Jobert G. Barin

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.

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.

Quantitative Analysis of Myocardial Inflammation by Flow Cytometry in Murine Autoimmune Myocarditis: Correlation with Cardiac Function

Inflammation has been increasingly recognized as an important pathological component of heart failure. Existing methods of assessing myocardial infiltrate are labor-intensive and provide data that are difficult to quantify and not representative of the whole heart. As a result, little effort has been made to systematically assess the components of myocardial inflammation. We established an alternative method of quantitative assessment of myocardial inflammation by flow cytometry after enzymatic digestion of hearts to characterize the infiltrate and study the association between inflammation and cardiac function in murine experimental autoimmune myocarditis. The severity of acute myocarditis uniquely correlated with the proportion of neutrophils, but not T cells, B cells, or macrophages. Both acute and chronic phases were characterized by the presence of CD44high (activated) T cells in the heart, whereas T cells trafficking through normal hearts exhibited CD44low phenotype. During the chronic phase, the proportion of CD4+ T cells was associated with increased left-ventricular volumes and deterioration of systolic function, the hallmarks of dilated cardiomyopathy. We conclude that flow cytometry on uniformly digested mouse hearts provides sensitive and reproducible assessment of myocardial infiltrate and can be used to dissect out the specific role of individual immune components from the overall inflammatory response in the heart.

Antibodies to selected minor target antigens in patients with anti-neutrophil cytoplasmic antibodies (ANCA).

In patients with anti‐neutrophil cytoplasmic antibodies (ANCA)‐associated vasculitis, indirect immunofluorescence (IF) distinguishes between cytoplasmic (C‐ANCA) and perinuclear (P‐ANCA) neutrophil staining patterns. In patients with primary systemic vasculitis such as Wegeners granulomatosis, microscopic polyangiitis and Churg–Strauss syndrome, these IF staining patterns correspond broadly with antibodies to the two major antigens: the C‐ANCA pattern is associated generally with antibodies to serine protease 3 (PR3) and the P‐ANCA pattern with antibodies to myeloperoxidase (MPO). However, some sera positive for ANCA by IF are negative for anti‐PR3 and anti‐MPO antibodies, suggesting the presence of antibodies to minor antigens of PMN granules. We tested sera from a previously well‐defined clinical cohort of patients for antibodies to four possible minor antigens: bactericidal permeability increasing protein, elastase, cathepsin G and lactoferrin. IF‐positive (+) sera had significantly higher antibody frequencies to the minor antigens than did the IF‐negative (–) sera (P < 0·01). Patients with IF+ PR3‐MPO‐ sera showed the most varied reactivity to the minor antigens. Among the IF+ groups, the IF+ PR3+/MPO‐ sera showed the lowest reactivity to the minor antigens. Patients with well‐defined ANCA specificities, e.g. the PR3‐ANCA response associated with Wegeners granulomatosis, are less likely than are other patient subsets to have antibodies to minor antigen targets. Autoantibodies to these minor antigens contribute to the overall pattern of ANCA identified by IF and help to explain why the correlation between IF and enzyme immunoassays show discrepancies. While the pathophysiological significance of antibodies to minor target antigens needs further evaluation, they may be markers of inflammation associated with disease processes.

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.

Iodination of murine thyroglobulin enhances autoimmune reactivity in the NOD.H2h4 mouse

Autoimmune thyroiditis in humans has been linked to excess iodine intake. A causative relationship between dietary iodine and thyroiditis has been clearly established in animal models of thyroiditis, including the NOD.H2h4 mouse strain, which develops enhanced thyroiditis spontaneously after supplementation of drinking water with sodium iodide. To assess the mechanisms by which iodine may contribute to disease pathogenesis, we have purified hypoiodinated thyroglobulin (Lo‐I Tg) from the thyroids of mice fed methimazole and potassium perchlorate. This preparation contained only a trace of iodine and was poorly reactive to monoclonal antibody 42C3, which has been shown previously to distinguish hypoiodinated from normal Tg. A cloned T cell line 2D11 from a diseased NOD.H2h4 mouse proliferated in response to normal Tg, but not to Lo‐I Tg. Serum antibodies from NOD.H2h4 mice with thyroiditis were poorly reactive to Lo‐I Tg. To determine that these changes were due specifically to iodine content, Lo‐I Tg was reiodinated in vitro. Reiodination of Lo‐I Tg partially re‐established the reactivity of NOD.H2h4 serum antibodies. The data demonstrate that the reactivity of thyroglobulin‐specific antibodies and certain T cells are dependent on the iodine content of thyroglobulin. These findings suggest that iodine contributes to autoimmune thyroiditis in the NOD.H2h4 mouse by directly enhancing the antigenicity of thyroglobulin.

Thyroid-Specific Expression of IFN-γ Limits Experimental Autoimmune Thyroiditis by Suppressing Lymphocyte Activation in Cervical Lymph Nodes

The role of IFN-γ in the pathogenesis of autoimmune disease is controversial, being described as immunostimulatory in some studies and immunosuppressive in others. To determine the contribution of local expression of IFN-γ, we derived NOD.H-2h4 transgenic mice overexpressing IFN-γ in a thyroid-restricted manner. Transgenic mice, which had serum IFN-γ levels similar to wild-type littermates, showed up-regulation of MHC class II on thyrocytes, but did not develop spontaneous thyroiditis. Upon immunization with murine thyroglobulin, transgenic mice developed milder disease and reduced IgG1 responses compared with wild type. The milder disease was associated with decreased frequency of activated CD44+ lymphocytes in the cervical lymph nodes. This suppressive effect was confirmed by showing that blockade of systemic IFN-γ with mAb enhanced disease and increased IgG1 responses. The study supports a disease-limiting role of IFN-γ in autoimmune thyroiditis. Furthermore, it provides the first evidence that local IFN-γ activity in the thyroid is sufficient for disease suppression.

Macrophage diversity in cardiac inflammation: A review

Cardiac inflammatory disease represents a significant public health burden, and interesting questions of immunopathologic science and clinical inquiry. Novel insights into the diverse programming and functions within the macrophage lineages in recent years have yielded a view of these cells as dynamic effectors and regulators of immunity, host defense, and inflammatory disease. In this review, we examine and discuss recent investigations into the complex participation of mononuclear phagocytic cells in the pathology of animal models of myocarditis.

Fatal Eosinophilic Myocarditis Develops in the Absence of IFN-γ and IL-17A

CD4+ T cells play a central role in inflammatory heart disease, implicating a cytokine product associated with Th cell effector function as a necessary mediator of this pathophysiology. IFN-γ–deficient mice developed severe experimental autoimmune myocarditis (EAM), in which mice are immunized with cardiac myosin peptide, whereas IL-17A–deficient mice were protected from progression to dilated cardiomyopathy. We generated IFN-γ−/−IL-17A−/− mice to assess whether IL-17 signaling was responsible for the severe EAM of IFN-γ−/− mice. Surprisingly, IFN-γ−/−IL-17A−/− mice developed a rapidly fatal EAM. Eosinophils constituted a third of infiltrating leukocytes, qualifying this disease as eosinophilic myocarditis. We found increased cardiac production of CCL11/eotaxin, as well as Th2 deviation, among heart-infiltrating CD4+ cells. Ablation of eosinophil development improved survival of IFN-γ−/−IL-17A−/− mice, demonstrating the necessity of eosinophils in fatal heart failure. The severe and rapidly fatal autoimmune inflammation that developed in the combined absence of IFN-γ and IL-17A constitutes a novel model of eosinophilic heart disease in humans. This is also, to our knowledge, the first demonstration that eosinophils have the capacity to act as necessary mediators of morbidity in an autoimmune process.

IL-33 Independently Induces Eosinophilic Pericarditis and Cardiac Dilation: ST2 Improves Cardiac Function

Background— IL-33 through its receptor ST2 protects the heart from myocardial infarct and hypertrophy in animal models but, paradoxically, increases autoimmune disease. In this study, we examined the effect of IL-33 or ST2 administration on autoimmune heart disease. Methods and Results— We used pressure-volume relationships and isoproterenol challenge to assess the effect of recombinant (r) IL-33 or rST2 (eg, soluble ST2) administration on the development of autoimmune coxsackievirus B3 myocarditis and dilated cardiomyopathy in male BALB/c mice. The rIL-33 treatment significantly increased acute perimyocarditis (P=0.006) and eosinophilia (P=1.3×10−5), impaired cardiac function (maximum ventricular power, P=0.0002), and increased ventricular dilation (end-diastolic volume, P=0.01). The rST2 treatment prevented eosinophilia and improved heart function compared with rIL-33 treatment (ejection fraction, P=0.009). Neither treatment altered viral replication. The rIL-33 treatment increased IL-4, IL-33, IL-1&bgr;, and IL-6 levels in the heart during acute myocarditis. To determine whether IL-33 altered cardiac function on its own, we administered rIL-33 to undiseased mice and found that rIL-33 induced eosinophilic pericarditis and adversely affected heart function. We used cytokine knockout mice to determine that this effect was due to IL-33-mediated signaling but not to IL-1&bgr; or IL-6. Conclusions— We show for the first time to our knowledge that IL-33 induces eosinophilic pericarditis, whereas soluble ST2 prevents eosinophilia and improves systolic function, and that IL-33 independently adversely affects heart function through the IL-33 receptor.