Key Points FAP-α is involved in a nonapoptotic signaling pathway in neutrophils. FAP-α is activated in parallel with RIPK–MLKL signaling. FAP-α could be a potential drug target in neutrophilic inflammatory responses. Cytokine-primed neutrophils can undergo a nonapoptotic type of cell death using components of the necroptotic pathway, including receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase-like (MLKL) and NADPH oxidase. In this report, we provide evidence for a potential role of serine proteases in CD44-mediated necroptotic death of GM-CSF–primed human neutrophils. Specifically, we observed that several inhibitors known to block the enzymatic function of fibroblast activation protein-α (FAP-α) were able to block CD44-mediated reactive oxygen species production and cell death, but not FAS receptor–mediated apoptosis. To understand how FAP-α is involved in this nonapoptotic death pathway, we performed immunoblotting experiments in the presence and absence of inhibitors of RIPK3, MLKL, p38 MAPK, PI3K, and FAP-α. The results of these experiments suggested that FAP-α is active in parallel with RIPK3, MLKL, and p38 MAPK activation but proximal to PI3K and NADPH oxidase activation. Interestingly, neutrophils isolated from the joints of patients suffering from rheumatoid arthritis underwent a GM-CSF–independent necroptosis following CD44 ligation; this effect was also blocked by both FAP-α and MLKL inhibitors. Taken together, our evidence shows that the RIPK3–MLKL pathway activates NADPH oxidase but requires, in addition to p38 MAPK and PI3K, a serine protease activity, whereby FAP-α is the most likely candidate. Thus, FAP-α could be a potential drug target in neutrophilic inflammatory responses to avoid exaggerated nonapoptotic neutrophil death, leading to tissue damage.

中文翻译：

---

RIPK3-MLKL-介导的中性粒细胞死亡需要同时激活成纤维细胞激活蛋白-α

关键点 FAP-α 参与中性粒细胞的非凋亡信号通路。FAP-α 与 RIPK-MLKL 信号同时激活。FAP-α 可能是中性粒细胞炎症反应的潜在药物靶点。细胞因子引发的中性粒细胞可以使用坏死性凋亡途径的成分进行非凋亡类型的细胞死亡，包括受体相互作用蛋白激酶-3 (RIPK3)、混合谱系激酶样 (MLK​​L) 和 NADPH 氧化酶。在本报告中，我们提供了丝氨酸蛋白酶在 GM-CSF 引发的人类中性粒细胞的 CD44 介导的坏死性凋亡中的潜在作用的证据。具体而言，我们观察到几种已知阻断成纤维细胞活化蛋白-α (FAP-α) 酶促功能的抑制剂能够阻断 CD44 介导的活性氧产生和细胞死亡，但不能阻断 FAS 受体介导的细胞凋亡。为了了解 FAP-α 如何参与这种非凋亡性死亡途径，我们在存在和不存在 RIPK3、MLKL、p38 MAPK、PI3K 和 FAP-α 抑制剂的情况下进行了免疫印迹实验。这些实验的结果表明 FAP-α 的活性与 RIPK3、MLKL 和 p38 MAPK 激活平行，但接近 PI3K 和 NADPH 氧化酶激活。有趣的是，从类风湿性关节炎患者的关节中分离出的中性粒细胞在 CD44 结扎后发生了不依赖于 GM-CSF 的坏死性凋亡。这种作用也被 FAP-α 和 MLKL 抑制剂阻断。总之，我们的证据表明 RIPK3-MLKL 途径激活 NADPH 氧化酶，但除了 p38 MAPK 和 PI3K 外，还需要丝氨酸蛋白酶活性，其中 FAP-α 是最有可能的候选者。因此，