Daniela Cihakova

Alternatively activated macrophages in infection and autoimmunity

Macrophages are innate immune cells that play an important role in activation of the immune response and wound healing. Pathogens that require T helper-type 2 (Th2) responses for effective clearance, such as parasitic worms, are strong inducers of alternatively activated or M2 macrophages. However, infections such as bacteria and viruses that require Th1-type responses may induce M2 as a strategy to evade the immune system. M2 are particularly efficient at scavenging self tissues following injury through receptors like the mannose receptor and scavenger receptor-A. Thus, M2 may increase autoimmune disease by presenting self tissue to T cells. M2 may also exacerbate immune complex (IC)-mediated pathology and fibrosis, a hallmark of autoimmune disease in women, due to the release of profibrotic factors such as interleukin-1beta, transforming growth factor-beta, fibronectin and matrix metalloproteinases. We have found that M2 comprise anywhere from 30% to 70% of the infiltrate during acute viral or experimental autoimmune myocarditis, and shifts in M2 populations correlate with increased IC deposition, fibrosis and chronic autoimmune pathology. Thus, women may be at an increased risk of M2-mediated autoimmunity due to estrogens ability to increase Th2 responses.

Interleukin-17A Is Dispensable for Myocarditis but Essential for the Progression to Dilated Cardiomyopathy

Rationale: One-third of myocarditis cases progresses to dilated cardiomyopathy (DCM), but the mechanisms controlling this process are largely unknown. CD4+ T helper (Th)17 cells have been implicated in the pathogenesis of autoimmune diseases, but the role of Th17-produced cytokines during inflammation-induced cardiac remodeling has not been previously studied. Objective: We examined the importance of interleukin (IL)-17A in the progression of myocarditis to DCM using a mouse model. Methods and Results: Immunization of mice with myocarditogenic peptide in complete Freunds adjuvant induced the infiltration of IL-17A–producing Th17 cells into the inflamed heart. Unexpectedly, IL-17A–deficient mice developed myocarditis with similar incidence and severity compared to wild-type mice. Additionally, IL-17A deficiency did not ameliorate the severe myocarditis of interferon (IFN)&ggr;-deficient mice, suggesting that IL-17A plays a minimal role during acute myocarditis. In contrast, IL-17A–deficient mice were protected from postmyocarditis remodeling and did not develop DCM. Flow cytometric and cytokine analysis revealed an important role for IL-17A in heart-specific upregulation of IL-6, TNF&agr;, and IL-1&bgr; and the recruitment of CD11b+ monocyte and Gr1+ granulocyte populations into the heart. Furthermore, IL-17A–deficient mice had reduced interstitial myocardial fibrosis, downregulated expression of matrix metalloproteinase-2 and -9 and decreased gelatinase activity. Treatment of BALB/c mice with anti–IL-17A monoclonal antibody administered after the onset of myocarditis abrogated myocarditis-induced cardiac fibrosis and preserved ventricular function. Conclusions: Our findings reveal a critical role for IL-17A in postmyocarditis cardiac remodeling and the progression to DCM. Targeting IL-17A may be an attractive therapy for patients with inflammatory dilated cardiomyopathy.

Chapter 4 Pathogenesis of Myocarditis and Dilated Cardiomyopathy

Myocarditis is a disease with a variable clinical presentation, ranging from asymptomatic to a fatal outcome. Among the recognized causes of myocarditis are mutations in multiple genes; infection by bacterial, rickettsial, mycotic, protozoan, and viral agents; and exposure to drugs, toxins, and alcohol. Some subtypes of myocarditis, such as giant cell myocarditis or eosinophilic necrotizing myocarditis, are suspected to be caused by an autoimmune inflammation. Several lines of evidence support the involvement of autoimmunity in myocarditis. These include the production of antibodies against relevant self-antigens, the fact that myocarditis symptoms can be relieved by immunosuppressive therapy in some patients, and a co-occurrence of myocarditis with other autoimmune diseases. Most of the evidence that myocarditis is an autoimmune disease comes from animal models. In this chapter, we discuss coxsackievirus B3-induced myocarditis and myosin-induced myocarditis as models of both viral and autoimmune inflammation in the heart. The latest advances in the study of autoimmunity have been concentrated on T helper cells, particularly the newly discovered subset, Th17 cells. Experimental autoimmune myocarditis (EAM), a mouse model of myocarditis induced by cardiac myosin, is partly an IL-17-driven disease. However, we have shown recently in IL-13 knockout mice that the disease can be driven through other pathways, and that the Th1 helper cells also lead to severe heart inflammation. Most importantly, IL-17A knockout mice are not fully protected against EAM and still develop mild myocarditis. The most abundant cells in heart infiltrate in human giant cell myocarditis or EAM are monocyte/macrophages, and there is now evidence that macrophages play a decisive role in the course of EAM.

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.

Interleukin-13 Protects Against Experimental Autoimmune Myocarditis by Regulating Macrophage Differentiation

We report here that interleukin (IL)-13 protects BALB/c mice from myocarditis, whether induced by peptide immunization or by viral infection. In contrast to mild disease in IL-4 knockout (KO) BALB/c mice, IL-13 KO BALB/c mice developed severe coxsackievirus B3 (CVB3)-induced autoimmune myocarditis and myocarditogenic peptide-induced experimental autoimmune myocarditis. Such severe disease was characterized by increased cardiac inflammation, increased total intracardiac CD45(+) leukocytes, elevated anti-cardiac myosin autoantibodies, and increased cardiac fibrosis. Echocardiography revealed that IL-13 KO mice developed severe dilated cardiomyopathy with impaired cardiac function and heart failure. Hearts of IL-13 KO mice had increased levels of the proinflammatory and profibrotic cytokines IL-1beta, IL-18, interferon-gamma, transforming growth factor-beta1, and IL-4 as well as histamine. The hallmark of the disease in IL-13 KO mice was the up-regulation of T-cell responses. CD4(+) T cells were increased in IL-13 KO hearts both proportionally and in absolute number. Splenic T cells from IL-13 KO mice were highly activated, and myosin stimulation additionally increased T-cell proliferation. CD4(+)CD25(+)Foxp3(+) regulatory T-cell numbers were decreased in the spleens of IL-13 KO mice. IL-13 deficiency led to decreased levels of alternatively activated CD206(+) and CD204(+) macrophages and increased levels of classically activated macrophages. IL-13 KO mice had increased caspase-1 activation, leading to increased production of both IL-1beta and IL-18. Therefore, IL-13 protects against myocarditis by modulating monocyte/macrophage populations and by regulating their function.

Sjögren syndrome: Advances in the pathogenesis from animal models

Sjögren syndrome is an autoimmune disease characterized by hyposecretion of the lacrimal and salivary glands, resulting in dryness of the eyes and mouth. Individuals may experience primary Sjögren syndrome or a secondary form accompanying another rheumatic autoimmune disease, such as rheumatoid arthritis or systemic lupus erythematosus. The pathogenic mechanisms of Sjögren syndrome remain largely unknown, in part a consequence of the heterogeneity of the disease. Animal models have shed light on the connections between specific pathways and symptoms, but an ideal system is wanting. Improved disease models will enable a better understanding of Sjögren syndrome, including how immune tolerance is lost and potential therapeutic interventions. Most importantly, an optimal model will enable detection of disease biomarkers, since injury to the salivary glands may precede lymphocytic infiltration. This review aims to characterize available mice models of Sjögren syndrome, including advantages and disadvantages, from the researchers perspective.

The varying faces of IL-6: From cardiac protection to cardiac failure

IL6 is a pleiotropic cytokine that is made in response to perturbations in homeostasis. IL6 becomes elevated in the acute response to host injury and can activate immune cells, direct immune cell trafficking, signal protective responses in local tissue, initial the acute phase response or initiate wound healing. In the short term this proinflammatory response is protective and limits host damage. It is when this acute response remains chronically activated that IL6 becomes pathogenic to the host. Chronically elevated IL6 levels lead to chronic inflammation and fibrotic disorders. The heart is a tissue where this temporal regulation of IL6 is very apparent. Studies from myocardial infarction show how short-term IL6 signaling can protect and preserve the heart tissue in response to acute damage, where long term IL6 signaling or an over-production of IL6R protein plays a causal role in cardiovascular disease. Thus, IL6 can be both protective and pathogenic, depending on the kinetics of the host response.

Cardiac fibroblasts mediate IL-17A–driven inflammatory dilated cardiomyopathy

IL-17A stimulates cardiac fibroblasts to produce inflammatory mediators critical for the recruitment and differentiation of myeloid cells during inflammatory dilated cardiomyopathy.

Animal Models for Autoimmune Myocarditis and Autoimmune Thyroiditis

This chapter describes four murine models of autoimmune diseases: two related to autoimmune myocarditis and two related to autoimmune thyroiditis. The first model, Coxsackie virus B3 (CB3)-induced myocarditis, results in the development of acute myocarditis in susceptible as well as resistant mouse strains, whereas chronic myocarditis develops only in genetically susceptible mice. CB3-induced myocarditis closely resembles the course of human myocarditis, which is believed to be initiated by viral infection. Mouse cardiac myosin heavy chain has been identified as the major antigen associated with the late chronic phase of viral myocarditis. The second model is cardiac myosin-induced experimental autoimmune myocarditis (EAM) and, in a modification, cardiac alpha-myosin heavy chain peptide-induced myocarditis. In the EAM model, cardiac myosin or the relevant peptide in Freunds complete adjuvant (FCA) is injected subcutaneously into mice. The immune response, the histological changes, and the genetic susceptibility seen in EAM are similar to those of CB3-induced myocarditis. The third model is experimental autoimmune thyroiditis (EAT). EAT can be induced in genetically susceptible strains of mice by immunization with mouse thyroglobulin in FCA or lipopolysaccharide. Mice susceptible to EAT have the H-2A(k), H-2A(s), or H-2A(q) alleles. We describe here a standard technique for the induction of EAT; it was developed in our laboratory and is widely used as a model for studying Hashimotos thyroiditis. The fourth model presented in this chapter is that of spontaneous autoimmune thyroiditis in NOD.H2h4 mice. These mice express the H-2A(k) allele on an NOD genetic background and develop spontaneous thyroiditis, which is exacerbated with dietary iodine.