Several studies report on the role of the immune system and ECM remodelling in the progression of cardiac diseases (Levine et al., 1990; Spinale, 2002; Anker and von Haehling, 2004; Spinale, 2007; Chen et al., 2008; Graham et al., 2008).
Myocarditis, but also myocardial infarction, inflammatory DCM as well as heart failure with reduced or normal ejection fraction are associated with inflammatory processes in the myocardium (Levine et al., 1990; Anker and von Haehling, 2004;
Maron et al., 2006; Dobaczewski et al., 2010; Frangogiannis, 2012). It is commonly believed that besides an activation of neurohormones, pro-inflammatory cytokines contribute to the progression of heart failure, therefore, the latter is now regarded as a state of chronic inflammation (Pagani et al., 1992; Anker and von Haehling, 2004;
Chen et al., 2008; von Haehling et al., 2009; Hedayat et al., 2010; Tamariz and Hare, 2010). Cardiac inflammation is associated with myocardial infiltration of inflammatory cells, the production of cytokines, endothelial cell activation and myocardial cell damage and degeneration (Mann, 2002; Anker and von Haehling, 2004; Wei, 2011;
Frangogiannis, 2012). In people with DCM and congestive heart failure (CHF), activation of endothelial cells with upregulation of endothelial cell adhesion molecules induces recruitment of inflammatory cells, such as macrophages, neutrophils and lymphocytes, into the myocardium (Devaux et al., 1997; Noutsias et al., 1999; Noutsias et al., 2002; Noutsias et al., 2003; Anker and von Haehling, 2004;
Wei, 2011). These cells are involved in initiating cardiac regeneration and repair.
Alongside damaged myocardial cells, cells of the ECM, i.e. fibroblast, mast cells and infiltrating inflammatory cells, contribute to the production of cytokines and growth factors (Nag, 1980; Stewart et al., 2003; Souders et al., 2009; Wei, 2011;
Frangogiannis, 2012). However, control of the inflammatory state by anti-inflammatory and pro-fibrotic factors is crucial.
Different populations of T helper (Th) subpopulations are reported to play a role in progression of cardiac diseases. Four major lineages, Th1, Th2, Th17 and T regulatory (Treg) cells are known (Mosmann et al., 1986; Aggarwal et al., 2003; Zhu et al., 2010). These T cell subsets are involved in fibrosis in chronic cardiac injury, while Th2 polarised responses promote fibrosis, Th1 cells might be anti-fibrogenic (Marra et al., 2009; Wei, 2011). Inflammatory events trigger a Th1 response and Th1 cytokines are believed to participate in the initiation of fibrosis, whereas the chronic
disease processes are usually driven by a Th2 response, resulting in fibroblast activation and proliferation, myofibroblast differentiation and ECM accumulation. In human heart failure patients, an immune response of Th1 predominance and Th1/Th2 imbalance has been reported (Cheng et al., 2009). Th17 cells are involved in the initiation or progression of inflammatory diseases, promoting degradation of type I and type III collagens and contributing to myocardial fibrosis (Feng et al., 2009; Wei, 2011). On the other hand, Treg cells constitute an anti-inflammatory and pro-fibrotic lineage of T cells that secrete IL-10 and TGF-β (Huber et al., 2006; Wei, 2011; Tang et al., 2012). However, Treg cells might also be involved in limitation of fibrogenesis, since they can suppress an excessive immune activation (Kvakan et al., 2009). Depending on the primary insult, the underlying condition and the activated cell populations cardiac regeneration and repair or a progressive cardiac disease can develop.
2.3.1 Cytokines
In cardiac disease, cytokines are produced by resident myocardial cells, such as damaged cardiomyocytes, fibroblasts, mast cells, and infiltrating inflammatory cells, for example macrophages and T cells (Kuhl et al., 1996; Noutsias et al., 2002).
Cytokines are highly potent endogenous mediators of intercellular communication (Klesius, 1982; Trotta, 1991). They bind to specific receptors and act as gene-regulatory proteins through the activation of transcription factors, such as NF-κB, sma- and mad-related proteins (Smad) and signal transduction and activators of transcription (STAT) proteins (Schutze et al., 1992; Briscoe and Guschin, 1994;
Nakao et al., 1997; Anker and von Haehling, 2004).
Based on their effect, cytokines were originally classified as pro- and anti-inflammatory. The pro-inflammatory cytokines of highest relevance in the progression of CHF in humans and in animal models are interleukin (IL)-1, IL-6, IL-8 and tumour necrosis factor (TNF)-α, whereas IL-10 and transforming growth factor (TGF)-β, for example, are anti-inflammatory cytokines and considered to be cardioprotective (Pagani et al., 1992; TorreAmione et al., 1996; Bolger et al., 2002;
Chen et al., 2008; von Haehling et al., 2009). Their different cytokine expression patterns characterise Th subpopulations, Th1 cells produce IFN-γ, whereas Th2 cells produce IL-4, IL-5 and IL-13 and Th17 cells produce IL-17, IL-21, IL-22 (Mosmann et
al., 1986; Aggarwal et al., 2003; Zhu et al., 2010). Treg cells can be differentiated into Treg type 1 cells which secrete IL-10, and Th3 cells which secrete TGF-β (Huber and Schramm, 2006; Wei, 2011; Tang et al., 2012).
2.3.1.1 Pro-inflammatory cytokines
The role of IL-1, IL-6 and TNF-α in human cardiac diseases has been studied extensively. Elevated circulating and myocardial levels were found in patients with various cardiac diseases and in CHF and have been correlated to disease severity and mortality (TorreAmione et al., 1996; Deswal et al., 2001b; Torre-Amione, 2005;
von Haehling et al., 2009). Pro-inflammatory cytokines have negative inotropic effects, which are mediated through down-regulation of cardiac beta-receptors, myocardial nitric oxide production and impaired myocardial energy production (Finkel et al., 1992; Pagani et al., 1992; Zell et al., 1997). These cytokines play a role in cardiac remodelling by activating transcription factors and stimulating enzymes involved in ECM degradation and deposition (Sivasubramanian et al., 2001;
Bradham et al., 2002b; Siwik and Colucci, 2004). Furthermore, they stimulate the expression of adhesion molecules, activate inflammatory cells and contribute to the production of cytokines and reactive oxygen species, which maintain the inflammatory process (Kukielka et al., 1993; Braun et al., 1995; Kukielka et al., 1995;
Deswal et al., 2001a; Yndestad et al., 2003; Torre-Amione, 2005; Castellano et al., 2008; Chen et al., 2008; Hedayat et al., 2010; Zhang et al., 2011). The resulting myocardial impairment, cardiac remodelling and persistent inflammatory response are suspected to contribute to the progression of CHF. The harmful effects of pro-inflammatory cytokines and the increased levels in patients with CHF were the rationale behind several clinical trials that blocked TNF-α in human heart failure patients (reviewed by Pagani et al., 1992; Mann, 2002). However, the results of these trials were disappointing, since no improvement of clinical signs and reduced survival times were observed (Anker and von Haehling, 2004; Torre-Amione, 2005;
Chen et al., 2008). This suggests that pro-inflammatory cytokines do not only exert detrimental effects, but might also be involved in cardiac regeneration in patients with CHF (Chen et al., 2008; Hedayat et al., 2010).
2.3.1.2 Anti-inflammatory cytokines
Anti-inflammatory cytokines, such as IL-10 and TGF-β, inhibit pro-inflammatory cytokines (Tsunawaki et al., 1988; de Waal Malefyt et al., 1991; Bolger et al., 2002;
Anker and von Haehling, 2004; Kaur et al., 2009). IL-10 is capable of down-regulating numerous inflammatory pathways by suppressing cytokine, adhesion molecule and matrix metalloproteinase production and by regulating growth and differentiation of lymphocytes (Fiorentino et al., 1991; Mosmann, 1994; Song et al., 1997; Moore et al., 2001; Anker and von Haehling, 2004; Frangogiannis, 2012).
TGF-β is a powerful immunosuppressive and profibrotic cytokine (Edwards et al., 1987; Dobaczewski et al., 2011; Kapur, 2011). It inhibits pro-inflammatory cytokine and endothelial cell adhesion molecule expression and thereby reduces the rolling and emigration of neutrophils and lymphocytes (Gamble and Vadas, 1988, 1991;
Smith et al., 1996; Frangogiannis, 2012). TGF-β has three isoforms, TGF-β1-3. Of these, TGF-β1 and TGF-β3 are major stimulators of fibroblasts during normal ECM homoeostasis and tissue repair (Lim and Zhu, 2006; Creemers and Pinto, 2011;
Dobaczewski et al., 2011). In cardiac diseases, they are considered to play a role in the resolution of inflammation and the transition to fibrosis. However, by inducing fibrosis and cardiomyocyte hypertrophy TGF-β promotes the structural remodelling of the heart and might therefore contribute to systolic and diastolic dysfunction in cardiac diseases (Nakao et al., 1997; Song et al., 1997; Lim and Zhu, 2006;
Creemers and Pinto, 2011; Dobaczewski et al., 2011; Westermann et al., 2011;
Frangogiannis, 2012). TGF-β2 is important for the fetal development of the heart and might be involved in activation of the fetal gene programme in the failing myocardium (Lim and Zhu, 2006).
2.3.2 Growth differentiation factor 15
Growth differentiation factor 15 (GDF-15), a member of the TGF-β superfamily, regulates inflammatory and apoptotic pathways needed for tissue development, differentiation and repair (Kempf and Wollert, 2009). A weak basal expression of GDF-15 is present in most tissues, with a marked increase in its expression in response to tissue injury and inflammation (Kempf and Wollert, 2009). In the heart, GDF-15 is produced by cardiomyocytes, endothelial cells, smooth muscle cells, adipocytes and macrophages (Kempf and Wollert, 2009) and is used as a biomarker
for disease progression and prognosis in people with acute and chronic heart failure (Kempf et al., 2007; Kempf and Wollert, 2009; Lok et al., 2012). In myocardial ischaemia, GDF-15 is considered to exert a protective role, because of its anti-apoptotic and anti-inflammatory effects. It might also be involved in cardiac fibrosis, as increased GDF-15 levels in cardiac diseases appear to correlate with the degree of myocardial fibrosis (Kempf et al., 2006; Kempf and Wollert, 2009; Lok et al., 2012). Nonetheless, people with DCM have been shown to exhibit only weak myocardial GDF-15 mRNA and protein production (Lok et al., 2012). Canine GDF-15 has been isolated from and characterised in an osteosarcoma cell line (Yamaguchi et al., 2008), but its transcription in the myocardium of healthy dogs and dogs with cardiac diseases has so far not been investigated.