Preliminary Study On The Initiation Mechanism Of M1-type Polarization Of Macrophage Induced By CVB3 To Promote Inflammation

Background:Coxsackievirus group B type 3(CVB3)was the most common pathogenic pathogens causing Viral Myocarditis(VMC),accounting for 50 percent of the causes of patients with Viral Myocarditis,for which there is currently no specific remedy.CVB3 infection is divided into three stages in the study model of VMC.In the first stage,CVB3 directly infects cardiomyocytes,promoting the replication and release of a large number of virus,destroying the function of cardiomyocytes,and infiltrating a variety of innate immune cells such as NK cells and monocytes.This stage is also the most widely studied.In the second stage,the virus replicates so much that it is further sensed by the innate immune cells in the tissue that cytokine cascades are released to activate the adaptive immune response and cause acute myocarditis.In the third stage,the adaptive immune response results in a decrease in viral titer,the damage is repaired by fibrosis,which manifests as chronic dilated cardiomyopathy and other diseases.Among the above three stages,the second stage induced myocarditis is the most serious stage in the pathological process of VMC caused by CVB3.However,the inflammatory activation mechanism of viral myocarditis in not yet clear.Therefore,elucidating the initiation mechanism of inflammatory activation of CVB3 in viral myocarditis is expected to provide new ideas for its targeted therapy.Objective:To clarify the cell types of CVB3-mediated macrophage polarization and explore the mechanism of CVB3-mediated macrophage polarization.Methods:1.In macrophage models from different sources,the polarization types of macrophages affected by CVB3 were identified as follows:1)THP-1 cells were induced by 75 ng/m L PMA for 24 hours to construct human M0-type macrophage model,which was identified by flow cytometry.Murine bone marrow mesenchymal stem cells were induced by 20 ng/m L GM-CSF for 7 days to construct the primary mouse bone marrow derived(BMDM)macrophage model,which was identified by flow cytometry.2)RT-q PCR was used to detect whether CVB3 infected macrophages affected replication.3)RT-q PCR was used to detect m RNA expression of cell marker molecules(IL-6,IL-1β,IL-10,CD86,CD163,CD206)of macrophages(RAW264.7,THP-1,BMDM)incubated with CVB3(MOI=20)at different times(0,1,3,6,12,24 hours)in order to determine the polarization type of macrophages preliminarily.4)Flow cytometry was used to detect the positive cell types of macrophages(RAW264.7,THP-1,BMDM)incubated with CVB3(MOI=20)at different times(0,1,3,6,12,24 hours)to further determine the polarization types of macrophages.5)The effects of CVB3(MOI=20)incubated macrophages(RAW264.7,THP-1,BMDM)at different times(0,1,3,6,12,24 hours)on cytokine release(TNF-α,IL-12p35,IL-12p40,IL-23p19,CXCL9,CXCL10)were detected by RT-q PCR to evaluate the effect of CVB3 on the biological function of macrophages.6)Tissue immunofluorescence was used to detect the polarization pattern of cardiac macrophages in CVB3-induced viral myocarditis.2.In macrophage models from different sources,the reprogramming process of urea cycle metabolism caused by CVB3 to macrophage polarization was identified as follows:1)Western blot was used to detect the changes of urea cycle related enzymes(ASS1,ASL,ARG1)in macrophages(RAW264.7,THP-1,BMDM)incubated with CVB3(MOI=20)at different times(0,1,2,3,4,5 hours).2)The changes of urea cycle related enzymes(ASS1,ASL,ARG1,OTC)in macrophages(RAW264.7,THP-1,BMDM)incubated with CVB3(MOI=20)were detected by RT-q PCR at different times(0,1,2,3,4,5 hours),so as to have an overall grasp of the reprogramming of urea cycle metabolism.3)The expression of ASS1 in cardiac macrophages in viral myocarditis caused by CVB3 was detected by tissue immunofluorescence.4)Metabolomics was used to analyze the absolute content changes of about 30amino acids in macrophages(RAW264.7 and THP-1)incubated with CVB3(MOI=20)for 5 h,so as to determine the changes in amino acid metabolism during the treatment of CVB3,further evidence of metabolic reprogramming in the urea cycle.3.To preliminarily explore the molecular mechanism of CVB3 leading to the urea cycle metabolic reprogramming of macrophages and then leading to polarization,as follows:1)The eukaryotic overexpressed plasmids(pc DNA3.1(-)-3FLAG-ASS1,pc DNA3.1(-)-3HA-ASS1,pc DNA3.1(-)-3FLAG-VP2)required by the experiment were constructed by one-step cloning method for subsequent mechanism exploration.2)Protein-protein interactions were detected by Co-IP(co-immunoprecipitation)after the eukaryotic expression plasmids of structural proteins VP1,VP2,VP3 and VP4 were respectively co-transfected into 293T cells for 48 hours with the eukaryotic expression plasmids of ASS1,a key enzyme of urea cycle.3)After pc DNA3.1(-)-3HA-ASS1 and pc DNA3.1(-)-3FLAG-VP2co-transfected into He La cells of CVB3 mode cells,the co-localization of ASS1 and structural protein VP2 in He La cells was observed by laser confocal microscopy.4)Western blot analysis of ASS1 protein expression in He La cells after transfection with 1.5μg pc DNA3.1(-)-3FLAG-VP2 at 12,24,36,and 48 hours indicated that there was a relationship between VP2 of CVB3 structural protein and host protein.5)Western blot analysis of the ubiquitination protein of RAW264.7 cells incubated with CVB3(MOI=20)showed that the ubiquitination degradation of ASS1was inhibited,and 10μM MG132 was used as positive control to clarify the mechanism of the increased expression of ASS1 protein under the effect of CVB3.6)Western blot analysis showed that 1.5μg pc DNA3.1(-)-3flag-VP2transfected He La cells for 48 h inhibited ASS1 ubiquitination degradation,and 10μM MG132 was used as positive control to elucidate the mechanism of the increased expression of ASS1 protein under VP2.Results:1.The human M0-type macrophage model can be successfully constructed by inducing THP-1 cells with 75 ng/m L PMA for 24 hours.Murine bone marrow mesenchymal stem cells induced by 20 ng/m L GM-CSF in 7 days could successfully construct the primary murine-derived macrophage model.CVB3 does not replicate in macrophages,but high concentrations of CVB3 incubate macrophages with long-lasting stimulation.2.After incubation of macrophages(RAW264.7,THP-1,BMDM)at high concentrations of CVB3(MOI=20)for different times(0,1,3,6,12,24 hours),the levels of M1 macrophage cell marker molecules IL-6,IL-1β,CD86 were significantly elevated(P<0.05).The m RNA level of IL-10 was also increased in M2-type macrophages,but the m RNA levels of CD163 and CD206 were significantly decreased(P<0.05).It was preliminarily concluded that macrophages incubated with CVB3 could induce polarization of M1 type macrophages.Flow cytometry showed that the proportion of CD86~+CD206~-M1 macrophages increased and that of CD86~-CD206~+M2 macrophages decreased under the effect of CVB3,which further confirmed that the macrophages incubated by CVB3 can promote the polarization of M1 macrophages.In the CVB3-induced viral myocarditis model,M1-type macrophage infiltration was also dominant.3.The expression levels of TNF-α,IL-12p35,IL-12p40,IL-23p19,CXCL9 and CXCL10 m RNA in macrophages incubated with high concentration of CVB3(MOI=20)were significantly increased(P<0.05).It has been confirmed that CVB3can release a large number of inflammatory cytokines and chemokines to play a pro-inflammatory role after promoting the polarization of macrophages towards M1type.4.High concentration of CVB3 promoted the protein level of ASS1 in macrophages(RAW264.7,THP-1,BMDM)at the beginning of polarization to be significantly up-regulated(P<0.05),while the transcriptional level was not significantly changed.CVB3 had no effect on other metabolic enzymes in the urea cycle,including ARG1,ASL and OTC.In CVB3-induced viral myocarditis,ASS1expression in cardiac macrophages was significantly up-regulated.Targeted amino acid metabolomics analysis of macrophages(RAW264.7,THP-1)at the beginning of the polarization stage showed that the content of citcitline in ASS1 substrate was significantly decreased(P<0.05),which fully confirmed that CVB3 induced the polarization of macrophages at the beginning of the polarization stage by causing the metabolic reprogramming of urea cycle.5.Through Co-IP identification of different structural proteins of CVB3 and ASS1,it was found that structural protein VP2 of CVB3 interacts with ASS1,and both of them colocalized in the cytoplasm of cells,while other structural proteins do not interact with ASS1.Further experiments showed that the eukaryotic expression plasmid of CVB3 structural protein VP2 could up-regulate the expression of ASS1protein in He La cells of CVB3 mode cells at different times,and this up-regulation was achieved by inhibiting the ubiquitination degradation of ASS1.Conclusions:1.High concentration of CVB3(MOI=20)can promote M1-type polarization of macrophages after incubation.2.CVB3 may promote the polarization of macrophages by inducing reprogramming of urea cycle metabolism in macrophages.3.CVB3 structural protein VP2 interacts with ASS1,a key enzyme in urea cycle,and both of them are colocalized in the cytoplasm of cells,which can up-regulate the expression of ASS1 protein by inhibiting the ubiquitination degradation of ASS1.