Genetics, Pathophysiology, and Treatment of Dominant Autoinflammatory Diseases

在本报告所述期间，我们专注于以下项目： 第一章 RIPK 1基因突变引起的一种新的自身炎性疾病的研究 三个家庭的成员提出了一个以前未描述的自身炎症性疾病的特点是早发性周期性发热发作，严重的间歇性淋巴结病，器官肿大，腹痛。 在一些个体中，这种疾病对托珠单抗的治疗性IL-6抑制有选择性反应。外显子组测序显示，RIPK 1是唯一一个来自所有三个家族的变异体满足筛选标准的基因。在一个家族中，唯一受影响的个体具有新生杂合突变p.Asp324Asn，第二个家族在5个受影响的个体中表现出p.Asp324His杂合突变的3代遗传，第三个家族在唯一受影响的个体中具有新生杂合突变p.Asp324Tyr。这三个突变导致在相同的关键残基上的不同取代，所述相同的关键残基是胱天蛋白酶-8切割RIPK 1所特别需要的。这种天冬氨酸在物种间高度保守，所有三种突变都不存在于人类变异数据库中。 这些突变在体外损害了caspase-8和caspase-6对RIPK 1的切割，尽管人类细胞中的切割似乎主要由caspase-8介导。我们将这种临床状况命名为“抗裂解RIPK 1诱导的自身炎症（CRIA）”综合征。 为了确定这种疾病的机制，我们与澳大利亚墨尔本Walter and Eliza Hall研究所的John Silke实验室建立了合作关系，以生成适当的小鼠模型。应当注意，小鼠序列中的残基325与人类中的残基324同源。Ripk 1-/-小鼠出生后死于全身性炎症，而Ripk 1 D325 A/D325 A小鼠在胚胎发育期间死亡。通过Casp 8和Ripk 3的组合损失而不是通过Ripk 3或Mlkl单独的损失完全防止胚胎致死。此外，RIPK 1激酶活性的丧失也防止了RIPK 1 D325 A/D325 A胚胎致死，然而小鼠在以RIPK 3依赖性方式脱离多器官炎症之前死亡。Ripk 1 D325 A/D325 A和Ripk 1 D325 A/+细胞对RIPK 3依赖的TNF诱导的凋亡和坏死性凋亡高度敏感。杂合子Ripk 1 D325 A/+小鼠存活，但对体内炎症刺激物反应过度。这些结果证明了caspase介导的RIPK 1裂解在胚胎发育过程中的重要性，并表明caspase裂解RIPK 1不仅抑制坏死性凋亡，而且在整个生命过程中维持炎症稳态。 描述这些发现的手稿已被《自然》杂志接受发表。 （二） PAPA综合征的神经生物学 化脓性关节炎伴坏疽性脓疡和痤疮（PAPA综合征）是由PSTPIP 1突变引起的，PSTPIP 1编码一种芘结合蛋白。患者出现严重的嗜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，我们将12例PAPA患者的样本提供给NIAMS合作者，他们是评估中性粒细胞功能的专家。他们的研究表明，循环低密度粒细胞（LDG）在PAPA受试者中升高。PAPA血清表现出受损的中性粒细胞胞外陷阱（NET）降解，这是纠正与外源性DNA酶1。重组人IL-1 β在PAPA中性粒细胞中诱导NET形成，但在健康对照中性粒细胞中不诱导。在健康对照中性粒细胞中NET形成由PAPA血清诱导，并且该作用被IL-1受体拮抗剂阿那白滞素抑制。来自PAPA中性粒细胞和LDG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，其与组织IL-1 β、IL-8和IL-17的增加相关。此外，在PAPA皮肤中检测到LDG基因签名。这些数据强调了中性粒细胞NET形成在PAPA综合征的病理生理学中的重要性，并为这种严重且通常难治性的疾病提出了新的治疗策略。 描述本研究结果的手稿在本报告期内发表在《风湿病年鉴》上。

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.