Genetics, Pathophysiology, and Treatment of Dominant Autoinflammatory Diseases

显性自身炎症性疾病的遗传学、病理生理学和治疗

• 批准号: 10027215
• 负责人: Daniel Kastner
• 金额: $ 179.37万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10027215
• 关键词: Abdominal Pain; Acne; Acute suppurative arthritis due to bacteria; Affect; Animal Model; Animals; Apoptosis; Aspartate; Australia; Binding Proteins; Biology; Biopsy; CASP6 gene; CASP8 gene; Caspase; Cell Line; Cells; Clinical; Collaborations; Data; Databases; Development; Disease; Embryo; Embryonic Development; Exhibits; Family; Fever; Functional disorder; Generations; Genes; Genetic; Goals; Homeostasis; Human; Hypersensitivity; IL8 gene; Impairment; In Vitro; Individual; Inflammation; Inflammatory; Inherited; Institutes; Interleukin-1 Receptors; Interleukin-17; Interleukin-6; Joints; Laboratories; Lead; Life; Lymphatic Diseases; Manuscripts; Mediating; Molecular; Mus; Mutation; National Institute of Arthritis and Musculoskeletal and Skin Diseases; Nature; Organ; Patients; Periodicity; Phosphotransferases; Proteins; Publications; Publishing; Pyoderma Gangrenosum; RIPK1 gene; RIPK3 gene; Recombinants; Refractory; Reporting; Resistance; Rheumatism; Role; Sampling; Sampling Studies; Serum; Skin; Stimulus; Syndrome; TNF gene; Therapeutic; Tissues; Variant; Visceromegaly; Weaning; anakinra; autoinflammatory; density; disease-causing mutation; early onset; exome sequencing; extracellular; genetic signature; granulocyte; in vivo; knockin animal; knockout animal; macrophage; marenostrin; member; mouse model; neutrophil; new technology; novel therapeutic intervention; postnatal; prevent

During the current reporting period we have focused on the following projects: 1) Studies of a New Autoinflammatory Disease Caused by Mutations in RIPK1 Members of three families presented with a previously undescribed autoinflammatory disorder characterized by early-onset periodic fever episodes, severe intermittent lymphadenopathy, organomegaly, and abdominal pain. In some individuals this disease was selectively responsive to therapeutic IL-6 inhibition with tocilizumab. Exome sequencing revealed that RIPK1 was the only gene in which a variant from all three families satisfied filtering criteria. In one family the sole affected individual had a de novo heterozygous mutation, p.Asp324Asn, the second family demonstrated 3-generation inheritance of the p.Asp324His heterozygous mutation in 5 affected individuals, and the third family had a de novo heterozygous mutation, p.Asp324Tyr, in the sole affected individual. These three mutations result in different substitutions at the same critical residue that is specifically required for RIPK1 cleavage by caspase-8. This aspartate is highly conserved across species, and all three mutations are absent from human variant databases. The mutations impaired RIPK1 cleavage by caspase-8 as well as by caspase-6 in vitro, though cleavage in human cells appeared to be mediated predominantly by caspase-8. We designate this clinical condition 'cleavage-resistant RIPK1-induced autoinflammatory (CRIA)' syndrome. To define the mechanism for this disease, we established a collaboration with John Silke's laboratory at the Walter and Eliza Hall Institute in Melbourne, Australia to generate appropriate mouse models. It should be noted that residue 325 in the mouse sequence is homologous to residue 324 in humans. Whereas Ripk1-/- mice die postnatally from systemic inflammation, Ripk1 D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by combined loss of Casp8 and Ripk3 but not by loss of Ripk3 or Mlkl alone. In addition, loss of RIPK1 kinase activity also prevented Ripk1 D325A/D325A embryonic lethality, however the mice died before weaning from multi organ inflammation in a RIPK3 dependent manner. Consistently, Ripk1 D325A/D325A and Ripk1 D325A/+ cells were hypersensitive to RIPK3 dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1 D325A/+ mice were viable, but hyper-responsive to inflammatory stimuli in vivo. These results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but maintains inflammatory homeostasis throughout life. A manuscript describing these findings has been accepted for publication in Nature. 2) Neutrophil Biology in PAPA Syndrome Pyogenic arthritis with pyoderma gangrenosum and acne (PAPA syndrome) is caused by mutations in PSTPIP1, which encodes a pyrin-binding protein. Patients develop severe neutrophil-mediated inflammation of the skin and joints. To study the role of neutrophils in PAPA, we provided samples from 12 PAPA patients to NIAMS collaborators expert in assessing neutrophil function. Their studies demonstrated that circulating low density granulocytes (LDGs) are elevated in PAPA subjects. PAPA sera exhibit impaired neutrophil extracellular trap (NET) degradation and this is corrected with exogenous DNase1. Recombinant human IL-1beta induces NET formation in PAPA neutrophils but not healthy control neutrophils. NET formation in healthy control neutrophils is induced by PAPA serum and this effect is inhibited by the IL-1 receptor antagonist, anakinra. NETs from PAPA neutrophils and LDGs stimulate IL-6 release from healthy macrophages. NETs are detected in skin biopsies of patients with PAPA syndrome in association with increased tissue IL-1beta, IL-8, and IL-17. Furthermore, LDG gene signatures are detected in PAPA skin. These data underscore the importance of neutrophil NET formation in the pathophysiology of PAPA syndrome, and suggest new therapeutic strategies for this severe and often treatment-refractory condition. A manuscript describing the results of this study was published during the current reporting period in the Annals of the Rheumatic Diseases.

在本报告所述期间，我们侧重于以下项目： 1)RIPK1基因突变引起的一种新的自身炎症性疾病的研究 三个家族的成员出现了一种以前未被描述的自体炎症疾病，其特征是早发性周期性发热，严重的间歇性淋巴结病，器官肿大和腹痛。在一些人中，这种疾病对tocilizumab的治疗性IL-6抑制有选择性的反应。外显子组测序显示，RIPK1是唯一一个三个家族的变异体都满足筛选标准的基因。在一个家系中，唯一受影响的个体具有新生杂合突变P.Asp324Asn，第二个家系在5个受累个体中表现出p.Asp324His杂合突变的3代遗传，第三个家系在唯一受影响个体中有一个新生杂合突变p.Asp324Tyr。这三个突变导致了caspase-8切割RIPK1所需的同一关键残基上的不同替换。这种天冬氨酸在物种间高度保守，所有这三个突变都不存在于人类变异数据库中。尽管人类细胞中的切割似乎主要是由caspase-8介导的，但这些突变在体外也损害了caspase-8和caspase-6对RIPK1的切割。我们将这种临床症状命名为RIPK1诱导的自身炎症(CRIA)综合征。 为了确定这种疾病的发病机制，我们与澳大利亚墨尔本沃尔特和伊莱扎·霍尔研究所的约翰·西尔克的实验室建立了合作，以产生合适的小鼠模型。应当注意，小鼠序列中的残基325与人类中的残基324同源。Ripk1-/-小鼠出生后死于全身炎症，而Ripk1 D325A/D325A小鼠在胚胎发育过程中死亡。Casp8和RIPK3的联合缺失可完全防止胚胎死亡，但单独缺失RIPK3或MLKL并不能完全防止胚胎死亡。此外，RIPK1激酶活性的丧失也阻止了Ripk1 D325A/D325A胚胎的死亡，但小鼠在断奶前死于RIPK3依赖的多器官炎症。Ripk1 D325A/D325A和Ripk1 D325A/+细胞对RIPK3依赖的肿瘤坏死因子诱导的细胞凋亡和坏死下垂高度敏感。杂合子Ripk1 D325A/+小鼠是存活的，但在体内对炎症刺激具有高反应性。这些结果证明了caspase介导的RIPK1裂解在胚胎发育中的重要性，并表明caspase裂解RIPK1不仅抑制坏死性下垂，而且在整个生命过程中维持炎症动态平衡。 描述这些发现的手稿已被接受发表在《自然》杂志上。 2)Papa综合征的中性粒细胞生物学 化脓性关节炎伴坏疽脓皮病和痤疮(Papa综合征)是由PSTPIP1基因突变引起的，PSTPIP1编码一种比林结合蛋白。患者会出现严重的中性粒细胞介导的皮肤和关节炎症。为了研究中性粒细胞在PAPA中的作用，我们提供了12例PAPA患者的样本给NIAMS合作者评估中性粒细胞功能的专家。他们的研究表明，PAPA受试者循环低密度粒细胞(LDG)升高。Papa血清表现出中性粒细胞胞外陷阱(NET)降解受损，这一点可用外源DNase1纠正。重组人IL-1β可诱导Papa中性粒细胞形成网状结构，但不能诱导健康对照中性粒细胞。健康对照中性粒细胞中的Net形成是由Papa血清诱导的，这一作用可被IL-1受体拮抗剂Anakinra抑制。来自Papa中性粒细胞和LDGs的Net刺激健康巨噬细胞释放IL-6。在Papa综合征患者的皮肤活检中检测到Net与组织IL-1β、IL-8和IL-17的升高有关。此外，在Papa皮肤中检测到LDG基因特征。这些数据强调了中性粒细胞网络形成在Papa综合征的病理生理学中的重要性，并为这种严重且往往难以治疗的疾病提出了新的治疗策略。 在本报告所述期间，在《风湿病年鉴》上发表了一份描述这项研究结果的手稿。