Background & Aims

The enteric nervous system (ENS) exists in close proximity to luminal bacteria. Intestinal microbes regulate ENS development, but little is known about their effects on adult enteric neurons. We investigated whether intestinal bacteria or their products affect the adult ENS via toll-like receptors (TLRs) in mice.

Methods

We performed studies with conventional C57/BL6, germ-free C57/BL6, Nestin-creER^T2:tdTomato, Nestin-GFP, and ChAT-cre:tdTomato. Mice were given drinking water with ampicillin or without (controls). Germ-free mice were given drinking water with TLR2 agonist or without (controls). Some mice were given a blocking antibody against TLR2 or a TLR4 inhibitor. We performed whole gut transit, bead latency, and geometric center studies. Feces were collected and analyzed by 16S ribosomal RNA gene sequencing. Longitudinal muscle myenteric plexus (LMMP) tissues were collected, analyzed by immunohistochemistry, and levels of nitric oxide were measured. Cells were isolated from colonic LMMP of Nestin-creER^T2:tdTomato mice and incubated with agonists of TLR2 (receptor for gram-positive bacteria), TLR4 (receptor for gram-negative bacteria), or distilled water (control) and analyzed by flow cytometry.

Results

Stool from mice given ampicillin had altered composition of gut microbiota with reduced abundance of gram-positive bacteria and increased abundance of gram-negative bacteria, compared with mice given only water. Mice given ampicillin had reduced colon motility compared with mice given only water, and their colonic LMMP had reduced numbers of nitrergic neurons, reduced neuronal nitric oxide synthase production, and reduced colonic neurogenesis. Numbers of colonic myenteric neurons increased after mice were switched from ampicillin to plain water, with increased markers of neurogenesis. Nestin-positive enteric neural precursor cells expressed TLR2 and TLR4. In cells isolated from the colonic LMMP, incubation with the TLR2 agonist increased the percentage of neurons originating from enteric neural precursor cells to approximately 10%, compared with approximately 0.01% in cells incubated with the TLR4 agonist or distilled water. Mice given an antibody against TLR2 had prolonged whole gut transit times; their colonic LMMP had reduced total neurons and a smaller proportion of nitrergic neurons per ganglion, and reduced markers of neurogenesis compared with mice given saline. Colonic LMMP of mice given the TLR4 inhibitor did not have reduced markers of neurogenesis. Colonic LMMP of germ-free mice given TLR2 agonist had increased neuronal numbers compared with control germ-free mice.

Conclusions

In the adult mouse colon, TLR2 promotes colonic neurogenesis, regulated by intestinal bacteria. Our findings indicate that colonic microbiota help maintain the adult ENS via a specific signaling pathway. Pharmacologic and probiotic approaches directed towards specific TLR2 signaling processes might be developed for treatment of colonic motility disorders related to use of antibiotics or other factors.

中文翻译：

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肠道细菌通过Toll样受体2诱导的小鼠神经维持成年肠道神经系统和硝能神经元。

背景与目标

肠神经系统（ENS）紧邻腔内细菌存在。肠道微生物调节ENS的发育，但对它们对成年肠神经元的影响知之甚少。我们调查了肠道细菌或其产物是否通过通行费样受体（TLRs）影响小鼠的成年ENS。

方法

我们使用常规C57 / BL6，无菌C57 / BL6，Nestin -creER ^T2：tdTomato，Nestin -GFP和ChAT-cre：tdTomato进行了研究。给小鼠喝有或没有氨苄西林的饮用水（对照）。给无细菌的小鼠喝含或不含TLR2激动剂的饮用水（对照）。一些小鼠被给予针对TLR2或TLR4抑制剂的封闭性抗体。我们进行了整个肠道运输，珠子潜伏期和几何中心研究。收集粪便并通过16S核糖体RNA基因测序进行分析。收集纵向肌肉肌神经丛（LMMP）组织，通过免疫组织化学分析，并测量一氧化氮的水平。从Nestin -creER ^T2的结肠LMMP中分离细胞：tdTomato小鼠，与TLR2（革兰氏阳性细菌的受体），TLR4（革​​兰氏阴性细菌的受体）或蒸馏水（对照）的激动剂一起孵育，并通过流式细胞仪进行分析。

结果

与仅饮水的小鼠相比，给予氨苄西林的小鼠的粪便改变了肠道菌群的组成，减少了革兰氏阳性菌的丰度，增加了革兰氏阴性菌的丰度。与仅喝水的小鼠相比，给予氨苄西林的小鼠结肠运动减少，其结肠LMMP的硝化神经元数量减少，神经元一氧化氮合酶生成减少，结肠神经发生减少。小鼠从氨苄西林转入白开水后，结肠神经元的神经元数量增加，神经发生标志物增多。巢蛋白阳性肠神经前体细胞表达TLR2和TLR4。在从结肠LMMP分离的细胞中，与TLR2激动剂一起孵育可将源自肠道神经前体细胞的神经元的百分比增加到大约10％，与用TLR4激动剂或蒸馏水孵育的细胞中约0.01％的细胞相比。接受针对TLR2抗体的小鼠延长了整个肠道的运输时间；与给予盐水的小鼠相比，它们的结肠LMMP减少了每个神经节的总神经元，减少了硝化神经元的比例，并且减少了神经发生的标志物。给予TLR4抑制剂的小鼠结肠LMMP的神经发生标记没有减少。与对照无菌小鼠相比，给予TLR2激动剂的无菌小鼠结肠LMMP的神经元数量增加。与给予生理盐水的小鼠相比，神经发生的标记物减少。给予TLR4抑制剂的小鼠结肠LMMP的神经发生标记没有减少。与对照无菌小鼠相比，给予TLR2激动剂的无菌小鼠结肠LMMP的神经元数量增加。与给予生理盐水的小鼠相比，神经发生的标记物减少。给予TLR4抑制剂的小鼠结肠LMMP的神经发生标记没有减少。与对照无菌小鼠相比，给予TLR2激动剂的无菌小鼠结肠LMMP的神经元数量增加。

结论

在成年小鼠结肠中，TLR2促进肠道细菌调节的结肠神经发生。我们的发现表明，结肠菌群可通过特定的信号途径帮助维持成人ENS。可以开发针对特定TLR2信号传导过程的药理和益生菌方法，以治疗与使用抗生素或其他因素有关的结肠动力性疾病。