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

在先天性巨结肠症模型中神经与微生物组和免疫系统的相互作用产生炎症

基本信息

批准号：
10283370
负责人：
Kristen Michelle Smith-Edwards
金额：
$ 9.15万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2022-07-31
项目状态：
已结题

项目摘要

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.

抽象的肠道 (ENS) 和自主神经系统 (ANS) 在维持肠道健康方面的重要性是在先天性巨结肠症 (HSCR) 中表现得很明显，该病的定义是远端肠中缺乏 ENS，并导致由许多突变（例如，Ednrb）引起的，可以通过手术切除“异常”肠道来控制。许多患者患有持续的功能性和炎症性肠道并发症（例如先天性巨结肠）相关小肠结肠炎 (HAEC)，表明近端肠的 ENS 也受到 HSCR 相关的影响尽管在解剖学上看起来正常，但我们的实验室使用 Ednrb 生成了一种新型 HSCR 小鼠模型。表达 GCaMP（一种基因编码的钙指示剂）的突变，可测量突触连接性 ENS 存在的近端肠道通过使用保持内在的体内和离体结肠制剂。和外在神经通路完好无损，我们之前已经展示了 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 小鼠具有特定的功能。产后近端结肠肌间神经元活动（自发和突触诱发）的变化这可能导致 HSCR 的慢性问题，年龄匹配的 Ednrb+/- (het)-GCaMP 小鼠。尽管整个肠道都有 ENS 神经支配，但仍表现出相似的功能表型，并且他们继续这些小鼠成年后与野生型同窝小鼠有显着不同。固有层中的免疫细胞计数，似乎更容易受到实验诱导的影响因此，HSCR 相关基因的“亚临床”突变（即不会引起 HSCR 的基因）。诊断）似乎足以损害 ENS 电路发展的各个方面，重要的是，Ednrb-/- 和 Ednrb+/-。 GCaMP 小鼠模拟 HSCR 的不同方面：新生儿的“临床”HSCR 并发症和“亚临床” 最近的几项 HSCR 研究发现，HSCR 问题一直持续到儿童期和成年期。微生物组和免疫力受损可能是炎症和 HAEC 增加的原因，但因果作用根据对神经系统的了解，微生物组在产生 HSCR 相关功能障碍中的作用尚不清楚。免疫-微生物群相互作用，我认为 ENS/ANS 会间接导致免疫功能障碍通过运动障碍引起的微生物组变化，但也可能通过直接神经免疫的变化为了检验这一假设，目标 1 (K99) 中的实验将定义免疫细胞特征和信息。产后年龄的“临床”和“亚临床”HSCR 小鼠模型中的微生物组，并确定是否 “HSCR 微生物组”对于产生 ENS、ANS 或免疫功能障碍实验是必要和/或充分的。目标 2 (R00) 将测试 ENS/ANS 中与 HSCR 相关的功能障碍是否会损害响应能力炎症免疫挑战，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，建议研究计划将广泛影响人类健康。