TLR4 Antagonist Research Articles

The Role of Aeromonas-Goblet Cell Interactions in Melatonin-Mediated Improvements in Sleep Deprivation-Induced Colitis.

Background Our previous studies demonstrated that melatonin could effectively ameliorate sleep deprivation- (SD-) caused oxidative stress-mediated gut microbiota disorder and colitis. The research further clarified the mechanism of melatonin in improving colitis from the perspective of the interaction between Aeromonas and goblet cells. Methods A seventy-two hours SD mouse model with or without melatonin intervention and fecal microbiota transplantation (FMT) to explore the vital position of Aeromonas-goblet cell interactions in melatonin improving SD-induced colitis. Moreover, Aeromonas or LPS-supplied mice were assessed, and the influence of melatonin on Aeromonas-goblet cell interactions-mediated oxidative stress caused colitis. Furthermore, in vitro experiment investigated the regulation mechanism of melatonin.Results Our study showed that SD induced colitis, with upregulation of Aeromonas and LPS levels and reductions in goblet cells number and MUC2 protein. Similarly, FMT from SD mice, Aeromonas veronii colonization, and LPS treatment restored the SD-like goblet cells number and MUC2 protein decrease and colitis. Moreover, LPS treatment downregulated the colonic antioxidant capacity. Yet, melatonin intervention reversed all consequence in SD, A.veronii colonization, and LPS-treated mice. In vitro, melatonin reversed A. veronii- or LPS-induced MUC2 depletion in mucus-secreting human HT-29 cells via increasing the expression level of Villin, Tff3, p-GSK-3β, β-catenin, and melatonin receptor 2 (MT2) and decreasing the level of p-IκB, p-P65, ROS, TLR4, and MyD88 proteins, while the improvement effect was blocked with pretreatment with a MT2 antagonist but were mimicked by TLR4 and GSK-3β antagonists and ROS scavengers. Conclusions Our results demonstrated that melatonin-mediated MT2 inhibits Aeromonas-goblet cell interactions to restore the level of MUC2 production via LPS/TLR4/MyD88/GSK-3β/ROS/NF-κB loop, further improving colitis in SD mice.

Cimifugin Ameliorates Lipotoxicity-Induced Hepatocyte Damage and Steatosis through TLR4/p38 MAPK- and SIRT1-Involved Pathways.

Objective Hepatic metabolic disorder induced by lipotoxicity plays a detrimental role in metabolic fatty liver disease pathogenesis. Cimifugin (Cim), a coumarin derivative extracted from the root of Saposhnikovia divaricata, possesses multiple biological properties against inflammation, allergy, and oxidative stress. However, limited study has addressed the hepatoprotective role of Cim. Here, we investigate the protective effect of Cim against lipotoxicity-induced cytotoxicity and steatosis in hepatocytes and clarify its potential mechanisms. Methods AML-12, a nontransformed mouse hepatocyte cell line, was employed in this study. The cells were incubated with palmitate or oleate to imitate hepatotoxicity or steatosis model, respectively. Results Cim significantly reversed palmitate-induced hepatocellular injury in a dose-dependent manner, accompanied by improvements in oxidative stress and mitochondrial damage. Cim pretreatment reversed palmitate-stimulated TLR4/p38 MAPK activation and SIRT1 reduction without affecting JNK, ERK1/2, and AMPK pathways. The hepatoprotective effects of Cim were abolished either through activating TLR4/p38 by their pharmacological agonists or genetical silencing SIRT1 via special siRNA, indicating a mechanistic involvement. Moreover, Cim treatment improved oleate-induced hepatocellular lipid accumulation, which could be blocked by either TLR4 stimulation or SIRT1 knockdown. We observed that SIRT1 was a potential target of TLR4 in palmitate-treated hepatocytes, since TLR4 agonist LPS aggravated, whereas TLR4 antagonist CLI-095 alleviated palmitate-decreased SIRT1 expression. SIRT1 knockdown did not affect palmitate-induced TLR4. In addition, TLR4 activation by LPS significantly abolished Cim-protected SIRT1 reduction induced by palmitate. These results collaboratively indicated that TLR4-regulated SIRT1 pathways was mechanistically involved in the protective effects of Cim against lipotoxicity. Conclusion In brief, we demonstrate the protective effects of Cim against lipotoxicity-induced cell death and steatosis in hepatocytes. TLR4-regulated p38 MAPK and SIRT1 pathways are involved in Cim-protected hepatic lipotoxicity. Cim is a potential candidate for improving hepatic metabolic disorders mediated by lipotoxicity.

Low-dose naltrexone, an opioid-receptor antagonist, is a broad-spectrum analgesic: a retrospective cohort study.

Aim: To evaluate the use of low-dose naltrexone (LDN) as a broad-spectrum analgesic. Methods: Retrospective cohort study from a single pain management practice using data from 2014 to2020. Thirty-six patients using LDN for ≥2months were matched to 42 controls. Pain scores were assessed at initial visit and at most recent/final documented visit using a 10-point scale. Results: Cases reported significantly greater pain reduction (-37.8%) than controls (-4.3%; p<0.001). Whole sample multivariate modeling predicts 33% pain reduction with LDN, with number needed to treat (for 50% pain reduction) of 3.2. Patients with neuropathic pain appeared to benefit even more than those with 'nociceptive'/inflammatory pain. Conclusion: LDN is effective in a variety of chronic pain states, likely mediated by TLR-4 antagonism.

Polysaccharides From the Aerial Parts of Tetrastigma Hemsleyanum Diels et Gilg Induce Bidirectional Immunity and Ameliorate LPS-Induced Acute Respiratory Distress Syndrome in Mice.

Tetrastigma hemsleyanum Diels et Gilg (Sanyeqing, SYQ) has traditionally been used to treat inflammation, high fever and improve immune function of patients. Polysaccharides have been proved to be one of the important components of SYQ. Previous studies have confirmed the antipyretic and antitumor effects of polysaccharides from SYQ (SYQP), and clarified that SYQP could enhance immunity through TLR4 signalling pathway. However, there were more possibilities for the mechanism by which SYQP exerted immunomodulatory effects and the role of SYQP in acute respiratory distress syndrome (ARDS) is elusive. The purpose of this study was further to explain the bidirectional modulation of immunity mechanism of SYQP in vitro and its effect in LPS-induced ARDS in vivo. Experimental results showed that SYQP significantly stimulated gene expressions of TLR1, TLR2 and TLR6 and secretion of cytokines in RAW264.7 cells. Individual or combined application of TLR2 antagonist C29 and TLR4 antagonist TAK-242 could reduce SYQP-mediated stimulation of cytokine secretion in RAW264.7 cells and mouse peritoneal macrophages (MPMs) to varying degrees. On the other hand, SYQP markedly inhibited the expression levels of inflammatory cytokines, NO, iNOS and COX-2 in LPS-treatment RAW264.7 cells. Moreover, in vivo results indicated that SYQP significantly reduced LPS-induced damage in ARDS mice through alleviating LPS-induced pulmonary morphological damage, inhibiting myeloperoxidase (MPO) expression levels, ameliorating the inflammatory cells in bronchoalveolar lavage fluid (BALF) and improving hematological status. Meanwhile, SYQP evidently reduced IL-6, TNF-α and IFN-γ secretion, the overexpression levels of TLR2 and TLR4, as well as the phosphorylation of NF-κB p65. In addition, SYQP reduced the phosphorylation of JAK2 and STAT1 and the overexpression of NLRP3, caspase-1, caspase-3 and caspase-8 in lung tissues of ARDS mice. In summary, our study confirmed that SYQP induced bidirectional immunity and ameliorated LPS-induced acute respiratory distress syndrome in mice through TLR2/TLR4-NF-κB, NLRP3/caspase and JAK/STAT signaling pathways, which provided a theoretical basis for further use of SYQP.

Molecular mechanism underlying the TLR4 antagonistic and antiseptic activities of papiliocin, an insect innate immune response molecule

SignificanceSimilar to mammalian TLR4/MD-2, the Toll9/MD-2-like protein complex in the silkworm, Bombyx mori, acts as an innate pattern-recognition receptor that recognizes lipopolysaccharide (LPS) and induces LPS-stimulated expression of antimicrobial peptides such as cecropins. Here, we report that papiliocin, a cecropin-like insect antimicrobial peptide from the swallowtail butterfly, competitively inhibits the LPS-TLR4/MD-2 interaction by directly binding to human TLR4/MD-2. Structural elements in papiliocin, which are important in inhibiting TLR4 signaling via direct binding, are highly conserved among insect cecropins, indicating that its TLR4-antagonistic activity may be related to insect Toll9-mediated immune response against microbial infection. This study highlights the potential of papiliocin as a potent TLR4 antagonist and safe peptide antibiotic for treating gram-negative sepsis.

Proteinase-activated Receptor-1 Antagonist Attenuates Brain Injury via Regulation of FGL2 and TLR4 after Intracerebral Hemorrhage in Mice

Proteinase-activated receptor-1 (PAR1) antagonist plays a protective effect in brain injury. We investigated the potential function and mechanisms of PAR1 antagonist in ICH-induced brain injury. Results showed that PAR1 antagonist protected against neurobehavior deficits, brain edema and blood–brain barrier integrity in ICH mice via the JNK/ERK/p38 MAPK signaling pathway at 24 h after ICH. In addition, ICH resulted in the increase of FGL2 and TLR4 expression over time, and phosphorylated (p-) JNK, ERK, p38 MAPK and IKKα expression. Suppression of FGL2 or TLR4 alleviated brain injury and decreased the expression of p-JNK, p-ERK, p-p38 MAPK and p-IKKα at 24 h after ICH, while overexpression of them showed the opposite result. Moreover, the protective effect of PAR1 antagonist on ICH-induced brain injury was blocked by FGL2 or TLR4 overexpression, and the levels of p-JNK, p-ERK and p-p38 MAPK were increased. Furthermore, PAR1 antagonist combined with TLR4 antagonist markedly alleviated brain injury after ICH at 72 h. Overall, PAR1 antagonist protected against short-term brain injury, and the effect of PAR1 antagonist on ICH-induced brain injury was mediated by FGL2 or TLR4.

Toll-like receptor 2 and 6 agonist fibroblast-stimulating lipopeptide increases expression and secretion of CXCL1 and CXCL2 by uveal melanocytes

Fibroblast-stimulating lipopeptide (FSL-1) can activate Toll-like receptor 2 and 6 (TLR2/6), which recognize relevant molecules from gram-positive pathogens, fungus, and mycoplasma, and elevates the expression of CXCL1 and CXCL2, neutrophil chemoattractants, in certain types of cells. This effect has not previously been reported in the uveal melanocytes (UM). This study was designed to test the hypothesis that FSL-1 can induce the expression and secretion of CXCL1 and CXCL2 via activation of TLR2/6 in cultured human UM and producing an acute non-infectious uveitis reaction in the mouse. Flow cytometry and fluorescent immunostaining were used to measure the effect of FSL-1 on the expression of TLR2/6 in UM. Real time PCR and ELISA analysis were used to assess the ability of FSL-1 to elevate CXCL1/CXCL2 levels in cell lysates and conditioned media of UM, respectively. Flow cytometry measured phosphorylated MAPK and activated NF-κB signals in UM, with and without FSL-1 treatment. ELISA analysis tested the impact of various signal inhibitors (NF-κB, p38 MAPK, JNK1/2 and ERK1/2) and TLR2/6 antagonists on FSL-1-induced CXCL1/CXCL2 levels in cultured UM. The effects of neutralizing antibodies to TLR2 on FSL-1-induced mouse uveitis were tested in an experimental animal model. FSL-1 induced the expression of TLR2/6 proteins in cultured UM. FSL-1 significantly elevated the CXCL1 and CXCL2 proteins and mRNA levels in cultured UM time- and dose-dependently. FSL-1 mainly activated NF-κB, JNK, and expression of TLR2. FSL-1-induced expression of CXCL1 and CXCL2 was blocked by NF-κB, JNK, ERK inhibitors and TLR2 antagonists. Intravitreal injection of FSL-1 induced acute non-infectious mouse uveitis, which was significantly reduced in severity by a TLR2 antagonist. These results suggest that UM may play a role in the immune reaction, which targets invading pathogens, especially gram-positive bacteria. On the other hand, an excessive reaction to molecules from gram-positive bacteria may promote an inflammatory state of non-infectious uveitis.

Human-Based Immune Responsive In Vitro Infection Models for Validation of Novel TLR4 Antagonists Identified by Computational Discovery.

Infectious diseases are still a major problem worldwide. This includes microbial infections, with a constant increase in resistance to the current anti-infectives employed. Toll-like receptors (TLRs) perform a fundamental role in pathogen recognition and activation of the innate immune response. Promising new approaches to combat infections and inflammatory diseases involve modulation of the host immune system via TLR4. TLR4 and its co-receptors MD2 and CD14 are required for immune response to fungal and bacterial infection by recognition of microbial cell wall components, making it a prime target for drug development. To evaluate the efficacy of anti-infective compounds early on, we have developed a series of human-based immune responsive infection models, including immune responsive 3D-skin infection models for modeling fungal infections. By using computational methods: pharmacophore modeling and molecular docking, we identified a set of 46 potential modulators of TLR4, which were screened in several tests systems of increasing complexity, including immune responsive 3D-skin infection models. We could show a strong suppression of cytokine and chemokine response induced by lipopolysacharide (LPS) and Candida albicans for individual compounds. The development of human-based immune responsive assays provides a more accurate and reliable basis for development of new anti-inflammatory or immune-modulating drugs.

Pathophysiological Role of TLR4 in Chronic Relapsing Itch Induced by Subcutaneous Capsaicin Injection in Neonatal Rats.

Despite the high prevalence of chronic dermatitis and the accompanied intractable itch, therapeutics that specifically target itching have low efficacy. Increasing evidence suggests that TLRs contribute to immune activation and neural sensitization; however, their roles in chronic itch remain elusive. Here, we show that the RBL-2H3 mast cell line expresses TLR4 and that treatment with a TLR4 antagonist opposes the LPS dependent increase in mRNA levels of Th2 and innate cytokines. The pathological role of TLR4 activation in itching was studied in neonate rats that developed chronic itch due to neuronal damage after receiving subcutaneous capsaicin injections. Treatment with a TLR4 antagonist protected these rats with chronic itch against scratching behavior and chronic dermatitis. TLR4 antagonist treatment also restored the density of cutaneous nerve fibers and inhibited the histopathological changes that are associated with mast cell activation after capsaicin injection. Additionally, the expression of IL-1β, IL-4, IL-5, IL-10, and IL-13 mRNA in the lesional skin decreased after TLR4 antagonist treatment. Based on these data, we propose that inhibiting TLR4 alleviated itch in a rat model of chronic relapsing itch, and the reduction in the itch was associated with TLR4 signaling in mast cells and nerve fibers.

697 Binding of gamma-glutamyl transferase to toll-like receptor-4 allows tissue factor activation in monocytes

Abstract Aims Gamma-glutamyl transferase (GGT) is well known to play a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of tissue factor (TF) gene expression, the principal initiator of the clotting cascade. GGT may potentially modulate TF expression, an assumption verified by previous studies carried out in human peripheral blood mononuclear cell (PBMC). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic properties since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation site. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. To assess whether GGT-induced TF stimulation is a consequence of binding to toll-like receptor (TLR)-4 and activation of NFkB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from an acute monocytic leukaemia patient, were incubated with hrGGT (0.5 ng/μL for PBMCs and 1 ng/µL for THP-1). LPS-Rs (0.5 ng/µL for PBMCs and 1 ng/µL for THP-1), CLI-095 (3 × 10−6M) and BAY-11-7082 (10−5 M) were used to block TLR-4 receptors, TLR4 signalling and NFkB respectively. TF protein concentration was determined by western blot analysis. TF pro-coagulant activity (PCA) was assessed through the use of Start Max coagulometer and results were expressed in pg/mL after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110 ± 70 to 510 ± 43, n = 7, P &amp;lt; 0.01) and THP-1 cells (PCA from 170 ± 64 to 460 ± 80, n = 15, P &amp;lt; 0.001), result confirmed by western blot. In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72 ± 17% n = 4, P &amp;lt; 0.01) a TLR-4 antagonist, CLI-095 (PCA: −74 ± 34%, n = 7, P &amp;lt; 0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71 ± 32%, n = 7, P &amp;lt; 0.001), a NF-kB inhibitor. Similar results were obtained in THP-1 cells (LPS-Rs: −76 ± 15%, n = 6, P &amp;lt; 0.01; CLI-095: −100 ± 6,6 %, n = 6, P &amp;lt; 0.01; BAY-11-7082: −100 ± 2,1%, n = 6, P &amp;lt; 0.01). hrGGT activates NFkB in hTLR4-positive HEK cell lines while does not induces effect in TLR4-negative HEK cell lines. Conclusions These data confirm the cytokine-like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells. Furthermore, it is highlighted for the first time the possible role of TLR-4 as the receptor of GGT and NFkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the consistent association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the silent atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque.

300 PCSK9 induces a prothrombotic response by activation of NFKB signalling pathway in mononuclear cells

Abstract Aims Strong evidence both experimental and clinical studies point out to an involvement of proprotein convertase subtilisin/kexin 9 (PCSK9), as an important player in hypercholesterolemia and atherosclerosis pathophysiology, identifying it, as a key molecule in the development of new cholesterol-lowering drugs and therapeutic target for atherosclerosis and related diseases. Emerging evidence shown that PCSK9 is straight implicated in inflammatory process where it may directly influence the activity of various cell types through NFkB activation, a key transcription factor involved in the induction of both pro-inflammatory cytokines and Tissue Factor (TF) expression, a major regulator of haemostasis and thrombosis in monocytes. Deductive reasoning makes therefore plausible to investigate a mechanistic link between circulation PCSK9 levels and TF expression, both in monocytes as well as on the surface of microparticles (MPs) generated from the same cells, with NFkB acting as the chain in the link. To investigate the involvement of NFkB signalling pathway in PCSK9-induced TF expression. Methods THP-1 cell line were stimulated with human (h) PCSK9 (5 μg/mL) or pre-incubated with BAY-117082 (BAY, 10−5M) an NFκB inhibitor, CLI-095 (3 × 10−6M), a highly TLR-4 signalling specific inhibitor and LPS-RS(5 μg/mL) a TLR-4 antagonist. TF procoagulant activity (PCA) was assessed by one-stage clotting assay using a STart Max semi-automated coagulation analyser. Concentration of TF-bearing MPs was determined using the Zymuphen MP-activity kit. Results hPCSK9 stimulated TF activity in THP-1 (PCA: from 50 ± 20 to 120 ± 20 ρg/mL, n = 10, P &amp;lt; 0.01). BAY, an NFkB inhibitor (PCA: −71 ± 23%, n = 5, P &amp;lt; 0.01) and CLI-095, a TLR-4 signalling inhibitor, (PCA: −86 ± 26%, n = 3, P &amp;lt; 0.05) and LPS-RS (PCA: −71 ± 23%, n = 5, P &amp;lt; 0.01) down-regulated PCSK9-induced TF activity completely. Furthermore THP-1stimulation with hPCSK9 causes the release of prothrombotic MPs-TF+ (MPs release from: 0.13 ± 0.07 to 0.42 ± 0.1 nM PS, n = 7, P &amp;lt; 0.05; PCA-MP+ from: 14 ± 3 to 44 ± 28 ρg/mL, n = 5, P &amp;lt; 0.05). Conclusions These data confirm the pivotal role of monocytes in the response PCSK9-induced TF-expression as well as the involvement of PCSK9 in inflammatory-thrombotic diseases through a direct stimulation of TF procoagulant activity and by the release of TF-bearing MPs. The possible mechanism of action involves recognition of PCSK9 by TLRs 2 and/or 4, on monocytes membrane surface, leading to activation of the transcription factor NFκB via an intracellular signalling cascade. Further studies will be needed to better understand the regulatory mechanisms underlying this complex set of biological responses that bind PCSK9, and coagulation events.

Neuroinflammation induced by amyloid beta 1-42 oligomers contributes to neuronal loss in an in vitro model of Alzheimer's disease.

Alzheimer's disease is characterized by the extracellular accumulation of senile plaques composed of amyloid beta and the intracellular deposition of neurofibrillary tangles composed of hyperphosphorylated tau. Soluble oligomers of Aβ 1-42 peptide (AβO) are neurotoxic and participate directly in the pathophysiology of Alzheimer's disease. Here, we studied the contribution of microglial activation in the human 1-42 AβO-induced neurotoxicity, in an in vitro model of Alzheimer's disease. Using primary mixed cultures of rat embryonic cortical neurons and microglial cells, AβO toxicity was investigated in presence or absence of TLR4 antagonist (TAK-242). Survival of cortical neurons, integrity of their neurite network and activation of microglial cells were studied by immunostaining. In addition, proteomic changes were investigated using an immuno-based protein assay covering 1300 proteins including inflammatory related proteins, immune cell marker, checkpoint molecules as well as mediators and signaling molecules related to apoptosis and necroptosis among many others. We showed that AβO application led to an activation of microglial cells, a release of pro-inflammatory cytokines and a chronic neurotoxicity. Neuronal loss was significantly reduced but not abolished in presence of an inhibitor of TLR4. In addition, protein expression analysis revealed major enrichments in protein pathways related to cytokine signaling. Altogether, these findings showed that the toxicity of AβO is partially due to the activation of microglial cells and the release of pro-inflammatory cytokines. It highlights the close pathological connection between AβO microglial cells, neuroinflammation and neuronal death in Alzheimer's disease.

Decrease in the inflammatory cytokines of LPS-stimulated PBMCs of patients with atherosclerosis by a TLR-4 antagonist in the co-culture with HUVECs

Toll-like receptors (TLRs) are among the players of inflammation during atherosclerosis. We assessed the effects of Eritoran, a TLR-4 antagonist, on lipopolysaccharide (LPS)-induced cytokines production by Peripheral Blood Mononuclear Cells (PBMCs) of patients with high-stenosis (HS) (n = 6) and healthy controls (HCs) (n = 6) co-cultured with Human Umbilical Vein Endothelial Cells (HUVECs). LPS stimulation significantly increased the levels of IL-6 (P = 0.007 and P = 0.005), TNF-α (P = 0.006 and P = 0.005), IL-2 (P = 0.007 and P = 0.002), IFN-γ (P = 0.006 and P = 0.003), IL-17A (P = 0.004 and P = 0.003), IL-17F (P = 0.005 and P = 0.003), IL-5 (P = 0.007 and P = 0.005), IL-13 (P = 0.006 and P = 0.005), IL-9 (P = 0.005 and P = 0.005) and IL-21 (P = 0.007 and P = 0.005) in HUVECs co-cultured with HC and HS PBMCs as compared with un-stimulated co-culture condition, respectively. Eritoran treatment (50 μg/mL and 100 μg/mL) significantly reduced the levels of LPS-induced IL-6 (P = 0.007 and P = 0.006; P = 0.007 and P = 0.007), TNF-α (P = 0.005 and P = 0.003; P = 0.007 and P = 0.005), IL-2 (P = 0.007 and P = 0.005; P = 0.005 and P = 0.004), IFN-γ (P = 0.007 and P = 0.005; P = 0.005 and P = 0.004), IL-17A (P = 0.005 and P = 0.002; P = 0.005 and P = 0.002), IL-17F (P = 0.006 and P = 0.006; P = 0.005 and P = 0.005), IL-5 (P = 0.007 and P = 0.006; P = 0.007 and P = 0.007), IL-9 (P = 0.005 and P = 0.005; P = 0.005 and P = 0.005) and IL-21 (P = 0.007 and P = 0.007; P = 0.005 and P = 0.005) in stimulated HUVECs co-cultured with HC and HS PBMCs, compared to un-treated condition, respectively. Our results demonstrate that attenuating effect of Eritoran on the inflammatory responses to LPS is higher in PBMCs of patients with high stenosis, suggesting its potential role in ameliorating inflammatory conditions in atherosclerosis.

TLR4 antagonism ameliorates persistent post-operative cognitive decline

Collagen cross-linking is mediated by lysyl oxidase (LOX) enzyme in the extracellular matrix (ECM) of mitral valve leaflets. Alterations in collagen content and LOX protein expression in the ECM of degenerative mitral valve may enhance leaflet expansion and disease severity.Twenty posterior degenerative mitral valve leaflets from patients with severe mitral regurgitation were obtained at surgery. Five normal posterior mitral valve leaflets procured during autopsy served as controls. Valvular interstitial cells (VICs) density was quantified by immunohistochemistry, collagen Types I and III by picro-sirius red staining and immunohistochemistry, and proteoglycans by alcian blue staining. Protein expression of LOX and its mediator TGFβ1 were quantified by immunofluorescence and gene expression by PCR.VIC density was increased, structural Type I collagen density was reduced, while reparative Type III collagen and proteoglycan densities were increased (P<.0001) with an increase in spongiosa layer thickness in myxomatous valves. These changes were associated with a reduction in LOX (P<.0001) and increase in TGFβ1 protein expression (P<.0001). However, no significant change was seen in gene expression. Linear regression analysis identified a correlation between Type I collagen density and LOX grade (R2=0.855; P<.0001).Reduced Type I collagen density with a simultaneous increase in Type III collagen and proteoglycan densities possibly contributes to spongiosa layer expansion resulting in incompetent mitral valve leaflets. Observed changes in Type I and III collagen densities in Degenerative Mitral Valve Disease may be secondary to alterations in LOX protein expression, contributing to disorganization of ECM and disease severity.

Binding of gamma-glutamyl transferase to TLR-4 allows the activation of tissue factor expression in human monocytes

Abstract Background Gamma-glutamyl transferase (GGT) plays a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the initiator of the clotting cascade. GGT may modulate TF expression, an assumption verified by previous studies carried out in human Peripheral Blood Mononuclear Cells (PBMCs). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic activity since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. Purpose To assess whether GGT-induced TF stimulation is a consequence of binding to Toll-Like Receptor (TLR)-4 and activation of NF-κB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from a leukemia patient, were incubated with hrGGT (0.5 ng/μl for PBMCs and 1ng/μl for THP-1). LPS-Rs (0.5 ng/μl for PBMCs and 1 ng/μl for THP-1), CLI-095 (3x10–6 M) and BAY-11-7082 (10–5 M) were used to block TLR-4 receptors, TLR4 signaling and NF-κB respectively. TF pro-coagulant activity (PCA) was assessed using of StartMax coagulometer and results were expressed in pg/ml after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110±70 to 510±43, n=7, p&amp;lt;0.01) and THP-1 cells (PCA from 170±64 to 460±80, n=15, p&amp;lt;0.001).In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72±17% n=4, p&amp;lt;0.01) a TLR-4 antagonist, CLI-095 (PCA:-74±34%, n=7, p&amp;lt;0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71±32%, n=7, p&amp;lt;0.001), a NF-κB inhibitor. Similar results were obtained in THP-1 cells [LPS-Rs: −76±15%, n=6, p&amp;lt;0.01; CLI-095: −100±6,6%, n=6, p&amp;lt;0.01; BAY-11-7082: −100±2,1%, n=6, p&amp;lt;0.01]. hrGGT activates NF-κB in hTLR4-positive HEK cell lines while doesn't induces effect in TLR4-negative HEK cells. Conclusions Besides confirming the cytokine-Like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells, these data identify for the first time the possible role of TLR-4 as the receptor of GGT and NfkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque. Funding Acknowledgement Type of funding sources: None.

Phosphorylated \u03b1-synuclein aggregated in Schwann cells exacerbates peripheral neuroinflammation and nerve dysfunction in Parkinson\u2019s disease through TLR2/NF-\u03baB pathway

To investigate the mechanism of peripheral neuropathy in Parkinson’s disease (PD), we prepared a PD mice model by long-term exposure of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mimic PD pathology in humans and the sciatic nerves were taken for further research. It turned out that phosphorylated α-synuclein (p-α-syn) was significantly deposited in Schwann cells (SCs) of sciatic nerves possibly contributing to degenerated myelin SCs and atrophied axons in MPTP group. Further analysis confirmed that toll-like receptors (TLRs) were implicated with PD peripheral neuropathy, in which TLR2 exhibits the predominant expression. Increased expression of inflammatory factors about TLR2/nuclear factor kappa-B (NF-κB) pathway was noted in MPTP group compared to saline group, with proteins on other pathways showing no changes. Moreover, MPTP-challenged mice exhibited worse motor ability and damaged nerve conduction, implicating that p-α-syn neurotoxicity might be relevant to impairments of motor and sensory nerves. After the treatment of CU-CPT22, a TLR2 antagonist, p-α-syn accumulation, motor and sensory function were ameliorated in CU-CPT22 combined with MPTP group. Thus, we demonstrated that pathological p-α-syn might combine TLR2 to affect SCs activation, inflammatory response as well as motor and sensory function through TLR2/nuclear factor kappa-B (NF-κB) signaling pathway. This study firstly demonstrates a novel mechanism of p-α-syn accumulated in SCs of peripheral nerves, which extends our understanding on SCs-mediated peripheral neuroinflammation related to TLR2/NF-κB signaling pathway and sheds light on potential new therapeutic avenues for PD.

Converging purinergic and immune signaling pathways drive IL-6 secretion by Fragile X cortical astrocytes via STAT3

The symptoms of Fragile X syndrome (FXS) are driven in part by abnormal glial-mediated function. FXS astrocytes release elevated levels of immune-related factors interleukin-6 (IL-6) and tenascin C (TNC), and also demonstrate increased purinergic signaling, a pathway linked to signaling factor release. Here, in cortical astrocytes from the Fmr1 knockout (KO) FXS mouse model, purinergic agonism enhanced TNC secretion and STAT3 phosphorylation, two processes linked to elevated IL-6 secretion in FXS, while STAT3 knockdown and TLR4 antagonism normalized Fmr1 KO IL-6 release. We therefore suggest that purinergic signaling and immune regulatory pathways converge to drive FXS cortical pro-inflammatory responses.

Blockade of TLR2 and TLR4 Attenuates Inflammatory Response and Parasite Load in Cutaneous Leishmaniasis.

Human cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a pronounced inflammatory response associated with ulcer development. Monocytes/macrophages, the main cells harboring parasites, are largely responsible for parasite control. Toll-like receptor (TLR) signaling leads to the transcription of inflammatory mediators, such as IL-1β and TNF during innate immune response. TLR antagonists have been used in the treatment of inflammatory disease. The neutralization of these receptors may attenuate an exacerbated inflammatory response. We evaluated the ability of TLR2 and TLR4 antagonists to modulate host immune response in L. braziliensis-infected monocytes and cells from CL patient skin lesions. Following TLR2 and TLR4 neutralization, decreased numbers of infected cells and internalized parasites were detected in CL patient monocytes. In addition, reductions in oxidative burst, IL-1β, TNF and CXCL9 production were observed. TNF production by cells from CL lesions also decreased after TLR2 and TLR4 neutralization. The attenuation of host inflammatory response after neutralizing these receptors suggests the potential of TLR antagonists as immunomodulators in association with antimonial therapy in human cutaneous leishmaniasis.

Role of hypothalamic-pituitary adrenal-axis, toll-like receptors, and macrophage polarization in pre-atherosclerotic changes induced by social isolation stress in mice

It has been well documented that chronic stress can induce atherosclerotic changes, however, the underlying mechanisms is yet to be established. In this regard, this study aimed to elucidate the relation between hypothalamic-pituitary adrenal-axis (HPA-axis), toll-like receptors (TLRs), as well as M1/M2 macrophage ratio and pre-atherosclerotic changes in social isolation stress (SIS) in mice. We used small interfering RNA against the glucocorticoid receptor (GR) to evaluate the relation between HPA-axis and TLRs. C57BL/6J mice were subjected to SIS and RT-PCR, ELISA, flow cytometry, and immunohistochemistry were used to assess the relations between pre-atherosclerotic changes and TLRs, macrophage polarization, pro-inflammatory cytokines, and cell adhesion molecules in aortic tissue. We used TAK-242 (0.3 mg/kg, intraperitoneally), a selective antagonist of TLR4, as a possible prophylactic treatment for atherosclerotic changes induced by SIS. We observed that isolated animals had higher serum concentration of corticosterone and higher body weight in comparison to normal animals. In isolated animals, results of in vitro study showed that knocking-down of the GR in bone marrow–derived monocytes significantly decreased the expression of TLR4. In vivo study suggested higher expression of TLR4 on circulating monocytes and higher M1/M2 ratio in aortic samples. Pathological study showed a mild pre-atherosclerotic change in isolated animals. Finally, we observed that treating animals with TAK-242 could significantly inhibit the pre-atherosclerotic changes. SIS can possibly increase the risk of atherosclerosis through inducing abnormal HPA-axis activity and subsequently lead to TLR4 up-regulation, vascular inflammation, high M1/M2 ratio in intima. Thus, TLR4 inhibitors might be a novel treatment to decrease the risk of atherosclerosis induced by chronic stress.

A TLR7 antagonist restricts interferon-dependent and -independent immunopathology in a mouse model of severe influenza.

Cytokine-mediated immune-cell recruitment and inflammation contribute to protection in respiratory virus infection. However, uncontrolled inflammation and the "cytokine storm" are hallmarks of immunopathology in severe infection. Cytokine storm is a broad term for a phenomenon with diverse characteristics and drivers, depending on host genetics, age, and other factors. Taking advantage of the differential use of virus-sensing systems by different cell types, we test the hypothesis that specifically blocking TLR7-dependent, immune cell-produced cytokines reduces influenza-related immunopathology. In a mouse model of severe influenza characterized by a type I interferon (IFN-I)-driven cytokine storm, TLR7 antagonist treatment leaves epithelial antiviral responses unaltered but acts through pDCs and monocytes to reduce IFN-I and other cytokines in the lung, thus ameliorating inflammation and severity. Moreover, even in the absence of IFN-I signaling, TLR7 antagonism reduces inflammation and mortality driven by monocyte-produced chemoattractants and neutrophil recruitment into the infected lung. Hence, TLR7 antagonism reduces diverse types of cytokine storm in severe influenza.