Study On The Mechanism Of PTX3 In Inflammation Induced By Viral Infection

BackgroundHand-foot-mouth disease(HFMD)is an infectious disease caused by a variety of enteroviruses.Enterovirus A71(EV-A71),coxsackievirus A16(CA16)and coxsackievirus A10(CA10)are the main pathogenic viruses.In order to further investigate the pathogenesis of HFMD,we analyzed the gene expression,protein level and phosphorylation site changes in blood,muscle,spleen and thymus of infected mice by transcriptome,proteome and phosphoproteome,which were comprehensively evaluated to study the common change factors of immune response caused by three kinds of virus infection.Meanwhile,we used the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection mouse model to analyze the key molecules involved in COVID-19 in multiple organs of mice after SARS-CoV-2 infection through proteome and phosphoproteome.Finally,we found that PTX3(Pentraxin 3),a protein involved in innate immunity,has a special role in the immune system against viral infection.PTX3 is a soluble recognition molecule,which can be rapidly produced by a variety of cells in response to inflammatory factors and exogenous microorganisms,and plays an important role in innate immunity triggered by bacterial,fungal and viral infections.In view of the diversity of PTX3 to inflammatory response,in addition to focusing on its key role in myositis caused by EV-A71 infection in mice,this study also explored the important role of PTX3 in severe pneumonia caused by SARS-CoV-2 infection in mice.Methods1)Seven-day-old Balb/c mice were infected by intraperitoneal with 100 p.1 100 LD50 EV-A71 virus,muscles were collected for proteomic and phosphoproteomic analysis at 2 and 5 days post infection.2)Seven-day-old ICR mice were infected by intraperitoneal with 100 μl 100 LD50 EV-A71,CA16,CA10 viruses,blood,muscle,spleen and thymus were taken at 2 day post infection to detect the viral load and perform RNA sequencing analysis.3)Eight 10-week-old K18-hACE2 mice were challenged with SARS-CoV-2 at a dose of 1×105 TCID50 by intranasal inoculation,mice were dissected at 5 dpi to collect the cortex,hippocampus,thalamus,lung,and kidney to screen for proteomic and phosphoproteomic profiling.4)Ten-day-old C57 mice were infected by intraperitoneal with 100 μl 100 LD50 EV-A71 virus,and 6-12-week-old hACE2 mice were inoculated intranasally with 50 μ 1×105 TCID50 SARS-CoV-2,qRT-PCR and ELISA were used to determine the level of PTX3 in diseased tissue(EV-A71:muscle;SARS-CoV-2:lung)after viral infection,and the source of PTX3 production was determined by immunofluorescence.5)In vitro ELISA detected PTX3 binding to EV-A71;Cell neutralization experiments to investigate the protective effect of PTX3 treatment on cells.6)Ten-day-old C57 and PTX3-/-mice were infected by intraperitoneal with 100 μl 100 LD50 EV-A71 virus,the C57 treatment group received daily intraperitoneal injections of 200 μg/kg PTX3 protein and the PTX3-/-treatment group received daily intraperitoneal injections of 400 μg/kg PTX3 protein,the clinical manifestations were observed and recorded.Blood and hindlimb skeletal muscles were taken at 3,5 and 7 days post infection,viral load was detected by qRT-PCR;H&E,immunohistochemistry,flow cytometry,MPO content detection and Luminex assay were used to explore the occurrence of inflammation.C3 deposition and FH aggregation in muscle tissue were determined by immunofluorescence and qRT-PCR,and the mechanism of PTX3 interaction with complement in regulating inflammatory response during viral infection was investigated.7)6-12-week-old hACE2 and hACE2&PTX3-/-mice were infected with 50 μl of 1×105 TCID50 SARS-CoV-2 by intranasal instillation,and the body weight was recorded daily.Blood and lung tissues were collected at 3,5 and 7 days after infection for detection of viral load by qRT-PCR and for observation of pathological damage by H&E.To explore the role of PTX3 in SARS-CoV-2 infection.8)10-day-old C57,PTX3-/-and PSGL-1-/-mice were intraperitoneally infected with 100 μl 100 LD50 EV-A71 virus,respectively.P-Selectin antagonist(2mg/kg)was intraperitoneally injected daily from day 0 of infection.The clinical scores,body weight and survival rate of the mice were recorded.Blood and hindlimb skeletal muscles were collected an 3,5,and 7 days post infection.The viral load was detected by qRT-PCR,and the inflammation was observed by H&E and MPO content detection.To explore the mechanism of interaction between PTX3 and PSGL-1 in viral infection.Result1)Based on the HFMD mouse infection model,we analyzed the differentially expressed proteins and phosphorylation sites at different time points after the infection of EV-A71 by proteome and phosphorylation proteome.The differentially expressed proteins and the phosphorylation sites were divided into six trends by FC values according to a fuzzy Cmeans clustering method.Among them,162 differentially expressed proteins and 110 differentially expressed phosphorylation sites were detected in the trend of continuous up-regulation,while PTX3 protein was found to be continuously up-regulated after virus infection.Transcriptome analysis showed that there were 554 differentially expressed genes in blood,2027 in muscle,2623 in spleen and 1348 in thymus.The expression of Ptx3 gene was up-regulated in the muscle tissues infected by all of the three viruses.These results suggested that PTX3 might be a common up-regulated factor in HFMD.2)Based on the SARS-CoV-2 mouse infection model,the differentially expressed proteins and phosphorylation sites in the cerebral cortex,hippocampus,thalamus,lung and kidney of mice infected with SARS-CoV-2 were analyzed by proteome and phosphoproteome analysis.We found that compared with the three brain regions,the peripheral lung and kidney showed relatively apparent antigen processing and presentation,complement and coagulation cascades.3)Based on EV-A71 mouse infection model,we detected the distribution,source and change trend of PTX3 in mice after infection.It was found that the expression of PTX3 in the blood and skeletal muscle began to increase on the third day after infection,and decreased on the seventh day after infection.The results of multicolor immunofluorescence staining showed that neutrophils and macrophages were the main sources of PTX3 in muscle tissues.PTX3 expression was also found to be upregulated in the lungs of SARS-CoV-2 infected hACE2 mice.4)According to in vitro binding experiments of PTX3 protein to EV-A71 virus,we found that PTX3 protein could directly bind to EV-A71 virus based on ELISA results;PTX3 could inhibit the replication of EV-A71 and SARS-CoV-2 with a protective effect on cells based on the cell neutralization experiments.5)According to in vivo experiment with PTX3-/-mice to verify the function of PTX3,we found that compared with wild-type C57 mice,PTX3/-mice had significantly higher viral loads in blood and muscle,with more inflammatory cell infiltration in muscle tissues as well as significantly higher levels of inflammatory cytokines in blood after EV-A71 infection.When treated with PTX3 protein,wild-type C57 mice showed alleviated phenotype after infection with EV-A71.including the improvement of weight loss,clinical symptoms and survival rate,and significant reduction of viral loads in hindlimb skeletal muscle and blood as well as the decrease of inflammatory cytokines in serum.In addition,an increase in C3 deposition and a decrease in FH content were found in the muscle of PTX3-/-mice.There were no significant differences in clinical parameters,viral loads,pathological changes and MPO content in serum among C57 mice,PTX3-/-mice and PSGL-1-/-mice treated with Pselectin antagonist.6)By the use of hACE2&PTX3-/-mice,we investigated the role of PTX3 against SARS-CoV-2 infection.It was found that the lung viral loads of SARSCoV-2 in hACE2&PTX3-/-mice were significantly higher than that in hACE2 mice,while the pathological manifestations were more serious.Conclusion:1)Based on the analysis of transcriptome,proteome and phosphoproteome,we found that EV-A71 infection can lead to the up-regulation of pentraxins family proteins.In vivo validation experiments in mice showed that PTX3 expression was up-regulated after EVA71 and SARS-CoV-2 virus infection,indicating that PTX3 plays an important role in the body’s innate immunity against viral invasion.2)PTX3 could bind to EV-A71 and inhibit the replication of EV-A71 and SARS-CoV-2 in vitro,indicating that PTX3 might have a direct antiviral effect on virus infection.In vivo functional studies with knockout mice have shown that PTX3 deficiency promoted EV-A71 and SARS-CoV-2 replication,exacerbated inflammatory responses,and encouraged inflammatory cell migration.Conversely,treatment with PTX3 protein on wild-type mice inhibited viral replication and reduced inflammation levels.Therefore,in vivo and in vitro experiments altogether verified that PTX3 had antiviral effects on EV-A71 infection as well as SARS-CoV-2 infection.3)The study on the mechanism of PTX3 against virus infection showed that PTX3 inhibited complement activation by regulating the inhibitory factor H.Meanwhile,PTX3 antagonized the migration of inflammatory cells into tissues which mediated by PSGL-1/P-selectin pathway through competitive binding with P-selectin.