Influence of Prebiotic Activity of Agave salmiana Fructans on Mucus Production and Morphology Changes in Colonic Epithelium Cell of Healthy Wistar Rats

The beneficial health of evaluating prebiotic effect by the consumption of Agave salmiana fructans (A. salmiana fructans) was assessed in the epithelium of the cecum and proximal colon of Wistar rats fed at different doses for 35 days with regards to mucus production, morphological cell changes, and the serum concentration of tumor necrosis factor-α (TNF-α). Results showed a significant increase in mucus-secreting cells (P < 0.05) and a normal structure with preserved crypts, without morphological damage to colonic cells for a dose of 12.5% (w/w) with respect to the control and the other doses evaluated. The concentration of pro-inflammatory cytokine TNF-α was decreased significantly (P < 0.05) in the groups with doses of 10 and 12.5% (w/w) at 7 and 35 days, respectively. This effect was positively correlated with the reduction of inflammation in epithelial cells. This study provides direct evidence of the effects of the A. salmiana fructans on the colonic epithelium, demonstrating that a diet supplemented with 12.5% of fructans for 35 days exerts health benefits through the strengthening of the mucosa layer, which favors the adherence of the bacterial population and suppresses inflammation.

YfiB and the tripartite signaling system YfiBNR is involved in virulence of the gut invasive pathogen Shigella flexneri.

Shigella flexneri is one of the principal cause of bacillary dysentery and contributes significantly to the worldwide implication of diarrheal infections. The presence and upsurge of multidrug resistance amongst Shigella strains, demands additional genetic analyses, advancement of new/improved drugs, and finding vaccine candidates against the pathogen. Whilst many features about the invasion of colonic cells by Shigella have been identified, fundamental gaps in information concerning in what way the bacteria transit, survive, and control gene expression, remain. Present study aims to illustrate the role of yfiB gene in Shigella virulence, which is a part of the periplasmic YfiBNR tripartite signaling system. This system is responsible for regulating cyclic-di-GMP levels inside the bacterial cells, which is a vital messenger molecule impacting varied cellular processes involving biofilm formation, cytotoxicity, motility, synthesis of exopolysaccharide, and other virulence mechanisms like adhesion and invasion of the bacteria. Through a combination of genetic, biochemical, and virulence assays, we show how knocking out the yfiB gene can disrupt the entire YfiBNR system and affect biofilm formation, bacterial invasion, surface attachment, and the virulence of Shigella. We then show how targeted mutagenesis of the significant amino acids of the YfiB protein can affect the proper functioning of the protein. This study eventually improves our understanding of the in-vivo persistence and survival of Shigella and provides a prospective new target to design anti-infective drugs and vaccines against S. flexneri and other bacterial pathogens.

Colonic Epithelial PHLPP2 Deficiency Promotes Colonic Epithelial Pyroptosis by Activating the NF-κB Signaling Pathway

Background. Ulcerative colitis (UC) is a chronic inflammatory disease with increasing prevalence worldwide. Barrier defect in intestinal epithelial cells (IECs) is one of the main pathogeneses in UC. Pyroptosis is a programmed lytic cell death and is triggered by inflammatory caspases, while little is known about its role in UC. Methods. Differentially expressed genes (DEGs) were identified by comparing UC patients with healthy controls from the GEO datasets. The candidate genes involved in pyroptosis were obtained, and the underlying molecular mechanism in the progression of UC was explored in vivo and in vitro. Results. Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2), a protein phosphatase, was downregulated and involved in regulating inflammation-induced IEC pyroptosis by modulating the NF-κB signaling in UC through bioinformatics analysis. Moreover, we demonstrated that PHLPP2 was downregulated in UC patients and UC mice. Besides, we found that PHLPP2 depletion activated the NF-κB signaling and increased the expressions of caspase-1 P20, Gasdermin N, IL-18, and IL-1β contributing to IEC pyroptosis and inflammation in UC mice. Furthermore, we found that PHLPP2-/- mice developed hypersensitivity to dextran sulfate sodium (DSS) treatment toward colitis showing activated NF-κB signaling and dramatically induced expressions of caspase-1 P20, Gasdermin N, IL-18, and IL-1β. Mechanically, this inflammation-induced downregulation of PHLPP2 was alleviated by an NF-κB signaling inhibitor in intestinal organoids of PHLPP2-/- mice and fetal colonic cells. Conclusions. PHLPP2 downexpression activated the NF-κB signaling and promoted the IEC pyroptosis, leading to UC progression. Therefore, PHLPP2 might be an attractive candidate therapeutic target for UC.

P679 The keystone bacterium Christensenella minuta improves colitis in in vivo preclinical Inflammatory Bowel Disease models

Background There is increasing evidence that microbiome-based therapies can correct dysbiosis and reduce inflammation associated with Inflammatory Bowel Diseases (IBD). In particular, the human commensal family of Christensenellaceae bacteria has been reported as missing in several cohorts of Crohn’s disease patients, indicating that it may have a role in maintaining microbial symbiosis in this population. To assess its potential for IBD management, we used the reference type species Christensenella minuta DSM 22607 to examine its anti-inflammatory properties. Methods We first performed two distinct independent preclinical colitis models: i) a moderate DNBS-induced colitis model in mice and ii) a severe TNBS-induced colitis model in rats. To decipher the mechanisms of action of C. minuta underlying the observed effects, we determined its ability to produce short chain fatty acids (SCFA) at different growth phases and we assessed its capacity to modulate the inflammatory response of human colonic cells. Results Our results showed that in both rodent models, C. minuta prevented intestinal damages by decreasing macroscopic scores, reduced colonic inflammation by limiting neutrophils infiltration in the colon and stimulated mucosal healing. We also confirmed that C. minuta is a high acetate and moderate butyrate producer. Finally, we showed that C. minuta displayed potent anti-inflammatory properties by decreasing the secretion of the pro-inflammatory cytokine IL-8 through NF-kB inhibition in HT-29 cells. Conclusion Together, these results revealed for the first time the strong anti-inflammatory properties of C. minuta and confirm its high potential as an innovative microbiome-based biotherapy for IBD.

Immunohistochemical expression of β-catenin, Ki67, CD3 and CD18 in canine colorectal adenomas and adenocarcinomas

Background Inflammation is believed to influence human colorectal carcinogenesis and may have an impact on prognosis and survival. The mucosal immunophenotype in dogs with colorectal cancer is poorly described. The aim of this study was to investigate whether the density, distribution and grade of tumor-infiltrating immune cells (TIIs) are different in normal colonic tissue vs benign stages (adenomas) and malignant stages (adenocarcinomas) of canine colorectal carcinogenesis, and thus, whether they can be considered as prognostic factors in dogs. This retrospective case-control study was performed on formalin-fixed, paraffin-embedded tissue samples from dogs with histologically confirmed colorectal adenoma (n = 18) and adenocarcinoma (n = 13) collected from archived samples. The samples had been collected by colonoscopy, surgery or during postmortem examination. Healthy colonic tissue obtained post mortem from dogs euthanized for reasons not involving the gastrointestinal tract served as control tissue (n = 9). Results The tumor samples had significantly lower numbers of CD3+ T-cells in the epithelium compared to controls (adenocarcinoma vs control, Kruskal-Wallis test, p = 0.0004, and adenoma vs control, p = 0.002). Adenomas had a significantly lower number of CD18+ cells in the lamina propria, compared to control samples (Kruskal-Wallis test, p = 0.008). Colonic samples from control dogs had uniform staining of β-catenin along the cell membrane of epithelial cells. Compared to normal colonic cells, the expression levels of cytoplasmic β-catenin were significantly higher in adenomas and adenocarcinomas (adenoma vs control Kruskal-Wallis test, p = 0.004, and adenocarcinoma vs control, p = 0.002). None of the control samples showed positive staining of β-catenin in the nucleus of colonic cells. In contrast, adenocarcinomas and adenomas showed moderate to strong staining of the cell nucleus. The nuclear β-catenin expression (signal strength and distribution) was significantly higher in adenomas compared to adenocarcinomas (Kruskal-Wallis test, p < 0.05). Conclusions β-catenin and Ki67 were not useful markers for demonstrating tumor progression from adenomas to adenocarcinomas. The lower presence of CD18 and CD3+ cells in colorectal tumors compared to controls indicates a reduced presence of histiocytes and T-cells, which may have implications for the pathogenesis and progression of colorectal cancer in dogs.

Immunohistochemical Expression of β-catenin, Ki67, CD3 and CD18 in Canine Colorectal Adenomas and Adenocarcinomas

Background Inflammation is believed to influence the human colorectal carcinogenesis and may have impact upon prognosis and survival. High presence of tumour-infiltrating CD3+ T-cells, is associated with a better outcome in humans with colorectal cancer. The mucosal immunophenotype in dogs with colorectal cancer is poorly described. The aim of this study was to investigate whether the density, distribution and grade of tumour-infiltrating immune cells (TIIs) in canine colorectal tumours is associated with histologic indicators of malignancy and can be considered a prognostic factor in dogs. This retrospective case-control study was performed on formalin-fixed, paraffin-embedded tissue samples from dogs with histologically confirmed colorectal adenoma (n=18) and adenocarcinoma (n=5) collected from archived samples. The samples had been collected by colonoscopy, surgery or during postmortem examination. Healthy colonic tissue obtained post mortem from dogs euthanized of reasons not involving the gastrointestinal tract, served as control tissue (n=9). Results: The tumour samples had significantly lower numbers of CD3+ T- cells in the epithelium compartment (Wilcoxon test, p=0,0006), as well as significantly lower number of CD18+ cells in the lamina propria, compared to control samples (Wilcoxon test, p=0,001). The Ki67 positive cells showed a strong signal in adenomas and adenocarcinomas. There was no clear distinction with regards to expression levels of the markers for tumour progression (β-catenin, and Ki67) between adenomas and adenocarcinomas. Colonic samples from control dogs had uniform staining of β-catenin along the cell membrane of epithelial cells. When compared to normal colonic cells, the expression levels of cytoplasmic β-catenin were significantly higher in adenomas and adenocarcinomas (Wilcoxon test, p=0,0002). None of the control samples showed positive staining of β-catenin in the nucleus of colonic cells. In contrast, adenocarcinoma and adenoma showed moderate to strong staining of the cell nucleus. Conclusions: β-catenin and Ki67 were not useful markers in distinguishing adenomas from adenocarcinomas. The lower presence of CD18- and CD3+ cells in tumours compared to controls, indicates a reduced presence of histiocytes and T-cells which may have implications for the defense against cancer progression.