Proinflammatory effects of respiratory syncytial virus-induced epithelial HMGB1 on human innate immune cell activation

Abstract

High mobility group box 1 (HMGB1) is a multifunctional nuclear protein that translocates to the cytoplasm, and is subsequently released to the extracellular space during infection and injury. Once released it acts as a damage-associated molecular pattern and regulates immune and inflammatory responses. Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in infants and elderly, for which no effective treatment or vaccine is currently available. This study investigated the effects of HMGB1 on cytokine secretion, as well as the involvement of NF-κB and TLR4 pathways in RSV-induced HMGB1 release in human airway epithelial cells (AECs) and its proinflammatory effects on several human primary immune cells. Purified HMGB1 was incubated with AECs (A549 and small alveolar epithelial cells) and various immune cells, and measured the release of proinflammatory mediators, and the activation of NF-κB and p38 MAPK. HMGB1 treatment significantly increased the phosphorylation of NF-κB and p38 MAPK but did not induce the release of cytokines/chemokines from AECs. However, addition of HMGB1 to immune cells did significantly induce the release of cytokines/chemokines and activated the NF-κB and p38 MAPK pathways. We found that activation of NF-κB accounted for RSV-induced HMGB1 secretion in AECs in a TLR4-dependent manner. These results indicated that HMGB1 secreted from AECs can facilitate the secretion of proinflammatory mediators from immune cells in a paracrine mechanism, thus promoting the inflammatory response that contributes to RSV pathogenesis. Therefore, blocking the proinflammatory function of HMGB1 may be an effective approach for developing novel therapeutics.

Introduction

Respiratory syncytial virus (RSV) is a ubiquitous, single-stranded, negative sense, enveloped RNA virus of the family Pneumoviridae and is an important cause of severe upper and lower respiratory tract infections during infancy and early childhood, worldwide. RSV infection also causes severe morbidity and mortality in the elderly and the immunocompromised (1,2). Currently, there is no effective treatment or vaccine available for RSV. Efforts to develop a safe vaccine for RSV have been hindered due to the complex nature of the infectivity and warrants investigations into the development of new vaccine and therapeutic strategies to treat and prevent respiratory infections caused by RSV. It is very critical to examine the inflammation and host immune response to RSV infection in order to understand its pathogenesis, as this response determines the severity of the disease and cellular immune responses are vital for the clearance of the virus. RSV infection has been shown to upregulate the expression of host genes involved in antiviral and cell-mediated immune responses (3–5). Nuclear factor-kappa B (NF-κB)/REL family of transcription factors plays an important role in coordinating the expression of a wide variety of genes that control immune responses (6). Moreover, Toll-like receptors (TLRs) are key molecules to innate and adaptive immune responses and contribute to early recognition of pathogens (7–9).

High mobility group box 1 (HMGB1) is a ubiquitous, highly conserved redox-sensitive non-histone chromatin-binding nuclear protein in the alarmins family, whose members alerts the immune system to damage and trigger an immediate response (10–15). HMGB1 was first identified as a facilitator of gene transcription, replication, DNA repair and recombination (16). In recent years, extracellular HMGB1 has been identified as a proinflammatory mediator that promotes immune responses by binding to pattern recognition receptors including TLRs and the receptor for advanced glycation end products (RAGE) (17–23), which are involved in inflammatory processes and have the ability to activate a common signaling pathway that culminates in the activation of NF-κB transcription factors. HMGB1 mediates cellular signaling through RAGE, TLR2 and TLR4 receptors to activate the intracellular signal of mitogen-activated protein kinases (MAPKs) and NF-κB (17–19). The interaction of HMGB1 with TLR2 or TLR4 mediates HMGB1’s proinflammatory actions whereas its interaction with RAGE activates NF-κB. As one of the most important downstream molecules in TLR signaling pathways, NF-κB is required for the gene expression of many inflammatory mediators, such as IL-1β, tumor necrosis factor-α, and IL-6 (18,24–26). HMGB1 is actively secreted by a variety of innate immune cells, and is passively released by necrotic cells, and drives inflammation and/or repair (10–15). Once HMGB1 is released into the extracellular milieu it acts as a proinflammatory mediator by activating a wide range of inflammatory responses including the robust release of cytokines (27,28). Recently, we showed that RSV-induced oxidative stress promotes HMGB1 extracellular release and triggers an inflammatory response, implicating the involvement of HMGB1 in RSV pathogenesis (29).

In order to better understand the early pathways of RSV pathogenesis and the immune response to this virus, we explored the proinflammatory activity of HMGB1 on various immune cells in the context of RSV infection and the mechanisms underlying HMGB1 release. We further studied the proinflammatory effects of HMGB1 on immune cells via activation of NF-κB and P38 MAPK signaling pathways. Herein, we demonstrate that RSV-induced HMGB1 release from airway epithelial cells (AECs) (A549 and small alveolar epithelial cells) is mediated in part by NF-κB and TLR-4. Human primary immune cells [(peripheral blood mononuclear cells (PBMCs), PBMC-derived monocytes, macrophages (MΦs), dendritic cells (DCs), and eosinophils (EOS) as well as THP-1 monocytes, THP-1 monocyte-derived MΦs, and EOL1 cells)] stimulated in vitro with purified HMGB1 [recombinant HMGB1 (rHMGB1) and secreted HMGB1 (sHMGB1)] induces the secretion of proinflammatory cytokines and chemokines, which involves the activation of P38 MAPK and NF-κB pathways. These results suggested that HMGB1 acts as a signaling molecule to directly activate immune cells, and its interaction with signaling pathways contributes to the inflammatory response to RSV infection. This study uncovers a hitherto underappreciated role for HMGB1 in driving inflammatory responses during RSV infection that will facilitate discovery of novel therapeutic strategies for the treatment of RSV-induced human diseases.

Materials and Methods:

Reagents

F12K medium, EDTA and HBSS without Mg ²⁺ or Ca ²⁺ were purchased from Gibco-BRL (Grand Island, NY). Novex 10%, 12%, 4–12% and 4–20% mini gels and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Invitrogen (Carlsbad, CA). HEPES and RPMI-1640 were from Cellgro (Manassas, VA). Small airway epithelial cell (SAEC) growth medium was from Lonza (Houston, TX). Dextran, Bay 11–7085, Phorbol 12-myristate 13-Acetate (PMA), Lipopolysaccharides (LPS), and Trypan blue, were obtained from Sigma-Aldrich (St. Louis, MO). The 10X Tris glycine buffer, 10X Tris glycine-SDS electrophoresis buffer, RC DC protein assay kit, human 27-Plex Bio-Plex kit and BioRad protein assay reagent were from BioRad (Hercules, CA). Amersham Full-Range Rainbow Molecular Weight Markers and hybond-polyvinylidene difluoride membrane (PVDF) were from GE Healthcare (Piscataway, NJ). Immobilon Western horseradish peroxidase (HRP) substrate and ProteoExtract Subcellular Proteome Extraction kit was obtained from EMD Millipore (Billerica, MA). Recombinant human HMGB1 and mouse monoclonal human RAGE antibody were purchased from R&D Systems (Minneapolis, MN). The recombinant human cytokines GM-CSF and IL-4 were from PeproTech (Rocky Hill, NJ). VarioMACS separation columns, MACS Separator (magnetic), and CD16 and CD14 MicroBeads were from Miltenyi Biotec (Auburn, CA). Rabbit polyclonal anti-human HMGB1 antibody and mouse monoclonal anti-TLR4 antibody were from Abcam (Cambridge, MA). Mouse monoclonal anti-β-actin antibody was from Sigma-Aldrich (St. Louis, MO). Phospho-NF-κB p65 (Ser536) rabbit monoclonal antibody, phospho-p38 MAPK (Thr180/Tyr182) rabbit mAb, goat anti-mouse IgG HRP and goat anti-rabbit IgG-HRP were from Cell signaling Technologies (Danvers, MA), and the FITC-conjugated secondary antibody was from Southern Biotech (Birmingham, AL).

Ethical Statement.

The use of plasma and PBMC samples in this study was conducted with the approval of the Institutional Review Board at the University of Texas Medical Branch in Galveston in accordance with its guidelines for the protection of human subjects (IRB# 04–371). All participants gave written informed consent to participate in the study.

RSV preparation.

For RSV stock preparation, the RSV Long strain was grown in HEp-2 cells and purified by centrifugation on discontinuous sucrose gradients as described previously (30,31). The virus titer of the purified RSV pools was 8–9 log₁₀ plaque forming units (PFU)/mL using a methylcellulose plaque assay (32). No contaminating cytokines were found in these sucrose-purified viral preparations. Lipopolysaccharide (LPS) was not detected using the limulus hemocyanin agglutination assay. Virus pools were aliquoted, quick-frozen on dry ice/alcohol, and stored at −80^oC until used.

Cell culture and RSV infection of airway epithelial cells.

The human airway epithelial cell line A549 (human alveolar type II-like epithelial cell line-American Type Culture Collection, Manassas, VA) and small alveolar epithelial (SAE) cells, which are primary human airway epithelial cells derived from terminal bronchioli of cadaveric donors (Lonza, San Diego, CA), were grown according to the manufacturer’s instructions. A549 cells were cultured and maintained in F12K medium containing 10% FBS, 2 mM glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin. SAE cells were maintained in small airway epithelial cell (SAEC) growth medium containing 7.5 mg/mL bovine pituitary extract, 0.5 mg/mL hydrocortisone, 0.5 μg/mL human epidermal growth factor, 0.5 mg/mL epinephrine, 10 mg/mL transferrin, 5 mg/mL insulin, 0.1 μg/mL retinoic acid, 0.5 μg/mL triiodothyronine, 50 mg/mL gentamicin and 50 mg/mL bovine serum albumin. Monolayers of undifferentiated SAE cells were cultured in 25-cm² flasks at 37⁰C and 5% CO₂ with an SAEC basal medium supplied with growth factors. Cells were used in the experiments at passage three (31). When SAE cells were used for RSV infection, the cells were changed to basal medium and not supplemented with growth factors 6 h prior to and throughout the length of the experiment. At 80–90% confluency, cell monolayers were infected with RSV at multiplicity of infection (MOI) of 1 (unless otherwise stated), as previously described (33). An equivalent amount of a 30% sucrose solution was added to uninfected A549 and SAE cells, as a control. In some experiments, cells were pretreated with Bay 11–7085, phorbol myristate acetate (PMA), recombinant HMGB1 (rHMGB1), secreted HMGB1 (sHMGB1), TLR-4 monoclonal antibody (mAb), or RAGE mAb for 1 h and then infected with RSV in the presence of the selected compound. The total number of cells and cell viability were measured by trypan blue exclusion. There was no significant change in cell viability when cells were incubated in the presence of sHMGB1 or rHMGB1 or Bay 11 or PMA.

Isolation of human immune cells from human peripheral blood and viral infection.

Peripheral blood (60 ml) was drawn from healthy, non-smoking individuals (18–50 years old) as we previously reported under a research protocol approved by the IRB committee at the University of Texas Medical Branch (29). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Hypaque and monocytes were purified using magnetic CD14 Microbeads (Miltenyi Biotec, Sunnyvale, CA). Primary monocyte-derived macrophages (MΦs) were obtained after 6 days of differentiation in RPMI-1640 medium supplemented with 2 mM L-glutamine, 10% FBS, 50 μM 2-mercaptoethanol, 1,000 U/L penicillin-streptomycin and 10ng/mL M-CSF. Eosinophils (EOS) were obtained by sedimentation in 4–6% dextran for 30 min at room temperature (RT), followed by centrifugation in a Ficoll-Hypaque gradient as described previously (34). Following centrifugation at 500 × g, upper layers of plasma and mononuclear cells were removed and saved for further analysis. Eosinophil isolation used hypotonic lysis for elimination of erythrocytes and negative selection using a combination of anti-CD16, anti-CD3, anti-CD235, and anti-CD14 microbeads to remove neutrophils and other contaminating cells using the MACS system 9 (Miltenyi Biotec). Eosinophil purity was consistently monitored by Hansel staining and typically ranged above 98%. Monocyte contamination was virtually undetectable and activated eosinophils were ≤ 1%. Primary monocyte-derived dendritic cells were obtained after 5 days of differentiation in RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine containing 1500 U/ml IL-4 and 1600 U/ml GM-CSF. THP-1 monocytes-derived MΦs were obtained by culturing the cells with 10 ng/mL PMA for 72 hours. Cells (5 × 10⁵) were pretreated with 100 μL of rHMGB1 or sHMGB1 at 100 ng/mL for 1 h followed by infection with RSV at an MOI of 3 for 1–2 h at 37°C and then washed twice with RPMI. For the remaining time of infection, the cells were placed in a 24-well plate in a total volume of 1 mL of RPMI. Cytoplasmic and nuclear fractions were prepared from MΦs using ProteoExtract Subcellular Proteome Extraction kit from EMD Millipore (Billerica, MA).

Purification of RSV-infected A549 cells-secreted HMGB1 (sHMGB1).

We purified sHMGB1 from RSV-infected A549 cells to compare with rHMGB1 and evaluate biological activity. A549 cells were cultured as described above and infected with RSV at an MOI of 1 in serum-free medium. Cell culture supernatants were centrifuged (12,000 x g, 10 min, 4˚C) and stored at −80˚C until further use. Western blot (WB) analysis with an anti-HMGB1 antibody preparation described above was used to determine the presence of sHMGB1 in the cell culture medium. The cell culture medium (~ 1.5 L) was thawed and purified using a combination of anion exchange chromatography and cation exchange chromatography. Eluted fractions were evaluated for the presence of HMGB1 by WB analysis with anti-HMGB1 antibody. The sHMGB1 corresponding fractions were combined and was further purified by an anion exchange chromatography step. The eluted fractions were evaluated for sHMGB1 using immunologic dot blotting with anti-HMGB1 antibody. The sHMGB1 peak was pooled and further purified by cation exchange chromatography.

Measurement of cytokines.

After 24 hours, cell-free supernatants were collected and tested for multiple cytokines using the Bio-Plex Human Cytokine 27-Plex panel (Bio-Rad Laboratories, Hercules, CA), according to the manufacturer’s instructions. The panel included the following cytokines: IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 p70, IL-13, IL-15, IL-17, Eotaxin, FGF-basic, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-bb, RANTES, TNF-α and VEGF.

Western blotting and densitometric analyses.

Total cell lysates were prepared from uninfected, RSV-infected, TLR-4 mAb, RAGE mAb, Bay 11, PMA and HMGB1-treated A549, SAE cells, PBMCs, monocytes, macrophages, eosinophils, dendritic cells, THP-1 monocytes, THP-1 monocyte-derived macrophages and EOL1 cells by adding ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1mM EGTA, 0.25% sodium deoxycholate, 1 mM Na₃VO₄, 1 mM NaF, 1% Triton X-100 and 1 μg/mL of aprotinin, leupeptin and pepstatin. After incubation on ice for 10 min, the lysates were centrifuged at 4^oC at 14,000 g to remove the detergent insoluble cell debris. Proteins (10 to 20 μg per sample) were boiled in 2X Laemmli buffer and fractionated by SDS-PAGE. Proteins were then transferred onto a hybond-polyvinylidene difluoride membrane (Amersham, Piscataway, NJ), and the membranes were incubated for 30 min in 10 mM Tris-buffered saline-Tween (TBST, Tris-HCl, pH 7.6, containing 150 mM NaCl, 0.05% Tween-20) and 5% bovine serum albumin. After blocking the non-specific binding sites, membranes were washed with TBST and then incubated sequentially with the primary antibody overnight at 4^oC and then anti-rabbit peroxidase-conjugated secondary antibody (1:10,000 in TBST) at RT for 30 min. Membranes were washed and signal was detected using Enhanced Chemilluminescence (ECL) substrate (Amersham) according to the manufacturer`s protocol. The primary antibodies for Western blots were anti-HMGB1 rabbit polyclonal antibody, Phospho-NF-κB p65 (Ser536) rabbit monoclonal antibody (mAb), phospho-p38 MAPK (Thr180/Tyr182) rabbit mAb and anti-β-actin monoclonal antibody. The secondary antibodies were goat anti-mouse IgG HRP and goat anti-rabbit IgG-HRP. Densitometric analysis of WB band intensities was performed using Image J (NIH) software.

Immunocytochemistry.

Cells were cultured in LabTek II chambers (Nalge Nunc, Penfield, NY) and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at RT for 30 min. The cells were then washed with PBS and incubated at 4^oC for 10 min with permeabilization buffer (PBS containing 0.1% Triton X-100). After blocking with 5% BSA in PBS for 1 h, cells were incubated with anti-HMGB1 antibody (Abcam), followed by incubation for 1 h with Alexa Flour 488-conjugated or FTIC-conjugated secondary antibody (Invitrogen). The cells were coverslip-mounted using mounting medium containing the fluorescent nuclear stain 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen) and signals were analyzed using a fluorescence microscope (Nikon, Japan).

Statistics.

A two-tailed Student’s t-test using 95% confidence levels was performed in all experiments using Graph PAD prism 5.02. Significance is presented as * p<0.05, ** p<0.005, and *** p<0.0005.

Results:

HMGB1 activates the NF-κB and p38 mitogen-activated protein kinase (MAPK) pathway in air way epithelial cells but does not induce release of proinflammatory mediators:

Airway epithelial cells (AECs) are the major targets of RSV infection, and have been shown to secrete a variety of proinflammatory molecules that regulate the migration and activation of leukocytes and play a key role in inflammatory and infectious processes in the lung (35,36). We previously showed that RSV infection induces significant HMGB1 extracellular release by AECs and addition of recombinant HMGB1 (rHMGB1) did not by itself induce the release of cytokines/chemokines from A549 cells (29). In this study, we further confirmed these results with the addition of secreted HMGB1 (sHMGB1) purified from RSV-infected A549 cell culture supernatants. A549 cells were incubated with sHMGB1 and/or rHMGB1 at 100 ng/mL followed by infection with RSV at an MOI of 1 and proinflammatory mediator release was measured after 24 h post infection (p.i.) in the cell-free supernatants using a Bio-Plex assay. Total cell lysates were utilized to measure phosphorylation of NF-κB (pP65 ser536) and p38 MAPK (Thr180/Tyr182) by Western blot (WB). A549 cells did not release proinflammatory mediators with the addition of purified HMGB1 (Figs. S1 A and B) (29). However, both sHMGB1 and rHMGB1 treatment of the A549 cells in the presence or absence of RSV resulted in a significant increase in the release of IL-8 and MCP-1, compared to untreated/RSV-infected cells, suggesting that HMGB1 provides danger signal to the cells in eliciting proinflammatory gene expression in RSV-infected A549 cells (Fig. S1 A). To confirm these results, we also examined the effect of purified HMGB1 on primary human small alveolar epithelial (SAE) cells, which are derived from terminal bronchioles of a normal individual. Similar findings were observed in SAE cells in which HMGB1 did not have any effect on the release of proinflammatory mediators (Fig. S1 B).

NF-κB is a multisubunit molecule that belongs to the REL family of transcription factors involved in the regulation of large number of genes that control various aspects of the immune and inflammatory response, and its activation is triggered by variety of stimuli including RSV (36,37). The P38 MAP kinase signaling pathway plays a central role in regulating cellular inflammatory and stress responses, protein synthesis, and in the production of proinflammatory cytokines (38). RSV is a potent inducer of NF-κB and p38 MAPK phosphorylation in A549 and SAE cells (33,39–42). In this study, NF-κB and p38 MAPK activation was determined in AECs after purified HMGB1 stimulation by measurement of pP65 and pP38 levels. Addition of rHMGB1 activated NF-κB and p38 MAPK signaling pathways in A549 and SAE cells, and was significantly enhanced in the presence of RSV (Figs. 1 A, B, C and D). Densitometric analysis of the WB showed that rHMGB1 treatment of A549 cells increased the pP65 expression by 2.1-fold and RSV induced 5.5-fold when compared to uninfected cells, whereas pP38 MAPK expression was increased by 2.9-fold with rHMGB1 treatment and RSV infection induced 3.4-fold compared to uninfected cells. There was a significant increase in both pP65 and pP38 levels in RSV-infected and rHMGB1-treated cells. Similar findings were observed in SAE cells in which rHMGB1 treatment alone increased pP65 levels by 1.5-fold and with RSV infection by 2.7-fold, whereas pP38 levels increased by 1.5-fold with rHMGB1 alone and by 3.4-fold in the presence of RSV. These results indicated that HMGB1 activates the p38 MAPK and NF-κB signaling pathways to promote the inflammatory response.

FIG 1.

The effect of purified HMGB1 on NF-κB (P65) and P38 MAPK activation in human airway epithelial cells. A549 (A and C) and SAE (B and D) cells were treated with recombinant HMGB1 (rHMGB1) (100 ng/mL) and mock-infected or infected with RSV at an MOI of 1. Cell-free supernatants and total cell lysates were prepared after 24 h post-infection (p.i.) to measure HMGB1, pP65 and pP38 by Western Blot (WB) (A and B). Densitometric analysis of WB band intensities were performed using Image J software and were normalized to internal loading control β-actin for lysates (C and D). The figure is representative of three independent experiments and quantified results are shown in bar graphs. * p<0.05, ** p<0.005, and *** p<0.0005.

RSV-induced HMGB1 translocation and extracellular release in airway epithelial cells was mediated via NF-κB:

RSV infection causes significant secretion of HMGB1 from AECs (29) and is a potent inducer of NF-κB in lung epithelial cells (33,39,40). Several studies have demonstrated that HMGB1 activates the NF-κB signal transduction pathway and facilitates several immunosuppressive mechanisms (43–45). In this study, we determined that HMGB1 secretion from AECs in response to RSV infection occurs via the NF-κB pathway. We investigated the effect of BAY 11–7085, a small molecule IKKα/β inhibitor of NF-κB activation, and phorbol 12-myristate 13-Acetate (PMA), a potent activator of NF-κB signaling, on RSV-induced HMGB1 release in A549 cells. A549 cells were pretreated either with Bay 11 (250 ng/mL) or PMA (1 μg/mL) and infected with RSV at an MOI of 1 and HMGB1 level was measured in the cell-free supernatants and total cell lysates. Inhibition of NF-κB signaling by Bay 11 significantly blocked HMGB1 release with a concomitant increase in the cellular HMGB1. We observed a decrease of 13.7-fold compared to RSV infection alone, which was 56.7-fold higher compared to uninfected cells (Figs. 2 A and C). NF-κB activation by PMA significantly enhanced RSV-induced HMGB1 release compared to each stimulus alone, as we noticed an increase of 36.4-fold compared to RSV infection alone, which was only 23.8-fold higher compared to control cells (Figs. 2 B and D). These findings confirm the involvement of NF-κB signaling in RSV-induced HMGB1 release from AECs.

FIG 2.

The effect of NF-κB inhibition and activation on RSV-induced HMGB1 release. A549 cells were pretreated either with Bay 11 (soluble inhibitor) at 250 ng/mL (A and C) or PMA (activator) at 1 μg/mL (B and D) in the presence or absence of RSV, and cell-free supernatants as well as total cell lysates were prepared at 24 h p.i. to measure HMGB1 by WB using anti-HMGB1 antibody (A and B). β-actin was an internal loading control. Densitometric analysis of HMGB1 WB band intensity of supernatants and lysates were performed using Image J software (C and D). Band intensities for lysates were normalized to internal control β-actin. The figure is representative of three independent experiments. * p<0.05 and ** p<0.005.

Effects of TLR4 or RAGE monoclonal antibodies on RSV-induced HMGB1 release and NF-κB activation:

Although our findings indicated that RSV infection induces HMGB1 secretion, the molecular basis of its nuclear translocation, mechanism of its release into the extracellular space, and the pathways governing its release are unclear. However, a possible mechanism involved reactive oxygen species generated in RSV-infected AECs due to increased oxidative stress (29). In order to determine the involvement of TLR4 and RAGE, receptors of HMGB1 signaling, we investigated the effect of blockage of these receptors using monoclonal antibodies (mAb). A549 cells were pretreated either with TLR4 mAb (2 μg/mL) or RAGE mAb (2 μg/mL) in the presence or absence of RSV and HMGB1 levels measured by WB. Our results showed that pretreating RSV-infected AECs with a TLR4 mAb significantly inhibited RSV-induced HMGB1 release by 0.37-fold (37%) compared to RAGE mAb treatment, suggesting the involvement of TLR4 signaling in RSV-induced HMGB1 secretion (Figs. 3 A and C). A slight, but not significant, reduction of RSV-induced HMGB1 release was observed with RAGE mAb pretreatment (Figs. 3 B and D). Activation of NF-κB was also slightly, but not significantly decreased with TLR4 blockage whereas blocking RAGE significantly enhanced NF-κB activation (Figs. 3 A, B, C and D). These results suggested that RSV-induced HMGB1 secretion in AECs was mediated through the TLR4 pathway whereas RAGE blockade had no effect on RSV-induced HMGB1 release.

FIG 3. Effect of TLR4 and RAGE monoclonal antibody (mAb) on RSV-induced HMGB1 release.

A549 cells were pretreated with TLR4 mAb (2 μg/mL) (A) and RAGE mAb (B) (μg/mL) and infected with RSV at an MOI of 1 and measured HMGB1 and pP65 levels by WB (A and B). Membrane was stripped and reprobed for β-actin as an internal control for protein integrity and loading. Densitometric analysis of HMGB1 WB band intensity of supernatants (upper panel) and lysates (lower panel) were performed using Image J software (C and D). Band intensities for lysates were normalized to internal control β-actin. The figure is representative of three independent experiments. Student t-test * p<0.05 and ** p<0.005 compared with control and RSV-infected cells.

HMGB1 activated peripheral blood mononuclear cells to induce an inflammatory response:

Peripheral blood mononuclear cells (PBMCs) is a population of immune cells consisting of 70–90% lymphocytes (T cells, B cells and NK cells), 10–30% monocytes, and 1–2% dendritic cells. In order to determine the effects of RSV infection on PBMCs in terms of HMGB1 release, we infected PBMCs with RSV at an MOI of 3 and measured HMGB1 levels in the cell-free supernatants and total cell lysates. RSV did not induce the release of HMGB1 from PBMCs, indicating that RSV-induced HMGB1 release was cell specific (Fig. 4 A). To understand the mechanisms associated with immune cell activation by HMGB1, we next examined the levels of NF-κB and p38 MAPK phosphorylation in PBMCs in response to purified HMGB1 treatment and RSV infection. PBMCs were incubated with rHMGB1 and sHMGB1 at 100 ng/mL in the presence or absence of RSV and we measured NF-κB and p38 MAPK activation by WB. Addition of purified HMGB1 alone significantly activated the NF-κB and p38 MAPK signaling pathways, but activation was diminished in the presence of RSV (Fig. 4 A). Densitometric analysis of the WB showed that rHMGB1 and sHMGB1 treatment of PBMCs increased pP65 level by ~1.6-fold and 4.1-fold, respectively. RSV alone caused about a 2.7-fold increase in pP65 levels whereas in the presence of rHMGB1 phosphorylation was decreased by 0.6-fold and in the presence of sHMGB1 it was decreased by 0.2-fold. pP38 MAPK levels were increased by 4.6-fold with rHMGB1 treatment and a 10.6-fold increase was observed in response to sHMGB1 treatment versus untreated cells. RSV infection alone induced a 1.1-fold compared to uninfected cells whereas RSV in combination with sHMGB1, increased P38 MAPK phosphorylation by 2.8-fold (Fig. 4 B). These results showed that HMGB1 activates both the p38 MAPK and NF-κB signaling pathways to promote the inflammatory responses in PBMCs. Our studies showed that RSV-induced HMGB1 secretion from AECs did not induce proinflammatory mediator release by A549 (29) and SAE cells (Figs. S1 A and B), but instead activated human monocytes to promote an inflammatory response (29). Here, we tested the effects of purified HMGB1 on proinflammatory mediator release in PBMCs. Primary human PBMCs were pretreated with purified HMGB1 (100 ng/mL) in the presence or absence of RSV, and cell-free supernatants were used to measure cytokine/chemokine release by a Bio-Plex assay (Fig. 5). Our results show that RSV infection alone induced a significant level of secretion of IL-6, FGF-basic, IP-10, and VEGF (Fig. 5-I); RSV infection of cultured human PBMCs plus rHMGB1 or sHMGB1 treatment significantly induced the release of proinflammatory mediators including IL-8, MCP-1, MIP-1α, MIP-1β, PDGF and RANTES (Fig. 5-II). Both RSV and HMGB1 synergistically increased IL-1RA, IL-10, G-CSF and TNF-α secretion (Fig. 5-III). We also observed an antagonistic effect of HMGB1 on IP-10 and MCP-1. These results indicate that HMGB1 released from AECs can provide danger signals to neighboring immune cells in the airways and promote inflammation, and also provide a synergistic signal in eliciting proinflammatory gene expression in RSV-infected human PBMCs.

FIG 4.

The effect of purified HMGB1 on NF-κB and P38 MAPK activation in human peripheral blood mononuclear cells (PBMCs). Human PBMCs were cultured with rHMGB1 or sHMGB1 (100 ng/mL) and mock-infected or infected with RSV at an MOI of 3. Cell-free supernatants and total cell lysates were prepared after 24 h p.i. to measure HMGB1, pP65 and pP38 by WB (A). Densitometric analysis of WB band intensities were performed using Image J software and were normalized to internal loading control β-actin for lysates (B). The figure is representative of three independent experiments and quantified results are shown in bar graphs. * p<0.05, ** p<0.005, and *** p<0.0005.

FIG 5.

Effect of purified rHMGB1and sHMGB1 on proinflammatory mediator release in human PBMCs. Cell-free supernatants from uninfected and RSV-infected PBMCs in the presence or absence of rHMGB1 or sHMGB1(100 ng/mL) were harvested after 24 h p.i. to measure the concentrations of different proinflammatory mediators by Bio-Plex. n = 3 independent experiments run in triplicate. Panel I: proinflammatory mediators stimulated by RSV. Panel II: proinflammatory mediators stimulated by RSV as well as HMGB1. Panel III: proinflammatory mediators stimulated in combination with HMGB1 and RSV infection. * p<0.05, ** p<0.005, and *** p<0.0005 compared with uninfected, RSV-infected, and HMGB1-treated cells.

HMGB1 activated NF-κB and MAPK signaling pathway in primary human monocytes to promote an inflammatory response:

Monocytes are leukocytes made in the bone marrow that then travel through blood to tissues where they can differentiate into macrophages (MΦs) and dendritic cells (DCs), which play an important role in the defense against invading microbes and in inflammation. Our previous studies showed that RSV did not induce HMGB1 release from human monocytes but activated these cells to induce release of proinflammatory mediators (29). In the present study, we examined the effects of HMGB1 on NF-κB and p38 MAPK signaling pathway activation in human monocytes to understand HMGB1’s proinflammatory role in inducing an inflammatory response to RSV infection. Addition of purified HMGB1 to monocytes significantly activated both signaling pathways (Figs. 6 A and B). Densitometric analysis of the WB showed that rHMGB1 treatment of human primary monocytes increased pP65 levels by 2.11-fold, whereas RSV infection alone induced 2.87-fold when compared to uninfected cells (Fig. 6 B). pP38 MAPK levels were increased by 3.8-fold with rHMGB1 treatment while RSV infection alone induced 4.64-fold compared to uninfected cells (Fig. 6 B). There was a significant decrease in both pP65 (22%) and pP38 (21%) levels in RSV-infected and rHMGB1-treated cells (Figs. 6 A and B). To confirm these results, we also examined the effect of purified HMGB1 on THP-1 monocytes and observed similar findings (Figs. S2 A and B), suggesting the activation of p38 MAPK and NF-κB signaling pathway by HMGB1 in monocytes. Addition of rHMGB1 (100 ng/mL) to human primary monocytes in the presence or absence of RSV induced a significant release of cytokines/chemokines [(Fig. 7 (29)]. Our results show that RSV infection alone induced a significant level of secretion of IL1-RA, IL-10, FGF-basic, and VEGF (Fig. 7-I), whereas RSV infection as well as rHMGB1 treatment of cultured human monocytes significantly induced the release of proinflammatory mediators including IL-1β, IL-6, IL-8, TNF-α, IP-10, GMCSF, MCP-1, MIP-1α, MIP-1β, IFN-γ and RANTES (Fig. 7-II). Both RSV and HMGB1 synergistically increased IL-12 and G-CSF secretion (Fig. 7-III). We observed similar results with THP-1 monocytes (Fig. S3).

FIG 6.

The effect of purified HMGB1 on NF-κB and P38 MAPK activation in human primary monocytes. Monocytes were treated with rHMGB1 or LPS (100 ng/mL) and mock-infected or infected with RSV at an MOI of 3. Cell-free supernatants and total cell lysates were prepared after 24 h p.i. to measure HMGB1, pP65 and pP38 by WB (A). Densitometric analysis of WB band intensities were performed using Image J software and were normalized to internal loading control β-actin for lysates (B). The figure is representative of three independent experiments and quantified results are shown in bar graphs. * p<0.05 and ** p<0.005

FIG 7.

Effect of purified rHMGB1 on proinflammatory mediator release by primary human monocytes. Cell-free supernatants from uninfected and RSV-infected monocytes in the presence or absence of rHMGB1 (100 ng/mL) were harvested after 24 h p.i. to measure the concentrations of different proinflammatory mediators by Bio-Plex. n = 3 independent experiments run in triplicate. Panel I: proinflammatory mediators stimulated by RSV. Panel II: proinflammatory mediators stimulated by RSV as well as HMGB1. Panel III: proinflammatory mediators stimulated in combination with HMGB1 and RSV infection. * p<0.05, ** p<0.005, and *** p<0.0005 compared with uninfected, RSV-infected, and HMGB1-treated cells.

Effect of RSV infection on HMGB1 release in human macrophages:

Monocytes/ MΦs play a central role in orchestrating the immune and inflammatory responses to infection and injury. Our previous studies have shown that RSV infection causes the translocation of HMGB1 from the nucleus to the cytoplasm and subsequently to the extracellular space in AECs (29). Since WB results did not detect the release of HMGB1 in monocyte-derived human MΦs in response to RSV infection (Figs. 8 A and B), we performed immunofluorescence microscopic studies to determine the localization of HMGB1. MΦs were immunostained with HMGB1 antibodies after 24 h p.i. in the presence or absence of RSV. As a positive control, cells were treated with LPS (100 ng/mL). HMGB1 was predominantly present in the nucleus as indicated by nuclear localization in uninfected cells, however in RSV-infected cells HMGB1 accumulated at the periphery within the cytoplasm. In LPS-treated cells, HMGB1 levels were significantly reduced as a result of its increased extracellular release (Fig. 8 C). These results were further confirmed by WB analysis of uninfected, RSV-infected and LPS-treated MΦs, where we observed a decreased nuclear HMGB1 levels with a concomitant increase in the cytoplasmic levels of RSV-infected cells; whereas with LPS treatment both nuclear and cytoplasmic HMGB1 levels were significantly decreased confirming its extracellular release (Fig. 8 D).

FIG 8.

The effect of purified HMGB1 on NF-κB and P38 MAPK activation in human primary macrophages (MΦs). Human primary MΦs were treated with rHMGB1 or sHMGB1 (100 ng/mL) and mock-infected or infected with RSV at an MOI of 3. Cell-free supernatants and total cell lysates were prepared after 24 h p.i. to measure HMGB1, pP65 and pP38 by WB (A). Densitometric analysis of WB band intensities were performed using Image J software and were normalized to internal loading control β-actin for lysates (B). The figure is representative of three independent experiments and quantified results are shown in bar graphs. * p<0.05 and ** p<0.005. (C) The effect of RSV infection on HMGB1 localization in primary human macrophages. Human MΦs were mock-infected or infected with RSV at an MOI 3 or LPS (100 ng/mL) and the cells were incubated after 24 h p.i. with rabbit anti-HMGB1 polyclonal antibody followed by Alexa Flour 488-conjugated secondary antibody. Nuclei were labeled with DAPI and analyzed by confocal immunofluorescence microscopy (original magnification 400X). Representative fluorescence microscopic images (HMGB1 and DAPI) and merged images (overlay) are shown. Scale bar, 20 μm. (D). Nuclear and cytoplasmic fractions were prepared from uninfected, RSV-infected and LPS-treated MΦs and probed with HMGB1 antibody. Membranes were stripped and reprobed for β-actin and Lamin B antibodies as an internal control for cytoplasmic and nuclear fractions, respectively.

Effects of HMGB1 on NF-κB and p38 MAPK signaling pathway activation and proinflammatory cytokine/chemokine release in human primary macrophages:

Alveolar MΦs play a central role in the host defense against respiratory pathogens by initiating innate and adaptive immune responses, which are also a primary source of proinflammatory cytokines and chemokines. In this study, we investigated the effects of HMGB1 on NF-κB and P38 MAPK signaling pathway activation and on proinflammatory mediator release in primary human MΦs to further explore the regulatory mechanisms associated with MΦs activation by HMGB1. Our results showed that HMGB1 activated the NF-ΚB signaling pathway in MΦs when added at a concentration of 100 ng/mL which is equivalent to the amount secreted by AECs after RSV infection (29) (Figs. 8 A and B; S2 C and D). P38 MAPK activation was observed only in the presence of RSV. Densitometric analysis of the WB showed that rHMGB1 and sHMGB1 treatment of monocyte-derived MΦs increased pP65 levels by 1.8-fold and 0.8-fold, respectively and RSV infection alone induced 1.14-fold compared to uninfected cells. Addition of HMGB1 to RSV-infected cells did not change the phosphorylation of P65 when compared to RSV infection alone. Addition of HMGB1 to human MΦs decreased the pP38 MAPK level significantly compared to uninfected cells, but in the presence of RSV sHMGB1 increased the pP38 levels by 34% compared to RSV alone (Figs. 8 A and B). Similar findings were observed with THP-1 monocyte-derived MΦs (Figs. S2 C and D). These results demonstrated that HMGB1 activated both p38 MAPK and NF-κB signaling pathways in human macrophages.

Our results also showed that addition of rHMGB1 (100 ng/mL) to human MΦs in the presence or absence of RSV induced significant release of cytokines/chemokines (Fig. 9). RSV infection alone induced significant secretion of MCP-1 (Fig. 9-I), whereas RSV infection as well as rHMGB1 treatment to cultured human MΦs significantly induced the release of proinflammatory mediators including IL1-RA, IL-6, IL-8, TNF-α, IP-10, GM-CSF, MIP-1α, MIP-1β and RANTES (Fig. 9-II). Both RSV and HMGB1 synergistically increased IL-12, G-CSF and IFN-γ secretion (Fig. 9-III). We observed similar results with THP-1 monocyte-derived MΦs (Fig. S4).

FIG 9.

Effect of purified rHMGB1 and sHMGB1 on proinflammatory mediator release in primary human macrophages. Cell-free supernatants from uninfected and RSV-infected human macrophages in the presence or absence of rHMGB1 or sHMGB1 (100 ng/mL) were harvested after 24 h p.i. to measure the concentrations of different proinflammatory mediators by Bio-Plex. n = 3 independent experiments run in triplicate. Panel I: proinflammatory mediators stimulated by RSV. Panel II: proinflammatory mediators stimulated by RSV as well as HMGB1. Panel III: proinflammatory mediators stimulated in combination by HMGB1 and RSV infection. * p<0.05, ** p<0.005, and *** p<0.0005 compared with uninfected, RSV-infected, and HMGB1-treated cells.

HMGB1 activates NF-κB and p38 MAPK signaling pathways and triggers the release of proinflammatory cytokines/chemokines in human primary eosinophils:

Eosinophils (EOS) are the leukocytes produced in the bone marrow that migrate to tissues throughout the body in order to fight against infections. In this study, we examined the effects of HMGB1 on NF-κB and p38 MAPK signaling pathway activation and proinflammatory mediator release in primary human EOS. RSV infection did not induce the release of HMGB1 in EOS, similar to that observed in other immune cells; however, HMGB1 (100 ng/mL) activated NF-ΚB and P38 MAPK in EOS (Figs. 10 A and B). Addition of rHMGB1 to EOS increased the phosphorylation of P65 and P38 MAPK significantly by 7 and 3.3-fold, respectively, compared to uninfected cells. In the presence of RSV infection, rHMGB1 increased pP65 levels by 42% compared to RSV alone, which was 2.1-fold compared to uninfected cells (Figs. 10 A and B). Similar findings were observed with EOL1 cells (Figs. S2 E and F). These results confirmed that HMGB1-induced activation of both p38 MAPK and NF-κB signaling pathways in human EOS.

FIG 10.

The effect of purified HMGB1 on NF-κB, P38 MAPK activation and proinflammatory mediator release in primary human eosinophils (EOS). EOS were treated with rHMGB1 (100 ng/mL) and mock-infected or infected with RSV at an MOI of 3. Cell-free supernatants and total cell lysates were prepared after 24 h p.i. to measure HMGB1, pP65 and pP38 by WB (A). Densitometric analysis of WB band intensities were performed using Image J software and were normalized to internal loading control β-actin for lysates (B). The figure is representative of three independent experiments and quantified results are shown in bar graphs. * p<0.05 and ** p<0.005. The concentrations of different proinflammatory mediators in cell-free supernatants were measured by Bio-Plex (C). n = 2 independent experiments run in triplicate. Proinflammatory mediators stimulated in combination by HMGB1 and RSV infection. * p<0.05 and ** p<0.005 compared with uninfected, RSV-infected, HMGB1-treated cells.

Addition of purified HMGB1 to human primary EOS in the presence of RSV significantly induced the release of IL-8 and PDGF (Fig. 10 C). Addition of HMGB1 to EOL1 cells significantly induced cytokine and chemokine release (Fig. S2 G).

HMGB1 activates NF-κB and p38 MAPK and triggers proinflammatory mediator release in human primary monocyte-derived dendritic cells

Dendritic cells (DCs) are the antigen-presenting cells that function to process antigens and present them to T cells to promote immunity to foreign antigens. They also secrete cytokines to regulate immune responses. In this study, we tested the effect of HMGB1 on NF-κB and P38 MAPK signaling pathway activation as well as proinflammatory mediator release in primary human DCs. RSV infection did not induce the release of HMGB1 in DCs, whereas purified HMGB1 activated NF-ΚB and P38 MAPK signaling pathway (Figs. 11 A and B). Addition of rHMGB1 to human primary DCs increased the phosphorylation of P65 and P38 MAPK significantly by 0.94 and 1.5-fold, respectively, compared to uninfected cells. Yet, in the presence of RSV, rHMGB1 increased the P65 and P38 MAPK phosphorylation by 33% and 37%, respectively, compared to RSV alone which was 1.26 and 5.2-fold increase, respectively, compared to uninfected cells (Figs. 11 A and B). RSV infection alone significantly induced the release of IL1–1β, IL-1ra, IL-4, IL-6, IL-10, IL-12, IL-17, Eotaxin, FGF basic, G-CSF, MCP-1, and MIP-1β from human DCs (Fig. 12-I). Addition of purified HMGB1 to human DCs as well as RSV infection induced significant release of IL-8, GM-CSF, IP-10, IFN-γ, MIP-1α, RANTES and TNF-α (Fig. 12-II). Both RSV and HMGB1 synergistically increased PDGF and VEGF (Fig. 12-III).

FIG 11.

The effect of purified HMGB1 on NF-κB and P38 MAPK activation in primary human dendritic cells (DCs). Human DCs were treated with rHMGB1 (100ng/mL) and mock-infected or infected with RSV at an MOI of 3. Cell-free supernatants and total cell lysates were prepared after 24 h p.i. to measure HMGB1, pP65 and pP38 by WB (A). Densitometric analysis of WB band intensities were performed using Image J software and were normalized to internal loading control β-actin for lysates (B). The figure is representative of three independent experiments and quantified results are shown in bar graphs. * p<0.05 and ** p<0.005.

FIG 12.

Effect of rHMGB1 on proinflammatory mediator release in human primary dendritic cells (DCs). Cell-free supernatants from uninfected and RSV-infected human DCs in the presence or absence of rHMGB1(100 ng/mL) were harvested after 24 h p.i. to measure the concentrations of different proinflammatory mediators by Bio-Plex. n = 2 independent experiments run in triplicate. Panel I: proinflammatory mediators stimulated by RSV. Panel II: proinflammatory mediators stimulated by RSV as well as HMGB1. Panel III: proinflammatory mediators stimulated in combination with HMGB1 and RSV infection. * p<0.05, ** p<0.005, and *** p<0.0005 compared with uninfected, RSV-infected, HMGB1-treated cells.

Discussion

HMGB1 is a novel inflammatory signaling molecule that contributes to the pathogenesis of many inflammatory diseases (24,46–48). In this study, we demonstrated that purified HMGB1 induced a proinflammatory response in human immune cells in vitro, that was characterized by activation of NF-κB and p38 MAPK signaling pathways, increased production of proinflammatory cytokines and chemokines, and the involvement of multiple intracellular mechanisms in RSV-induced HMGB1 secretion. Altogether, this evidence points to HMGB1 as an important proinflammatory mediator that contributes to RSV pathogenesis.

The first cells encountered by RSV are typically AECs, subsequent cellular contact is then with innate immune cells such as monocytes, alveolar MΦs, and DCs in the airways, which produce significant levels of proinflammatory cytokines; a response to viral infection that helps to control adaptive immunity by interaction with T helper cells (49). RSV primarily affects AECs, but immune cells play a role in the immunopathology of RSV infection and are important components for the antiviral response. RSV infection induces a range of proinflammatory cytokine/chemokine and growth factor released by A549 and SAE cells (33,40,50), and have also been detected in nasopharyngeal secretions in RSV-infected infants (29). Although RSV-induced proinflammatory cytokines have been studied in AECs, there are no extensive studies in immune cells or on the proinflammatory role of extracellular HMGB1 in various immune cell activation. In this study, we evaluated the proinflammatory effects of HMGB1 on RSV infection in AECs (A549 and SAE) and multiple types of immune cells (PBMCs, monocytes, MΦs, EOS and DCs). HMGB1’s effects on NF-κB and P38 MAPK signaling pathway activation and production of cytokines and chemokines was also examined. Herein, we show that purified human recombinant and secreted HMGB1 treatment caused phosphorylation of NF-κB and p38 MAPK in AECs, and several immune cells (Figs. 1, 4, 6, 8, 10 and 11). The present study also demonstrated that RSV-induced HMGB1 extracellular release by AECs is mediated by NF-κB as inhibition of NF-κB signaling significantly blocked HMGB1 release (Fig. 2 A) whereas activation of NF-κB enhanced the extracellular release of HMGB1 in RSV-infected A549 cells (Fig. 2 B), suggesting the involvement of NF-κB signaling in RSV-induced HMGB1 release from AECs. Previous studies have also shown the involvement of NF-κB signaling in HMGB1 release (51,52). HMGB1 functions via interactions with TLRs and RAGE (19,53). TLR4, crucial innate immune pattern recognition receptor, plays an important role in regulating the immune response and inflammatory reaction (54). Moreover, HMGB1 is an important ligand of TLR4 that induces the release of proinflammatory cytokines (55,56). HMGB1-TLR4 signaling induces the activation of MAPK and NF-κB (57), and the activation of NF-κB leads to the increased release of various proinflammatory cytokines, including IL-1β, IL-6 and TNF-α (58–60). The release of proinflammatory cytokines can lead to the recruitment of inflammatory cells into the pulmonary microvasculature (61,62). In this study, using neutralizing Abs, we demonstrated that HMGB1 secretion from AECs was TLR4 dependent (Fig. 3 A) and that blocking TLR4 signaling decreased the RSV-induced NF-κB phosphorylation (Fig. 3 A), compared to RAGE (Fig. 3 B), suggesting a role for TLR4 in RSV-induced HMGB1 secretion, which is mediated through the NF-κB pathway. Our results showed that the molecular mechanism of HMGB1 release from AECs in response to RSV infection was mediated by both the NF-κB and TLR4 pathways, suggesting an important role of TLR4/NF-κB pathway in RSV-induced HMGB1 release. The underlying mechanism involves, at least in part, inhibition of TLR4/NF-κB-dependent signaling pathway, which provide the new evidence for therapeutic application of HMGB1 release blockade to target inflammatory processes in respiratory viral infections.

In this study, we used various immune cells including human primary cells and cell lines as well as AECs to investigate the proinflammatory effects of HMGB1. We compared cytokine release from AECs (A549 and SAE), PBMCs, PBMC-derived monocytes, MΦs, DCs, and EOS as well as THP-1 monocytes, THP-1 monocyte-derived MΦs, and EOL1 cells after treatment with purified human rHMGB1 and sHMGB1 in the presence or absence of RSV. We show that purified HMGB1 is a proinflammatory mediator and induces activation of the NF-κB and p38 MAPK signaling pathways and the release of inflammatory cytokines and chemokines. Our previous and current studies show that RSV induces HMGB1 release from AECs and that treatment with purified HMGB1 did not have any effect on these cells (Figs. 1–3 and S1 A and B) (29). The present study demonstrates that RSV infection does not induce the release of HMGB1 from any of the immune cells but that addition of LPS promotes HMGB1 translocation from the nucleus to the extracellular space (Figs. 4, 6, 8, 10 and 11). Although the addition of HMGB1 to AECs did not itself induce the release of cytokines/chemokines compared to various immune cells (Figs. S1 A and B) (29), it did activate NF-κB and MAPK signaling pathways (Fig. 1). Addition of purified HMGB1 to various immune cells induced the release of proinflammatory cytokines and activated NF-κB and MAPK signaling pathways (Figs. 4, 6, 8, 10 and 11). HMGB1 in the presence of RSV, either additively or synergistically induced IL-1RA, IL-8, IL-10, TNF-α, G-CSF, MCP-1, MIP-1α, MIP-1β, RANTES, and PDGF release by PBMCs, whereas HM GB1 induced IL-6, IL-8, IL-12, GMCSF, G-CSF, IFN-γ, TNF-α, IP-10, MIP-1α, MIP-1β, and RANTES release in both monocytes and MΦs (Figs. 5, 7 and 9). Addition of purified HMGB1 to human EOS in the presence of RSV, synergistically induced the release of IL-8 and PDGF whereas in DCs HMGB1 induced the release of IL-8, GM-CSF, IP-10, IFN-γ, MIP-1α, RANTES, TNF-α, PDGF, and VEGF secretion (Figs. 10 C and 12). HMGB1 induced IL-8, TNF-α, G-CSF, RANTES, IP-10, and MIP-1α release either additively or synergistically by monocytes, MΦs and DCs, suggesting that HMGB1 secreted by AECs in response to RSV infection potentially signal the monocytes/MΦs/DCs to release proinflammatory cytokines and chemokines, and contributes to RSV pathogenesis. These results also demonstrated that monocytes, MΦs and DCs are the cellular targets for the HMGB1’s proinflammatory function, suggesting that RSV-induced epithelial HMGB1 can initiate and amplify inflammatory responses in the airways during infection or injury caused by RSV. Previous studies have shown the release of HMGB1 by monocytes/MΦs with addition of LPS or proinflammatory cytokines and induces the release of proinflammatory mediators (63). Others and we have shown that once HMGB1 is released into the extracellular space, it triggers local inflammatory responses by activating neighboring immune cells in the airways to release more proinflammatory cytokines (28,29,53,64). Previous studies have also shown the activation of signaling pathways and cytokine release by rHMGB1 in human microvascular endothelial cells and mesenchymal stem cells (27,65). Upon release, HMGB1 has been shown to stimulate macrophage function and induce inflammatory response (66,67). Our study also demonstrated that RSV-induced HMGB1 secretion activated immune cells to induce not only proinflammatory mediators but also anti-inflammatory cytokines including IL-1RA, IL-4, IL-10 and IFN-γ. Previous studies have shown that in addition to the production of proinflammatory mediators such as TNF-α and IL-1β, and HMGB1, activated MΦs also produce anti-inflammatory cytokines such as IL-10 and TGF-β1, which have suppressive or enhancing effects on the immune and inflammatory responses (68) including pulmonary inflammation (69). Our study also showed that RSV infection as well as purified HMGB1 significantly induced IFN-γ-induced protein-10 (IP-10) production in AECs as well as in immune cells (Figs. S 1 A and B, 5, 7, 9, 12 and S2-S4). Studies have shown the increased release of IP-10 in RSV/influenza and bacterial co-infection of human monocyte-derived MΦs (55).

This is the first study to show the proinflammatory effect of purified HMGB1 on activation of several immune cells and multiple signaling pathways, suggesting the involvement of multiple intracellular mechanisms responsible for RSV pathogenesis, with a role for HMGB1 secretion in response to viral challenge. The in vitro cell culture model of RSV infection along with purified HMGB1 treatment of various human immune cells, demonstrate that RSV induces significant HMGB1 release in AECs, and that secreted HMGB1 activates immune cells to induce an inflammatory response, suggesting that HMGB1 acts as a signaling molecule to alert the immune system to the potential damage and promote inflammation. Our studies also demonstrate that RSV and HMGB1 synergistically induce significant release of proinflammatory cytokines and chemokines from immune cells, suggesting that human blood leukocytes contribute to the immunopathology of RSV. Additionally, the inflammatory pathways that are activated during RSV infection and HMGB1 treatment provided the significance related to the synergistic induction of proinflammatory mediators. Our observations are in consistent with the previous study reported (27). By focusing on the intracellular mechanisms that regulate inflammatory pathways, we demonstrated a proinflammatory role for HMGB1 in the activation of immune cells.

In summary, our results provide a mechanistic pathway of RSV-induced HMGB1 release in AECs, and that secreted HMGB1 alerts the host immune system by activating PBMCs, monocytes and MΦs in a TLR4 and RAGE dependent manner, via NF-κB and P38 MAPK signaling pathway activation, to induce proinflammatory mediator release (Fig. 13). Our studies indicate important cross-talk between the HMGB1, NF-κB, TLR, RAGE, and MAPK signaling pathways in the course of RSV infection, with increasing extracellular HMGB1 levels able to induce inflammation, which was mediated through NF-κB and TLR4. The potential involvement of multiple intracellular mechanisms suggests that our findings may be relevant to other respiratory viruses. We hypothesize that RSV-induced NF-κB and TLR4-dependent HMGB1 secretion from AECs, activates immune cells to promote the production of proinflammatory mediators, and contribute to RSV pathogenesis. Therefore, blocking HMGB1 proinflammatory activities may be an effective therapeutic strategy, not only against RSV-induced lower respiratory tract infections, but also other viruses responsible for human infections.

FIG 13.

RSV-induced secreted HMGB1 from airway epithelial cells (AECs) activates immune cells to promote an inflammatory response. RSV induces NF-κB-mediated HMGB1 release and activates NF-κB/P38 MAPK signaling pathways in AECs to induce proinflammatory mediator release. Secreted HMGB1 from AECs activates immune cells via NF-κB/P38 MAPK activation and induces the release of proinflammatory mediators and promotes inflammation.