Macrophages play a crucial role in host innate immune defense against infection and tissue injury. Macrophages are highly plastic cells and their subtypes have been characterized as M1 (also termed classically activated) and M2 (alternatively activated). Although the M1/M2 paradigm has been well documented, less is known regarding the role of macrophage activation/polarization in inflammation-associated necrotic cell death. To address this gap in current knowledge, we prepared bone marrow-derived macrophages, induced them to M1 or M2 subtypes, and then investigated the expression of necroptosis signaling molecules and macrophage subtype-dependent responses to different necroptosis inducers. We found that necroptosis effector mixed lineage kinase domain-like protein (MLKL) and the key necroptosis regulator Z-DNA/RNA binding protein 1 were predominantly induced in M1 but not M2 macrophages. Interestingly, the protein but not mRNA levels of receptor-interacting protein kinase-3 (RIPK3) were also upregulated in M1 macrophages. We further found that macrophage necrotic cell death, the releases of lactate dehydrogenase and dead cell proteases as well as MLKL phosphorylation at Ser345 in response to various necroptosis inducers were greatly augmented in M1 but not M2 macrophages, and the accelerated effects were blocked by two structurally distinct specific RIPK3 inhibitors GSK872 or GSK843. Thus, our findings demonstrate that M1 but not M2 subtypes of macrophages are more susceptible to inflammation-related lytic cell death in an RIPK3 kinase activity-dependent manner.

巨噬细胞在宿主对感染和组织损伤的先天免疫防御中起着至关重要的作用。巨噬细胞是高度可塑性的细胞，它们的亚型被表征为 M1（也称为经典激活）和 M2（替代激活）。尽管 M1/M2 范式已被充分证明，但关于巨噬细胞激活/极化在炎症相关坏死细胞死亡中的作用知之甚少。为了解决当前知识中的这一差距，我们制备了骨髓来源的巨噬细胞，将它们诱导为 M1 或 M2 亚型，然后研究了坏死性凋亡信号分子的表达和巨噬细胞亚型依赖性对不同坏死性凋亡诱导剂的反应。我们发现坏死性凋亡效应混合谱系激酶结构域样蛋白 (MLK​​L) 和关键的坏死性凋亡调节因子 Z-DNA/RNA 结合蛋白 1 主要在 M1 而非 M2 巨噬细胞中诱导。有趣的是，受体相互作用蛋白激酶-3 (RIPK3) 的蛋白质而非 mRNA 水平也在 M1 巨噬细胞中上调。我们进一步发现巨噬细胞坏死细胞死亡、乳酸脱氢酶和死细胞蛋白酶的释放以及响应各种坏死性凋亡诱导剂的 Ser345 位点 MLKL 磷酸化在 M1 而非 M2 巨噬细胞中大大增加，并且加速作用被两个结构上的阻断不同的特异性 RIPK3 抑制剂 GSK872 或 GS​​K843。因此，