INITIATION OF THE IMMUNE RESPONSE TO ASPERGILLUS FUMIGATUS

 描述（由申请方提供）：烟曲霉是人类每天吸入的致病真菌。呼吸道先天免疫系统阻止吸入的分生孢子（营养孢子）萌发成组织侵入性菌丝的能力代表了关键的免疫检查点。由于医疗技术的进步，生活在免疫受损条件下的患者显著增加，导致每年估计有20万例曲霉病。在高危人群中，尽管采用了当代联合抗真菌治疗，死亡率仍为25-50%。这些趋势突出了需要更好地了解杀菌免疫的分子和细胞基础，以推进基于免疫的预防方法。在该项目的最初资助期间，我们引入了一种新的真菌生物报告基因，称为荧光曲霉菌报告基因（FLARE），以追踪 分生孢子在与肺部免疫细胞的细胞相互作用过程中的作用。使用这种方法，我们确定了脾酪氨酸激酶（Syk）为肺先天防御所必需的，并在初步研究中，表明Syk以细胞外源性方式调节中性粒细胞杀真菌活性。虽然Syk可以整合来自髓样细胞中的C型凝集素受体、Fc受体和整合素的信号，但其在呼吸道真菌挑战期间的基本细胞来源和真菌诱导的激活信号仍然不清楚。除了中性粒细胞，我们的研究表明，-CC-趋化因子受体2（CCR 2）-表达炎症单核细胞（IM）形成第二个必不可少的先天效应细胞在呼吸挑战。在肺中，IM分化成单核细胞衍生的树突状细胞（Mo-DC），与直接分生孢子摄取和杀伤一致，并参与与肺中性粒细胞的细胞间串扰以增强肺中的抗真菌活性。在初步研究中，IM/Mo-DC的转录谱分析和肺蛋白质组学分析鉴定了细胞间串扰和呼吸爆发的五种候选介质作为候选效应机制。在下一个项目期间，我们建议更深入地了解IM/MoDC -中性粒细胞间串扰，Syk活性对其的诱导和调节，其相关的分子递质及其在肺中的抗孢子防御中的作用。在目的1中，我们通过鉴定Syk信号传导的基本细胞来源和确定介导肺中分生孢子摄取和杀伤的基本上游衔接蛋白和受体来鉴定Syk信号传导的保护机制。在目标2中，我们将通过建立细胞和运输要求、诱导信号、细胞间串扰的定向性质来定义IM/Mo-DC和中性粒细胞抗真菌活性的相互调节。在目标3中，我们将定义IM/Mo-DC-中性粒细胞串扰的分子递质和效应器系统。提出的研究是重要的和创新的，因为他们确定了IM和中性粒细胞之间的先天免疫串扰作为一种新的机制，以调节肺中的抗真菌免疫。了解先天免疫串扰的诱导，调节和参与者解决了一个关键的知识缺口，这将为弱势患者群体的免疫增强策略提供信息。

 DESCRIPTION (provided by applicant): Aspergillus fumigatus is pathogenic fungus that humans inhale on a daily basis. The ability of the respiratory innate immune system to prevent the germination of inhaled conidia (vegetative spores) into tissue-invasive hyphae represents a critical immunologic checkpoint. Due to advances in medical technology, there has been a significant rise in patients that live in immune compromised conditions, leading to an estimated 200,000 cases of aspergillosis annually. In high-risk groups, mortality rates remain 25-50% despite contemporary combination antifungal therapies. These trends highlight the need for an improved understanding of the molecular and cellular basis of sterilizing immunity to advance immune-based adjunctive approaches. In the initial funding period of this project we introduced a novel fungal bioreporter, termed fluorescent Aspergillus reporter (FLARE), to trace the viability of conidia during cellular interactions with immune cells in the lung. Using this approach, we identified spleen tyrosine kinase (Syk) as essential for innate defense in the lung, and in pilot studies, show that Syk acts in a cell-extrinsic manner to regulate neutrophil fungicidal activity. Although Syk can integrate signals from C-type lectin receptors, Fc receptors, and integrins in myeloid cells, its essential cellular source and fungus-induced activation signals during respiratory fungal challenge remain poorly defined. Beyond neutrophils, our studies show that -CC- chemokine receptor 2 (CCR2)-expressing inflammatory monocytes (IMs) form a second essential innate effector cell during respiratory challenge. In the lung, IMs differentiate into monocyte-derived dendritic cells (Mo-DCs), coincident with direct conidial uptake and killing, and engage in intercellular crosstalk with lung neutrophils to boost antifungal activity in the lun. In pilot studies, transcriptional profiling of IM/Mo-DCs and lung proteomic analyses identified fiv candidate mediators of intercellular crosstalk and the respiratory burst as a candidate effector mechanism. During the next project period, we propose to gain a deeper understanding of IM/MoDC - neutrophil intercellular crosstalk, its induction and regulation by Syk activity, its relevant molecular transmitters, and its role in anticonidial defense in the lung. In Aim 1, we identify protective mechanisms of Syk signaling by identifying its essential cellular source and by determining essential upstream adaptor proteins and receptors that mediate conidial uptake and killing in the lung. In Aim 2, we will define reciprocal regulation of IM/Mo-DC and neutrophil antifungal activity by establishing the cellular and trafficking requirements, induction signals, ad directional nature of intercellular crosstalk. In Aim 3, we will define molecular transmitters and effector systems of IM/Mo-DC-neutrophil crosstalk. The proposed studies are significant and innovative because they identify innate immune crosstalk between IMs and neutrophils as a novel mechanism to regulate antifungal immunity in the lung. Understanding the induction, regulation, and participants of innate immune crosstalk addresses a critical knowledge gap that will inform immune-enhancing strategies in vulnerable patient groups.