TLR4-dependent Signaling Pathways Research Articles

Short-term use of ceftriaxone sodium leads to intestinal barrier disruption and ultrastructural changes of kidney in SD rats

Objective Antibiotic treatments are known to disturb gut microbiota, but their effects on the mucosal barrier and extraintestinal diseases are rarely discussed. The aim of this study was to evaluate and visualize the impact of antibiotics on colonic mucus and the microbial community, and to assess whether intestinal dysbacteriosis is involved in the pathogenesis and progression of extraintestinal diseases in vivo. Materials and Methods Twenty-one SD rats were randomly assigned into three groups followed by different experimental treatments. The albumin–creatinine ratio, urinary protein and occult blood semi-quantified test were tested. Fecal samples were collected at different time points (0,4, and 12 weeks) for 16S rRNA gene sequencing. Colon and kidney specimens were examined using light microscopy and transmission electron microscopy (TEM) to identify morphological changes. Results Ceftriaxone intervention for one week did not cause any symptoms of diarrhea or weight loss, but the alpha and beta diversities of gut microbiota decreased quickly and significantly, a lower Firmicutes/Bacteroidetes (F/B) ratio was observed. At week 12, although the alpha and beta diversities increased to a level similar to that of the control (CON) group, LEfSe analysis indicated that the microbial community composition still differed significantly in each group. In addition, KEGG metabolic prediction revealed different metabolic functions in each group. TEM examination of colon revealed that dramatic morphological changes were observed in the ceftriaxone (Cef) group, wherein microvilli were misaligned and shortened significantly and morphologically intact bacteria were seen on the epithelial cell surface. TEM examination of kidneys from the Cef group showed characteristic glomerular changes in the form of widely irregularly thickened glomerular basement membrane (GBM) and foot process fusion or effacement; mild thickening of the GBM and foot process fusion was detected when ceftriaxone and Resatorvid (TAK242, an inhibitor of TLR4 signaling) are used together in the ceftriaxone + TAK242 (TAK) group. Conclusions Short-term use of ceftriaxone induced dynamic changes of gut microbiota and lead to intestinal barrier disruption and ultrastructural changes of kidneys in the SD rats. Moreover, interference with the TLR4-dependent signaling pathway can alleviate the damage to the intestinal barrier and kidney.

Immune-enhancing effects of Hibiscus syriacus roots in RAW264.7 macrcophages

ABSTRACT In this study, we evaluated whether extracts of the roots of Hibiscus syriacus (HSR) exert immune activation activities and elucidated its potential mechanism in macrophages. The HSR dose-dependently increased the production of immunomodulators such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor (TNF-α) activated phagocytosis in macrophages. Inhibition of toll-like receptors 4 (TLR4) reduced the production of immunomodulators induced by HSR. Inhibition of mitogen-activated protein kinase (MAPKs), nuclear factor-κB (NF-κB) and phosphoinositide-3 kinase (PI3K) signaling attenuated the production of immunomodulators induced by HSR. Based on the results of this study, HSR was thought to activate macrophages through the production of immunomodulators and phagocytosis activation through TLR4-dependent MAPKs, NF-κB and PI3K signaling pathways. Therefore, it is thought that the HSR has the potential to be used as agents for enhancing immunity.

Heat shock protein 40 of Streptococcus pneumoniae induces immune response of human dendritic cells via TLR4-dependent p38 MAPK and JNK signaling pathways.

Heat shock protein 40 (HSP40) is a vaccine adjuvant candidate for Streptococcus pneumoniae. The mechanism by which HSP40 activates the human dendritic cells (DCs) is unclear. DCs were isolated from human peripheral blood and their markers (HLA-DR, CD86, CD83, and CD80) were detected by flow cytometry. The messengerRNA (mRNA) and secretion levels of inflammary cytokines were measured after DCs were stimulated with recombinant HSP40 (rHSP40). Short hairpin RNAs were used to knock down toll-like receptor 2 (TLR2) and TLR4. The TLR2- or TLR4-deficient DCs were treated with lipopolysaccharides, rHSP40, or peptidoglycan, and then the secretion levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured. Moreover, the secretion levels of TNF-α and IL-6 were measured after DCs were treated with mitogen-activated protein kinase (MAPK) inhibitors including SB203580, SP600125, and U0126. In addition, the phosphorylation levels of p38 MAPK and Jun N-terminal kinase (JNK) in DC cells were determined using western blot analysis after treatment with rHSP40 for different times. DCs were successfully isolated and cultured. rHSP40 treatment significantly increased cytokine levels in a concentration-dependent manner. TLR4 deficiency, but not TLR2 deficiency, significantly suppressed the rHSP40-induced secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). SB203580 and SP600125 significantly inhibited the rHSP40-induced secretion of TNF-α and IL-6. rHSP40 significantly enhanced the phosphorylation of p38 MAPK and JNK. HPS40 stimulates the immune response of DCs via the p38 MAPK and JNK signaling pathways, which depend on TLR4.

Alhagi pseudalhagi Extract Exerts Protective Effects Against Intestinal Inflammation in Ulcerative Colitis by Affecting TLR4-Dependent NF-κB Signaling Pathways.

Alhagi pseudalhagi Desv. Extract (APE) is the major active fraction extracted from the aerial part of Alhagi pseudalhagi Desv. In view of its application in Uyghur medicine, it may be beneficial for the treatment of ulcerative colitis (UC). The aim of the present study was to investigate the possible beneficial effects of APE on UC mice and detect the possible mechanisms underlying these effects. Methods: An acute UC model was established in mice using dextran sulfate sodium. Sixty mice were randomly divided into six groups: normal, UC model, sulfasalazine (200 mg/kg), high-dose APE (APE-H, 2.82 g/kg), middle-dose APE (APE-M, 1.41 g/kg), and low-dose APE (APE-L, 0.70 g/kg) groups. Drugs were administered by gavage for 10 days after the induction of colitis. Serum and colon tissue samples were collected from the mice during the experiment, and survival signs, body weight changes, disease activity index (DAI), colon length, and colon wet weight in mice were determined after the treatment. UC-induced damage, including inflammation and ulceration of colon mucosa, were observed by the naked eye as well as using hematoxylin and eosin staining (H&E) and scanning electron microscopy and scored according to Wallace and Keean’s criteria. We measured the levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, and IL-10 in the serum and colon tissues using ELISA. Additionally, the relative protein levels of toll-like receptor 4 (TLR4), nuclear factor-kappa B p65 (NF-κB p65), phosphorylated NF-κB p65 at Ser536 (p-p65 Ser536), inhibitor kappa B-kinase ß (IK-Kβ), and phosphorylated IK-Kβ (Ser176/180) (p-IK-Kβ) in colonic mucosal epithelial tissues were detected using western blotting. The main functional components of APE were analyzed and confirmed by UPLC-MS/MS. Results: APE treatment repaired the UC-induced colon mucosa injury, reduced the weight loss, attenuated DAI, colon macroscopic damage index, and histological inflammation, and significantly downregulated the levels of inflammatory markers, including TNF-α, IL-1β, and IL-6, in the serum and colon tissues. Additionally, APE treatment reduced the levels of TLR4 and phosphorylation of p-NF-κB and p-IK-Kβ. The main components of APE are taxifolin, 3,5-dihydroxy-2-(4-hydroxyphenyl)-7-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) oxan-2-yl] oxychromen-4-one, hyperoside, rutin, kaempferol, isorhamnetin, 7,8-dihydroxyflavone, and kaempferide. Conclusions: To the best of our knowledge, the present study is first to demonstrate that APE exerts a protective effect against intestinal inflammation in UC by affecting TLR4-dependent NF-κB signaling pathways.

Interplay between NOD1 and TLR4 Receptors in Macrophages: Nonsynergistic Activation of Signaling Pathways Results in Synergistic Induction of Proinflammatory Gene Expression.

Interactions between pattern-recognition receptors shape innate immune responses to pathogens. NOD1 and TLR4 are synergistically interacting receptors playing a pivotal role in the recognition of Gram-negative bacteria. However, mechanisms of their cooperation are poorly understood. It is unclear whether synergy is produced at the level of signaling pathways downstream of NOD1 and TLR4 or at more distal levels such as gene transcription. We analyzed sequential stages of human macrophage activation by a combination of NOD1 and TLR4 agonists (N-acetyl-d-muramyl-l-alanyl-d-isoglutamyl-meso-diaminopimelic acid [M-triDAP] and LPS, respectively). We show that events preceding or not requiring activation of transcription, such as activation of signaling kinases, rapid boost of glycolysis, and most importantly, nuclear translocation of NF-κB, are regulated nonsynergistically. However, at the output of the nucleus, the combination of M-triDAP and LPS synergistically induces expression of a subset of M-triDAP- and LPS-inducible genes, particularly those encoding proinflammatory cytokines (TNF, IL1B, IL6, IL12B, and IL23A). This synergistic response develops between 1 and 4 h of agonist treatment and requires continuous signaling through NOD1. The synergistically regulated genes have a lower basal expression and higher inducibility at 4 h than those regulated nonsynergistically. Both gene subsets include NF-κB-inducible genes. Therefore, activation of the NF-κB pathway does not explain synergistic gene induction, implying involvement of other transcription factors. Inhibition of IKKβ or p38 MAPK lowers agonist-induced TNF mRNA expression but does not abolish synergy. Thus, nonsynergistic activation of NOD1- and TLR4-dependent signaling pathways results in the synergistic induction of a proinflammatory transcriptional program.

Cinnamaldehyde Treatment of Prostate Cancer-Associated Fibroblasts Prevents Their Inhibitory Effect on T Cells Through Toll-Like Receptor 4.

IntroductionCancer-associated fibroblasts (CAFs) promote tumor progression; thus, drugs that can modify CAFs need to be identified.MethodsTo test the effect of cinnamaldehyde on prostate CAFs, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-2H-tetrazolium bromide assay was used to determine their survival. When spleen cells were treated with CAF supernatant, the proliferation of T cells was inhibited as determined by flow cytometry. After cinnamaldehyde treatment, this immunosuppressive function of CAFs was partially reversed. To explore the molecular mechanism, Western blotting and the quantitative real-time polymerase chain reaction were applied, and TLR4-dependent signaling pathway-related protein and mRNA levels were quantified.ResultsCinnamaldehyde acted on the TLR4-dependent signaling pathway, altering the function of CAFs such that its supernatant no longer inhibited the proliferation of T cells.ConclusionThese data indicate that cinnamaldehyde can modify the functions of CAFs, which may be helpful for treating tumors. Cinnamaldehyde can suppress CAF T-cell inhibition.

Bivalent therapeutic vaccine against HPV16/18 genotypes consisting of a fusion protein between the extra domain A from human fibronectin and HPV16/18 E7 viral antigens.

BackgroundIn vivo targeting of human papillomavirus (HPV) derived antigens to dendritic cells might constitute an efficient immunotherapeutic strategy against cervical cancer. In previous works, we have shown that the extra domain A from murine fibronectin (mEDA) can be used to target antigens to toll-like receptor 4 (TLR4) expressing dendritic cells and induce strong antigen-specific immune responses. In the present study, we have produced a bivalent therapeutic vaccine candidate consisting of the human EDA (hEDA) fused to E7 proteins from HPV16 and HPV18 (hEDA-HPVE7-16/18) and evaluate its potential as a therapeutic vaccine against cervical cancer.Materials and methodsRecombinant fusion proteins containing HPV E7 proteins from HPV16 and HPV18 virus subtypes fused to hEDA were produced and tested in vitro on their capacity to bind TLR4 and induce the production of tumor necrosis factor-α or interleukin (IL)-12 by human monocytes and dendritic cells. The immunogenicity and potential therapeutic activity of the vaccine in combination with cisplatin or with the TLR3 agonist molecules polyinosinic‐polycytidylic acid (Poly IC) or Poly ICLC was evaluated in mice bearing subcutaneous or genital orthotopic HPV16 TC-1 tumors.ResultshEDA-HPVE7-16/18 prototype vaccine binds human TLR4 and stimulate TLR4-dependent signaling pathways and IL-12 production by human monocyte-derived dendritic cell. Vaccination with hEDA-HPVE7-16/18 induced strong HPVE7-specific Cytotoxic T lymphocyte (CTL) responses and eliminated established tumors in the TC-1-based tumor model. The antitumor efficacy was significantly improved by combining the fusion protein with cisplatin or with the TLR-3 ligand Poly IC and especially with the stabilized analog Poly ICLC. Moreover, hEDA-HPVE7-16/18+Poly ICLC induced full tumor regression in 100% of mice bearing orthotopic genital HPV tumors.ConclusionOur results suggest that this therapeutic vaccine formulation may be an effective treatment for cervical tumors that do not respond to current therapies.

PPAR γ agonist, pioglitazone, suppresses melanoma cancer in mice by inhibiting TLR4 signaling.

Although previous studies demonstrated an anticancer effect for the ligands of peroxisome proliferator-activated receptor gamma (PPARγ) through activation of its anti-inflammatory responses, nevertheless the anti-tumor mechanism of PPARγ has not been intensively investigated. One of the molecules involved in cancer progression is toll-like receptor 4 (TLR4). B16F10 melanoma cells were cultured with or without LPS for 24 hr. The cells were subcutaneously injected to two groups of C57BL/6 mice. After the development of palpable tumors each group of animals were divide to four sub-groups and received pioglitazone in different dose ranges (0,10,50,100 mg/kg/day) for 10 days. At the end of the study, the expression ofTlr4, Myd-88, Nf-kb1 genes was evaluated by qRT-PCR in different groups in mice tumor. The TLR-4 protein expression was evaluated by IHC. TNF-α level in mice tumor and serum were measured byELISAkits. Tumor volume was measured with Vernier calipers. We observed that activation of PPARγ by its agonist, pioglitazone, reduces tumor volume, Tlr-4, Myd-88, Nf-kb1 mRNA expression, TLR4 protein expression and TNF-α production in melanoma tumor especially in groups that were injected with LPS -stimulated cells. Moreover, treatment of melanoma cells with pioglitazone showed that the inhibitory effects of pioglitazone on LPS-induced inflammatory responses were TLR4 dependent. The results indicate that pioglitazone, a PPARγ agonist, has a beneficial protective effect against melanoma via interfering with the TLR4-dependent signaling pathways.

The effect of fenofibrate, a PPARα activator on toll-like receptor-4 signal transduction in melanoma both in vitro and in vivo.

The anti-cancer effect of peroxisome proliferator-activated receptor (PPAR) α ligands on growth and metastatic potential of melanoma cells has been shown previously. However, themechanismunderlying these effects remains to be elucidated. Here, we investigated the effects of fenofibrate (PPAR ligand) on Toll-like receptor-4 (TLR-4) signaling in mice melanoma. Mice melanoma cells (B16F10) were treated with fenofibrate or LPS or LPS + fenofibrate or pre-treated with CLI-095 (a TLR4 inhibitor), followed by fenofibrate. In in vivo model, C57BL/6 mice were subcutaneously injected with B16F10 cells (with/without LPS pre-treatment), and fenofibrate was administrated after development of palpable tumors. Cell proliferation, the expression level of Tlr4, Myd88, Nf-κb1 genes, TLR-4 protein expression, TNF-α levels, and tumor volume were measured. Our results indicated that fenofibrate significantly inhibited the Tlr-4, Myd-88, and Nf-kb1 mRNA expression and TNF-α concentration in B16F10 LPS-stimulated cells. In addition, blocking TLR-4 signaling increased the anti-inflammatory potential of fenofibrate. Also fenofibrate can reduce LPS-induced tumor volume, Tlr-4, Myd-88, Nf-kb1 mRNA, and TLR-4 protein expression in tumor tissue and also TNF-α level in tumor tissue lysate. Our data indicate that fenofibrate may exert its anti-melanoma effects via interaction with TLR4-dependent signaling pathway (TLR-4/MyD-88/ NF-kB).

FimH as a mucosal adjuvant enhances persistent antibody response and protective efficacy of the anti-caries vaccine

ObjectiveTo investigate whether the recombinant FimH-S.T protein could modulate immune response to anti-caries vaccine in vitro and in vivo. DesignRecombinant FimH protein derived from Salmonella was constructed and purified. The expression of dendritic cell maturation markers and cytokines release were performed by flow cytometry, Real-time PCR and ELISA. In addition, BALB/c mice were administered with anti-caries PAc vaccine plus FimH-S.T, antibody responses were evaluated by ELISA. Splenocytes of immunized mice were detected for their proliferative ability in response to in vitro retreatment with PAc antigen by flow cytometry. Caries protection against dental caries formation was also investigated. ResultsThe purified FimH-S.T induced phenotypic maturation of DC2.4 by up-regulating the expression of costimulatory molecules and MHC II, provoked the production and secretion of cytokines via TLR4-dependent signaling pathway in vitro. Furthermore, the mice immunized with the mixture of FimH-S.T and PAc significantly enhanced the PAc-specific antibodies in the serum along with saliva and promoted splenocyte proliferation. Our results also confirmed that PAc+FimH-S.T decreased the caries lesions formation which provided high protective efficacy against dental caries. ConclusionOur study demonstrates that recombinant FimH-S.T could enhance specific IgA responses and protection of anti-caries vaccine, possessing mucosal adjuvant ability by activating DC2.4 via TLR4 signaling pathway.

Epigallocatechin-3-gallate ameliorates lipopolysaccharide-induced acute lung injury by suppression of TLR4/NF-κB signaling activation.

Acute lung injury (ALI) is a serious clinical syndrome with a high rate of mortality. The activation of inflammation is well-recognized as a vital factor in the pathogenesis of lipopolysaccharide (LPS)-induced ALI. Therefore, suppression of the inflammatory response could be an ideal strategy to prevent ALI. Epigallocatechin-3-gallate (EGCG), mainly from green tea, has been shown to have an anti-inflammatory effect. The aim of the study was to explore whether EGCG alleviates inflammation in sepsis-related ALI. Male BALB/C mice were treated with EGCG (10 mg/kg) intraperitoneally (ip) 1 h before LPS injection (10 mg/kg, ip). The results showed that EGCG attenuated LPS-induced ALI as it decreased the changes in blood gases and reduced the histological lesions, wet-to-dry weight ratios, and myeloperoxidase (MPO) activity. In addition, EGCG significantly decreased the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the lung, serum, and bronchoalveolar lavage fluid, and alleviated the expression of TLR-4, MyD88, TRIF, and p-p65 in the lung tissue. In addition, it increased the expression of IκB-α and had no influence on the expression of p65. Collectively, these results demonstrated the protective effects of EGCG against LPS-induced ALI in mice through its anti-inflammatory effect that may be attributed to the suppression of the activation of TLR 4-dependent NF-κB signaling pathways.

Biglycan is a new high-affinity ligand for CD14 in macrophages

Sterile inflammation is a therapeutic target in many diseases where it represents an important initiator of disease progression. However, the detailed mechanisms underlying its evolution and biological relevance are not yet completely elucidated. Biglycan, a prototype extracellular matrix-derived damage-associated molecular pattern, mediates sterile inflammation in macrophages through Toll-like receptor (TLR) 2 and/or TLR4-dependent signaling pathways. Here we discovered that soluble biglycan is a novel high-affinity ligand for CD14, a well-known GPI-anchored co-receptor for TLRs. CD14 is required for all biglycan-mediated TLR2/4 dependent inflammatory signaling pathways in macrophages. By binding to CD14 and choosing different TLR signaling branches, biglycan induced TNF-α and CCL2 via TLR2/4, HSP70 through TLR2, and CCL5 via TLR4. Mechanistically, biglycan evoked phosphorylation and subsequent nuclear translocation of p38, p44/42, and NF-κB, and these effects were due to a specific, high-affinity interaction between biglycan protein core and CD14. Finally, we provide proof-of-principle for the requirement of CD14, by transiently overexpressing biglycan in a mouse model of renal ischemia/reperfusion injury performed in Cd14−/− mice. Lack of Cd14 prevented biglycan-mediated cytokine expression, recruitment of macrophages, M1 macrophage polarization as well as mitigated the tubular damage and serum creatinine levels, thereby improving renal function. Thus, CD14 inhibition could lead to the reduction in the activation of biglycan-TLR2/4 signaling pathways and could be a novel therapeutic approach in inflammatory kidney diseases.

Compound A attenuates toll-like receptor 4-mediated paclitaxel resistance in breast cancer and melanoma through suppression of IL-8

BackgroundPaclitaxel (PTX) is a potent anti-cancer drug commonly used for the treatment of advanced breast cancer (BCA) and melanoma. Toll-like receptor 4 (TLR4) promotes the production of pro-inflammatory cytokines associated with cancer chemoresistance. This study aims to explore the effect of TLR4 in PTX resistance in triple-negative BCA and advanced melanoma and the effect of compound A (CpdA) to attenuate this resistance.MethodsBCA and melanoma cell lines were checked for the response to PTX by cytotoxic assay. The response to PTX of TLR4-transient knockdown cells by siRNA transfection was evaluated compared to the control cells. Levels of pro-inflammatory cytokines, IL-6 and IL-8, and anti-apoptotic protein, XIAP were measured by real-time PCR whereas the secreted IL-8 was quantitated by ELISA in TLR4-transient knockdown cancer cells with or without CpdA treatment. The apoptotic cells after adding PTX alone or in combination with CpdA were detected by caspase-3/7 assay.ResultsPTX could markedly induce TLR4 expression in both MDA-MB-231 BCA and MDA-MB-435 melanoma cell lines having a basal level of TLR4 whereas no significant induction in TLR4-transient knockdown cells occurred. The siTLR4-treated BCA cells revealed more dead cells after PTX treatment than that of mock control cells. IL-6, IL-8 and XIAP showed increased expressions in PTX-treated cells and this over-production effect was inhibited in TLR4-transient knockdown cells. Apoptotic cells were detected higher when PTX and CpdA were combined than PTX treatment alone. Isobologram exhibited the synergistic effect of CpdA and PTX. CpdA could significantly decrease expressions of IL-6, XIAP and IL-8, as well as excreted IL-8 levels together with reduced cancer viability after PTX treatment.ConclusionsThe acquired TLR4-mediated PTX resistance in BCA and melanoma is explained partly by the paracrine effect of IL-6 and IL-8 released into the tumor microenvironment and over-production of anti-apoptotic protein, XIAP, in BCA cells and importantly CpdA could reduce this effect and sensitize PTX-induced apoptosis in a synergistic manner. In conclusion, the possible impact of TLR4-dependent signaling pathway in PTX resistance in BCA and melanoma is proposed and using PTX in combination with CpdA may attenuate TLR4-mediated PTX resistance in the treatment of the patients.

Alterations in the gut barrier and involvement of Toll-like receptor 4 in murine postoperative ileus.

The changes in the gut barrier (GB) and associated mechanisms in postoperative ileus (POI) are still unclear. Toll-like receptor 4 (TLR4) is involved in inflammation, which may cause GB dysfunction and POI. Here, the roles of the GB in POI in relation to TLR4-dependent signaling pathways were explored. POI was induced by small bowel manipulation in wild-type (WT) and TLR4-knockout (TLR4-/-) mice. Twenty-four hours after manipulation, indices of gastrointestinal (GI) transit, GB structure and function, inflammation, and related signaling pathways were analyzed. Normal GI motility and GB function were not affected by TLR4 knockout. Compared with WT POI mice, TLR4-/-POI mice showed milder GI transit retardation, GB dysfunction, and inflammatory responses. In WT mice, GB disorder was characterized by colonic goblet cells depletion, increased gut claudin-2 expression, and decreased CD4+/CD8+ ratios in intestinal Peyer's patches. Green fluorescent protein-tagged Escherichia coli in the gut was detected in plasma and extraintestinal organs, followed with increased plasma lipopolysaccharide. These changes displayed in WT POI mice were less severe in TLR4-/-POI mice. Furthermore, the mRNA and protein expression of interleukin-6, monocyte chemotactic protein-1, pp38 and pJNK in the intestine, and TNF-α level in plasma were significantly increased in WT, but not TLR4-/-POI mice. These results indicate that GB is impaired in the experimental POI, with inflammation being involved in this pathological process. TLR4 deficiency alleviated GB dysfunction and suppressed inflammation by disrupting the activation of mitogen-activated protein kinase signaling pathways, thereby ameliorating POI.

TLR2 activation induced by H. pylori LPS promotes the differential expression of claudin-4, -6, -7 and -9 via either STAT3 and ERK1/2 in AGS cells

Gastric carcinogenesis has been associated to H. pylori virulence factors that induce a chronic inflammation process. Lipopolysaccharides play a role in chronic inflammatory responses via TLR2- and TLR4-dependent signaling pathways. Similarly, cellular invasiveness, metastatic potential and prognosis are usually associated to claudin-4, -6, -7 and -9 expression in gastric carcinogenesis. Therefore, the aim of this study was to determine if H. pylori LPS exerts an influence on carcinogenesis-related claudin expression and if it was directly regulated through the TLR2 pathway. Human antrum gastric adenocarcinoma AGS cells exposed or not to H. pylori LPS were used. Polyclonal anti-claudin-4, -6, -7 and -9, anti-TLR2, anti-pERK1/2 as well as rabbit monoclonal anti-pNFκB p65 and mouse monoclonal anti-CdX2 were used. ERK1/2 inhibitor UO126 and STAT3 inhibitor Stattic were also used. Western blot, immunofluorescence and confocal experiments were performed in whole cells as well as total protein, nuclear and cell membrane fractions. The results showed that H. pylori LPS increased the expression of TLR2 in a time dependent bi-phasic manner (<12 and >12h exposure). Immunofluorescence using AGS monolayers corroborated the double phase TLR2 expression mainly on the cell membrane but a detectable signal was also determined in the cytoplasm of the cells. Activation of NFkB was downstream and depended on TLR2 expression as a statistically significant increase in pNFkB, that followed a pattern highly similar to the TLR2 expression was observed on the cell membrane fraction. The increase in TLR2 expression was accompanied by dramatically increased claudin-4 expression in cultures exposed from 30m to 8h to LPS. Increased expression of claudin-6, -7 and -9 also increases in >12h LPS exposure times. The increase in claudins expression was also dependent on NFkB activation. The results also showed an increase in pSTAT3 that followed a bi-phasic pattern that began 30min after stimulation and was compatible with the increase in TLR2 expression. The expression of the claudin-4 related CDX2 transcription factor did not followed the biphasic pattern. The results also showed that claudin-4 expression was STAT3 dependent whereas claudin-6, 7 and 9 expressions was ERK1/2 dependent. Our results suggest that H. pylori LPS induces TLR2 expression in the AGS cells, and that the longer the exposure to LPS, the greater the expression of TLR2 in the cell membrane. Consequently the expression of claudin-4, -6, -7 and -9 also increases.

Core fucose is critical for CD14-dependent Toll-like receptor 4 signaling.

Core fucosylation, a posttranslational modification of N-glycans, modifies several growth factor receptors and impacts on their ligand binding affinity. Core-fucose-deficient mice generated by ablating the α1,6 fucosyltransferase enzyme, Fut8, exhibit severe pulmonary emphysema, partly due to impaired macrophage function, similar to aged Toll-like receptor 4 (Tlr4)-deficient mice. We therefore suspect that a lack of core fucose affects the TLR4-dependent signaling pathway. Indeed, upon lipopolysaccharide stimulation, Fut8-deficient mouse embryonic fibroblasts (MEFs) produced similar levels of interleukin-6 but markedly reduced levels of interferon-β (IFN-β) compared with wild-type MEFs. Lectin blot analysis of the TLR4 signaling complex revealed that core fucosylation was specifically found on CD14. Even though similar levels of TLR4/myeloid differentiation factor 2 (MD2) activation and dimerization were observed in Fut8-deficient cells after lipopolysaccharide stimulation, internalization of TLR4 and CD14 was significantly impaired. Given that internalized TLR4/MD2 induces IFN-β production, impaired IFN-β production in Fut8-deficient cells is ascribed to impaired TLR4/MD2 internalization. These data show for the first time that glycosylation critically regulates TLR4 signaling.

Oxymatrine attenuates CCl4-induced hepatic fibrosis via modulation of TLR4-dependent inflammatory and TGF-β1 signaling pathways

Oxymatrine (OMT) is able to effectively protect against hepatic fibrosis because of its anti-inflammatory property, while the underlying mechanism remains incompletely understood. In this study, forty rats were randomly divided into five groups: control group, model group (carbon tetrachloride, CCl4) and three OMT treatment groups (30, 60, 120mg/kg). After CCl4 alone, the fibrosis score was 20.2±0.8, and the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hydroxyproline content, and collagen I expression was elevated, but OMT blunted these parameters. Treatment with OMT prevented CCl4-induced increases in expression of pro-inflammatory and pro-fibrotic cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α, meanwhile OMT promoted the expression of anti-inflammatory and anti-fibrotic factors such as interleukin (IL)-10 and bone morphogenetic protein and activin membrane-bound inhibitor (Bambi). Moreover, lipopolysaccharides (LPS) and high mobility group box-1 (HMGB1), which activates Toll-like receptor 4 (TLR4) and modulate hepatic fibrogenesis through hepatic stellate cells (HSCs) or Kupffer cells, were significantly decreased by OMT treatment. These results were further supported by in vitro data. First, OMT suppressed the expression of TLR4 and its downstream pro-inflammatory cytokines, lowered the level of HMGB1, TGF-β1 in macrophages. Then, OMT promoted Bambi expression and thereby inhibited activation of HSCs mediated by transforming growth factor (TGF)-β1. In conclusion, this study showed that OMT could effectively attenuate the CCl4-induced hepatic fibrosis, and this effect may be due to modulation of TLR4-dependent inflammatory and TGF-β1 signaling pathways.

Linoleic acid and stearic acid elicit opposite effects on AgRP expression and secretion via TLR4-dependent signaling pathways in immortalized hypothalamic N38 cells

The regulation of food intake is a promising way to combat obesity. It has been implicated that various fatty acids exert different effects on food intake and body weight. However, the underlying mechanism remains poorly understood. The aim of the present study was to investigate the effects of linoleic acid (LA) and stearic acid (SA) on agouti-related protein (AgRP) expression and secretion in immortalized mouse hypothalamic N38 cells and to explore the likely underlying mechanisms. Our results demonstrated that LA inhibited, while SA stimulated AgRP expression and secretion of N38 cells in a dose-dependent manner. In addition, LA suppressed the protein expression of toll-like receptor 4 (TLR4), phosphorylation levels of JNK and IKKα/β, suggesting the inhibition of TLR4-dependent inflammation pathway. However, the above mentioned inhibitory effects of LA were eliminated by TLR4 agonist lipopolysaccharide (LPS). In contrast, SA promoted TLR4 protein expression and activated TLR4-dependent inflammation pathway, with elevated ratio of p-JNK/JNK. While TLR4 siRNA reversed the stimulatory effects of SA on AgRP expression and TLR4-dependent inflammation. Moreover, we found that TLR4 was also involved in LA-enhanced and SA-impaired leptin/insulin signal pathways in N38 cells. In conclusion, our findings indicated that LA elicited inhibitory while SA exerted stimulatory effects on AgRP expression and secretion via TLR4-dependent inflammation and leptin/insulin pathways in N38 cells. These data provided a better understanding of the mechanism underlying fatty acids-regulated food intake and suggested the potential role of long-chain unsaturated fatty acids such as LA in reducing food intake and treating obesity.

TLR4‑dependent signaling pathway modulation: A novel mechanism by which pioglitazone protects against nutritional fibrotic steatohepatitis in mice.

Activation of the innate immune system is involved in the development of chronic liver diseases, including nonalcoholic steatohepatitis. Toll‑like receptor 4 (TLR4) is one of the sensors of the innate immune system. The aim of the present study was to elucidate the role of the TLR4‑dependent signaling pathway, and examine the effect of pioglitazone on hepatic fibrosis, through modulation of the TLR4 pathway in a mouse model of nutritional fibrotic steatohepatitis. Male C57BL/6J mice were fed a methionine‑choline deficient (MCD) diet for 8 weeks to induce nonalcoholic fibrotic steatohepatitis. The PPARγ agonist, pioglitazone, and PPARγ inhibitor, GW9662, were administered to the mice, respectively. The effects of the induction of PPARγ on liver biochemistry and histology, the modulation of TLR4 and its downstream pathway, and the expression levels of inflammatory and fibrogenic genes were assessed using reverse transcription‑quantitative polymerase chain reaction and Western blot analyses. The MCD‑fed mice exhibited progressive hepatic steatosis, necrotic inflammation and fibrosis, along with increase levels of serum alanine aminotransferase and aspartate aminotransferase, accompanied by the upregulation of TLR4, the TLR4‑myeloid differentiation primary response gene 88‑dependent pathway and downstream genes, and proinflammatory and profibrotic genes; and downregulation of basic membrane protein and activin membrane‑bound inhibitor. The administration of pioglitazone was found to reverse hepatic nutritional fibrosis via restoration of the expression levels of proinflammatory and profibrotic genes in the MCD‑fed mice. The results of the present study provide novel evidence supporting the protective role of pioglitazone in ameliorating nutritional fibrotic steatohepatitis, through modulation of the TLR4‑mediated signaling pathway.

Asian sand dust increases MUC8 and MUC5B expressions via TLR4-dependent ERK2 and p38 MAPK in human airway epithelial cells.

Asian sand dust (ASD) is a natural phenomenon and originates from the deserts of China and is known to contain various chemical and biomolecular components that enhance airway inflammation. The overproduction of airway mucins is an important pathologic finding in inflammatory airway diseases. However, the mechanism of ASD on mucin production of airway epithelial cells has not been elucidated. To investigate the effect and signaling pathway of ASD on mucin expressions in human airway epithelial cells. In the NCI-H292 cells and the primary cultures of human nasal epithelial cells, the effect and signaling pathway of ASD on MUC8 and MUC5B expressions were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). ASD increased MUC8 and MUC5B expressions and activated the phosphorylations of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). U0126 (ERK1/2 MAPK inhibitor) and SB203580 (p38 MAPK inhibitor) inhibited ASD-induced MUC8 and MUC5B expressions. In addition, knockdowns of ERK2 and p38 MAPK by siRNA blocked ASD-induced MUC8 and MUC5B mRNA expressions. Toll-like receptor 4 (TLR4) mRNA expression was increased after treatment with ASD. Knockdown of TLR4 by siRNA blocked ASD-induced MUC8 and MUC5B mRNA expressions. Furthermore, the phosphorylations of ERK1/2 and p38 MAPK were blocked by knockdown of TLR4. These results show that ASD induces MUC8 and MUC5B expressions via TLR4-dependent ERK2 and p38 MAPK signaling pathway in human airway epithelial cells.