**4. Key players in immunity**

#### **4.1 Pathogen recognition receptors**

Cardiotropic viruses can be cytopathic, killing off host cells, yet their viral RNA is detected and tends to persist in cardiac muscle. Viral persistence in the myocardium can then lead to chronic inflammatory cardiomyopathy. Pathogens such as viruses are recognized by Tolllike receptors (TLRs) and other pattern recognition receptors of the innate immune response. The recognition of a pathogenic insult releases proinflammatory cytokines that serve as protectors from infection and perpetrators of chronic inflammatory disease (Lane et al. 1993; Kawai et al. 2006). Activation of Toll-like receptors by pathogen associated molecular patterns and the subsequent production of proinflammatory cytokines can lead to protection as well as the exacerbation of an autoimmune response.

Pathogen-associated molecular patterns from viruses like double-stranded RNA are sensed by Toll-like receptor 3 (TLR3) (Schnare et al. 2001; Pasare et al. 2003). Infections with coxsackievirus B in TLR3 knock out mice have demonstrated an important role for TLR3 in host defense. The innate antiviral response is mediated, at least in part, by nucleic acidsensing receptors such as TLR3, retinoic acid inducible gene I (RIG-I), and melanoma differentiation-associated protein-5 (MDA-5). The activation of RIG-I/MDA5 receptor pathways is thought to evoke type I IFN responses. TLR3, which recognizes double stranded RNA, is critical in the antiviral immune response against coxsackievirus B3. TLR3 deficient mice are highly susceptible to coxsackievirus B3, where impaired antiviral responses and acute myocarditis ensue. Increased disease in TLR knock out mice is associated with decreased production of IL-12p40, IFNγ and IL-1β post infection. Mice deficient in the TLR3 adaptor protein, Trif, have a similar disease course as TLR3 knock out mice suggesting that the TLR3-Trif pathway is also important in the host response to coxsackievirus B3 infection. Research from Negishi et al reveals a critical cooperation between the RIG-I/MDA5-type I IFN and TLR3-type II IFN signaling axes for efficient innate antiviral immune responses (Negishi et al. 2008). Importantly, a rare TLR3 variant has been identified in patients diagnosed with enteroviral myocarditis (Gorbea et al. 2010). These patients also held a greatly increased incidence of a common polymorphism. Gorbea et al also demonstrated that induction of the TLR3 variant or the TLR3 possessing the common polymorphism with synthetic double stranded RNA hindered proper TLR3 mediated signaling. Also, with coxsackievirus B3 infected cell lines, mutated TLR3 impaired type I IFN signalling and production and failed to control viral replication (Gorbea et al. 2010). The Gorbea et al study thus suggests that individuals who possess these particular TLR3 variants may have an ineffective innate anti-enteroviral response that fails to clear the virus and in turn, elevates the associative risk for cardiac disease. Interestingly, human cardiac myosin pathogenic epitopes can directly stimulate other human Toll-like receptors such as Toll-like receptors 2 and 8 (TLR2, TLR8). Stimulation of these receptors allows for the production of proinflammatory cytokines from human monocytes. TLR8, found within

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

Cardiotropic viruses can be cytopathic, killing off host cells, yet their viral RNA is detected and tends to persist in cardiac muscle. Viral persistence in the myocardium can then lead to chronic inflammatory cardiomyopathy. Pathogens such as viruses are recognized by Tolllike receptors (TLRs) and other pattern recognition receptors of the innate immune response. The recognition of a pathogenic insult releases proinflammatory cytokines that serve as protectors from infection and perpetrators of chronic inflammatory disease (Lane et al. 1993; Kawai et al. 2006). Activation of Toll-like receptors by pathogen associated molecular patterns and the subsequent production of proinflammatory cytokines can lead to

Pathogen-associated molecular patterns from viruses like double-stranded RNA are sensed by Toll-like receptor 3 (TLR3) (Schnare et al. 2001; Pasare et al. 2003). Infections with coxsackievirus B in TLR3 knock out mice have demonstrated an important role for TLR3 in host defense. The innate antiviral response is mediated, at least in part, by nucleic acidsensing receptors such as TLR3, retinoic acid inducible gene I (RIG-I), and melanoma differentiation-associated protein-5 (MDA-5). The activation of RIG-I/MDA5 receptor pathways is thought to evoke type I IFN responses. TLR3, which recognizes double stranded RNA, is critical in the antiviral immune response against coxsackievirus B3. TLR3 deficient mice are highly susceptible to coxsackievirus B3, where impaired antiviral responses and acute myocarditis ensue. Increased disease in TLR knock out mice is associated with decreased production of IL-12p40, IFNγ and IL-1β post infection. Mice deficient in the TLR3 adaptor protein, Trif, have a similar disease course as TLR3 knock out mice suggesting that the TLR3-Trif pathway is also important in the host response to coxsackievirus B3 infection. Research from Negishi et al reveals a critical cooperation between the RIG-I/MDA5-type I IFN and TLR3-type II IFN signaling axes for efficient innate antiviral immune responses (Negishi et al. 2008). Importantly, a rare TLR3 variant has been identified in patients diagnosed with enteroviral myocarditis (Gorbea et al. 2010). These patients also held a greatly increased incidence of a common polymorphism. Gorbea et al also demonstrated that induction of the TLR3 variant or the TLR3 possessing the common polymorphism with synthetic double stranded RNA hindered proper TLR3 mediated signaling. Also, with coxsackievirus B3 infected cell lines, mutated TLR3 impaired type I IFN signalling and production and failed to control viral replication (Gorbea et al. 2010). The Gorbea et al study thus suggests that individuals who possess these particular TLR3 variants may have an ineffective innate anti-enteroviral response that fails to clear the virus and in turn, elevates the associative risk for cardiac disease. Interestingly, human cardiac myosin pathogenic epitopes can directly stimulate other human Toll-like receptors such as Toll-like receptors 2 and 8 (TLR2, TLR8). Stimulation of these receptors allows for the production of proinflammatory cytokines from human monocytes. TLR8, found within

protection as well as the exacerbation of an autoimmune response.

subsequent adaptive immune response.

**4.1 Pathogen recognition receptors** 

**4. Key players in immunity** 

endosomes, detects single stranded RNA, such as the coxsackievirus B3 genome and instigates inflammation (Triantafilou et al. 2005; Zhang et al. 2009).

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

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 sensors (antigen presenting cells) that remain poorly understood.

#### **4.2 Antigen presenting cells (APCs)**

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

The Key Players of Coxsackievirus-Induced Myocarditis 255

with cardioprotective properties. In the hearts of healthy wild type mice, tissue-resident dendritic cells take up and present endogenous heart-specific peptides (Eriksson et al. 2003). Activated and self-antigen loaded dendritic cells induce myocarditis and heart failure in genetically susceptible mice. The mechanism by which dendritic cells instigate damage to the myocardium is likely a combined effect from tissue damage and innate immunity activation that causes dendritic cells to activate autoreactive T cells and target the myocardium (Marty et al. 2006). This proposed mechanism was supported by Eriksson et al who have shown that injection of dendritic cells loaded with cardiac myosin peptide induces CD4+ T-cell-mediated autoimmune myocarditis (Eriksson et al. 2003). Interestingly, the dendritic cell-induced autoimmunity observed by Eriksson et al resulted only with TLR and CD40 stimulation. They demonstrated how TLR signalling following the onset and resolution of acute myocarditis instigates the reoccurrence of inflammatory infiltrates in the heart and the onset of autoimmunity. TLR signalling activation was also important for myocarditis induction in mice injected with damaged, immune stimulating cardiomyocytes (Eriksson et al. 2003). These few studies provide an insight into the possible role of dendritic cells in the induction of myocarditis and they offer an alternative, complete Freund's adjuvant-free method of inducing experimental induced

The work done by Eriksson et al suggests targetting TLR signalling pathways may be needed as a therapeutic avenue to protect from heart-specific autoimmunity. In a scenario where microbial infections are acting concurrently with myocardial damage, such as with coxsackievirus B3 infection and myocarditis onset, self peptide–loaded dendritic cells might respond to the various pathogen associated molecular patterns in the environment that stimulate different TLRs and induce tolerance rather than act in antigenic mimicry. The end result may not be antigenic mimicry to instigate autoimmunity, but downregulation of autoreactive T cells and induction of tolerance (Eriksson et al. 2003; Blyszczuk et al. 2008). With this in mind, innate activation pathways such as TLR signaling, may be attractive

Macrophages, another type of antigen presenting cell, play a critical role in the immune response to coxsackievirus B3 infection and have been implicated in the pathogenesis of coxsackievirus B3-induced autoimmune myocarditis. There are two groups of macrophages, type I or type II that are defined by their activation markers and cytokine production. Type II macrophages have been linked to the cardiac repair stage following acute myocarditis (Nahrendorf et al. 2007). The significance of macrophages in coxsackievirus B3 myocarditis was demonstrated in previous work by Richer et al and Horwitz et al, where a transgenic TGF-β mouse model demonstrated a protective role for TGF-β against autoimmune disease. This protection coincided with a reduction in macrophage maturation suggesting the important involvement of macrophage inflammatory properties (Horwitz et al. 2006; Richer et al. 2006). It has been suggested therefore, that a balance between the inflammatory macrophages that are necessary for defense against viruses and the macrophages necessary for the resolution of an immune response and tissue healing is critical for an appropriate antiviral immune response that avoids autoimmunity (Heath et al. 2004). Interestingly, macrophage phenotype can differ between male and female mice with coxsackievirus B3 induced myocarditis. Since coxsackievirus B3 infection induces severe myocarditis only in male mice it is possible that myocardial infiltrating macrophages detected in female mice will have a distinct functional phenotype that contributes to their protection from coxsackievirus B3-induced myocarditis. Li et al observed myocardial infiltrating

autoimmune myocarditis (Afanasyeva et al. 2004).

targets for autoimmune myocarditis therapy.

response to viral infection such as CD4+ T cells, CD8+ T cells, γδ T cells, B cells, macrophages, mast cells, neutrophils, NK cells and DC cells are all detected in the hearts of mice post coxsackievirus B3 infection and with experimental induced autoimmune myocarditis induction (Afanasyeva et al. 2004; Cooper 2009; Kemball et al. 2010).

Antigen presenting cells play a pivotal role in the stimulation of acquired immunity and can be manipulated by cytokines and environmental factors. The manipulation of antigen presenting cells modulates the T cell response and results in changes in tolerance to specific antigens. Antigen presenting cells influence lymphocyte responses by 1) promoting helper T-cell 1 (Th1), Th2 or Th17 responses, 2) inducing peripheral tolerance, and 3) activating regulatory T cells (Chatenoud et al. 2005; Ait-Oufella et al. 2006; Blyszczuk et al. 2008). From autoimmunity studies it has been curiously determined that regulatory T cells and Th17 cells have opposing functions during autoimmunity (Langrish et al. 2005). Th17 cells are an important pro-inflammatory T cell lineage during heightened tissue inflammation and autoimmunity, whereas regulatory T cells cells function to suppress immune responses (Richer et al. 2008; Korn et al. 2009; Marchant et al. 2010; Wing et al. 2010; Zou et al. 2010). Antigen presenting cells trigger changes in regulatory T cells and other T cell populations, which alters disease outcome and may be a promising therapeutic avenue to further investigate in viral-induced autoimmune diseases such as viral myocarditis.

Damage to the myocardium and the onset of coxsackievirus B3-induced acute myocarditis in mice is attributable to many immune factors including activated antigen-specific T cell activity. Two signals are required for activating T cells: first through the T-cell receptor engaging with antigen loaded MHC on antigen presenting cells and next through costimulatory molecules on antigen presenting cells such as CD40 and B7. CD40L on T cells engages CD40 on antigen presenting cells activating them to secrete cytokines and express adhesion molecules. Signalling from both the T cell receptor and costimulatory molecules promotes the proliferation of the antigen-specific T cells and stimulates an anti-antigen immune response. Early work with CD40/CD40L revealed enhanced CD40 expression on cardiac myocytes of coxsackievirus B3-infected mice and reduced myocardial inflammation with anti-CD40L/B7-1 monoclonal antibody treatment (Seko et al. 1998). Increased expression of CD40 and the B7 family of costimulatory molecules has also been observed in myocardial tissue from patients with dilated cardiomyopathy and acute myocarditis (Seko et al. 1998). Recently, CD40-Ig treatment was used post-coxsackievirus B3 infection to block the interaction between CD40/CD40L in male Balb/c mice and notably, this treatment reduced inflammation and coxsackievirus B3 transcription. CD40-Ig treatment also skewed the Th1/Th2 response in favour of Th2 cytokines rather than Th1 (Bo et al. 2010). This work and studies with myocardial tissue from patients has important implications not only for the role of CD40, but also for potential therapeutic options that downregulate the inflammatory Th1 response in coxsackievirus B3-mediated acute myocarditis.

Dendritic cells are highly specialized antigen presenting cells that upon encountering a pathogen undergo maturation. This process involves antigen processing, upregulation of major histocompatibility class (MHC) class II molecules, induction of costimulatory activity and migration to lymph nodes, where they prime antigen-specific T cells. With their antigen processing capability, dendritic cells can trigger activation of autoreactive T cells (Eriksson et al. 2003). Dendritic cells may also be involved in both host defense and maintenance of peripheral tolerance. Dendritic cells may also play an important role in autoimmune myocarditis (Marty et al. 2006). Dendritic cells from infected susceptible mice produce lower levels of cytokines and chemokines, particularly IP-10, a chemokine

response to viral infection such as CD4+ T cells, CD8+ T cells, γδ T cells, B cells, macrophages, mast cells, neutrophils, NK cells and DC cells are all detected in the hearts of mice post coxsackievirus B3 infection and with experimental induced autoimmune

Antigen presenting cells play a pivotal role in the stimulation of acquired immunity and can be manipulated by cytokines and environmental factors. The manipulation of antigen presenting cells modulates the T cell response and results in changes in tolerance to specific antigens. Antigen presenting cells influence lymphocyte responses by 1) promoting helper T-cell 1 (Th1), Th2 or Th17 responses, 2) inducing peripheral tolerance, and 3) activating regulatory T cells (Chatenoud et al. 2005; Ait-Oufella et al. 2006; Blyszczuk et al. 2008). From autoimmunity studies it has been curiously determined that regulatory T cells and Th17 cells have opposing functions during autoimmunity (Langrish et al. 2005). Th17 cells are an important pro-inflammatory T cell lineage during heightened tissue inflammation and autoimmunity, whereas regulatory T cells cells function to suppress immune responses (Richer et al. 2008; Korn et al. 2009; Marchant et al. 2010; Wing et al. 2010; Zou et al. 2010). Antigen presenting cells trigger changes in regulatory T cells and other T cell populations, which alters disease outcome and may be a promising therapeutic avenue to further

Damage to the myocardium and the onset of coxsackievirus B3-induced acute myocarditis in mice is attributable to many immune factors including activated antigen-specific T cell activity. Two signals are required for activating T cells: first through the T-cell receptor engaging with antigen loaded MHC on antigen presenting cells and next through costimulatory molecules on antigen presenting cells such as CD40 and B7. CD40L on T cells engages CD40 on antigen presenting cells activating them to secrete cytokines and express adhesion molecules. Signalling from both the T cell receptor and costimulatory molecules promotes the proliferation of the antigen-specific T cells and stimulates an anti-antigen immune response. Early work with CD40/CD40L revealed enhanced CD40 expression on cardiac myocytes of coxsackievirus B3-infected mice and reduced myocardial inflammation with anti-CD40L/B7-1 monoclonal antibody treatment (Seko et al. 1998). Increased expression of CD40 and the B7 family of costimulatory molecules has also been observed in myocardial tissue from patients with dilated cardiomyopathy and acute myocarditis (Seko et al. 1998). Recently, CD40-Ig treatment was used post-coxsackievirus B3 infection to block the interaction between CD40/CD40L in male Balb/c mice and notably, this treatment reduced inflammation and coxsackievirus B3 transcription. CD40-Ig treatment also skewed the Th1/Th2 response in favour of Th2 cytokines rather than Th1 (Bo et al. 2010). This work and studies with myocardial tissue from patients has important implications not only for the role of CD40, but also for potential therapeutic options that downregulate the inflammatory

Dendritic cells are highly specialized antigen presenting cells that upon encountering a pathogen undergo maturation. This process involves antigen processing, upregulation of major histocompatibility class (MHC) class II molecules, induction of costimulatory activity and migration to lymph nodes, where they prime antigen-specific T cells. With their antigen processing capability, dendritic cells can trigger activation of autoreactive T cells (Eriksson et al. 2003). Dendritic cells may also be involved in both host defense and maintenance of peripheral tolerance. Dendritic cells may also play an important role in autoimmune myocarditis (Marty et al. 2006). Dendritic cells from infected susceptible mice produce lower levels of cytokines and chemokines, particularly IP-10, a chemokine

myocarditis induction (Afanasyeva et al. 2004; Cooper 2009; Kemball et al. 2010).

investigate in viral-induced autoimmune diseases such as viral myocarditis.

Th1 response in coxsackievirus B3-mediated acute myocarditis.

with cardioprotective properties. In the hearts of healthy wild type mice, tissue-resident dendritic cells take up and present endogenous heart-specific peptides (Eriksson et al. 2003). Activated and self-antigen loaded dendritic cells induce myocarditis and heart failure in genetically susceptible mice. The mechanism by which dendritic cells instigate damage to the myocardium is likely a combined effect from tissue damage and innate immunity activation that causes dendritic cells to activate autoreactive T cells and target the myocardium (Marty et al. 2006). This proposed mechanism was supported by Eriksson et al who have shown that injection of dendritic cells loaded with cardiac myosin peptide induces CD4+ T-cell-mediated autoimmune myocarditis (Eriksson et al. 2003). Interestingly, the dendritic cell-induced autoimmunity observed by Eriksson et al resulted only with TLR and CD40 stimulation. They demonstrated how TLR signalling following the onset and resolution of acute myocarditis instigates the reoccurrence of inflammatory infiltrates in the heart and the onset of autoimmunity. TLR signalling activation was also important for myocarditis induction in mice injected with damaged, immune stimulating cardiomyocytes (Eriksson et al. 2003). These few studies provide an insight into the possible role of dendritic cells in the induction of myocarditis and they offer an alternative, complete Freund's adjuvant-free method of inducing experimental induced autoimmune myocarditis (Afanasyeva et al. 2004).

The work done by Eriksson et al suggests targetting TLR signalling pathways may be needed as a therapeutic avenue to protect from heart-specific autoimmunity. In a scenario where microbial infections are acting concurrently with myocardial damage, such as with coxsackievirus B3 infection and myocarditis onset, self peptide–loaded dendritic cells might respond to the various pathogen associated molecular patterns in the environment that stimulate different TLRs and induce tolerance rather than act in antigenic mimicry. The end result may not be antigenic mimicry to instigate autoimmunity, but downregulation of autoreactive T cells and induction of tolerance (Eriksson et al. 2003; Blyszczuk et al. 2008). With this in mind, innate activation pathways such as TLR signaling, may be attractive targets for autoimmune myocarditis therapy.

Macrophages, another type of antigen presenting cell, play a critical role in the immune response to coxsackievirus B3 infection and have been implicated in the pathogenesis of coxsackievirus B3-induced autoimmune myocarditis. There are two groups of macrophages, type I or type II that are defined by their activation markers and cytokine production. Type II macrophages have been linked to the cardiac repair stage following acute myocarditis (Nahrendorf et al. 2007). The significance of macrophages in coxsackievirus B3 myocarditis was demonstrated in previous work by Richer et al and Horwitz et al, where a transgenic TGF-β mouse model demonstrated a protective role for TGF-β against autoimmune disease. This protection coincided with a reduction in macrophage maturation suggesting the important involvement of macrophage inflammatory properties (Horwitz et al. 2006; Richer et al. 2006). It has been suggested therefore, that a balance between the inflammatory macrophages that are necessary for defense against viruses and the macrophages necessary for the resolution of an immune response and tissue healing is critical for an appropriate antiviral immune response that avoids autoimmunity (Heath et al. 2004). Interestingly, macrophage phenotype can differ between male and female mice with coxsackievirus B3 induced myocarditis. Since coxsackievirus B3 infection induces severe myocarditis only in male mice it is possible that myocardial infiltrating macrophages detected in female mice will have a distinct functional phenotype that contributes to their protection from coxsackievirus B3-induced myocarditis. Li et al observed myocardial infiltrating

The Key Players of Coxsackievirus-Induced Myocarditis 257

decreasing viral replication and virus-mediated damage. Resolution of inflammation and progressive remodeling are associated with high levels of another cytokine transforming

TGF-β is a pleiotropic, immunomodulating cytokine that greatly contributes to myocardial repair and remodelling (Khan et al. 2006; Rubtsov et al. 2007). Cardiac fibroblasts are the predominant source of secreted TGF-β within the heart. Secretion of TGF-β drives differentiation of cardiac fibroblasts into their more active myofibroblast form (Lijnen et al. 2002). It is with this active connective tissue cell form and stimulation by TGF-β that copious amounts of collagen can be secreted (Petrov et al. 2002). Fibrillar collagen is a leading contributor to extensive fibrosis. With disproportionate amounts of secreted collagen, ventricles tighten, restricting proper diastolic function (Kania et al. 2009). TGF-β contributes to the secretion of collagen via the TGF-β-Smad pathway that promotes collagen gene activation and translation (Khan et al. 2006). TGF-β also enhances the production of adhesion molecules that in turn, promote the longevity of myofibroblasts (Vaughan et al. 2000). Macrophages also secrete TGF-β in the heart (Riemann et al. 1994). They colocalize with myofibroblasts in fibrotic heart tissue and act as either initiating or supplemental

Coxsackievirus B3 first targets and replicates in the pancreas before reaching the heart. To inhibit coxsackievirus B3 spread to the heart and initiation of chronic disease, Horwitz et al developed a transgenic TGF-β mouse model where TGF-β is overexpressed in the pancreas. The expression of TGF-β in the pancreatic beta cells recruited macrophages into the pancreas, reduced viral replication, and inhibited the onset of coxsackievirus B3-induced autoimmune myocarditis. This study also demonstrated that the protective effect was strictly attributed to TGF-β and not IL-4, which has been linked to both autoimmunity suppression and antigen-presenting cell activation (Horwitz et al. 2006). Later on, Richer et al demonstrated that LPS from *Salmonella minnesota* and signalling through TLR-4 was capable of bypassing the protective effect provided by TGF-β in coxsackievirus B3-mediated autoimmune myocarditis (Richer et al. 2006). The authors also showed that neither antibody isotype switching, the extent of viral replication, nor the expression of CD40 was modulated with LPS induced TLR-4 signalling, rather the circumventing effect was due to failed APC expression of CD40 and inherent TLR-4 signalling effects such as the production of pro-

Though over-expression of TGF-β in the pancreas can protect from coxsackievirus B3 induced autoimmune myocarditis, there is still evidence that increased levels of TGF-β1 in the heart enhance, rather than protect from chronic disease. Elevated levels of TGF-β have also been tied to dilated, ischemic and hypertrophic cardiomyopathies (Khan et al. 2006). As mentioned previously, TGF-β can enhance collagen secretion and thus promote extensive tissue fibrosis. A recent study examined the effect of astragaloside IV, a Chinese medical herb that has anti-myocardial injury and immunoregulatory properties, to inhibit myocardial fibrosis in Balb/c mice inoculated with coxsackievirus B3 (Chen et al. 2011). Interestingly, astragaloside IV exhibited a protective effect alike TGF-β against myocardial fibrosis and significantly ameliorated survival in coxsackievirus B3-infected mice that developed dilated cardiomyopathy. The authors also suggest that the protective role exerted by astragaloside IV is likely due to its ability to interfere with TGF-β-Smad signalling

Lipopolysaccharide injection at the time of coxsackievirus B3 infection helps overcome genetic resistance in susceptible mice. This investigation also identified interleukin (IL)-1 as

through the direct downregulation of Smad2/3 and Smad 4 (Chen et al. 2011).

growth factor-β (TGF-β) in the myocardium.

inflammatory cytokines (Richer et al. 2006).

sources of TGF-β1 (Hinglais et al. 1994; Kuwahara et al. 2004).

macrophages from coxsackievirus B3-infected male mice expressing high levels of classically activated macrophages (type I) markers, such as inducible nitric oxide synthase, IL-12, TNFα, and CD16/32, whereas macrophages from females had increased expression of arginase 1, IL-10, macrophage mannose receptor and macrophage galactose type C-type lectin that are typically associated with alternatively activated macrophages (type II) (Li et al. 2009). Li et al also demonstrated a distinct myocardial-derived cytokine signature that is sex-biased and contributes to differential macrophage polarization after coxsackievirus B3 infection. With adoptive transfer experiments using *ex vivo* programmed M1 macrophages, Li et al observed significantly increased myocarditis in both male and female mice. However, the transfer of M2 macrophages into susceptible male mice protected mice from myocardial inflammation (Li et al. 2009). This protection was postulated to be the result of a modulated local cytokine profile that contributed to the promotion of peripheral regulatory T cells differentiation. This work has helped our understanding of a possible mechanism that underlies the gender bias in coxsackievirus B3 myocarditis susceptibility. Developing therapeutic strategies that manipulate macrophage polarization may be a promising avenue for the treatment of inflammatory heart diseases.

#### **4.3 Cytokines and chemokines**

Cytokines and chemokines also play critical roles in the detection of pathogens and the response by the innate immune system. Unfortunately, they are also actively involved in the pathogenesis and progression of viral myocarditis. Transgenic mouse models expressing cytokines have facilitated our understanding of interplay between cytokines at the sites of infection and the development of autoimmune disease.

During experimental induced autoimmune myocarditis, it has been thought that CD4+ Th cells differentiate into IL-2- and IFNγ-producing Th1 and IL-4-, IL-10- and IL-13-producing Th2 cell subsets and that the balance in T-helper cytokines can influence susceptibility and outcome of myocarditis (Horwitz et al. 2000). In recent research, it has been revealed that IL-1, IL-6, and IL-23 promote the differentiation of a distinct CD4+ T cell population that produces IL-17 and develops independently of Th1 and Th2 lineages (Blyszczuk et al. 2008). This new population denoted Th17, plays an important role for various models of immunemediated tissue injury, including organ-specific autoimmunity diseases like myocarditis (Horwitz et al. 2000).

Work from our laboratory has established a critical link between IL-6 and disease severity. Work done by Poffenberger and colleagues has shown a significant increase in disease severity with the absence of IL-6 after coxsackievirus B3 infection in mice. An increase in inflammatory mediators associated with the progression of myocarditis such as TNF-α and MCP1 was observed in concordance with the increase in disease severity (Poffenberger et al. 2009). Without IL-6 to regulate the early immune response after infection, the early inflammatory response leads to increased chronic myocarditis severity as the disease progresses (Poffenberger et al. 2009).

An important factor affecting the immune response to the virus and viral clearance is the pro-inflammatory cytokine interferon-γ (IFNγ). Coxsackievirus B3 infection in mice deficient in IFNγ results in increased disease severity and increased viral replication in the heart (Eriksson et al. 2001; Fairweather et al. 2005). Expression of IFNγ in the pancreas can control viral replication as well as the virus-mediated damage in the heart and ensuing autoimmune disease (Horwitz et al. 2000). This cytokine also controls disease severity in an adjuvant induction disease model. In essence, IFNγ likely limits myocarditis pathology by

macrophages from coxsackievirus B3-infected male mice expressing high levels of classically activated macrophages (type I) markers, such as inducible nitric oxide synthase, IL-12, TNFα, and CD16/32, whereas macrophages from females had increased expression of arginase 1, IL-10, macrophage mannose receptor and macrophage galactose type C-type lectin that are typically associated with alternatively activated macrophages (type II) (Li et al. 2009). Li et al also demonstrated a distinct myocardial-derived cytokine signature that is sex-biased and contributes to differential macrophage polarization after coxsackievirus B3 infection. With adoptive transfer experiments using *ex vivo* programmed M1 macrophages, Li et al observed significantly increased myocarditis in both male and female mice. However, the transfer of M2 macrophages into susceptible male mice protected mice from myocardial inflammation (Li et al. 2009). This protection was postulated to be the result of a modulated local cytokine profile that contributed to the promotion of peripheral regulatory T cells differentiation. This work has helped our understanding of a possible mechanism that underlies the gender bias in coxsackievirus B3 myocarditis susceptibility. Developing therapeutic strategies that manipulate macrophage polarization may be a promising avenue

Cytokines and chemokines also play critical roles in the detection of pathogens and the response by the innate immune system. Unfortunately, they are also actively involved in the pathogenesis and progression of viral myocarditis. Transgenic mouse models expressing cytokines have facilitated our understanding of interplay between cytokines at the sites of

During experimental induced autoimmune myocarditis, it has been thought that CD4+ Th cells differentiate into IL-2- and IFNγ-producing Th1 and IL-4-, IL-10- and IL-13-producing Th2 cell subsets and that the balance in T-helper cytokines can influence susceptibility and outcome of myocarditis (Horwitz et al. 2000). In recent research, it has been revealed that IL-1, IL-6, and IL-23 promote the differentiation of a distinct CD4+ T cell population that produces IL-17 and develops independently of Th1 and Th2 lineages (Blyszczuk et al. 2008). This new population denoted Th17, plays an important role for various models of immunemediated tissue injury, including organ-specific autoimmunity diseases like myocarditis

Work from our laboratory has established a critical link between IL-6 and disease severity. Work done by Poffenberger and colleagues has shown a significant increase in disease severity with the absence of IL-6 after coxsackievirus B3 infection in mice. An increase in inflammatory mediators associated with the progression of myocarditis such as TNF-α and MCP1 was observed in concordance with the increase in disease severity (Poffenberger et al. 2009). Without IL-6 to regulate the early immune response after infection, the early inflammatory response leads to increased chronic myocarditis severity as the disease

An important factor affecting the immune response to the virus and viral clearance is the pro-inflammatory cytokine interferon-γ (IFNγ). Coxsackievirus B3 infection in mice deficient in IFNγ results in increased disease severity and increased viral replication in the heart (Eriksson et al. 2001; Fairweather et al. 2005). Expression of IFNγ in the pancreas can control viral replication as well as the virus-mediated damage in the heart and ensuing autoimmune disease (Horwitz et al. 2000). This cytokine also controls disease severity in an adjuvant induction disease model. In essence, IFNγ likely limits myocarditis pathology by

for the treatment of inflammatory heart diseases.

infection and the development of autoimmune disease.

**4.3 Cytokines and chemokines** 

(Horwitz et al. 2000).

progresses (Poffenberger et al. 2009).

decreasing viral replication and virus-mediated damage. Resolution of inflammation and progressive remodeling are associated with high levels of another cytokine transforming growth factor-β (TGF-β) in the myocardium.

TGF-β is a pleiotropic, immunomodulating cytokine that greatly contributes to myocardial repair and remodelling (Khan et al. 2006; Rubtsov et al. 2007). Cardiac fibroblasts are the predominant source of secreted TGF-β within the heart. Secretion of TGF-β drives differentiation of cardiac fibroblasts into their more active myofibroblast form (Lijnen et al. 2002). It is with this active connective tissue cell form and stimulation by TGF-β that copious amounts of collagen can be secreted (Petrov et al. 2002). Fibrillar collagen is a leading contributor to extensive fibrosis. With disproportionate amounts of secreted collagen, ventricles tighten, restricting proper diastolic function (Kania et al. 2009). TGF-β contributes to the secretion of collagen via the TGF-β-Smad pathway that promotes collagen gene activation and translation (Khan et al. 2006). TGF-β also enhances the production of adhesion molecules that in turn, promote the longevity of myofibroblasts (Vaughan et al. 2000). Macrophages also secrete TGF-β in the heart (Riemann et al. 1994). They colocalize with myofibroblasts in fibrotic heart tissue and act as either initiating or supplemental sources of TGF-β1 (Hinglais et al. 1994; Kuwahara et al. 2004).

Coxsackievirus B3 first targets and replicates in the pancreas before reaching the heart. To inhibit coxsackievirus B3 spread to the heart and initiation of chronic disease, Horwitz et al developed a transgenic TGF-β mouse model where TGF-β is overexpressed in the pancreas. The expression of TGF-β in the pancreatic beta cells recruited macrophages into the pancreas, reduced viral replication, and inhibited the onset of coxsackievirus B3-induced autoimmune myocarditis. This study also demonstrated that the protective effect was strictly attributed to TGF-β and not IL-4, which has been linked to both autoimmunity suppression and antigen-presenting cell activation (Horwitz et al. 2006). Later on, Richer et al demonstrated that LPS from *Salmonella minnesota* and signalling through TLR-4 was capable of bypassing the protective effect provided by TGF-β in coxsackievirus B3-mediated autoimmune myocarditis (Richer et al. 2006). The authors also showed that neither antibody isotype switching, the extent of viral replication, nor the expression of CD40 was modulated with LPS induced TLR-4 signalling, rather the circumventing effect was due to failed APC expression of CD40 and inherent TLR-4 signalling effects such as the production of proinflammatory cytokines (Richer et al. 2006).

Though over-expression of TGF-β in the pancreas can protect from coxsackievirus B3 induced autoimmune myocarditis, there is still evidence that increased levels of TGF-β1 in the heart enhance, rather than protect from chronic disease. Elevated levels of TGF-β have also been tied to dilated, ischemic and hypertrophic cardiomyopathies (Khan et al. 2006). As mentioned previously, TGF-β can enhance collagen secretion and thus promote extensive tissue fibrosis. A recent study examined the effect of astragaloside IV, a Chinese medical herb that has anti-myocardial injury and immunoregulatory properties, to inhibit myocardial fibrosis in Balb/c mice inoculated with coxsackievirus B3 (Chen et al. 2011). Interestingly, astragaloside IV exhibited a protective effect alike TGF-β against myocardial fibrosis and significantly ameliorated survival in coxsackievirus B3-infected mice that developed dilated cardiomyopathy. The authors also suggest that the protective role exerted by astragaloside IV is likely due to its ability to interfere with TGF-β-Smad signalling through the direct downregulation of Smad2/3 and Smad 4 (Chen et al. 2011).

Lipopolysaccharide injection at the time of coxsackievirus B3 infection helps overcome genetic resistance in susceptible mice. This investigation also identified interleukin (IL)-1 as

The Key Players of Coxsackievirus-Induced Myocarditis 259

IL-13 is another cytokine that can protect mice from both viral and adjuvant induced myocarditis. Cihakova et al demonstrated that IL-13 knock out BALB/c mice develop severe autoimmune myocarditis and their pathology is characterized by increased cardiac inflammation, increased total intracardiac CD45+ leukocytes, elevated anti-cardiac myosin autoantibodies, and increased cardiac fibrosis, with impaired cardiac function and heart failure. Hearts of IL-13 knock out mice showed elevated levels of the proinflammatory and profibrotic cytokines including IL-1β, IL-18, IFN-γ, TGF-β, and IL-4. CD4+ T cells were also highly increased in IL-13 knock out hearts. Splenic T cells from the knock out mice were greatly activated and with myosin stimulation, immensely proliferated. Regulatory T-cells harvested from spleens were also affected in knock out mice, where they showed a decrease in numbers compared to wild type mice. IL-13 knock out also reduced alternatively activated CD206(+) and CD204(+) macrophages and hightened levels of classically activated macrophages. Caspase-1 activation was increased, which then likely increased production of both IL-1β and IL-18. This study exemplified IL-13 as another important immunomodulating cytokine that protects against myocarditis by manipulating T cell and

Examining T cells subsets is another avenue investigated to determine pathogenic or protective factors in myocarditis development. Mice deficient in T-bet, a T-box transcription factor required for Th1 cell differentiation and IFN-γ production, develop severe autoimmune heart disease. T-bet can also regulate autoimmunity by controlling nonspecific CD8+ T cell bystander functions in the inflamed target organ such as the heart. CD4+ Th1 cells producing INFγ are protective in experimental induced autoimmune myocarditis. This protection is likely attributed to regulation of IL-17 production by Th17 cells. Th17 cells produce IL-17, a proinflammatory cytokine that activates T cells and other immune cells to produce a variety of cytokines, chemokines and cell adhesion molecules. Rangachari et al have shown that Th17 cells are involved in acute viral myocarditis and enhance humoral responses (Rangachari et al. 2006). The relationship between Th17 cells and coxsackievirus B3 replication still remained unclear so they infected BALB/c mice with the virus and observed increased viral replication, expression of splenic Th17 cells, serum IL-17, and cardiac IL-17 mRNA that were all accompanied by progressive cardiac injury. Interestingly, Th1 and CD8+ T cell expression was elevated and the neutralization of IL-17 further upregulated splenic Th1 and CD8+ T cell numbers and levels of cardiac IFN-γ mRNA. Cardiac pathology was improved after IL-17 neutralization and correlated with reduced viral replication and decreases in cardiac inflammatory cytokines IL-17, TNF-α, and IL-1β. This study implicates Th17 cells in contributing coxsackeivirus B3 replication in viral myocarditis, and implicates IL-17 as a target for regulating antiviral immune responses (Yuan et al. 2010). Th17 cells are activated by IL-23, a cytokine likely produced by activated macrophages and dendritic cells, through receptor interactions made of IL-12Rβ1 and IL-23 receptor (Langrish et al. 2005). IL-23 is a heterodimeric cytokine like IL-12 that is composed of a p19 and p40 subunit similar to IL-12. IL-23–mediated immune responses have a different gene expression pattern than IL-12–driven T cell responses and IL-23 does not promote the development of IFN-γ-producing Th1 cells as does IL-12. IL-23 is however, one of the many contributors to pathogenic CD4+ T cell expansion. With its anti-IFN-γ and Th1 cell activity, IL-23 helps establish and maintain organ-specific inflammatory autoimmune diseases such as myocarditis (Langrish et al. 2005). Understanding the molecular basis for the differential gene expression pattern observed with IL-23–dependent T cell populations and investigating IL-23's cellular mechanism of action in autoimmunity could provide additional therapeutic targets for the treatment of inflammatory autoimmune diseases.

macrophage populations (Cihakova et al. 2008).

the mediator responsible for causing the change in disease course. Injection of IL-1 alone overcomes the genetic resistance to induced myocarditis similarly to lipopolysaccharide treatment. The change in disease susceptibility is likely due to increased IL-1 production in the heart. Production of IL-1 begins during the acute stage of disease but persists into the chronic phase of disease. Interestingly, the levels of IL-1 in the heart correlate with the degree of fibrotic lesions during disease. Eriksson et al demonstrated that injection of an IL-1 receptor agonist prior to infection sufficiently decreases viral titres in the heart and reduced chances of mortality. They also showed that IL-1 receptor stimulation is required for efficient dendritic cell activation, the subsequent induction of autoreactive CD4+ T cells, and resulting autoimmune disease (Eriksson et al. 2003).

Another important immunomodulating cytokine that contributes to viral-induced myocarditis and controls macrophage activation is IL-10. IL-10 is produced in the myocardium during both the acute and chronic stages of virus-induced myocarditis. Chronic coxsackievirus B3-induced myocarditis features viral RNA persistence and chronic inflammation that is primarily mediated by macrophages and T cells therefore, cytokines like IL-10 that control these critical immune cells are important to investigate. IL-10 genedeficient mice have been used to confirm the regulatory role of IL-10 in the outcome of coxsackievirus B3 myocarditis. Mice deficient in IL-10 have uncontrolled nitric oxide synthase production, which likely contributes to their ongoing myocardial injury (Szalay et al. 2006). IL-10 in experimental induced autoimmune myocarditis mice hearts is mainly detected in non-cardiomyocytic non-inflammatory cells (ie. fibroblasts, smooth muscle cells, and endothelial cells) and IL-10-targeting cells. The IL-10-targetting cells, which express both IL-10 receptors 1 and 2, are mainly T cells expressing αβT cell antigen receptors (αβT cells) and CD11b+ cells such as macrophages, dendritic cells, and granulocytes. Several studies have demonstrated a therapeutic effect for IL-10 in autoimmune and inflammatory diseases. In myocarditis models, IL-10 has been shown to inhibit the secretion of proinflammatory cytokines such as TNF-α, IFN-γ, iNOS, IL-2 and IL-12 and has displayed major effects on immune cells (Horwitz et al. 2000). These few studies suggest that the role of IL-10 in disease development could be predominantly protective.

IL-12, another key cytokine, is comprised of p40 and p35 subunits. IL-12 signals through a heterodimeric receptor composed of two units, IL-12Rβ1 and IL-12Rβ2. p40 and IL-12Rβ1 deficient mice show less or no myocardial disease with adjuvant-induction and treatment of wild type mice with IL-12, exacerbates disease suggesting IL-12 as a driving force in disease development similar to the actions of IL-1β and TNFα (Eriksson et al. 2001; Afanasyeva et al. 2004). However, Fairweather et al have found that IL-12 deficiency does not prevent myocarditis, rather viral replication significantly increases, causing more myocardial tissue damage. A decrease in inflammatory infiltrates was also observed and corresponded to with reduced TNF-α and IFN-γ levels in the heart. IL-12 and IFNg positively regulate each other and type I inflammatory responses. Type I inflammatory responses are believed to be responsible for tissue damage in autoimmune diseases. Eriksson et al further investigated the role of the IL-12/IFN-γ (Th1) axis in the development of autoimmune myocarditis. They observed resistance to disease in IL-12p40-deficient mice that were bred on a susceptible background. In the absence of IL-12, they suggested that autospecific CD4+ T cells proliferated poorly and exerted Th2 cytokine responses. IFN-γ-deficient mice developed fatal autoimmune disease. Interestingly, blocking IL-4R signalling did not confer susceptibility to myocarditis in IL-12p40-deficient mice. This suggests that IL-12 triggers autoimmunity in a manner independent of the cytokines IFN-γ and IL-4 (Eriksson et al. 2001).

the mediator responsible for causing the change in disease course. Injection of IL-1 alone overcomes the genetic resistance to induced myocarditis similarly to lipopolysaccharide treatment. The change in disease susceptibility is likely due to increased IL-1 production in the heart. Production of IL-1 begins during the acute stage of disease but persists into the chronic phase of disease. Interestingly, the levels of IL-1 in the heart correlate with the degree of fibrotic lesions during disease. Eriksson et al demonstrated that injection of an IL-1 receptor agonist prior to infection sufficiently decreases viral titres in the heart and reduced chances of mortality. They also showed that IL-1 receptor stimulation is required for efficient dendritic cell activation, the subsequent induction of autoreactive CD4+ T cells, and

Another important immunomodulating cytokine that contributes to viral-induced myocarditis and controls macrophage activation is IL-10. IL-10 is produced in the myocardium during both the acute and chronic stages of virus-induced myocarditis. Chronic coxsackievirus B3-induced myocarditis features viral RNA persistence and chronic inflammation that is primarily mediated by macrophages and T cells therefore, cytokines like IL-10 that control these critical immune cells are important to investigate. IL-10 genedeficient mice have been used to confirm the regulatory role of IL-10 in the outcome of coxsackievirus B3 myocarditis. Mice deficient in IL-10 have uncontrolled nitric oxide synthase production, which likely contributes to their ongoing myocardial injury (Szalay et al. 2006). IL-10 in experimental induced autoimmune myocarditis mice hearts is mainly detected in non-cardiomyocytic non-inflammatory cells (ie. fibroblasts, smooth muscle cells, and endothelial cells) and IL-10-targeting cells. The IL-10-targetting cells, which express both IL-10 receptors 1 and 2, are mainly T cells expressing αβT cell antigen receptors (αβT cells) and CD11b+ cells such as macrophages, dendritic cells, and granulocytes. Several studies have demonstrated a therapeutic effect for IL-10 in autoimmune and inflammatory diseases. In myocarditis models, IL-10 has been shown to inhibit the secretion of proinflammatory cytokines such as TNF-α, IFN-γ, iNOS, IL-2 and IL-12 and has displayed major effects on immune cells (Horwitz et al. 2000). These few studies suggest that the role

IL-12, another key cytokine, is comprised of p40 and p35 subunits. IL-12 signals through a heterodimeric receptor composed of two units, IL-12Rβ1 and IL-12Rβ2. p40 and IL-12Rβ1 deficient mice show less or no myocardial disease with adjuvant-induction and treatment of wild type mice with IL-12, exacerbates disease suggesting IL-12 as a driving force in disease development similar to the actions of IL-1β and TNFα (Eriksson et al. 2001; Afanasyeva et al. 2004). However, Fairweather et al have found that IL-12 deficiency does not prevent myocarditis, rather viral replication significantly increases, causing more myocardial tissue damage. A decrease in inflammatory infiltrates was also observed and corresponded to with reduced TNF-α and IFN-γ levels in the heart. IL-12 and IFNg positively regulate each other and type I inflammatory responses. Type I inflammatory responses are believed to be responsible for tissue damage in autoimmune diseases. Eriksson et al further investigated the role of the IL-12/IFN-γ (Th1) axis in the development of autoimmune myocarditis. They observed resistance to disease in IL-12p40-deficient mice that were bred on a susceptible background. In the absence of IL-12, they suggested that autospecific CD4+ T cells proliferated poorly and exerted Th2 cytokine responses. IFN-γ-deficient mice developed fatal autoimmune disease. Interestingly, blocking IL-4R signalling did not confer susceptibility to myocarditis in IL-12p40-deficient mice. This suggests that IL-12 triggers autoimmunity in a manner

resulting autoimmune disease (Eriksson et al. 2003).

of IL-10 in disease development could be predominantly protective.

independent of the cytokines IFN-γ and IL-4 (Eriksson et al. 2001).

IL-13 is another cytokine that can protect mice from both viral and adjuvant induced myocarditis. Cihakova et al demonstrated that IL-13 knock out BALB/c mice develop severe autoimmune myocarditis and their pathology is characterized by increased cardiac inflammation, increased total intracardiac CD45+ leukocytes, elevated anti-cardiac myosin autoantibodies, and increased cardiac fibrosis, with impaired cardiac function and heart failure. Hearts of IL-13 knock out mice showed elevated levels of the proinflammatory and profibrotic cytokines including IL-1β, IL-18, IFN-γ, TGF-β, and IL-4. CD4+ T cells were also highly increased in IL-13 knock out hearts. Splenic T cells from the knock out mice were greatly activated and with myosin stimulation, immensely proliferated. Regulatory T-cells harvested from spleens were also affected in knock out mice, where they showed a decrease in numbers compared to wild type mice. IL-13 knock out also reduced alternatively activated CD206(+) and CD204(+) macrophages and hightened levels of classically activated macrophages. Caspase-1 activation was increased, which then likely increased production of both IL-1β and IL-18. This study exemplified IL-13 as another important immunomodulating cytokine that protects against myocarditis by manipulating T cell and macrophage populations (Cihakova et al. 2008).

Examining T cells subsets is another avenue investigated to determine pathogenic or protective factors in myocarditis development. Mice deficient in T-bet, a T-box transcription factor required for Th1 cell differentiation and IFN-γ production, develop severe autoimmune heart disease. T-bet can also regulate autoimmunity by controlling nonspecific CD8+ T cell bystander functions in the inflamed target organ such as the heart. CD4+ Th1 cells producing INFγ are protective in experimental induced autoimmune myocarditis. This protection is likely attributed to regulation of IL-17 production by Th17 cells. Th17 cells produce IL-17, a proinflammatory cytokine that activates T cells and other immune cells to produce a variety of cytokines, chemokines and cell adhesion molecules. Rangachari et al have shown that Th17 cells are involved in acute viral myocarditis and enhance humoral responses (Rangachari et al. 2006). The relationship between Th17 cells and coxsackievirus B3 replication still remained unclear so they infected BALB/c mice with the virus and observed increased viral replication, expression of splenic Th17 cells, serum IL-17, and cardiac IL-17 mRNA that were all accompanied by progressive cardiac injury. Interestingly, Th1 and CD8+ T cell expression was elevated and the neutralization of IL-17 further upregulated splenic Th1 and CD8+ T cell numbers and levels of cardiac IFN-γ mRNA. Cardiac pathology was improved after IL-17 neutralization and correlated with reduced viral replication and decreases in cardiac inflammatory cytokines IL-17, TNF-α, and IL-1β. This study implicates Th17 cells in contributing coxsackeivirus B3 replication in viral myocarditis, and implicates IL-17 as a target for regulating antiviral immune responses (Yuan et al. 2010). Th17 cells are activated by IL-23, a cytokine likely produced by activated macrophages and dendritic cells, through receptor interactions made of IL-12Rβ1 and IL-23 receptor (Langrish et al. 2005). IL-23 is a heterodimeric cytokine like IL-12 that is composed of a p19 and p40 subunit similar to IL-12. IL-23–mediated immune responses have a different gene expression pattern than IL-12–driven T cell responses and IL-23 does not promote the development of IFN-γ-producing Th1 cells as does IL-12. IL-23 is however, one of the many contributors to pathogenic CD4+ T cell expansion. With its anti-IFN-γ and Th1 cell activity, IL-23 helps establish and maintain organ-specific inflammatory autoimmune diseases such as myocarditis (Langrish et al. 2005). Understanding the molecular basis for the differential gene expression pattern observed with IL-23–dependent T cell populations and investigating IL-23's cellular mechanism of action in autoimmunity could provide additional therapeutic targets for the treatment of inflammatory autoimmune diseases.

The Key Players of Coxsackievirus-Induced Myocarditis 261

Recent research has demonstrated that coxsackievirus B3-infected Balb/c mice have increased levels of cardiac CXCL10 and this level fluctuates in a time and dose-dependent manner (Yue et al. 2011). The same research group treated coxsackievirus B3-infected mice with a CXCL10 mutant that lacks the critical chemo-attractant part. This mutant effectively blocked endogenous CXCL10 activity and protected coxsackievirus B3-infected mice from developing myocarditis (Yue et al. 2011). The CXCL10 mutant expressing mice had greater survival, less changes in body weight, less inflammation and necrosis in the heart. Cardiac Th1 cytokines IFN-γ, IL-12, TNF-α, were also found to be significantly reduced with CXCL10/CXCR3 signalling inhibition. This suggests that dampening the Th1 response to coxsackievirus B3 infection through blocking CXCL10 activity may present as an effective strategy to suppress immune inflammation and myocardial damage in coxsackievirus B3-

The production of immunomodulating mediators such as cytokines and chemokines affects not only antigen presenting cell and T cell populations acting at the site of injury, but greatly influences the outcome of disease. The expression of particular cytokines and chemokines after a pathogenic insult, such as coxsackievirus B3 infection, influences the balance of effector versus regulatory responses that ensue. Once the balance between effector versus regulatory responses tips in a particular direction, chronic autoimmune-type disease or the prevention of disease will materialize (Rouse et al. 2010; Wing et al. 2010) (Figure 3). It is therefore critical to bear in mind the possibility of tipping the immunity balance when

Fig. 3. Immunomodulation of the immune balance and the effects on disease outcome. With coxsackievirus B3 infection, populations of regulatory and effector cells are produced. It is with particular immunomodulating mediators such as cytokines interferon (IFN) and tumor necrosis factor (TNF) that enhanced production and proliferation of effector T cells (Teff) is

myocarditis disease. Pro-regulatory cytokines transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) are examples of immune modulators that promote the enhanced production of regulatory T cells (regulatory T cells) and the prevention of autoimmune

promoted and leads to the persistent destruction of the myocardium and chronic

manipulating key immune players in coxsackievirus-induced myocarditis.

mediated myocarditis (Yue et al. 2011).

myocarditis disease.

Experimental and preliminary human studies have demonstrated that TNF-α plays a crucial role in viral-induced myocarditis. Calabrese et al investigated the expression of TNF-α and both its receptors (TNFRI and TNFRII) in both viral and non viral myocarditis. Expression of TNF-α was significantly enhanced in viral myocarditis compared to non viral myocarditis. Importantly, cardiac myocytes express TNFα receptors TNFR1 (TNFRp55) and TNFR2 (TNFRp75), implicating the possible importance of enhance TNF-α in the myocardium. Histological analysis revealed that myocardial necrosis and cellular infiltration are more prominent in TNF-α-positive cases further supporting the notion that the expression of TNF-α significantly contributes to the pathogenesis of viral myocarditis and including the severity of cardiac dysfunction (Calabrese et al. 2004).

Chemokines act as chemotactic mediators in leukocyte trafficking to sites of infection (Groom et al. 2011). In addition to their chemo-attractant activity, chemokines can influence disease severity by modifying immune response strength and polarity. This immune modulating capability thus makes them attractive targets for viral myocarditis therapeutics. Chemokines apart of the CC chemokine family such as CCL2, CCL4, and CCL19 have been shown to mediate mononuclear cell migration to the heart in coxsackievirus B3-induced myocarditis (Chen et al. 2009). In fact, a recent gene therapy approach using a CCL2 mutant that lacked chemo-attractant activity in a Balb/c coxsackievirus B3-infection model impaired appropriate Th1 immune responses and significantly controlled myocardial disease (Yue et al. 2011). Blocking CCL2 with expression of the mutant did not assist in viral clearance. The potential therapeutic effect of blocking CCL2 lies in the CCL2 mutant's ability to weaken the pro-inflammatory Th1 immune response (Yue et al. 2011).

The CDC family of chemokines also contribute to myocarditis pathogenesis. CDC chemokines act on monoculear CXCR3 expressing cells and include such members as IFN-inducible protein 10 (IP10/CXCL10), monokine induced by IFN-γ (Mig/CXCL9), and IFN-inducible Tcell a chemoattractant (I-TAC/CXCL11) (Groom et al. 2011). CXCL10 and its interaction with its receptor CXCR3 have been implicated in many virus disease models. CXCL10 is a key contributor in the innate immune response to viral infection. By interacting with its receptor CXCR3, CXCL10 manipulates natural killer cell trafficking and their production of IFN-γ. Yuan et al recently found CXCL10 levels in the heart to be inversely related to viral titers after coxsackievirus B3 infection and a massive infiltration of CXCR3+, CD4+, and CD8+ cells (Yuan et al. 2009). The production of associated inflammatory cytokines followed with the infiltration of leukocytes though the anti-viral response was not effective in clearing the virus or ensuring survival in coxsackievirus B3-infected CXCL10 transgenic mice (Yuan et al. 2009). CXCL10 did assist viral clearance and protect myocytes from damage in the early stages of infection by robustly attracting NK cells and enhancing IFN-γ production to infection sites (Yuan et al. 2009). Though CXCL10 appears to improve cardiac pathology and reduce viral persistence during initial infection stages, the caveat with using this chemokine as a potential coxsackievirus B3-induced myocarditis therapy lies with its inherent actions as a Th1-type chemeokine. Th1 immune responses escalate detrimental cardiac fibrosis during the reclamation stage of disease so enhancing important contributors to this repair process may acceleration rather than prevent the development of chronic disease.

Determining the effect of CXCL10 on viral replication and recruitment of innate immune cells in other organs than the heart such as the liver and pancreas must still be pursued. As seen with another immunomodulatory factor, TGF-β, the immune responses in other organs than the heart can affect susceptibility to severe myocardial injury and the development of chronic disease.

Experimental and preliminary human studies have demonstrated that TNF-α plays a crucial role in viral-induced myocarditis. Calabrese et al investigated the expression of TNF-α and both its receptors (TNFRI and TNFRII) in both viral and non viral myocarditis. Expression of TNF-α was significantly enhanced in viral myocarditis compared to non viral myocarditis. Importantly, cardiac myocytes express TNFα receptors TNFR1 (TNFRp55) and TNFR2 (TNFRp75), implicating the possible importance of enhance TNF-α in the myocardium. Histological analysis revealed that myocardial necrosis and cellular infiltration are more prominent in TNF-α-positive cases further supporting the notion that the expression of TNF-α significantly contributes to the pathogenesis of viral myocarditis

Chemokines act as chemotactic mediators in leukocyte trafficking to sites of infection (Groom et al. 2011). In addition to their chemo-attractant activity, chemokines can influence disease severity by modifying immune response strength and polarity. This immune modulating capability thus makes them attractive targets for viral myocarditis therapeutics. Chemokines apart of the CC chemokine family such as CCL2, CCL4, and CCL19 have been shown to mediate mononuclear cell migration to the heart in coxsackievirus B3-induced myocarditis (Chen et al. 2009). In fact, a recent gene therapy approach using a CCL2 mutant that lacked chemo-attractant activity in a Balb/c coxsackievirus B3-infection model impaired appropriate Th1 immune responses and significantly controlled myocardial disease (Yue et al. 2011). Blocking CCL2 with expression of the mutant did not assist in viral clearance. The potential therapeutic effect of blocking CCL2 lies in the CCL2 mutant's ability to weaken the

The CDC family of chemokines also contribute to myocarditis pathogenesis. CDC chemokines act on monoculear CXCR3 expressing cells and include such members as IFN-inducible protein 10 (IP10/CXCL10), monokine induced by IFN-γ (Mig/CXCL9), and IFN-inducible Tcell a chemoattractant (I-TAC/CXCL11) (Groom et al. 2011). CXCL10 and its interaction with its receptor CXCR3 have been implicated in many virus disease models. CXCL10 is a key contributor in the innate immune response to viral infection. By interacting with its receptor CXCR3, CXCL10 manipulates natural killer cell trafficking and their production of IFN-γ. Yuan et al recently found CXCL10 levels in the heart to be inversely related to viral titers after coxsackievirus B3 infection and a massive infiltration of CXCR3+, CD4+, and CD8+ cells (Yuan et al. 2009). The production of associated inflammatory cytokines followed with the infiltration of leukocytes though the anti-viral response was not effective in clearing the virus or ensuring survival in coxsackievirus B3-infected CXCL10 transgenic mice (Yuan et al. 2009). CXCL10 did assist viral clearance and protect myocytes from damage in the early stages of infection by robustly attracting NK cells and enhancing IFN-γ production to infection sites (Yuan et al. 2009). Though CXCL10 appears to improve cardiac pathology and reduce viral persistence during initial infection stages, the caveat with using this chemokine as a potential coxsackievirus B3-induced myocarditis therapy lies with its inherent actions as a Th1-type chemeokine. Th1 immune responses escalate detrimental cardiac fibrosis during the reclamation stage of disease so enhancing important contributors to this repair process may

Determining the effect of CXCL10 on viral replication and recruitment of innate immune cells in other organs than the heart such as the liver and pancreas must still be pursued. As seen with another immunomodulatory factor, TGF-β, the immune responses in other organs than the heart can affect susceptibility to severe myocardial injury and the development of

and including the severity of cardiac dysfunction (Calabrese et al. 2004).

pro-inflammatory Th1 immune response (Yue et al. 2011).

acceleration rather than prevent the development of chronic disease.

chronic disease.

Recent research has demonstrated that coxsackievirus B3-infected Balb/c mice have increased levels of cardiac CXCL10 and this level fluctuates in a time and dose-dependent manner (Yue et al. 2011). The same research group treated coxsackievirus B3-infected mice with a CXCL10 mutant that lacks the critical chemo-attractant part. This mutant effectively blocked endogenous CXCL10 activity and protected coxsackievirus B3-infected mice from developing myocarditis (Yue et al. 2011). The CXCL10 mutant expressing mice had greater survival, less changes in body weight, less inflammation and necrosis in the heart. Cardiac Th1 cytokines IFN-γ, IL-12, TNF-α, were also found to be significantly reduced with CXCL10/CXCR3 signalling inhibition. This suggests that dampening the Th1 response to coxsackievirus B3 infection through blocking CXCL10 activity may present as an effective strategy to suppress immune inflammation and myocardial damage in coxsackievirus B3 mediated myocarditis (Yue et al. 2011).

The production of immunomodulating mediators such as cytokines and chemokines affects not only antigen presenting cell and T cell populations acting at the site of injury, but greatly influences the outcome of disease. The expression of particular cytokines and chemokines after a pathogenic insult, such as coxsackievirus B3 infection, influences the balance of effector versus regulatory responses that ensue. Once the balance between effector versus regulatory responses tips in a particular direction, chronic autoimmune-type disease or the prevention of disease will materialize (Rouse et al. 2010; Wing et al. 2010) (Figure 3). It is therefore critical to bear in mind the possibility of tipping the immunity balance when manipulating key immune players in coxsackievirus-induced myocarditis.

Fig. 3. Immunomodulation of the immune balance and the effects on disease outcome. With coxsackievirus B3 infection, populations of regulatory and effector cells are produced. It is with particular immunomodulating mediators such as cytokines interferon (IFN) and tumor necrosis factor (TNF) that enhanced production and proliferation of effector T cells (Teff) is promoted and leads to the persistent destruction of the myocardium and chronic myocarditis disease. Pro-regulatory cytokines transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) are examples of immune modulators that promote the enhanced production of regulatory T cells (regulatory T cells) and the prevention of autoimmune myocarditis disease.

infection.

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