| Virus myocarditis(VMC) is an inflammation of the myocardium as a result of viral infection characteristic of diffuse infiltration and myocardial necrosis in the myocardium. The incidence and prevalence of myocarditis is increasing by years accounting to 0.12%-12% worldwide, and about 2%-15% in China. Patients with VMC have an increased risk of development of dilated cardiomyopathy(DCM), the 5-year survival rate of which is less than 50% and heart failure; and VMC is one of the leading causes of sudden cardiac death among adolescents. However, diagnosis of VMC is most challenging and is often under-recognized due to heterogeneity of its clinical presentation. Among various aetiologies of viral myocarditis including enteroviruses, adenoviruses, influenza viruses, parvoviruses and cytomegaloviruses, prominent infectious causes for VMC are enterovirus like Coxsackievirus serotype B3(CVB3). CVB3 infection is an important predisposing factor for the development of DCM and heart transplantation, for serocally, enteroviral genome can be detected in up to 70% of DCM patients and CVB3-reactive antibodies are found in 50% of DCM patients.CVB3 is a positive sense single-stranded RNA viruse belonging to the Enterovirus genus within the Picornaviridae family. CVB3 entry into host gastrointestinal tracts via fecal-oral route before spreading into multiple target tissues such as heart, pancreas, brain, liver, lung and spleen; with heart and pancreas as the dominant target tissues. CVB3 infects epithelial cells and lymphocyte via two receptors: DAF(decayaccelerating factor) and CAR(coxsackievirus and adenovirus receptor). Studies from experimental susceptible murine models(male BALB/c) of CVB3 infection suggest that the disease course of myocarditis assumes 3 distinct stages. The first acute viremic phase(day0-4~7) is characterized by vivid cardiac viral replication(peak on day 3-4) and innate immune cells infiltration, causing direct cardiomyocyte damage and production of inflammatory cytokines including IL-1b, IL-6, IL-18, TNF-a and type I/II interferons(IFNs) by innate cells, cardiac myocytes and fibroblasts. In the subacute phase(day 7-2 or 3 weeks), in which the virus is present in very low level, release of cardiomyocyte antigen activates Ag presentation by APC cells and induce specific T and B responses. Infiltration of T cells of Th1 and Th17 type typically peaks at 7–14 days with cardiac inflammation reaches its peak. The third stage(chronic phase, months to years) is characterized by resolution of immune response, cardiac repair and remodeling. However, viral genome persistence by Th2 response causes, fibrotic scar and cardiac dilation leading to left ventricular dysfunction and DCM.CVB3-induced acute myocarditis is a result of an early direct viral-lysis-induced cardiomyocyte necrosis and the subsequent inflammatory immune infiltration involving both innate and adaptive cells. Since the primary cardiac infiltrates consist mainly innate immune cell types such as NK, NKT, gdT and macrophages, each cells types could influence the disease outcome. Innate immunity plays very critical roles in the infectious inflammatory diseases by direct anti-microbial defense, vivid production of inflammatory cytokines as well as type I interferon and modulation of types of helper T(Th) response. When encountering microbial component, a panel of pattern recognition receptors(PRRs) on or within innate cells such as toll-like receptors(TLRs), could recognize pathogen associated molecular patterns(PAMPs) and transmit a cascade of signals to activate innate inflammatory cytokine and type I interferon response to quickly defense and restrict microbial infection.,Meanwhile, in terms of CVB3 myocarditis, mammalian hearts could use innate immunity to respond to cardiac injury caused by CVB3 lysis at very early phase(day 0-3) by detecting damage-associated molecular pattern(DAMP) via PRRs, then trigger signal cascades that activate NF-κB and interferon regulatory transcriptional factors that in turn regulate target genes encoding pro-inflammatory cytokines, chemokines and interferons in the heart.Neutrophil, a conventional polymorphonuclear granucyte famous for the very quick response and first recruitment to the local tissue where bacterial infection occurs, falls into our research focus for the reason that this innate cell is a good candidate for the cardiac innate responder cells sensing both PAMPs and DAMPs. Neutrophils are recognized as one of the major players during acute bacterial inflammation, and primary effector cells against bacterial and fungal infections. The role of neutrophils in viral infections remain poorly characterized. Intensive neutrophil infiltration(up to 80% of infiltrated leucocytes) has been detected in the airways and lungs during human respiratory syncytial virus(RSV) and herpes simplex virus(HSV) infections. Data from limited experimental and clinical settings show that, upon infection, neutrophils can be induced to express genes encoding key inflammatory mediators including complement components, chemokines and cytokines, and anti-microbial peptides(AMPs). Meanwhile, infiltrated neutrophils can automatically induce apoptotic cell bodies, naming neutrophil excellular traps(NETs) consisting cellular components such as DNA, histone and AMPs and ROS, to restrict microbial dissemination. We speculate that neutrophils, first innate responder cells to cardiac CVB3 infection, may play very important roles in the development of acute VMC by sensing and responding to both PAMPs and DAMPs in the heart.During CVB3-induced acute VMC, virus replication within the cardiomyocyte causes cell lysis, which prompts the release of a panel of DAMPs including high-mobility group box 1 protein(HMGB1), extracellular matrix proteins, metabolites, and genomic DNA. HMGB1 could be released by injured tissue cells, activated monocytes, macrophages and neutrophils, and is considered to amplify auto antigen-antibody complex related monocyte inflammatory response through interaction with Toll-like receptor 4(TLR4) and Receptor for advanced glycation end-products(RAGE). HMGB1-RAGE interaction in physiological conditions gives “find me” signal to activate phagocytosis, repair function of immune cells and tissue-regeneration function of stem cells by enhancing their interaction with vessel endothelial cells and moving. While under infection, HMGB1 dominantly use TLR2/TLR4 to trigger inflammatory response and immune infiltration.Our preliminary data demonstrated that 1) A very early infiltration(as early as day 1.5 post CVB3 infection) of neutrophils could be detected in the heart upon CVB3 infection, before infiltration of most of other innate cells like macrophages; 2) During this very early phase, high levels of HMGB1 proteins were released into the heart which peak on day 3 post infection; 3) When examining the expression of HMGB1 receptors on neutrophils upon CVB3 infection, we found that TLR4 expression was more dominant than RAGE; however, we found another non-TLRs receptor for HMGB1, TREM-1(triggering receptors expressed on myeloid cells-1), has very high background expression level and significantly enhanced expression upon CVB3 infection, being the most dominantly up-regulated HMGB1 receptors on neutrophils. 4) In vitro HMGB1 stimulation of murine neutrophils could significantly increase the production of IL-17 A, TNF-a and IFN-g. All these preliminary data indicate that neutrophils may play important role in the course of CVB3 infection.Taken together, in this study, we focus the role of neutrophil early cardiac infiltration in the development of acute CVB3 myocarditis, we expect the highly up-regulated TREM1 expression on neutrophils could sense both viral PAMPs and cardiac DAMPs---HMGB1, this HMGB1/TREM-1 axis could activate multi-function of neutrophils and amplify the innate inflammation in the cause of acute VMC.Part one: Pro-inflammatory role of early-infiltrated neutrophils in CVB3-induced acute myocarditis1. Establishment of murine model of CVB3-induced myocarditisTo investigate the role of neutrophils in the pathogenesis of VMC, we first established BALB/c mice model of VMC in BALB/c(H-2d) mice by intraperitoneal injection of 1000 TCID50 dosage of CVB3 in 0.1ml PBS. Typical acute VMC symptom including viral replication peak on day 3 p.i., weight loss amounting to 20% and 50%-80% survival rate of mice on day 7 p.i. was observed. Hematoxylin/eosin(HE) staining of paraffin section of heart tissue of CVB3-infected mice showed massive inflammatory infiltration and myocytes necrosis on day 7 p.i. compared to intact cardiomyocytes seen in non-treated mice, which is consistent with the significantly enhanced cardiac inflammatory cytokine levels in CVB3-infected mice.2. CVB3 infection induced bone marrow and peripheral blood neutrophils amplificationTo see the dynamic change of neutrophils in mice following CVB3 infection, we first detected the frequency of CD11b+Ly6G+ neutrophils within the bone marrow and the blood by Flow cytometry. It was found that upon CVB3 i.p. infection, similar to the situation in acute bacterial infection, a massive propagation of neutrophils was seen in both BM and blood, with numbers increasing more than 10 times as compared to those in mice without infection.3. Cardiac infiltration of neutrophils was quick and peak on day 1.5 upon CVB3 infectionTo see the dynamic change of neutrophils infiltrated into the heart, we then detected the frequency of neutrophils, macrophages, monocytes and gdT cells at 0, 36, 72 hours post-infection. It was found that during the initial 3 days of CVB3 infection, cardiac infiltrated neutrophils and monocytes increased, peaking at day 1.5 and declined by day 3; while macrophages, NK and gdT cells increased their cardiac infiltration continuously from day 0 to day 3. Neutrophils and monocytes represent the most abundant immune cells in the heart at day 3 p.i..In the spleen, frequency and numbers of almost all lymphocytes and mononuclear phagocytes significantly increased during the course of day0-day3. Within those cells, significantly enhanced infiltration of neutrophils(14%-46% in CD11b+ myeloid cells, 1.5×105/spleen/day3) was confirmed, more than numbers of splenic macrophages and monocytes.4. Depletion of neutrophils of mice led to significantly reduced myocardial inflammationTo characterize the role of neutrophils in the course of CVB3 acute infection, we inject mice with anti-Ly6G–specific m Ab intravenously to deplete neutrophils at day-3 and day-1 before CVB3 infection. Then the viral replication, the cardiac immune infiltration and inflammatory CKs levels were evaluated. It was found that 1) 2 injection of anti-Ly6 G m Ab results in up to 95% neutrophil depletion in blood; 2) Neutrophil depletion led to slightly increased mortality of mice; 3)Significantly ameliorated viral myocarditis was seen in neutrophil-depleted mice; 4) The numbers of splenic infiltrated CD11b+, neutrophils, monocytes as well as CD3+T cells were all significantly reduced; 5) Viral replication seemed not influenced by neutrophil depletion. These data indicate that neutrophils play a detrimental role in the development of VMC by promoting cardiac immune infiltration.Above all, during the acute infection of CVB3 in BALB/c mice, neutrophils in BM and blood were quickly motivated and propagated which moved to the heart and the spleen before macrophages and γδ T cells did; and the infiltration reached peak at very early phase(day 2-3) before slight declining. Specific depletion of neutrophils before CVB3 infection significantly alleviated CVB3-induced myocarditis indicaiting early-cardiac-infiltrated neutrophils play pro-inflammatory roles in CVB3 infection.Part two: Neutrophils sense cardiomyocyte-released HMGB1 to amplify cardiac inflammationWe postulate that early cardiac-infiltrated neutrophils may sense both viral PAMPs and cardiac DAMPs in the early phase of CVB3 infection, thus performing its pro-inflammatory role, Then, what kind of DAMP could be sensed by neutrophils is studied.1. Cardiac HMGB1 expression dynamics following CVB3 infectionDuring CVB3 acute infection, direct virus-caused cytolysis of cardiomyocytes was seen before inflammatory cell infiltration in the heart. Lysed cardiomyocytes would release most of their cellular components as DAMPs in which HMGB1, a proinflammatory mediator involved in the pathogenesis ofautoimmune diseases, is the most famous one. We first examined HMGB1 level in the heart of mice at day 0, 3, 7 post CVB3 infection and found that HMGB1 protein expression level was significantly elevated in cardiac tissue and peaked on day 3 post-infection, then down-regulated by day 7. The dynamics of HMGB1 level in the heart is in parallel with the replication dynamics of CVB3 and that of neutrophil infiltration in the heart following CVB3 infection.2. CVB3 infection of primary cardiomyocytes increased HMGB1 releaseTo confirm the HMGB1 release upon CVB3 lysis of the heart tissue, we infected primary murine cardiomyocytes and macrophages with CVB3 and detected the HMGB1 level. It was found that HMGB1 had background expression in the primary murine cardiomyocytes, and significantly increased its level upon CVB3 infection from 0-24 hrs; similar results were obtained from macrophages.3. Glycyrrhizin inhibition of HMGB1 led to significantly alleviated VMCTo explore the role of heart tissue-released HMGB1 in the development of CVB3-inducedmyocarditis, HMGB1 inhibitor, glycyrrhizin was injected to mice 1 h after CVB3 challenge, once every 2 days for 3 times. It was found that: 1) glycyrrhizin administration significantly enhanced survival of mice, and reduced body weight loss; 2) Compared to PBS-treated mice, glycyrrhizin markedly attenuated CVB3-induced cardiac inflammation; 3) FACS assay revealed that frequency of CD11b+Ly6Chigh monocytes and CD11b+Ly6G+ neutrophils in the spleen and monocyte in blood were significantly reduced in GZA-treated mice(p<0.05). 4) glycyrrhizin significantly reduced virus load and replication in the heart. These data indicated that blockade of HMGB1 could decrease VMC development via reducing viral replication and monocytes/neutrophils recruitment to the heart.4. Adoptive transfer of HMGB1-pretreated murine neutrophils significantly increased myocarditis and viral replicationTo see if neutrophils could sense heart-released HMGB1 and perform pro-inflammatory role, we isolated murine neutrophils fron the BM(90% purity)and transfer 5*105 WT-derived neutrophil, CVB3-infected mice-derived neutrophils、and HMGB1-pretreated WT neutrophils into mice infected with CVB3 1 day before. It was found that: 1) VMC: compared to CVB3-infected mice, WT neutrophil transfer did not change survival but slightly increased vardiac inflammation; compared to WT-neutrophil, CVB3-neutrophil transfer significantly reduced survival rate of mice and increased VMC severity(p<0.05); while HMGB1 pre-treated WT neutrophil transfer significantly increased VMC as compared to WT neutrophil transfer, but no difference with CVB3-neutrophil transfer; 2) viral replication: WT neutrophil transfer had no effect on CVB3 replication as compared to no neutrophil transfer; CVB3-neutrophil transfer significantly increased cardiac viral replication(25 times, p<0.01); HMGB1 pre-treated WT neutrophil significantly increased viral level as compared to WT neutrophil transfer,but had no difference with CVB3-neutrophil transfer; 3) Splenic and peripheral blood lymphoid and myeloid cell composition: CVB3 infection significantly increased splenic lymphoid and myeloid cell numbers; WT neutrophil transfer had no role on cell numbers; while CVB3-neutrophil transfer significantly reduced all immune cell numbers(especially neutrophils, macrophages, monocytes and CD4+T cells), the same effect as obtained by HMGB1 pre-treated WT neutrophil transfer; Blood immune cell change had almost the same situation.Taken together, early and quick replication of CVB3 in the heart tissue released large amounts of DAMPs---HMGB1 which peaked on day3 post CVB3 infection; at the same time, cardiac infiltrated neutrophils sensed this DAMP, significantly increased viral replication systemically and cardiac immune infiltration, accounting for its detrimental role in the development of VMC.Part three: Role of TREM-1 in acute VMCHMGB1 act on a variety of receptors on innate cells such as TLR2, TLR4 and RAGE which may be used by neutrophils to sense HMGB1. We are interested in another HMGB1 potential receptor, TREM-1. We tried to investigate that if HMGB1/TREM-1 axis was employed by neutrophils upon CVB3 infection and involved in the pathogenesis of acute VMC.1. TREM1 was the most significantly up-regulated HMGB1 receptor on neutrophils upon CVB3 infectionWhen examining the expression of HMGB1 receptors on neutrophils upon CVB3 infection, we found that TLR4 expression was more dominant than RAGE; however, we found another non-TLRs receptor for HMGB1, TREM-1(triggering receptors expressed on myeloid cells-1), has very high background expression level and significantly enhanced expression upon CVB3 infection, being the most dominantly up-regulated HMGB1 receptors on neutrophils.2. TREM-1 expression was significantly increased on neutrophils upon CVB3 infectionQuantitative real-time PCR analysis revealed that TREM-1 expression in the heart was 10-fold up-regulated upon CVB3 infection and peaked at day 6. FACS analysis revealed that TREM-1 expression was significantly up-regulated on neutrophils, monocytes and macrophages at day 3 post infection.3. HMGB1 axis activated neutrophil production of inflammatory CKsTo investigate role of TREM1-HMGB1 axis on neutrophils, r HMGB1 or heart homogenate of day 3 was added to murine neutrophils with or without CVB3, multi-function of neutrophil including phagocytosis, NET, AMP synthesis and inflammatory CK secretion were studied. Some experiment was still in performance(data not yet obtained). It was found that HMGB1 addition significantly increase the production of IL-17 A, TNF-α, IFN-γ and chemokine CXCL1 by neutrophils. It indicate that HMGB1 could enhance the pro-inflammatory role of neutrophils in vitro. To investigate role of TREM-1 on the inflammatory CK production by macrophages, we screened a TREM1-si RNA with 70% knockdown efficiency and transfected into RAW264.7 cells before CVB3 infection, it was found that TREM1 knockdown significantly reduced levels of IL-1β and IL-6, but not TNF-α by macrophages.4. Blockade of TREM-1 significantly decreased VMC and viral replicationTo assess the role of TREM-1 in the pathogenesis of VMC, TREM-1 specific inhibitor LP17 was administered to mice at day-1 and day 3, and CVB3 infection induced VMC was evaluated. It was found that LP17 inhibition of TREM1’s function significantly enhanced survival of mice, decreased cardiac immune infiltration and cardiac inflammatory CKs levels, indicating inflammation-amplifying role of TREM1 in the course of CVB3 infection.5. TREM1-HMGB1 axis was dominantly employed by neutrophils to amplify cardiac inflammation and viral replicationTo confirm the direct link between HMGB1/TREM-1 axis and neutrophils, we purified and injected WT-neutrophil, CVB3-neutrophil, HMGB1-pretreated WT-neutrophil and HMGB1-pretreated WT-neutrophil plus LP17 inhibition into mice receiving CVB3 1 day before. It was found that: 1) VMC: compared to HMGB1-pretreated WT-neutrophil transfer-induced worse of VMC, LP17 blockade significantly decreased disease severity indicating therapeutic role; 2) Viral replication: compared to HMGB1-pretreated WT-neutrophil transfer-induced viral replication robustly promotion, LP17 blockade significantly decreased CVB3 replication(back to effect of WT neutrophil transfer); 3) Splenic and blood lymphoid and myeloid cell composition: compared to HMGB1-pretreated WT-neutrophil transfer-induced all immune cell numbers robust reduction, LP17 blockade significantly enhanced most of the immune cell numbers to the levels of CVB3 infection control group except macrophage(p>0.05, vs HMGB1-neutrophil).Taken together, focusing role of neutrophils in the course of CVB3 infection and development of VMC, we found an significant inflammation-amplifying and viral-enhancing role of neutrophils via TREM1-HMGB1 axis during the early phase of CVB3 infection(day 0-3), suggesting neutrophils as an important innate patrolling cell sensing both PAMPs and DAMPs during viral early infection and an important immune-modulatory cell during viral-mediated inflammatory diseases; and indicating noval molecular target for the future treatment of voral myocarditis. |
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