STAT4 mediated immunity to Pneumocystis

描述（由申请人提供）：由于与HIV流行密切相关，肺孢子虫已上升到致死性机会性肺部感染的最前沿，但这种非典型真菌病原体越来越多地与药理学驱动的免疫抑制以及慢性肺病（如COPD）患者相关。 CD 4 + T细胞的丢失是肺孢子虫肺炎的一个已知诱发因素，但CD 4 + T细胞如何特异性控制肺孢子虫感染尚不清楚。缺乏体外培养肺孢子虫的能力，加上高种属特异性，限制了对人CD 4 T细胞介导的肺孢子虫应答的研究。辅助性T细胞免疫肺孢子虫是复杂的，在小鼠缺乏辅助性T细胞1型（Th 1）的签名细胞因子IFN-？或Th 2特征性细胞因子IL-4并不比野生型动物更易感染。虽然一些数据表明Th 2应答具有保护性，但在许多实验模型中，证据大多支持Th 1应答在介导对鼠疟原虫的保护性免疫中的作用。延迟（在2-3周较高的负担相比，野生型），但完整的（在4-5周的负担相比，野生型没有差异）肺孢子虫清除已观察到在CD 4-能力IFN-？R缺陷型小鼠、IL-12 p35缺陷型小鼠和IL-23 p19缺陷型小鼠。这些细胞因子之间的一个共性是IFN-？，IL-12 p35和IL-23 p19各自激活pro-Th 1转录因子信号转导和转录激活因子4（STAT 4）。因此，为了更正式地阐述T辅助细胞发育在鼠疟原虫宿主防御中的作用，我们将重点放在驱动Th 1和Th 2应答的特异性转录因子上。在初步研究中，我们表明，CD 4-有能力的小鼠缺乏STAT 4，但不是pro-Th 2转录因子STAT 6，是无法清除P.鼠肺后4周的挑战。在攻毒后2周，当所有组之间的鼠疟原虫负荷相似时，STAT 4-/-小鼠表现出肺IL-1？水平降低，IL- 1？，CXCL 1、CCL 2和CCL 5以及肺巨噬细胞中总CD 3+、CD 3 +/CD 4+和MHC II+细胞的减少。在抗CD 3刺激后，STAT 4-/-CD 4 + T细胞显示IL-2和IFN-？产生受损。作为鼠肺吸虫在IFN-？R、IL-12 p35和IL-23 p19缺陷型小鼠在感染早期受损，而不是在STAT 4-/-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-γ协同作用，IL-12 p35或IL-23 p19以最大化STAT 4活化和随后的抗肺孢子虫免疫应答。为此，我们表明，STAT 4激活剂IL-18，以及Th 1促进细胞因子IL-27，诱导在肺中的鼠疟原虫感染。令人惊讶的是，我们发现Th 1相关转录因子T-bet或STAT 1缺陷的小鼠在攻击后4周未显示出对鼠肺吸虫的清除受损，表明在鼠肺吸虫的肺清除中对STAT 4的严格要求。因此，这项R21探索性/发展性研究资助的重点是确定STAT 4诱导的机制和STAT 4介导的适应性免疫防御途径的机制，这些途径介导肺清除鼠疟原虫。我们的中心假设是，通过STAT 4信号介导的保护性肺免疫肺孢子虫。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）确定IL-18和IL-27在STAT 4信号传导和宿主防御鼠肺孢子虫肺部感染中的作用和（2）阐明肺孢子虫肺部感染期间的STAT 4依赖性CD 4 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.