Pre- and post-treatment lung microbiota, metabolome and immune signatures at the site of disease in patients with active pulmonary tuberculosis

总结 人类微生物组对于传染病的发病机制很重要。然而，我们对 微生物组在结核病（TB）中的作用，可以说是世界上最重要的肺部疾病， 极其有限。在撒哈拉以南非洲，结核病因艾滋病毒而恶化，即使使用抗逆转录病毒治疗， 降低肺部免疫力。活动性肺结核（支气管肺泡腔）的发病部位是一个独特的 环境和免疫生态位，但其微生物群令人惊讶地研究不足。我们不知道如何 分类群，包括那些对肺健康很重要分类群（口腔厌氧发酵菌），与细菌发酵有关 终产物，如短链脂肪酸（SCFA），可能影响结核病和组织的免疫控制 修复.此外，结核病治疗方案由数千剂抗生素组成，但其对结核病的长期影响仍然存在。 肺微生物群迄今尚未被表征。因此，我们缺乏关键的基础知识， 将肺部微生物群作为潜在的诊断或治疗靶点进行研究，以改善结核病的预后。 我们将测试我们的中心假设，即疾病部位的口腔厌氧发酵菌与升高的 肺SCFA和受损的炎症和组织修复生物标志物在TB病例（n=50），并在治疗 最后，这些分类群和生物标志物仍然受到干扰，但一年后有所改善。我们将招募同等数量的 我们位于开普敦的高结核病-艾滋病毒负担地点的艾滋病毒阳性患者。我们将测试我们的中心假设， 三个目标。目的1将使用改良的支气管肺泡灌洗（BAL）程序，比较疾病部位 在治疗前，将微生物群中的微生物群与对侧非患病肺组织中的微生物群进行比较。目标2将描述，在每个 治疗前的肺部位，特定分类群，SCFAs，炎症和组织修复之间的关联 生物标志物，并研究向离体刺激的BAL细胞中加入SCFA是否会损害免疫标志物 以剂量依赖的方式释放。Aim 3将在治疗结束时通过支气管镜检查对患者进行重新采样， 一年后，重复测量每个肺部部位的微生物群、SCFA和宿主生物标志物。 如果疾病部位与微生物群紊乱相关，通过SCFA与肺功能受损相关， 免疫和组织修复，包括治疗后，它将证明研究的微生物群和长期结核病临床 结果（例如，进展、治疗失败、复发），这需要大型且昂贵的试验。它将使 研究针对微生物群的测试或治疗干预措施（抗生素，药物，益生元，疫苗）。 实现我们目标的关键是改良支气管镜检查和BAL微生物采样的转移 程序（要求尽量减少低微生物丰度下呼吸道标本的交叉污染）和 尖端计算专业知识（需要结合生物标志物和 临床数据）从纽约大学到斯泰伦博斯大学（SU）。南非临床医生和科学家 将通过研究和培训访问在每个领域进行培训，长期目标是建立一个研究 在SU的肺部微生物群和呼吸系统健康计划。

SUMMARY The human microbiome is important for infectious disease pathogenesis. However, our understanding of the microbiome’s role in tuberculosis (TB), which is arguably the most important lung disease in the world, is extremely limited. In sub-Saharan Africa, TB is exacerbated by HIV which, even with antiretroviral therapy, results in reduced pulmonary immunity. The site-of-disease in active TB (bronchoalveolar space) is a unique environmental and immunological niche but its microbiota is surprisingly understudied. We do not know how taxa, including those important for lung heath (oral anaerobic fermenters), correlate with bacterial fermentation end-products like short chain fatty acids (SCFAs), which may influence immunological control of TB and tissue repair. Furthermore, the TB regimen is comprised of thousands of doses of antibiotics yet its long-term effect on the lung microbiota is hitherto uncharacterized. We hence lack key foundational knowledge that precludes research on the lung microbiota as a potential diagnostic or therapeutic target to improve TB outcomes. We will test our central hypothesis that site-of-disease oral anaerobic fermenters are associated with elevated pulmonary SCFAs and impaired inflammation and tissue repair biomarkers in TB cases (n=50) and, at treatment end, these taxa and biomarkers remain perturbed but improve a year later. We will recruit an equal number of HIV-positive patients at our high TB-HIV burden site in Cape Town. We will test our central hypothesis using three aims. Aim 1 will, using a modified bronchoalveolar lavage (BAL) procedure, compare the site-of-disease microbiota to that in contralateral non-diseased lung tissue before treatment. Aim 2 will characterize, at each lung site before treatment, the association between specific taxa, SCFAs, inflammation and tissue repair biomarkers, and investigate whether SCFA addition to ex vivo stimulated BAL cells impairs immune marker release in a dose-dependent manner. Aim 3 will re-sample patients by bronchoscopy at treatment end and a year later, and repeat measurements of the microbiota, SCFAs, and host biomarkers at each lung site. If the site-of-disease is associated with a perturbed microbiota, linked via SCFAs, to impaired pulmonary immunity and tissue repair, including after treatment, it will justify study of the microbiota and long-term TB clinical outcomes (e.g., progression, treatment failure, relapse), which requires large and expensive trials. It will enable research on tests or therapeutic interventions (antibiotics, drugs, prebiotics, vaccines) that target the microbiota. Key to achieving our aims are the transfer of the modified bronchoscopy and BAL microbiota sampling procedure (required to minimize cross contamination in low microbial abundance lower airway specimens) and leading-edge computational expertise (required to co-analyze sequence data in conjunction with biomarker and clinical data) from New York University to Stellenbosch University (SU). South African clinicians and scientists will train in each area by through research and training visits with the long-term aim of establishing a research program on the lung microbiota and respiratory health at SU.