Identification and Characterization of Signaling Pathways and Mediators Regulating Mast Cell-Related Disorders

调节肥大细胞相关疾病的信号通路和介质的鉴定和表征

• 批准号: 10014234
• 负责人: Dean D Metcalfe
• 金额: $ 146.83万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014234
• 关键词: Adhesions; Affinity; Alkaline Phosphatase; Alleles; Allergic; Allergic Reaction; Allergic inflammation; Cell Proliferation; Cell Surface Receptors; Cells; Characteristics; Collaborations; Complex; Critical Pathways; Data; Dermis; Disease; Disease Management; Disease Marker; Disease Progression; Effector Cell; Event; Exhibits; Fc epsilon RI; Flow Cytometry; G-Protein-Coupled Receptors; Genetic Transcription; Genotype; Goals; Hepatic Stellate Cell; Hepatomegaly; Histamine; Human; IgE; IgE Receptors; Immune; Immunofluorescence Immunologic; Individual; Inflammation; Inherited; Interleukin-6; Investigation; JAK2 gene; Knowledge; Link; Liver; Liver Fibrosis; MEKs; Mast Cell Neoplasm; Mechanics; Mediating; Mediator of activation protein; Missense Mutation; Mus; Nature; Oncogenic; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Process; Production; Receptor Protein-Tyrosine Kinases; Regulation; Reporting; Resolution; Role; SPHK1 enzyme; STAT3 gene; Serum; Severity of illness; Signal Pathway; Signal Transduction; Skin; Source; Stains; Stat5 protein; Stimulus; Systemic Mastocytosis; Therapeutic Intervention; Tissues; Tryptase; Tyrosine Kinase Inhibitor; Urticaria; Variant; antigen binding; autocrine; base; cancer cell; cell behavior; cell growth; cytokine; extracellular; extracellular vesicles; genetic variant; human disease; in vivo; intercellular communication; liver function; mast cell; mastocytosis; neoplastic; neoplastic cell; patient response; receptor; response; sphingosine 1-phosphate; stellate cell; vibration

Mast cells are effector and regulatory cells involved in the pathogenesis of allergic inflammation, generally through the activation of the high affinity IgE receptor (Fc-epsilon-RI). Binding of an antigen to IgE/Fc-epsilon-RI complexes initiates intracellular signaling events that drive mast cell responses. In addition, signaling by other cell surface receptors can activate mast cells or modify their responses to the IgE receptor. A deeper understanding of the signaling mechanisms initiated by receptors expressed in mast cells and their crosstalk can give clues on the regulation of allergic reactions and treatment options for human disease. Recently, we found that an adhesion G-protein coupled receptor, ADGRE2 (EMR2) is abundantly expressed in human mast cells and that a missense variant (p.C492Y) of ADGRE2 identified in patients with autosomal dominant vibratory urticaria associates with disease presentation. Patients with this variant exhibit mast cell hyperreactivity upon mechanical stimuli of repetitive nature resulting in localized hives, increased histamine levels in serum and increased extracellular tryptase staining in the dermis compared to normal subjects. The p.C492Y variant destabilizes a non-covalent interaction between the alpha and beta subunits of ADGRE2, rendering the receptor more prone to dissociating the alpha subunit after vibration and resulting in its activation. In fiscal year (FY) 2019, in collaboration with Drs. Schwartz, Lyons, and Milner we have unveiled another mechanism for the activation of ADGRE2 by proteolytic cleavage of the alpha ADGRE2 subunit mediated by an uncommon heterotretramer of mast cell tryptase. Heterotetramers of alpha and beta tryptase are present in patients with hereditary alpha-tryptasemia, but not in other individuals that usually have beta homotetramers. This mechanism links their genotype with the higher responsiveness of these patients to vibratory stimuli in the skin. Another focus in our lab has also been the study of mast cell proliferative disorders. In systemic mastocytosis (SM), neoplastic accumulation of mast cells associates with genetic variants, particularly the missense mutation D816V, in the tyrosine kinase receptor KIT, rendering it constitutively active. Normal KIT signaling is important for the differentiation, proliferation and survival of mast cells and also enhances mast cell responses to allergic stimulation. However, oncogenic signaling due to D816V or other KIT variants results in abnormal mast cell responses. We have previously implicated the sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) axis as a critical pathway in oncogenic KIT signaling leading to abnormal mast cell growth, a knowledge that can be helpful when considering alternatives approaches to the treatment of aggressive SM, where tyrosine kinase inhibitors have shown limited long-term improvement. In FY 2019, we have demonstrated a role for aberrant signaling of mast cells with D816V-KIT in the persistent IL-6 production in SM. Patients with SM have increased serum levels of IL-6 in association with disease severity and progression. However, the cell source and mechanisms involved are not well understood. We found that intracellular IL-6 staining by flow cytometry and immunofluorescence was primarily associated with mast cells and suggested a higher percentage of IL-6 positive mast cells in patients with higher D816V allelic burden. Among the aberrant signals from D816V-KIT, we identified increased JAK2 activity and MEK/ERK- and PI3K-derived signals as drivers of IL-6 transcription and consequent IL-6 release, the former two by regulating the activation/expression of STAT5. The involvement of STAT5 in persistent IL-6 production in D816V-KI mast cells contrasts with mechanisms described in other malignant cells where autocrine feed-forward loops involving IL-6 and STAT3 are common. The study provides the first clues into mechanisms leading to persistent IL-6 production in mastocytosis and potential target molecules for therapeutic intervention. During FY 2019 we have also completed studies on the identification of other vehicles of intercellular communication released by mast cells that may contribute to the pathology of mast cell proliferative diseases. We demonstrated that serum of patients with SM contains elevated concentrations of extracellular vesicles (EVs) with a mast cell signature and which associate with markers of disease severity. Based on the associations of the number of EVs in SM with hepatomegaly and alkaline phosphatase levels, a marker of liver function, we asked whether these EVs could contribute to the abnormal liver pathology associated with mastocytosis. In FY 2019, we reported that KIT contained in these EVs in macrocytosis was transferred into hepatic stellate cells eliciting proliferation, cytokine production, and differentiation, processes known to occur in hepatic fibrosis pathologies. Confirming these conclusions, the effects were reduced by KIT inhibition or neutralization and recapitulated by enforced expression of KIT or constitutively active D816V-KIT. Our data also indicated that stellate cells were activated in vivo in livers from mice injected with SM-EVs. This study suggests that EVs in SM have the potential to induce a fibrotic phenotype in stellate cells, a characteristic change associated with liver pathology, by introducing functionally active KIT into stellate cells. The identification of EVs from neoplastic cells in mastocytosis has opened the possibility that their specific cargos can be shuttled into cells other than stellate cells, dysregulating their function and thus contributing to some pathological aspects associated with mastocytosis. Studies of SM-EVs on other cell targets is a subject of further investigation during the current FY2019.

肥大细胞通常是通过高亲和力IgE受体（FC-EPSILON-RI）激活过敏性炎症发病机理的效应细胞和调节细胞。抗原与IgE/FC-EPSILON-RI复合物的结合会启动驱动肥大细胞反应的细胞内信号传导事件。另外，其他细胞表面受体的信号传导可以激活肥大细胞或改变其对IgE受体的反应。对在肥大细胞中表达的受体及其串扰发起的信号传导机制的更深入了解可以为调节人类疾病的过敏反应和治疗选择提供线索。 最近，我们发现在人类肥大细胞中大量表达了一种粘附G蛋白偶联受体ADGRE2（EMR2），并且在与疾病表现的常染色体支配性振动尿素辅助膜的患者中，ADGRE2的错义变体（P.C492Y）鉴定出ADGRE2。与正常受试者相比，这种变体的患者在重复性性质的机械刺激上表现出肥大细胞的过度反应性，导致局部蜂巢，血清中的组胺水平升高以及真皮的细胞外胰蛋白酶染色增加。 P.C492Y变体破坏了ADGRE2的Alpha和Beta亚基之间的非共价相互作用，使受体更容易振动后解离Alpha亚基并导致其激活。在2019财政年度（FY），与Drs合作。 Schwartz，Lyons和Milner我们通过肥大细胞胰蛋白酶的不常见的异质体介导的alpha adgre2亚基的蛋白水解裂解揭示了ADGRE2激活ADGRE2的另一种机制。 α和β-胰蛋白酶的异驱精酶存在于遗传性α-触及症患者中，但在其他通常患有β同型酯剂的个体中不存在。 这种机制将其基因型与这些患者对皮肤振动刺激的反应性更高。 我们实验室中的另一个重点也是对肥大细胞增殖疾病的研究。在全身肥大症（SM）中，肥大细胞与遗传变异的肿瘤积累，尤其是酪氨酸激酶受体受体试剂盒中的遗传变异，尤其是错义突变D816V，使其具有宪法上的活性。正常的试剂盒信号传导对于肥大细胞的分化，增殖和存活至关重要，还可以增强肥大细胞对过敏性刺激的反应。但是，由于D816V或其他套件变体引起的致癌信号导致肥大细胞反应异常。 我们以前已将鞘氨醇激酶1（SPHK1）/鞘氨醇1-磷酸（S1P）轴作为致癌套件信号传导的关键途径，导致肥大细胞生长异常，在考虑对攻击性SM的替代方案进行治疗的方法时，这种知识可能会有所帮助，在该治疗中，侵袭性抑制剂抑制剂限制了侵袭性改进，这一知识是有用的。 在2019财年，我们在SM中持续的IL-6产生中证明了具有D816V-KIT的肥大细胞的异常信号传导的作用。 SM患者与疾病严重程度和进展有关的IL-6血清水平升高。但是，涉及的细胞来源和机制尚不清楚。我们发现，细胞内IL-6通过流式细胞术和免疫荧光染色主要与肥大细胞相关，并提出较高的D816V等位基因负担较高的患者的IL-6阳性肥大细胞比例较高。在来自D816V-KIT的异常信号中，我们确定了JAK2活性的增加，MEK/ERK和PI3K衍生的信号是IL-6转录的驱动因素以及随之而来的IL-6释放，前者是通过调节STAT5的激活/表达来调节的。 D816V-KI肥大细胞中STAT5参与持续的IL-6产生与其他恶性细胞中描述的机制形成对比，在其他恶性细胞中，涉及IL-6和STAT3的自分泌前馈回路很常见。这项研究为导致肥大细胞增多症中持续的IL-6产生的机制提供了第一批线索，并进行了治疗干预的潜在靶标分子。 在2019财年期间，我们还完成了有关鉴定肥大细胞释放的其他细胞间通信车辆的研究，这些车辆可能有助于肥大细胞增殖性疾病的病理。 我们证明，SM患者的血清包含具有肥大细胞特征的细胞外囊泡（EV）浓度升高，并且与疾病严重程度的标记相关。基于SM中电动汽车数量与肝肿大和碱性磷酸酶水平的关联，这是肝功能的标志物，我们询问这些EV是否可以有助于与乳腺吞噬相关的异常肝脏病理学。在2019财年，我们报道说，这些EV中包含的大细胞增多症中包含的试剂盒被转移到肝标细胞中，引发了增殖，细胞因子产生和分化，这是肝纤维化病理中已知的过程。确认了这些结论，通过试剂盒抑制或中和降低了效果，并通过强制表达试剂盒或组成型活性D816V-kit进行了概括。我们的数据还表明，从注射SM-EV的小鼠的肝脏中，在体内激活了星状细胞。这项研究表明，SM中的EV具有通过将功能活性试剂盒引入星状细胞中的星状细胞中的纤维化表型，这是与肝脏病理相关的特征变化。 从肥大细胞增多症中鉴定出来自肿瘤细胞的电动汽车的鉴定可以使其特异性尸体可以将其特异性尸体穿梭到星状细胞以外的其他细胞中，从而使其功能失调，从而有助于与肥大细胞增多症相关的某些病理方面。对其他细胞靶标的SM-EV的研究是当前2019财年进一步研究的主题。