HMGB1 Participates In Myocardial Fibrosis In EAM And Influences The Function Of Innate Immune Cells

BackgroundHigh mobility group box 1 (HMGB1), an important inflammatory mediator, is actively secreted by immune cells and some non-immune cells or passively released by damaged cells and necrotic cells. HMGB1 has been implicated in many inflammatory diseases. Our previous published data demonstrated that HMGB1 was up-regulated in heart tissue or serum in experimental autoimmune myocarditis (EAM); HMGB1 blockade could ameliorate cardiac fibrosis at the last stage of EAM. And yet, until now, no data directly showed that HMGB1 was associated with cardiac fibrosis. Therefore, the aims of the present work were to assess whether (I) up-regulated HMGB1 could directly lead to cardiac fibrosis in EAM; (Ⅱ) cardiac fibroblast/ myofibroblasts could secrete HMGB1 as another source of high-level HMGB1 in EAM; and (Ⅲ) HMGB1 blockade could effectively prevent cardiac fibrosis at the last stage of EAM. At the same time, the preliminary discussion on the immune imbalance which may be involved in autoimmune myocarditis was carried out, it included the studies on Toll-like (TLR) receptors and Runt-related transcription factor 3 (Runx3)/CpG-ODN. These immune molecules may be associated with the secretion or release of HMGB1.MethodStandard experimental procedure involving flow cytometry, qRT PCR, Western blotting, ELISA, cell culture and proliferation assays were employed in the study.1. Establishment of Experimental Autoimmune Myocarditis modelsBALB/c mice,6-8 weeks old, were purchased from the Animal Center of Yangzhou University and maintained in the Animal Center of Jiangsu University. All animal procedures in this study were in compliance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No.85-23, revised 1996).Establishment of experimental autoimmune myocarditis models was carried out by inoculated with 100 μg of MyHC-α (MyHC-α614-629; Ac-SLKLMATLFSTYASAD-OH), emulsified at 1:1 ratio in PBS/CFA at day 0 and day 7. On day 54, the mice were anaesthetized with pentobarbital sodium via intra peritoneal and killed by cervical dislocation. All experiments were approved by the Institutional Committee on the Use of Animals for Research and Teaching, Jiangsu University.2. Flow cytometry analysisFifteen chronic diabetics, fourteen gestational diabetics and seventeen preeclampsia patients whose age ranged from 19 to 41 years consented to be included in this study. Flow cytometry analysis was used to count the frequency of circulating Th17 cells in peripheral blood mononuclear cells (PBMCs) from the patients.3. Fluorescence quantitative PCR method (qRT-PCR)TLR2, TLR4, TLR9, RAGE, HMGB1, Collagen type Ⅰ/Ⅲ (Col 1/3), Osteopontin (OPN), Hlx, Runx3, MMP1/2 and TIMP1 messenger levels were assessed by RT-qPCR.4. Western blotWestern blot analysis was used to detect the expression levels of Collagen Ⅰ, Collagen Ⅲ, P-PKC-P, P-ErK-1/2, F-ErK-1/2 and HMGB1 proteins in cardiac fibroblests/myofibroblatsts, and Runx3 protein in A549 cells or CpG-ODN-induced A549 cells.5. ELISA for Cytokine measurementsEnzyme-linked immunosorbent assay (ELISA) was used for quantitative detection of IFN-γ, IL-17, IL-1β and high mobility group box 1 (HMGB1) in plasma from patients with chronic diabetics, gestational diabetics and preeclampsia.6. Cell culture and proliferation assaysCardiac fibroblasts were isolated from 1-to 2-week-old BALB/c mice according to previous reports [31,32]. The cell culture medium, DMEM (contain 10% foetal bovine serum) was used to culture cardiac fibroblasts. Cardiac fibroblast/myofibroblast proliferation was evaluated by MTT assay, and MTT assay was also used to detect the effects of CpG-ODN on Runx3 siRNA transfected cell growth.7. Migration experimentsThe migration experiment was performed by using 6-well transwell plates with an 8-lm-pore-size polycarbonate filter. The effect of cardiac fibroblast/myofibroblast migration was analyzed by migration experiment under the condition of with or without exogenous HMGB1.8. HistopathologyHE staining was used for analyzing histopathology in experimental autoimmune myocarditis models; cardiac fibrosis was evaluated by Sirius red staining. And immunofluorescence staining was used to analyze the ability of cardiac fibroblast/myofibroblast to secrete HMGB1 under LPS challenge.9. Statistical analysisAll data were shown as means±standard deviation (SD). The statistical significance of differences between groups was determined by the Student’s test or two-way analysis of variance using GraphPad Prism 5.0 and/or SPSS11.5 software. P value<0.05 was considered significant.ResultsOur results clearly demonstrated that:1. HMGB1 could directly lead to cardiac collagen deposition, which was associated with PKCβ/Erkl/2 signalling pathway; furthermore, cardiac fibroblast/myofibroblasts could actively secrete HMGB1 under external stress; and HMGB1 secreted by cardiac fibroblasts/myofibroblasts led to cardiac fibrosis via PKCβ activation by autocrine means; HMGB1 blockade could efficiently ameliorate cardiac fibrosis in EAM mice.2. HMGB1 was shuttled from nucleus to cytoplasm in cardiac fibroblasts/myofibroblasts under external stress. This study has explored that except for immune cells, endothelial cells, cardiomyocytes and necrosis cells, whether cardiac fibroblasts/myofibroblasts, as another important component of heart, were also the critical sources of high-level HMGB1 in EAM.3. A higher possibility that the increased plasma HMGB1 in pregnancy associated diseases can lead to ILCs differentiation and subsequently production of IL-17. Again the increased IL-17 in pregnancy associated diseases including preeclampsia and gestational diabetes may be produced by ILC3 and may pose a threat to fetus leading to spontaneous abortions if they are triggered to elicit uncontrolled inflammatory responses.4. CpG-ODN significantly inhibited the proliferation of A549 cells. The expression of Runx3 in the mRNA and protein level was increased in A549 cells stimulated by CpG-ODN. The CpG-ODN-stimulated cell proliferation was significantly inhibited in Runx3 siRNA-transfected A549 cells. This result indicated that there was a connection between TLR9 activation and Runx3 expression, and it may relate to HMGB1 secretion, which need to be further confirmed.ConclusionOur works clearly demonstrated that HMGB1 could directly lead to cardiac collagen deposition by a PKCβ/Erk1/2-dependent signalling pathway. HMGB1, secreted by cardiac fibroblasts/myofibroblasts under external stress, led to cardiac fibrosis via PKCβ activation through autocrine, and HMGB1 blockade could efficiently ameliorate cardiac fibrosis in EAM mice. In addition, increased plasma HMGB1 in pregnancy associated diseases can lead to ILCs differentiation and subsequently production of IL-17. TLR9 activation connected with Runx3 expression in inhibiting A549 cell proliferation, it may relate to HMGB1 secretion.