Role And Mechanism Of Monocytes In Cytokine Storm Caused By SARS-CoV-2

COVID-19,caused by a novel coronavirus（SARS-Co V-2）,is an acute respiratory infection caused by an infection that,while causing illness in healthy individuals,is more likely to threaten older people with underlying medical conditions.The cytokine storm caused by excessive release of cytokines by the body in response to viral infection is emerging as one of the mechanisms leading to acute respiratory distress syndrome and acute systemic organ dysfunction in COVID-19.Although the mechanisms of SARS-Co V-2 induced infection are complex,inflammation and abnormal coagulation may promote the development and progression of COVID-19 and induce activation of monocytes/macrophages.Understanding the role of monocytes in inflammatory cytokine storms is essential to mitigate tissue damage caused by this response.Based on monocyte function and cytokine secretion,we explored the role and mechanisms of novel coronaviruses in cytokine storms to provide important guidance for the treatment of COVID-19.Studies have shown that angiotensin converting enzyme 2（ACE-2）is the main binding receptor of SARS coronavirus and SARS-Co V-2.In this study,flow cytometry and western blotting were used to examine the high expression of ACE-2protein on the surface of Calu-1 cells.SARS-Co V-2 spike protein S stimulated Calu-1cells,and the expression of GM-CSF and CD40 L was significantly increased after i TRAQ proteome analysis compared with human protein database,which may lead to lung epithelial cells regulating monocyte activation and inflammatory cytokine secretion through CD40L/CD40 pathway.Western blotting and q RT-PCR were used to confirm the results of i TRAQ protein.Next,peripheral blood mononuclear cells（PBMC）were isolated from human concentrated leukocytes provided by healthy volunteers,The co-culture experiment of lung epithelial cells and monocytes was established to study the effect and mechanism of activation of monocytes by SARS-Co V-2.Calu-1 cells were stimulated by different concentrations of SARS-Co V-2-S.The secretion and expression of GMCSF were analyzed by ELISA and PCR,and the expression of CD40 L was analyzed by flow cytometry.The results showed that the monocyte phenotype was not altered by the co-culture of calu-1 cells with monocytes,however,the expression of CD86 and HLA-DR in monocytes increased significantly after SARS-Co V-2-S was added to the co-culture system,indicating that monocytes acquired the inflammatory phenotype of M1-type macrophages.The results showed that the co-culture of calu-1cells with Monocytes did not cause a change in monocyte phenotype,but when SARS-Co V-2-S was added to the co-culture system,the expression of CD86 and HLA-DR in Monocytes was significantly increased,suggesting that Monocytes acquired an inflammatory phenotype of M1-type macrophages.The results of intracellular staining and ELISA showed that SARS-Co V-2-S stimulated monocytes to secrete IL-18 and IL-1β through Calu-1 cells,suggesting that SARS-Co V-2-S activated monocytes by acting on lung epithelial cells and promoted the differentiation of monocytes into M1 inflammatory phenotype.Western blotting results showed that SARS-Co V-2-S induced the secretion of GM-CSF and up-regulated the expression of CD40 L by activating PI3K/AKT in Calu-1 cells.In SARS-Co V-2-S-stimulated Calu-1 cells co-cultured with monocytes,the expression of CD86 and HLA-DR in monocytes was significantly decreased with the addition of blocking antibody to CD40 or neutralizing antibody to GM-CSF,and IL-18 and IL-1β in cell culture supernatant were significantly decreased.These results suggest that SARS-Co V-2-S activates Calu-1 cells,participates in monocyte activation through CD40L/CD40 and GM-CSF,and induces monocyte inflammatory phenotype and secretion of inflammatory cytokines.In summary,we have explored the role and mechanisms of monocytes in the novel coronavirus-induced cytokine storm.And the results demonstrated that with the binding of S protein to ACE-2 receptor on the surface of Calu-1 cells,SARS-Co V-2virus could activate PI3K/AKT pathway,induce GM-CSF secretion from lung epithelial cells and up-regulate CD40 L expression.Further,the inflammatory phenotype of monocytes and the secretion of inflammatory cytokines were triggered by the activation of Calu-1 cells through the involvement of CD40L/CD40 and GMCSF in the activation of monocytes.Thus,we infer that monocyte phenotype activation has a promoting role in the cytokine storm triggered by SARS-Co V-2.Results above provide a preliminary reference base for the mechanisms by which SARS-Co V-2 infects the lung and triggers inflammation,and provide promising evidence for the treatment of COVID-19 by targeting monocytes and inflammatory cytokines.