TLR4 mRNA Levels Research Articles

Vitamin K2 can suppress the expression of Toll-like receptor 2 (TLR2) and TLR4, and inhibit calcification of aortic intima in ApoE-/- mice as well as smooth muscle cells.

Background and objectives Vascular calcification is a common complication in atherosclerosis. Accumulating evidence showed that Toll-like receptors (TLRs) mediate pro-inflammatory and atherosclerosis. Recent studies demonstrated that vascular calcification is one of the detrimental effects of vitamin K (Vit K) antagonists. However, the effects of Vit K on the expression of TLR2 and 4 and intimal calcification in artery remained unidentified. Methods and results Eighteen ApoE-/- mice were randomly divided into model group, Vit K-treated group, and control group. The mice of model and Vit K-treated group were fed with high-fat diet, while control group mice were fed with normal diet. Mice of Vit K-treated group were administered orally with vitamin K2 (40 mg.kg-1.day-1) for 12 weeks. Twelve weeks later the aortic sections of mice were acquired and stained with hematoxylin and eosin and von Kossa, respectively. Calcium content and activity of alkaline phosphatase (ALP) at aortic tissues were measured. The expression levels of TLR2 and TLR4 in aorta sections were detected by immunohistochemisty and RT-PCR, respectively. The effects of Vit K on cellular calcification were further studied in A7r5 SMCs. Results demonstrated that high-fat diet induced typical atherosclerosis with intimal calcification in ApoE-/- mice, while in Vit K-treated group atherosclerosis and calcium deposits were not serious; Vit K2 also inhibited cellular calcification in A7r5 SMCs. Quantitative analysis showed that calcium and ALP activity at aortic tissues in the Vit K-treated mice were significantly lower than that of the model group ( P < 0.01); Compared to the control group, the expression levels of TLR2 and TLR4 in the model group were significantly higher ( P < 0.05), while in Vit K-treated group the levels of TLR2 and 4 were significantly lower than that in the model group. Furthermore, the content of calcium was positively related to the expression levels of TLR2 and TLR4 mRNA at aortic tissues ( r = 0.77 and r = 0.79, respectively, both P < 0.001). Conclusion VitK2 can inhibit intimal calcification of aortic artery induced by high-fat diet in ApoE-/- mice and A7r5 SMCs calcification induced by β-sodium glycerophosphate, and meanwhile can reduce the expression of TLR2 and TLR4. These results suggested that the effects of VitK2 on vascular calcification may be associated with the expression of TLR2 and TLR4.

Exosomes from NSC-34 Cells Transfected with hSOD1-G93A Are Enriched in miR-124 and Drive Alterations in Microglia Phenotype.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder affecting motor neurons (MNs). Evidences indicate that ALS is a non-cell autonomous disease in which glial cells participate in both disease onset and progression. Exosomal transfer of mutant copper-zinc superoxide dismutase 1 (mSOD1) from cell-to-cell was suggested to contribute to disease dissemination. Data from our group and others showed that exosomes from activated cells contain inflammatory-related microRNAs (inflamma-miRNAs) that recapitulate the donor cell. While glia-derived exosomes and their effects in neurons have been addressed by several studies, only a few investigated the influence of motor neuron (MN)-derived exosomes in other cell function, the aim of the present study. We assessed a set of inflamma-miRs in NSC-34 MN-like cells transfected with mutant SOD1(G93A) and extended the study into their derived exosomes (mSOD1 exosomes). Then, the effects produced by mSOD1 exosomes in the activation and polarization of the recipient N9 microglial cells were investigated. Exosomes in coculture with N9 microglia and NSC-34 cells [either transfected with either wild-type (wt) human SOD1 or mutant SOD1(G93A)] showed to be transferred into N9 cells. Increased miR-124 expression was found in mSOD1 NSC-34 cells and in their derived exosomes. Incubation of mSOD1 exosomes with N9 cells determined a sustained 50% reduction in the cell phagocytic ability. It also caused a persistent NF-kB activation and an acute generation of NO, MMP-2, and MMP-9 activation, as well as upregulation of IL-1β, TNF-α, MHC-II, and iNOS gene expression, suggestive of induced M1 polarization. Marked elevation of IL-10, Arginase 1, TREM2, RAGE, and TLR4 mRNA levels, together with increased miR-124, miR-146a, and miR-155, at 24 h incubation, suggest the switch to mixed M1 and M2 subpopulations in the exosome-treated N9 microglial cells. Exosomes from mSOD1 NSC-34 MNs also enhanced the number of senescent-like positive N9 cells. Data suggest that miR-124 is translocated from the mSOD1 MNs to exosomes, which determine early and late phenotypic alterations in the recipient N9-microglial cells. In conclusion, modulation of the inflammatory-associated miR-124, in mSOD1 NSC-34 MNs, with potential benefits in the cargo of their exosomes may reveal a promising therapeutic strategy in halting microglia activation and associated effects in MN degeneration.

Ameliorating the Effect of Pioglitazone on LPS-Induced Inflammation of Human Oligodendrocyte Progenitor Cells.

Oligodendrocyte progenitor cells (OPCs) are appropriate model cells for studying the progress of neurodegenerative disorders and evaluation of pharmacological efficacies of small molecules for treatment of these disorders. Here, we focused on the therapeutic role of Pioglitazone, which is a selective agonist of peroxisome proliferator-activated receptor gamma (PPARγ), a respective nuclear receptor in inflammatory responses. Human embryonic stem cell-derived OPCs were pretreated by Pioglitazone at differing concentrations. Pretreated OPCs were further examined after induction of inflammation by LPS. Interestingly, Pioglitazone reversed the inflammatory conditions and enhanced OPC viability. Data showed that Pioglitazone reduced Nitric Oxide (NO) production. Moreover, Pioglitazone enhanced cell viability through distinct mechanisms including reduction of apoptosis and regulation of cell cycle markers. This study demonstrated that NO induces apoptosis through FOXO1 and degradation of β-catenin, while the presence of Pioglitazone inhibited these effects in rescuing human OPCs from apoptosis. Also, Pioglitazone did not show a significant influence on mRNA levels of TLR2, TRL4, and TNFα. Furthermore, simultaneous treatment of Pioglitazone with CHIR, a GSKβ inhibitor, facilitated anti-apoptotic responses of OPCs. Taken together, therapy with Pioglitazone represents a novel potential drug in alleviating the loss of OPCs in neurodegenerative conditions.

Identification of a TLR2-regulated gene signature associated with tumor cell growth in gastric cancer

Toll-like receptors (TLRs) are key regulators of innate immune responses, and their dysregulation is observed in numerous inflammation-associated malignancies, including gastric cancer (GC). However, the identity of specific TLRs and their molecular targets which promote the pathogenesis of human GC is ill-defined. Here, we sought to determine the clinical utility of TLR2 in human GC. TLR2 mRNA and protein expression levels were elevated in >50% of GC patient tumors across multiple ethnicities. TLR2 was also widely expressed among human GC cell lines, and DNA microarray-based expression profiling demonstrated that the TLR2-induced growth responsiveness of human GC cells corresponded with the up-regulation of six anti-apoptotic (BCL2A1, BCL2, BIRC3, CFLAR, IER3, TNFAIP3) and down-regulation of two tumor suppressor (PDCD4, TP53INP1) genes. The TLR2-mediated regulation of these anti-apoptotic and tumor suppressor genes was also supported by their increased and reduced expression, respectively, in two independent genetic GC mouse models (gp130F/F and Gan) characterized by high tumor TLR2 expression. Notably, enrichment of this TLR2-regulated gene signature also positively correlated with augmented TLR2 expression in human GC tumors, and served as an indicator of poor patient survival. Furthermore, treatment of gp130F/F and cell line-derived xenograft (MKN1) GC mouse models with a humanized anti-TLR2 antibody suppressed gastric tumor growth, which was coincident with alterations to the TLR2-driven gene signature. Collectively, our study demonstrates that in the majority of GC patients, elevated TLR2 expression is associated with a growth-potentiating gene signature which predicts poor patient outcomes, thus supporting TLR2 as a promising therapeutic target in GC.

Influence of the total saponin fraction from Dioscorea nipponica Makino on TLR2/4-IL1R receptor singnal pathway in rats of gouty arthritis

Ethnopharmacological relevanceDioscorea nipponica Makino have been extensively used in traditional medicine for the treatment of arthritic diseases, particularly gouty arthritis (GA). Materials and methodsSixty male Wistar rats were divided into six groups: the normal group, model group, colchicine group (COL) and three total saponin groups (RDN) (high dose [160mg/kg], middle dose [80mg/kg] and low dose [40mg/kg]). The mRNA and protein expression levels of TLR2, TLR4, IRAK1, TRAF6, TAK1, IKKα, IκBα and NF-κB in the synovial tissue of joint were detected by realtime PCR and WB methods respectively. The contents of IL-1β, IL-6 and TNF-α in the blood serum were measured by Elisa method. The activation of NF-κB was measured by EMSA method. ResultsOur study showed that RDN decreased both the mRNA and protein expressions of TLR2, TLR4, IRAK1, TRAF6, TAK1, IKKα, IκBα and NF-κB of the synovial tissue of joint of rats induced with monosodium urate crystal (MSU). They could also reduce the levels of IL-1β, IL-6 and TNF-α in the blood serum. Further, EMSA results showed that RDN reduced the DNA binding ability of NF-κB p65 of model group. ConclusionRDN has the effect of anti-inflammation in MSU-induced GA model. This is realised by influencing an important inflammatory signal pathway which is called TLR2/4-IL1R receptor signal pathway. It highlights the potential utility of RDN for the management of GA.

PU.1 serves a critical role in the innate defense against Aspergillusfumigatus via dendritic cell-associated C-type lectin receptor-1 and toll-like receptors-2 and 4 in THP-1-derived macrophages.

Aspergillus fumigatus (A. fumigatus) is one of the most common fungal pathogens of invasive pulmonary aspergillosis (IPA), which may be life threatening in immunocompromised individuals. The dendritic cell-associated C-type lectin receptor (Dectin-1), toll-like receptor (TLR)-2 and TLR-4 are major pattern recognition receptors in alveolar macrophages that recognize A. fumigatus components. The PU.1 transcription factor is known to be important for the transcriptional control of these three receptors in mature macrophages. The present study investigated whether alterations of PU.1 expression may affect the innate defense against A. fumigatus in the human monocyte THP-1 cell line. THP-1-derived macrophages were transduced with PU.1 adenoviral vectors and transfected with PU.1 small interfering RNA, and the mRNA and protein expression levels of Dectin-1, TLR-2 and TLR-4 were measured. In addition, the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were ascertained, and fungal phagocytosis and killing were assessed. The results demonstrated that overexpression of PU.1 by recombinant adenoviral vectors resulted in a significant upregulation of Dectin-1, TLR-2 and TLR-4 at the transcriptional and translational levels. In response to A. fumigatus stimulation, PU.1 overexpression increased TNF-α and IL-1β production. In addition, Dectin-1, TLR-2 and TLR-4 upregulation may have enhanced the phagocytosis and killing ability of THP-1-derived macrophages. As expected, silencing of PU.1 led to downregulation of Dectin-1, TLR-2, TLR-4 and the expression of pro-inflammatory cytokines, as well as decreased phagocytosis and the killing ability of THP1-derived macrophages. In conclusion, the results indicate that PU.1 may be a critical factor for the innate defense against A. fumigatus, and may therefore be a potential target for the prophylaxis and treatment of IPA.

The immune mechanism of intestinal tract Toll-like receptor in mediating EV71 virus type severe hand-foot-and-mouth disease and the MAPK pathway.

Immunological response is thought to play a crucial role in the development of a severe hand-foot-and-mouth disease (HFMD) infection in children, but the mechanisms remain largely unknown. This study was designed to help in elucidating the immunopathological pathways involved in the disease by quantifying Toll-like receptor (TLR) mRNAs, MAPK factors and cytokine levels in children experiencing the disease. A total of 86 enterovirus 71 (EV71)-infected HFMD children (49 with mild and 27 with severe disease), along with 30 healthy children were involved in the study. Peripheral vein blood samples were collected from each individual, and used to isolate peripheral blood mononuclear cells (PBMCs) for mRNA extraction and sera for measuring levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-6 and IL-10. The average expression levels of TLR3, TLR4, TLR7 and TLR8 mRNA in PBMCs of children with severe HFMD were significantly higher than those in the other children, the lowest values were found in the healthy control group (P<0.05). The expression levels of TLR2 and TLR9 mRNA were not significantly different (P>0.05) among the groups. Additionally, the expression levels of TNF-α, IFN-γ, IL-6 and IL-10 in the serum of the children in the severe group were significantly higher than those in the other two groups, and the lowest values were again found in the control group (P<0.05). Pearson correlation analysis showed that the TLR3, TLR4, TLR7 and TLR8 mRNA levels in PBMCs were positively correlated with the TNF-α, IFN-γ, IL-6 and IL-10 levels in the serum (P<0.05). Furthermore, the expression levels of the ERK, JNK and p38 mRNA in PBMCs of children in the severe group were significantly higher than those in the other two groups, with the lowest values being in the control group (P<0.05). Pearson correlation analysis showed that the TLR3, TLR4, TLR7 and TLR8 mRNA levels in PBMCs were positively correlated with ERK, JNK and p38 mRNA levels (P<0.05). The results of our study seem to indicate that the high expression levels of TLR3, TLR4, TLR7 and TLR8 induced in severe EV71 HFMD regulate the expression of cytokines by MAPK signaling pathway and negatively affect the ability of the organism to resolve the infection. Further studies are needed to test the hypothesis that immuno-modulation would be an effective treatment approach in pediatric cases of severe HFMD.

The Effects of Agaricus blazei Murill Polysaccharides on Cadmium-Induced Apoptosis and the TLR4 Signaling Pathway of Peripheral Blood Lymphocytes in Chicken.

In this study, we investigated the effects of Agaricus blazei Murill polysaccharides (ABP) on cadmium (Cd)-induced apoptosis and the TLR4 signaling pathway of chicken peripheral blood lymphocytes (PBLs). Seven-day-old healthy chickens were randomly divided into four groups, and each group contained 20 males. The cadmium-supplemented diet group (Cd group) was fed daily with full feed that contained 140mg cadmium chloride (CdCl2)/kg and 0.2mL saline. The A. blazei Murill polysaccharide diet group (ABP group) was fed daily with full feed with 0.2mL ABP solution (30mg/mL) by oral gavage. The cadmium-supplemented plus A. blazei Murill polysaccharide diet group (Cd + ABP group) was fed daily with full feed containing 140mgCdCl2/kg and 0.2mL ABP solution (30mg/mL) by gavage. The control group was fed daily with full feed with 0.2mL saline per day. We measured the apoptosis rate and messenger RNA (mRNA) levels of apoptosis genes (caspase-3, Bax, and Bcl-2), the mRNA levels of TLR4 and TLR4 signaling pathway-related factors (MyD88, TRIF, NF-κB, and IRF3), the TLR4 protein expression, and the concentrations of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in chicken PBLs. The results showed that the PBL apoptosis rate was significantly increased, the mRNA levels of caspase-3 and Bax were significantly increased, while that of Bcl-2 was significantly reduced. The Bax/Bcl-2 ratio was significantly increased in the Cd group at 20, 40, and 60days after treatment compared with that in the control group. After treatment with ABP, the above changes were clearly suppressed. At the same time, ABP reduced the concentrations of IL-1β, IL-6, and TNF-α induced by Cd. We also found that ABP inhibited the TLR4 mRNA level and protein expression and inhibited the mRNA levels of MyD88, TRIF, NF-κB, and IRF3. The results demonstrated that Cd could induce apoptosis, activate the TLR4 signaling pathway, and induce the expression of inflammatory cytokines in chicken PBLs, and that the administration of ABP clearly inhibited Cd-induced effects on chicken PBLs.

A20 Attenuates Liver Fibrosis in NAFLD and Inhibits Inflammation Responses.

We previously reported A20 was able to inhibit lipid accumulation in nonalcoholic steatohepatitis. We want to investigate whether A20 influences liver fibrosis in this study. Liver tissues from patients with hepatic fibrosis (n = 9) and healthy individuals (n = 7) were studied for A20 protein level by immunohistochemistry. A20 messenger RNA (mRNA) and protein level were also analyzed in two murine hepatic fibrosis models: methionine- and choline-deficient (MCD) diet and extrahepatic bile duct ligation (BDL) operation by real-time PCR and western blot. In vitro, the LX-2 human hepatic stellate cell line was treated by LPS at 0, 0.001, 0.01, 0.1, and 1μg/mL for 6h or at the concentration of 0.1μg/mL for 0, 6, 12, and 24h, then A20 expression levels were detected by western blot and PCR. The mRNA level of α-SMA, collagen I, collagen III, TGF-β, IL-6, MCP-1, and TLR4 was also examined by PCR. We then overexpressed A20 in LX-2 cells using adenovirus technique. Levels of α-SMA, collagen I, collagen III, TGF-β, IL-6, MCP-1, and TLR4 were examined in A20-overexpression LX-2 cells. Patients with hepatic fibrosis showed significantly higher A20 protein level compared with healthy controls. A20 mRNA and protein levels were also increased in livers from MCD feeding or BDL operation mice in comparison to normal controls. In LX-2 cells, LPS induced A20 protein in a concentration-dependent manner. The mRNA levels of α-SMA, collagen I, collagen III, TGF-β, IL-6, MCP-1, and TLR4 were increased after LPS treatment. Overexpression of A20 in LX-2 cells inhibited α-SMA deposition and collagen I, collagen III secretion. TGF-β, IL-6, MCP-1, and TLR4 mRNA levels were also reduced in A20-overexpression LX-2 cells in response to LPS stimulation. A20 overexpression inhibits hepatic stellate cell activation, which could be the mechanism for high A20 expression protected livers from fibrosis. Enhancement of A20 expression seems to be rational therapeutic strategies for liver fibrosis.

Monoclonal antibody against Toll-like receptor4 attenuates ventilator-induced lung injury in rats by inhibiting MyD88- and NF-κB-dependent signaling.

The mechanisms through which mechanical ventilation causes non-infectious inflammatory diseases and lung injury are poorly understood. Animals models of this type of injury suggest that it involves signaling mediated by Toll‑like receptor (TLR)4and9. In this study, in order to gain further insight into the involvement of TLR4 in this type of injury, we performed invivo and invitro experiments to determine the mechanisms through which TLR4 triggers inflammation. We also examined whether the use of TLR4 monoclonal antibody(mAb) can alleviate this type of injury. For this purpose, rats were tracheotomized and administered intratracheal injections of anti‑TLR4 mAb or saline, and then ventilated for 4 h at a high tidal volume(HTV) of 40 ml/ kg or allowed to breathe spontaneously for the same period of time (controls). Alveolar macrophages(AMs) were isolated from the bronchoalveolar lavage fluid(BALF) of the rats and stimulated for 16h with tumor necrosis factor(TNF)‑α in the presence or absence of anti‑TLR4 mAb. Lung injury was assessed by examining lung histopathology, lung wet/dry weight ratio, BALF total protein and cytokine levels in BALF and plasma. The mRNA and protein expression levels of TLR4, TLR9, myeloid differentiation factor88(Myd88) and nuclear factor(NF)‑κB were measured in cultured macrophages. Compared to the controls (spontaneous breathing), the ventilated rats exhibited greater pulmonary permeability, more severe inflammatory cell infiltration/lung edema, and higher levels of interleukin(IL)‑1β, IL‑6 and TNF‑α in BALF and plasma. The AMs from the ventilated rats expressed higher mRNA and protein levels of TLR4, TLR9, Myd88 and NF‑κB compared with the macrophages from the spontaneously breathing rats. The ventilated rats pre‑treated with anti‑TLR4 mAb exhibited markedly attenuated signs of ventilation‑induced injury, such as less lung inflammation and pulmonary edema, fewer cells in BALF, and lower levels of ILs and TNF‑α in BALF and plasma. Similarly, the TNF‑α‑dependent increases in the mRNA and protein expression of TLR4, Myd88 and NF‑κB in AMs were attenuated when TNF‑α was co‑administered with anti‑TLR4 mAb than when TNF-α was administered alone. Co‑administering anti-TLR4 mAb also reduced the TNF‑α‑dependent secretion of ILs. On the whole, our data demonstrate that TLR4 contributes significantly to ventilation‑induced lung injury by activating the Myd88/NF‑κB pathway, and pre‑treating rats with anti‑TLR4 mAb partially protects them against this type of injury by inhibiting Myd88/NF-κB signaling.

Effect of adenosine A2A receptor agonist combined with bone marrow mesenchymal stem cells transplantation on the negative immune regulation in mice with acute liver failure

Objective To investigate the therapeutic effect and possible mechanism of adenosine A2A receptor agonist (CGS21680) combined with bone marrow mesenchymal stem cells (BMMSC) transplantation in acute liver failure (ALF). Methods Fifty male C57BL/6 mice, 6—8 weeks old, were fed with standard diet for 1 week and randomly divided into 5 groups according to random number table: healthy control group (n=6), model group (n=11), BMMSC group (n=11), CGS21680/BMMSC group (n=11) and CGS21680 group (n=11). Except healthy control group, the other mice were injected with D-GalN and lipopolysaccharide (LPS) to establish ALF model. Ten hours later, CGS21680/BMMSC group and CGS21680 group were injected intraperitoneally with adenosine A2A receptor agonist CGS21680 (2.1 mg/kg). In addition, the BMMSC group and CGS21680/BMMSC group were injected BMMSC (1×106) through tail vein. After 24 hours, pathological changes of liver tissue was observed by hematoxylin and eosin staining. The change of proportion of mouse splenic Treg among CD4+ T lymphocytes was detected by flow cytometry. Toll-like receptor (TLR)4 and nuclear factor (NF)-κB expression levels in liver tissue were detected by real-time fluorescence quantitative polymerase chain reaction (PCR) and Western blot. One-way analysis of variance (one-way ANOVA) and SNK-q test was conducted for data analysis. Results Serum IL-6 levels were (23.67±2.97) pg/mL in healthy control group, (151.47±6.03) pg/mL in model control group, (72.10±3.74) pg/mL in BMMSC group, (53.35±2.50) pg/mL in CGS21680/BMMSC group and (84.85±3.25) pg/mL in CGS21680 group. The differences between healthy control group and the other 4 groups were all statistically significant (t=46.02, 25.51, 19.58 and 34.03, respectively, all P<0.01). Serum TNF-ɑ levels were (24.62±3.19) pg/mL in healthy control group, (102.25±2.10) pg/mL in model control group, (54.71±2.23) pg/mL in BMMSC group, (42.20±4.72) pg/mL in CGS21680/BMMSC group and (81.76±3.50) pg/mL in CGS21680 group. The differences between healthy control group and the other 4 groups were all statistically significant (t=46.49, 19.97, 7.72 and 29.57, respectively, all P<0.01). The differences of spleen Treg proportion in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=51.67, 12.22, 5.91 and 18.21, respectively, all P<0.01). The differences of TLR4 mRNA levels of liver tissue in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=26.31, 21.33, 13.24 and 27.14, respectively, all P<0.05). The differences of NF-κB mRNA level of liver tissue in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=16.56, 16.34, 7.83 and 13.11, respectively, all P<0.05). The differences of TLR4 protein level in liver tissue of healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=35.60, 10.38, 6.05 and 18.02, respectively, all P<0.05). The differences of liver NF-κB protein level in the healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=10.80, 7.30, 4.61 and 13.24, respectively, all P<0.05). Conclusions Adenosine A2A receptor agonist combined with BMMSC can significantly up-regulate the proportion of Treg cells in acute liver failure mice and inhibit the TLR4/NF-κB pathway activation, with both coordinated regulation, and further inhibit the liver inflammation. Key words: Mesenchymal stem cells; Liver failure, acute; Receptor, adenosine A2A; TLR4/ NF-κB signal pathway

Hepatitis B Surface Antigen Promotes the Invasion of Hepatitis B Virus-Related Hepatocellular Carcinoma Cells by Upregulation of Toll-Like Receptor 2.

Hepatitis B virus (HBV) infection is one of the major risk factors leading to the development of hepatocellular carcinoma (HCC). Hepatitis B surface antigen (HBsAg) plays a pivotal role in HBV-related HCC pathogenesis, and Toll-like receptor (TLR) 2 is also considered to mediate tumor progression. However, the interaction between HBsAg and TLR2 in HCC progression remains unclear. Thus, the aim of the study was to explore the effect of HBsAg-TLR2 pathway on growth and invasion of HBV-related HCC cells and examine the potential mechanisms been involved. The expression of TLR2 was measured in two different HCC cell lines (HepG2 and HepG2.2.15) with or without recombinant HBsAg by real-time reverse polymerase chain reaction and Western blot. Cellular proliferation, invasion, cytokine productions, and downstream signaling pathways were also measured in TLR2-silencing HepG2.2.15 cells in response to HBsAg stimulation. The mRNA and protein levels of TLR2 were significantly elevated in HepG2.2.15 cells than those in HepG2 cells. HBsAg simulation increased proinflammatory cytokine production and invasion of HepG2.2.15 cells, while this process was inhibited by TLR2 silence. However, TLR2 siRNA transfection alone did not affect the bioactivities of tumor cells. Moreover, HBsAg increased expression of MyD88 and phosphorylation of NF-κB p50 and p38MAPK. Downregulation of TLR2 inhibited HBsAg-induced MyD88 and p-NF-κB, but not p-p38MAPK in HepG2.2.15 cells. In conclusion, HBsAg stimulation promotes the invasion of HBV-related HCC cells. TLR2/MyD88/NF-κB signaling pathway may be involved in this procession by upregulation of cytokine production. The interaction between TLR2 and HBsAg may contribute to the poor prognosis of HBV-related HCC.

Involvement of the Toll-Like Receptor/Nitric Oxide Signaling Pathway in the Pathogenesis of Cervical Cancer Caused by High-Risk Human Papillomavirus Infection.

Human papillomavirus (HPV) can activate Toll-like receptor (TLR)/nitric oxide (NO) signaling pathways; however, whether the TLR/NO pathway is involved in cervical cancer caused by high-risk HPV (HR-HPV) remains unclear. In this study, 43 HR-HPV-positive patients with cervical cancer (CC group), 39 HR-HPV-positive patients with a healthy cervix (HR-HPV group), and 33 HR-HPV-negative controls were recruited. NO concentration in cervical canal and expression of inducible NO synthase (iNOS) in cervical tissues were detected. Expressions of key TLR/NO pathway genes (TLR3/4/7/8, NF-κB p65, and iNOS) in cervical epithelial cells were detected by quantitative reverse transcription PCR. Expressions of TLR4, NF-κB p65, and iNOS in CaSki, HeLa, and C33a cells were determined by Western blot. NO concentration in cervical canal of CC group was significantly higher than in other groups (P < 0.05). Positive rates of iNOS in cervical tissues were 72.1%, 28.2%, and 3.1% in the CC group, HR-HPV group, and controls, respectively (P < 0.05). Levels of TLR3, TLR4, TLR7, TLR8, NF-κB p65, and iNOS in cervical epithelial cells were higher in CC group than in other groups (P < 0.05). Both mRNA and protein levels of TLR4, NF-κB p65, and iNOS were higher in HPV-positive HeLa and CaSki cells than in HPV-negative C33a cells (P < 0.05). Together, these results suggest that TLR/NO signaling pathway may be involved in pathogenesis of cervical cancer caused by HR-HPV.

Effect of Kangshuanyihao Formula on the Inflammatory Reaction and SIRT1/TLR4/NF-κB Signaling Pathway in Endothelial Injury.

Endothelial injury plays an important role in atherosclerosis (AS). Kangshuanyihao formula uses therapeutic principles from Chinese medicine to supplement Qi, thereby promoting blood circulation, and remove blood stasis. The mechanism by which the formula inhibits endothelial injury was examined in a rat model of 1,25-dihydroxyvitamin D3 (VD3) intraperitoneal injection and high-fat-induced endothelial injury. Rats were randomly divided into the model, high-dose, middle-dose, low-dose, positive drug (rosuvastatin), and combination (positive drug + middle-dose) groups; 10 Sprague-Dawley rats served as the blank group. The aortic endothelium was stained with hematoxylin and eosin and the levels of blood lipids and inflammation markers (mRNA and protein) were measured. Endothelial injury, lipid levels, and inflammation were increased in the model. Kangshuanyihao formula reduced endothelial injury, improved lipid levels, and downregulated inflammation, as shown by significant reduction of the protein levels of SIRT1, TLR4, and NF-κB and mRNA levels of SIRT1, TLR4, NF-κB, IL-1β, IL-6, and IL-12. Thus, we conclude that Kangshuanyihao formula can inhibit the inflammatory reaction in the rat model of high-fat-induced endothelial injury after intraperitoneal injection of VD3. This mechanism may be attributed to regulating the SIRT1/TLR4/NF-κB signaling pathway.

P2.05-001 α7-nAchR Agonist GTS-21 Reduces Radiation-Induced Lung Injury by Inhibiting HMGB1/TLR-4/NF-κB Pathway: Topic: Biology

The cholinergic anti-inflammatory signaling pathway allows the autonomic nervous system to modulate immunologic stimuli and inflammatory processes α7 nicotinic acetylcholine receptor (α7-nAChR) is a major component in this pathway. GTS-21, a selective α7 nicotinic acetylcholine receptor agonist, has been demonstrated as a promising treatment for inflammation. So, the aim of this study is to determine whether treatment GTS-21 can mitigate the radiation induced lung injury. C57BL/6 mice were randomly divided into three groups: a control group, a 12 Gy thoracic irradiation group, a 12 Gy thoracic irradiation group treated with 4mg/kg GTS-21 immediately after irradiation. Each of group were sacrificed at 1, 3, 7, 14, 21d and 3m, 6m post-irradiation, and the sections were respectively stained with hematoxylin and eosin (HE) and Masson’s trichrome to assess the degree of inflammation and fibrosis. Serum concentrations of TNF-α, IL-1β and IL-6 were quantitatively measured by Cytometric Bead Array (CBA) kit. Real-time PCR and Western blot were used to detect the mRNA and protein levels of HMGB1, TLR-4, NF-κB, MyD88 and TGF-β in lung tissue from GTS-21 group and irradiation control group at different time after radiation. The result from HE and Masson staining showed that GTS-21 could dramatically reduce radiation-induced lung inflammation and following mitigate lung fibrosis. Then, we found that radiation-induced TNF-α, IL-1β and IL-6 in serum were also inhibited by GTS-21. Comparing to the control group, the mRNA levels of HMGB1,TLR-4 and NF-κB were decreased at the early time of radiation pneumonitis, and the most significant difference was observed at 21d post-irradiation(P<0.05). the mRNA levels of TGF-β was decrease in GTS-21group at 3m and 6m post -irradiation when compared to control (P<0.05). However, there did not have any different on MyD88 between GTS-21and control groups. The result from western blot showed that the protein levels of HMGB1, TLR-4 and NF-κB in GTS-21 group were also significantly decreased at 21d after radiation. After 3m and 6m from radiation, the protein level of TGF-β was decreased dramatically at GTS-21 group. GTS-21 can reduce radiation pneumonitis and fibrosis by inhibiting HMGB1/TLR-4/NF-κB pathway which subsequently decrease TGF-β expression.

Porphyromonas gingivalis promotes low-density lipoprotein oxidation and atherosclerosis

ObjectivesRecent studies have reported that periodontal disease increases the risk of atherosclerosis. We previously reported that Porphyromonas gingivalis (Pg) accelerated atherosclerotic plaque formation in hyperlipidemic apoE-/- mice by initiating inflammation. Because the oxidative modification of lipoprotein plays a major role in atherosclerosis, we characterized the reactive oxygen species (ROS) produced by Pg and its ability to oxidize low-density lipoprotein (LDL). MethodsAtherosclerotic plaque formation in the aortic sinuses of Apoeshl mice injected intravenously with Pg 381 was assessed by Oil Red O staining. Anti-mouse antibodies to HOCl-oxidized LDL and 4-HNE, PLA2, MPO, and CD36 were used for immunohistochemistry. Intracellular ROS detection was performed using 2′,7′-dichlorodihydrofluorescein diacetate. Quantitative reverse transcription polymerase chain reaction was performed using primers specific for TLR-2, TLR-4, TLR-9, NOD-1, LOX-1, NOX-2, NOX-4, p22phox, and p47phox. ResultsPg challenge significantly induced ox-LDL and 4-HNE-, PLA2-, MPO-, and CD36-positive areas in proximal aortic lesions. TLR-2, TLR-4, NOD-1, LOX-1, and NADPH oxidase subunit-specific mRNA levels in the aorta were significantly increased. Furthermore, Pg significantly induced ROS production in monocytes. ConclusionsThese results suggest that Pg promotes LDL oxidation and contributes to atheroma development.

Study on the susceptibility of human corneal epithelial cells to Acanthamoeba in a hypoxia condition

Objective: To investigate the regulation mechanism of Acanthamoeba-induced immune responses of human corneal epithelial cells in a hypoxia condition. Methods: Telomerase-immortalized human epithelial cells (THCEs) challenged with Acanthamoeba (1×106/ml) were incubated under normoxic (21% O2) (control group) or hypoxic (1% O2) (experiment group) conditions respectively. The mRNA of toll-like receptor (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor kappa-B (NF-κB), tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), interferon-β (IFN-β), and the protein level of TLR4, inhibitor of nuclear factor kappa-Bα (IκBα), phosphorylated extracellular-signal related kinase 1/2 (p-ERK1/2) and phosphorylated IκBα (p-IκBα) were detected. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines (TNF-α, IL-8 and IFN-β). Results: The mRNA levels of Acanthamoeba-induced TLR4, MyD88, NF-κB, TNF-α, IFN-β and IL-8 in THCEs under hypoxia was down-regulated by 47%, 41%, 45%, 53%, 36% and 50% respectively, compared to control group. And the protein levels of p-IκBα and p-ERK1/2 were significantly down-regulated by 56% and 55%, respectively, while the protein expression of IκBα was increased. The secretions of TNF-α, IL-8 and IFN-β under hypoxia were reduced by 46%, 28% and 35%, respectively. Conclusion: Hypoxia might attenuated the inflammatory response of the human corneal epithelial cells against Acanthamoeba infection by suppressing TLR4 signaling.

Glycyrrhizin Reduces HMGB1 and Bacterial Load in Pseudomonas aeruginosa Keratitis.

PurposeHigh mobility group box 1 (HMGB1) contributes to poor disease outcome in Pseudomonas aeruginosa keratitis. This study tests the prophylactic effect of treatment with HMGB1 inhibitors, glycyrrhizin (GLY) and its derivative, carbenoxolone (CBX), for Pseudomonas keratitis.MethodsWe treated C57BL/6 (B6) mice subconjunctivally with GLY or CBX, infected with a noncytotoxic clinical isolate (KEI 1025) or a cytotoxic strain (ATCC 19660) of P. aeruginosa, and injected intraperitoneally with either agent. Clinical score, photography with a slit lamp, real-time RT-PCR, ELISA, myeloperoxidase (MPO) assay, bacterial plate count, histopathology, and absorbance assays were used to assess treatment efficacy and bacteriostatic activity.ResultsAfter KEI 1025 infection, GLY treatment reduced HMGB1 (mRNA and protein levels) and improved disease outcome with significant reduction in mRNA levels of IL-1β, TLR4, CXCL2, and IL-12; protein expression (IL-1β, CXCL2); neutrophil infiltrate; and bacterial load. Treatment with GLY enhanced antimicrobial proteins, including CRAMP and mBD2, but not mBD3. Glycyrrhizin also reduced clinical scores and improved disease outcome in corneas infected with strain 19660. However, neither HMGB1 mRNA or protein levels were reduced, but rather, CXCL2 expression (mRNA and protein), neutrophil infiltrate, and bacterial load were reduced statistically. Treatment with GLY initiated 6 hours after infection reduced plate count; GLY also was bacteriostatic for KEI 1025 and ATCC 19660.ConclusionsGlycyrrhizin reduces HMGB1 and is protective against P. aeruginosa–induced keratitis with a clinical isolate that is noncytotoxic. It was similar, but less effective when used after infection with a cytotoxic strain, which did not reduce HMGB1.

AMPK activation by metformin inhibits local innate immune responses in the isolated rat heart by suppression of TLR 4-related pathway

Toll like receptors (TLRs) are key players in the innate immune responses. The energy sensing enzyme, AMPK, has been implicated in the modulation of immunity. The present study investigated whether AMPK activation by metformin could contribute to the regulation of immune responses in the isolated heart via suppression of TLR4 activity, independent of circulatory immunity. Isolated Wistar rat hearts were perfused with Krebs-Henseleit buffer in the absence or presence of lipopolysaccharide (LPS; 0.2μM), LPS+metformin (10mM), and LPS+metformin+compound C (10μM). Following measurement of hemodynamic parameters, TLR4-activation related changes and TLR4 mRNA level in the heart was examined by western blotting and real-time PCR. The activation of AMPK was evaluated by measuring the ratio of p-AMPKα and p-ACC to their non-phosphorylated forms. The effluent and cardiac levels of TNF-α and IL6 were assayed by ELISA. LPS profoundly increased the levels of TLR4 mRNA, MyD88 (TLR4 adaptor protein), and NF-κB and also the release of TNF-α and IL6 from the heart. The enhancement in the TLR4 activity was associated with a significant depression of myocardial function. Metformin clearly augmented the phosphorylation of both AMPKα and ACC and in addition to improvement of cardiac performance, markedly suppressed the TLR4 activity. Antagonizing AMPK by compound C which is a selective inhibitor of AMPK pathway, considerably reversed the protective effects of metformin against the TLR4-related activity. The results of the study demonstrated the importance of TLR4-involved local immune responses in the LPS-induced myocardial dysfunction and indicated a clear link between AMPK and TLR4.

Up-regulated TLR2 and TLR4 expressions in liver and spleen during acute murine T. gondii infection.

Toll-like receptors (TLRs) play a central role in the pathogen clearance and pathological processes. The liver is an important innate immune organ, in which Kupffer cells and hepatocytes are important innate immune cells. However, the role of TLR2 and TLR4 in the liver caused by Toxoplasma gondii infection remains less clear. In this study, mice were infected with T. gondii RH strain and the grades of liver and spleen injuries were histopathologically evaluated. TLR2+ and TLR4+ cells in the livers and spleens were detected by immunohistochemistry, and their messenger RNA (mRNA) expressions were detected using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The pathological severities in the livers and spleens were increased with time in T. gondii-infected mice. Compared with uninfected controls, obvious TLR2+ and TLR4+ cells were observed in the livers and spleens infected with T. gondii at 8days post-infection, accompanied with significantly over-expressed mRNA levels of TLR2 and TLR4 in the livers and spleens after infection. Our data indicated that increased levels of TLR2 and TLR4 in the liver and spleen may play an important role during acute T. gondii infection.