The Pathogenesis And Regulation Mechanism Of HMGB1 In Myocarditis

High mobility group box 1 (HMGB1), a non-histone nuclear protein, has been functionally characterized as an alarmin or damage associated molecular protein (DAMP). It is constitutively expressed in quiescent cells and stored preformed in the nucleus. HMGB1 is the most evolutionarily conserved protein in eukaryotes. HMGB1 was merely a structural protein that resided in the nucleus where it functions to stabilize DNA structure and modulate transcriptional activity. The major structural features of HMGB1 are its two DNA-binding domains, termed the A and B boxes, and a C-terminal domain. HMGB1 was actively secreted by activated macrophages and dendritic cells or passively secreted by necrosis or apotosis cells. HMGB1 widely involved in the inflammatory and cancer, sepesis, autoimmune disease.Autoimmune myocarditis, caused by viral myocarditis, was mediated by Th17 cells. Because of virus infected cardiomyocytes, myosin was released and led to autoimmune myocarditis. Autoimmune myocarditis also was a main factor of dilated cardiomyopathy and heart failure. Th17 cells involved in the pathogenesis of myocarditis. Experimental autoimmune myocarditis (EAM) is a mouse model of postinfectious myocarditis, characterized by inflammatory infiltration of the myocardium and cardiomyocytes necrosis. EAM can be induced by inoculation with the coxsackie B virus or cardiac myosin. And we also found that HMGB1 level was increased in the plasma and Th17 cells infiltrated the heart. Therefore, we aimed to clarify whether Thl7 cells are involved in EAM pathogenesis; whether HMGB1 contributes to the progression of EAM; whether HMGB1 expression is associated with Th17 cell activity in EAM. And whether cardiomyocytes could actively secrete HMGB1. Objective(1) HMGB1 released into the extracellular milieu, binds to the receptor for advanced glycation endproducts (RAGE) or toll like receptor 2/4/7 (TLR2/4/7), and has restorative effects on CD4+ T helper cell regulation. And we also found that HMGB1 level was increased in the plasma and Thl7 cells infiltrated the heart. Therefore, we aimed to clarify the HMGB1 effects on Th17 cells and the pathogenesis on EAM.(2) HMGB1 has been functionally characterized as an alarmins, was actively secreted by activated macrophages and dendritic cells or passively secreted by necrosis or apotosis cells. Whether cardiomyocytes could actively secrete HMGB1 on the milieu stress. Therefore, we want to clarity the mechanism of cardiomyocytes actively secreted HMGB1 and affect on heart.(3) HMGB1 has recently been shown to also be associated with inflammatory diseases. Therefore whether inhibition of HMGB1 could ameliorate the degree of myocardial injury in this experimental model. And we produced the mAb of HMGB1 B box.Methods(1) HMGB1 B-box expression and producing of monoclonal antibodies The plasmid of HMGB1 B-box provided by Prof FengTian He from the third military medical university. The plasmid was transformed DH5a, cultivating 5～7h, keeping OD value (A600) for 1～1.5, then IPTG added. After 6h, bacteria were collected. The protein was extracted and purified. The polymyxin B column was used to inhibit endotoxin activity.15μg protein with complete freund'adjuvant was immunized BALB/c mice by i.p. every two weeks. After one week of second immunization, immunizaed again by vein. Cell fusion, cloning and mAb producing by conventional method. (2) The myocarditis induction and pathological analysisMyHCα614-629 with complete freund' adjuvant (CFA) immunized BALB/c mouse on day 0 and day 7. And control group immunized by PBS with CFA. After three weeks, the serum was collected and the heart was fixed in 10% formalin. Serum HMGB1, TNFα, IL-1β, IFN-γand IL-17 were detected by ELISA. The HMGB1, IFN-γ, IL-17, T-bet, RORγT mRNA were analyzed by RT-qPCR. HE staining were used to analysis myocardial pathology, Th17 cells infiltrated the myocardial tissue by immunofluorescence.(3) HMGB1 promotes Th17 cell expansion and HMGB1 blockade decreases Th17 cell infiltration and ameliorates myocardial injuryCD4+ T-cells were isolated by magnetic activated cell sorting (MACS) using CD4 antibodies according to the manufacturer's instructions. Isolated T-cells were plated onto a 96-precoated-well plate with anti-CD3 mAb and anti-CD28 mAb. Cultures were supplemented with 1μg/ml HMGB1, and also included control group and unassociated group. For HMGB1 blockade, neutralizing monoclonal antibody against HMGB1 (100μg/mouse i.p.) was administered every other day according to our lab protocol.(4) LPS-induced HMGB1 production by viable cardiomyocytesHearts were harvested, minced and digested with CollagenaseⅡ(160 U/ml). After a washing step, the cells were suspended in M199 with 10% FCS. The myocytes were enriched by a preplating approach (to remove contaminating cells) before being seeded into cell culture plates. Endotoxemia was induced by i.p. injection of 0.5 ml of normal saline (NS) containing LPS (10 mg/kg) to 3-month-old mice. This dosing regimen is in accord with previous studies assessing LPS-induced cardiac function. Control mice received saline. As an in vitro correlate of endotoxemia, the myocytes were exposed to 10μg/ml LPS, a dosing regimen previously to address LPS-induced signaling in isolated cardiomyocytes. And the signaling pathway was analyzed by immunofluorescence, western-blot and TLR4-/-, PI3Kγ-/-(5) LPS decreases myocardial contractility in vivo via a mechanism that involves HMGB1Endotoxemia was induced and intervented on WT, PI3Kγ-/- C57BL/6 mouse. Millar tip transducer catheter (Model SPR-893,1.4 Fr.) was advanced into the left ventricle via the right carotid artery. Pressure-volume loops were generated by occlusion of the inferior caval vein using a PowerLab system connected to theMillar catheter. Left ventricular end systolic elastance (Ees) was considered to be the best index of myocardial contractile function and was determined from pressure-volume loop data.Results(1) The plasmid was sequenced again. HMGB1 B box proteins were expressed, the molecular weight was identified by SDS-PAGE. Two cell lines were isolated namely 1D2F4E3 and 2D4E3A2. And the mAbs were IgG2a,κtype, valence was 106.(2) Myocarditis model was induced by MyHCα614-629. Monocyte, neutrophil, and lymphocyte infiltration in the myocardium was observed. Th17 cell infiltration was identified by immunofluorescence. Serum IL-17 levels by ELISA were significantly increased compared with controls (667.09±120.44 pg/ml vs.68±31.11 pg/ml, p<0.05). However, serum IFN-y levels were unchanged. In the mice spleens, RORyt and IL-17 mRNA expression were significantly increased; T-bet and IFN-γdid not show any change in mRNA expression.(3) HMGB1 blockade resulted in a significant reduction of cardiac myocytes as well as a significant decrease in monocyte, neutrophil, and lymphocyte infiltration of the myocardium on HE. Th17 cells infiltration significantly decrease compared to the untreated group. Serum IL-17 was also significantly decrease (212.11±67.30 pg/ml) compared to the untreated group (617.82±146.00 pg/ml, p<0.01). HMGB1 blockade did not affect IFN-γsecretion.(4) HMGB1 was noted in the circulation at 12-24h after LPS administration; much later than the increase in circulating TNF-α; In control mice, HMGB1 was localized predominantly to the nuclei of the myocytes, whereas the LPS-induced increase in myocardial expression of HMGB1 is apparent in both the cytoplasm and nuclei. Challenge of cardiomyocyte monolayers with 10μg/ml LPS did not affect myocyte viability. Challenge of cardiomyocytes with LPS, resulted in a transient increase in phosphorylation of PI3Kγwithin cardiomyocytes, peaking at 5-15min after the challenge. LPS administration to mice decreased myocardial contractility(5) Injection of HMGB1 did not affect myocardial contractility until circulating evels of HMGB1 were increased; at which point decreases in ardiac contractility. The LPS-induced increase in myocardial HMGB1 protein levels and decrease in myocardial contractility.Conclusions(1) Two cell lines were isolated namely 1D2F4E3 and 2D4E3A2. And the mAbs were IgG 2a,κtype, valence was 106.(2) EAM was induced using MyHC-α614-629 peptides in wild-type male BALB/c mice and found that cardiac myocyte necrosis was accompanied by lymphocyte infiltration into the myocardium. These were confirmed to be Th17 cells by immunofluorescence. Similarly, HMGB1 was increased in both cardiac tissue and serum; HMGB1 blockade resulted in a significant reduction in cardiac myocyte necrosis and a significant decrease in Th17 cell myocardial infiltration. Serum IL-17 was also noticeably decreased after treatment. Furthermore, gross severity scores decreased.(3) HMGB1 secreted by injured cardiac myocytes, fibroblasts, and activated macrophages and monocytes was associated with Th17 cell polarization, differentiation, and proliferation in the EAM model.(4) HMGB1 should be extended to include active release of HMGB1 by " stressed," but still viable, cardiomyocytes. the current study provide the first demonstration that viable cardiac myocytes can produce the HMGB1, when challenged by LPS in vivo and in vitro. In addition, some of the components of the cardiomyocyte signaling pathway have been identified and sequentially positioned (i.e., LPS activates a TLR4/PI3Ky pathway to induce HMGB1 secretion).(5) LPS can induce HMGB1 secretion by viable cardiac myocytes through a TLR4/PI3Ky signaling pathway, and HMGB1 plays a role in the LPS-induced myocardial contractile dysfunction. The results of the current study also have broader implications.