Manipulation of macrophage polarization by a fungal meningitis pathogen

真菌性脑膜炎病原体对巨噬细胞极化的操纵

基本信息

批准号：
10652653
负责人：
Hiten D Madhani
金额：
$ 64.28万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-23 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10652653
关键词：
Alveolar Macrophages B-Lymphocytes Binding Biochemical Bone Marrow CD14 Antigen Cells ChIP-seq Chimera organism Confocal Microscopy Coupled Cryptococcus Cryptococcus neoformans Cryptococcus neoformans infection Cytokine Signaling Data Diagnosis Down-Regulation Flow Cytometry Fungal Meningitis Goals Growth Hand Image Immune Immune response Immune system Immunity Immunology In Vitro Incubated Infection Inflammatory Innate Immune System Interleukin-13 Interleukin-4 Knock-out Lead Ligands Lung Macrophage Mediating Mediator Methods Modality Modeling Molecular Mus Mycoses Myeloid Cells Natural Immunity Pathogenesis Pathway interactions Population Predisposition Protein Secretion Proteins Recombinants Reporter Role STAT3 gene STAT6 Transcription Factor STAT6 gene Salmonella Signal Transduction Site Structure of germinal center of lymph node Surface T cell response TLR4 gene Testing Thick Tissues Up-Regulation Virulence Work arginase cytokine effector T cell experimental study extracellular forward genetics in vivo innate immune mechanisms interstitial monocyte neutrophil pathogen pathogenic fungus predictive modeling programs recruit response

项目摘要

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.

抽象的我们对侵入性真菌病原体如何克服宿主感染障碍知之甚少，但此类感染是诊断和治疗最具挑战性的疾病之一。我们关注的焦点是真菌性脑膜炎的最常见原因，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反应。在隐球菌感染的最初部位，肺，多个髓系细胞群存在细胞，包括肺泡巨噬细胞 (AM)、间质巨噬细胞 (IM)、单核细胞和中性粒细胞。重要的是，2 型免疫反应是由新型隐球菌感染引发的，并介导增加对隐球菌感染的易感性。我们观察到新型隐球菌与小鼠骨髓的孵育衍生巨噬细胞 (BMDM) 会诱导精氨酸酶 1 (Arg1)，这是 2 型反应的标志物。通过通过正向遗传学，我们鉴定了一种新型隐球菌分泌蛋白 CPL1，它是 Arg1 诱导所需的，并且体内毒力。值得注意的是，重组 CPL1 蛋白足以诱导 BMDM 中 Arg1 的表达。体外并增强 M2 对 2 型细胞因子 IL-4 的真实反应。相应地，CPL1 对于 C 至关重要。新型隐球菌在体内激活间质巨噬细胞中的 Arg1。 TLR4，最出名的是 LPS 受体，但它也是一种我们知之甚少的 2 型反应调节因子，它是 CPL1 作用于 BMDM 所必需的。正如 TLR4 所做的那样由于 STAT6 不直接与关键的 2 型转录因子 STAT6 偶联，因此我们寻求间接的作用机制。沙门氏菌通过分泌效应器 steE 促进 M2 极化，steE 通过模仿细胞因子激活 STAT3 发信号。事实上，我们发现 STAT3 也是 CPL1 诱导 Arg1 所必需的。这些数据导致以下工作模型：1）CPL1通过微弱激活TLR4间接促进巨噬细胞极化产生激活 STAT3 的细胞因子，2) STAT3 和关键的 2 型调节因子 STAT6 之间的合作导致巨噬细胞极化增强以驱动毒力。为了测试这个模型，我们组装了一个具有真菌发病机制和免疫学专业知识的协作团队。通过目标 1，我们将阐明 CPL1 在体外促进巨噬细胞极化的机制。我们将测试CPL1提升的模型 M2 通过 STAT3-STAT6 合作做出反应，我们将研究 CPL1 如何通过 TLR4 发挥作用。通过目标2，我们将检验CPL1在体内极化肺间质巨噬细胞的假设，从而产生可复制的生态位。我们将研究 TLR4 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 CPL1 局部作用模型的预测促进巨噬细胞极化以实现病原体复制。这些研究有潜力改变我们对侵袭性真菌病原体如何重新编程先天免疫系统以使其适应其功能的理解优势。