Exploring Determinants of Intrinsic DNA Load Towards Inflammaging

探索导致炎症的内在 DNA 负荷的决定因素

• 批准号: 10576391
• 负责人: Yuk Yuen Lan
• 金额: $ 20.46万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10576391
• 关键词: Abate; Acceleration; Aging; Algorithms; Ataxia; Ataxia Telangiectasia; Autoimmune; Autoimmunity; Automobile Driving; Autophagocytosis; Biological; Biological Assay; Biological Process; Biology; Cell Aging; Cell Nucleus; Cells; Cellular Morphology; Chromatin; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Cyclic GMP; Cytosol; DNA; DNA Damage; DNA Virus Infections; Diabetes Mellitus; Disease; Exhibits; Family; Foundations; Future; Genomic Instability; Heart Diseases; Homeostasis; Human; Immune; Immune response; Immunity; Immunofluorescence Immunologic; Impaired cognition; Infection; Inflammaging; Inflammation; Inflammatory; Interferon Type I; Interferons; Intrinsic factor; Knock-out; Knockout Mice; Ligands; Malignant Neoplasms; Mediating; Mitochondrial DNA; Modeling; Mus; Nuclear; Optics; Pathologic; Pathology; Pathway interactions; Patients; Pattern; Phenotype; Ploidies; Polyarthritides; Premature aging syndrome; Production; Progeria; Property; Retrotransposon; Risk; Role; Signal Transduction; Sterility; Stimulator of Interferon Genes; System; Tissues; cell growth; clinically relevant; ds-DNA; endonuclease; extranuclear DNA; immune activation; immunogenic; immunoregulation; improved; insight; member; novel; novel strategies; novel therapeutic intervention; nuclease; overexpression; patient engagement; response; senescence; sensor; systemic inflammatory response; theories; transcriptomics

PROJECT SUMMARY Sterile inflammation is exhibited in aging‐related conditions, including heart disease, cancer, diabetes, and cognitive decline. Such hyperinflammation typically displays an altered immune response comprising a type I interferon response and downstream senescence-associated secretory phenotype (SASP). The intrinsic factors that induce this chronic low-grade inflammation are not well understood. In studying the initiation of autoimmune polyarthritis in mice lacking the lysosomal endonuclease Dnase2a, we discovered a cell‐autonomous pathway through which damaged nuclear DNA is trafficked to the cytosol where it is recognized by the innate cytosolic DNA sensing cGAS-STING axis, triggering an inflammatory cascade. In healthy cells, nuclear DNA found in the cytosol is removed via autophagy for lysosomal degradation by DNASE2A, which degrades double-stranded DNA (dsDNA)—ligand for the DNA sensor cGAS. Consistent with this notion that damaged or irreparable nuclear DNA is a trigger of immunity when mis- localized and accumulated in the cytosol, elevated loads of extranuclear DNA in replicative senescent cells and cells from ataxia (AT) and progeria (HGPS) patients engage the same DNA sensing pathway leading to persistent inflammation. Controlling the intrinsic DNA load is thus critical to avoid sterile inflammation. While nucleases can clear DNA, unknown DNA export factors may facilitate DNA exit to the cytosol. By targeting curated sets of potential nucleases and DNA export factors, we propose to identify unknown intrinsic DNA controlling factors in a single-cell based CRISPR knockout optical screen by assaying dsDNA content using immunofluorescence. Hits will be validated for their DNA export or degradative function and ranked for their involvement in senescence and STING activation downstream of cGAS. Selected tops hits, along with DNASE2A, will be evaluated for their capacity in suppressing inflammation and SASP in senescent cells and AT and HGPS patient cells. In Dnase2a knockout mice, we will investigate immune cell phenotype in tissues to understand the mechanisms underlying the systemic senescent pathology. The identification and characterization of new determinants in inflammation and senescence will add new insights to the basic biology of DNA-mediated immunity and open up novel strategies in modulating inflammation to benefit aging-associated pathologies, including interferonopathy and laminopathy, autoimmunity and cancer.

项目概要 无菌性炎症表现在与衰老相关的疾病中，包括心脏病、癌症、 糖尿病和认知能力下降。这种过度炎症通常表现出免疫改变 反应包括 I 型干扰素反应和下游衰老相关反应 分泌表型（SASP）。诱发这种慢性低度炎症的内在因素 没有被很好地理解。在研究缺乏自身免疫性多关节炎的小鼠中 溶酶体核酸内切酶 Dnase2a，我们发现了一条细胞自主途径，通过该途径 受损的核 DNA 被运输至细胞质，并被先天细胞质识别。 DNA 感应 cGAS-STING 轴，引发炎症级联反应。在健康细胞中，核 细胞质中发现的 DNA 通过 DNASE2A 的溶酶体降解作用通过自噬去除， 它可以降解双链 DNA (dsDNA)——DNA 传感器 cGAS 的配体。持续的 根据这一观点，受损或不可修复的核 DNA 是免疫的触发因素，当错误 定位并积累在细胞质中，复制过程中核外 DNA 的负载量增加 衰老细胞与共济失调 (AT) 和早衰 (HGPS) 患者的细胞具有相同的 DNA 导致持续炎症的传感通路。因此，控制内在 DNA 负载是 对于避免无菌性炎症至关重要。虽然核酸酶可以清除 DNA，但未知的 DNA 输出因子 可能有助于 DNA 离开细胞质。通过针对潜在核酸酶和 DNA 的精选组 输出因素，我们建议识别单细胞中未知的内在 DNA 控制因素 通过使用免疫荧光测定 dsDNA 含量进行基于 CRISPR 敲除光学筛选。 将验证命中的 DNA 输出或降解功能，并根据其参与程度进行排名 衰老和 cGAS 下游的 STING 激活。精选热门热门歌曲，以及 DNASE2A，将评估其抑制炎症和 SASP 的能力 衰老细胞以及 AT 和 HGPS 患者细胞。我们将在 Dnase2a 敲除小鼠中进行研究 组织中的免疫细胞表型，以了解系统性免疫细胞的潜在机制 衰老病理学。炎症新决定因素的鉴定和表征 衰老将为 DNA 介导的免疫的基础生物学增添新的见解，并开放 制定调节炎症的新策略，以有益于与衰老相关的病理，包括 干扰素病和核纤层病、自身免疫和癌症。