CD4 T CELLS: LESSONS LEARNED FROM ASTHMA

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 进行性阻塞性肺疾病是CF发病率和死亡率的主要原因。 肺部感染在CF中是常见的，并且大多数患者将成为铜绿假单胞菌的慢性定殖，尽管免疫系统完整且免疫应答强烈。 铜绿假单胞菌早期定植和转化为粘液型与肺功能更快丧失和死亡率增加相关。 一旦CF患者发展为铜绿假单胞菌的慢性定植，即使使用强化抗生素，他们通常也无法清除感染。 铜绿假单胞菌已经开发出通过形成生物膜和释放多种毒力因子来逃避先天性和适应性免疫应答的方法。 然而，对铜绿假单胞菌产生的CD 4 T细胞适应性免疫应答的性质可能会显著改变病原体清除和组织损伤诱导之间的平衡。 虽然Th 1（IFNg）应答对于通过巨噬细胞和嗜中性粒细胞活化的细菌清除是重要的，但Th 2（IL-4）应答主要导致抗体产生，特别是IgE，并且在细菌清除中无效。 在慢性铜绿假单胞菌感染的小鼠模型中，与具有Th 2免疫应答的小鼠相比，具有Th 1免疫应答的小鼠具有更好的细菌清除、更温和的肺部炎症和改善的存活率，并且IFNg（Th 1）被证明是慢性铜绿假单胞菌肺部感染的大鼠模型中气道炎症的有效治疗。 与未感染CF患者的PBMC相比，来自长期定植有铜绿假单胞菌的CF患者的总外周血单核细胞（PBMC）响应于假单胞菌抗原产生更高水平的IL-4（Th 2）和更低水平的IFNg（Th 1）。 慢性感染铜绿假单胞菌的CF儿童的全血培养物比未感染CF患者的培养物产生更多的IL-4。 此外，最近的一项研究表明，与未感染的CF患者和健康对照相比，长期定植有铜绿假单胞菌的CF患者在支气管肺泡灌洗（BAL）中具有较高水平的Th 2细胞和IL-4以及较低水平的IFNg。 此外，对铜绿假单胞菌的Th 2偏倚也与CF患者的肺功能降低相关，这增加了将免疫应答转向Th 1表型并远离Th 2表型可能导致慢性铜绿假单胞菌感染患者结局改善的可能性。 CF患者还具有哮喘和ABPA的高患病率，这两种疾病都是引起破坏性气道炎症并由Th 2淋巴细胞活化介导的疾病。 CF患者中Th 2免疫应答的优势进一步得到两项研究的支持，这两项研究表明，与健康对照相比，CF患者的血清和痰液阳离子蛋白水平升高，这是Th 2细胞诱导的嗜酸性粒细胞活化的指标。这些数据表明，Th 2免疫应答不仅与铜绿假单胞菌的无效气道清除相关，还可能导致破坏性气道炎症和更快的肺功能下降。 CF中Th 2免疫应答的调节可能提供新的治疗靶点，特别是在铜绿假单胞菌慢性定殖的患者中。 上皮细胞中CFTR的突变不能轻易解释CF肺病的过度炎症特征。 CD 4 T细胞在适应性免疫应答的产生中是重要的，可以调节慢性炎症，并且大量存在于CF气道周围的上皮下空间中。 CD 4 T细胞表达CFTR，因此可能受到该基因突变的影响。 CD 4 T细胞可以分化成几种效应子亚型，包括产生IFNg的Th 1细胞和产生IL-4的Th 2细胞。 CFTR突变如何影响CD 4 T细胞功能尚不清楚。 我们的实验室最近表明，来自CF小鼠的活化幼稚CD 4 T细胞产生比野生型（WT）小鼠更大量的IL-4（Th 2），而IFNg（Th 1）的产生是相当的，这表明CFTR正在调节CD 4 T细胞分化。 我们还表明，与WT相比，CF小鼠的活化CD 4 T细胞中细胞内钙水平（T细胞受体活化产生的重要信号）增加，并且IL-4转录因子NFAT的核结合增加。 钙是NFAT细胞质向核转位所需的，通过激活钙调磷酸酶，进而使NFAT去磷酸化。 我们已经证实了这些发现的体内意义，表明CF小鼠与WT小鼠相比，在暴露于A.并增加了A.烟曲霉和铜绿假单胞菌暴露。 这些结果暗示CF中存在原发性CD 4 T细胞免疫缺陷，并强烈表明CFTR调节CD 4 T细胞分化（Th 1与Th 2）。 此外，鉴于Th 2免疫应答与慢性铜绿假单胞菌定殖和肺功能差相关的数据越来越多，这些发现具有显著的临床意义。 越来越多的证据表明CF中存在Th 2偏向的免疫应答，并且这种偏向与铜绿假单胞菌感染的无效清除相关。 虽然CFTR在上皮细胞中的功能一直受到关注，但我们提出了CFTR在CD 4 T细胞中作用的新机制。 在CF小鼠模型中，我们已经表明CFTR可以调节CD 4 T细胞分化，使得突变有利于Th 2偏好。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Progressive obstructive lung disease is the leading cause of morbidity and mortality in CF. Pulmonary infections are common in CF and the majority of patients will become chronically colonized with Pseudomonas aeruginosa despite an intact immune system and a vigorous immune response. Early colonization and conversion of the P. aeruginosa to its mucoid form are associated with more rapid loss of lung function and increased mortality. Once CF patients develop chronic colonization with P. aeruginosa they are often unable to clear the infection even with use of intensive antibiotics. P. aeruginosa has developed ways to escape both the innate and adaptive immune response by forming biofilms and releasing a variety of virulence factors. However, the nature of the CD4 T cell adaptive immune response generated to P. aeruginosa may dramatically alter the balance between clearance of the pathogen and induction of tissue damage. While Th1 (IFNg) responses are important for bacterial clearance through macrophage and neutrophil activation, Th2 (IL-4) responses primarily result in antibody production, particularly IgE, and are not effective in the clearance of bacteria. In mouse models of chronic P. aeruginosa infection, mice with a Th1 immune response had better bacterial clearance, milder lung inflammation and improved survival compared to mice with Th2 immune responses and IFNg (Th1) was shown to be an effective treatment of airway inflammation in a rat model of chronic P. aeruginosa lung infection. Total peripheral blood mononuclear cells (PBMCs) from CF patients chronically colonized with P. aeruginosa produced higher levels of IL-4 (Th2) and lower levels of IFNg (Th1) in response to Pseudomonal antigens than PBMCs from uninfected CF patients. Whole blood cultures from CF children chronically infected with P. aeruginosa had greater IL-4 production than did cultures from noninfected CF patients. Additionally, a recent study showed that CF patients chronically colonized with P. aeruginosa had higher levels of Th2 cells and IL-4 and lower levels of IFNg in bronchial alveolar lavage (BAL) when compared to noninfected CF patients and healthy controls. Additionally, Th2 bias to P. aeruginosa has also been associated with lower lung function in CF patients raising the possibility that shifting immune responses toward a Th1 phenotype, and away from a Th2 phenotype could potentially lead to improved outcomes in chronically P. aeruginosa-infected patients. CF patients also have a high prevalence of asthma and ABPA, both of which are diseases that cause damaging airway inflammation and are mediated by activation of Th2 lymphocytes. The predominance of Th2 immune responses in CF patients is further supported by two studies showing that CF patients, compared to healthy controls, had increased levels of serum and sputum cationic protein, an indicator of Th2 cell induced eosinophilic activation. These data suggest that not only is a Th2 immune response associated with ineffective airway clearance of P. aeruginosa, it may also contribute to damaging airway inflammation and a more rapid lung function decline. Modulation of Th2 immune responses in CF may provide new therapeutic targets especially in patients chronically colonized with P. aeruginosa. Mutations of CFTR in epithelial cells do not readily explain the exaggerated inflammation characteristic of CF lung disease. CD4 T cells are important in the generation of an adaptive immune response, can regulate chronic inflammation and are present in large numbers in the subepithelial space surrounding the CF airway. CD4 T cells express CFTR and therefore may be affected by mutations in this gene. CD4 T cells can differentiate into several effector subtypes including Th1 cells that make IFNg and Th2 cells that make IL-4. How CFTR mutations affect CD4 T cell function is poorly understood. Our lab has recently shown that activated naive CD4 T cells from CF mice produce greater amounts of IL-4 (Th2) than wild type (WT) mice while IFNg (Th1) production is equivalent, suggesting that CFTR is regulating CD4 T cell differentiation. We have also shown that intracellular calcium levels, an important signal generated by T cell receptor activation, are increased in activated CD4 T cells from CF mice when compared to WT and that there is increased nuclear binding of the IL-4 transcription factor, NFAT. Calcium is required for NFAT cytoplasmic to nuclear translocation by activation of calcineurin which in turn dephosphorylates NFAT. We have confirmed the in vivo significance of these findings by showing that CF mice, when compared to WT mice, generate an exaggerated Th2 immune response following exposure to A. fumigatus and have increased CD4 T cell IL-4 production (Th2) following A. fumigatus and P. aeruginosa exposure. These results implicate a primary CD4 T cell immune defect in CF and strongly suggest that CFTR regulates CD4 T cell differentiation (Th1 versus Th2). Further, these findings have significant clinical implications given the growing data that Th2 immune responses are associated with chronic P. aeruginosa colonization and poor lung function. There is growing evidence that there is a Th2 biased immune response in CF and that this bias is associated with ineffective clearance of P. aeruginosa infection. While much focus has been on CFTR function in epithelial cells, we propose a novel mechanism for the role of CFTR in CD4 T cells. In a CF mouse model we have shown that CFTR may regulate CD4 T cell differentiation such that mutations favor a Th2 bias.