The role of T regulatory cells in cigarette smoke-induced susceptibility to tuberculosis

香烟烟雾(CS)损害宿主对结核分枝杆菌(MTB)的保护性免疫反应。 然而，一个被忽视的方面是，CS或尼古丁如何可能促使免疫抑制细胞增加 对结核分枝杆菌的敏感性。T调节细胞(Tregs)是一种免疫抑制细胞，可使实验室 动物和人类对结核病的反应。CS或尼古丁不仅增加了Tregs进入肺部的量，而且还诱导了 细胞表面信号分子程序性死亡-1与细胞毒性T淋巴细胞相关蛋白 4(CTLA-4)-增强其免疫抑制活性。我们的中心假设是CS增强了 Treg通过诱导PD-1和CTLA-4的免疫抑制功能，以及Treg的这种上调 活动性在影响结核分枝杆菌感染的控制中起着关键作用。我们将采取双管齐下的方式， 使用体内小鼠模型和使用原代人类细胞的体外模型。首先，我们将采用 将同等数量的空气或CS暴露的Tregs转移到CS-naive小鼠中，然后用 结核分枝杆菌确定转移CS暴露的Tregs是否比转移暴露于空气的Tregs更加重结核分枝杆菌感染 特雷格斯。第二，我们将利用原代人肺泡巨噬细胞(AM)+Tregs的共培养，仅与 Tregs单独接触介质、CS或尼古丁，以及PD-1和CTLA-4的药物拮抗剂 建立CS/尼古丁增加Treg活性以削弱抗结核免疫的机制。 目的1：在活体小鼠模型中建立CS暴露的Treg在MTB易感性中的作用。 假设：我们预测CS诱导的Treg活性上调是CS 易患肺结核。因此，与接受空气暴露Tregs的小鼠相比，接受CS暴露Tregs的小鼠 将更容易感染结核病，表现为宿主保护性M1巨噬细胞和TH1细胞较少，较大 结核分枝杆菌的负担，更糟糕的肺部病理，和降低的存活率。方法：我们将采用相同的转让方式 从空气或CS暴露的供体小鼠到CS幼稚小鼠的Tregs数量，感染MTB的受体小鼠， 并确定微生物学和免疫学结果。 目的2：建立CS暴露的Tregs在损害人AM对结核分枝杆菌控制中的作用。假设： CS或尼古丁会增加Tregs上PD-1和CTLA-4的表达，在与AM共同培养时，会增加Tregs上PD-1和CTLA-4的表达 Tregs对AM的抑制作用，表现为免疫抑制细胞因子水平的增加， 宿主保护性细胞因子水平降低，吞噬小体-溶酶体(P-L)融合和自噬减少，以及 结核分枝杆菌的更大恢复。阻断PD-1或CTLA-4将消除这些影响。方法：AIR-、CS-或 尼古丁暴露的Tregs将与朴素AM共同培养，感染MTB±PD-1或CTLA-4抑制剂， 并对结核分枝杆菌载量、细胞因子、P-L融合和自噬进行分析。 该项目将阐明Tregs在介导CS诱导的结核病易感性中所起的作用。调查结果来自 这些研究可以为开发独特的免疫调节方法治疗结核病提供基础。

Cigarette smoke (CS) impairs host-protective immune responses to Mycobacterium tuberculosis (MTB). However, an overlooked aspect is how CS or nicotine may drive immunosuppressive cells to increase susceptibility to MTB. T regulatory cells (Tregs) are immunosuppressive cells that predispose laboratory animals and humans to TB. CS or nicotine not only increases the influx of Tregs to the lungs but also induces cell surface signaling molecules – programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) – that enhance their immunosuppressive activity. Our central hypothesis is that CS enhances the immunosuppressive function of Tregs via induction of PD-1 and CTLA-4, and that this upregulation of Treg activity plays a pivotal role in impairing control of MTB infection. We will undertake a two-pronged approach, using both an in vivo murine model and an ex vivo model using primary human cells. First, we will adoptively transfer equal number of air- or CS-exposed Tregs into CS-naïve mice and then infect the recipient mice with MTB to determine if transfer of CS-exposed Tregs aggravates MTB infection more than transfer of air-exposed Tregs. Second, we will utilize co-cultures of primary human alveolar macrophages (AM) + Tregs with only the Tregs exposed to medium alone, CS, or nicotine, and pharmacologic antagonists to PD-1 and CTLA-4 to establish the mechanism by which CS/nicotine increases Treg activity to impair anti-TB immunity. Aim 1: Establish the role CS-exposed Tregs play in predisposing to MTB in an in vivo murine model. Hypothesis: We predict that CS-induced upregulation of Treg activity is a pivotal mechanism by which CS predisposes to MTB. Thus, compared to mice receiving air-exposed Tregs, mice receiving CS-exposed Tregs will be more susceptible to TB, demonstrated by fewer host-protective M1 macrophages and TH1 cells, greater burden of MTB, worse lung pathology, and reduced survival. Approach: We will adoptively transfer the same number of Tregs from air- or CS-exposed donor mice to CS-naïve mice, infect the recipient mice with MTB, and determine microbiological and immunological outcomes. Aim 2: Establish the role CS-exposed Tregs play in impairing human AM control of MTB. Hypothesis: CS or nicotine will increase PD-1 and CTLA-4 expression on Tregs, and in co-cultures with AM, augment the suppressive effect of Tregs on AM, demonstrated by increased levels of immunosuppressive cytokines, decreased levels of host-protective cytokines, reduced phagosome-lysosome (P-L) fusion and autophagy, and greater recovery of MTB. Blocking PD-1 or CTLA-4 will abrogate these effects. Approach: Air-, CS-, or nicotine-exposed Tregs will be co-cultured with naïve AM, infected with MTB ± inhibitors to PD-1 or CTLA-4, and analyzed for MTB burden, cytokines, P-L fusion, and autophagy. This project will clarify the role played by Tregs in mediating CS-induced susceptibility to TB. Findings from these studies can provide the foundation for developing unique immunomodulatory approaches to treat TB.