INITIATION OF THE IMMUNE RESPONSE TO ASPERGILLUS FUMIGATUS

 描述(申请人提供)：烟曲霉是人类每天吸入的致病真菌。呼吸先天免疫系统阻止吸入的分生孢子(营养孢子)萌发成组织侵入菌丝的能力是一个关键的免疫学检查点。由于医疗技术的进步，生活在免疫受损条件下的患者大幅增加，每年估计导致20万例曲霉病。在高危人群中，尽管现代联合抗真菌疗法，死亡率仍保持在25%-50%。这些趋势突出表明，需要更好地了解绝育免疫的分子和细胞基础，以推进基于免疫的辅助方法。在这个项目的初始资金阶段，我们引入了一种新的真菌生物报告程序，称为荧光曲霉报告程序(Flare)，以追踪其活性。 分生孢子在与肺内免疫细胞的细胞相互作用过程中。利用这种方法，我们确定了脾酪氨酸激酶(Syk)对于肺的先天防御是必不可少的，并在初步研究中表明，Syk以细胞-外在方式调节中性粒细胞的杀菌活性。虽然Syk可以整合髓系细胞中C型凝集素受体、Fc受体和整合素的信号，但其基本的细胞来源和真菌在呼吸道真菌攻击时诱导的激活信号仍未得到很好的确定。除了中性粒细胞外，我们的研究表明，在呼吸攻击过程中，表达CC-趋化因子受体2(CCR2)的炎性单核细胞(IMS)形成了第二个必要的先天效应细胞。在肺中，IMS分化为单核细胞来源的树突状细胞(Mo-DC)，与分生孢子的直接摄取和杀伤一致，并与肺中性粒细胞进行细胞间串扰，以增强肺部的抗真菌活性。在先导研究中，IM/Mo-DC的转录图谱和肺蛋白质组分析确定了五个候选的细胞间串扰和呼吸爆发的介体作为候选效应机制。在接下来的项目期间，我们建议更深入地了解IM/MoDC-中性粒细胞之间的串扰，它通过Syk活性的诱导和调节，它的相关分子递质，以及它在肺部抗分生孢子防御中的作用。在目标1中，我们通过确定Syk信号的基本细胞来源以及通过确定介导肺内分生孢子摄取和杀死的必要上游适配蛋白和受体来确定Syk信号的保护机制。在目标2中，我们将通过确定细胞和运输要求、诱导信号和细胞间串扰的方向性来定义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发射器和效应系统。所提出的研究具有重要意义和创新性，因为他们发现IMS和中性粒细胞之间的天然免疫串扰是调节肺内抗真菌免疫的一种新机制。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鸿沟，这将为脆弱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.