Anna‐Maria Müller

Autoimmune response to cardiac troponin I (TnI) induces inflammation and fibrosis in the myocardium. High-mobility group box 1 (HMGB1) is a multifunctional protein that exerts proinflammatory activity by mainly binding to receptor for advanced glycation end products (RAGE). The involvement of the HMGB1-RAGE axis in the pathogenesis of inflammatory cardiomyopathy is yet not fully understood. Using the well-established model of TnI-induced experimental autoimmune myocarditis (EAM), we demonstrated that both local and systemic HMGB1 protein expression was elevated in wild-type (wt) mice after TnI immunization. Additionally, pharmacological inhibition of HMGB1 using glycyrrhizin or anti-HMGB1 antibody reduced inflammation in hearts of TnI-immunized wt mice. Furthermore, RAGE knockout (RAGE-ko) mice immunized with TnI showed no structural or physiological signs of cardiac impairment. Moreover, cardiac overexpression of HMGB1 using adeno-associated virus (AAV) vectors induced inflammation in the hearts of both wt and RAGE-ko mice. Finally, patients with myocarditis displayed increased local and systemic HMGB1 and soluble RAGE (sRAGE) expression. Together, our study highlights that HMGB1 and its main receptor, RAGE, appear to be crucial factors in the pathogenesis of TnI-induced EAM, because inhibition of HMGB1 and ablation of RAGE suppressed inflammation in the heart. Moreover, the proinflammatory effect of HMGB1 is not necessarily dependent on RAGE only. Other receptors of HMGB1 such as Toll-like receptors (TLRs) may also be involved in disease pathogenesis. These findings could be confirmed by the clinical relevance of HMGB1 and sRAGE. Therefore, blockage of one of these molecules might represent a novel therapeutic strategy in the treatment of autoimmune myocarditis and inflammatory cardiomyopathy.

Background: Immune checkpoint inhibitor (ICI) therapy is often accompanied by immune-related pathology, with an increasing occurrence of high-risk ICI-related myocarditis. Understanding the mechanisms involved in this side effect could enable the development of management strategies. In mouse models, immune checkpoints, such as PD-1 (programmed cell death protein 1), control the threshold of self-antigen responses directed against cardiac TnI (troponin I). We aimed to identify how the immunoproteasome, the main proteolytic machinery in immune cells harboring 3 distinct protease activities in the LMP2 (low-molecular-weight protein 2), LMP7 (low-molecular-weight protein 7), and MECL1 (multicatalytic endopeptidase complex subunit 1) subunit, affects TnI-directed autoimmune pathology of the heart. Methods: TnI-directed autoimmune myocarditis (TnI-AM), a CD4 + T-cell–mediated disease, was induced in mice lacking all 3 immunoproteasome subunits (triple-ip −/− ) or lacking either the gene encoding LMP2 and LMP7 by immunization with a cardiac TnI peptide. Alternatively, before induction of TnI-AM or after establishment of autoimmune myocarditis, mice were treated with the immunoproteasome inhibitor ONX 0914. Immune parameters defining heart-specific autoimmunity were investigated in experimental TnI-AM and in 2 cases of ICI-related myocarditis. Results: All immunoproteasome-deficient strains showed mitigated autoimmune-related cardiac pathology with less inflammation, lower proinflammatory and chemotactic cytokines, less interleukin-17 production, and reduced fibrosis formation. Protection from TnI-directed autoimmune heart pathology with improved cardiac function in LMP7 −/− mice involved a changed balance between effector and regulatory CD4 + T cells in the spleen, with CD4 + T cells from LMP7 − /− mice showing a higher expression of inhibitory PD-1 molecules. Blocked immunoproteasome proteolysis, by treatment of TLR2 (Toll-like receptor 2)–engaged and TLR7 (Toll-like receptor 7)/TLR8 (Toll-like receptor 8)–engaged CD14 + monocytes with ONX 0914, diminished proinflammatory cytokine responses, thereby reducing the boost for the expansion of self-reactive CD4 + T cells. Correspondingly, in mice, ONX 0914 treatment reversed cardiac autoimmune pathology, preventing the induction and progression of TnI-AM when self-reactive CD4 + T cells were primed. The autoimmune signature during experimental TnI-AM, with high immunoproteasome expression, immunoglobulin G deposition, interleukin-17 production in heart tissue, and TnI-directed humoral autoimmune responses, was also present in 2 cases of ICI-related myocarditis, demonstrating the activation of heart-specific autoimmune reactions by ICI therapy. Conclusions: By reversing heart-specific autoimmune responses, immunoproteasome inhibitors applied to a mouse model demonstrate their potential to aid in the management of autoimmune myocarditis in humans, possibly including patients with ICI-related heart-specific autoimmunity.

Cyclophilins are a group of highly conserved cytosolic enzymes that have a peptidylprolyl cis/trans isomerase activity. Cyclophilin A (CyPA) can be secreted in the extracellular space by inflammatory cells and upon cell death. The presence of CyPA in patients with non-ischemic cardiomyopathy is associated with poor clinical prognosis. Here, we investigated the inhibition of extracellular CyPA in a mouse model of troponin I-induced autoimmune myocarditis using the strictly extracellular CyPA-inhibitor MM284. Since A/J mice develop severe inflammation and fibrosis after immunization with murine cardiac troponin I (mcTn I), we used this model to analyze the effects of an extracellular CyPA inhibition. As extracellular CyPA-inhibitor we used the recently described CsA-derivate MM284. In vitro studies confirmed that MM284 inhibits CyPA-induced monocytic migration and adhesion. A/J mice immunized with mcTnI were treated with MM284 or vehicle every second day. After 28 days, we found a considerable reduction of myocardial injury and fibrosis. Further analysis revealed a reduced myocardial presence of T-cells and macrophages compared to control treated animals. Whereas MMP-9 expression was reduced significantly by MM284, we observed no significant reduction of inflammatory cytokines such as IL-6 or TNFα. Extracellular CyPA plays an important role in autoimmune myocarditis for myocardial damage and fibrosis. Our data suggest a new pharmacological approach for the treatment of myocardial inflammation and reduction of cardiac fibrosis by inhibition of extracellular CyPA.

Cardiovascular diseases are the leading cause of death in industrialized nations worldwide. Of all deaths resulting from cardiovascular diseases, 2% are caused by inflammatory heart disease; specifically, myocarditis. The etiology causing myocarditis still remains unclear. Both infectious and non-infectious factors are capable of triggering myocarditis. Acute myocarditis manifests itself in a variety of ways ranging from subclinical disease to sudden heart failure, as well as the occurrence of chest pain, palpitations, and syncope. Myocarditis can lead to dilated cardiomyopathy, this being the most frequent cause for heart transplantation. Since the underlying mechanism and the pathways behind the disease initiation and progression still need to be elucidated, the need for mouse models simulating the human disease is evident. Various mouse models are frequently used to study myocarditis. Inflammation of the myocardium as a result of infectious agents can be investigated with a widely used animal model where mice are infected with coxsackievirus B3. For autoimmune (non-viral) myocarditis, several mouse models (including induction with myosin or troponin I) have been established to better understand the role of autoantibodies and their influence on disease progression. With these different models, various phases of the disease can be investigated and these findings are used to develop more specific therapies that can be translated into the clinic as a "bench-to-bedside" approach.

BACKGROUND: Overexpression of interleukin-10 (IL-10) in murine CD11b(+) monocytes/macrophages via GMP-adapted mRNA-nucleofection was expected to improve clinical outcome and reduce adverse side effects in autoimmune myocarditis. This study represents the proof of principle for a novel anti-inflammatory therapy using overexpression of IL-10 in murine monocytes/macrophages by mRNA-nucleofection for the treatment of autoimmune myocarditis. METHODS AND RESULTS: Autoimmune myocarditis was induced in A/J mice by subcutaneous immunization with troponin I. CD11b(+) monocytes/macrophages were isolated from the peritoneum and IL-10 was overexpressed by mRNA-nucleofection. These cells were injected intravenously. Myocardial inflammation was assessed via histological and immunohistochemical examinations. Myocardial fibrosis was analyzed with Masson's trichrome staining. Antitroponin I antibodies were determined within the serum. Physical performance was evaluated using a running wheel and echocardiography. In vitro overexpression of IL-10 in CD11b(+) monocytes/macrophages resulted in a 7-fold increased production of IL-10 (n=3). In vivo higher levels of IL-10 and less inflammation were detected within the myocardium of treated compared with control mice (n=4). IL-10-treated mice showed lower antitroponin I antibodies (n=10) and a better physical performance (n=10). CONCLUSIONS: Application of IL-10-overexpressing CD11b(+) monocytes/macrophages reduced inflammation and improved physical performance in a murine model of autoimmune myocarditis. Thus, the use of genetically modified monocytes/macrophages facilitated a targeted therapy of local inflammation and may reduce systemic side effects. Because the nucleofection technique is GMP adapted, an in vivo use in humans seems basically feasible and the transfer to other inflammatory diseases seems likely.