第一部分 白塞病中性粒细胞转录组学研究

研究目的

白塞病（Behçet's disease, BD）是一种病因未明的慢性复发性自身免疫/自身炎症性疾病，其发病机制复杂且尚未完全明晰。多形核中性粒细胞（Polymorphonuclear neutrophil, PMN）的过度活化和浸润是核心致病因素之一，参与 BD 急性炎症的反复发作。因此，本研究旨在深度探究 BD 患者外周血 PMN 的基因表达谱特征，剖析 PMN 活化参与 BD 发病的具体机制，进而为全面理解 BD 的发病机制提供全新的视角与理论依据。

研究方法

本研究纳入了10例未经治疗的活动期BD患者以及10例与之性别、年龄相匹配的健康对照（Healthy controls, HC）。采用Ficoll-Hypaque密度梯度离心技术分离获取BD和HC的外周血PMN，运用流式细胞术测定分离纯度，后使用Trizol试剂提取总RNA。通过Illumina Novaseq平台开展Bulk RNA测序，分析差异表达基因（Differentially expressed genes, DEGs），并运用R语言进行京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析以及转录因子调控网络分析，精准挖掘潜在的关键调控因子及信号通路。同时，运用定量实时聚合酶链反应（Quantitative Reverse Transcription Polymerase Chain Reaction, qRT-PCR）和蛋白质免疫印迹（Western Blot, WB）实验，对趋化相关的DEGs和关键转录因子的差异表达情况进行验证。

研究结果

通过Bulk RNA测序研究发现BD外周血PMN呈现出转录高度活跃的状态，其基因表达谱与 HC 的 PMN 存在显著差异。本研究共鉴定出了567个DEGs，其中有 520 个基因表达显著上调，47 个基因表达显著下调。KEGG通路富集分析显示BD患者PMN（BD-PMN）中NF-κB信号通路和趋化因子信号通路显著激活，呈现出明显的上调趋势。转录因子调控网络分析进一步揭示，ETS1 极有可能是调控趋化相关基因（例如 CCR6、CCL5 等）表达，进而增强 BD-PMN 趋化功能的关键转录因子。

此外，qRT-PCR 实验证实了BD-PMN 中 ETS1、CCR6 和 CCL5 的 mRNA 表达水平相较于 HC 显著增高。WB实验也验证了BD-PMN趋化因子受体CCR6的蛋白表达高于HC。

研究结论

本研究首次针对 BD 患者外周血 PMN 开展全面的 Bulk 转录组学研究，成功揭示了 BD-PMN 转录活跃的基因表达特征。研究明确了 PMN 的过度活化和趋化性增强是参与 BD 发病的重要机制，关键转录因子 ETS1 介导的趋化相关分子 CCR6 和 CCL5 的上调，在 BD-PMN 趋化和炎症反应进程中发挥着核心作用。这一研究成果为深入理解 BD 的发病机制提供了新的理论依据，同时也为后续探索潜在的治疗靶点和创新治疗策略奠定了坚实基础。

第二部分 TNF抑制剂靶向中性粒细胞甲羟戊酸代谢物/TRPM2/钙信号通路抑制白塞病血管炎症

研究目的

白塞病（BD）是一种病因未明的复发性自身免疫/自身炎症性疾病，发病机制复杂。前期研究显示过度活化的多形核中性粒细胞（PMN）在BD发病机制中至关重要。血管炎是BD的基本病理表现，血管内皮细胞（Vascular endothelial cell, VEC）是PMN的关键靶细胞。尽管肿瘤坏死因子（Tumor necrosis factor, TNF）抑制剂已被广泛用于BD的治疗，但其具体作用机制仍有待深入探究。异常代谢及其产物已被证实参与多种自身免疫/自身炎症性疾病的发生发展。由于PMN极易坏死，是循环中代谢物的重要来源。本研究旨在全面揭示BD的免疫代谢特征，筛选介导BD患者PMN（BD-PMN）过度活化的关键代谢物及其潜在分子机制，并进一步探究TNF抑制剂治疗BD的新机制。

研究方法

本研究聚焦免疫代谢，对BD患者和健康对照组（HC）血清代谢组学和外周血各免疫细胞的转录组学数据进行了系统的整合分析。通过使用甲羟戊酸（Mevalonate, MVA）途径各关键酶的抑制剂，筛选出其中参与BD-PMN过度活化的关键代谢物--法尼基焦磷酸（Farnesyl pyrophosphate, FPP）。通过靶向质谱检测了BD 患者血清和 PMN 中的 FPP 水平，并通过队列随访分析了其与 BD 疾病活动指标的相关性。

此外，我们在体外生物合成了FPP，并通过流式细胞术、免疫荧光、ELISA、qRT-PCR等实验检测了FPP诱导的PMN钙离子流、促炎细胞因子产生、中性粒细胞胞外陷阱（Neutrophil extracellular trap, NET）形成（NETosis），及其上清对VEC的活化和损伤的作用。我们通过全面分析BD-PMN的转录组学数据，筛选出了其中表达升高的关键钙离子通道--受体电位离子通道蛋白M亚家族成员2（Transient Receptor Potential Melastatin 2, TRPM2）‌并通过WB加以验证。为探究BD-PMN中TRPM2高表达的原因，我们分别使用活动BD血清或促炎细胞因子孵育HC-PMN，筛选到了BD血清微环境中介导TRPM2表达上调的关键细胞因子TNF。同时，我们还检测了 TNF 抑制剂治疗前后 BD-PMN 中 TRPM2 的表达变化。

此外，本研究构建了髓系细胞特异性FPP合成酶敲除小鼠和TRPM2全敲除小鼠，并诱导建立了中性粒细胞相关性血管炎和自身免疫性葡萄膜炎的疾病模型。分别通过H&E染色、免疫组化、免疫荧光以及眼部CT和眼底镜检查等方法，对疾病的患病率和严重程度进行评估；并通过流式细胞术检测各组小鼠外周血中性粒细胞的活化情况。最后，我们对公共数据库中多种自身免疫和自身炎症性疾病PMN的转录组学数据进行了再分析，进一步验证和扩展了我们的实验结论。

研究结果

本研究揭示了BD患者的免疫代谢特征，其中以PMN的为主的类固醇生物合成途径上调最为突出。进一步分析发现BD-PMN中MVA途径上调，提示其代谢产物FPP增多。靶向质谱证实，未经治疗且处于疾病活动期的BD患者血清和PMN样本中FPP水平显著高于HC，且与BD疾病活动性指标CRP和ESR呈正相关。随访结果显示，BD患者病情缓解后PMN和血清中FPP水平均较基线显著下降。

研究发现，FPP能够以剂量依赖性方式特异性促进PMN产生促炎细胞因子和NETs，且BD-PMN对FPP应答更强，其上清液对VEC的活化和损伤作用更强。机制方面，FPP通过BD-PMN中高表达的TRPM2通道-钙离子依赖性机制介导BD-PMN过度活化。BD血清中高水平的TNF，而非IL-6、IL8、IL-18和IL-1β，是上调TRPM2表达的关键细胞因子。体内联合体外实验显示，TNF抑制治疗可能通过降低TRPM2表达，缓解FPP介导的BD-PMN过度活化，从而改善BD病情。

动物实验显示，髓系特异性FPP合成酶敲除小鼠和TRPM2基因敲除小鼠均表现出显著减轻的血管炎症和损伤，主要表现为中性粒细胞浸润减少、血管通透性降低和 NETosis 减轻。此外，髓系特异性FPP合成酶敲除小鼠诱导的 BD 相关葡萄膜炎和中性粒细胞活化程度也显著轻于对照小鼠。

最后，我们通过公共数据库分析发现，TRPM2在类风湿关节炎、抗中性粒细胞胞浆抗体相关性血管炎等多种有TNF抑制剂临床应用的自身免疫/自身炎症性疾病的PMN中的表达均显著增加，这进一步扩展了我们研究结果的普适性。

研究结论

本研究首次通过多组学整合分析，全面揭示了 BD 的免疫代谢特征，明确了 MVA 代谢物 FPP 显著增加，并强调了 FPP-TRPM2-TNF/NETs 正反馈环路在 BD 发病中的关键作用，提出了 TNF 抑制剂可能通过调节该环路发挥治疗作用的新机制。这项研究不仅为理解 BD 的发病机制提供了新的免疫代谢视角，为 TNF 抑制剂在 BD 及其他自身免疫性 / 自身炎症性疾病中的治疗效果提供了新的理论依据，也为开发更具针对性的治疗策略提供了重要的理论支撑。

Part I: The Transcriptional Study of Neutrophils from Patients with Behçet's Disease

Objective:

Behçet's disease (BD) is a chronic relapsing systemic vasculitis of unknown etiology. Neutrophil hyperactivation is a major cause of recurrent acute inflammation in BD. This study aims to provide new insights into the disease mechanism by investigating the gene expression profiles of neutrophils in BD patients and to further explore the involvement and molecular mechanisms of neutrophils in the pathogenesis of BD.

Methods:

This study included 10 treatment-naive active BD patients and 10 sex- and age-matched healthy controls (HC). Neutrophils were isolated from peripheral blood of BD patients and HC by Ficoll-Hypaque density gradient centrifugation. The purity of the isolated neutrophils was assessed by flow cytometry, and total ribonucleic acid (RNA) was extracted using Trizol reagent. Bulk RNA sequencing was performed on the Illumina NovaSeq platform to identify differentially expressed genes (DEGs), followed by KEGG pathway enrichment analysis and transcriptional regulatory network analysis using the R programming language. Expression levels of chemotaxis-related DEGs and associated transcription factors were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot (WB) assays.

Results:

Bulk RNA sequencing revealed significant gene expression differences between neutrophils from BD patients and healthy controls (HC). A total of 567 differentially expressed genes (DEGs) were identified, including 520 upregulated and 47 downregulated genes. KEGG pathway enrichment analysis revealed significant enrichment of NF-κB and chemokine signaling pathways in BD neutrophils. Transcription factor regulatory network analysis suggested that ETS1 might be a key transcription factor that regulates the expression of chemotaxis-related genes (such as CCR6 and CCL5), thereby enhancing the chemotaxis of BD neutrophils.

Furthermore, qRT-PCR experiments confirmed that the mRNA expression levels of ETS1, CCR6, and CCL5 were significantly higher in BD neutrophils than in HC. The upregulated protein expression level of the chemokine receptor CCR6 was confirmed by WB in neutrophils from BD than from HC. In addition, we found that the expression level of ETS1 in neutrophils was significantly higher in BD patients.

Conclusions:

This study represents the first comprehensive transcriptomic analysis of neutrophils in BD. We elucidated the gene expression profiles of BD neutrophils and demonstrated that neutrophil hyperactivation and enhanced chemotaxis are key mechanisms underlying BD pathogenesis. Our finding revealed that upregulation of the chemotaxis-related molecules ETS1, CCR6, and CCL5 might play a critical role in BD inflammation. This study not only offers new insights into the molecular mechanisms underlying BD, but also highlights potential therapeutic targets for developing potential treatments.

Part II: TNF Inhibitors Target a Mevalonate Metabolite/TRPM2/Calcium Signaling Axis in Neutrophils to Dampen Vasculitis in Behçet's Disease

Objective:

Behçet's disease (BD) is a chronic systemic vasculitis of unknown etiology.  Previous studies have shown that overactivated polymorphonuclear neutrophils (PMNs) play a significant role in the pathogenesis of BD. Vasculitis is the fundamental pathological manifestation of BD and vascular endothelial cells (VEC) are important target cells for PMNs. Although tumor necrosis factor (TNF) inhibitors have been widely used to treat BD, their precise mechanisms of action remain to be elucidated. Aberrant metabolism and metabolites have been implicated in the development of several autoimmune/inflammatory diseases. PMNs are highly prone to necrosis and are a significant source of metabolites in circulation. This study aims to elucidate the immunometabolic profile of BD, to identify key metabolites inducing the hyperactivation of PMN in BD patients (BD-PMN) and their underlying molecular mechanisms, and to further investigate the therapeutic mechanisms of TNF inhibitors.

Methods:

We conduct an integrative analysis of serum metabolomics and peripheral immune cell transcriptomics of BD patients compared to healthy controls (HC). Inhibitors targeting key enzymes of the mevalonate (MVA) pathway were employed and identified farnesyl pyrophosphate (FPP) as the critical metabolite involved in BD-PMN hyperactivation. The clinical relevance of serum and neutrophil FPP levels was validated by targeted mass spectrometry and cohort follow-up analyses.

Additionally, we synthesized FPP and conducted flow cytometry (FC), immunofluorescence (IF), ELISA, and qRT-PCR to further assess its role in BD-PMN calcium influx, proinflammatory cytokine production, and the formation of neutrophil extracellular traps (NETs).  We comprehensively analyzed the transcriptomics of calcium channels in BD-PMN and validated with Western blotting. In addition, HC-PMN was incubated with active BD serum or proinflammatory cytokines to identify the key cytokine promoting TRPM2 expression. The expression level of TRPM2 in PMN from BD patients was measured pre- and post-therapy with TNF inhibitors.

We employed myeloid cell-specific FPP synthase (FPPS) knockout and TRPM2 knockout mice to induce neutrophil-mediated vasculitis and experimental autoimmune uveitis (EAU). The morbidity, severity, and PMN activation were assessed by HE, immunohistochemistry, IF, ocular computed tomography, funduscopy, and FC.

Finally, we reanalyzed neutrophil transcriptomic data from public databases of several autoimmune and autoinflammatory diseases.

Results:

Our study reveals that an upregulation of the steroid biosynthesis pathway in PMN is a prominent immunometabolic feature in BD patients. Further analysis identified a dysregulated MVA pathway in BD-PMN, indicating an increase in metabolite FPP. FPP levels were significantly higher in serum and PMN from treatment-naïve, active BD patients compared to HC, and positively correlated with disease activity markers CRP and ESR. Follow-up studies showed markedly decreased FPP levels in both PMN and serum after BD patients achieved remission.

Notably, we found that FPP stimulated PMN to produce proinflammatory cytokines in a dose-dependent manner, and high concentrations of FPP induce PMN death, primarily through NETosis, with BD-PMN being hyperresponsive to FPP. Supernatants from FPP-stimulated BD-PMN induced activation and damage in VEC. Mechanistically, FPP activates PMN via a TRPM2-calcium-dependent pathway, with higher TRPM2 expression in BD-PMN. TNF, but not IL-6, IL-8, IL-18, or IL-1β, upregulates TRPM2 on BD-PMN, and TNF inhibitors treatment could mitigate this effect in BD patients.

Animal studies demonstrated that neutrophil-specific FPPS knockout mice and TRPM2 knockout mice exhibited significantly reduced vasculitis, with decreased neutrophil infiltration, vascular permeability, and NETosis. Neutrophil-specific FPPS knockout mice also showed milder EAU and neutrophil activation.

Finally, analysis of public databases revealed increased TRPM2 expression in PMNs from various autoimmune/autoinflammatory diseases with clinical applications of TNF inhibitors, such as rheumatoid arthritis and antineutrophil cytoplasmic antibody-associated vasculitis, thereby extending the generalizability of our findings.

Conclusions:

This study is the first to reveal the immunometabolic profile of BD through integrated multi-omic analyses, identifying significantly increased metabolite FPP.  Our findings highlight the critical role of the FPP-TRPM2-TNF/NETs positive feedback loop in BD and propose a novel mechanism by which TNF inhibitors exert their therapeutic effects by modulating this pathway.  This study not only provides new insights into the pathogenesis of BD but also offers a novel immunometabolic mechanism for the therapeutic efficacy of TNF inhibitors in BD and other autoimmune/autoinflammatory diseases, contributing to the development of more targeted therapeutic strategies.