Faculty Opinions recommendation of A Crohn's disease variant in Atg16l1 enhances its degradation by caspase 3.

Abstract Background In patients operated due to active Crohn’s disease (CD), the negative effect of steroids and biologics on septic complications is known. Until now, azathioprine (AZA) is considered to be safe. The aim of our study was to assess the impact of AZA on intestinal epithelial cells damage in patients with active CD as a surrogate marker of healing. Methods Intestinal specimens taken from macroscopically healthy surgical margins of all consecutive CD patients operated due to active isolated ileocecal disease during the study period (2014–2016) were evaluated. Expression of caspase-3, p-53 and Ki-67 as markers of cell apoptosis, DNA damage and proliferation were immunohistochemically tested and assessed using a confocal microscope and microimage for in-tissue-cytometry analysis. Western-blot analysis was performed for the evaluation of cellular integrity using ZO-1 and E-cadherin as a markers. 30-day clinical outcomes were assessed. The study was approved by the institutional Ethics Committee. Results From 61 operated due to active CD patients, 35 met the inclusion criteria. Patients were divided accordingly to preoperative treatment: treated with no immunomodulators (N-9 patients), on steroids (S-14 patients), on AZA (A-6 patients), and on combination therapy, AZA + steroids (AS-6 patients). There were no substantial differences between groups. We found statistically significant increase of apoptosis in A group compared with N (5.33 ± 1.05 vs. 1.29 ± 0.51, p = 0.011), but also S group (1.58 ± 0.68, p = 0.014) and increase of DNA damage in A, AS and S groups compared with N group (p =0.01; p = 0.032; p = 0.035, respectively) (Figure 1a–c). P53-mediated cell cycle arrest and apoptosis through a caspase-3-dependent pathway in response to DNA damage was the most intensive in A group (Figure 2a). Reduction of cell proliferative activity in group A did not reach statistical significance (p = 0,057). A reduction of ZO-1 in A group and increased level of E-cadherin in S group were found. The effect of the decreased number of tight junctions and disintegration of the mucosa layer was observed in A group (Figure 2b). Clinically, in 30-day postoperative follow-up, six wound healing complications and one anastomotic leak were found, all in patients treated with immunomodulators. Conclusion We found that in epithelial cells of the small and large intestine of patients treated with AZA, apoptotic activity and DNA damage processes are increased when regeneration processes and mucosal integrity are significantly disturbed. These abnormalities of intestinal epithelial regeneration may be a surrogate marker of impaired mucosal healing.

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P073 An increased autophagy and decreased apoptosis is detected in intestinal fibroblasts from Crohn’s Disease patients

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjab076.202 ◽

2021 ◽

Vol 15 (Supplement_1) ◽

pp. S176-S177

Author(s):

L Gisbert-Ferrandiz ◽

M Queralt ◽

J Cosín-Roger ◽

S Coll ◽

C Bauset ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Lamina Propria ◽

Caspase 3 ◽

Statistical Significance ◽

Intestinal Tissue ◽

Protein Ratio ◽

Protein Levels ◽

Mtor Protein ◽

Significant Increment

Abstract Background Fibrosis is a complication commonly present in Crohn’s disease (CD) patients with a structuring (B2) or penetrating (B3) phenotype, with no effective treatment. This process is characterized by a disequilibrium between the production and degradation of the extracellular matrix (ECM), mainly regulated by myofibroblasts. We aim to analyse here, the expression of markers of autophagy, apoptosis and proliferation in intestinal fibroblasts from CD patients. Methods Fibroblasts were isolated from the damaged intestinal mucosa of CD patients with a penetrating and stenotic behaviour. Control cells were obtained from the non-damaged intestine of patients with colorectal cancer. Protein levels of markers of autophagy and apoptosis were determined by Western Blot in isolated fibroblasts. The proliferation marker Ki67 was analysed by immunohistochemistry (IHC) in 5 µm slides of intestinal tissue from control or CD patients. Statistical significance was measured by t-test. Results In fibroblasts from CD patients, we detected a significant decrease in the ratio phospho-mTOR / mTOR (Fig. A) in parallel with a non-significant increment in the LC3 II / LC3 I protein ratio (174% ± 46.5), and a decrease in p62 protein levels (84.8% ± 5.5). When compared between CD behaviours, a significant decreased in the phospho-mTOR / mTOR protein ratio was detected in fibroblasts from B2- compared to that obtained in cells from B3-CD patients (Fig. B). The analysis of the expression of an apoptosis marker, Caspase 3, revealed a decreased of cleaved caspase 3 protein levels in CD fibroblasts compared to levels detected in control cells (Fig C). Finally, we observed in the lamina propria of the intestine from CD patients an increased number of Ki67 positive cells, compared to that detected in control tissue. Conclusion Our data show an increased autophagy and decreased apoptosis in isolated intestinal fibroblasts from CD patients; the high number of cells proliferating in the lamina propria of the intestinal tissue of these patients, strongly suggests a higher viability of these cells in the fibrotic context.

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Effects of Dietary Oat Beta-Glucans on Colon Apoptosis and Autophagy through TLRs and Dectin-1 Signaling Pathways—Crohn’s Disease Model Study

Nutrients ◽

10.3390/nu13020321 ◽

2021 ◽

Vol 13 (2) ◽

pp. 321

Author(s):

Łukasz Kopiasz ◽

Katarzyna Dziendzikowska ◽

Małgorzata Gajewska ◽

Michał Oczkowski ◽

Kinga Majchrzak-Kuligowska ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Epithelial Cells ◽

Caspase 3 ◽

Disease Model ◽

Time Dependent ◽

Sprague Dawley ◽

Beta Glucan ◽

High Molar Mass ◽

Beta Glucans

Background: Crohn’s disease (CD) is characterized by chronic inflammation of the gastrointestinal tract with alternating periods of exacerbation and remission. The aim of this study was to determine the time-dependent effects of dietary oat beta-glucans on colon apoptosis and autophagy in the CD rat model. Methods: A total of 150 Sprague–Dawley rats were divided into two main groups: healthy control (H) and a TNBS (2,4,6-trinitrobenzosulfonic acid)-induced colitis (C) group, both including subgroups fed with feed without beta-glucans (βG−) or feed supplemented with low- (βGl) or high-molar-mass oat beta-glucans (βGh) for 3, 7, or 21 days. The expression of autophagy (LC3B) and apoptosis (Caspase-3) markers, as well as Toll-like (TLRs) and Dectin-1 receptors, in the colon epithelial cells, was determined using immunohistochemistry and Western blot. Results: The results showed that in rats with colitis, after 3 days of induction of inflammation, the expression of Caspase-3 and LC3B in intestinal epithelial cells did not change, while that of TLR 4 and Dectin-1 decreased. Beta-glucan supplementation caused an increase in the expression of TLR 5 and Dectin-1 with no changes in the expression of Caspase-3 and LC3B. After 7 days, a high expression of Caspase-3 was observed in the colitis-induced animals without any changes in the expression of LC3B and TLRs, and simultaneously, a decrease in Dectin-1 expression was observed. The consumption of feed with βGl or βGh resulted in a decrease in Caspase-3 expression and an increase in TLR 5 expression in the CβGl group, with no change in the expression of LC3B and TLR 4. After 21 days, the expression of Caspase-3 and TLRs was not changed by colitis, while that of LC3B and Dectin-1 was decreased. Feed supplementation with βGh resulted in an increase in the expression of both Caspase-3 and LC3B, while the consumption of feed with βGh and βGl increased Dectin-1 expression. However, regardless of the type of nutritional intervention, the expression of TLRs did not change after 21 days. Conclusions: Dietary intake of βGl and βGh significantly reduced colitis by time-dependent modification of autophagy and apoptosis, with βGI exhibiting a stronger effect on apoptosis and βGh on autophagy. The mechanism of this action may be based on the activation of TLRs and Dectin-1 receptor and depends on the period of exacerbation or remission of CD.

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