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

转录辅助调节因子 CITED1 对巨噬细胞抗真菌活性的调节

• 批准号: 10727860
• 负责人: David Edward Nelson
• 金额: $ 38.97万
• 依托单位: MIDDLE TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-31 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10727860
• 关键词: Ablation; Adopted; Adoption; Affect; Age; Alveolar Macrophages; Antifungal Agents; Binding; Biochemical; Biological Assay; Cell Line; Cells; Cellular Assay; Cessation of life; Chromatin; Complement 3d Receptors; Complex; Cryptococcosis; Cryptococcus; Cryptococcus neoformans; Cryptococcus neoformans infection; Data; Development; Drug resistance; EP300 gene; Enzymes; Exposure to; Family member; Feedback; Funding; Fungal Meningitis; Gene Expression; Gene Expression Profiling; Genes; Genetic Transcription; Goals; Growth; IRF1 gene; Immune response; Immune system; Immunocompetent; In Vitro; Individual; Infection; Inflammatory; Ingestion; Inhalation; Integration Host Factors; Interferon Type II; Life; Lung; Macrophage; Measures; Molecular; Mus; Mycoses; Nuclear Translocation; Outcome; Pathogenicity; Persons; Phagosomes; Phenotype; Phosphorylation; Protein Family; Proteins; Reactive Nitrogen Species; Regulation; Reporter; Research; STAT1 gene; Shapes; Signal Pathway; Signal Transduction; Yeasts; antimicrobial; combat; cytokine; effective therapy; enhancing factor; fungicide; gene function; gene regulatory network; genome-wide; histone acetyltransferase; imaging approach; improved; insight; latent infection; live cell imaging; mortality; mutant; novel therapeutic intervention; novel therapeutics; pathogen; polarized cell; prevent; programs; resistant strain; response; side effect; transcription factor; transcriptome; transcriptome sequencing; transcriptomic profiling; urban setting; whole genome

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.

抽象的 加密新外身（CN）是一种在城市环境中发现的无处不在的致病酵母菌 人生的头几年。感染通常是通过CN繁殖的吸入而发生的 免疫能力的个体这些人被肺泡巨噬细胞有效摄入和杀死。但是，如果 CN避免破坏，它利用巨噬细胞作为生长生长位并从肺部传播，导致 真菌性脑膜炎，即使没有治疗（约82％的死亡率），每年造成约181,000人死亡。这 抗药菌株的出现和现有抗真菌剂的有毒副作用必要 制定对抗CN感染的新策略。为了促进这一点，改善了对如何 CN避免巨噬细胞破坏。因此，我们研究的总体目标 程序是了解CN感染如何影响巨噬细胞杀真菌活动并识别宿主 增强它的因素。 M1极化增加了巨噬细胞的抗癌活性，这就是 受IFNγ暴露刺激。这触发了> 1000个基因的表达改变，主要由Stat1指导， 包括编码INOS的NOS2，这是产生抗菌反应性氮种所需的酶。 在以前的资金期间，我们表明，虽然细胞内CN感染部分逆转基因 与M1极化相关的表达变化，iNOS水平增加。这是由 Cusit1的表达，我们表明它是STAT1调节基因的共激活因子。作为一步 为了实现我们的长期目标，我们将调查引用1如何增强IFNγ响应和 影响CN感染的巨噬细胞的极化和真菌活性。具体来说，我们将：确定 引用1如何影响细胞内CN感染的M1转录组和结果。我们将（a）确定 使用转录组和染色质分析的组合，引用了1个调节的基因，（b）确定 引用1对M1表型的影响，通过测量其对促炎性细胞因子分泌的影响和 这些细胞的iNOS依赖性抗晶活性。 2）确定引用1如何调节IFNγ- 刺激基因（ISG）表达。正如我们的初步数据表明积极的反馈关系 在STAT1和引用1之间，我们将（a）研究引用1增加的机制 通过测量引用1对CBP的影响：STAT1染色质复合物，表达了STAT1调节的ISGS （b）确定STAT1如何使用记者和基于剪切和运行的测定法调节引用1的转录。 此外，我们将研究如何使用活的IFNγ刺激引用1蛋白的核转运 细胞成像方法。总的来说，这些研究将提供有关细胞内CN刺激的新见解 引用1的表达在宿主巨噬细胞中改变了ISG的表达，这可能如何影响其 表型和杀菌活动。