Blockade Of Toll-like Receptor 4 Research Articles

Human platelet interaction with E. coli O111 promotes tissue-factor-dependent procoagulant activity, involving Toll like receptor 4.

Platelets have a major role in clotting activation and contribute to the innate immune response during systemic infections. Human platelets contain tissue factor (TF) and express functional Toll-like receptor 4 (TLR4). However, the role of TLR4 in triggering the procoagulant properties of platelets, upon challenge with bacteria, is yet unknown. Our hypothesis is that E. coli O111-TLR4 interaction activates platelets and elicits their procoagulant activity. We demonstrated that the strain, but not ultrapure LPS, increased surface P-selectin expression, platelet dependent TF procoagulant activity (TF-PCA) and prompted a faster thrombin generation (TG). Blockade of TLR4 resulted in decreased platelet activation, TF-PCA and TG, revealing the participation of this immune receptor on the procoagulant response of platelets. Our results provide a novel mechanism by which individuals with bacterial infections would have an increased incidence of blood clots. Furthermore, the identification of platelet TF and TLR4 as regulators of the effect of E. coli O111 might represent a novel therapeutic target to reduce the devastating consequences of the hemostatic disorder during sepsis.

Gut-homing Δ42PD1+Vδ2 T cells promote innate mucosal damage via TLR4 during acute HIV type 1 infection.

The innate immune cells underlying mucosal inflammatory responses and damage during acute HIV-1 infection remain incompletely understood. Here, we report a Vδ2 subset of gut-homing γδ T cells with significantly upregulated Δ42PD1 (a PD1 isoform) in acute (~20%) HIV-1 patients compared to chronic HIV-1 patients (~11%) and healthy controls (~2%). The frequency of Δ42PD1+Vδ2 cells correlates positively with plasma levels of pro-inflammatory cytokines and fatty-acid-binding protein before detectable lipopolysaccharide in acute patients. The expression of Δ42PD1 can be induced by in vitro HIV-1 infection and is accompanied by high co-expression of gut-homing receptors CCR9/CD103. To investigate the role of Δ42PD1+Vδ2 cells in vivo, they were adoptively transferred into autologous humanized mice, resulting in small intestinal inflammatory damage, probably due to the interaction of Δ42PD1 with its cognate receptor Toll-like receptor 4 (TLR4). In addition, blockade of Δ42PD1 or TLR4 successfully reduced the cytokine effect induced by Δ42PD1+Vδ2 cells in vitro, as well as the mucosal pathological effect in humanized mice. Our findings have therefore uncovered a Δ42PD1-TLR4 pathway exhibited by virus-induced gut-homing Vδ2 cells that may contribute to innate immune activation and intestinal pathogenesis during acute HIV-1 infection. Δ42PD1+Vδ2 cells may serve as a target for the investigation of diseases with mucosal inflammation.

The role of RNase 7 in innate cutaneous defense against Pseudomonas aeruginosa.

The ribonuclease RNase 7 is a major skin-derived human antimicrobial protein expressed in keratinocytes. Here we show that the gram-negative pathogen Pseudomonas aeruginosa secretes factor(s) that induced RNase 7 gene and protein expression in human primary keratinocytes. The metalloprotease inhibitor marimastat, the epidermal growth factor receptor (EGFR) inhibitor AG1478 and the EGFR blocking antibody cetuximab significantly attenuated this induction, indicating an important role of the EGFR for the P.aeruginosa-mediated RNase 7 induction. In line with this, siRNA-mediated downregulation of ADAM17, a metalloprotease known to proteolytically mediate the release of soluble EGFR ligands, decreased the P.aeruginosa-mediated RNase 7 induction in keratinocytes. The impact of the EGFR was also demonstrated in a human 3D skin equivalent where blockade of the EGFR diminished induction of RNase 7 by P.aeruginosa. Blockade of Toll-like receptor 5 (TLR5), a pattern recognition receptor (PRR) known to be activated by P.aeruginosa, only moderately reduced the P.aeruginosa-mediated RNase 7 induction in keratinocytes. The functional relevance of RNase 7 to participate in cutaneous defense against P.aeruginosa was demonstrated by antibodies that neutralized the antimicrobial activity of RNase 7. These antibodies significantly inhibited the capacity of human stratum corneum skin extracts to control growth of P.aeruginosa. Taken together, our results indicate that P.aeruginosa induces the expression of RNase 7 in keratinocytes in an EGFR-dependent manner. Enhanced release of RNase 7 contributes to control cutaneous growth of P.aeruginosa.

Cardiac troponin I exacerbates myocardial ischaemia/reperfusion injury by inducing the adhesion of monocytes to vascular endothelial cells via a TLR4/NF-κB-dependent pathway

Cardiac troponin I (cTnI), a biomarker for myocardial damage and risk stratification, may be involved in the pathogenesis of cardiovascular diseases, which was ascribed to the effect of cTnI auto-antibodies. Whether or not cTnI itself has a direct impact on acute myocardial injury is unknown. To exclude the influence of cTnI antibody on the cardiac infarct size, we studied the effect of cTnI shortly after myocardial ischaemia-reperfusion (I/R) injury when cTnI antibodies were not elevated. Pretreatment with cTnI augmented the myocardial infarct size caused by I/R, accompanied by an increase in inflammatory markers in the blood and myocardium. Additional experiments using human umbilical vein endothelial cells (HUVECs) showed that the detrimental effect of cTnI was related to cTnI-induced increase in vascular cell adhesion molecule-1 (VCAM-1) expression and VCAM-1 mediated adhesion of human monocytes (THP-1) to HUVECs, which could be neutralized by VCAM-1 antibody. Both toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) were involved in the signalling pathway, because blockade of either TLR4 or NF-κB inhibited the cTnI's effect on VCAM-1 expression and adhesion of monocytes to endothelial cells. Moreover, TLR4 inhibition reduced cTnI-augmented cardiac injury in rats with I/R injury. We conclude that cTnI exacerbates myocardial I/R injury by inducing the adhesion of monocytes to vascular endothelial cells via activation of the TLR4/NF-κB pathway. Inhibition of TLR4 may be an alternative strategy to reduce cTnI-induced myocardial I/R injury.

A specific anti-citrullinated protein antibody profile identifies a group of rheumatoid arthritis patients with a toll-like receptor 4-mediated disease.

BackgroundIncreased expression of toll-like receptor 4 (TLR4) and its endogenous ligands, is characteristic of rheumatoid arthritis (RA) synovitis. In this study, we evaluated how these TLR4 ligands may drive pathogenic processes and whether the fine profiling of anti-citrullinated protein antibodies (ACPA) based on their target specificity might provide a simple means to predict therapeutic benefit when neutralizing TLR4 in this disease.MethodsThe capacity of RA synovial fluids (RASF) to stimulate cytokine production in monocytes from patients with RA was analyzed by ELISA. The presence of TLR4 activators in RASF was determined by measuring the levels of ACPA, ACPA subtypes with reactivity to specific citrullinated peptides and other TLR4 ligands. Neutralization of TLR4 signaling was investigated using NI-0101, a therapeutic antibody that targets TLR4.ResultsRASF exhibited a heterogeneous capacity to induce production of proinflammatory cytokines by monocytes isolated from patients with RA. Such cytokine responses were significantly modified by TLR4 blockade achieved using NI-0101. The analysis of the content of RASF and matched sera demonstrated that ACPA fine specificities in patient samples predict cellular response to anti-TLR4 exposure in vitro.ConclusionTLR4 represents a possible therapeutic target in RA. Our study demonstrates that TLR4 inhibition in an ex vivo model of RA pathogenesis can significantly modulate cytokine release and does so in specific subgroups of RA patient-derived samples. It also suggests that ACPA fine profiling has the potential to identify RA patients with a predominantly TLR4-driven pathotype that could be used to predict preferential response to TLR4 antagonism.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1128-5) contains supplementary material, which is available to authorized users.

The Vascular Endothelial Growth Factor Inhibitors Ranibizumab and Aflibercept Markedly Increase Expression of Atherosclerosis-Associated Inflammatory Mediators on Vascular Endothelial Cells.

IntroductionRecent studies have suggested that the VEGF inhibitors, Ranibizumab and Aflibercept may be associated with an excess of cardiovascular events, potentially driven by increasing atheroma instability, leading to plaque rupture and clinical events. Inflammation plays a key role in the progression of atherosclerotic plaque and particularly conversion to an unstable phenotype. Here, we sought to assess the in vitro effects of these drugs on the expression of key inflammatory mediators on endothelial cells.MethodsHuman coronary artery endothelial cells were co-incubated for 16h with Ranibizumab (0.11nM) or Aflibercept (0.45nM), as determined by each drug’s peak serum concentration (Cmax). Expression at protein (ELISA) and gene (RT-PCR) level of inflammatory chemokines CCL2, CCL5 and CXC3L1 as well as gene expression for the cell adhesion molecules VCAM-1, ICAM-1 and the key NF-κb protein p65 was assessed. VEGF-A protein levels were also determined.ResultsBoth drugs significantly increased chemokine, cell adhesion molecule (CAM) and p65 expression, while decreasing VEGF-A protein secretion. At equivalent Cmax concentrations, Aflibercept was significantly more pro-inflammatory than Ranibizumab. Reduction of secreted VEGF-A levels significantly attenuated inflammatory effects of both drugs, whereas blockade of the VEGF-A receptor or silencing of VEGF-A gene synthesis alone had no effect, suggesting that binding of drug to secreted VEGF-A is crucial in promoting inflammation. Finally, blockade of Toll-like receptor 4 significantly reduced inflammatory effects of both drugs.ConclusionWe demonstrated here, for the first time, that both drugs have potent pro-inflammatory effects, mediated via activation of Toll-like receptor 4 on the endothelial cell surface by drug bound to VEGF-A. Further studies are required to investigate whether these effects are also seen in vivo.

Targeting the Toll of Drug Abuse: The Translational Potential of Toll-Like Receptor 4.

There is growing recognition that glial proinflammatory activation importantly contributes to the rewarding and reinforcing effects of a variety of drugs of abuse, including cocaine, methamphetamine, opioids, and alcohol. It has recently been proposed that glia are recognizing, and becoming activated by, such drugs as a CNS immunological response to these agents being xenobiotics; that is, substances foreign to the brain. Activation of glia, primarily microglia, by various drugs of abuse occurs via toll like receptor 4 (TLR4). The detection of such xenobiotics by TLR4 results in the release of glial neuroexcitatory and neurotoxic substances. These glial products of TLR4 activation enhance neuronal excitability within brain reward circuitry, thereby enhancing their rewarding and reinforcing effects. Indeed, selective pharmacological blockade of TLR4 activation, such as with the non-opioid TLR4 antagonist (+)-naltrexone, suppresses a number of indices of drug reward/reinforcement. These include: conditioned place preference, self-administration, drugprimed reinstatement, incubation of craving, and elevations of nucleus accumbens shell dopamine. Notably, TLR4 blockade fails to alter self-administration of food, indicative of a selective effect on drugs of abuse. Genetic disruption of TLR4 signaling recapitulates the effects of pharmacological TLR4 blockade, providing converging lines of evidence of a central importance of TLR4. Taken together, multiple lines of evidence converge to raise TLR4 as a promising therapeutic target for drug abuse.

(+)-Naltrexone is neuroprotective and promotes alternative activation in the mouse hippocampus after cardiac arrest/cardiopulmonary resuscitation.

Despite dramatic improvement in cardiopulmonary resuscitation (CPR) and other techniques for cardiac arrest (CA), the majority of survivors continue to show signs of decreased memory or executive cognitive function. Such memory impairment may be due to hippocampal CA1 neuronal death, which is delayed by several days after CA/CPR. Classical microgliosis in the CA1 region may contribute to neuronal death, yet the role of a key activation receptor Toll Like Receptor 4 (TLR4) has not been previously investigated for such neuronal death after CA/CPR. We show that (+)-naltrexone was neuroprotective after CA/CPR. TLR4 blockade was associated with decreased expression of markers for microglial/macrophage activation and T cell and B cell infiltration, as well as decreased pro-inflammatory cytokine levels. Notably, IL-10 expression was elevated in response to CA/CPR, but was not attenuated by (+)-naltrexone, suggesting that the local monocyte/microglial phenotype had shifted towards alternative activation. This was confirmed by elevated expression of Arginase-1, and decreased expression of NFκB p65 subunit. Thus, (+)-naltrexone and other TLR4 antagonists may represent a novel therapeutic strategy to alleviate the substantial burden of memory or executive cognitive function impairment after CA/CPR.

Isobavachalcone attenuates lipopolysaccharide-induced ICAM-1 expression in brain endothelial cells through blockade of toll-like receptor 4 signaling pathways

Inflammation has been implicated in the pathogenesis of various cerebral diseases. Thus, control of brain inflammation is regarded as one of the important therapeutic strategies for the treatment of neurodegenerative diseases such as Alzheimer׳s disease and stroke. Isobavachalcone, a flavonoid from Psoralea corylifolia, is known to possess a wide spectrum of biological activities and is expected to be useful in preventing or treating neurodegenerative diseases. However, very little is known regarding its effects on cerebral inflammation. In this study, we examined the effect of isobavachalcone on leukocyte adhesion and intercellular adhesion molecule-1 (ICAM-1) expression in brain endothelial cells activated with lipopolysaccharide (LPS) and explored the possible mechanisms involved. Isobavachalcone significantly down-regulated LPS-induced ICAM-1 expression and leukocyte–endothelial cell adhesion and suppressed NF-κB activity which is implicated in the expression of ICAM-1. It attenuated ICAM-1 expression as well as NF-κB transcriptional activity induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or polyriboinosinic polyribocytidylic acid (poly[I:C]). Isobavachalcone also down-regulated LPS or poly[I:C]-induced expression of IFN-β, which can indirectly activate NF-κB. These data imply that isobavachalcone can modulate both MyD88-dependent and TRIF-dependent signaling of toll-like receptor 4 (TLR4). Taken together, our data suggest that isobavachalcone inhibits LPS-induced ICAM-1 expression and leukocyte adhesion to brain endothelial cell by blocking TLR4 signaling and thus, has the potential to ameliorate neuronal injury in brain diseases associated with inflammation.

Enhancement of islet engraftment and achievement of long-term islet allograft survival by Toll-like receptor 4 blockade.

Toll-like receptors are key players in sterile inflammation phenomena and can link the innate and adaptive immune systems by enhancing graft immunogenicity. They are also considered mediators of types 1 and 2 diabetes development. The aim of the present study was to assess the role of Toll-like receptor-4 (TLR4) in mediating the inflammatory and immune responses to pancreatic islets, thereby promoting inflammatory destruction and immune rejection of islet grafts. Experiments were conducted in murine and human in vitro systems and in vivo murine islet transplant models, using species-specific anti-TLR4 monoclonal antibodies. In vitro, mixed lymphocyte-islet reaction experiments were performed to assess T-cell activation and proliferation. In vivo, both a syngeneic (B6-to-B6) marginal mass islet transplant model to assess the impact of TLR4 blockade on islet engraftment and an allogeneic (DBA1-to-B6) model were used. In vitro TLR4 blockade decreased lipopolysaccharide-mediated β-cell apoptosis and T-cell activation and proliferation against allogeneic islets. In vivo, TLR4 blockade resulted in significantly better syngeneic marginal mass islet engraftment and in indefinite allogeneic islet graft survival. Tolerance was not observed because donor-specific skin graft rechallenge in nonrejecting animals resulted in rejection of both skin and islets, but without accelerated rejection as compared to naive animals. Taken together, our data indicate that TLR4 blockade leads to a significant improvement of syngeneic islet engraftment and of allogeneic islet graft survival. A mechanism of graft accommodation with concurrent inhibition of donor-specific immune memory is likely to be involved.

Toll-like receptor 4 inhibition reduces vascular inflammation in spontaneously hypertensive rats

AimsHypertension is associated with increased levels of circulating cytokines and recent studies have shown that innate immunity contributes to hypertension. The mechanisms which hypertension stimulates immune response remain unclear, but may involve formation of neo-antigens that activate the immune system. Toll like receptor 4 (TLR4) is an innate immune receptor that binds a wide spectrum of exogenous (lipopolysaccharide) and endogenous ligands. TLR4 signaling leads to activation of nuclear factor kappa B (NFκB) and transcription of genes involved in inflammatory response. We previously demonstrated that TLR4 blockade reduces blood pressure and the augmented vascular contractility in spontaneously hypertensive rats (SHR). Here we hypothesized that inhibition of TLR4 ameliorates the vascular inflammatory process by a NFκB signaling pathway. Main methodsSHR and Wistar rats were treated with anti-TLR4 antibody (1μg/day) or unspecific IgG for 15days (i.p.). Key findingsAnti-TLR4 treatment decreased production of reactive oxygen species and expression of IL-6 cytokine in mesenteric resistance arteries from SHR, when compared with IgG-treated SHR. Anti-TLR4 treatment also abolished the increased vascular reactivity to noradrenaline observed in IgG-treated SHR, as described before, and inhibition of NFκB decreased noradrenaline responses only in IgG-treated SHR. Mesenteric arteries from SHR treated with anti-TLR4 displayed decreased expression of MyD88, but not TRIF, key molecules in TLR4 signaling. Phosphorylation of p38 and NF-κB p65 were decreased in arteries from anti-TLR4-treated SHR versus IgG-treated SHR. SignificanceTogether, these results suggest that TLR4 is a key player in hypertension and vascular inflammatory process by a NFκB signaling pathway.

Involvement of de novo ceramide synthesis in pro-inflammatory adipokine secretion and adipocyte–macrophage interaction

Interaction between adipocytes and macrophages has been suggested to play a central role in the pathogenesis of obesity. Ceramide, a sphingolipid de novo synthesized from palmitate, is known to stimulate pro-inflammatory cytokine secretion from multiple types of cells. To clarify whether de novo synthesized ceramide contributes to cytokine dysregulation in adipocytes and macrophages, we observed cytokine secretion in mature 3T3-L1 adipocytes (L1) and RAW264.7 macrophages (RAW) cultured alone or co-cultured under the suppression of de novo ceramide synthesis.Palmitate enhanced ceramide accumulation and stimulated the expression and secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in L1. The suppression of serine-palmitoyl transferase, a rate-limiting enzyme of de novo ceramide synthesis, by myriocin or siRNA attenuated those palmitate-induced alterations, and a ceramide synthase inhibitor fumonisin B1 showed similar results. In contrast, the inhibitor of sphingosine kinase or a membrane-permeable ceramide analogue augmented the cytokine secretion. Myriocin effects on the palmitate-induced changes were not abrogated by toll-like receptor-4 blockade. Although palmitate stimulated RAW to secrete tumor necrosis factor-α (TNF-α), it did not significantly increase ceramide content, and neither myriocin nor fumonisin B1 attenuated the TNF-α hypersecretion. The co-culture of L1 with RAW markedly augmented IL-6 and MCP-1 levels in media. Myriocin or fumonisin B1 significantly lowered these cytokine levels and suppressed the gene expression of TNF-α and MCP-1 in RAW and of IL-6 and MCP-1 in L1.In conclusion, de novo synthesized ceramide partially mediates the palmitate effects on pro-inflammatory adipokines and is possibly involved in the interaction with macrophages.

Role of TREM-1 in response to Aspergillus fumigatus infection in corneal epithelial cells.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a cell surface receptor that is highly expressed in inflammatory lesions caused by infectious agents such as bacteria and fungi and amplifies immune responses. The aim of the current study was to investigate TREM-1 expression in corneal epithelial cells infected by Aspergillus fumigatus (A. fumigatus) and evaluate its role. In this study, infection with A. fumigatus upregulated TREM-1 expression in corneal epithelial cells both in vitro and in vivo. Furthermore, treatment with the antagonistic peptide of TREM-1 decreased the levels of inflammatory cytokines that were enhanced by the fungal infection. We speculated that cross-talk occurs between TREM-1 and Toll-like receptor-4 (TLR-4) in cornea fungal infection. Inhibitors of TLR-4 and myeloid differentiation factor 88 (MyD88) could partially inhibit the upregulation of TREM-1 induced by A. fumigatus respectively. In addition, TLR-4 blockade enhanced the inhibitory effect of the antagonistic peptide of TREM-1 on A. fumigatus-induced inflammation. These findings suggest that TREM-1 plays critical roles in fungal infection, and targeting it may represent a novel therapeutic strategy for patients with fungal keratitis.

Oral microbiome link to neurodegeneration in glaucoma.

BackgroundGlaucoma is a progressive optic nerve degenerative disease that often leads to blindness. Local inflammatory responses are implicated in the pathology of glaucoma. Although inflammatory episodes outside the CNS, such as those due to acute systemic infections, have been linked to central neurodegeneration, they do not appear to be relevant to glaucoma. Based on clinical observations, we hypothesized that chronic subclinical peripheral inflammation contributes to neurodegeneration in glaucoma.MethodsMouthwash specimens from patients with glaucoma and control subjects were analyzed for the amount of bacteria. To determine a possible pathogenic mechanism, low-dose subcutaneous lipopolysaccharide (LPS) was administered in two separate animal models of glaucoma. Glaucomatous neurodegeneration was assessed in the retina and optic nerve two months later. Changes in gene expression of toll-like receptor 4 (TLR4) signaling pathway and complement as well as changes in microglial numbers and morphology were analyzed in the retina and optic nerve. The effect of pharmacologic blockade of TLR4 with naloxone was determined.FindingsPatients with glaucoma had higher bacterial oral counts compared to control subjects (p<0.017). Low-dose LPS administration in glaucoma animal models resulted in enhancement of axonal degeneration and neuronal loss. Microglial activation in the optic nerve and retina as well as upregulation of TLR4 signaling and complement system were observed. Pharmacologic blockade of TLR4 partially ameliorated the enhanced damage.ConclusionsThe above findings suggest that the oral microbiome contributes to glaucoma pathophysiology. A plausible mechanism by which increased bacterial loads can lead to neurodegeneration is provided by experiments in animal models of the disease and involves activation of microglia in the retina and optic nerve, mediated through TLR4 signaling and complement upregulation. The finding that commensal bacteria may play a role in the development and/or progression of glaucomatous pathology may also be relevant to other chronic neurodegenerative disorders.

Abstract 299: Blockade of Toll-like Receptor 4 During Acute Lipopolysaccharide Treatment Preserves Afferent Arteriolar Autoregulatory Behavior.

Lipopolysaccharide (LPS), a component of the bacterial wall of gram-negative bacteria is a ligand for toll-like receptor 4 (TLR4). Immune system activation and generation of reactive oxygen species (ROS) is linked to blunted renal autoregulatory behavior. Accordingly, we hypothesize that blockade of TLR4 during acute LPS treatment preserves afferent arteriolar autoregulatory behavior. Rats received a bolus injection of LPS (1 mg/kg i.p.) and were treated with either anti-TLR4 antibody (1μg; i.p.) or TLR4 receptor antagonist (LPS-RS; 5mg; i.p.). Kidneys were harvested 4 hours later for juxtamedullary nephron studies. Autoregulatory behavior was assessed in these groups by increasing perfusion pressure in 15 mmHg increments from 65 to 170 mmHg. In control kidneys, afferent arteriolar diameter decreased by 29 ± 5% when perfusion pressure was increased from 65 to 170 mmHg. LPS alone (4 hrs.) significantly blunted the autoregulatory response such that afferent arteriolar diameter remains essentially unchanged between 65 (12.4 ± 0.9 μm) and 170 mmHg (12.7 ± 1.2 μm). In kidneys co-treated with LPS and anti-TLR4 antibody, afferent arteriolar diameter decreased by 26 ± 6% (P &lt; 0.05; n=6) during increments in perfusion pressure from 65 to 170mmHg. In kidneys co-treated with LPS and the TLR4 receptor antagonist, LPS-RS, afferent arteriolar diameter decreased by 24 ± 5% (P &lt; 0.05; n=4) over the same pressure range, indicating preservation of pressure-induced vasoconstriction. Reactive oxygen species play a role in blunting rat afferent arteriolar autoregulatory resistance adjustments. Therefore, we assessed autoregulatory behavior in LPS treated kidneys during acute exposure to the superoxide dismutase mimetic, Tempol (10-6 mol/L). In these kidneys, afferent arteriolar diameter decreased by 28 ± 3% (P &lt; 0.05; n=6) indicating restoration of autoregulatory capability. These data indicate that LPS treatment induces TLR4 activation, and stimulates ROS generation. Blocking TLR4 with anti-TLR4 antibody or LPS-RS or scavenging ROS with Tempol preserves and restores afferent arteriolar autoregulatory behavior suggesting that blockade of TLR4 may open novel therapeutic targets for inflammatory kidney disease.

FibronectinEDA promotes chronic cutaneous fibrosis through Toll-like receptor signaling.

Scleroderma is a progressive autoimmune disease affecting multiple organs. Fibrosis, the hallmark of scleroderma, represents transformation of self-limited wound healing into a deregulated self-sustaining process. The factors responsible for maintaining persistent fibroblast activation in scleroderma and other conditions with chronic fibrosis are not well understood. Toll-like receptor 4 (TLR4) and its damage-associated endogenous ligands are implicated in immune and fibrotic responses. We now show that fibronectin extra domain A (Fn(EDA)) is an endogenous TLR4 ligand markedly elevated in the circulation and lesional skin biopsies from patients with scleroderma, as well as in mice with experimentally induced cutaneous fibrosis. Synthesis of Fn(EDA) was preferentially stimulated by transforming growth factor-β in normal fibroblasts and was constitutively up-regulated in scleroderma fibroblasts. Exogenous Fn(EDA) was a potent stimulus for collagen production, myofibroblast differentiation, and wound healing in vitro and increased the mechanical stiffness of human organotypic skin equivalents. Each of these profibrotic Fn(EDA) responses was abrogated by genetic, RNA interference, or pharmacological disruption of TLR4 signaling. Moreover, either genetic loss of Fn(EDA) or TLR4 blockade using a small molecule mitigated experimentally induced cutaneous fibrosis in mice. These observations implicate the Fn(EDA)-TLR4 axis in cutaneous fibrosis and suggest a paradigm in which aberrant Fn(EDA) accumulation in the fibrotic milieu drives sustained fibroblast activation via TLR4. This model explains how a damage-associated endogenous TLR4 ligand might contribute to converting self-limited tissue repair responses into intractable fibrogenesis in chronic conditions such as scleroderma. Disrupting sustained TLR4 signaling therefore represents a potential strategy for the treatment of fibrosis in scleroderma.

Effects of Central Injection of Anti-LPS Antibody and Blockade of TLR4 on GnRH/LH Secretion during Immunological Stress in Anestrous Ewes

The present study was designed to examine the effect of intracerebroventricular (icv) administration of antilipopolysaccharide (LPS) antibody and blockade of Toll-like receptor 4 (TLR4) during immune stress induced by intravenous (iv) LPS injection on the gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) secretion in anestrous ewes. Injection of anti-LPS antibody and TLR4 blockade significantly (P < 0.01) reduced the LPS dependent lowering amount of GnRH mRNA in the median eminence (ME). Moreover, blockade of TLR4 caused restoration of LH-β transcription in the anterior pituitary decreased by the immune stress. However, there was no effect of this treatment on reduced LH release. The results of our study showed that the blockade of TLR4 receptor in the hypothalamus is not sufficient to unblock the release of LH suppressed by the immune/inflammatory challenges. This suggests that during inflammation the LH secretion could be inhibited directly at the pituitary level by peripheral factors such as proinflammatory cytokines and circulating endotoxin as well.

Blockade of Toll-like receptor 4 attenuates morphine tolerance and facilitates the pain relieving properties of morphine.

The ventrolateral periaqueductal gray (vlPAG) is an integral locus for morphine action. Although it is clear that glia contribute to the development of morphine tolerance, to date, the investigation of their role has been limited to spinal and medullary loci. Opioids induce a neuroinflammatory response that opposes acute and long-term analgesia, thereby limiting their efficacy as therapeutic agents. Recent data suggest that the innate immune receptor Toll-like receptor 4 (TLR4), along with its coreceptor myeloid differentiation factor-2 (MD-2), mediates these effects. To date, the brain loci through which TLR4 modulates morphine tolerance have not been identified. We have previously demonstrated that chronic subcutaneous morphine results in tolerance that is accompanied by increases in vlPAG glial cell activity. Using in vivo pharmacological manipulations of vlPAG glia and TLR4 in the adult male rat, we show that intra-vlPAG administration of the general glial cell metabolic inhibitor propentofylline or the astrocyte activity inhibitor fluorocitrate attenuate tolerance to morphine. Characterization of MD-2 expression within the PAG revealed dense MD-2 expression throughout the vlPAG. Further, antagonizing vlPAG TLR4 dose dependently prevented the development of morphine tolerance, and vlPAG microinjections of TLR4 agonists dose dependently produced a "naive" tolerance to subsequent challenge doses of morphine. Finally, using a model of persistent inflammatory pain and pharmacological manipulation of TLR4 we demonstrate that systemic antagonism of TLR4 potentiated acute morphine antihyperalgesia. These results, together, indicate that vlPAG glia regulate morphine tolerance development via TLR4 signaling, and implicate TLR4 as a potential therapeutic target for the treatment of pain.

Lipopolysaccharide-induced toll-like receptor 4 signaling enhances the migratory ability of human esophageal cancer cells in a selectin-dependent manner

Esophageal cancer is an aggressive malignancy, and emerging data suggest that postoperative infections may promote cancer progression. Systemic exposure to lipopolysaccharide (LPS), a Gram-negative bacterial antigen involved in such infections, has been shown to increase cancer cell adhesion to the hepatic sinusoids invivo. We investigated the direct impact of LPS on the migratory ability of esophageal cancer cells via the LPS receptor toll-like receptor 4 (TLR4). Human esophageal squamous carcinoma cell lines and immortalized normal esophageal mucosa cells were tested for TLR4 surface expression by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. TLR4 signaling in response to LPS stimulation was tested in these cells by measuring p38 MAP kinase phosphorylation on Western blot. The impact of TLR4 signaling was measured by static adhesion assays invitro and on early invivo migration by intravital microscopy of the liver. Upon LPS stimulation, phosphorylation of p38 was detected in the human esophageal cancer cells HKESC-2. Also, LPS-stimulated HKESC-2 cells showed a twofold increased adhesion to fibronectin and to hepatic sinusoidal endothelium. These effects were abolished by TLR4 inhibition using the small-molecule inhibitor eritoran. Adhesion to fibronectin and hepatic sinusoidal endothelium was also diminished by blockade of p38 phosphorylation and inhibitors of selectin-selectin ligand binding. LPS can increase the migratory ability of human esophageal cancer cells by increasing their adhesive properties through TLR4 signaling and selectin ligands. TLR4, p38, and selectin blockade may therefore prove to be a new therapeutic strategy for this aggressive malignancy.

PORF5 plasmid protein of Chlamydia trachomatis induces MAPK-mediated pro-inflammatory cytokines via TLR2 activation in THP-1 cells

Infection with Chlamydia trachomatis induces inflammatory pathologies in the urogenital tract that can lead to infertility and ectopic pregnancy. Pathogenesis of infection has been mostly attributed to excessive cytokine production. However, precise mechanisms on how C. trachomatis triggers this production, and which protein(s) stimulate inflammatory cytokines remains unknown. In the present study, the C. trachomatis pORF5 protein induced tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-8 (IL-8) in dose- and time-dependent manners in the THP-1 human monocyte cell line. We found that intracellular p38/mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)/MAPK signaling pathways were required for the induction of TNF-α, IL-1β and IL-8. Blockade of toll-like receptor 2 (TLR2) signaling reduced induction levels of TNF-α, IL-8 and IL-1β. We concluded that the C. trachomatis pORF5 protein might contribute to the inflammatory processes associated with chlamydial infections.