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，其具有NADPH氧化酶的各种缺陷，使其功能失调，并容易发生侵袭性和危及生命的细菌和真菌感染。虽然缺陷氧化酶在免疫缺陷中的作用是无可争议的，但疾病中的紊乱炎症（例如，伤口愈合不良、阻塞性肉芽肿和自身免疫），这可能与感染有关，也可能与感染无关，仍然无法解释。据推测，CGD中的紊乱性炎症是由于缺陷的磷脂酰丝氨酸（PS）暴露和聚集在吞噬CGD的中性粒细胞上，导致吞噬细胞的识别和吞噬受损。初步数据表明，在中性粒细胞凋亡过程中，需要适当的PS暴露的NADPH氧化酶的功能，这需要协调激活磷脂（PL）争先恐后地暴露PS，和失活的氨基磷脂转位酶返回PS的质膜内小叶。该提案旨在通过评估PL混乱和氨基磷脂移位酶活性来确定CGD中性粒细胞上PS暴露的缺陷。假设去活化CGD中性粒细胞将具有减少的PL混乱和持续的氨基磷脂移位酶活性。PS暴露于凋亡细胞上是吞噬细胞识别所必需的，并且通过以立体特异性方式识别PS的受体PSR介导。PSR沿着与其他束缚受体的结合通过免疫学“沉默”机制介导凋亡中性粒细胞的摄取，导致TGF β产生和抑制吞噬细胞的促炎细胞因子产生。因此，我们假设PS缺陷的凋亡CGD中性粒细胞将显示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细胞溶解，释放有害蛋白。提出了体外吞噬细胞吞噬试验和体内小鼠模型。据预测，相对于凋亡的正常中性粒细胞，将凋亡的CGD中性粒细胞滴注到野生型小鼠的发炎的腹膜中将显示延迟的清除、TGF β产生的抑制和促炎细胞因子产生的增强; CGD中性粒细胞表面上PS的恢复将逆转这一点。最后，在PS脂质体存在下用无菌真菌菌丝注射CGD小鼠将导致积聚的嗜中性粒细胞的清除增强，TGF β产生增强和炎症消退。预计来自本研究的数据将确定新的潜在治疗靶点，以解决CGD的紊乱性炎症。

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.