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

摘要 新生隐球菌(Cn)是一种广泛存在于城市环境中的致病酵母菌。 人们在生命的最初几年。感染通常通过吸入CN传播物而发生，在 具有免疫能力的个体，这些物质被肺泡巨噬细胞有效地摄取和杀死。但是，如果 CN避免破坏，它利用巨噬细胞作为生长利基，并从肺部传播，导致 真菌性脑膜炎，如果不治疗是致命的(死亡率约为82%)，每年造成约181,000人死亡。这个 耐药菌株的出现和现有抗真菌药物的毒副作用使 制定抗击CN感染的新战略。为了促进这一点，更好地理解 CN避免巨噬细胞的破坏是必要的。因此，我们研究的首要目标是 该计划旨在了解CN感染如何影响巨噬细胞杀菌活性并识别宿主 增强它的因素。巨噬细胞的抗隐球菌活性通过M1极化而增加，这是 受干扰素γ刺激。这触发了主要由STAT1调控的&gt；1000基因的表达变化。 包括编码iNOS的NOS2，iNOS是一种产生抗微生物活性氮物种所需的酶。 在之前的资助期间，我们发现，当细胞内CN感染时，基因部分逆转 与M1极化相关的表达变化，iNOS水平升高。随之而来的是 CITED1的表达，我们发现CITED1是STAT1调节基因的共激活因子。作为一个步骤 为了实现我们的长期目标，我们将研究CITED1如何增强干扰素γ反应和 影响CN感染的巨噬细胞的极化和杀菌活性。具体来说，我们将：确定 CITED1如何影响细胞内CN感染的M1转录组和结局。我们将(A)确定 使用转录组和染色质组合分析和(B)确定CITED1调节的基因 CITED1通过检测其对促炎细胞因子分泌的影响而对M1表型的影响 这些细胞的iNOS依赖的抗隐球菌活性。2)确定CITED1如何调节干扰素γ- 刺激基因(ISG)表达。由于我们的初步数据表明存在正反馈关系 在STAT1和CITED1之间，我们将(A)研究CITED1增加 通过检测CITED1对CBP：STAT1染色质复合体的影响来表达STAT1调节的ISGs 以及(B)使用报告和基于切割和运行的分析来确定STAT1如何调节Cited1转录。 此外，我们还将研究干扰素γ如何刺激CITED1蛋白的核转位。 细胞成像方法。总的来说，这些研究将为细胞内CN如何刺激提供新的见解 CITED1的表达改变宿主巨噬细胞的ISG表达及其可能的影响 表型和杀菌活性。

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.