In previous studies we found that IL-12 Rβ1 was essential for the development of autoimmune myocarditis. On the other hand, IFN-γ, the prototypic cytokine of Th1 responses, actually downregulated the severity of disease. We had to consider, therefore, that another cytokine operating through the same receptor may be responsible for the abrogation of disease. At the time of the studies, it became clear that IL-23 also acts on the IL-12 receptor. The 2 cytokines, IL-12 and IL-23, share the same P40 subunit, but IL-12 has a P35 subunit, whereas IL-23 possesses a unique P19 subunit. The following experiments were carried out by a graduate student, Christian Baldeviano. To separate the relative contribution of IL-12 and IL-23 to myocarditis, he immunized BALB/c mice deficient in P40 subunit and found that the dilution completely prevented myocarditis (Baldeviano and others 2010
). On the other hand, mice deficient in the P35 subunit were comparable to the wild-type controls. We concluded, therefore, that IL-23 is probably required for autoimmune myocarditis. To make sure that IL-23 is capable of inducing autoimmune myocarditis in P40 KO mice, Christian reconstituted the animals with either IL-12 or IL-23. After immunization with the cardiac myosin peptide in complete Freund adjuvant, we found that IL-23 reconstituted P40 KO mice developed typical cardiac muscle inflammation and necrosis. In contrast, reconstitution with IL-12 resulted in only low-grade disease. Thus, we concluded that IL-23 is sufficient for the development of severe autoimmune myocarditis.
To assess the relative contributions of Th1 and Th17 cells in cardiac inflammation, we quantitated the subpopulation of CD4+ T lymphocytes taken directly from the heart on day 21, the peak time of myocarditis. About equal numbers of IL-17A and IFN-γ producing cells were present in the hearts, indicating that both lineages contributed to the final picture of cardiac inflammation. To estimate how much each subset of T cells contributed to cardiac pathology, Christian used an adaptive transfer model of autoimmune myocarditis. Th1 and Th17 cell lines were generated separating from a CD4+ precursor and stimulated with the myocarditogenic peptide. The cells were then transferred to irradiated syngeneic recipients. Th1 cells produced only mild disease in the recipients, whereas the IL-17-polarized cells induced severe myocarditis with extensive infiltration and disruption of the normal myocardial architecture. In a head-to-head comparison, therefore, IL-17 cells are more pathogenic in myocarditis than IL-1 cells.
The hallmark of TH17 cells is the secretion of cytokines of the IL-17 family, including IL-17A, IL-17F, and IL-22. These cytokines were tested by injecting the respective specific monoclonal antibodies to groups of mice immunized with the myocardidogenic peptide in complete Freund adjuvant (Baldeviano and others 2010
). Surprisingly, treatment with antibody to IL-17A produced little or no reduction in the incidence or severity of myocarditis and antibody to IL-17F resulted only in a modest decrease in disease. In contrast, neutralization of IL-22 by monoclonal antibody significantly enhanced the severity of myocarditis when compared with control mice, indicating that this cytokine has a protective role in the disease. We concluded that none of the 3 prototypic Th17-cytokines tested, IL-17A, IL-17F, and IL-22, is essential for the development of the inflammatory phase of autoimmune myocarditis.
To investigate further the role of IL-17A in cardiac inflammation, Christian immunized IL-17A genetically deficient mice with the myocarditogenic peptide in CFA. Although the IL-17A mice developed myocarditis with an incidence and severity similar to controls on day 21, we found that the IL-17A-deficient animals did not develop the severe, life-threatening dilated cardiomyopathy seen in wild-type mice. In fact, IL-17A KO animals were completely protected from the heart failure that lead to the death of many of the controls.
To study the mechanism of protection from dilated cardiomyopathy in IL-17A-deficient mice, we measured cytokines in heart infiltrates after immunization with the myocarditogenic peptide CFA. The most striking finding was a substantial reduction in levels of IL-6 on days 14 and 21 after immunization (Cihakova and Rose 2008
). These results suggested, but did not establish, that IL-6 is critical in the progression from inflammatory myocarditis to fibrotic dilated cardiomyopathy. This protection from dilated cardiomyopathy in IL-17A-deficient mice is associated with decreased myocardial fibrosis even as early as 18 days after immunization. Further, we found a significant reduction in matrix metalloproteinase 2 (MMP2) and MMP9, 2 matrix metalloproteins that are associated with cardiac remodeling. Conversely, expression of the tissue inhibitors of the 2 metalloproteinases (TIMP1 and TIMP4) was increased in the IL-17A-deficient animals.
A final series of experiments was carried out to determine if neutralization of IL-17A with monoclonal antibody could be used to prevent the development of dilated cardiomyopathy and the concordant heart failure. We found that treatment with anti-IL-17A monoclonal antibody significantly reduced the interstitial fibrosis and inflammation and reversed the cardinal signs of cardiac failure (Baldeviano and others 2010