Dual contribution of the spleen tyrosine kinase (SYK) to epidermolysis bullosa acquisita pathogenesis

获得性大疱性表皮松解症(EBA)是一种典型的器官特异性自身免疫性疾病，由针对III型胶原(COL7)的自身抗体引起。在过去的几年里，EBA动物模型的使用对了解该病的发病机制做出了重大贡献。相比之下，开发新疗法的高度医疗需求到目前为止还没有实现。在EBA发病的传入阶段，在给定的遗传背景下，抗原提呈细胞诱导依赖于CD4的B细胞反应，导致产生抗COL7自身抗体。我们实验室最近的证据表明，中性粒细胞和GM-CSF也参与了这一过程。脾酪氨酸激酶(SYK)是B细胞和中性粒细胞激活的中枢。因此，SYK是一个潜在的药物靶点，用于调节实验性EBA中自身抗体的产生(本提案的目标1)。传出相由自身抗体与COL7结合而开始，主要位于皮肤的真皮-表皮交界处。这种免疫复合体(IC)的形成在DEJ建立了一个促炎环境，导致依赖CD18的Gr-1阳性效应细胞重新聚集到皮肤中。在皮肤中，这些效应白细胞通过特定的激活Fcγ受体(Fcgr)与DEJ处的IC结合。通过涉及PI3Kβ、AKT、p38和ERK1/2的信号级联反应，释放出活性氧(ROS)和蛋白水解酶，最终导致表皮下起泡。由于SYK介导Fcgr信号转导，靶向SYK很可能会削弱IC诱导的实验性EBA中Gr-1阳性效应白细胞的激活。EBA小鼠模型中的这些发现也将在新建立的大疱性类天疱疮小鼠模型中得到验证。潜在的治疗相关性将在已经建立(免疫诱导的)EBA的小鼠身上进行评估。作为一个探索性的目标，其他信号分子在实验性EBA发病机制中的作用将在体外和体内得到验证。最终，小鼠模型的发现将在患者样本中得到(至少在形态上)验证。为了解决这些研究问题，将使用成熟的EBA和BP模型系统。在项目相关的前期工作中，我们已经确定了SYK在中性粒细胞体外激活中的关键作用。此外，使用SYK-FLOXED和靶细胞-cre小鼠进行的试验性小鼠实验表明，SYK对EBA中水泡的形成具有细胞类型特异性的贡献。鉴于我们的假设可以得到验证，SYK--以及可能的其他信号分子--可能成为EBA患者潜在的新治疗选择，也可能成为BP患者的新治疗选择。

Epidermolysis bullosa acquisita (EBA) is a prototypical, organ-specific autoimmune disease caused by autoantibodies directed against type VII collagen (COL7). Over the past years, use of EBA animal models has significantly contributed to the understanding of the disease's pathogenesis. In contrast, the high medical need to develop novel treatments, has not been achieved so far. In the afferent phase of EBA pathogenesis, within a given genetic context, antigen-presenting cells induce a CD4-dependent B cell response, leading to the production of anti-COL7 autoantibodies. Recent evidence from our laboratory indicated that neutrophils and GM-CSF are also involved. The spleen tyrosine kinase (SYK) is central for both, B cell and neutrophil activation. Hence, SYK is a potential pharmaceutical target to modulate autoantibody production in experimental EBA (aim 1 of this proposal). The efferent phase is initiated by the binding of the autoantibodies to COL7, mainly located at the dermal-epidermal junction (DEJ) in the skin. This formation of immune-complexes (IC) at the DEJ establishes a pro-inflammatory milieu, resulting in the CD18-dependent recruitment of Gr-1 positive effector cells into the skin. In the skin, these effector leukocytes bind to the IC at the DEJ via specific, activating Fc gamma receptors (Fcgr). Through a signaling cascade involving PI3Kbeta, AKT, p38 and ERK1/2, reactive oxygen species (ROS) and proteases are released, ultimately causing subepidermal blistering. As SYK mediates Fcgr signaling, targeting SYK will most likely impair IC-induced activation of Gr-1 positive effector leukocytes in experimental EBA. These findings in the EBA mouse model will also be validated in a newly established mouse model of bullous pemphigoid. The potential therapeutic relevance will be evaluated in mice with already established (immunization-induced) EBA. As an exploratory aim, the contribution of other signaling molecules in experimental EBA pathogenesis will be tested both in vitro and in vivo. Ultimately, findings from mouse models will be (at least morphologically) validated in patient samples. To address these research questions, well-established models systems of EBA and BP will be used. In project-related preliminary work, we already established a key role of SYK for in vitro activation of neutrophils. Furthermore, pilot mouse experiments, using SYK-floxed and target cell-cre mice, indicate a cell type-specific contribution of SYK to blister formation in EBA. Given, our assumptions can be validated, SYK -and possibly additional signaling molecules- could emerge as potential new treatment options for patients with EBA, and possibly also BP.