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.

肥大细胞是参与过敏性炎症发病机制的效应和调节细胞，通常通过激活高亲和力IgE受体（Fc-IgE-RI）。抗原与IgE/Fc-IgM-RI复合物的结合启动驱动肥大细胞应答的细胞内信号传导事件。此外，其他细胞表面受体的信号传导可以激活肥大细胞或改变它们对IgE受体的反应。更深入地了解肥大细胞中表达的受体及其相互作用引发的信号传导机制，可以为过敏反应的调节和人类疾病的治疗提供线索。 最近，我们发现粘附G蛋白偶联受体ADGRE 2（EMR 2）在人肥大细胞中大量表达，并且在常染色体显性遗传振动性荨麻疹患者中鉴定出ADGRE 2的错义变体（p.C492Y）与疾病表现相关。与正常受试者相比，具有这种变体的患者在重复性质的机械刺激下表现出肥大细胞高反应性，导致局部荨麻疹，血清中组胺水平升高和真皮中细胞外类胰蛋白酶染色增加。p.C492Y变体使ADGRE 2的α和β亚基之间的非共价相互作用不稳定，使受体更易于在振动后解离α亚基并导致其活化。在2019财年（FY），我们与Schwartz、里昂和米尔纳博士合作，通过肥大细胞类胰蛋白酶的一种罕见异源四聚体介导的α ADGRE 2亚基的蛋白水解裂解，揭示了ADGRE 2活化的另一种机制。遗传性α-类胰蛋白酶血症患者中存在α和β类胰蛋白酶的异四聚体，但在其他通常具有β同四聚体的个体中不存在。 这种机制将他们的基因型与这些患者对皮肤中振动刺激的较高反应性联系起来。 我们实验室的另一个重点也是肥大细胞增殖性疾病的研究。在系统性肥大细胞增多症（SM）中，肥大细胞的肿瘤性蓄积与酪氨酸激酶受体KIT中的遗传变异相关，特别是错义突变D816 V，使其具有组成性活性。正常的KIT信号传导对于肥大细胞的分化、增殖和存活是重要的，并且还增强肥大细胞对过敏性刺激的应答。然而，由于D816 V或其他KIT变体引起的致癌信号传导导致异常肥大细胞应答。 我们以前曾暗示鞘氨醇激酶1（SPHK 1）/鞘氨醇-1-磷酸（S1 P）轴作为致癌KIT信号传导的关键途径，导致肥大细胞生长异常，这一知识在考虑治疗侵袭性SM的替代方法时可能是有帮助的，其中酪氨酸激酶抑制剂显示出有限的长期改善。 在2019财年，我们已经证明了D816 V-KIT肥大细胞的异常信号传导在SM中持续产生IL-6中的作用。SM患者血清IL-6水平升高与疾病严重程度和进展相关。然而，所涉及的细胞来源和机制还不清楚。我们发现，细胞内IL-6染色的流式细胞仪和免疫荧光主要与肥大细胞，并建议在患者中具有较高的D816 V等位基因负荷的IL-6阳性肥大细胞的百分比较高。在来自D816 V-KIT的异常信号中，我们鉴定了增加的JAK 2活性和MEK/ERK-和PI 3 K-衍生的信号作为IL-6转录和随后的IL-6释放的驱动因子，前两者通过调节STAT 5的活化/表达。STAT 5参与D816 V-KI肥大细胞中持续的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STAT 3的自分泌前馈回路是常见的。该研究提供了导致肥大细胞增多症持续产生IL-6的机制的第一条线索，以及用于治疗干预的潜在靶分子。 在2019财年，我们还完成了关于鉴定肥大细胞释放的其他细胞间通讯载体的研究，这些载体可能有助于肥大细胞增殖性疾病的病理学。 我们证明，SM患者的血清中含有浓度升高的细胞外囊泡（EV），具有肥大细胞特征，并且与疾病严重程度的标志物相关。基于SM中EV数量与肝肿大和碱性磷酸酶水平（肝功能标志物）的相关性，我们询问这些EV是否会导致与肥大细胞增多症相关的异常肝脏病理学。在2019财年，我们报告了这些大红细胞症EV中包含的KIT转移到肝星状细胞中，引发增殖，细胞因子产生和分化，这些过程已知发生在肝纤维化病理学中。重申这些结论，KIT抑制或中和降低了这种作用，并通过KIT或组成型活性D816 V-KIT的强化表达重现了这种作用。我们的数据还表明，注射SM-EV的小鼠肝脏中的星状细胞在体内被激活。这项研究表明，SM中的EV有可能通过将功能活性KIT引入星状细胞而诱导星状细胞中的纤维化表型，这是与肝脏病理学相关的特征性变化。 从肥大细胞增多症的肿瘤细胞中鉴定出EV已经开启了这样的可能性，即它们的特定货物可以穿梭到星状细胞以外的细胞中，使它们的功能失调，从而导致与肥大细胞增多症相关的一些病理学方面。SM-EV对其他细胞靶点的研究是当前2019财年进一步研究的主题。