Genetics, Pathophysiology, and Treatment of Dominant Autoinflammatory Diseases

在本报告所述期间，我们重点研究了RIPK 1基因突变引起的一种新的自身炎症性疾病。RIPK 1基因编码的蛋白质是先天免疫信号通路的关键调节因子。为了确保最佳的炎症反应，RIPK 1通过充分表征的泛素化和磷酸化事件以及caspase-8介导的切割在炎症后进行调节。这种切割事件的生理相关性尚不清楚，尽管它被认为抑制RIPK 3的激活和坏死性凋亡。在这个项目中，我们发现杂合错义突变D324 N，D324 H和D324 Y阻止了人类RIPK 1的半胱天冬酶切割，并导致早发性周期性发热综合征和严重的间歇性淋巴结病。 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（D324 N），第二个家族在5个受影响的个体中证实了p.Asp324His（D324 H）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（D324 Y）。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激酶活性的丧失也阻止了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依赖性TNF诱导的凋亡和坏死性凋亡高度敏感。杂合子Ripk 1 D325 A/+小鼠存活且大体正常，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在本报告所述期间发表在《自然》杂志上。

During the current reporting period we focused on studies of a new autoinflammatory disease caused by mutations in the RIPK1 gene. The protein encoded by the RIPK1 gene is a key regulator of innate immune signaling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage. The physiological relevance of this cleavage event has been unclear, although it has been thought to inhibit activation of RIPK3 and necroptosis. In this project we found that the heterozygous missense mutations D324N, D324H, and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy. 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 (D324N), the second family demonstrated 3-generation inheritance of the p.Asp324His (D324H) heterozygous mutation in 5 affected individuals, and the third family had a de novo heterozygous mutation, p.Asp324Tyr (D324Y), 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 and grossly normal, 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 was published in Nature during the current reporting period.