Jing-Hui Zhang

Th17 Cells Contribute to Viral Replication in Coxsackievirus B3-Induced Acute Viral Myocarditis

Acute viral myocarditis (AVMC) is characterized by virus-triggered myocardial inflammation, and Coxsackievirus B3 (CVB3) is the primary pathogen. We previously proved that Th17 cells, besides having proinflammatory effects, were involved in AVMC by enhancing humoral response. However, the relationship between Th17 cells and CVB3 replication remains unknown. In this experiment, we infected BALB/c mice with CVB3 for establishing AVMC models and then found that, with the increase of viral replication, the expressions of splenic Th17 cells, serum IL-17, and cardiac IL-17 mRNA were elevated significantly, accompanied by the progressive cardiac injuries of AVMC. Furthermore, on day 5, the peak time for viral replication, correlation was positive between cardiac IL-17 mRNA and CVB3 RNA (correlation index = 0.835; p < 0.01). Although the expressions of Th1 and CD8+ T cells, which could secrete the antiviral cytokine IFN-γ and damage the heart, were also elevated, along with Th17 cells, in AVMC, the neutralization of IL-17 further upregulated the percentages of splenic Th1 and CD8+ T cells and the levels of cardiac IFN-γ mRNA. The cardiac pathological changes were obviously improved after neutralization, with reduced viral replication followed by decreases in the cardiac inflammatory cytokines IL-17, TNF-α, and IL-1β. These data suggest that Th17 cells contribute to CVB3 replication in AVMC, and that IL-17 might be an important target for regulating the balance of antiviral immunities.

Th17 Cells Facilitate the Humoral Immune Response in Patients with Acute Viral Myocarditis

BackgroundRecently, the Th17 cell, a newly determined CD4+Th subset, was reported to participate in the inflammation of myocarditis combined with Th1 cells, and this study aimed to explore whether it was involved in the Th2 cell-mediated humoral immunity in viral myocarditis.MethodsA total of 34 patients, including 16 acute viral myocarditis (AVMC) and 18 dilated cardiomyopathy (DCM) having a history of AVMC, were enrolled for this study besides 18 healthy volunteers.ResultsThe frequencies of Th17 and Th1 cells, especially Th17 cells in AVMC patients, while those of Th1 and Th2 cells, especially Th2 cells in DCM group, were all increased significantly compared with those in healthy volunteers (P < 0.01), with no changes of Th2 cells in AVMC and Th17 cells in DCM groups. The similar results were also observed in Th cell cytokines (IL-17, INF-γ, and IL-4) and key transcript factors (RORγt, T-bet, and GATA-3). Meanwhile, antiheart antibodies (AHA) of IgG type were found in 15 (93.8%) patients with AVMC and ten (55.6%) cases with DCM, accompanied by the higher expression of IL-17R on B cells and the frequencies of B cells than those in healthy controls (P < 0.01 in AVMC and P < 0.05 in DCM, respectively) who had no AHA. Furthermore, both of the B cell activities in AVMC and DCM groups were elevated and positively correlated to serum IL-17 (R = 0.66, P < 0.01) and IL-4 (R = 0.47, P < 0.05) respectively, with no correlation to INF-γ.ConclusionsIt was Th17 cells but not Th2 cells that helped the B cells to produce AHA in AVMC and not until at the late phase of viral myocarditis could Th2 cells play the important role in mediating humoral response.

Neutralization of IL-17 inhibits the production of anti-ANT autoantibodies in CVB3-induced acute viral myocarditis

Anti-adenine nucleotide translocator (ANT) autoantibodies are related to the development of Coxsackievirus B3 (CVB3)-triggered acute viral myocarditis (AVMC). Recently, studies suggested that IL-17 especially produced by a novel CD4(+) Th-cell subset Th17 cells contributed to the production of pathogenic autoantibodies in some autoimmune diseases. However, the pathogenic role of IL-17 in AVMC remains largely unknown. In this study, we investigated whether IL-17 was associated with the disease progression and the production of anti-ANT autoantibodies in AVMC mouse model. The results showed that IL-17 monoclonal antibody (mAb)-treated AVMC mice had decreased HW/BW, reduced serum CK-MB activity and improved pathological score of heart sections along with the reduction of circulating IL-17 level and serum anti-ANT autoantibodies. The correlation index of serum IL-17 concentration and anti-ANT-autoantibody level was 0.874, p<0.01. In addition, the experimental results in vitro further proved that IL-17mAb could inhibit the proliferation of CD19(+) B lymphocytes and the secretion of anti-ANT autoantibodies. Our data suggested that IL-17 was related to the disease progression in AVMC mouse model by regulating the production of autoantibodies and blocking IL-17 might represent a promising novel therapeutic approach.

TLR4 signaling induced TLR2 expression in the process of mimic cerebral ischemia/reperfusion in vitro

Both TLR4 and TLR2 participated in the mediation of the inflammatory injury in the process of partial cerebral ischemia/reperfusion. However, it still remains unclear whether a crosstalk exists between TLR2 and TLR4 in ischemic cerebral damage. In the present study, we investigated the effect of TLR4 signaling on TLR2 expression during mimic cerebral I/R in vitro. BV-2 cells were cultured and treated with ischemia/reperfusion, then transfected with the plasmid pEGFP-H1/TLR4-siRNA, the plasmid pEGFP-H1/control sequence-siRNA and the blank plasmid, respectively. Interestingly, the expression of TLR2 and TLR4 mRNA and protein, NF-κB p65 mRNA and supernatant TNF-α level were significantly higher in ischemia/reperfusion treated cells than those lack of ischemia/reperfusion treatment, and as compared with those in ischemia/reperfusion treated cells without transfection, no significant differences about the above mentioned gene and protein expression were found in the blank plasmid tranfected cells and the plasmid pEGFP-H1/control sequence-siRNA transfected cells respectively, while the expression levels in the plasmid pEGFP-H1/TLR4-siRNA transfected cells were significantly lower. Additionally, in order to determine the effects of pyrrolidinediethyldithiocarbamate (PDTC), an NF-κB inhibitor, on the TLR4-induced TLR2 expression in BV-2 cells treated with ischemia/reperfusion, it was found that TLR4 and TLR2 mRNA expressions in PDTC pretreated cells were significantly lower in comparison with normal saline pretreated cells and non-pretreated cells. The data suggested that TLR2 activation, signaled by TLR4 and regulated by NF-κB, might be directly involved play an important role in ischemia/reperfusion induced brain damage.

Infectious Tolerance to ADP/ATP Carrier Peptides Induced by Anti-L3T4 Monoclonal Antibody in Dilated Cardiomyopathy Mice

CD4 T cells are suspected to play an important role in the pathogenesis of dilated cardiomyopathy (DCM). This study sought to evaluate whether anti-L3T4 monoclonal antibody (McAb) could induce the infectious tolerance to the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) carrier peptides to protect mice from DCM. BALB/c mice (n = 16) were immunized with the peptides derived from human ADP/ATP carrier on the 1st, 14th, 28th, 49{th}, and 79th days, and some of them (n = 6) were also injected with anti-L3T4 McAb on the −1{st}, 0, and 1st days. On the 180th day, the splenocytes (SC) from the McAb-treated group were transferred into the syngeneic recipients (n = 6) who were also immunized with the peptides in the same manner. The sham-immunized mice were taken as the controls (n = 10). Results showed that the serum antibody against the ADP/ATP carrier examined with ELISA was positive in all mice only immunized with the peptides (DCM group), while negative in the McAb-treated, the SC-transferred, and the Control groups. The mRNA expression of IFN-γ, IL-2, and IL-4, especially IL-4 in T cells investigated using real-time quantitative PCR and the percentages of T helper 1 (Th1) and Th2 subsets, especially Th2 subset detected with Flow Cytometry were all increased in DCM group, accompanied by the cardiac histopathological changes like those in DCM. Such findings were not seen in the other three groups. It concluded that anti-L3T4 McAb could inhibit the occurrence of DCM induced by the ADP/ATP carrier peptides in mice, and this immune tolerance could be transferred to the syngeneic recipients.

Treatment and Prevention of Experimental Autoimmune Myocarditis with CD28 Superagonists

Objectives: Experimental autoimmune myocarditis (EAM), a rodent model of human dilated cardiomyopathy (DCM), is mediated by an autoimmune mechanism. We investigated whether a CD28 superagonistic antibody selectively targeting CD4+CD25+ regulatory T cells (Tregs) provides effective therapy for EAM. Methods: Four groups of 5 rats were used. The normal control group was immunized with PBS. The EAM group was immunized with porcine myosin. The experimental group was immunized with myosin and superagonistic CD28 antibody JJ316. The final group was immunized with myosin and an unrelated rat IgG. Autoantibody and IL-10 production, CD4+CD25+ cell levels, Foxp3 expression and cardiac histology were analyzed. Results: Anti-myosin autoantibody levels were higher in the EAM and isotype control groups than the normal control group (p < 0.05), and reduced in the CD28-JJ316 group (p < 0.05). The levels of CD25+CD4+ cells, IL-10 and splenocyte Foxp3 expression were significantly lower in the EAM and isotype control groups versus the CD28-JJ316 group (p < 0.05). Infiltration of inflammatory cells was observed in the EAM and isotype control groups, whereas CD28-JJ316 ameliorated myocarditis. Conclusion: CD28 superagonists could be effective in EAM treatment by up-regulating Foxp3 expression and contributing to CD4+CD25+ Treg activation and expansion. The enhancement in IL-10 by CD28 superagonists also ameliorated the disease.

Effects of propofol on the mRNA expression of toll-like receptor 4 in BV-2 cells during mimic ischemia-reperfusion injury in vitro

SummaryThis study investigated the effects of propofol on the mRNA expression of Toll-like receptor-4 (TLR4) in BV-2 cells during mimic ischemia-reperfusion (I/R) injury in vitro. BV-2 cells, a mouse microglia line, were cultured and divided into 4 groups at random: control group (group C), ischemia/reperfusion group (group I/R), low-dose propofol (25 μmol/L) intervention group (group PF25) and high-dose propofol (100 μmol/L) intervention group (group PF100). The mRNA expression of TLR4 and NF-κB was measured by means of RT-PCR. TNF-α levels in the supernatants of BV-2 cells were detected by ELISA. The results showed that the mRNA expression of TLR4 and NF-κB was significantly higher in groups I/R, PF25 and PF100 than in group C (P<0.01). And the TNF-α level in the supernatants was elevated in groups I/R, PF25 and PF100 as compared with that in group C (P<0.01). After pre-treatment with propofol, the mRNA expressions of TLR4 and NF-κ B and the TNF-α level were significantly decreased in groups PF25 and PF100 in comparison to those in group I/R (P<0.01). And the decrease in those indicators was more significant in group PF100 than in group PF25 (P<0.01). It was concluded that propofol exerted brain-protecting effects during I/R injury by suppressing the mRNA expressions of TLR4 and NF-κB and deceasing the TNF-α level.This study investigated the effects of propofol on the mRNA expression of Toll-like receptor-4 (TLR4) in BV-2 cells during mimic ischemia-reperfusion (I/R) injury in vitro. BV-2 cells, a mouse microglia line, were cultured and divided into 4 groups at random: control group (group C), ischemia/reperfusion group (group I/R), low-dose propofol (25 μmol/L) intervention group (group PF25) and high-dose propofol (100 μmol/L) intervention group (group PF100). The mRNA expression of TLR4 and NF-κB was measured by means of RT-PCR. TNF-α levels in the supernatants of BV-2 cells were detected by ELISA. The results showed that the mRNA expression of TLR4 and NF-κB was significantly higher in groups I/R, PF25 and PF100 than in group C (P<0.01). And the TNF-α level in the supernatants was elevated in groups I/R, PF25 and PF100 as compared with that in group C (P<0.01). After pre-treatment with propofol, the mRNA expressions of TLR4 and NF-κ B and the TNF-α level were significantly decreased in groups PF25 and PF100 in comparison to those in group I/R (P<0.01). And the decrease in those indicators was more significant in group PF100 than in group PF25 (P<0.01). It was concluded that propofol exerted brain-protecting effects during I/R injury by suppressing the mRNA expressions of TLR4 and NF-κB and deceasing the TNF-α level.

Analysis of specific Th1/Th2 helper cell responses and IgG subtype antibodies in anti-CD4 monoclonal antibody treated mice with autoimmune cardiomyopathy

SummaryThe cytokine repertoire of ADP/ATP carrier-specific humoral immune responses and the cytokine-dependent anti-ADP/ATP carrier antibody IgG subclasses were examined in a cohort of ADP/ATP carrier-immunized BALB/c mice treated with anti-CD4 monoclonal antibody. Eighteen male BALB/c mice (6–8 weeks old) were randomized into 3 groups: dilated cardiomyopathy (DCM) group, DCM-tolerance (Tol) group and control group. The mice in DCM group were immunized with the peptides derived from human ADP/ATP carrier protein for 6 months and mice in the control group were sham-immunized, while the mice in DCM-Tol group were immunized with ADP/ATP carrier protein and anti-CD4 McAb simultaneously. Serum autoantibody against ADP/ATP carrier and IgG subclasses were measured by ELISA, intracellular cytokines IFN-γ and IL-4 of Th cells were monitored with flow cytometry, and splenic T cell cytokines IFN-γ, IL-2, IL-4 and IL-6 were detected by using real-time fluorescent quantitative PCR. The results showed that the autoantibody against ADP/ATP carrier was found in all mice in DCM group, and the antibody level, serum IgG1 and IgG2a subclasses, cytokines in T cells and Th cells were all elevated in DCM group, as compared with those in control group (P<0.01). On the other hand, in DCM-Tol group, the autoantibody level and contents of all the cytokines were significantly different from those in DCM group (P<0.01), and were close to those in control group. And the levels of IgG1, IgG2a, IgG2b and IgG3 were influenced, to varying degrees, by anti-CD4 McAb as compared with those in DCM group. All these four types of IgG subclasses were substantially decreased in DCM-Tol group as compared with DCM group. It is concluded that the treatment with anti-CD4 McAb could prevent the activation of T cells, reverse the abnormal secretion of cytokines and the imbalance between Th1/Th2 cell subsets and abnormal production of autoantibody against ADP/ATP carrier, and eventually avoid myocardial injuries.The cytokine repertoire of ADP/ATP carrier-specific humoral immune responses and the cytokine-dependent anti-ADP/ATP carrier antibody IgG subclasses were examined in a cohort of ADP/ATP carrier-immunized BALB/c mice treated with anti-CD4 monoclonal antibody. Eighteen male BALB/c mice (6–8 weeks old) were randomized into 3 groups: dilated cardiomyopathy (DCM) group, DCM-tolerance (Tol) group and control group. The mice in DCM group were immunized with the peptides derived from human ADP/ATP carrier protein for 6 months and mice in the control group were sham-immunized, while the mice in DCM-Tol group were immunized with ADP/ATP carrier protein and anti-CD4 McAb simultaneously. Serum autoantibody against ADP/ATP carrier and IgG subclasses were measured by ELISA, intracellular cytokines IFN-γ and IL-4 of Th cells were monitored with flow cytometry, and splenic T cell cytokines IFN-γ, IL-2, IL-4 and IL-6 were detected by using real-time fluorescent quantitative PCR. The results showed that the autoantibody against ADP/ATP carrier was found in all mice in DCM group, and the antibody level, serum IgG1 and IgG2a subclasses, cytokines in T cells and Th cells were all elevated in DCM group, as compared with those in control group (P<0.01). On the other hand, in DCM-Tol group, the autoantibody level and contents of all the cytokines were significantly different from those in DCM group (P<0.01), and were close to those in control group. And the levels of IgG1, IgG2a, IgG2b and IgG3 were influenced, to varying degrees, by anti-CD4 McAb as compared with those in DCM group. All these four types of IgG subclasses were substantially decreased in DCM-Tol group as compared with DCM group. It is concluded that the treatment with anti-CD4 McAb could prevent the activation of T cells, reverse the abnormal secretion of cytokines and the imbalance between Th1/Th2 cell subsets and abnormal production of autoantibody against ADP/ATP carrier, and eventually avoid myocardial injuries.

Analysis of IgG subclass antibodies and expression of T-Cell receptor signaling molecules in anti-CD4 monoclonal antibody treated mice with autoimmune cardiomyopathy

T-cell immune abnormality in patients of dilated cardiomyopathy has been intensively studied over the past 10 years. In this study, we aim to focus on the molecular mechanism of T-cells in autoimmune cardiomyopathy mouse model by detecting the expression of three T-cell signaling molecules. Balb/C mice (n = 12) were immunized with the peptides derived from human ADP/ATP carrier on the 1st, 14th, 28th, 49th and 79th days, and half of them were also injected with anti-L3T4 McAb on the − 1st, 0 and 1st days. The sham-immunized mice were taken as the controls (n = 6). The main result shows that the antibody response of IgG subclasses such as IgG1, IgG2b and IgG3 were definitely blocked except IgG2a in CD4+ cell-depleted Balb/C mice. In addition, the average mRNA expression of p56lck, p59fyn and zap-70 were all found to be dramatically higher in the mice immunized with only ADP/ATP carrier peptides than in the control-group. At meantime, reduced levels of the protein kinases p56lck, p59fyn and zap-70 were clearly observed in anti-CD4 McAb immunized group compared with DCM group. We propose that the proliferation of T-cells was significantly inhibited in anti-CD4 treated mice and CD4+ T-cells may play a critical role in ADP/ATP carrier caused mouse DCM.