| Objective:Viral myocarditis(VMC)is a viral-induced inflammation of myocardium characterized with intensive immune infiltration and impaired cardiac systolic and conduction.20% of patients suffering from acute VMC develop into dilated cardiomyopathy(DCM),which leads to repeating heart failure and is the major causeof heart transplantation accounting for 10% of sudden cardiac deaths in young people.Among all causative viruses,Coxsackievirus B type 3(CVB3)is still considered as the most prevalent causing virus for VMC especially DCM.The pathogenesis of VMC is still not fully understood,effective and specific therapies for VMC is lacking.Hence it is of significance to clarify the pathogenesis of VMC.CVB3 is non-enveloped,positive sense single-stranded RNA belonging to the family of Picornaviridae.It primarily propagates in gastrointestinal tracts before releasing into the bloodstream,theninfects multi organs including heart,pancreas,brain and liver,causing viral myocarditis,pancreatitis and hepatitis.Both virus-mediated myocyte injury and cardiac immune injury contribute to the pathogenesis of viral myocarditis and cardiac fibrosis.In the acute phase of CVB3 infection(day0~3-7),direct cardiac damage by virus,cardiac lymphoid cell infiltration and Toll-like receptors(TLRs)-triggered inflammatory cytokine(IL-1b,IL-6,IL-18,TNF-a,IL-17)response,play important roles in early antiviral defense and cardiac injury.In the subacute phase(day7-14),adaptive T and B response are activated and recruited into the heart.T cells are considered as main pathogenic cells for the development of VMC and autoimmune Abs accelerate this process.In the chronic phase,Tregs and alternatively activated macrophages as well as TGF-b and IL-10 contribute to cardiac repair and cardiac fibrosis.Our preliminary data suggest that CVB3 infection induces a very early and quick peripheral neutrophil amplification and intensive influx into the hearts of mice during early infection(day0~3)which is associated with the severity of myocarditis.Neutrophils differentiate in bone marrow and are most abundant leukocytes in the blood.Neutrophils are critical effectors in anti-bacterial or anti-fungi immunity via quick infiltration and production of antimicrobial peptides,cytokines and reactive oxygen species(ROS).Our previous data indicate that peripheral neutrophils play dispensable roles in CVB3-induced myocarditis since peripheral depletion of neutrophils by anti-Ly6 G neutralizing m Abs has no effect on severity of myocarditis and viral replication.However,this neutralizing m Ab is unable to deplete BM-differentiating neutrophils and tissue-infiltrated neutrophils,thus is not ideal to evaluate role of cardiac-resident neutrophils in myocarditis.Neutrophil tracking to the site of inflammation requires adhesion and transmigration through blood vessels,which is orchestrated by molecules on leukocytes(?2 integrins)and on endothelial cells(ICAM-1).Annexin A1(Anx A1),are well documented to counter-regulate excessive neutrophil accumulation,thus possess an anti-inflammatory action.Thereafter,we try to use Anx A1 injection as an indirect means,,to investigate what will happen to VMC when recruitment of neutrophils is mostly blocked.Annexin A1(Anx A1),a glucocorticoid-regulated protein that has anti-inflam matory and pro-resolving actions,is expressed in circulating leucocytes,macrophages,fibroblasts mast cells and is particularly abundant in neutrophils.Anx A1 regulates neutrophil rolling,tissue recruitment and neutrophil apoptosis via interaction with formylated peptide receptor(FPR2)on vesicular endothelial cells..In addition to that,apoptotic neutrophil-released Anx A1 binds to PS,inducing phagocytosis of neutrophils by macrophages,a process called efferocytosis.This removal induces macrophage reprogramming toward a resolving phenotype,another key event to restore tissue homeostasis.Both Anx A1 and Anx A1-active N-terminal peptide Ac2-26 exert anti-inflammatory and pro-resolving activities.Neutrophils contribute to pathogen clearance by producing neutrophil extracellular traps(NETs),which are genomic DNA-based net-like structures that capture bacteria and fungi.Although NETs also express antiviral factors,such as myeloperoxidase and ?-defensin,the involvement of NETs in antiviral responses remains unclear.Neutrophils also contribute to bacterial killing by ROS production.In this study,we propose that early cardiac neutrophil influx may play roles in the host antiviral defense and pathogenesis of VMC via their effector intermediates(such as cytokines,ROS)and NET structure(or abnormal necroptosis).First,we use Anx A1/Ac2-26 injection strategies during CVB3 infection to see the disease outcome after blocking cardiac influx of neutrophils.Then we would in vitro study whether abnormal death form of neutrophils might cause cardiomyocyte injury and increase cardiac inflammation through direct effect or macrophage-modulatory mechanism.Methods:1.Establishment of BALB/c model of CVB3-induced myocarditis.BALB/c mice were intraperitoneally injected with 1000 TCID50 CVB3.During a 7 days period,mice succumbed a 25% weight loss and 20% mortality.Viral replication peaks on day 3 mounting to105 PFU/g heart.Hematoxylin/eosin(HE)staining of heart sections showed myocytes necrosis and massive inflammatory infiltration.2.Immunohistochemical staining of myocarditis.The cardiac tissue was fixed and dehydrated by paraformaldehyde,Embedded and maked ultrathin slices,H&E staining indicates the degree of inflammation of heart tissue.3.Purification of Bone marrow neutrophils and flow cytometry analysis.The neutrophils was obtained from mouse femur and tibia,and obtained by Percoll density gradient centrifugation,with a purity of 95%.Neutrophil phenotype is CD11b+Ly6G+ or CD11b+Gr1high.Blood neutrophils are 4~10% in CD45+ lymphocyte.4.Detection of neutrophil-produced ROS.Synthesis of ROS was induced by PMA or CVB3,detected by probe H2 DCFDA and flow cytometry analysis.5.Fluorescence confocal detection of Neutrophil extracellular traps(NETs): 1)Laser confocal microscopy: formation of NETs was induced by PMA,citrullinated histone(Cit-H3)was detected by specific m Ab and then an anti-rabbit Alexa fluor 488 m Ab,DNA was stained with DAPI.2)Western blot detection of citrullinated histone(Cit-H3)by anti-Cit-H3 m Ab.6.Treatment of CVB3-infected mice with Ac2-26 injection.BALB/c male mice were injected with Ac2-26(2.5mg/kg/d)on day-1/1/3/5,and infected with CVB3 n day0.Viral Load and acute VMC were analyzed at 7 days post infection.7.Purification and flow cytometry analysis of peripheral and cardiac immune cells.Immune cells were purified from peripheral blood and hearts of mice by 40% to 75% discontinuous Percoll gradient centrifugation.The composition of myeloid cells were identified by 5 m Abs staining: CD45,CD11 b,Ly6C,Ly6 G,F4/80 and T cells by 4 m Abs staining: CD45,CD3,CD4,CD8.8.Cardiac function detection by small animal ultrasound imaging system: left ventricular end systolic diameter and left ventricular end-diastolic diameter were measured by small animal ultrasonic imaging system(Vevo 2100)to get the left ventricular ejection fraction(EF %)and left ventricular fractional shortening(FS%).9.ELISA and Real-time PCR detection of chemokines and inflammatory cytokines in peripheral blood and heart.10.Effect of neutrophil ROS or NET effect on primary cardiac myocytes:BM-neutrophils were stimulated with PMA or CVB3 for 4 hrs to induce ROS roduction and NET formation,in the presence of MPO inhibitor(ABAH),NET inhibitor(Cl-amidine)or Ac2-26,then incubated with primary cardiomyocyte for 4 hrs,cell apoptosis was detected by PI-Annexin V staining and flow cytometry.11.Statistical analysis: the data is represented by mean plus standard deviation.Statistical analysis was conducted with Graphpad Prism 5 statistical software;Variance analysis was used for comparison between groups.P<0.05 was considered to be statistically different.Results:1.CVB3 infection up-regulated neutrophil-chemokines expression in hearts of mice.RT-PCR analysis of heart sample revealed that after CVB3 infection,cardiac neutrophil chemokine(CXCL1,CXCL2,CXCL3,CXCL5,IL-8)expression was significantly up-regulated at 3 dpi.2.CVB3 infection induced peripheral blood neutrophil amplification and cardiac neutrophil influx.FACS assay demonstrated a massive expansion of peripheral blood neutrophils peaking on 3 dpi(65% in CD45+ cells)after CVB3 infection.In accordance with that,we found a robust influx of neutrophil into the hearts of mice(45% in CD45+ cells)at 3 dpi while the absolute numbers reached peak at 7 dpi(3~5?104/g heart).Ly6 Chigh monocytes showed a similar kinetics while the percentage and numbers were much less then neutrophils.3.CVB3 infection activated neutrophil production of MPO and ROS in vivo and in vitro.To evaluate the activation status of neutrophils during CVB3 infection,their functional intermediates,MPO and ROS,were detected.Immunohistochemistry and Western blot assay demonstrated that CVB3 infection up-regulated level of MPO in hearts of mice during day 0-7.Meanwhile,neutrophil activation by CVB3 led to vigorous production of ROS,in hearts and in primary cardiomyocytes.4.CVB3 up-regulated cardiac endogenous Annexin A1 expression.Before interfering the neutrophil recruitment by annexin A1,we detected endogenous Anx A1 expression by immunohistochemistry.It was found that the cardiac Anx A1 level was very low which was significantly increased by CVB3 infection during 0-7 dpi.5.Injection of Anx A1 peptides Ac2-26 decreased viral replication and the severity of VMC.Anx A1 mimetic peptide Ac2-26(2.5 mg / kg)were i.p.administrated to mice on day-1,1,3 and 5 when mice were infected with CVB3 on day 0.Compared with CVB3 group,Treatment of mice with Ac2-26 significantly reduced CVB3-induced mortality and body weight loss.It also decreased IL-1? and IL-6 production,myocardial necrosis and immune injury and improved the cardiac histopathological score.Ultrasound diagnosis showed improved LVIDs and LVIDd of heart indicating an improved cardiac function.And,RT-PCR and TCID50 assay revealed that cardiac viral replication and load were also significantly reduced.All data demonstrated that Ac2-26 administration significantly reduced CVB3-induced cardiac inflammation and injury.6.Annexin A1-mediated resolution of VMC was associated with reduced blood neutrophil expansion and cardiac neutrophil accumulation.To evaluate the mechanism of Anx A1-mediated VMC resolution,we studied changes of leukocyte composition in blood and heart after CVB3 infection.1)Anx A1 reduced peripheral neutrophil expansion: Flow cytometry revealed that proportion and number of blood CD11b+ myeloid cells and CD11b+Ly6G+ neutrophils were significantly reduced after injection of Anx A1(neutrophil: 90% to 63%,1.4?106/ml to 0.4?106/ml).2)Anx A1 reduced cardiac neutrophil chemokine expression.Cardiac neutrophil chemokine(CXCL1/2/3/5)levels on 7 dpi were significantly decreased by Anx A1 treatment.3)Anx A1 reduced cardiac neutrophils and monocytes infiltration.By cytometry analyzing cardiac infiltrated CD45+ lymphoid cells on day 7,it was found that cardiac CD11b+ myeloid cells,neutrophils as well Ly6 Chigh monocytes were all significantly reduced in proportion and in numbers after Anx A1 treatment.Among myeloid cells,neutrophils were most effectively reduced(45% to 33%,3?104/g heart to 1.2?104/g heart).7.CVB3 induced neutrophil ROS production in vitro which promoted cardiomyocytes apoptosis and could be inhibited by Anx A1.To investigate mechanism of cardiac-neutrophil-mediated injury,bone marrow-derived neutrophils were treated with PMA or CVB3 and ROS production was increased.Co-incubation of supernatant ROS with primary cardiomyocytes might promote apoptosis which was impaired by Anx A1.8.PMA–induced NETs promoted cardiomyocyte apoptosis while CVB3 was unable to induce NET formation.By Fluorescence confocal assay,we found that PMA incubation induced BM-neutrophils to form classical NET structure,which could be blocked by Anx A1.Further,incubation of NET with primary cardiomyocytes increased myocyte apoptosis,which could be blocked by NET inhibitor,MPO inhibitor and Ac2-26.We could not detect NET formation by CVB3.We plan to further evaluate whether CVB3 induce neutrophils to form necroptosis,another form of death.9.In vivo NET inhibitor(Cl-amidine)injection reduced CVB3 myocarditis.We injected(day-1/1/3/5)mice with 4 doses of PAD4 inhibitor(Cl-amidine)to block NET formation during CVB3 infection,and found the the percent survival of mice was significanty increased indicating in vivo neutrophil NET increased CVB3-induced myocarditis injury.Conclusion:1.CVB3 infection induced peripheral blood amplification and cardiac infiltration of neutrophils at early stage of viral infection,which was associated with the severity of myocarditis.2.CVB3 up-regulated endogenous Annexin A1 expression.3.Injection of Anx A1 peptides Ac2-26 reduced viral replication and the severity of CVB3-induced VMC.4.Anx A1-mediated resolution of VMC was associated with significantly reduced blood neutrophil expansion and cardiac neutrophil influx.5.CVB3 activated neutrophils to produce large amounts of ROS,which might promote cardiomyocytes apoptosis and could be impaired by Anx A1.6.PMA but not CVB3 induced NET in vitro which promoted cardiomyocyte apoptosis.In vivo abnormal NETs(or necroptosis)induced by CVB3 exacerbated susceptibility of mice to CVB3-myocarditis(experiment undergoing). |
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