Project Grant F31HD123619

DEVELOPMENT OF TISSUE DERIVED INTESTINAL ORGANOIDS AS A MODEL SYSTEM FOR MECHANISTIC TESTING IN CHILDREN WITH AUTISM-ASSOCIATED ENTEROCOLITIS - PROJECT SUMMARY IT IS WELL-ESTABLISHED THAT GASTROINTESTINAL (GI) SYMPTOMS OCCUR MORE FREQUENTLY IN CHILDREN WITH AUTISM SPECTRUM DISORDER (ASD) THAN IN TYPICALLY DEVELOPING (TD) CHILDREN, AND RECENT PUBLICATIONS REPORT THAT THE SEVERITY OF GI DYSFUNCTION IS POSITIVELY CORRELATED WITH THE SEVERITY OF ASD-RELATED BEHAVIORS AND COGNITIVE CHALLENGE. DESPITE THESE STRONG ASSOCIATIONS, THE BIOLOGICAL MECHANISMS LINKING INTESTINAL PATHOPHYSIOLOGY TO NEURODEVELOPMENTAL OUTCOMES REMAIN POORLY UNDERSTOOD. ONE COMPELLING POSSIBILITY INVOLVES SEROTONIN SIGNALING WITHIN THE GUT-BRAIN AXIS. EXCESS SEROTONIN CAN STIMULATE IMMUNE ACTIVATION, PROMOTE INTESTINAL INFLAMMATION, AND DRIVE DIARRHEA; SYMPTOMS COMMONLY OBSERVED IN CHILDREN WITH ASD. OUR DATA SUGGESTS THAT CHILDREN WITH ASD HAVE DECREASED EXPRESSION OF SERT IN THE TERMINAL ILEUM AND INCREASED EXPRESSION OF THE SEROTONIN RECEPTORS HTR3A; SUGGESTING A MECHANISM BY WHICH LOCAL SEROTONIN CLEARANCE IS IMPAIRED. IMPORTANTLY, SERT EXPRESSION IS STRONGLY MODULATED BY THE GUT MICROBIOTA. SEVERAL FLAGELLATED MICROBES SUPPRESS SERT MRNA AND PROTEIN LEVELS, WHILE BENEFICIAL COMMENSALS ENHANCE SERT EXPRESSION. CONSISTENT WITH THIS, OUR WORK IDENTIFIES DISTINCT MICROBIAL SHIFTS IN THE TERMINAL ILEUM OF CHILDREN WITH ASD, INCLUDING INCREASED ABUNDANCE OF LACHNOSPIRACEAE AND UNCLASSIFIED CLOSTRIDIUM. THESE ORGANISMS ALSO HAVE FLAGELLA AND COULD ACTIVATE TLR5 WHICH MIGHT LEAD TO DECREASED SERT AND EXCESS SEROTONIN. WE ALSO IDENTIFIED THAT HTR3A EXPRESSION WAS ELEVATED IN CHILDREN WITH ASD COMPARED TO TD CONTROL. HTR3A HAS BEEN ASSOCIATED WITH PRO-INFLAMMATORY SIGNALING, SUGGESTING THAT EXCESS SEROTONIN IN THE GUT MAY FURTHER AMPLIFY EPITHELIAL INFLAMMATION AND CONTRIBUTE TO GI DYSFUNCTION IN ASD. WE HYPOTHESIZE THAT CHILDREN WITH ASD HARBOR CLOSTRIDIAL AND LACHNOSPIRACEAE SPECIES WITH HIGHLY IMMUNOSTIMULATORY FLAGELLA THAT ACTIVATE TLR5 ON TERMINAL ILEUM ENTEROCYTES, LEADING TO SUPPRESSION OF EPITHELIAL SERT EXPRESSION. REDUCED SERT RESULTS IN EXCESS EXTRACELLULAR SEROTONIN, WHICH ACTIVATES THE UPREGULATED HTR3A RECEPTOR TO DRIVE MUCOSAL INFLAMMATION AND GUT DYSFUNCTION, THEREBY CONTRIBUTING TO GI AND POTENTIALLY NEUROBEHAVIORAL SYMPTOMS IN ASD. TO TEST THE HYPOTHESIS, WE PROPOSE THE FOLLOWING TWO AIMS. AIM 1. DEFINE HOW FLAGELLATED CLOSTRIDIAL AND LACHNOSPIRACEAE SPECIES ENRICHED IN ASD ACTIVATE TLR5 AND SUPPRESS SERT EXPRESSION IN TISSUE-DERIVED HUMAN INTESTINAL ORGANOIDS. AIM 2. DEFINE HOW SEROTONIN EXCESS ACTIVATES HTR3A TO PROMOTE EPITHELIAL INFLAMMATION AND DYSFUNCTION IN HUMAN ORGANOIDS FROM ASD AND TD CHILDREN. THIS PROPOSAL DIRECTLY ADDRESSES A CRITICAL AND LONG-STANDING GAP BY IDENTIFYING SPECIFIC MICROBIAL AND EPITHELIAL PATHWAYS THAT DISRUPT SEROTONIN HOMEOSTASIS IN THE TERMINAL ILEUM, AN ANATOMIC SITE UNIQUELY AFFECTED IN ASD. BY LEVERAGING HUMAN-DERIVED ILEAL AND COLONIC ORGANOIDS AND MICROBIAL ISOLATES, THE PROPOSED RESEARCH MOVES BEYOND ASSOCIATIVE MICROBIOME STUDIES TO ESTABLISH CAUSAL MECHANISMS IN ASD.