Exploration of the Activity of DNA Located Outside of Cellular Nucleus to Amplify Inflammation in Inflammatory Bowel Disease in Children Through Biological Pathway Cyclic GMP-AMP Synthase (cGAS) - Stimulator of Interferon Genes (STING)

Study Overview
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Brief Summary
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Frequency of Inflammatory Bowel Diseases in children (IBD)-Crohn's disease (CD), Ulcerative colitis (UC) is constantly increasing. Pediatric-onset IBD represent a different nosological entity (from adult IBD) because of their major inflammatory activity, their significant anatomical extent and their stenotic and/or fistulizing character sometimes from diagnosis. Intestinal lesions are due to dysregulation of the intestinal immune system but the cause is unknown. The investigators hypothesize that extranuclear DNA participates in the amplification of the inflammatory response at the intestinal and blood levels during pediatric IBD through the cGAS-STING pathway. The investigators will analyse blood and fecal samples, and colonic biopsies issued from ill children and control participants on age of 6 to 17 years. The investigators think that this study will provide a better understanding of the mechanisms involved in pediatric IBD, assess the place of the cGAS-STING pathway, identify potential biomarkers of pediatric IBD and new potential therapeutic targets based in particular on the inhibition of the cGAS-STING pathway.

Detailed Description
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Inflammatory Bowel Diseases in children (IBD)-Crohn's disease (CD), ulcerative colitis (UC) are severe pathology that can affect the entire digestive tract. Their annual incidence is however constantly increasing. IBD are complex multifactorial pathologies whose cause is still unknown today. IBD occurs on a predisposing genetic background in the presence of exogenous factors and alteration of the intestinal microbiota. Intestinal lesions are due to dysregulation of the intestinal immune system with increased secretion of pro-inflammatory cytokines at the expense of anti-inflammatory cytokines. Pediatric-onset IBD represent a different nosological entity (from adult IBD) because of their major inflammatory activity, their significant anatomical extent and their stenotic and/or fistulizing character sometimes from diagnosis. Their impact is not only individual (growth retardation, puberty delay, psychological disorders) but also family/parental, school and social. These particularities justify that biomedical research focuses on it in a more specific way. Extracellular and extranuclear DNA (enDNA) play a major role in innate immunity by stimulating pro-inflammatory responses and activating type I interferon production. The pro-inflammatory action of enDNA is mediated by enzyme cGAS, protein STING, toll-like receptor 9 (TLR9), and the inflammasome complex NLRP3. The investigators hypothesize that enDNA participates in the amplification of the inflammatory response at the intestinal and blood levels during pediatric IBD through the cGAS-STING pathway. They also hypothesize that there are links between the cGAS-STING pathway and other pathways involved in pediatric IBD such as NOD2 and Autophagy. The investigators will analyse blood and fecal samples, and colonic biopsies issued from ill children and controls on age of 6 to 17 years. The investigators think that this study will provide a better understanding of the mechanisms involved in pediatric IBD, assess the place of the cGAS-STING pathway, identify potential biomarkers of pediatric IBD and new potential therapeutic targets based in particular on the inhibition of the cGAS-STING pathway.

Official Title
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Pro-inflammatory Role of Extracellular DNA in Inflammatory Bowel Disease in Children: Study of the cGAS-STING Pathway

Conditions
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Inflammatory Bowel Diseases, Crohn Disease, Ulcerative Colitis

Intervention / Treatment
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* Biological: Blood and fecal samples
* Procedure: Coolonic biopsies


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Participants aged from 6 years inclusive to 17 years inclusive Boys and girls Presenting an IBD or suspicion of IBD Requiring a colonoscopy for diagnosis or follow-up or other reason (abdominal pain, diarrhoea, rectal bleeding, weight loss) not confirming the diagnosis of Crohn's disease or Ulcerative Colitis Active IBD if: CD: PCDAI score >5 and CRP>20mg/L and faecal calprotectin> 400 µg/g UC: PUCAI score>10 and faecal calprotectin>250 µg/g IBD in remission if: CD: PCDAI score<5 and CRP<20mg/L and faecal calprotectin<400 µg/g UC: PUCAI score <10 and faecal calprotectin < 250 µg/g Patients and their parents who gave their consent to participate in the study Exclusion Criteria: Refusal of the participant and/or one of his two parents Body weight less than or equal to 20 kg Blood hemoglobin level less than or equal to 9 g/dl Refusal or contraindication to general anesthesia Co-existing severe chronic pathology and/or treatment that could interfere with the results of the study; example: trisomy 21, treatment with growth hormone etc. Protected person (under guardianship or curatorship) Person under legal protection Person not affiliated to a social security scheme Pregnant or breastfeeding woman

Ages Eligible for Study
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Minimum Age: 6 Years
Maximum Age: 17 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Health Services Research
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Other: Patients with samples<br>Blood and fecal samples, and colonic biopsies will be analysed and compared between 3 groups of participants :1/ Active IBD; 2/Inactive IBD; 3/Controls non-IBD. | Biological: Blood and fecal samples<br>* Blood and fecal samples will be performed<br>Procedure: Coolonic biopsies<br>* colonic biopsies will be analysed<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Amount of mRNA specific for colonic cGAS | the comparison, between the 3 groups of patients, of the quantity of specific mRNA of colonic cGAS expressed as the number of reads during RNA sequencing (RNAseq), by a Mann-Whitney U test. It is planned from the outset to compare the groups 2 by 2, regardless of the result of an overall test such as a Kruskal-Wallis test. | Baseline | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| quantitative difference of amount of circulating mtDNA | to find quantitative differences between the 3 groups concerning the circulating mtDNA, the circulating total DNA, by the qPCR technique: by specific primer sequences to identify the origin of the DNA (mitochondrial or nuclear). Results will be expressed by the -2∆∆Ct method (fold increase) compared to the control. | Baseline | 
| Cytokine response | to find quantitative differences between the 3 groups concerning the cytokine response by Luminex®: in pg/ml or in ng/ml depending on the cytokines | Baseline | 
| inflammatory / dysimmune response of cytokines | to find quantitative differences between the 3 groups concerning the inflammatory / dysimmune response of cytokines, components of the cGAS-STING pathways autophagy, NOD2, intestinal mucins, integrins, cadherins by RNAseq type transcriptomic analysis in Transcript Per Million (TPM). This outcome wil be mesured at the blood level abd the colonic level. | Baseline | 
| Difference of DNA methylation by Methyl-Seq between the 3 groups | to find quantitative differences between the 3 groups concerning the study of DNA methylation by Methyl-Seq. Results expessed in pourcentage of methylation. This outcome will be mesured at the bllod level and the colonic level. | Baseline | 
| Activation (phosphorylation) of the components of the cGAS-STING pathway | Activation (phosphorylation) of the components of the cGAS-STING pathway (proteins cGAS, p-CGAS, STING,p-STING,TBK1, p-TBK1,IRF-3, p-IRF-3) by Western-Blot | Baseline | 
| plasma DNase activity | plasma DNase activity in Kunitz unitz | baseline | 
| differences in microbial distribution | search for quantitative differences in microbial distribution between the 3 groups by pyrosequencing RNA 16s of the microbiota at the fecal level | Baseline | 
| quantity (number of reads) of colonic STING-specific mRNA | quantitative differences between the 3 groups concerning the quantity (number of reads) of colonic STING-specific mRNA at the colonic level | Baseline | 
| extracellular DNA by qPCR | We will use specific primer sequences to identify the origin of the DNA (mitochondrial or nuclear). Results will be expressed by the -2∆∆Ct method (fold increase) compared to the control. This outcome will be mesured at the colonic level. | Baseline | 
| Amount of STING in the cytoplasm | cytoplasmic presence of STING by histology, immunohistochemical staining and optical microscopy analysis. Results will be qualitative (present/absent; localization) and quantitative based on optical density (pourcentage of positive cells) | Baseline | 
| Amount of cGAS in the cytoplasm | cytoplasmic presence of cGAS by histology, immunohistochemical staining and optical microscopy analysis. Results will be qualitative (present/absent; localization) and quantitative based on optical density (pourcentage of positive cells) | Baseline | 
| Amount of nuclear DNA in teh cytoplasm | cytoplasmic presence of nuclear DNA by histology, immunohistochemical staining and optical microscopy analysis. Results will be qualitative (present/absent; localization) and quantitative based on optical density (pourcentage of positive cells) | Baseline | 
| Amount of mitochondrial DNA in the cytoplasm | cytoplasmic presence of mitochondrial DNA by histology, immunohistochemical staining and optical microscopy analysis. Results will be qualitative (present/absent; localization) and quantitative based on optical density (pourcentage of positive cells) | Baseline | 
| Amount of colonic DNase activity | Measurement of colonic DNase activity in Kunitz unitz | Baseline | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Crohn disease, Ulcerative colitis, cell-free DNA, cGAS, STING, Child, intestinal inflammation, Inflammatory Bowel disease