Sylvia Frisancho-Kiss

Interferon-γ Protects against Chronic Viral Myocarditis by Reducing Mast Cell Degranulation, Fibrosis, and the Profibrotic Cytokines Transforming Growth Factor-β1, Interleukin-1β, and Interleukin-4 in the Heart

Inflammatory fibrosis is a characteristic feature of myocarditis, dilated cardiomyopathy (DCM), and congestive heart failure. Th1-type immune responses, mediated by interleukin (IL)-12-induced interferon (IFN)-γ, are believed to exacerbate autoimmune diseases including myocarditis. In this study, we examined the effect of IL-12Rβ1 and IFN-γ deficiency on the development of chronic CB3-induced myocarditis using knockout mice. We found increased chronic CB3-induced myocarditis (14.1 to 43.1%, P < 0.001); pericarditis (1.5 to 7.6%, P < 0.001); fibrosis (9.7 to 27.4%, P < 0.05); and the profibrotic cytokines transforming growth factor-β1, IL-1β, and IL-4 in the hearts of IFN-γ-deficient mice. All mice infected with CB3 developed DCM, but IFN-γ-deficient mice developed a fibrous, adhesive pericarditis associated with increased numbers of degranulating mast cells (MCs) in the pericardium (26.6 to 45.9%, P < 0.01), increased histamine levels (716 to 1930 ng/g of heart, P < 0.01), and reduced survival (100 to 43%). In contrast, IL-12Rβ1 deficiency did not significantly alter the development of chronic myocarditis. Thus, IFN-γ protects against the development of severe chronic myocarditis, pericarditis, and DCM after CB3 infection by reducing MC degranulation, fibrosis, and the profibrotic cytokines transforming growth factor-β1, IL-1β, and IL-4 in the heart.

Cutting Edge: Cross-Regulation by TLR4 and T cell Ig Mucin-3 Determines Sex Differences in Inflammatory Heart Disease

Recent clinical studies have reinforced the importance of sex-related differences in the pathogenesis of cardiovascular diseases, with an increased incidence and mortality in men. Similar to humans, male BALB/c mice infected with coxsackievirus B3 (CVB3) develop more severe inflammation in the heart even though viral replication is no greater than in females. We show that TLR4 and IFN-γ levels are significantly elevated and regulatory T cell (Treg) populations significantly reduced in the heart of males following CVB3 infection, whereas females have significantly increased T cell Ig mucin (Tim)-3, IL-4 and Treg. Blocking Tim-3 in males significantly increases inflammation and TLR4 expression while reducing Treg. In contrast, defective TLR4 signaling significantly reduces inflammation while increasing Tim-3 expression. Cross-regulation of TLR4 and Tim-3 occurs during the innate and adaptive immune response. This novel mechanism may help explain why inflammatory heart disease is more severe in males.

Cutting Edge: T Cell Ig Mucin-3 Reduces Inflammatory Heart Disease by Increasing CTLA-4 during Innate Immunity

Autoimmune diseases can be reduced or even prevented if proinflammatory immune responses are appropriately down-regulated. Receptors (such as CTLA-4), cytokines (such as TGF-β), and specialized cells (such as CD4+CD25+ T regulatory cells) work together to keep immune responses in check. T cell Ig mucin (Tim) family proteins are key regulators of inflammation, providing an inhibitory signal that dampens proinflammatory responses and thereby reducing autoimmune and allergic responses. We show in this study that reducing Tim-3 signaling during the innate immune response to viral infection in BALB/c mice reduces CD80 costimulatory molecule expression on mast cells and macrophages and reduces innate CTLA-4 levels in CD4+ T cells, resulting in decreased T regulatory cell populations and increased inflammatory heart disease. These results indicate that regulation of inflammation in the heart begins during innate immunity and that Tim-3 signaling on cells of the innate immune system critically influences regulation of the adaptive immune response.

IL-12 Protects against Coxsackievirus B3-Induced Myocarditis by Increasing IFN-γ and Macrophage and Neutrophil Populations in the Heart

Th1-type immune responses, mediated by IL-12-induced IFN-γ, are believed to exacerbate certain autoimmune diseases. We recently found that signaling via IL-12Rβ1 increases coxsackievirus B3 (CVB3)-induced myocarditis. In this study, we examined the role of IL-12 on the development of CVB3-induced myocarditis using mice deficient in IL-12p35 that lack IL-12p70. We found that IL-12 deficiency did not prevent myocarditis, but viral replication was significantly increased. Although there were no changes in the total percentage of inflammatory cells in IL-12-deficient hearts compared with wild-type BALB/c controls by FACS analysis, macrophage and neutrophil populations were decreased. This decrease corresponded to reduced TNF-α and IFN-γ levels in the heart, suggesting that macrophage and/or neutrophil populations may be a primary source of TNF-α and IFN-γ during acute CVB3 myocarditis. Increased viral replication in IL-12-deficient mice was not mediated by reduced TNFRp55 signaling, because viral replication was unaltered in TNFRp55-deficient mice. However, STAT4 or IFN-γ deficiency resulted in significantly increased viral replication and significantly reduced TNF-α and IFN-γ levels in the heart, similar to IL-12 deficiency, indicating that the IL-12/STAT4 pathway of IFN-γ production is important in limiting CVB3 replication. Furthermore, STAT4 or IFN-γ deficiency also increased chronic CVB3 myocarditis, indicating that therapeutic strategies aimed at reducing Th1-mediated autoimmune diseases may exacerbate common viral infections such as CVB3 and increase chronic inflammatory heart disease.

Mast cells and innate cytokines are associated with susceptibility to autoimmune heart disease following coxsackievirus B3 infection.

The development of autoimmune disease involves a combination of genetic and environmental factors. Many autoimmune diseases are believed to be triggered by viral infections. Since the early, natural immune response to infection can determine the later development of the adaptive immune response, innate immunity likely influences the progression from viral immunity to autoimmunity. To investigate the role of the innate immune response on susceptibility to autoimmune disease, we compared the early cytokine response of mice susceptible or resistant to the development of autoimmune heart disease following viral infection. We found that susceptible BALB/c mice produced elevated levels of TNF-α, IL-1β, and IL-4 within hours of Coxsackievirus B3 (CB3) infection. These cytokines are known to be critical for the development of autoimmune heart disease, and are also rapidly produced from activated mast cells (MC). Degranulating MC were observed as early as 6 h following CB3 infection in the heart, and significantly higher numbers of MC were found in the spleen of susceptible BALB/c mice at this time. Thus, susceptibility to autoimmune heart disease can be determined as early as 6 h following viral infection in susceptible strains of mice.

Sex differences in coxsackievirus B3-induced myocarditis: IL-12Rβ1 signaling and IFN-γ increase inflammation in males independent from STAT4

Cardiovascular disease is the number one killer of men and women in North America. Male BALB/c mice infected with coxsackievirus B3 (CVB3) develop more severe inflammatory heart disease compared to female mice, similar to the increased heart disease that occurs in men. We show here that increased inflammation in male mice is not due to increased viral replication in the heart, but associated with increased proinflammatory cytokines IL-1β, IL-18 and IFN-γ. We have previously reported that IL-12Rβ1 signaling increases CVB3-induced myocarditis and IL-1β/IL-18 levels in males, while IL-12(p35)/STAT4-induced IFN-γ does not alter the severity of acute disease. However, whether differences exist between males and females in these two cytokine signaling pathways is unknown. In this study, we examined sex differences in 1) IL-12Rβ1 signaling or 2) STAT4/IFN-γ pathways following CVB3 infection in BALB/c mice. We found that male and female mice deficient in IL-12Rβ1 had decreased inflammation and viral replication in the heart, indicating that IL-12Rβ1 signaling increases myocarditis in both sexes. In contrast, STAT4 deficiency did not alter the sex difference in myocarditis, with males maintaining increased inflammation over females. IFN-γ deficient males, however, had decreased myocarditis and viral replication compared to females. Thus, IFN-γ increases inflammation in males independent from STAT4. These results demonstrate that sex differences greatly influence viral replication and the severity of acute CVB3-induced myocarditis.

Complement Receptor 1 and 2 Deficiency Increases Coxsackievirus B3-Induced Myocarditis, Dilated Cardiomyopathy, and Heart Failure by Increasing Macrophages, IL-1β, and Immune Complex Deposition in the Heart

Complement and complement receptors (CR) play a central role in immune defense by initiating the rapid destruction of invading microorganisms, amplifying the innate and adaptive immune responses, and mediating solubilization and clearance of immune complexes. Defects in the expression of C or CR have been associated with loss of tolerance to self proteins and the development of immune complex-mediated autoimmune diseases such as systemic lupus erythematosus. In this study, we examined the role of CR on coxsackievirus B3 (CVB3)-induced myocarditis using mice deficient in CR1/2. We found that CR1/2 deficiency significantly increased acute CVB3 myocarditis and pericardial fibrosis resulting in early progression to dilated cardiomyopathy and heart failure. The increase in inflammation was not due to increased viral replication, which was not significantly altered in the hearts of CR1/2-deficient mice, but was associated with increased numbers of macrophages, IL-1β levels, and immune complex deposition in the heart. The complement regulatory protein, CR1-related gene/protein Y (Crry), was increased on cardiac macrophage populations, while immature B220low B cells were increased in the spleen of CR1/2-deficient mice during acute CVB3-induced myocarditis. These results show that expression of CR1/2 is not necessary for effective clearance of CVB3 infection, but prevents immune-mediated damage to the heart.

Mast Cells and Inflammatory Heart Disease: Potential Drug Targets

Inflammation underlies the pathogenesis of many common cardiovascular diseases (CVD) such as myocardial infarction, atherosclerosis, myocarditis and dilated cardiomyopathy. Allergic disorders like allergic rhinitis and asthma, both chronic inflammatory conditions, have recently been linked to increased CVD and death. Studies have found that increased IgE levels, eosinophilia, positive skin-prick tests, self-reported asthma and enzymes that regulate leukotriene synthesis (5-lipoxygenase) predict a high risk for atherosclerosis, stroke and myocardial infarction. Mast cells (MCs), cells involved in the pathogenesis of allergy and asthma, are emerging as key players in the regulation of inflammation and fibrosis in the heart and vasculature. Our laboratory has found that MC numbers are increased in mice susceptible to developing chronic dilated cardiomyopathy. The fibrosis associated with chronic heart disease is increased by MC degranulation. MCs can also act as antigen-presenting cells increasing inflammation in the heart through Toll-like receptor-4 signaling and increased proinflammatory cytokine production. Similar inflammatory mechanisms are observed for myocarditis and atherosclerosis. Many of the drugs currently used to reduce heart disease act on mediators/ pathways downstream of MC degranulation. An improved understanding of the role of MCs in regulating inflammation and fibrosis will enable researchers and clinicians to better treat heart disease.