Askari Kasra, Johnson Jennifer L, Shukla Aparna, Meneses-Salas Elsa, Kiosses William B, Yu Juan, Catz Sergio D
Department of Molecular and Cellular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States.
Division of Inflammation Biology, La Jolla Institute for Immunology, 9420 Athena Cir, La Jolla, CA 92037, United States.
J Leukoc Biol. 2025 Apr 23;117(4). doi: 10.1093/jleuko/qiaf012.
Regulated sequential exocytosis of neutrophil granules is essential for orchestrating the innate immune response, while uncontrolled secretion causes inflammation. We developed and characterized Nexinhib20, a small-molecule inhibitor that targets azurophilic granule exocytosis in neutrophils by blocking the interaction between the small GTPase Rab27a and its effector JFC1. Its therapeutic potential has been demonstrated in several preclinical models of inflammatory disease. Here, using neutrophils from Jfc1-KO mice, we show that JFC1 regulates the mobilization of a small subpopulation of CD11b+ granules. Nexinhib20 inhibits the mobilization of β2-integrins from a subset of CD11b+ granules to the plasma membrane in human and mouse neutrophils. The putative impact of Nexinhib20 on integrin activation is caused by decreased avidity, secondary to its effect on β2-integrin mobilization. CD11b mobilization and integrin activation were unaffected by pharmacological inhibition or activation of Rac1. Using quantitative 3D enhanced resolution microscopy, we show that neutrophil activation induces the recruitment of JFC1 to CD11b+ granules. Nexinhib20 decreased the localization of JFC1 at CD11b+ granules without affecting the association of Rac1 with CD11b. Nexinhib20 inhibits JFC1 recruitment but not endogenous Rac1 activation in living cells. Using orthogonal analyses of Rac1 activity consisting of a sensitive, time-resolved fluorescence energy transfer, Rac1-PAK1-binding assay, and endogenous Rac1-GTP examination, we show that Nexinhib20 does not interfere with Rac1 activation. Instead, we confirm its molecular mode of action as the inhibition of the Rab27a-JFC1 binding. Thus, Nexinhib20 limits β2-integrin mobilization to the cell surface, decreasing avidity and affecting active integrin availability in a JFC1-dependent but Rac1-independent manner.
中性粒细胞颗粒的有序顺序胞吐作用对于协调先天免疫反应至关重要，而不受控制的分泌会导致炎症。我们开发并鉴定了Nexinhib20，这是一种小分子抑制剂，通过阻断小GTP酶Rab27a与其效应器JFC1之间的相互作用，靶向中性粒细胞中的嗜天青颗粒胞吐作用。其治疗潜力已在多种炎症性疾病的临床前模型中得到证实。在这里，我们使用来自Jfc1基因敲除小鼠的中性粒细胞，表明JFC1调节一小部分CD11b+颗粒的动员。Nexinhib20抑制人源和小鼠中性粒细胞中β2整合素从一部分CD11b+颗粒向质膜的动员。Nexinhib20对整合素激活的假定影响是由于其对β2整合素动员的作用导致亲和力降低所致。CD11b的动员和整合素激活不受Rac1的药理学抑制或激活的影响。使用定量3D增强分辨率显微镜，我们表明中性粒细胞激活诱导JFC1募集到CD11b+颗粒。Nexinhib20降低了JFC1在CD11b+颗粒上的定位，而不影响Rac1与CD11b的结合。Nexinhib20抑制活细胞中JFC1的募集，但不抑制内源性Rac1的激活。使用由灵敏的时间分辨荧光能量转移、Rac1-PAK1结合测定和内源性Rac1-GTP检测组成的Rac1活性正交分析，我们表明Nexinhib20不干扰Rac1的激活。相反，我们证实其分子作用模式是抑制Rab27a-JFC1结合。因此，Nexinhib20限制β2整合素向细胞表面的动员，降低亲和力并以JFC1依赖但Rac1独立的方式影响活性整合素的可用性。
