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

肥大细胞是参与过敏性炎症发病的效应和调节细胞，通常通过激活高亲和力的IgE受体(Fc-epsilon-RI)。抗原与IgE/Fc-epsilon-RI复合体结合可启动细胞内信号事件，从而驱动肥大细胞反应。此外，其他细胞表面受体的信号可以激活肥大细胞或改变它们对IgE受体的反应。深入了解肥大细胞中表达的受体启动的信号机制及其串扰，可以为人类疾病的过敏反应调节和治疗选择提供线索。 最近，我们发现粘附性G蛋白偶联受体ADGRE2(EMR2)在人类肥大细胞中大量表达，ADGRE2的错义突变(p.C492Y)与常染色体显性遗传性振荡性荨麻疹患者的疾病表现有关。与正常受试者相比，患有这种变异体的患者表现出肥大细胞对重复性机械刺激的高反应性，导致局部蜂窝，血清组胺水平增加，真皮细胞外类胰蛋白酶染色增加。P.C492Y变异体破坏了ADGRE2的α和β亚基之间的非共价相互作用，使受体在振动后更容易解离α亚单位并导致其激活。在2019财年(FY)，我们与Schwartz、Lyons和Milner博士合作，公布了另一种激活ADGRE2的机制，该机制通过肥大细胞类胰蛋白酶的一种不常见的异三聚体介导的αADGRE2亚单位的蛋白水解性切割来激活ADGRE2。遗传性α-类胰蛋白酶血症患者中存在α-和β-类胰蛋白酶异构体，而其他通常具有β-同型异构体的个体中则不存在。这种机制将他们的基因与这些患者对皮肤振动刺激的较高反应性联系在一起。 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，肥大细胞的肿瘤性积累与遗传变异有关，特别是酪氨酸激酶受体试剂盒中的错义突变D816V，使其具有结构性活性。正常的KIT信号对肥大细胞的分化、增殖和存活非常重要，也能增强肥大细胞对过敏刺激的反应。然而，D816V或其他试剂盒变异导致的致癌信号会导致肥大细胞反应异常。 我们此前曾暗示，鞘氨醇激酶1(SPHK1)/鞘氨醇-1-磷酸(S1P)轴是致癌KIT信号转导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的替代方法时可能有帮助，在侵袭性SM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D816V-KIT对肥大细胞的异常信号在SM持续产生IL-6中的作用。系统性红斑狼疮患者血清IL-6水平升高，与疾病严重程度和进展有关。然而，所涉及的细胞来源和机制还不是很清楚。我们通过流式细胞仪和免疫荧光检测发现，IL-6细胞内染色主要与肥大细胞相关，提示在D816V等位基因载量较高的患者中，IL-6阳性的肥大细胞比例较高。在来自D816V-KIT的异常信号中，我们发现JAK2活性增加和MEK/ERK-和PI3K衍生信号是IL-6转录和随后IL-6释放的驱动因素，前两者是通过调节STAT5的激活/表达来实现的。在D816V-Ki肥大细胞中，STAT5参与持续产生IL-6，这与在其他恶性肿瘤细胞中描述的机制形成了鲜明对比，在其他恶性细胞中，涉及IL-6和STAT3的自分泌前馈循环是常见的。这项研究首次提供了导致肥大细胞增多症持续产生IL-6的机制和潜在的治疗干预靶分子的线索。 在2019财年，我们还完成了肥大细胞释放的其他细胞间通讯载体的识别研究，这些载体可能有助于肥大细胞增生性疾病的病理。我们证明SM患者的血清含有高浓度的细胞外小泡(EVS)，具有肥大细胞的特征，并与疾病严重程度的标志物相关。根据SM中EVS的数量与肝脏肿大和碱性磷酸酶水平的关系，我们询问这些EVS是否参与了与肥大细胞增多症相关的肝脏异常病理。在2019财年，我们报道了这些大细胞增多的EVS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，这是已知在肝纤维化病理中发生的过程。证实了这些结论，KIT抑制或中和降低了这种作用，而强制表达KIT或组成活性的D816V-KIT则重现了这种作用。我们的数据还表明，注射SM-EVS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EVS通过将功能活性试剂盒引入星状细胞，有可能在星状细胞中诱导纤维化表型，这是与肝脏病理相关的特征变化。 在肥大细胞增生症中从肿瘤细胞中识别EV可能打开了一种可能性，即它们的特定货物可以穿梭到星状细胞以外的细胞中，从而导致它们的功能失调，从而导致与肥大细胞增多症相关的一些病理方面的变化。SM-EVS在其他细胞目标上的研究是本财年2019财年进一步研究的主题。

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.