Proinflammatory High-mobility Group Box Research Articles

HMGB1 neuroimmune signaling and REST-G9a gene repression contribute to ethanol-induced reversible suppression of the cholinergic neuron phenotype.

Adolescent binge drinking increases Toll-like receptor 4 (TLR4), receptor for advanced glycation end products (RAGE), the endogenous TLR4/RAGE agonist high-mobility group box 1 (HMGB1), and proinflammatory neuroimmune signaling in the adult basal forebrain in association with persistent reductions of basal forebrain cholinergic neurons (BFCNs). In vivo preclinical adolescent intermittent ethanol (AIE) studies find anti-inflammatory interventions post-AIE reverse HMGB1-TLR4/RAGE neuroimmune signaling and loss of BFCNs in adulthood, suggesting proinflammatory signaling causes epigenetic repression of the cholinergic neuron phenotype. Reversible loss of BFCN phenotype in vivo is linked to increased repressive histone 3 lysine 9 dimethylation (H3K9me2) occupancy at cholinergic gene promoters, and HMGB1-TLR4/RAGE proinflammatory signaling is linked to epigenetic repression of the cholinergic phenotype. Using an ex vivo basal forebrain slice culture (FSC) model, we report EtOH recapitulates the in vivo AIE-induced loss of ChAT+IR BFCNs, somal shrinkage of the remaining ChAT+ neurons, and reduction of BFCN phenotype genes. Targeted inhibition of EtOH-induced proinflammatory HMGB1 blocked ChAT+IR loss while disulfide HMBG1-TLR4 and fully reduced HMGB1-RAGE signaling decreased ChAT+IR BFCNs. EtOH increased expression of the transcriptional repressor RE1-silencing transcription factor (REST) and the H3K9 methyltransferase G9a that was accompanied by increased repressive H3K9me2 and REST occupancy at promoter regions of the BFCN phenotype genes Chat and Trka as well as the lineage transcription factor Lhx8. REST expression was similarly increased in the post-mortem human basal forebrain of individuals with alcohol use disorder, which is negatively correlated with ChAT expression. Administration of REST siRNA and the G9a inhibitor UNC0642 blocked and reversed the EtOH-induced loss of ChAT+IR BFCNs, directly linking REST-G9a transcriptional repression to suppression of the cholinergic neuron phenotype. These data suggest that EtOH induces a novel neuroplastic process involving neuroimmune signaling and transcriptional epigenetic gene repression resulting in the reversible suppression of the cholinergic neuron phenotype.

Soluble CD93 lectin-like domain sequesters HMGB1 to ameliorate inflammatory diseases.

Rationale: CD93, a C-type lectin-like transmembrane glycoprotein, can be shed in a soluble form (sCD93) upon inflammatory stimuli. sCD93 effectively enhances apoptotic cell clearance and has been proposed as an inflammatory disease biomarker. The function of sCD93 involved directly in inflammation remains to be determined. Herein, we attempted to examine the hypothesis that sCD93 might sequester proinflammatory high-mobility group box 1 protein (HMGB1), exerting anti-inflammatory properties. Methods: Different forms of soluble recombinant human CD93 (rCD93) were prepared by a mammalian protein expression system. rCD93-HMGB1 interaction was assessed using co-immunoprecipitation and solid-phase binding assays. Effects of soluble rCD93 were evaluated in HMGB1-induced macrophage and vascular smooth muscle cells (VSMC) activation and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, CaCl2-induced and angiotensin II-infused abdominal aortic aneurysm (AAA) formation and ovariectomized-induced osteoporosis in mice. Results: Protein binding studies revealed that soluble rCD93, via the lectin-like domain (D1), can bind to HMGB1 and intercept HMGB1-receptor interaction. Soluble rCD93 containing D1 inhibited HMGB1-induced proinflammatory cytokine production and intracellular mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB activation in macrophages and VSMCs, thereby attenuating CaCl2-induced and angiotensin II-infused AAA models. During osteoclastogenesis, RANKL stimulated HMGB1 secretion that promoted RANKL-induced osteoclastogenesis in return. Soluble rCD93 containing D1 impeded RANKL-induced osteoclastogenic marker gene expression and intracellular MAPK/NF-κB signaling, thereby mitigating ovariectomized-induced osteoporosis. Conclusion: These findings demonstrate the therapeutic potential of soluble recombinant CD93 containing D1 in inflammatory diseases. Our study highlights a novel anti-inflammatory mechanism, i.e., sequestration of HMGB1, through which sCD93 prevents HMGB1-receptor interaction on effector cells and alleviates inflammation.

MLKL Regulates Rapid Cell Death-independent HMGB1 Release in RSV Infected Airway Epithelial Cells.

Respiratory syncytial virus (RSV)-induced bronchiolitis is a significant contributor to infant morbidity and mortality. Previously, we identified that necroptosis, a pro-inflammatory form of cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3, and mixed lineage kinase domain like protein (MLKL), occurs in RSV-infected human airway epithelial cells (hAECs), mediating the release of the alarmin high mobility group box 1 (HMGB1). Here, we show that RSV infection of hAECs induces the biphasic release of HMGB1 at 6 (“early”) and 24 (“late”) hours post infection (hpi). The early phase of HMGB1 release at 6 hpi is cell death-independent, however, this release is nonetheless attenuated by inhibition of MLKL (primarily associated with necroptosis). The early release of HMGB1 promotes the late phase of HMGB1 release via the activation of RAGE (receptor for advanced glycation endproducts) and occurs with cell death. Treatment of hAECS with exogenous HMGB1 combined with a pan-caspase inhibitor induces hAEC necroptosis, and is attenuated by the RAGE antagonist, FPS-ZM1. Together, these findings demonstrate that RSV infection of hAECs leads to the early release of HMGB1, followed by a paracrine feed-forward amplification loop that further increases HMGB1 levels and promotes cell death. As the inhibition of MLKL or targeting of HMGB1/RAGE pathway attenuates the release of pro-inflammatory HMGB1 and decreases viral load, this suggests that the pharmacological targeting of these pathways may be of benefit for the treatment of severe RSV bronchiolitis.

The Autophagosomes Containing Dengue Virus Proteins and Full-Length Genomic RNA Are Infectious.

Autophagic machinery is involved in selective and non-selective recruitment as well as degradation or exocytosis of cargoes, including pathogens. Dengue virus (DENV) infection induces autophagy that enhances virus replication and vesicle release to evade immune system surveillance. This study reveals that DENV2 induces autophagy in lung and liver cancer cells and showed that DENV2 capsid, envelope, NS1, NS3, NS4B and host cell proinflammatory high mobility group box 1 (HMGB1) proteins associated with autophagosomes which were purified by gradient centrifugation. Capsid, NS1 and NS3 proteins showing high colocalization with LC3 protein in the cytoplasm of the infected cells were detected in the purified double-membrane autophagosome by immunogold labeling under transmission electron microscopy. In DENV infected cells, the levels of capsid, envelope, NS1 and HMGB1 proteins are not significantly changed compared to the dramatic accumulation of LC3-II and p62/SQSTM1 proteins when autophagic degradation was blocked by chloroquine, indicating that these proteins are not regulated by autophagic degradation machinery. We further demonstrated that purified autophagosomes were infectious when co-cultured with uninfected cells. Notably, these infectious autophagosomes contain DENV2 proteins, negative-strand and full-length genomic RNAs, but no viral particles. It is possible that the infectivity of the autophagosome originates from the full-length DENV RNA. Moreover, we reveal that DENV2 promotes HMGB1 exocytosis partially through secretory autophagy. In conclusion, we are the first to report that DENV2-induced double-membrane autophagosomes containing viral proteins and full-length RNAs are infectious and not undergoing autophagic degradation. Our novel finding warrants further validation of whether these intracellular vesicles undergo exocytosis to become infectious autophagic vesicles.

Oxidative Stress Induced Damage and Early Senescence in Preterm Placenta.

Introduction Senescent cells have been demonstrated to release High Mobility Group Box 1 (HMGB1) which induces labor through an inflammatory pathway. This research is aimed at demonstrating whether telomere shortening, proinflammatory HMGB1, and oxidative damage marker 8-OHdG play a role in the placenta of preterm birth in comparison to term birth. Method A cross-sectional study on 67 full thickness of the placenta obtained from mothers with term and preterm birth. Mothers with clinical signs of infection (fever > 38°C, leukocytosis > 18000/μL, or abnormal vaginal discharge) and other pregnancy complications were excluded. Real-time polymerase chain reaction was performed to measure T/S ratio and ELISA quantification to measure the amount of HMGB1 and 8-OHdG. Result A total of 34 placentas from preterm and 33 placentas from term birth were examined. Maternal characteristics were comparable between the two groups. There were no statistical difference of T/S ratio (p = 0.181), HMGB1 (p = 0.119), and 8-OHdG (p = 0.144) between the preterm and term groups. HMGB1 was moderately correlated with 8-OHdG (r = 0.314). Telomere T/S ratio of the placenta did not differ between preterm and term labor despite difference in gestational age, suggesting earlier shortening in the preterm group. It is possible that critical telomere length has been achieved in both term and preterm placenta that warrants labor through senescence process. The result of our study also showed that HMGB1 was not correlated to telomere length, due to the fact that HMGB1 is not upregulated until the critical length of telomere for senescence is exhibited. Conclusion Similar telomere length might be exhibited due to early telomere shortening in preterm birth that mimics the term placenta. The relationship between placental telomere shortening and HMGB1 release remains to be uncovered. Further research is needed to discover the factors leading to early telomere shortening in the placenta of preterm birth.

Anti-HMGB1 auto-Abs influence fatigue in patients with Crohn's disease.

Fatigue is common in all chronic inflammatory and autoimmune diseases. A conceptual model for understanding the biological basis of fatigue describes it as being a part of the sickness behaviour response generated by pro-inflammatory cytokines and other mediators. We hypothesised that the pro-inflammatory high mobility group box 1 (HMGB1) protein is a fatigue-inducing molecule and that auto-Abs against HMGB1 reduce fatigue. We measured Abs against disulphide (ds) HMGB1 and fully reduced (fr) HMGB1 in plasma from 57 patients with Crohn’s disease. Fatigue was rated using the fatigue visual analogue scale (fVAS) and disease activity with faecal calprotectin, C-reactive protein and the Simple Endoscopic Score for Crohn’s disease. Multivariable regression models identified anti-dsHMGB1 and anti-frHMGB1 Abs as the strongest contributing factors for fVAS scores (B = −29.10 (P = 0.01), R2 = 0.17, and B = −17.77 (P = 0.01), R2 = 0.17, respectively). Results indicate that anti-HMGB1 auto-Abs alleviate fatigue possibly by down-regulating HMGB1-induced sickness behaviour.

High levels of endothelial and platelet microvesicles in patients with type 1 diabetes irrespective of microvascular complications

IntroductionPatients with type 1 diabetes have high risk of developing microvascular complications, and microangiopathy contributes to premature cardiovascular disease in this population. The role that microvesicles (MVs) may play in the development of microangiopathy in type 1 diabetes remains unclear. Materials and methodsPlasma levels of endothelial MVs (EMVs) and platelet MVs (PMVs) in 130 patients with type 1 diabetes without microangiopathy, 106 patients with microangiopathy and 100 matched healthy controls were analyzed using flow cytometry. MV expression of procoagulant phosphatidylserine (PS) and proinflammatory high mobility group box-1 protein (HMGB1) was also assessed. ResultsPatients with type 1 diabetes had markedly elevated levels of EMVs and PS+ EMVs as well as PMVs and PS+ PMVs compared to healthy controls (p < .001 for all). Furthermore, HMGB1+ EMVs and HMGB1+ PMVs were significantly increased in patients (p < .001 for all). After adjusting for potential confounders, there were no clear differences between patients with or without microvascular complications for any of the MV parameters. ConclusionType 1 diabetes is a prothrombotic and proinflammatory disease state that, regardless of the presence of clinical microangiopathy, is associated with elevated levels of plasma MVs, in particular those of an endothelial origin. We have for the first time demonstrated that patients with type 1 diabetes have higher levels of HMGB1+ MVs. HMGB1 is an alarmin with potent proinflammatory effects that drive endothelial dysfunction, and it would therefore be of interest to further study the role of HMGB1+ MVs in the development of macrovascular complications in type 1 diabetes.

Using HMGB1 Protein as a Surrogate Marker for Lung Cancer in High‐Risk Populations

Lung cancer is the leading cause of cancer‐related death, independent of gender or ethnicity, with a 5‐year overall survival of 17.1%. Although screening protocols for early detection exist, lung cancer is often diagnosed at an advanced stage. This is due, in part, to many patients experiencing barriers to healthcare access which contribute to delays in diagnosis. Supra‐normal biosynthesis of saturated (SFA) and unsaturated fatty (UFA) acids is a hallmark of cancer. In fact, several lines of evidence demonstrate that increased lipid desaturation by stearoyl‐CoA desaturase (SCD1) promotes lung cancer growth. We hypothesize that increased intracellular UFA, such as oleate (OA), promotes cancer cell growth by reducing cancer associated inflammation. To test this hypothesis we evaluated the effect of OA and inhibition of OA synthesis on proinflammatory High Mobility Group Box Protein 1 (HMGB1). Decreasing OA synthesis reduced intracellular HMGB1 expression, but increased its release from cells. Given our data, alterations in OA levels appear to have a substantial effect on localization of HMGB1. Further work is necessary to define the connections between SCD1, OA and HMGB1 release in lung cancer cells. This knowledge will provide insight into how fat regulates cancer development.Support or Funding InformationThe University of Minnesota School of Medicine New Faculty Start up and College of Saint Scholastica McNair Scholars Program.

Specific Sialoforms Required for the Immune Suppressive Activity of Human Soluble CD52.

Human CD52 is a small glycopeptide (12 amino acid residues) with one N-linked glycosylation site at asparagine 3 (Asn3) and several potential O-glycosylation serine/threonine sites. Soluble CD52 is released from the surface of activated T cells and mediates immune suppression via its glycan moiety. In suppressing activated T cells, it first sequesters the pro-inflammatory high mobility group Box 1 (HMGB1) protein, which facilitates its binding to the inhibitory sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) receptor. We aimed to identify the features of CD52 glycan that underlie its bioactivity. Analysis of native CD52 purified from human spleen revealed extensive heterogeneity in N-glycosylation and multi-antennary sialylated N-glycans with abundant polyLacNAc extensions, together with mainly di-sialylated O-glycosylation type structures. Glycomic (porous graphitized carbon-ESI-MS/MS) and glycopeptide (C8-LC-ESI-MS) analysis of recombinant soluble human CD52-immunoglobulin Fc fusion proteins revealed that CD52 bioactivity was correlated with a high abundance of tetra-antennary α-2,3/6 sialylated N-glycans. Removal of α-2,3 sialylation abolished bioactivity, which was restored by re-sialylation with α-2,3 sialyltransferases. When glycoforms of CD52-Fc were fractionated by anion exchange MonoQ-GL chromatography, bioactive fractions displayed mainly tetra-antennary, α-2,3 sialylated N-glycan structures and a lower relative abundance of bisecting GlcNAc structures compared to non-bioactive fractions. In addition, O-glycan core type-2 di-sialylated structures at Ser12 were more abundant in bioactive CD52 fractions. Understanding the structural features of CD52 glycan required for its bioactivity will aid its development as an immunotherapeutic agent.

Dyspigmented hypertrophic scars: Beyond skin color.

Although pigment synthesis is well understood, relevant mechanisms of psychologically debilitating dyspigmentation in nascent tissue after cutaneous injuries are still unknown. Here, differences in genomic transcription of hyper- and hypopigmented tissue relative to uninjured skin were investigated using a red Duroc swine scar model. Transcription profiles differed based on pigmentation phenotypes with a trend of more upregulation or downregulation in hyper- or hypopigmented scars, respectively. Ingenuity Pathway Analysis of significantly modulated genes in both pigmentation phenotypes showed pathways related to redox, metabolic, and inflammatory responses were more present in hypopigmented samples, while those related to stem cell development differentiation were found mainly in hyperpigmented samples. Cell-cell and cell-extracellular matrix interactions and inflammation responses were predicted (z-score) active in hyperpigmented and inactive in hypopigmented. The proinflammatory high-mobility group box 1 pathway showed the opposite trend. Analysis of differentially regulated mutually exclusive genes showed an extensive presence of metabolic, proinflammatory, and oxidative stress pathways in hypopigmented scars, while melanin synthesis, glycosaminoglycans biosynthesis, and cell differentiation pathways were predominant in hyperpigmented scar. Several potential therapeutic gene targets have been identified.

What's New in SHOCK, September 2018?

What's New in SHOCK, September 2018?

The HMGB1/RAGE Pro-Inflammatory Axis in the Human Placenta: Modulating Effect of Low Molecular Weight Heparin.

We evaluated whether physiological and pre-eclamptic (PE) placentae, characterized by exacerbated inflammation, presented alterations in pro-inflammatory High Mobility Group Box 1 (HMGB1) and its Receptor of Advanced Glycation End products (RAGE) expression. Moreover, we investigated, in physiological placental tissue, the ability of Low Molecular Weight Heparin (LMWH) to modify HMGB1 structural conformation thus inhibiting RAGE binding and HMGB1/RAGE axis inflammatory activity. HMGB1, RAGE, IL-6 and TNFα (HMGB1/RAGE targets) mRNA expression were assessed by Real Time PCR. HMGB1, RAGE protein levels were assessed by western blot assay. Physiological term placental explants were treated by 0.5 U LMWH for 24 or 48 h. HMGB1 and RAGE expression and association were evaluated in LMWH explants by RAGE immunoprecipitation followed by HMGB1 immunoblot. HMGB1 spatial localization was evaluated by immuofluorescent staining (IF). HMGB1 expression was increased in PE relative to physiological placentae while RAGE was unvaried. 24 h LMWH treatment significantly up-regulated HMGB1 expression but inhibited HMGB1/RAGE complex formation in physiological explants. RAGE expression decreased in treated relative to untreated explants at 48 h. IF showed HMGB1 localization in both cytoplasm and nucleus of mesenchymal and endothelial cells but not in the trophoblast. IL-6 and TNFα gene expression were significantly increased at 24 h relative to controls, while they were significantly down-regulated in 48 h vs. 24 h LMWH explants. Our data depicted a new molecular mechanism through which LMWH exerts its anti-inflammatory effect on PE placentae, underlying the importance of HMGB1/RAGE axis in PE inflammatory response.

Recombinant adeno-associated virus vector carrying the thrombomodulin lectin-like domain for the treatment of abdominal aortic aneurysm

Background and aimsThrombomodulin (TM), through its lectin-like domain (TMD1), sequesters proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that sustains inflammation and tissue damage. Our previous study demonstrated that short-term treatment with recombinant TM containing all the extracellular domains (i.e., rTMD123) inhibits HMGB1-RAGE signaling and confers protection against CaCl2-induced AAA formation. In this study, we attempted to further optimize TM domains, as a potential therapeutic agent for AAA, using the recombinant adeno-associated virus (AAV) vector. MethodsThe therapeutic effects of recombinant TMD1 (rTMD1) and recombinant AAV vectors carrying the lectin-like domain of TM (rAAV-TMD1) were evaluated in the CaCl2-induced AAA model and angiotensin II-infused AAA model, respectively. ResultsIn the CaCl2-induced model, treatment with rTMD1 suppressed the tissue levels of HMGB1 and RAGE, macrophage accumulation, elastin destruction and AAA formation, and the effects were comparable to a mole-equivalent dosage of rTMD123. In the angiotensin II-infused model, a single intravenous injection of rAAV-TMD1 (1011 genome copies), which resulted in a persistently high serum level of TMD1 for at least 12 weeks, effectively attenuated AAA formation with suppression of HMGB1 and RAGE levels and inhibition of proinflammatory cytokine production, macrophage accumulation, matrix metalloproteinase activities and oxidative stress in the aortic wall. ConclusionsThese findings corroborate the therapeutic potential of the TM lectin-like domain in AAA. The attenuation of angiotensin II-infused AAA by one-time delivery of rAAV-TMD1 provides a proof-of-concept validation of its application as potential gene therapy for aneurysm development.

Myeloid thrombomodulin lectin-like domain inhibits osteoclastogenesis and inflammatory bone loss.

Osteoclastogenesis is an essential process during bone metabolism which can also be promoted by inflammatory signals. Thrombomodulin (TM), a transmembrane glycoprotein, exerts anti-inflammatory activities such as neutralization of proinflammatory high-mobility group box 1 (HMGB1) through TM lectin-like domain. This study aimed to identify the role of myeloid TM (i.e., endogenous TM expression on the myeloid lineage) in osteoclastogenesis and inflammatory bone loss. Using human peripheral blood mononuclear cells and mouse bone marrow-derived macrophages, we observed that the protein levels of TM were dramatically reduced as these cells differentiated into osteoclasts. In addition, osteoclastogenesis and extracellular HMGB1 accumulation were enhanced in primary cultured monocytes from myeloid-specific TM-deficient mice (LysMcre/TMflox/flox) and from TM lectin-like domain deleted mice (TMLeD/LeD) compared with their respective controls. Micro-computerized tomography scans showed that ovariectomy-induced bone loss was more pronounced in TMLeD/LeD mice compared with controls. Finally, the inhibiting effects of recombinant TM lectin-like domain (rTMD1) on bone resorption in vitro, and bone loss in both the ovariectomized model and collagen antibody-induced arthritis model has been detected. These findings suggested that the myeloid TM lectin-like domain may inhibit osteoclastogenesis by reducing HMGB1 signaling, and rTMD1 may hold therapeutic potential for inflammatory bone loss.

Stimulation of Central α2 Receptors Attenuates Experimental Necrotizing Pancreatitis.

Severe necrotizing pancreatitis (SNP) is a disease with relevant morbidity and mortality until today. No specific therapy is in sight. Central α2 agonists such as clonidine and dexmedetomidine are known to have anti-inflammatory effects though the cholinergic anti-inflammatory pathway and are implemented in the clinical routine as adjunct sedative drugs. Their potential effect on SNP has not yet been tested. Severe necrotizing pancreatitis was induced in male Wistar rats. Four treatment groups received either clonidine or dexmedetomidine before (prophylactic) or after induction of SNP (therapeutic). After 12 hours, pancreatic morphologic injury, systemic proinflammatory high-mobility group box 1 protein, and pancreatic and pulmonary myeloperoxidase levels were evaluated. Severe necrotizing pancreatitis was fully established 12 hours after induction. "Prophylactic" and "therapeutic" administration of clonidine and dexmedetomidine reduced pancreatic morphologic injury (P < 0.05 vs SNP), serum proinflammatory high-mobility group box 1 protein (P < 0.0001 vs SNP), as well as pancreatic and pulmonary myeloperoxidase levels (P < 0.01 vs SNP). Prophylactic and therapeutic applications of the central α2 agonists clonidine and dexmedetomidine are effective to attenuate local and systemic injury in experimental SNP and should be evaluated in the clinical setting.

CO2 pneumoperitoneum preserves β-arrestin 2 content and reduces high mobility group box-1 (HMGB-1) expression in an animal model of peritonitis.

Laparoscopy (LS) has been shown to decrease the inflammatory sequelae of endotoxemia. β-arrestin 2 plays an important function in signal transduction pathway of TLR4. High mobility group box-1 (HMGB-1) is involved in the delayed systemic inflammatory response. We investigated the effects of CO2 insufflation on liver, lung, and kidney expression of both β-arrestin 2 and HMGB-1 during sepsis. Cecal ligation and puncture (CLP) was performed in male rats and 6 h later the animals were randomly assigned to receive a CO2 pneumoperitoneum or laparotomy. Animals were euthanized; liver, lung, and kidney were removed for the evaluation of β-arrestin 2 and HMGB-1 expression. Immunohistochemical detection of myeloperoxidase (MPO) was investigated in lung and liver and bacterial load was determined in the peritoneal fluid. CO2 pneumoperitoneum reduced peritoneal bacterial load, increased the expression of β-arrestin 2, and blunted the expression of the potent proinflammatory HMGB-1 in liver, lung, and kidney compared with laparotomy. Liver and lung MPO was markedly reduced in rats subjected to LS compared with laparotomy. We believe that CO2 exerts an early protective effect by reducing bacterial load and likely toll-like receptor activation which in turn leads to a preserved β-arrestin 2 expression and a reduced HMGB-1 expression.

The NLRP3/ASC inflammasome promotes T-cell-dependent immune complex glomerulonephritis by canonical and noncanonical mechanisms

Interleukin (IL)-1β contributes to renal injury in immune complex glomerulonephritis. However, production of mature IL-1β depends on activation of the inflammasome that cleaves pro-IL-1β into its secretable form. A functional role of the NLRP3-containing inflammasome, which responds to various endogenous danger signals, was found in tubulointerstitial nephropathies, but its function in glomerular disease has not been established. To determine whether NLRP3 and its adapter molecule ASC contribute to glomerulonephritis, we induced T-cell-dependent autologous nephrotoxic serum nephritis in Nlrp3- and Asc-deficient mice. Renal expression of NLRP3/ASC inflammasome components and pro-IL-1β increased during nephrotoxic serum nephritis and was abundant in renal dendritic cells. This was associated with renal production of mature IL-1β, indicating inflammasome activation. Nlrp3 and Asc deficiency significantly attenuated glomerular injury, renal leukocyte infiltration, and T-cell activation. Production of mature IL-1β was abrogated in Asc-deficient mice, consistent with a loss of inflammasome-dependent IL-1β activation. Surprisingly, renal IL-1β secretion remained intact in Nlrp3-deficient mice, indicating noncanonical pro-inflammatory effects of NLRP3 in autologous nephrotoxic serum nephritis. This may include NLRP3-mediated glomerular release of pro-inflammatory high-mobility group box 1 protein as a noncanonical function of NLRP3/ASC in glomerulonephritis. Thus, therapeutic blockade of the NLRP3/ASC/IL-1β axis may be beneficial in glomerulonephritis.

Recombinant Human Thrombomodulin Suppresses Experimental Abdominal Aortic Aneurysms Induced by Calcium Chloride in Mice

To investigate whether recombinant thrombomodulin containing all the extracellular domains (rTMD123) has therapeutic potential against aneurysm development. The pathogenesis of abdominal aortic aneurysm (AAA) is characterized by chronic inflammation and proteolytic degradation of extracellular matrix. Thrombomodulin, a transmembrane glycoprotein, exerts anti-inflammatory activities such as inhibition of cytokine production and sequestration of proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that may sustain inflammation and tissue damage. The in vivo effects of treatment and posttreatment with rTMD123 on aortic dilatation were measured using the CaCl2-induced AAA model in mice. Characterization of the CaCl2-induced model revealed that HMGB1 and RAGE, both localized mainly to macrophages, were persistently upregulated during a 28-day period of AAA development. In vitro, rTMD123-HMGB1 interaction prevented HMGB1 binding to macrophages, thereby prohibiting activation of HMGB1-RAGE signaling in macrophages. In vivo, short-term treatment with rTMD123 upon AAA induction suppressed the levels of proinflammatory cytokines, HMGB1, and RAGE in the aortic tissue; reduced the infiltrating macrophage number; and finally attenuated matrix metalloproteinase production, extracellular matrix destruction, and AAA formation without disturbing vascular calcification. Consistently, posttreatment with rTMD123 seven days after AAA induction alleviated vascular inflammation and retarded AAA progression. These data suggest that rTMD123 confers protection against AAA development. The mechanism of action may be associated with reduction of proinflammatory mediators, blockade of macrophage recruitment, and suppression of HMGB1-RAGE signaling involved in aneurysm formation and downstream macrophage activation.

Thrombomodulin: protectorate God of the vasculature in thrombosis and inflammation.

Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated activation of protein C. Recently, we conducted a multicentre, double-blind, randomized trial to evaluate the efficacy and safety of recombinant human soluble thrombomodulin (rhsTM, also known as ART-123) for the treatment of disseminated intravascular coagulation (DIC), and found that rhsTM therapy is more effective and safer than low-dose heparin therapy. Thus, in 2008, rhsTM (Recomodulin) was approved for the treatment of DIC in Japan. Here we re-evaluate the therapeutic basis of this drug from the view of its anticoagulant, anti-inflammatory, and cytoprotective properties. Structurally, the extracellular portion of TM is composed of three domains: an N-terminal lectin-like domain (TM-D1), followed by an epidermal growth factor (EGF)-like domain (TM-D2), and an O-glycosylation-rich domain (TM-D3). TM-D2 and TM-D3 are important for the protein's anticoagulant cofactor activities, i.e. inhibition of thrombin and activation of protein C. TM-D1 plays an important role in attenuation of inflammatory responses, through inhibition of leukocyte adhesion to endothelial cells, inhibition of complement pathways, neutralization of lipopolysaccharide (LPS), and sequestration and degradation of pro-inflammatory high-mobility group box 1 protein (HMGB1). Thus, TM on the surface of endothelial cells prevents dissemination of pro-coagulant and pro-inflammatory molecules, and by doing so, allows these molecules to act locally at the site of injury. In patients with sepsis and DIC, TM expression is down-regulated, which may result in dissemination of pro-coagulant and pro-inflammatory molecules throughout the systemic circulation. Replacement with rhsTM may offer therapeutic value in such conditions.

Inhibitor of NF-κB Kinases α and β Are Both Essential for High Mobility Group Box 1-Mediated Chemotaxis

Inhibitor of NF-kappaB kinases beta (IKKbeta) and alpha (IKKalpha) activate distinct NF-kappaB signaling modules. The IKKbeta/canonical NF-kappaB pathway rapidly responds to stress-like conditions, whereas the IKKalpha/noncanonical pathway controls adaptive immunity. Moreover, IKKalpha can attenuate IKKbeta-initiated inflammatory responses. High mobility group box 1 (HMGB1), a chromatin protein, is an extracellular signal of tissue damage-attracting cells in inflammation, tissue regeneration, and scar formation. We show that IKKalpha and IKKbeta are each critically important for HMGB1-elicited chemotaxis of fibroblasts, macrophages, and neutrophils in vitro and neutrophils in vivo. By time-lapse microscopy we dissected different parameters of the HMGB1 migration response and found that IKKalpha and IKKbeta are each essential to polarize cells toward HMGB1 and that each kinase also differentially affects cellular velocity in a time-dependent manner. In addition, HMGB1 modestly induces noncanonical IKKalpha-dependent p52 nuclear translocation and p52/RelB target gene expression. Akin to IKKalpha and IKKbeta, p52 and RelB are also required for HMGB1 chemotaxis, and p52 is essential for cellular orientation toward an HMGB1 gradient. RAGE, a ubiquitously expressed HMGB1 receptor, is required for HMGB1 chemotaxis. Moreover, IKKbeta, but not IKKalpha, is required for HMGB1 to induce RAGE mRNA, suggesting that RAGE is at least one IKKbeta target involved in HMGB1 migration responses, and in accord with these results enforced RAGE expression rescues the HMGB1 migration defect of IKKbeta, but not IKKalpha, null cells. Thus, proinflammatory HMGB1 chemotactic responses mechanistically require the differential collaboration of both IKK-dependent NF-kappaB signaling pathways.