FGL2 Exacerbates Immunocoagulation Caused By SARS-CoV-2 Infection

ObjectiveNovel coronavirus infection often causes coagulation and fibrinolytic system abnormalities in severe patients.Patients have elevated levels of D-dimer and fibrinogen,complicated by pulmonary embolism and deep vein thrombosis,and microthrombosis of lungs and extrapulmonary organs.The above suggests that a high risk of thrombosis in COVID-19 severe/critical patients.The COVID-19 autopsy showed focal hemorrhage and necrosis in the kidney and lungs.Therefore,it is important to investigate the mechanism of immune coagulation abnormalities in severe patients.Fibrinogen-like protein 2（FGL2）,belonging to the fibrinogen-related protein superfamily,includes two forms:the membrane bound FGL2（m FGL2）and the soluble FGL2（s FGL2）.m FGL2,also known as FGL2prothrombinase,can directly cleave prothrombin into thrombin to promote coagulation and thrombosis,while s FGL2 mainly plays an immunomodulatory role.Therefore,we focused on FGL2 to explore its possible role in thrombosis and tissue damage in patients with severe COVID-19.MethodsFirst,the FFPE of COVID-19 renal autopsy samples was sequenced by spatial transcriptome technology to obtain cell spatial location distribution and to study the expression of genes in situ cells.Then,Single-cell transcriptome sequencing datasets,ELISA,IHC,IF,H&E and Masson staining were used to analyze the expression of FGL2 in acutely infected patients and severe patients.Next,two infection models of mouse adaptations to the novel coronavirus were established:MA17 and MASCp36.Mice were infected with the MA17 strain,body weight changes were recorded daily and mortality was counted 14 days post-infection.Pulmonary virus titers were detected by q RT-PCR.A variety of staining methods,including H&E,IHC and Masson staining,were used to identify mouse lung histopathological changes,FGL2expression,fibrinogen deposition,and collagen fiber formation.After the MASCp36 strain infected mice,the changes in Fgl2 transcription levels in lung were identified by RNA sequencing,and the expression of FGL2 protein and fibrinogen deposition were identified by IHC.Finally,the mechanism of upregulation of FGL2 expression in severe patients was studied.On the one hand,in order to study the regulatory effect of SARS-CoV-2 on Fgl2expression,different viral proteins were co-transfected with the core promoter of h Fgl2 to293T cells.The transcriptional regulatory activity of viral proteins on the Fgl2 promoter region was analyzed by a dual-luciferase reporter system.On the other hand,in order to study the effect of cytokine storm on Fgl2 expression in severe patients,monocytes in peripheral blood mononuclear cells（PBMCs）were induced to differentiate into macrophages（MDMs）and dendritic cells（Mo DCs）.After stimulation of MDMs,Mo DCs,lung epithelial cells and renal tubular epithelial cells by different cytokines in vitro,the expression level of Fgl2 was detected by q RT-PCR.ResultsSpatial transcriptome sequencing of kidney tissue from COVID-19 autopsy showed that the proportion of fibroblasts and endothelial cell subsets in the kidneys of severe patients increased.Differential gene analysis found that up-regulated genes expressed in these cell subsets positively correlated with disease severity.At the same time,we found that the receptors of the SARS-CoV-2 are widely distributed in kidney tissue and expressed in different cell subtypes.More importantly,FGL2 in the control group was mainly expressed in podocytes,while in severe patients,it was mainly expressed in renal tubular cells and vascular endothelial cells.The above suggests that SARS-CoV-2 infection may lead to altered cell subsets of FGL2 expression in kidney tissue.ELISA analysis showed that SARS-CoV-2 can significantly increase plasma s FGL2levels in acutely infected and severe patients.The data analysis of sc RNA-seq,IHC and IF results showed that the expression of FGL2 was much higher in severe patients than in healthy donors and mild patients,mainly expressed in macrophages,dendritic cells,lung epithelial cells,renal tubular epithelial cells and endothelial cells.In addition,there were a large amount of fibrinogen deposition in the interstitium and perivascular tissues.Masson staining showed that the normal structure of the lungs and kidneys of the COVID-19autopsy had been destroyed and capillary microthrombes appeared.The above results suggest that FGL2 may be involved in thrombosis and tissue destruction caused by SARS-CoV-2 infection.The infection of MA17 strain increased the expression of FGL2 in the lung of WT mice,while the survival rate of Fgl2^-/-mice was significantly improved,inflammatory cell infiltration of lung tissue,the fibrinogen deposition and the formation of thrombosis and collagen fibers was reduced.The results showed that the deletion of Fgl2 could antagonize lung damage caused by MA17 virus infection.Similarly,MASCp36-infected elderly C57BL/6 mice had higher levels of transcription of Fgl2 and expression of FGL2 protein in the lungs.These results confirm that FGL2 deficiency reduces tissue damage caused by SARS-CoV-2 infection and improves survival in mice.Finally,the mechanistic study in vitro of the upregulation of Fgl2 expression found that the S protein of the SARS-CoV-2 Omicron strain could increase the transcriptional activity of the h Fgl2 promoter.A variety of cytokines can promote the expression of Fgl2 in MDMs and Mo DCs,and IL-1βand IL-6 are the main inflammatory factors that promote the expression of Fgl2 in lung epithelial cells.The above data confirm that the upregulation of Fgl2 expression in severe patients are affected by SARS-CoV-2 and inflammatory factor storm.ConclusionIn severe/critically ill elderly patients caused by SARS-CoV-2,viral infection and cytokine storm jointly upregulate the expression of Fgl2,leading to thrombosis and tissue damage.Thus,Specific inhibition of FGL2 activity may become a new strategy for the clinical treatment of critically ill patients.