DataSheet_1_SARS-CoV-2/ACE2 Interaction Suppresses IRAK-M Expression and Promotes Pro-Inflammatory Cytokine Production in Macrophages.pdf

The major cause of death in SARS-CoV-2 infected patients is due to de-regulation of the innate immune system and development of cytokine storm. SARS-CoV-2 infects multiple cell types in the lung, including macrophages, by engagement of its spike (S) protein on angiotensin converting enzyme 2 (ACE2) receptor. ACE2 receptor initiates signals in macrophages that modulate their activation, including production of cytokines and chemokines. IL-1R-associated kinase (IRAK)-M is a central regulator of inflammatory responses regulating the magnitude of TLR responsiveness. Aim of the work was to investigate whether SARS-CoV-2 S protein-initiated signals modulate pro-inflammatory cytokine production in macrophages. For this purpose, we treated PMA-differentiated THP-1 human macrophages with SARS-CoV-2 S protein and measured the induction of inflammatory mediators including IL6, TNFα, IL8, CXCL5, and MIP1a. The results showed that SARS-CoV-2 S protein induced IL6, MIP1a and TNFα mRNA expression, while it had no effect on IL8 and CXCL5 mRNA levels. We further examined whether SARS-CoV-2 S protein altered the responsiveness of macrophages to TLR signals. Treatment of LPS-activated macrophages with SARS-CoV-2 S protein augmented IL6 and MIP1a mRNA, an effect that was evident at the protein level only for IL6. Similarly, treatment of PAM3csk4 stimulated macrophages with SARS-CoV-2 S protein resulted in increased mRNA of IL6, while TNFα and MIP1a were unaffected. The results were confirmed in primary human peripheral monocytic cells (PBMCs) and isolated CD14+ monocytes. Macrophage responsiveness to TLR ligands is regulated by IRAK-M, an inactive IRAK kinase isoform. Indeed, we found that SARS-CoV-2 S protein suppressed IRAK-M mRNA and protein expression both in THP1 macrophages and primary human PBMCs and CD14+ monocytes. Engagement of SARS-CoV-2 S protein with ACE2 results in internalization of ACE2 and suppression of its activity. Activation of ACE2 has been previously shown to induce anti-inflammatory responses in macrophages. Treatment of macrophages with the ACE2 activator DIZE suppressed the pro-inflammatory action of SARS-CoV-2. Our results demonstrated that SARS-CoV-2/ACE2 interaction rendered macrophages hyper-responsive to TLR signals, suppressed IRAK-M and promoted pro-inflammatory cytokine expression. Thus, activation of ACE2 may be a potential anti-inflammatory therapeutic strategy to eliminate the development of cytokine storm observed in COVID-19 patients.

SARS-CoV-2感染患者死亡的主要原因在于固有免疫系统的功能失调及细胞因子风暴的形成。SARS-CoV-2通过其刺突（S）蛋白与血管紧张素转换酶2（ACE2）受体结合，感染肺部多种细胞类型，包括巨噬细胞。ACE2受体在巨噬细胞中启动信号，调节其活化，包括细胞因子和趋化因子的产生。IL-1R相关激酶（IRAK）-M是调节炎症反应的关键调节因子，它调节Toll样受体（TLR）反应的强度。本研究旨在探讨SARS-CoV-2 S蛋白启动的信号是否调节巨噬细胞中促炎细胞因子的产生。为此，我们对PMA分化的THP-1人巨噬细胞进行了SARS-CoV-2 S蛋白处理，并测量了包括IL6、TNFα、IL8、CXCL5和MIP1a在内的炎症介质的诱导。结果显示，SARS-CoV-2 S蛋白诱导了IL6、MIP1a和TNFα mRNA的表达，而对IL8和CXCL5 mRNA水平无影响。我们进一步检查了SARS-CoV-2 S蛋白是否改变了巨噬细胞对TLR信号的敏感性。用SARS-CoV-2 S蛋白处理LPS激活的巨噬细胞，可增强IL6和MIP1a mRNA的表达，但这种效应仅在IL6的蛋白质水平上显现。类似地，用PAM3csk4刺激的巨噬细胞处理SARS-CoV-2 S蛋白，导致IL6 mRNA增加，而TNFα和MIP1a则无影响。这些结果在初级人外周单核细胞（PBMCs）和分离的CD14+单核细胞中得到证实。巨噬细胞对TLR配体的敏感性受非活性IRAK激酶同型IRAK-M的调节。事实上，我们发现SARS-CoV-2 S蛋白在THP1巨噬细胞、初级人PBMCs和CD14+单核细胞中均抑制了IRAK-M mRNA和蛋白质的表达。SARS-CoV-2 S蛋白与ACE2的结合导致ACE2的内化和活性抑制。先前研究表明，ACE2的激活可诱导巨噬细胞的抗炎反应。用ACE2激活剂DIZE处理巨噬细胞，可抑制SARS-CoV-2的促炎作用。我们的结果表明，SARS-CoV-2/ACE2相互作用使巨噬细胞对TLR信号过度敏感，抑制IRAK-M并促进促炎细胞因子的表达。因此，激活ACE2可能是一种潜在的抗炎治疗策略，以消除COVID-19患者中观察到的细胞因子风暴的发展。