Sex differences in autoimmune disease from a pathological perspective.

Autoimmune diseases affect approximately 8% of the population, 78% of whom are women. The reason for the high prevalence in women is unclear. Women are known to respond to infection, vaccination, and trauma with increased antibody production and a more T helper (Th)2-predominant immune response, whereas a Th1 response and inflammation are usually more severe in men. This review discusses the distribution of autoimmune diseases based on sex and age, showing that autoimmune diseases progress from an acute pathology associated with an inflammatory immune response to a chronic pathology associated with fibrosis in both sexes. Autoimmune diseases that are more prevalent in males usually manifest clinically before age 50 and are characterized by acute inflammation, the appearance of autoantibodies, and a proinflammatory Th1 immune response. In contrast, female-predominant autoimmune diseases that manifest during the acute phase, such as Graves' disease and systemic lupus erythematosus, are diseases with a known antibody-mediated pathology. Autoimmune diseases with an increased incidence in females that appear clinically past age 50 are associated with a chronic, fibrotic Th2-mediated pathology. Th17 responses increase neutrophil inflammation and chronic fibrosis. This distinction between acute and chronic pathology has primarily been overlooked, but greatly impacts our understanding of sex differences in autoimmune disease.

Gonadectomy of male BALB/c mice increases Tim-3(+) alternatively activated M2 macrophages, Tim-3(+) T cells, Th2 cells and Treg in the heart during acute coxsackievirus-induced myocarditis.

The incidence of cardiovascular disease, including inflammatory heart diseases like myocarditis, is increased in men. Similarly, male BALB/c mice infected with coxsackievirus B3 (CVB3) develop more severe acute inflammation in the heart compared to females. To better understand the effect of male sex hormones on cardiac inflammation, we gonadectomized (Gdx) male BALB/c mice and examined acute CVB3-induced myocarditis compared to sham controls. Viral replication in the heart was not significantly altered between Gdx and sham mice. However, gonadectomy significantly reduced testosterone levels and inflammation in the heart. FACS analysis of cell populations isolated from the heart revealed that CD11b(+) cells were significantly reduced in Gdx males. However, a GR1(+)F4/80(+) subset of CD11b(+) cells was significantly increased. Because this subset also expressed the interleukin (IL)-4R and IL-10, we refer to these cells as "alternatively activated" or M2 macrophages. A greater percentage of M2 macrophages in Gdx males expressed the inhibitory receptor Tim-3, while fewer expressed IL-1beta and IL-10. Only M2 macrophages upregulated TLR4 and Tim-3, whereas GR1(-)IL-4R(lo) macrophages did not. Additionally, IL-4(+)CD4(+) Th2 cells, Foxp3(+) regulatory T (Treg) cells and Tim-3(+)CD4(+) T cells were significantly increased in the heart following Gdx. Thus, we report for the first time that the inhibitory receptor Tim-3 is expressed on M2 macrophages. Our findings show that sex hormones and/or other mediators released from the testes inhibit anti-inflammatory populations in the heart including Tim-3(+) M2, Tim-3(+)CD4(+) T cells, Th2 and Treg resulting in more severe acute cardiac inflammation in males following CVB3 infection.

Sex differences in coxsackievirus B3-induced myocarditis: IL-12Rbeta1 signaling and IFN-gamma 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-1beta, IL-18 and IFN-gamma. We have previously reported that IL-12Rbeta1 signaling increases CVB3-induced myocarditis and IL-1beta/IL-18 levels in males, while IL-12(p35)/STAT4-induced IFN-gamma 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-12Rbeta1 signaling or 2) STAT4/IFN-gamma pathways following CVB3 infection in BALB/c mice. We found that male and female mice deficient in IL-12Rbeta1 had decreased inflammation and viral replication in the heart, indicating that IL-12Rbeta1 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-gamma deficient males, however, had decreased myocarditis and viral replication compared to females. Thus, IFN-gamma 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.

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-alpha, IL-1beta, 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.

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.

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-gamma 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.

Complement receptor 1 and 2 deficiency increases coxsackievirus B3-induced myocarditis, dilated cardiomyopathy, and heart failure by increasing macrophages, IL-1beta, 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-1beta 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 B220(low) 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.

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-beta), 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.

Viruses as adjuvants for autoimmunity: evidence from Coxsackievirus-induced myocarditis.

Adjuvants historically are considered to stimulate immune responses 'non-specifically'. Recently, a renewed understanding of the critical role of innate immunity in influencing the development of an adaptive immune response has led researchers to a better understanding of 'the adjuvant effect'. Although innate immune cells do not respond to specific antigenic epitopes on pathogens, they do produce restricted responses to particular classes of pathogens via pattern recognition receptors such as Toll-like receptors (TLR). Coxsackievirus infection was found to upregulate TLR4 on mast cells and macrophages immediately following infection. Although both susceptible and resistant mice produce a mixture of Th1 and Th2 cytokines, susceptible mice have increased levels of key proinflammatory cytokines, increased numbers of mast cells, and go on to develop chronic autoimmune heart disease. TLR4 signalling also increases acute myocarditis and proinflammatory cytokines in the heart. Many similarities are described in the pathogenesis of Coxsackievirus and the adjuvant-induced model of myocarditis including upregulation of particular TLRs and cytokines soon after inoculation. Recent findings suggest that mast cell activation by viruses or adjuvants may be important in initiating autoimmune disease.

A novel model of drug hapten-induced hepatitis with increased mast cells in the BALB/c mouse.

Clinical evidence suggests that idiosyncratic hepatitis following administration of halogenated volatile anesthetics is mediated by autoimmune responses. No murine model to study mechanisms of anesthetic-induced or any other form of drug-induced idiosyncratic hepatitis exists. Anesthetics are believed to trigger hepatitis by covalently linking a trifluoroacetyl (TFA) chloride hapten to hepatic proteins, forming haptenated self-proteins. To test this hypothesis, we developed a hapten-induced model of hepatitis by immunization with syngeneic S100 liver proteins covalently coupled to TFA (TFA-S100). We found that TFA-S100 induced hepatitis was more severe than disease induced by S100 plus adjuvants or by the adjuvant alone and was characterized by neutrophil, mast cell, and eosinophil infiltration. TFA-specific IgG1 antibodies directly correlated with hepatitis, whereas S100 autoantibodies did not. TNF-alpha, IL-1beta, and IL-6 released from splenocytes collected 2 weeks after TFA-S100 inoculation were increased resembling the elevated serum cytokines reported in patients with autoimmune hepatitis (AIH). Three weeks after inoculation, the peak of hepatitis, we noted decreased numbers of Kupffer cells and lower levels of IL-6 and IL-10 in the liver, cytokines produced by Kupffer cells. This is the first report, to our knowledge, of a hapten-induced model of hepatitis with immune and autoimmune features.

IL-12 protects against coxsackievirus B3-induced myocarditis by increasing IFN-gamma and macrophage and neutrophil populations in the heart.

Th1-type immune responses, mediated by IL-12-induced IFN-gamma, are believed to exacerbate certain autoimmune diseases. We recently found that signaling via IL-12Rbeta1 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-alpha and IFN-gamma levels in the heart, suggesting that macrophage and/or neutrophil populations may be a primary source of TNF-alpha and IFN-gamma 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-gamma deficiency resulted in significantly increased viral replication and significantly reduced TNF-alpha and IFN-gamma levels in the heart, similar to IL-12 deficiency, indicating that the IL-12/STAT4 pathway of IFN-gamma production is important in limiting CVB3 replication. Furthermore, STAT4 or IFN-gamma 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.

Interferon-gamma protects against chronic viral myocarditis by reducing mast cell degranulation, fibrosis, and the profibrotic cytokines transforming growth factor-beta 1, interleukin-1 beta, 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)-gamma, are believed to exacerbate autoimmune diseases including myocarditis. In this study, we examined the effect of IL-12R beta 1 and IFN-gamma 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-beta(1), IL-1 beta, and IL-4 in the hearts of IFN-gamma-deficient mice. All mice infected with CB3 developed DCM, but IFN-gamma-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-12R beta 1 deficiency did not significantly alter the development of chronic myocarditis. Thus, IFN-gamma 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-beta(1), IL-1 beta, and IL-4 in the heart.