Immunological and microbial shifts in the aging rhesus macaque lung during nontuberculous mycobacterial infection - scRNA

Nontuberculous mycobacteria (NTM) are environmentally ubiquitous organisms that predominately cause pulmonary disease (NTMPD) primarily in those over the age of 65. The incidence of NTMPD has steadily increased in the US, now exceeding that of Mycobacterium tuberculosis (M. tb), making it a disease of high consequence. However, the mechanisms leading to higher susceptibility and severity of NTMPD with aging are poorly defined. Here, we compared host response following inoculation of young and aged rhesus macaque in the right caudal lobe with M. avium subsp. hominissuis (MAH) to address this knowledge gap. Bacterial load, microbial community compositions, and host responses were monitored longitudinally using bacterial culture, immunological assays, 16S amplicon sequencing, and single cell genomics assays. Unilateral infection resulted in comparable, albeit low bacterial load in both young and aged animals that was confined to the infected side. A robust inflammatory response was predominantly in the inoculated lung, however, immune cell infiltration and antigen-specific T-cell responses were detected in both lungs. Computed tomography, gross pathology and histopathology examination revealed increased disease severity in aged versus young animals. Nasal, oral and fecal swabs, and BAL samples showed decompartmentalization of the lower respiratory microbiome and translocation of bacterial DNA typically found in the gut and oral-pharyngeal cavity. Finally, single-cell RNA sequencing indicated that aged animals generated a heightened inflammatory response to MAH infection. Collectively these data suggest that increased disease severity in the aged is mediated by a dysregulated macrophage response rather than higher bacterial burden.

非结核分枝杆菌（Nontuberculous mycobacteria, NTM）是广泛存在于环境中的病原微生物，主要在65岁以上人群中引发肺部疾病（NTMPD）。当前美国的NTMPD发病率持续攀升，现已超过结核分枝杆菌（Mycobacterium tuberculosis, M. tb）感染，成为具有高临床危害性的疾病。然而，衰老导致NTMPD易感性升高与病情加重的具体分子机制仍未明确。为填补这一认知空白，本研究对比分析了年轻与老年恒河猴在右尾叶接种鸟分枝杆菌亚种人型亚种（M. avium subsp. hominissuis, MAH）后的宿主免疫应答情况。研究通过细菌培养、免疫学检测、16S扩增子测序以及单细胞基因组学检测等技术手段，纵向监测了实验动物的细菌载量、微生物群落组成与宿主应答特征。结果显示：单侧肺部感染后，年轻与老年恒河猴的感染侧肺部细菌载量虽处于较低水平，但两组细菌载量相当，且细菌仅局限于感染侧肺部；接种侧肺部出现了显著的炎症应答，而双侧肺部均检测到免疫细胞浸润与抗原特异性T细胞应答。计算机断层扫描、大体病理与组织病理学检查结果表明，老年恒河猴的感染病情严重程度显著高于年轻个体。对鼻腔、口腔、粪便拭子及支气管肺泡灌洗液（BAL）样本的分析显示，下呼吸道微生物群落出现分区破坏，肠道与口咽腔定植的典型细菌DNA发生了跨区域移位。最后，单细胞RNA测序结果证实，老年恒河猴对MAH感染产生了更为强烈的炎症应答。综上，本研究数据提示，老年群体NTMPD病情严重程度升高并非由细菌负荷增加所介导，而是由巨噬细胞应答失调所致。