小鼠肺移植早期再灌注损伤中NETs作用的可视化研究

Reperfusion injury (RI) is mainly attributed to complement-mediated immune attack, but various attempts to inhibit complement have so far been disappointing. We hypothesize that complement activation is thriving as Neutrophil Extracellular Traps (NETs) cannot be removed in time by impaired donor alveolar macrophages, and fading as more and more recipient -derived monocytes are developing into alveolar macrophages in graft. Firstly, the correlation between NETs overstocking and persistent complement activation is detected in B6=>B6 isogenic grafts with varying damage. In group A1, A2 and A3, The donor lungs suffer from 1h, 24h or 72h of cold ischemia (CI) respectively. Recipients will be sampled at 2h, 4h, 8h, 24h, 3d and 7d after reperfusion. The dynamics of macrophages and neutrophils near NETs will be measured by intraviatl multiphoton imaging,FACS and ELISA。Secondly, the consequence on NETs and complement activation will be observed by changing macrophage phagocytic function or local nuclease level. In group B1 (1h CI+24h reperfusion), apoptosis is induced in donor alveolar macrophages 4d and 1d before lung harvest. In group B2 (72h CI+24h reperfusion), 5×105/100ul alveolar macrophages will be adoptively transferred to recipients immediately after reperfusion.I n group B3 (72h CI+24h reperfusion), 50ug/50ul of DNase 1 will be instilled into chest cavity before closing. The behaviour of macrophages and neutrophils near NETs will be observed. Finally, the feasibility of targeting NETs will be tested in a model of lung retransplantation. RI in the second recipient is expected to be ameliorated greatly by preconditioning in the first recipient, a mimic of Ex vivo lung perfusion device. Our study will answer if impaired NETs degradation is a critical upstream event in RI, and if NETs is a potential target to prevent complement from being over activated.

补体主导的免疫攻击是再灌注损伤主因，但抑制补体的诸多尝试差强人意。我们假设，补体激活因供者肺泡巨噬细胞受损、中性粒细胞外陷阱(NETs)滞留而盛，随受者单核细胞分化为巨噬细胞、NETs被清除而衰。在B6=>B6肺移植，首先确定NETs滞留与持续补体激活的关联。A1-A3组供肺分别经历1h、24h、72h冷缺血，再灌注不同时间点获取标本。活体多光子荧光显微成像观察NETs附近巨噬细胞与中性粒细胞动态变化。其次，改变巨噬细胞吞噬功能或局部核酸酶水平，观察其对NETs和补体激活的影响。B1组取肺前诱导供者巨噬细胞凋亡，B2组受者再灌注后立即输注巨噬细胞。B3组关胸前胸内滴注DNase 1。最后用肺再移植模型模仿体外灌流装置，受损肺在第一受者调理24h，再植入第二受者后损伤有望减轻。本研究将明确"肺巨噬细胞损伤-NETs滞留-补体持续亢进"的关键机制，NETs有望成为肺再灌注损伤治疗靶。

原发性移植物功能障碍（PGD）是肺移植术后危及生命的严重并发症，缺血再灌注损伤（IRI）通常被认为是PGD的主要原因。中性粒细胞胞外陷阱（NETs）可促进无菌炎症。我们假设，补体激活因供者肺泡巨噬细胞受损、中性粒细胞外陷阱 (NETs)滞留而盛随受者单核细胞分 化为巨噬胞、NETs被清除而衰。.首先，我们确定了B6 小鼠同基因移植肺中NETs累积和持续补体激活之间的相关性。冷缺血（CI）1h，72h或96h的供肺在再灌注后24h发生不同程度的损伤。 RNA测序证明补体系统在IRI中被激活。双光子成像显示,随着IRI严重程度的增加，NETs积压越来越多。蛋白质印迹分析表明肺泡巨噬细胞功能失常。.其次，我们确定了DNase对NETs的影响。双光子成像显示，在静脉注射DNase后NETs溶解，但更多的中性粒细胞募集。组织学显示，NETs溶解处的肺泡结构严重受损，并有大量炎性细胞浸润。 DNase治疗似乎释放了NETs中的蛋白酶，导致局部的肺损伤。 这个现象在用DNase治疗的肺囊性纤维化患者中也有报道。进一步研究表明，NETs片段通过激活募集的单核细胞刺激先天免疫反应。.第三，确定了NETs在IRI中的作用。长时间的冷缺血会在再灌注后诱导坏死性凋亡（necroptosis）。96 h冷缺血24h再灌注之后，移植肺显示典型的坏死性凋亡的病理：气道上皮细胞肿胀、呈半透明，但胞核和细胞膜保持完整。免疫组化p-MLKL染色确定，受累的细胞主要是气道上皮细胞，而血管内皮并未明显受累。冷缺血诱导的IRI可导致气道上皮受损，通过pMLKL多聚体形成的孔隙释出。通过抑制PAD4阻止NETs的正常释放，结果上皮损伤加重，大量胞浆内容为被释放。由于NETs碎片的过量累积，C3a和C5a的激活被上调，导致非经典Ly6C - 单核细胞的募集。 Ly6C -）单核细胞转化为促炎巨噬细胞（M1），招募更多的经典单核细胞和中性粒细胞。.总之，NET是一柄双刃剑。一方面，NETs可以封堵IRI中凋亡性坏死引起的细胞渗漏；另一方面，如果NETs的碎片积压，将持续激活补体系统，导致固有免疫反应的激活。 外源性DNase会溶解DNA纤维，释放有毒的蛋白酶和细胞因子，从而加剧移植肺损伤。