肺炎衣原体感染小鼠模型的CD4+ T细胞凋亡的研究

Chlamydia (C.), an omnipresent obligate intracellular bacterial pathogen, infects a wide range of host species. Infections with C. pneumoniae，causing respiratory infections such as bronchitis and pneumonia, have also been consistently associated with chronic inflammatory diseases such as atherosclerosis, Alzheimer's disease and asthma. These associations promote the analysis of mechanisms that regulate disease in response to C. pneumoniae infections. Our previous research showed that reduced early T cell responses associate with enhanced disease in C57BL/6 mice as compared to A/J mice. A possible reason for the delayed T cell responses is increased activation-induced cell death (AICD) of CD4+ T cells as suggested by modeling of T helper cell regulation. We stipulate that suppression of T cell apoptosis increases the Th cell population size, and shifts the response towards Th2 because of greater resistance of Th2 cells than Th1 cells against AICD-mediated apoptosis..In this study, we aim to test the concept of apoptosis-mediated regulation of T helper cells, and its consequences on C. pneumoniae-induced lung disease. We will first evaluate if the naturally protected A/J mice have a lower apoptotic rate of CD4+ T cells in infected lungs than in susceptible C57BL/6 mice. Subsequently, we attempt to reduce the high apoptotic rate of CD4+ T cells in C57BL/6 mice by the use of two types of apoptosis inhibitors (Q-VD-OPh as a wide-spectrum caspase inhibitor and H-Val-Pro-Met-Leu-Lys-OH as a Bax-Inhibiting-Peptide). In addition, we will use 34 one-step duplex reverse-transcription PCRs on genes for apoptosis (caspase-3, caspase-8, caspase-9, Bcl-2, Bax, Fas, FasL, TNFR1, TNFR2), genes for immune cells (neutrophils, macrophages，natural killer cells，pan-T cells，Th1 T cells，Th2 T cells，CD45RB，CD45RO，CD8+, B cells）, and genes for inflammatory regulators (Arginase-1, Arginase-2, NOS2，Cybb, Ptgs2，IL-6，IL-4，TNF-α，IFN-γ，IL-10，Cxcl2, CRP, Ccl2, Serpine1). A multifactorial statistical analysis will be performed on the associations of CD4+ T cell apoptotic rates and other factors [genetics, infection or not, mRNA levels for apoptosis-associated genes/immune cells/inflammatory regulators, data from flow cytometry and TUNEL assay, and usage of different apoptosis inhibitors] to further identify the mechanisms of CD4+ T cells apoptosis in this murine model of C. pneumoniae pulmonary infections.

肺炎衣原体感染主要引起人的非典型性肺炎，而且和冠心病等慢性炎症性疾病相关。CD4+ T细胞的调控是肺炎衣原体致病机理的关键，但具体机制不详。我们在预试验中发现，肺炎衣原体感染的C57BL/6小鼠比A/J有着显著低下的T细胞反应和更严重的肺脏疾病,以及更高的CD4+T细胞凋亡率。我们推测"抑制AICD介导的CD4+ T细胞凋亡会增加Th细胞数量，同时促进Th1反应向Th2转换"。我们首先观察细胞凋亡抑制剂能否降低C57BL/6小鼠的高CD4+T细胞凋亡率。然后用统计学方法综合分析如下因子和CD4+ T细胞凋亡的相关性：小鼠基因组成,衣原体感染与对照，肺脏疾病，肺脏衣原体清除率，33个目标基因（9个细胞凋亡基因，10个T/Th1/Th2细胞和14个炎症调控因子）的mRNA水平，流式细胞和TUNEL试验结果和不同细胞凋亡抑制剂的使用。此研究将为衣原体感染的Th1/Th2极化和疫苗研制提供新思路。

衣原体是一类严格细胞内寄生,具有独特发育周期的革兰氏阴性原核细胞型微生物，含11个种。肺炎衣原体感染主要引起人的非典型性肺炎，而且和冠心病等慢性炎症性疾病相关。在此项国家自然基金的资助下，我们开展了CD4+ T 细胞凋亡和肺炎衣原体致病机理相关性的研究。研究发现，肺炎衣原体感染的 C57BL/6 小鼠比 A/J 有着显著低下的 T 细胞反应和更严重的肺脏疾病,以及更高的 CD4+T 细胞凋亡率；此外，我们建立了基于A/J、C57BL/6、C3H、C57BL/6的IL-17KO小鼠的肺炎衣原体感染模型。检测9 个细胞凋亡基因，10 个 T/Th1/Th2 细胞和 14 个炎症调控因子的 mRNA 水平的CD4+ T 细胞凋亡的相关性。初步研究表明，抑制 AICD 介导的 CD4+ T 细胞凋亡是调控肺炎衣原体致病机理的关键。此外，在此项国家自然基金的资助下，我们建立了检测所有衣原体种的FRET-PCR技术，开展了我国家禽和牛的衣原体分子流行病学研究。和此项课题相关的科研成果包括：13篇SCI论文，3件国家发明专利和2项科研奖励。此研究将为衣原体感染的 Th1/Th2 极化和疫苗研制提供新思路。