scholarly journals Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

2020 ◽  
Author(s):  
Lina Y Alkaissi ◽  
Martin E Winberg ◽  
Stéphanie DS Heil ◽  
Staffan Haapaniemi ◽  
Pär Myrelid ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Ussing Chambers ◽  
E Coli ◽  
Follicle Associated Epithelium ◽  
Vitro Model ◽  
Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

scholarly journals Enhanced E. coli LF82 Translocation through the Follicle-associated Epithelium in Crohn’s Disease is Dependent on Long Polar Fimbriae and CEACAM6 expression, and Increases Paracellular Permeability

2019 ◽  
Vol 14 (2) ◽  
pp. 216-229 ◽  
Author(s):  
Åsa V Keita ◽  
Lina Yakymenko Alkaissi ◽  
Elin B Holm ◽  
Stéphanie D S Heil ◽  
Benoit Chassaing ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Paracellular Permeability ◽  
Ileal Mucosa ◽  
Clinical Recurrence ◽  
Ussing Chambers ◽  
E Coli ◽  
Follicle Associated Epithelium ◽  
Vitro Model

Abstract Background and Aims Patients with Crohn’s disease [CD] harbour an increased number of adherent-invasive E. coli [AIEC]. The strain LF82, identified in the ileal mucosa of CD patients, has been extensively studied for pathogenic mechanisms. However, understanding of the interaction of LF82 with the intestinal mucosa of CD patients is lacking. Methods Here, we investigated the importance of long polar fimbriae [LPF] type 1 pili and the carcinoembryonic antigen-related cell-adhesion molecule 6 [CEACAM6] for translocation of LF82 in an in vitro model of follicle-associated epithelium [FAE], and in the FAE and villus epithelium [VE] of patients with CD and controls, using Ussing chambers. Results Significantly greater LF82 passage occurred in the FAE model compared with in the VE Caco-2cl1 mono-culture. Moreover, bacterial translocation was inhibited by either LPF disruption or pre-incubation with anti-CEACAM6 antibody. Tissue mounted in Ussing chambers showed significantly higher LF82 passage in FAE from patients with CD compared with control FAE, that was diminished in LF82 lacking LPF and by blocking host CEACAM6. Interestingly, addition of LF82 to the CD FAE tissues significantly increased paracellular permeability [of 51Chromium-EDTA] compared with baseline, and the increase was inhibited by anti-CEACAM6. Immunofluorescence and immunoblots showed higher expression of CEACAM6 in FAE of patients with CD compared with in FAE from controls. Conclusions These data suggest that the FAE of CD patients is a site of vulnerability for invasion by LF82 via a mechanism that requires both bacterial LPF and host CEACAM6. Further, LF82 has the ability to increase paracellular passage through the FAE of patients with CD. These data can help define novel therapeutic targets in CD for the prevention of clinical recurrence.

ACTIVATED INTESTINAL MUSCLE CELLS PROMOTE PREADIPOCYTE MIGRATION: A NOVEL MECHANISM OF CREEPING FAT FORMATION IN CROHN’S DISEASE

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S34-S34
Author(s):  
Ren Mao ◽  
Genevieve Doyon ◽  
Ilyssa Gordon ◽  
Jiannan Li ◽  
Sinan Lin ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Muscularis Propria ◽  
Dominant Factor ◽  
Stricture Formation ◽  
Intestinal Tissue ◽  
Mesenteric Fat

Abstract Background and Aims Creeping fat, the wrapping of mesenteric fat around the bowel wall, is a typical feature of Crohn’s disease, and is associated with stricture formation and bowel obstruction. How creeping fat forms is unknown, and we interrogated potential mechanisms using novel intestinal tissue and cell interaction systems. Methods Tissues from normal, ulcerative colitis, non-strictured and strictured Crohn’s disease intestinal specimens were obtained. Fresh and decellularized tissue, mesenteric fat explants, primary human adipocytes, pre-adipocytes, muscularis propria cells, and native extracellular matrix were used in multiple ex vivo and in vitro systems involving cell growth, differentiation and migration, proteomics, and integrin expression. Results Crohn’s disease muscularis propria cells produced an extracellular matrix scaffold which is in direct spatial and functional contact with the immediately overlaid creeping fat. The scaffold contained multiple proteins, but only fibronectin production was singularly upregulated by TGF-b1. The muscle cell-derived matrix triggered migration of pre-adipocytes out of mesenteric fat, fibronectin being the dominant factor responsible for their migration. Blockade of α5β1 on the pre-adipocyte surface inhibited their migration out of mesenteric fat and on 3D decellularized intestinal tissue extracellular matrix. Conclusion Crohn’s disease creeping fat appears to result from the migration of pre-adipocytes out of mesenteric fat and differentiation into adipocytes in response to an increased production of fibronectin by activated muscularis propria cells. These new mechanistic insights may lead to novel approaches for prevention of creeping fat-associated stricture formation.

scholarly journals Extracellular vesicles package dsDNA to aggravate Crohn’s disease by activating the STING pathway

2021 ◽  
Vol 12 (9) ◽  
Author(s):  
Fan Zhao ◽  
Tao Zheng ◽  
Wenbin Gong ◽  
Jie Wu ◽  
Haohao Xie ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Extracellular Vesicles ◽  
Intestinal Inflammation ◽  
Therapeutic Effects ◽  
Murine Colitis ◽  
Sting Pathway ◽  
Deficient Mice

AbstractCrohn’s disease (CD) is an intestinal immune-dysfunctional disease. Extracellular vesicles (EVs) are membrane-enclosed particles full of functional molecules, e.g., nuclear acids. Recently, EVs have been shown to participate in the development of CD by realizing intercellular communication among intestinal cells. However, the role of EVs carrying double-strand DNA (dsDNA) shed from sites of intestinal inflammation in CD has not been investigated. Here we isolated EVs from the plasma or colon lavage of murine colitis and CD patients. The level of exosomal dsDNA, including mtDNA and nDNA, significantly increased in murine colitis and active human CD, and was positively correlated with the disease activity. Moreover, the activation of the STING pathway was verified in CD. EVs from the plasma of active human CD triggered STING activation in macrophages in vitro. EVs from LPS-damaged colon epithelial cells were also shown to raise inflammation in macrophages via activating the STING pathway, but the effect disappeared after the removal of exosomal dsDNA. These findings were further confirmed in STING-deficient mice and macrophages. STING deficiency significantly ameliorated colitis. Besides, potential therapeutic effects of GW4869, an inhibitor of EVs release were assessed. The application of GW4869 successfully ameliorated murine colitis by inhibiting STING activation. In conclusion, exosomal dsDNA was found to promote intestinal inflammation via activating the STING pathway in macrophages and act as a potential mechanistic biomarker and therapeutic target of CD.

scholarly journals Interaction of Enteropathogenic and Shiga Toxin-Producing Escherichia coli and Porcine Intestinal Mucosa: Role of Intimin and Tir in Adherence

2005 ◽  
Vol 73 (9) ◽  
pp. 6005-6016 ◽  
Author(s):  
Francis Girard ◽  
Isabelle Batisson ◽  
Gad M. Frankel ◽  
Josée Harel ◽  
John M. Fairbrother
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Actin Polymerization ◽  
Animal Species ◽  
Ex Vivo ◽  
Epec Strain ◽  
E Coli ◽  
In Vitro Organ Culture

ABSTRACT The ileal in vitro organ culture (IVOC) model using tissues originating from colostrum-deprived newborn piglets has proven to be an effective way to study the attaching and effacing (A/E) phenotype of porcine enteropathogenic Escherichia coli (EPEC) ex vivo. The aim of this study was to investigate the role of intimin subtype and Tir in the adherence of EPEC and Shiga-toxin-producing E. coli (STEC), isolated from different animal species, to porcine intestinal IVOC. Moreover, the role of intimin in Tir-independent adherence of the human EPEC strain E2348/69 was investigated using intimin and Tir-deficient derivatives. Our results demonstrated that A/E E. coli strains (AEEC) from various animal species and humans induce the A/E phenotype in porcine ileal IVOC and that intimin subtype influences intestinal adherence and tropism of AEEC strains. We also showed that a tir mutant of EPEC strain E2348/69 demonstrates close adherence to the epithelial cells of porcine ileal IVOC segments, with microvillous effacement but with no evidence of actin polymerization or pedestal formation, and that intimin seems to be involved in this phenotype. Overall, this study provides further evidence for the existence of one or more host-cell-encoded intimin receptor(s) in the pig gut.

Effect of MCAM on the aggressive phenotype in human prostate cancer.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e23135-e23135
Author(s):  
Marianna Kruithof-de Julio ◽  
Eugenio Zoni ◽  
Letizia Astrologo ◽  
Janine Melsen ◽  
Irena Klima ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Cells ◽  
Metastatic Cancer ◽  
Perivascular Niche ◽  
Hematopoietic Stem ◽  
Aggressive Phenotype ◽  
Vitro Model ◽  
Set Up

e23135 Background: Prostate Cancer (PCa) is the most common cancer in males and the second leading cause of death from cancer in men. Understanding the factors that regulate homing and survival of metastatic cancer cells in the bone is important for the identification of new therapeutic targets. High MCAM expression has been detected in the stroma of lytic and blastic lesions in preclinical models of PCa bone metastasis. The objective of this study is to characterize the role of MCAM in the maintenance of the aggressive phenotype in human PCa. Methods: We knocked and down MCAM in the lytic PC-3M-Pro4Luc2_dTomato and in the blastic C4-2B_dTomato PCa cell lines. Validation was done at both protein and RNA level. We performed functional assays such as migration and proliferation. RT-qPCR was used to test MCAM knockdown on EMT markers. The effect of the knockdown on the maintenance of cancer stem/progenitor-like cells was measured by ALDEFLUOR. Results: MCAM knockdown reduced proliferation in PC-3M-Pro4Luc2_dTomato PCa cells and resulted in increased E-Cadherin expression. Metastatic human PCa cells target the hematopoietic stem cell (HSC) niche in the bone marrow at the level of an “endosteal/osteoblast” niche and a “vascular/perivascular” niche. We set-up an in vitro model of “osteoblast niche” to study the prostate cancer cells upon co-culture with osteoblasts and to determine the effects on cancer stem/progenitor-like markers. We found that MCAM is required for the osteoblast-mediated induction of ALDH activity on PCa cells and MCAM knockdown prevented the increase in the size of the ALDHhigh subpopulation in PC-3M-Pro4Luc2_dTomato, mediated by human osteoblasts. Additionally, MCAM knockdown in PCa cells co-culture with osteoblast, prevented the induction of MCAM expression by osteoblasts. Finally, MCAM is significantly increased in the ALDHhigh cells and identifies a new subset of ALDHhigh / MCAMhigh cells which could be depleted upon MCAM knockdown. Conclusions: We detected a new subset of ALDHhigh/MCAMhigh cells and demonstrated the MCAM influences the maintenance of an aggressive-mesenchymal phenotype in human PCa. Therefore, MCAM represent an interesting target molecule to modulate the behavior of aggressive PCa cells.

scholarly journals IBD Serology and Disease Outcomes in African Americans With Crohn’s Disease

2017 ◽  
Vol 24 (1) ◽  
pp. 209-216 ◽  
Author(s):  
Madeline Bertha ◽  
Arthi Vasantharoopan ◽  
Archana Kumar ◽  
Beau B Bruce ◽  
Jarod Prince ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
African Americans ◽  
Crohn's Disease ◽  
Multivariable Analysis ◽  
Missing At Random ◽  
Serological Markers ◽  
Disease Phenotype ◽  
E Coli ◽  
The Relationship

Abstract Backgrounds Recent studies have identified the role of serologic markers in characterizing disease phenotype, location, complications, and severity among Northern Europeans (NE) with Crohn’s disease (CD). However, very little is known about the role of serology in CD among African Americans (AA). Our study explored the relationship between serology and disease phenotype in AA with CD, while controlling for genetic ancestry. Methods AAs with CD were enrolled as participants through multicenter collaborative efforts. Serological levels of IgA anti-Saccharomyces cervisiae antibody (ASCA), IgG ASCA, E. coli outermembrane porin C, anti-CBir1, and ANCA were measured using enzyme-linked immunosorbent assays. Genotyping was performed using Illumina immunochip technology; an admixture rate was calculated for each subject. Multiple imputation by chained equations was performed to account for data missing at random. Logistic regression was used to calculate adjusted odds ratio (OR) for associations between serological markers and both complicated disease and disease requiring surgery. Results A total of 358 patients were included in the analysis. The majority of our patients had inflammatory, noncomplicated disease (58.4%), perianal disease (55.7%), and documented colonic inflammation (86.8%). On multivariable analysis, both IgG ASCA and OmpC were associated with complicated disease (OR, 2.67; 95% CI, 1.67–4.28; OR, 2.23; 95% CI, 1.41–3.53, respectively) and disease requiring surgery (OR, 2.51; 95% CI, 1.49–4.22; OR, 3.57; 95% CI, 2.12–6.00). NE admixture to the African genome did not have any associations or interactions in relation to clinical outcome. Conclusions Our study comprises the largest cohort of AAs with CD. The utility of serological markers for the prognosis of CD in NE applies equally to AA populations.

scholarly journals Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohn’s disease

2020 ◽  
Author(s):  
Rasa Elmentaite ◽  
Alexander Ross ◽  
Kylie R. James ◽  
Daniel Ortmann ◽  
Tomas Gomes ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Single Cell ◽  
Ex Vivo ◽  
Cell Types ◽  
Gut Development ◽  
Human Gut ◽  
Single Cell Sequencing ◽  
Computational Analyses

SummaryHuman gut development requires the orchestrated interaction of various differentiating cell types. Here we generate an in-depth single-cell map of the developing human intestine at 6–10 weeks post-conception, a period marked by crypt-villus formation. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells, which are distinct from LGR5-expressing cells. We use computational analyses to show that these cells contribute to differentiated cell subsets directly and indirectly via the generation of LGR5-expressing stem cells and receive signals from the surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNAseq profiles from paediatric Crohn’s disease epithelium alongside matched healthy controls to reveal disease associated changes in epithelial composition. Contrasting these with the fetal profiles reveals re-activation of fetal transcription factors in Crohn’s disease epithelium. Our study provides a unique resource, available at www.gutcellatlas.org, and underscores the importance of unravelling fetal development in understanding disease.

scholarly journals Role of Meprins to Protect Ileal Mucosa of Crohn's Disease Patients from Colonization by Adherent-Invasive E. coli

PLoS ONE ◽  
2011 ◽  
Vol 6 (6) ◽  
pp. e21199 ◽  
Author(s):  
Emilie Vazeille ◽  
Marie-Agnès Bringer ◽  
Aurélie Gardarin ◽  
Christophe Chambon ◽  
Christoph Becker-Pauly ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ileal Mucosa ◽  
E Coli

scholarly journals Mechanism of glucocorticoid regulation of the intestinal tight junction barrier

2007 ◽  
Vol 292 (2) ◽  
pp. G590-G598 ◽  
Author(s):  
Michel A. Boivin ◽  
Dongmei Ye ◽  
John C. Kennedy ◽  
Rana Al-Sadi ◽  
Chris Shepela ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Protein Expression ◽  
Tight Junction ◽  
Barrier Function ◽  
In Vitro Model ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Vitro Model

A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-α-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-α-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-α-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-α-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-α increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-α-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-α-induced activation. Glucocorticoids inhibit the TNF-α-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-α-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier.

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