Neural interactions with the microbiome and immune system that produce inflammation in Hirschsprung disease models

摘要 肠道（ENS）和自主神经系统（ANS）在维持肠道健康中的重要性是 在先天性巨结肠（HSCR）中很明显，其定义为远端肠中ENS的缺失， 通过许多突变（例如，Ednrb）。HSCR通过手术切除“异常”的肠道来管理， 许多患者患有持续的功能性和炎性肠并发症（例如，巨结肠 相关性小肠结肠炎，HAEC），表明近端肠中的ENS也受到HSCR相关性结肠炎的影响。 尽管在解剖学上看起来正常我们的实验室用Ednrb产生了一种新的HSCR小鼠模型 表达GCaMP的突变，GCaMP是一种遗传编码的钙指示剂，用于测量突触连接， ENS存在的近端肠。通过使用体内和离体结肠制剂， 和外部神经通路完整，我们以前已经表明如何ENS，ANS（交感神经和 副交感神经），感觉输入通常影响结肠功能。Ednrb-/-（null）-GCaMP小鼠具有特异性 出生后近端结肠肌间神经元活动（自发和突触诱发）的变化 这可能会导致HSCR中的慢性问题。有趣的是，年龄匹配的Ednrb+/-（het）-GCaMP小鼠 显示出相似的功能表型，尽管在整个肠道中有ENS神经支配， 与野生型同窝仔的成年个体有显著差异。这些小鼠表现出运动障碍， 固有层中的免疫细胞计数，并且似乎更容易受到实验诱导的 炎症因此，HSCR相关基因中的“亚临床”突变（即，那些不会引起HSCR的 诊断）似乎足以损害ENS电路开发的各个方面。Ednrb-/-和Ednrb+/- GCaMP小鼠模型HSCR的不同方面：新生儿中的“临床”HSCR并发症和“亚临床”HSCR并发症 HSCR问题持续到童年和成年。最近的几项HSCR研究发现， 微生物组和免疫力受损可能是炎症和HAEC增加的基础，但因果作用 微生物组在产生HSCR相关功能障碍中的作用尚不清楚。根据我们对神经系统的了解， 免疫-微生物群相互作用，我假设ENS/ANS间接导致免疫功能障碍 通过动力障碍引起的微生物组变化，但也可能通过直接神经免疫系统的变化， 通信为了检验这一假设，目标1（K99）中的实验将定义免疫细胞谱， 在出生后年龄的“临床”和“亚临床”HSCR小鼠模型中， “HSCR微生物组”是产生ENS、ANS或免疫功能障碍所必需的和/或足够的。实验 目标2（R 00）将测试ENS/ANS中的HSCR相关功能障碍是否会损害对 炎症免疫挑战，并确定“HSCR微生物组”在解决炎症中的作用。 由于许多消化系统疾病表现出神经、微生物组和/或免疫失调，因此提出了 研究计划将广泛影响人类健康。

ABSTRACT The importance of the enteric (ENS) and autonomic nervous systems (ANS) in maintaining intestinal health is made evident in Hirschsprung disease (HSCR), defined by the absence of the ENS in distal bowel and caused by a number of mutations (e.g., Ednrb). HSCR is managed with surgery to remove the ‘abnormal’ bowel, but many patients suffer from persistent functional and inflammatory bowel complications (e.g., Hirschsprung Associated Enterocolitis, HAEC), indicating that the ENS in proximal bowel is also affected by HSCR-related mutations despite appearing anatomically normal. Our lab generated a novel HSCR mouse model with Ednrb mutations that expresses GCaMP, a genetically-encoded calcium indicator, to measure synaptic connectivity in proximal bowel where the ENS is present. By using in vivo and ex vivo colon preparations that keep intrinsic and extrinsic nerve pathways intact, we have previously shown how ENS, ANS (sympathetic and parasympathetic), and sensory inputs normally influence colon function. Ednrb-/- (null)-GCaMP mice had specific changes in myenteric neuron activity (spontaneous and synaptically-evoked) in proximal colon at post-natal ages that likely contribute to the chronic issues in HSCR. Interestingly, age-matched Ednrb+/- (het)-GCaMP mice displayed a similar functional phenotype, despite having ENS innervation in the entire bowel, and they continued to be significantly different from wild-type littermates as adults. These mice exhibited dysmotility, had altered immune cell counts in the lamina propria, and appeared to be more vulnerable to experimentally-induced inflammation. Therefore, ‘subclinical’ mutations in HSCR-related genes (i.e., those that would not cause a HSCR diagnosis) appear to be sufficient to impair aspects of ENS circuit development. Importantly, Ednrb-/- and Ednrb+/- GCaMP mice model different aspects of HSCR: ‘clinical’ HSCR complications in newborns and ‘subclinical’ HSCR issues that persist into childhood and adulthood. Several recent HSCR studies have identified shifts in the microbiome and impaired immunity that may underlie increased inflammation and HAEC, but the causal role of the microbiome in producing HSCR-related dysfunction is unknown. Based on what is known about neuro- immune-microbiota interactions, I hypothesize that the ENS/ANS contributes to immune dysfunction indirectly via microbiome changes due to dysmotility, but also potentially through changes in direct neuro-immune communication. To test this hypothesis, experiments in Aim 1 (K99) will define the immune cell profile and microbiome in ‘clinical’ and ‘subclinical’ HSCR mouse models at post-natal ages, and determine whether the ‘HSCR microbiome’ is necessary and/or sufficient to produce ENS, ANS, or immune dysfunction. Experiments in Aim 2 (R00) will test whether HSCR-related dysfunction in the ENS/ANS impairs the ability to respond to inflammatory immune challenge, and determine the role of the ‘HSCR microbiome’ in resolving inflammation. As numerous digestive disorders exhibit neural, microbiome and/or immune dysregulation, the proposed research program will broadly impact human health.