Integrin-dependent mechanism of T cell-mediated pulmonary fibrosis

描述(由申请人提供)： 肺纤维化是慢性炎症过程和环境暴露的常见后果。现有的抗肝纤维化治疗效果不佳。数据表明，T细胞普遍存在于患者的肺中，与纤维化相关，并表达整合素，这些整合素可能是定义T细胞促纤维化或抗纤维化表型的主开关。这项研究的总体目标是阐明整合素表达的T细胞在肺内促纤维化和抗纤维化调节中的机制作用。本研究的特殊假设是，肺T细胞上整合素1V23、1V25、22和1E的表达和功能活性决定了它们的调节表型(与Th3和Tregs类似)、在肺中的持久性和对胶原产生的刺激。我们进一步假设，肺内不表达整合素的T细胞的渗透导致活化的转化生长因子-2减少，肿瘤坏死因子-1和干扰素-3的表达增加，从而减少胶原的产生。将实现以下具体目标。1.在间质性肺病患者中，确定肺T细胞上整合素的高表达是否与T细胞的转化生长因子-2相关的调节表型和肺功能降低有关，而T淋巴细胞的非整合素表达与炎症表型和肺功能的升高相关。2.检测CCL18体内过表达模型中肺T淋巴细胞上整合素的表达水平和功能活性是否与其在肺内持续存在时间延长有关，是否与肺组织中转化生长因子-2的产生、活化和细胞表面结合增加有关，以及是否与胶原蛋白堆积有关。确定CCL18过表达对博莱霉素诱导的胶原沉积的部分中和作用是否依赖于肺T淋巴细胞上1V整合素表达的降低、活化的转化生长因子-2的表达降低以及肿瘤坏死因子-1和干扰素-3的升高。确定这些整合素介导的促纤维化和抗纤维化机制是否可以在治疗上进行调节，以调节实验动物肺中胶原的积累。3.确定含1V、22或1E整合素的整合素在体外是否促进肺T细胞和原代成纤维细胞之间的细胞-细胞相互作用、增加转化生长因子-2的产生和/或激活、T细胞与细胞表面的转化生长因子-2的结合以及成纤维细胞在细胞培养中刺激胶原的产生。确定用siRNA中和T细胞上整合素的表达或用中和抗体中和整合素的功能是否消除了它们的调节表型和在共培养中的促纤维化作用。确定健康志愿者来源的肺T细胞中1V整合素的过度表达是否会导致调节性纤维化表型的改变。研究CCL18通过刺激转化生长因子-2的表达、活化和细胞表面结合，以及含1V和22整合素的表达，直接调节正常人肺T细胞向纤维化转化的能力。 公共卫生相关性： 拟议的研究与退伍军人健康的相关性。过度的疤痕或纤维化在肺、肝和肾脏的各种疾病、伤口肉芽组织、动脉硬化和所有器官的慢性炎症中起着关键作用。关于一个器官或疾病中纤维化机制的新知识可能广泛适用于发生不必要的过度瘢痕的各种疾病。不同疾病的瘢痕形成机制可能具有某些关键的病理成分。例如，硬皮病、类风湿性关节炎、过敏性肺炎、结节病和特发性肺纤维化等疾病会导致肺部结疤。与肺纤维化相关的死亡率在老年和男性人群以及那些遭受军事暴露的人群中甚至更高。这些群体在退伍军人中有大量的代表。这一建议广泛适用于肺部医学、炎症和免疫异常、自身免疫和免疫介导的纤维化。因此，这项研究适用于VA患者护理任务，指定的肺部疾病、自身免疫性疾病和伤口愈合的优先领域，以及指定的慢性病研究领域。

DESCRIPTION (provided by applicant): Pulmonary fibrosis is a frequent consequence of chronic inflammatory processes and environmental expo- sures. Available antifibrotic therapies are inefficient. Data suggest that T cells are commonly present in the lungs of patients in association with fibrosis and express integrins, and that these integrins may be a master switch that defines a profibrotic or antifibrotic phenotype of T cells. The Overall Goal of this study is to clarify the mechanistic role of integrin-expressing T cells in the pro- and antifibrotic regulation in the lungs. The Spe- cific Hypothesis of this study is that the expression and functional activity of integrins 1V23, 1V25, 22- containing and 1E-containing integrins, on pulmonary T cells defines their regulatory phenotype (TGF-2+ simi- lar to Th3 and Tregs acting through membrane-bound TGF-2), persistence in the lung, and stimulation of col- lagen production. We further hypothesize that pulmonary infiltration of T cells that do not express integrins leads to a decrease in active TGF-2, elevated expression of TNF-1 and IFN-3, and subsequently to attenuation of collagen production. The following Specific Aims will be addressed. 1. Determine whether elevated expres- sion of integrins on pulmonary T cells is associated with a TGF-2-associated regulatory phenotype of T cells and with lower pulmonary function, whereas infiltration of T lymphocytes not expressing integrins is associated with inflammatory phenotype and higher pulmonary function in patients with interstitial lung disease. 2. Deter- mine whether elevated expression levels and functional activity of integrins on pulmonary T lymphocytes in the CCL18 overexpression model in vivo define a) their prolonged persistence in the lungs, b) the increased pro- duction, activation, and cell surface binding of TGF-2, and c) collagen accumulation. Determine whether partial neutralization of bleomycin-induced collagen accumulation by CCL18 overexpression is dependent on de- creased expression of 1V-containing integrins on pulmonary T lymphocytes, their decreased expression of ac- tive TGF-2, and elevation of TNF-1 and IFN-3. Determine whether these integrin-mediated pro- and antifibrotic mechanisms can be therapeutically modulated to regulate collagen accumulation in the lungs of experimental animals. 3. Determine whether 1V-, 22-, or 1E-containing integrins facilitate cell-cell interactions between pul- monary T cell and primary fibroblasts in vitro, the increase in TGF-2 production and/or activation, cell surface binding of TGF-2 by T cells, and stimulation of collagen production by fibroblasts in cell culture. Determine whether neutralization of the expression of integrins on T cells with siRNA or of the integrin function with neu- tralizing antibodies abrogate their regulatory phenotype and the profibrotic effect in co-cultures. Determine whether overexpression of 1V-containing integrins in pulmonary T cells derived from healthy volunteers causes changes toward regulatory profibrotic phenotype. Investigate the ability of CCL18 to directly modulate the phe- notype of normal human pulmonary T cells toward profibrotic, by stimulating the expression, activation, and cell surface binding of TGF-2, and expression of 1V-containing and 22-containing integrins. PUBLIC HEALTH RELEVANCE: Relevance of the Proposed Research to Veterans Health. Excessive scarring, or fibrosis, plays a pivotal role in various diseases of the lungs, liver, and kidneys, in wound granulation, arteriosclerosis and in chronic inflammation in all organs. New knowledge about mechanisms of fibrosis in one organ or disease may be broadly applicable to various diseases in which unwanted excessive scarring occurs. Scarring mechanisms in different diseases may share certain key pathologic components. For example, scarring of the lungs occurs in such diseases as scleroderma, rheumatoid arthritis, hypersensitivity pneumonitis, sarcoidosis, and idiopathic pulmonary fibrosis. Rates of lung fibrosis-associated deaths are even higher in the elderly and male populations, and those subjected to military exposures. These groups are abundantly represented in the veteran population. This proposal is broadly applicable to pulmonary medicine, inflammatory and immune abnormalities, autoimmunity, and immune-mediated fibrosis. Therefore, this research is applicable to the VA patient care mission, the designated priority areas of pulmonary disorders, autoimmune disorders, and wound healing and to the designated research area of chronic diseases.