STAT4 mediated immunity to Pneumocystis

描述（由申请人提供）：由于肺囊虫与HIV流行密切相关，它已经上升到致死性机会性肺部感染的前沿，然而这种非典型真菌病原体正越来越多地与药理学驱动的免疫抑制以及慢性肺部疾病（如COPD）患者相关。CD4+ T细胞的丢失是已知的肺囊虫肺炎的易感因素，但CD4+ T细胞如何特异性控制肺囊虫感染尚不清楚。缺乏体外培养肺囊虫的能力，加上高物种特异性，限制了对人CD4 T细胞介导的肺囊虫反应的研究。肺囊虫的辅助性T免疫是复杂的，因为小鼠缺乏辅助性T型1 （Th1）特征细胞因子IFN-？或Th2标志细胞因子IL-4并不比野生型动物更容易感染。尽管一些数据表明Th2反应具有保护作用，但在许多实验模型中，证据大多支持Th1反应在介导对鼠弓形虫的保护性免疫中的作用。延迟（2-3周负担比野生型高），但完整（4-5周负担与野生型相比无差异）肺囊虫清除已在cd4激活的IFN-？R缺陷小鼠，IL-12p35缺陷小鼠和IL-23p19缺陷小鼠。这些细胞因子的一个共同点是IFN-？IL-12p35和IL-23p19分别激活前th1转录因子信号换能器和转录激活器4 （STAT4）。因此，为了更正式地解决辅助性T发育在鼠p.m urina宿主防御中的作用，我们将重点放在驱动Th1和Th2反应的特定转录因子上。在初步研究中，我们发现缺乏STAT4（而不是前th2转录因子STAT6）的cd4胜任小鼠在攻击后4周无法从肺部清除鼠假体。在攻击后2周，当所有组间鼠p.m urina负荷相似时，STAT4-/-小鼠表现出肺IL-1？- 1？以及肺消化系统中CD3+、CD3+/CD4+和MHC II+细胞总数的减少。在抗cd3刺激下，STAT4-/- CD4+ T细胞显示IL-2和IFN-?的产生受损。小鼠肺中IFN-？R， IL-12p35和IL-23p19缺陷小鼠在感染早期受损，而不是在STAT4-/-小鼠中观察到的晚期（4周），这些结果表明额外的介质可能与IFN-？， IL-12p35或IL-23p19最大化STAT4激活和随后的抗肺囊虫免疫反应。为此，我们发现在P. murina感染期间，STAT4激活因子IL-18和Th1促进细胞因子IL-27在肺部被诱导。令人惊讶的是，我们发现缺乏th1相关转录因子T-bet或STAT1的小鼠在攻击后4周并没有表现出对鼠肺泡的清除受损，这表明肺对鼠肺泡的清除严格要求STAT4。因此，本次R21探索性/发展性研究基金的重点是明确STAT4诱导的机制，以及STAT4介导的适应性免疫防御通路介导鼠假体肺清除的机制。我们的中心假设是通过STAT4信号传导介导肺对肺囊虫的保护性免疫。为了解决这一假设，我们提出了以下两个独立的，相互关联的目的：(1)建立IL-18和IL-27在STAT4信号传导和宿主防御鼠肺假体感染中的作用；(2)阐明肺囊虫感染期间STAT4依赖性CD4 T细胞表型。

DESCRIPTION (provided by applicant): Pneumocystis has risen to the forefront of lethal, opportunistic lung infections as a result of its close association with the HIV epidemic, yet this atypical fungal pathogen is becoming increasingly associated with pharmacologic-driven immunosuppression as well as patients with chronic lung diseases such as COPD. Loss of CD4+ T cells is a known predisposing factor to Pneumocystis pneumonia, yet how CD4+ T cells specifically control Pneumocystis infection is not known. The lack of the ability to culture Pneumocystis in vitro coupled with high species specificity has limited investigations into human CD4 T cell mediated responses to Pneumocystis. T helper immunity to Pneumocystis is complicated, in that mice deficient in the T helper type 1 (Th1) signature cytokine IFN-? or the Th2 signature cytokine IL-4 are not more susceptible to infection than wild-type animals. Although some data points to Th2 responses being protective, in many experimental models, evidence mostly supports a role for Th1 responses in mediating protective immunity to P. murina. Delayed (higher burden at 2-3 weeks compared to wild-type), but intact (no differences in burden at 4-5 weeks compared to wild-type) clearance of Pneumocystis has been observed in CD4-competent IFN-?R deficient mice, IL-12p35 deficient mice and IL-23p19 deficient mice. One commonality between these cytokines is that IFN-?, IL-12p35 and IL-23p19 each activate the pro-Th1 transcription factor Signal Transducer and Activator of Transcription 4 (STAT4). Therefore, to more formally address the role of T helper development in P. murina host defense, we focused on specific transcription factors that drive Th1 and Th2 responses. In preliminary studies, we show that CD4-competent mice deficient in STAT4, but not the pro-Th2 transcription factor STAT6, are unable to clear P. murina from the lungs 4 weeks post-challenge. At 2 weeks post-challenge, when P. murina burden was similar between all groups, STAT4-/- mice demonstrated reduced lung levels of IL-1?, IL- 1?, CXCL1, CCL2 and CCL5 as well as reductions in total CD3+, CD3+/CD4+ and MHC II+ cells in lung digests. Upon anti-CD3 stimulation, STAT4-/- CD4+ T cells displayed impaired production of IL-2 and IFN-?. As P. murina lung clearance in IFN-?R, IL-12p35 and IL-23p19 deficient mice was impaired early in infection, rather than late (4 weeks) as observed in STAT4-/- mice, these results suggest that an additional mediator may synergize with IFN-?, IL-12p35 or IL-23p19 to maximize STAT4 activation and the subsequent anti- Pneumocystis immune response. To this end, we show that the STAT4 activator IL-18, as well as the Th1 promoting cytokine IL-27, are induced in the lungs during P. murina infection. Surprisingly, we found that mice deficient in the Th1-associated transcription factors T-bet or STAT1 did not display impaired clearance of P. murina 4 weeks post-challenge, indicating a strict requirement for STAT4 in lung clearance of P. murina. Therefore, the focus of this R21 Exploratory/Developmental Research Grant is to define mechanisms of STAT4 induction and mechanisms of STAT4-mediated adaptive immune defense pathways that mediate lung clearance of P. murina. Our central hypothesis is that signaling through STAT4 mediates protective lung immunity against Pneumocystis. To address this hypothesis, we have proposed the following two independent, interrelated Aims: (1) Establish roles for IL-18 and IL-27 in STAT4 signaling and host defense against P. murina lung infection and (2) Elucidate the STAT4-dependent CD4 T cell phenotype during Pneumocystis lung infection. PUBLIC HEALTH RELEVANCE: Our data has uncovered a new understanding of the lung immune response to Pneumocystis, a significant cause of pneumonia in immunosuppressed individuals. We have discovered that CD4+ T cells require the transcription factor STAT4 to mediate host defense against Pneumocystis. Studies in this proposal will define mechanisms of STAT4 induction and mechanisms of STAT4-mediated adaptive immune defense pathways that mediate lung clearance of Pneumocystis.