Interactions of pDCs with Aspergilus

描述(申请人提供)：烟曲霉是侵袭性霉菌感染的最常见原因。即使在治疗和早期诊断方面取得了进步，死亡率仍然很高。浆细胞样树突状细胞(PDCs)是一种功能不同的树突状细胞，在病毒刺激下能迅速产生大量的I型干扰素(IFN)，主要是通过依赖于病毒核酸感应的机制。PDCs通过分泌细胞因子和启动T细胞与先天获得性免疫联系在一起。PDCs和真菌之间的相互作用还没有得到很好的描述。该应用建立在令人信服的初步数据基础上，证明：1)人pDC直接抑制真菌生长的机制包括：1)烟曲霉菌诱导pDC死亡；2)烟曲霉菌丝刺激后pDC释放细胞因子，包括I型IFN；3)pDC枯竭使小鼠对肺部和静脉注射烟曲霉菌容易过敏，其机制似乎至少部分归因于免疫反应失调；4)肺部感染烟曲霉菌导致PDC流入肺部。我们假设pDC在宿主抵抗侵袭性曲霉病中发挥重要作用，它通过直接介导抗真菌活性和调节先天和获得性免疫反应。为了解决这一假设，我们将使用体外和体外实验来确定pDC在防御烟曲霉菌中的作用。 活体模型。在目标1中，我们将探索我们观察到的pDC与烟曲霉菌体外孵育导致真菌识别、抗真菌活性和细胞因子释放的机制基础。PDC识别分生孢子和菌丝形态所需的受体将被探索。烟曲霉菌诱导PDC死亡的机制将被阐明。将使用完整的pDC和pDC裂解物来表征pDC如何介导抗真菌活性。最后，将研究pDC对烟曲霉菌的细胞因子和趋化因子的反应。在目标2中，我们将通过阐明发生曲霉病的机制来继续我们的论证，即pDC在宿主对曲霉病的防御中具有非多余的作用。在侵袭性曲霉病小鼠模型中，将确定PDC耗竭对死亡率、真菌负担、免疫细胞招募、病理和细胞因子反应的影响。PDC介导的保护所需的免疫系统的手臂将通过共耗尽PDCs和特定的白细胞亚群以及基因敲除小鼠进行检查。最后，我们将利用流式细胞仪和显微镜来确定pDCs在体内是否与分生孢子和菌丝结合。在资助期内完成这些研究将在真菌发病机制、真菌免疫学和PDC生物学方面取得重大概念性进展。此外，所获得的知识可能会导致预防和治疗侵袭性真菌病的新策略。

DESCRIPTION (provided by applicant): Aspergillus fumigatus is the most common cause of invasive mold infections. Even with advances in therapy and early diagnostics, mortality rates remain high. Plasmacytoid dendritic cells (pDCs) comprise a functionally distinct lineage of DCs that rapidly produce copious amounts of type I interferons (IFNs) upon stimulation with viruses, predominantly via mechanisms dependent on sensing of viral nucleic acids. pDCs link innate to adaptive immunity by secreting cytokines and by priming T cells. The interplay between pDCs and fungi has not been well characterized. The application builds upon compelling preliminary data demonstrating that: 1) human pDCs directly inhibit fungal growth via a mechanism that involves A. fumigatus-induced pDC death; 2) following stimulation with A. fumigatus hyphae, pDCs release cytokines, including type I IFNs; 3) depletion of pDCs renders mice hypersusceptible to pulmonary and intravenous challenge with A. fumigatus by a mechanism that appears to be due, at least in part, to a dysregulated immune response; and 4) pulmonary infection with A. fumigatus results in pDC influx into the lungs. We hypothesize that pDCs play a major role in host defenses against invasive aspergillosis by mediating direct antifungal activity and by modulating the innate and adaptive immune response. To address this hypothesis, we will define the contribution of pDCs in the defense against A. fumigatus using both in vitro and in vivo models. In Aim 1, we will explore the mechanistic basis of our observations that incubation of pDCs with A. fumigatus in vitro results in fungal recognition, antifungal activity and cytokine release. The receptors required for pDC recognition of the conidial and hyphal fungal morphotypes will be explored. The mechanism of pDC death induced by A. fumigatus will be elucidated. How pDCs mediate antifungal activity will be characterized using both intact pDCs and pDC lysates. Finally, the cytokine and chemokine response of pDCs to A. fumigatus will be investigated. In aim 2, we will follow up our demonstration that pDCs have a non- redundant role in host defenses against aspergillosis by illuminating the mechanisms by which this occurs. The effect of pDC depletion on mortality, fungal burden, immune cell recruitment, pathology and cytokine response will be determined in murine models of invasive aspergillosis. The arms of the immune system required for pDC-mediated protection will be examined by co-depleting pDCs and specific leukocyte subsets and with knockout mice. Lastly, we will determine whether pDCs associate with conidia and hyphae in vivo using flow cytometry and microscopy. Completion of these studies over the funding period will result in major conceptual advances in fungal pathogenesis, fungal immunology and pDC biology. Moreover, the knowledge gained may lead to novel strategies to prevent and treat invasive mycoses.