Receptor for advanced glycation end products (RAGE) on iNKT cells mediates lung ischemia-reperfusion injury.
ABSTRACT: Activation of invariant natural killer T (iNKT) cells and signaling through receptor for advanced glycation end products (RAGE) are known to independently mediate lung ischemia-reperfusion (IR) injury. This study tests the hypothesis that activation of RAGE specifically on iNKT cells via alveolar macrophage-produced high mobility group box 1 (HMGB1) is critical for the initiation of lung IR injury. A murine in vivo hilar clamp model was utilized, which demonstrated that RAGE(-/-) mice were significantly protected from IR injury. Treatment of WT mice with soluble RAGE (a decoy receptor), or anti-HMGB1 antibody, attenuated lung IR injury and inflammation, whereas treatment with recombinant HMGB1 enhanced IR injury in WT mice but not RAGE(-/-) mice. Importantly, lung dysfunction, cytokine production and neutrophil infiltration were significantly attenuated after IR in J?18(-/-) mice reconstituted with RAGE(-/-) iNKT cells (versus WT iNKT cells). In vitro studies demonstrated that, after hypoxia-reoxygenation, alveolar macrophage-derived HMGB1 augmented IL-17 production from iNKT cells in a RAGE-dependent manner. These results suggest that HMGB1-mediated RAGE activation on iNKT cells is critical for initiation of lung IR injury and that a crosstalk between macrophages and iNKT cells via the HMGB1/RAGE axis mediates IL-17 production by iNKT cells causing neutrophil infiltration and lung IR injury.
Project description:Staphylococcus (S.) aureus has emerged as an important cause of necrotizing pneumonia. Lung injury during S. aureus pneumonia may be enhanced by local release of damage associated molecular patterns such as high-mobility group box 1 (HMGB1). In the current study we sought to determine the functional role of HMGB1 and its receptors, toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE), in the injurious host response to S. aureus pneumonia.Pneumonia was induced in wild type (Wt), TLR4 deficient (tlr4-/-) and RAGE deficient (rage-/-) mice by intranasal inoculation of 1?×?107 colony-forming units (CFU) of a USA300 S. aureus. In a separate set of experiments, Wt mice were injected intraperitoneally with a monoclonal anti-HMGB1 antibody or an isotype matched control antibody immediately before and every 24 hours after intranasal infection of S. aureus. Mice were sacrificed at 6, 24, 48 or 72 hours after infection for harvesting of blood and organs.S. aureus pneumonia was associated with HMGB1 release in the bronchoalveolar compartment peaking after 24 hours. Anti-HMGB1 attenuated lung pathology and protein leak and reduced interleukin-1? release 6 hours after infection, but not at later time points. RAGE deficiency more modestly attenuated lung pathology without influencing protein leak, while TLR4 deficiency did not impact on lung injury.These data suggest that HMGB1 and RAGE, but not TLR4, contribute to lung injury accompanying the early phase of S. aureus pneumonia.
Project description:The effects of acute hyperglycemia on lung ischemia-reperfusion (IR) injury and the role of receptor for advanced glycation end-products (RAGE) signaling in this process are unknown. The objective of this study was twofold: (1) evaluate the impact of acute hyperglycemia on lung IR injury; and (2) determine if RAGE signaling is a mechanism of hyperglycemia-enhanced IR injury. We hypothesized that acute hyperglycemia worsens lung IR injury through a RAGE signaling mechanism. C57BL/6 wild-type (WT) and RAGE knockout (RAGE (-/-)) mice underwent sham thoracotomy or lung IR (1-h left hilar occlusion and 2-h reperfusion). Acute hyperglycemia was established by dextrose injection 30 minutes before ischemia. Lung injury was assessed by measuring lung function, cytokine expression in bronchoalveolar lavage fluid, leukocyte infiltration, and microvascular permeability via Evans blue dye. Mean blood glucose levels doubled in hyperglycemic mice 30 minutes after dextrose injection. Compared with IR in normoglycemic mice, IR in hyperglycemic mice significantly enhanced lung dysfunction, cytokine expression (TNF-?, keratinocyte chemoattractant, IL-6, monocyte chemotactic protein-1, regulated upon activation, normal T cell expressed and secreted), leukocyte infiltration, and microvascular permeability. Lung injury and dysfunction after IR were attenuated in normoglycemic RAGE (-/-) mice, and hyperglycemia failed to exacerbate IR injury in RAGE (-/-) mice. Thus, this study demonstrates that acute hyperglycemia exacerbates lung IR injury, whereas RAGE deficiency attenuates IR injury and also prevents exacerbation of IR injury in an acute hyperglycemic setting. These results suggest that hyperglycemia-enhanced lung IR injury is mediated, at least in part, by RAGE signaling, and identifies RAGE as a potential, novel therapeutic target to prevent post-transplant lung IR injury.
Project description:Sepsis-evoked acute lung injury (ALI) and its extreme manifestation, acute respiratory distress syndrome (ARDS), constitute a major cause of mortality in intensive care units. High levels of the long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) have been positively correlated with increased severity and unfavorable prognoses in patients with sepsis. Nevertheless, the function and molecular mechanism of NEAT1 in ALI remain elusive. In the current study, high levels of NEAT1 were confirmed in lipopolysaccharide- (LPS-) induced ALI mice models and in LPS-stimulated cells from the alveolar epithelial A549 cell line. Intriguingly, cessation of NEAT1 led to increased cell viability and decreased lactate dehydrogenase release, apoptosis, and caspase-3/9 activity, which conferred protection against LPS-induced injury in these cells. NEAT1 inhibition also restrained LPS-evoked transcripts and production of inflammatory cytokines IL-6, IL-1?, and TNF-?. A mechanism analysis corroborated the activation of high-mobility group box1 (HMGB1)/receptors for advanced glycation end products (RAGE) and NF-?B signaling in LPS-treated A549 cells. NEAT1 suppression reversed the activation of this pathway. Notably, reactivating HMGB1/RAGE signaling via HMGB1 overexpression blunted the anti-injury and anti-inflammation effects of NEAT1 knockdown. These findings suggest that NEAT1 may aggravate the progression of ALI and ARDS by inducing alveolar epithelial cell injury and inflammation via HMGB1/RAGE signaling, implying a promising treatment target for these conditions.
Project description:Traumatic brain injury (TBI) results in systemic inflammatory responses that affect the lung. This is especially critical in the setting of lung transplantation, where more than half of donor allografts are obtained postmortem from individuals with TBI. The mechanism by which TBI causes pulmonary dysfunction remains unclear but may involve the interaction of high-mobility group box-1 (HMGB1) protein with the receptor for advanced glycation end products (RAGE). To investigate the role of HMGB1 and RAGE in TBI-induced lung dysfunction, RAGE-sufficient (wild-type) or RAGE-deficient (RAGE(-/-)) C57BL/6 mice were subjected to TBI through controlled cortical impact and studied for cardiopulmonary injury. Compared to control animals, TBI induced systemic hypoxia, acute lung injury, pulmonary neutrophilia, and decreased compliance (a measure of the lungs' ability to expand), all of which were attenuated in RAGE(-/-) mice. Neutralizing systemic HMGB1 induced by TBI reversed hypoxia and improved lung compliance. Compared to wild-type donors, lungs from RAGE(-/-) TBI donors did not develop acute lung injury after transplantation. In a study of clinical transplantation, elevated systemic HMGB1 in donors correlated with impaired systemic oxygenation of the donor lung before transplantation and predicted impaired oxygenation after transplantation. These data suggest that the HMGB1-RAGE axis plays a role in the mechanism by which TBI induces lung dysfunction and that targeting this pathway before transplant may improve recipient outcomes after lung transplantation.
Project description:We recently implicated a role for CD4(+) T cells and demonstrated elevated IL-17A expression in lung ischemia-reperfusion (IR) injury. However, identification of the specific subset of CD4(+) T cells and their mechanistic role in IR injury remains unknown.We tested the hypothesis that invariant natural killer T (iNKT) cells mediate lung IR injury via IL-17A signaling.Mice underwent lung IR via left hilar ligation. Pulmonary function was measured using an isolated lung system. Lung injury was assessed by measuring edema (wet/dry weight) and vascular permeability (Evans blue dye). Inflammation was assessed by measuring proinflammatory cytokines in lungs, and neutrophil infiltration was measured by immunohistochemistry and myeloperoxidase levels.Pulmonary dysfunction (increased airway resistance and pulmonary artery pressure and decreased pulmonary compliance), injury (edema, vascular permeability), and inflammation (elevated IL-17A; IL-6; tumor necrosis factor-?; monocyte chemotactic protein-1; keratinocyte-derived chemokine; regulated upon activation, normal T-cell expressed and secreted; and neutrophil infiltration) after IR were attenuated in IL-17A(-/-) and Rag-1(-/-) mice. Anti-IL-17A antibody attenuated lung dysfunction in wild-type mice after IR. Reconstitution of Rag-1(-/-) mice with wild-type, but not IL-17A(-/-), CD4(+) T cells restored lung dysfunction, injury, and inflammation after IR. Lung dysfunction, injury, IL-17A expression, and neutrophil infiltration were attenuated in J?18(-/-) mice after IR, all of which were restored by reconstitution with wild-type, but not IL-17A(-/-), iNKT cells. Flow cytometry and enzyme-linked immunosorbent spot assay confirmed IL-17A production by iNKT cells after IR.These results demonstrate that CD4(+) iNKT cells play a pivotal role in initiating lung injury, inflammation, and neutrophil recruitment after IR via an IL-17A-dependent mechanism.
Project description:High mobility group box 1 (HMGB1) is tightly connected to the process of tissue organization upon tissue injury. Here we show that HMGB1 controls epithelium and connective tissue regeneration both in vivo and in vitro during palatal wound healing. Heterozygous HMGB1 (Hmgb1+/-) mice and Wild-type (WT) mice were subjected to palatal injury. Maxillary tissues were stained with Mallory Azan or immunostained with anti-HMGB1, anti-proliferating cell nuclear antigen (PCNA), anti-nuclear factor-?B (NF-?B) p50 and anti-vascular endothelial growth factor (VEGF) antibodies. Palatal gingival explants were cultured with recombinant HMGB1 (rHMGB1) co-treated with siRNA targeting receptor for advanced glycation end products (RAGEs) for cell migration and PCNA expression analysis. Measurement of the wound area showed differences between Hmgb1+/- and WT mice on Day 3 after wounding. Mallory Azan staining showed densely packed of collagen fibers in WT mice, whereas in Hmgb1+/- mice weave-like pattern of low density collagen bundles were present. At three and seven days post-surgery, PCNA, NF-?B p50 and VEGF positive keratinocytes of WT mice were greater than that of Hmgb1+/- mice. Knockdown of RAGE prevents the effect of rHMGB1-induced cell migration and PCNA expression in gingival cell cultures. The data suggest that HMGB1/RAGE axis has crucial roles in palatal wound healing.
Project description:High-mobility group box-1 (HMGB1) is a damage-associated molecular pattern (DAMP) increased in response to liver injury. Because HMGB1 is a ligand for the receptor for advanced glycation endproducts (RAGE), we hypothesized that induction of HMGB1 could participate in the pathogenesis of liver fibrosis though RAGE cell-specific signaling mechanisms. Liver HMGB1 protein expression correlated with fibrosis stage in patients with chronic hepatitis C virus (HCV) infection, primary biliary cirrhosis (PBC), or alcoholic steatohepatitis (ASH). Hepatic HMGB1 protein expression and secretion increased in five mouse models of liver fibrosis attributed to drug-induced liver injury (DILI), cholestasis, ASH, or nonalcoholic steatohepatitis (NASH). HMGB1 was up-regulated and secreted mostly by hepatocytes and Kupffer cells (KCs) following CCl4 treatment. Neutralization of HMGB1 protected, whereas injection of recombinant HMGB1 promoted liver fibrosis. Hmgb1 ablation in hepatocytes (Hmgb1?Hep ) or in myeloid cells (Hmgb1?Mye ) partially protected, whereas ablation in both (Hmgb1?Hep?Mye ) prevented liver fibrosis in vivo. Coculture with hepatocytes or KCs from CCl4 -injected wild-type (WT) mice up-regulated Collagen type I production by hepatic stellate cells (HSCs); yet, coculture with hepatocytes from CCl4 -injected Hmgb1?Hep or with KCs from CCl4 -injected Hmgb1?Mye mice partially blunted this effect. Rage ablation in HSCs (Rage?HSC ) and RAGE neutralization prevented liver fibrosis. Last, we identified that HMGB1 stimulated HSC migration and signaled through RAGE to up-regulate Collagen type I expression by activating the phosphorylated mitogen-activated protein kinase kinase (pMEK)1/2, phosphorylated extracellular signal-regulated kinase (pERK)1/2 and pcJun signaling pathway. Conclusion: Hepatocyte and KC-derived HMGB1 participates in the pathogenesis of liver fibrosis by signaling through RAGE in HSCs to activate the pMEK1/2, pERK1/2 and pcJun pathway and increase Collagen type I deposition.
Project description:The receptor for advanced glycation end products (RAGE), a cell membrane receptor, recognizes ligands produced by cigarette smoke (CS) and has been implicated in the pathogenesis of COPD. We demonstrate that deletion or pharmacologic inhibition of RAGE prevents development of CS-induced emphysema. To identify molecular pathways by which RAGE mediates smoking related lung injury we performed unbiased gene expression profiling of alveolar macrophages (AM) obtained from RAGE null and C57BL/6 WT mice exposed to CS for one week or four months. Pathway analysis of RNA expression identified a number of genes integral to the pathogenesis of COPD impacted by the absence of RAGE. Altered expression of antioxidant response genes and lung protein 4-HNE immunostaining suggest attenuated oxidative stress in the RAGE null mice despite comparable CS exposure and lung leukocyte burden as the WT mice. Reduced endoplasmic reticulum stress in response to CS exposure also was observed in the AM from RAGE null mice. These findings provide novel insight into the sources of oxidative stress, macrophage activation, and the pathogenesis of lung disease due to CS exposure.
Project description:Genetic or pharmacological ablation of toll-like receptor 2 (TLR2) protects against myocardial ischemia/reperfusion injury (MI/R). However, the endogenous ligand responsible for TLR2 activation has not yet been detected. The objective of this study was to identify HMGB1 as an activator of TLR2 signalling during MI/R. C57BL/6 wild-type (WT) or TLR2(-/-)-mice were injected with vehicle, HMGB1, or HMGB1 BoxA one hour before myocardial ischemia (30 min) and reperfusion (24 hrs). Infarct size, cardiac troponin T, leukocyte infiltration, HMGB1 release, TLR4-, TLR9-, and RAGE-expression were quantified. HMGB1 plasma levels were measured in patients undergoing coronary artery bypass graft (CABG) surgery. HMGB1 antagonist BoxA reduced cardiomyocyte necrosis during MI/R in WT mice, accompanied by reduced leukocyte infiltration. Injection of HMGB1 did, however, not increase infarct size in WT animals. In TLR2(-/-)-hearts, neither BoxA nor HMGB1 affected infarct size. No differences in RAGE and TLR9 expression could be detected, while TLR2(-/-)-mice display increased TLR4 and HMGB1 expression. Plasma levels of HMGB1 were increased MI/R in TLR2(-/-)-mice after CABG surgery in patients carrying a TLR2 polymorphism (Arg753Gln). We here provide evidence that absence of TLR2 signalling abrogates infarct-sparing effects of HMGB1 blockade.
Project description:The receptor for advanced glycation end-products (RAGE) is involved in inflammatory response during acute respiratory distress syndrome (ARDS). Growing body of evidence support strategies of RAGE inhibition in experimental lung injury, but its modalities and effects remain underinvestigated. Anesthetised C57BL/6JRj mice were divided in four groups; three of them underwent orotracheal instillation of acid and were treated with anti-RAGE monoclonal antibody (mAb) or recombinant soluble RAGE (sRAGE), acting as a decoy receptor. The fourth group served as a control. Lung injury was assessed by the analysis of blood gases, alveolar permeability, histology, AFC, and cytokines. Lung expression and distribution epithelial channels ENaC, Na,K-ATPase, and aquaporin (AQP)-5 were assessed. Treatment with either anti-RAGE mAb or sRAGE improved lung injury, arterial oxygenation and decreased alveolar inflammation in acid-injured animals. Anti-RAGE therapies were associated with restored AFC and increased lung expression of AQP-5 in alveolar cell. Blocking RAGE had potential therapeutic effects in a translational mouse model of ARDS, possibly through a decrease in alveolar type 1 epithelial cell injury as shown by restored AFC and lung AQP-5 expression. Further mechanistic studies are warranted to describe intracellular pathways that may control such effects of RAGE on lung epithelial injury and repair.