中性粒细胞丝氨酸蛋白酶在1型糖尿病自身免疫启动中的作用机制及干预靶点研究

Type 1 diabetes mellitus (T1DM) is an autoimmune disease with an increasing annual global incidence. However, its etiology remains poorly characterized. Our previous research found that circulating protein levels together with enzymatic activities of neutrophil serine proteases (NSP) including neutrophil elastase (NE) and proteinase 3 (PR3), stored in neutrophil primary granules, were markedly elevated in T1DM patients, especially in those with diabetic duration of less than 1 year. Our animal studies showed neutrophil infiltration in pancreas was observed and the level of circulating NE/PR3 enzymatic activities was markedly elevated as early as within one month in diabetic NOD mice. Furthermore, treatment of NOD mice with Sivelestat (a selective NE inhibitor) significantly attenuated insulitis and macrophage polarization from M1 to M2. Our in vitro study showed that recombinant NE was able to directly induce inflammation in MIN6 β cells and promote macrophage migration. Above findings indicate neutrophil-released NE and PR3 is an early mediator of autoimmune diabetes. However, the underlying mechanisms how NE and PR3 activate acquired immunity-mediated destruction of insulin-producing β-cells in T1DM are largely unknown. Based on these findings, we hypothesize that during the early postnatal stage, neutrophils are recruited to the islets possibly by the chemokines CXCL1 and CXCL2 released from β cells and macrophages and subsequently activated, leading to NETosis and release of NE and PR3, which in turn act as pro-inflammatory factors to further augment macrophage infiltration, increase autoantigen release, or exacerbate macrophage-mediated diabetogenic T cell activation, thereby leading to insulitis and β cell destruction. In this study, we will determine whether NE and PR3 are the early mediators of insulitis and autoimmune diabetes in NOD mice; we will elucidate the molecular mechanisms by which NE and PR3 triggers insulitis and pancreatic β cell destruction by mediating immune cell crosstalk; we will explore the therapeutic potential of NE/PR3 inhibitors for treating T1DM; we will conduct a clinical investigation to evaluate the dynamic changes of NE and PR3 in the course of healthy autoantibody-positive subjects with high risk for developing diabetes and patients with T1DM, to evaluate whether or not elevated serum NE and PR3 is causally associated with T1DM in Chinese. This study will provide scientific evidences for the detailed role of NE and PR3 in the development of T1DM and the application of NE and PR3 as predictors and drug targets for T1DM.

目前1型糖尿病（T1DM）病因未明。我们前期发现中性粒细胞分泌的中性粒细胞丝氨酸蛋白酶NE和PR3的变化是中国人群T1DM的早期事件；出生后1月内的NOD小鼠可检测到胰岛中性粒细胞浸润及循环NE/PR3酶活性的增加，NE抑制剂可显著改善NOD小鼠的胰岛炎，重组NE可直接促进β细胞炎症和巨噬细胞迁移，提示NE/PR3激活是T1DM发病中的早期关键事件。然而NE/PR3如何造成β细胞损伤的机制仍不清楚。因此，我们提出中性粒细胞在胰岛中的浸润及激活，释放NE/PR3造成β细胞的早期损伤，介导β细胞释放自身抗原诱发自身免疫反应；阻断NE/PR3在胰岛中的激活是防治T1DM的新途径。本项目将揭示NE/PR3在T1DM发病过程中的激活机制，探讨NE/PR3在启动胰岛炎及自身免疫反应中的作用机理，发展针对NE/PR3为靶点的防治T1DM的新策略，评估利用NE/PR3筛查及诊断T1DM的临床价值。

先天免疫细胞和适应性免疫细胞在T1D的胰岛β细胞自身免疫破坏中都起着关键作用。然而，触发胰岛中先天免疫细胞的招募和激活的早期致病事件仍不清楚。临床研究表明，中性粒细胞和中性粒细胞弹性蛋白酶（NE）与1型糖尿病患者的β细胞自身免疫密切相关。本研究发现新生NOD小鼠胰腺浸润中性粒细胞逐渐增多，胰腺及循环中性粒细胞弹性蛋白酶（NE）水平及活性升高。给予NE抑制剂Sivelestat后，NOD小鼠30周龄时发病率相较于对照组显著降低，且显著推迟了发病时间。机制上，抑制剂给药显著减少了NOD小鼠早期中性粒细胞及巨噬细胞浸润，继而减少了糖尿病致病性CD8+T细胞、炎性Th1及Th17 CD4+T细胞胰岛浸润，体外研究表明，NE直接诱导Min6β细胞和RAW 264.7巨噬细胞产生炎症反应，并促进巨噬细胞迁移。因此，我们得出T1D免疫发病机制新理论—胰岛“二次打击”学说：遗传与环境互相作用诱发机体天然免疫细胞如中性粒细胞在胰岛浸润及激活，引起β细胞初期炎症损伤，造成第一轮打击；释放胰岛β细胞抗原，诱发获得免疫攻击，继发第二轮打击，最终导致β细胞彻底破坏，发生T1D。基于揭示的T1D免疫发病新机制，局部组织升高的中性粒细胞蛋白酶可进入血循环，有望成为预测诊断疾病风险的重要血清指标。