Renate Öttl

Critical Role for Monocyte Chemoattractant Protein-1 and Macrophage Inflammatory Protein-1α in Induction of Experimental Autoimmune Myocarditis and Effective Anti–Monocyte Chemoattractant Protein-1 Gene Therapy

Background— Autoimmune myocarditis is a principal cause of heart failure among young adults and is often a precursor of dilated cardiomyopathy. Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α) are potent chemotactic factors for mononuclear cells. The inflammatory infiltrate observed in myocardial lesions of myocarditis consists of >70% mononuclear cells. To determine their critical role in the pathogenesis of myocarditis, we inhibited mononuclear cell activation and migration to see if it would affect disease severity and disease prevalence in experimental autoimmune myocarditis (EAM). Methods and Results— In this report, we demonstrated that blockade of MCP-1 or MIP-1α with monoclonal antibodies significantly reduced severity of myocarditis in BALB/c mice immunized with cardiac myosin. Similar results were obtained when CCR2−/− and CCR5−/− mice were used. In CCR2−/− mice, not only disease severity but also disease prevalence was reduced. To further inhibit mononuclear cell activation and migration, we transfected the mice before inducing EAM with a dominant-negative inhibitor of MCP-1 gene (7ND). This transfection significantly reduced the disease severity, decreased mRNA expression levels, especially of the chemokines RANTES, MIP-2, IP-10, MCP-1, T-cell activation gene 3, and eotaxin in the myocardium, and resulted in a reduction in cardiac myosin-induced interleukin-1 and interleukin-4 and in an increase in interferon-γ and interleukin-10 cytokine production by splenocytes. Conclusions— Overall, these findings suggest that the chemokines MCP-1 and MIP-1α, acting through their receptors CCR2 and CCR5, are important in the induction of EAM and that inhibition of MCP-1 with 7ND gene transfection significantly reduced disease severity. This strategy may be a new feasible form of gene therapy against autoimmune myocarditis.

Absence of auto-antibodies against cardiac troponin I predicts improvement of left ventricular function after acute myocardial infarction

AIMS Application of antibodies against cardiac troponin I (cTnI-Ab) can induce dilation and dysfunction of the heart in mice. Recently, we demonstrated that immunization with cTnI induces inflammation and fibrosis in myocardium of mice. Others have shown that auto-antibodies to cTnI are present in patients with acute coronary syndrome, but little is known about the clinical relevance of detected cTnI-Ab. METHODS AND RESULTS First, anti-cTnI and anti-cTnT antibody titres were measured in sera from 272 patients with dilated- (DCM) and 185 with ischaemic- (ICM) cardiomyopathy. Secondly, 108 patients with acute myocardial infarction (AMI) were included for a follow-up study. Heart characteristics were determined by magnetic resonance imaging 4 days and 6-9 months after AMI. Altogether in 7.0% of patients with DCM and in 9.2% with ICM, an anti-cTnI IgG antibody titre >/=1:160 was measured. In contrast, only in 1.7% of patients with DCM and in 0.5% with ICM, an anti-cTnT IgG antibody titre >/=1:160 was detected. Ten out of 108 patients included in the follow-up study were tested positive for cTnI-Ab with IgG Ab titres >/=1:160. TnI-Ab negative patients showed a significant increase in left ventricular ejection fraction (LVEF) and stroke volume 6-9 months after AMI. In contrast, there was no significant increase in LVEF and stroke volume in TnI-Ab positive patients. CONCLUSION We demonstrate for the first time that the prevalence of cTnI-Abs in patients with AMI has an impact on the improvement of the LVEF over a study period of 6-9 months.

Identification of Cardiac Troponin I Sequence Motifs Leading to Heart Failure by Induction of Myocardial Inflammation and Fibrosis

Background— Despite the widespread use of cardiac troponins for diagnosis of myocyte injury and risk stratification in acute cardiac disorders, little is known about the long-term effects of the released troponins on cardiac function. Recently, we showed that an autoimmune response to cardiac troponin I (cTnI) induces severe inflammation and subsequent fibrosis in the myocardium. This autoimmune disorder predisposes to heart failure and cardiac death in mice. Methods and Results— To investigate the role of cTnI-specific T cells, T cells were isolated from splenocytes of mice immunized with murine cTnI (mcTnI). Wild-type mice that received mcTnI-specific T cells showed high mcTnI-specific antibody titers, increased production of the proinflammatory cytokines interleukin-1&bgr; and tumor necrosis factor-&agr;, severe inflammation and fibrosis in the myocardium, and reduced fractional shortening. To identify the antigenic determinants of troponin I responsible for the observed inflammation, fibrosis, and heart failure, 16 overlapping 16mer to 18mer peptides covering the entire amino acid sequence of mcTnI (211 residues) were synthesized. Only mice immunized with residues 105 to 122 of mcTnI developed significant inflammation and fibrosis in the myocardium, with increased expression of the inflammatory chemokines RANTES, monocyte chemotactic protein-1, macrophage inflammatory protein-1&agr;, macrophage inflammatory protein-1&bgr;, macrophage inflammatory protein-2, T-cell activation-3, and eotaxin and the chemokine receptors CCR1, CCR2, and CCR5. Mice immunized with the corresponding human cTnI residues 104 to 121 and the mcTnI residues 131 to 148 developed milder disease. Conclusions— Transfer of troponin I–specific T cells can induce inflammation and fibrosis in wild-type mice, which leads to deterioration of contractile function. Furthermore, 2 sequence motifs of cTnI that induce inflammation and fibrosis in the myocardium are characterized.

Role of the Cholinergic Antiinflammatory Pathway in Murine Autoimmune Myocarditis

Rationale: This study was performed to gain insights into novel therapeutic approaches for the treatment of autoimmune myocarditis. Objective: Chemical stimulation of the efferent arm of the vagus nerve through activation of nicotinic acetylcholine receptor subtype-7&agr; (&agr;7-nAChR) has been shown to be protective in several models of inflammatory diseases. In the present study, we investigated the potentially protective effect of vagus nerve stimulation on myocarditis. Methods and Results: A/J mice were immunized with cardiac troponin I (TnI) to induce autoimmune myocarditis. Mice were exposed to drinking water that contained nicotine in different concentrations and for different time periods (for 3 days at 12.5 mg/L; 3 days at 125 mg/L; 21 days at 12.5 mg/L; and 21 days at 125 mg/L after first immunization). TnI-immunized mice with no pharmacological treatment showed extensive myocardial inflammation and fibrosis and significantly elevated levels of interleukin-6 and tumor necrosis factor-&agr;. Furthermore, elevated levels of mRNA transcripts of proinflammatory chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1&bgr;, and RANTES) and chemokine receptors (CCR1, CCR2, and CCR5) were found. Oral nicotine administration reduced inflammation within the myocardium, decreased the production of interleukin-6 and tumor necrosis factor-&agr;, and downregulated the expression of monocyte chemoattractant protein-1, macrophage inflammatory protein-1&bgr;, RANTES, CCR1, CCR2, and CCR5. In addition, nicotine treatment resulted in decreased expression of matrix metalloproteinase-14, natriuretic peptide precursor B, tissue inhibitor of metalloproteinase-1, and osteopontin, proteins that are commonly involved in heart failure. Finally, we found that nicotine reduced levels of pSTAT3 (phosphorylated signal transducer and activator of transcription 3) protein expression within the myocardium. Neostigmine treatment did not affect the progression of myocarditis. Conclusions: We showed that activation of the cholinergic antiinflammatory pathway with nicotine reduces inflammation in autoimmune myocarditis. Our results may open new possibilities in the therapeutic management of autoimmune myocarditis.

Autoimmunity against cardiac troponin I in ischaemia reperfusion injury

Autoimmunity against cardiac troponin I (cTnI) has deleterious effects on the infarcted myocardium early after onset of ischaemia. Here, we explored the impact of cTnI‐autoimmunity in the long term. Furthermore, we studied the effects of cTnI‐autoimmunity on the infarcted myocardium following revascularization measures in terms of ischaemia reperfusion injury (IRI), which resembles clinical reality more closely.

Comparison of IL-10 and MCP-1 7ND Gene Transfer with AAV9 Vectors for Protection from Murine Autoimmune Myocarditis

AIMS Overexpression of therapeutic genes with potential disease-limiting effects, specifically at the site of inflammation, remains a major clinical challenge. In this study, we investigate the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of the anti-inflammatory mediators interleukin (IL)-10 and a dominant-negative inhibitor of monocyte chemoattractant protein-1 (MCP1-7ND) on prevention of autoimmune myocarditis. METHODS AND RESULTS Autoimmune myocarditis was induced by immunizing A/J mice with subcutaneous injection of 120 µg cardiac troponin I (cTnI) on Days 0, 7, and 14. Two weeks prior to initial immunization, each mouse received a single systemic dose of 10(12) AAV9 vectors carrying the coding sequence of IL-10 or MCP1-7ND transcriptionally targeted to the heart. Mice were sacrificed 28 days after initial immunization for further analysis. Only expression of IL-10 resulted in a highly significant decrease in myocardial inflammation and fibrosis, as well as an increased ejection fraction compared with controls. Further analyses of cytokine profiles of cTnI-stimulated splenocytes from IL-10 and MCP1-7ND-treated mice revealed significant alterations compared with controls. In addition, transcript levels of chemokine receptor CCR4 and T-cell activation gene were significantly reduced in hearts of IL-10-treated mice as determined by quantitative real-time PCR. CONCLUSION Our study suggests that cardiac expression of IL-10 with AAV9 vectors is a promising therapeutic approach for autoimmune myocarditis.

Mucosal tolerance induction in autoimmune myocarditis and myocardial infarction.

BACKGROUND Antigen-specific therapy is a compelling approach for the treatment of autoimmune conditions. Primary goal is to induce the specific tolerization of self-reactive immune cells without altering host immunity against pathogens. We studied the effects of mucosal tolerance induction on cTnI-induced experimental autoimmune myocarditis (EAM) and post-infarct remodeling. METHODS Mucosal tolerance was induced by intranasal application of cTnI, alternatively anti-CD3 p.o. Protocols varied in frequency, dosage and time point of application before EAM. We then applied the most effective regimen to mice undergoing myocardial infarction in order to verify its effectiveness in post-infarct cardiac remodeling. The myocardium was evaluated on histological slides and for the cytokine secretion pattern, while echocardiography determined cardiac function. RESULTS A single dose of 100 μg of cTnI 7 days prior to myocarditis appeared to be most effective in suppressing inflammation and fibrosis (p = 0.03), while improving fractional shortening (p = 0.02). Treatment with intranasal cTnI upregulated IL-10 expression. On the other hand, frequent intranasal application of high doses of cTnI increased myocardial inflammation. Anti-CD3 p.o. showed the propensity to reduce myocardial inflammation and improve cardiac function. The single dose regimen of i.n. cTnI applied 7 days before a myocardial infarction reduced inflammation by trend (p=0.07) and improved heart function (p=0.002). Moreover, expression of matrix metalloproteinases 9 and 14 significantly decreased when treated with intranasal cTnI (p<0.01). CONCLUSIONS Depending on the optimal amount, the time period and the choice of antigen, effective mucosal tolerance can be achieved and represents an appealing therapeutic approach in the inflammatory process of cardiac remodeling.

AUTOANTIBODY PROFILING IN CARDIOVASCULAR DISEASES AND IDENTIFICATION OF NEW TARGETS

Heart diseases are the leading causes of death worldwide. Dilated cardiomyopathy (DCM), myocarditis and ischemic cardiomyopathy (ICM) can be caused by various factors. After disease onset a dysregulation of the immune system is believed to play a central role in disease progression. Autoantibodies

MICROARRAY ANALYSIS REVEALS NOVEL TARGET AUTOANTIGENS IN DILATED CARDIOMYOPATHY

Dilated cardiomyopathy (DCM) is the leading cause of heart failure. It can be caused by various factors but dysregulation of the immune system is believed to play a central role in disease progression. Autoantibodies directed against various antigens present in cardiac tissue are found in DCM

Response to Letter Regarding Article, “Cardiac Troponin I but Not Cardiac Troponin T Induces Severe Autoimmune Inflammation in the Myocardium”

BACKGROUND Cardiac troponins in blood are the most preferred markers of myocardial damage. The fact that they are normally not found in the circulation provides a high level of clinical sensitivity and specificity even when cardiac lesions are small. After myocardial injury, the troponins enter the circulation, where they can be used for diagnosis of acute coronary syndromes. Thus, the cardiac troponins are paramount for disease classification and risk stratification. However, little is known about the long-term effects of the released troponins on cardiac function. METHODS AND RESULTS In this study we prepared recombinant murine cardiac troponin I (mc-TnI) and murine cardiac troponin T and used them to immunize mice. We report that A/J mice immunized with mc-TnI developed severe inflammation of the myocardium with increased expression of inflammatory chemokines RANTES (regulated on activation normal T cell expressed and secreted), monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, T-cell activation gene 3, and eotaxin and chemokine receptors CCR1, CCR2, and CCR5. The inflammation was followed by cardiomegaly, fibrosis, reduced fractional shortening, and 30% mortality over 270 days. In contrast, mice immunized with murine cardiac troponin T or with the control buffer showed little or no inflammation and no death. Furthermore, we demonstrate that mice preimmunized with mc-TnI before left anterior descending coronary artery ligation showed greater infarct size, more fibrosis, higher inflammation score, and reduced fractional shortening. CONCLUSIONS Overall, our results show for the first time that provocation of an autoimmune response to mc-TnI induces severe inflammation in the myocardium followed by fibrosis and heart failure with increased mortality in mice.