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Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith, 8, e73204 (2013); S. Kozar , 13, 626-633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.
The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery (blood in stool) worldwide every year, resulting in more than 200,000 deaths. A major challenge in combating bacillary dysentery is the lack of a small-animal model that recapitulates the symptoms observed in infected individuals, including bloody diarrhea. Here, we show that similar to humans, infant rabbits infected with S. flexneri experience severe inflammation, massive ulceration of the colonic mucosa, and bloody diarrhea. T3SS-dependent invasion of epithelial cells is necessary and sufficient for mediating immune cell infiltration and vascular lesions. However, massive ulceration of the colonic mucosa, bloody diarrhea, and dramatic weight loss are strictly contingent on the ability of the bacteria to spread from cell to cell. The infant rabbit model features bacterial dissemination as a critical determinant of S. flexneri pathogenesis and provides a unique small-animal model for research and development of therapeutic interventions.
There is great interest in safe and effective alternative therapies that could benefit patients with inflammatory bowel diseases (IBD). L-arginine (Arg) is a semi-essential amino acid with a variety of physiological effects. In this context, our aim was to investigate the role of dietary Arg in experimental colitis. We used two models of colitis in C57BL/6 mice, the dextran sulfate sodium (DSS) model of injury and repair, and infection. Animals were given diets containing (1) no Arg (Arg), 6.4 g/kg (Arg), or 24.6 g/kg Arg (Arg); or (2) the amino acids downstream of Arg: 28 g/kg L-ornithine (Orn) or 72 g/kg L-proline (Pro). Mice with DSS colitis receiving the Arg diet had increased levels of Arg, Orn, and Pro in the colon and improved body weight loss, colon length shortening, and histological injury compared to Arg and Arg diets. Histology was improved in the Arg vs. Arg group. Orn or Pro diets did not provide protection. Reduction in colitis with Arg diet also occurred in -infected mice. Diversity of the intestinal microbiota was significantly enhanced in mice on the Arg diet compared to the Arg or Arg diets, with increased abundance of Bacteroidetes and decreased Verrucomicrobia. In conclusion, dietary supplementation of Arg is protective in colitis models. This may occur by restoring overall microbial diversity and Bacteroidetes prevalence. Our data provide a rationale for Arg as an adjunctive therapy in IBD.
In IBD patients, integration between a hyper-activated immune system and epithelial cell plasticity underlies colon cancer development. However, molecular regulation of such a circuity remains undefined. Claudin-1 (Cld-1), a tight-junction integral protein deregulation alters colonic epithelial cell (CEC) differentiation, and promotes colitis severity while impairing colitis-associated injury/repair. Tumorigenesis is a product of an unregulated wound-healing process and therefore we postulated that upregulated Cld-1 levels render IBD patients susceptible to the colitis-associated cancer (CAC). Villin Cld-1 mice are used to carryout overexpressed studies in mice. The role of deregulated Cld-1 expression in CAC and the underlying mechanism was determined using a well-constructed study scheme and mouse models of DSS colitis/recovery and CAC. Using an inclusive investigative scheme, we here report that upregulated Cld-1 expression promotes susceptibility to the CAC and its malignancy. Increased mucosal inflammation and defective epithelial homeostasis accompanied the increased CAC in Villin-Cld-1-Tg mice. We further found significantly increased levels of protumorigenic M2 macrophages and β-cateninSer552 (β-CatSer552) expression in the CAC in Cld-1Tg vs. WT mice. Mechanistic studies identified the role of PI3K/Akt signaling in Cld-1-dependent activation of the β-CatSer552, which, in turn, was dependent on proinflammatory signals. Our studies identify a critical role of Cld-1 in promoting susceptibility to CAC. Importantly, these effects of deregulated Cld-1 were not associated with altered tight junction integrity, but on its noncanonical role in regulating Notch/PI3K/Wnt/ β-CatSer552 signaling. Overall, outcome from our current studies identifies Cld-1 as potential prognostic biomarker for IBD severity and CAC, and a novel therapeutic target.
Exomeres are a recently discovered type of extracellular nanoparticle with no known biological function. Herein, we describe a simple ultracentrifugation-based method for separation of exomeres from exosomes. Exomeres are enriched in Argonaute 1-3 and amyloid precursor protein. We identify distinct functions of exomeres mediated by two of their cargo, the β-galactoside α2,6-sialyltransferase 1 (ST6Gal-I) that α2,6- sialylates N-glycans, and the EGFR ligand, amphiregulin (AREG). Functional ST6Gal-I in exomeres can be transferred to cells, resulting in hypersialylation of recipient cell-surface proteins including β1-integrin. AREG-containing exomeres elicit prolonged EGFR and downstream signaling in recipient cells, modulate EGFR trafficking in normal intestinal organoids, and dramatically enhance the growth of colonic tumor organoids. This study provides a simplified method of exomere isolation and demonstrates that exomeres contain and can transfer functional cargo. These findings underscore the heterogeneity of nanoparticles and should accelerate advances in determining the composition and biological functions of exomeres.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.
The myeloid translocation gene family member MTG16 is a transcriptional corepressor that relies on the DNA-binding ability of other proteins to determine specificity. One such protein is the ZBTB family member Kaiso, and the MTG16:Kaiso interaction is necessary for repression of Kaiso target genes, such as matrix metalloproteinase-7. Using the azoxymethane and dextran sodium sulfate (AOM/DSS) murine model of colitis-associated carcinoma, we previously determined that MTG16 loss accelerates tumorigenesis and inflammation. However, it was unknown whether this effect was modified by Kaiso-dependent transcriptional repression. To test for a genetic interaction between MTG16 and Kaiso in inflammatory carcinogenesis, we subjected single and double knockout (DKO) mice to the AOM/DSS protocol. Mtg16 mice demonstrated increased colitis and tumor burden; in contrast, disease severity in Kaiso mice was equivalent to wild-type controls. Surprisingly, Kaiso deficiency in the context of MTG16 loss reversed injury and pro-tumorigenic responses in the intestinal epithelium following AOM/DSS treatment, and tumor numbers were returned to near to wild-type levels. Transcriptomic analysis of non-tumor colon tissue demonstrated that changes induced by MTG16 loss were widely mitigated by concurrent Kaiso loss, and DKO mice demonstrated downregulation of metabolism and cytokine-associated gene sets with concurrent activation of DNA damage checkpoint pathways as compared with Mtg16. Further, Kaiso knockdown in intestinal enteroids reduced stem- and WNT-associated phenotypes, thus abrogating the induction of these pathways observed in Mtg16 samples. Together, these data suggest that Kaiso modifies MTG16-driven inflammation and tumorigenesis and suggests that Kaiso deregulation contributes to MTG16-dependent colitis and CAC phenotypes.
BACKGROUND & AIMS - Patients with inflammatory bowel diseases who have postinflammatory polyps (PIPs) have an increased risk of colorectal neoplasia (CRN). European guidelines propose that patients with PIPs receive more frequent surveillance colonoscopies, despite limited evidence of this increased risk. We aimed to define the risk of CRN and colectomy in patients with inflammatory bowel diseases and PIPs.
METHODS - We conducted a multicenter retrospective cohort study of patients with inflammatory bowel diseases who underwent colonoscopic surveillance for CRN, from January 1997 through January 2017, at 5 academic hospitals and 2 large nonacademic hospitals in New York or the Netherlands. Eligible patients had confirmed colonic disease with duration of at least 8 years (or any duration, if they also had primary sclerosing cholangitis) and no history of advanced CRN (high-grade dysplasia or colorectal cancer) or colectomy. The primary outcome was occurrence of advanced CRN according to PIP status; secondary outcomes were occurrence of CRN (inclusive of low-grade dysplasia) and colectomy.
RESULTS - Of 1582 eligible patients, 462 (29.2%) had PIPs. PIPs were associated with more severe inflammation (adjusted odds ratio 1.32; 95% confidence interval [CI] 1.13-1.55), greater disease extent (adjusted odds ratio 1.92; 95% CI 1.34-2.74), and lower likelihood of primary sclerosing cholangitis (adjusted odds ratio 0.38; 95% CI 0.26-0.55). During a median follow-up period of 4.8 years, the time until development of advanced CRN did not differ significantly between patients with and those without PIPs. PIPs did not independently increase the risk of advanced CRN (adjusted hazard ratio 1.17; 95% CI 0.59-2.31). The colectomy rate was significantly higher in patients with PIPs (P = .01).
CONCLUSIONS - In a retrospective analysis of data from 2 large independent surveillance cohorts, PIPs were associated with greater severity and extent of colon inflammation and higher rates of colectomy, but were not associated with development of any degree of CRN. Therefore, intervals for surveillance should not be shortened based solely on the presence of PIPs.
Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.
An imbalance in the colonic microbiota might underlie many human diseases, but the mechanisms that maintain homeostasis remain elusive. Recent insights suggest that colonocyte metabolism functions as a control switch, mediating a shift between homeostatic and dysbiotic communities. During homeostasis, colonocyte metabolism is directed toward oxidative phosphorylation, resulting in high epithelial oxygen consumption. The consequent epithelial hypoxia helps to maintain a microbial community dominated by obligate anaerobic bacteria, which provide benefit by converting fiber into fermentation products absorbed by the host. Conditions that alter the metabolism of the colonic epithelium increase epithelial oxygenation, thereby driving an expansion of facultative anaerobic bacteria, a hallmark of dysbiosis in the colon. Enteric pathogens subvert colonocyte metabolism to escape niche protection conferred by the gut microbiota. The reverse strategy, a metabolic reprogramming to restore colonocyte hypoxia, represents a promising new therapeutic approach for rebalancing the colonic microbiota in a broad spectrum of human diseases.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.