The Role Of Galectin-9 In CVB3 Induced Myocarditis Of BALB/c Mice And Possible Mechanisms

The incidence and severity of heart disease, including myocarditis, is higher among men than women. Viral myocarditis (VMC), or inflammation of the heart, is a principal cause of heart failure in young adults often leading to chronic heart disease and dilated cardiomyopathy (DCM). Viral infection is one of the important reasons for VMC. Among the viruses, Coxsackievirus group B type 3 (CVB3) infection accounts for more than half of VMC cases. CVB3 is a member of the picomavirus family of small, non-enveloped, positive sense, RNA viruses, and are major etiological agents in clinical viral myocarditis. A murine model of CVB3 induced myocarditis has been established which mimicks many of the characteristics of the clinical disease. Although the pathogenesis of CVB3 infection has been studied for decades, it is only recently recognized that many variables, including viral genome structure, host genetic background, the age, and the immune status of the host, interact with each other to determine the initiation, occurrence and progression of viral myocarditis.The bias of CD4+ Th immune response appears to influence the severity of myocarditis. On the one hand, the Thl immune response, rather than the Th2 immune response, is considered to be detrimental to heart tissue. Opavsky et al. found that the severity of disease is attenuated in CD4 knockout mice, confirming the role of CD4+ T cells in CVB3 induced myocarditis. Huber et al. found CD4+Thl cell responses are essential to CVB3 induced myocarditis susceptibility. On the other hand, regulatory T cells play a major role in protection against inflammation in the heart, and their alteration by viral infection may contribute substantially to the outcome of myocarditis. Recent studies have shown that autoimmune myocarditis and multiorgan inflammation are controlled by Foxp3+ T cells highly expressing the glucocorticoid-induced TNF receptor familyrelated protein (GITR). Depletion of the GITR+ T regulatory cells allowed activation of autoimmune heart disease. Huber and colleagues described myocarditis could be overcome by a coxsackievirus variant which maintained and induced regulatory T cells function.Furthermore, cytokines play a key role in CVB3 induced myocarditis. In susceptible mice IL-1βand IL-18 play a significant role in the pathogenesis of CVB3 induced myocarditis. IL-1 or TNF-a can promote myocarditis in resistant B10.A mice. Our previous study indicated that exogenous administration of IL-4 is found to improve myocardial inflammation and the severity of myocarditis in mice infected with CVB3. Thus, therapeutic strategies of CVB3 induced myocarditis should consider regulation of regulatory T cells (Tregs) differentiation as well as inhibition of pro-inflammatory cells and Thl cytokines.To shift the cytokine balance to anti-inflammatory cytokines and to delete pro-inflammatory Thl cells that closely associate with etiology of CVB3 induced myocarditis, it is necessary to discover novel biological substances that can selectively suppress the function or delete activated Thl cells, at the same time expanding Tregs. Galectin-9, one of the (3-galactoside binding animal lectins belonging to the galectin family, induces apoptosis of eosinophils, cancer cells, and T cells. Galectin-9 preferentially induces apoptosis of activated CD4+ T cells through Ca+ influx-calpain-caspasel pathway. Zhu et al. have recently demonstrated that galectin-9 is a ligand of T cell immunoglobulin- and mucin domain-containing molecule3 (TIM-3) that was expressed selectively on terminally differentiated Thl cells, Th17, Tregs, and that galectin-9 induces apoptosis of TIM-3-expressing cells in vitro and in vivo. Furthermore, exogenous administration of galectin-9 ameliorates experimental allergic encephalitis, an autoimmune disease of the central nervous system. More recently it has been shown that galectin-9 ameliorates a mouse collagen-induced arthritis (CIA) model and HSV induced lesions by suppressing the generation of Th17, promoting the induction of regulatory T cells.In this study, we compared galectin-9 expression in different organs of males before and after CVB3 infection. Then we examined the effects of in vivo treatment with recombinant galectin-9 on the development of myocardial inflammation in male mice infected with CVB3. Meanwhile, we analyzed the modulation of immune cells in the heart and spleen of CVB3 infected male mice in vivo with or without recombinant galectin-9 treatment and explored the mechanisms involved.The purpose of the present study was to clarify the role of galectin-9 in the development of murine acute myocarditis induced by CVB3 and its possible mechanisms involved. 1. Change of galectin-9 expression before and after CVB3 infectionGalectins are a family of animal lectins that bindβ-galactosides. Current research indicates that galectins play important roles in diverse physiological and pathological processes, including immune and inflammatory responses, tumour development and progression, neural degeneration, atherosclerosis, diabetes. Among these, galectin-1,-3,-9 are the key regulators of immune response and inflammation. To better understand the mechanisms responsible for increased heart disease in male mice, we first compared galectins expression in the heart from male BALB/c mice before and after CVB3 infection. We found galectin-9 mRNA expression as well as galectin-1 in the heart from males was up-regulated after CVB3 infection. Then we further focused on galectin-9 mRNA expression in different organs, and found it was significantly higher in the heart as well as spleen and mesenteric lymph node (MLN) after CVB3 infection by RT-PCR assessment. Subsequently we confirmed the higher expression of galectin-9 protein in the heart by immunohistochemistry after CVB3 infection.2. Differential severity of CVB3 induced myocarditis before and after galectin-9 treatmentViral myocarditis is characterized by inflammatory cells infiltration in the interstitial tissue of myocardium with the result of myocyte focal necrosis or fibrosis and finally the loss of heart function. It well mimics the characteristics of clinical viral myocarditis using CVB3 infected myocarditis murine model with the indices of serology, pathology and body weight reduced rate as well as mortality. From day 3 post-infection, the body weight of PBS treated mice decreased obviously. On day 7 post-infection, the body weight decreased 15%. While in galectin-9 treated mice, on day 3 post-infection, the body weight also reduced but then recovered on day 5. The activity of serum Creatine Kinase (CK) and MB isoenzyme of Creatine Kinase (CK-MB) was significantly down-regulated and the level of Cardiac Troponin I (cTnl) was also decreased in galectin-9 treated BALB/c mice on day 7 post-infection. The pathological studies, the most direct and important diagnosis criteria revealed that in PBS treated mice, on day 7 post-infection, H.E showed the focal infiltration of inflammatory cells and the massive necrosis of myocytes. While in galectin-9 treated mice, there was no obvious inflammatory cells infiltration and no myocyte necrosis. Moreover, lower frequency of CD45+ immune cells in the heart of galectin-9 treated mice than PBS treated mice confirmed the above histological findings. As far as mortality was concerned, the mortality in CVB3 infected male mice was 70%, which was much higher than that in galectin-9 treated mice with the mortality of 15%. From the above results, the conclusion could be drawn that galectin-9 alleviated the severity of viral myocarditis after CVB3 infection.3. Effects of galectin-9 treatment on inflammatory cytokines productionInflammatory cytokines, such as TNF-α, IFN-γ, IL-4 and IL-10 play a role in the initiation of inflammatory cell infiltration in the lesions. To clarify whether galectin-9 treatment regulates cytokines production, levels of inflammatory cytokines (TNF-α, IFN-γ, IL-4, IL-10) in myocardial tissue and serum from CVB3 infected mice were assessed with or without galectin-9 administration. CVB3 infection resulted in the increased levels of TNF-αand IFN-γin the mice. Galectin-9 treatment led to significant decreased levels of TNF-αand IFN-γin both myocardial tissue and serum of the infected mice, whereas galectin-9 increased the levels of IL-4 and IL-10. PartⅡPossible mechanisms underlies galectin-9 functions1. Effects of galectin-9 treatment on CVB3 replicationCVB3 can infect myocytes and replicate in cells, which is the prerequisite to induce VMC. To exclude the possibility that galectin-9 could effectively rescue mice from lethal myocarditis was due to decreased CVB3 replication, the viral load in heart tissues was also assessed by real-time PCR and plaque assay, and it was found that galectin-9 treatment does not significantly change myocardial viral burden, suggesting that the alleviation of viral myocarditis by galectin-9 is not due to the direct down-regulation of viral replication.2. Effects of galectin-9 treatment on immune responseSince alleviated inflammation in galectin-9 treated mice was not due to decreased viral replication in the heart, we next performed experiments to clarify whether galectin-9 modulates the balance of T immune response and influences the number of Tregs. Results showed that galectin-9 administration had significantly decreased percentage of CD4+ T cells, whereas it had remarkably increased percentage of Tregs as well as CDllb+ or Gr-1+ cells in heart and spleen from CVB3 infected mice. We further compared the level of Tim-3 (T cell Ig and mucin-3, the receptor of galectin-9) and TLR4 on immune cells isolated from the heart of males with or without galectin-9 treatment. Tim-3 expression was significantly reduced on CD4+ T cells but increased on macrophages from heart of males on day 7 p.i., while TLR4 expression was significantly reduced on CDllb+ cells and macrophages.Since administration of galectin-9 alleviated CVB3 induced myocarditis, we addressed to analyze cellular mechanisms and whether the effects are entirely due to loss of pathogenic T cells and increased Tregs.3. Influence of galectin-9 treatment on the apoptosis and proliferation of CD4+ T cellsPrior in vitro studies have indicated that galectin-9 triggering of pro-inflammatory CD4+ T cells causes them to undergo apoptosis. In the model of CVB3 induced myocarditis, since inflammation appeared to be mainly orchestrated by IFN-y producing CD4+ T cells, so we detected the effects of galectin-9 on the induction of apoptosis of CD4+ T cells. As shown in results, about 50% CD4+ T cells underwent apoptosis after galectin-9 treatment, perhaps accounting in part for the anti-inflammatory effect of galectin-9. Furthermore, while about 90% of CD4+CD25low/int effector T cells underwent apoptosis,10% of CD4+CD25high regulatory T cells were annexin V+We further explored whether in vivo galectin-9 treatment would influence the proliferation of CD4+ T cells. As shown, BrdU+ proliferating CD4+ T cells increased in heart on day 7 after CVB3 infection, whereas it significantly decreased in mice receiving galectin-9 treatment. The decreased proliferative response mainly existed in the CD4+CD25low/int effector T cells.These results indicated that in vivo galectin-9 administration efficiently down-regulated CD4+ T cell responses. The differential regulation of apoptosis and proliferation of effctor and regulatory T cells by galectin-9 maybe due to the differential expression of Tim-3.4. Effects of galectin-9 treatment on the differentiation of regulatory T cells In prior experiments have established that the severity of CVB3 induced myocarditis can be modulated by regulatory T cells. To determine whether galectin-9 administration to CVB3 infected mice had any influence on the Tregs response, experiments were done to clarify whether galectin-9 is involved in the differentiation of CD4+CD25+Foxp3+ Tregs. Thus, we transferred naive CD4+CD25-T cells into CVB3 infected nude mice on day 3 post-infection and galectin-9 was i.p. injected everyday till day 7. These mice were sacrificed on day 7 post-infection and heart infiltrated cells and splenocytes were separated, then the percentage of CD4+CD25+ Foxp3+ Tregs was determined by FACS. Results showed that there was a significant increase of CD4+ CD25+ Foxp3+ Tregs in the heart from galectin-9 treated mice compared to non-treated mice. In addition, the percentage of CD4+CD25+Foxp3+ Tregs in spleen from galectin-9 treated mice was significantly higher than non-treated mice. From the data described above, we clarified that galectin-9 promoted the induction of Tregs during CVB3 induced myocarditis in vivo.5. Influence of galectin-9 treatment on expansion of myeloid suppressor cellsGalectin-9 treated animals, as compared with control animals, showed significantly expanded populations of CDllb+ and Gr-1+ cells, which phenotypes possessed by myeloid suppressor cells. So we detected the influence of galectin-9 treatment on expansion of CDllb+Gr-l+ cells. We found that galectin-9 promoted the expansion of CDllb+Gr-1+ cells, which expressed low level of F4/80, CD80,CD86 and IAd, a phenotypes possessed by myeloid suppressor cells. They also could secret IL-10 and TGF-β. Moreover, CDllb+Gr-1+ cells increased the number of regulatory T cells by expansion of previously existed cells, which partly depended on IL-10 production.In conclusion, the present study demonstrated that galectin-9 may play a crucial role in CVB3 induced myocarditis and it may be one of the novel therapeutic candidates that can suppress autoimmune inflammation by regulating the T cell differentiation and the balance of pathogenic and regulatory T cells, such that pro-inflammatory cytokines production are inhibited and anti-inflammatory cytokines are enhanced.