DataSheet_1_Beneficial or detrimental activity of regulatory T cells, indoleamine 2,3-dioxygenase, and heme oxygenase-1 in the lungs is influenced by the level of virulence of Mycobacterium tuberculosis strain infection.docx

Tuberculosis (TB) caused by the complex Mycobacterium tuberculosis (Mtb) is the main cause of death by a single bacterial agent. Last year, TB was the second leading infectious killer after SARS-CoV-2. Nevertheless, many biological and immunological aspects of TB are not completely elucidated, such as the complex process of immunoregulation mediated by regulatory T cells (Treg cells) and the enzymes indoleamine 2,3-dioxygenase (IDO) and heme oxygenase 1 (HO-1). In this study, the contribution of these immunoregulatory factors was compared in mice infected with Mtb strains with different levels of virulence. First Balb/c mice were infected by intratracheal route, with a high dose of mild virulence reference strain H37Rv or with a highly virulent clinical isolate (strain 5186). In the lungs of infected mice, the kinetics of Treg cells during the infection were determined by cytofluorometry and the expression of IDO and HO-1 by RT-PCR and immunohistochemistry. Then, the contribution of immune-regulation mediated by Treg cells, IDO and HO-1, was evaluated by treating infected animals with specific cytotoxic monoclonal antibodies for Treg cells depletion anti-CD25 (PC61 clone) or by blocking IDO and HO-1 activity using specific inhibitors (1-methyl-D,L-tryptophan or zinc protoporphyrin-IX, respectively). Mice infected with the mild virulent strain showed a progressive increment of Treg cells, showing this highest number at the beginning of the late phase of the infection (28 days), the same trend was observed in the expression of both enzymes being macrophages the cells that showed the highest immunostaining. Animals infected with the highly virulent strain showed lower survival (34 days) and higher amounts of Treg cells, as well as higher expression of IDO and HO-1 one week before. In comparison with non-treated animals, mice infected with strain H37Rv with depletion of Treg cells or treated with the enzymes blockers during late infection showed a significant decrease of bacilli loads, higher expression of IFN-g and lower IL-4 but with a similar extension of inflammatory lung consolidation determined by automated morphometry. In contrast, the depletion of Treg cells in infected mice with the highly virulent strain 5186 produced diffuse alveolar damage that was similar to severe acute viral pneumonia, lesser survival and increase of bacillary loads, while blocking of both IDO and HO-1 produced high bacillary loads and extensive pneumonia with necrosis. Thus, it seems that Treg cells, IDO and HO-1 activities are detrimental during late pulmonary TB induced by mild virulence Mtb, probably because these factors decrease immune protection mediated by the Th1 response. In contrast, Treg cells, IDO and HO-1 are beneficial when the infection is produced by a highly virulent strain, by regulation of excessive inflammation that produced alveolar damage, pulmonary necrosis, acute respiratory insufficiency, and rapid death.

由结核分枝杆菌复合体（Mycobacterium tuberculosis complex, Mtb）引起的结核病（Tuberculosis, TB）是单一细菌病原体致死的首要病因。去年，结核病仍是仅次于新型冠状病毒（SARS-CoV-2）的第二大传染性致死疾病。然而，结核病的诸多生物学与免疫学机制尚未完全阐明，例如调节性T细胞（Treg cells）介导的复杂免疫调控过程，以及吲哚胺2,3-双加氧酶（indoleamine 2,3-dioxygenase, IDO）和血红素氧合酶1（heme oxygenase 1, HO-1）的相关作用机制。本研究针对不同毒力水平的结核分枝杆菌感染小鼠模型，对比了上述免疫调控因子的作用效果。首先通过气管内途径（intratracheal route）感染BALB/c小鼠，分别接种高剂量的弱毒参考菌株H37Rv，或是高毒力临床分离株（菌株5186）。通过流式细胞术（cytofluorometry）检测感染小鼠肺部中调节性T细胞的感染动态变化，并通过逆转录聚合酶链反应（RT-PCR）与免疫组织化学（immunohistochemistry）技术检测IDO与HO-1的表达水平。随后，通过使用针对调节性T细胞耗竭的特异性细胞毒性单克隆抗体（monoclonal antibodies）抗CD25（PC61克隆），或是使用特异性抑制剂分别阻断IDO与HO-1的活性（分别为1-甲基-D,L-色氨酸（1-methyl-D,L-tryptophan）与锌原卟啉IX（zinc protoporphyrin-IX）），评估调节性T细胞、IDO及HO-1介导的免疫调控作用。感染弱毒菌株的小鼠体内调节性T细胞数量呈进行性升高，在感染晚期初始阶段（第28天）达到峰值；两种酶的表达趋势与之一致，巨噬细胞为免疫染色阳性信号最强的细胞类型。感染高毒力菌株的小鼠生存率更低（中位生存时间34天），且体内调节性T细胞数量更高，同时IDO与HO-1的表达水平较前者提前一周升高。与未处理组小鼠相比，感染弱毒株H37Rv且接受调节性T细胞耗竭治疗，或在感染晚期接受酶活性阻断剂治疗的小鼠，其肺部杆菌负荷显著降低，干扰素γ（IFN-γ）表达水平升高，白细胞介素4（IL-4）表达水平降低，但通过自动化形态计量学（morphometry）检测发现，肺部炎性实变范围与未处理组无显著差异。与之相反，感染高毒力菌株5186的小鼠经调节性T细胞耗竭后，出现了类似于重症急性病毒性肺炎的弥漫性肺泡损伤，生存率进一步降低且杆菌负荷升高；而同时阻断IDO与HO-1活性则会导致极高的杆菌负荷与伴有坏死的广泛性肺炎。由此可见，在弱毒结核分枝杆菌诱导的晚期肺结核中，调节性T细胞、IDO及HO-1的活性是有害的，这可能是因为上述因子削弱了Th1应答（Th1 response）介导的免疫保护作用。与之相反，当感染由高毒力菌株引发时，调节性T细胞、IDO及HO-1则通过调控过度炎症反应发挥有益作用，避免肺泡损伤、肺部坏死、急性呼吸衰竭与快速死亡的发生。