Induction of cytokine formation by human intestinal bacteria in gut epithelial cell lines

Aims: To investigate the effects of human gut micro‐organisms on cytokine production by human intestinal cell lines. Methods and Results: Quantitative real‐time PCR assays were developed to measure the production of pro‐inflammatory (IL‐1α, IL‐6, IL‐18 and TNFα) and anti‐inflammatory (TGF‐β1, TGF‐β2, TGF‐β3, IL‐4 and IL‐10) cytokines in HT‐29 and Caco‐2 cell lines. They were co‐cultured with a range of mucosal bacteria isolated from ulcerative colitis patients, together with lactobacilli and bifidobacteria obtained from healthy people. HT‐29 cells were also co‐cultured with Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli and Salmonella typhimurium. The majority of commensal bacteria tested suppressed the expression of anti‐inflammatory cytokine mRNA, increased IL‐18, reduced IL‐1α, and with the exception of nonpathogenic E. coli, reduced TNF‐α. All overtly pathogenic species increased both pro‐inflammatory and anti‐inflammatory cytokine mRNA. Conclusion: Commensal and pathogenic species induced fundamentally different cytokine responses in human intestinal epithelial cell lines. Significance and Impact of the Study: Interactions between commensal bacteria tested in this study and the innate immune system were shown to be anti‐inflammatory in nature, in contrast to the pathogenic organisms investigated. These data contribute towards our understanding of how potential probiotic species can be used to suppress the pro‐inflammatory response in inflammatory bowel disease.     

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.     

A Product of Heme Catabolism Modulates Bacterial Function and Survival

Bilirubin is the terminal metabolite in heme catabolism in mammals. After deposition into bile, bilirubin is released in large quantities into the mammalian gastrointestinal (GI) tract. We hypothesized that intestinal bilirubin may modulate the function of enteric bacteria. To test this hypothesis, we investigated the effect of bilirubin on two enteric pathogens; enterohemorrhagic E. coli (EHEC), a Gram-negative that causes life-threatening intestinal infections, and E. faecalis, a Gram-positive human commensal bacterium known to be an opportunistic pathogen with broad-spectrum antibiotic resistance. We demonstrate that bilirubin can protect EHEC from exogenous and host-generated reactive oxygen species (ROS) through the absorption of free radicals. In contrast, E. faecalis was highly susceptible to bilirubin, which causes significant membrane disruption and uncoupling of respiratory metabolism in this bacterium. Interestingly, similar results were observed for other Gram-positive bacteria, including B. cereus and S. aureus. A model is proposed whereby bilirubin places distinct selective pressure on enteric bacteria, with Gram-negative bacteria being protected from ROS (positive outcome) and Gram-positive bacteria being susceptible to membrane disruption (negative outcome). This work suggests bilirubin has differential but biologically relevant effects on bacteria and justifies additional efforts to determine the role of this neglected waste catabolite in disease processes, including animal models.     

Homologous high-throughput expression and purification of highly conserved <Emphasis Type="Italic">E coli</Emphasis> proteins

Background  

Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD). Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s) involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies.     

Modeling the Infection Dynamics of Bacteriophages in Enteric Escherichia coli: Estimating the Contribution of Transduction to Antimicrobial Gene Spread

Animal-associated bacterial communities are infected by bacteriophages, although the dynamics of these infections are poorly understood. Transduction by bacteriophages may contribute to transfer of antimicrobial resistance genes, but the relative importance of transduction among other gene transfer mechanisms is unknown. We therefore developed a candidate deterministic mathematical model of the infection dynamics of enteric coliphages in commensal Escherichia coli in the large intestine of cattle. We assumed the phages were associated with the intestine and were predominantly temperate. Model simulations demonstrated how, given the bacterial ecology and infection dynamics, most (>90%) commensal enteric E. coli bacteria may become lysogens of enteric coliphages during intestinal transit. Using the model and the most liberal assumptions about transduction efficiency and resistance gene frequency, we approximated the upper numerical limits (“worst-case scenario”) of gene transfer through specialized and generalized transduction in E. coli by enteric coliphages when the transduced genetic segment is picked at random. The estimates were consistent with a relatively small contribution of transduction to lateral gene spread; for example, generalized transduction delivered the chromosomal resistance gene to up to 8 E. coli bacteria/hour within the population of 1.47 × 10⁸ E. coli bacteria/liter luminal contents. In comparison, the plasmidic bla_CMY-2 gene carried by ∼2% of enteric E. coli was transferred by conjugation at a rate at least 1.4 × 10³ times greater than our generalized transduction estimate. The estimated numbers of transductants varied nonlinearly depending on the ecology of bacteria available for phages to infect, that is, on the assumed rates of turnover and replication of enteric E. coli.     

Regulatory effects of Spirulina complex polysaccharides on growth of murine RSV‐M glioma cells through Toll‐like receptor 4

This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down‐regulating angiogenesis via a Toll‐like receptor 4 signal. Murine RSV‐M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV‐M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)‐17 in both C3H/HeN and C3H/HeJ tumor‐bearing mice. Treatment with E. coli LPS induced much greater IL‐17 production in tumor‐bearing C3H/HeN mice than in tumor‐bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re‐transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti‐cluster of differentiation (CD)8, anti‐CD4, anti‐CD8 antibodies, and anti‐asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti‐interferon‐γ antibodies had no effect on glioma cell growth, anti‐IL‐17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS‐treated mice than in those from saline‐ or E. coli LPS‐treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down‐regulating angiogenesis, and that this down‐regulation is mediated in part by regulating IL‐17 production.     

Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation

Vibrio vulnificus is a foodborne pathogen causing septicemia with high mortality rate. In this study, we explored how Escherichia coli, one of the commensal bacteria in the human gastrointestinal tract, can interact with V. vulnificus. Our study results show that the amount of biofilm produced by V. vulnificus was reduced in the presence of E. coli ATCC 35218, although the growth of V. vulnificus L-180 remained unaffected. We also detected an antibiofilm effect of E. coli culture supernatant against V. vulnificus, which could not be reduced even after heat treatment. These findings indicate that E. coli and its culture supernatant may be suitable to prevent biofilm formation by V. vulnificus. By contrast, live cells of V. vulnificus could reduce the amount of preformed E. coli biofilm, but its culture supernatant could not. This suggests that the cell-associated factors contribute toward reduction in E. coli biofilm. Therefore, we speculate that ingestion of an infectious dose of V. vulnificus might induce dislodging of the commensal bacteria from the intestinal epithelia and thus can colonize to initiate the infection.     

Effect of heme oxygenase‐1 transduced bone marrow mesenchymal stem cells on damaged intestinal epithelial cells in vitro

In this study, we explored the effects of mesenchymal stem cells (MSCs) from bone marrow overexpressing heme oxygenase‐1 (HO‐1) on the damaged human intestinal epithelial barrier in vitro. Rat MSCs were isolated from bone marrow and transduced with rat HO‐1 recombinant adenovirus (HO‐MSCs) for stable expression of HO‐1. Colorectal adenocarinoma 2 (Caco2) cells were treated with tumor necrosis factor‐α (TNF‐α) to establish a damaged colon epithelial model. Damaged Caco2 were cocultured with MSCs, Ad‐MSCs, Ad‐HO + MSCs or HO‐MSCs. mRNA and protein expression of Zona occludens‐1 (ZO‐1) and human HO‐1 and the release of cytokines were measured. ZO‐1 and human HO‐1 in Caco2 were significantly decreased after treatment with TNF‐α; and this effect was reduced when coculture with MSCs from bone marrow. Expression of ZO‐1 was not significantly affected by Caco2 treatment with TNF‐α, Ad‐HO, and MSCs. In contrast, ZO‐1 and human HO‐1 increased significantly when the damaged Caco2 was treated with HO‐MSCs. HO‐MSCs showed the strongest effect on the expression of ZO‐1 in colon epithelial cells. Coculture with HO‐MSCs showed the most significant effects on reducing the expression of IL‐2, IL‐6, IFN‐γ and increasing the expression of IL‐10. HO‐MSCs protected the intestinal epithelial barrier, in which endogenous HO‐1 was involved. HO‐MSCs play an important role in the repair process by reducing the release of inflammatory cytokines and increasing the release of anti‐inflammatory factors. These results suggested that HO‐MSCs from bone marrow were more effective in repairing the damaged intestinal epithelial barrier, and the effectiveness of MSCs was improved by HO‐1 gene transduction, which provides favorable support for the application of stem cell therapy in the intestinal diseases.     

Effects of ceftriaxone‐induced intestinal dysbacteriosis on dendritic cells of small intestine in mice

Intestinal microflora plays a pivotal role in the development of the innate immune system and is essential in shaping adaptive immunity. Dysbacteriosis of intestinal microflora induces altered immune responses and results in disease susceptibility. Dendritic cells (DCs), the professional antigen‐presenting cells, have gained increasing attention because they connect innate and adaptive immunity. They generate both immunity in response to stimulation by pathogenic bacteria and immune tolerance in the presence of commensal bacteria. However, few studies have examined the effects of intestinal dysbacteriosis on DCs. In this study, changes of DCs in the small intestine of mice under the condition of dysbacteriosis induced by ceftriaxone sodium were investigated. It was found that intragastric administration of ceftriaxone sodium caused severe dysteriosis in mice. Compared with controls, numbers of DCs in mice with dysbacteriosis increased significantly (P = 0.0001). However, the maturity and antigen‐presenting ability of DCs were greatly reduced. In addition, there was a significant difference in secretion of IL‐10 and IL‐12 between DCs from mice with dysbacteriosis and controls. To conclude, ceftriaxone‐induced intestinal dysbacteriosis strongly affected the numbers and functions of DCs. The present data suggest that intestinal microflora plays an important role in inducing and maintaining the functions of DCs and thus is essential for the connection between innate and adaptive immune responses.     

HIF‐1α mediates the induction of IL‐8 and VEGF expression on infection with Afa/Dr diffusely adhering E. coli and promotes EMT‐like behaviour

Microbes regulate a large panel of intracellular signalling events that can promote inflammation and/or enhance tumour progression. Indeed, it has been shown that infection of human intestinal cells with the Afa/Dr diffusely adhering E. coli C1845 strain induces expression of pro‐angiogenic and pro‐inflammatory genes. Here, we demonstrate that exposure of cryptic‐like intestinal epithelial cells to C1845 bacteria induces HIF‐1α protein levels. This effect depends on the binding of F1845 adhesin to the membrane‐associated DAF receptor that initiates signalling cascades promoting translational mechanisms. Indeed, inhibition of MAPK and PI‐3K decreases HIF‐1α protein levels and blocks C1845‐induced phosphorylation of the ribosomal S6 protein. Using RNA interference we show that bacteria‐induced HIF‐1α regulates the expression of IL‐8, VEGF and Twist1, thereby pointing to a role for HIF‐1 in angiogenesis and inflammation. In addition, infection correlates with a loss of E‐cadherin and cytokeratin 18 and a rise in fibronectin, suggesting that bacteria may induce an epithelial to mesenchymal transition‐like phenotype. Since HIF‐1α silencing results in reversion of bacteria‐induced EMT markers, we speculate that HIF‐1α plays a key role linking bacterial infection to angiogenesis, inflammation and some aspects of cancer initiation.     

In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration

Aims: The behaviour of an Escherichia coli isolate of broiler origin harbouring a bla_TEM‐52‐carrying plasmid (lactose‐negative mutant of B1‐54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration. Methods and Results: Fresh faeces from a healthy volunteer, negative for cephalosporin‐resistant E. coli, were selected to prepare inocula. The microbiota was monitored by plating on diverse selective media, and a shift in the populations of bacteria was examined by 16S rDNA PCR denaturing gradient gel electrophoresis. Escherichia coli transconjugants were verified by plasmid and pulsed‐field gel electrophoresis profiles (PFGE). The avian extended‐spectrum β‐lactamase‐positive E. coli was able to proliferate without selective pressure of cefotaxime, and E. coli transconjugants of human origin were detected 24 h after inoculation of the donor strain. Upon administration of cefotaxime to the fresh medium, an increase in the population size of E. coli B1‐54 and the transconjugants was observed. PFGE and plasmid analysis revealed a limited number of human E. coli clones receptive for the bla_TEM‐52‐carrying plasmid. Conclusions: These observations provide evidence of the maintenance of an E. coli strain of poultry origin and the horizontal gene transfer in the human commensal bowel microbiota even without antimicrobial treatment. Significance and Impact of the Study: The fact that an E. coli strain of poultry origin might establish itself and transfer its bla gene to commensal human E. coli raises public health concerns.     

The ability of an attaching and effacing pathogen to trigger localized actin assembly contributes to virulence by promoting mucosal attachment

Enterohaemorrhagic Escherichia coli (EHEC) colonizes the intestine and causes bloody diarrhoea and kidney failure by producing Shiga toxin. Upon binding intestinal cells, EHEC triggers a change in host cell shape, generating actin ‘pedestals’ beneath bound bacteria. To investigate the importance of pedestal formation to disease, we infected genetically engineered mice incapable of supporting pedestal formation by an EHEC‐like mouse pathogen, or wild type mice with a mutant of that pathogen incapable of generating pedestals. We found that pedestal formation promotes attachment of bacteria to the intestinal mucosa and vastly increases the severity of Shiga toxin‐mediated disease.     

Toll‐like receptor 4 regulates spontaneous intestinal tumorigenesis by up‐regulating IL‐6 and GM‐CSF

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll‐like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%–90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The Apc^Min/+ (multiple intestinal neoplasia) model mouse is a well‐utilized model of FAP, an inherited form of intestinal cancer. In this study, Apc^Min/+ intestinal adenoma mice were generated on TLR4‐sufficient and TLR4‐deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of Apc^Min/+ WT and Apc^Min/+ TLR4^?/? mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high‐throughput RNA‐Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine‐cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM‐CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of Apc^Min/+ TLR4^?/? mice compared with Apc^Min/+ WT mice. In the functional study of core down‐regulation factors, it was found that IL6, GM‐CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.     

Physiological activity of E. coli engineered to produce butyric acid

Faecalibacterium prausnitzii (F. prausnitzii) is one of the most abundant bacteria in the human intestine, with its anti-inflammatory effects establishing it as a major effector in human intestinal health. However, its extreme sensitivity to oxygen makes its cultivation and physiological study difficult. F. prausnitzii produces butyric acid, which is beneficial to human gut health. Butyric acid is a short-chain fatty acid (SCFA) produced by the fermentation of carbohydrates, such as dietary fibre in the large bowel. The genes encoding butyryl-CoA dehydrogenase (BCD) and butyryl-CoA:acetate CoA transferase (BUT) in F. prausnitzii were cloned and expressed in E. coli to determine the effect of butyric acid production on intestinal health using DSS-induced colitis model mice. The results from the E. coli Nissle 1917 strain, expressing BCD, BUT, or both, showed that BCD was essential, while BUT was dispensable for producing butyric acid. The effects of different carbon sources, such as glucose, N-acetylglucosamine (NAG), N-acetylgalactosamine (NAGA), and inulin, were compared with results showing that the optimal carbon sources for butyric acid production were NAG, a major component of mucin in the human intestine, and glucose. Furthermore, the anti-inflammatory effects of butyric acid production were tested by administering these strains to DSS-induced colitis model mice. The oral administration of the E. coli Nissle 1917 strain, carrying the expression vector for BCD and BUT (EcN-BCD-BUT), was found to prevent DSS-induced damage. Introduction of the BCD expression vector into E. coli Nissle 1917 led to increased butyric acid production, which improved the strain’s health-beneficial effects.     

Citrobacter rodentium induces rapid and unique metabolic and inflammatory responses in mice suffering from severe disease

The mouse pathogen Citrobacter rodentium is used to model infections with enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC). Pathogenesis is commonly modelled in mice developing mild disease (e.g., C57BL/6). However, little is known about host responses in mice exhibiting severe colitis (e.g., C3H/HeN), which arguably provide a more clinically relevant model for human paediatric enteric infection. Infection of C3H/HeN mice with C. rodentium results in rapid colonic colonisation, coinciding with induction of key inflammatory signatures and colonic crypt hyperplasia. Infection also induces dramatic changes to bioenergetics in intestinal epithelial cells, with transition from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and higher abundance of SGLT4, LDHA, and MCT4. Concomitantly, mitochondrial proteins involved in the TCA cycle and OXPHOS were in lower abundance. Similar to observations in C57BL/6 mice, we detected simultaneous activation of cholesterol biogenesis, import, and efflux. Distinctly, however, the pattern recognition receptors NLRP3 and ALPK1 were specifically induced in C3H/HeN. Using cell‐based assays revealed that C. rodentium activates the ALPK1/TIFA axis, which is dependent on the ADP‐heptose biosynthesis pathway but independent of the Type III secretion system. This study reveals for the first time the unfolding intestinal epithelial cells' responses during severe infectious colitis, which resemble EPEC human infections.     

Live Faecalibacterium prausnitzii induces greater TLR2 and TLR2/6 activation than the dead bacterium in an apical anaerobic co‐culture system

Inappropriate activation of intestinal innate immune receptors, such as toll‐like receptors (TLRs), by pathogenic bacteria is linked to chronic inflammation. In contrast, a “tonic” level of TLR activation by commensal bacteria is required for intestinal homeostasis. A technical challenge when studying this activation in vitro is the co‐culturing of oxygen‐requiring mammalian cells with obligate anaerobic commensal bacteria. To overcome this, we used a novel apical anaerobic co‐culture system to successfully adapt a TLR activation assay to be conducted in conditions optimised for both cell types. Live Faecalibacterium prausnitzii, an abundant obligate anaerobe of the colonic microbiota, induced higher TLR2 and TLR2/6 activation than the dead bacterium. This enhanced TLR induction by live F. prausnitzii, which until now has not previously been described, may contribute to maintenance of gastrointestinal homeostasis. This highlights the importance of using physiologically relevant co‐culture systems to decipher the mechanisms of action of live obligate anaerobes.     

Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> Isolated from Patients Affected by Crohn’s Disease

The etiopathogenesis of Crohn’s disease (CD) is still controversial: several genetic, immunologic, and environmental factors, including some bacteria, have been implicated. This study has been devised to assess the involvement of Escherichia coli in CD. Seven E. coli strains were isolated from 14 biopsies obtained from ileocolic ulcers of patients affected by inflammatory bowel disease (IBD), including six with ulcerative colitis and eight with CD. Five strains, exclusively isolated from CD patients, were found inside mucosal cells. Different PCR techniques (for chuA, yjaA, TspE4.C2, escV, and bfpB genes) were performed and PFGE was carried out to characterize these bacteria in comparison with other E. coli strains isolated from non-IBD specimens. The correlation of these characters with bacterial invasiveness on intestinal (Caco-2) and phagocytic (U937) cells was assessed. Overall our pilot data suggest that five among eight strains isolated from CD patients belonged to the adherent-invasive E. coli (AIEC) group, and were invasive on Caco-2 cells and resistant to phagocytosis. These findings suggest that these bacteria could be considered target organisms whose elimination could reduce the intestinal inflammatory process and CD progression.