**9. The role of STAT proteins in Chagas' cardiomyopathy**

There is growing evidence that not only NF-kB but also STAT transcription factors are engaged in *T. cruzi* infection. Recently, we have demonstrated that serine phosphorylation of STAT1 at position 727 is targeted by *T. cruzi*, suggesting that the parasite inhibits the antiparasitic effects of STAT1 [87] (**Figure 5**). Ponce and coworkers reported that the secretion of endogenous IL-6 or the addition of recombinant IL-6 protects cardiomyocytes from cell death by apoptosis during an infection with *T. cruzi* [88]. Furthermore, the authors showed the phosphorylation of STAT3 at tyrosine 705 by IL-6 in response to infection with *T. cruzi*. In cardiac tissues, the expression of the STAT3-regulated antiapoptotic factor Bcl-2 was increased, suggesting that, during the acute phase of infection with *T. cruzi*, tyrosine-phosphorylated STAT3 acts as a mediator of cell survival. In summary, the results of this important study suggest that STAT3 executes pro-survival effects in cardiac muscle cells evoked by the parasite.

In addition, Ponce et al. demonstrated that, in *T. cruzi*-infected cells, the release of IL-6 via a TLR2-dependent pathway is required to induce survival of cardiomyocytes [88]. The enzymatic activity of cruzipain, the main cysteine protease secreted by this parasite, critically interferes with IL-6-mediated STAT3 phosphorylation by means of cleavage of the ectodomain of glycoprotein gp130, which is the shared receptor of several IL-6-type cytokines [89]. The parasite cysteine protease inhibitor chagasin inhibits cruzipain-induced gp130 cleavage,

**Figure 5.** Illustration of a cardiac myocyte showing replicating intracellular *T. cruzi* parasites that evade the protective role of IFN-γ signaling by targeting essential components of the JAK-STAT pathway such as the dephosphorylation of STAT1 at serine 727. While phosphorylation of STAT1 serine 727 is required for full transcriptional activity, its dephosphorylation inhibits antiparasitic effects of STAT1.

suggesting that the pro-inflammatory IL-6 response in *T. cruzi*-infected cells is modified by cysteine protease activity. In addition, it has been well established that STAT transcription factors activate genes whose products have been identified as *s*uppressors *o*f *c*ytokine *s*ignaling (SOCS) which act as inhibitors of the JAK-STAT pathway in a negative feedback loop [90].

Previous studies have described how STAT3 is activated by the two cytokines IL-6 or IL-10 [91, 92] and how the expression of SOCS3 is upregulated by the anti-inflammatory IL-10 in *T. cruzi*-infected cardiomyocytes [71]. During chronic infection with *T. cruzi*, the expression of SOCS2 is upregulated and most probably plays a significant role in the etiopathogenesis of Chagas' heart disease by influencing heart muscle damage [93].

Another member of the STAT family, the transcription factor STAT4, is activated in response to the cytokine IL-12, which acts as a pro-inflammatory cytokine and drives Th cells along a Th1 lineage. STAT6 is activated by receptor binding of two cytokines with anti-inflammatory properties, IL-4 and IL-13, which provide an alternative signal for the development along a Th2 lineage. Tarleton and coworkers demonstrated that STAT4-deficient mice were highly susceptible to infection with *T. cruzi*, whereas STAT6-deficient mice showed enhanced resistance with lower parasitemia and little or no evidence of inflammatory processes in the heart muscle as compared to their wild-type littermates [94]. The apparent absence of disease in chronically infected STAT6-deficient mice is remarkable despite their inability to achieve entire parasite clearance. The findings in this investigation suggest that the severity of inflammation critically depends on STAT4- and STAT6-mediated cytokine-driven immune reactions which modulate tissue parasite load [94]. Finally, the authors infer that the clearance of intracellular parasites may not be required to prevent the progression of the disease to cardiomyopathy.

### **10. Concluding remarks**

showed that the cysteine protease cruzipain on the surface of *T. cruzi* appears to have a protective effect on the host cell and serves as a survival factor supporting the propagation of the parasites [80]. Our group demonstrated apoptotic rat cardiomyocytes upon infection with either trypomastigote or amastigote stages of *T. cruzi* [81]. Petersen et al. showed that both *T. cruzi* infection and activation of NF-κB prevented apoptotic cell death in isolated neonatal rat cardiomyocytes [82]. Another study reported that amastigotes presented higher rates of

The JAK-STAT-signaling pathway has an important role in cardiomyopathy, myocarditis, and myocardial infarction [84]. Cardiomyocytes express various receptors for cytokines and growth factors (among others, TNFα and EGF) on their surface. Secreted cytokines or growth factors may be involved in the apoptotic cell death of cardiomyocytes and chronic cardiomyopathy. Specifically, the balance in the activation state of the two related transcription factors STAT1 and STAT3 may determine the outcome between cell death and survival of cardiac

In the context of chronic Chagas' disease, which can develop up to 25 years after parasitic infection, the question arises as to how the parasite can persist and replicate for such a long period of time in the host without causing an exacerbating immune response. The most obvious explanation is that the parasite has developed effective mechanisms to circumvent the immune response which affects the steady balance between parasite load and apoptosisinduced destruction of host cells. Various parasitological studies highlight the dogma that the replication of parasites in the cardiac myocytes is required to initiate the complete picture of Chagas' heart disease ranging from acute myocarditis to chronic cardiomyopathy [86].

There is growing evidence that not only NF-kB but also STAT transcription factors are engaged in *T. cruzi* infection. Recently, we have demonstrated that serine phosphorylation of STAT1 at position 727 is targeted by *T. cruzi*, suggesting that the parasite inhibits the antiparasitic effects of STAT1 [87] (**Figure 5**). Ponce and coworkers reported that the secretion of endogenous IL-6 or the addition of recombinant IL-6 protects cardiomyocytes from cell death by apoptosis during an infection with *T. cruzi* [88]. Furthermore, the authors showed the phosphorylation of STAT3 at tyrosine 705 by IL-6 in response to infection with *T. cruzi*. In cardiac tissues, the expression of the STAT3-regulated antiapoptotic factor Bcl-2 was increased, suggesting that, during the acute phase of infection with *T. cruzi*, tyrosine-phosphorylated STAT3 acts as a mediator of cell survival. In summary, the results of this important study suggest that STAT3

In addition, Ponce et al. demonstrated that, in *T. cruzi*-infected cells, the release of IL-6 via a TLR2-dependent pathway is required to induce survival of cardiomyocytes [88]. The enzymatic activity of cruzipain, the main cysteine protease secreted by this parasite, critically interferes with IL-6-mediated STAT3 phosphorylation by means of cleavage of the ectodomain of glycoprotein gp130, which is the shared receptor of several IL-6-type cytokines [89]. The parasite cysteine protease inhibitor chagasin inhibits cruzipain-induced gp130 cleavage,

apoptosis-like cell death as compared to trypomastigotes [83].

**9. The role of STAT proteins in Chagas' cardiomyopathy**

executes pro-survival effects in cardiac muscle cells evoked by the parasite.

muscle cells during infection with *T. cruzi* [85].

224 Cardiomyopathies - Types and Treatments

In summary, the pathogenic protozoan *T. cruzi* has evolved complex and still undefined mechanisms to circumvent an effective immune response in the human myocardium. The bypassing of protective signaling pathways by the parasite such as the JAK-STAT pathway may account for the intracellular multiplication and long-lasting persistence of *T. cruzi* in the host. Amastigotes of *T. cruzi* proliferating in the cytoplasm of infected cardiomyocytes have developed effective strategies to counteract the attack executed by STAT proteins, which are crucial for an effective immune defense against the protozoan. Further research efforts are required to elucidate the role of cytokine-driven gene expression in the fight against the parasite.

### **Acknowledgement**

The research on this subject was funded by grants from Deutsche Forschungsgemeinschaft (DFG), Deutsche Gesellschaft für Kardiologie (DGK), and Deutsches Zentrum für Herz- und Kreislaufforschung (DZHK).
