Chen Yiliang, Huang Wenxin, Yang Moua, Xin Gang, Cui Weiguo, Xie Zijian, Silverstein Roy L
From the Blood Research Institute, Blood Center of Wisconsin, Milwaukee (Y.C., W.H., M.Y., G.X., W.C., R.L.S.); Department of Cell Biology, Neurobiology and Anatomy (M.Y., R.L.S.) and Department of Medicine (R.L.S.), Medical College of Wisconsin, Milwaukee; and Departments of Medicine, Pharmacology and Surgery, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV (Z.X.).
Arterioscler Thromb Vasc Biol. 2017 Aug;37(8):1462-1469. doi: 10.1161/ATVBAHA.117.309444. Epub 2017 Jun 15.
Circulating levels of cardiotonic steroids (CTS) are elevated in various chronic inflammatory conditions, but the role of CTS in inflammation remains largely unknown. We have previously shown that the CTS ouabain stimulates proinflammatory responses in murine macrophages. In this study, we aim to explore the mechanism how CTS induce proinflammatory responses in primary murine and human macrophages.
Using both murine peritoneal macrophages and human monocyte-derived macrophages, we demonstrated that ouabain activated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), leading to proinflammatory cytokine (eg, MCP-1 [monocyte chemotactic protein 1], TNF-α [tumor necrosis factor-α], IL-1β [interleukin-1β], and IL-6) production. By applying siRNA techniques and murine peritoneal macrophages isolated from genetically modified mice, we showed that macrophages partially deficient in Na/K-ATPase, the receptor for CTS, or fully deficient in the scavenger receptor CD36 or TLR4 (Toll-like receptor) were resistant to ouabain-induced NF-κB activation, suggesting an indispensable role of these 3 receptors in this pathway. Mechanistically, this effect of ouabain was independent of the ion transport function of the Na/K-ATPase. Instead, ouabain stimulated a signaling complex, including Na/K-ATPase, CD36, and TLR4. Subsequently, TLR4 recruited MyD88 adaptor protein for NF-κB activation. Furthermore, intraperitoneal injection of ouabain into mice specifically recruited Ly6CCCR2 monocyte subtypes to the peritoneal cavities, indicating that the CTS ouabain triggers inflammation in vivo.
CTS activate NF-κB leading to proinflammatory cytokine production in primary macrophages through a signaling complex, including CD36, TLR4, and Na/K-ATPase. These findings warrant further studies on endogenous CTS in chronic inflammatory diseases, such as atherosclerosis.
强心甾体（CTS）的循环水平在各种慢性炎症状态下会升高，但CTS在炎症中的作用仍 largely未知。我们之前已表明CTS哇巴因可刺激小鼠巨噬细胞中的促炎反应。在本研究中，我们旨在探究CTS在原代小鼠和人巨噬细胞中诱导促炎反应的机制。
使用小鼠腹腔巨噬细胞和人单核细胞衍生的巨噬细胞，我们证明哇巴因激活了NF-κB（活化B细胞的核因子κ轻链增强子），导致促炎细胞因子（如MCP-1 [单核细胞趋化蛋白1]、TNF-α [肿瘤坏死因子-α]、IL-1β [白细胞介素-1β]和IL-6）的产生。通过应用小干扰RNA技术和从基因改造小鼠分离的小鼠腹腔巨噬细胞，我们表明Na/K-ATP酶（CTS的受体）部分缺陷或清道夫受体CD36或Toll样受体4（TLR4）完全缺陷的巨噬细胞对哇巴因诱导的NF-κB激活具有抗性，表明这3种受体在该途径中起不可或缺的作用。从机制上讲，哇巴因的这种作用独立于Na/K-ATP酶的离子转运功能。相反，哇巴因刺激了一种信号复合物，包括Na/K-ATP酶、CD36和TLR4。随后，TLR4募集MyD88衔接蛋白以激活NF-κB。此外，向小鼠腹腔内注射哇巴因可特异性地将Ly6C+CCR2单核细胞亚型募集到腹腔，表明CTS哇巴因在体内引发炎症。
CTS通过包括CD36、TLR4和Na/K-ATP酶的信号复合物激活NF-κB，导致原代巨噬细胞中促炎细胞因子的产生。这些发现值得对慢性炎症性疾病（如动脉粥样硬化）中的内源性CTS进行进一步研究。
