Targeting Siglec-8/Siglec-F Reduce Allergic Responses In Vitro and In Vivo

描述（由申请人提供）：Siglec-8是CD33唾液酸结合免疫球蛋白样凝集素（Siglec）家族的成员，仅在人肥大细胞、嗜酸性粒细胞和嗜碱性粒细胞中发现。这种跨膜蛋白具有识别特定碳水化合物分子（聚糖）的胞外结构域，以及基于酪氨酸的胞内抑制基元，据推测可将受体结合转化为免疫抑制。小鼠siglece - f和人类siglece -8在功能上是趋同的。小鼠siglece - f主要在小鼠嗜酸性粒细胞上表达。用特异性siglece -8抗体孵育人嗜酸性粒细胞，或用siglece - f抗体孵育小鼠嗜酸性粒细胞，可诱导细胞凋亡，用siglece - f抗体给药小鼠可减少嗜酸性粒细胞数量。相反，在体外，抗体与人肥大细胞的siglece -8结合不会诱导细胞凋亡，但会抑制通过FceRI激活诱导的介质释放。siglece -8和siglece - f都结合了一种独特的聚糖配体，称为6'-sulfo-sLex，或NeuAca2-3(6- o- sulfo)Ga1S1-4[Fuca1-3]G1cNAc。因此，通过其天然聚糖配体，或通过抗体或拟糖激动剂激活siglece -8/ siglece - f，可能提供一种特异性抑制和/或消耗嗜酸性粒细胞和肥大细胞的新方法，从而减少过敏性炎症反应。因此，我们假设：(i)过敏性炎症细胞表面的siglece -8/ siglece - f与其在组织上表达的天然碳水化合物配体结合，通过激活负信号通路来限制过敏性炎症；（ii）体内变应性炎症可通过调节组织中天然siglece -8配体的表达来控制；（iii） siglece -8/ siglece - f单克隆抗体或基于其配体结构的合成分子（糖仿制品）将能够阻断过敏性炎症。这些概念将通过四个具体目标来探讨。目的1将表征天然siglece -8/ siglece - f配体并确定其组织表达。目的2将确定Siglec- 8/Siglec- f配体模拟物在体外限制嗜酸性粒细胞和肥大细胞过敏反应的能力。Aim 3将探索单克隆抗体或配体模拟物在体内限制过敏反应的能力，Aim 4将确定siglece -8参与诱导嗜酸性粒细胞凋亡和抑制肥大细胞介质释放的机制。我们提出的关于siglece -8/ siglece - f的研究应该为以嗜酸性粒细胞、嗜碱性粒细胞和肥大细胞数量增加或介质增加为特征的疾病的治疗找到新的治疗方法。

DESCRIPTION (provided by applicant): Siglec-8 is a member of the CD33 sialic acid-bind immunoglobulin-like lectin (Siglec) family found only on human mast cells, eosinophils and basophils. This transmembrane protein has an extracellular domain that recognizes specific carbohydrate molecules (glycans), and intracellular tyrosine-based inhibition motifs that putatively converts receptor binding into immune suppression. Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs. Mouse Siglec-F is expressed predominantly on mouse eosinophils. Incubation of human eosinophils with specific Siglec-8 antibodies, or mouse eosinophils with Siglec-F antibodies, induces apoptosis, and dosing of mice with Siglec-F antibodies reduces eosinophil numbers. In contrast, Siglec-8 engagement on human mast cells by antibodies in vitro does not induce apoptosis but does inhibit mediator release induced via FceRI activation. Both Siglec-8 and Siglec-F bind a unique glycan ligand referred to as 6'-sulfo-sLex, or NeuAca2-3(6-O-sulfo)Ga1S1-4[Fuca1-3]G1cNAc. Therefore, activation of Siglec-8/Siglec-F through its natural glycan ligand, or through antibodies or glycomimetic agonists, may provide a novel means to specifically inhibit and/or deplete eosinophils and mast cells, thereby reducing allergic inflammatory responses. We thus hypothesize that: (i) Siglec-8/Siglec-F on the surface of allergic inflammatory cells binds to its natural carbohydrate ligands, expressed on tissues, to limit allergic inflammation by activating a negative signaling pathway; (ii) Allergic inflammation in vivo can be controlled by regulating expression of natural Siglec-8 ligands in tissues; and (iii) Siglec-8/Siglec-F mAbs or synthetic molecules based on the structure of their ligands (glycomimetics) will be capable of interrupting allergic inflammation. These concepts will be explored through four specific aims. Aim 1 will characterize natural Siglec-8/Siglec-F ligands and determine their tissue expression. Aim 2 will determine the ability of Siglec- 8/Siglec-F ligand mimetics to limit allergic responses in eosinophils and mast cells in vitro. Aim 3 will explore the ability of mAbs or ligand mimetics to limit allergic responses in vivo, and Aim 4 will identify mechanisms by which Siglec-8 engagement induces eosinophil apoptosis and inhibits mast cell mediator release. Our proposed work on Siglec-8/Siglec-F should identify new therapeutic approaches for the treatment of diseases characterized by increased numbers of, or mediators from, eosinophils, basophils and mast cells.