Manipulation of macrophage polarization by a fungal meningitis pathogen

摘要 我们对侵入性真菌病原体如何克服宿主障碍感染知之甚少，但这种感染 是最具挑战性的诊断和治疗。我们的重点是真菌性脑膜炎最常见的原因， 新型隐球菌。巨噬细胞和其他骨髓细胞对于杀死病原体和促进免疫至关重要。 免疫反应。在隐球菌感染的初始部位，肺，多个群体的骨髓 细胞，包括肺泡巨噬细胞（AM）、间质巨噬细胞（IM）、单核细胞和嗜中性粒细胞。 重要的是，2型免疫反应是由C。新生儿感染和介导增加 隐球菌感染的易感性。我们观察到C.新生儿与小鼠骨髓- 衍生的巨噬细胞（BMDM）导致2型应答的标志物--精氨酸酶-1（Arg 1）的诱导。通过 我们发现了一个C新生儿分泌蛋白CPL 1，Arg 1诱导所需， 体内毒力值得注意的是，重组CPL 1蛋白足以诱导Arg 1在BMDM中的表达， 体外和提高真正的M2反应2型细胞因子IL-4。相应地，CPL 1对C. 新生儿体内激活间质巨噬细胞中的Arg 1。TLR 4，最为人所知的是LPS受体， 也是2型反应的一个不太清楚的介体，是CPL 1作用于BMDM所必需的。与TLR 4一样， 不直接耦合到关键的2型转录因子STAT 6，我们寻求间接的作用机制。 沙门氏菌通过分泌的效应物steE促进M2极化，steE通过模拟细胞因子激活STAT 3 信号事实上，我们发现STAT 3也需要通过CPL 1的Arg 1诱导。这些数据导致 以下工作模型：1）CPL 1通过弱激活TLR 4间接促进巨噬细胞极化， 产生激活STAT 3的细胞因子，以及2）STAT 3与关键的2型调节因子STAT 6之间的合作 导致增强的巨噬细胞极化以驱动毒力。为了测试这个模型，我们组装了一个 在真菌发病机理和免疫学方面具有专业知识的协作团队。通过目标1，我们将阐明 CPL 1促进巨噬细胞体外极化的机制。我们将测试CPL 1增强的模型 M2的反应通过STAT 3-STAT 6的合作，我们将研究如何通过TLR 4 CPL 1的功能。 通过目标2，我们将检验CPL 1在体内极化肺间质巨噬细胞的假设，从而 产生一个复制的小生境。我们将研究TLR 4在巨噬细胞/单核细胞区室中的作用， 我们将使用厚切片成像和其他方式来测试CPL 1局部作用模型的预测 以促进巨噬细胞极化从而使病原体能够复制。这些研究有可能 改变了我们对侵袭性真菌病原体如何重新编程先天免疫系统的理解， 优势

ABSTRACT We understand very little of how invasive fungal pathogens overcome host blocks to infection, yet such infections are among the most challenging to diagnose and treat. Our focus is the most common cause of fungal meningitis, Cryptococcus neoformans. Macrophages and other myeloid cells are critical for killing pathogens and promoting immune responses. Within the initial site of Cryptococcal infection, the lung, multiple populations of myeloid cells exist, including alveolar macrophages (AMs), interstitial macrophages (IMs), monocytes, and neutrophils. Importantly, a type 2 immune response is triggered by C. neoformans infection and mediates increased susceptibility to Cryptococcal infection. We observed that incubation of C. neoformans with murine bone marrow- derived macrophages (BMDMs) results in induction of arginase-1 (Arg1) a marker for type 2 responses. Through forward genetics, we identified a C. neoformans secreted protein, CPL1, required for Arg1 induction and virulence in vivo. Remarkably, recombinant CPL1 protein is sufficient to induce Arg1 expression in BMDMs in vitro and to boost the bona fide M2 response to the type 2 cytokine IL-4. Correspondingly, CPL1 is critical for C. neoformans to activate Arg1 in interstitial macrophages in vivo. TLR4, known best as an LPS receptor but which is also a poorly-understood mediator of type 2 responses, is required for CPL1 to act on BMDMs. As TLR4 does not directly couple to the key type 2 transcription factor STAT6, we sought indirect mechanisms of action. Salmonella promotes M2 polarization via a secreted effector, steE, that activates STAT3 by mimicking cytokine signaling. Indeed, we found STAT3 to also be required for Arg1 induction by CPL1. These data lead to the following working model: 1) CPL1 promotes macrophage polarization indirectly by weakly activating TLR4 to produce cytokines that activate STAT3, and 2) cooperation between STAT3 and the key type 2 regulator STAT6 leads to enhanced macrophage polarization to drive virulence. To test this model, we have assembled a collaborative team with expertise in fungal pathogenesis and immunology. Through Aim 1, we will elucidate mechanisms by which CPL1 promotes macrophage polarization in vitro. We will test the model that CPL1 boosts the M2 response via STAT3-STAT6 cooperation, and we will investigate how CPL1 functions through TLR4. Through Aim 2, we will test the hypothesis that CPL1 polarizes lung interstitial macrophages in vivo, thereby producing a replicative niche. We will investigate the role of TLR4 in the macrophage/monocyte compartment, and we will use thick section imaging and other modalities to test predictions of the model that CPL1 acts locally to promote macrophage polarization to enable pathogen replication. These studies have the potential to transform our understanding how an invasive fungal pathogen reprogram the innate immune system to its advantage.