Immunopathology of the Pneumocystis Life Cycle Stages

描述（由申请人提供）： 肺孢子虫属的真菌生物首先在临床上被注意到，因为它们在免疫状态受损的人类和其他哺乳动物中引起常常致命的肺炎（PCP）。今天，这些真菌的生态位已经扩大到包括患有慢性阻塞性肺病等潜在慢性疾病的患者，其中它们的存在被认为是一种共病因素。在了解肺孢子虫的生命周期，传播和自然史方面取得了有限的进展，这在很大程度上是由于缺乏连续的体外培养系统。这些生物体在完整或免疫衰弱宿主中生长和生存所使用的策略在很大程度上是未知的。感染的真正媒介，即传播形式，还没有被确定，也没有确定其传播方式，通常认为是通过空气传播的孢子。肺孢子虫的生命周期包括营养型（trophs），被认为是营养的无性分裂生命周期阶段;以及其他几个发育阶段，包括含有8个子囊孢子的包囊形式。孢囊与营养体的不同之处在于，它含有主要由B-1，3葡聚糖组成的厚细胞壁;较大（5- 8 μ m vs 1- 4 μ m）;并且用真菌壁染色剂乌洛托品银染色。孢囊的形成被认为是营养体交配的结果，但无性和有性阶段之间的相互作用尚不清楚。 在上一次Merit审查期间，我们发现用棘白菌素治疗肺孢子虫感染抑制了B-1，3-葡聚糖的形成，从而使免疫抑制小鼠和大鼠中的混合包囊和营养体种群急剧转变为几乎完全由营养体组成的种群。引人注目的是，这些小鼠的死亡率显著降低，但它们的肺中存在大量的营养体。此外，棘白菌素治疗的小鼠不能传播感染。在本提案中，我们将使用棘白菌素作为分子工具，以研究人员以前从未使用过的方式剖析生命周期。 我们的中心假设是，无性和性周期协同作用，以维持哺乳动物肺部的平衡，从而允许长期和可持续的感染，确保持续传播而不会导致宿主死亡。我们认为包囊是传播媒介，包囊形成过程需要B-1，3-葡聚糖。然而，通过B-1，3葡聚糖形成的囊肿会在宿主中诱导有害的炎症反应，从而增加疾病的严重程度。在目前的建议中，我们将确定每个不同的人口引起的免疫反应，并使用棘白菌素解剖肺孢子虫的生命周期。为此，我们提出了以下具体目标：（1）以棘白菌素和阿尼芬净为分子工具，通过转录分析和体内研究来分析肺孢子虫的生活史，以评估肺孢子虫感染的传播和识别感染因子;（2）通过一系列结合和吞噬试验来表征鼠肺孢子虫包囊和营养型与肺泡巨噬细胞的相互作用;和（3）通过评估囊充满和囊耗尽的鼠肺吸虫种群的肺中的细胞因子和细胞谱，研究鼠肺吸虫囊在免疫重建后有害的肺过度炎症反应中的作用。

DESCRIPTION (provided by applicant): Fungal organisms in the genus Pneumocystis, were first clinically noticed because they caused an oftentimes lethal pneumonia (PCP) in humans and other mammals with compromised immune status. Today, the niche of these fungi have expanded to include patients with underlying chronic diseases like COPD, where their presence has been suggested to be a co-morbidity factor. Limited progress has been made in understanding the life cycle, transmission, and natural history of Pneumocystis, due in large part to the absence of a continuous in vitro culture system. The strategies used by these organisms to grow and survive in the context of an intact or immune-debilitated host are largely unknown. The actual agent of infection, the transmissive form, has not been identified, nor has its mode of travel, generally thought to be via an airborne spore. The life cycle of Pneumocystis involves trophic forms (trophs), thought to be the vegetative, asexually dividing life cycle stage; as well as several other developmental stages including the cyst form, which contains 8 ascospores. The cyst is distinct from the trophs in that it contains a thick cell wall comprised mostly of B-1,3 glucan; is larger (5-8um vs 1-4um); and stains with the fungal wall stain, methenamine silver. Cyst formation is thought to result from mating of trophs, but the interplay between the asexual and sexual phases is not clear. During the tenure of the previous Merit Review, we found that the treatment of Pneumocystis infections with echinocandins suppressed the formation of B-1,3-glucan thereby dramatically shifting the mixed cyst and troph populations in immunosuppressed mice and rats to one made almost exclusively of trophic forms. Strikingly, the mortality was markedly reduced in these mice, yet large populations of trophs were present in their lungs. Moreover, echinocandin treated mice were unable to transmit the infection. In the present proposal, we will use the echinocandins as molecular tools to dissect the life cycle in a manner that has never been available to investigators before. It is our central hypothesis that the asexual and sexual cycles act in concert to maintain a balance within the mammalian lung that permits a long and sustainable infection that ensures sustained transmission without demise of the host. We posit that cysts are the agents of transmission and the process of cyst formation requires B-1,3-glucan. However, the presence of cysts via B-1,3 glucan induces a detrimental inflammatory response in the host that increases severity of disease. In the present proposal, we will identify the immune responses evoked by each different population and dissect the life cycle of Pneumocystis using the echinocandins. Towards these goals, we propose the following specific aims: (1) Dissect the Pneumocystis life cycle using the echinocandin, anidulafungin, as a molecular tool by transcriptional analyses and in vivo studies to assess transmission and identify the infectious agent of Pneumocystis infection; (2) Characterize the P. murina cyst and trophic form interactions with alveolar macrophages through a series of binding and phagocytosis assays; and (3) Investigate the role of P. murina cysts in the deleterious pulmonary hyperinflammatory response following immune reconstitution by evaluating cytokine and cellular profiles in the lungs of cyst replete and cyst depleted populations of P. murina.