中性粒细胞通过NETs释放LL37活化浆细胞样树突状细胞在ANCA相关性血管炎中的作用

Neutrophils are effector and target cells of anti-neutrophil cytoplasm antibodies (ANCA) associated vasculitis (AAV) and play a key role in the immune mechanism of AAV. Neutrophil extracellular net (NETs) is a unique way of death which neutrophils extrude large amounts of nuclear DNA into the extracellular space in the form of web-like DNA structures (the NETs).NETs contain DNA as well as large amounts of antimicrobial peptide LL37,a neutrophil protein that facilitate the uptake and recognition of self DNA by plasmacytoid DCs (pDCs).. This research based on our prior findings: 1. The appearance of NETs increase in AAV patients; 2. ANCA can promote the formation of NETs, the release of intracellular autoantigen and antimicrobial peptide LL37;3. BDCA2, the marker of pDCs, and LL37 are co-deposed at the inflamed tissue of AAV patients. According to recent studies on NETs, we speculate that neutrophils may release LL37 through the formation of NETs, activate pDCs by LL37-DNA complex crosslink with TLR9, then break immune tolerance. Herein ,we Investigate the influence of NETs on pDCs activation and maturation in vitro and vivo experiments; then observe the pDCs activation, maturation, antigen presented effects, and immune pathological injury effect by interventing LL37-DNA-TLR9 ligand receptor model. In this case, we may find a clue for the treatment of AAV by interventing the crosslinking of LL37 and TLR9 and rebuild immune tolerance.

中性粒细胞是抗中性粒细胞胞浆抗体（ANCA）相关性血管炎（AAV）发病的核心。中性粒细胞胞外捕网（NETs）是一种独特的死亡方式。本项目前期发现AAV患者外周血NETs形成增加，肾组织内存在抗微生物肽LL37与pDCs特异性标志BDCA2共沉积；体外发现ANCA可诱导NETs形成释放自身抗原及LL37。结合NETs研究进展推测：中性粒细胞可能通过NETs释放内源性危险信号LL37-DNA复合物，以配体-受体结合模式与TLR-9交联，活化pDCs导致抗原呈递，打破自身耐受。本项目拟通过体外LL37-DNA荧光示踪系统及流式细胞术观察pDCs对抗原的吞噬及活化状态；进而通过ODN-TTAGGG特异性抑制及人工TLR-9配体干预LL37-DNA复合物与TLR9交联，体外及AAV模型观察pDCs抗原递呈能力及免疫效应，以期通过干预这种配体-受体结合，重树免疫耐受，为寻找治疗AAV的新靶点提供线索。

中性粒细胞是抗中性粒细胞胞浆抗体（ANCA）相关性血管炎（AAV）发病的核心。中性粒细胞胞外捕网（NETs）是一种独特的死亡方式。本项目前期发现AAV患者外周血NETs形成增加，肾组织内存在抗微生物肽LL37与pDCs特异性标志BDCA2共沉积；体外发现ANCA可诱导NETs形成释放自身抗原及LL37。本项目采用流式活细胞分选、激光共聚焦、免疫荧光、流式细胞检测、酶联免疫吸附测定等方法，初步明确明确NETs介导pDCs 异常活化，在AAV 免疫机制中的地位。主要结果：1.AAV患者外周血中性粒细胞通过NETs形成释放抗菌肽LL37及暴露ANCA靶抗原LAMP-2、MPO、PR3；2.AAV 患者外周血中性粒细胞通过NETs 释放LL37，可刺激体外分离的健康人外周血pDC细胞活化、成熟并产生IFN-α，而打破自身耐受；3.拮抗LL37或TLR9功能后， AAV患者NETs刺激pDC细胞活化、成熟及IFN-α产生明显下降。4.成功采用琼脂糖明胶皮下埋植法富集、分离具有自发NETs倾向的小鼠中性粒细胞，可为中性粒细胞及NETs的进一步研究提供数量充足稳定的细胞来源。结论：AAV患者体内外周血及募集至受累肾脏局部炎症损伤组织的中性粒细胞NETs形成增加，释放大量LL37与自身DNA形成LL37-DNA复合物，通过TLR9途径活化pDC细胞，产生大量IFN-α，参与AAV炎症反应及免疫应答提示调节LL37-TLR9 途径可影响pDCs 免疫应答而成为干预AAV 进展的新靶点。