中文摘要

背景和目的

IgG4相关性疾病（IgG4 related disease, IgG4-RD）是慢性炎症伴纤维化的疾病，临床表现为累及脏器肿瘤样增大变硬，血清IgG4升高，病理大量淋巴细胞、浆细胞浸润，尤其是IgG4+浆细胞。最常见的累及器官为颌下腺，泪腺，腮腺，胰腺，肺，腹膜后/主动脉等。IgG4-RD合并过敏患者比例约为60%，过敏IgG4-RD患者更容易累及颌面部及颈部，且外周血嗜酸性粒细胞比例较非过敏患者升高。由于受累器官纤维化可导致不可逆性损伤，针对该病发病机制研究尤为重要。IgG4-RD血浆来源外泌体蛋白质组学研究尚属空白。因此本研究首先通过蛋白质组学技术探索IgG4-RD患者血浆来源外泌体同健康对照相比是否存在蛋白表达谱差异。通过IgG4-RD合并过敏患者血浆来源外泌体蛋白质组学，探究具有过敏免疫背景的IgG4-RD患者血浆来源外泌体蛋白表达以及是否参与调控IgG4-RD器官特异性损伤。颌下腺作为IgG4-RD患者常用研究样本用于组织的蛋白质谱验证。本研究最后探究IgG4-RD患者颌下腺蛋白质组学，揭示IgG4-RD组织损伤特征及探究外泌体是否参与IgG4-RD组织损伤。

材料和方法

本研究纳入134例初治IgG4-RD患者及150例性别、年龄匹配的健康对照（Healthy control, HC）。本研究通过伦理委员会审核，入组患者及HC均签署知情同意书。本研究通过超速离心及外泌体提取试剂盒的方式分别提取IgG4-RD及HC血浆来源外泌体。采用TMT标记提取的血浆外泌体蛋白，通过液相色谱-串联质谱（LC-MS/MS）技术进行检测。筛选差异蛋白进行ELISA及WB验证。流式细胞术技术检测IgG4-RD外泌体处理健康B细胞后B亚群分化水平及活性氧自由基（Reactive oxygen species, ROS）表达水平。收集IgG4-RD外泌体处理后的B细胞进行蛋白质组学检测。同时，IgG4-RD有无过敏患者各15例，采用LC-MS/MS技术检测血浆来源外泌体蛋白质组学。最后本研究收集4例病理确诊IgG4-RD患者及4例病理确诊的慢性非特异性涎腺炎患者颌下腺，采用稳定同位素标记蛋白质组学技术（Isobaric tags for relative and absolute, iTRAQ）检测IgG4-RD受累颌下腺蛋白质组学变化。

结果

与HC相比，IgG4-RD血浆来源外泌体质谱共检测出178个差异表达蛋白，差异蛋白主要富集于补体级联通路。外泌体来源补体C3及C5与IgG4-RD临床指标相关。IgG4-RD外泌体刺激HC的B细胞，流式检测发现记忆性B细胞（Memory B）及浆母细胞（Plasmablast）升高，而初始B细胞（Naïve B）下降。B细胞蛋白质组学检测发现，IgG4-RD外泌体刺激后B细胞细胞色素酶C（Cytochrome c, somatic, CYCS）相对丰度升高，且激活下游的补体通路。IgG4-RD过敏分型蛋白质组发现，相较于无过敏的IgG4-RD患者，合并过敏的IgG4-RD患者外泌体蛋白的整合素通路和补体通路被抑制。颌下腺蛋白质组学结果提示，与慢性非特异性涎腺炎比较，IgG4-RD患者颌下腺共发现269个差异表达蛋白，其中141个上调蛋白及128个下调蛋白。细胞浸润富集分析提示，T细胞、B细胞、巨噬细胞及血小板呈现明显活化，且免疫调节相关蛋白表达异常。GO（Gene ontology）分析提示差异表达蛋白富集在信号转导、细胞通讯及免疫反应。KEGG（Kyoto encyclopedia of genes and genomes）通路富集分析提示差异蛋白富集于免疫通路活化、细胞毒性、Fcγ受体介导的细胞吞噬及代谢异常。PPI（Protein-protein interaction）分析提示Lck/Yes相关新蛋白酪氨酸激酶（Lck/Yes-related novel protein tyrosine kinase, LYN），补体受体类型2（Complement receptor type 2, CR2），脾酪氨酸激酶（Spleen tyrosine kinase, SYK），CD72及酪氨酸蛋白磷酸酶非受体6型（Tyrosine-protein phosphatase non-receptor type 6, PTPN6）是节点蛋白，侧面暗示B细胞受体（B cell receptor, BCR）通路活化。同时我们发现，颌下腺的差异蛋白也主要参与外泌体组成并且补体通路出现活化。

结论

IgG4-RD患者血浆来源外泌体补体通路活化。IgG4-RD血浆来源外泌体可促进B细胞分化，在该过程中B细胞CYCS通路活化及补体通路激活。IgG4-RD合并过敏的患者血浆外泌体蛋白质组学表明补体和整合素通路较非过敏组显著下调，提示补体通路和整合素通路与过敏患者的疾病进展相关。IgG4-RD颌下腺蛋白质组学结果发现BCR通路活化、组织浸润外泌体增多、补体通路激活，提示外泌体或可参与IgG4-RD组织损伤。

Abstract

Background and objective:

IgG4 related disease (IgG4-RD) is a chronic fibroinflammatory disease characterized by tufecive swelling of involved organs, elevated serum IgG4 and lymphocytes, plasma cells, especially IgG4+ plasma cells infiltration in involved organs. The most commonly affected organs are submandibular glands, lacrimal glands, parotid gland, pancreas, bile ducts, lung, retroperitoneum/aorta. Allergic symptoms are frequently present in about 60% of IgG4-RD, patients with allergy have higher tendency of head-neck involvement and eosinophila. Irreversible organ function damage occurs due to persistent tissue fibrosis. So, to comprehensively investigate the pathogenesis of IgG4-RD is of urgent need. Protein profiling of plasma-derived exosomes is not elucidated in IgG4-RD. Therefore, the aim of this study is to investigate the protein profiling of plasma-derived exosomes by Liquid chromatography-tandem mass spectrometry (LC-MS/MS). Next, proteomics of IgG4-RD patients with/without allergy were also investigated in order to demonstrate the immune background, relationship and mechanism of allergy to organ specific damage. Submandibular glands (SMGs) are commonly used as the research specimens in patients with IgG4-RD for tissue protein mass spectrometry verification. Finally, the protein expression profiling of submandibular glands in IgG4-RD were evaluated to define whether exosome participated a role in organ damage.

Materials and methods

134 untreated patients with IgG4-RD and 150 gender-age matched healthy controls were enrolled in our study. This study was approved by the ethnic committee, written informed consent were obtained for all patients and controls enrolled. Exosomes were extracted by ultra-centrifuge and exosome extract kit. Exosome proteins were labled by TMT and detected by LC-MS/MS. Differentially expressed proteins (DEPs) were validated by ELISA and WB. B cell differentiation and ROS expression level were detected by flowcytometry analysis. In addition, B cells cultured by IgG4-RD and HC exosomes were detected by LC-MS/MS. Besides, plasma protein profiling of 15 IgG4-RD patients with allergy, 15 IgG4-RD without allergy and 15 gender-age matched were detected. Finally, proteomics expression was detected in 4 biopsy-proven IgG4-RD and 4 biopsy-proven chronic sialadentis by isobaric tags for relative and absolute quantitation (iTRAQ).

Results

A total of 178 differentially expressed proteins were identified in plasma derived exosomes in IgG4-RD patients compared with HCs, and these proteins were enriched predominantly in the complement cascade pathway. Furthermore, reduced expression levels of complement components C3 and C5 in IgG4-RD were correlated with clinical parameters. Following stimulation with IgG4-RD plasma derived exosomes, the percentages of naïve B cells decreased, while those of memory B cells and plasmablasts increased; the levels of cytochrome c, somatic (CYCS) and downstream complement system activation also increased. The proteome revealed integrin pathway and complement cascade pathway were downregulated (inhibited) of IgG4-RD patients with allergy compared with those without allergy, indicating complement system and integrin pathway participated in pathogenesis of IgG4-RD with allergy. A total of 269 DEPs were identified in IgG4-RD SMGs compared with controls, including 141 upregulated proteins and 128 downregulated proteins. In terms of cell subsets infiltration, T cells, B cells, macrophages and platelets were activated significantly. Immune related proteins were altered tremendously in IgG4-RD compared with controls. GO analysis indicated altered proteins enriched in signal transduction, cell communication and immune response in biological process. KEGG pathway analysis showed DEPs mainly enriched in immune pathway activation, cytotoxicity, Fc gamma R-mediated phagocytosis, and metabolic abnormalities. According to disease correlation analysis, altered proteins associated with immune system disease, lymphoproliferative disorders, immunologic deficiency syndromes, homological malignancies. LYN, CR2, SYK, CD72 and PTPN6 were identified as hub proteins, indicating BCR signaling pathway activation. In addition, exosomes were also enriched in SMGs of IgG4-RD.

Conclusion

Proteomic profiling revealed that complement cascade pathway activated plasma derived exosome proteins, which may participate in the pathogenesis of IgG4-RD through complement activation and may be involved in B cell differentiation and activation of the B cell auto-oxidative damage pathway. Inhibition of complement and integrin pathways in plasma exosome of IgG4-RD patients with allergy, indicating complement and integrin involved in disease progression. Exosome enriched in SMGs which may indicate exosome parcipated in tissue damage in IgG4-RD. Further mechanism needs to be evaluated.