Characterization and therapeutic targeting of neutrophil serine proteases in ANCA vasculitis

抗中性粒细胞细胞质抗体（ANCA）相关血管炎（AAV）是一种坏死性小血管炎症性疾病，可影响各个器官，常在肾脏表现为坏死性新月形肾小球肾炎伴快速进展性肾功能衰竭。ANCA结合中性粒细胞和单核细胞表面的蛋白酶3 （PR3）或髓过氧化物酶（MPO），这些细胞只表达这些自身抗原。ANCA激活这些粘附并破坏内皮的细胞，导致全身性血管炎。我们描述了几种损伤机制，其中一些涉及酶活性中性粒细胞丝氨酸蛋白酶（NSPs）。NSP家族包括PR3、人中性粒细胞弹性酶（HNE）和组织蛋白酶G （CatG）以及低丰度的NSP4。PR3是一种独特的NSP，它提供了主要的ANCA抗原。nsp是由组织蛋白酶C （CatC）去除n端二肽而产生的非活性酶原形式。CatC的功能缺失突变阻止了NSP的成熟并导致酶原降解。药理CatC抑制剂有可能取消NSP成熟，从而消除NSP蛋白和蛋白水解活性。因此，药物抑制CatC可能为AAV提供了一种新的治疗策略。根据这一建议，我们将(i)系统地表征人类和小鼠中性粒细胞和单核细胞中的NSP蛋白和蛋白水解活性，（ii）通过药物CatC抑制靶向NSP，利用干细胞分化模型和内皮细胞来表征NSP介导的损伤机制，以及（iii）利用小鼠AAV疾病模型验证药物CatC抑制可以防止anca诱导的血管炎的假设。我们的发现将有助于理解anca诱导的将NSPs与血管炎联系起来的疾病机制。此外，该研究可以鼓励临床试验探索药物CatC抑制作为AAV的新治疗方法。

Anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) are necrotizing small-vessel inflammatory diseases that can affect every organ, frequently manifesting in the kidneys as necrotizing crescentic glomerulonephritis with rapidly-progressive renal failure. ANCA bind to either proteinase 3 (PR3) or myeloperoxidase (MPO) on the surface of neutrophils and monocytes that exclusively express these autoantigens. ANCA activate these cells that adhere to and damage the endothelium leading to systemic vasculitis. We characterized several injury mechanisms some of which involve enzymatically active neutrophil serine proteases (NSPs). The NSP family consist of PR3, human neutrophil elastase (HNE) and cathepsin G (CatG) and low-abundant NSP4. PR3 is a unique NSP for it provides the major ANCA antigen. NSPs are generated from inactive zymogen proforms by cathepsin C (CatC) that removes an N-terminal dipeptide. Loss-of-function mutations in CatC prevents NSP maturation and leads to zymogen degradation. Pharmacological CatC inhibitors have the potential to abrogate NSP maturation thereby eliminating NSP proteins and proteolytic activity. Thus, pharmacological CatC inhibition provides possibly a novel treatment strategy in AAV. With this proposal, we will (i) systematically characterize NSP proteins and proteolytical activity in human and murine neutrophils and monocytes, (ii) target NSPs by pharmacological CatC inhibition to characterize NSP-mediated damage mechanisms using stem cell differentiation models and endothelial cells, and (iii) test the hypothesis that pharmacological CatC inhibition protects from ANCA-induced vasculitis using murine AAV disease models. Our findings will improve understanding ANCA-induced disease mechanisms that link NSPs to vasculitis. In addition, the study could encourage clinical trials exploring pharmacological CatC inhibition as a new treatment for AAV.