Immune modulation by metagenomic DNA from the gastrointestinal microbiota of newborn infants

胃肠道微生物区系和宿主免疫系统之间相互作用紊乱，通常被称为生物失调，与一系列疾病有关，例如炎症性肠病、抗生素相关性腹泻和肥胖症。对于正在建立微生物区系的新生儿来说，控制胃肠道免疫反应尤其重要，因为他们需要避免过度炎症，以应对潜在病原菌的瞬时定植。尽管越来越多的证据表明胃肠道微生物区系在微生物区系失调中发挥了作用，但很难确定在微生物区系水平上明确定义微生物区系失调的参数。人类宿主可以通过先天免疫受体检测微生物，如Toll样受体组中的TLR9。TLR9识别特定的微生物DNA短寡聚体，参与宿主免疫反应的促炎激活和抗炎调节。通过元基因组序列分析来量化TLR9刺激和TLR9调节序列基序，结合依赖TLR9的实验免疫分析来表征粪便微生物区系，对于研究宿主-微生物相互作用的动力学是有吸引力的，因为它具有识别胃肠道失调的基于序列的诊断生物标记物的潜力。我们的初步数据表明，与成人相比，新生儿粪便微生物区系诱导TLR9依赖的免疫激活的能力降低，并且相对于已知TLR9结合序列基序的相对丰度，元基因组序列组成发生了变化。因此，我们建议使用新生儿粪便微生物区系作为研究依赖DNA的宿主-微生物免疫相互作用的模型。相应地，这项建议的目的是收集婴儿和成人的横断面队列，在体外测量和比较对粪便元基因组DNA的免疫反应(目标I)，对样本进行元基因组测序，分别计算免疫刺激和免疫调节TLR9结合低聚体的相对丰度，并建立基于元基因组序列的免疫激活预测模型(目标II)，以及在无菌小鼠模型中测量粪便元基因组DNA对胃肠道免疫刺激的影响，以及新生粪便元基因组DNA在化学诱导结肠炎小鼠模型中减轻炎症的能力(目标III)。

Disturbed interactions between the gastrointestinal microbiota and the host immune system, commonly referred to as dysbiosis, have been associated with a diverse set of diseases, including for example inflammatory bowel disease, antibiotic-associated diarrhea and obesity. Control over gastrointestinal immune responses is especially important in newborn infants that are in the process of establishing their microbiota, as they need to avoid excessive inflammation in response to transient colonization with potential pathogenic bacteria. In spite of growing evidence for a functional role of the gastrointestinal microbiota for dysbiosis, it has been difficult to identify parameters that would clearly define dysbiosis on the microbiota level. The human host can detect microbes via innate immune receptors such as TLR9 from the group of Toll-like receptors. TLR9 recognizes specific short oligomers of microbial DNA and is involved both in the pro-inflammatory activation and anti-inflammatory modulation of host immune responses. Fecal microbiota characterization by metagenomic sequence analysis to quantify TLR9-stimulating and TLR9-regulating sequence motifs, in combination with TLR9-dependent experimental immune assays, is attractive to study the dynamics of host-microbe interactions, as it has the potential to identify sequence-based diagnostic biomarkers for gastrointestinal dysbiosis.Our preliminary data indicate that the newborn infant fecal microbiota has a reduced capacity to induce TLR9-dependent immune activation, compared to that of adults, as well as an altered metagenomic sequence composition with respect to the relative abundances of known TLR9-binding sequence motifs. We therefore propose to use the newborn fecal microbiota as a model to study DNA-dependent host-microbe immune interactions. Correspondingly, the aims of this proposal are to assemble a cross-sectional cohort of infants and adults and to measure and compare immune responses to fecal metagenomic DNA in vitro (Objective I), to perform metagenomic sequencing on the samples, calculate relative abundances of immunostimulatory and immunoregulatory TLR9-binding oligomers, respectively, and to develop predictive models for metagenomic sequence-based immune activation (Objective II), and to measure the effects of gastrointestinal immune stimulation with fecal metagenomic DNA in a germfree mouse model, as well as the ability of newborn fecal metagenomic DNA to attenuate inflammation in a murine model of chemically induced colitis (Objective III).