Defective PS Exposure in Neutrophil Apoptosis in CGD

描述（由申请人提供）：慢性肉芽肿病（CGD）是一种罕见的遗传性疾病，由NADPH氧化酶的各种缺陷导致其功能失调，易导致侵袭性和危及生命的细菌和真菌感染。虽然氧化酶缺陷在免疫缺陷中的作用是无可争议的，但疾病中的无序炎症（如伤口愈合不良、肉芽肿梗阻和自身免疫）可能与感染有关，也可能与感染无关，仍未得到解释。据推测，CGD中的紊乱炎症是由于磷脂酰丝氨酸（PS）暴露缺陷和凋亡的CGD中性粒细胞积累导致吞噬细胞识别和吞噬受损。初步数据表明，在中性粒细胞凋亡过程中，适当的PS暴露需要NADPH氧化酶的功能，这一事件需要磷脂（PL）的协调激活以暴露PS，以及将PS返回质膜内小叶的氨基磷脂转位酶的失活。本研究旨在通过评估PL置乱和氨基磷脂转位酶活性来确定PS暴露在CGD中性粒细胞上的缺陷。据推测，凋亡的CGD中性粒细胞会减少PL乱序和维持氨基磷脂转位酶活性。PS暴露在凋亡细胞上是吞噬细胞识别所必需的，并通过受体介导，PSR以立体特异性的方式识别PS。PSR与其他栓系受体的结合通过免疫“沉默”机制介导凋亡中性粒细胞的摄入，导致tgf - β的产生和抑制吞噬细胞的促炎细胞因子的产生。因此，我们假设PS缺陷的凋亡的CGD中性粒细胞会表现出受损的吞噬细胞识别和吞噬，并促进促炎细胞因子的产生。此外，未吞噬的CGD中性粒细胞将经历凋亡后的细胞溶解，释放有害蛋白。提出了体外吞噬吞噬试验和体内小鼠模型。我们预测，相对于凋亡的正常中性粒细胞，注入野生型小鼠炎症腹膜后，凋亡的CGD中性粒细胞会出现清除延迟，抑制tgf - β的产生，增强促炎细胞因子的产生；CGD中性粒细胞表面PS的恢复将逆转这种情况。最后，在PS脂质体存在的情况下，给CGD小鼠注射无菌真菌菌丝，可以增强对积累的中性粒细胞的清除，增强tgf - β的产生，并缓解炎症。预计这项研究的数据将确定新的潜在治疗靶点，以解决CGD的紊乱炎症。

DESCRIPTION (provided by applicant): Chronic granulomatous disease (CGD) is a rare, inherited disorder of various defects of the NADPH oxidase rendering it dysfunctional and predisposing to invasive and life-threatening bacterial and fungal infection. While the role of the defective oxidase in immunodeficiency is undisputed, disordered inflammation in the disease (e.g., poor wound healing, obstructing granulomata and autoimmunity), which may or may not be associated with infection, remains unexplained. It is hypothesized that disordered inflammation in CGD results from defective phosphatidylserine (PS) exposure and accumulation on apoptosing CGD neutrophils that leads to impaired recognition and engulfment by phagocytes. Preliminary data suggest that the functioning NADPH oxidase is required for appropriate PS exposure during neutrophil apoptosis, an event that requires coordinate activation of phospholipid (PL) scrambling to expose PS, and inactivation of the aminophospholipid translocase which returns PS to the plasma membrane inner leaflet. This proposal seeks to define the defect in PS exposure on CGD neutrophils by assessing both PL scrambling and aminophospholipid translocase activity. It is hypothesized that apoptosing CGD neutrophils will have diminished PL scrambling and sustained aminophospholipid translocase activity. Exposure of PS on apoptotic cells is required for recognition by phagocytes and is mediated through a receptor, the PSR, that recognizes PS in a stereospeciflc manner. Engagement of the PSR along with other tethering receptors mediates the ingestion of apoptotic neutrophils by an immunologically "silent" mechanism that results in TGFbeta production and suppression of pro-inflammatory cytokine production from the phagocyte. Thus, we hypothesized that PS deficient apoptotic CGD neutrophils will show impaired phagocytic recognition and engulfment, and promote pro-inflammatory cytokine production. Furthermore, unengulfed CGD neutrophils will undergo post-apoptotic cytolysls releasing injurious proteins. Both in vitro phagocytic engulfment assays and in vivo murine models are proposed. It is predicted that apoptotic CGD neutrophils, relative to apoptotic normal neutrophils, instilled into the inflamed peritonea of wild type mice will show delayed clearance, suppression of TGFbeta production and enhanced pro-inflammatory cytokine production; restoration of PS on the surface of CGD neutrophils will reverse this. Finally, injection of CGD mice with sterile fungal hyphae in the presence of PS liposomes will lead to enhanced clearance of accumulating neutrophils, enhanced TGFbeta production and resolution of inflammation. It is expected that data from this investigation will identify novel potential therapeutic targets to address disordered inflammation of CGD.