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 新型隐球菌 (Cn) 是城市环境中普遍存在的致病酵母，可感染大多数人 人们在生命的最初几年内通常通过吸入 Cn 繁殖体而发生感染。 然而，免疫功能正常的个体会被肺泡巨噬细胞有效地摄取并杀死。 Cn避免了破坏，它利用巨噬细胞作为生长环境并从肺部传播，从而导致 真菌性脑膜炎，如果不治疗则致命（死亡率约 82%），每年导致约 181,000 人死亡。 耐药菌株的出现以及现有抗真菌药物的毒副作用使得有必要 制定对抗 CN 感染的新策略，以促进这一目标，更好地了解如何进行。 因此，Cn 避免被巨噬细胞破坏是必要的。 计划的目的是了解 CN 感染如何影响巨噬细胞杀菌活性并识别宿主 M1 极化可增强巨噬细胞抗隐球菌活性，即 M1 极化。 受到 IFNγ 暴露的刺激，这会触发超过 1000 个基因的表达改变，主要由 STAT1 指导， 其中包括编码 iNOS 的 Nos2，iNOS 是一种产生抗菌活性氮所需的酶。 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 与 M1 极化相关的表达变化，伴随着 iNOS 水平的增加。 CITED1 的表达，我们证明它作为 STAT1 调节基因的共激活剂。 为了实现我们的长期目标，我们将研究 CITED1 如何增强 IFNγ 反应并实现 具体来说，我们将： 确定。 CITED1 如何影响 M1 转录组和细胞内 Cn 感染的结果我们将 (A) 确定。 使用转录组和染色质分析相结合的 CITED1 调节基因，并且 (B) 确定 通过测量 CITED1 对促炎细胞因子分泌的影响以及 CITED1 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 CITED1 如何调节 IFNγ-。 我们的初步数据表明，刺激基因（ISG）表达呈正反馈关系。 在 STAT1 和 CITED1 之间，我们将 (A) 研究 CITED1 增加 通过测量 CITED1 对 CBP:STAT1 染色质复合物的影响来检测 STAT1 调节的 ISG 的表达 (B) 使用报告基因和基于 CUT&RUN 的检测确定 STAT1 如何调节 Cited1 转录。 此外，我们将研究 IFNγ 如何使用活细胞刺激 CITED1 蛋白的核转位 总的来说，这些研究将为细胞内 Cn 刺激提供新的见解。 CITED1 的表达改变宿主巨噬细胞中的 ISG 表达，以及这如何影响它们 表型和杀菌活性。