Modulation of Macrophage Antifungal Activity by the Transcriptional Co-regulator CITED1

摘要 新型隐球菌（Cn）是一种普遍存在于城市环境中的致病性酵母菌， 人在生命的最初几年。感染通常通过吸入Cn繁殖体而发生，并且在 在免疫活性个体中，这些细胞被肺泡巨噬细胞有效地摄取和杀死。但如果 Cn避免破坏，它利用巨噬细胞作为生长龛，并从肺部传播，导致 真菌性脑膜炎，如果不治疗是致命的（死亡率约82%），每年造成约181，000人死亡。的 耐药菌株的出现和现有抗真菌剂的毒副作用需要 制定新的战略来对抗Cn感染。为了促进这一点，更好地了解如何 Cn避免被巨噬细胞破坏是必要的。因此，我们研究的首要目标 该项目旨在了解Cn感染如何影响巨噬细胞的杀真菌活性， 巨噬细胞的抗隐球菌活性通过M1极化而增加， IFNγ暴露刺激。这触发了超过1000个基因的表达改变，主要由STAT 1指导， 包括编码iNOS的Nos 2，iNOS是产生抗微生物活性氮物质所需的酶。 在之前的资助期间，我们发现，虽然细胞内Cn感染部分逆转了基因， 表达变化与M1极化相关，iNOS水平升高。这伴随 CITED 1的表达，我们证明它作为STAT 1调节基因的共激活因子发挥作用。的方向迈出了一步 为了实现我们的长期目标，我们将研究CITED 1如何增强IFNγ应答， 影响Cn感染的巨噬细胞的极化和杀真菌活性。具体而言，我们将：确定 CITED 1如何影响M1转录组和细胞内Cn感染的结果。我们将（A）识别 使用转录组和染色质谱分析的组合来确定CITED 1调节的基因，以及（B）确定CITED 1调节的基因的表达。 通过测量CITED 1对促炎细胞因子分泌的影响以及CITED 1对M1表型的影响， 这些细胞的iNOS依赖性抗隐球菌活性。2)确定CITED 1如何调节IFNγ- 刺激基因（ISG）表达。正如我们的初步数据表明，正反馈关系 在STAT 1和CITED 1之间，我们将（A）研究CITED 1增加 通过测量CITED 1对CBP：STAT 1染色质复合物的影响来表达STAT 1调节的ISG 和（B）使用报告基因和基于CUT& RUN的测定法确定STAT 1如何调节Cited 1转录。 此外，我们将研究IFNγ如何刺激CITED 1蛋白质的核转位，使用活的 细胞成像方法。总的来说，这些研究将提供新的见解，细胞内的Cn刺激 CITED 1的表达改变了宿主巨噬细胞中ISG的表达，以及这可能如何影响它们的功能。 表型和杀真菌活性。

ABSTRACT Cryptococcus neoformans (Cn) is a ubiquitous pathogenic yeast found in urban environments that infects most people within the first few years of life. Infection typically occurs through the inhalation of Cn propagules, and in immunocompetent individuals these are efficiently ingested and killed by alveolar macrophages. However, if Cn avoids destruction, it utilizes macrophages as a growth niche and disseminates from the lungs, resulting in fungal meningitis, which is lethal if not treated (~82% mortality rate), causing ~181,000 deaths annually. The emergence of drug-resistant strains and the toxic side-effects of existing antifungal agents necessitates the development of new strategies to combat Cn infections. To facilitate this, an improved understanding of how Cn avoids destruction by macrophages is necessary. Therefore, the overarching goal of our research program is to understand how Cn infection impacts macrophage fungicidal activity and identify host factors that enhance it. Macrophage anticryptococcal activity is increased by M1 polarization, which is stimulated by IFNγ exposure. This triggers the altered expression of >1000 genes, largely directed by STAT1, including Nos2, which encodes iNOS, an enzyme required to produce antimicrobial reactive nitrogen species. During the prior funding period, we showed that while intracellular Cn infection partially reversed gene expression changes associated with M1 polarization, iNOS levels were increased. This was accompanied by expression of CITED1, which we showed to function as a co-activator of STAT1-regulated genes. As a step towards achieving our long-term goal, we will investigate how CITED1 enhances the IFNγ response and affects the polarization and fungicidal activity of Cn-infected macrophages. Specifically, we will: Determine how CITED1 impacts the M1 transcriptome and outcome of intracellular Cn infection. We will (A) identify CITED1-regulated genes using a combination of transcriptome and chromatin profiling and (B) Determine the effect of CITED1 on the M1 phenotype by measuring its impact on proinflammatory cytokine secretion and the iNOS-dependent anticryptococcal activity of these cells. 2) Determine how CITED1 modulates IFNγ- stimulated gene (ISG) expression. As our preliminary data indicates a positive feedback relationship between STAT1 and CITED1, we will (A) investigate the mechanism by which CITED1 increases the expression of STAT1-regulated ISGs by measuring the effect of CITED1 on CBP:STAT1 chromatin complexes and (B) determine how STAT1-regulates Cited1 transcription using reporter and CUT&RUN-based assays. Additionally, we will investigate how IFNγ stimulates the nuclear translocation of CITED1 proteins using a live cell imaging approach. Collectively, these studies will provide new insights into how intracellular Cn-stimulated expression of CITED1 alters ISG expression in host macrophages and how this might influence their phenotype and fungicidal activity.