Overexpression Of MyD88 Research Articles

N-acetylcysteine alleviates PCB52-induced hepatotoxicity by repressing oxidative stress and inflammatory responses.

Polychlorinated biphenyls (PCBs), particularly low chlorinated congeners in our environment, can induce human hepatotoxicity. However, the mechanisms by which PCBs cause hepatotoxicity remain elusive. Moreover, there are no effective treatments for this condition. In this study, 40 μM PCB52 was administered to rat (Brl-3A) and human hepatocytes (L-02) for 48 h following the N-acetylcysteine (NAC)/saline pretreatment. A significant decrease in cell viability was observed in PCB52-treated cells relative to the control. Besides, PCB52 significantly increased reactive oxygen species (ROS) levels and malondialdehyde (MDA) contents, suggesting induction of oxidative stress. The expression of Traf6, MyD88, and Tnf in Brl-3A cells and that of MYD88, TNF, and IL1B in L-02 cells were significantly upregulated by PCB52. Consistently, overexpression of TLR4, MyD88, Traf6, and NF-κB p65 proteins was observed in PCB52-treated cells, indicating activation of inflammatory responses. Nevertheless, no changes in kelch-like ECH-associated protein 1 (keap1), nuclear factor-erythroid 2-related factor (nrf2), and heme oxygenase-1 proteins were observed in PCB52-treated cells, indicating non-activation of the keap1/nrf2 pathway. Pretreatment with NAC significantly ameliorated PCB52 effects on cell viability, ROS levels, MDA contents and expression of inflammatory elements at both RNA and protein levels. However, no changes in keap1, nrf2 and HO-1 protein levels were detected following NAC pretreatment. Taken together, with non-activated keap1/nrf2 pathway, PCB52-induced oxidative stress and inflammatory responses could be responsible for its hepatotoxicity. These effects were effectively attenuated by NAC pretreatment, which scavenges ROS and dampens inflammatory responses. This study might provide novel strategies for the treatment of the PCBs-associated hepatotoxic effects.

A small molecule inhibitor of MyD88 exhibits broad spectrum antiviral activity by up regulation of type I interferon

Recent studies highlight that infection with Coxsackievirus B3, Venezuelan equine encephalitis virus (VEEV), Marburg virus, or stimulation using poly I:C (dsRNA), upregulates the signaling adaptor protein MyD88 and impairs the host antiviral type I interferon (IFN) responses. In contrast, MyD88 deficiency (MyD88−/−) increases the type I IFN and survivability of mice implying that MyD88 up regulation limits the type I IFN response. Reasoning that MyD88 inhibition in a virus-like manner may increase type I IFN responses, our studies revealed lipopolysaccharide stimulation of U937 cells or poly I:C stimulation of HEK293-TLR3, THP1 or U87 cells in the presence of a previously reported MyD88 inhibitor (compound 4210) augmented IFN-β and RANTES production. Consistent with these results, overexpression of MyD88 decreased IFN-β, whereas MyD88 inhibition rescued IFN-β production concomitant with increased IRF3 phosphorylation, suggesting IRF-mediated downstream signaling to the IFN-β response. Further, compound 4210 treatment inhibited MyD88 interaction with IRF3/IRF7 indicating that MyD88 restricts type I IFN signaling through sequestration of IRF3/IRF7. In cell based infection assays, compound 4210 treatment suppressed replication of VEEV, Eastern equine encephalitis virus, Ebola virus (EBOV), Rift Valley Fever virus, Lassa virus, and Dengue virus with IC50 values ranging from 11 to 42 μM. Notably, administration of compound 4210 improved survival, weight change, and clinical disease scores in mice following challenge with VEEV TC-83 and EBOV. Collectively, these results provide evidence that viral infections responsive to MyD88 inhibition lead to activation of IRF3/IRF7 and promoted a type I IFN response, thus, raising the prospect of an approach of host-directed antiviral therapy.

The suppressive role of miR-362-3p in epithelial ovarian cancer

Ovarian cancer is a common cancer worldwide. Epithelial ovarian cancer (EOC) is the most common subtype of ovarian cancer. This study was designed to explore the function of miR-362-3p in EOC. QRT-PCR analysis was used to test miR-362-3p levels in EOC tissues and cell lines. Cell viability was tested via MTT assay. Transwell systems were applied to assay cell migration. The target gene of miR-362-3p was evaluated using dual luciferase reporter assays. The MyD88 protein in EOC cells was tested via western blot. Our data showed that miR-362-3p was expressed at low levels in EOC tissues and cells. miR-362-3p inhibited cell proliferation and migration, bound the 3′-untranslated region (UTR) of MyD88, and inhibited MyD88 expression. MyD88 was inversely correlated with miR-362-3p in EOC, and MyD88 overexpression partly reduced the anti-proliferative effect of miR-362-3p in EOC cells. In conclusion, our data showed that miR-362-3p has an anti-proliferative effect on EOC.

TB-02 NF-KB CANONICAL PATHWAY ACTIVATION DRIVES GLYCOLYSIS AND TUMOR PROGRESSION IN PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA

Recent genomic analyses have identified highly recurrent genetic alterations in PCNSL. However, due to the lack of clinically representative PCNSL preclinical models, the pathogenic mechanisms of these alterations remains largely unknown. Here, we established the largest panel of 12 clinically relevant PCNSL patient-derived orthotopic xenografts retained the histopathologic phenotype, lymphoma expression subtype, copy number alterations and 90% of the non-synonymous mutations of primary tumors, with 100% concordance of MYD88 and CD79B mutations, which are highly recurrent in PCNSL. Patient tumor regression with high-dose methotrexate correlated with in vitro sensitivity to methotrexate in corresponding PCNSL models. By knocking down canonical NF-kB pathway genes, we found that successful orthotopic xenograft formation was dependent on NF-kB canonical pathway activation induced by MYD88 mutation or overexpression of EBV-related LMP1. Metabolically, PCNSL xenografts phenocopied the high 18F-fluorodeoxyglucose uptake observed in patients and demonstrated glycolytic dependence, revealing new potential therapeutic strategies in PCNSL. Collectively, we found NF-kB canonical pathway activation as a crucial driver of PCNSL xenograft progression and found that NF-kB canonical pathway induced an addiction to glycolysis, revealing a novel potential therapeutic strategy. Our PCNSL xenograft panel represents a valuable and reproducible preclinical tool that has the potential to help decipher how genetic and/or epigenetic alterations contributes to lymphomagenesis and tumor maintenance and enhance the development of novel therapeutic strategies in PCNSL.

Protective effects of the combination Bifidobacterium longum plus lactoferrin against NSAID-induced enteropathy.

Nonsteroidal anti-inflammatory drugs can exert detrimental effects in the lower digestive tract. The aim of this study was to examine the protective effects of a combination of the probiotic Bifidobacterium longum BB536 (Bifidobacterium) with the prebiotic lactoferrin in a rat model of diclofenac-induced enteropathy. Enteropathy was induced in 40-wk-old male rats by intragastric diclofenac (4 mg/kg twice daily for 14 d). Lactoferrin (100 mg/kg twice daily), Bifidobacterium (2.5 × 106 CFU/rat twice daily) or their combination were administered 1 h before diclofenac. At the end of treatments, the ileum was processed for the evaluation of histologic damage, myeloperoxidase (MPO) and malondialdehyde (MDA) levels, as well as the expression of Toll-like receptors 2 and 4 (TLR-2/-4) and the activation of downstream signaling molecules (MyD88 and nuclear factor [NF]-κB p65). Blood hemoglobin and fecal calprotectin were also assessed. Diclofenac induced intestinal damage, along with increments of MPO and MDA, overexpression of TLR-2, TLR-4, MyD88, and NF-κB p65, increased fecal calprotectin and decreased blood hemoglobin levels. Lactoferrin or Bifidobacterium alone prevented diclofenac-induced enteric damage, and the changes in blood hemoglobin, MPO, MDA, fecal calprotectin, and NF-κB p65. Bifidobacterium, but not lactoferrin, decreased TLR-4 expression, although none of them affected MyD88 overexpression. TLR-2 expression was slightly enhanced by all treatments. The combined administration of lactoferrin and Bifidobacterium reduced further the intestinal damage, and restored MPO and blood hemoglobin levels. Diclofenac induced ileal mucosal lesions by activation of inflammatory and pro-oxidant mechanisms. These detrimental actions were prevented by the combination of lactoferrin with Bifidobacterium likely through the modulation of TLR-2/-4/NF-κB proinflammatory pathways.

MyD88 overexpression deteriorates Ang-II-induced ED via upregulating MPO and COX2 and downregulating eNOS in the corpus cavernosum of rats.

Erectile dysfunction (ED) is a common sexual problem for men and the exploration of its treatment is still in mire demand. We aim to investigate the role of the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88) signaling pathway in the pathogenesis of angiotensin II (Ang-II) induced ED. Male Sprague-Dawlay rats were treated with Ang-II and intracavernous pressure (ICP) was measured to confirm the occurrence of ED. The corpus cavernosum penises of rats were transfected with plasmids to overexpressed MyD88. Inflammatory and vascular parameters including myeloperoxidase (MPO), cyclooxygenase2 (COX2), endothelial nitric oxide synthase (eNOS), malondialdehyde (MDA), superoxide dismutase (SOD), reactive oxygen species (ROS), and cytokines in treated and untreated ED rats were measured. Flow cytometry was used to determine the apoptosis of endothelial cells of corpus cavernosum penises of rats. Ang-II-induced ED rats were found to contain upregulated TLR4, MyD88, MPO, and COX2, and downregulated eNOS. MyD88 overexpression deteriorates cavernous structural damage, reduces ICP and ICP/MAP values and reverses the therapeutic effect of anti-TLR4 antibodies in rats with Ang-II-induced ED. Moreover, overexpression of MyD88 further upregulated MPO and COX2, downregulated eNOS, promoted oxidative stress, inflammation, and cell apoptosis rate via positively regulating the TLR4/MyD88 signaling pathway, while anti-TLR4 antibodies downregulated MPO and COX2, upregulated eNOS, suppressed oxidative stress, inflammation, and cell apoptosis rate via inactivating the TLR4/MyD88 signaling pathway in the rat corpus cavernosum penises. Furthermore, MyD88 overexpression promotes oxidative stress and inflammation and reverses the effect of anti-TLR4 antibodies in the penis of ED rats. MyD88 overexpression deteriorates Ang-II-induced ED via upregulating MPO and COX2 and downregulating eNOS in the corpus cavernosum rats.

Modified Genomic Self-DNA Influences In Vitro Survival of HT29 Tumor Cells via TLR9- and Autophagy Signaling.

In relation of immunobiology, the consequence of the crosstalk between TLR9-signaling and autophagy is poorly documented in HT29 cancer cells. To assess the TLR9-mediated biologic effects of modified self-DNA sequences on cell kinetics and autophagy response HT29 cells were incubated separately with intact genomic (g), hypermethylated (m), fragmented (f), and hypermethylated/fragmented (m/f) self-DNAs. Cell viability, apoptosis, cell proliferation, colonosphere-formation were determined. Moreover, the relation of TLR9-signaling to autophagy response was assayed by real-time RT-PCR, immunocytochemistry and transmission electron microscopy (TEM). After incubation with g-, m-, and m/f-DNAs cell viability and proliferation decreased, while apoptosis increased. F-DNA treatment resulted in an increase of cell survival. Methylation of self-DNA resulted in decrease of TLR9 expression, while it did not influence the positive effect of DNA fragmentation on MyD88 and TRAF6 overexpression, and TNFα downregulation. Fragmentation of DNA abrogated the positive effect of methylation on IRAK2, NFκB and IL-8 mRNA upregulations. In case of the autophagy genes and proteins, g- and f-DNAs caused significant upregulation of Beclin1, Atg16L1, and LC3B. According to TEM analyses, autophagy was present in each group of tumor cells, but to a varying degree. Incubation with m-DNA suppressed tumor cell survival by inducing features of apoptotic cell death, and activated mitophagy. F-DNA treatment enhanced cell survival, and activated macroautophagy and lipophagy. Colonospheres were only present after m-DNA incubation. Our data provided evidence for a close existing interplay between TLR9-signaling and the autophagy response with remarkable influences on cell survival in HT29 cells subjected to modified self-DNA treatments.

Immune and Antioxidant Enzyme Response of Longfin Yellowtail (Seriola rivoliana) Juveniles to Ultra-diluted Substances Derived from Phosphorus, Silica and Pathogenic Vibrio.

This research aimed to observe the effect of homeopathic treatments prepared from Vibrio parahaemolyticus and V. alginolyticus (H1) and commercial homeopathic medication Phosphoricum acidum and Silicea terra (H2) on the immune and antioxidant response in Seriola rivoliana juveniles under usual culture conditions and challenged with V. parahaemolyticus. Quantitative polymerase chain reaction analysis was used to study changes in the expression of key genes related to immune response, cytokines (interleukin-1β [IL-1β]), adapter protein for cytokine release (MyD88) and piscidin and spectrophotometric techniques to analyze the activity of antioxidant superoxide dismutase (SOD) and catalase (CAT) enzymes in Seriola rivoliana juveniles at 30 (weaning stage [WS]) and 60 (early juveniles [EJ]) days post-hatching. The H1 treatment led to over-expression of the IL-1β and MyD88 genes in fish at WS and EJ with respect to control, contrary to the H2 treatment that led to under-expression of the IL-1β, MyD88 and piscidin genes at the EJ stage. In fish challenged with V. parahaemolyticus, both H1 and H2 led to over-expression of IL-1β and MyD88; H2 caused an over-expression of piscidin. The SOD activity was higher in H1 with respect to H2 and the control group. CAT remained relatively stable with both H1 and H2 treatments. The results suggest that the overall effect of H1 was due to the presence of unknown antigens in low concentrations, while the response to H2-specifically during challenge-may have been due to a stimulating effect of nano-structures, prevailing from mother tincture after sequential dilution/succussion, in a pathway similar to that attributed to nano-vaccines.

Analysis of Genomic Alteration in Primary Central Nervous System Lymphoma and the Expression of Some Related Genes

Primary central nervous system lymphoma (PCNSL) is a rare and special type of non-Hodgkin lymphoma. The treatment of PCNSL is comprehensive, combining surgery, radiotherapy, and chemotherapy. However, the outcome is poor because of its high invasiveness and rate of recurrence. We analyzed 22 cases of PCNSL using next-generation sequencing (NGS) to detect 64 candidate genes. We used immunohistochemical methods to analyze gene expression in 57 PCNSL samples. NGS showed that recurrent mutations in KMT2D and CD79B, components of the NF-κB pathway, accounted for 65% of total mutations in PCNSL samples. The most frequent mutated gene was PIM1 (77.27%, 17/22), followed by MYD88 (63.64%, 14/22), CD79B (69.09%, 13/22), and KMT2D (50.00%, 11/22). Mutations of the CD79B gene were associated with an inferior progression-free survival (PFS), and GNA13 gene mutations were associated with a shorter PFS and overall survival (OS) in PCNSL patients (P < .05). PIM1 and MYD88 were highly expressed in PCNSL patients and were related to their OS time. MYD88 overexpression might be an independent and poor prognostic predictor of OS time. In summary, we identified highly recurrent genetic lesions in CD79B and KMT2D, components of the NF-κB pathway, in PCNSL and validated the expression of PIM1 and MYD88 related to poor survival, thereby providing novel insights into the pathogenesis and precision medicine of PCNSL.

Gene Expression Profile of Toll-Like Receptor/Adaptor/Interferon Regulatory Factor/Cytokine Axis During Liver Regeneration After Partial Ischemia-Reperfusion Injury

Toll-like receptors and downstream signal transduction pathways play pivotal roles in induction of inflammation, which is crucial for liver injury and regeneration. Using a mouse model of partial hepatic ischemia-reperfusion injury followed by a 28-day time course for liver repair and regeneration, we assessed gene expression levels for Toll-like receptors, myeloid differentiation primary response 88, TIR-domain-containing adapter-inducing interferon-β, nuclear factor κB, interferon regulatory factors, tumor necrosis factor-α, and interleukins 1β and 6 at days 1, 4, 7, 14, and 28 after reperfusion in liver and blood cells by quantitative polymerase chain reaction. Mouse liver was gradually injured until 24 hours after reperfusion, and necrotic areas remained for 7 days. Concurrent with liver necrosis, overexpression of hepatocyte growth factor in blood cells (days 1-14), transient overexpression of cyclin D1 at day 7 in hepatic cells, and overexpression of transforming growth factor-β1 at days 7 and 14 in blood cells were used to characterize the priming, proliferative, and termination phases of liver regeneration. Liver regeneration was associated with significant up-regulation of Toll-like receptor 4, p65, interferon regulatory factors 1, 3, 9, tumor necrosis factor-α, and interleukin 1β at 24 hours. Liver regeneration was also associated with persistent overexpression of MyD88 (days 1-28) and with delayed TIR-domain-containing adapter-inducing interferon-β (days 4-28) in hepatic cells. In peripheral blood cells, Toll-like receptor 2 and MyD88 were up-regulated at 24 hours and Toll-like receptor 4 (days 1-14) and interferon regulatory factor 1 (days 1-7) showed persistent overexpression concomitant with interferon regulatory factor 5 (days 7-14); interleukin 1β (days 1-28) and interleukin 6 (day 4-28) also showed persistent expression. We depict for the first time a prospective view of cooperative transcriptional activation of Toll-like receptors/adaptors/interferon regulatory factors/cytokines in both liver and blood cells during different phases of liver repair after ischemia-reperfusion injury.

Mutational and expressional analyses of MYD88 gene in common solid cancers.

Myeloid differentiation primary response gene 88 (MYD88) is a protein involved in hematopoietic differentiation and innate immunity. Recent studies revealed MYD88 mutation in hematological malignancies and MYD88 overexpression in some solid cancers. The aim of this study was to see whether alterations of MYD88 protein expression and somatic mutation of MYD88 gene are features of common solid cancers. We analyzed MYD88 mutation in 45 gastric, 45 colorectal, 45 breast, 45 hepatocellular, 45 prostate and 45 lung carcinomas by single-strand conformation polymorphism (SSCP). We also analyzed MYD88 protein expression in 60 gastric, 60 coloretal and 107 prostate carcinomas by immunohistochemistry. In the immunohistochemistry results, MYD88 protein was highly expressed in gastric (75%), colorectal (80%) and prostate (83%) cancers. However, MYD88 expression was significantly different among normal tissues (gastric: 58%, colon: 100%, prostate: 86%). MYD88 expression was significantly increased in gastric cancer cells compared with normal cells, whereas it was decreased in colorectal cancer cells compared with normal cells. There were no somatic mutations of the MYD88 gene in gastric, colorectal, breast, hepatocellular, prostate and lung carcinomas. Our data indicate that MYD88 overexpression might be a feature of many solid cancers, but MYD88 expression in normal cells differs depending on the organs. The data suggest that a gain of MYD88 expression in gastric cancers might play a role in cancer pathogenesis by activating oncogenic functions of MYD88.

Phosphatidylinositide 3-Kinase Contributes to the Anti-Inflammatory Effect of Abutilon crispum L. Medik Methanol Extract.

Abutilon crispum L. Medik, better known as bladdermallow, is used as a traditional remedy in India, for its anti-inflammatory effect due to its high content of flavonoids. However, research about its anti-inflammatory effect at the molecular level has not been performed. In this study, we aimed to investigate the mechanism of Abutilon crispum methanol extract (Ac-ME) in inhibiting the inflammatory response by conducting several experiments including cellular and molecular assays. Ac-ME inhibited the production of nitric oxide (NO) in RAW264.7 cells during treatment of LPS and Pam3CSK4 without exhibiting cytotoxicity. Ac-ME also suppressed the mRNA expression of inducible nitric oxide (iNOS) and proinflammatory cytokines such as interleukin (IL)-1β and IL-6. Moreover, Ac-ME was shown to inhibit the NF-κB pathway, according to the luciferase reporter gene assay performed with a NF-κB-Luc construct containing NF-κB-binding promoter regions under MyD88 and TRIF overexpression conditions, and immunoblotting analysis by determining the phospho-form levels of IκBα, IKKα/β, and p85, a regulatory domain of phosphatidylinositide 3-kinase (PI3K). Finally, we observed that the level of phospho-p85 induced by the overexpression of spleen tyrosine kinase (Syk) and Src was decreased by Ac-ME at 200 μg/ml. Therefore, these results suggest that Ac-ME has an anti-inflammatory effect by targeting PI3K in the NF-κB signaling pathway.

Effects of moroxydine hydrochloride and ribavirin on the cellular growth and immune responses by inhibition of GCRV proliferation

Moroxydine hydrochloride (Mor) and ribavirin (Rib) are known for their multi-antiviral activities against DNA and RNA viruses but little information is available about the pharmacological impact in aquaculture. The present study was undertaken to investigate the response of host cells to antiviral compounds during the anti-GCRV treatment. The scanning electron microscope results showed that Ctenopharyngodon idella kidney (CIK) cells have a higher death rate at 72h post virus infection. At the concentration of 40μgmL−1, Mor and Rib had a significant protective effect on virus-infected cells. Moreover, the gene expressions of vp5, vp6 and NS66 were significantly inhibited by treatment with Mor or Rib, especially gene expression of the vp5. For the immunoregulatory action, no distinct induction of the expression of immune genes was observed after the addition of Mor and Rib to the virus-free cells. However, the compounds significantly decreased the virus-induced gene overexpression of Myd88, Mx1, IL-1β, IL-8, I-IFN and TNFα in CIK cells. Moreover, Mor and Rib significantly inhibited the immune genes upregulation which was induced by GCRV in kidney, liver, muscle and gill of grass carp, despite greater partial gene expressions were detected than the virus-free control group. Besides, Mor and Rib blocked cell cycle changes, cytopathic effects, cellular death and virus proliferation in CIK cells thereby maintaining normal morphological structure. Overall, Mor and Rib as antiviral compounds are effective for the control of GCRV replication and the indirectly regulation of cellular immune response.

Pellino-3 promotes endotoxin tolerance and acts as a negative regulator of TLR2 and TLR4 signaling.

Development of endotoxin tolerance in macrophages during sepsis reprograms Toll-like receptor 4 signaling to inhibit proinflammatory cytokines without suppressing anti-inflammatory and antimicrobial mediators and protects the host from excessive inflammation and tissue damage. However, endotoxin tolerance renders septic patients immunocompromised and unable to control secondary infections. Although previous studies have revealed the importance of several negative regulators of Toll-like receptor signaling in endotoxin tolerance, the role of Pellino proteins has not been addressed. The present report shows that the induction of endotoxin tolerance in vivo in mice and in vitro in human monocytes and THP-1 and MonoMac-6 macrophages increases the expression of Pellino-3. Overexpression of Pellino-3 in human embryonic kidney 293/Toll-like receptor 2 or 293/Toll-like receptor 4/myeloid differentiation factor-2 cells inhibited Toll-like receptor 2/4-mediated activation of nuclear factor-κB and induction of CXCL-8 mRNA, and Pellino-3 ablation increased these responses. Pellino-3-deficient THP-1 cells had elevated Toll-like receptor 2/4-driven tumor necrosis factor-α, interleukin-6 mRNA, and Toll-like receptor 4-driven CCL5 gene expression in response to Toll-like receptor agonists and heat-killed Escherichia coli and Staphylococcus aureus, cytokines controlled by the MyD88 and Toll-interleukin-1R domain-containing protein inducing interferon-β-mediated pathways, respectively. In addition, deficiency in Pellino-3 slightly increased phagocytosis of heat-killed bacteria. Transfected Pellino-3 inhibited nuclear factor-κB activation driven by overexpression of MyD88, TIR domain-containing adapter inducing interferon-β, interleukin-1R-associated kinase-1, and tumor necrosis factor receptor activator of nuclear factor-κB-binding kinase-1, TGF-β-activated kinase 1, and tumor necrosis factor receptor-associated factor-6, and inhibited interleukin-1R-associated kinase 1 modifications and tumor necrosis factor receptor activator of nuclear factor-κB-binding kinase 1 phosphorylation. Finally, Pellino-3 ablation in THP-1 decreased the extent of endotoxin tolerization. Thus, Pellino-3 is involved in endotoxin tolerance and functions as a negative regulator of Toll-like receptor 2/4 signaling.

Fructose-1,6-bisphosphate suppresses lipopolysaccharide-induced expression of ICAM-1 through modulation of toll-like receptor-4 signaling in brain endothelial cells.

Fructose-1,6-bisphosphate (FBP) is a glycolytic intermediate with salutary effects in various brain injury models, but its neuroprotective mechanism is incompletely understood. In this study, we examined the effects of FBP on the expression of adhesion molecules in cerebrovascular endothelial cells and explored the possible mechanisms therein involved. FBP significantly down-regulated lipopolysaccharide (LPS)-induced expression of adhesion molecules and leukocyte adhesion to brain endothelial cells and inhibited NF-κB activity, which is implicated in the expression of adhesion molecules. FBP abrogated ICAM-1 expression and NF-κB activation induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or overexpression of MyD88 or TRAF6. FBP suppressed TRAF6-induced phosphorylation of TAK1, IKKβ and IκBα, but fail to affect NF-κB activity induced by ectopic expression of IKKβ. In addition, LPS-induced IRAK-1 phosphorylation was inhibited by FBP, suggesting the presence of multiple molecular targets of FBP in MyD88-dependent signaling pathway. FBP significantly attenuated ICAM-1 expression and NF-κB activity induced by poly[I:C] or overexpression of TRIF or TBK1. FBP significantly repressed the expression of interferon-β (IFN-β) and the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C] or overexpression of TRIF or TBK1, but fail to affect IRF3 activity induced by ectopic expression of constitutively active IRF3. Overall, our results demonstrate that FBP modulates both MyD88- and TRIF-dependent signaling pathways of TLR4 and subsequent inflammatory responses in brain endothelial cells, providing insight into its neuroprotective mechanism in brain injury associated with inflammation.

Poly(I:C) induces antiviral immune responses in Japanese flounder (Paralichthys olivaceus) that require TLR3 and MDA5 and is negatively regulated by Myd88.

Polyinosinic:polycytidylic acid (poly(I:C)) is a ligand of toll-like receptor (TLR) 3 that has been used as an immunostimulant in humans and mice against viral diseases based on its ability to enhance innate and adapt immunity. Antiviral effect of poly(I:C) has also been observed in teleost, however, the underling mechanism is not clear. In this study, we investigated the potential and signaling mechanism of poly(I:C) as an antiviral agent in a model of Japanese flounder (Paralichthys olivaceus) infected with megalocytivirus. We found that poly(I:C) exhibited strong antiviral activity and enhanced activation of head kidney macrophages and peripheral blood leukocytes. In vivo studies showed that (i) TLR3 as well as MDA5 knockdown reduced poly(I:C)-mediated immune response and antiviral activity to significant extents; (ii) when Myd88 was overexpressed in flounder, poly(I:C)-mediated antiviral activity was significantly decreased; (iii) when Myd88 was inactivated, the antiviral effect of poly(I:C) was significantly increased. Cellular study showed that (i) the NF-κB activity induced by poly(I:C) was upregulated in Myd88-overexpressing cells and unaffected in Myd88-inactivated cells; (ii) Myd88 overexpression inhibited and upregulated the expression of poly(I:C)-induced antiviral genes and inflammatory genes respectively; (iii) Myd88 inactivation enhanced the expression of the antiviral genes induced by poly(I:C). Taken together, these results indicate that poly(I:C) is an immunostimulant with antiviral potential, and that the immune response of poly(I:C) requires TLR3 and MDA5 and is negatively regulated by Myd88 in a manner not involving NK-κB. These results provide insights to the working mechanism of poly(I:C), TLR3, and Myd88 in fish.

MyD88 expression is associated with paclitaxel resistance in lung cancer A549 cells.

The purpose of the present study was to investigate the relationship between myeloid differentiation primary response gene88 (MyD88) expression and the resistance to paclitaxel of A549 lung cancer cells. In order to achieve MyD88 gene overexpression or knockdown in the A549 cell line, the cells were infected with lentivirus carrying MyD88 gene or shRNA to MyD88. MyD88 mRNA and protein expression was measured by RT-qPCR, immunohistochemistry and western blotting after infection for confirmation. Cell prolife-ration was detected by the WST-1 assay. Flow cytometry was used to measure the cell cycle and apoptosis. The transwell migration assay was used to observe the change of migration of transfected cells. The results showed that the overexpression of MyD88 increased the resistance of lung cancer A549 cells to paclitaxel, while the suppression of MyD88 increased the sensitivity of A549 lung cancer cells to paclitaxel. Following the paclitaxel treatment, a decreased apoptosis and G2 phase ratio, an increased cell migration ratio, and an increased production of IL-8 were found in MyD88‑overexpressed A549 cells. The western blot analysis revealed that Myd88 overexpression resulted in an increased level of Bcl-2 but a decreased level of Bax in A549 cells. These findings suggested that the expression level of MyD88 is closely associated with paclitaxel resistance in A549 lung cancer cells. Thus, the downregulation of MyD88 in A549 cells increased its sensitivity to paclitaxel treatment, whereas the upregulation of MyD88 substantiates its paclitaxel resistance.

Human Parainfluenza Virus Type 2 V Protein Inhibits TRAF6-Mediated Ubiquitination of IRF7 To Prevent TLR7- and TLR9-Dependent Interferon Induction

Paramyxovirus V proteins block Toll-like receptor 7 (TLR7)- and TLR9-dependent signaling leading to alpha interferon production. Our recent study has provided evidence that interaction of the V proteins with IRF7 is important for the blockade. However, the detailed mechanisms still remain unclear. Here we reexamined the interaction of the human parainfluenza virus type 2 (HPIV2) V protein with signaling molecules involved in TLR7/9-dependent signaling. Immunoprecipitation experiments in HEK293T cells transfected with V protein and one of the signaling molecules revealed that the V protein interacted with not only IRF7 but also TRAF6, IKKα, and MyD88. Whereas overexpression of TRAF6 markedly enhanced the level of V protein associating with IRF7, IKKα, and MyD88 in HEK293T cells, the level of V protein associating with TRAF6 was little affected by overexpression of IRF7, IKKα, and MyD88. Moreover, knockdown or knockout of endogenous TRAF6 in HEK293T or mouse embryonic fibroblast cells resulted in dissociation of the V protein from IRF7, IKKα, and MyD88. These results demonstrate that binding of the V protein to IRF7, IKKα, and MyD88 is largely indirect and mediated by endogenous TRAF6. It was found that the V protein inhibited TRAF6-mediated lysine 63 (K63)-linked polyubiquitination of IRF7, which is prerequisite for IRF7 activation. Disruption of the tryptophan-rich motif of the V protein significantly affected its TRAF6-binding efficiency, which correlated well with the magnitude of inhibition of K63-linked polyubiquitination and the resultant activation of IRF7. Taken together, these results suggest that the HPIV2 V protein prevents TLR7/9-dependent interferon induction by inhibiting TRAF6-mediated K63-linked polyubiquitination of IRF7.

MYD88 expression and L265P mutation in diffuse large B-cell lymphoma

Activated B-cell-like subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by chronic active B-cell receptor signaling and a constitutive activation of the nuclear factor κB pathway. As a driving force of nuclear factor κB overactivity, myeloid differentiation primary response gene 88 (MYD88) L265P mutation occurs in activated B-cell-like DLBCL. However, the MYD88 L265P mutation has not yet been studied in conjunction with MYD88 protein expression in DLBCL cases. Thus, we investigated MYD88 expression in 124 DLBCL specimens by immunohistochemistry. In addition, the MYD88 L265P mutation was examined by polymerase chain reaction and direct DNA sequencing. MYD88 was overexpressed in 38.7% (48/124) of DLBCL cases. MYD88 was expressed in the cytoplasm of lymphoma cells. MYD88 overexpression was associated with older age (P = .016) and tumor recurrence (P = .007). In univariate analysis, MYD88 overexpression was correlated with shortened disease-free survival (DFS) of DLBCL (P = .013) and non-germinal center B-cell-like DLBCL (P = .034). In multivariate analysis, MYD88 overexpression was an independent factor for reduced DFS (P = .025). MYD88 L265P mutation was found in 6.5% (8/124) of DLBCL cases. All but 1 case were of non-germinal center B-cell-like subtype. The MYD88 L265P mutation was not associated with MYD88 expression (Spearman ρ = -0.074) and clinicopathologic parameters of DLBCL. The data indicate that high MYD88 expression is significantly associated with tumor recurrence and shortened DFS in patients with DLBCL. In addition, the incidence of MYD88 L265P mutation in Korea is much lower than previously reported. Thus, MYD88 may be crucial for lymphoma progression, independent of MYD88 L265P mutation.

Mutational and Expressional Analyses of MYD88 Gene in Common Solid Cancers

Myeloid differentiation primary response gene 88 (MYD88) is a protein involved in hematopoietic differentiation and innate immunity. Recent studies revealed MYD88 mutation in hematological malignancies and MYD88 overexpression in some solid cancers. The aim of this study was to see whether alterations of MYD88 protein expression and somatic mutation of MYD88 gene are features of common solid cancers. We analyzed MYD88 mutation in 45 gastric, 45 colorectal, 45 breast, 45 hepatocellular, 45 prostate and 45 lung carcinomas by single-strand conformation polymorphism (SSCP). We also analyzed MYD88 protein expression in 60 gastric, 60 coloretal and 107 prostate carcinomas by immunohistochemistry. In the immunohistochemistry results, MYD88 protein was highly expressed in gastric (75%), colorectal (80%) and prostate (83%) cancers. However, MYD88 expression was significantly different among normal tissues (gastric: 58%, colon: 100%, prostate: 86%). MYD88 expression was significantly increased in gastric cancer cells compared with normal cells, whereas it was decreased in colorectal cancer cells compared with normal cells. There were no somatic mutations of the MYD88 gene in gastric, colorectal, breast, hepatocellular, prostate and lung carcinomas. Our data indicate that MYD88 overexpression might be a feature of many solid cancers, but MYD88 expression in normal cells differs depending on the organs. The data suggest that a gain of MYD88 expression in gastric cancers might play a role in cancer pathogenesis by activating oncogenic functions of MYD88.