Study Of The Role And Mechanism Of High Mobility Group Box Protein 1 In The Asthmatic Airway Remodeling

BACKGROUND AND AIM:Bronchial asthma (asthma) is one of the most prevalent chronic diseases characterized by chronic airway inflammation, mucus hypersecretion and airway hyperresponsiveness, which has a great harmful impact on health of roughly 300 million people in the world. Although most asthmatic patients can be effectively treated,asthma still gives an unacceptable burden on society and health care systems as well as asthmatic patients around the world.Early treatment of airway remodelling has the potential to decrease disease severity, improve control and prevent disease exacerbation,but the mechanism among the airway remodeling is still unclear.It has been proved that cytokines play a important role in orchestrating the chronic airway inflammation and structural changes of in asthma.High mobility group box 1 (HMGB1) has been found as a new proinflammatory cytokines.High mobility group box protein 1 (HMGB1) is a nonhistone DNA-binding nuclear protein that acts as a potent endogenous "danger signal" for the initiation and enlargement of innate immunity in many diseases.Many studies of clinical patients and animal models have supported that HMGB1 acts as a pro-inflammatory mediator when it is localized in the extracellular space In fact, high levels of HMGB1 have been found in clinical inflammatory conditions, such as sepsis,rheumatoid arthritis, atherosclerosis, chronic kidney disease, systemic lupus erythematosus and so on. Recently, we reported that HMGB1 levels in sputum and serum increased in patients with asthma and COPD and were significantly negatively correlated with the pulmonary function index and positively correlated with neutrophil counts. We also demonstrated that the expression of HMGB 1 increased in the lung and bronchial alveolar lavage fluid (BALF) of ovalbumin (OVA)-induced acute asthmatic mice. A study by Shim et al. showed that HMGB1 plays an important role in the pathogenesis of asthma characterized by eosinophilic airway inflammation. More interestingly, HMGB1 has been shown to play an important role in pulmonary fibrosis, partly through reduction of fibroblast proliferation and down regulation of interleukin (IL)-1β levels in the BALF. In addition, some studies have shown that HMGB1 increases the secretion of vascular endothelial growth factor (VEGF) and that treatment with anti-HMGB1 antibodies suppresses the activity of matrix metalloproteinase (MMP)-9; both of these targets are involved in airway remodeling, the important pathophysiological feature of chronic asthma.Based on this background, we hypothesized that HMGB1 released in the airway may play an important role in the pathogenesis of airway remodeling in a mouse model of chronic asthma. To test this, we measured the levels of HMGB1 in lung tissue and BALF in these mice and then investigated the effects of anti-HMGB1 antibody treatment by examining respiratory mechanics, OVA-specific immunoglobulin (Ig)E, collagen deposition, smooth muscle thickness, epithelial mucus, and inflammation mediators. In addition, we investigated whether HMGB1 had direct effects on human fetal lung fibroblast proliferation, migration, and collagen synthesis in vitro to elucidate the mechanisms involved in these processes. Finally, we examined the cellular source of transforming growth factor (TGF)-β1, VEGF, and MMP-9 in the asthmatic response to HMGB1 or HMGB1/IL-1β complexes.METHODSAnimal experiments1.Balb/c female mice(Eight mice per group) were divided into the PBS control group, ovalbumin (OVA) group, OVA+isotype antibody group, OVA+anti-HMGB1 antibody group. Mice were sensitized with OVA in aluminum hydroxide by intraperitoneal injections and challenged with OVA intranasally for 6 wk. Anti-HMGB1 neutralizing antibody was injected intraperitoneally before challenge.2.Twenty-four hours after the final challenge, airway hyperresponsiveness (AHR) to methacholine (MCh; Sigma) was determined by body plethysmography and invasive method.3.Bronchoalveolar lavage fluid and lung tissue collection:Mice were sacrificed 48 h after the last allergen challenge, and bronchoalveolar lavage fluid (BALF) was collected after three washes with PBS. Total and differential cell counts were determined by hematoxylin and eosin (H&E), and BALF supernatants were stored for further evaluation by enzyme-linked immu-nosorbent assay (ELISA). The right lung was removed and frozen for collagen content (upper lobe) and and Western blotting determination (lower lobe). The left lung was fixed and paraffin-embedded, were stained with IHC,hematoxylin and eosin (H&E), Masson Trichrome, and Periodic Acid-Schiff (PAS).4.Determination of Lung Collagen ContentThe collagen assay was performed according to the Sirius Red Collagen Detection Kit manual. Results were expressed as ug of collagen per g lung.5.ImmunohistochemistryLeft lung was fixed and stained with Hematoxylin & Eosin to evaluate the histological inflammation. Lung tissues were exposed to anti-a-SMA antibody or anti-HMGB1 antibody to analyze the expression of both of them.6.Airway remodeling analysisAirway mucus production, peribronchial fibrosis and peribronchial smooth muscle layer area were analyzed in both large and small airways. To detect airway mucus production, lung sections were stained with periodic acid-Schiff s (PAS) reagent. Masson’s trichrome staining was used for assessment of subepithelial fibrosis. a-SMA immunostaining was used to evaluate airway smooth muscle hypertrophy/hyperplasia.In vitro tests1.HMGB1 impact on Human fetal lung fibroblast(MRC-5)Human fetal lung fibroblast(MRC-5) were cultured in DMEM1.1 Fibroblast Migration AssayMigration was determined using the "scratch-wound" assay.1.2 Fibroblast Proliferation AssayFibroblasts (5*104/well) were seeded in 12-well plate in DMEM with HMGB1 or without HMGB1on Day 0. On Days 1,2, and 3, cells were trypsinized and the number of viable cells was determined.1.3 Expression of a-SMA Detected by Real-Time PCR and Western blotting1.4 Collagen Synthesis by Lung FibroblastsThe cell supernatants treated with recombinant HMGBlwere used to determined the collagen secretion according to Sirius Red Collagen Detection Kit manual.2.The cellular source of TGF-β1, VEGF, and MMP-9 in response to HMGB1 or HMGB1/IL-1β complexes2.1 Primary culture of human bronchial epithelial cells (PBECs)The primary human bronchial epithelial cells were seeded in 12-well plates at 2 ×105 cells per well before being challenged with HMGB1 or HMGB1/IL-1(3 complexes for 24 h.2.2 Cell lines, culture, and differentiationA549,16-HBE, U937, and THP-1 cells were cultured in RPMI-1640. For differentiation of THP-1 and U937 cells into a monocyte/macrophage-like phenotype, the cells were treated with 20 ng/ml phorbol-12-myristate-13-acetate (PMA) for 48 h (THP-1) or 72 h (U937) at a density of 1 ×106 cells/ml. For each experiment,2 ×105 epithelial cells or 1 ×106 U937 or THP-1 cells were seeded into 12-well plates were stimulated with HMGB1, IL-1β\or HMGB1/IL-1β complexes. After 24 h, the cell culture supernatants, cell RNA, and cell lysates were collected for further analyses.2.3 Isolation and culture of eosinophils Peripheral blood eosinophils were purified using a negative immunomagnetic procedure.The purity was assessed by H&E staining and was at least 95%.A total of 5 ×105 cells/ml were plated in 24-well plates and were treated with HMGB1 or HMGB1/IL-1β complexes for 24 h.2.4 Neutrophil isolation and cultureNeutrophils were isolated using a standard protocol from the blood of patients by Percoll density gradient centrifugation. After incubation for 1 h in at 37℃, neutrophils were treated with HMGB1 or HMGB1/IL-1β complexes for 30 min. Untreated cells were used as controls.2.5 Preparation of HMGB1-IL-1β complexesHMGB1 diluted in PBS was incubated with IL-1β respectively, in different ratios to give the indicated final concentrations in cell cultures. Solutions were incubated at 4℃ for 16 h.2.6 Quantitative polymerase chain reaction (PCR)Quantification of the PCR signals was performed by comparing the cycle threshold (Ct) value of the gene of interest with the Ct value of the reference gene.2.7 Western blotting analysis.The protein level of β-actin,β-Tubulin,VEGF, MMP-9,HMGB1 and TGF β1 were detected using Western blotting analysis. Bands were subsequently analyzed using Quantity One.2.8 Enzyme-linked immunosorbent assay.Levels of total and OVA-specific IgE, IL-1β,IL-4, IL-5, IL-6, IL-13, IFN-y, TNF-a,VEGF,total MMP-9 and active TGF-β1 were determined in BAL fluid,lung lysates or cell culture supernatants with enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions.Statistical analysisResults are expressed as the mean±SE. Either one-way or two-way Analysis of Variance (ANOVA) followed by Bonferroni post test was performed to compare groups. P-values<0.05 were considered statistically significant.RESULTS1.HMGB1 expression was elevated in the chronic asthma mouse modelHMGB1 expression in lung tissues and HMGB1 levels in the BALF was higher in OVA-induced asthmatic mice than in control mice.HMGB1 expression was detected in bronchiolar epithelial cells and alveolar macrophages,The numbers of HMGB1-positive cells obviously increased in asthmatic mice compared to control mice.2.Effects of anti-HMGBl on OVA-induced chronic airway remodelingThe mice in the anti-HMGB1 antibody group showed reduced total cell numbers and numbers of eosinophils as compared to mice from the asthmatic group.As expected, OVA-induced asthmatic mice had a significant increase in AHR.However, anti-HMGB1 antibody administration to OVA-induced mice led to a reduction in AHR.Anti-HMGB1 antibody administration to asthmatic mice reduced collagen deposition in the bronchovascular regions compared with that in asthmatic mice. The anti-HMGB1 antibody administration mice resulted in a reduction in the percentage of PAS-positive airway epithelium and SMA-stained peribronchial area as compared with asthmatic mice.Levels of OVA-specific IgE,IL-1β, IL-4, IL-5, IL-13, tumor necrosis factor (TNF)-a,VEGF, active-TGF-(31 and total MMP-9 were significantly reduced in the BALF from mice in the anti-HMGB 1 antibody group compared with mice asthmatic group. The changes in VEGF, total TGF-β1, and MMP-9 expression levels in the lung tissues were further confirmed by western blot. In addition, the high MMP-9 activity detected by gelatin zymography in the BALF from asthmatic mice was decreased after administration of anti-HMGB1 antibody3 Effects of HMGB1 on human fetal lung fibroblasts (MRC-5 cells)HMGB1 at concentrations of 250 and 1000 ng/ml increased the migration capacity of MRC-5 cells in a concentration-dependent manner compared with the untreated control.Additionally 250 or 1000 ng/ml HMGB1 significantly increased the number of MRC-5 cells as compared with the control. A time course experiment using 1000 ng/ml HMGB1 showed that the maximal proliferation response was observed at day 2.HMGB1 concentrations of 250 and 1000 ng/ml significantly increased a-SMA mRNA expression and a-SMA protein expression compared with control cells.while 1000 ng/ml HMGB1 increased collagen levels to as much as 2-3 times that of untreated control cellsHMGB1 slightly increased the level of TGF-β1 secretionHMGB1 or IL-1β alone, for 24 h resulted in a slight increase in TGF-β1 levels in supernatants only from eosinophils.this increase was not detected in supernatants from other cells.HMGB1 increased the expression and activity of MMP-9The significant MMP-9 production was observed in 16-HBE cells, PBECs, A549 cells, and neutrophils pre-incubated with 0.5 or 2 ng/ml IL-1β followed by treatment with 250 or 1000 ng/ml HMGB1 as compared to cells treated with HMGB1 or IL-1β alone.HMGB1 increased the expression and secretion of VEGFThe treatment with HMGB1/DL-1β complexes resulted in upregulation of VEGF secretion by 2.5-4fold in VEGF secretion froml6-HBE cells, PBEC, A549 cells, undifferentiated U937 cells, undifferentiated and differentiated THP-1 cells, or alveolar macrophages.CONCLUSIONThus Blocking HMGB1 activity may reverse airway remodeling through suppressing airway inflammation and modulating lung fibroblast phenotype and activation.