Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn’s Disease

AbstractObjectiveIntestinal epithelial cell (IEC) barrier dysfunction is critical to the development of Crohn’s disease (CD). However, the mechanism is understudied. We recently reported increased microRNA-31-5p (miR-31-5p) expression in colonic IECs of CD patients, but downstream targets are unknown.DesignMiR-31-5p target genes were identified by integrative analysis of RNA- and small RNA-sequencing data from colonic mucosa and confirmed by qPCR in colonic IECs. Functional characterization of Activin Receptor-Like Kinase 1 (ACVRL1 or ALK1) in IECs was performed ex vivo using 2 dimensional-cultured human primary colonic IECs. The impact of altered colonic ALK1 signaling in CD for the risk of surgery and endoscopic relapse was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator.ResultsALK1 was identified as a target of miR-31-5p in colonic IECs of CD patients and confirmed using a 3’-UTR reporter assay. Activation of ALK1 restricted the proliferation of colonic IECs in an EdU proliferation assay and down-regulated the expression of stemness-related genes. Activated ALK1 signaling directed the fate of colonic IEC differentiation toward colonocytes. Down-regulated ALK1 signaling was associated with increased stemness and decreased colonocyte-specific marker expression in colonic IECs of CD patients compared to healthy controls. Activation of ALK1 enhanced epithelial barrier integrity in a trans-epithelial electrical resistance permeability assay. Lower colonic ALK1 expression was identified as an independent risk factor for surgery and associated with a higher risk of endoscopic relapse in CD patients.ConclusionDecreased colonic ALK1 disrupted colonic IEC barrier integrity and associated with deteriorated clinical outcomes in CD patients.

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P149 DECREASED COLONIC ACTIVIN RECEPTOR-LIKE KINASE 1 DISRUPTS EPITHELIAL BARRIER INTEGRITY AND IS ASSOCIATED WITH A POOR CLINICAL OUTCOME IN CROHN’S DISEASE

Gastroenterology ◽

10.1053/j.gastro.2019.11.133 ◽

2020 ◽

Vol 158 (3) ◽

pp. S46 ◽

Cited By ~ 1

Author(s):

Takahiko Toyonaga ◽

Benjamin Keith ◽

Jasmine Barrow ◽

Matthew Schaner ◽

Elizabeth Wolber ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Clinical Outcome ◽

Epithelial Barrier ◽

Poor Clinical Outcome ◽

Activin Receptor ◽

Barrier Integrity ◽

Receptor Like Kinase

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Butyrate-producing bacteria supplemented in vitro to Crohn’s disease patient microbiota increased butyrate production and enhanced intestinal epithelial barrier integrity

Scientific Reports ◽

10.1038/s41598-017-11734-8 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 102

Author(s):

Annelies Geirnaert ◽

Marta Calatayud ◽

Charlotte Grootaert ◽

Debby Laukens ◽

Sarah Devriese ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Disease Patient ◽

Epithelial Barrier ◽

Intestinal Epithelial ◽

Intestinal Epithelial Barrier ◽

Barrier Integrity ◽

Butyrate Production

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Olaparib: A Clinically Applied PARP Inhibitor Protects from Experimental Crohn’s Disease and Maintains Barrier Integrity by Improving Bioenergetics through Rescuing Glycolysis in Colonic Epithelial Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/7308897 ◽

2021 ◽

Vol 2021 ◽

pp. 1-17

Author(s):

Dominika Kovács ◽

Viola Bagóné Vántus ◽

Eszter Vámos ◽

Nikoletta Kálmán ◽

Rudolf Schicho ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Epithelial Cells ◽

Intestinal Permeability ◽

Parp Inhibitor ◽

Epithelial Barrier ◽

Extracellular Acidification ◽

Barrier Integrity ◽

Lymphocyte Ratio ◽

Extracellular Acidification Rate

Crohn’s disease (CD) is an inflammatory disorder of the intestines characterized by epithelial barrier dysfunction and mucosal damage. The activity of poly(ADP-ribose) polymerase-1 (PARP-1) is deeply involved in the pathomechanism of inflammation since it leads to energy depletion and mitochondrial failure in cells. Focusing on the epithelial barrier integrity and bioenergetics of epithelial cells, we investigated whether the clinically applied PARP inhibitor olaparib might improve experimental CD. We used the oral PARP inhibitor olaparib in the 2,4,6-trinitrobenzene sulfonic acid- (TNBS-) induced mouse colitis model. Inflammatory scoring, cytokine levels, colon histology, hematological analysis, and intestinal permeability were studied. Caco-2 monolayer culture was utilized as an epithelial barrier model, on which we used qPCR and light microscopy imaging, and measured impedance-based barrier integrity, FITC-dextran permeability, apoptosis, mitochondrial oxygen consumption rate, and extracellular acidification rate. Olaparib reduced the inflammation score, the concentration of IL-1β and IL-6, enhanced the level of IL-10, and decreased the intestinal permeability in TNBS-colitis. Blood cell ratios, such as lymphocyte to monocyte ratio, platelet to lymphocyte ratio, and neutrophil to lymphocyte ratio were improved. In H2O2-treated Caco-2 monolayer, olaparib decreased morphological changes, barrier permeability, and preserved barrier integrity. In oxidative stress, olaparib enhanced glycolysis (extracellular acidification rate), and it improved mitochondrial function (mitochondrial coupling efficiency, maximal respiration, and spare respiratory capacity) in epithelial cells. Olaparib, a PARP inhibitor used in human cancer therapy, improved experimental CD and protected intestinal barrier integrity by preventing its energetic collapse; therefore, it could be repurposed for the therapy of Crohn’s disease.

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S1184 Myosin IXB Gene Variants Are Associated with Crohn's Disease: Evidence for a Primary Epithelial Barrier Defect

Gastroenterology ◽

10.1016/s0016-5085(09)60934-3 ◽

2009 ◽

Vol 136 (5) ◽

pp. A-208

Author(s):

Katrin Wanner ◽

Carsten Buning ◽

Tahir Durmus ◽

Tamas Molnar ◽

Dirk J. De Jong ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Epithelial Barrier ◽

Gene Variants ◽

Barrier Defect

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Protection of Epithelial Barrier Function by the Crohn's Disease Associated Gene Protein Tyrosine Phosphatase N2

Gastroenterology ◽

10.1053/j.gastro.2009.07.078 ◽

2009 ◽

Vol 137 (6) ◽

pp. 2030-2040.e5 ◽

Cited By ~ 65

Author(s):

Michael Scharl ◽

Gisela Paul ◽

Achim Weber ◽

Barbara C. Jung ◽

Michael J. Docherty ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Protein Tyrosine Phosphatase ◽

Barrier Function ◽

Tyrosine Phosphatase ◽

Epithelial Barrier ◽

Epithelial Barrier Function ◽

Protein Tyrosine

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P149 DECREASED COLONIC ACTIVIN RECEPTOR-LIKE KINASE 1 DISRUPTS EPITHELIAL BARRIER INTEGRITY AND IS ASSOCIATED WITH A POOR CLINICAL OUTCOME IN CROHN’S DISEASE

Inflammatory Bowel Diseases ◽

10.1093/ibd/zaa010.069 ◽

2020 ◽

Vol 26 (Supplement_1) ◽

pp. S29-S29

Author(s):

Takahiko Toyonaga ◽

Benjamin Keith ◽

Jasmine Barrow ◽

Matthew Schaner ◽

Elizabeth Wolber ◽

...

Keyword(s):

Gene Expression ◽

Clinical Outcome ◽

Rna Sequencing ◽

Small Rna ◽

Integrative Analysis ◽

Epithelial Barrier ◽

Small Rna Sequencing ◽

Poor Clinical Outcome ◽

Barrier Integrity ◽

Receptor Like Kinase

Abstract Background and Aims Intestinal epithelial cell (IEC) barrier dysfunction is critical to the development of Crohn’s disease (CD). Mechanisms controlling colonocyte differentiation and barrier defects are understudied in CD. We recently reported increased expression of microRNA-31-5p (miR-31-5p) in colonic IECs of CD patients compared to non-IBD (NIBD) controls. In this study we identify and characterize the miR-31-5p target gene Activin Receptor-Like Kinase 1 (ALK1) as a key regulator of colonocyte maturation and IEC barrier integrity, specifically the functional impact of aberrant ALK1 signaling on colonocyte differentiation, barrier integrity, and clinical disease outcomes in CD. Methods MiR-31-5p target genes were identified in CD patients by integrative analysis of RNA- and small RNA-sequencing data from colonic mucosa and confirmed by quantitative RT-PCR (qPCR) in isolated colonic IECs. Functional characterization of ALK1 in colonic IECs was performed ex vivo using 2- or 3-dimensional cultured human primary colonic IECs. The impact of altered colonic ALK1 signaling for the risk of surgery and endoscopic relapse was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator among CD patients. Results Integrative analysis of RNA- and small RNA-sequencing and follow-up qPCR identified ALK1 as a candidate target of miR-31-5p in the colonic IECs of CD patients. A 3’-UTR reporter assay with site-directed mutagenesis confirmed the direct regulation of ALK1 expression by miR-31-5p in HEK293T cells. Increased activation of ALK1 restricted the proliferation of primary colonic IECs in an EdU proliferation assay and down-regulated the expression of stemness-related genes, such as LGR5 and ASCL2. Activated ALK1 signaling directed the fate of human colonic IEC differentiation toward colonocytes. Down-regulated ALK1 signaling was associated with increased stemness-related gene expression and decreased colonocyte-specific gene expression in the isolated colonic IECs of CD patients compared to non-IBD controls. Activation of ALK1 did not affect colonic IEC migration in a wound healing assay, but enhanced epithelial barrier integrity in a trans-epithelial electrical resistance (TEER)-based permeability assay. Lower colonic ALK1 expression was associated with higher risks of surgery and endoscopic relapse in CD patients. Conclusion Decreased colonic ALK1 signaling disrupts colonic IEC barrier integrity and is associated with a poor clinical outcome in CD patients.

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S1698 Increased Expression of Epithelial TLR5 and Activation of the TLR5 Pathway Leads to Epithelial Barrier Dysfunction in Experimental Crohn's Disease

Gastroenterology ◽

10.1016/s0016-5085(08)61169-5 ◽

2008 ◽

Vol 134 (4) ◽

pp. A-252

Author(s):

Ruo Jia ◽

Xiao Ming Wang ◽

Li-Guo Jia ◽

Fabio Cominelli ◽

Theresa T. Pizarro

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Epithelial Barrier ◽

Barrier Dysfunction ◽

Epithelial Barrier Dysfunction

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P681 Higher in vitro mucin degradation, but no increased paracellular permeability by faecal water from Crohn’s disease patients

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjab076.801 ◽

2021 ◽

Vol 15 (Supplement_1) ◽

pp. S601-S601

Author(s):

H Becker ◽

N Kameli ◽

A Rustichelli ◽

H Britt ◽

F Stassen ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Barrier Function ◽

Disease Onset ◽

Paracellular Permeability ◽

Epithelial Barrier ◽

Rrna Gene ◽

Relative Abundances ◽

The Impact

Abstract Background Crohn’s disease (CD) is a chronic inflammatory gastro-intestinal condition with a variable disease course. Impaired intestinal integrity and microbial dysbiosis are associated with disease onset and exacerbations. We hypothesized that a perturbed microbial activity in CD patients may contribute to the impaired barrier function. Therefore, this study aimed to examine the impact of faecal bacterial products of active CD patients, CD patients in remission, and healthy controls (HC) on mucin degradation and intestinal epithelial barrier function in vitro. Methods Six HC and twelve CD patients were included. Faecal samples were obtained within one week prior to endoscopy processed within 6 hours after collection. Disease activity was determined by the short endoscopic score for CD (SES-CD). Faecal water (FW) and bacterial membrane vesicles (MVs) were applied on mucin agar to determine mucin degradation. Further, differentiated Caco-2 cell monolayers were exposed to FW and MVs to assess transepithelial electrical resistance (TEER) and paracellular junction stability using permeation of fluorescein isothiocyanate-labelled dextran of 4 kDa. Relative abundances of faecal bacterial genera were evaluated by 16S rRNA gene amplicon sequencing. Results FW-induced mucin degradation was higher in CD samples as compared to HC (p<0.01), but was not linked to specific bacterial relative abundances. FW resulted in 78–87% decrease of TEER in three of the remissive (p<0.001) but not the active CD or HC samples. The decrease of TEER was not linked to increased paracellular permeability. MVs did not induce mucin degradation or epithelial barrier disruption. Conclusion The higher mucin degradation capacity of CD patient-derived FW might indicate contributions of microbial products to CD pathophysiology and warrants further investigation. Moreover, the altered epithelial resistance in some individuals is not due to paracellular alterations.

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