Induction Of Experimental Autoimmune Myocarditis Research Articles

Lupeol alleviates autoimmune myocarditis by suppressing macrophage pyroptosis and polarization via PPARα/LACC1/NF-κB signaling pathway

BackgroundAutoimmune myocarditis, with increasing incidence and limited therapeutic strategies, is in urgent need to explore its underlying mechanisms and effective drugs. Pyroptosis is a programmed cell death that may contribute to the pathogenesis of myocarditis. Nonetheless, no direct evidence validated the role of pyroptosis in autoimmune myocarditis. Lupeol (Lup), a pentacyclic triterpene, possesses various biological activities such as antidiabetic properties. However, the effects of Lup on autoimmune myocarditis and pyroptosis remain unelucidated. PurposeThis study aimed to reveal the role of pyroptosis in autoimmune myocarditis and explore the protective effects of Lup, and its engaged mechanisms. MethodsThe experimental autoimmune myocarditis (EAM) mouse model was established by immunization with a fragment of cardiac myosin in Balb/c mice. Lup and MCC950 were administered after EAM induction. The protective effects were assessed by inflammation score, cardiac injury, chronic fibrosis, and cardiac function. Mechanistically, the effects of Lup on the M1 polarization and pyroptosis of macrophages were evaluated. Transcriptome sequencing and molecular docking were subsequently employed, and the underlying mechanisms of Lup were further explored in vitro with small interfering RNA and adenovirus. ResultsAdministration of Lup and MCC950 alleviated EAM progression. Western blotting and immunofluorescence staining identified macrophages as the primary cells undergoing pyroptosis. Lup inhibited the expression of pyroptosis-associated proteins in macrophages during EAM in a dose-dependent manner. Furthermore, Lup suppressed pyroptosis in both bone marrow-derived macrophages (BMDMs) and THP-1-derived macrophages in vitro. In addition, Lup inhibited the M1 polarization of macrophages both in vivo and in vitro. Mechanistically, the protective effects of Lup were demonstrated via the suppression of the nuclear factor-κΒ (NF-κB) signaling pathway. Transcriptome sequencing and molecular docking revealed the potential involvement of peroxisome proliferator-associated receptor α (PPARα). Subsequently, we demonstrated that Lup activated PPARα to reduce the expression level of LACC1, thereby inhibiting the NF-κB pathway and pyroptosis. ConclusionOur findings indicated the crucial role of macrophage pyroptosis in the pathogenesis of EAM. Lup ameliorated EAM by inhibiting the M1 polarization and pyroptosis of macrophages through the PPARα/LACC1/NF-κB signaling pathway. Thus, our results provided a novel therapeutic target and agent for myocarditis.

Effect of SARS-CoV-2 mRNA-Vaccine on the Induction of Myocarditis in Different Murine Animal Models.

In the course of the SARS-CoV-2 pandemic, vaccination safety and risk factors of SARS-CoV-2 mRNA-vaccines were under consideration after case reports of vaccine-related side effects, such as myocarditis, which were mostly described in young men. However, there is almost no data on the risk and safety of vaccination, especially in patients who are already diagnosed with acute/chronic (autoimmune) myocarditis from other causes, such as viral infections, or as a side effect of medication and treatment. Thus, the risk and safety of these vaccines, in combination with other therapies that could induce myocarditis (e.g., immune checkpoint inhibitor (ICI) therapy), are still poorly assessable. Therefore, vaccine safety, with respect to worsening myocardial inflammation and myocardial function, was studied in an animal model of experimentally induced autoimmune myocarditis. Furthermore, it is known that ICI treatment (e.g., antibodies (abs) against PD-1, PD-L1, and CTLA-4, or a combination of those) plays an important role in the treatment of oncological patients. However, it is also known that treatment with ICIs can induce severe, life-threatening myocarditis in some patients. Genetically different A/J (most susceptible strain) and C57BL/6 (resistant strain) mice, with diverse susceptibilities for induction of experimental autoimmune myocarditis (EAM) at various age and gender, were vaccinated twice with SARS-CoV-2 mRNA-vaccine. In an additional A/J group, an autoimmune myocarditis was induced. In regard to ICIs, we tested the safety of SARS-CoV-2 vaccination in PD-1-/- mice alone, and in combination with CTLA-4 abs. Our results showed no adverse effects related to inflammation and heart function after mRNA-vaccination, independent of age, gender, and in different mouse strains susceptible for induction of experimental myocarditis. Moreover, there was no worsening effect on inflammation and cardiac function when EAM in susceptible mice was induced. However, in the experiments with vaccination and ICI treatment, we observed, in some mice, low elevation of cardiac troponins in sera, and low scores of myocardial inflammation. In sum, mRNA-vaccines are safe in a model of experimentally induced autoimmune myocarditis, but patients undergoing ICI therapy should be closely monitored when vaccinated.

LNA oligonucleotide mediates an anti-inflammatory effect in autoimmune myocarditis via targeting lactate dehydrogenase B.

Treatment of myocarditis is often limited to symptomatic treatment due to unknown pathomechanisms. In order to identify new therapeutic approaches, the contribution of locked nucleic acid antisense oligonucleotides (LNA ASOs) in autoimmune myocarditis was investigated. Hence, A/J mice were immunized with cardiac troponin I (TnI) to induce experimental autoimmune myocarditis (EAM) and treated with LNA ASOs. The results showed an unexpected anti‐inflammatory effect for one administered LNA ASO MB_1114 by reducing cardiac inflammation and fibrosis. The target sequence of MB_1114 was identified as lactate dehydrogenase B (mLDHB). For further analysis, mice received mLdhb‐specific GapmeR during induction of EAM. Here, mice receiving the mLdhb‐specific GapmeR showed increased protein levels of cardiac mLDHB and a reduced cardiac inflammation and fibrosis. The effect of increased cardiac mLDHB protein level was associated with a downregulation of genes of reactive oxygen species (ROS)‐associated proteins, indicating a reduction in ROS. Here, the suppression of murine pro‐apoptotic Bcl‐2‐associated X protein (mBax) was also observed. In our study, an unexpected anti‐inflammatory effect of LNA ASO MB_1114 and mLdhb‐specific GapmeR during induction of EAM could be demonstrated in vivo. This effect was associated with increased protein levels of cardiac mLDHB, mBax suppression and reduced ROS activation. Thus, LDHB and LNA ASOs may be considered as a promising target for directed therapy of myocarditis. Nevertheless, further investigations are necessary to clarify the mechanism of action of anti‐inflammatory LDHB‐triggered effects.

Sex-Specific Differences of the Inflammatory State in Experimental Autoimmune Myocarditis.

Increasing evidence suggests male sex as a potential risk factor for a higher incidence of cardiac fibrosis, stronger cardiac inflammation, and dilated cardiomyopathy (DCM) in human myocarditis. Chronic activation of the immune response in myocarditis may trigger autoimmunity. The experimental autoimmune myocarditis (EAM) model has been well established for the study of autoimmune myocarditis, however the role of sex in this pathology has not been fully explored. In this study, we investigated sex differences in the inflammatory response in the EAM model. We analyzed the cardiac function, as well as the inflammatory stage and fibrosis formation in the heart of EAM male and female rats. 21 days after induction of EAM, male EAM rats showed a decreased ejection fraction, stroke volume and cardiac output, while females did not. A significantly elevated number of infiltrates was detected in myocardium in both sexes, indicating the activation of macrophages following EAM induction. The level of anti-inflammatory macrophages (CD68+ ArgI+) was only significantly increased in female hearts. The expression of Col3A1 and fibrosis formation were more prominent in males. Furthermore, prominent pro-inflammatory factors were increased only in male rats. These findings indicate sex-specific alterations in the inflammatory stage of EAM, with a pro-inflammatory phenotype appearing in males and an anti-inflammatory phenotype in females, which both significantly affect cardiac function in autoimmune myocarditis.

Single-Cell RNA Sequencing to Dissect the Immunological Network of Autoimmune Myocarditis.

Myocarditis can develop into dilated cardiomyopathy, which may require heart transplantation. The immunological network of myocarditis phases remains unknown. This study aimed to investigate the immunological network during the transition from myocarditis to cardiomyopathy and to identify the genes contributing to the inflammatory response to myocarditis. Mice were treated with myosin heavy chain-α peptides to generate an experimental autoimmune myocarditis (EAM) model. We performed single-cell RNA sequencing analysis of Cd45+ cells extracted from mouse hearts during different EAM phases, including normal control, acute inflammatory, subacute inflammatory, and myopathy phases. Human heart tissues were collected from the surgically removed hearts of patients who had undergone heart transplantation. We identified 26 cell subtypes among 34 665 cells. Macrophages constituted the main immune cell population at all disease phases (>60%), and an inflammation-associated macrophage cluster was identified in which the expression of Hif1a-regulated genes was upregulated. The neutrophil population was increased after the induction of EAM, and neutrophils then released Il-1 to participate in the EAM process. T cells were observed at the highest percentage at the subacute inflammatory phase. T-helper 17 cells, in which the expression of Hif1a-regulated genes was upregulated, constituted the main T-cell population detected at the acute inflammatory phase, whereas regulatory T cells were the main T-cell population detected at the subacute inflammatory phase, and γδ T cells releasing Il-17 were the main T-cell population observed at the myopathy phase. Moreover, the Hif1a expression level correlated with the extent of inflammation. In addition, PX-478 could alleviate the inflammatory responses of the different EAM phases. Last, HIF1A was expressed at higher levels in patients with acute autoimmune myocarditis than in patients with dilated cardiomyopathy and healthy control subjects. We present here a comprehensive single-cell landscape of the cardiac immune cells in different EAM phases. In addition, we elucidate the contribution of Hif1a to the inflammatory response through the regulation of immune cell activity, particularly of macrophage cluster 2 and T-helper 17 cells. Moreover, an Hif1a inhibitor alleviated inflammatory cell infiltration of the EAM model and may serve as a potential therapeutic target in the clinic.

Myocarditis Elicits Dendritic Cell and Monocyte Infiltration in the Heart and Self-Antigen Presentation by Conventional Type 2 Dendritic Cells.

Autoimmune myocarditis often leads to dilated cardiomyopathy (DCM). Although T cell reactivity to cardiac self-antigen is common in the disease, it is unknown which antigen presenting cell (APC) triggers autoimmunity. Experimental autoimmune myocarditis (EAM) was induced by immunizing mice with α-myosin loaded bone marrow APCs cultured in GM-CSF. APCs found in such cultures include conventional type 2 CD11b+ cDCs (GM-cDC2s) and monocyte-derived cells (GM-MCs). However, only α-myosin loaded GM-cDC2s could induce EAM. We also studied antigen presenting capacity of endogenous type 1 CD24+ cDCs (cDC1s), cDC2s, and MCs for α-myosin-specific TCR-transgenic TCR-M CD4+ T cells. After EAM induction, all cardiac APCs significantly increased and cDCs migrated to the heart-draining mediastinal lymph node (LN). Primarily cDC2s presented α-myosin to TCR-M cells and induced Th1/Th17 differentiation. Loss of IRF4 in Irf4fl/fl.Cd11cCre mice reduced MHCII expression on GM-cDC2s in vitro and cDC2 migration in vivo. However, partly defective cDC2 functions in Irf4fl/fl.Cd11cCre mice did not suppress EAM. MCs were the largest APC subset in the inflamed heart and produced pro-inflammatory cytokines. Targeting APC populations could be exploited in the design of new therapies for cardiac autoimmunity.

Galectin-3 deficiency enhances type 2 immune cell-mediated myocarditis in mice.

Experimental autoimmune myocarditis (EAM) is a mouse model of immune-mediated myocarditis and cardiomyopathy. The role of Galectin-3 (Gal-3), a β-galactoside-binding lectin, in autoimmune myocarditis has not been studied. Therefore, the aim of this study was to delineate the role of Gal-3 in myosin peptide-induced autoimmune myocarditis in mice. EAM was induced in relatively resistant C57BL/6J mice (wild type, WT) and in mice with a targeted deletion of Gal-3 gene (Gal-3KO) by immunization with myosin peptide MyHCα334-352. Gal-3KO mice developed more severe myocarditis and more pronounced heart hypertrophy than WT mice. Increased infiltration of CD45+ leucocytes, CD3+ T cells, F4/80+ macrophages, and eosinophils was observed in hearts of Gal-3KO mice compared to WT mice on day 21 after EAM induction. Moreover, hearts of Gal-3KO mice had more T helper type 2 (Th2) cells, alternatively activated M2 macrophages, higher amounts of IgG deposits, and higher serum levels of IL-4 and IL-33 than WT mice. Ablation of Gal-3 in Th1-dominant C57BL/6J mice that are relatively resistant to EAM resulted in more severe disease characterized by type 2 cardiac inflammation. The complex effects of Gal-3 on EAM progression might be important in the consideration of therapeutic options for the treatment of EAM.

Cardiosphere-Derived Cells Attenuate Inflammation, Preserve Systolic Function, and Prevent Adverse Remodeling in Rat Hearts With Experimental Autoimmune Myocarditis.

Cardiosphere-derived cells (CDCs) have yielded promising efficacy signals in early-phase clinical trials of ischemic and nonischemic cardiomyopathy. The potential efficacy of CDCs in acute myocarditis, an inflammatory cardiomyopathy without effective therapy, remains unexplored. Given that CDCs produce regenerative, cardioprotective, anti-inflammatory, and anti-fibrotic effects (all of which could be beneficial in acute myocarditis), we investigated the efficacy of intracoronary delivery of CDCs in a rat model of experimental autoimmune myocarditis. Lewis rats underwent induction of experimental autoimmune myocarditis by subcutaneous footpad injection of purified porcine cardiac myosin supplemented with Mycobacterium tuberculosis on days 1 and 7. On day 10, rats were randomly assigned to receive global intracoronary delivery of 500 000 CDCs or vehicle. Global intracoronary delivery was performed by injection of cells or vehicle into the left ventricular (LV) cavity during transient occlusion of the aortic root. Rats were euthanized 18 days after infusion. Cardiac volumes and systolic function were assessed by serial echocardiography, performed on days 1, 10, and 28. Myocardial inflammation, T-cell infiltration, and cardiac fibrosis were evaluated by histology. Experimental autoimmune myocarditis was successfully induced in 14/14 rats that completed follow-up. Left ventricular ejection fraction (LVEF) and volumes were comparable on days 1 and 10 between groups. CDC infusion resulted in increased LVEF (81.5% ± 3% vs 65.4% ± 8%, P < .001) and decreased LV end-systolic volume (43 ± 15 vs 100 ± 24 μL, P < .001) compared to placebo administration at 18 days post-infusion. Cardiosphere-derived cell infusion decreased myocardial inflammation (7.4% ± 7% vs 20.7% ± 4% of myocardium, P = .007), cardiac fibrosis (16.6% ± 13% vs 38.1% ± 3% of myocardium, P = .008), and myocardial T-cell infiltration (30.4 ± 29 vs 125.8 ± 49 cells per field, P = .005) at 18 days post-infusion compared to placebo administration. Intracoronary delivery of CDCs attenuates myocardial inflammation, T-cell infiltration, and fibrosis while preventing myocarditis-induced systolic dysfunction and adverse remodeling in rats with experimental autoimmune myocarditis.

Sympathetic nerve blocks promote anti-inflammatory response by activating the JAK2-STAT3–mediated signaling cascade in rat myocarditis models: A novel mechanism with clinical implications

Left stellectomy has become an important therapeutic option for patients with potentially fatal arrhythmias. However, the antiarrhythmic mechanism of left stellectomy is not well known. The cholinergic anti-inflammatory pathway (CAIP) is a complex immune mechanism that regulates peripheral inflammatory responses. The purpose of this study was to evaluate the effect of left stellectomy on CAIP using rat experimental autoimmune myocarditis (EAM) models. EAM was produced by injecting 2 mg of porcine cardiac myosin into the footpads of rats. Left stellectomy was performed before EAM induction. We evaluated the effect of left stellectomy on arrhythmic events, survival, inflammation, and CAIP in rats without and with EAM. Left stellectomy prevented arrhythmia and improved survival in EAM rats. Left stellectomy decreased the levels of tumor necrosis factor α, interleukin 6, and high mobility group box 1 (P< .05 vs EAM) in serum and heart tissues from EAM rats. In heart rate variability analysis, high-frequency peaks of the power spectrumdensities, reflecting parasympathetic cardiovagal tone, were significantly decreased in EAM rats, but increased after left stellectomy. The ratios of phosphorylated STAT3/STAT3 (signal transducer and activator of transcription 3) and phosphorylated JAK2/JAK2 (Janus kinase 2) decreased in cell lysates of the spleen, liver, and heart in EAM rats. However, the same ratios significantly increased after left stellectomy. Nuclear factor κB in cell lysates of the spleen, liver, and heart increased in EAM rats, but decreased after left stellectomy. In EAM models, left stellectomy increased survival of the rats while showing antiarrhythmic effects with reduced inflammation via activation of the JAK2-STAT3-mediated signaling cascade. Our findings suggest an exciting opportunity to develop new and novel therapeutics to attenuate cardiac inflammation.

Adult murine cardiomyocytes exhibit regenerative activity with cell cycle reentry through STAT3 in the healing process of myocarditis

Mammalian cardiomyocytes substantially lose proliferative capacity immediately after birth, limiting adult heart regeneration after injury. However, clinical myocarditis appears to be self-limiting with tissue-reparative properties. Here, we investigated the molecular mechanisms underlying the recovery from myocarditis with regard to cardiomyocyte proliferation using an experimental autoimmune myocarditis (EAM) model. Three weeks after EAM induction (EAM3w), cardiac tissue displayed infiltration of inflammatory cells with cardiomyocyte apoptosis. However, by EAM5w, the myocardial damage was remarkably attenuated, associated with an increase in cardiomyocytes that were positively stained with cell cycle markers at EAM3w. Cardiomyocyte fate mapping study revealed that the proliferating cardiomyocytes primarily derived from pre-existing cardiomyocytes. Signal transducer and activator of transcription 3 (STAT3) was robustly activated in cardiomyocytes during inflammation, accompanied by induction of interleukin-6 family cytokines. Cardiomyocyte-specific ablation of STAT3 gene suppressed the frequency of cycling cardiomyocytes in the recovery period without influencing inflammatory status, resulting in impaired tissue repair and cardiac dysfunction. Finally, microarray analysis revealed that the expression of regeneration-related genes, metallothioneins and clusterin, in cardiomyocytes was decreased by STAT3 gene deletion. These data show that adult mammalian cardiomyocytes restore regenerative capacity with cell cycle reentry through STAT3 as the heart recovers from myocarditis-induced cardiac damage.

Eosinophil-derived IL-4 drives progression of myocarditis to inflammatory dilated cardiomyopathy.

Inflammatory dilated cardiomyopathy (DCMi) is a major cause of heart failure in children and young adults. DCMi develops in up to 30% of myocarditis patients, but the mechanisms involved in disease progression are poorly understood. Patients with eosinophilia frequently develop cardiomyopathies. In this study, we used the experimental autoimmune myocarditis (EAM) model to determine the role of eosinophils in myocarditis and DCMi. Eosinophils were dispensable for myocarditis induction but were required for progression to DCMi. Eosinophil-deficient ΔdblGATA1 mice, in contrast to WT mice, showed no signs of heart failure by echocardiography. Induction of EAM in hypereosinophilic IL-5Tg mice resulted in eosinophilic myocarditis with severe ventricular and atrial inflammation, which progressed to severe DCMi. This was not a direct effect of IL-5, as IL-5TgΔdblGATA1 mice were protected from DCMi, whereas IL-5-/- mice exhibited DCMi comparable with WT mice. Eosinophils drove progression to DCMi through their production of IL-4. Our experiments showed eosinophils were the major IL-4-expressing cell type in the heart during EAM, IL-4-/- mice were protected from DCMi like ΔdblGATA1 mice, and eosinophil-specific IL-4 deletion resulted in improved heart function. In conclusion, eosinophils drive progression of myocarditis to DCMi, cause severe DCMi when present in large numbers, and mediate this process through IL-4.

The neuropeptide cortistatin attenuates experimental autoimmune myocarditis via inhibition of cardiomyogenic T cell-driven inflammatory responses.

Myocarditis is an inflammatory and autoimmune cardiovascular disease that causes dilated myocardiopathy and is responsible for high morbidity and mortality worldwide. Cortistatin is a neuropeptide produced by neurons and cells of the immune and vascular systems. Besides its action in locomotor activity and sleep, cortistatin inhibits inflammation in different experimental models of autoimmune diseases. However, its role in inflammatory cardiovascular disorders is unexplored. Here, we investigated the therapeutic effects of cortistatin in a well-established preclinical model of experimental autoimmune myocarditis (EAM). We induced EAM by immunization with a fragment of cardiac myosin in susceptible Balb/c mice. Cortistatin was administered i.p. starting 7, 11 or 15days after EAM induction. At day 21, we evaluated heart hypertrophy, myocardial injury, cardiac inflammatory infiltration and levels of serum and cardiac inflammatory cytokines, cortistatin and autoantibodies. We determined proliferation and cytokine production by heart draining lymph node cells in response to cardiac myosin restimulation. Systemic injection of cortistatin during the effector phase of the disease significantly reduced its prevalence and signs of heart hypertrophy and injury (decreased the levels of brain natriuretic peptide) and impaired myocardial inflammatory cell infiltration. This effect was accompanied by a reduction in self-antigen-specific T-cell responses in lymph nodes and in the levels of cardiomyogenic antibodies and inflammatory cytokines in serum and myocardium. Finally, we found a positive correlation between cardiac and systemic cortistatin levels and EAM severity. Cortistatin emerges as a new candidate to treat inflammatory dilated cardiomyopathy.

Abstract 403: Critical Role for Dipeptidyl Peptidase-4 Activity in the Pathogenesis of Experimental Autoimmune Myocarditis

Autoimmune myocarditis is the most common cause of heart failure among young adults and is a major precursor of dilated cardiomyopathy. We have shown that dipeptidyl peptidase-4 (DPP-4) plays a detrimental role in the pathogenesis of experimental autoimmune myocarditis (EAM) in mice. To elucidate how DPP-4 induces cardiac dysfunction in the EAM heart, mouse EAM models were given normal diet (CONT-group, N = 6), or a diet mixed with linagliptin, a potent DPP-4 inhibitor (83 mg/kg chow corresponding to around 3 mg/kg oral dosing) (LINA-group, N = 6). After 21-day of EAM induction, left ventricular ejection fraction was significantly higher in LINA-group than in CONT-group (72.6 ± 6.6%* vs. 60.1 ± 9.2%, * P &lt; 0.05). Immunostaining demonstrated that the number of RORγt-positive Th17 cells, a subset of T-lymphocytes expressing high level of enzymatically active DPP-4, infiltrated to the EAM hearts was significantly smaller in LINA-group than in CONT-group (46.9 ± 2.4 RFU* vs. 116.8 ± 8.6 RFU, * P &lt; 0.05). Consistently, the activity of DPP-4 in the EAM hearts was significantly lower in LINA-group than in CONT-group. Mass spectrometry analysis using lysates from the EAM hearts co-immunoprecipitated with Flag-DPP-4 recombinant protein demonstrated that DPP-4 interacts with cathepsin-G (CTSG), a plasma membrane-bound serine protease, in the EAM hearts. Co-immunoprecipitation assay verified the physical interaction between CTSG and DPP-4. The CTSG activity in the EAM hearts was markedly elevated, and treatment with linagliptin effectively suppressed the CTSG activity in the EAM hearts. We also found that DPP-4 significantly suppressed the activity of α1-antichymotrypsin, a protease which can catalyzes CTSG and is activated in response to EAM. Finally, we demonstrated that the level of angiotensin II, a major product catalyzed by CTSG, in the EAM hearts was significantly decreased in LINA-group than in CONT-group. Thus, these results suggest that DPP-4 expressing on the surface of Th17 cells physically interacts with CTSG, thereby enhancing CTSG activity by suppressing α1-antichymotrypsin, which, in turn, promoting the accumulation of angiotensin II, in the EAM hearts. In conclusion, DPP-4 derived from Th17 cells aggravates cardiac dysfunction during EAM.

The eotaxin-CCR3 pathway is required for eosinophil trafficking to the heart in eosinophilic myocarditis

Abstract Cardiac manifestations are a major cause of morbidity and mortality in patients with eosinophil-associated diseases. Eosinophils have been proposed to play a pathologic role in the heart. Nevertheless, the pathways that recruit eosinophils to the heart have not been described. In IFNγ−/− IL-17A−/− mice, induction of experimental autoimmune myocarditis results in a Th-2-driven, eosinophilic inflammation. IFNγ−/− IL-17A−/− mice had much higher expression of the eosinophil-attracting chemokines eotaxin-1 (Ccl11) and eotaxin-2 (Ccl24) in the heart than wildtype mice. Genetic ablation of the eotaxin receptor CCR3 resulted in a dramatic decrease in heart infiltrating eosinophils. Adoptive transfer experiments with CCR3+/+ or CCR3−/− eosinophils into eosinophil-deficient ΔdblGATA1 or IFNγ−/− IL-17A−/− ΔdblGATA1 recipients showed that two conditions have to be met for efficient eosinophil trafficking to the inflamed heart: high eotaxin expression in the heart and expression of CCR3 by eosinophils. We identified the source of cardiac eotaxins by RT-PCR of FACS sorted heart cells and by immunohistochemistry. Eotaxin-1 was mainly produced by cardiac fibroblasts with interstitial localization in the heart. In vitro culture of cardiac fibroblasts with IL-4 and IL-13 induced eotaxin-1 expression. In contrast, eotaxin-2 was expressed by multiple inflammatory cell types, located at inflammatory foci, and substantial expression was only found in mice lacking IFNγ and IL-17A during myocarditis. In conclusion, eosinophil trafficking to the heart is dependent on the eotaxin-CCR3 pathway in mice with eosinophilic myocarditis. Blockade of this pathway may be a useful therapeutic approach to prevent eosinophil-mediated heart damage.

Abstract 14911: Dipeptidyl Peptidase-4 Deteriorates Cardiac Function During Autoimmune Myocarditis by Facilitating Cathepsin-G Activity

Post-myocarditis dilated cardiomyopathy is the most common cause of non-hereditary heart failure developing at a young age. Our colleagues have shown previously that dipeptidyl peptidase-4 (DPP-4) worsens the development of experimental autoimmune myocarditis (EAM) by promoting expression of inflammatory cytokines and cardiac fibrosis in mice. In order to clarify how DPP-4 mediates this detrimental effect in the EAM heart, mouse EAM model generated by immunizing with α-MHC peptide in BALB/c mice were given a diet mixed with linagliptin, a selective DPP-4 inhibitor (83 mg/kg chow corresponding to around 3 mg/kg oral dosing) (LINA group, N = 6), or normal diet (CONT group, N = 6). After 21 days of EAM induction, lung weight / body weight were significantly smaller in LINA group than in CONT group (7.7 ± 0.1 mg/g* vs. 8.3 ± 0.1 mg/g, *P &lt; 0.05). Left ventricular ejection fraction after induction of EAM was significantly higher in LINA group than in CONT group (72.6 ± 6.6%* vs. 60.1 ± 9.2%, *P &lt; 0.05). Immunostaining demonstrated that the number of RORγt-positive Th17 cells infiltrated to inflamed myocardium was significantly smaller in LINA group than in CONT group. Consistent with this result and the previous finding that Th17 cells express high level of enzymatically active DPP-4, the DPP-4 activity in inflamed myocardium was significantly lower in LINA group than in CONT group (46.9 ± 2.4 RFU* vs. 116.8 ± 8.6 RFU, *P &lt; 0.05). Mass spectrometry analysis using lysates from inflamed myocardium co-immunoprecipitated with Flag-DPP-4 recombinant protein revealed that DPP-4 bound to cathepsin-G (CTSG), a plasma membrane-bound serine protease, in the EAM heart. Co-immunoprecipitation - western blot analysis verified the physical interaction between DPP-4 and CTSG. The activity of CTSG in inflamed myocardium was significantly higher in EAM mice compared to those in untreated mice, and CTSG activity in the EAM heart was significantly lower in LINA group than in CONT group. The amount of angiotensin II, a major product catalyzed by CTSG, in the EAM heart was also significantly lower in LINA group than in CONT group. Collectively, these results suggest that DPP-4 derived from Th17 cells aggravates cardiac dysfunction during EAM, possibly through enhancing CTSG activity in the heart.

Dipeptidyl Peptidase-4 Suppression Leads to Attenuation of Cardiac Dysfunction Caused by Autoimmune Myocarditis Through Inhibiting Cathepsin-G Activity

We have shown previously that dipeptidyl peptidase-4 (DPP-4) plays a critical role in the development of experimental autoimmune myocarditis(EAM). Here we show that the mechanism how DPP-4 promotes the progression of EAM in mice. Mouse EAM model were given a diet mixed with linagliptin, a DPP-4 inhibitor (3mg/kg/day)(LINA-group), or normal diet (CONT-group). Left ventricular ejection fraction after 21 days of EAM induction was significantly higher in LINAgroup than in CONT group. Immunostaining showed that the number of Th17 cells infiltrated to the EAM heart was significantly smaller in LINA-group than in CONT-group. Consistent with this finding and the fact that Th17 cells express high level of active DPP-4, the activity of DPP-4 in the EAM heart was significantly lower in LINA-group than in CONT-group. Mass spectrometry and co-immunoprecipitation-western blot analyses revealed that DPP-4 physically interacts with cathepsin-G(CTSG) in inflamed myocardium. The CTSG activity in the EAM heart was significantly higher in EAM mice compared to those in untreated mice, and the activity of CTSG in inflamed myocardium was significantly lower in LINA-group than in CONT-group. The amount of angiotensin II, a product catalyzed by CTSG, in inflamed myocardium was also significantly lower in LINA-group than in CONT-group. In conclusion, suppression of DPP-4 causes a salutary effect in the cardiac function during EAM by inhibiting CTSG activity in the heart.

Commensal microbiota regulate inflammatory cardiac remodeling (BA6P.126)

Abstract In this study, we sought to understand how the microbiota regulates inflammatory mechanisms underlying cardiac autoimmunity. We employ an autoimmunization model of heart disease in mice, termed experimental autoimmune myocarditis (EAM). This disease model depends on CD4+ T cells for pathogenesis, immediately implying a cytokine product of these cells as a critical mediator of disease pathogenesis, and an attractive target for therapeutic intervention. Our laboratory has previously reported that Th17 cells are critical for the disease process. In order to establish a role for the commensal microbiota in autoimmune heart disease, we treated animals with a comprehensive polyantibiotic cocktail to induce antibiotic dysbiosis. Treating BALB/cJ animals with antibiotic limited the development of myocarditis, and furthermore prevented their progression to later heart failure. Segmented filamentous bacteria (SFB) represent a clade of Clostridia that have been found to elicit intestinal Th17 differentiation upon colonization, and subsequently modulate susceptibility to Th17-associated autoimmune disease. We colonized BALB/cJ mice with SFB and observed that these animals progress to diminished cardiac function following the induction of EAM. Microbiota manipulations were associated with differences in canonical Th17-associated programs, pointing to these pathways as an important mechanism in coupling commensal sensing to the progression of heart disease.

Novel link between angiotensin II and Wnt signalling in myocardial fibrogenesis in inflammatory cardiomyopathy

Rationale: Identification of genetic or epigenetic factors regulating the pathophysiology of inflammatory dilated cardiomyopathy (iDCM) might result in novel treatment strategies against inflammatory heart diseases. Angiotensin (Ang) II contributes to fibrogenesis through the production of pro-fibrotic factors and has been implicated in the pathogenesis of diverse cardiovascular diseases. Wnt signalling controls heart development, but on the other hand - plays a pivotal role in adult cardiac remodelling, mainly in the fibrosis formation. However, the link between Ang II and Wnt pathways and their particular roles in the progression of the iDCM remain unknown yet. Methods and results: Experimental autoimmune myocarditis (EAM), mimicking human iDCM, was induced in BALB/c wild-type (wt) and Agtr1-/- mice using alpha-myosin heavy chain peptide/complete Freund's adjuvant immunization. Healthy heart fibroblasts (HF) or heart-infiltrating inflammatory CD133+ progenitors (representing main source of cardiac myofibroblasts) were isolated at the peak of EAM, expanded and treated with Transforming Growth Factor-beta (TGF-β), Wnt proteins or their inhibitors. We demonstrated for the first time that Wnt signalling is activated in wt myocardium (within infiltrating inflammatory cells) in the acute phase and persist in TGF-β-mediated fibrotic phase of the EAM (mainly within myofibroblasts). Wt HF or heart-infiltrating inflammatory CD133+ progenitors upon stimulation with TGF-β differentiated into pathogenic myofibroblasts. This process is regulated by Wnt signalling. Blocking of Wnt activity with natural or chemical inhibitors abrogated myofibroblast differentiation of both, wt HF or inflammatory CD133+ progenitors in vitro, and in vivo- that prevented myocardial fibrosis formation. Moreover, we showed that Agtr1-/- mice develop similar extents of myocarditis, but with significantly reduced post-inflammatory fibrosis compared to wt animals. Specific chimera mice revealed that bone marrow cell compartment is indeed responsible for the fibrosis formation. Importantly, Wnt signalling was not activated in Agtr1-/- heart following the EAM induction. Furthermore, heart-infiltrating inflammatory CD133+ progenitors derived from Agtr1-/- inflamed heart showed significantly reduced TGF-β-mediated myofibroblast differentiation and lack of Wnt signalling activation. Conclusion: Our results clearly define Ang II and Wnt signalling as main mediators of myocardial fibrogenesis in the EAM model. We assume that specific targeting of Ang II/Wnt signalling components might be suitable for therapeutic interventions.

Abstract 296: Targeting Macrophage Subsets Attenuates Cardiac Remodeling and Dysfunction Following Experimental Myocarditis in Rat

Introduction: The mechanism of myocarditis is complex and poorly understood. Monocytes and macrophages comprise the majority of infiltrates in myocarditis. Hypothesis: We aimed to test the hypothesis that macrophage subsets have a key role in the pathogenesis and progression of myocarditis, and that targeting macrophages would modulate disease progression and outcome. Methods and Results: Experimental autoimmune myocarditis (EAM) was induced in 67 male Lewis rats. Of them, 27 were sacrificed at different time points to assess M1 and M2- macrophage subsets by flow cytometry. The remaining rats were randomized either to early macrophage depletion (from day 8-14 after EAM induction), by intravenous injections of clodronate-liposomes (CL), or late depletion (from day 15-35 after induction of EAM), or PBS-liposome injections (control). Left ventricula (LV) remodeling and function were assessed by echocardiography; before induction of EAM, before CL treatment, and 35 days after induction of EAM. Macrophages in the heart peaked at 21 days after induction of EAM (25±7%,p=0.0008).The number of M1 macrophages in the heart was highest at 14 days after EAM induction (16.3±2%), while M2 macrophages peaked at day 21 (15.5±2.7%,p=0.003). Early macrophage depletion (8-14d) decreased LV diastolic dimension and volume by 5.3±3.9% and 10.4±8% (p=0.04). Late macrophage depletion (15-35d) increased LV wall thickness and decreased LV systolic dimension and volume by 10.2±4% and 21±8% (p=0.03). Furthermore, late macrophage depletion increased LV ejection fraction and fractional shortening by 14.6±6% (p=0.01) and 22±10% (p=0.02) 35 days after EAM. Conclusions: Our study describes, for the first time, the kinetics of M1 and M2 macrophages in ocarditis and shows that macrophages contribute to the progression of adverse remodeling and dysfunction. Thus, targeting macrophages could be a new therapeutic strategy to improve cardiac remodeling and function in myocarditis.

Cathelicidin antimicrobial peptide inhibits fibroblast migration via P2X7 receptor signaling

Fibrosis is one of the most common pathological alterations in heart failure, and fibroblast migration is an essential process in the development of cardiac fibrosis. Experimental autoimmune myocarditis (EAM) is a model of inflammatory heart disease characterized by inflammatory cell infiltration followed by healing without residual fibrosis. However, the precise mechanisms mediating termination of inflammation and nonfibrotic healing remain to be elucidated. Microarray analysis of hearts from model mice at multiple time points after EAM induction identified several secreted proteins upregulated during nonfibrotic healing, including the anti-inflammatory cathelicidin antimicrobial peptide (CAMP). Treatment with LL-37, a human homolog of CAMP, activated MAP kinases in fibroblasts but not in cardiomyocytes, indicating that fibroblasts were the target of CAMP activity. In addition, LL-37 decreased fibroblast migration in the in vitro scratch assay. P2X7 receptor (P2X7R), a well-known receptor for LL-37, was involved in LL-37 mediated biological effect on cardiac fibroblasts. Stimulation of BzATP, a P2X7R agonist, activated MAPK in fibroblasts, whereas the P2X7R antagonist, BBG, as well as P2X7R deletion abolished both LL-37-mediated MAPK activation and LL-37-induced reduction in fibroblast migration. These results strongly suggest that CAMP upregulation during myocarditis prevents myocardial fibrosis by restricting fibroblast migration via activation of the P2X7R−MAPK signaling pathway.