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细胞和调节细胞，通常通过激活高亲和力 IgE 受体 (Fc-epsilon-RI)。抗原与 IgE/Fc-ε-RI 复合物的结合启动驱动肥大细胞反应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启动的信号传导机制及其串扰可以为过敏反应的调节和人类疾病的治疗选择提供线索。 最近，我们发现粘附 G 蛋白偶联受体 ADGRE2 (EMR2) 在人类肥大细胞中大量表达，并且在常染色体显性振动性荨麻疹患者中发现的 ADGRE2 错义变体 (p.C492Y) 与疾病表现相关。与正常受试者相比，患有这种变异的患者在重复性机械刺激下表现出肥大细胞高反应性，导致局部荨麻疹、血清中组胺水平增加以及真皮中细胞外类胰蛋白酶染色增加。 p.C492Y 变体破坏了 ADGRE2 的 α 和 β 亚基之间的非共价相互作用的稳定性，使受体更容易在振动后解离 α 亚基并导致其激活。在 2019 财年 (FY)，与 Drs 合作。 Schwartz、Lyons 和 Milner 揭示了另一种激活 ADGRE2 的机制，该机制是通过肥大细胞类胰蛋白酶不常见的异四聚体介导的 α ADGRE2 亚基的蛋白水解裂解来激活的。 α和β类胰蛋白酶异四聚体存在于遗传性α-类胰蛋白酶血症患者中，但不存在于其他通常具有β同四聚体的个体中。 这种机制将他们的基因型与这些患者对皮肤振动刺激的更高反应性联系起来。 我们实验室的另一个重点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，肥大细胞的肿瘤性积累与酪氨酸激酶受体 KIT 中的遗传变异相关，尤其是错义突变 D816V，使其持续活跃。正常的 KIT 信号传导对于肥大细胞的分化、增殖和存活很重要，并且还增强肥大细胞对过敏刺激的反应。然而，D816V 或其他 KIT 变体引起的致癌信号传导会导致肥大细胞反应异常。 我们之前曾指出，鞘氨醇激酶 1 (SPHK1)/1-磷酸鞘氨醇 (S1P) 轴是致癌 KIT 信号传导中导致肥大细胞异常生长的关键途径，这一知识在考虑治疗侵袭性 SM 的替代方法时很有帮助，因为酪氨酸激酶抑制剂在侵袭性 SM 方面的长期改善效果有限。 2019 财年，我们证明了 D816V-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，细胞来源和所涉及的机制尚不清楚。我们发现，通过流式细胞术和免疫荧光进行的细胞内 IL-6 染色主要与肥大细胞相关，并表明在 D816V 等位基因负荷较高的患者中，IL-6 阳性肥大细胞的百分比较高。在来自 D816V-KIT 的异常信号中，我们发现 JAK2 活性增加以及 MEK/ERK 和 PI3K 衍生信号作为 IL-6 转录和随后的 IL-6 释放的驱动因素，前两者通过调节 STAT5 的激活/表达。 STAT5 参与 D816V-KI 肥大细胞中持续产生 IL-6 的过程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 的自分泌前馈循环很常见。该研究为肥大细胞增多症中导致持续产生 IL-6 的机制和治疗干预的潜在靶分子提供了第一条线索。 在 2019 财年期间，我们还完成了对肥大细胞释放的其他细胞间通讯媒介的识别研究，这些媒介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)，其具有肥大细胞特征，并且与疾病严重程度的标志物相关。根据 SM 中 EV 数量与肝肿大和碱性磷酸酶水平（肝功能标志物）的关联，我们询问这些 EV 是否可能导致与肥大细胞增多症相关的异常肝脏病理。在 2019 财年，我们报道了这些大红细胞 EV 中包含的 KIT 被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。证实这些结论的是，KIT 抑制或中和可降低效果，并可通过强制表达 KIT 或组成型活性 D816V-KIT 来重述效果。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，通过将功能活跃的 KIT 引入星状细胞，SM 中的 EV 有可能在星状细胞中诱导纤维化表型，这是一种与肝脏病理学相关的特征性变化。 从肥大细胞增多症中的肿瘤细胞中鉴定出 EVs 开启了一种可能性，即它们的特定货物可以穿梭到星状细胞以外的细胞中，从而失调其功能，从而导致与肥大细胞增多症相关的一些病理学方面。 SM-EV 对其他细胞靶标的研究是 2019 财年进一步研究的主题。