第一部分：NLRP12基因突变引起自身炎症性疾病的发病机制研究

摘要

研究目的：NLRP12相关自身炎症性疾病（NLRP12 associated autoinflammatory disease，NLRP12-AID）是一种由于NLRP12基因突变引起的常染色体显性遗传的单基因自身炎症性疾病。在固有免疫中，NLRP12蛋白可作为胞内模式识别受体，通过抑制NF-κB信号通路及调节炎性小体活性来调控炎症反应，同时在适应性免疫中参与T细胞活化与调节。然而，NLRP12基因在NLRP12-AID中的致病机制仍然不详。本研究旨在基于NLRP12-AID患者的临床特征，从固有免疫和适应性免疫角度分别探究该病的发病机制。

研究方法：收集本中心前瞻性队列中所有诊断为NLRP12-AID患者的临床资料和基因信息，在此基础上探究发病机制。一方面，利用患者原代单核细胞及HEK293T细胞，通过体外功能实验和细胞转染等方法研究NLRP12在炎性小体通路中的作用；另一方面，利用流式细胞术检测患者CD4+ T细胞中各亚群水平，并探讨其亚群异常的潜在机制。

研究结果：总结描述本中心NLRP12-AID队列患者的临床表型和基因型特征，发现除自身炎症相关症状外，半数患者还存在过敏反应异常活化或免疫球蛋白E（immunoglobulin E，IgE）水平升高，根据这两方面临床特征对发病机制进行探究。固有免疫方面，在两名携带截短突变和重复变异的NLRP12-AID患者的单核细胞中，发现炎性小体通路过度活化，白介素-1β（interleukin-1β，IL-1β）水平显著升高。通过HEK293T细胞构建炎性小体活化模型显示，NLRP12不直接参与炎性小体组装，但可与NLRP3结合抑制其活性。此外，携带LRR结构域突变的NLRP12-AID患者外周血中炎性小体活性高于非LRR结构域突变患者及健康对照。适应性免疫方面，对一例携带NLRP12剪接突变导致蛋白表达水平降低患者的CD4+ T细胞进行转录组测序，发现辅助型T细胞（T helper cell，Th）2相关因子表达水平升高，而Th17细胞相关因子表达水平降低。采用流式细胞术检测5例NLRP12-AID患者CD4+ T细胞亚群的比例，证明NLRP12-AID患者外周血中IL-4+ CD4+ T细胞比例升高，IL-17+ CD4+ T细胞比例降低。共培养实验表明，CD4+ T细胞亚群异常并非由于单核细胞的直接作用或继发效应引起。在初始CD4+ T细胞中过表达NLRP12基因后诱导其向Th2和Th17方向分化，发现Th2相关的IL4表达水平降低，Th17相关IL17A、IL23、RORC表达水平升高。

研究结论：NLRP12蛋白并不直接参与炎性小体组装，而是与NLRP3结合抑制其活性。NLRP12基因发生功能丧失性突变破坏这种抑制作用，进而导致NLRP3的过度活化可能是NLRP12-AID发病的主要机制。NLRP12-AID患者外周血中存在IL-4+ CD4+ T细胞比例升高及IL-17+ CD4+ T细胞比例降低，该现象可能是由于NLRP12基因参与调控初始CD4+ T细胞的分化所致，这可能与临床上该病患者常出现过敏反应异常活化或IgE水平显著升高密切相关。

第二部分：TRAF3IP2基因突变引起一种新型自身炎症性皮肤脓疱病发病机制研究

摘要

研究目的：TRAF3IP2基因编码接头蛋白NF-κB激活因子1（NF-κB activator 1，ACT1），在IL-17信号通路的传导及B细胞活化过程中发挥重要作用。该基因的功能丧失性突变与寻常型银屑病和慢性皮肤黏膜念珠菌病的发生密切相关。我们在临床上发现了一例以自身炎症性皮肤脓疱病为主要表现但诊断不明的患者，经全基因组测序发现患者携带TRAF3IP2基因两个新位点的复合杂合突变，但目前尚无关于该基因与皮肤脓疱病的相关报道。本研究旨在探究TRAF3IP2基因在这一新型自身炎症性皮肤脓疱病中的致病性以及发病机制。

研究方法：本研究从患者的基因型入手，通过体外功能实验验证其携带突变位点的致病性。进一步探究患者体内是否存在与该表型密切相关的中性粒细胞异常及IL-36信号通路的过度活化。最后，利用人永生化角质形成细胞（HaCat细胞系）研究TRAF3IP2基因是否能引起信号通路活性的变化及其潜在机制，对基因型与表型之间的关联进行讨论。

研究结果：在基因型研究中，双荧光素酶报告实验和免疫共沉淀结果表明，突变可影响NF-κB和AP-1的转录活性，并干扰ACT1与IL-17RA之间的相互作用，原代样本刺激实验显示IL-17信号通路的传导完全受损，提示患者携带的突变为功能丧失型突变。基于临床表型的研究发现，患者中性粒细胞产生中性粒细胞胞外诱捕网（neutrophil extracellular traps，NETs）的水平显著增加，血浆中IL-36炎性因子浓度明显高于正常人。在外源性IL-36刺激下，成纤维细胞能产生更多IL-8，表明患者体内存在中性粒细胞及IL-36信号通路的过度活化。为进一步探究基因型与表型的关联，我们在HaCat细胞中敲降TRAF3IP2后进行了转录组测序，结果显示中性粒细胞趋化相关基因表达水平显著增加，且患者中性粒细胞产生的NETs可促进HaCat细胞表达更多IL-36相关炎性因子。此外，TRAF3IP2基因本身也可导致HaCat细胞中IL-36信号通路过度活化，从而促进更多IL-8的产生。该现象可能与基因对IL-8启动子活性的调控、外源性IL-17A和IL-36等炎性因子的反馈效应无关，而与敲降所导致的IL36RN基因表达水平降低有关。淋巴细胞初步研究表明，患者体内Th17细胞亚群增加且B细胞过度活化，但IL-22水平未见增加。TRAF3IP2基因的异常可能还影响HaCat细胞的增殖、凋亡和分化等功能。

研究结论：TRAF3IP2基因的突变不仅与寻常型银屑病和慢性皮肤黏膜念珠菌病相关，还可能引起一种新型自身炎症性脓疱病的临床表型。该病的发病机制可能与TRAF3IP2功能丧失引起的中性粒细胞过度趋化和NETs水平增加，以及IL36RN表达水平降低导致的IL-36信号通路过度活化密切相关。本研究拓宽了TRAF3IP2基因突变相关疾病谱及自身炎症性皮肤脓疱病的表型谱，提示自身免疫与自身炎症之间可能存在密切联系。

Research on the Pathogenesis of Systemic Autoinflammatory Diseases Induced by NLRP12 Gene Mutations

Abstract

Objectives: NLRP12-associated autoinflammatory disease (NLRP12-AID) is an autosomal dominant autoinflammatory disorder caused by variants of NLRP12 gene. The NLRP12 protein functions as an intracellular pattern recognition receptor in the innate immune system, modulating inflammatory responses by inhibiting the NF-κB signaling pathway and regulating inflammasome activity. Additionally, it is involved in the activation and regulation of T cells in the adaptive immune response. Despite its multiple biological effects, the role of NLRP12 in the pathogenesis of NLRP12-AID remains controversial. This study aims to elucidate the pathogenesis of NLRP12-AID based on the clinical characteristics exhibited by patients, focusing on both innate and adaptive immune mechanisms.

Methods: Clinical phenotypes and genetic information were collected from all patients diagnosed with NLRP12-AID in a prospective cohort at our center, serving as the foundation for exploring the pathogenesis of the disease. On one hand, the role of NLRP12 in the inflammasome pathway was investigated using monocytes from patients and HEK293T cells, employing in vitro functional assays and cell transfection techniques. On the other hand, flow cytometry was used to analyze the levels of different CD4+ T cell subsets in patients, and the underlying mechanisms were further explored.

Results: Analysis of clinical phenotypic and genotypic characteristics of NLRP12-AID patients in our cohort revealed that in addition to autoinflammatory symptoms, half of the patients displayed a hyper-responsiveness to allergic reactions or elevated immunoglobin E (IgE) levels. The pathogenic mechanisms related to these characteristics were investigated. Excessive activation of the inflammasome pathway and significantly elevated interleukin 1β (IL-1β) levels were observed in the monocytes of two NLRP12-AID patients with truncation and repeat variants. In a HEK293T cell model of inflammasome activation, it was demonstrated that NLRP12 does not directly participate in the assembly of the inflammasome but can inhibit NLRP3 activity through direct interaction. Moreover, NLRP3 inflammasome activity was found to be higher in NLRP12-AID patients with LRR domain mutations compared to those without such mutations and healthy controls. In the context of adaptive immunity, transcriptome sequencing of CD4+ T cells from a patient with a splice mutation resulting in reduced NLRP12 expression revealed elevated levels of T helper 2 (Th2) -related factors and decreased levels of Th17-related factors. Flow cytometry analysis of CD4+ T cell subsets in five NLRP12-AID patients corroborated these findings. Co-culture experiments indicated that the abnormal distribution of CD4+ T cell subsets was not due to direct or secondary effects from monocytes. Additionally, upon overexpressing NLRP12 in naive CD4+ T cells and inducing polarization toward Th2 and Th17 lineages, an increase in Th17-related gene expression, including IL17A, IL23 and RORC, alongside a decrease in Th2-related IL4 expression were observed.

Conclusion: Although mutations in the NLRP12 gene can lead to the onset of autoinflammatory disease, NLRP12 itself does not participate in the assembly of the inflammasome. Instead, it exerts its inhibitory effects on NLRP3 activation through direct interaction. Loss-of-function mutations in the NLRP12 gene disrupt this inhibitory role, resulting in excessive NLRP3 activation, which may represent one of the pathogenic mechanisms underlying NLRP12-AID. Additionally, NLRP12-AID patients exhibit elevated levels of IL-4+CD4+ T cells and reduced levels of IL-17+CD4+ T cell. This phenomenon is likely related to the role of the NLRP12 gene in the polarization of naive CD4+ T cells and may be associated with the hyper-responsiveness observed in allergic reactions and elevated IgE levels.

Research on the Pathogenesis of a Novel Autoinflammatory Pustular Skin Disease Induced by TRAF3IP2 Gene Mutations

Abstract

Objectives: TRAF3IP2 gene encodes the adaptor protein ACT1 (NF-κB activator 1，ACT1), which plays a critical role in the IL-17 signaling pathway and the activation of B cells. Loss-of-function mutations in this gene are closely associated with the pathogenesis of psoriasis vulgaris and chronic mucocutaneous candidiasis. We identified a patient presenting primarily with an autoinflammatory pustular skin disease characterized by an undefined clinical diagnosis. Whole-genome sequencing revealed the presence of two novel compound heterozygous mutations in the TRAF3IP2 gene.

Methods: We utilized in vitro functional assays to validate the pathogenicity of these two mutation sites in TRAF3IP2, based on the patient's genotype. Further investigations focused on assessing abnormalities in neutrophil function and the excessive activation of the IL-36 signaling pathway, which are hypothesized to be associated with the clinical phenotype. Additionally, we employed the immortalized human keratinocyte cells (HaCat cells) to evaluate whether alterations observed in the patient could be induced by the TRAF3IP2 gene and the underlying mechanisms, thereby discussing the correlation between genotype and phenotype.

Results: Genotypic studies, including dual-luciferase reporter assays and co-immunoprecipitation, indicated that the identified mutations could significantly impair the transcriptional activity of NF-κB and AP-1, and disrupt the interaction between ACT1 and IL-17RA. Stimulation experiments with primary patient samples demonstrated a complete impairment of IL-17 signaling transduction, suggesting that the mutations are indeed loss-of-function. Phenotypic investigations revealed significantly elevated levels of neutrophil extracellular trap (NETs) produced by the patient's neutrophils, alongside markedly higher plasma concentrations of IL-36 inflammatory factors compared to healthy controls. Following external IL-36 stimulation, fibroblasts from the patient exhibited increased production of IL-8, confirming the overactivation of the IL-36 signaling pathway. To further investigate the genotype-phenotype correlation, we knocked down the TRAF3IP2 gene in HaCat cells and conducted transcriptomic sequencing, revealing a significant upregulation of genes related to neutrophil chemotaxis. The NETs generated by the patient's neutrophils also enhanced the expression of IL-36 related inflammatory factors in HaCat cells. Furthermore, we observed that the TRAF3IP2 gene could induce excessive activation of the IL-36 signaling pathway in HaCat cells, leading to increased IL-8 levels. This phenomenon is likely associated with reduced IL36RN expression resulting from the knockdown, rather than with the regulation of IL-8 promoter activity by TRAF3IP2 or feedback effects from external inflammatory mediators such as IL-17A and IL-36. Preliminary studies of the patient's lymphocytes revealed an increased Th17 subset and enhanced B cell activation, while IL-22 levels did not show elevation. Additionally, abnormalities in the TRAF3IP2 gene may also affect the proliferation, apoptosis, and differentiation of HaCat cells.

Conclusion: Mutations in the TRAF3IP2 gene are not only associated with psoriasis vulgaris and chronic mucocutaneous candidiasis but may also contribute to a novel clinical phenotype of autoinflammatory pustular skin disease. The pathogenesis of this condition appears to be closely related to the overactivation of neutrophils and elevated NETs levels resulting from TRAF3IP2 loss of function, as well as the excessive activation of the IL-36 signaling pathway due to decreased IL36RN expression. This study expands the spectrum of diseases associated with TRAF3IP2 gene mutations, enhancing our understanding of the phenotypic characteristics of autoinflammatory pustular skin diseases and suggesting a significant interplay between autoimmunity and autoinflammation.