HMGB1 is upregulated in the airways in asthma and potentiates airway smooth muscle contraction via TLR4 (2017)

Asthma is characterized by variable airflow obstruction, airway hyperresponsiveness, and inflammation. Airway smooth muscle (ASM) contributes to asthma pathophysiology via hypercontractility, increased mass, and inflammatory mediator release.1Doeing D.C. Solway J. Airway smooth muscle in the pathophysiology and treatment of asthma.J Appl Physiol (1985). 2013; 114: 834-843Crossref PubMed Scopus (91) Google Scholar Clinical studies and animal models demonstrate a role for high-mobility group box 1 (HMGB1) and its receptors in airway inflammation and asthma.2Hou C. Zhao H. Liu L. Li W. Zhou X. Lv Y. et al.High mobility group protein B1 (HMGB1) in asthma: comparison of patients with chronic obstructive pulmonary disease and healthy controls.Mol Med. 2011; 17: 807-815Crossref PubMed Scopus (112) Google Scholar, 3Hou C. Kong J. Liang Y. Huang H. Wen H. Zheng X. et al.HMGB1 contributes to allergen-induced airway remodeling in a murine model of chronic asthma by modulating airway inflammation and activating lung fibroblasts.Cell Mol Immunol. 2015; 12: 409-423Crossref PubMed Scopus (58) Google Scholar HMGB1's activity and receptor interactions is determined by its redox state, with oxidation rendering HMGB1 inactive.4Magna M. Pisetsky D.S. The role of HMGB1 in the pathogenesis of inflammatory and autoimmune diseases.Mol Med. 2014; 20: 138-146Crossref PubMed Scopus (222) Google Scholar We have investigated the redox state of airway HMGB1 and the role of HMGB1 in ASM function. HMGB1 expression and/or redox state was investigated in sputum by ELISA, nonreducing electrophoresis, and Western blotting; in bronchial tissue by immunohistochemistry; and in ASM cells (ASMCs) by quantitative PCR, immunofluorescence, and flow cytometry. The effect of HMGB1 on ASMC reactive oxygen species (ROS) production, migration, proliferation, apoptosis, and contraction was evaluated. Leicestershire Research Ethics Committee approved the study, with informed consent obtained from all subjects. Statistical analysis was performed using GraphPad Prism 6.0. For detailed Methods, see this article's Online Repository at www.jacionline.org. Sputum HMGB1 concentration was increased in those with severe asthma but not in those with mild to moderate asthma versus healthy controls (controls) (Fig 1, A; see Table E1 in this article's Online Repository at www.jacionline.org), correlating significantly with total cell counts and nonviable cell counts/g sputum (see Fig E1, A and B, in this article's Online Repository at www.jacionline.org), but not sputum differential cell counts (ie, % eosinophils, neutrophils, macrophages, lymphocytes, or epithelial cells) nor lung function (data not shown). HMGB1 concentration in sputum from those with severe asthma was unaffected by oral corticosteroid (OCS) treatment, and did not correlate with OCS dose (data not shown). Both disulphide and reduced HMGB1 were significantly increased in sputum from those with severe asthma versus controls (Fig 1, B). In sputum with detectable HMGB1, the proportion of reduced versus disulphide HMGB1 was increased in those with severe asthma versus controls (Fig 1, C). Sputum endogenous secretory receptor for advanced glycosylation end products (endogenous secretory RAGE), measured in a subset of sputum samples, was not different between groups (Fig E1, C; see Table E2 in this article's Online Repository at www.jacionline.org). HMGB1 expression was significantly increased in ASM in bronchial biopsies from those with severe asthma versus controls (Fig 1, D; see Table E3 in this article's Online Repository at www.jacionline.org), with no effect of OCS observed. No differences in ASM RAGE or epithelial HMGB1/RAGE expression in bronchial biopsies were observed (Fig E1, D-F; Table E3). Representative photomicrographs of HMGB1/RAGE staining are shown in Fig E1, G-L, in this article's Online Repository. HMGB1 expression was investigated in primary ASMCs. Although there was no differential expression of HMGB1 mRNA (Fig 2, A), HMGB1 protein expression was significantly reduced in ASMCs from those with asthma versus controls, assessed by flow cytometry (Fig 2, B) and immunofluorescence (see Fig E2, A and B, in this article's Online Repository at www.jacionline.org). Release of HMGB1 extracellularly by ASMCs from those with asthma and/or the absence in culture of proinflammatory mediators present in asthmatic airways could explain the anomalous in vitro and in vivo data. Indeed HMGB1 protein expression was significantly upregulated in ASMCs from those with asthma but not controls following stimulation with TNF-α, IL-1β and IFN-γ, or poly(I:C) (Fig 2, C); however, HMGB1 expression poststimulation was not different between asthma and health. HMGB1 in ASMC supernatants was below the limit of detection. Reduced recombinant HMGB1, at concentrations equivalent to those in sputum, caused a concentration-dependent increase in intracellular ROS production in ASMCs from controls, but not in ASMCs from those with asthma (Fig 2, D), which was reduced by the RAGE decoy receptor soluble RAGE (sRAGE) and the Toll-like receptor (TLR) 4 antagonist LPS from Rhodobacter sphaeroides (LPS-RS) (Fig 2, E). This defective response in ASMCs from those with asthma was not due to impaired ROS generation capacity; ROS production was similar at baseline, and was increased in response to hydrogen peroxide stimulation of ASMCs from those with asthma versus controls.5Sutcliffe A. Hollins F. Gomez E. Saunders R. Doe C. Cooke M.S. et al.Increased nicotinamide adenine dinucleotide phosphate oxidase 4 expression mediates intrinsic airway smooth muscle hypercontractility in asthma.Am J Respir Crit Care Med. 2012; 185: 267-274Crossref PubMed Scopus (85) Google Scholar In addition, ASMC RAGE and TLR4 expression was not different between those with asthma and controls (Fig E2, C-E). HMGB1 activity and receptor binding is dependent on its oxidation state, and is regulated by several binding partners.4Magna M. Pisetsky D.S. The role of HMGB1 in the pathogenesis of inflammatory and autoimmune diseases.Mol Med. 2014; 20: 138-146Crossref PubMed Scopus (222) Google Scholar The complex interplay between these factors affects the cellular response to HMGB1 and might affect ROS production in ASM from those with asthma. In the absence of bradykinin, contraction of collagen gels impregnated with ASMCs from those with asthma was not significantly different from controls (see Fig E3, A, in this article's Online Repository at www.jacionline.org), nor was it affected by HMGB1 (100, 300 [data not shown], and 1000 ng/mL [Fig E3, B and C]). However bradykinin-mediated contraction of collagen gels impregnated with ASMCs was potentiated by 1000 ng/mL HMGB1, resulting in a decrease in the area under the curve at 60 minutes (Fig E3, D and E), but not 100 to 300 ng/mL HMGB1 (data not shown). In the presence of HMGB1, bradykinin-mediated contraction of collagen gels impregnated with ASMCs from those with asthma was potentiated to a greater extent compared with controls (Fig E3, F) and was significantly inhibited by LPS-RS, but not by sRAGE (Fig 2, F-H). sRAGE and LPS-RS had no significant effect in the absence of HMGB1. HMGB1 and/or sRAGE or LPS-RS (10 μg/mL) had no effect on ASMC migration (wound healing assay: HMGB1 [100-1000 ng/mL] ± CXCL12 [10-100 ng/mL], data not shown, ORIS assay: 3-1000 ng/mL HMGB1, Fig E3, G), proliferation in the presence/absence of serum (Fig E3, H), and apoptosis or necrosis (Fig E3, I and J). Our data support and extend previous studies suggesting an imbalance between HMGB1 and endogenous secretory RAGE in the asthmatic airways might have implications for HMGB1 in asthma pathophysiology.6Watanabe T. Asai K. Fujimoto H. Tanaka H. Kanazawa H. Hirata K. Increased levels of HMGB-1 and endogenous secretory RAGE in induced sputum from asthmatic patients.Respir Med. 2011; 105: 519-525Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Because the increased HMGB1 we see in the sputum in asthma correlates with sputum total cell and nonviable cell counts, we propose that HMGB1 can be upregulated in the airways in asthma because of inflammatory and stress stimuli that can result in HMGB1 secretion actively by activated immune cells and passively by necrotic cells.7Shim E.-J. Chun E. Lee H.-S. Bang B.-R. Cho S.-H. Min K.-U. et al.Eosinophils modulate CD4+ T cell responses via high mobility group box-1 in the pathogenesis of asthma.Allergy Asthma Immunol Res. 2015; 7: 190-194Crossref PubMed Scopus (13) Google Scholar, 8Ito I. Fukazawa J. Yoshida M. Post-translational methylation of high mobility group box 1 (HMGB1) causes its cytoplasmic localization in neutrophils.J Biol Chem. 2007; 282: 16336-16344Crossref PubMed Scopus (169) Google Scholar We propose that the ROS produced in response to HMGB1 in ASMCs from controls, in a RAGE/TLR4-dependent manner, terminally oxidize HMGB1, rendering it inactive4Magna M. Pisetsky D.S. The role of HMGB1 in the pathogenesis of inflammatory and autoimmune diseases.Mol Med. 2014; 20: 138-146Crossref PubMed Scopus (222) Google Scholar or alter Ca2+ homeostasis, leading to reduced contractility via a TLR4/ROS-dependent mechanism as in murine cardiomyocytes,9Zhang C. Mo M. Ding W. Liu W. Yan D. Deng J. et al.High-mobility group box 1 (HMGB1) impaired cardiac excitation-contraction coupling by enhancing the sarcoplasmic reticulum (SR) Ca(2+) leak through TLR4-ROS signaling in cardiomyocytes.J Mol Cell Cardiol. 2014; 74: 260-273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar thus limiting the potentiation of contraction of collagen gels impregnated with ASMCs from controls. Because of the ROS-generating capacity of ASMCs in response to HMGB1 being defective in asthma, these ROS-mediated responses would be reduced. Therefore, HMGB1 can potentiate contraction of collagen gels impregnated with ASMCs from those with asthma, in a TLR4-dependent manner, to a greater extent than those impregnated with ASMCs from controls. Thus, HMGB1 could contribute to ASM dysfunction and airway hyperresponsiveness in asthma, as supported by animal models,3Hou C. Kong J. Liang Y. Huang H. Wen H. Zheng X. et al.HMGB1 contributes to allergen-induced airway remodeling in a murine model of chronic asthma by modulating airway inflammation and activating lung fibroblasts.Cell Mol Immunol. 2015; 12: 409-423Crossref PubMed Scopus (58) Google Scholar, 10Lee C.C. Lai Y.T. Chang H.T. Liao J.W. Shyu W.C. Li C.Y. et al.Inhibition of high-mobility group box 1 in lung reduced airway inflammation and remodeling in a mouse model of chronic asthma.Biochem Pharmacol. 2013; 86: 940-949Crossref PubMed Scopus (55) Google Scholar possibly representing a potential therapeutic target. We thank Mitesh Pancholi, Vijay Mistry, and all the clinical staff for helping with collecting samples and patient details, and the patients who participated in this study. In addition, we thank Natasha Johnson, Jelizaveta Lisova, Naima Khalifa, and Adelina Gavrila for their technical assistance. Subjects were recruited from Glenfield Hospital, Leicester, United Kingdom. Asthma severity was defined according to the Global Initiative for Asthma (GINA) treatment stepsE1Global Strategy for Asthma Management and Prevention. Global Initiative for Asthma (GINA) 2014. Available at: http://www.ginasthma.org. Updated 2016. Accessed May 16, 2016.Google Scholar: mild to moderate asthma, GINA 1 to 3; severe asthma, GINA 4 to 5. Healthy subjects had no history of respiratory disease and normal spirometry. The study was approved by the Leicestershire Research Ethics Committee. Informed consent was obtained from all subjects (UHL 10613). Sputum was induced using inhaled incremental concentrations of nebulized hypertonic saline at 3%, 4%, and 5%, each administered for 5 minutes. Sputum plugs were selected and diluted in 8 volumes (weight/volume) of PBS. Samples were rocked on ice for 15 minutes and then spun at 790g for 10 minutes at 4°C. Four volumes of the PBS supernatants were collected and kept at −80°C until further use (eg, HMGB1 ELISA and Western blotting). The remaining cell and mucus pellets were resuspended in 4 volumes of 0.2% dithiothreitol (DTT) and rocked on ice for 15 minutes to disperse the cells. The cell suspension was then filtered through a 48-μm nylon gauze and the total cell number was determined using a hemocytometer. The DTT supernatant was obtained by centrifugation as before, removed, and stored at –80°C. Cells were resuspended in PBS at a density of 0.5 to 0.75 × 106 per mL and 75 μL of the cell suspension was adhered onto glass slides using cytospins (450 rpm for 6 minutes). Slides were air-dried for at least 15 minutes, fixed with 90% methanol for 10 minutes, and then stained with the Romanowsky method for differential cell counts to measure the percentage of sputum neutrophils, eosinophils, macrophages, epithelial cells, and lymphocytes.E2Pavord I.D. Pizzichini M.M. Pizzichini E. Hargreave F.E. The use of induced sputum to investigate airway inflammation.Thorax. 1997; 52: 498-501Crossref PubMed Scopus (353) Google Scholar HMGB1 was detected using a kit from Oxford Biosystems (Milton Park, Oxfordshire, United Kingdom) according to manufacturer's instructions. All redox forms of HMGB1 are detected. Endogenous secretory RAGE was detected using a kit from B-Bridge International (distributed by Metachem Diagnostics Ltd, Northampton, United Kingdom) according to manufacturer's instructions. An equivalent amount of each PBS-diluted sputum sample was loaded on gels. SDS-PAGE was performed on a 12% gel under nonreducing conditions to preserve the redox status of HMGB1 and proteins transferred to nitrocellulose membranes, which were blocked with 5% skimmed milk in Tris-buffered saline, pH 7.0, containing 0.1% Tween 20 (TBS-T). Blocked membranes were probed with rabbit anti-HMGB1 (1:1000) in TBS-T plus 5% milk overnight at 4°C, washed several times with TBS-T, and incubated for 1 hour with antirabbit peroxidase-conjugated antibody (1:10,000). Western blots were visualized using an enhanced chemiluminescence kit according to the manufacturer's instructions (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Recombinant HMGB1 was run as a standard in a subset of samples to assess the amount of HMGB1 in healthy controls versus samples with asthma. Data were analyzed by densitometry and normalized to a recombinant HMGB1 standard for differences between sputa from healthy controls and patients with asthma, or in samples with detectable HMGB1 the amount of reduced HMGB1 was calculated as a percentage of total HMGB1 in the sample. Human bronchial biopsies were embedded into glycol methacrylate (Polysciences, Northampton, United Kingdom). Two-micrometer sections were probed with a mouse monoclonal alpha-smooth muscle actin antibody (clone 1A4, Dako, Cambridge, United Kingdom) or appropriate isotype control, to identify ASM. Sequential sections were stained with a rabbit monoclonal anti-HMGB1 antibody (20 μg/mL, clone EPR3507, Abcam, Cambridge, United Kingdom), a mouse monoclonal anti-RAGE antibody (Millipore, Watford, United Kingdom, clone DD/A11, 10 μg/mL), or appropriate isotype controls (Dako). For RAGE staining, an amplification step was performed with a mouse LINKER for 15 minutes at room temperature. The Envision FLEX kit (Dako) was used. Nuclei were identified with Mayer's hematoxylin (blue staining was per smooth muscle or epithelial area (HMGB1) or by for RAGE no staining, 1 staining, moderate staining, by a ASM were from bronchial ASM cells were in with with 100 100 100 and 1 United Kingdom). Cells were characterized for expression by flow cytometry. ASM cells from patients with asthma from a of GINA treatment were with no differences in response to HMGB1 stimulation are for was performed to investigate the of RAGE by ASM cells, using a C. J. expression of the RAGE receptor and its soluble result of regulated PubMed Scopus Google Scholar to soluble and RAGE RAGE and RAGE from United Kingdom. was as a were from and products were run on an gel and of Leicester, Leicester, United Kingdom). was for of mRNA expression of HMGB1 in ASM cell culture from normal and asthma total cellular was from ASM cells using total kit and on treatment according to manufacturer's instructions. and were assessed using a United Kingdom) and 1 of total from each ASM cell culture was using kit of 10 of per in a of 20 μL was performed using the in a Watford, United Kingdom). All samples were in and was for HMGB1 were from United Kingdom), HMGB1 and HMGB1 were as for The was using the method and as For each the was calculated from the HMGB1 The is the of the healthy controls. Thus, the of each of each products were run on an gel and of ASM cells were fixed and stained with a mouse monoclonal anti-HMGB1 antibody (10 μg/mL, clone United Kingdom). antibody was rabbit Cells were with United Kingdom) to identify An with an was to least of with at least cells per of were assessed per HMGB1 staining in ASM cells was the number of HMGB1 cells was by the number of as a percentage of ASM identified by protein expression was measured in ASM cells using mouse anti-RAGE μg/mL, clone and μg/mL, clone United Kingdom). antibody for RAGE was rabbit (Dako). HMGB1 was measured in ASM cells with PBS containing and 0.1% using the anti-HMGB1 antibody μg/mL, clone EPR3507, and a antirabbit antibody United Kingdom). controls were rabbit mouse and mouse For ASM cells were incubated in plus or proinflammatory TNF-α, IFN-γ, 10 United Kingdom) or poly(I:C) μg/mL, United Kingdom) for flow cytometry was performed with a United Kingdom) and analysis performed with ROS production was measured using for or reduced recombinant HMGB1 in 1 1 pH United Kingdom, per 1 of protein were in was measured with an Cambridge, United Kingdom). For ASM cells were with sRAGE (10 μg/mL, Cambridge, United Kingdom) or LPS from Rhodobacter sphaeroides 10 μg/mL, for 1 HMGB1 binding to 10 sRAGE not at 1000 ng/mL R. H. J. Liu K. Y. et of in vitro binding of high mobility group box-1 and to receptor for advanced effect on Google Scholar and an of shown to HMGB1 activity by H. R. J. et for advanced end of in Full Text Full Text PDF PubMed Scopus Google Scholar LPS-RS was at a of that shown to HMGB1 of F. group receptor 4 mediates the of cells in 2015; PubMed Scopus Google Scholar, J. and as of Mol 2015; Google Scholar gel contraction was assessed as L. A. Saunders R. D. et cells airway smooth muscle cell via of Immunol. PubMed Scopus Google Scholar The collagen gel μL μL 10 μL ASM cell suspension in 20 μL was impregnated with ASM cells in the presence of or reduced recombinant HMGB1 in the presence or absence of sRAGE or LPS-RS at 10 μg/mL) and to for minutes. were from the using a and gel contraction measured in the absence or presence of the United Kingdom). were a and gel area was measured as a percentage of area using ASM migration was assessed using the cell migration kit United Kingdom). were performed for control, control, reduced recombinant HMGB1 sRAGE (10 μg/mL), or LPS-RS (10 μg/mL), which were for in Cells were fixed and stained with were with an United Kingdom) and the number of cells in the migration compared with the was ASM cells were to in with 10 to cell serum for and then using a μL in a ASM cells were washed with the of HMGB1 ± CXCL12 were then at and The number of cells that had into the was by a ± 1000 ng/mL reduced recombinant HMGB1 was to ASM cells for The with the United was then to each according to manufacturer's instructions and the at on an The percentage of and necrotic ASM cells to ± 1000 ng/mL reduced recombinant HMGB1 for was identified by staining with μL binding ± μg/mL, on a flow and analysis using Statistical analysis was performed using GraphPad Prism data were analyzed with or or by appropriate comparison data were analyzed using by were used. data were analyzed with was HMGB1 protein expression in ASM cells was assessed by B, Representative staining of cell HMGB1 and and HMGB1 ASM from subjects with asthma and subjects was shown to different of RAGE by products were by gel and identified as RAGE or soluble of RAGE healthy control, A asthma. Representative flow cytometry RAGE and TLR4 expression in ASM cells. isotype and are RAGE or cells, ASM receptor expression was as for healthy subjects and subjects with asthma for RAGE or TLR4 and healthy patient with asthma, of contraction of collagen gels impregnated with ASM cells from healthy controls and subjects with asthma in the absence of bradykinin, with the area under the curve a as an and a of contraction of collagen gels impregnated with ASM from subjects with asthma in the absence of bradykinin, following with HMGB1 of contraction of collagen gels impregnated with ASM cells in the presence of following with HMGB1 ASM HMGB1 versus control, at each a of contraction of collagen gels impregnated with ASM cells in the presence of following with HMGB1 healthy asthma in at 60 minutes in gels impregnated with ASM from healthy controls and subjects with asthma, versus with the significant ASM cell migration, measured by cell in the migration was but was not significantly different following treatment for with 1000 ng/mL HMGB1, or by the activity of HMGB1 with 10 sRAGE or 10 Cell proliferation as measured by the Cell was unaffected following treatment with 1000 ng/mL HMGB1 for in the presence or absence of The percentage of cells apoptosis as detected by staining and necrosis as detected by was not significantly different following treatment with 1000 ng/mL HMGB1. was as a for the HMGB1 1, 2, ± ± ± ± healthy healthy mild % healthy Sputum ± ± ± ± Sputum healthy mild Sputum epithelial capacity; inhaled ± healthy mild asthma. in a Table for 1, 2, ± ± ± ± healthy mild % healthy Sputum ± ± ± ± Sputum healthy mild Sputum epithelial are of those for the HMGB1 endogenous secretory inhaled capacity; ± healthy mild asthma. in a Table for ± ± ± healthy % healthy Sputum ± ± ± Sputum healthy inhaled capacity; ± healthy in a capacity; inhaled Samples are of those for the HMGB1 endogenous secretory inhaled capacity; inhaled capacity;