Lactoferrin suppresses LPS-induced expression of HMGB1, microRNA 155, 146, and TLR4/MyD88/NF-кB pathway in RAW264.7 cells.

OBJECTIVE: This current study evaluated the underlying mechanisms of LF against the inflammatory microRNAs (miRNAs), HMGB1 expression, and TLR4-MyD88-NF-кB pathway in LPS-activated murine RAW264.7 cells. METHODS: MTT assay was used to assess cell metabolism and the cell culture levels of the cytokines (TNF-α, IL-6) were evaluated by Enzyme-linked immunosorbent assay (ELISA). The expression of miRNAs was quantified by using qPCR and the expression of HMGB1, TLR4, MyD88, and phosphorylated NF-κB (P-p65) were determined with Western blot and qPCR, respectively. RESULTS: The results indicated that LF downregulates IL-6 and TNF-α expression. LF exhibited the degradation of P-p65 and reduced the production of HMGB1, TLR4, and MyD88 in LPS-induced inflammatory response. Importantly, in parallel with the suppression of cytokines and HMGB1-TLR4-MyD88-NF-кB pathway, LF could induce a decrease in inflammatory selected miRNAs, mmu-mir-155, and mmu-mir-146a expression. CONCLUSIONS: Altogether, these findings provide LF as a prominent anti-inflammatory agent that could modulate HMGB1, mmu-mir-155, mmu-mir-146a, and TLR4/MyD88/NF-кB pathway.

Etidronate down-regulates Toll-like receptor 2 ligand-induced chemokine production by inhibiting MyD88 expression and NF-κB activation.

OBJECTIVE: Pretreatment of J774.1 cells with etidronate, a non-nitrogen-containing bisphosphonate (non-NBP) used as an antibone resorptive drug, was previously reported to inhibit Toll-like receptor (TLR) 2 agonist-induced proinflammatory cytokine production. The present study aimed to examine the effects of etidronate on chemokine production by human monocytic U937 cells incubated with Pam3Cys-Ser-(Lys)4 (Pam3CSK4, a TLR2 ligand) and lipid A (a TLR4 ligand). METHODS: U937 cells were pretreated with or without etidronate, and then incubated with or without Pam3CSK4 or lipid A. Levels of secreted human interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) in culture supernatants and activation of nuclear factor-κB (NF-κB) p65 were measured by enzyme-linked immunosorbent assay (ELISA). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) activity in supernatants. Expression of intracellular adhesion molecule (ICAM)-1 and MyD88 was analyzed by flow cytometry and Western blot analysis, respectively. RESULTS: Etidronate down-regulated IL-8 and MCP-1 production and NF-κB p65 activation induced by Pam3CSK4, but not lipid A, in U937 cells. Etidronate also inhibited MyD88 expression in U937 cells incubated with Pam3CSK4. CONCLUSION: Etidronate down-regulates IL-8 and MCP-1 production in U937 cells by inhibiting both the expression of MyD88 and activation of NF-κB p65 in the TLR2, but not TLR4, pathway.

Sevoflurane Post-treatment Upregulated miR-203 Expression to Attenuate Cerebral Ischemia-Reperfusion-Induced Neuroinflammation by Targeting MyD88.

To investigate the expression of miR-203 by sevoflurane treatment and its effect on neuroinflammation induced by cerebral ischemia-reperfusion. Rats were randomly divided into sham operation group (C), cerebral ischemia-reperfusion group (I/R), and sevoflurane treatment group (S). The neurological function score was evaluated. The area of cerebral infarction was evaluated by TTC staining. The expression of inflammatory factor in brain tissue was detected by ELISA. The apoptosis of neurons was detected by TUNEL. A miR-203 agonist and inhibitor treated the cerebral ischemia-reperfusion model. The luciferase assay verified whether miR-203 targeted MyD88. To further verify the relationship between miR-203 and MyD88, the I/R group was treated with MyD88 activator and inhibitor, and the mRNA expressions of miR-203 and MyD88 in brain tissue were detected by RT-PCR. Western blot was used to detect the expression of MyD88 protein in brain tissue, and the above experiment was repeated. Compared with the I/R group, miR-203 mRNA was significantly increased in brain tissue and the neurological function score, the area of cerebral infarction, the expression of inflammatory factor, and MyD88 mRNA were decreased in the S group (P < 0.05). After treatment of miR-203 agonist and inhibitor in the I/R group, overexpression of miR-203 could alleviate cerebral ischemia-reperfusion injury, and miR-203 inhibitor could aggravate cerebral ischemia-reperfusion injury. The miR-203 agonist could enhance the action of sevoflurane, and the miR-203 inhibitor could reverse the action of sevoflurane. miR-203 agonist treatment could inhibit the expression of MyD88 gene and protein and reduce the neuroinflammation induced by cerebral ischemia-reperfusion. The treatment of sevoflurane upregulated miR-203 expression, which targeted MyD88 and attenuate neuroinflammation induced by cerebral ischemia-reperfusion.

MiR-146a alleviates inflammation of acute gouty arthritis rats through TLR4/MyD88 signal transduction pathway.

OBJECTIVE: The aim of this study was to explore the effect of micro-ribonucleic acid (miR)-146a on acute gouty arthritis rats through Toll-like receptor-4/myeloid differentiation factor 88 (TLR4/MyD88) signal transduction pathway. MATERIALS AND METHODS: A total of 30 clean-grade Sprague-Dawley rats were divided into three groups, including agomiR-146a group (n=10), antagomiR-146a group (n=10) and negative control group (NC, n=10). The model was successfully established via a one-time injection of sodium urate into ankle joint cavity. Subsequently, agomiR-146a (10 µL), antagomiR-146a (10 µL) and normal saline (10 µL) were intrathecally injected into rats in the three groups at 1 h before injection and 12 h, 24 h, 48 h and 72 h after injection, respectively. The ankle joint swelling index, joint dysfunction index and joint inflammation index of rats in the three groups were closely monitored. After 72 h of observation, the rats were euthanized, and synovial tissues were collected from the knee joint. The expression and distribution of nuclear factor-κB (NF-κB) in synovial tissues were detected using the immunohistochemical method. Meanwhile, the expression levels of inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6) were detected via enzyme-linked immunosorbent assay. Furthermore, the mRNA and protein expression levels of TLR4 and MyD88 were detected via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blotting, respectively. RESULTS: No statistically significant differences in the joint swelling index, joint dysfunction index, joint inflammation index, TLR4 and MyD88 and related inflammatory factors were found between the NC group and antagomiR-146a group. Compared with the NC group, agomiR-146a group showed markedly reduced ankle joint swelling index (p<0.05). Meanwhile, joint landing behavior and inflammatory swelling were significantly relieved in the agomiR-146a group (p<0.05). The mRNA and protein expression levels of TLR4 and MyD88 were remarkably decreased as well (p<0.05). Furthermore, the expression and distribution of NF-κB in synovial tissues of agomiR-146a group was markedly reduced when compared with the NC group (p<0.05). In addition, agomiR-146a group exhibited significantly lower expression levels of inflammatory factors (TNF-α, IL-1 and IL-6) in synovial tissues (p<0.05). CONCLUSIONS: MiR-146a alleviates joint inflammation of acute arthritis in rats through the TLR4/MyD88/NF-κB signaling pathway, which may become a new therapeutic target.

MyD88 Regulates LPS-induced NF-ĸB/MAPK Cytokines and Promotes Inflammation and Malignancy in Colorectal Cancer Cells.

BACKGROUND/AIM: Inflammation may play a role in cancer initiation and progression. The molecular mechanisms by which inflammation causes colorectal cancer, remains unclear. The present study investigated a signaling pathway that affects inflammation in colorectal cancer. MATERIALS AND METHODS: SW480 cells, HCT116 cells, and cells with knockdown of myeloid differentiation 88 (MyD88), and forced expression of MyD88 were treated with lipopolysaccharide (LPS; 1 µg/ml). Inflammation-related mRNA expression was analyzed by the quantitative reverse transcription polymerase chain reaction and inflammatory cytokines were detected by western blotting. The enzyme-linked immunosorbent assay (ELISA) was used to quantify inflammation-related cytokines in colorectal cancer cells. Cancer cell properties were evaluated using the wound-healing assay, transwell migration assay, transwell invasion assay, colony-formation assay, and CCK-8 assay. RESULTS: LPS up-regulated mRNA and protein levels of inflammatory factors in colorectal cancer cells. Knockdown of MyD88 inhibited LPS-induced mRNA expression and inflammatory protein expression in colorectal cancer cells. Similarly, silencing of MyD88 expression suppressed LPS-induced changes in the biological behavior of colorectal cancer cells. Silencing of MyD88 expression down-regulated expression of proteins of the LPS/nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-ĸB)/mitogen-activated protein kinase (MAPK) signaling pathway. Restoration of the expression of MyD88 reversed the effects in LPS-treated HCT116 cells. CONCLUSION: MyD88-regulated LPS/NF-ĸB/MAPK signaling pathway affects the inflammatory and biological behavior of LPS-induced colorectal cancer cells.

Effects of guluronic acid (G2013) on gene expression of TLR2, TLR4, MyD88, TNF-α and CD52 in multiple sclerosis under in vitro conditions.

Context: Multiple sclerosis (MS) is an autoimmune and chronic inflammatory disease of CNS. The α-L-guluronic acid (G2013) as novel NSAID with immunomodulatory effects has shown its positive effects in various investigations.Objective: Present research aimed to study the potency of G2013 on gene expression of TLR2, TLR4, MyD88, TNF-α and CD52 in PBMCs of MS patients under in vitro conditions. Materials and methods: 24 blood samples from MS patients and healthy controls were considered for RT-PCR and flow cytometry techniques under two different doses of G2013.Results: Our research indicated that this drug could significantly decrease the gene expression of TLR2, TLR4 and TNF-α compared to untreated group. Conclusion: Data demonstrated that the guluronic acid is able to modify the expression levels of TLR2, TLR4 and TNF-α genes to less than the pathogenic boarder line level, which it might be recommended for reducing the pathological process in multiple sclerosis.

TLR2/MyD88/NF-κB signaling pathway regulates IL-1ß production in DF-1 cells exposed to Mycoplasma gallisepticum LAMPs.

Mycoplasma gallisepticum (M. gallisepticum) is one of the most important pathogens that cause chronic respiratory disease in chickens. M. gallisepticum-derived lipid-associated membrane proteins (LAMPs) are thought to be one of the major factors in mycoplasma pathogenesis and are potent inducers of the host innate immune response. However, the interaction of pathogenic M. gallisepticum-derived LAMPs with Toll-like receptors (TLRs) and the signaling pathways responsible for activating inflammation and NF-κB have not been fully elucidated. In this study, we found that IL-1ß expression was induced in DF-1 cells stimulated with M. gallisepticum LAMPs. Subcellular localization experiments using immunofluorescence assays (IFAs) showed p65 translocation from the cytoplasm to the nucleus in DF-1 cells following stimulation with M. gallisepticum LAMPs. Phosphorylation of p65 was detected in LAMP-stimulated DF-1 cells. Treatment with an NF-κB-specific inhibitor showed that NF-κB is required for M. gallisepticum LAMP-induced IL-1ß expression. In addition, the results indicated that TLR2 and myeloid differentiation primary-response protein 88 (MyD88)-dependent signaling pathways were involved in the activation of NF-κB by M. gallisepticum LAMPs. Together, these results provide evidence that M. gallisepticum LAMPs activate IL-1ß production through the NF-κB pathway via TLR2 and MyD88.

Pam2CSK4 and Pam3CSK4 induce iNOS expression via TBK1 and MyD88 molecules in mouse macrophage cell line RAW264.7.

OBJECTIVE: The aim of this study was to investigate the involvement of TLR adaptor molecules, such as TRIF, MyD88, and TBK1 in the induction of iNOS and nitric oxide (NO) production in Pam2CSK4 and Pam3CSK4-treated mouse macrophages. METHOD: Mouse macrophage cell line (RAW264.7) was transfected with trif, myd88, and tbk1 siRNAs before stimulated with Pam2CSK4 and Pam3CSK4. The iNOS gene and protein expression were determined by RT-PCR and immunoblotting, respectively. The NO production was determined by Griess reaction assay. RESULTS: The results showed that the induction of iNOS expression and NO production by Pam2CSK4 and Pam3CSK4 were diminished in tbk1 and myd88-depleted mouse macrophages but not trif-depleted cells. CONCLUSION: These results suggested that the TBK1 and MyD88 molecules were essential for the induction of iNOS expression and NO production by both Pam2CSK4 and Pam3CSK4 via TLR2 signaling.

LncRNA CRNDE triggers inflammation through the TLR3-NF-κB-Cytokine signaling pathway.

Colorectal neoplasia differentially expressed (CRNDE), an oncogene, is highly expressed in many tumor cells and affects cellular proliferation, migration, invasion, and apoptosis. Its function and mechanism of action is a research hotspot. In this study, microarray analysis was performed to discover the differentially expressed genes in CRNDE over-expression cells. RT² Profiler PCR Array was used to study the expression of genes related to the toll-like receptor (TLR) pathway. We found that over-expression of CRNDE in astrocytes increased the expression of key factors in the toll-like receptor signaling pathway, especially toll-like receptor-3-mediated MyD88-independent pathway. Furthermore, it up-regulated expression levels of downstream transcription factor such as nuclear factor kappa B and numerous cytokines. In contrast, CRNDE knockdown in glioma U87MG cell line showed an opposite trend in the expression of the above-mentioned genes. We speculated that CRNDE might trigger inflammation to regulate tumorigenesis and tumor development through the toll-like receptor pathway.

Myeloid differentiation factor 88 is up-regulated in epileptic brain and contributes to experimental seizures in rats.

Accumulating evidence supports that activation of inflammatory pathways is a crucial factor contributing to the pathogenesis of seizures. In particular, the activation of interleukin-1 beta (IL-1ß) system exerts proconvulsant effects in a large variety of seizure models. Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein in the signaling cascade elicited by IL-1ß. The present study aimed to investigate the expression pattern of MyD88 in rat models of seizures and in patients with refractory temporal lobe epilepsy (TLE), and to study the role of MyD88 in epileptic seizures. Our results revealed that MyD88 was up-regulated in the hippocampus of rats in the lithium-pilocarpine model of acute seizures. Importantly, MyD88 overexpression was also significantly present in the brain from chronic epileptic rats and the temporal neocortex specimens from drug-resistant TLE patients. In the acute seizure model, both the behavioral and electrographic seizure activities were record and analyzed in rats for 90min, starting immediately after pilocarpine injection. ST2825, a synthetic MyD88 inhibitor, was administered intracerebroventricularly (2.5-5.0-10µg in 2µl) 20min before pilocarpine injection. We found that ST2825 at doses of 5µg and 10µg significantly inhibited the pilocarpine-induced behavioral and electrographic seizures. Moreover, 10µg ST2825 prevented the proconvulsant actions of IL-1ß. As previous evidence suggested that IL-1ß proconvulsant effects was mediated by enhancing the phosphorylation level of the NR2B subunit of N-methyl-d-aspartate (NMDA) receptor, we then probed whether this molecular was involved in the effect of the pharmacological inhibition. Our results revealed that 10µg ST2825 markedly reversed the increased Tyr1472-phosphorylation of the NR2B subunit of NMDA receptor observed in the proconvulsant conditions of IL-1ß and in seizures induced by pilocarpine alone. These findings indicate that altered expression of MyD88 might contribute to the pathogenesis of seizures and targeting of this adaptor protein might represent a novel therapeutic strategy to suppress seizure activities.

Kaempferol ameliorates H9N2 swine influenza virus-induced acute lung injury by inactivation of TLR4/MyD88-mediated NF-κB and MAPK signaling pathways.

Kaempferol, a very common type of dietary flavonoids, has been found to exert antioxidative and anti-inflammatory properties. The purpose of our investigation was designed to reveal the effect of kaempferol on H9N2 influenza virus-induced inflammation in vivo and in vitro. In vivo, BALB/C mice were infected intranasally with H9N2 influenza virus with or without kaempferol treatment to induce acute lung injury (ALI) model. In vitro, MH-S cells were infected with H9N2 influenza virus with or without kaempferol treatment. In vivo, kaempferol treatment attenuated pulmonary edema, the W/D mass ratio, pulmonary capillary permeability, myeloperoxidase (MPO) activity, and the numbers of inflammatory cells. Kaempferol reduced ROS and Malondialdehyde (MDA) production, and increased the superoxide dismutase (SOD) activity. Kaempferol also reduced overproduction of TNF-α, IL-1ß and IL-6. In addition, kaempferol decreased the H9N2 viral titre. In vitro, ROS, MDA, TNF-α, IL-1ß and IL-6 was also reduced by kaempferol. Moreover, our data showed that kaempferol significantly inhibited the upregulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphorylation level of IκBα and nuclear factor-κB (NF-κB) p65, NF-κB p65 DNA binding activity, and phosphorylation level of MAPKs, both in vivo and in vitro. These results suggest that kaempferol exhibits a protective effect on H9N2 virus-induced inflammation via suppression of TLR4/MyD88-mediated NF-κB and MAPKs pathways, and kaempferol may be considered as an effective drug for the potential treatment of influenza virus-induced ALI.

The effect of n-3/n-6 polyunsaturated fatty acids on acute reflux esophagitis in rats.

BACKGROUND: Polyunsaturated fatty acids (PUFAs) play various roles in inflammation. However, the effect of PUFAs in the development of reflux esophagitis (RE) is unclear. This study is to investigate the potential effect of n-3/n-6 PUFAs on acute RE in rats along with the underlying protective mechanisms. METHODS: Forty Sprague Dawley rats were randomly divided into four groups (n = 10 in each group). RE model was established by pyloric clip and section ligation. Fish oil- and soybean oil-based fatty emulsion (n-3 and n-6 groups), or normal saline (control and sham operation groups) was injected intraperitoneally 2 h prior to surgery and 24 h postoperatively (2 mL/kg, respectively). The expressions of interleukin (IL)-1ß, IL-8, IL-6 and myeloid differentiation primary response gene 88 (MyD88) in esophageal tissues were evaluated by Western blot and immunohistochemistry after 72 h. The malondialdehyde (MDA) and superoxide dismutase (SOD) expression in the esophageal tissues were determined to assess the oxidative stress. RESULTS: The mildest macroscopic/microscopic esophagitis was found in the n-3 group (P < 0.05). The expression of IL-1ß, IL-8, IL-6 and MyD88 were increased in all RE groups, while the lowest and highest expression were found in n-3 and n-6 group, respectively (P < 0.05). The MDA levels were increased in all groups (P < 0.05), in an ascending trend from n-3, n-6 groups to control group. The lowest and highest SOD levels were found in the control and n-3 group, respectively (P < 0.05). CONCLUSION: n-3 PUFAs may reduce acute RE in rats, which may be due to inhibition of the MyD88-NF-kB pathway and limit oxidative damage.

Lipopolysaccharide and IL-1ß coordinate a synergy on cytokine production by upregulating MyD88 expression in human gingival fibroblasts.

Both lipopolysaccharide (LPS) and interleukin (IL)-1ß activate the MyD88-dependent signaling pathways to stimulate proinflammatory cytokine expression. However, it remains unknown how LPS and IL-1ß interact with each other to coordinate the stimulation. In this study, we sought to investigate the interaction between LPS and IL-1ß on MyD88-dependent signaling pathways in human gingival fibroblasts (HGFs). Results showed that LPS derived from Porphyromonas gingivalis (Pg LPS) and IL-1ß cooperatively stimulated mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) signaling pathways, and subsequent expression of proinflammatory cytokine expression. Furthermore, our results showed that Pg LPS and IL-1ß exerted a synergy on MyD88 expression and knockdown of MyD88 expression by small interfering RNA diminished the synergistic effect of Pg LPS and IL-1ß on IL-6 expression, suggesting that upregulation of MyD88 is involved in the coordinated stimulation by Pg LPS and IL-1ß of proinflammatory cytokine expression. Finally, our results showed that pharmacological inhibitors for MAPK and NFκB significantly reduced IL-6 secretion stimulated by Pg LPS and IL-1ß, indicating that the MyD88-dependent MAPK and NFκB signaling pathways are essential for the upregulation of proinflammatory cytokine expression by Pg LPS and IL-1ß. Taken together, this study showed that LPS and IL-1ß coordinate a synergy on cytokine production by upregulating MyD88 expression in HGFs.

B-cell-specific conditional expression of Myd88p.L252P leads to the development of diffuse large B-cell lymphoma in mice.

The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

Cloning, Characterization, and Expression Analysis of MyD88 in Rana dybowskii.

The myeloid differentiation factor 88 (MyD88) is the most common adaptor protein in toll-like receptor (TLR) signaling pathways and plays an important role in the innate immune system. In this report, we conducted rapid amplification of complementary DNA (cDNA) ends (RACE), multiple sequence alignment, conserved domain search, phylogenetic tree construction, and quantitative real-time PCR to obtain and analyze the full-length cDNA sequence, the amino acid sequential structures, and the expression patterns of Rana dybowskii (Rd) MyD88. The full-length cDNA of RdMyD88 is 1472 bp, with an open reading frame of 855 bp, encoding a protein of 285 amino acid residues. The RdMyD88 amino acid sequence contains a death domain (DD) and a Toll/interleukin-1 receptor (TIR) domain. RdMyD88 was calculated as a hydrophilic protein with predicted molecular mass and pI of 32.79 kDa and 6.00, respectively. Eighteen possible phosphorylation sites including eight serine residues, six tyrosine residues, and four threonine residues are predicted. Analysis of multiple sequence alignment and phylogenetic tree revealed that the predicted RdMyD88 protein is closest to its Xenopus counterparts. The PCR result showed that RdMyD88 is expressed in various tissues of R. dybowskii. Quantitative real-time PCR (qPCR) was used to examine the expression of RdMyD88 in the heart, liver, and kidney. After Rana grylio virus (RGV) exposure, the expression of RdMyD88 in the heart, liver, and kidney were significantly upregulated and reached peak levels at 48, 48, and 72 h post-infection (hpi), respectively. Meanwhile, in response to Aeromonas hydrophila (AH) infection, clear upregulation of RdMyD88 was observed in the heart, liver, and kidney and reached its peak at 48, 6, and 12 hpi, respectively. The highest levels of induction were found in the kidney after both RGV and AH infections. These findings indicate that RdMyD88 has a conserved structure and is probably an important component of the innate immunity in R. dybowskii. This report firstly characterized one adaptor molecule of the TLR signaling pathways in R. dybowskii, thereby providing reference for further researches on the amphibian innate immune system.

Anti-neuroinflammatory effects of citreohybridonol involving TLR4-MyD88-mediated inhibition of NF-кB and MAPK signaling pathways in lipopolysaccharide-stimulated BV2 cells.

In the course of searching for anti-neuroinflammatory metabolites from marine fungi, citreohybridonol was isolated from marine-derived fungal strain Toxicocladosporium sp. SF-5699. Citreohybridonol inhibited production of nitric oxide (NO) and prostaglandin E2 (PGE2) in BV2 cells stimulated by lipopolysaccharide (LPS). Citreohybridonol also suppressed the expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and other pro-inflammatory cytokines including interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the LPS-stimulated cells. In the further study, citreohybridonol disturbed nuclear translocation of nuclear factor-kappa B (NF-κB) in LPS-stimulated BV2 cells by inhibiting the phosphorylation of the inhibitor kappa B-α (IκB-α). Citreohybridonol also had inhibitory effect on the LPS-stimulated phosphorylation of p38 mitogen-activated protein kinase (MAPK). Finally, citreohybridonol suppressed the protein expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in LPS-induced BV2 cells. These results suggest that citreohybridonol has anti-neuroinflammatory effect in LPS-stimulated BV2 cells by modulating TLR4-mediated several inflammatory pathways such as NF-κB and p38 MAPK pathways.

Kaposi's Sarcoma-Associated Herpesvirus Reduces Cellular Myeloid Differentiation Primary-Response Gene 88 (MyD88) Expression via Modulation of Its RNA.

UNLABELLED: Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a human gammaherpesvirus associated with several human malignancies. The replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. Interleukin 1 (IL-1) is a major mediator for inflammation and plays an important role in both innate and adaptive immunity. Myeloid differentiation primary response gene 88 (MyD88) is an essential adaptor molecule for IL-1 as well as most Toll-like receptor signaling. In this study, we identified a novel mechanism by which KSHV interferes with host inflammation and immunity. KSHV RTA specifically reduces the steady-state protein levels of MyD88, and physiological levels of MyD88 are downregulated during KSHV lytic replication when RTA is expressed. The N-terminal region of RTA is required for the reduction of MyD88. Additional studies demonstrated that RTA targets MyD88 expression at the RNA level, inhibits RNA synthesis of MyD88, and may bind MyD88 RNA. Finally, RTA inhibits IL-1-mediated activation of NF-κB. Because IL-1 is abundant in the KS microenvironment and inhibits KSHV replication, this work may expand our understanding of how KSHV evades host inflammation and immunity for its survival in vivo. IMPORTANCE: MyD88 is an important molecule for IL-1-mediated inflammation and Toll-like receptor (TLR) signaling. This work shows that KSHV inhibits MyD88 expression through a novel mechanism. KSHV RTA may bind to MyD88 RNA, suppresses RNA synthesis of MyD88, and inhibits IL-1-mediated signaling. This work may expand our understanding of how KSHV evades host inflammation and immunity.

TLR4-MyD88-TRAF6-TAK1 Complex-Mediated NF-κB Activation Contribute to the Anti-Inflammatory Effect of V8 in LPS-Induced Human Cervical Cancer SiHa Cells.

The synthetic compound 7-4-[Bis-(2-hydroxyethyl)-amino]-butoxy-5-hydroxy-8-methoxy-2-phenylchromen-4-one (V8) is a novel flavonoid-derived compound. In this study, we investigated the effects of V8 on Toll-like receptor 4 (TLR4)-mediated inflammatory reaction in human cervical cancer SiHa cells and lipopolysaccharide (LPS)-induced TLR4 activity in cervical cancer SiHa (HPV16+) cells, but not in HeLa (HPV18+) and C33A (HPV-) cells. In addition, V8 inhibited LPS-induced expression of TLR4, MyD88, TRAF6 and phosphorylation of TAK1, and their interaction with TLR4 in SiHa cells, resulting in an inhibition of TLR4-MyD88-TRAF6-TAK1 complex. Moreover, V8 blocked LPS-induced phosphorylation of IκB and IKK, resulting in inhibition of the nuclear translocation of P65-NF-κB in SiHa cells. We also found that V8 reduced the expression of NF-κB target genes, such as those for COX-2, iNOS, IL-6, IL-8, CCL-2, and TNF-α in LPS-stimulated SiHa cells. These results suggested that V8 exerted an anti-inflammatory effect on SiHa cells by inhibiting the TLR4-MyD88-TRAF6-TAK1 complex-mediated NF-κB activation.

Expression and significance of myeloid differentiation factor 88 in non-small cell lung carcinoma and normal paracancerous tissues.

We studied the expression level of myeloid differentiation factor 88 (MyD88) in non-small cell lung carcinoma (NSCLC) and normal paracancerous tissues, to determine its relationship with clinical pathological characteristics and prognosis. In total, 82 NSCLC patients who had received surgical treatment in our hospital between September 2008 and December 2013 were selected for this study. Another 82 normal paracancerous lung tissue samples were used as controls. All patients had complete clinical records, and they were followed-up for 5 years. The expression level of MyD88 protein was detected by immunohistochemical assay. The positive expression rate of MyD88 in NSCLC tissues (62.2%) was markedly higher than that in normal tissues (10.9%), and was independent of patient characteristics such as age, gender, pathological pattern, history of smoking, and tumor size (P > 0.05). However, MyD88 expression was significantly correlated with degree of differentiation, clinical staging, and lymphatic metastasis (P < 0.05), and was negatively correlated with prognosis. The 5-year survival rate of patients with positive MyD88 expression was significantly lower than that of patients without positive expression (P < 0.05). MyD88 was expressed at a higher level in NSCLC tissues and was closely associated with poor prognosis. MyD88 may be a novel eligible target for treating NSCLC.

Fibronectin fragment-induced expression of matrix metalloproteinases is mediated by MyD88-dependent TLR-2 signaling pathway in human chondrocytes.

INTRODUCTION: Fibronectin fragments (FN-fs) are increased in the cartilage of patients with osteoarthritis (OA) and have a potent chondrolytic effect. However, little is known about the cellular receptors and signaling mechanisms that are mediated by FN-fs. We investigated whether the 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) regulates cartilage catabolism via the Toll-like receptor (TLR)-2 signaling pathway in human chondrocytes. METHODS: Small interfering RNA was used to knock down TLR-2 and myeloid differentiation factor 88 (MyD88). TLR-2 was overexpressed in chondrocytes transfected with a TLR-2 expression plasmid. The expression levels of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 were analyzed using quantitative real-time reverse transcription polymerase chain reactions, immunoblotting, or enzyme-linked immunosorbent assay. The effect of TLR-2 on 29-kDa FN-f-mediated signaling pathways was investigated by immunoblotting. RESULTS: TLR-2, TLR-3, TLR-4, and TLR-5 mRNA were significantly overexpressed in OA cartilage compared with normal cartilage, whereas no significant difference of TLR-1 mRNA expression was found. 29-kDa FN-f significantly increased TLR-2 expression in human chondrocytes in a dose- and time-dependent manner. Knockdown of TLR-2 or MyD88, the latter a downstream adaptor of TLR-2, significantly inhibited 29-kDa FN-f-induced MMP production at the mRNA and protein levels. Conversely, TLR-2 overexpression led to enhanced MMP production by 29-kDa FN-f. In addition, TLR-2 knockdown apparently inhibited 29-kDa FN-f-mediated activation of phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, and p38, but not of c-Jun N-terminal kinase or extracellular signal-regulated kinase. Exposure to synovial fluid (SF) from affected joints of patients with OA elevated MMP-1, MMP-3, and MMP-13 expression markedly in primary chondrocytes without reducing cell viability. However, TLR-2 knockdown in chondrocytes significantly suppressed SF-induced MMP induction. CONCLUSIONS: Our data demonstrate that the MyD88-dependent TLR-2 signaling pathway may be responsible for 29-kDa FN-f-mediated cartilage catabolic responses. Our results will enhance understanding of cartilage catabolic mechanisms driven by cartilage degradation products, including FN-f. The modulation of TLR-2 signaling activated by damage-associated molecular patterns, including 29-kDa FN-f, is a potential therapeutic strategy for the prevention of cartilage degradation in OA.