Mechanistic human studies to prevent Hirschsprung-associated enterocolitis

Project Summary: Our goal is to find new ways to prevent and treat Hirschsprung disease-associated enterocolitis (HAEC), a form of bowel inflammation. HAEC is a life-threatening manifestation of Hirschsprung disease (HSCR) characterized by explosive (often bloody) diarrhea, abdominal distension, fever, and risk of bacterial translocation from the gut lumen to the blood, causing sepsis. Sepsis is the most common cause of death in children with HSCR. HSCR is a first trimester developmental anomaly defined by absence of enteric nervous system (ENS) at the end of the bowel. Bowel lacking ENS is called “aganglionic”. Aganglionic bowel tonically contracts and lacks propagating contractions causing functional obstruction. Because ENS also controls blood flow, epithelial, and immune cell activity in the bowel, HAEC mechanisms are complicated, and incompletely understood. HSCR treatment (devised 75 years ago) is surgical removal of aganglionic bowel and re-attachment of “good” ENS-containing bowel near the anal verge (called “pull-through surgery”). In theory, pull-through surgery should result in a perfect cure, but ~35% of children have HAEC after surgery and ~10% undergo “re-do” surgery because of persistent problems. If we understood why some children with HSCR appear healthy after pull-through surgery and others have recurrent HAEC, we might then be able to devise new ways to treat or prevent HAEC. Interestingly, HSCR symptoms vary dramatically before any treatment. Some children are critically ill within days of birth. Others appear well for months or years before HSCR symptoms become clear. For example, recently a 53-year-old man was newly diagnosed with HSCR in Japan. Because HSCR is always present at birth, variable symptom onset and severity suggests HSCR symptoms (including HAEC) are influenced by factors beyond ENS biology. Serendipitously, we discovered in an inbred HSCR mouse model (Piebald lethal, sl/sl) that diet alters survival 3.4-fold within a single mouse colony. Exploiting this observation, we discovered the “Protective” diet reduces Enterobacteriaceae (bowel inflammation-associated bacteria), increases stool butyrate (energy source for colonocytes and a histone deacetylate inhibitor), increases epithelial production of anti-microbial peptides and fucosyltransferases (which protect from inflammation), and reduces gut epithelial oxygen levels (facilitating growth of beneficial obligate anaerobes near colon epithelium). These mechanistic observations suggest new therapies (e.g., butyrate supplementation or elimination of Enterobacteriaceae), but we need to know which mechanisms in mice are relevant for humans with HSCR. To do this we will analyze stool metabolites, stool microbes, and gut epithelial biology in bowel removed from children during pull-through surgery (Aims 1 and 2). We will also examine ENS anatomy near the proximal resection margin using a new tissue clearing and 3D imaging method we invented that provides exceptional data about ENS anatomy (Aim 3). Our studies test specific mechanistic hypotheses and may suggest potentially effective HAEC prevention or treatment strategies.