**3. Experimental autoimmune myocarditis**

As described in the previous section, mouse models greatly aid the analysis of autoimmune diseases. To set apart the autoimmune phase of disease from acute infection an experimental induced autoimmune myocarditis model was developed (Blyszczuk et al. 2008). Experimental induced autoimmune myocarditis mimics the typical chronic phase of disease observed in genetically susceptible mice infected with coxsackievirus B and different stages of disease severity observed with experimental induced autoimmune myocarditis models are graded according to the extent of inflammatory infiltrates at the peak of inflammation. Autoimmune myocarditis can be induced by the injection of cardiac myosin with complete Freund's adjuvant and pertussis toxin. Mice injected with this combination of self- antigen and adjuvants are able to generate cardiac myosin-specific autoantibodies and present with

The Key Players of Coxsackievirus-Induced Myocarditis 249

results to be reproducible and facilitate detailed analysis of the autoimmune disease cellular

Other species such as rats and Guinea pigs have also greatly contributed to our understanding of myocarditis. Immunization of different animal models with heart homogenates derived from various species has not been, unfortunately, successful at inducing myocarditis, where the results were often controversial (Neu et al. 1987). In most of those models, mild lesions appeared in the heart. However, out of the differing species models, 2 reports: the guinea pig model by Hosenpud et al and the murine model by Neu et al, showed enlargement of the heart with autoimmune myocarditis (Hosenpud et al. 1985; Neu et al. 1987). The authors also noted discoloration of cardiac surfaces, pericardial effusion and lethal clinical course that were conditions not previously observed in experimental induced autoimmune myocarditis. Kodama et al were also able to produce such pathological and clinical conditions using the Lewis rat model (Kodama et al. 1992) though the cardiac myosin immunization murine model seems to have had the greatest

There are many ways in which autoimmune myocarditis can been induced in animal models. Immunization of susceptible mice with cardiac myosin or with a myocardiogenic peptide derived from the alpha cardiac heavy chain emulsified in complete Freund's adjuvant induces myocarditis in mice with a peak of inflammation in the heart around day 21. This inflammation observed with induced autoimmune disease is similar to that seen in the coxsackievirus B3 induced autoimmune myocarditis model during the chronic phase, however in this case, does not include the earlier complication of a pathogen infection. The immunization with cardiac myosin is associated with production of cardiac myosin-specific autoantibodies and cardiac myosin-specific T cells (Kodama et al. 1992; Godsel et al. 2001; Leuschner et al. 2009; Rose 2010). It has been demonstrated that the induction of disease by immunization with cardiac myosin can only be successful in genetically susceptible mice (Neu et al. 1987). We suggest that the complex genetics of the host therefore determines whether an infection will resolve or proceed to an adverse autoimmune outcome (Poffenberger et al. 2010). Identifying particular traits that favour susceptibility or resistance to an autoimmune incidence helps us understand how infectious diseases culminate in autoimmune disease. Other methods used to induce autoimmune myocarditis include administration of 2 early, critical proinflammatory cytokines, IL1b and TNF-α and injection of mice with myosin in combination with CFA and additional lipopolysaccharide. Some groups have reported the induction of autoimmune myocarditis using immunization with porcine cardiac myosin (Wang et al. 1999). Interestingly, Wittner et al. produced myosin autoantibodies and myocarditis in rabbits using immunization with bovine heart myosin (Wittner et al. 1983). Daniels et al reported the development of a recombinant model of experimental induced autoimmune myocarditis, induced by immunization with a 68kDa fragment of cardiac myosin referred to as Myo4 (Daniels et al. 2008). Myo4 induces severe autoimmune myocarditis in A/J mice 21 days post immunization. The immune response to Myo4 immunization is characterized by Th1 and Th17 features. Myo4-experimental induced autoimmune myocarditis has advantages over other models in terms of immunogen production and the ability to measure antigen-specific functional immunity in *ex vivo* assays. Myo4 and other immunoantigen experimental induced autoimmune myocarditis provide a means to investigate epitope spreading and the effect on disease pathology as well as prophylactic and therapeutic treatment studies aimed at developing therapeutics to alleviate

and molecular components (Neu et al. 1987).

impact on our understanding of autoimmune myocarditis.

acute and chronic autoimmune myocarditis (Daniels et al. 2008).

heart pathology similar to coxsackievirus B3-induced disease 3 weeks post-injection (Figure 2) (Kodama et al. 1992).

Fig. 2. Experimental autoimmune myocarditis versus coxsackievirus B3 induced myocarditis. Experimental autoimmune myocarditis involves the induction of autoimmunity by the injection of self-protein such as cardiac myosin or troponin I with adjuvants or without or injections of other cardiomyogenic peptides to induce chronic myocarditis as seen with coxsackievirus B3 infection in susceptible mice.

Neu et al suggest that autoimmune myocarditis is induced indirectly by viral infection and that one causative factor may be an autoimmune response to the cardiac myosin released or exposed after the virus-mediated myocyte damage (Neu et al. 1987). If this is the case, autoimmune disease should be induced by immunization with cardiac myosin alone and susceptibility to coxsackievirus B3-induced autoimmune myocarditis should be as likely as susceptibility to the myosin-induced autoimmune disease (Neu et al. 1987). Troponin I has also been used as an autoantigen for induced autoimmune myocarditis (Leuschner et al. 2009). Programmed cell death-1 receptor deficient mice develop cardiomyopathy with production of high-titered autoantibodies against cardiac troponin I (Okazaki et al. 2003). Cardiac troponin I induces a robust autoimmune response that encompasses both humoral and cellular responses that leads to severe inflammation and fibrosis in the myocardium of mice. Mice induced with cardiac troponin I have a genetic and sex biased susceptibility for myocardial inflammation compared to other autoimmune disease models (Leuschner et al. 2009). The key to reproducing ideal autoimmune disease conditions for studying myocarditis is to use a defined immunogen, ie cardiac myosin, troponin I etc. obtained from the same species as the study's model. Moreover, genetically defined inbred strains of susceptible and resistant mice allow

heart pathology similar to coxsackievirus B3-induced disease 3 weeks post-injection (Figure

Fig. 2. Experimental autoimmune myocarditis versus coxsackievirus B3 induced myocarditis. Experimental autoimmune myocarditis involves the induction of

myocarditis as seen with coxsackievirus B3 infection in susceptible mice.

autoimmunity by the injection of self-protein such as cardiac myosin or troponin I with adjuvants or without or injections of other cardiomyogenic peptides to induce chronic

Neu et al suggest that autoimmune myocarditis is induced indirectly by viral infection and that one causative factor may be an autoimmune response to the cardiac myosin released or exposed after the virus-mediated myocyte damage (Neu et al. 1987). If this is the case, autoimmune disease should be induced by immunization with cardiac myosin alone and susceptibility to coxsackievirus B3-induced autoimmune myocarditis should be as likely as susceptibility to the myosin-induced autoimmune disease (Neu et al. 1987). Troponin I has also been used as an autoantigen for induced autoimmune myocarditis (Leuschner et al. 2009). Programmed cell death-1 receptor deficient mice develop cardiomyopathy with production of high-titered autoantibodies against cardiac troponin I (Okazaki et al. 2003). Cardiac troponin I induces a robust autoimmune response that encompasses both humoral and cellular responses that leads to severe inflammation and fibrosis in the myocardium of mice. Mice induced with cardiac troponin I have a genetic and sex biased susceptibility for myocardial inflammation compared to other autoimmune disease models (Leuschner et al. 2009). The key to reproducing ideal autoimmune disease conditions for studying myocarditis is to use a defined immunogen, ie cardiac myosin, troponin I etc. obtained from the same species as the study's model. Moreover, genetically defined inbred strains of susceptible and resistant mice allow

2) (Kodama et al. 1992).

results to be reproducible and facilitate detailed analysis of the autoimmune disease cellular and molecular components (Neu et al. 1987).

Other species such as rats and Guinea pigs have also greatly contributed to our understanding of myocarditis. Immunization of different animal models with heart homogenates derived from various species has not been, unfortunately, successful at inducing myocarditis, where the results were often controversial (Neu et al. 1987). In most of those models, mild lesions appeared in the heart. However, out of the differing species models, 2 reports: the guinea pig model by Hosenpud et al and the murine model by Neu et al, showed enlargement of the heart with autoimmune myocarditis (Hosenpud et al. 1985; Neu et al. 1987). The authors also noted discoloration of cardiac surfaces, pericardial effusion and lethal clinical course that were conditions not previously observed in experimental induced autoimmune myocarditis. Kodama et al were also able to produce such pathological and clinical conditions using the Lewis rat model (Kodama et al. 1992) though the cardiac myosin immunization murine model seems to have had the greatest impact on our understanding of autoimmune myocarditis.

There are many ways in which autoimmune myocarditis can been induced in animal models. Immunization of susceptible mice with cardiac myosin or with a myocardiogenic peptide derived from the alpha cardiac heavy chain emulsified in complete Freund's adjuvant induces myocarditis in mice with a peak of inflammation in the heart around day 21. This inflammation observed with induced autoimmune disease is similar to that seen in the coxsackievirus B3 induced autoimmune myocarditis model during the chronic phase, however in this case, does not include the earlier complication of a pathogen infection. The immunization with cardiac myosin is associated with production of cardiac myosin-specific autoantibodies and cardiac myosin-specific T cells (Kodama et al. 1992; Godsel et al. 2001; Leuschner et al. 2009; Rose 2010). It has been demonstrated that the induction of disease by immunization with cardiac myosin can only be successful in genetically susceptible mice (Neu et al. 1987). We suggest that the complex genetics of the host therefore determines whether an infection will resolve or proceed to an adverse autoimmune outcome (Poffenberger et al. 2010). Identifying particular traits that favour susceptibility or resistance to an autoimmune incidence helps us understand how infectious diseases culminate in autoimmune disease. Other methods used to induce autoimmune myocarditis include administration of 2 early, critical proinflammatory cytokines, IL1b and TNF-α and injection of mice with myosin in combination with CFA and additional lipopolysaccharide. Some groups have reported the induction of autoimmune myocarditis using immunization with porcine cardiac myosin (Wang et al. 1999). Interestingly, Wittner et al. produced myosin autoantibodies and myocarditis in rabbits using immunization with bovine heart myosin (Wittner et al. 1983). Daniels et al reported the development of a recombinant model of experimental induced autoimmune myocarditis, induced by immunization with a 68kDa fragment of cardiac myosin referred to as Myo4 (Daniels et al. 2008). Myo4 induces severe autoimmune myocarditis in A/J mice 21 days post immunization. The immune response to Myo4 immunization is characterized by Th1 and Th17 features. Myo4-experimental induced autoimmune myocarditis has advantages over other models in terms of immunogen production and the ability to measure antigen-specific functional immunity in *ex vivo* assays. Myo4 and other immunoantigen experimental induced autoimmune myocarditis provide a means to investigate epitope spreading and the effect on disease pathology as well as prophylactic and therapeutic treatment studies aimed at developing therapeutics to alleviate acute and chronic autoimmune myocarditis (Daniels et al. 2008).

The Key Players of Coxsackievirus-Induced Myocarditis 251

autoimmune myocarditis, a disease likely directed at multiple self-antigens, NOD mice were tolerized to cardiac myosin using a covalently coupled antigen approach and subsequently challenged with coxsackievirus B3. Cardiac myosin tolerance to effectively prevent coxsackievirus B3-mediated autoimmune myocarditis was not observed, possibly reprimanding cardiac myosin as a single autoantigen player in viral-mediated disease

Another possible player in viral-mediated disease as demonstrated with experimental induced autoimmune myocarditis may be multinucleated giant cells. Wang et al showed that the cardiac lesions in experimental induced autoimmune myocarditis are histologically similar to human myocarditis, with myocyte swelling, necrosis, and fibrosis that are accompanied by mononuclear cell infiltration consisting of granulocytes, macrophages, CD4+, CD8+ T cells, B cells and multinucleated giant cells (Wang et al. 1999; Blyszczuk et al. 2008; Daniels et al. 2008). Also, the histological features observed in myocarditis induced by cardiac myosin are very similar to the description of active giant-cell myocarditis in humans (Wang et al. 1999; Blyszczuk et al. 2008). Therefore, it has been inferred that macrophagederived multinucleated giant cells, together with other inflammatory cells, are important mediators of myocyte destruction. The presence of a heterogeneous population of cellular components in the cardiac infiltrate also implies the existence of a complex, cytokine-rich microenvironment that may contribute to the pathogenesis of autoimmune myocarditis. TNF-α, IL-1β, IL-2, IFN-γ, IL-4 and IL-10 are cytokines that have been detected in culture supernatants of splenocytes derived from mice at the peak of myocardial disease. Their detected levels were higher in mice that developed myocarditis than mice immunized but not developing disease. Furthermore, it has been previously suggested that TNF-α contributes to myocarditis pathogenesis by either causing direct injury of cardiomyocytes or, together with IL1b, triggering the production of nitric oxide. The expression of E-selectin, VCAM-1 and ICAM-1 has also been found to be markedly increased in inflamed hearts during induced disease. The increased expression of these adhesion molecules on the vascular endothelium may contribute to the extravasation and accumulation of inflammatory cells observed in the myocardium (Wang et al. 1999; Blyszczuk et al. 2008). There are limitations of using experimental induced autoimmune myocarditis as a model to separate the mechanisms of biphasic (acute infection and chronic) viral disease, where the model relies too heavily on the inoculation of a single antigen. There are undoubtedly other key immune players that contribute to the onset of autoimmune disease following virus infection with the innate immune system being one of the major contributors having a dual role: controlling and perpetuating disease. Despite the fact that the experimental induced autoimmune myocarditis models are rather artificial, they offer the advantage of studying disease pathogenesis and autoimmune mechanisms *in vivo* in the absence of an infective agent. Immunization with myosin peptide-loaded activated dendritic cells offers a useful tool to dissect the role of antigen-presenting cells (APCs) and effector cells while studying disease mechanisms. From the experimental induced autoimmune myocarditis model we can not only learn about autoimmune mechanisms contributing to disease development, but we can study the pathophysiology of inflammatory heart disease and design novel immunomodulating treatment strategies. In addition, the experimental induced autoimmune myocarditis model might offer a potential tool to improve the diagnostic accuracy of currently available and future imaging technologies (Blyszczuk et al. 2008). Work from our lab using the experimental induced autoimmune myocarditis model has helped focus our attention to the role of innate immunity in response to an infectious agent

(Horwitz et al. 2005).

Experimental induced autoimmune myocarditis is mediated by a CD4+ T cell immune response. Homing of cardiac myosin-specific CD4+ T cells into the myocardium is the first pathologic event observed with experimental induced autoimmune myocarditis. Subsequently, neurohumoral factors such as cytokines and chemokines are released in the myocardium. This then recruits various bystander inflammatory cells to cross vascular endothelial cell walls and enter the myocardium. In effect, blocking the recruitment of inflammatory mediators to the myocardium may present as a critical target for myocarditis therapy (Tanaka et al. 2011). Experimental induced autoimmune myocarditis development is also typically associated with elevated titers of heart-specific autoantibodies, though in rats and susceptible mouse strains the response depends on the induction and expansion of heart specific, autoreactive CD4+ T cells. In BALB/c mice, CD4+ T cells belong to a specific subset of IL-17 producing helper cells, i.e. Th17 cells (Chang et al. 2008). With experimental induced autoimmune myocarditis, heart-infiltrating alpha-myosin-specific CD4+ Th17 cells are pathogenic and cause ongoing inflammation in the myocardium (Blyszczuk et al. 2008). Notably, autoimmune myocarditis induced by myosin can be reiterated in mice adoptively transferred with pathogenic CD4+ T lymphocytes (Sukumaran et al. 2011). In addition to T cells, passive administration of antimyosin monoclonal antibodies induces myocarditis in DBA/2 but not in BALB/c mice. DBA/2 mice are susceptible to passively induced disease due to the presence of myosin or a myosin like protein in their extracellular matrix. In addition to antibodies and T cells contributing to the pathogenesis of inflammatory myocardial lesions (Leuschner et al. 2009), injection of activated bone marrow (BM)-derived dendritic cells loaded with heart-specific self peptide can instigate disease pathogenesis (Kania et al. 2009).

Autoimmune myocarditis can be induced experimentally by several means as is the case for protection from induced disease. Amelioration of disease has been demonstrated with oral administration of specific antigens in several autoimmune models (Gonnella et al. 2009). Protection from experimental induced autoimmune myocarditis has also been observed with many studies from Godsel et al (Godsel et al. 2001). They successfully administered syngeneic splenocytes covalently coupled with ethylene carbodiimide (ECDI) by intravenous injections to prevent and treat a number of autoimmune diseases in animal models. Initial application of this approach in humans has been fortunately successful. Godsel et al essentially demonstrate in animal models that coupled-cell tolerance is an effective approach for the prevention of myocarditis and may present as a useful antigenspecific immunotherapy for treating myocarditis in humans (Godsel et al. 2001). Antigenspecific peripheral tolerance induction also represents a powerful tool for dissecting the mechanisms involved in cardiac autoimmunity. Although myosin-specific tolerization is commonly used to prevent experimental induced autoimmune myocarditis, it is also important to note that tissue homogenates are equally useful. This approach has yet to be tested in other models of myocarditis including coxsackievirus myocarditis (Godsel et al. 2001). Horwitz et al's investigation in to cardiac myosin tolerance and protection from experimental induced autoimmune myocarditis conflicted with results presented in the Godsel et al studies (Horwitz et al. 2005). Since cardiac myosin is a major autoantigen in virus-induced myocarditis the question was asked whether inhibition of this autoantibody response to cardiac myosin could prevent destructive autoimmunity. In NOD mice, cardiac myosin-specific antibodies develop following both coxsackievirus B3 infection and experimental induced autoimmune myocarditis induction. To ask whether the induction of peripheral tolerance to a single self-antigen could be used to prevent coxsackieviral-induced

Experimental induced autoimmune myocarditis is mediated by a CD4+ T cell immune response. Homing of cardiac myosin-specific CD4+ T cells into the myocardium is the first pathologic event observed with experimental induced autoimmune myocarditis. Subsequently, neurohumoral factors such as cytokines and chemokines are released in the myocardium. This then recruits various bystander inflammatory cells to cross vascular endothelial cell walls and enter the myocardium. In effect, blocking the recruitment of inflammatory mediators to the myocardium may present as a critical target for myocarditis therapy (Tanaka et al. 2011). Experimental induced autoimmune myocarditis development is also typically associated with elevated titers of heart-specific autoantibodies, though in rats and susceptible mouse strains the response depends on the induction and expansion of heart specific, autoreactive CD4+ T cells. In BALB/c mice, CD4+ T cells belong to a specific subset of IL-17 producing helper cells, i.e. Th17 cells (Chang et al. 2008). With experimental induced autoimmune myocarditis, heart-infiltrating alpha-myosin-specific CD4+ Th17 cells are pathogenic and cause ongoing inflammation in the myocardium (Blyszczuk et al. 2008). Notably, autoimmune myocarditis induced by myosin can be reiterated in mice adoptively transferred with pathogenic CD4+ T lymphocytes (Sukumaran et al. 2011). In addition to T cells, passive administration of antimyosin monoclonal antibodies induces myocarditis in DBA/2 but not in BALB/c mice. DBA/2 mice are susceptible to passively induced disease due to the presence of myosin or a myosin like protein in their extracellular matrix. In addition to antibodies and T cells contributing to the pathogenesis of inflammatory myocardial lesions (Leuschner et al. 2009), injection of activated bone marrow (BM)-derived dendritic cells loaded with heart-specific self peptide can instigate disease pathogenesis

Autoimmune myocarditis can be induced experimentally by several means as is the case for protection from induced disease. Amelioration of disease has been demonstrated with oral administration of specific antigens in several autoimmune models (Gonnella et al. 2009). Protection from experimental induced autoimmune myocarditis has also been observed with many studies from Godsel et al (Godsel et al. 2001). They successfully administered syngeneic splenocytes covalently coupled with ethylene carbodiimide (ECDI) by intravenous injections to prevent and treat a number of autoimmune diseases in animal models. Initial application of this approach in humans has been fortunately successful. Godsel et al essentially demonstrate in animal models that coupled-cell tolerance is an effective approach for the prevention of myocarditis and may present as a useful antigenspecific immunotherapy for treating myocarditis in humans (Godsel et al. 2001). Antigenspecific peripheral tolerance induction also represents a powerful tool for dissecting the mechanisms involved in cardiac autoimmunity. Although myosin-specific tolerization is commonly used to prevent experimental induced autoimmune myocarditis, it is also important to note that tissue homogenates are equally useful. This approach has yet to be tested in other models of myocarditis including coxsackievirus myocarditis (Godsel et al. 2001). Horwitz et al's investigation in to cardiac myosin tolerance and protection from experimental induced autoimmune myocarditis conflicted with results presented in the Godsel et al studies (Horwitz et al. 2005). Since cardiac myosin is a major autoantigen in virus-induced myocarditis the question was asked whether inhibition of this autoantibody response to cardiac myosin could prevent destructive autoimmunity. In NOD mice, cardiac myosin-specific antibodies develop following both coxsackievirus B3 infection and experimental induced autoimmune myocarditis induction. To ask whether the induction of peripheral tolerance to a single self-antigen could be used to prevent coxsackieviral-induced

(Kania et al. 2009).

autoimmune myocarditis, a disease likely directed at multiple self-antigens, NOD mice were tolerized to cardiac myosin using a covalently coupled antigen approach and subsequently challenged with coxsackievirus B3. Cardiac myosin tolerance to effectively prevent coxsackievirus B3-mediated autoimmune myocarditis was not observed, possibly reprimanding cardiac myosin as a single autoantigen player in viral-mediated disease (Horwitz et al. 2005).

Another possible player in viral-mediated disease as demonstrated with experimental induced autoimmune myocarditis may be multinucleated giant cells. Wang et al showed that the cardiac lesions in experimental induced autoimmune myocarditis are histologically similar to human myocarditis, with myocyte swelling, necrosis, and fibrosis that are accompanied by mononuclear cell infiltration consisting of granulocytes, macrophages, CD4+, CD8+ T cells, B cells and multinucleated giant cells (Wang et al. 1999; Blyszczuk et al. 2008; Daniels et al. 2008). Also, the histological features observed in myocarditis induced by cardiac myosin are very similar to the description of active giant-cell myocarditis in humans (Wang et al. 1999; Blyszczuk et al. 2008). Therefore, it has been inferred that macrophagederived multinucleated giant cells, together with other inflammatory cells, are important mediators of myocyte destruction. The presence of a heterogeneous population of cellular components in the cardiac infiltrate also implies the existence of a complex, cytokine-rich microenvironment that may contribute to the pathogenesis of autoimmune myocarditis. TNF-α, IL-1β, IL-2, IFN-γ, IL-4 and IL-10 are cytokines that have been detected in culture supernatants of splenocytes derived from mice at the peak of myocardial disease. Their detected levels were higher in mice that developed myocarditis than mice immunized but not developing disease. Furthermore, it has been previously suggested that TNF-α contributes to myocarditis pathogenesis by either causing direct injury of cardiomyocytes or, together with IL1b, triggering the production of nitric oxide. The expression of E-selectin, VCAM-1 and ICAM-1 has also been found to be markedly increased in inflamed hearts during induced disease. The increased expression of these adhesion molecules on the vascular endothelium may contribute to the extravasation and accumulation of inflammatory cells observed in the myocardium (Wang et al. 1999; Blyszczuk et al. 2008).

There are limitations of using experimental induced autoimmune myocarditis as a model to separate the mechanisms of biphasic (acute infection and chronic) viral disease, where the model relies too heavily on the inoculation of a single antigen. There are undoubtedly other key immune players that contribute to the onset of autoimmune disease following virus infection with the innate immune system being one of the major contributors having a dual role: controlling and perpetuating disease. Despite the fact that the experimental induced autoimmune myocarditis models are rather artificial, they offer the advantage of studying disease pathogenesis and autoimmune mechanisms *in vivo* in the absence of an infective agent. Immunization with myosin peptide-loaded activated dendritic cells offers a useful tool to dissect the role of antigen-presenting cells (APCs) and effector cells while studying disease mechanisms. From the experimental induced autoimmune myocarditis model we can not only learn about autoimmune mechanisms contributing to disease development, but we can study the pathophysiology of inflammatory heart disease and design novel immunomodulating treatment strategies. In addition, the experimental induced autoimmune myocarditis model might offer a potential tool to improve the diagnostic accuracy of currently available and future imaging technologies (Blyszczuk et al. 2008).

Work from our lab using the experimental induced autoimmune myocarditis model has helped focus our attention to the role of innate immunity in response to an infectious agent

The Key Players of Coxsackievirus-Induced Myocarditis 253

endosomes, detects single stranded RNA, such as the coxsackievirus B3 genome and

Signalling through another critical receptor, Toll-like receptor 4 (TLR4) also leads to the expression of proinflammatory cytokines, but has been implicated as a cardiomyopathy etiological factor. Satoh et al have suggested that myocardial expression of TLR4 is linked to coxsackievirus B3 replication in human cardiomyopathy and that TLR4 may be directly involved in the pathogenesis of disease (Satoh et al. 2004). Viral proteins have actually been found to co-localize with TLR4 in infected cardiac tissue. In coxsackievirus B infected mice, TLR4 deficiency reduces viral pathogenesis and the production of several cytokines

Another major player in host defense is the critical adaptor protein for TLR signaling myeloid differentiation primary response gene (MyD88). MyD88 signaling has been associated with several aspects of the pathogenesis of chronic autoimmune myocarditis. MyD88 activates self-antigen presenting cells and promotes autoreactive CD4+ T-cell expansion in experimental induced autoimmune myocarditis. To determine the role of MyD88 in the progression of acute myocarditis to an end-stage heart failure, Blyszczuk et al used alpha-myosin heavy chain peptide (MyHC-alpha)-loaded activated dendritic cells (Blyszczuk et al. 2008). They induced myocarditis in wild-type and MyD88 knock out mice and observed comparable heart-infiltrating cell subsets and CD4+ T-cell responses. Injection of complete Freund's adjuvant or MyHC-alpha/complete Freund's adjuvant into diseased mice caused cardiac fibrosis, ventricular dilation, and disrupted heart function in wild-type but not MyD88 knock out mice (Pasare et al. 2003; Marty et al. 2006; Blyszczuk et al. 2008). The protection of MyD88 knock out mice from the induction of experimental induced autoimmune myocarditis is likely from the impairment of other key players of autoimmunity such as antigen presenting cells. The role of MyD88 in cardiac fibrosis has been demonstrated with chimeric mice, where the origin of fibroblasts that replace inflammatory infiltrates was determined to be from the bone marrow. MyD88 has thus been suggested to be critical for the development of cardiac fibrosis during progression to heart failure (Pasare et al. 2003; Marty et al. 2006). Fuse et al observed elevated MyD88 cardiac protein levels in the hearts of wild-type mice after exposure to coxsackievirus B3 and MyD88 knock out mice have a greater survival rate (86%) compared to wild type mice (35%) after coxsackievirus B3 exposure (Fuse et al. 2005). MyD88 is implicated not only in cardiac inflammation and mediating cytokine production, but is also associated with skewing the Th1/Th2 cytokine balance, increasing the expression of coxsackie-adenoviral receptor important for virus entry and viral titers after coxsackievirus B3 incidence. In the absence of MyD88, protection from virus infection and disease is observed and is suggested to be associated with IRF-3 and IFN-β activation (Fuse et al. 2005). From the above mentioned MyD88 work, it is fair to infer that MyD88 could be a useful target for preventative heartspecific autoimmunity and cardiomyopathy treatments (Marty et al. 2006). TLR signalling may be a major contributor to the initiation and progression of autoimmune myocarditis though there are many additional players such as the cells that express viral and self antigen

instigates inflammation (Triantafilou et al. 2005; Zhang et al. 2009).

including IL-1β and IL-18 (Pasare et al. 2003; Pasare et al. 2004).

sensors (antigen presenting cells) that remain poorly understood.

Following viral infection, a cellular immune response is needed to completely clear the virus. However these same cells can drive chronic inflammation and autoimmune responses. Antigen presenting cells and other cell types critical in activating the cellular

**4.2 Antigen presenting cells (APCs)** 

and as a driving force in the development of autoimmune disease. The innate immune system includes many key players like pathogen recognition receptors that recognize highly conserved pathogen-associated molecular patterns on microbial invaders. These receptors include the Toll-like receptors and expression of these receptors on antigen presenting cells such as macrophages and dendritic cells determine not only innate immunity but the subsequent adaptive immune response.
