Neuro-Immune Interactions in the Gut

Study Overview
=================
Brief Summary
-----------------
Hirschsprung's disease (HD) is diagnosed shortly after birth and is characterized by the presence of megacolon. HD is caused when ganglion cells of the enteric nervous system (ENS) in the wall of the large intestine do not develop before birth. This results in a lack of gastrointestinal motility and leads to stool obstruction. It is known that ablation of enteric nerves is associated with intestinal infection and inflammation. Indeed the most severe complication in HD is Hirschsprung's associated enterocolitis (HAEC), characterized by explosive diarrhea, abdominal distension, fever and impending septic shock. Bacteria overgrowth and changes in colonic mucosal immune cell populations during HAEC suggest a possible defect in mucosal immune homeostasis. Under steady state conditions, the mucosal immune system must be tightly controlled to avoid harmful reactions against commensal flora and food antigens, while allowing protective immune responses against invading pathogens. This balance between tolerance and defense is influenced by the mucosal microenvironment, which in turn determines the phenotype and stability of mucosal immune cell populations. The goal of this project is to understand if the enteric nervous system plays a role in regulating mucosal immunity and how this might contribute to the development of HAEC.

Detailed Description
-----------------
Hirschsprung's disease (HD) is diagnosed shortly after birth and is characterized by the absence of enteric nerves in parts of colon [Amiel et al.]. Following surgical correction many patients develop HD-associated enterocolitis (HAEC), a condition distinguished by intestinal inflammation resulting in abdominal distension, severe diarrhea, fever and sepsis [Demehri et al.]. The underlying factors leading to HAEC remain poorly understood and likely involve a defect in epithelial barrier, including decreased mucin production and insufficient immunoglobulin translocation. The establishment of the epithelial barrier is dependent on epithelial recognition of microbial products by innate immune receptors, like toll-like receptors (TLRs) [Peterson et al.]. TLR-dependent epithelial recognition of microflora also coordinates the immune response away from harmless commensal bacteria and towards pathogenic invaders. Both innate and adaptive effector cell functions are influenced by epithelial-derived signals. Under homeostatic conditions commensal bacteria induce anti-inflammatory cytokines in epithelial cells which trigger a tolerogenic phenotype in mucosal antigen presenting cells (APC) resulting in generation of commensal-specific regulatory T cells (Tregs) [Curotto de Lafaille et al.]. During infection, recognition of pathogenic organisms by epithelial cells leads to secretion of inflammatory cytokines thereby inducing an inflammatory APC phenotype which promotes T effector cell (Th1, Th17) generation. The enteric nervous system is directly located underneath the epithelium and controls epithelial cell function. Ablation of enteric glia cells, one of the two cell types of the ENS, in mice is associated with inflammation and enterocolitis [Cornet et al.]. In a study from 2011 Flamant and co-workers demonstrate that enteric glia cells protect from a shigella flexneri invasion by preventing lesions in the epithelial barrier mediated by the glia cell derived neurothrophic factor S-nitrosoglutathione (GSNO) [Flamant et al.]. We hypothesize that the lack of an enteric nervous system in HD patients modulates the microbial recognition of epithelial cells and thereby the phenotype of underlying mucosal APCs and effector T cells; this might be associated with the manifestation of HAEC.

Official Title
-----------------
The Enteric Nervous System as Modulator of Mucosal Immune Cells

Conditions
-----------------
Hirschsprung's Disease Associated Enterocolitis

Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion Criteria: Informed consent Exclusion Criteria: No signed informed consent No blood from patients with weak general state of health

Ages Eligible for Study
-----------------
Minimum Age: 0 Months
Maximum Age: 18 Years

Sexes Eligible for Study
-----------------
All

Accepts Healthy Volunteers
-----------------
Accepts Healthy Volunteers

Study Plan
=================
How is the study designed?
-----------------

Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Hirschsprung's disease patients<br>Children diagnosed with Hirschsprung's disease or Hirschsprung's disease associated enterocolitis | |
| control patients<br>Children diagnosed and treated for miscellaneous bowel diseases | |

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Phenotypic analysis of immune and nervous cell populations | Determining cell frequencies and subtypes using fluorescence-activated cell sorting (FACS) and FlowJo software | 5 years | 
| Expression profil | RNA expression profile of whole colon tissue and single cell populations | 5 years | 
| Histological analysis | Microscopic analysis of colonic tissue using immunofluorescence and immunohistochemistry | 5 years | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Microbial metagenomics sequencing | 16S/18S/ITS Amplicon | 5 years | 
| Identifying genetic defect | Targeted Sanger sequencing of known Hirschsprung's disease associated genes | 5 years | 

 Terms related to the study
=================
Keywords Provided by Centre Hospitalier Valida
-----------------
enteric neuropathies, Immune cells, Nervous cells, Microbiom