新型冠状病毒肺炎 （COVID-19）是由新型冠状病毒SARS-CoV-2感染引起的，是以肺炎为主要临床症状的急性呼吸道传染病。大多数新冠感染患者表现为无症状感染或轻度呼吸道感染症状，但有40%左右的重症感染患者会出现缺氧、呼吸衰竭等多器官衰竭。新冠肺炎流行的回顾性数据表明，对 SARS-CoV-2 的过度炎症反应被认为是 COVID-19 患者疾病严重程度和死亡的主要原因，高水平的促炎细胞因子与疾病严重程度和死亡率成正相关。SARS-CoV-2 ORF8蛋白已被发现是一种调节宿主炎症反应的毒力因子，感染ORF8蛋白缺失毒株的新冠患者都有着较轻微的临床表现，重症概率和死亡率都为零，并且促炎细胞因子和趋化因子表达水平都较低；感染ORF8敲除的SARS-CoV-2病毒可以使小鼠的肺部炎症反应减弱。这些结果表明ORF8 在调控机体炎症反应中发挥了重要作用。肺泡是SARS-CoV-2侵染的主要部位，肺泡巨噬细胞是定居在肺泡表面最丰富的先天免疫细胞，它们在清除病原体和促进组织修复的过程中发挥重要作用。然而有研究显示，在重症新冠肺炎患者的支气管肺泡灌洗液中巨噬细胞大量富集，并且这些细胞表现为过度激活状态。失调的巨噬细胞大量分泌IL1β、IL6、TNFα 和CCL2、CCL4等细胞因子，引起过度炎症反应导致病情加重。目前关于被感染的巨噬细胞过度激活的原因尚不清楚。因此，探究SARS-CoV-2 ORF8蛋白对巨噬细胞功能的调控作用以及具体机制，有助于更加了解SARS-CoV-2引起的过度炎症反应的致病机制以及为临床重症感染患者的治疗策略提供新思路。

本论文主要的研究内容如下：

未激活的巨噬细胞中，ORF8 不影响促炎细胞因子的表达

比较新冠肺炎轻症患者与重症患者的炎性因子表达水平，在 GEO 数据库 （GSE207015 与 GSE227341）中分析了炎症细胞因子 IL6，IL1β 和 TNFα 的表达 情况。结果显示，新冠肺炎重症患者的炎症因子 IL6，IL1β 和 TNFα 的转录水平 表达显著升高，与患者的临床特征一致。

在本课题中,我们首先对 SARS-CoV-2 ORF8 在几个 SARS-CoV-2 变异株中的 保守性进行了比对分析,发现 ORF8 在这些变异株中相对保守。接下来，我们选 摘要 II 择了 Wuhan-Hu-1 原始毒株的 ORF8 蛋白作为研究对象，将 SARS-CoV-2 ORF8 蛋白的表达载体转染到 RAW264.7细胞中，并观察 ORF8蛋白对细胞增殖和分化 的影响。发现 ORF8不改变 RAW264.7细胞的形态，细胞仍呈未分化的圆形，同 时对表达 ORF8 蛋白的 RAW264.7 细胞进行细胞计数，结果显示 ORF8 蛋白对 RAW264.7 细胞的增殖无显著影响。之后提取了对照组和表达 ORF8 蛋白的 RAW264.7 细胞的 RNA，通过 q-PCR（Quantitative real time PCR）方法检测促炎 细胞因子 Il6、Tnfα 和抗炎细胞因子 Il10、Tgfβ 在细胞样本中的转录水平表达量。 结果表明，促炎 Il6、Tnfα 和抗炎 Il10、Tgfβ 细胞因子的转录水平表达量均没有 变化，这说明 ORF8在未激活 RAW264.7巨噬细胞中，对细胞的增殖以及促炎和 抗炎细胞因子的表达都没有影响。

LPS 诱导激活的 RAW264.7 细胞中，ORF8 上调促炎细胞因子的表达

由于巨噬细胞未被 LPS 激活时，既不表现促炎状态也不表现抗炎状态，但 是新冠患者体内的巨噬细胞表现为过度激活的促炎状态。我们利用非特异性炎 症诱导剂 LPS 诱导巨噬细胞分化为促炎表型，然后观察巨噬细胞的形态变化并 提取对照组和 ORF8 表达组的细胞样本的 RNA，通过 q-PCR 方法检测促炎细胞 因子 Il6、Tnfα 和抗炎细胞因子 Il10、Tgfβ 在细胞样本中的转录水平表达量。结 果表明，ORF8 的表达显著上调了激活后的巨噬细胞中促炎细胞因子 Il6 和 Tnfα 转录水平的表达量，而对抗炎细胞因子 Il10、Tgfβ 的转录水平没有显著影响。 与 LPS 单独处理组相比，ORF8 蛋白与 LPS 的共刺激使促炎因子 Il6 和 Tnfα 转录 水平的上调更显著。ORF8 在激活 RAW264.7 巨噬细胞中，显著增强了对促炎细 胞因子的表达。

ORF8 通过上调 Piezo1 来增强 RAW264.7 细胞促炎细胞因子的表达

Piezo1 是巨噬细胞特异性高表达的机械感知离子通道，在调节巨噬细胞炎 症反应中发挥重要作用。Piezo1 可以调控巨噬细胞的Ca2+内流，转录因子NFκB 的激活依赖于 Ca2+ ，细胞内 Ca2+ 浓度的增加可以增强 NFκB 的磷酸化。于是我 们在 RAW264.7 巨噬细胞系中表达 ORF8 蛋白，通过 Western Blot 方法检测 RAW264.7 巨噬细胞中 Piezo1 的蛋白水平表达量以及 NF-κB 的磷酸化水平。结 果显示 ORF8 可以显著上调 RAW264.7 细胞中 Piezo1 的蛋白表达量，且这种上 调现象在 LPS 激活的细胞显著高于未激活的 RAW264.7 细胞。进一步，我们通 过 Western Blot 方法检测了炎症信号通路相关的关键转录因子 NF-κB 的激活状 态，结果显示，在 LPS 激活 RAW264.7 细胞中，ORF8 显著上调了 NF-κB 的磷 酸化水平。这些数据显示 ORF8 可能是通过上调 Piezo1 的表达来增强 NFκB 的 磷酸化水平，从而上调促炎细胞因子 Il6 和 Tnfα 的表达。

Piezo1 相关抑制剂 GsMTx4 抑制 ORF8 介导的 RAW264.7 细胞过度炎症反应

抑制剂 GsMTx4 处理 RAW264.7 细胞系，采用台盼蓝染色法进行细胞计数， 据此绘细胞增殖曲线，并计算 GsMTx4 对 RAW264.7 细胞系的半抑制剂浓度 （half maximal inhibitory concentration, IC50），数据表明该细胞系的 IC50 为 11.62 μM。

采用 q-PCR 方法评估 GsMTx4 对 RAW264.7 细胞系促炎因子表达的调控作 用，结果表明 GsMTx4 能够显著下调 ORF8 介导的 RAW264.7 细胞系的 Il6 和 Tnfα 转录水平的表达，GsMTx4 不影响 RAW264.7 细胞系抗炎细胞因子 Il10、 Tgfβ 的表达。最后，采用 WB 方法检测，经 GsMTx4 处理后 RAW264.7 细胞系 中 Piezo1 蛋白的表达水平以及 NF-κB 的磷酸化水平，结果显示 GsMTx4 在蛋白 水平不影响 Piezo1 的表达，但是能够下调炎症信号通路 NF-κB 蛋白的磷酸化， 这说明 GsMTx4 是通过抑制离子通道蛋白 Piezo1 的活性来抑制促炎细胞因子的 表达。

综上所述，本论文的研究发现，ORF8 是新冠肺炎重症患者出现细胞因子风 暴的促成因素之一，ORF8 增强了新型冠状病毒的毒力，ORF8 的表达促进了活 化的巨噬细胞促炎细胞因子的表达。过表达 ORF8 能够上调促炎细胞因子 Il6 和 Tnfα 转录水平的表达，进而使巨噬细胞表现为过度激活状态。本论文首次将 ORF8 与巨噬细胞过度炎症激活联系起来，并寻找其调控的下游靶点基因的分子 机制，揭示了 ORF8 通过上调机械感知通道蛋白 Piezo1 的表达从而促进巨噬细 胞激活的分子机制。重要的是，本论文使用 Piezo1 的抑制剂 GsMTx4，其可以 逆转 ORF8 导致的促炎细胞因子 Il6 和 Tnfα 的上调表达，还能够抑制炎症信号通 路 NF-κB 的磷酸化，从而能抑制巨噬细胞的过度炎症反应，GsMTx4 有可能成 为新冠肺炎重症患者细胞因子风暴治疗的候选药物。

Novel coronavirus pneumonia (COVID-19) is an acute respiratory infectious disease caused by the novel coronavirus SARS-CoV-2 infection, which is mainly characterized by pneumonia. Most COVID-19 patients show asymptomatic infection or mild respiratory symptoms, but about 40 percent of severely infected patients will experience multiple organ failure such as hypoxia and respiratory failure. Retrospective data from the COVID-19 epidemic suggest that excessive inflammatory response to SARS-CoV-2 is considered to be a major cause of disease severity and death in COVID-19 patients, and high levels of proinflammatory cytokines are positively associated with disease severity and mortality. SARS-CoV-2 ORF8 protein has been found to be a virulence factor that regulates host inflammatory response. COVID-19 patients infected with ORF8 protein-deficient strains have relatively mild clinical manifestations, with zero probability of severe disease and zero mortality, and lower expression levels of proinflammatory cytokines and chemokines. Infection with ORF8-knockout SARS-CoV-2 virus reduced lung inflammation in mice. These results suggest that ORF8 plays an important role in the regulation of inflammation in the body. Alveoli are the main site of SARS-CoV-2 infection, and alveolar macrophages are the most abundant innate immune cells that reside on the surface of the alveoli, and they play an important role in the process of clearing pathogens and promoting tissue repair. However, studies have shown that macrophages are highly enriched in bronchoalveolar lavage fluid of patients with severe novel coronavirus pneumonia, and these cells appear to be over-activated. Dysregulated macrophages secrete a large amount of IL1β, IL6, TNFα, CCL2, CCL4 and other cytokines, which cause excessive inflammatory response and aggravate the disease. The reason for the overactivation of infected macrophages remains unclear. Therefore, exploring the regulatory effect of SARS-CoV-2 ORF8 protein on macrophage function and the specific mechanism will help to better understand the pathogenic mechanism of excessive inflammation caused by SARS-CoV-2 and provide new ideas for treatment strategies for patients with severe infection in clinic.

The main research contents of this paper are as follows:

ORF8 did not affect the expression of proinflammatory cytokines in unactivated macrophages

Expressions of inflammatory cytokines IL6, IL1β and TNFα were analyzed in GEO database (GSE207015 and GSE227341) to compare the expression levels of inflammatory cytokines in mild and severe COVID-19 patients. The results showed that the transcription levels of inflammatory factors IL6, IL1β and TNFα were significantly increased in patients with severe COVID-19, which was consistent with the clinical characteristics of patients.

In this study, we first analyzed the conserved properties of SARS-CoV-2 ORF8 in several SARS-CoV-2 variants, and found that ORF8 was relatively conserved in these variants. Next, we selected the ORF8 protein of the original strain of Wuhan-Hu-1 as the research object, transfected the expression vector of SARS-CoV-2 ORF8 protein into RAW264.7 cells, and observed the effect of ORF8 protein on cell proliferation and differentiation. It was found that ORF8 did not change the morphology of RAW264.7 cells, and the cells were still undifferentiated state. Meanwhile, the cell count of RAW264.7 cells expressing ORF8 protein showed that ORF8 protein had no significant effect on the proliferation of RAW264.7 cells. After that, RNA from the control group and RAW264.7 cells expressing ORF8 protein were extracted, and Quantitative q-PCR (real time PCR) was used to detect the transcriptional expression levels of proinflammatory cytokines Il6, Tnfα and anti-inflammatory cytokines Il10 and Tgfβ in the cell samples. The results showed that the expression levels of pro-inflammatory Il6, Tnfα, anti-inflammatory Il10 and Tgfβ cytokines were not changed, indicating that ORF8 had no effect on cell proliferation and the expression of pro-inflammatory and anti-inflammatory cytokines in RAW264.7 macrophages.

ORF8 up-regulated the expression of pro-inflammatory cytokines in RAW264.7 cells induced and activated by LPS

Because macrophages exhibit neither a pro-inflammatory nor an antiinflammatory state when they are not activated by LPS, macrophages in COVID-19 patients exhibit an overactivated pro-inflammatory state. We used LPS, a non-specific inflammatory inducer, to induce macrophages to differentiate into pro-inflammatory phenotypes, and then observed the morphological changes of macrophages and extracted RNA from cell samples of control group and ORF8 expression group. The expression levels of proinflammatory cytokines Il6, Tnfα and anti-inflammatory cytokines Il10 and Tgfβ in cell samples were detected by q-PCR. The results showed that ORF8 significantly up-regulated the transcription levels of pro-inflammatory cytokines Il6 and Tnfα in activated macrophages, but had no significant effect on the transcription levels of anti-inflammatory cytokines Il10 and Tgfβ. The co-stimulation of ORF8 protein with LPS significantly upregulated the transcription levels of proinflammatory factors Il6 and Tnfα compared with LPS alone. ORF8 significantly enhanced the expression of proinflammatory cytokines in RAW264.7 macrophages.

ORF8 enhances the expression of pro-inflammatory cytokines in RAW264.7 cells by up-regulating Piezo1

Piezo1 is a mechanosensory ion channel that is specifically and highly expressed in macrophages and plays an important role in regulating macrophage inflammatory response. Piezo1 regulates Ca2+ influx in macrophages. Activation of the transcription factor NFκB depends on Ca2+, and increased intracellular Ca2+ concentration enhances the phosphorylation of NFκB. We then expressed ORF8 protein in RAW264.7 macrophages and detected the expression of Piezo1 protein and the phosphorylation of NF-κB in RAW264.7 macrophages by Western Blot. The results showed that ORF8 significantly up-regulated the protein expression of Piezo1 in RAW264.7 cells, and this up-regulation was significantly higher in LPS-activated cells than in unactivated RAW264.7 cells. Further, we detected the activation status of NF-κB, a key transcription factor associated with inflammation signaling pathway, by Western Blot analysis. The results showed that ORF8 significantly up-regulated the phosphorylation level of NF-κB in RAW264.7 cells activated by LPS. These data suggest that ORF8 may enhance the phosphorylation of NFκB by up-regulating the expression of Piezo1, thereby up-regulating the expression of pro-inflammatory cytokines Il6 and Tnfα.

GsMTx4, a PIEZO1-related inhibitor, inhibits ORF8-mediated excessive inflammatory responses in RAW264.7 cells

RAW264.7 cell line was treated with inhibitor GsMTx4, and cell count was performed by Trypan blue staining method, according to which cell proliferation curve was plotted. The half maximal inhibitory concentration (IC50) of GsMTx4 on RAW264.7 cell line was calculated, and the data showed that the IC50 of the cell line was 11.62 μM

q-PCR was used to evaluate the regulatory effect of GsMTx4 on the expression of pro-inflammatory factors in RAW264.7 cell line. The results showed that GsMTx4 could significantly down-regulate the expression of Il6 and Tnfα transcription levels in ORF8-mediated RAW264.7 cell line. GsMTx4 did not affect the expression of antiinflammatory cytokines Il10 and Tgfβ in RAW264.7 cell line. Finally, the expression level of Piezo1 protein and the phosphorylation level of NF-κB in RAW264.7 cell line after treatment with GsMTx4 were detected by WB method. The results showed that GsMTx4 did not affect the expression of Piezo1 at the protein level, but could downregulate the phosphorylation of NF-κB protein in the inflammatory signaling pathway. This suggests that GsMTx4 inhibits the expression of pro-inflammatory cytokines by inhibiting the activity of the ion channel protein Piezo1.

In summary, the study in this paper found that ORF8 is one of the contributing factors to cytokine storm in severe patients with COVID-19, ORF8 enhances the virulence of the novel coronavirus, and the expression of ORF8 promotes the expression of activated pro-inflammatory cytokines in macrophages. Overexpression of ORF8 can up-regulate the transcription levels of the pro-inflammatory cytokines Il6 and Tnfα, and then make macrophages appear to be over-activated. In this paper, for the first time, ORF8 was linked to the excessive inflammatory activation of macrophages, and the molecular mechanism of downstream target genes regulated by ORF8 was investigated. The molecular mechanism of ORF8 promoting the activation of macrophages was revealed by up-regulating the expression of Piezo1, a mechanical sensing channel protein. Importantly, GsMTx4, an inhibitor of Piezo1, was used in this paper to reverse the up-regulated expression of the pro-inflammatory cytokines Il6 and Tnfα induced by ORF8, as well as to inhibit the phosphorylation of the inflammatory signaling pathway NF-κB, thereby inhibiting the excessive inflammatory response of macrophages. GsMTx4 may be a candidate for cytokine storm therapy in severe COVID-19 patients.