Regulation of Innate and Acquired Immunity in Human COPD and Emphysema

描述(由申请人提供)： 吸烟的长期免疫影响，特别是对肺部的影响，仍然是一个知之甚少的研究领域。我们已经证明，肺气肿吸烟者肺中的抗原提呈细胞(APC)高度激活，暴露于烟雾中的小鼠增加了肺中致病的APC，这对T辅助细胞(Th)1、Th17炎症和肺气肿的发展至关重要。烟草烟雾如何激活APC以及导致肺组织损伤的下游分子机制仍不清楚。为了寻找促进肺适应不良免疫反应的内源性分子，我们发现肺气肿吸烟者血浆补体蛋白3(C3)浓度升高，并发现中性粒细胞弹性蛋白酶(NE)和MMP12裂解并释放其活性片段。我们还发现，暴露在香烟烟雾中的C3-/-小鼠可以减少肺部炎症和减轻肺气肿。有趣的是，C3-/-小鼠的这种表型的特点是肺APC不成熟，并保留了另一种补体蛋白C1q的表达，这种补体蛋白的缺乏与自身免疫性炎症性疾病有关。此外，用C1q治疗小鼠可以减少肺部炎症和肺气肿，而用siRNA抑制C1q则提高了它们诱导Th17效应细胞的能力。总而言之，这些新的关键观察指出了补体蛋白在肺气肿中的免疫调节作用；我们拟议的研究的临床意义包括发现新的、有效的非免疫损害补体为基础的疗法来治疗吸烟引起的肺部疾病。本研究的三个目的是：1.研究吸入香烟烟雾肺组织中激活补体和刺激致病抗原前体细胞的作用机制(S)。假设：C3a和/或C5a是有效激活肺部致病APC以响应香烟烟雾所必需的。这项工作将通过：1)研究C3介导的APC激活的机制(S)和慢性吸烟暴露的野生型、C3aR-/-和C5aR-/-小鼠的肺免疫表型；2)利用相同的模型，在体内检测C3抑制剂(如CR2-Fh)的有效性；2)在使用siRNA的人类翻译研究中，将探索C3a和C5a信号在APC介导的CD4T细胞激活中的作用。目的2.探讨C1q对香烟烟雾致肺炎症时肺内APC的影响。假设：暴露在烟雾中的小鼠APC中C1q的减少刺激Th1/Th17介导的炎症并加重肺气肿，而APC产生C1q则减少导致肺气肿的炎症反应。我们将利用C1q-/-小鼠和APC(CD11ccre/C1qflx/flx小鼠)特异性C1q缺乏在烟雾诱导的肺气肿模型中探讨C1q的作用。使用功能丧失(SiRNA)和功能获得(添加回C1q蛋白)的补充研究将被用来进一步确定C1q调节在人类肺气肿APC中的作用。目的3.探讨吸烟小鼠肺APC中C3和C1q之间串扰的机制(S)。假设：C1q促进耐受性肺APC的发展。利用在Foxp3启动子下表达GFP的转基因小鼠(Foxp3GFP)，研究C3、C3aR和C5aR在C1q介导的T细胞耐受诱导中的作用，以及香烟烟雾对抑制细胞因子的产生的影响。这一建议是基于我们的人类翻译研究，重点是补体蛋白在促进肺部自身反应性免疫反应发展中的作用。这些目标的成功完成将为使用新的和有效的非免疫损害补体疗法治疗吸烟引起的肺部疾病提供强有力的理论基础。

DESCRIPTION (provided by applicant): The long-term immune effects of cigarette smoking especially in the lung remain a poorly understood area of research. We have demonstrated that antigen presenting cells (APCs) are highly activated in the lungs of smokers with emphysema, and mice exposed to smoke increase pathogenic APCs in the lungs that is critical for T helper (Th) 1, and Th17 inflammation and emphysema development. How tobacco smoke activates APCs and the downstream molecular mechanisms responsible for lung tissue injury remain unknown. In search of endogenous molecules that could promote maladaptive lung immune responses, we found elevated plasma concentrations of complement protein 3 (C3) in smokers with emphysema, and show that neutrophil elastase (NE) and MMP12 cleave and release its active fragments. We have also found that C3-/- mice exposed to cigarette smoke have reduced lung inflammation and attenuated emphysema. Interestingly this phenotype in C3-/- mice was marked by immature lung APCs and preserved expression of another complement protein C1q, lack of which has been associated with autoimmune inflammatory diseases. Further, treatment of mice with C1q reduced lung inflammation and emphysema while inhibition of C1q with siRNA increased their ability to induce Th17 effector cells. Collectively, these new critical observations point to an immune modulatory role for complement proteins in emphysema; the clinical significance of our proposed studies includes discovering novel and effective non-immune compromising complement-based therapeutics for treatment of smoke- induced lung diseases. Our three Aims are: Aim 1. Identify mechanism(s) activating complement and stimulating pathogenic APCs in lungs exposed to inhale cigarette smoke. Hypothesis: C3a and/or C5a are required for efficient activation of pathogenic APCs in the lungs in response to cigarette smoke. This work will be accomplished by i) investigating the mechanism(s) for C3 mediated APC activation and lung immune phenotype of wild type, C3aR-/-, and C5aR-/- mice exposed to chronic smoke; using the same model, the efficacy of C3 inhibitor (e.g. CR2-fH) will be examined in vivo ii) In human translational studies using siRNA the function of C3a and C5a signaling in APC-mediated activation of CD4 T cells will be explored. Aim 2. Determine the effect of C1q on lung APCs in cigarette smoke-induced lung inflammation. Hypothesis: Reduction of C1q in APCs of mice exposed to smoke stimulates Th1/Th17 mediated inflammation and worsens emphysema whereas production of C1q by APC reduces inflammatory responses that result in emphysema. We will explore the role of C1q using C1q-/- mice and specific C1q deficiency in APCs (CD11ccre/C1qflx/flx mice) in a model of smoke induced emphysema. Complementary studies using loss of function (siRNA) and gain of function (add back C1q protein) will be used to further determine the role of C1q modulation in APCs in human emphysema. Aim 3. Determine the mechanism(s) for the crosstalk between C3 and C1q in lung APCs of smoke exposed mice. Hypotheses: C1q promotes development of tolerogenic lung APCs. Using transgenic mice expressing GFP under Foxp3 promoter (Foxp3GFP) the role of C3, C3aR and C5aR in C1q mediated induction of T cell tolerance, and production of inhibitory cytokines will be examined in response to cigarette smoke. This proposal is based on our human translational studies that are focused on the role of complement proteins in promoting development of autoreactive immune responses in the lungs. Successful completion of the objectives should provide a strong rationale for the use of novel and effective non-immune compromising complement-based therapeutics for treatment of smoke-induced lung diseases.