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.

结核病（TB），由复杂的分枝杆菌属（Mycobacterium tuberculosis，简称Mtb）引起，是单一细菌感染导致死亡的主要原因。去年，结核病位居SARS-CoV-2之后，成为第二大致死性传染病。尽管如此，许多关于结核病的生物学和免疫学方面尚未完全阐明，例如由调节性T细胞（Treg细胞）介导的免疫调节复杂过程，以及吲哚胺2,3-双加氧酶（IDO）和血红素加氧酶1（HO-1）等酶的作用。在本研究中，比较了这些免疫调节因素在感染不同致病力水平的Mtb菌株的鼠类中的贡献。首先，通过气管内途径感染Balb/c小鼠，使用高剂量低致病力参考菌株H37Rv或高度致病力的临床分离株（菌株5186）。通过细胞流式光度术测定感染小鼠肺中的Treg细胞感染动力学，并通过RT-PCR和免疫组织化学测定IDO和HO-1的表达。随后，通过给予感染动物针对Treg细胞的特异性细胞毒性单克隆抗体（抗CD25，PC61克隆）或使用特异性抑制剂（1-甲基-D,L-色氨酸或锌原卟啉-IX）阻断IDO和HO-1的活性，评估了由Treg细胞、IDO和HO-1介导的免疫调节的贡献。感染低致病力菌株的小鼠显示出Treg细胞的逐渐增加，在感染的晚期阶段（28天）达到最高值，同样，酶的表达趋势也一致，巨噬细胞显示出最高的免疫染色。感染高度致病力菌株的小鼠显示出较低的存活率（34天）和较高的Treg细胞数量，以及IDO和HO-1在一周前的较高表达。与未治疗动物相比，感染H37Rv菌株的小鼠在晚期感染期间进行Treg细胞耗竭或酶阻断治疗，显示出明显的细菌负荷减少，IFN-g表达升高，IL-4降低，但由自动化形态计量确定的炎症肺实变范围相似。相反，在高度致病力菌株5186感染小鼠中耗竭Treg细胞产生了与严重急性病毒性肺炎相似的弥漫性肺泡损伤，生存率较低，细菌负荷增加，而阻断IDO和HO-1则产生了高细菌负荷和广泛的肺炎伴有坏死。因此，似乎Treg细胞、IDO和HO-1在由低致病力Mtb引起的晚期肺结核中具有破坏性，可能因为这些因素降低了由Th1反应介导的免疫保护。相反，当感染由高度致病力菌株引起时，Treg细胞、IDO和HO-1是有益的，通过调节过度炎症，这种炎症产生了肺泡损伤、肺坏死、急性呼吸衰竭和快速死亡。