Autoimmune Myocarditis In Rats Research Articles

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).

Protein Kinase C-Mediated Hyperphosphorylation and Lateralization of Connexin 43 Are Involved in Autoimmune Myocarditis-Induced Prolongation of QRS Complex.

Myocarditis is a serious and potentially life-threatening disease, which leads to cardiac dysfunction and sudden cardiac death. An increasing number of evidence suggests that myocarditis is also a malignant complication of coronavirus pneumonia, associated with heart failure and sudden cardiac death. Prolonged QRS complexes that are related to malignant arrhythmias caused by myocarditis significantly increase the risk of sudden cardiac death in patients. However, the molecular mechanisms are not fully known at present. In this study, we identify protein kinase C (PKC) as a new regulator of the QRS complex. In isolated hearts of normal rats, the PKC agonist, phorbol-12-myristate-13-acetate (PMA), induced prolongation of the QRS complex. Mechanistically, hyperphosphorylation and lateralization of connexin 43 (Cx43) by PKC induced depolymerization and internalization of Cx43 gap junction channels and prolongation of the QRS duration. Conversely, administration of the PKC inhibitor, Ro-32-0432, in experimental autoimmune myocarditis (EAM) rats after the most severe inflammation period still significantly rescued the stability of the Cx43 gap junction and alleviated prolongation of the QRS complex. Ro-32-0432 reduced phosphorylation and blocked translocation of Cx43 in EAM rat heart but did not regulate the mRNA expression level of ventricular ion channels and the other regulatory proteins, which indicates that the inhibition of PKC might have no protective effect on ion channels that generate ventricular action potential in EAM rats. These results suggest that the pharmacological inhibition of PKC ameliorates the prolongation of the QRS complex via suppression of Cx43 hyperphosphorylation, lateralization, and depolymerization of Cx43 gap junction channels in EAM rats, which provides a potential therapeutic strategy for myocarditis-induced arrhythmias.

Cardiac apoptosis caused by elevated cholesterol level in experimental autoimmune myocarditis

It has been reported that cholesterol-lowing agents can ameliorate severity of myocarditis. However, the beneficial effect of the agents has been claimed to be independent of cholesterol reduction as there is no significant change in the plasma cholesterol level in myocarditis. In the present study, using experimental autoimmune myocarditis (EAM) rats as an animal model, we demonstrated that EAM induced elevation of cholesterol level and impaired cholesterol efflux capacity in the cardiac tissue. Moreover, serum high-density lipoprotein (HDL) content was reduced and HDL function associated protein Paraoxonase 1 (PON1) activity was decreased. Besides, the major structural protein within HDL, Apolipoprotein A1 (ApoA1) expression in the cardiac tissues was significantly reduced while the level of serum ApoA1 was not significantly altered. Importantly, cholesterol depleting agent methyl-β-cyclodextrin (MβCD) alleviated the development of EAM, as monitored by decreased ratio of heart weight to body weight (HW/BW), decreased infiltration of inflammatory cells and collagen deposition, improved cardiac function, reduced expression of apoptosis-related protein Bax, Fas, FasL and caspase-3 and increased level of anti-apoptotic protein Bcl-2. These results suggest that reduction of cholesterol level in cardiac tissue could suppress EAM-induced cardiac apoptosis through both intrinsic and extrinsic apoptotic pathways.

Silencing of STAT4 Protects Against Autoimmune Myocarditis by Regulating Th1/Th2 Immune Response via Inactivation of the NF-κB Pathway in Rats.

Signal transducer and activator of transcription 4 (STAT4) has been implicated in the progression of myocarditis. The aim of the current study was to investigate the role by which STAT4 influences autoimmune myocarditis in an attempt to identify a theoretical therapeutic perspective for the condition. After successful establishment of an autoimmune myocarditis rat model, the expression patterns of STAT4, NF-κB pathway-related genes, Th1 inflammatory cytokines (IFN-γ and IL-2), and Th2 inflammatory cytokines (IL-6 and IL-10) were subsequently determined. The rats with autoimmune myocarditis were treated with oe-STAT4 or sh-STAT4 lentiviral vectors to evaluate the role of STAT4 in autoimmune myocarditis, or administrated with 1mL 10μmol/L of BAY11-7082 (the NF-κB pathway inhibitor) via tail vein to investigate the effect of the NF-κB pathway on autoimmune myocarditis. Finally, cell apoptosis was evaluated. The serum levels of IFN-γ and IL-2, extent of IκBα and P65 phosphorylation, and the expression of STAT4 were elevated, while the serum levels of IL-6 and IL-10 as well as the expression of IκBα were reduced among the rats with autoimmune myocarditis, which was accompanied by an increase in the apoptotic cells. More importantly, the silencing of STAT4 or the inhibition of the NF-κB pathway was detected to result in a decrease in the serum levels of IFN-γ and IL-2 and an elevation of the serum levels of IL-6 and IL-10, and inhibited myocardial cell apoptosis in rats with autoimmune myocarditis. Moreover, STAT4 silencing was also observed to decrease the extent of IκBα and P65 phosphorylation while acting to elevate the expression of IκBα. Taken together, silencing of STAT4 could hinder the progression of autoimmune myocarditis by balancing the expression of Th1/Th2 inflammatory cytokines via the NF-κB pathway, which may provide a novel target for experimental autoimmune myocarditis (EAM) treatment.

PPARα suppresses Th17 cell differentiation through IL-6/STAT3/RORγt pathway in experimental autoimmune myocarditis

Family members of peroxisome proliferator-activated receptors (PPARs), such as PPARγ, have been shown to be effective in regulating T helper 17 (Th17) cell differentiation. However, whether PPARα, another important family member of PPARs, contributes to Th17 cell differentiation remains controversial. In the present study, we show that PPARα may be a negative regulator of Th17 cell differentiation. In CD4+ T cells from PPARα knockout mice, PPARα deficiency enhances IL-17 and IL-6 levels and promotes Th17 cell differentiation. In contrast, in CD4+ T cells from wild type mice, PPARα activation suppresses Th17 cell differentiation. Furthermore, IL-6 neutralizing antibody dose-dependently reduces the activity of STAT3 and down-regulates the protein expression of RORγt in CD4+ T cells from PPARα knockout mice but has no effect on that of wild type mice. On the other hand, in isolated CD4+ T cells from experimental autoimmune myocarditis (EAM) rats, PPARα agonist Fenofibrate decreased the expression of IL-17 and RORγt, increased the expression of Foxp3, while PPARα antagonist MK886 reversed these effects. Importantly, in vivo activation of PPARα ameliorates EAM by suppressing Th17 cell differentiation through reducing the expression of RORγt and phosphorylated STAT3 that are upregulated in EAM hearts. These results imply that PPARα suppresses Th17 cell differentiation through IL-6/STAT3/RORγt signaling pathway and suggest that PPARα may become a molecular target for treating autoimmune myocarditis.

Small molecule disruption of G protein βγ subunit signaling reprograms human macrophage phenotype and prevents autoimmune myocarditis in rats.

The purpose of this study was to determine whether blocking of G protein βγ (Gβγ) signaling halts heart failure (HF) progression by macrophage phenotype manipulation. Cardiac Gβγ signaling plays a crucial role in HF pathogenesis. Previous data suggested that inhibiting Gβγ signaling reprograms T helper cell 1 (Th1) and Th2 cytokines, suggesting that Gβγ might be a useful drug target for treating HF. We investigated the efficacy of a small molecule Gβγ inhibitor, gallein, in a clinically relevant, experimental autoimmune myocarditis (EAM) model of HF as well as in human macrophage phenotypes in vitro. In the myocardium of HF patients, we observed that G protein coupled receptor kinase (GRK)2 levels were down-regulated compared with healthy controls. In rat EAM, treatment with gallein effectively improved survival and cardiac function, suppressed cardiac remodeling, and further attenuated myocardial protein expression of GRK2 as well as high mobility group box (HMGB)1 and its cascade signaling proteins. Furthermore, gallein effectively inhibited M1 polarization and promoted M2 polarization in vivo in the EAM heart and in vitro in human monocyte-derived macrophages. Taken together, these data suggest that the small molecule Gβγ inhibitor, gallein, could be an important pharmacologic therapy for HF as it can switch the phenotypic reprogramming from M1 to M2 phenotype in a rat model of EAM heart and in human macrophages.

Urinary candidate biomarkers in an experimental autoimmune myocarditis rat model

Urine is a better source than plasma for biomarker studies, as it can accumulate all changes in the body. Various candidate urinary biomarkers of physiological condition, kidney disease and even brain dysfunction, have been detected in urine; however, urine has rarely been used to reflect cardiac diseases. In this study, urine at day 0, 14, 21 and 28 were collected from the myosin-induced autoimmune myocarditis rat models. The candidate urinary biomarkers were then characterized using the isobaric tandem mass tag labeling approach coupled with offline two-dimensional reverse-phase liquid chromatography and high-resolution mass spectrometry. Compared with controls, forty-six urinary proteins were significantly changed in the myocarditis rats; among them, ten had previously been associated with myocarditis, twelve corresponding gene products had annotated as mainly cardiovascular network genes by the Ingenuity Pathway Analysis, four urinary proteins were validated by western blot, thirteen were reported in previous urine proteome studies of other diseases and twenty-six were reported the first time to be related to myocarditis. SignificanceThis is the first study to use isobaric tandem mass tag labeling approach in the urine proteome analysis of experimental autoimmune myocarditis. These findings may provide clues for the pathogenesis of myocarditis. And the study showed that urine can be a good source of myocarditis biomarkers.

FGL2 knockdown improves heart function through regulation of TLR9 signaling in the experimental autoimmune myocarditis rats.

Fibrinogen-like protein 2 (FGL2) is an important immune regulator of both innate and adaptive response. It is present on the surface of macrophages and endothelial cells, and can be constitutively secreted by CD4+CD8+ T cells. Previous studies showed that FGL2 is a potential target for the treatment of experimental autoimmune myocarditis. However, the molecular mechanism of the roles of FGL2 in experimental autoimmune myocarditis is poorly understood. Here, we silenced FGL2 gene by using FGL2-RNAi lentivirus to reveal the heart function in experimental autoimmune myocarditis rats. We found that the cardiac myosin of pigs' hearts induced Lewis rats to come into being as autoimmune myocarditis. TLR9 was upregulated in the heart of experimental autoimmune myocarditis rats. After primary immunization (21day), the cardiac function of the myocarditis model group improved (P<0.05). Significantly, the levels of INF-α and NF-κB in the FGL2-RNAi-treated group were lower compared to those in the myocarditis model (EAM) group (P<0.05). Notably, the inflammation score correspondence with the protein and mRNA levels of TLR9 in myocardial tissues was markedly reduced compared to that in the EAM group (P<0.05). These results support a role of FGL2 to alleviate inflammatory situation in the myocardium through regulation of the TLR9 signaling pathway in the experimental autoimmune myocarditis rats.

Fibrinogen-like protein-2 causes deterioration in cardiac function in experimental autoimmune myocarditis rats through regulation of programmed death-1 and inflammatory cytokines.

Programmed death-1 (PD-1) plays an important role in protecting against inflammation and myocyte damage in T-cell-mediated myocarditis. To understand whether fibrinogen-like protein-2 (FGL2) can affect the role of the PD-1/PD-L1 pathway in experimental autoimmune myocarditis (EAM), we investigated cardiac function in EAM rats over-expressing FGL2. Over-expression of FGL2 significantly decreased PD-1 and deteriorated cardiac function in rats with autoimmune myocarditis. Histopathology revealed increased inflammatory cell infiltrate in EAM-FGL2 rats compared with the control groups (EAM, EAM-GFP and NC). Notably, transcription factor forkhead box P3 (Foxp3) and retinoic acid-related orphan receptor γt (RORγt) protein and mRNA levels were statistically (P<0·05) increased in EAM rats. We also found that interferon-γ, interleukin-6, interleukin-17 and brain natriuretic peptide levels were profoundly increased in serum of FGL2 over-expressing EAM rats. Hence, FGL2 plays an important role in the pathogenesis of autoimmune myocarditis that also involves the PD-1/PD-L1 pathway. Our findings may provide novel therapeutic targets for the treatment of immune-induced heart injury.

Effect of QiShenYiQi pill on myocardial collagen metabolism in experimental autoimmune myocarditis rats.

To observe the effect of QiShenYiQi pill (QSYQ) on myocardial collagen metabolism in experimental autoimmune myocarditis rats, and to explore its mechanism of action. Lewis rats underwent the injection of myocardial myosin mixed with freund's complete adjuvant were randomized into three groups: model, valsartan and QSYQ groups. And we treated rats which were injected phosphate buffered saline (PBS) mixed with freund's complete adjuvant as control group. Rats were intervened and euthanized at 4 and 8 weeks. We use alkaline hydrolysis to detect the content of myocardial hydroxyproline (HYP), and ELISA to detect the level of serum procollagen type I carboxyterminal peptide (PICP), procollagen type III amino-terminal peptide (PIIINP), and collagen C telopeptide type I (CTX-I). Myocardial MMP-1 and TIMP-1 protein expression was detected by immunohistochemistry, and myocardial MMP-1 and TIMP-1 mRNA expression was detected by real-time qPCR. QSYQ reduced the content of myocardial HYP, and this reduction was greater over time. QSYQ also reduced the serum concentration of PICP, PIIINP, CTX-I and the PICP/PIIINP ratio, which further reduced over time, whereas its effect on lowering PICP was significantly greater than that of valsartan at 4 and 8 weeks, and lowering CTX-I was significantly greater than that of valsartan at 8 weeks. In addition, after 4 weeks, QSYQ enhanced the protein and mRNA expression of MMP-1 and TIMP-1, and its effect on highering TIMP-1 was significantly greater than that of valsartan, whereas there was no significant difference in the expression of myocardial MMP-1 or TIMP-1 at 8 weeks. QSYQ reduced the ratio of MMP-1/TIMP-1, which further reduced over time, and the effect of QYSQ was significantly greater than that of valsartan after 4 weeks. This study provides evidence that QSYQ can reduce the rate of myocardial collagen synthesis and degradation. It also effectively improved the degree of myocardial fibrosis in experimental autoimmune myocarditis rats and it had a tendency to have a greater effect with longer treatment duration, which is related to the mechanism of regulation of MMP-1 and TIMP-1 expression in the myocardial rat.

GW27-e0383 The mechanism of C/EBPβ in the procedure of myosin induced myocardial fibrosis and inflammation in experimental autoimmune myocarditis rats

GW27-e0383 The mechanism of C/EBPβ in the procedure of myosin induced myocardial fibrosis and inflammation in experimental autoimmune myocarditis rats

Effect and Mechanism of QiShenYiQi Pill on Experimental Autoimmune Myocarditis Rats

BackgroundTo observe the effect of QiShenYiQi pill (QSYQ) on experimental autoimmune myocarditis rats, and to explore its mechanism of action.Material/methodsLewis rats underwent the injection of myocardial myosin mixed with Freund’s complete adjuvant were randomized into 3 groups: model, valsartan, and QSYQ groups. Rats injected with phosphate-buffered saline (PBS) mixed with Freund’s complete adjuvant were used as the control group. Rats were euthanized at 4 and 8 weeks, and we weighed rat body mass, heart mass, and left ventricular mass. Myocardium sections were stained with hematoxylin and eosin (H&E) and Masson trichrome. Myocardial TGF-β1 and CTGF protein expression was detected by immunohistochemistry, and myocardial TGF-β1 and CTGF mRNA expression was detected by real-time qPCR.ResultsQSYQ reduced HMI and LVMI, as well as the histological score of hearts and CVF, which further decreased over time, and its effect was significantly greater than that of valsartan at 4 and 8 weeks. After 4 weeks, QSYQ inhibited the protein and mRNA expression of TGF-β1 and CTGF, and its effect on lowering CTGF was significantly greater than that of valsartan. In addition, after 8 weeks, QSYQ also inhibited the protein and mRNA expression of CTGF, whereas there was no significant difference in the expression of myocardial TGF-β1.ConclusionsThis study provides evidence that QSYQ can improve cardiac remodeling of experimental autoimmune myocarditis rats. It also effectively improved the degree of myocardial fibrosis, which is related to the mechanism of regulation of TGF-β1 CTGF.

Anti-inflammatory role of obestatin in autoimmune myocarditis.

Obestatin is a popular endogeneous peptide, known to have an autoimmune regulatory effect on energy metabolism and the gastrointestinal system. Studies regarding the anti-inflammatory effects of obestatin are scarce. The aim of this study was to show the anti-inflammatory effect of obestatin in an experimental model of autoimmune myocarditis in rats. Experimental autoimmune myocarditis was induced in Lewis rats by immunization with subcutaneous administration of porcine cardiac myosin, twice at 7-day intervals. Intraperitoneal pretreatment with obestatin (50μg/kg) was started before the induction of myocarditis and continued for 3weeks. The severity of myocarditis was evidenced by clinical, echocardiographic and histological findings. In addition, by-products of neutrophil activation, lipid peroxidation, inflammatory and anti-inflammatory cytokines were measured in serum. Obestatin significantly ameliorated the clinical and histopathological severity of autoimmune myocarditis. Therapeutic effects of obestatin in myocarditis were associated with reduced lipid peroxidation, suppression of polymorphonuclear leukocyte infiltration and enhancement of glutathione synthesis, inhibition of serum inflammatory and activation of anti-inflammatory cytokines. Histopathologically, the left ventricle was significantly dilated, and its wall thickened, along with widespread lymphocytic and histocytic infiltration. The myocardium was severely infiltrated with relatively large mononuclear cells. These histopathological changes were observed in lesser degrees in obestatin-treated rats. This study demonstrated a novel anti-inflammatory effect of obestatin in an experimental model of autoimmune myocarditis. Consequently, obestatin administration may represent a promising therapeutic approach for myocarditis and dilated cardiomyopathy in the future.

Curcumin ameliorates experimental autoimmune acute myocarditis in rats as evidenced by decrease in thioredoxin immunoreactivity.

This study was performed to investigate the effect of curcumin on cardiac myosin-induced autoimmune myocarditis in rats and the change in thioredoxin (TRX) immunoreactivity in cardiomyocytes following curcumin treatment. Twenty-four six-week-old male Wistar rats were randomly allocated into 4 groups of 6 rats each. Group I received neither curcumin nor myosin. Group II received an oral solution of 1 g/kg/day of curcumin daily, from day 1 to day 21. To induce myocarditis, animals of both group III and group IV were injected by 1 mg of porcine cardiac myosin on days 1 and 8. In addition, animals of group IV received an oral solution of 1 g/kg/day of curcumin daily, from day 1 to day 21. Serum levels of creatine phosphokinase, troponin-T, tumour necrosis factor-alpha and interleukin-6 were estimated. Hearts were processed for histopathological and immunohistochemical studies. Serum biomarkers levels were significantly increased in myocarditis group as compared to other groups. The intake of curcumin significantly reduced the deviation in these markers. Sections of the wall of the heart from myocarditis group were characterised by inflammatory cell infiltration. Most of cardiomyocytes showed pyknotic nuclei and increased sarcoplasmic eosinophilia with strong immunoreactivity for TRX. Sections from myocarditis-curcumin group showed normal architecture with moderate immunoreactivity for TRX. The present study demonstrated that curcumin ameliorates acute myocarditis in rats and encouraged the estimation of serum level of TRX as a relevant indicator for the evaluation of the progress of acute myocarditis.

Overexpression of Fgl2 deteriorates the heart function of experimenta l autoimmune myocarditis rats

Studies have demonstrated that fibrinogen-like protein 2 (Fgl2) has been involved in immune and inflammatory responses which might contribute to the pathogenesis of immune-mediated diseases. Experimental autoimmune myocarditis (EAM) provided a model that mimics the pathophysiology of human giant cell myocarditis. We observed that the effect of Fgl2 overexpression on the heart function of autoimmune myocarditis rats using echocardiography and detected the level of inflammatory cytokines using enzyme-linked immunosorbent assay. Fgl2 overexpression decreased the heart function of EAM model rat, increased the inflammatory cell infiltrations, and increased the level of brain natriuretic peptide (BNP). Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-17 (IL-17). These results indicate that Fgl2 is a potent target for the treatment of EAM. In conclusion, Fgl2 is a potent target to treat inflammatory dysfunctions of the heart.

Comparison of pre- and post-contrast myocardial T1 with histology findings in Experimental autoimmune myocarditis in rats

Background Magnetic resonance imaging as a noninvasive method offers diagnostic and prognostic information in myocarditis. T1 mapping techniques aim to overcome the limitations of late gadolinium enhancement to assess diffuse fibrosis and inflammatory infiltrates. Using an established animal model of myocarditis, the aim of this study was to measure myocardial T1 before the onset, and in the acute and chronic stages of the disease and to compare its course with histological and immunohistochemistry findings.

Dysregulated Overexpression of S100A8 and S100A9 Calgranulin Family Proteins in IFNγR-/- Allogeneic T Cells Is Associated with Reduced Graft Versus Host Disease in Vivo

The therapeutic benefit of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for treatment of hematologic malignancies is primarily derived from the anti-leukemia effect mediated by T cells contained in the donor graft. Unfortunately, these T cells also mediate graft-versus-host disease (GvHD), the major complication of allo-HSCT. We and others have demonstrated that interferon gamma receptor deficient (IFNγR-/-) allogeneic donor grafts, when transplanted into wild-type (WT) recipients (IFNγR-/- donors → WT recipients), reduce GvHD compared with WT donor grafts (WT donors → WT recipients) whilst maintaining a robust Graft vs. Leukemia effect (GvL).The aim of the present study was to elucidate the mechanism by which IFNyR deficient donor T cells reduce GvHD. To achieve this we performed RNA expression analyses on allo-activated T cells. To obtain allo-activated T cells, we co-cultured B6 CD4+CD25- T cells, isolated from WT and IFNγR-/- mice, with irradiated Balb/c antigen presenting cells (APCs). After six days of co-culture, CD4+CD25+ allo-activated T cells were sorted and total RNA extracted. Subsequent RNA profiling was performed using Mouse Genome 430 2.0 array (Affymetrix). From the numerous genes found to be differentially regulated between WT and IFNyR-/- T cells we sought to identify, for further analysis, those genes meeting the following criteria: (i.) genes with known human orthologs, (ii.) genes exhibiting the most pronounced differential expression between WT and IFNγR-/- T cells, (iii.) genes for which knockout or transgenic mice are available and (iv) genes with products that can be targeted by small molecule inhibitors. Two candidates were identified by applying all mentioned criteria: S100A8 and S100A9. Expression levels of S100A8 and S100A9 were 22 and 26 fold higher in APC activated IFNγR-/- T cells than WT T cells, respectively, which was also confirmed using qRT-PCR (Figure 1).It has been reported that S100A8 and S100A9, two members of the calgranulin family of proteins, form a heterodimer complex and may modulate/mitigate several inflammatory diseases by potentially binding NFKB intracellularly and inflammatory cytokines such as IL-6 and TNF when excreted extracellularly.1,2 Overexpression of S100A9 alone is sufficient to upregulate the expression of S100A8 and vice versa.3 In addition, S100A9-/- mice also lack expression of S100A8.4 We hypothesized that S100A8/S100A9 expression in allo-reactive donor T cells functions as a suppressor of GvHD. To test this hypothesis, we performed allo-HSCT with donor T cells isolated from either S100A9 overexpressing transgenic or WT B6 mice transplanted into Balb/c recipients. S100A9 overexpressing donor T cells induced significantly less GvHD than WT T cells. Recipients of S100A9 overexpressing T cells survived significantly longer (p=0.049, n=5), had reduced weight loss and higher percentages of B220+ B cells (indicative of less severe clinical GvHD) than recipients of WT T cells. This suggests that S100A9 might indeed function as a suppressor of GvHD and could account, at least in part, for the diminished GvHD potential of IFNyR-/- T cells. Modulation of the S100A9/A8 complex therefore represents a novel therapeutic target for the treatment of GvHD. [Display omitted] 1. Otsuka K, Terasaki F, Ikemoto M, et al. Suppression of inflammation in rat autoimmune myocarditis by S100A8/A9 through modulation of the proinflammatory cytokine network. Eur J Heart Fail. 2009;11(3):229-237.2. Ikemoto M, Murayama H, Itoh H, Totani M, Fujita M. Intrinsic function of S100A8/A9 complex as an anti-inflammatory protein in liver injury induced by lipopolysaccharide in rats. Clin Chim Acta. 2007;376(1-2):197-204.3. Cheng P, Corzo CA, Luetteke N, et al. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein. J Exp Med. 2008;205(10):2235-2249.4.Manitz MP, Horst B, Seeliger S, et al. Loss of S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants invitro. Mol Cell Biol. 2003;23(3):1034-1043. DisclosuresNo relevant conflicts of interest to declare.

Mortality and morbidity in different immunization protocols for experimental autoimmune myocarditis in rats

We aimed to investigate the histological and clinical presentations of experimental autoimmune myocarditis (EAM) induced by different immunization schemes. Male young Lewis rats were divided into five groups immunized by porcine myocardial myosin: subcutaneously (SC) 2 mg (in two 1-mg doses on day 0 and 7), 0 mg (sham group) subcutaneously into rear footpads (RF), 0.25 mg RF, 0.5 mg RF or 1 mg RF (all RF once on day 0). On day 21, left ventricular (LV) function was assessed by cardiac magnetic resonance imaging and cardiac catheterization. The type and degree of myocardial inflammatory infiltrates were determined by conventional histology and immunohistochemistry. In the SC immunized rats and in the RF sham group, we observed 0% mortality, while in the actively RF immunized rats, mortality was 20, 20 and 44% for the 0.25 mg, 0.5 mg and 1 mg myosin doses respectively. Morbidity as defined by inflammatory infiltrates on haematoxylin and eosin (HE) staining was 22% in the SC immunized rats, 0% in the RF sham group and 100% in all actively RF immunized groups. We observed augmented relative ventricle weight and spleen weight, increased LV end-diastolic pressure, reduced LV developed pressure and reduced LV ejection fraction in all with myosin-immunized RF groups without any systematic dose effect. Subcutaneous immunization to the neck and flanks did not induce a reproducible EAM, while RF myosin administration reliably led to EAM. Lower myosin doses seem to induce the complete histological and clinical picture of EAM while being associated with lower mortality, non-specific symptoms and animal distress.

Clinical and histological presentations of experimental autoimmune myocarditis in rats using different immunization schemes

Background Myocarditis is an inflammatory cardiac disease with a wide range of symptoms and various etiologies. The diagnostic workup and the treatment of myocarditis remain highly controversial. Therefore, there is a high demand for reliable animal models. Experimental autoimmune myocarditis (EAM) represents a model of human myocarditis and heart failure. In this study we investigated the histological, functional and clinical presentations of EAM induced by different immunization schemes. Methods Male young Lewis rats were divided into 5 groups immunized by porcine myocardial myosin: 1 mg subcutaneously (SC) on day 0 and 7; 0.25 mg subcutaneously into rear footpads (RF) on day 0; 0.5 mg RF on day 0; and 1 mg RF on day 0; and a sham group, injected with a vehicle solution without myosin into the RF on day 0. Rats were sacrificed on day 21 after assessment of left ventricular (LV) function by cardiac magnetic resonance imaging and cardiac catheterization under isoflurane anesthesia. The ventricle weight was measured and the location, type and degree of myocardial inflammatory infiltrates were determined by conventional histology and immunohistochemistry (CD 68). Results Rats immunized in the RF showed a mortality of 20%, 20% and 44% for the 0.25 mg, 0.5 mg and 1 mg myosin doses respectively, while the SC group and the RF sham group yielded a mortality of 0%. Morbidity as defined by inflammatory infiltrates on HE staining was 22% in the SC immunized rats, 0% in the RF sham group and 100% in all actively RF immunized groups. Histologically we observed macrophage-rich inflammatory infiltrates in both ventricles. All RF groups immunized with myosin showed augmented relative ventricle weight and spleen weight, increased LV end-diastolic pressure, reduced LV developed pressure and reduced LV ejection fraction, without any dose-dependend effect. Conclusions

Non-invasive assessment of experimental autoimmune myocarditis in rats using a 3 T clinical MRI scanner

The aim of this study was to assess the use of a 3 T clinical cardiac magnetic resonance (CMR) scanner to detect injury to the heart in experimental autoimmune myocarditis (EAM). The use of 3 T CMR for the detection of cardiac injury was assessed in EAM (n = 55) and control (n = 10) male Lewis rats. Animals were evaluated with serial CMR imaging studies, using a 3 T scanner, and with 2D echocardiography before, and at 2 and 5 weeks after EAM induction. By CMR, regional wall motion abnormalities were noted in seven out of eight rats with myocarditis 5 weeks after induction. Subsequently, the rats developed significant left ventricular (LV) dilatation, wall thickening, and pericardial effusion. Average LV systolic and diastolic volumes increased from 131 ± 10 to 257 ± 20 µL (P = 0.0008), and from 309 ± 14 to 412 ± 24 µL (P < 0.0001), and ejection fraction markedly deteriorated (from 58 ± 2 to 37 ± 5%; P = 0.0003). Areas of fibrosis were located by late gadolinium enhancement (LGE) CMR at the subepicardium, mainly within the anterior, lateral, and inferior walls. The extent and location of LGE were highly correlated (r = 0.94; P < 0.0001) with areas of myocardial fibrosis by histopathology, with 85% sensitivity and 86% specificity. A clinical 3 T CMR scanner enables accurate detection, quantification, and monitoring of experimental myocarditis in rats, and could be used for translational research to study the pathophysiology of the disease and evaluate novel therapies.