Knockdown Of TLR4 Research Articles

Cross-Talk between HLA Class I and TLR4 Mediates P-Selectin Surface Expression and Monocyte Capture to Human Endothelial Cells.

Donor-specific HLA Abs contribute to Ab-mediated rejection (AMR) by binding to HLA molecules on endothelial cells (ECs) and triggering intracellular signaling, leading to EC activation and leukocyte recruitment. The molecular mechanisms involving donor-specific HLA Ab-mediated EC activation and leukocyte recruitment remain incompletely understood. In this study, we determined whether TLRs act as coreceptors for HLA class I (HLA I) in ECs. We found that human aortic ECs express TLR3, TLR4, TLR6, and TLR10, but only TLR4 was detected on the EC surface. Consequently, we performed coimmunoprecipitation experiments to examine complex formation between HLA I and TLR4. Stimulation of human ECs with HLA Ab increased the amount of complex formation between HLA I and TLR4. Reciprocal coimmunoprecipitation with a TLR4 Ab confirmed that the crosslinking of HLA I increased complex formation between TLR4 and HLA I. Knockdown of TLR4 or MyD88 with small interfering RNAs inhibited HLA I Ab-stimulated P-selectin expression, von Willebrand factor release, and monocyte recruitment on ECs. Our results show that TLR4 is a novel coreceptor for HLA I to stimulate monocyte recruitment on activated ECs. Taken together with our previous published results, we propose that HLA I molecules form two separate signaling complexes at the EC surface, that is, with TLR4 to upregulate P-selectin surface expression and capture of monocytes to human ECs and integrin β4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection.

Guben Tongluo Formula Protects LPS-induced Damage in Lamina Propria B Lymphocytes Through TLR4/MyD88/NF-κB Pathway.

The main pathological feature of immunoglobulin A nephropathy (IgAN), an autoimmune kidney disease, is the deposition of IgA immune complexes, accompanied by mesangial cell proliferation and elevated urine protein. The Guben Tongluo formula (GTF) is a traditional Chinese medicine prescription, which has predominant protective effects on IgAN. However, the therapeutic mechanism of the GTF in IgAN remains elusive. The present study aimed to determine the effects of GTF in treating IgAN via regulating the TLR4/MyD88/NF-κB pathway. In the present study, lamina propria B lymphocytes were treated with different concentrations of lipopolysaccharide (LPS) (0, 1, 5, 10 and 20 ng/mL). Flow cytometry was used to define positive CD86+CD19+ cells. CCK-8 assay was used to examine cell proliferation. RNAi was used to induce TLR4 silencing. qRT-PCR and Western blotting were used to determine gene expression. It was found that the LPS dose-dependently increased the content of IgA and galactose-deficient IgA1 (Gd-IgA), the levels of TLR4, Cosmc, MyD88 and phosphorylated (p)-NF-κB, and the ratio of CD86+CD19+ and IgA-producing B cells. However, the TLR4 knockdown reversed the role of LPS. This suggests that TLR4 mediates the effects of LPS on lamina propria B lymphocytes. Furthermore, the GTF could dose-dependently counteract the effects of LPS and TLR4 overexpression on lamina propria B lymphocytes through the TLR4/MyD88/NF-κB pathway. Collectively, these results demonstrate that the GTF can regulate the TLR4/MyD88/NF-κB pathway to treat IgAN model lamina propria B lymphocytes stimulated by LPS.

Phenotypic Alteration of BMDM In Vitro Using Small Interfering RNA.

Autologous macrophage transfer is an emerging platform for cell therapy. It is anticipated that conventional macrophage reprogramming based on ex vivo polarization using cytokines and ligands of TLRs may enhance the therapeutic effect. We describe an alternative approach based on small interfering RNA (siRNA) knockdown of selected molecular cues of macrophage polarization, namely EGR2, IRF3, IRF5, and TLR4 in Raw264.7 monocyte/macrophage cell line and mouse-bone-marrow-derived macrophages (BMDMs). The impact of IRF5 knockdown was most pronounced, curtailing the expression of other inflammatory mediators such as IL-6 and NOS2, especially in M1-polarized macrophages. Contrary to IRF5, EGR2 knockdown potentiated M1-associated markers while altogether abolishing M2 marker expression, which is indicative of the principal role of EGR2 in the maintenance of alternative phenotypes. IRF3 knockdown suppressed M1 polarization but upregulated Arg 1, a canonical marker of alternative polarization in M1 macrophages. As anticipated, the knockdown of TLR4 also attenuated the M1 phenotype but, akin to IRF3, significantly induced Arginase 1 in M0 and M1, driving the phenotype towards M2. This study validates RNAi as a viable option for the alteration and maintenance of macrophage phenotypes.

Abstract P1071: Concurrent Stat1 And Stat3 Signaling Modulates Tlr4/nfkb-mediated Immunomodulatory And Antifibrotic Effects Of Cardiomyocyte-damaged Released S100a1 Protein In Cardiac Fibroblasts

Background: We previously showed that S100A1 released from damaged cardiomyocytes can accelerate post-myocardial infarction in vivo healing. The action of S100A1 as a damage-associated molecular pattern seemed to occur via an immunomodulatory (e.g., IL10 increase) and antifibrotic impact (e.g., COL1A1 and ACTA2 downregulation) in cardiac fibroblasts (CFs) via a TRL4/NKFB pathway. The signaling network in CFs conveying this effect has not been characterized yet. Methods and Results: We performed a 48h time-resolved transcriptome analysis on cultured adult rat CF stimulated with rec. human S100A1 protein (10 -6 M). Bioinformatic GSEA yielded an instant and almost exclusive significant upregulation of the chemokine activity GO-term with a delayed suppression of ECM genes. A transcription factor enrichment analysis for the differentially regulated transcripts indicated an immediate activation of NFKB/RELA with a concurrent temporary STAT1 but lasting STAT3 signaling. A secretome LC-MS/MS-based analysis confirmed the shift of S100A1-treated CFs to mostly chemokine secreting cells. With CCL2 as most secreted chemokine and COL1A1 and ACTA2 as established antifibrotic marker genes for released S100A1, we used a RT-PCR readout for these genes in response to siRNA-based knock-down (KD) of STAT1 and STAT3 in S100A1-treated CFs, while TLR4 KD was used to abrogate NFKB/RELA signaling due to known mechanisms (n=10 each group). S100A1 treatment upregulated CCL2 (16-fold*) and downregulated COL1A1 (3.1-fold*) and ACTA2 (2.9-fold*) mRNA expression vs control (*P<0.01) that was blunted by TLR4 KD. STAT3 KD doubled the CCL2 mRNA increase (32.9-fold*), attenuated COL1A1 mRNA downregulation by S100A1 treatment to 2-fold* but left ACTA2 downregulation unaffected vs contr. (*P<0.05). Conversely, STAT1 KD completely blocked the S100A1 mediated downregulation of ACTA2 but neither impacted S100A1-mediated changes in CCL2 nor COL1A1. Conclusion: Our study identifies concurrent STAT1 and STAT3 activation as a modulator of S100A1-mediated TLR4-NFKB signaling in CFs. As S100A1-activated CFs express e.g., IL-10, we speculate that IL-gp130 trans signaling may convey this effect guiding further studies on S100A1’s extracellular effects in the diseased heart.

MicroRNA-140-5p Suppresses Hypoxia-induced Nephrotoxicity and Renal Fibrogenesis in in vitro Conditions

Background: miR-140-5p is frequently dysregulated in different types of tumors and has been found to suppress inflammation through TLR4 in lung injury. In the current investigation, we try to decipher the possible function of miR-140-5p in hypoxia-induced nephrotoxicity and renal fibrogenesis in human renal cells. Methods: Hypoxia in HK-2 cells was produced using cobalt chloride (CoCl2). Hypoxia-induced cellular viability and apoptosis were determined by cell viability assay and ELISA-based apoptosis assay, respectively. Western blotting was carried out to measure expression levels of pro-fibrotic and apoptosis-related proteins. Result: The present study observed that upregulation of miR-140-5p significantly decreases apoptosis and increases cell viability in hypoxia challenged HK-2 cells. Furthermore, miR-140-5p over expression decreased expression of pro-apoptotic proteins like Bad and Bax and pro-fibrotic proteins like Collagen I, CTGF and fibronectin. While as, expression of anti-apoptotic protein, namely Bcl-2, was increased in hypoxia-insulted HK-2 cells. Moreover, the luciferase reporter assay confirmed that miR-140-5p targets TLR4. TLR4 knockdown prevents miR-140-5p from performing its suppressive role in hypoxia linked to apoptosis and fibrosis.

PCV2 infection aggravates OTA-induced immunotoxicity in vivo and in vitro

Ochratoxin A (OTA), frequently existing in the food and feeds, could induce immunotoxicity. Porcine circovirus type 2 (PCV2), as a primary causative agent of porcine circovirus–associated disease, also could induce immunosuppression. However, it is still unknown whether PCV2 infection impacts OTA-induced immunotoxicity. The pigs and porcine alveolar macrophages (PAMs) were used as the model in the present experiment. The results in vivo indicated that PCV2 infection exacerbated OTA-induced immunotoxicity, NF-κB p65 phosphorylation, and TLR4 and MyD88 mRNA and protein expression in spleen. The results in vitro showed that OTA at 7.0 and 9.0 μM decreased cell viability and increased LDH release of PAMs without PCV2 infection. However, with PCV2 infection, OTA at 5.0, 7.0 and 9.0 μM significantly decreased cell viability and increased LDH release compared with absence of PCV2 infection. In addition, OTA at 5.0 and 7.0 μM significantly increased Annexin V/PI-positive rate, apoptosis of nuclear, γ-H2AX foci, IL-1α and TNF-α expression in PAMs with PCV2 infection compared with absence of PCV2 infection. In addition, PCV2 infection enhanced OTA-induced TLR4 and MyD88 mRNA and protein expression and NF-κB p65 phosphorylation. Knockdown of TLR4 alleviated the exacerbating effects of PCV2 infection on OTA-induced cytotoxicity, apoptosis and DNA damage in PAMs. These results indicated that PCV2 infection aggravated OTA-induced immunotoxicity and reduced the dose of OTA-induced immunotoxicity via TLR4/NF-κB p65 signaling pathway, which could provide basis for establishing limits for OTA.

E.coli JM83 damages the mucosal barrier in Ednrb knockout mice to promote the development of Hirschsprung‑associated enterocolitis via activation of TLR4/p‑p38/NF‑κB signaling.

Hirschsprung-associated enterocolitis (HAEC) is characterized by intestinal mucosal damage and an imbalance in the intestinal microbiota. Recent studies have indicated that the TLR4/p-p38/NF-κB signaling pathway in the intestine is of great importance to intestinal mucosal integrity. The present study aimed to investigate the role of TLR4/phosphorylated (p-)38/NF-κB signaling in the pathogenesis of HAEC in E. coli JM83-infected endothelin receptor B (Ednrb)−/− mice. Ednrb−/− mice were infected with E. coli JM83 by oral gavage to establish the HAEC model. Wild-type and Ednrb−/− mice were randomly divided into uninfected and E. coli groups. The role of TLR4/p-p38/NF-κB signaling was further evaluated by in vivo and in vitro analyses. The activation of the TLR4/p-p38/NF-κB signaling pathway induced by E. coli JM83 resulted in HAEC in Ednrb−/− mice, which was evidenced by a significant increase in the expression of TNF-α, TGF-β and IL-10, and a decreased density of F-actin protein expression. TLR4 knockdown reduced the severity of enterocolitis and attenuated the expression of IL-10, TNF-α and TGF-β, whilst increasing the density of F-actin protein in Ednrb−/− mice after E. coli infection. These results indicated that E. coli JM83 activates TLR4/p-p38/NF-κB signaling in Ednrb−/− to promote the development of HAEC. Thus, inhibition of this signaling pathway may benefit the treatment and prevention of HAEC.

Qishen Granule (QSG) Inhibits Monocytes Released From the Spleen and Protect Myocardial Function via the TLR4-MyD88-NF-κB p65 Pathway in Heart Failure Mice.

Preliminary clinical and basic researches have proved that Qishen granule (QSG) is an effective prescription for treating heart failure (HF) in China, with a characteristic of regulating the ratio of M1/M2 macrophage in the myocardium. However, the regulative mechanism of monocytes targeting the cardio-splenic axis has not been fully elucidated. This study aimed to investigate the effects and mechanism of QSG inhibiting the release of splenic monocytes and the recruitment of myocardial tissue both in vivo and in vitro. Experiments in mice with acute myocardial infarction (AMI)-induced HF demonstrated that QSG could exert anti-inflammatory effects by inhibiting splenic monocytes release and phenotypic changes. Moreover, in vitro experiments indicated QSG could inhibit LPS-stimulated macrophage-conditioned medium (CM)-induced H9C2 cardiomyocyte injury by upregulating the key proteins in TLR4-MyD88-NF-κB p65 pathway. In addition, knockdown or overexpression of TLR4 in H9C2 cells further confirmed that QSG could attenuate inflammatory injury in cardiomyocytes via the TLR4-MyD88-NF-κB p65 pathway. Overall, these data suggested that QSG could improve cardiac function and reduce the inflammatory response in AMI-induced HF by inhibiting splenic monocytes release, and protecting myocardial function via the TLR4-MyD88-NF-κB pathway in heart failure mice.

Inhibition of HMGB1/TLR4 Signaling Pathway by Digitoflavone: A Potential Therapeutic Role in Alcohol-Associated Liver Disease.

Digitoflavone (DG) is a natural flavonoid abundant in many fruits, vegetables, and medicinal plants. We investigated whether DG inhibits lipid accumulation and inflammatory responses in alcoholic liver disease (ALD) in vivo and in vitro. The mouse ALD model was established by chronically feeding male C57BL/6 mice an ethanol-containing Lieber-DeCarli liquid diet. In vitro, mouse peritoneal macrophages (MPMs) and mouse bone marrow-derived macrophages (BMDMs) were stimulated with LPS/ATP, whereas HepG2 cells and mouse primary hepatocytes were treated with ethanol. DG reduced the serum levels of transaminase and serum and hepatic levels of triglycerides and malondialdehyde in ALD mice. DG downregulated SREBP1 and its target genes and upregulated PPARα and its target genes in the liver of mice with ALD. DG inhibited TLR4-mediated NLRP3 inflammasome activation, consequently reversing the inflammatory response, including the production of HMGB1, IL-1β, and IL-36γ, as well as the infiltration of macrophages and neutrophils. DG blocked NLRP3/ASC/caspase-1 inflammasome activation and HMGB1 release in LPS/ATP-stimulated MPMs. When Tlr4 was knocked in LPS/ATP-stimulated BMDMs, HMGB1 production and release were blocked, and NLRP3-mediated cleavage and release of IL-1β was suppressed in Hmgb1-silenced BMDMs. DG amplified these inhibitory effects in Tlr4 or Hmgb1 knockdown BMDMs. In ethanol-exposed hepatocytes, DG reduced lipogenesis and promoted lipid oxidation by inhibiting the HMGB1-TLR4 signaling pathway while suppressing the inflammatory response induced by ethanol exposure. Our data demonstrated that DG inhibited the occurrence of lipid accumulation and the inflammatory response via the HMGB1-TLR4 axis, underscoring a promising approach and utility of DG for the treatment of ALD.

The protective effect of Escherichia coli Nissle 1917 on the intestinal barrier is mediated by inhibition of RhoA/ROCK2/MLC signaling via TLR-4

AimsThis study investigated the protective effect of Escherichia coli Nissle 1917 (EcN) on intestinal barrier and the mechanism in the context of acute severe inflammation. Materials and methodsIn this study, mice received lipopolysaccharide (LPS) intraperitoneal injection with or without EcN administration to construct a mouse model of endotoxemia. Clinical scores, intestinal permeability, inflammatory cytokines and histopathological analysis of four main organs from different groups were assessed. The expression of tight junction proteins and activation of RhoA/ROCK2/MLC signaling were examined using western blotting. The localization of tight junction proteins was examined by immunofluorescence. Caco-2 monolayers with or without TLR-4 knockdown were incubated with EcN or TNF-α/IFN-γ and the monolayer barrier function was assessed by transepithelial electrical resistance (TER) and FITC-dextran 4000 Da (FD-4) flux. The expression of tight junction proteins and activation of RhoA/ROCK2/MLC signaling were examined by western blotting. The localization of tight junction proteins was examined by immunofluorescence. Key findingsWe found that EcN downregulated the RhoA/ROCK2/MLC signaling pathway to preserve barrier function and alleviated systemic inflammation in mouse model. And EcN also protected barrier function of Caco-2 monolayers by inhibiting the activation of RhoA/ROCK2/MLC signaling via TLR-4. SignificanceThe results indicated that EcN protected the intestinal barrier function in endotoxemia through inhibiting the activation of RhoA/ROCK2/MLC signaling via TLR-4.

Toll-Like Receptor 4 Exacerbates Mycoplasma pneumoniaevia Promoting Transcription Factor EB-Mediated Autophagy.

Mycoplasma pneumoniae (M. pneumoniae) is the most common cause of community-acquired pneumonia. Toll-like receptors (TLRs) play an essential role in pneumonia. The purpose of this study was to investigate the roles of TLR4 in M. pneumoniae. Mice were administrated with 100 μl (1 × 107 ccu/ml) of M. pneumoniae. HE staining was applied for histological analysis. The protein expression was determined by western blot. The cytokine level was detected by ELISA. The results showed that TLR4-deficient mice were protected from M. pneumoniae. However, downregulation of TLR4 inhibited inflammatory response and autophagy. Moreover, transcription factor EB (TFEB) participated in M. pneumoniae-induced inflammatory response and autophagy, while knockdown of TLR4 downregulated TFEB and its nuclear translocation.

A comprehensive gene expression profile analysis of prostate cancer cells resistant to paclitaxel and the potent target to reverse resistance.

Background: Paclitaxel resistance is the major clinical obstacle in the chemotherapy of prostate cancer (PCa), but the resistant mechanism is less investigated.Purpose: To establish two paclitaxel-resistant PCa cells, provide a comprehensive gene expression profile analysis of resistant cells and the potential target to reverse resistance.Methods: Two Paclitaxel-resistant PCa cells (PC3/PR, LNcap/PR) were established by gradually increasing drug concentration. MTT and transwell assays were performed to detect drug sensitivity, cell proliferation and migration abilities. RNA-Sequencing (RNA-seq) and bioinformatic analyses were performed to identify abnormally expressed genes (AEGs) in resistant cells, and annotate the biological functions of AEGs. The role of the candidate AEG, TLR-4, on the resistant phenotypes was further investigated.Results: The resistance index of resistant cells was 2-3, and they showed a slower proliferation and increased migration ability. 4741 AEGs were screened out (Log2fold change absolute: log2FC(abs) > 1) in the resistant cells, and they were enriched in 2'-5'-oligoadenylate synthetase activity and chemical carcinogenesis. A number of AEGs, CCND2, IGFBP3, FOS, SHH, ZEB2, and members of FGF, FGFR and WNT families were also identified to be involved in cancer- and resistant phenotype-related processes. Finally, TLR-4 was validated significantly increased in resistant cells, and knockdown of TLR-4 increased drug-sensitivity, inhibited the proliferation and migration abilities.Conclusions: The study provided a comprehensive gene expression profile of paclitaxel-resistant PCa cells, and TLR-4 could be a potential target to reverse paclitaxel resistance.

The Roll of Toll-like Receptors in the Stem Cell Inflammatory Response

Background: Toll-like receptors serve as ligands for LPS and other inflammatory mediators. Previous studies have shown that TLR4 is deleterious, while TLR9 is beneficial in the setting of inflammation and ischemia. Umbilical stem cells (USCs) have shown promise in the acute treatment of inflammation but their response to inflammatory mediators has not been fully elucidated. We hypothesized that knockdown of TLR4 in USCs would result in lower levels of IL-6 and VEGF production, while knockdown of TLR9 would produce higher levels of these cytokines when cells were exposed to LPS or hypoxia. 
 
 Methods: USCs were cultured in polystyrene flasks in Mesenpro media at 37C in 5% CO2 in air. Cells were plated into 12-well plates at a concentration of 100,000 cells/well. Cells were transfected for 24h with siRNA to knockdown TLR4 and TLR9, respectively. Knockdown was confirmed by PCR. Experimental groups were: 1) Control, 2) Scramble siRNA for negative control, 3) TLR4 siRNA and 4) TLR9 siRNA. After 24 hours the media was changed and cells were exposed to either LPS (200ng/ml) or 5% oxygen for 24 hours. The supernatant was then collected and analyzed with ELISA for VEGF and IL-6. Data were analyzed by Mann Whitney test and p<0.05 was significant. 
 
 Results: TLR4 and TLR9 were effectively knocked down by the transfection process. However, no significant levels of VEGF or IL-6 were detected from any of the experimental groups. 
 
 Conclusion and Implications: Although no significant levels of VEGF or IL-6 were detected in the ELISA after exposure to inflammatory agents, there is still indication that TLR4 and TLR9 play critical roles in inflammation. The experiment should be run again and tested for more inflammatory cytokines. Positive results from this study can translate to an in vivo model, in which engineered TLR4KD stem cells have the potential to minimize tissue inflammation and beyond standard cell therapy. 
 
 Acknowledgement: This project was funded, in part, with support from the Indiana Clinical and Translational Sciences Institute funded, in part by UL1TR002529 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 

Melanin of Sporothrix globosa affects the function of THP-1 macrophages and modulates the expression of TLR2 and TLR4

BackgroundMelanin is an important virulence factor for Sporothrix globosa, the causative agent of sporotrichosis, a subcutaneous mycosis that occurs worldwide. Although previous research suggests that melanin is involved in the pathogenesis of sporotrichosis, little is known about its influence on the macrophages that represent the frontline components of innate immunity. ObjectivesTo evaluate the effects of melanin on phagocytic activity and the expression of Toll-like receptor (TLR)2 and TLR4 during S. globosa infection of macrophages in vitro. MethodsTo compare phagocytic activity and survival rates, THP-1 macrophages and primary mouse peritoneal macrophages were co-cultured with a wild-type S. globosa strain (Mel+), an albino mutant strain (Mel˗), a tricyclazole-treated Mel + strain (TCZ-Mel+), or melanin ghosts extracted from S. globosa conidia. Reactive oxygen species (ROS), nitric oxide (NO) generation, tumor necrosis factor (TNF)-α and interleukin (IL)-6 were assayed in THP-1 cells infected with S. globosa conidia. Quantitative PCR and western blotting were used to observe the effect of melanin on TLR2 and TLR4 expression. Knockdown of TLR2/4 expression with small interfering RNA was performed to further verify the role of these receptors during infection. ResultsMacrophages infected with Mel + conidia showed a lower phagocytosis index and a higher survival rate than TCZ-Mel+ and Mel˗ in vitro. After incubation with S. globosa, the release of ROS, NO, TNF-α and IL-6 by THP-1 were decreased in the presence of melanin. Increased mRNA and protein expression of TLR2 and TLR4 occurred upon S. globosa infection in THP-1, whereas the presence of melanin suppressed TLR2 and TLR4. Moreover, TLR2 or TLR4 knockdown showed a trend toward reducing the pernicious effect of S. globosa conidia on THP-1 cells in vitro. ConclusionsCollectively, our results indicated that melanin inhibits the phagocytosis of S. globosa and guards against macrophage attack by providing protection from oxygen- and nitrogen-derived radicals, as well as suppressing the host pro-inflammatory cytokine response (TNF-α and IL-6). Melanin was also involved in modulating TLR2 and TLR4 receptor expression, weakening the killing efficiency of S. globosa.

Toll-like receptor 4 is a master regulator for colorectal cancer growth under high-fat diet by programming cancer metabolism

Although high-fat diet (HFD) has been implicated in the development of colorectal cancer (CRC), the critical signaling molecule that mediates the cancer growth is not well-defined. Identifying the master regulator that controls CRC growth under HFD can facilitate the development of effective therapeutics for the cancer treatment. In this study, the global lipidomics and RNA sequencing data show that, in the tumor tissues of CRC-bearing mouse models, HFD not only increases tumor weight, but also the palmitic acid level and TLR4 expression, which are reduced when HFD is replaced by control diet. These concomitant changes suggest the roles of palmitic acid and TLR4 in CRC growth. Subsequent studies show that palmitic acid regulates TLR4 expression in PU.1-dependent manner. Knockdown of PU.1 or mutations of PU.1-binding site on TLR4 promoter abolish the palmitic acid-increased TLR4 expression. The role of palmitic acid/PU.1/TLR4 axis in CRC growth is further examined in cell model and animal models that are fed either HFD or palmitic acid-rich diet. More importantly, iTRAQ proteomics data show that knockdown of TLR4 changes the metabolic enzyme profiles in the tumor tissues, which completely abolish the HFD-enhanced ATP production and cancer growth. Our data clearly demonstrate that TLR4 is a master regulator for CRC growth under HFD by programming cancer metabolism.

Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells via Src and Sirt1/3 Pathway.

Oxidized low-density lipoprotein (oxLDL) induced a foam-cell-like phenotype of the vascular smooth muscle cells (VSMCs), leading to the inflammatory responses incorporating Toll-like receptor- (Tlr-) mediated cellular alterations. However, the role of Tlr4 in foam cell formation and underlying molecular pathways has not been comprehensively elucidated. To further investigate the mechanism, VSMCs were incubated with different doses of oxLDL, and then, the lipid, reactive oxygen species (ROS) accumulation, Tlr family genes, and the foam cell phenotype were explored. We observed that oxLDL induced foam cell-like phenotype in VSMCs and led to lipid and ROS accumulation in a dose-dependent manner. Furthermore, in the Tlr family, Tlr4 demonstrated the strongest upregulation under oxLDL stimulation. Simultaneously, oxLDL induced activation of Src, higher expression of Nox2, and lower expression of Mnsod, Sirt1, and Sirt3. By interfering the TLR4 expression, the phenotype alteration, lipid accumulation in VSMCs, and Src kinase activation induced by oxLDL were abolished. After interfering Src activation, the oxLDL-induced lipid accumulation and foam cell phenotype in VSMCs were also alleviated. Furthermore, the ROS accumulation, upregulated Nox2 expression, downregulated Sirt1, Sirt3, and Mnsod expression in VSMCs under oxLDL stimulation were also relieved after the knockdown of Tlr4. Additionally, overexpression of Sirt1 and Sirt3 ameliorated the ROS accumulation and foam cell-like marker expression in VSMCs. These results demonstrated that beyond its familiar role in regulating inflammation response, Tlr4 is a critical regulator in oxLDL-induced foam cell formation in VSMCs via regulating Src kinase activation as well as Sirt1 and Sirt3 expression.

Hydroxysafflor Yellow A Inhibits Staphylococcus aureus-Induced Mouse Endometrial Inflammation via TLR2-Mediated NF-kB and MAPK Pathway.

The present study is designed to investigate the effect of hydroxysafflor yellow A (HYA) on Staphylococcus aureus (S. aureus)-induced mouse endometrial inflammation and to explore its molecular mechanism. We established a mouse endometritis model by intrauterine injection of S. aureus and intrauterine injection of HYA for treatment. Immunohistochemistry, immunofluorescence, and Western blot were used to detect protein expression in uterine tissue, and qPCR was used to measure mRNA expression. HYA could significantly weak uterine pathological changes caused by S. aureus and reduce MPO activity, CD45, CD3, and ED-1 protein expression in uterine tissues of S. aureus-infected mice. Similarly, HYA also significantly decreased S. aureus induced the increase in TNF-α, IL-1β, and IL-6 in uterine tissue. In vivo, we found that knockdown of TLR2 was very important could significantly reduce S. aureus induced the elevated expression of TNF-α, IL-1β, and IL-6 in mEECs. Importantly, in terine tissues of S. aureus-infected mice, HYA significantly decreased the ratio of p-p65/p65, p-IKBα/IKBα, p-p38/p38, p-Erk/Erk, and p-JNK/JNK expression. HYA displays anti-inflammatory effects on S. aureus mouse endometrial inflammation, and this effect might be related to HYA which could block TLR2-mediated NF-kB and MAPK pathway.

Ganglioside GT1b increases hyaluronic acid synthase 2 via PI3K activation with TLR2 dependence in orbital fibroblasts from thyroid eye disease patients

Thyroid eye disease (TED) is a complex autoimmune disease with a spectrum of signs. we previously reported that trisialoganglioside (GT)1b is significantly overexpressed in the orbital tissue of TED patients, and that exogenous GT1b strongly induced HA synthesis in orbital fibroblasts. However, the signaling pathway in GT1b-induced hyaluronic acid synthase (HAS) expression in orbital fibroblasts from TED patients have rarely been investigated. Here, we demonstrated that GT1b induced phosphorylation of Akt/mTOR in a dose-dependent manner in orbital fibroblasts from TED patients. Both co-treatment with a specific inhibitor for PI3K and siRNA knockdown of TLR2 attenuated GT1b-induced Akt phosphorylation. GT1b significantly induced HAS2 expression at both the transcriptional and translational level, which was suppressed by specific inhibitors of PI3K or Akt/mTOR, and by siRNA knockdown of TLR2. In conclusion, GT1b induced HAS2 in orbital fibroblasts from TED patients via activation of the PI3K-related signaling pathway, dependent on TLR2.

Serum Amyloid A Aggravates Lipopolysaccharide-Induced Injury of BEAS-2B Cells by Activating Toll-Like Receptor 2/Activator Protein-1 Signaling

The serum amyloid A (sAA) is a common sensitive indicator for the diagnosis of infectious diseases, and sAA levels are increased in pneumonia. However, the detailed molecular mechanism is unknown. Previous studies have demonstrated the participation of Toll-like receptor (TLR) 2 and its downstream protein activator protein-1 (AP-1) in inflammatory lung injury. This study aimed to investigate the effect of sAA on LPS-induced BEAS-2B cells injury and uncover the possible mechanism. The human bronchial epithelial cell line BEAS-2B was exposed to sAA with or without lipopolysaccharide (LPS) treatment, then cell viability, inflammation and apoptosis were evaluated. The effects of TLR2 knockout on sAA + LPS-treated BEAS-2B cells were also determined. Results revealed that sAA treatment reduced cell viability in a concentration-dependent manner and the effect of 500 nM sAA on cell viability was approximately equivalent to LPS. The levels of inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, monocyte chemotactic protein (MCP)-1 and IL-6 as well as cell apoptosis and expression of proteins related to apoptosis were significantly increased upon sAA or LPS stimulation. The expression of TLR2 and AP-1 was also elevated in cells challenged with sAA or LPS. Besides, sAA and LPS co-treatment further enhanced the actions of LPS. However, the knockdown of TLR2 obviously blunted the effects of LPS and sAA co-treatment on cell viability, inflammation and apoptosis. Taken together, our results revealed that sAA could exert an enhanced effect on LPS-induced BEAS-2B cells injury via promoting TLR2/AP-1 expression.

TLR4-MyD88-NF-κB signaling pathway contributes to the progression of secondary hepatic injury and fibrosis in hepatolithiasis

This paper focused on evaluating the effect of TLR4-MyD88-NF-κB signaling pathway in the progression of secondary hepatic injury and fibrosis in hepatolithiasis. The levels of inflammatory factors (IL-1β, IL-6, TNF-α) and serum biochemical values (ALT, AST, Tbil, Dbil, ALP, GGT) were detected by ELISA. IHC was used to detected the expression level of TLR4 in liver tissues of hepatolithiasis patients and mice. The pathological changes of liver tissue were observed by HE staining. The levels of MyD88, NF-κB, IκB, Laminin (LN), and chitosan enzyme 3-like protein 1 (CHI3L1) were detected by western blotting. In hepatolithiasis patients, the levels of TNF-α, IL-1β, and IL-6 were distinctly raised and proteins associated with TLR4-MyD88-NF-κB signaling pathway (such as TLR4, MyD88, NF-κB, and IκB) in liver tissues were significantly up-regulated. In Bile duct ligation (BDL) model of mice, the results showed that in addition to the significant increase of inflammatory factors, liver function indexes, and fibrosis indexes in BDL mice were also significantly up-regulated. Additionally, TLR4-MyD88-NF-κB signaling pathway was activated in BDL mice. After TLR4 knockdown in BDL mice, inflammatory factors, liver function indexes, and fibrosis indexes were significantly down-regulated. TLR4-MyD88-NF-κB signaling pathway proteins were restrained. TLR4-MyD88-NF-κB signaling pathway took part in the progression of secondary hepatic injury and fibrosis in hepatolithiasis. Inhibition of TLR4-MyD88-NF-κB signaling pathway can reduce the progression of secondary hepatic injury and fibrosis in hepatolithiasis.