Chenyang Wang

Dysbiosis of Gut Fungal Microbiota is Associated With Mucosal Inflammation in Crohn’s Disease

Goals: We aim to characterize the fungal microbiota in the intestinal mucosa and feces in patients with Crohn’s disease (CD). Background: Fungi represent a diverse microbial community in the human intestine and might play a role in the pathogenesis of CD; however, little is known about the structure and composition of the fungal microbiota especially adhering to the intestinal mucosa in CD patient. Study: Nineteen patients with active CD and 7 healthy individuals were recruited in this study. The mucosa-associated and fecal fungal microbiotas in CD patients were analyzed using culture-independent community fingerprint techniques. Results: The fungal richness and diversity were significantly elevated in the inflamed mucosa compared with the noninflamed mucosa. The predominant fungal composition in the inflamed mucosa was strikingly altered, mainly characterized by expansion in the proportions of Candida spp., Gibberella moniliformis, Alternaria brassicicola, and Cryptococcus neoformans. The fecal fungal community was perturbed in CD patients as accompanied by increased fungal diversity and prevalence in Candida albicans, Aspergillus clavatus, and C. neoformans. The species richness and diversity of the mucosal fungal community were associated with the expression of TNF-&agr;, IFN-&ggr;, or IL-10 (P<0.05). The diversity of the fecal fungal microbiota positively correlated with serum C-reactive protein and CD activity index (P<0.05). Conclusions: This study first demonstrates that the fungal microbiota in the inflamed mucosa is distinguishable from that of the noninflamed area. Shifts of gut fungal microbiota composition may be associated with mucosal inflammation and disease activity of CD. Our data would provide novel insights into understanding the potential of gut fungal microbiota in the pathogenesis of CD.

Effect of n-3 polyunsaturated fatty acids on membrane microdomain localization of tight junction proteins in experimental colitis.

Ulcerative colitis (UC) is a gastrointestinal disorder characterized by an inflammatory process associated with mucosal damage. Many studies have shown that n‐3 polyunsaturated fatty acids (PUFAs) possess anti‐inflammatory effects in inflammatory bowel disease. The aim of this study was to investigate whether n‐3 PUFAs could alleviate intestinal damage in experimental UC. In the present study, we found that in 2,4,6‐trinitrobenzenesulfonic acid‐induced colitic rats, the damage to the intestinal mucosa was accompanied by a disrupted tight junction (TJ) structure. In accordance with these changes, the distribution and expression of TJ proteins, including occludin, claudin‐1, claudin‐3, claudin‐5, claudin‐8 and ZO‐1, in membrane microdomains was altered. The distribution of flotillin‐1, a lipid raft marker protein, was also changed. Moreover, we found for the first time that n‐3 PUFAs prevented redistribution of TJ proteins from Triton X‐100‐insoluble raft‐like membrane microdomains to Triton X‐100‐soluble fractions. The expression of ZO‐1, claudin‐1, claudin‐5 and claudin‐8 was significantly elevated by n‐3 PUFAs. n‐3 PUFAs also attenuated the disruption of TJ structure and improved the histological score. Our results demonstrate that the expression and distribution of TJ proteins in TJ membrane microdomains might be affected in UC, and that such altered expression of TJ proteins in membrane microdomains in experimental UC is affected by n‐3 PUFAs. These findings may have therapeutic potential in intestinal inflammation.

Disruption of tight junctions during polymicrobial sepsis in vivo

The disruption of intestinal epithelial tight junctions may result in barrier function dysfunction during polymicrobial sepsis. The pathophysiology of sepsis involves breakdown of barrier integrity, which correlates with adverse outcome during sepsis. However, the mechanisms underlying loss of barrier function in sepsis remain unknown. In the present study in mice, tight junction (TJ) structure was analysed by transmission electron microscopy; intestinal permeability was assessed using molecular tracer measurement; and the distribution of TJ proteins was investigated by immunofluorescence microscopy. The membrane microdomains of TJs were isolated using discontinuous sucrose density gradients and the expression of TJ proteins in these was determined by western blot. Immunofluorescence microscopy revealed that claudins 1, 3, 4, 5, and 8 were present predominantly in the microvillous surface of epithelial cells and along the lateral membranes of the cells; in sepsis, however, labelling of these proteins was present diffusely within cells and was no longer focused at the lateral cell boundaries. Moreover, the expression of claudin‐2 was markedly up‐regulated in sepsis. Using western blot analysis, we found that occludin and claudins were displaced from raft fractions to non‐raft fractions in membrane microdomains of TJs in sepsis. In addition, the disruption of TJ structure was accompanied by increased intestinal permeability. Our results demonstrate for the first time that redistribution of TJ proteins in TJ membrane microdomains and redistribution of claudins in epithelial cells of the colon lead to alteration of TJ architecture and TJ barrier dysfunction during the development of polymicrobial sepsis. Copyright

Molecular-Phylogenetic Characterization of the Microbiota in Ulcerated and Non-Ulcerated Regions in the Patients with Crohn's Disease

Background The dysbiosis of intestinal microbiota has been established in Crohns disease (CD), but the molecular characterization of this dysbiosis in Chinese subjects with CD remains unclear. This study aims to investigate the predominant bacterial composition of the faecal and mucosal-associated microbiota in Chinese CD patients using culture-independent techniques. Methods/Principal Findings Eighteen patients with CD and 9 healthy controls were included in this study. The faeces and the intestinal mucosal tissues from the ulcerated and nonulcerated sites were subjected to bacterial community fingerprinting using denaturing gradient gel electrophoresis (DGGE). The predominant bacterial composition in the faeces and mucosa was determined with DNA sequencing and BLAST. We showed that the bacterial diversity in the faeces of CD patients was reduced compared with that in healthy controls (p<0.01). The faecal bacterial dysbiosis of the patients was characterized by an elevated abundance of γ-Proteobacteria (especially Escherichia coli and Shigella flexneri) and a reduced proportion of Bacteroidetes and Firmicutes. Five bacterial species defined the microbiota imbalance of the ulcerated mucosa in CD, including an increase in Escherichia coli, a decrease in Faecalibacterium prausnitzii, Lactobacillus coleohominis, Bacteroides sp and Streptococcus gallolyticus in the bacterial community as compared with the nonulcerated (p<0.01). Conclusions/Significance This is the first description of intestinal microbiota dysbiosis in Chinese CD patients. These results allow a better understanding of the faecal and mucosal microbiota in CD, showing a predominance of some opportunistic pathogenic bacteria and a decrease in beneficial bacterial species. The findings may provide novel insights into the pathogenesis of CD in Chinese population.

Invasion of enteropathogenic Escherichia coli into host cells through epithelial tight junctions

Enteropathogenic Escherichia coli (EPEC) has been shown to disrupt the barrier function of host intestinal epithelial tissues through entering tight junctions. However, the mechanism by which this occurs remains poorly understood. In this study, we determined that EPEC invades host cells through tight junctions as it initiates infection. Immunofluorescence microscopy revealed redistribution of the tight‐junction proteins occludin and ZO‐1 from an intercellular to a cytoplasmic location after EPEC invasion. Flotillin‐1 was recruited to sites of EPEC entry. EPEC entered host cells through tight‐junction membrane microdomains. Tight‐junction ultrastructure was disrupted following EPEC infection, accompanied by loss of barrier function. EPEC infection caused a time‐dependent decrease in trans‐epithelial electrical resistance. Subcellular fractionation using discontinuous sucrose density gradients demonstrated a decline in raft‐associated occludin following exposure to EPEC. These results indicate the important role of host membrane tight‐junction microdomains in EPEC invasion.

Altered distribution of tight junction proteins after intestinal ischaemia/reperfusion injury in rats

Tight junction (TJ) disruptions have been demonstrated both in vitro and more recently in vivo in infection. However, the molecular basis for changes of TJ during ischaemia‐reperfusion (I/R) injury is poorly understood. In the present study, intestinal damage was induced by I/R in an animal model. As assessed by TUNEL and propidium iodide uptake, we showed that I/R injury induced apoptosis as well as necrosis in rat colon, and the frequency of apoptotic and necrotic cells reached the maximum at 5 hrs of reperfusion. Immunofluorescence microscopy revealed that claudins 1, 3 and 5 are strongly expressed in the surface epithelial cells of the colon; however, labelling of all three proteins was present diffusely within cells and no longer focused at the lateral cell boundaries after I/R. Using Western blot analysis, we found that distribution of TJ proteins in membrane microdomains of TJ was markedly affected in I/R injury rats. Occludin, ZO‐1, claudin‐1 and claudin‐3 were completely displaced from TX‐100 insoluble fractions to TX‐100 soluble fractions, and claudin‐5 was partly displaced. The distribution of lipid raft marker protein caveolin‐1 was also changed after I/R. I/R injury results in the disruption of TJs, which characterized by relocalization of the claudins 1, 3 and 5 and an increase in intestinal permeability using molecular tracer measurement. I/R injury altered distribution of TJ proteins in vivo that was associated with functional TJ deficiencies.

Successful treatment of severe sepsis and diarrhea after vagotomy utilizing fecal microbiota transplantation: a case report

IntroductionDysbiosis of intestinal microbiota likely plays an important role in the development of gut-derived infections, making it a potential therapeutic target against sepsis. However, experience with fecal microbiota transplantation (FMT) in the treatment of sepsis and knowledge of the underlying mechanisms are extremely lacking. In this article, we describe a case of a patient who developed sepsis after a vagotomy and later received an infusion of donor feces microbiota, and we report our findings.MethodsA 44-year-old woman developed septic shock and severe watery diarrhea 4 days after undergoing a vagotomy. Antibiotics, probiotics and supportive treatment strategies were used for about 30 day after surgery, but the patient’s fever, bacteremia and watery diarrhea persisted. Considering the possibility of intestinal dysbiosis, we evaluated the structure and composition of the patient’s fecal microbiota using 16S rDNA-based molecular techniques. As expected, the gut microbiota was extensively disrupted; therefore, a donor fecal suspension was delivered into the patient by nasoduodenal tube. The patient’s clinical outcomes and shifts of the gut microbiota following the treatment were also determined.ResultsDramatically, the patient’s septic symptoms and severe diarrhea were successfully controlled following FMT. Her stool output markedly declined after 7 days and normalized 16 days after FMT. A significant modification in her microbiota composition was consistently seen, characterized by a profound enrichment of the commensals in Firmicutes and depletion of opportunistic organisms in Proteobacteria. Furthermore, we identified a reconstituted bacterial community enriched in Firmicutes and depleted of Proteobacteria members that was associated with fecal output, plasma markers of inflammation and T helper cells.ConclusionsIn this report, we describe our initial experience with FMT, in which we successfully used it in the treatment of a patient with sepsis and severe diarrhea after a vagotomy. Our data indicate an association between repaired intestinal microbiota barrier and improvement of clinical outcomes. Our patient’s surprising clinical benefits from FMT demonstrate the role of intestinal microbiota in modulating immune equilibrium. It represents a breakthrough in the clinical management of sepsis and suggests new therapeutic avenues to pursue for microbiota-related indications.

Fish Oil Enhances Recovery of Intestinal Microbiota and Epithelial Integrity in Chronic Rejection of Intestinal Transplant

Background The intestinal chronic rejection (CR) is the major limitation to long-term survival of transplanted organs. This study aimed to investigate the interaction between intestinal microbiota and epithelial integrity in chronic rejection of intestinal transplantation, and to find out whether fish oil enhances recovery of intestinal microbiota and epithelial integrity. Methods/Principal Findings The luminal and mucosal microbiota composition of CR rats were characterized by DGGE analysis at 190 days after intestinal transplant. The specific bacterial species were determined by sequence analysis. Furthermore, changes in the localization of intestinal TJ proteins were examined by immunofluorescent staining. PCR-DGGE analysis revealed that gut microbiota in CR rats had a shift towards Escherichia coli, Bacteroides spp and Clostridium spp and a decrease in the abundance of Lactobacillales bacteria in the intestines. Fish oil supplementation could enhance the recovery of gut microbiota, showing a significant decrease of gut bacterial proportions of E. coli and Bacteroides spp and an increase of Lactobacillales spp. In addition, CR rats showed pronounced alteration of tight junction, depicted by marked changes in epithelial cell ultrastructure and redistribution of occuldin and claudins as well as disruption in TJ barrier function. Fish oil administration ameliorated disruption of epithelial integrity in CR, which was associated with an improvement of the mucosal structure leading to improved tight junctions. Conclusions/Significance Our study have presented novel evidence that fish oil is involved in the maintenance of epithelial TJ integrity and recovery of gut microbiota, which may have therapeutic potential against CR in intestinal transplantation.

Enteropathogenic Escherichia coli changes distribution of occludin and ZO-1 in tight junction membrane microdomains in vivo

Diarrhea is a disease caused by enteropathogenic Escherichia coli (EPEC) infection, which caused the deaths of several hundred thousand children each year. However, the molecular mechanisms underlying EPEC infection in vivo are not fully understood. In the present study, we used the C57BL/6J mouse as an in vivo model of EPEC infection and investigated the effect of EPEC on tight junction (TJ) structure and barrier function. TJ ultrastructure was studied by transmission electron microscopy and a small molecule tracer biotin was used to examine the paracellular permeability of the colon. The distribution of TJ proteins occludin and ZO-1 in the epithelium was investigated by immunofluorescence microscopy. Our results demonstrated that TJ structure was disrupted following EPEC infection. And the morphological changes of TJ were accompanied by increased paracellular permeability which led to impairment of TJ barrier function. Immunofluorescency analysis revealed that occludin and ZO-1 were translocated from villous membrane to the cytoplasm in intestinal epithelial cells during EPEC invasion. Moreover, wild-type EPEC and the mutant EPEC strain, DeltaespF, had similar effects on barrier function and TJ protein localization at 5 days postinfection. Our findings demonstrate that EPEC infection in vivo led to disruption of tight junction barrier function. These results may provide insights into the molecular mechanism of the pathogenesis of EPEC infection.

Bacteremia in patients with acute pancreatitis as revealed by 16S ribosomal RNA gene-based techniques*.

Objectives:To define the characteristic of bacteremia in patients with acute pancreatitis and determine its possible association with the disease severity. Design:A prospective controlled study. Setting:ICU of Jinling Hospital, China. Patients:A total of 48 patients with mild or severe acute pancreatitis were enrolled in the study. Interventions:None. Measurements and Main Results:Samples of peripheral blood were collected from the patients at 4 or 5 and 9 or 10 days after acute pancreatitis was definitely diagnosed. Resulting DNA from the blood was analyzed using denaturing gradient gel electrophoresis, and separated fragments were sequenced for identification of bacterial species. Bacterial DNA was detected in peripheral blood from 68.8% of patients with acute pancreatitis, and more than half (60.4%) of the patients encountered polymicrobial flora. Translocated bacteria in patients with acute pancreatitis were primarily constituted of opportunistic pathogens derived from the gut, including Escherichia coli, Shigella flexneri, Enterobacteriaceae bacterium, Acinetobacter lwoffii, Bacillus coagulans, and Enterococcus faecium. The species of circulating bacteria shifted remarkably among the patients with different severity. The presence of the bacteremia correlated positively with the Acute Physiology and Chronic Health Evaluation-II scores of patients with acute pancreatitis (r = 0.7918, p < 0.0001). Conclusions:This study provides a detailed description on the prevalence of bacteremia and characteristic of bacterial species in patients with acute pancreatitis. We demonstrate an association between the bacteremia and the disease severity, which enables us to better understand a potential role of bacterial translocation in the pathogenesis of septic complication in acute pancreatitis.