Laboratory of Clinical Chemistry, Medical School, University of Crete, Heraklion, Greece.
Department of Pediatrics, Medical School, University of Crete, Heraklion, Greece.
Front Immunol. 2021 Jun 23;12:683800. doi: 10.3389/fimmu.2021.683800. eCollection 2021.
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.
严重急性呼吸综合征冠状病毒 2 感染患者的主要死亡原因是先天免疫系统失调和细胞因子风暴的发展。严重急性呼吸综合征冠状病毒 2 通过其刺突 (S) 蛋白与血管紧张素转换酶 2 (ACE2) 受体结合，感染肺部的多种细胞类型，包括巨噬细胞。ACE2 受体在巨噬细胞中启动信号，调节其激活，包括细胞因子和趋化因子的产生。白细胞介素 1 受体相关激酶 (IRAK)-M 是调节 TLR 反应幅度的炎症反应的中央调节剂。这项工作的目的是研究严重急性呼吸综合征冠状病毒 2 S 蛋白引发的信号是否调节巨噬细胞中促炎细胞因子的产生。为此，我们用严重急性呼吸综合征冠状病毒 2 S 蛋白处理 PMA 分化的 THP-1 人巨噬细胞，并测量包括白细胞介素 6 (IL6)、肿瘤坏死因子 α (TNFα)、白细胞介素 8 (IL8)、CXCL5 和 MIP1a 在内的炎症介质的诱导。结果表明，严重急性呼吸综合征冠状病毒 2 S 蛋白诱导 IL6、MIP1a 和 TNFα mRNA 的表达，而对 IL8 和 CXCL5 mRNA 水平没有影响。我们进一步研究了严重急性呼吸综合征冠状病毒 2 S 蛋白是否改变了巨噬细胞对 TLR 信号的反应性。用严重急性呼吸综合征冠状病毒 2 S 蛋白处理脂多糖激活的巨噬细胞，增加了白细胞介素 6 和 MIP1a mRNA 的水平，这种作用在蛋白质水平上仅对白细胞介素 6 明显。同样，用严重急性呼吸综合征冠状病毒 2 S 蛋白处理 PAM3csk4 刺激的巨噬细胞，导致白细胞介素 6 的 mRNA 增加，而肿瘤坏死因子 α和 MIP1a 不受影响。这些结果在原代人外周单核细胞 (PBMC) 和分离的 CD14+单核细胞中得到了证实。IRAK-M，一种无活性的 IRAK 激酶同工型，调节巨噬细胞对 TLR 配体的反应性。事实上，我们发现严重急性呼吸综合征冠状病毒 2 S 蛋白在 THP1 巨噬细胞和原代人 PBMC 和 CD14+单核细胞中均抑制 IRAK-M mRNA 和蛋白的表达。严重急性呼吸综合征冠状病毒 2 S 蛋白与 ACE2 的结合导致 ACE2 的内化和其活性的抑制。ACE2 的激活先前已被证明在巨噬细胞中诱导抗炎反应。用 ACE2 激活剂 DIZE 处理巨噬细胞可抑制严重急性呼吸综合征冠状病毒 2 的促炎作用。我们的结果表明，严重急性呼吸综合征冠状病毒 2/ACE2 相互作用使巨噬细胞对 TLR 信号超敏，抑制 IRAK-M 并促进促炎细胞因子的表达。因此，ACE2 的激活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。
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