Autoimmune Myocarditis Research Articles

Single cell RNA sequencing to dissect transcriptional signature of cardiac fibroblasts activation in experimental autoimmune myocarditis

Abstract Introduction Dilated cardiomyopathy (DCM) is a condition that evolves from inflammation of the myocardium, and it is often associated with a poor prognosis and fatal outcome. Experimental autoimmune myocarditis (EAM) represents a CD4+ T cell-dependent animal model of acute myocarditis followed by the development of post-inflammatory DCM. Purpose The aim of the study was to investigate the transcriptional changes in cardiac fibroblasts during disease progression in a mouse model of EAM at the single cell level. Methods 6–8-week-old BALB/c mice (or reporter mice expressing EGFP under the collagen type I promoter) were immunized with αMyHC peptide together with Complete Freunds’ Adjuvant. Live cardiac cells were sorted from healthy hearts (d0), at acute myocarditis (d19), during resolution of inflammation (d25), and at the DCM stage (d35) using the BD FACSAria II Cell Sorter. Single-cell cDNA libraries were prepared using 10x Genomics technology, and the sequencing was performed on an Illumina NovaSeq 6000 platform, targeting 50000 reads per cell. Single cell RNA sequencing data was analyzed using RStudio software and Seurat packages. At d0 and d19 of EAM, bulk RNAseq was performed on live cardiac EGFP+CD45-CD34- fibroblasts. Results Following a quality control check, we included in the analysis 25058 cells. We identified 13 distinct main cell populations, including fibroblasts, lymphoid and myeloid immune cells, endothelial cells, smooth muscle cells, and cardiomyocytes (Fig. 1A). Differential expression analysis revealed that fibroblasts in acute myocarditis (d19) showed activation of genes related to immune processes such as cytokine-cytokine receptor interaction, IL-17, and TNF-alpha signaling pathways. Subset analysis on 4901 cardiac fibroblasts resulted in 5 distinct fibroblast subsets, FB1–FB5 (Fig. 1B). A major fibroblast subset found in the heart (FB1) expanded during the course of EAM (Fig. 1C). Of note, cells expressing the myofibroblastic signature (FB4) were identified at d19 only (Fig. 1C). Fibroblast subset FB1 activated expression of proinflammatory genes, such as Ly6a, Ly6c1, Cxcl14, Ccl2, and Ccl7, particularly during the inflammatory phase of EAM (d19, Fig. 1D). Surprisingly, the expression of profibrotic genes, including Col1a1, Col3a1, and Postn in FB1, was downregulated during the progression of EAM (Fig. 1E). Bulk RNA sequencing analysis confirmed that cardiac fibroblasts in acute myocarditis (d19) activated immune processes (chemokine production), but also showed dysregulated expression of genes involved in cytoskeletal re-organization and extracellular matrix turnover. Conclusions Our data indicated that acute inflammation in the heart activated a pro-inflammatory transcriptional response in cardiac fibroblasts and induced a fibroblast-to-myofibroblast transition. Understanding changes in transcriptomic signatures on a single cell level can help to develop new therapies in inflammatory heart diseases.

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.

Abstract 16249: A Case of Myocarditis and Pneumonitis Associated With Pembrolizumab Therapy

Introduction: Immune checkpoint inhibitors (ICI) are a novel therapy that activates antitumor immunity but can have varied immune-related adverse effects. As ICI therapy is used to treat malignancy, cases of autoimmune myocarditis and heart failure increase, thought to be due to PD-1 inhibition, which may be potentially fatal. Case presentation: A 37 yo female with history of autoimmune hepatitis and stage III melanoma on Pembrolizumab (ninth cycle 1.5 months prior) presented with chest pain, tachycardia, fatigue, dyspnea, and fever. Lab studies were notable for elevated troponin, lactic acidosis, and disseminated intravascular coagulation (DIC). CT thorax showed asymmetric ground glass opacities, concerning for ICI pneumonitis. Echocardiogram showed an LVEF of 15% with global hypokinesis. She was treated for severe sepsis, DIC, and respiratory failure, temporarily requiring intra-aortic balloon pump. Heart failure guideline-directed medical therapy (GDMT) was started. Clinical presentation was concerning for acute myocarditis related to ICI therapy as a cause for new cardiomyopathy. A cardiac MRI (CMR) revealed subepicardial late gadolinium enhancement (LGE) along the basal inferior/inferolateral wall segments concerning for myocarditis. Corticosteroids with a prolonged taper were started. Endomyocardial biopsy of the septum (EMB) was negative. Her clinical status improved and repeat echo showed LVEF recovery up to 45%. Discussion: Endomyocardial biopsy (EMB) provides definitive diagnosis of myocarditis and is recommended (Class I) for new-onset heart failure with hemodynamic compromise and/or arrhythmias. Although our patient’s EMB was negative (likely due to limitation of sample location), she met the 2013 ESC guideline diagnostic criteria for myocarditis with elevated troponin, functional and structural abnormalities on cardiac imaging. With increased utilization of ICI therapy in the management of aggressive malignancies, it is important to monitor for potentially adverse effects such as autoimmune myocarditis. Standard treatment is prompt initiation of corticosteroids, though newer studies report successful treatment with immunomodulators, such as abatacept and ruxolitinib, if steroids are unsuccessful.

Abstract 13808: Investigating Autophagy in Experimental Autoimmune Myocarditis Using Single Cell RNA Sequencing and Spatial Transcriptomics

Background: Autophagy has emerged as an important process in the context of heart failure. Experimental autoimmune myocarditis (EAM) represents an animal model of acute myocarditis (d19) and post-inflammatory dilated cardiomyopathy (d35). Aim: To explore autophagy dynamics in a mouse model of EAM at the single cell level. Methods: 6-8-week-old BALB/c mice were immunized with αMyHC peptide and complete Freund’s adjuvant. Single-cell RNA (scRNA) libraries were prepared using 10x Genomics technology and sequenced, targeting 50’000 reads/cell. For spatial transcriptomics, snap-frozen hearts embedded in OTC were sectioned into 10 μm-thick fragments and placed on 10x Genomics Visium Gene Expression Slide and processed using 10x Genomics Visium protocols. The data were analyzed using RStudio software. LC3B and Beclin-1 proteins were detected by IHC. Results: After quality control check, we included in the RNAseq analysis 25’058 cells clustering in 11 distinct cell populations (Fig. 1A). Expression of autophagic genes Atg7 and Becn1 was relatively consistent across all cell populations, whereas Atg3 and Map1lc3b (encoding LC3B) showed high expression specifically in monocytes. Analysis of monocytes indicated up-regulation of Atg3, Atg7 and Becn1 during the progression of EAM. Spatial transcriptomics analysis identified 5911 spots under the tissue and results showed an up-regulation of Atg3, Atg7, Becn1 and Map1lc3b in the hearts during the inflammatory phase of EAM. This increased expression was found throughout the entire heart section (Fig. 1B). Moreover, we detected the presence of LC3B and Beclin-1 proteins specifically localized within the inflammatory compartment of inflamed hearts (d19). Conclusions: In the EAM model, acute inflammation in the heart is associated with activated autophagy. Targeting this autophagic response could potentially improve disease outcomes. Financed by the National Science Centre (Poland), grant 2019/35/B/NZ5/00551

ET-1 receptor type B (ETBR) overexpression associated with ICAM-1 downregulation leads to inflammatory attenuation in experimental autoimmune myocarditis.

An experimental autoimmune myocarditis rat model was established by subcutaneous injection of porcine myocardial myosin (PCM). The effect of ET-1 receptor type B (ETBR) overexpression on autoimmune myocarditis was observed via tail vein injection of ETBR overexpression lentivirus in rats. We further investigated the mechanisms involved in the regulation of autoimmune myocarditis by ETBR overexpression. Six rats were randomly selected from 24 male Lewis rats as the NC group, and the remaining 18 rats were injected with PCM on Day 0 and Day 7, to establish the experimental autoimmune myocarditis (EAM) rat model. The 18 rats initially immunized were randomly divided into three groups: the EAM group, ETBR-oe group, and GFP group. On Day 21 after the initial immunization of rats, cardiac echocardiography and serum brain natriuretic peptide (BNP) analysis were performed to evaluate cardiac function, myocardial tissue HE staining was performed to assess myocardial tissue inflammatory infiltration and the myocarditis score, and mRNA expression of IFN-γ, IL-12, and IL-17 was detected by qRT-PCR. Subsequently, immunohistochemical analysis was performed to detect the localization and expression of the ETBR and ICAM-1 proteins, and the expression of ETBR and ICAM-1 was verified by qRT-PCR and western blotting methods. On Day 21 after initial immunization, left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVEDs), and serum BNP concentrations increased in the hearts of rats in the EAM group compared with the NC group (P < 0.01), and ejection fraction (EF) and fractional shortening (FS) decreased compared with those of the normal control (NC) group (P < 0.01). LVEDd, LVEDs, and serum BNP concentrations decreased in the ETBR-oe group compared with the EAM group, while EF and FS increased significantly (P < 0.01). HE staining showed that a large number of inflammatory cell infiltrates, mainly lymphocytes, were observed in the EAM group, and the myocarditis score was significantly higher than that of the NC group (P < 0.01). Compared with that of the EAM group, myocardial tissue inflammatory cell infiltration was significantly reduced in the ETBR-oe group, and the myocarditis scores were significantly lower (P < 0.01). The mRNAs of the inflammatory factors IFN-γ, IL-12 and IL-17 in myocardial tissue of rats in the EAM group exhibited elevated levels compared with those of the NC group (P < 0.01) while the mRNAs of IFN-γ, IL-12 and IL-17 were significantly decreased in the ETBR-oe group compared with the EAM group (P < 0.01). Immunohistochemistry showed that the staining depth of ETBR protein in myocardial tissue was greater in the EAM group than in the NC group, and significantly greater in the ETBR-oe group than in the EAM group, while the staining depth of ICAM-1 was significantly greater in the EAM group than in the NC group, and significantly lower in the ETBR-oe group than in the EAM group. The ICAM-1 expression level was significantly higher in the EAM group than in the NC group (P < 0.01), and was significantly lower in the ETBR-oe groupthan in the EAM group (P < 0.01).

MicroRNAs in Myocarditis-Review of the Preclinical In Vivo Trials.

Myocarditis is an inflammatory heart disease with viruses as the most common cause. Regardless of multiple studies that have recently been conducted, the diagnostic options still need to be improved. Although endomyocardial biopsy is known as a diagnostic gold standard, it is invasive and, thus, only sometimes performed. Novel techniques of cardiac magnetic resonance are not readily available. Therapy in viral infections is based mainly on symptomatic treatment, while steroids and intravenous immunoglobulins are used in autoimmune myocarditis. The effectiveness of neither of these methods has been explicitly proven to date. Therefore, novel diagnostic and therapeutic strategies are highly needed. MiRNAs are small, non-coding molecules that regulate fundamental cell functions, including differentiation, metabolism, and apoptosis. They present altered levels in different diseases, including myocarditis. Numerous studies investigating the role of miRNAs in myocarditis have already been conducted. In this review, we discussed only the original preclinical in vivo research. We eventually included 30 studies relevant to the discussed area. The altered miRNA levels have been observed, including upregulation and downregulation of different miRNAs in the mice models of myocarditis. Furthermore, the administration of mimics or inhibitors of particular miRNAs was shown to significantly influence inflammation, morphology, and function of the heart and overall survival. Finally, some studies presented prospective advantages in vaccine development.

PCSK9 inhibition ameliorates experimental autoimmune myocarditis by reducing Th17 cell differentiation through LDLR/STAT-3/ROR-γt pathway

Proprotein convertase subtilisin kexin type 9 (PCSK9) was characterized as a protein regulating circulating cholesterol metabolism; however, recent studies demonstrated a role for PCSK9 in inflammatory and autoimmune diseases unrelated to cholesterol alterations. The implication of PCSK9 in myocarditis is unclear and we aim at investigating the roles and mechanisms of PCSK9 in myocarditis. Male BALB/c mice received subcutaneous immunization with MyHC-α peptide on days 0 and 7 to establish the experimental autoimmune myocarditis (EAM) model. PCSK9 inhibitor, evolocumab, was administered subcutaneously once a week starting on day 0 and all mice were euthanized on day 21. Our results showed that PCSK9 inhibition ameliorated the cardiac inflammation of EAM mice. PCSK9 inhibition reduced both the levels of cardiac and peripheral blood PCSK9. We found that CD4+ T cells, CD8+ T cells, macrophages, and cardiomyocytes in the heart of EAM mice could express PCSK9. PCSK9 inhibition decreased the differentiation of cardiac Th17 cells by lowering ROR-γt levels but had no effects on Th1, Th2, and Treg cell differentiation. In vitro experiments of CD4+ T cells, we found that PCSK9 directly promoted Th17 cell differentiation through LDLR/STAT3/ROR-γt pathway. Collectively, we demonstrated that PCSK9 inhibition ameliorated the severity of EAM mice by reducing Th17 cell differentiation. PCSK9 is a promising target for treating myocarditis.

ANGPTL2 promotes immune checkpoint inhibitor-related murine autoimmune myocarditis

Use of immune checkpoint inhibitors (ICIs) as cancer immunotherapy advances rapidly in the clinic. Despite their therapeutic benefits, ICIs can cause clinically significant immune-related adverse events (irAEs), including myocarditis. However, the cellular and molecular mechanisms regulating irAE remain unclear. Here, we investigate the function of Angiopoietin-like protein 2 (ANGPTL2), a potential inflammatory mediator, in a mouse model of ICI-related autoimmune myocarditis. ANGPTL2 deficiency attenuates autoimmune inflammation in these mice, an outcome associated with decreased numbers of T cells and macrophages. We also show that cardiac fibroblasts express abundant ANGPTL2. Importantly, cardiac myofibroblast-derived ANGPTL2 enhances expression of chemoattractants via the NF-κB pathway, accelerating T cell recruitment into heart tissues. Our findings suggest an immunostimulatory function for ANGPTL2 in the context of ICI-related autoimmune inflammation and highlight the pathophysiological significance of ANGPTL2-mediated cardiac myofibroblast/immune cell crosstalk in enhancing autoimmune responses. These findings overall provide insight into mechanisms regulating irAEs.

Background and roles: myosin in autoimmune diseases.

The myosin superfamily is a group of molecular motors. Autoimmune diseases are characterized by dysregulation or deficiency of the immune tolerance mechanism, resulting in an immune response to the human body itself. The link between myosin and autoimmune diseases is much more complex than scientists had hoped. Myosin itself immunization can induce experimental autoimmune diseases of animals, and myosins were abnormally expressed in a number of autoimmune diseases. Additionally, myosin takes part in the pathological process of multiple sclerosis, Alzheimer's disease, Parkinson's disease, autoimmune myocarditis, myositis, hemopathy, inclusion body diseases, etc. However, research on myosin and its involvement in the occurrence and development of diseases is still in its infancy, and the underlying pathological mechanisms are not well understood. We can reasonably predict that myosin might play a role in new treatments of autoimmune diseases.

First-line cemiplimab monotherapy and continued cemiplimab beyond progression plus chemotherapy for advanced non-small-cell lung cancer with PD-L1 50% or more (EMPOWER-Lung 1): 35-month follow-up from a mutlicentre, open-label, randomised, phase 3 trial

Cemiplimab provided significant survival benefit to patients with advanced non-small-cell lung cancer with PD-L1 tumour expression of at least 50% and no actionable biomarkers at 1-year follow-up. In this exploratory analysis, we provide outcomes after 35 months' follow-up and the effect of adding chemotherapy to cemiplimab at the time of disease progression. EMPOWER-Lung 1 was a multicentre, open-label, randomised, phase 3 trial. We enrolled patients (aged ≥18 years) with histologically confirmed squamous or non-squamous advanced non-small-cell lung cancer with PD-L1 tumour expression of 50% or more. We randomly assigned (1:1) patients to intravenous cemiplimab 350 mg every 3 weeks for up to 108 weeks, or until disease progression, or investigator's choice of chemotherapy. Central randomisation scheme generated by an interactive web response system governed the randomisation process that was stratified by histology and geographical region. Primary endpoints were overall survival and progression free survival, as assessed by a blinded independent central review (BICR) per Response Evaluation Criteria in Solid Tumours version 1.1. Patients with disease progression on cemiplimab could continue cemiplimab with the addition of up to four cycles of chemotherapy. We assessed response in these patients by BICR against a new baseline, defined as the last scan before chemotherapy initiation. The primary endpoints were assessed in all randomly assigned participants (ie, intention-to-treat population) and in those with a PD-L1 expression of at least 50%. We assessed adverse events in all patients who received at least one dose of their assigned treatment. This trial is registered with ClinicalTrials.gov, NCT03088540. Between May 29, 2017, and March 4, 2020, we recruited 712 patients (607 [85%] were male and 105 [15%] were female). We randomly assigned 357 (50%) to cemiplimab and 355 (50%) to chemotherapy. 284 (50%) patients assigned to cemiplimab and 281 (50%) assigned to chemotherapy had verified PD-L1 expression of at least 50%. At 35 months' follow-up, among those with a verified PD-L1 expression of at least 50% median overall survival in the cemiplimab group was 26·1 months (95% CI 22·1-31·8; 149 [52%] of 284 died) versus 13·3 months (10·5-16·2; 188 [67%] of 281 died) in the chemotherapy group (hazard ratio [HR] 0·57, 95% CI 0·46-0·71; p<0·0001), median progression-free survival was 8·1 months (95% CI 6·2-8·8; 214 events occurred) in the cemiplimab group versus 5·3 months (4·3-6·1; 236 events occurred) in the chemotherapy group (HR 0·51, 95% CI 0·42-0·62; p<0·0001). Continued cemiplimab plus chemotherapy as second-line therapy (n=64) resulted in a median progression-free survival of 6·6 months (6·1-9·3) and overall survival of 15·1 months (11·3-18·7). The most common grade 3-4 treatment-emergent adverse events were anaemia (15 [4%] of 356 patients in the cemiplimab group vs 60 [17%] of 343 in the control group), neutropenia (three [1%] vs 35 [10%]), and pneumonia (18 [5%] vs 13 [4%]). Treatment-related deaths occurred in ten (3%) of 356 patients treated with cemiplimab (due to autoimmune myocarditis, cardiac failure, cardio-respiratory arrest, cardiopulmonary failure, septic shock, tumour hyperprogression, nephritis, respiratory failure, [n=1 each] and general disorders or unknown [n=2]) and in seven (2%) of 343 patients treated with chemotherapy (due to pneumonia and pulmonary embolism [n=2 each], and cardiac arrest, lung abscess, and myocardial infarction [n=1 each]). The safety profile of cemiplimab at 35 months, and of continued cemiplimab plus chemotherapy, was generally consistent with that previously observed for these treatments, with no new safety signals INTERPRETATION: At 35 months' follow-up, the survival benefit of cemiplimab for patients with advanced non-small-cell lung cancer was at least as pronounced as at 1 year, affirming its use as first-line monotherapy for this population. Adding chemotherapy to cemiplimab at progression might provide a new second-line treatment for patients with advanced non-small-cell lung cancer. Regeneron Pharmaceuticals and Sanofi.

STING regulates the transformation of the proinflammatory macrophage phenotype by HIF1A into autoimmune myocarditis.

Macrophages play an essential role in the pathogenesis of autoimmune myocarditis, but the molecular mechanism remains largely unknown. Here, the role of Stimulator of interferon gene (Sting) in autoimmune myocarditis was investigated. Six-week-old male BALB/c mice received two subcutaneous injections of 250μg α-MyHC peptide to establish experimental autoimmune myocarditis (EAM). With single-cell RNA sequencing analysis of cardiac immune (Cd45+) cells, Sting was found to initiate proinflammatory macrophage differentiation related to the acute EAM phase. Furthermore, proinflammatory macrophages contribute to the pathogenesis of EAM via hypoxia-inducible factor-1α (Hif1α). A higher expression level of Sting was detected in macrophages from myocarditis, which was positively correlated with Hif1α expression. Single-stranded DNA (ssDNA) accumulation in macrophages in myocarditis was observed in the hearts of EAM mice. Pharmacological blockade of STING by C-176 (a specific inhibitor) ameliorated the inflammatory response of EAM and reduced proinflammatory molecule (Ifn-β, Tnf-α, Ccl2, and F4/80) expression and Hif1α expression. In vitro studies revealed that ssDNA activated the expression of Sting; in turn, Sting accelerated proinflammatory molecule expression in mouse macrophages. Inhibition of Hif1α expression could reduce Sting-associated cardiac inflammation and proinflammatory molecule expression. In addition, the expression of STING and ssDNA accumulation in macrophages were observed in human autoimmune myocarditis heart samples. STING activated proinflammatory macrophage via HIF1A, promoting the development of autoimmune myocarditis. The STING signaling pathway might provide a novel mechanism of autoimmune myocarditis and serve as a potential therapeutic target for autoimmune myocarditis patients.

Modulation of TLR4/NFκB Pathways in Autoimmune Myocarditis.

Myocarditis is an inflammatory and oxidative disorder characterized by immune cell recruitment in the damaged tissue and organ dysfunction. In this paper, we evaluated the molecular pathways involved in myocarditis using a natural compound, Coriolus versicolor, in an experimental model of autoimmune myocarditis (EAM). Animals were immunized with an emulsion of pig cardiac myosin and complete Freund's adjuvant supplemented with mycobacterium tuberculosis; thereafter, Coriolus versicolor (200 mg/Kg) was orally administered for 21 days. At the end of the experiment, blood pressure and heart rate measurements were recorded and the body and heart weights as well. From the molecular point of view, the Coriolus versicolor administration reduced the activation of the TLR4/NF-κB pathway and the levels of pro-inflammatory cytokines (INF-γ, TNF-α, IL-6, IL-17, and IL-2) and restored the levels of anti-inflammatory cytokines (IL-10). These anti-inflammatory effects were accompanied with a reduced lipid peroxidation and nitrite levels and restored the antioxidant enzyme activities (SOD and CAT) and GSH levels. Additionally, it reduced the histological injury and the immune cell recruitment (CD4+ and CD68+ cells). Moreover, we observed an antiapoptotic activity in both intrinsic (Fas/FasL/caspase-3) and extrinsic (Bax/Bcl-2) pathways. Overall, our data showed that Coriolus versicolor administration modulates the TLR4/NF-κB signaling in EAM.

Ursolic acid reduces oxidative stress injury to ameliorate experimental autoimmune myocarditis by activating Nrf2/HO-1 signaling pathway.

Background: Oxidative stress is crucial in experimental autoimmune myocarditis (EAM)-induced inflammatory myocardial injury. Ursolic acid (UA) is an antioxidant-enriched traditional Chinese medicine formula. The present study aimed to investigate whether UA could alleviate inflammatory cardiac injury and determine the underlying mechanisms. Methods: Six-week-old male BALB/c mice were randomly assigned to one of the three groups: Sham, EAM group, or UA intervention group (UA group) by gavage for 2weeks. An EAM model was developed by subcutaneous injection of α-myosin heavy chain derived polypeptide (α-MyHC peptide) into lymph nodes on days 0 and 7. Echocardiography was used to assess cardiac function on day 21. The inflammation level in the myocardial tissue of each group was compared using hematoxylin and eosin staining (HE) of heart sections and Interleukin-6 (IL-6) immunohistochemical staining. Masson staining revealed the degree of cardiac fibrosis. Furthermore, Dihydroethidium staining, Western blot, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used to determine the mechanism of cardioprotective effects of UA on EAM-induced cardiac injury, and the level of IL-6, Nrf2, and HO-1. Results: In EAM mice, UA intervention significantly reduced the degree of inflammatory infiltration and myocardial fibrosis while improving cardiac function. Mechanistically, UA reduced myocardial injury by inhibiting oxidative stress (as demonstrated by a decrease of superoxide and normalization of pro- and antioxidant enzyme levels). Interestingly, UA intervention upregulated the expression of antioxidant factors such as Nrf2 and HO-1. In vitro experiments, specific Nrf2 inhibitors reversed the antioxidant and antiapoptotic effects of ursolic acid, which further suggested that the amelioration of EAM by UA was in a Nrf2/HO-1 pathway-dependent manner. Conclusion: These findings indicate that UA is a cardioprotective traditional Chinese medicine formula that reduces EAM-induced cardiac injury by up-regulating Nrf2/HO-1 expression and suppressing oxidative stress, making it a promising therapeutic strategy for the treatment of EAM.

Aggravation of TGFβ1-Smad Pathway and Autoimmune Myocarditis by Fungicide (Tebuconazole) Exposure.

Myocarditis is an inflammatory cardiac disorder and the primary cause of heart failure in young adults. Its origins can be attributed to various factors, including bacterial or viral infections, exposure to toxins or drugs, endocrine disruptors (EDs), and autoimmune processes. Tebuconazole (TEB), which is a member of the triazole fungicide family, is utilized to safeguard agricultural crop plants against fungal pathogens. Although TEB poses serious threats to mammal health, the information about how it induces toxic effects through various pathways, particularly in autoimmune diseases, are still limited. Thus, the aim of this paper was to evaluate the effect of TEB exposure in autoimmune myocarditis (AM). To induce AM, rats were immunized with porcine cardiac myosin and exposed to TEB for 21 days. Thereafter, animals were sacrificed, and histological, biochemical, and molecular analyses were performed. TEB exposure increased heart weight, systolic blood pressure and heart rate already augmented by AM. Additionally, it significantly increased creatine phosphokinase heart (CK-MB), creatine phosphokinase (CPK), cardiac troponin T (cTnT), and cardiac troponin I (cTnI), as compared to the control. From the histological perspective, TEB exacerbates the histological damage induced by AM (necrosis, inflammation and cell infiltration) and increased fibrosis and collagen deposition. TEB exposure strongly increased pro-inflammatory cytokines and prooxidant levels (O2-, H2O2, NO2-, lipid peroxidation) and reduced antioxidant enzyme levels, which were already dysregulated by AM. Additionally, TEB increased NOX-4 expression and the TGFβ1-Smads pathway already activated by AM. Overall, our results showed that TEB exposure strongly aggravated the cardiotoxicity induced by AM.

Assessment of Cardiac Functions in Children Suffering from Celiac Disease

Background: Celiac disease (CD) is an autoimmune disorder that occurs in genetically predisposed individuals. Among the complications of celiac disease, idiopathic dilated cardiomyopathy and autoimmune myocarditis are popular causes of considerable mortality and morbidity. The objective of this study is to evaluate the heart functioning in CD children.&#x0D; Methods: This cross-sectional study was conducted out on 40 children (twenty Patients diagnosed as having celiac disease and twenty healthy, age and sex matched children send as control group). All participants underwent basic laboratory investigations. Every child had an echocardiogram, which comprised both traditional echocardiography and tissue Doppler imaging.&#x0D; Results: The conventional echocardiographic assessment parameters (LVEED, LVESD, septal thickness, EF% FS%, E/A and TAPSE) were insignificantly different between both groups. There was no statistically substantial variation in mitral anulus velocity (S) and E/A among both groups. According to mean value of myocardial performance index (MPI) it was statistically significant greater in celiac disease of the affected group contrasted to the control group (P value 0.002).&#x0D; Conclusions: The tissue doppler echocardiogram can identify subclinical early stage of cardiac involvement in CD patients.

No Evidence for Myocarditis or Other Organ Affection by Induction of an Immune Response against Critical SARS-CoV-2 Protein Epitopes in a Mouse Model Susceptible for Autoimmunity.

After Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) developed into a global pandemic, not only the infection itself but also several immune-mediated side effects led to additional consequences. Immune reactions such as epitope spreading and cross-reactivity may also play a role in the development of long-COVID, although the exact pathomechanisms have not yet been elucidated. Infection with SARS-CoV-2 can not only cause direct damage to the lungs but can also lead to secondary indirect organ damage (e.g., myocardial involvement), which is often associated with high mortality. To investigate whether an immune reaction against the viral peptides can lead to organ affection, a mouse strain known to be susceptible to the development of autoimmune diseases, such as experimental autoimmune myocarditis (EAM), was used. First, the mice were immunized with single or pooled peptide sequences of the virus's spike (SP), membrane (MP), nucleocapsid (NP), and envelope protein (EP), then the heart and other organs such as the liver, kidney, lung, intestine, and muscle were examined for signs of inflammation or other damage. Our results showed no significant inflammation or signs of pathology in any of these organs as a result of the immunization with these different viral protein sequences. In summary, immunization with different SARS-CoV-2 spike-, membrane-, nucleocapsid-, and envelope-protein peptides does not significantly affect the heart or other organ systems adversely, even when using a highly susceptible mouse strain for experimental autoimmune diseases. This suggests that inducing an immune reaction against these peptides of the SARS-CoV-2 virus alone is not sufficient to cause inflammation and/or dysfunction of the myocardium or other studied organs.

Th17/Treg balance is regulated by myeloid-derived suppressor cells in experimental autoimmune myocarditis.

Autoimmune myocarditis is caused by both innate and adaptive immune responses. Many studies have found that myeloid-derived suppressor cells (MDSCs) suppress T-cell responses and reduce immune tolerance, while MDSCs may serve as a key player in inflammatory responses and pathogenesis in variety of autoimmune diseases. However, research into the role of MDSCs in experimental autoimmune myocarditis (EAM) remains lacking. We discovered that the expansion of MDSCs in EAM was closely related to the severity of myocardial inflammation. At an early stage of EAM, both adoptive transfer (AT) and selective depletion of MDSCs could inhibit the expression of IL-17 in CD4+ cells and downregulate the Th17/Treg ratio, alleviating excessive inflammation of EAM myocarditis. In another experiment, in addition, MDSCs transferred after selective depletion could increase IL-17 and Foxp3 expressions in CD4+ cells, as well as the Th17/Treg ratio, contributing to the aggravation of myocardial inflammation. MDSCs promoted the Th17 cell induction under Th17-polarizing conditions in vitro but suppressed Treg expansion. These findings suggest that MDSCs play a plastic role in sustaining mild inflammation in EAM by shifting Th17/Treg balance.

IgG4 Antibodies Induced by Repeated Vaccination May Generate Immune Tolerance to the SARS-CoV-2 Spike Protein.

Less than a year after the global emergence of the coronavirus SARS-CoV-2, a novel vaccine platform based on mRNA technology was introduced to the market. Globally, around 13.38 billion COVID-19 vaccine doses of diverse platforms have been administered. To date, 72.3% of the total population has been injected at least once with a COVID-19 vaccine. As the immunity provided by these vaccines rapidly wanes, their ability to prevent hospitalization and severe disease in individuals with comorbidities has recently been questioned, and increasing evidence has shown that, as with many other vaccines, they do not produce sterilizing immunity, allowing people to suffer frequent re-infections. Additionally, recent investigations have found abnormally high levels of IgG4 in people who were administered two or more injections of the mRNA vaccines. HIV, Malaria, and Pertussis vaccines have also been reported to induce higher-than-normal IgG4 synthesis. Overall, there are three critical factors determining the class switch to IgG4 antibodies: excessive antigen concentration, repeated vaccination, and the type of vaccine used. It has been suggested that an increase in IgG4 levels could have a protecting role by preventing immune over-activation, similar to that occurring during successful allergen-specific immunotherapy by inhibiting IgE-induced effects. However, emerging evidence suggests that the reported increase in IgG4 levels detected after repeated vaccination with the mRNA vaccines may not be a protective mechanism; rather, it constitutes an immune tolerance mechanism to the spike protein that could promote unopposed SARS-CoV2 infection and replication by suppressing natural antiviral responses. Increased IgG4 synthesis due to repeated mRNA vaccination with high antigen concentrations may also cause autoimmune diseases, and promote cancer growth and autoimmune myocarditis in susceptible individuals.

A combination of Sophoraflavescens alkaloids and Panaxquinquefolium saponins attenuates coxsackievirus B3‑induced acute myocarditis in mice via NF‑κB signaling.

Timely treatment of viral myocarditis (VMC), a form of cardiac inflammation caused by viral infections, can reduce the occurrence of dilated cardiomyopathy and sudden death. Our previous study demonstrated the anti-inflammatory and anti-fibrotic effects of KX, a combination of Sophora flavescens alkaloids and Panax quinquefolium saponins, on an autoimmune myocarditis model in vivo. The present study explored the effects of KX on coxsackievirus B3 (CVB3)-induced acute VMC in mice. Mice were randomly divided into four groups: Control, VMC, KX-high (275 mg/kg) and KX-low (138 mg/kg). Mice in the VMC, KX-high and KX-low groups received injections of CVB3 to establish the VMC model, and those in the KX-high and KX-low groups also received KX by gavage (10 ml/kg) 2 h after virus injection until euthanasia was performed on day 7 or 21. Mice in the control group received an equal KX volume of purified water. The levels of lactate dehydrogenase (LDH), creatine kinase-myocardial band (CK-MB), cardiac troponin I (cTn-I), IL-1β, IL-6, TNF-α and high-sensitive C-reactive protein (hs-CRP) in mouse serum was measured using ELISA. Myocardial tissue structure and degree of injury were observed using hematoxylin and eosin staining. Western blotting and reverse transcription-quantitative PCR were performed to detect the expression levels of NF-κB pathway-related mRNA and protein in myocardial tissue. The results showed that the inflammation and myocardial damage levels of the mice in the VMC group were higher at 7 days than those at 21 days. At both 7 and 21 days, KX decreased the serum CK-MB, LDH, cTn-I, IL-6, TNF-α and hs-CRP levels, and inhibited NF-κB pathway-related mRNA and protein expression in the myocardium of mice. These findings indicated that KX may reduce the inflammatory response and attenuate the pathological damage in the acute and subacute phases of CVB3-induced VMC through the NF-κB pathway.

Nursing of a patient with Sintilimab-induced autoimmune myocarditis

Nursing of a patient with Sintilimab-induced autoimmune myocarditis