, developing enterocolitis without the typical requirement for antibiotic based removal of competing commensal microbes. Strikingly, the exaggerated antimicrobial responses seen in Sigirr −/− mice were found to cause a rapid and dramatic loss of commensal microbes from the infected intestine. This depletion appears to reduce the ability of the microbiota to compete for space and nutrients (colonization resistance) with the invading pathogens, leaving the intestine highly susceptible to pathogen colonization. Thus, SIGIRR expression by IEC reflects a strategy that sacrifices maximal innate responsiveness by IEC in order to promote commensal microbe based colonization resistance against bacterial pathogens.
Despite being in close contact with billions of commensal bacteria, the epithelial cells that line the intestine develop very weak innate inflammatory responses to bacterial products. The goal of this study was to explore why these cells respond so poorly, and how increasing their innate responsiveness would impact on host defense against invading bacterial pathogens. We show that a negative regulator of innate signaling called SIGIRR, limits the inflammatory responses of the intestine to bacteria. Following infection by the bacterial pathogen Citrobacter rodentium, the intestines of mice lacking SIGIRR showed exaggerated inflammatory, antimicrobial and proliferative responses. Through transplantation studies, we showed it was SIGIRR expression by intestinal epithelial cells that limits these responses, and that the exaggerated responses were driven by cytokine signaling through the interleukin-1 receptor. Despite their exaggerated responses, SIGIRR deficient mice proved extremely susceptible to infection by C. rodentium and other intestinal bacterial pathogens. We found the exaggerated inflammatory responses rapidly depleted intestinal commensal microbes, reducing their ability to outcompete invading pathogens for space and nutrients (colonization resistance). Our study thus clarifies that the hypo-responsiveness of epithelial cells plays an unexpected but critical role in host defense, by promoting commensal microbe based competition against enteric pathogens.
Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn’s lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.
Dysfunction of the intestinal epithelium is believed to result in excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn's disease; an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum1. Beside the physical barrier established by the tight contact of cells, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides which hamper access and survival of bacteria adjacent to the epithelium2. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a pathogenic mechanism driving Crohn's disease (CD) in humans3. However, the regulation of epithelial cell death and its role in intestinal homeostasis remains poorly understood.
Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IEC) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8ΔIEC) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8ΔIEC mice lacked Paneth cells and showed reduced numbers of goblet cells suggesting dysregulated anti-microbial immune cell functions of the intestinal epithelium. Casp8ΔIEC mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumor necrosis factor (TNF) -α, was associated with increased expression of receptor-interacting protein 3 (RIP3) and could be inhibited upon blockade of necroptosis. Finally, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohn's disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Taken together, our data demonstrate a critical function of caspase-8 in regulating intestinal homeostasis and in protecting IEC from TNF-α induced necroptotic cell death.
The intestinal epithelium maintains a state of controlled inflammation despite continuous contact with Gram-negative commensal bacteria and lipopolysaccharide (LPS) on its luminal surface. Recognition of LPS by the TLR4/MD-2 complex results in proinflammatory gene expression and cytokine secretion in intestinal epithelial cells (IEC). We have shown that IEC express low levels of MD-2 and TLR4 and are poorly responsive to LPS. In this study, we did a comprehensive analysis to understand the immune-mediated and epigenetic mechanisms by which IEC down-regulate MD-2 expression. Expression of MD-2 and TLR4 mRNA was examined in human inflammatory bowel disease and intestinal epithelial cell lines (T84, HT-29, Caco-2). Nuclear factor-κB transcriptional activation was used as a measure of LPS responsiveness. Intestinal epithelial cellsin patients with IBD exhibited increased expression of MD-2 and TLR4 mRNA. Lipopolysaccharide responsiveness in IEC was polarized to the basolateral membrane. Bisulfite sequencing of the MD-2 promoter demonstrated methylation of CpG dinucleotides. Inhibition of methylation by 5-azacytidine and histone deactylation by trichostatin A, two forms of epigenetic silencing, resulted in increased mRNA expression of MD-2 in IEC. These results demonstrate various molecular mechanisms by which IEC down-regulate MD-2 and thereby protect against dysregulated inflammation to commensal bacteria in the intestinal lumen.
MD-2; Toll-like Receptor 4 (TLR4); Intestinal Epithelial Cells; Lipopolysaccharide; Methylation; Histone deacetylation
The human intestinal microbiota plays an important role in modulation of mucosal immune responses. To study interactions between intestinal epithelial cells (IECs) and commensal bacteria, a functional metagenomic approach was developed. One interest of metagenomics is to provide access to genomes of uncultured microbes. We aimed at identifying bacterial genes involved in regulation of NF-κB signaling in IECs. A high throughput cell-based screening assay allowing rapid detection of NF-κB modulation in IECs was established using the reporter-gene strategy to screen metagenomic libraries issued from the human intestinal microbiota.
A plasmid containing the secreted alkaline phosphatase (SEAP) gene under the control of NF-κB binding elements was stably transfected in HT-29 cells. The reporter clone HT-29/kb-seap-25 was selected and characterized. Then, a first screening of a metagenomic library from Crohn's disease patients was performed to identify NF-κB modulating clones. Furthermore, genes potentially involved in the effect of one stimulatory metagenomic clone were determined by sequence analysis associated to mutagenesis by transposition.
The two proinflammatory cytokines, TNF-α and IL-1β, were able to activate the reporter system, translating the activation of the NF-κB signaling pathway and NF-κB inhibitors, BAY 11-7082, caffeic acid phenethyl ester and MG132 were efficient. A screening of 2640 metagenomic clones led to the identification of 171 modulating clones. Among them, one stimulatory metagenomic clone, 52B7, was further characterized. Sequence analysis revealed that its metagenomic DNA insert might belong to a new Bacteroides strain and we identified 2 loci encoding an ABC transport system and a putative lipoprotein potentially involved in 52B7 effect on NF-κB.
We have established a robust high throughput screening assay for metagenomic libraries derived from the human intestinal microbiota to study bacteria-driven NF-κB regulation. This opens a strategic path toward the identification of bacterial strains and molecular patterns presenting a potential therapeutic interest.
The gastrointestinal tract is the largest reservoir of commensal bacteria in the human body, providing nutrients and space for the survival of microbes while concurrently operating mucosal barriers to confine the microbial population. The epithelial cells linked by tight junctions not only physically separate the microbiota from the lamina propria, but also secrete proinflammatory cytokines and reactive oxygen species in response to pathogen invasion and metabolic stress and serve as a sentinel to the underlying immune cells. Accumulating evidence indicates that commensal bacteria are involved in various physiological functions in the gut and microbial imbalances (dysbiosis) may cause pathology. Commensal bacteria are involved in the regulation of intestinal epithelial cell turnover, promotion of epithelial restitution and reorganization of tight junctions, all of which are pivotal for fortifying barrier function. Recent studies indicate that aberrant bacterial lipopolysaccharide-mediated signaling in gut mucosa may be involved in the pathogenesis of chronic inflammation and carcinogenesis. Our perception of enteric commensals has now changed from one of opportunistic pathogens to active participants in maintaining intestinal homeostasis. This review attempts to explain the dynamic interaction between the intestinal epithelium and commensal bacteria in disease and health status.
Intestinal barrier; Commensal bacteria; Enterocytes; Tight junctions; Lipopolysaccharide; CD14/TLR4; Inflammatory bowel disease; Colorectal cancer
BACKGROUND AND AIM—Intestinal epithelial cells (IEC) are thought to participate in the mucosal defence against bacteria and in the regulation of mucosal tissue homeostasis. Reactivity of IEC to bacterial signals may depend on interactions with immunocompetent cells. To address the question of whether non-pathogenic bacteria modify the immune response of the intestinal epithelium, we co-cultivated enterocyte-like CaCO-2 cells with human blood leucocytes in separate compartments of transwell cultures.
METHODS—CaCO-2/PBMC co-cultures were stimulated with non-pathogenic bacteria and enteropathogenic Escherichia coli. Expression of tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-8, monocyte chemoattracting protein 1 (MCP-1), and IL-10 was studied by enzyme linked immunosorbent assays (cytokine secretion) and by semiquantitative reverse transcription-polymerase chain reaction.
RESULTS—Challenge of CaCO-2 cells with non-pathogenic E coli and Lactobacillus sakei induced expression of IL-8, MCP-1, IL-1β, and TNF-α mRNA in the presence of underlying leucocytes. Leucocyte sensitised CaCO-2 cells produced TNF-α and IL-1β whereas IL-10 was exclusively secreted by human peripheral blood mononuclear cells. CaCO-2 cells alone remained hyporesponsive to the bacterial challenge. Lactobacillus johnsonii, an intestinal isolate, showed reduced potential to induce proinflammatory cytokines but increased transforming growth factor beta mRNA in leucocyte sensitised CaCO-2 cells. TNF-α was identified as one of the early mediators involved in cellular cross talk. In the presence of leucocytes, discriminative activation of CaCO-2 cells was observed between enteropathogenic E coli and non-pathogenic bacteria.
CONCLUSION—The differential recognition of non-pathogenic bacteria by CaCO-2 cells required the presence of underlying leucocytes. These results strengthen the hypothesis that bacterial signalling at the mucosal surface is dependent on a network of cellular interactions.
Keywords: CaCO-2 cells; leucocytes; enteropathogenic E coli; Lactobacilli; tumour necrosis factor; interleukin 1β; interleukin 10; chemokines
The gastrointestinal tract harbors a diverse microbiota that has co-evolved with mammals for eons. Though most associations are symbiotic or commensal, some resident bacteria (termed pathobionts) have the potential to cause disease. Type VI secretion systems (T6SSs) are a novel mechanism for forging host-microbial interactions. We reveal a unique protective role for the T6SS of Helicobacter hepaticus, a Gram-negative bacterium of the intestinal microbiota. T6SS mutants display increased intracellular numbers within intestinal epithelial cells (IECs) and during intestinal colonization. Remarkably, T6SS directs an anti-inflammatory gene expression profile in IECs, and CD4+ T cells from T6SS mutant-colonized animals produce increased interleukin-17 (IL-17) in response to IECs presenting H. hepaticus antigens. These data demonstrate that H. hepaticus interacts with IECs and employs the T6SS to establish a balanced host relationship by limiting colonization and intestinal inflammation. We propose that altering host-bacterial equilibriums contribute to human disorders such as inflammatory bowel disease and colon cancer.
Clostridium difficile (Cd) is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotic-treated individuals. Commensal bacteria are known to have a significant role in the intestinal accumulation of Cd after antibiotic treatment, but little is known about how they affect host immunity during Cd infection. Here we report that Cd infection results in translocation of commensals across the intestinal epithelial barrier that is critical for neutrophil recruitment through the induction of an IL-1β-mediated positive feedback loop. Mice lacking ASC, an essential mediator of IL-1β and IL-18 processing and secretion, were highly susceptible to Cd infection. ASC−/− mice exhibited enhanced translocation of commensals to multiple organs after Cd infection. Notably, ASC−/− mice exhibited impaired CXCL1 production and neutrophil influx into intestinal tissues in response to Cd infection. The impairment in neutrophil recruitment resulted in reduced production of IL-1β and CXCL1, but not IL-18. Importantly, translocated commensals were required for ASC/Nlrp3-dependent IL-1β secretion by neutrophils. Mice lacking IL-1β were deficient in inducing CXCL1 secretion, suggesting that IL-1β is the dominant inducer of ASC-mediated CXCL1 production during Cd infection. These results indicate that translocated commensals play a crucial role in CXCL1-dependent recruitment of neutrophils to the intestine through an IL-1β/NLRP3/ASC-mediated positive feedback mechanism that is important for host survival and clearance of translocated commensals during Cd infection.
The intestinal microbiota plays an important role in modulation of mucosal immune responses. To seek interactions between intestinal epithelial cells (IEC) and commensal bacteria, we screened 49 commensal strains for their capacity to modulate NF-κB. We used HT-29/kb-seap-25 and Caco-2/kb-seap-7 intestinal epithelial cells and monocyte-like THP-1 blue reporter cells to measure effects of commensal bacteria on cellular expression of a reporter system for NF-κB. Bacteria conditioned media (CM) were tested alone or together with an activator of NF-κB to explore its inhibitory potentials. CM from 8 or 10 different commensal species activated NF-κB expression on HT-29 and Caco-2 cells, respectively. On THP-1, CM from all but 5 commensal strains stimulated NF-κB. Upon challenge with TNF-α or IL-1β, some CM prevented induced NF-κB activation, whereas others enhanced it. Interestingly, the enhancing effect of some CM was correlated with the presence of butyrate and propionate. Characterization of the effects of the identified bacteria and their implications in human health awaits further investigations.
The intestinal immune system is crucial for the maintenance of mucosal homeostasis and has evolved under the dual pressure of protecting the host from pathogenic infection and coexisting with the dense and diverse commensal organisms in the lumen. Intestinal intraepithelial lymphocytes (iIELs) are the first element of the host T cell compartment available to respond to oral infection by pathogens. This study demonstrated that oral infection by Salmonella enterica serovar Typhimurium promoted the expansion of iIELs, particularly CD8+ TCRγδ+ IELs, enhanced expression of NKG2D on iIELs, increased expression of MULT1, and decreased expression of Qa-1 by intestinal epithelial cells (IECs), leading to activation of, particularly, CD8+ TCRγδ+ iIELs and cytolytic activity against S. Typhimurium-infected IECs. Blockade of NKG2D recognition or depletion of TCRγδ+ cells using a depleting monoclonal antibody significantly attenuated the clearance of S. Typhimurium in the intestine and other tissues. This study suggests that iIELs, particularly CD8+ TCRγδ+ iIELs, play important roles in the detection of pathogenic bacteria and eradication of infected epithelial cells and, thus, provide protection against invading pathogens. These data further our understanding of the mechanisms by which the immune system of the intestinal mucosa discriminates between pathogenic and commensal organisms.
Intestinal epithelial cells (IECs) respond to lipopolysaccharide (LPS) from gram-negative bacteria in the presence of the soluble form of CD14 (sCD14), a major endotoxin receptor. Since sCD14 is also known to interact with gram-positive bacteria and their components, we looked at whether sCD14 could mediate their effects on human IECs. To this end, we examined the production of proinflammatory cytokines following exposure of the IECs to specific gram-positive bacteria or their lipoteichoic acids (LTAs) in the absence and presence of human milk as a source of sCD14. In contrast to LPS from Escherichia coli or Salmonella enteritidis, neither the gram-positive bacteria Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 nor their LTAs stimulated IECs, even in the presence of sCD14. However, both LTAs inhibited the sCD14-mediated LPS responsiveness of IECs. We have previously hypothesized that sCD14 in human milk is a means by which the neonate gauges the bacterial load in the intestinal lumen and liberates protective proinflammatory cytokines from IECs. The present observations suggest that gram-positive organisms, via their LTAs, temper this response and prevent an exaggerated inflammatory response.
Interleukin 2 (IL-2)- and IL-10-knockout mice develop spontaneous colitis under conventional but not germ-free conditions, suggesting that commensal bacteria play an important role in the pathogenesis of colitis. However, interactions between commensal bacteria and colonic epithelial cells have not been fully investigated. We therefore assessed the ability of various commensal bacteria and probiotics to adhere to and invade colonic epithelial cells. Effects of the bacteria on production of proinflammatory cytokines were also measured. Commensal bacteria, including mucosal organisms isolated from ulcerative colitis (UC) patients, such as Fusobacterium varium, reported as a possible pathogen in UC, Bacteroides vulgatus, Escherichia coli and Clostridium clostridioforme, as well as their type strains and probiotics, were assessed for their ability to adhere to and invade colonic epithelial cells using two cell lines, SW-480 and HT-29. Our experiments employed co-incubation, a combination of scanning and transmission electron microscopy and recovery of bacteria from infected-cell lysates. F. varium and several other commensal bacteria, but not probiotics, adhered to colonic epithelial cells and invaded their cytoplasm. ELISA and real-time PCR revealed that the host cells, particularly those invaded by F. varium, showed significant increases in IL-8 and TNF-α concentrations in supernatants, with elevation of IL-8, TNF-α, MCP-1 and IL-6 mRNAs. Furthermore, IL-8 and TNF-α expression and nuclear phosphorylated NF-κB p65 expression could be immunohistochemically confirmed in inflamed epithelium with cryptitis or crypt abscess in UC patients. Certain commensal bacteria can invade colonic epithelial cells, activating early intracellular signalling systems to trigger host inflammatory reactions.
Soluble factors released by Bifidobacterium breve C50 (Bb) alleviate the secretion of pro-inflammatory cytokines by immune cells, but their effect on intestinal epithelium remains elusive. To decipher the mechanisms accounting for the cross-talk between bacteria/soluble factors and intestinal epithelium, we measured the capacity of the bacteria, its conditioned medium (Bb-CM) and other Gram(+) commensal bacteria to dampen inflammatory chemokine secretion.
TNFα-induced chemokine (CXCL8) secretion and alteration of NF-κB and AP-1 signalling pathways by Bb were studied by EMSA, confocal microscopy and western blotting. Anti-inflammatory capacity was also tested in vivo in a model of TNBS-induced colitis in mice.
Bb and Bb-CM, but not other commensal bacteria, induced a time and dose-dependent inhibition of CXCL8 secretion by epithelial cells driven by both AP-1 and NF-κB transcription pathways and implying decreased phosphorylation of p38-MAPK and IκB-α molecules. In TNBS-induced colitis in mice, Bb-CM decreased the colitis score and inflammatory cytokine expression, an effect reproduced by dendritic cell conditioning with Bb-CM.
Bb and secreted soluble factors contribute positively to intestinal homeostasis by attenuating chemokine production. The results indicate that Bb down regulate inflammation at the epithelial level by inhibiting phosphorylations involved in inflammatory processes and by protective conditioning of dendritic cells.
The intestinal epithelial cells (IECs) form a selective permeability barrier separating luminal content from underlying tissues. Upon injury, the intestinal epithelium undergoes a wound healing process. Intestinal wound healing is dependent on the balance of three cellular events; restitution, proliferation, and differentiation of epithelial cells adjacent to the wounded area. Previous studies have shown that various regulatory peptides, including growth factors and cytokines, modulate intestinal epithelial wound healing. Recent studies have revealed that novel factors, which include toll-like receptors (TLRs), regulatory peptides, particular dietary factors, and some gastroprotective agents, also modulate intestinal epithelial wound repair. Among these factors, the activation of TLRs by commensal bacteria is suggested to play an essential role in the maintenance of gut homeostasis. Recent studies suggest that mutations and dysregulation of TLRs could be major contributing factors in the predisposition and perpetuation of inflammatory bowel disease. Additionally, studies have shown that specific signaling pathways are involved in IEC wound repair. In this review, we summarize the function of IECs, the process of intestinal epithelial wound healing, and the functions and mechanisms of the various factors that contribute to gut homeostasis and intestinal epithelial wound healing.
Intestinal epithelial cell; Wound healing; Restitution; Growth factors; Toll-like receptor
Background: Probiotic bacteria have a beneficial effect on intestinal inflammation. In this study, we have examined the effect of lactic acid and commensal Gram positive (+) bacteria conditioned media (CM) on tumour necrosis factor α (TNF-α) release and the mechanisms involved.
Methods: Lipopolysaccharide (LPS) induced TNF-α secretion by peripheral blood mononuclear cells or the THP-1 cell line was monitored in the presence or absence of bacteria CM obtained from two probiotic strains, Bifidobacterium breve (Bb) and Streptococcus thermophilus (St), and three commensal bacterial strains (Bifidobacterium bifidum, Ruminococcus gnavus, and unidentified Streptococcus). Bb and St bacteria CM were allowed to cross filter grown intestinal epithelial cell monolayers (HT29-19A) to assess intestinal transport of active bacterial products. These products were characterised and their effect on LPS binding to THP-1 cells and nuclear factor κB (NFκB) activation assessed.
Results: Dose dependent inhibition of LPS induced TNF-α secretion was noted for both probiotic bacteria CM (64% and 71% inhibition for Bb and St, respectively) and to a lesser extent commensal bacteria CM (21–32% inhibition). Active products from Bb and St were resistant to digestive enzymes and had a molecular mass <3000 Da. Their inhibitory effect was preserved after transepithelial transport across intestinal cell monolayers, mainly in inflammatory conditions. LPS-FITC binding to THP-1 cells and NFκB activation were significantly inhibited by Bb and St CM.
Conclusion: B breve and S thermophilus release metabolites exerting an anti-TNF-α effect capable of crossing the intestinal barrier. Commensal bacteria also display a TNF-α inhibitory capacity but to a lesser extent. These results underline the beneficial effect of commensal bacteria in intestinal homeostasis and may explain the role of some probiotic bacteria in alleviating digestive inflammation.
probiotics; inflammatory cytokines; intestinal barrier
The intestinal epithelium not only provides a vital physical barrier between the host and environment but is also required for uptake of nutrients and the induction of tolerance against commensals. Deregulation of any of these functions leads to several disease states including chronic infection, inflammatory bowel disease, and cancer. Here, we describe a novel role for the complement regulator CD46 in the regulation of intestinal epithelial cell (IEC) barrier function. We found that CD46 directly interacts in IECs with the cytoplasmic kinase SPAK and with transmembrane E-cadherin, both proteins necessary for epithelial cell junction and barrier formation. Further, CD46 activation on Caco-2 cells induced rapid and significant decrease in transepithelial resistance with concomitant increase in paracellular permeability. Importantly, though CD46 activation of IEC layers allowed for increased transgression of pathogenic E. coli, it also increased epithelial cell proliferation and accelerated wound repair. These data suggest a previously unappreciated role for CD46 in the maintenance of epithelial cell barrier integrity as well as barrier repair. However, this role for CD46 as “gate keeper” of the epithelium could also provide reason as to why so many pathogens bind to CD46 as such event would facilitate infection.
CD46; complement; mucosal immunity; epithelial cells; wound healing
Cells of the innate immune system regulate immune responses through the production of antimicrobial peptides, chemokines, and cytokines, including human beta-defensins (hBDs) and CCL20. In this study, we examined the kinetics of primary human oral epithelial cell (HOEC) production of CCL20 and hBDs in response to Fusobacterium nucleatum, a commensal bacterium of the oral cavity, which we previously showed promotes HOEC induction of hBDs. HOECs secrete maximal levels of CCL20 at 6 h, following stimulation by F. nucleatum cell wall (FnCW). The kinetics of CCL20 release is distinct from that of hBD-2 and -3, which peaks after 24 h and 48 h of FnCW stimulation, respectively. FnCW-induced release of CCL20 by HOECs requires both transcriptional and translational activation. Release of CCL20 by HOECs is inhibited by brefeldin A, suggesting that it is secreted through a vesicle transport pathway. Other epithelium-derived agents that FnCW induces, such as hBD-2, hBD-3, tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), are also able to release CCL20. By focusing on mitogen-activated protein kinases, we show that both extracellular signal-regulated kinase 1/2 and p38, but not JNK, are required for hBD-, TNF-α-, and IL-1β-induced secretion of CCL20 by HOECs. The ability of FnCW and its induced hBDs to produce proinflammatory cytokines and CCL20 suggests the broad role of F. nucleatum and human antimicrobial peptides in primary immune responses elicited by oral epithelium.
Bifidobacteria belong to one of the predominant bacterial groups in the intestinal microbiota of infants and adults. Several beneficial effects on the health status of their human hosts have been demonstrated making bifidobacteria interesting candidates for probiotic applications. Adhesion of probiotics to the intestinal epithelium is discussed as a prerequisite for colonisation of and persistence in the gastrointestinal tract.
In the present study, 15 different strains of bifidobacteria were tested for adhesion. B. bifidum was identified as the species showing highest adhesion to all tested intestinal epithelial cell (IEC) lines. Adhesion of B. bifidum S17 to IECs was strongly reduced after treatment of bacteria with pronase. These results strongly indicate that a proteinaceous cell surface component mediates adhesion of B. bifidum S17 to IECs. In silico analysis of the currently accessible Bifidobacterium genomes identified bopA encoding a lipoprotein as a B. bifidum-specific gene previously shown to function as an adhesin of B. bifidum MIMBb75. The in silico results were confirmed by Southern Blot analysis. Furthermore, Northern Blot analysis demonstrated that bopA is expressed in all B. bifidum strains tested under conditions used to cultivate bacteria for adhesion assays. The BopA gene was successfully expressed in E. coli and purified by Ni-NTA affinity chromatography as a C-terminal His6-fusion. Purified BopA had an inhibitory effect on adhesion of B. bifidum S17 to IECs. Moreover, bopA was successfully expressed in B. bifidum S17 and B. longum/infantis E18. Strains overexpressing bopA showed enhanced adhesion to IECs, clearly demonstrating a role of BopA in adhesion of B. bifidum strains.
BopA was identified as a B. bifidum-specific protein involved in adhesion to IECs. Bifidobacterium strains expressing bopA show enhanced adhesion. Our results represent the first report on recombinant bifidobacteria with improved adhesive properties.
Adhesion; Bifidobacteria; Intestinal epithelial cells; Probiotics
Intestinal epithelial cells can respond to certain bacteria by producing an array of cytokines and chemokines which are associated with host immune responses. Lactobacillus acidophilus NCFM is a characterized probiotic, originally isolated from human feces. This study aimed to test the ability of L. acidophilus NCFM to stimulate cytokine and chemokine production in intestinal epithelial cells and to elucidate the mechanisms involved in their upregulation. In experiments using intestinal epithelial cell lines and mouse models, we observed that L. acidophilus NCFM could rapidly but transiently upregulate a number of effector genes encoding cytokines and chemokines such as interleukin 1α (IL-1α), IL-1β, CCL2, and CCL20 and that cytokines showed lower expression levels with L. acidophilus NCFM treatment than chemokines. Moreover, L. acidophilus NCFM could activate a pathogen-associated molecular pattern receptor, Toll-like receptor 2 (TLR2), in intestinal epithelial cell lines. The phosphorylation of NF-κB p65 and p38 mitogen-activated protein kinase (MAPK) in intestinal epithelial cell lines was also enhanced by L. acidophilus NCFM. Furthermore, inhibitors of NF-κB (pyrrolidine dithiocarbamate [PDTC]) and p38 MAPK (SB203580) significantly reduced cytokine and chemokine production in the intestinal epithelial cell lines stimulated by L. acidophilus NCFM, suggesting that both NF-κB and p38 MAPK signaling pathways were important for the production of cytokines and chemokines induced by L. acidophilus NCFM.
The oral cavity harbors a diverse and complex microbial community. Bacteria accumulate on both the hard and soft oral tissues in sessile biofilms and engage the host in an intricate cellular dialog, which normally constrains the bacteria to a state of commensal harmony. Dendritic cells (DCs) are likely to balance tolerance and active immunity to commensal microorganisms as part of chronic inflammatory responses. While the role played by DCs in maintaining intestinal homeostasis has been investigated extensively, relatively little is known about DC responses to oral bacteria.
In this study, we pulsed human monocyte-derived immature DCs (iDCs) with cell wall extracts from pathogenic and commensal gram-positive or gram-negative oral bacteria.
Although all bacterial extracts tested induced iDCs to mature and produce cytokines/chemokines including interleukin-12p40, tumor necrosis factor-α, and monocyte chemoattractant protein-1 (MCP-1), the most important factor for programming DCs by oral bacteria was whether they were gram-positive or gram-negative, not whether they were commensal or pathogenic. In general, gram-negative oral bacteria, except for periodontopathic Porphyromonas gingivalis, stimulated DC maturation and cytokine production at lower concentrations than gram-positive oral bacteria. The threshold of bacteria needed to stimulate chemokine production was 100-fold to 1000-fold lower than that needed to induce cytokines. In addition, very low doses of oral commensal bacteria triggered monocytes to migrate toward DC-derived MCP-1.
Oral commensal and pathogenic bacteria do not differ qualitatively in how they program DCs. DC-derived MCP-1 induced in response to oral commensal bacteria may play a role, at least in part, in the maintenance of oral tissue integrity by attracting monocytes.
commensal bacteria; dendritic cells; MCP-1
This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a+CD11R1high and CD4+ cells from ileal Peyer's patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response.
Inflammatory bowel disease (IBD) has been attributed to aberrant mucosal immunity to the intestinal microbiota. The transcription factor XBP1, a key component of the endoplasmic reticulum (ER) stress response, is required for development and maintenance of secretory cells and linked to JNK activation. We report that XBP1 deletion in intestinal epithelial cells (IEC) results in spontaneous enteritis and increased susceptibility to induced colitis secondary to both Paneth cell deficiency and overactive responses of the intestinal epithelial cell (IEC) to the IBD-inducers, TNFα and flagellin. An association of XBP1 variants with human IBD was identified and replicated (rs35873774, P-value 1.6×10−5) with novel, private hypomorphic variants identified as susceptibility factors. Hence, intestinal inflammation can originate solely from XBP1 abnormalities in IEC thus linking cell-specific ER stress to the induction of organ-specific inflammation. We report the first mouse model of spontaneous intestinal inflammation arising from alterations in a genetic risk factor for human IBD.
Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals.
We show the identification of a Lactobacillus plantarum LM3 cell surface protein (48 kDa), which specifically binds to human fibronectin (Fn), an extracellular matrix protein. By means of mass spectrometric analysis this protein was identified as the product of the L. plantarum enoA1 gene, coding the EnoA1 alfa-enolase. Surface localization of EnoA1 was proved by immune electron microscopy. In the mutant strain LM3-CC1, carrying the enoA1 null mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay. Moreover, by an adhesion assay we show that LM3-CC1 cells bind to fibronectin-coated surfaces less efficiently than wild type cells, thus demonstrating the significance of the surface displaced EnoA1 protein for the L. plantarum LM3 adhesion to fibronectin.
Adhesion to host tissues represents a crucial early step in the colonization process of either pathogens or commensal bacteria. We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying LM3 and LM3-CC1 surface proteins. Isolation of LM3-CC1 strain was possible for the presence of expressed enoA2 gene in the L. plantarum genome, giving the possibility, for the first time to our knowledge, to quantitatively compare adhesion of wild type and mutant strain, and to assess doubtless the role of L. plantarum Eno A1 as a fibronectin binding protein.
Breast milk is known to protect the infant against infectious and immuno-inflammatory diseases, but the mechanisms of this protection are poorly understood. Objectives: We hypothesized that transforming growth factor-β2 (TGF-β2), an immunoregulatory cytokine abundant in breast milk, may have a direct anti-inflammatory effect on immature human intestinal epithelial cells (IECs).
Human fetal ileal organ culture, primary human fetal IECs, and the human fetal small intestinal epithelial cell line H4 were stimulated with interleukin 1β (IL-1β) with or without TGF-β2. Pro-inflammatory cytokine secretion and mRNA expression were measured by ELISA and quantitative real-time polymerase chain reaction, respectively. Alterations in ERK signalling were detected from IECs by immunoblotting and in fetal ileal tissue culture by immunohistochemistry. SMAD6 knockdown was performed by transfecting the cells with SMAD6 siRNA.
TGF-β2 significantly attenuated IL-1β-induced pro-inflammatory cytokine production in fetal intestinal organ culture and the cell culture models. In addition, TGF-β2 reduced the IL-1β-induced IL-8 and IL-6 mRNA response in H4 cells. TGF-β2 markedly inhibited IL-1β-induced phosphorylation of ERK, which was necessary for the cytokine response. The inhibitory effect of TGF-β2 on IL-1β-induced cytokine production was completely abrogated by SMAD6 siRNA knockdown.
TGF-β2 attenuates IL-1β-induced pro-inflammatory cytokine production in immature human IECs by inhibiting ERK signalling. The anti-inflammatory effect of TGF-β2 is dependent on SMAD6. Breast milk TGF-β2 may provide the neonate with important immunoregulatory support. TGF-β2 might provide a novel means to improve intestinal immunophysiology in premature neonates.
Transforming growth factor-β; Immature intestinal epithelium; Modulation of immune response