Molecular Analysis Of Leukocyte Activation By Chemoattractants

该项目的目的是确定血液白细胞迁移到发炎或感染的特定组织部位的分子机制和生物学背景。我们已经集中在介导这一过程的趋化蛋白，并确定了一个大家族的趋化受体，部署在白细胞表面的成员。我们还鉴定了由病毒（包括疱疹病毒、痘病毒和HIV）产生的不同化学引诱物和化学引诱物受体模拟物。我们使用基因组学、分子生物学、细胞生物学和流行病学作为分析这些分子的主要方法。一个主要的目标是确定具体的疾病协会个别化学引诱物和化学引诱物受体，以确定潜在的新的治疗靶点。 一个关键的策略是分析疾病模型中基因敲除小鼠的表型以及人类化学引诱物系统基因突变患者的表型。 1.）在2022财年，我们报告了一例6岁男孩的重度中性粒细胞减少症病例。 患者具有与WHIM综合征一致的经典CXCR 4突变。 该病例说明了这种疾病的表现的异质性。 2.）在FY 22中，我们报告了内毒素对WHIM综合征模型小鼠的临床和血液学影响。 疣、低丙种球蛋白血症、感染和骨髓增生异常综合征（WHIM）免疫缺陷是由常染色体显性遗传的功能获得性CXCR 4突变引起的，该突变促进了由成熟白细胞的骨髓滞留引起的重度泛白细胞减少症。因此，WHIM患者会发生复发性细菌感染;然而，败血症并不常见。为了研究这种临床二分法，我们用LPS攻击WHIM模型小鼠。WHIM和野生型（WT）小鼠的LD 50相似，LPS诱导WT小鼠的急性淋巴细胞减少症与Cxcr 4无关。相比之下，在WHIM小鼠中，LPS不影响循环T细胞水平，但B细胞水平由于Cxcr 4 high晚期前B细胞的选择性、细胞内在性和Cxcr 4 WHIM等位基因依赖性出现而恶性增加，这是大肠杆菌感染的表型模式。在WT和WHIM小鼠中，CXCR 4拮抗剂AMD 3100迅速增加循环淋巴细胞水平，然后在随后的LPS处理后迅速收缩。因此，LPS诱导的淋巴细胞减少症是CXCR 4独立的，WHIM突变不会增加临床LPS敏感性。LPS对晚期前B细胞动员的异常WT Cxcr 4非依赖性，但Cxcr 4 WHIM依赖性的促动员作用揭示了变体受体的不同信号传导途径。 3.）第三章在2022财年，我们回顾了实验室的一项新发现，涉及趋化因子与磷脂酰丝氨酸结合以清除凋亡细胞。这是我们2021年在PLoS Biology上发表的论文的后续。 4.）在2022财年，我们还参与了利用基因组学识别免疫缺陷患者新表型和疾病的合作工作。 特别是，如21财年年度报告所述，这一努力导致发现SASH 3缺陷是一种新的先天性免疫缺陷。 在2022财年，该部门还推进了其在克氏锥虫发病机制、小鼠巨细胞病毒发病机制、WHIM综合征的新型治疗、WHIM综合征的基因治疗、允许通用移植的新型临床前骨髓预处理方案以及关于COVID-19发病机制的新工作方面的工作;然而，这些项目正在进行中，在本报告期内没有发表工作。

The aim of this project is to define the molecular mechanisms and biological contexts for blood leukocyte migration to specific tissue sites that are inflamed or infected. We have focused on chemoattractant proteins that mediate this process and have identified members of a large family of chemoattractant receptors that are deployed on the leukocyte cell surface. We have also identified members of a diverse group of chemoattractant and chemoattractant receptor mimics made by viruses, including herpesviruses, poxviruses and HIV. We use genomics, molecular biology, cell biology and epidemiology as the principle methods for analyzing these molecules. A major goal is to identify specific disease associations of individual chemoattractants and chemoattractant receptors, in order to identify potential new therapeutic targets. A key strategy is to analyze phenotypes of gene knockout mice in disease models as well as phenotypes in patients with mutations in human chemoattractant system genes. 1.) In FY22, we reported a case of severe neutropenia in a 6-year-old boy. The patient has a classic CXCR4 mutation consistent with WHIM syndrome. The case illustrates the heterogeneity of presentation in this disorder. 2.) In FY22, we reported the clinical and hematologic effects of endotoxin in WHIM Syndrome model mice. Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome immunodeficiency is caused by autosomal dominant gain-of-function CXCR4 mutations that promote severe panleukopenia caused by bone marrow retention of mature leukocytes. Consequently, WHIM patients develop recurrent bacterial infections; however, sepsis is uncommon. To study this clinical dichotomy, we challenged WHIM model mice with LPS. The LD50 was similar in WHIM and wild-type (WT) mice, and LPS induced acute lymphopenia in WT mice that was Cxcr4 independent. In contrast, in WHIM mice, LPS did not affect circulating T cell levels, but the B cell levels anomalously increased because of selective, cell-intrinsic, and Cxcr4 WHIM allele-dependent emergence of Cxcr4high late pre-B cells, a pattern that was phenocopied by Escherichia coli infection. In both WT and WHIM mice, the CXCR4 antagonist AMD3100 rapidly increased circulating lymphocyte levels that then rapidly contracted after subsequent LPS treatment. Thus, LPS-induced lymphopenia is CXCR4 independent, and a WHIM mutation does not increase clinical LPS sensitivity. Anomalous WT Cxcr4-independent, but Cxcr4 WHIM-dependent, promobilizing effects of LPS on late pre-B cell mobilization reveal a distinct signaling pathway for the variant receptor. 3.) In FY22, we reviewed a new discovery from the lab involving chemokine binding to phosphatidylserine for apoptotic cell clearance. This is a follow up to our 2021 paper in PLoS Biology. 4.) In FY22, we also contributed to collaborative work using genomics to identify novel phenotypes and diseases in immunodeficiency patients. In particular, as detailed in the FY21 annual report, this effort led to the discovery of SASH3 deficiency as a novel inborn error of immunity. In FY22, the section also advanced its work on T.cruzi pathogenesis, Mouse cytomegalovirus pathogenesis, novel treatment for WHIM syndrome, gene therapy for WHIM syndrome, a novel preclinical bone marrow conditioning protocol that allows universal transplantation, and new work on covid-19 pathogenesis; however, these projects are ongoing and did not result in published work in this reporting period.