How Does Nicotine Impair Macrophage Killing of Mycobacterium Tuberculosis?

生理浓度尼古丁损害巨噬细胞对结核分枝杆菌(MTB)的杀伤作用 导致细菌负荷增加2-5倍。我们还发现，防止尼古丁结合- 无论是从药理上还是通过尼古丁受体的基因破坏--显著减少吸烟 烟雾提取物对结核分枝杆菌巨噬细胞杀伤的损伤作用。但尼古丁的作用机制 结核分枝杆菌对巨噬细胞控制的损害尚不清楚。我们的假设是尼古丁破坏巨噬细胞 通过核因子κB介导的抑制自噬和凋亡以及直接抗结核杆菌(I)的作用 通过Dird/microRNA介导的抑制宿主保护性细胞因子的产生和(Ii)间接通过 增加CTLA-4的产生和T调节细胞(Tregs)的激活。 目的1：探讨尼古丁直接损伤巨噬细胞杀伤结核分枝杆菌的机制。 方法：我们将分离尼古丁原始的人肺泡巨噬细胞(AM)和单核细胞来源的 巨噬细胞(MDM)，用MTB±尼古丁感染它们，并量化自噬、凋亡、细胞内负荷 而抑制NFκB的程度可减轻尼古丁的影响。我们还将敲除特定的NFκB 确定它们在尼古丁介导的抗结核分枝杆菌免疫抑制中的作用。假设： 尼古丁诱导AM和MDM中的NFκB将抑制自噬和细胞凋亡，导致负荷增加 是MTB的。抑制NFκB的激活将消除尼古丁的这些作用，并恢复对巨噬细胞的杀伤 山地车。敲除Dier基因将逆转尼古丁对MTB诱导的细胞因子的抑制。 目的2：探讨尼古丁间接损伤巨噬细胞杀伤结核分枝杆菌的机制。 Subaim A.使用原代人类细胞的体外模型。方法：我们将培养幼稚的人类MDM 用天真与尼古丁暴露的Tregs，并用MTB感染细胞。要确定CTLA-4的表达是否 负责尼古丁诱导的Treg活性增加和继发性MDM抑制，细胞还将 与抗CTLA-4中和抗体孵育。假设：尼古丁会增加CTLA-4的表达 在树上，增加其IL-10和转化生长因子β的产生。这将抑制结核杆菌感染的MDM的自噬。 并且与与尼古丁-天真Tregs共培养的MDM相比，导致更大的细菌负担。的对抗性 CTLA-4将消除尼古丁的这些免疫抑制作用。 苏巴伊姆B.活体小鼠模型。方法：我们将从未暴露的或尼古丁中采用Tregs- 将B6.PL(Thy1.1)小鼠暴露于Treg耗竭的Foxp3+GFP+DTR+(Thy1.2)小鼠，感染受体小鼠 并量化结核杆菌负荷、巨噬细胞和T细胞表型、肺组织病理学和存活率。 假设：接受尼古丁暴露的小鼠将有更少的宿主保护性M1肺巨噬细胞和 Th1和TH17细胞与接受尼古丁天真Tregs的小鼠进行比较。结果，接受尼古丁的小鼠- 暴露的树将更容易感染结核杆菌，表现为更大的结核杆菌负担，更少的宿主保护 巨噬细胞和辅助性T细胞表型，更严重的肺部病理，并降低存活率。 项目的潜在影响：美国退伍军人尼古丁暴露的高流行率(通过使用 香烟烟雾和无烟尼古丁产品)，以及由于海外部署在结核病中而导致更大的结核病风险 在一些地方病流行的国家，无家可归者相对较多，而且还在不断高龄。而尼古丁会损害 人类巨噬细胞对结核分枝杆菌的控制，目前还没有工作来确定它是如何发生的；说明 这些机制将提供科学动力，帮助提醒医疗和公共卫生界 退伍军人和非退伍军人同时接触尼古丁和结核杆菌的危险。来自这些的发现 研究还可以为开发免疫调节治疗方法提供基础，即使在 没有尼古丁暴露；例如，使用临床上可用的抗CTLA-4药物来使Tregs失活 尝试优化宿主对结核病的免疫力。

Nicotine at physiologic concentrations impairs macrophage killing of Mycobacterium tuberculosis (MTB), resulting in a 2-5 fold increase in bacterial burden. We have also found that preventing nicotine binding – either pharmacologically or by genetic disruption of the nicotinic receptor – significantly mitigated cigarette smoke extract-induced impairment of macrophage killing of MTB. But the mechanisms by which nicotine impairs macrophage control of MTB are not known. Our hypothesis is that nicotine sabotages macrophage function against MTB (i) directly through NFκB-mediated inhibition of autophagy and apoptosis as well as through DICER/microRNA-mediated inhibition of host-protective cytokine production and (ii) indirectly through increased production of CTLA-4 and activation of T regulatory cells (Tregs). Aim 1: Determine the mechanisms by which nicotine directly impairs macrophage killing of MTB. Approach: We will isolate nicotine-naïve human alveolar macrophages (AM) and monocyte-derived macrophages (MDM), infect them with MTB ± nicotine, and quantify autophagy, apoptosis, intracellular burden of MTB, and the extent NFκB inhibition mitigates the effects of nicotine. We will also knockdown specific NFκB subunits and DICER to determine their roles in nicotine-mediated inhibition of anti-MTB immunity. Hypothesis: Nicotine induction of NFκB in AM and MDM will inhibit autophagy and apoptosis, resulting in increased burden of MTB. Inhibition of NFκB activation will abrogate these effects of nicotine and restore macrophage killing of MTB. Knockdown of DICER will reverse the suppression of MTB-induced cytokines by nicotine. Aim 2: Determine the mechanisms by which nicotine indirectly impairs macrophage killing of MTB. Subaim A. An ex vivo model using primary human cells. Approach: We will culture naïve human MDM with naïve vs. nicotine-exposed Tregs and infect the cells with MTB. To determine if CTLA-4 expression is responsible for nicotine-induced increase in Treg activity and secondary MDM suppression, the cells will also be incubated with anti-CTLA-4 neutralizing antibody. Hypothesis: Nicotine will increase CTLA-4 expression on Tregs, augmenting their production of IL-10 and TGFβ. This will inhibit autophagy in MTB-infected MDM and result in greater bacterial burden compared to MDM co-cultured with nicotine-naïve Tregs. Antagonism of CTLA-4 will abrogate these immunosuppressive effects of nicotine. Subaim B. In vivo murine model. Approach: We will adoptively transfer Tregs from unexposed or nicotine- exposed B6.PL(Thy1.1) mice into Treg-depleted Foxp3+GFP+DTR+(Thy1.2) mice, infect the recipient mice with MTB, and quantify MTB burden, macrophage and T cell phenotypes, lung histopathology, and survival. Hypothesis: Mice receiving nicotine-exposed Tregs will have fewer host-protective M1 lung macrophages and TH1 and TH17 cells compared to mice receiving nicotine-naïve Tregs. As a result, mice receiving nicotine- exposed Tregs will be more susceptible to MTB, demonstrated by greater MTB burden, fewer host-protective macrophages and T helper phenotypes, more severe lung pathology, and decreased survival. Potential impact of project: U.S. Veterans have a high prevalence of nicotine exposure (through use of cigarette smoke and smokeless nicotine products) and a greater risk for TB due to overseas deployment in TB endemic countries, relatively high prevalence of homelessness, and advancing age. While nicotine impairs human macrophage control of MTB, no work has been conducted to determine how this occurs; illuminating these mechanisms will provide the scientific impetus to help alert the medical and public health communities of this danger of concomitant nicotine and MTB exposures in veterans and non-veterans. Findings from these studies can also provide the foundation for developing immunomodulatory approaches to treatment even in the absence of nicotine exposure; e.g., use of clinically available anti-CTLA-4 agents to deactivate Tregs in an attempt to optimize host immunity against TB.