Upregulation Of Toll-like Receptor Research Articles

Alzheimer’s Disease:Intracellular Beta Amyloid Completes the Irreversible Pathway from Spirochetes to Biofilms to Beta Amyloid to Hyperphosphorylated Tau Protein

In this histopathological study, we have identified beta amyloid (Aβ) intracellularly in hippocampal specimens of Alzheimer’s disease (AD) patients. This is a continuation of the same histopathological project in which we observed biofilms intracellularly in the same neuronal cells in the same brain samples. To demonstrate that these were intracellular biofilms, we utilized the same techniques that showed biofilms in senile plaques in AD, in occluded eccrine ducts in atopic dermatitis, and in tonsils of psoriasis patients. Lyme spirochetes have recently been cultured from AD brains, and those same cultivated organisms have been shown in vitro to make biofilms, beta amyloid precursor protein (AβPP), and Aβ. We believe these spirochetes (and others) make the in vivo biofilms, and we believe our finding of intracellular Aβ helps confirm the in vitro observations. The Aβ, in turn, has previously been shown to stimulate the production and accumulation of hyperphosphorylated tau protein which has been shown to result in axonal and dendritic disintegration. With neuronal cell deterioration, the biofilms, AβPP, Aβ, and neurofibrillary tangles that were once inside are now present outside the cells. Once in the tissue, biofilms lead to upregulation of Toll-like receptor 2 (TLR2) which by known pathways leads to further production of Aβ. Thus, the Aβ can be derived from two sources: one is the spirochetes themselves and the other is from the activation of the innate immune system. The two major components of AD (tau protein and Aβ) have consequently been shown to be created by the pathogenic spirochetes. The spirochetes themselves have been shown to be of Lyme disease and dental origin.

Relevance of megalin receptor injury with nuclear factor-kappa B upregulation in acute kidney injury induced in rats.

Proximal tubule protein take-up is interceded by 2 receptors, megalin and cubilin. These receptors rescue an assortment of filtered ligands including fundamental vitamins and hormones. The objective of this study was to investigate the potential relation of megalin receptor injury with nuclear factor-kappa B (NF-κB) upregulation in acute kidney injury rat model. Twenty four rats were allocated into two groups: control group received saline, while the second group was intoxicated with cadmium chloride (2.4mg Cd/kg/day i.p) for 30 days. Blood urea nitrogen, serum creatinine, tissue oxidant-antioxidant parameters (malondialdehyde [MDA] and reduced glutathione [GSH]) and expression levels for NF-κB, toll like receptor-2 (TLR2), toll like receptor-4 (TLR4), and megalin receptor were estimated. Noticeable downregulation of megalin receptor versus upregulation of NF-κB, TLR2, and TLR4 were observed in AKI rat model together with significant elevation in MDA as well as significant reduction in GSH. The study concluded that the oxidative stress in kidney tissue leads to megalin receptor damage, which indeed motivates upregulation of NF-κB through TLRs 2 and 4 pathways.

Down-regulation of inflammatory signaling pathways despite up-regulation of Toll-like receptors; the effects of corticosteroid therapy in brain-dead kidney donors, a double-blind, randomized, controlled trial

BackgroundThe brain death of a potential organ donor induces a systemic inflammatory response, resulting in inferior organ quality and function. Our study aimed to evaluate the effects of methylprednisolone (MPN) therapy on pattern recognition receptor (PRR) signaling in potential brain-dead (BD) kidney donors. Material and methodsTo evaluate the effects of MPN therapy on PRR signaling in BD kidney donors we performed a prospective randomized treatment-versus-control study. Fifty-one potential kidney donors were randomly divided into three groups: brain-dead donors (BDDs) who received 15 mg/kg/d of methylprednisolone (group T1, n = 17), BDDs who received 15 mg/kg/d of MPN at the time of filling consent for kidney donation and 100 mg/2 h until kidney harvest (group T2, n = 17), and normal donors as controls n = 17. Gene expression for Toll-like receptors (TLRs) 1–9 and their signaling pathway molecules including MYD88, TRIF, NF-KB1, IRAK, IRF3, and IRF7, as well as the inflammatory cytokines RANTES, IL-1β, TNF-α, IL-6, CXCL8, IL-18, IFN-α, and IFN-β was determined by PCR array. Due to the crucial role of TLRs 2 and 4 in pattern recognition, surface expression of these molecules was analyzed by flow cytometry. Plasma levels of inflammatory cytokines were measured by immunoassay. Finally, serum creatinine and cystatin C were measured in 100 kidney recipients one week and one, three, and six months after transplant. ResultPolymerase chain reaction (PCR) array gene expression revealed greater expression of TLRs and signaling molecules in group T1 than in the controls. Surface expression of TLRs 2 and 4 were significantly greater in group T2 than in group T1 (P < .05). Plasma concentrations of inflammatory cytokines were significantly greater in group T1 than in controls (P < .05). The recipients that received kidneys from group T1 had significantly higher levels of creatinine and cystatin C than the recipients of kidneys from both group T1 and controls (P<0.05). ConclusionAdministration of MPN to BDDs at specified periods until kidney harvest resulted in less systemic inflammation in the BDDs and improved renal function in kidney graft recipients compared with common MPN therapy.

Innately activated TLR4 signal in the nucleus accumbens is sustained by CRF amplification loop and regulates impulsivity

Cognitive impulsivity is a heritable trait believed to represent the behavior that defines the volition to initiate alcohol drinking. We have previously shown that a neuronal Toll-like receptor 4 (TLR4) signal located in the central amygdala (CeA) and ventral tegmental area (VTA) controls the initiation of binge drinking in alcohol-preferring P rats, and TLR4 expression is upregulated by alcohol-induced corticotropin-releasing factor (CRF) at these sites. However, the function of the TLR4 signal in the nucleus accumbens shell (NAc-shell), a site implicated in the control of reward, drug-seeking behavior and impulsivity and the contribution of other signal-associated genes, are still poorly understood. Here we report that P rats have an innately activated TLR4 signal in NAc-shell neurons that co-express the α2 GABAA receptor subunit and CRF prior to alcohol exposure. This signal is not present in non-alcohol drinking NP rats. The TLR4 signal is sustained by a CRF amplification loop, which includes TLR4-mediated CRF upregulation through PKA/CREB activation and CRF-mediated TLR4 upregulation through the CRF type 1 receptor (CRFR1) and the MAPK/ERK pathway. NAc-shell Infusion of a neurotropic, non-replicating herpes simplex virus vector for TLR4-specific small interfering RNA (pHSVsiTLR4) inhibits TLR4 expression and cognitive impulsivity, implicating the CRF-amplified TLR4 signal in impulsivity regulation.

Toll-like receptor 4 (TLR4) impairs nitric oxide contributing to Angiotensin II-induced cavernosal dysfunction

AimAngiotensin II (AngII), a corpus cavernosum (CC) constrictor peptide, modulates Toll like receptor (TLR) expression, a key element of the innate immune system, contributing to impaired vascular function in pathological conditions. However, it is unknown whether TLR4 is involved in AngII-induced erectile dysfunction. In this study, we investigated whether TLR4 plays a role in cavernosal dysfunction caused by AngII upregulation. Material and methodsCavernosal smooth muscle cells (CSMC) from C57/BL6 mice were treated with AngII (0.1μM) or bacterial LPS (50ng/ml) for 12–24h and TLR4 expression was assessed. Mice were infused with AngII (90ng/min, 28days) and treated with anti-TLR4 antibody (0.1mg/daily, i.p.) for the last 14days of the treatment. CC tissue was used for functional studies and for Western blotting. Nitric Oxide Synthase (NOS) activity was measured by conversion of [3H]-l-arginine to [3H]-l-citrulline, systemic TNF-α levels by ELISA, and reactive oxygen species (ROS) by immunofluorescence. Key findingsWe report upregulation of TLR4 in CSMC following AngII or LPS stimulation. In AngII-infused mice, chronic treatment with anti-TLR4 antibody (28±2.1%) attenuates adrenergic CC contraction, which also ameliorates nitrergic (68.90±0.21 vs. 51.07±0.63, 8Hz, AngII-infused mice treated vs. non-treated). Decreased endothelial NOS expression, reduced NOS activity, and augmented levels of TNF-α, and ROS were found following AngII-infusion. These alterations were prevented, or at least decreased by anti-TLR4 antibody treatment. SignificanceInhibition of TLR4 ameliorates AngII-impaired cavernosal relaxation, decreases TNF-α levels, and restores NO bioavailability, demonstrating that TLR4 partly mediates AngII-induced cavernosal dysfunction.

Ginkgolide B Suppresses Methamphetamine-Induced Microglial Activation Through TLR4-NF-κB Signaling Pathway in BV2 Cells.

Accumulating evidence suggests that microglial cells have altered morphology and proliferation in different brain regions of methamphetamine (Meth) abusers and Meth-abusing animal models. However, the possible mechanisms underlying Meth-induced microglial activation remain poorly understood. Meanwhile, Toll-like receptor4 (TLR4) is closely associated with inflammation. Therefore the aim of the present study was to assess whether Meth treatment affects TLR4 expression; in addition, we evaluated the effects of ginkgolide B (GB), a diterpene lactone extracted from Ginkgo biloba, on Meth-mediated inflammation. BV2 cells were treated with Meth. Interestingly, Meth treatment significantly increased TLR4 expression, activated the NF-κB signaling pathway, and promoted TNF-α, IL-6 and IL-1β excretion. These effects, however, were partially attenuated by GB pre-treatment. To further confirm the role of TLR4 in Meth-mediated inflammation, the siRNA technology was applied to knock down TLR4, which resulted in hampered Meth-mediated inflammatory responses, confirming the important role of TLR4 in this process. Taken together, our findings suggested that Meth exposure results in BV2 cell activation, in association with TLR4 upregulation. GB could attenuate Meth-induced inflammation, at least partially through TLR4-NF-κB signaling pathway, therefore, targeting TLR4 may constitute a potential intervention strategy for Meth mediated neuroinflammation.

The critical role of macrophages in the pathogenesis of hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a painful chronic inflammatory disease with a prevalence between 1 and 4% of general population. The pathogenesis of HS long eluded scientists, but growing evidence suggests that it is a consequence of inflammatory dysregulation. Recent studies suggest that dysregulated immune response to skin flora and overexpression of inflammatory cytokines leads to chronic skin inflammation seen in HS. Macrophages are the most numerous inflammatory cells found in HS infiltrates and release numerous pro-inflammatory cytokines such as IL-23, and IL-1β and TNF-α, exacerbating the inflammation and contributing to the pathogenesis of HS. Furthermore, in HS, there is dysregulated function of other immune players closely associated with macrophage function including: matrix metalloproteases (MMP) 2 and 9 overexpression, toll-like receptor upregulation, impaired Notch signalling, NLRP3 inflammasome upregulation, and dysregulated keratinocyte function.Lifestyle factors including obesity and smoking also contribute to macrophage dysfunction and correlate with HS incidence. The overexpression of pro-inflammatory cytokines and subsequent efficacy of anti-cytokine biologic therapies highlights the importance of managing macrophage dysfunction. Future therapies should target key molecular drivers of macrophage dysfunction such as TLR2 and NLRP3 overexpression.

Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells.

Acute kidney injury (AKI) leads to a worse prognosis in diabetic patients compared with prognoses in non-diabetic patients, but whether and how diabetes affects kidney repair after AKI remains unknown. Here, we used scratch-wound healing and transwell migration models to examine whether and how wound healing is affected by high glucose levels in cultured kidney proximal tubular cells (RPTC). The results show that scratch-wound healing and transwell migration were significantly slower in high-glucose-treated kidney tubular cells (30 mM glucose) than in low-glucose-treated cells (5.5 mM). Toll-like receptor 4 (TLR4), MyD88, phospho-protein kinase C (PKC), phospho-p38 MAPK and monocyte chemoattractant protein-1 (MCP-1) mRNA levels were upregulated after high glucose treatments. Staurosporine, a selective PKC inhibitor, inhibited TLR4, MyD88 and p-p38 upregulation in the high-glucose-treated cells, indicating the involvement of PKC in high-glucose-induced TLR4 upregulation. The pharmacological inhibition of TLR4 or shRNA-mediated TLR4 knockdown improved wound healing and transwell migration in high-glucose-treated RPTC. In contrast, the overexpression of TLR4 in low-glucose-treated RPTC suppressed wound healing, mimicking the effects of high glucose levels. These results suggest that the upregulation of TLR4 expression via PKC activation contributes to defective wound healing in high-glucose-treated kidney tubular cells.

Subcutaneous injection of dendritic cells aggravates atherosclerosis in ApoE‑knockout mice by activation of TLR4.

Dendritic cells (DCs) are specialized antigen-presenting cells which are important in immune diseases, in particular atherosclerosis, a chronic inflammatory disease, however their role in atherosclerosis-associated immunity is unclear. To evaluate the role of DCs in atherosclerosis, exogenous bone marrow-derived DCs were transferred into ApoE−/− mice in the present study. The extent of disease was measured in the aorta and was compared with mice treated with phosphate-buffered saline (PBS) or left untreated and fed a western diet. Mice receiving exogenous DCs demonstrated significantly larger atherosclerotic lesions compared with the mice treated with PBS, with increasing numbers of mature DCs in circulation and enhanced DC infiltration into plaque lesions, in addition to activation of circulating inflammatory components and atherosclerotic lesions. Furthermore, it was demonstrated that exogenous DCs upregulated the expression of Toll-like receptor 4 (TLR4) on DCs, which may be an important mechanism to activate DCs and aggravate atherosclerosis. Therefore the present study concluded that exogenous DCs may induce maturation of endogenous DCs via upregulation of TLR4, further increasing the inflammatory response and accelerating atherosclerosis.

Vitamin D attenuates hyperoxia-induced lung injury through downregulation of Toll-like receptor 4

With considerable morbidity and mortality, bron-chopulmonary dysplasia (BPD) is a focus of attention in neonatology. Hyperoxia-induced lung injury has long been used as a model of BPD. Among all the signaling pathways involved, Toll-like receptor 4 (TLR4) has been demonstrated to play an important role, and is known to be regulated by vitamin D. This study aimed at elucidating the effect of vitamin D on hyperoxia-induced lung injury and the role of TLR4 in the process. Vitamin D was administered to hyperoxia-treated neonatal rats to investigate changes in the morphology of lungs and expression of pro-inflammatory cytokines, apoptotic proteins and TLR4. Vitamin D attenuated hyperoxia-induced lung injury by protecting the integrity of the lung structure, decreasing extracellular matrix deposition and inhibiting inflammation. The upregulation of TLR4 by hyperoxia was ameliorated by vitamin D and apoptosis was reduced. Vitamin D administration antagonized the activation of TLR4 and therefore alleviated inflammation, reduced apoptosis and preserved lung structure.

Chronic hyperosmotic stress inhibits renal Toll-Like Receptors expression in striped catfish (Pangasianodon hypophthalmus, Sauvage) exposed or not to bacterial infection

Toll-like Receptors (TLRs) are the first innate receptors in recognizing pathogen-associated molecular patterns. In fish, upregulation of toll-like receptors during infection has been largely demonstrated while the effects of abiotic stressors on their expression remain poorly investigated. In this study, striped catfish were submitted during 20 days to three salinity profiles (freshwater, low saline water, saline water), followed by injection of a bacterial strain of Edwardsiella ictaluri. The expression of TLRs 1, 3, 4, 5, 7, 9, 19, 21, and 22 was measured in kidney at different time points in non infected and infected striped catfish. Infection induced overexpression of TLRs 1, 3, 4, 5, 7, 21 and 22. With elevated salinity, the expression of all TLRs, except TLR5, was severely decreased, particularly after bacterial infection. The TLRs responsiveness of striped catfish facing bacterial disease and salinity stress and possible consequences on striped catfish immune response's efficiency are discussed.

Resveratrol inhibits the IL-1β-induced expression of MMP-13 and IL-6 in human articular chondrocytes via TLR4/MyD88-dependent and -independent signaling cascades.

The natural polyphenolic compound, resveratrol, has been shown to exhibit anti-osteoarthritic activity. Therefore it is hypothesized that resveratrol may serve as a nutritional supplement to counteract osteoarthritis(OA). However, the mechanisms responsible for these anti-osteoarthritic effects have not yet been fully elucidated. The aim of this study was to determine whether the biological effects of resveratrol against interleukin(IL)-1β‑induced inflammation in human articular chondrocytes involved both Toll-like receptor4(TLR4)/myeloid differentiation factor88(MyD88)-dependent and-independent signaling pathways. Human articular chondrocytes derived from patients with OA were stimulated with IL-1β, and then co-treated with resveratrol. Cell viability was subsequently evaluated by MTS assays, and the concentrations of matrix metalloproteinase(MMP)-13 and the pro-inflammatory factor, IL-6, were detected in culture supernatants using ELISA. The mRNA and protein levels of downstream mediators of TLR4/MyD88-dependent and-independent signaling pathways were also assayed by RT-qPCR and western blot analysis, respectively. Our results revealed that resveratrol prevented the IL-1β-induced reduction in cell viability. Furthermore, stimulation of the chondrocytes with IL-1β resulted in a significant upregulation of TLR4 and downstream targets of both TLR4/MyD88-dependent and-independent signaling pathways that are associated with the synthesis of MMP-13 and IL-6. Correspondingly, IL-1β-induced catabolic and inflammatory responses were effectively reversed by resveratrol. Taken together, these data suggest that resveratrol exerted protective effects against matrix degradation and inflammation in OA-affected chondrocytes by inhibiting both TLR4/MyD88-dependent and-independent signaling pathways. Thus, resveratrol represents a potential treatment for OA and warrants further investigation.

Up-regulation of TLR2 and TLR4 in high mobility group Box1-stimulated macrophages in pulpitis patients.

Objective(s):High Mobility Group Box1 (HMGB1) is a nonhistone, DNA-binding protein that serves a crucial role in regulating gene transcription and is involved in a variety of proinflammatory, extracellular activities. The aim of this study was to explore whether HMGB1 stimulation can up-regulate the expression of Toll-like Receptor 2 (TLR2) and Toll-like Receptor 4 (TLR4) on macrophages from pulpitis and to clarify the subsequent events involving Th17 cells and Th17 cell-associated cytokine changes.Materials and Methods:Having prepared dental pulp tissues of pulpitis and healthy controls, macrophage were isolated and cultured. Macrophages were thereafter stimulated by HMGB1 time course. RT-QPCR, flowcytometer, immunofluorescence, Western blotting, and ELISA techniques were used in the present research.Results:Our results showed that the expression of TLR2 and TLR4 on macrophages stimulated with HMGB1 increased in pulpitis compared with controls (macrophages without HMGB1 stimulation) with a statistical significance (P<0.001). In addition, the levels of IL-17, IL-23, and IL-6 in supernatants from cultured macrophages stimulated with HMGB1 from pulpitis increased, and NF-kB, the downstream target of TLR2 and TLR4, also showed a marked elevation after macrophages’ stimulation by HMGB1.Conclusion:The evidence from the present study suggests that the enhanced TLR2 and TLR4 pathways and Th17 cell polarization may be due to HMGB1 stimulation in pulpitis.

Identifying genetic hypomethylation and upregulation of Toll-like receptors in Kawasaki disease.

Kawasaki disease (KD) is an acute febrile systemic vasculitis that occurs in children and is characterized by elevated levels of proinflammatory cytokines. Toll-like receptors (TLRs) serve as the sensor arm of the innate immune system and induce proinflammatory cytokine expressions.We recruited a total of 18 paired KD patients, before intravenous immunoglobulin (IVIG) and at least 3 weeks after IVIG treatment, 18 healthy controls, and 18 febrile controls. For TLR genes and their cytosine-phosphate-guanine (CpG) markers, we used Affymetrix GeneChip® Human Transcriptome Array 2.0 and Illumina HumanMethylation450 BeadChip to evaluate gene expression levels and methylation patterns, respectively.KD patients demonstrated a significantly differential expression of TLR mRNA levels compared to both the healthy and febrile controls, with only TLR 3 and 7 not differing between the KD patients and the controls. After patients underwent IVIG treatment, the TLR mRNA levels, except for TLR3, decreased significantly in KD patients. In contrast, the methylation status of the CpG sites of TLR1, 2, 4, 6, 8, and 9 demonstrated an opposite tendency between the two stages of both the KD samples and the controls.TLRs, particularly TLR1, 2, 4, 6, 8, and 9, may stimulate the immunopathogenesis of KD. These results are among the first to use TLRs to prove that a bacterial inflammatory response may trigger KD.

TLR4 Influences Hepatitis B Virus Related Hepatocellular Carcinoma by Regulating the Wnt/β-Catenin Pathway

Background/Aims: We investigated the correlation between toll-like receptor 4 (TLR4) and β-catenin for disclosing the potential pathogenesis of hepatocellular carcinoma (HCC). Methods: Immunohistochemical toolkit was implemented to measure the expression of TLR4 and β-catenin in 98 cases of HCC tissues and adjacent tissues. After setting up the HepG2.2.15 hepatitis B virus (HBV) related HCC cell line, we divided the cells into the control group, TLR4 siRNA group, β-catenin siRNA group, and pcDNA.3.1 TLR4 + β-catenin siRNA group. Western blot, CCK-8 method, Transwell and flow cytometry were used to detect protein expression, cell proliferation, cell migration and invasion as well as cell apoptosis, respectively. Nude mice tumor model was established to observe the effects of TLR4 and β-catenin on the progression of HBV-related HCC in vivo. Results:The positive rates of TLR4 and β-catenin were higher in HCC tissues compared with normal tissues. Both the TLR4 siRNA group and β-catenin siRNA group exhibited a decreased expression of β-catenin. The proliferation, migration and invasion of tumor cells in the above two groups were suppressed, while the cell apoptosis appeared to be stimulated. As suggested by the results from in vivo and in vitro experiments, the up-regulation of TLR4 could antagonize the corresponding effect of β-catenin siRNA. Conclusions: TLR4 can affect the expression of β-catenin and hence influence the progression of HBV-related HCC.

The Opening of ATP-Sensitive K+ Channels Protects H9c2 Cardiac Cells Against the High Glucose-Induced Injury and Inflammation by Inhibiting the ROS-TLR4-Necroptosis Pathway

Background/Aims: Hyperglycemia activates multiple signaling molecules, including reactive oxygen species (ROS), toll-like receptor 4 (TLR4), receptor-interacting protein 3 (RIP3, a kinase promoting necroptosis), which mediate hyperglycemia-induced cardiac injury. This study explored whether inhibition of ROS-TLR4-necroptosis pathway contributed to the protection of ATP-sensitive K⁺ (K_ATP) channel opening against high glucose-induced cardiac injury and inflammation. Methods: H9c2 cardiac cells were treated with 35 mM glucose (HG) to establish a model of HG-induced insults. The expression of RIP3 and TLR4 were tested by western blot. Generation of ROS, cell viability, mitochondrial membrane potential (MMP) and secretion of inflammatory cytokines were measured as injury indexes. Results: HG increased the expression of TLR4 and RIP3. Necrostatin-1 (Nec-1, an inhibitor of necroptosis) or TAK-242 (an inhibitor of TLR4) co-treatment attenuated HG-induced up-regulation of RIP3. Diazoxide (DZ, a mitochondrial K_ATP channel opener) or pinacidil (Pin, a non-selective K_ATP channel opener) or N-acetyl-L-cysteine (NAC, a ROS scavenger) pre-treatment blocked the up-regulation of TLR4 and RIP3. Furthermore, pre-treatment with DZ or Pin or NAC, or co-treatment with TAK-242 or Nec-1 attenuated HG-induced a decrease in cell viability, and increases in ROS generation, MMP loss and inflammatory cytokines secretion. However, 5-hydroxy decanoic acid (5-HD, a mitochondrial K_ATP channel blocker) or glibenclamide (Gli, a non-selective K_ATP channel blocker) pre-treatment did not aggravate HG-induced injury and inflammation. Conclusion: K_ATP channel opening protects H9c2 cells against HG-induced injury and inflammation by inhibiting ROS-TLR4-necroptosis pathway.

Epigallocatechin-3-gallate alleviates paraquat-induced acute lung injury and inhibits upregulation of toll-like receptors

AimsTo evaluate the detoxifying effect of epigallocatechin-3-gallate (EGCG) on paraquat (PQ)-induced acute lung injury in mice, and to explore the action mechanisms. Main methodsFollowing administration of PQ, the mice received a low, a medium or a high dose of EGCG daily for three days. Histopathology of the lungs were examined by H&E staining. The levels of inflammatory cytokines, such as TNF-α, IL-1β and IL-6, in the bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay. Activation of NF-κB was assessed by Western blot and electrophoretic mobility gel shift assay. The expression of toll-like receptor (TLR)-2, 4, 9 and TLR adaptors (MyD88 and TRAF6) was detected by Western blot and immunohistochemical staining. The protective effect of EGCG against PQ toxicity was validated in vitro using A549 lung cancer cell line. Key findingsTreatment with EGCG dose-dependently attenuated PQ-induced acute lung injury in mice by reducing alveolar edema, hemorrhage, inflammatory cell infiltration and production of inflammatory cytokines. EGCG inhibited the activation of NF-κB and the upregulation of TLR 2, 4 and 9 as well as their adaptors MyD88 and TRAF6 in the lungs following PQ challenge. In addition, EGCG significantly reduced PQ-induced cell death, cytokine production, activation of NF-κB, and upregulation of TLRs and adaptors in A549 cells. SignificanceOur data suggest that TLR-mediated activation of NF-κB in the non-immune pulmonary cells could be involved in PQ-induced acute lung injury, and it may serve as a target of EGCG against PQ pulmonary toxicity.

Preventive effects of dexmedetomidine on the development of cognitive dysfunction following systemic inflammation in aged rats.

In the present study, we examined whether and by what mechanisms dexmedetomidine (DMED) prevents the development of systemic inflammation (SI)-induced cognitive dysfunction in aged rats. Animals received a single intraperitoneal (i.p.) injection of either 5.0mg/kg lipopolysaccharide (LPS) or vehicle. LPS-treated rats were further divided into three groups: early DMED, late DMED, or midazolam (MDZ) treatment (n=12 each). Seven days after LPS injection, cognitive function was evaluated using a novel object recognition task, followed by measurement of hippocampal levels of proinflammatory cytokines and Toll-like receptor 4 (TLR-4) expression. For ex vivo experiments, microglia were isolated from the hippocampus for assessment of cytokine response to LPS. LPS-treated rats showed memory deficits, hippocampal neuroinflammation, and TLR-4 upregulation as compared to saline-treated animals. However, early DMED treatment was able to attenuate these SI-induced neurocognitive changes, whereas no benefits were observed in the MDZ and late DMED treatment groups. In ex vivo experiments, early DMED treatment prevented the development of SI-induced excessive microglial hyperactivation, which was blocked by the nonspecific α2-adrenergic receptor (AR) antagonist atipamezole or the specific α2A-AR antagonist BRL-44408, but not by the specific α2B/C-AR antagonist ARC-239. On the other hand, neither DMED nor MDZ had a direct effect on LPS-induced release of pro-inflammatory cytokines from hippocampal microglia at clinically relevant concentrations. Our findings highlight that treatment with DMED during, but not after, peripheral SI can prevent subsequent hippocampal neuroinflammation, overexpression of TLR-4 in microglia, and cognitive dysfunction, as mediated by the α2A-AR signaling pathway.

Abstract 598: Puerarin Inhibits Oxldl-mediated Proinflammatory and Proatherogenic Effects in Human Macrophages

Puerarin, a major isoflavonoid compound isolated from the Chinese herb Kudzu roots, has been a long history and widely used for treatment of cerebral and cardiovascular diseases in China and other Asian countries, including coronary artery disease, heart failure and myocardial infarction. However, the underlying mechanisms by which puerarin inhibits vascular atherogenic diseases are largely unknown. The activated macrophages release of inflammatory cytokines and uptake of oxLDL forming foam cells are critical for the development of atherosclerotic plaque. The present study investigated whether puerarin inhibited atherogenic lipid oxLDL-mediated macrophage activation and foam cell formation in human THP1 macrophage. The Cells were cultured and differentiated into macrophages by pre-incubation with 100 ng/ml PMA. Treatment with oxLDL (50 μg/ml) significantly increased the mRNA expression of proinflammatory cytokines tumor necrosis factor α(TNFα, 160%) and interleukin (IL)1β(13 fold) accompanied by upregulation of toll-like receptor 4 (TLR4, 165%) and the ratio of phospho-I κ Bα/I κ Bα in THP1 macrophage. Puerarin dose-dependently prevented an increase in oxLDL-induced proinflammatory gene expression with downregulation of TLR4 and the ratio of phospho-I κ Bα/I κ Bα. Furthermore, puerarin prevented oxLDL-mediated lipid deposition and foam cell formation associated with downregulation of scavenger receptor CD36. Flow cytometry analysis showed that puerarin reduced the number of early apoptotic cells of macrophages induced by oxLDL. Our results show that puerarin has anti-inflammatory and anti-atherogenic effects in vitro, the underling mechanisms may involve the inhibition of TLR4/NF κ B pathway and downregulation of CD36 expression. The results from the present study provide scientific evidence and may expand our armamentarium to use puerarin for prevention and treatment of cardiovascular and atherosclerotic diseases.

Associations of microbiota and toll-like receptor signaling pathway in esophageal adenocarcinoma.

BackgroundToll-like receptors (TLRs) recognize known molecules from microbes and have an established role in tumorigenesis. Using a rat model of esophageal adenocarcinoma, and human clinical samples, we investigated genes central to TLR-mediated signal transduction and characterized the esophageal microbiome across the spectrum of esophageal adenocarcinoma carcinogenesis.MethodsWe surgically induced bile/acid reflux in rats and their esophagi were harvested at 40 weeks post-surgery. Tissue samples from the model were selected for gene expression profiling. Additionally, for rat and human samples microbiome analysis was performed using PCR-ESI-MS-TOF technology with validation by fluorescence in situ hybridization.ResultsGene expression results in the rat model indicated a significant upregulation of TLRs 1-3, 6, 7 and 9 in EAC compared to normal epithelium. PCR-ESI-MS-TOF analysis revealed a prevalence of Escherichia coli in Barrett’s esophagus (60 %) and esophageal adenocarcinoma (100 %), which was validated by fluorescence in situ hybridization. In the human clinical samples, Streptococcus pneumonia was detected in high abundance in gastroesophageal reflux disease and Barrett’s esophagus (50–70 %) in comparison to tumor adjacent normal epithelium, dysplasia, and esophageal adenocarcinoma (20–30 %). E. coli was detected in the Barrett’s esophagus and esophageal adenocarcinoma groups but was absent in the tumor adjacent normal epithelium, dysplasia, and the gastroesophageal reflux disease groups.ConclusionsWe demonstrated an association between the TLR signaling pathway and E. coli hinting towards possible early molecular changes being mediated by microbes in the rat model of esophageal adenocarcinoma carcinogenesis. Studies on human clinical samples also corroborate results to some extent; however, a study with larger sample size is needed to further explore this association.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2093-8) contains supplementary material, which is available to authorized users.