The role of spleen tyrosine kinase (syk) in neutrophil responses to pathogenic fungi.

项目摘要：侵袭性真菌感染(IFI)在目前的实践中对患者构成越来越大的威胁 医药。现代内科医生使用强大的免疫调节疗法对复合体进行干预 疾病，如自身免疫性疾病、恶性肿瘤和移植。虽然这些因素的影响 治疗取得重大临床突破，其免疫抑制作用有深刻的一面 影响，往往使患者面临包括IFI在内的危及生命的感染并发症的高风险。致信地址 这种日益增长的未得到满足的需求，我们已经将注意力转向中性粒细胞，这是一种关键的第一反应先天免疫细胞 是快速清除真菌所必需的。事实上，中性粒细胞不足或功能障碍的患者处于 IFI的风险最高。在巨噬细胞中，另一种先天免疫细胞，识别复杂的多层 碳水化合物真菌的细胞壁部分是通过凝集素受体Dectin-1来完成的，它与β-1，3- 葡聚糖和激活脾酪氨酸激酶(SYK)导致促炎细胞因子上调 制作。虽然SYK活性对巨噬细胞的杀菌活性是必不可少的，但它在中性粒细胞中的作用尚未得到证实。 被定义。几个问题是确定SYK在中性粒细胞中的作用的主要障碍。第一, 中性粒细胞是短命的细胞(24小时)，第二，遗传操作是困难的，因为他们的终端 差异化。此外，SYK缺陷小鼠的产生是不可能的，因为SYK对 胚胎血管发育。尽管存在这些挑战，我们还是试图确定SYK在中性粒细胞中的作用。 通过以下关键观察，了解真菌的相互作用。首先，我们通过以下方式解决中性粒细胞的长寿问题 使用转录因子HoxB8的有条件激活。通过表达HoxB8，前体干细胞 细胞或成髓细胞保持无限分裂，在HoxB8失活后，成髓细胞分化 允许产生无限的中性粒细胞。第二，我们证明HoxB8系统是服从于 CRISPR/Cas9编辑允许生成缺乏SYK的中性粒细胞。第三，为了去卷曲 复杂的真菌表面，我们利用纯化的真菌碳水化合物来探测真菌样颗粒 中性粒细胞的特异性反应。第四，我们证明了HoxB8中性粒细胞可以在体内补充中性粒细胞。 在中性粒细胞减少和念珠菌感染的小鼠模型中的作用。以确定其分子机制 在中性粒细胞真菌天然免疫中，我们提出了三个独立的特异性目标：(1)测定中性粒细胞中的 SYK缺陷的HoxB8中性粒细胞对特定微生物刺激的体外反应，(2)确定SYK在 中性粒细胞效应器功能对酵母形态类型的响应，以及(3)描绘了SYK对 体内中性粒细胞-真菌免疫。在我们的研究结束时，我们将阐明亚细胞机制 负责中性粒细胞-真菌的发病机制，希望开发改进的杀菌策略来 应对侵袭性真菌病日益增长的临床负担，特别是在高风险患者中。 好了！

Project Summary: Invasive fungal infections (IFI) constitute a rising threat to patients in the current practice of medicine. The modern physician uses potent immunomodulatory therapeutics to intervene on complex illnesses such as autoimmune disorders, malignancy and transplantation. While the impact of these therapeutics have resulted in major clinical breakthroughs, their immune inhibitory effects have profound side effects, often leaving patients at high risk for life-threatening infectious complications including IFI. To address this growing unmet need, we have turned attention to neutrophils, a critical first-responder innate immune cell required for rapid fungal elimination. In fact, patients with insufficient or dysfunctional neutrophils are at the highest risk for IFI. In macrophages, another innate immune cell, the recognition of the complex multi-layered carbohydrate fungal cell wall is performed, in part, through the lectin receptor, Dectin-1, which binds to β-1,3- glucan and activates spleen tyrosine kinase (syk) resulting in upregulation of pro-inflammatory cytokine production. While syk activity is essential for fungicidal activity in macrophages, its role in neutrophils has yet to be defined. Several issues present major impediments towards defining the role of syk in neutrophils. First, neutrophils are short lived cells (<24hrs) and second, genetic manipulation is difficult owing to their terminal differentiation. Moreover, the generation of syk-deficient mice is not possible given syk contribution to embryonic vascular development. Despite these challenges, we sought to define the role of syk in neutrophil- fungal interactions through the following key observations. First, we address the longevity of neutrophils by using conditional activation of the transcription factor, HoxB8. Through expression of HoxB8, precursor stem cells or myeloblasts remain indefinitely dividing, and following HoxB8 inactivation, myeloblasts differentiate permitting generation of unlimited neutrophils. Second, we demonstrate that the HoxB8 system is amenable to CRISPR/Cas9 editing allowing generation of syk-deficient neutrophils. Third, in order to deconvolute the complex fungal surface, we developed fungal-like particles using purified fungal carbohydrates to probe specific neutrophil responses. Fourth, we demonstrate that HoxB8 neutrophils complement in vivo neutrophil function in a mouse model of neutropenia and Candida infection. To determine the molecular mechanism of syk in fungal innate immunity in neutrophils, we propose three independent specific aims: (1) Determine the in vitro response of syk-deficient HoxB8 neutrophils to defined microbial stimuli, (2) Define the role of syk in neutrophil effector function in response to yeast morphotype, and (3) Delineate the contribution of syk to neutrophil-fungal immunity in vivo. At the conclusion of our studies, we will clarify the subcellular mechanisms responsible for neutrophil-fungal pathogenesis with the hope to develop improved fungicidal strategies to address the growing clinical burden of invasive fungal disease, especially in the highest risk patients. !