Transcriptomic Profiling Reveals That HMGB1 Induces Macrophage Polarization Different from Classical M1.

Macrophages are key inflammatory immune cells that display dynamic phenotypes and functions in response to their local microenvironment. In different conditions, macrophage polarization can be induced by high-mobility group box 1 (HMGB1), a nuclear DNA-binding protein that activates innate immunity via the Toll-like receptor (TLR) 4, the receptor for advanced glycation end products (RAGE), and C-X-C chemokine receptor (CXCR) 4. This study investigated the phenotypes of murine bone-marrow-derived macrophages (BMDMs) stimulated with different HMGB1 redox isoforms using bulk RNA sequencing (RNA-Seq). Disulfide HMGB1 (dsHMGB1)-stimulated BMDMs showed a similar but distinct transcriptomic profile to LPS/IFNγ- and LPS-stimulated BMDMs. Fully reduced HMGB1 (frHMGB1) did not induce any significant transcriptomic change. Interestingly, compared to LPS/IFNγ- and LPS-, dsHMGB1-stimulated BMDMs showed lipid metabolism and foam cell differentiation gene set enrichment, and oil red O staining revealed that both dsHMGB1 and frHMGB1 alleviated oxidized low-density lipoprotein (oxLDL)-induced foam cells formation. Overall, this work, for the first time, used transcriptomic analysis by RNA-Seq to investigate the impact of HMGB1 stimulation on BMDM polarization. Our results demonstrated that dsHMGB1 and frHMGB1 induced distinct BMDM polarization phenotypes compared to LPS/IFNγ- and LPS- induced phenotypes.

Disulfide and Fully Reduced HMGB1 Induce Different Macrophage Polarization and Migration Patterns.

Macrophage plasticity enables cells to obtain different functions over a broad proinflammatory and repairing spectrum. In different conditions, macrophages can be induced by high-mobility group box 1 (HMGB1), a nuclear DNA-binding protein that activates innate immunity, to polarize towards a pro- (M1) or anti-inflammatory (M2) phenotype. In this study, we investigated the phenotypes of murine bone-marrow-derived macrophages (BMDMs) induced by different HMGB1 redox isoforms in depth. Our results demonstrate that disulfide HMGB1 (dsHMGB1) induces a unique macrophage phenotype that secretes pro-inflammatory cytokines, rather than inducing metabolic changes leading to nitric oxide production. Fully reduced HMGB1 (frHMGB1) did not induce macrophage polarization. The migrating function of BMDMs was measured by scratch assay after the stimulation with dsHMGB1 and frHMGB1. Both dsHMGB1 and frHMGB1 induced cell migration. We found that dsHMGB1 mediates cytokine secretion and cellular motility, mainly through toll-like receptor 4 (TLR4). Importantly, our data shows that dsHMGB1 and frHMGB1 induce distinct BMDM polarization phenotypes, and that dsHMGB1 induces a unique phenotype differing from the classical proinflammatory macrophage phenotype.

Redox modifications of cysteine residues regulate the cytokine activity of HMGB1.

BACKGROUND: High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is passively released during cell death and secreted by activated cells of many lineages. HMGB1 contains three conserved redox-sensitive cysteine residues: cysteines in position 23 and 45 (C23 and C45) can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. METHODS: Primary human macrophages or murine macrophage-like RAW 264.7 cells were activated in cell cultures by redox-modified or point-mutated (C45A) recombinant HMGB1 preparations or by lipopolysaccharide (E. coli.0111: B4). Cellular phosphorylated NF-κB p65 subunit and subsequent TNF-α release were quantified by commercial enzyme-linked immunosorbent assays. RESULTS: Cell cultures with primary human macrophages and RAW 264.7 cells demonstrated that fully reduced HMGB1 with all three cysteines expressing thiol side chains failed to generate phosphorylated NF-КB p65 subunit or TNF-α. Mild oxidation forming a C23-C45 disulfide bond, while leaving C106 with a thiol group, was required for HMGB1 to induce phosphorylated NF-КB p65 subunit and TNF-α production. The importance of a C23-C45 disulfide bond was confirmed by mutation of C45 to C45A HMGB1, which abolished the ability for cytokine induction. Further oxidation of the disulfide isoform also inactivated HMGB1. CONCLUSIONS: These results reveal critical post-translational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during inflammation.

Therapeutic administration of etoposide coincides with reduced systemic HMGB1 levels in macrophage activation syndrome.

BACKGROUND: Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. HMGB1 is a nuclear protein released extracellularly during proinflammatory lytic cell death or secreted by activated macrophages, NK cells, and additional cell types during infection or sterile injury. Extracellular HMGB1 orchestrates central events in inflammation as a prototype alarmin. TLR4 and the receptor for advanced glycation end products operate as key HMGB1 receptors to mediate inflammation. METHODS: Standard ELISA and cytometric bead array-based methods were used to examine the kinetic pattern for systemic release of HMGB1, ferritin, IL-18, IFN-γ, and MCP-1 before and during treatment of four children with critical MAS. Three of the patients with severe underlying systemic rheumatic diseases were treated with biologics including tocilizumab or anakinra when MAS developed. All patients required intensive care therapy due to life-threatening illness. Add-on etoposide therapy was administered due to insufficient clinical response with standard treatment. Etoposide promotes apoptotic rather than proinflammatory lytic cell death, conceivably ameliorating subsequent systemic inflammation. RESULTS: This therapeutic intervention brought disease control coinciding with a decline of the increased systemic HMGB1, IFN-γ, IL-18, and ferritin levels whereas MCP-1 levels evolved independently. CONCLUSION: Systemic HMGB1 levels in MAS have not been reported before. Our results suggest that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of HMGB1 antagonists. They also advocate therapeutic etoposide administration in severe MAS and provide a possible biological explanation for its mode of action.

Sex-dependent role of microglia in disulfide high mobility group box 1 protein-mediated mechanical hypersensitivity.

ABSTRACT: High mobility group box 1 protein (HMGB1) is increasingly regarded as an important player in the spinal regulation of chronic pain. Although it has been reported that HMGB1 induces spinal glial activation in a Toll-like receptor (TLR)4-dependent fashion, the aspect of sexual dimorphisms has not been thoroughly addressed. Here, we examined whether the action of TLR4-activating, partially reduced disulfide HMGB1 on microglia induces nociceptive behaviors in a sex-dependent manner. We found disulfide HMGB1 to equally increase microglial Iba1 immunoreactivity in lumbar spinal dorsal horn in male and female mice, but evoke higher cytokine and chemokine expression in primary microglial culture derived from males compared to females. Interestingly, TLR4 ablation in myeloid-derived cells, which include microglia, only protected male mice from developing HMGB1-induced mechanical hypersensitivity. Spinal administration of the glial inhibitor, minocycline, with disulfide HMGB1 also prevented pain-like behavior in male mice. To further explore sex difference, we examined the global spinal protein expression using liquid chromatography-mass spectrometry and found several antinociceptive and anti-inflammatory proteins to be upregulated in only male mice subjected to minocycline. One of the proteins elevated, alpha-1-antitrypsin, partially protected males but not females from developing HMGB1-induced pain. Targeting downstream proteins of alpha-1-antitrypsin failed to produce robust sex differences in pain-like behavior, suggesting that several proteins identified by liquid chromatography-mass spectrometry are required to modulate the effects. Taken together, the current study highlights the importance of mapping sex dimorphisms in pain mechanisms and point to processes potentially involved in the spinal antinociceptive effect of microglial inhibition in male mice.

The impact of damage-associated molecular patterns on the neurotransmitter release and gene expression in the ex vivo rat carotid body.

NEW FINDINGS: What is the central question of this study? Are carotid bodies (CBs) modulated by the damage-associated molecular patterns (DAMPs) and humoral factors of aseptic tissue injury? What are the main findings and their importance? DAMPs (HMGB1, S100 A8/A9) and blood plasma from rats subjected to tibia surgery, a model of aseptic injury, stimulate the release of neurotransmitters (ATP, dopamine) and TNF-α from ex vivo rat CBs. All-thiol HMGB1 mediates upregulation of immune-related biological pathways. These data suggest regulation of CB function by endogenous mediators of innate immunity. ABSTRACT: The glomus cells of carotid bodies (CBs) are the primary sensors of arterial partial O2 and CO2 tensions and moreover serve as multimodal receptors responding also to other stimuli, such as pathogen-associated molecular patterns (PAMPs) produced by acute infection. Modulation of CB function by excessive amounts of these immunomodulators is suggested to be associated with a detrimental hyperinflammatory state. We have hypothesized that yet another class of immunomodulators, endogenous danger-associated molecular patterns (DAMPs), released upon aseptic tissue injury and recognized by the same pathogen recognition receptors as PAMPs, might modulate the CB activity in a fashion similar to PAMPs. We have tested this hypothesis by exposing rat CBs to various DAMPs, such as HMGB1 (all-thiol and disulfide forms) and S100 A8/A9 in a series of ex vivo experiments that demonstrated the release of dopamine and ATP, neurotransmitters known to mediate CB homeostatic responses. We observed a similar response after incubating CBs with conditioned blood plasma obtained from the rats subjected to tibia surgery, a model of aseptic injury. In addition, we have investigated global gene expression in the rat CB using an RNA sequencing approach. Differential gene expression analysis showed all-thiol HMGB1-driven upregulation of a number of prominent pro-inflammatory markers including Il1α and Il1ß. Interestingly, conditioned plasma had a more profound effect on the CB transcriptome resulting in inhibition rather than activation of the immune-related pathways. These data are the first to suggest potential modulation of CB function by endogenous mediators of innate immunity.

Neuroinflammatory markers associate with cognitive decline after major surgery: Findings of an explorative study.

OBJECTIVE: Long-term cognitive decline is an adverse outcome after major surgery associated with increased risk for mortality and morbidity. We studied the cerebrospinal fluid (CSF) and serum biochemical inflammatory response to a standardized orthopedic surgical procedure and the possible association with long-term changes in cognitive function. We hypothesized that the CSF inflammatory response pattern after surgery would differ in patients having long-term cognitive decline defined as a composite cognitive z score of ≥1.0 compared to patients without long-term cognitive decline at 3 months postsurgery. METHODS: Serum and CSF biomarkers of inflammation and blood-brain barrier (BBB) integrity were measured preoperatively and up to 48 hours postoperatively, and cognitive function was assessed preoperatively and at 2 to 5 days and 3 months postoperatively. RESULTS: Surgery was associated with a pronounced increase in inflammatory biomarkers in both CSF and blood throughout the 48-hour study period. A principal component (PC) analysis was performed on 52 inflammatory biomarkers. The 2 first PC (PC1 and PC2) construct outcome variables on CSF biomarkers were significantly associated with long-term cognitive decline at 3 months, but none of the PC construct serum variables showed a significant association with long-term cognitive decline at 3 months. Patients both with and patients without long-term cognitive decline showed early transient increases of the astroglial biomarkers S-100B and glial fibrillary acidic protein in CSF, and in BBB permeability (CSF/serum albumin ratio). INTERPRETATION: Surgery rapidly triggers a temporal neuroinflammatory response closely associated with long-term cognitive outcome postsurgery. The findings of this explorative study require validation in a larger surgical patient cohort. Ann Neurol 2020;87:370-382.

Juvenile idiopathic arthritis patients have a distinct cartilage and bone biomarker profile that differs from healthy and knee-injured children.

OBJECTIVES: Joint destruction is a hallmark of juvenile idiopathic arthritis (JIA). Clinical evaluation and radiographic imaging are current methods to identify destruction. Biomarkers could aid an earlier and more sensitive diagnosis. Our aim was to investigate levels of bone and cartilage degradation biomarkers in JIA patients, compared to healthy children or juveniles with knee injuries. METHODS: Triple-paired synovial fluid, plasma and urine samples from 29 JIA patients were compared to 61 plasma samples from healthy children and synovial fluid from 41 knee-injured juveniles. Cartilage biomarkers ARGS neoepitope of aggrecan (ARGS), cartilage oligomeric matrix protein (COMP), type II collagen epitope (C2C), bone biomarkers N-terminal type I collagen cross-linked telopeptide (NTX-I) and tartrate-resistant acid phosphatase 5b (TRAP5b) were analysed by immunoassays. RESULTS: Plasma levels of ARGS, C2C, COMP and TRAP5b were increased in JIA compared to healthy children. Compared to knee-injured juveniles, synovial fluid C2C and TRAP5b were increased in JIA, while ARGS and COMP were decreased. For JIA patients, local (synovial fluid) and systemic (plasma/urine) levels of bone biomarkers correlated positively; age correlated negatively to plasma levels of C2C and TRAP5b; no correlation was found between biomarkers and gender, affected joint count, disease duration or medication. CONCLUSIONS: Elevated levels of destruction biomarkers in JIA compared to healthy children indicate a potential to serve as clinical tools for destructive joint disease. High levels of TRAP5b, NTX-I and collagen II in JIA in contrast to more pronounced aggrecan and COMP degradation in juvenile knee injuries, suggests that JIA patients have a unique biomarker pattern, different from healthy and knee-injured children.

Neuroinflammation in Response to Intracerebral Injections of Different HMGB1 Redox Isoforms.

BACKGROUND: Neuroinflammation triggered by infection or trauma is the cause of central nervous system dysfunction. High-mobility group box 1 protein (HMGB1), released from stressed and dying brain cells, is a potent neuroinflammatory mediator. The proinflammatory functions of HMGB1 are tightly regulated by post-translational redox modifications, and we here investigated detailed neuroinflammatory responses induced by the individual redox isoforms. METHODS: Male Dark Agouti rats received a stereotactic injection of saline, lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1, and were accessed for blood-brain barrier modifications using magnetic resonance imaging (MRI) and inflammatory responses by immunohistochemistry. RESULTS AND CONCLUSIONS: Significant blood-brain barrier disruption appeared 24 h after injection of lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1 compared to controls, as assessed in post-gadolinium T1-weighted MRI images and confirmed by increased uptake of FITC-conjugated dextran. Immunohistochemistry revealed that both HMGB1 isoforms also induced a local production of IL-1ß. Additionally, disulfide HMGB1 increased major histocompatibility complex class II expression and apoptosis. Together, the results demonstrate that extracellular, cerebral HMGB1 causes significant blood-brain barrier disruption in a redox-independent manner and activates several components of neuroinflammation. Blocking HMGB1 might potentially improve clinical outcome in conditions such as stroke and traumatic brain injury.

The immune response of the human brain to abdominal surgery.

OBJECTIVE: Surgery launches a systemic inflammatory reaction that reaches the brain and associates with immune activation and cognitive decline. Although preclinical studies have in part described this systemic-to-brain signaling pathway, we lack information on how these changes appear in humans. This study examines the short- and long-term impact of abdominal surgery on the human brain immune system by positron emission tomography (PET) in relation to blood immune reactivity, plasma inflammatory biomarkers, and cognitive function. METHODS: Eight males undergoing prostatectomy under general anesthesia were included. Prior to surgery (baseline), at postoperative days 3 to 4, and after 3 months, patients were examined using [11 C]PBR28 brain PET imaging to assess brain immune cell activation. Concurrently, systemic inflammatory biomarkers, ex vivo blood tests on immunoreactivity to lipopolysaccharide (LPS) stimulation, and cognitive function were assessed. RESULTS: Patients showed a global downregulation of gray matter [11 C]PBR28 binding of 26 ± 26% (mean ± standard deviation) at 3 to 4 days postoperatively compared to baseline (p = 0.023), recovering or even increasing after 3 months. LPS-induced release of the proinflammatory marker tumor necrosis factor-α in blood displayed a reduction (41 ± 39%) on the 3rd to 4th postoperative day, corresponding to changes in [11 C]PBR28 distribution volume. Change in Stroop Color-Word Test performance between postoperative days 3 to 4 and 3 months correlated to change in [11 C]PBR28 binding (p = 0.027). INTERPRETATION: This study translates preclinical data on changes in the brain immune system after surgery to humans, and suggests an interplay between the human brain and the inflammatory response of the peripheral innate immune system. These findings may be related to postsurgical impairments of cognitive function. Ann Neurol 2017;81:572-582.

Systemic HMGB1 Neutralization Prevents Postoperative Neurocognitive Dysfunction in Aged Rats.

Postoperative neurocognitive disorders are common complications in elderly patients following surgery or critical illness. High mobility group box 1 protein (HMGB1) is rapidly released after tissue trauma and critically involved in response to sterile injury. Herein, we assessed the role of HMGB1 after liver surgery in aged rats and explored the therapeutic potential of a neutralizing anti-HMGB1 monoclonal antibody in a clinically relevant model of postoperative neurocognitive disorders. Nineteen to twenty-two months Sprague-Dawley rats were randomly assigned as: (1) control with saline; (2) surgery, a partial hepatolobectomy under sevoflurane anesthesia and analgesia, + immunoglobulin G as control antibody; (3) surgery + anti-HMGB1. A separate cohort of animals was used to detect His-tagged HMGB1 in the brain. Systemic anti-HMGB1 antibody treatment exerted neuroprotective effects preventing postoperative memory deficits and anxiety in aged rats by preventing surgery-induced reduction of phosphorylated cyclic AMP response element-binding protein in the hippocampus. Although no evident changes in the intracellular distribution of HMGB1 in hippocampal cells were noted after surgery, HMGB1 levels were elevated on day 3 in rat plasma samples. Experiments with tagged HMGB1 further revealed a critical role of systemic HMGB1 to enable an access to the brain and causing microglial activation. Overall, these data demonstrate a pivotal role for systemic HMGB1 in mediating postoperative neuroinflammation. This may have direct implications for common postoperative complications like delirium and postoperative cognitive dysfunction.

A novel high mobility group box 1 neutralizing chimeric antibody attenuates drug-induced liver injury and postinjury inflammation in mice.

Acetaminophen (APAP) overdoses are of major clinical concern. Growing evidence underlines a pathogenic contribution of sterile postinjury inflammation in APAP-induced acute liver injury (APAP-ALI) and justifies development of anti-inflammatory therapies with therapeutic efficacy beyond the therapeutic window of the only current treatment option, N-acetylcysteine (NAC). The inflammatory mediator, high mobility group box 1 (HMGB1), is a key regulator of a range of liver injury conditions and is elevated in clinical and preclinical APAP-ALI. The anti-HMGB1 antibody (m2G7) is therapeutically beneficial in multiple inflammatory conditions, and anti-HMGB1 polyclonal antibody treatment improves survival in a model of APAP-ALI. Herein, we developed and investigated the therapeutic efficacy of a partly humanized anti-HMGB1 monoclonal antibody (mAb; h2G7) and identified its mechanism of action in preclinical APAP-ALI. The mouse anti-HMGB1 mAb (m2G7) was partly humanized (h2G7) by merging variable domains of m2G7 with human antibody-Fc backbones. Effector function-deficient variants of h2G7 were assessed in comparison with h2G7 in vitro and in preclinical APAP-ALI. h2G7 retained identical antigen specificity and comparable affinity as m2G7. 2G7 treatments significantly attenuated APAP-induced serum elevations of alanine aminotransferase and microRNA-122 and completely abrogated markers of APAP-induced inflammation (tumor necrosis factor, monocyte chemoattractant protein 1, and chemokine [C-X-C motif] ligand 1) with prolonged therapeutic efficacy as compared to NAC. Removal of complement and/or Fc receptor binding did not affect h2G7 efficacy. CONCLUSION: This is the first report describing the generation of a partly humanized HMGB1-neutralizing antibody with validated therapeutic efficacy and with a prolonged therapeutic window, as compared to NAC, in APAP-ALI. The therapeutic effect was mediated by HMGB1 neutralization and attenuation of postinjury inflammation. These results represent important progress toward clinical implementation of HMGB1-specific therapy as a means to treat APAP-ALI and other inflammatory conditions. (Hepatology 2016;64:1699-1710).

Antibodies against High Mobility Group Box protein-1 (HMGB1) versus other anti-nuclear antibody fine-specificities and disease activity in systemic lupus erythematosus.

INTRODUCTION: The non-histone nuclear protein high mobility group box protein-1 (HMGB1) is typically associated with nucleosomes, but may shuttle between the nucleus and the cytoplasm, and under some conditions also be released extracellularly and participate in systemic inflammation. Monoclonal HMGB1-targeting antibodies can ameliorate murine polyarthritis and lupus-like disease. Interestingly, autoantibodies against HMGB1 have also been described in patients with systemic lupus erythematosus (SLE), but their clinical implications remain elusive. The main aims of this study were to detect serum anti-HMGB1 antibodies in patients with SLE and relate them to other types of antinuclear antibodies (ANA), and to disease activity. METHODS: 188 Swedish SLE patients meeting the 1982 American College of Rheumatology classification criteria and/or the 2012 Systemic Lupus International Collaborating Clinics classification criteria participated in the study. Anti-HMGB1 antibody levels were analysed in patient and control (n = 112) sera by an in-house ELISA using recombinant histidine-tagged HMGB1. SLE sera were also analysed for ANA by immunofluorescence (IF) microscopy (IF-ANA) using fixed HEp-2 cells, and by a line-blot assay for antigen fine-specificities. To quantify antibodies to double-stranded DNA, a fluoroenzyme-immunoassay was employed. RESULTS: At inclusion, 23 % of the SLE patients were anti-HMGB1 antibody positive compared to 5 % of the controls. Anti-HMGB1 antibodies occurred in 49 % of the IF-ANA positive SLE patients, and in 34 % of IF-ANA negative cases (p = 0.004). Levels of anti-HMGB1 antibodies correlated with anti-dsDNA antibody levels (r = 0.49; p < 0.001). Significant, but less pronounced correlations were found regarding anti-HMGB1 and SLE disease activity index (SLEDAI-2K: r = 0.15; p = 0.04), classical complement function (r = -0.24; p = 0.002) and complement protein C4 (r = -0.23; p = 0.002). Average anti-HMGB1 antibody levels were significantly higher among patients with homogenous ± other IF-ANA staining patterns (median 180 AU) compared to IF-ANA negative cases (median 83 AU) (p = 0.004). Rabbit anti-HMGB1 antibodies gave rise to cytoplasmic, but not nuclear, staining of HEp-2 cells. CONCLUSIONS: We confirm that anti-HMGB1 antibodies are common in SLE and correlate with disease activity variables. Although anti-HMGB1 antibodies measured by ELISA often coincide with nuclear IF-ANA staining, our results indicate that anti-HMGB1 antibodies do not give rise to nuclear staining of the predominantly used commercial HEp-2 cell slides.

JAK/STAT1 signaling promotes HMGB1 hyperacetylation and nuclear translocation.

Extracellular high-mobility group box (HMGB)1 mediates inflammation during sterile and infectious injury and contributes importantly to disease pathogenesis. The first critical step in the release of HMGB1 from activated immune cells is mobilization from the nucleus to the cytoplasm, a process dependent upon hyperacetylation within two HMGB1 nuclear localization sequence (NLS) sites. The inflammasomes mediate the release of cytoplasmic HMGB1 in activated immune cells, but the mechanism of HMGB1 translocation from nucleus to cytoplasm was previously unknown. Here, we show that pharmacological inhibition of JAK/STAT1 inhibits LPS-induced HMGB1 nuclear translocation. Conversely, activation of JAK/STAT1 by type 1 interferon (IFN) stimulation induces HMGB1 translocation from nucleus to cytoplasm. Mass spectrometric analysis unequivocally revealed that pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 IFN-induced HMGB1 acetylation within the NLS sites. Together, these results identify a critical role of the JAK/STAT1 pathway in mediating HMGB1 cytoplasmic accumulation for subsequent release, suggesting that the JAK/STAT1 pathway is a potential drug target for inhibiting HMGB1 release.

TLR activation regulates damage-associated molecular pattern isoforms released during pyroptosis.

Infection of macrophages by bacterial pathogens can trigger Toll-like receptor (TLR) activation as well as Nod-like receptors (NLRs) leading to inflammasome formation and cell death dependent on caspase-1 (pyroptosis). Complicating the study of inflammasome activation is priming. Here, we develop a priming-free NLRC4 inflammasome activation system to address the necessity and role of priming in pyroptotic cell death and damage-associated molecular pattern (DAMP) release. We find pyroptosis is not dependent on priming and when priming is re-introduced pyroptosis is unaffected. Cells undergoing unprimed pyroptosis appear to be independent of mitochondrial involvement and do not produce inflammatory cytokines, nitrous oxide (NO), or reactive oxygen species (ROS). Nevertheless, they undergo an explosive cell death releasing a chemotactic isoform of the DAMP high mobility group protein box 1 (HMGB1). Importantly, priming through surface TLRs but not endosomal TLRs during pyroptosis leads to the release of a new TLR4-agonist cysteine redox isoform of HMGB1. These results show that pyroptosis is dominant to priming signals and indicates that metabolic changes triggered by priming can affect how cell death is perceived by the immune system.

Novel role of PKR in inflammasome activation and HMGB1 release.

The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1ß, IL-18 and high-mobility group box 1 (HMGB1). By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1ß, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.

Redox modification of cysteine residues regulates the cytokine activity of high mobility group box-1 (HMGB1).

High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is released passively during cell injury and necrosis, and secreted actively by immune cells. HMGB1 contains three conserved redox-sensitive cysteine residues: C23 and C45 can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. Using tandem mass spectrometric analysis, we now have established that the C106 thiol and the C23-C45 disulfide bond are required for HMGB1 to induce nuclear NF-κB translocation and tumor necrosis factor (TNF) production in macrophages. Both irreversible oxidation to sulphonates and complete reduction to thiols of these cysteines inhibited TNF production markedly. In a proof of concept murine model of hepatic necrosis induced by acetaminophen, during inflammation, the predominant form of serum HMGB1 is the active one, containing a C106 thiol group and a disulfide bond between C23 and C45, whereas the inactive form of HMGB1, containing terminally oxidized cysteines, accumulates during inflammation resolution and hepatic regeneration. These results reveal critical posttranslational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during pathogenesis.

Suppression of pain and joint destruction by inhibition of the proteasome system in experimental osteoarthritis.

Osteoarthritis is a degenerative joint disease with pain and loss of joint function as major pathological features. Recent studies show that proteasome inhibitors reduce pain in various pathological conditions. We evaluated the effects of MG132, a reversible proteasome inhibitor on pain and joint destruction in a rat model of osteoarthritis. Osteoarthritis was induced by intraarticular injection of monosodium iodoacetate into the rat knee. Knee joint stiffness was scored and nociception was evaluated by mechanical pressure applied to the respective hind paw. Knee joint destruction was assessed by radiological and histological analyses. Expression of matrix metalloproteinase-3 (MMP-3) was analyzed by quantitative reverse transcription polymerase chain reaction in the knee articular cartilage. Expression of substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in the dorsal root ganglia (L4-L6) by quantitative reverse transcription polymerase chain reaction and in the knee joints by immunohistochemistry. Our results indicate that daily treatment of osteoarthritic rats with MG132 significantly increases their mobility while the swelling, pain thresholds, and pathological features of the affected joints were reduced. Furthermore, the upregulated expression of MMP-3, SP, and CGRP in the arthritic rats was normalized by MG132 administration. We conclude that the proteasome inhibitor MG132 reduces pain and joint destruction, probably by involving the peripheral nervous system, and that changes in SP and CGRP expression correlate with alterations in behavioural responses. Our findings suggest that nontoxic proteasome inhibitors may represent a novel pharmacotherapy for osteoarthritis.