Bacterial and Fungal Dysbiosis in Hirschsprung-Associated Enterocolitis

 描述（由申请人提供）：肠远端部分完全缺乏肠神经节被称为先天性巨结肠病（HSCR）。患有HSCR的儿童表现出一系列症状（例如，严重的便秘、腹泻、肠道炎症），易患败血症，并可能危及生命。在大多数情况下，手术矫正可以根除这些症状。但在高达30%的病例中，手术未能治愈这种疾病，此后，儿童患有慢性小肠结肠炎，可导致菌血症，彻底败血症，甚至死亡。这种小肠结肠炎称为Hirschsprung相关性小肠结肠炎（HAEC），其潜在机制几乎一无所知。在我们对HSCR儿童发生HAEC的详细研究过程中，我们发现有HAEC病史的儿童粪便中真菌菌群多样性发生了显著变化， HAEC（但不是活性HAEC）的发生率与未发生HAEC的患者相比（Frykman et al. PloS One 10：e0124172，2015）。HAEC患者中真菌多样性的急剧变化可能是由于旨在治疗小肠结肠炎的多个疗程的抗生素导致的持续真菌大量繁殖。或者，可能有早期的变化，在细菌和 由于结肠无神经节细胞症而发生的真菌微生物组使儿童易患HAEC或潜在改变的先天免疫，使得白色念珠菌可能是致病物。无论哪种可能性是正确的，结肠细菌和真菌对先天性巨结肠患儿小肠结肠炎的发展所起的作用仍不清楚。内皮素受体B基因（Ednr B）突变导致小鼠和人类（先天性巨结肠病）的结肠无神经节细胞症，从而发展为严重的小肠结肠炎。导致HAEC的机制知之甚少，目前的治疗方法是针对控制症状，成功率非常有限。因此，抗真菌药物治疗可能是治疗HAEC儿童的新的靶向治疗，目前HAEC儿童的替代方案很少。我们的总体假设是，真菌有助于HSCR儿童发展HAEC的生态失调，并且发展小肠结肠炎的Ednrb缺失小鼠作为研究微生物变化的适当和临床相关模型。初步数据表明，患有小肠结肠炎的Ednrb基因敲除小鼠的肠道C。白色念珠菌我们的计划是首先全面表征患有结肠无神经节细胞症的Ednrb-/-小鼠中的细菌和真菌微生物组，特别是小肠结肠炎的发展;然后确定结肠细菌，真菌和先天免疫在小肠结肠炎中的功能作用。我们的最终目标是了解真菌在HAEC组织炎症中的作用，最好在小鼠模型中进行研究。这个R 03申请提出了一个详细的策略，以追求这些与HAEC相关的机理研究，同时仍然在他杰出的K 08指导团队的指导下。最终目标是申请人利用本次拟定研究期间获得的数据沿着人类数据，成功申请R 01。

 DESCRIPTION (provided by applicant): A total absence of enteric ganglia in the distal portions of the intestine is known as Hirschsprung disease (HSCR). Children with HSCR present with a constellation of symptoms (e.g., severe constipation, diarrhea, intestinal inflammation) that predispose to sepsis, and can be life-threatening. Surgical correction eradicates these symptoms in most cases. But in up to 30% of cases, surgery fails to cure the disorder, and thereafter, children suffer from chronic enterocolitis that can lead to bacteremia, outright sepsis, and even death. Almost nothing is known about the underlying mechanisms of this enterocolitis, known as Hirschsprung-associated enterocolitis (HAEC). In the course of our detailed studies of children with HSCR who developed HAEC, we discovered a striking shift in fungal microflora diversity with increased Candida albicans in the stool of children with a history of HAEC (but not active HAEC), compared with those who did not develop HAEC (Frykman et al. PloS One 10:e0124172, 2015). The dramatic shifts in fungal diversity in HAEC patients could potentially be persistent fungal blooms as a consequence of multiple courses of antibiotics aimed at treating the enterocolitis. Alternatively, there may be early changes in the bacterial and fungal microbiome that occur as a result of colonic aganglionosis that predispose children to HAEC or potentially altered innate immunity such that C albicans may be a pathobioant. Regardless of which possibility may be correct, the individual contributions made by colonic bacteria and fungi to the development of enterocolitis in children with Hirschsprung disease remains unclear. Endothelin Receptor B gene (Ednrb) mutations cause colonic aganglionosis in both mice and humans (Hirschsprung disease) that develop severe enterocolitis. The mechanisms that contribute to HAEC are poorly understood, and the current treatment approaches are directed towards control of symptoms, with very limited success. Hence, treatment with antifungal agents could be novel and targeted therapy to treat children with HAEC, who currently have few alternatives. Our overall hypothesis is that fungi contribute to the dysbiosis of HSCR children who develop HAEC, and the Ednrb-null mice that develops enterocolitis serves as an appropriate and clinically relevant model to study microbiomal changes. Preliminary data indicate that Ednrb-null mice with enterocolitis have increased gut C. albicans. Our plan is to first comprehensively characterize the bacterial and fungal microbiome, especially the development of enterocolitis, in Ednrb-/- mice with colonic aganglionosis; then to identify the functional role of colonic bacteria, fungi and innate immunity in enterocolitis. Our ultimate goal is to understand the role of fungi in tissue inflammation of HAEC, which can best be studied in a mouse model. This R03 application proposes a detailed strategy to pursue these mechanistic studies related to HAEC, while still under the guidance of his outstanding K08 mentoring team. The ultimate goal is for the applicant to leverage the data obtained during this proposed investigation along with human data, into a successful R01 application.