**3. Inflammatory regulation of myocarditis by the host: regulatory T cells and healthy vs pathological signalling**

#### **3.1 Mouse genetic determinants of immune infiltration**

Once virus infection has taken hold in the heart there is infiltration and onset of immune responses to clear infection and lay down provisional matrix in place of damaged tissue. As we discussed earlier in the chapter, the severity of the immune response varies broadly from patient to patient, from mild to massive immune infiltration that can lead to DCM and congestive heart failure. We know from the mouse models of CVB3 induced myocarditis that immune infiltration is correlated with the amount of virus inoculum, and therefore with the degree of damage that has been mediated by virus. However experiments comparing the susceptibility of various inbred strains of mice to CVB3 induced myocarditis showed that there could be a genetic predisposition to myocarditis severity (McManus et al., 1993). These data suggest that a genetic predisposition of the individual patient to mild or severe myocarditis, combined with viremia and cardiac viral load could act in concert to determine severity and number of myocarditic foci. The studies conducted with inbred mouse strains have implicated the MHC class II (H2) locus (Wolfgram et al., 1986), in addition to other genetic loci (Traystman et al., 1991) associated with the severity and susceptibility to CVB3 myocarditis, both acute, chronic and autoimmune. Not surprisingly, the H2 locus of the MHC II complex has been implicated in clearance of acute coxsackievirus infection

survival protein, Akt, is activated during the CVB3 replication cycle (Esfandiarei et al., 2007; Esfandiarei et al., 2004; Liu et al., 2008). It may appear counter-intuitive for a cytolytic virus to evolve to activate pro-survival pathways, particularly if the virus requires the cell to lyse at the end of the cycle. However, as we discussed in section 2.1 CVB3 replication is cytotoxic from the onset so pro-survival pathways may be selected evolutionarily to support the survival of the cell while viral protein synthesis and viral progeny assembly are taking place. These mechanisms of cell survival during virus infection act in favour of viral replication because they allow maximum amounts of virus progeny to be assembled prior to cell lysis and release of viral progeny. Subsequently, these same survival pathways must be inhibited to then permit cell death and lysis for release of assembled viral progeny virions.

It is now apparent that several cell death pathways and perhaps dozens of proteins are activated in a web of molecular interaction in order to short circuit normal death mechanisms, to cause rapid and aberrant rupture of the infected cell (Figure 1). New pathways of cell death, such as necroptosis, may as yet be shown to mediate death of the CVB3 infected cell. Nevertheless, there already exists a large web of seemingly disparate pathways of CVB3 induced cellular lysis. As we discussed earlier, it is difficult to determine the foundation pathway that is responsible for virally induced cell lysis of infected cardiomyocytes, and which ones are triggered, merely as a matter of coincident activation. New methods of data organisation and analysis are clearly needed as more pathways and mechanisms of CVB3 induced cell death are revealed. Knowledge of the truly pertinent and central pathways of cell death could lead to the most robust antivirals. Toward the end of this chapter we will discuss the use of systems biology to organise the myriad of pathways activated during CVB3 infection (proteolytic and kinase) and how it could be applied to

**3. Inflammatory regulation of myocarditis by the host: regulatory T cells and** 

Once virus infection has taken hold in the heart there is infiltration and onset of immune responses to clear infection and lay down provisional matrix in place of damaged tissue. As we discussed earlier in the chapter, the severity of the immune response varies broadly from patient to patient, from mild to massive immune infiltration that can lead to DCM and congestive heart failure. We know from the mouse models of CVB3 induced myocarditis that immune infiltration is correlated with the amount of virus inoculum, and therefore with the degree of damage that has been mediated by virus. However experiments comparing the susceptibility of various inbred strains of mice to CVB3 induced myocarditis showed that there could be a genetic predisposition to myocarditis severity (McManus et al., 1993). These data suggest that a genetic predisposition of the individual patient to mild or severe myocarditis, combined with viremia and cardiac viral load could act in concert to determine severity and number of myocarditic foci. The studies conducted with inbred mouse strains have implicated the MHC class II (H2) locus (Wolfgram et al., 1986), in addition to other genetic loci (Traystman et al., 1991) associated with the severity and susceptibility to CVB3 myocarditis, both acute, chronic and autoimmune. Not surprisingly, the H2 locus of the MHC II complex has been implicated in clearance of acute coxsackievirus infection

**2.5 Coxsackievirus induced cell death: many dimensions of complexity** 

elucidate the dominant pathways of virus induced cell death.

**3.1 Mouse genetic determinants of immune infiltration** 

**healthy vs pathological signalling** 

Fig. 1. Pathways of cell death in CVB3 infected cardiomyocytes. A. Coxsackievirus replication results in production of reactive oxygen species which promote the release of cytochrome c from mitochondria. The formation of Bak and Bax at the membrane surface mediate passage of cytochrome c into the cytosol. Overexpression of the anti-apoptotic proteins BCl 2 and BCl-XL during CVB3 infection significantly reduce cytochrome *c* release and activation of the caspase-9 pathway of apoptosis, which leads to caspase-3 death effector caspase activation. ROS = reactive oxygen species. B. The unfolded protein response (UPR) of the endoplasmic reticulum (ER) is activated during CVB3 infection of cardiomyocytes leading to activation of all 3 known pathways of the UPR: spliced XBP-1s, PERK and ATF6a ER stress sensors are all activated during CVB3 infection. Virus replication occurs on the surface of double membrane vesicles and membrane derived from the ER, activating the ER stress sensors. The result of XBP-1s activation is to slow the onset of cell death to prevent the cell from rupturing prior to the assembly of progeny virus virions. UPR = unfolded protein response. C and D. Glycogen synthase kinase -3β (GSK-3β) is activated during CVB3 infection resulting in phosphorylation and subsequent proteosomal degradation of its target, β –catenin, in the cytosol. C. Loss of β –catenin in the cytosol results in loss of connections between the actin cytoskeleton and cadherin receptors, via connections to α and β –catenins. D. Less β –catenin in the cytosol means that there will be less available to translocate across the nucleus to activate survival gene expression via TCF and TBP transcription factors.

unchecked.

for prevention of chronic virus replication.

Cellular and Immunological Regulation of Viral Myocarditis 279

myocarditis would suppress the immune response and allow virus replication to proceed

In a study by Shi et al (Shi et al., 2010) T-reg cells were adoptively transferred into CVB3 infected mice and the effect of the transferred cells upon viral load and signalling within the heart were investigated. Included in the study were PBS injection controls, but also naïve T cells. Decreased immune infiltration and enhanced Akt activation, as compared to the naïve CD4 T cell and PBS grafted controls, were notable in the T-reg adopted mice (Figure 2). Perhaps the most surprising of the results was the decreased viral load, not only in the heart but also in the pancreas and spleen, associated with lower expression of the Coxsackie-Adenovirus Receptor (CAR). The authors adoptively transferred, not only T-reg cells, but CD25 (-) CD4 T cells (naïve) as well (Figure 2A), into a separate group of mice. In fact, the naïve CD4 T cell treated control group fared worse than either the PBS or T-reg treated groups, related to less Akt activation and higher CAR receptor expression. As a result, the naive T cell population brought on a more severe myocarditis phenotype due to higher viral loads. Taken altogether, Shi *et al.* describe the protective role that T-reg cells play in immune infiltration of the heart during viral myocarditis, suggesting that there is a balance to be struck between clearance of infection and immune-associated damage to the myocardium. In fact, in this study there was observed decreased viral loads consonant with a decrease in immune infiltration due to reduced TNF-α release and lower CAR expression (Figure 2B). The authors found that though TNF-α could enhance CAR expression in mouse cardiomyocytes the co-addition of TGF-β eliminated TNF-α-induced CAR expression. To conclude, these findings suggest a more intimate link between inflammation and virus replication than previously posited. Thus, moderating the immune response may be critical

Although the dominant factor in virus suppression during T-reg treatment was most likely down-regulation of CAR expression, we propose that the alteration in signaling evoked by T-regs is also playing a significant role in modulating virus replication. The basis of our argument is that CAR expression was suppressed 3-fold *in vivo* and 5-fold in a dividing cardiomyocyte cell line *in vitro*, but the suppression of viral load in the Treg group was almost 2-logarithms reduced as compared to the control group. Although the correlation between cell receptor expression and virus infection may not be linear, the decreased levels of CAR receptor may not explain the entire antiviral effect of T-regs in the myocardium during virus infection. It is entirely possible that the signalling environment altered by the adoptive transfer of T-regs may have also contributed to a less favorable environment for virus replication. The authors reported activation of Akt in the myocardium of the T-reg treated groups, which suggests that the activation of other signalling proteins may have been altered by T-reg transfer. We have reported that Akt activation is required for successful CVB3 replication (Esfandiarei et al., 2007; Esfandiarei et al., 2004; Esfandiarei et al., 2006), a pro-survival protein required by the virus to optimize the longevity of the infected cell to promote optimal progeny virus production. On the other hand, the Akt activation reported by Shi *et al.* may merely be a reflection of a healthier environment created by allografted T-reg cells in the myocardium. It is quite clear that an unbraked immunologically active environment supports enhanced cellular signalling driven by viruses for successful replication (Esfandiarei et al., 2006; Marchant et al., 2009a; Marchant et al., 2008). For example, we have previously reported that the powerful immune-stimulating protein p38 is required for effective virus replication in a CVB3 myocarditis mouse model (Marchant et al., 2009a). The activation of p38 MAP kinase was not investigated by Shi *et al.*,

(Wolfgram et al., 1986). The presence of a strong adaptive response that includes robust neutralising antibodies is required to control CVB3 infection in mice (Wolfgram et al., 1986) and patients (McKinney et al., 1987; Misbah et al., 1992), as well as other picornaviruses, like polio (MacLennan et al., 2004). Therefore, the host's own genetic makeup will determine how quickly and how completely virus infection is cleared from the body. An inability to mount a broad immune response will lead to more virus replication, more immune infiltraton of the myocardium, more matrix deposition and longer recovery times. In addition to these host dependent factors there may also be environmental factors that play roles during infection and inflammation: extrinsic source stress, pollution/allergens and population density could also contribute to susceptibility and severity of viral myocarditis.

### **3.2 Chronic infection and the cycle of virus-immune mediated damage**

Over the last 3 decades it has become apparent that viral load is a primary determinant of viral myocarditis severity (Cheung et al., 2008; Chow et al., 1992; Lim et al., 2005; Marchant et al., 2009a; McManus et al., 1993), the virus causes extensive, early direct injury to the myocardium through its replication. The studies cited above all indicate significant injury to the myocardium prior to cardiac immune infiltration, supporting the view that considerable damage to the heart is directly mediated by the virus alone. An argument has been made previously for the importance of chronic virus replication in myocarditis and as a basis of dilated cardiomyopathy (Szalay et al., 2006) and chronic heart failure. Enhanced immunoproteasome expression in chronically CVB3 infected cardiomyocytes (Szalay et al., 2006) may increase the expression and presentation of auto-antigens, leading to more severe myocarditis through autoimmune avenues. Chronic virus replication will lead to consistent damage which will, in turn, result in provisional matrix turnover. This type of damagerepair loop will propagate the fibrosis that is evident during chronic myocarditis and DCM. As we covered in the apoptosis/death pathway sections of this chapter the release of cell constituents and debris, and progeny virus and proinflammatory cytokines, from an infected cell will lead to inflammation at the site of virus insult. Therefore, the damage inflicted by the virus itself will be exacerbated by the onset of cytolytic immune infiltration and fibrosis.

#### **3.3 Regulatory T cells**

Not all T cell infiltration and immunity are harmful as there are moderating arms of the immune response that solely act to moderate the strength of a response, preventing autoimmunity. Li et al recently reported that allografted M2 (anti-inflammatory) macrophages led to improvement of virus-induced myocarditis (Li et al., 2009) which was associated with enhanced levels of regulatory T cells (Treg). Huber et al have also reported decreased viral load and immune infiltration after adoptive transfer of a CD4+ CD25+ regulatory-like T cell population into a mouse model of CVB3 infection (Huber et al., 2006). Regulatory T cells are CD4 + cells that express the -subunit of the IL2 receptor CD25, and are negative for CD127. The hallmark protein expressed in these cells is the transcription factor FoxP3 and they circulate as a functionally distinct T cell subpopulation, preventing autoimmune and other aberrant responses to innocuous environmental antigens (Wing and Sakaguchi). The T-reg population prevents the proliferation of self-reactive effector T cell populations and were aptly named suppressor T cells when first discovered for their ability to secrete IL10 and moderate Th1 immunity (Wing and Sakaguchi). Thus, it would be reasonable to postulate that adoptive transfer of T-regs into a mouse model of viral

(Wolfgram et al., 1986). The presence of a strong adaptive response that includes robust neutralising antibodies is required to control CVB3 infection in mice (Wolfgram et al., 1986) and patients (McKinney et al., 1987; Misbah et al., 1992), as well as other picornaviruses, like polio (MacLennan et al., 2004). Therefore, the host's own genetic makeup will determine how quickly and how completely virus infection is cleared from the body. An inability to mount a broad immune response will lead to more virus replication, more immune infiltraton of the myocardium, more matrix deposition and longer recovery times. In addition to these host dependent factors there may also be environmental factors that play roles during infection and inflammation: extrinsic source stress, pollution/allergens and population density could also contribute to susceptibility and severity of viral myocarditis.

Over the last 3 decades it has become apparent that viral load is a primary determinant of viral myocarditis severity (Cheung et al., 2008; Chow et al., 1992; Lim et al., 2005; Marchant et al., 2009a; McManus et al., 1993), the virus causes extensive, early direct injury to the myocardium through its replication. The studies cited above all indicate significant injury to the myocardium prior to cardiac immune infiltration, supporting the view that considerable damage to the heart is directly mediated by the virus alone. An argument has been made previously for the importance of chronic virus replication in myocarditis and as a basis of dilated cardiomyopathy (Szalay et al., 2006) and chronic heart failure. Enhanced immunoproteasome expression in chronically CVB3 infected cardiomyocytes (Szalay et al., 2006) may increase the expression and presentation of auto-antigens, leading to more severe myocarditis through autoimmune avenues. Chronic virus replication will lead to consistent damage which will, in turn, result in provisional matrix turnover. This type of damagerepair loop will propagate the fibrosis that is evident during chronic myocarditis and DCM. As we covered in the apoptosis/death pathway sections of this chapter the release of cell constituents and debris, and progeny virus and proinflammatory cytokines, from an infected cell will lead to inflammation at the site of virus insult. Therefore, the damage inflicted by the virus itself will be exacerbated by the onset of cytolytic immune infiltration

Not all T cell infiltration and immunity are harmful as there are moderating arms of the immune response that solely act to moderate the strength of a response, preventing autoimmunity. Li et al recently reported that allografted M2 (anti-inflammatory) macrophages led to improvement of virus-induced myocarditis (Li et al., 2009) which was associated with enhanced levels of regulatory T cells (Treg). Huber et al have also reported decreased viral load and immune infiltration after adoptive transfer of a CD4+ CD25+ regulatory-like T cell population into a mouse model of CVB3 infection (Huber et al., 2006). Regulatory T cells are CD4 + cells that express the -subunit of the IL2 receptor CD25, and are negative for CD127. The hallmark protein expressed in these cells is the transcription factor FoxP3 and they circulate as a functionally distinct T cell subpopulation, preventing autoimmune and other aberrant responses to innocuous environmental antigens (Wing and Sakaguchi). The T-reg population prevents the proliferation of self-reactive effector T cell populations and were aptly named suppressor T cells when first discovered for their ability to secrete IL10 and moderate Th1 immunity (Wing and Sakaguchi). Thus, it would be reasonable to postulate that adoptive transfer of T-regs into a mouse model of viral

**3.2 Chronic infection and the cycle of virus-immune mediated damage** 

and fibrosis.

**3.3 Regulatory T cells** 

myocarditis would suppress the immune response and allow virus replication to proceed unchecked.

In a study by Shi et al (Shi et al., 2010) T-reg cells were adoptively transferred into CVB3 infected mice and the effect of the transferred cells upon viral load and signalling within the heart were investigated. Included in the study were PBS injection controls, but also naïve T cells. Decreased immune infiltration and enhanced Akt activation, as compared to the naïve CD4 T cell and PBS grafted controls, were notable in the T-reg adopted mice (Figure 2). Perhaps the most surprising of the results was the decreased viral load, not only in the heart but also in the pancreas and spleen, associated with lower expression of the Coxsackie-Adenovirus Receptor (CAR). The authors adoptively transferred, not only T-reg cells, but CD25 (-) CD4 T cells (naïve) as well (Figure 2A), into a separate group of mice. In fact, the naïve CD4 T cell treated control group fared worse than either the PBS or T-reg treated groups, related to less Akt activation and higher CAR receptor expression. As a result, the naive T cell population brought on a more severe myocarditis phenotype due to higher viral loads. Taken altogether, Shi *et al.* describe the protective role that T-reg cells play in immune infiltration of the heart during viral myocarditis, suggesting that there is a balance to be struck between clearance of infection and immune-associated damage to the myocardium. In fact, in this study there was observed decreased viral loads consonant with a decrease in immune infiltration due to reduced TNF-α release and lower CAR expression (Figure 2B). The authors found that though TNF-α could enhance CAR expression in mouse cardiomyocytes the co-addition of TGF-β eliminated TNF-α-induced CAR expression. To conclude, these findings suggest a more intimate link between inflammation and virus replication than previously posited. Thus, moderating the immune response may be critical for prevention of chronic virus replication.

Although the dominant factor in virus suppression during T-reg treatment was most likely down-regulation of CAR expression, we propose that the alteration in signaling evoked by T-regs is also playing a significant role in modulating virus replication. The basis of our argument is that CAR expression was suppressed 3-fold *in vivo* and 5-fold in a dividing cardiomyocyte cell line *in vitro*, but the suppression of viral load in the Treg group was almost 2-logarithms reduced as compared to the control group. Although the correlation between cell receptor expression and virus infection may not be linear, the decreased levels of CAR receptor may not explain the entire antiviral effect of T-regs in the myocardium during virus infection. It is entirely possible that the signalling environment altered by the adoptive transfer of T-regs may have also contributed to a less favorable environment for virus replication. The authors reported activation of Akt in the myocardium of the T-reg treated groups, which suggests that the activation of other signalling proteins may have been altered by T-reg transfer. We have reported that Akt activation is required for successful CVB3 replication (Esfandiarei et al., 2007; Esfandiarei et al., 2004; Esfandiarei et al., 2006), a pro-survival protein required by the virus to optimize the longevity of the infected cell to promote optimal progeny virus production. On the other hand, the Akt activation reported by Shi *et al.* may merely be a reflection of a healthier environment created by allografted T-reg cells in the myocardium. It is quite clear that an unbraked immunologically active environment supports enhanced cellular signalling driven by viruses for successful replication (Esfandiarei et al., 2006; Marchant et al., 2009a; Marchant et al., 2008). For example, we have previously reported that the powerful immune-stimulating protein p38 is required for effective virus replication in a CVB3 myocarditis mouse model (Marchant et al., 2009a). The activation of p38 MAP kinase was not investigated by Shi *et al.*,

Cellular and Immunological Regulation of Viral Myocarditis 281

Activation of p38 MAP kinase is required for virus replication to take place, such that inhibition of p38 MAP kinase is an effective antiviral strategy *in vitro* (Si et al., 2005) and *in vivo* (Marchant et al., 2009a). The activation of ERK MAP kinase was first reported in the context of CVB3 infection both *in vitro* (Luo et al., 2002) and *in vivo* (Opavsky et al., 2002). The activation of this molecule by virus binding the CAR receptor may be involved in triggering endocytosis uptake of CVB3 during entry (Marchant et al., 2009b). The activation of p38 MAP kinase was later shown to be required for cell lysis and release of progeny virion. Si et al reported that inhibition of p38 MAP kinase could block progeny virus production but not virus protein expression (Si et al., 2005). Underlining the importance of p38 during virus replication was a relatively recent study that investigated the role of p38 during the life cycle of several different viruses. The activation of p38 MAP kinase was demonstrated as a common and dominant signalling molecule during the replication of many different types of viruses (Marchant et al., 2010). This molecule's intimate link to the immune response is perhaps not surprising; one might expect that Akt activation, p38 inhibition and T-reg immune control lead to improved outcome and result in lower viral

With regard to new treatment strategies, this does not necessarily mean that we should be injecting T-reg cells into afflicted patients, but methods should be pursued that mediate the immune response to swing the pendulum in favor of viral clearance and reparation, and away from immune infiltration of the myocardium that favors higher viral loads. The work of several recent studies [(Li et al., 2009; Shi et al., 2010) reviewed in (Yajima and Knowlton, 2009)] have demonstrated the virus' evolved ability to bias immune activation and associated signaling queues for the benefit of virus replication. Given the results presented by Shi *et al.*, one might initially conclude that immune suppression would be a sound therapeutic strategy. However the Myocarditis Treatment Trial, wherein patients were treated with immunosuppressive agents (Mason et al., 1995), showed that such administration had no significant benefit for outcome of human myocarditis. As such, panimmune suppression is not the way forward and better targeted methods of immune modulation are needed. Shi *et al.* demonstrated that TGF-β secreted by T-reg cells may have been responsible for the decreased CAR expression and enhanced Akt activation (Figure 1). Such suggests that we need not inhibit the immune response entirely, but rather target, modulate and encourage the arm of the immune system that promotes virus clearance. The way forward may not be adoptive transfer of T-regs, but administration of a drug that can

The Matrix MetalloProteinases (MMPs) have been implicated in the immune response to CVB3 infection in addition to delaying matrix remodelling that takes place after acute

The MMPs are collectively termed 'matrixins' and were first discovered for their ability to degrade the tails of tadpoles during frog morphogenesis (Visse and Nagase, 2003). These enzymes are zinc endoproteinases and there have since been 24 vertebrate MMP genes discovered that play a large number of roles during immune and developmental processes. There are several gene knock-out strains of MMPs that have been used to elucidate the roles of these enzymes during development and immunity. For example, MMP-8-/- mice are more prone to skin cancers due to delayed neutrophil infiltration and an aberrant immune response (Balbin et al., 2003). Matrix MetalloProteinase-12-/- mice are more susceptible to

**3.4 ERK and p38 MAP kinases** 

load and immune infiltration.

promote T-reg differentiation and function.

**3.5 The matrix metalloproteinases** 

infection.

but we would predict less net activation of p38 in the presence of T-regs, and thus an environment that is less conducive to virus replication. Although p38 MAP kinase activation is required for suppressor (T-reg) T cell activity and function (Adler et al., 2007), we propose that adoptive transfer of T-regs may have decreased the immune infiltrate in the myocardium with less activation of p38 MAP kinase.

Fig. 2. Adoptive transfer of T cells into CVB3 infected mice has assisted in the elucidation of the beneficial arms of the immune response that aid in clearance of virus from the heart. A. Transfer of naïve T cells or control treatment with PBS, followed by infection with CVB3 results in high viral load in the heart followed by a large influx of T cells. An indication of myocarditis in the mouse model is the detection of decreased Akt activation, and MMP, TNF-α, and IFN-γ expression. Shi et al demonstrated that TNF- α induces coxsackieadenovirus receptor (CAR) expression in myocardium during CVB3 replication which may promote chronic virus replication which could lead to dilated cardiomyopathy and heart failure through successive rounds of provisional matrix deposition and degradation. B. Adoptive transfer of syngeneic regulatory T cells reduces the immunologically reactive environment which results in less MMP, TNF-α, and IFN-γ expression. The result is lower CAR expression, lower viral loads and enhanced Akt activation, leading to a healthier, less immunologically reactive environment.

#### **3.4 ERK and p38 MAP kinases**

280 Myocarditis

but we would predict less net activation of p38 in the presence of T-regs, and thus an environment that is less conducive to virus replication. Although p38 MAP kinase activation is required for suppressor (T-reg) T cell activity and function (Adler et al., 2007), we propose that adoptive transfer of T-regs may have decreased the immune infiltrate in the

> Adoptive transfer of T cell subsets

Fig. 2. Adoptive transfer of T cells into CVB3 infected mice has assisted in the elucidation of the beneficial arms of the immune response that aid in clearance of virus from the heart. A. Transfer of naïve T cells or control treatment with PBS, followed by infection with CVB3 results in high viral load in the heart followed by a large influx of T cells. An indication of myocarditis in the mouse model is the detection of decreased Akt activation, and MMP, TNF-α, and IFN-γ expression. Shi et al demonstrated that TNF- α induces coxsackie-

adenovirus receptor (CAR) expression in myocardium during CVB3 replication which may promote chronic virus replication which could lead to dilated cardiomyopathy and heart failure through successive rounds of provisional matrix deposition and degradation. B. Adoptive transfer of syngeneic regulatory T cells reduces the immunologically reactive environment which results in less MMP, TNF-α, and IFN-γ expression. The result is lower CAR expression, lower viral loads and enhanced Akt activation, leading to a healthier, less

**P** 

Resolved infection and immunity

CAR expression

MMPs, TNF-α, IFN-γ, IL-10, TGF-β

**Akt**

**A** TNaive **B** Treg

**P** 

**Akt**

MMPs, TNF-α, IFN-γ

CAR expression

Chronic infection

Chronic myocarditis – Dilated cardiomyopathy - Heart failure

immunologically reactive environment.

Provisional matrix turnover - fibrosis

myocardium with less activation of p38 MAP kinase.

Activation of p38 MAP kinase is required for virus replication to take place, such that inhibition of p38 MAP kinase is an effective antiviral strategy *in vitro* (Si et al., 2005) and *in vivo* (Marchant et al., 2009a). The activation of ERK MAP kinase was first reported in the context of CVB3 infection both *in vitro* (Luo et al., 2002) and *in vivo* (Opavsky et al., 2002). The activation of this molecule by virus binding the CAR receptor may be involved in triggering endocytosis uptake of CVB3 during entry (Marchant et al., 2009b). The activation of p38 MAP kinase was later shown to be required for cell lysis and release of progeny virion. Si et al reported that inhibition of p38 MAP kinase could block progeny virus production but not virus protein expression (Si et al., 2005). Underlining the importance of p38 during virus replication was a relatively recent study that investigated the role of p38 during the life cycle of several different viruses. The activation of p38 MAP kinase was demonstrated as a common and dominant signalling molecule during the replication of many different types of viruses (Marchant et al., 2010). This molecule's intimate link to the immune response is perhaps not surprising; one might expect that Akt activation, p38 inhibition and T-reg immune control lead to improved outcome and result in lower viral load and immune infiltration.

With regard to new treatment strategies, this does not necessarily mean that we should be injecting T-reg cells into afflicted patients, but methods should be pursued that mediate the immune response to swing the pendulum in favor of viral clearance and reparation, and away from immune infiltration of the myocardium that favors higher viral loads. The work of several recent studies [(Li et al., 2009; Shi et al., 2010) reviewed in (Yajima and Knowlton, 2009)] have demonstrated the virus' evolved ability to bias immune activation and associated signaling queues for the benefit of virus replication. Given the results presented by Shi *et al.*, one might initially conclude that immune suppression would be a sound therapeutic strategy. However the Myocarditis Treatment Trial, wherein patients were treated with immunosuppressive agents (Mason et al., 1995), showed that such administration had no significant benefit for outcome of human myocarditis. As such, panimmune suppression is not the way forward and better targeted methods of immune modulation are needed. Shi *et al.* demonstrated that TGF-β secreted by T-reg cells may have been responsible for the decreased CAR expression and enhanced Akt activation (Figure 1). Such suggests that we need not inhibit the immune response entirely, but rather target, modulate and encourage the arm of the immune system that promotes virus clearance. The way forward may not be adoptive transfer of T-regs, but administration of a drug that can promote T-reg differentiation and function.

#### **3.5 The matrix metalloproteinases**

The Matrix MetalloProteinases (MMPs) have been implicated in the immune response to CVB3 infection in addition to delaying matrix remodelling that takes place after acute infection.

The MMPs are collectively termed 'matrixins' and were first discovered for their ability to degrade the tails of tadpoles during frog morphogenesis (Visse and Nagase, 2003). These enzymes are zinc endoproteinases and there have since been 24 vertebrate MMP genes discovered that play a large number of roles during immune and developmental processes. There are several gene knock-out strains of MMPs that have been used to elucidate the roles of these enzymes during development and immunity. For example, MMP-8-/- mice are more prone to skin cancers due to delayed neutrophil infiltration and an aberrant immune response (Balbin et al., 2003). Matrix MetalloProteinase-12-/- mice are more susceptible to

Cellular and Immunological Regulation of Viral Myocarditis 283

mediated by kinases and phosphatases are used by viruses as they are crucial for pathogen replication, propagation, and evasion from host immune responses (Ribet and Cossart, 2010). Several studies have showed that multiple phosphorylated-proteins individually regulate the events that constitute virus replication [reviewed in (Esfandiarei and McManus, 2008)], yet the phospho-proteins are part of an intracellular signal-transduction network, composed of several pathways. Thus, systems perspective studies have enabled us to address network mechanisms active during host-virus interactions, with specific emphasis on elucidating key determinants of disease severity and the effective translation of these

Virus infection is equivalent to a network perturbation, in that viruses have evolved effective strategies to manipulate multiple signalling pathways and induce crosstalk and feedback loops among pathways to form a network (Garmaroudi et al., 2010). In fact, viruses activate signal-transduction networks through multiple independent events, which include viral docking to receptors, viral protein synthesis, viral progeny release and virusinduced inflammatory responses (Tam, 2006). Thus, to properly understand how signaltransduction networks are disrupted by viruses, a global multivariate approach is required

One of the major challenges of defining a signal-transduction network model in the context of a disease is to study a holistic picture of molecular structures, coming together to make complex and dynamic networks. New tools are required to systematically perturb and monitor signalling processes and functions within cells. Indeed, the emergence of high-throughput, extremely parallel technologies enable biologists to monitor multiple cellular components all together (Albeck et al., 2008). These tools have provided researchers the opportunity to collect comprehensive and large datasets. Nowadays, in the era of "new biology" researchers are 'drowning' in data, in that one emerging challenge is how to organize, interpret and extract pertinent information. The complex web of death pathways discussed in section 2.3 or the complex nature of the immune response discussed in section 3.0, all argue a need for higher level analysis through machine learning. Though this analysis requires skilled computational biologists, using mathematical, statistical and computational techniques to put together the biological components into functional molecular and cellular network models in a systematic

One work that has emerged from recent studies of networked systems in the virusinfected host cell revealed ~260 host cellular factors, affecting virus infection (Brass et al., 2008), however only a small subset of these proteins played a role in the early stage of virus infection. Interestingly, a complementary study showed how a multi-parameter approach could unravel host-virus protein interactions that likely act as a network to facilitate the early steps of HIV-1 infection (Konig et al., 2008). Recently, small-molecule inhibitor pairs were used to perturb pairs of phospho-proteins to reveal causal mechanisms within the signal-transduction network response of cardiomyocytes to coxsackievirus B3 (CVB3) infection. Hierarchical cluster analysis of the resulting dataset showed that paired-inhibitor data was required for accurate predictions of the network. In this study we also depicted a high-confidence network based on partial correlations, which identified phospho-IкBα as a central "hub" in a measured phosphorylation signature (Garmaroudi et al., 2010). Now, biologists are poised to understand the network mechanisms in virus infection, in that these networks might be used to improve patient

concepts into new systems-oriented therapeutic targets.

(Ideker et al., 2001; Kleppe et al., 2006).

fashion (Janes and Yaffe, 2006).

diagnosis, monitoring, and treatment.

bacterial infection due to an innate antimicrobial property of MMP-12 in intracellular compartments (Houghton et al., 2009). This MMP was shown to bind to bacterial membranes within the endosomes of macrophages and destabilise bacteria, thus acting as an antimicrobial.
