Objective To investigate the role and mechanism of RNA-Specific adenosine deaminase 3 (Adar3) in regulating macrophage polarization during Coxsackievirus B3(CVB3)-induced viral myocarditis (VM). Methods Bone marrow-derived macrophages (BMDM) from mice were cultured in vitro and induced into M1/M2 macrophages using interferon-gamma (IFN-γ)/lipopolysaccharide (LPS) or interleukin 4 (IL-4), respectively. The mRNA expression levels of Adar1, Adar2, and Adar3 in each group of cells were assessed by real-time quantitative PCR (qRT-PCR). Specific siRNAs targeting the Adar3 gene were designed, synthesized, and transiently transfected into M2 macrophages. The mRNA levels of M2 polarization-related marker genes-including arginase 1 (Arg1), chitinase 3-like molecule 3 (YM1/Chi3l3), and resistin-like molecule alpha (RELMα/FIZZ1)-were detected by qRT-PCR. RNA sequencing was performed to analyze the signaling pathways affected by Adar3. The expression levels of Wnt/β-catenin signaling pathway were further validated using qRT-PCR and Western blot. The adeno-associated virus overexpressing Adar3 was designed, synthesized, and injected into mice via tail vein. Three weeks later, a myocarditis mouse model was established. After an additional week, the phenotype and function of cardiac macrophages, as well as multiple indicators of VM (including echocardiography, body weight, histopathology and serology) were examined. Additionally, the protein levels of the Wnt/β-catenin signaling pathway were assessed. Results Compared to M0-type macrophages, the expression level of Adar3 was significantly increased in M2-type macrophages. After transfection of Adar3 siRNA, the mRNA levels of Arg1, YM1 and FIZZ1 in M2 macrophages were downregulated. RNA sequencing revealed 149 upregulated genes and 349 downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and subsequent validation experiments indicated that Adar3 modulated the Wnt/β-catenin signaling pathway. In vivo experiments demonstrated that Adar3 overexpression alleviated the cardiac dysfunction of VM mice. The proportion of M1 macrophages in the heart decreased, while the proportion of M2 macrophages increased. At the same time, the Adar3 overexpression activated the Wnt/β-catenin signaling pathway. Conclusion Adar3 promotes macrophage polarization toward the M2 phenotype by activating the Wnt/β-catenin signaling pathway, thereby alleviating VM.
Animals ; Adenosine Deaminase/metabolism* ; Macrophages/immunology* ; Wnt Signaling Pathway/genetics* ; Myocarditis/immunology* ; Mice ; Coxsackievirus Infections/metabolism* ; Male ; Mice, Inbred BALB C ; Enterovirus B, Human/physiology* ; beta Catenin/genetics*

OBJECTIVETo investigate the therapeutic effect of IL-6 mAb on experimental autoimmune myocarditis (EAM) in rats, and search the mechanism of the role of IL-6, helper T cells 17 (Th17) and regulative T cells (Treg) in EAM pathogenesis.

METHODSThirty-four Lewis rats were divided into three groups randomly, i.e. control group (n = 6), EAM group (n = 12), and IL-6 mAb intervention group (n = 16). Rats in EAM group and IL-6 mAb intervention group were injected intracutaneously with myosin to establish EAM model. Rats in IL-6 mAb intervention group were injected intraperitoneally with 1 mg IL-6 mAb on 1st, 7th to 20th day after cardiac myosin immune injection. Myocardial inflammation was examined by HE stain, Masson stain, and TdT assay (TUNEL reaction) on 21st and 84th day after IL-6 mAb therapy in order to assess the therapeutic role. Spleen cells were analyzed by flow cytometry to illustrate Th17 and Treg cells? number and function. The serum concentration of IL-6, IL-10, IL-17, and TGF-beta in each group was measured by ELISA, concentration of STAT3, RORgammat, and Foxp3 mRNA in each group was determined with RT-PCR. Spleen cells derived from EAM were stimulated by IL-6 mAb in vitro, and the concentration of IL-10, IL-17 and TGF-beta was measured by ELISA.

RESULTSInflammation score, fibrosis score, and apoptosis index in IL-6 mAb intervention group were significantly decreased as compared with those in EAM group (P < 0.01). The number of Th17 and Treg cells in EAM group on the 21st day (experimental acute peak stage) were increased, and those in intervention group on the 21st day were significantly inhibited (P < 0.01). The concentration of serum IL-6, IL-10, IL-17 and TGF-beta in intervention group on the 21st day was decreased dramatically in comparison with that in EAM group on the same day (P < 0.01). The levels of peripheral blood STAT3, RORgammat, Foxp3 mRNA in intervention group on the 21st day was decreased significantly as compared with that in EAM group (P < 0.01). The expression of IL-10, IL-17 and TGF-beta was increased significantly (P < 0.01) by stimulation of IL-6 mAb on spleen cells derived from EAM in vitro.

CONCLUSIONSIL-6 mAb could neutralize IL-6, and ameliorate myocarditis and reduce heart autoimmune responses. IL-6 mAb has significantly protective effects on EAM by suppressing Th17 and Treg cells.

Animals ; Antibodies, Monoclonal ; therapeutic use ; Autoimmune Diseases ; drug therapy ; immunology ; Disease Models, Animal ; Forkhead Transcription Factors ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-17 ; metabolism ; Interleukin-6 ; immunology ; Male ; Myocarditis ; drug therapy ; immunology ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; Rats ; Rats, Inbred Lew ; STAT3 Transcription Factor ; metabolism ; Th17 Cells ; immunology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo explore the effects of emodin in myocardial protection in mice with viral myocarditis (VMC) and explore molecular mechanisms.

METHODSFifty-five male 4-week-old BALB/c mice were randomly divided into control group (n=15), model group (n=20), and emodin group (n=20). The mice in model and emodin groups were inoculated with 0.1 ml Eagle's solution containing coxsackievirus B3 intraperitoneally, and those in the control group were given only 0.1 ml Eagle's solution. From the day of inoculation, the mice in emodin group received intragastric administration with 0.1 ml of 3 mg/ml emodin solution once daily for 21 consecutive days, and those in the control and model groups received 0.1 ml distilled water only. On day 7 after inoculation, 5 mice from each group were sacrificed to determine the viral titers in the cardiac tissues. All the mice were sacrificed on day 22 for measurement of the heart weight and histopathological inspection of the heart with HE staining. The mRNA and protein expression levels of myocardial interleukin-23 (IL-23) and interleukin-17 (IL-17) were detected by real-time quantitative PCR and Western blotting, respectively, and serum IL-23 and IL-17 levels were examined using enzyme linked immunosorbent assay (ELISA). Th17 cell frequencies were analyzed by flow cytometry. The expression levels of myocardial nuclear factor-κB (NF-κB) p65 in the cardiomyocyte nuclei were examined using Western blotting, and myocardial interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) contents were detected by ELISA.

RESULTSThe mortality, myocardial histopathologic scores and virus titers in emodin group were all significantly lower than those in the model group (P<0.05). The heart-to-body weight ratio, myocardial IL-23 and IL-17 expressions, serum IL-23 and IL-17 levels, Th17 cell frequencies, cardiomyocyte nuclear NF-κB p65 expression, and myocardial contents of IL-1β, IL-6 and TNF-α were all significantly increased in the model group as compared to the control group (P<0.01) but reduced significantly in emodin group as compared to model group (P<0.05).

CONCLUSIONEmodin can protect against VMC by inhibiting IL-23/IL-17 inflammatory axis, Th17 cell proliferation and viral replication in mice.

Animals ; Coxsackievirus Infections ; immunology ; Cytokines ; immunology ; Emodin ; pharmacology ; Enterovirus ; physiology ; Interleukin-17 ; immunology ; Interleukin-23 ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; immunology ; virology ; Th17 Cells ; cytology ; drug effects ; Transcription Factor RelA ; metabolism ; Virus Replication

OBJECTIVETo investigate the role and significance of cardiac mast cells and Toll-like receptor 4 (TLR4) in the development and progression of viral myocarditis (VMC).

METHODSForty-eight Balb/c mice were randomly divided into a control group (n=24) and a model group (n=24). Coxsackievirus B3 was intraperitoneally injected into the model group mice to establish a VMC model. In each group, cardiac tissues were collected from 8 mice at 7, 14 and 28 days after the model was established. The cardiac tissues were stained with hematoxylin and eosin as well as Masson trichrome to observe pathological changes in cardiac tissues. The number and degranulation of cardiac mast cells at each time point were measured and evaluated by toluidine blue staining and transmission electron microscopy. The mRNA and protein expression of TLR4 in cardiac tissues was measured by RT-PCR and immunohistochemistry. In the model group, the correlation between number of cardiac mast cells and mRNA expression of TLR4 at all time points was analyzed.

RESULTSThe model group had significantly higher pathological scores of cardiac tissues than the control group at all time points (P<0.05). The myocardial collagen volume fraction in the model group at 28 days was significantly higher than in the control group at all time points and higher than in the model group at 7 and 14 days (P<0.05). At each time point, the model group had a significantly increased number of mast cells (P<0.05), and significantly increased mRNA and protein expression of TLR4 (P<0.05) compared with the control group. In the model group, the number of cardiac mast cells was positively correlated with the mRNA expression of TLR4 at all time points (R²=0.877, P<0.05).

CONCLUSIONSMice with VMC have significantly increased numbers of cardiac mast cells and expression of TLR4 compared with control mice at all time points, suggesting that mast cells and TLR4 may play important roles in the inflammatory response and fibrosis of VMC.

Animals ; Coxsackievirus Infections ; immunology ; Enterovirus B, Human ; Female ; Mast Cells ; physiology ; Mice ; Mice, Inbred BALB C ; Myocarditis ; immunology ; Myocytes, Cardiac ; pathology ; Toll-Like Receptor 4 ; analysis ; genetics ; physiology

OBJECTIVEPrevious studies have shown that hydrogen sulfide (H2S) plays key roles in a number of biological processes, including vasorelaxation, inflammation, apoptosis, ischemia/reperfusion and oxidative stress, which are involved in the pathogenesis of myocarditis. This study aimed to examine the expression of cystathionine-γ-lyase(CSE)/H2S pathway in mice with viral myocarditis.

METHODSSix-week-old inbred male mice were randomly assigned to control (n=25) and myocarditis group (n=30). The myocarditis and the control groups were inoculated intraperitoneally with 0.1 mL 10-5.69TCID50/mL CVB3 or vehicle (PBS) alone respectively. Ten mice were sacrificed 4 and 10 days after injection. Blood and heart specimens were harvested for measuring the content of serum H2S and the H2S production rates in cardiac tissues. Heart sections were stained with hematoxylin and eosin. Immunohistochemisty was used to detect the CSE protein expression in the heart.

RESULTSIn the myocarditis group, the serum H2S content and H2S production rates in cardiac tissues were significantly higher than those in the control group 4 and 10 days after injection (P<0.05). The expression of CSE protein in the heart in the myocarditis group was also significantly higher than that in the control group (P<0.05).

CONCLUSIONSCSE and its downstream production H2S increase in mice with acute viral myocarditis. The increased expression of CSE/H2S pathway might be involved in the pathogenesis of viral myocarditis.

Animals ; Coxsackievirus Infections ; etiology ; Cystathionine gamma-Lyase ; analysis ; Enterovirus B, Human ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hydrogen Sulfide ; metabolism ; Killer Cells, Natural ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; etiology

OBJECTIVETo explore the effects of adenovirus vector-mediated gene transfer of ICOSIg fusion protein on experimental autoimmune myocarditis (EAM) in Lewis rats.

METHODSExpression vector containing ICOSIg (p-Adeno-ICOSIg) was constructed by fusion of human ICOS and IgGFc segment. Adenovirus vector was digested by PacI enzyme and transfected into HEK 293 cells. Adenovirus expressing ICOSIg was produced. EGFP was constructed into adenovirus vector and used as control. EAM was induced in Lewis rats by injection of porcine cardiac myosin. All immunized Lewis rats were divided into 4 groups. Group A (n = 15) and B (n = 15) received adenovirus containing ICOSIg on day 0 and day 14 respectively to study the effects of costimulatory molecules gene therapy on T cell activation and inflammation; group C (n = 10) and group D (n = 10) received adenovirus containing EGFP on day 0 and day 14 respectively as controls. Group E (n = 10) was normal controls that did not receive immunization. On day 28, all rats were killed after echocardiography examination. Histopathological examination was performed to observe myocardial inflammation. Protein levels of ICOS, ICOSL, B7-1 and B7-2 were detected by Western blot. INF-gamma, IL-2 and IL-4 mRNA were determined by realtime RT-PCR.

RESULTSOn day 28, cardiac function was significantly improved and myocardial inflammation significantly attenuated in group B compared to group A, C and D (all P < 0.05). B7-1 expression at protein level was significantly lower in group B than that of group C (P < 0.05). ICOS and ICOSL expressions at protein level were significantly decreased in both group A and B compared with group C and D (P < 0.05). IFN-gamma mRNA level significantly decreased and IL-4 mRNA significantly increased in group A and B compared to group C and D (P < 0.05).

CONCLUSIONSBlockade of costimulatory pathway with gene therapy of ICOSIg alleviated autoimmune inflammatory damage and improved cardiac function in Lewis rats with EAM. Down-regulated costimulatory molecules in the myocardium and reduced inflammatory cytokine secretion might be responsible for the beneficial effects of ICOSIg in this model.

Adenoviridae ; genetics ; Animals ; Antigens, Differentiation, T-Lymphocyte ; genetics ; Autoimmune Diseases ; immunology ; pathology ; therapy ; Disease Models, Animal ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Immunoglobulin Fc Fragments ; genetics ; Inducible T-Cell Co-Stimulator Protein ; Male ; Myocarditis ; immunology ; pathology ; therapy ; Rats ; Rats, Inbred Lew ; Recombinant Fusion Proteins ; genetics

BACKGROUNDExperimental autoimmune myocarditis (EAM) in rats is a T-cell-mediated disorder. The initiation and maintenance of autoimmune responses in EAM depend on the maturation state of dendritic cells. IL-10 is a pleiotrophic immunomodulatory cytokine that functions at different levels of the immune response, so it has emerged as a promising therapeutic factor for the treatment of autoimmune/inflammatory diseases. This study was designed to test the hypothesis that IL-10 gene modified bone marrow-derived immature dendritic cells (iDCs) ameliorate EAM and to explore the underlying mechanisms.

METHODSEAM was induced using the methods of cardiac myosin immunization on day 0 and day 7. Immature and mature bone marrow-derived dendritic cells (BMDCs) were generated without or with the stimulation by lipopolysaccharide (LPS) and the phenotype was analyzed by flow cytometry. Some of the iDCs were transfected by pcDNA3-IL-10 plasmid. 2 x 10(6)/per rat mature DC (mDC), immature DC (iDC), pcDNA3 transfected iDC, pcDNA3-IL-10 transfected iDC or phosphate buffered saline (PBS) were injected intravenously for treatment 5 days after the first immunization. On day 21, HE staining was performed to detect the myocardial inflammation and T lymphocyte proliferation assay was used to determine the effects of IL-10 gene transfected iDC on autoreactive T cell proliferation. Expression of IkappaB, the inhibitor of NF-kappaB pathway, was determined by Western blot.

RESULTSBMDCs generated in a medium supplemented with granulocyte-macrophage-colony-stimulating factor (GM-CSF) were relatively immature, as determined by flow cytometry. However, stimulation with LPS induced these cells to become mature (m) DCs with higher levels of surface major histocompatibility complex (MHC)-II and costimulatory molecules. Intravenous administration of iDCs, especially pcDNA3-IL-10 transfected iDC, ameliorated the histopathological severity of the myosin induced-EAM, and the effect was lost after the DCs underwent maturation induced by in vitro exposure to LPS. IL-10 gene modified iDC inhibited the antigen specific T cell responses towards cardiac myosin. IkappaB protein was up-regulated significantly in the IL-10 gene modified iDC group.

CONCLUSIONSIL-10 gene modified iDC induced antigen-specific tolerance in EAM. The underlying mechanisms may be related to costimulatory molecules down-regulation and NF-kappaB pathway inhibition.

Animals ; Autoimmune Diseases ; immunology ; Dendritic Cells ; physiology ; Immune Tolerance ; Interleukin-10 ; genetics ; Lymphocyte Activation ; Male ; Myocarditis ; immunology ; Myosins ; immunology ; NF-kappa B ; physiology ; Rats ; Rats, Inbred Lew ; Signal Transduction ; Transfection

OBJECTIVETo investigate whether IL-10 gene modification on immature dendritic cells (iDC) could induce autoimmune tolerance in rat experimental autoimmune myocarditis (EAM).

METHODSEAM was induced by cardiac myosin immunization on day 0 and day 7 in rats. A total of 2 x 10(6) mature DC (mDC), iDC, pcDNA3 transfected iDC, pcDNA3-IL-10 transfected iDC or PBS were injected intravenously at 5th immunization day. Three weeks later, echocardiography and HE staining were performed to observe the cardiac function and myocardial inflammation. Th1/Th2 cytokines were detected by ELISA and MHC-II molecules, costimulatory molecules were identified by flow cytometry. In vitro T lymphocyte proliferation assay and adoptive transfer of DCs were performed to determine the antigen specific tolerance induced by IL-10 gene modification on iDCs.

RESULTSEAM rats treated with pcDNA3-IL-10 transfected iDC showed improved cardiac function and reduced inflammatory cells infiltration into myocardium. Moreover, lower Th1 and higher Th2-type response was induced, MHC-II and costimulatory molecules down-regulated and antigen specific immunological responses towards cardiac myosin inhibited in pcDNA3-IL-10-iDC treated EAM rats.

CONCLUSIONTreatment with IL-10 gene modified iDCs could ameliorates EAM by inducing Th2 polarization and down-regulation of MHC-II molecules and costimulatory molecule expressions.

Animals ; Animals, Genetically Modified ; Autoimmune Diseases ; immunology ; Bone Marrow Cells ; Cell Line ; Dendritic Cells ; immunology ; Genetic Therapy ; Immune Tolerance ; Interleukin-10 ; genetics ; immunology ; Myocarditis ; immunology ; Rats ; Rats, Inbred Lew

OBJECTIVEViral myocarditis (VM) is one of the most common acquired myocardial diseases in children. However, its pathogenesis is not clear. Recent studies indicate that the cytotoxicity mediated by cytotoxic T lymphocyte (CTL) plays an important role in the development of myocardial injury involved in VM. Apoptosis mediated by Fas/FasL pathway is an essential mechanism of target cells damage by CTL. In this study, the authors investigated the regulatory effects of neutralizing anti-Fas ligand (anti-FasL) antibody on apoptosis and caspase-3 expression in experimental coxsackievirus B3 myocarditis and the role of the CTL mediated apoptosis in myocardium through Fas/FasL pathway in the development of VM.

METHODSA total of 80 BALB/c mice were used in the experiments. They were divided randomly into the following groups: normal control group (Gr1), CVB3 control group (Gr2), IgG control group (Gr3) and anti-FasL antibody therapy group (Gr4). The mice in Gr2, Gr3 and Gr4 were inoculated with 0.15 ml of TCID(50) 10(9)/ml coxsackie virus B3 (CVB3) and the mice in Gr1 with 0.15 ml of Eagle reagent. The mice in Gr3 and Gr4 were inoculated with IgG (0.1 mg/kg) and FasL antibody (0.1 mg/kg) on days 0 and days 3 after inoculation (p.i.), respectively. Eight mice in each group were sacrificed on day 10 p.i. Histopathological studies and terminal transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were used to quantify inflammation, necrosis and apoptosis in myocardium. The expression of active caspase-3 in myocardium was determined by immunohistochemistry. Caspase-3 mRNA and CVB3 mRNA were analyzed by reverse-transcription polymerase chain reaction (RT-PCR).

RESULTS(1) Caspase-3 activation and apoptosis were seen in the myocardium of mice with myocarditis. They had a significantly positive correlation with the changes of myocardial histopathologic scores (r = 0.81, P < 0.05; r = 0.73, P < 0.05). (2) The pathologic scores, average percentages of apoptotic cardiomyocytes, expression of active caspase-3 (protein and mRNA) and expression of CVB3 mRNA in myocardium of mice in Gr4, were significantly reduced compared to those in the myocardium of mice in Gr2 (P < 0.01, P < 0.01, P < 0.01, P < 0.01, and P < 0.05, respectively) and Gr3 (P < 0.01, P < 0.01, P < 0.05, P < 0.01, and P < 0.05, respectively).

CONCLUSIONMyocytic apoptosis is a key mechanism responsible for myocardial damage in viral myocarditis. Anti-FasL antibody can effectively reduce expression of active caspase-3 protein and mRNA, viral replication, cardiomyocytic apoptosis and myocardial injury in the experimental CVB3 myocarditis.

Animals ; Antibodies, Neutralizing ; therapeutic use ; Apoptosis ; Caspase 3 ; immunology ; Coxsackievirus Infections ; drug therapy ; immunology ; Enterovirus B, Human ; physiology ; Fas Ligand Protein ; immunology ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; immunology ; virology ; Myocardium ; immunology ; pathology ; Virus Replication

Endomyocardial biopsy often fails to show myocardial inflammation for patients with clinically suspected myocarditis. The serum isoforms of troponin T (cTnT) level is a very sensitive marker of myocardial injury and it is elevated even in the absence of myocardial inflammation. We investigated the correlations for myocardial injury, virus titers and inflammation in acute viral infection. Using the murine coxsackievirus group B3 (CVB3) myocarditis model, the histopathologic findings and virus titers in mouse hearts were compared with the serum cTnT levels measured by ELISA at various time points. Viable virus titers in the hearts peaked at 3 days after infection (8.22+/-0.13 log10 PFU/100 mg of heart); they decreased at day 7 and no viable virus was detected from day 14. Myocardial inflammation was minimal at day 3, peaked at day 7 and markedly decreased at day 14. The individual serum TnT levels were significantly increased at day 3 (7.37+/-1.46 ng/ml), persisted to day 7 (0.73+/-0.08 ng/ml), and normalized at day 14. Serum cTnT levels were correlatable with virus titers in the heart (r=0.744, P <0.01), but the serum cTnT levels were not correlated with the degrees of inflammation. Using the less myocarditic strain of CVB3, similar relationships were observed between the changes for the serum cTnT levels and the heart virus titers. During the course of viral infection, myocardial injury precedes the pathologic evidence of inflammation, and the elevated cTnT levels provide evidence of myocardial injury even in the absence of any histologic findings of myocarditis.
Acute Disease ; Animals ; Coxsackievirus Infections/*pathology ; Enterovirus B, Human/isolation & purification/pathogenicity/*physiology ; Female ; Heart/*virology ; Hela Cells ; Humans ; Inflammation/*immunology ; Mice ; Mice, Inbred BALB C ; Myocardial Infarction/immunology/*pathology ; Myocarditis/immunology/pathology/*virology ; *Myocardium/immunology/pathology ; Research Support, Non-U.S. Gov't ; Troponin T/blood ; Virus Replication