Neutrophil NAD(P)H oxidase is required for hemorrhagic shock-enhanced TLR2 up-regulation in alveolar macrophages in response to LPS

Alveolar macrophages (AMs) play an important role in the development of posttrauma lung inflammation through initiating polymorphonuclear neutrophil (PMN) migration by direct interactions with PMN, which is in turn mediated by the expression of chemokines and cytokines. We have recently reported that hemorrhagic shock-activated PMN sensitize AM to bacteria LPS for the up-regulation of Toll-like receptor (TLR)2; in turn, this TLR2 up-regulation results in the amplification of expression of cytokines and chemokines in the AM in response to the bacterial products LPS and peptidoglycan, associated with enhanced PMN sequestration in the lung. We sought to address the mechanism underlying the augmentation of TLR2 in AM by shock-activated PMN. We found that hemorrhagic shock/resuscitation (shock) followed by a low dose of i.t. LPS markedly increased TLR2 mRNA expression in AM in wild-type (WT) mice. In contrast, in mice lacking the gp91 subunit of nicotinamide adenine dinucleotide phosphate (reduced form) oxidase (gp91) or in neutropenic WT mice, the increase in TLR2 mRNA was attenuated. Coculture of AM with PMN derived from WT-shocked mice caused a significantly higher level of TLR2 expression in the AM in response to LPS. However, this increase in TLR2 expression was less evident when the AMs were cocultured with PMN derived from gp91 mice subjected to shock. The antioxidant polyethylene glycol catalase markedly decreased MyD88-dependent activation of IL-1 receptor associated kinase 4 and TLR2 expression in the AM in response to LPS. Thus, PMN nicotinamide adenine dinucleotide phosphate (reduced form) oxidase sensitizes hemorrhagic shock-primed AM to LPS, at least in part via enhancing IL-1 receptor associated kinase 4 activity.     

Toll-like receptor 4 deficiency causes pulmonary emphysema

TLRs have been studied extensively in the context of pathogen challenges, yet their role in the unchallenged lung is unknown. Given their direct interface with the external environment, TLRs in the lungs are prime candidates to respond to air constituents, namely particulates and oxygen. The mechanism whereby the lung maintains structural integrity in the face of constant ambient exposures is essential to our understanding of lung disease. Emphysema is characterized by gradual loss of lung elasticity and irreversible airspace enlargement, usually in the later decades of life and after years of insult, most commonly cigarette smoke. Here we show Tlr4(-/-) mice exhibited emphysema as they aged. Adoptive transfer experiments revealed that TLR4 expression in lung structural cells was required for maintaining normal lung architecture. TLR4 deficiency led to the upregulation of what we believe to be a novel NADPH oxidase (Nox), Nox3, in lungs and endothelial cells, resulting in increased oxidant generation and elastolytic activity. Treatment of Tlr4(-/- )mice or endothelial cells with chemical NADPH inhibitors or Nox3 siRNA reversed the observed phenotype. Our data identify a role for TLR4 in maintaining constitutive lung integrity by modulating oxidant generation and provide insights into the development of emphysema.     

Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly.

Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more responsive to other stimuli, a phenomenon known as "priming." Since the mechanism of LPS-dependent priming is incompletely understood, we investigated its effects on assembly and activation of the NADPH oxidase. LPS pretreatment increased superoxide (O2-) generation nearly 10-fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). In a broken-cell O2--generating system, activity was increased in plasma membrane-rich fractions and concomitantly decreased in specific granule-rich fractions from LPS-treated cells. Oxidation-reduction spectroscopy and flow cytometry indicated LPS increased plasma membrane association of flavocytochrome b558. Immunoblots of plasma membrane vesicles from LPS-treated PMNs demonstrated translocation of p47-phox but not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three- to sixfold increase (compared with either agent alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O2- generation by intact PMNs. LPS treatment caused limited phosphorylation of p47-phox, and plasma membrane-enriched fractions from LPS- and/or fMLP-treated cells contained fewer acidic species of p47-phox than did those from cells treated with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respiratory burst.     

TLR4 activation mediates kidney ischemia/reperfusion injury

Ischemia/reperfusion injury (IRI) may activate innate immunity through the engagement of TLRs by endogenous ligands. TLR4 expressed within the kidney is a potential mediator of innate activation and inflammation. Using a mouse model of kidney IRI, we demonstrated a significant increase in TLR4 expression by tubular epithelial cells (TECs) and infiltrating leukocytes within the kidney following ischemia. TLR4 signaling through the MyD88-dependent pathway was required for the full development of kidney IRI, as both TLR4(-/-) and MyD88(-/-) mice were protected against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. In vitro, WT kidney TECs produced proinflammatory cytokines and chemokines and underwent apoptosis after ischemia. These effects were attenuated in TLR4(-/-) and MyD88(-/-) TECs. In addition, we demonstrated upregulation of the endogenous ligands high-mobility group box 1 (HMGB1), hyaluronan, and biglycan, providing circumstantial evidence that one or more of these ligands may be the source of TLR4 activation. To determine the relative contribution of TLR4 expression by parenchymal cells or leukocytes to kidney damage during IRI, we generated chimeric mice. TLR4(-/-) mice engrafted with WT hematopoietic cells had significantly lower serum creatinine and less tubular damage than WT mice reconstituted with TLR4(-/-) BM, suggesting that TLR4 signaling in intrinsic kidney cells plays the dominant role in mediating kidney damage.     

Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts

Carbon monoxide (CO), a byproduct of heme catabolism by heme oxygenase (HO), confers potent antiinflammatory effects. Here we demonstrate that CO derived from HO-1 inhibited Toll-like receptor (TLR) 2, 4, 5, and 9 signaling, but not TLR3-dependent signaling, in macrophages. Ligand-mediated receptor trafficking to lipid rafts represents an early event in signal initiation of immune cells. Trafficking of TLR4 to lipid rafts in response to LPS was reactive oxygen species (ROS) dependent because it was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and in gp91(phox)-deficient macrophages. CO selectively inhibited ligand-induced recruitment of TLR4 to lipid rafts, which was also associated with the inhibition of ligand-induced ROS production in macrophages. TLR3 did not translocate to lipid rafts by polyinosine-polycytidylic acid (poly(I:C)). CO had no effect on poly(I:C)-induced ROS production and TLR3 signaling. The inhibitory effect of CO on TLR-induced cytokine production was abolished in gp91(phox)-deficient macrophages, also indicating a role for NADPH oxidase. CO attenuated LPS-induced NADPH oxidase activity in vitro, potentially by binding to gp91(phox). Thus, CO negatively controlled TLR signaling pathways by inhibiting translocation of TLR to lipid rafts through suppression of NADPH oxidase-dependent ROS generation.     

Nicotinamide adenine dinucleotide phosphate (reduced form) oxidase is important for LPS-induced endothelial cell activation

Activation of the endothelium plays an important role in the innate immune response. This process is associated with an increase in the production of superoxide (O2-) by nicotinamide adenine dinucleotide phosphate (reduced form; NADPH) oxidase. Our objective was to determine if O2- from NADPH oxidase contributes to activation of human umbilical vein endothelial cells by LPS as it does for TNF-alpha. We used the adhesion molecule intracellular adhesion molecule 1 and cytokine IL-8 as indicators of human umbilical vein endothelial cell activation and measured O2- production with chemiluminescence. LPS increased baseline and NADPH-stimulated O2- production. The increase was reduced by tiron, a protein kinase C inhibitor (bisindolylmaleimide I hydrochloride), the flavin inhibitor (diphenylene iodonium), and by a short interfering RNA against the p22phox component of NADPH oxidase. Inhibition of NADPH oxidase with the short interfering RNA reduced the induction by LPS of intracellular adhesion molecule 1 mRNA, protein, and IL-8 release (by enzyme-linked immunosorbent assay). The production of O2- by NADPH oxidase contributes to intracellular signaling by LPS in endothelial cells as it does for TNF-alpha and helps turn on the innate immune response in these cells.     

Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung

In models of acute lung injury, CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands, including CXCL1 and CXCL2/3, are chemotactic for PMNs, CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung, aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice, but not in littermate CXCR2-/- mice. Surprisingly, lethally irradiated wild-type mice reconstituted with CXCR2-/- BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium, but CXCR2-/- mice reconstituted with CXCR2-/- BM showed no PMN recruitment. Conversely, CXCR2-/- mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment, inconsistent with a role of CXCR2 on PMNs alone. Cell culture, immunohistochemistry, flow cytometry, and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury.     

RIP links TLR4 to Akt and is essential for cell survival in response to LPS stimulation

Receptor-interacting protein (RIP) has been reported to associate with tumor necrosis-associated factor (TRAF)2 and TRAF6. Since TRAF2 and TRAF6 play important roles in CD40 signaling and TRAF6 plays an important role in TLR4 signaling, we examined the role of RIP in signaling via CD40 and TLR4. Splenocytes from RIP(-/-) mice proliferated and underwent isotype switching normally in response to anti-CD40-IL-4 but completely failed to do so in response to LPS-IL-4. However, they normally up-regulated TNF-alpha and IL-6 gene expression and CD54 and CD86 surface expression after LPS stimulation. RIP(-/-) splenocytes exhibited increased apoptosis and impaired Akt phosphorylation after LPS stimulation. These results suggest that RIP is essential for cell survival after TLR4 signaling and links TLR4 to the phosphatidylinositol 3 kinase-Akt pathway.     

TLR ligand decreases mesenteric ischemia and reperfusion injury-induced gut damage through TNF-alpha signaling

Ischemic gut contributes to the development of sepsis and organ failure in critically ill patients. Toll-like receptors (TLRs) have been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We hypothesize that LPS, a ligand for TLR4, decreases mesenteric I/R injury-induced gut damage through tumor necrosis factor alpha (TNF-alpha) signaling. First, wild-type (WT) mice were fed with oral antibiotics for 4 weeks to deplete the intestinal commensal microflora. At week 3, drinking water was supplemented with LPS (10 microg/microL) to trigger TLRs. The intestinal mucosa was harvested for TLR4 protein, caspase 3 activity, and terminal deoxynucleotide transferase labeling assay. Second, WT and Tnfrsf1a mice received 30-min ischemia and 30-min reperfusion (30I-30R) or 30I-180R of the intestine; intestinal permeability and lipid peroxidation of the intestine were examined. Third, WT and Tnfrsf1a mice were fed with oral antibiotics with or without LPS and received 30I-180R of the intestine. The intestinal mucosa was harvested for lipid peroxidation; glutathione (GSH) level; nuclear factor kappaB (NF-kappaB) and AP-1 DNA-binding activity; Bcl-w, TNF-alpha, and CXCR2 mRNA expression; and HSP70 protein assay. Commensal depletion increased caspase 3 activity as well as villi apoptosis and decreased TLR4 expression of the intestinal mucosa. LPS increased TLR4 expression and decreased villi apoptosis. Commensal depletion augmented 30I-180R-induced intestine permeability as well as lipid peroxidation and decreased GSH level in WT mice but not in Tnfrsf1a mice. LPS decreased 30I-180R-induced intestinal permeability as well as lipid peroxidation and increased GSH level of the intestinal mucosa in WT mice but not in Tnfrsf1a mice. Commensal depletion with 30I-180R increased NF-kappaB and AP-1 DNA-binding activity, HSP70 protein expression, and decreased Bcl-w and TNF-alpha mRNA expression of the intestinal mucosa in WT mice but not in Tnfrsf1a mice. Collectively, commensal microflora induces TLR4 expression and decreases apoptosis of the intestinal mucosa. Commensal depletion enhances I/R-induced gut damage. LPS prevents I/R-induced intestinal permeability, lipid peroxidation, and decrease in GSH level. Given that the preventive effect of LPS on I/R-induced gut damage and NF-kappaB activity of the intestine is abolished in Tnfrsf1a mice, we conclude that TLR ligand decreases mesenteric I/R injury-induced gut damage through TNF-alpha signaling.     

Tumor necrosis factor-alpha blockade prevents neutrophil CD18 receptor upregulation and attenuates acute lung injury in porcine sepsis without inhibition of neutrophil oxygen radical generation.

Tumor necrosis factor (TNF alpha), both by direct action and by trafficking cells of the immune system, is implicated in cardiopulmonary derangements and PMN-mediated microvascular injury associated with gram-negative sepsis. We examined the effects of pretreatment with a monoclonal antibody to TNF alpha on PMN function, hemodynamic derangements, and alveolar capillary membrane damage in a septic porcine model. Anti-TNF alpha profoundly improved hemodynamic consequences in this model. Reduction in PMN CD11/18 receptor expression, lung myeloperoxidase activity, and attenuation of peripheral neutropenia (all P < 0.05) indicate that pretreatment significantly reduced lung sequestration of PMNs seen in septic controls. In contrast, PMN oxygen radical (O2-) generation was not significantly different from unprotected septic animals. Despite the presence of circulating PMNs primed for O2- burst, alveolar capillary membrane damage, assessed by bronchoalveolar lavage protein content and arterial PO2 was markedly attenuated in the treatment group (P < 0.05). We conclude that anti-TNF alpha suppresses systemic hemodynamic actions of TNF alpha. Further, it prevents upregulation of PMN adhesion receptors inhibiting PMN/endothelial cell interaction. This prevents formation of a "microenvironment," protected from circulating oxidant scavengers, into which sepsis-activated PMNs release their toxic products. Pretreatment with anti-TNF alpha monoclonal antibody thus affords global protection in porcine Gram-negative sepsis.     

Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling

Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8(+) T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8(+) T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand-containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8(+) T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.     

Toll4 (TLR4) expression in cardiac myocytes in normal and failing myocardium

Expression of innate immune response proteins, including IL-1beta, TNF, and the cytokine-inducible isoform of nitric oxide synthase (iNOS), have been documented in the hearts of humans and experimental animals with heart failure regardless of etiology, although the proximal events leading to their expression are unknown. Noting that expression of a human homologue of Drosophila Toll, a proximal innate immunity transmembrane signaling protein in the fly, now termed human Toll-like receptor 4 (hTLR4), appeared to be relatively high in the heart, we examined TLR4 mRNA and protein abundance in isolated cellular constituents of cardiac muscle and in normal and abnormal murine, rat, and human myocardium. TLR4 expression levels in cardiac myocytes and in coronary microvascular endothelial cells could be enhanced by either LPS or IL-1beta, an effect inhibited by the oxygen radical scavenger PDTC. Transfection of a constitutively active TLR4 construct, CD4/hTLR4, resulted in activation of a nuclear factor-kappaB reporter construct, but not of an AP-1 or an iNOS reporter construct, in cardiac myocytes. In normal murine, rat, and human myocardium, TLR4 expression was diffuse, and presumably cytoplasmic, in cardiac myocytes. However, in remodeling murine myocardium remote from sites of ischemic injury and in heart tissue from patients with idiopathic dilated cardiomyopathy, focal areas of intense TLR4 staining were observed in juxtaposed regions of 2 or more adjacent myocytes; this staining was not observed in control myocardium. Increased expression and signaling by TLR4, and perhaps other Toll homologues, may contribute to the activation of innate immunity in injured myocardium.     

Osteoclastogenesis through TLR/NOD signaling

Lipopolysaccharide (LPS) and muramyl dipeptide (MDP) are components of microbial cell walls that cause innate immune responses and inflammation. Toll-like receptor 4 (TLR4) is a receptor for LPS and transduces signals through myeloid differentiation factor 88 (MyD88), which plays essential roles in the TLR/IL-1R signaling and activates NF-kappaB and MAP kinase pathways to induce RANKL expression in osteoblasts. Osteoblasts express NOD2, an intracellular sensor for MDP, in response to LPS, IL-1 and TNF. NOD2 binds RIP2, a serine/threonine kinase which transduces NF-kappaB signaling. Thus MDP synergistically enhances osteoclast formation induced by LPS, IL-1 and TNF through RANK ligand up-regulation in osteoblasts. In summary, innate immune receptors, TLR4 and NOD2, recognize bacterial components on cell surfaces and inside cells, respectively, and these signals cross-talk to induce RANKL expression in osteoblasts, which results in enhancing osteoclast formation and function.     

Blood transfusion and the two-insult model of post-injury multiple organ failure

Neutrophils (PMNs) have been implicated in the pathogenesis of multiple organ failure (MOF). The two-insult model of MOF is based on the fundamental concept that two sequential and independent insults that are individually innocuous against the host can cause overwhelming inflammation. The in vitro PMN priming/activation sequence simulates the two-insult model. Our work has demonstrated that transfusion is an early consistent risk factor for post-injury MOF and lysophosphatidylcholines (lyso-PCs) are generated in stored blood components. Additionally, platelet-activating factor (PAF) is a key inflammatory agent produced in severely injured patients. We therefore hypothesize that two events, trauma and transfusion, enhance PMN cytotoxicity irrespective of the sequence. Superoxide (O2-) production was measured by reduction of cytochrome c, adherence to fibrinogen was assessed by the radioactivity of adherent Na2(51)CrO4 (51Cr)-labeled PMNs, and endothelial cell (EC) damage by measuring the radioactivity released from 51Cr-labeled human umbilical vein endothelial cells monolayers. Isolated PMNs were primed with buffer, PAF (2 microM), or lyso-PCs (4.5, 15, and 30 microM) followed by activation with buffer, N-formyl-methionyl-leucyl-phenylalanine (fMLP) (1 microM), PAF (2 microM), or lyso-PCs (4.5, 15, and 30 microM). Neither PAF nor lyso-PCs alone stimulated O2- production. While PAF alone caused PMN adherence, lyso-PCs alone did not allowed PMNs to adhere to fibrinogen. However, both combinations of PAF/lyso-PCs and lyso-PCs/PAF significantly augmented O2- production and PMN adherence. Furthermore, these enhanced PMN cytotoxic responses significantly caused EC damage. These findings suggest that in the scenario of the two-insult model, early or late transfusion administered following trauma can provoke PMN cytotoxicity via priming or activation, thereby increasing the risk of post-injury MOF.     

血小板对TLR4表达及活化在脂多糖诱发小鼠血小板减少中的作用

目的 探讨脂多糖(LPS)诱发小鼠血小板减少中血小板活化、血小板对Toll样受体4 (TLR4)的表达的变化及中性粒细胞对其影响.方法 中国医学科学院放射医学研究所ICR小鼠87只随机(随机数字法)分为健康对照(C)组、模型(M)组(3,6,12,24,48,72 h时点)及中性粒细胞减少症(NEP)组.M组尾静脉注射LPS 8 mg/kg后分别于相应时点取血,NEP组注射LPS 24 h前予尾静脉注射抗中性粒细胞单克隆抗体,注射LPS24h后取血.血细胞分析仪检测小鼠血小板参数:血小板计数(PC)、平均血小板体积( MPV)和血小板分布宽度(PDW);ELISA法检测小鼠血浆肿瘤坏死因子-α(TNF-α)、巨噬细胞集落刺激因子(M-CSF)和可溶性CD40配体(sCD40 L)浓度;流式细胞仪检测血小板TLR4阳性表达率.组间比较采用单因素方差分析.结果 尾静脉注射LPS3h后小鼠PC(×109 L-1)下降30％[(719.8±135.9) vs.(1013.1±136.6,P＜0.01],24h内达谷值[(374.7±115.1) vs.(1013.1±136.6),P＜0.01];MPV和PDW 12～48 h较C组增大(P＜0.01);LPS攻击后6～24h血浆TNF-α,M-CSF,sCD40L显著升高(与C组比较P ＜0.01),PC与血浆M-CSF(r=-0.746,P＜0.01),sCD40L(r=-0.573,P＜ 0.001)水平负相关;LPS刺激6h后血小板TLR4阳性表达(％)较C组增加[ (50.37±3.20) vs.(45.76±2.49) P＜0.01];NEP组血小板TLR4表达率(％)低于M组同时点[ (48.32±2.17) vs.(55.69±3.95,P＜0.01].结论 以sCD40L及M-CSF升高为代表的血小板过度活化与血小板TLR4表达中性粒细胞依赖性上调共同参与脂多糖诱发小鼠血小板减少.     

Mice that exclusively express TLR4 on endothelial cells can efficiently clear a lethal systemic Gram-negative bacterial infection

Recognition of LPS by TLR4 on immune sentinel cells such as macrophages is thought to be key to the recruitment of neutrophils to sites of infection with Gram-negative bacteria. To explore whether endothelial TLR4 plays a role in this process, we engineered and imaged mice that expressed TLR4 exclusively on endothelium (known herein as Endothelium^TLR4 mice). Local administration of LPS into tissue induced comparable neutrophil recruitment in Endothelium^TLR4 and wild-type mice. Following systemic LPS or intraperitoneal E. coli administration, most neutrophils were sequestered in the lungs of wild-type mice and did not accumulate at primary sites of infection. In contrast, Endothelium^TLR4 mice showed reduced pulmonary capillary neutrophil sequestration over the first 24 hours; as a result, they mobilized neutrophils to primary sites of infection, cleared bacteria, and resisted a dose of E. coli that killed 50% of wild-type mice in the first 48 hours. In fact, the only defect we detected in Endothelium^TLR4 mice was a failure to accumulate neutrophils in the lungs following intratracheal administration of LPS; this response required TLR4 on bone marrow–derived immune cells. Therefore, endothelial TLR4 functions as the primary intravascular sentinel system for detection of bacteria, whereas bone marrow–derived immune cells are critical for pathogen detection at barrier sites. Nonendothelial TLR4 contributes to failure to accumulate neutrophils at primary infection sites in a disseminated systemic infection.     

TLR4 links innate immunity and fatty acid-induced insulin resistance

TLR4 is the receptor for LPS and plays a critical role in innate immunity. Stimulation of TLR4 activates proinflammatory pathways and induces cytokine expression in a variety of cell types. Inflammatory pathways are activated in tissues of obese animals and humans and play an important role in obesity-associated insulin resistance. Here we show that nutritional fatty acids, whose circulating levels are often increased in obesity, activate TLR4 signaling in adipocytes and macrophages and that the capacity of fatty acids to induce inflammatory signaling in adipose cells or tissue and macrophages is blunted in the absence of TLR4. Moreover, mice lacking TLR4 are substantially protected from the ability of systemic lipid infusion to (a) suppress insulin signaling in muscle and (b) reduce insulin-mediated changes in systemic glucose metabolism. Finally, female C57BL/6 mice lacking TLR4 have increased obesity but are partially protected against high fat diet-induced insulin resistance, possibly due to reduced inflammatory gene expression in liver and fat. Taken together, these data suggest that TLR4 is a molecular link among nutrition, lipids, and inflammation and that the innate immune system participates in the regulation of energy balance and insulin resistance in response to changes in the nutritional environment.     

TLR4 stimulation by LPS enhances angiogenesis in a co‐culture system consisting of primary human osteoblasts and outgrowth endothelial cells

The development of new approaches leading to fast and successful vascularization of tissue‐engineered constructs is one of the most intensively studied subjects in tissue engineering and regenerative medicine. Recently, TLR4 activation and LPS stimulation of endothelial cells have been reported to promote angiogenesis in a variety of settings. In this study, we demonstrate that TLR4 activation by Ultrapure LPS Escherichia coli 0111:B4 (LPS‐EB) significantly enhances microvessel formation in a co‐culture system consisting of outgrowth endothelial cells (OECs) and primary human osteoblasts (pOBs). The precise modes of TLR4 action on the process of angiogenesis have also been investigated in this study. Using quantitative fluorescence microscopy in monocultures of OECs and pOBs, it was found that TLR4 activation through LPS‐EB upregulates the expression level of TLR4/MYD88 and enhances both angiogenesis and osteogenesis. Furthermore, ELISA and qRT–PCR have shown that the level of two adhesion molecules (ICAM‐1 and E‐selectin), two cytokines (IL‐6 and IL‐8) and two growth factors (VEGF and PDGF‐BB) related to angiogenesis increase significantly after LPS‐EB treatment. This increased understanding of the role of TLR4 in angiogenesis could be of value in various settings related to tissue repair and tissue engineering. Moreover, since LPS and TLR4 agonists improve angiogenesis and osteogenesis, TLR4 agonists (endogenous or synthetic) could be used for angiogenesis intervention in vivo and therefore could be tested for their potential clinical applications in promoting angiogenesis in bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.