Myeloid Differentiation Primary Response Gene Research Articles

Syk Plays a Critical Role in the Expression and Activation of IRAK1 in LPS-Treated Macrophages.

To address how interleukin-1 receptor-associated kinase 1 (IRAK1) is controlled by other enzymes activated by toll-like receptor (TLR) 4, we investigated the possibility that spleen tyrosine kinase (Syk), a protein tyrosine kinase that is activated at an earlier stage during TLR4 activation, plays a central role in regulating the functional activation of IRAK1. Indeed, we found that overexpression of myeloid differentiation primary response gene 88 (MyD88), an adaptor molecule that drives TLR signaling, induced IRAK1 expression and that piceatannol, a Syk inhibitor, successfully suppressed the MyD88-dependent upregulation of IRAK1 under LPS treatment conditions. Interestingly, in Syk-knockout RAW264.7 cells, IRAK1 activity was almost completely blocked after LPS treatment, while providing a Syk-recovery gene to the knockout cells successfully restored IRAK1 expression. According to our measurements of IRAK1 mRNA levels, the transcriptional upregulation of IRAK1 was induced by LPS treatment between 4 and 60 min, and this can be suppressed in Syk knockout cells, providing an effect similar that that seen under piceatannol treatment. The overexpression of Syk reverses this effect and leads to a significantly higher IRAK1 mRNA level. Collectively, our results strongly suggest that Syk plays a critical role in regulating both the activity and transcriptional level of IRAK1.

Astragaloside IV attenuates cognitive impairments induced by transient cerebral ischemia and reperfusion in mice via anti-inflammatory mechanisms

Astragaloside IV (AS-IV) is the main active component isolated from the traditional Chinese medicinal herb Astragalus membranaceus. Studies have demonstrated that AS-IV has neuroprotective effects in cerebral ischemic models. In this study, we aimed to investigate the effects of AS-IV on memory impairment induced by transient cerebral ischemia and reperfusion in mice, as well as the associated signaling mechanisms. Severe memory deficits were induced by bilateral common carotid artery occlusion (BCCAO) in mice as indicated in the Morris water maze test in this study. Oral administration of AS-IV (10 and 20mg/kg, once per day, started 7days before surgery and continued for 7days after surgery) significantly attenuated memory impairment and neuroinflammation. Moreover, AS-IV treatment significantly reduced the expression of toll-like receptor-4 (TLR4) and its downstream adaptor proteins, including myeloid differentiation primary response gene 88 (MyD88), toll/interleukin-1 receptor-domain containing adaptor-inducing interferon-β (TRIF) and tumour necrosis factor receptor associated factor-6 (TRAF6), and subsequently inhibited NF-κB phosphorylation. It is well-known that cerebral ischemia and reperfusion injury enhances the formation of reactive oxygen species (ROS) and further neuroinflammation. Importantly, we found that AS-IV suppressed NLRP3 inflammasome activation by controlling ROS production. In addition, AS-IV markedly reduced overactivation of microglia and the overexpression of inflammatory cytokines in the hippocampus compared with the transient cerebral ischemia and reperfusion group. These results suggest that AS-IV might possess neuroprotective effects against transient cerebral ischemia and reperfusion partly through its anti-inflammatory effects by inhibiting TLR4 signaling pathway and NLRP3 inflammasome overactivation.

TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology

Viral infection during fetal or neonatal stages increases the risk of developing neuropsychiatric disorders such as schizophrenia and autism spectrum disorders. Although neurons express several key regulators of innate immunity, the role of neuronal innate immunity in psychiatric disorders is still unclear. Using cultured neurons and in vivo mouse brain studies, we show here that Toll‐like receptor 3 (TLR3) acts through myeloid differentiation primary response gene 88 (MYD88) to negatively control Disrupted in schizophrenia 1 (Disc1) expression, resulting in impairment of neuronal development. Cytokines are not involved in TLR3‐mediated inhibition of dendrite outgrowth. Instead, TLR3 signaling suppresses expression of several psychiatric disorder‐related genes, including Disc1. The impaired dendritic arborization caused by TLR3 activation is rescued by MYD88 deficiency or DISC1 overexpression. In addition, TLR3 activation at the neonatal stage increases dendritic spine density, but narrows spine heads at postnatal day 21 (P21), suggesting a long‐lasting effect of TLR3 activation on spinogenesis. Our study reveals a novel mechanism of TLR3 in regulation of dendritic morphology and provides an explanation for how environmental factors influence mental health.

MV140, a sublingual polyvalent bacterial preparation to treat recurrent urinary tract infections, licenses human dendritic cells for generating Th1, Th17, and IL-10 responses via Syk and MyD88

Recurrent urinary tract infections (RUTIs) are one of the most common bacterial infectious diseases, especially in women. Antibiotics remain the mainstay of treatment, but their overuse is associated with antibiotic-resistant infections and deleterious effects in the microbiota. Therefore, alternative approaches are fully demanded. Sublingual immunization with MV140 (Uromune), a polyvalent bacterial preparation (PBP) of whole heat-inactivated bacteria, demonstrated clinical efficacy for the treatment of RUTIs, but the involved immunological mechanisms remain unknown. Herein, we demonstrated that MV140 endorses human dendritic cells (DCs) with the capacity to generate Th1/Th17 and IL-10-producing T cells by mechanisms depending on spleen tyrosine kinase (Syk)- and myeloid differentiation primary response gene 88 (MyD88)-mediated pathways. MV140-induced activation of nuclear factor κB (NF-κB) and p38 in human DCs is essential for the generated Th1/Th17 and IL-10 immune responses whereas c-Jun N-terminal Kinase (JNK) and extracellular-signal regulated kinase (ERK) contribute to Th1 and IL-10 responses, respectively. Sublingual immunization of BALB/c mice with MV140 also induces potent systemic Th1/Th17 and IL-10 responses in vivo. We uncover immunological mechanisms underlying the way of action of MV140, which might well also contribute to understand the rational use of specific PBPs in other clinical conditions with potential high risk of recurrent infections.

MTOR-Driven Metabolic Reprogramming Regulates Legionella pneumophila Intracellular Niche Homeostasis.

Vacuolar bacterial pathogens are sheltered within unique membrane-bound organelles that expand over time to support bacterial replication. These compartments sequester bacterial molecules away from host cytosolic immunosurveillance pathways that induce antimicrobial responses. The mechanisms by which the human pulmonary pathogen Legionella pneumophila maintains niche homeostasis are poorly understood. We uncovered that the Legionella-containing vacuole (LCV) required a sustained supply of host lipids during expansion. Lipids shortage resulted in LCV rupture and initiation of a host cell death response, whereas excess of host lipids increased LCVs size and housing capacity. We found that lipids uptake from serum and de novo lipogenesis are distinct redundant supply mechanisms for membrane biogenesis in Legionella-infected macrophages. During infection, the metabolic checkpoint kinase Mechanistic Target of Rapamycin (MTOR) controlled lipogenesis through the Serum Response Element Binding Protein 1 and 2 (SREBP1/2) transcription factors. In Legionella-infected macrophages a host-driven response that required the Toll-like receptors (TLRs) adaptor protein Myeloid differentiation primary response gene 88 (Myd88) dampened MTOR signaling which in turn destabilized LCVs under serum starvation. Inactivation of the host MTOR-suppression pathway revealed that L. pneumophila sustained MTOR signaling throughout its intracellular infection cycle by a process that required the upstream regulator Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and one or more Dot/Icm effector proteins. Legionella-sustained MTOR signaling facilitated LCV expansion and inhibition of the PI3K-MTOR-SREPB1/2 axis through pharmacological or genetic interference or by activation of the host MTOR-suppression response destabilized expanding LCVs, which in turn triggered cell death of infected macrophages. Our work identified a host metabolic requirement for LCV homeostasis and demonstrated that L. pneumophila has evolved to manipulate MTOR-dependent lipogenesis for optimal intracellular replication.

The TLR Adaptor Protein MyD88 Mediates Cell Sensitivity to HDAC Inhibitors through a Cytokine-Dependent Mechanism

IntroductionHistone deacetylase inhibitors (HDI) are promising new agents for the treatment of haematological malignancy including lymphoma. HDI have potent anti-proliferative effects in cell-based studies; in the clinical setting, however, the picture is quite variable, and the outcome of HDI therapy is complex and difficult to predict.The toll-like receptor (TLR) adaptor protein MyD88 (Myeloid Differentiation Primary Response Gene 88) is expressed at high levels in haematopoietic tissues and has recently been found to be the target for somatic mutation in lymphoid malignancies. In particular, substitution of the amino acid leucine to proline at position 265 (MyD88 L265P mutation) is estimated to be prevalent in over 90% of cases of Waldenstrom's macroglobulinaemia and around 37% of diffuse large B cell lymphoma (DLBCL) of the ABC subtype.Methods and ResultsUsing an unbiased genome-wide loss-of-function screen (Fotheringham et al, 2009), MyD88 was identified as a key regulator and candidate biomarker which mediates the susceptibility of cancer cells to HDI: We show here in mechanism of action studies that expression of MyD88 can influence sensitivity of various cancer cell lines to treatment with an HDI. Short-term siRNA silencing of MyD88 reduced HDI sensitivity of U2OS osteosarcoma cells (as assessed by immunoblotting and FACS/propidium iodide staining), whereas over-expression of MyD88 and MyD88 L265P led to enhanced sensitivity.Given its role in TLR-signalling, we measured NFκB activity and pro-inflammatory cytokine expression, which were shown to be increased (in particular IL6 levels) when MyD88 was stably or transiently overexpressed in U2OS cells; co-expression of mutated MyD88 L265P augmented this effect. In DLBCL lymphoma cell lines of the ABC subtype, those cells expressing endogenous wild-type MyD88 (RIVA) were found to be less sensitive to HDI treatment compared to those cells carrying a heterozygous MyD88 L265P mutation (HBL-1). MyD88-mutated cells again showed higher levels of IL6 expression on cytokine analysis. Transient knockdown of endogenous MyD88 in ABC DLBCL cells resulted in decreased HDI sensitivity, as did disruption of the TLR/MyD88/NFκB signalling pathway by blocking either the TLR receptor, MyD88 aggregation or IL6 secretion. Immunohistochemical analysis of tumour tissue microarrays from patients with DLBCL carried out in conjunction with this work showed higher expression of MyD88 compared to patients with prostate or colorectal carcinoma. Finally, using a mass spectroscopy approach, we were able to show that MyD88 is acetylated, and that it can be directly deacetylated by the cytoplasmic histone deacetylase HDAC6.Discussion and translational significanceCollectively our findings show that in cultured cells, high levels of MyD88 expression are associated with increased sensitivity to HDAC inhibition, whereas low level expression coincides with decreased sensitivity. We hypothesize that MyD88, in its role as adaptor protein in TLR signalling, plays an important role in regulating the expression and autocrine action of pro-inflammatory cytokines such as IL6 which contribute to the sensitization of cancer cells to HDI. The central role played by MyD88 in this mechanism highlights the importance of TLR signalling and innate immunity, and more generally the inflammatory properties of the extra-cellular tumour microenvironment in mediating the effects of HDAC inhibitors on malignant cells. Therefore, both expression level of MyD88 and MyD88 mutation status could serve as a potential biomarker for HDI sensitivity in patient selection. DisclosuresNo relevant conflicts of interest to declare.

Mechanisms of Suppressive Effects of MYD88 Inhibitors on the Growth of Lymphoma and Leukemia Cells

Introduction : Myeloid differentiation primary response gene 88 (MYD88) is an adaptor protein that binds to the Toll-like/interleukin-1 receptor to form a homodimer and activates NF-kB pathway. Activating MYD88 mutations are found in 90% of lymphoplasmacytic lymphoma (Waldenström’s macroglobulinemia) cases and 30% of activated B-cell type diffuse large B-cell lymphoma (DLBCL) cases. To investigate the role of MYD88 in the growth of lymphoma/leukemia cells, with or without MYD88mutations, and to examine whether MYD88 inhibitors can be used as novel molecular-targeted drugs, we studied their effects on the growth of lymphoma and leukemia cells in vitro.Methods : Seven lymphoma/leukemia cell lines (TMD8 derived from DLBCL with MYD88 mutations, Daudi from Burkitt lymphoma, NALM-6 from B-ALL, Jurkat and KOPT-K1 from T-ALL, THP-1 and TMD7 from AML without MYD88 mutations) and normal lymphocytes from healthy volunteers were obtained, following an informed consent. The effect of the MYD88 inhibitor, ST2825, on the in vitro growth of these cell lines was then examined using the WST-8 colorimetric assay. ST2825 is a synthetic peptide-mimetic compound, which inhibits MYD88 dimerization. A Bruton tyrosine kinase (BTK) inhibitor, ibrutinib, was used as the reference inhibitor. The effect of ST2825 on protein expression was examined by immunoblot analysis. Cell cycle analysis and Annexin V apoptosis assay were performed using flow cytometry. To comprehensively screen the changes in mRNA expression after ST2825 treatment, microarray analysis was performed for TMD8 and Jurkat cells. MYD88knockdown experiments using small interfering RNA were also performed.Results : The MYD88 protein was expressed in all cell lines. ST2825 (30 μM) suppressed the growth of all lymphoma/leukemia cell lines, but did not affect the viability of normal lymphocytes. The IC50 for TMD8 cells was similar to that for other cells, such as Daudi cells. Apoptosis assay revealed that ST2825 induces apoptosis in all the cell lines studied. In Daudi cells, ST2825 induced G2/M cell cycle arrest. Immunoblot analysis revealed that ST2825 suppresses the phosphorylation of certain NF-kB signaling components, such as RELA and IkB, in all cell lines. In TMD8, Daudi, and NALM-6 cells, ST2825 suppressed the phosphorylation of BTK, and MYD88 knockdown suppressed the phosphorylation of RELA and BTK. Microarray analysis revealed that ST2825 treatment downregulates the expression of various genes including MYD88, and upregulates the expression of other genes such as NGFR, FOSB, and HSP. Treatment with ibrutinib (1 nM) suppressed the growth of only TMD8 cells. Treatment with ST2825 plus ibrutinib additively suppressed the growth of TMD8 cells.Conclusions : The MYD88 inhibitor ST2825 suppresses the growth of various lymphoma and leukemia cells, suggesting that MYD88 is involved in regulating the growth of these cells; however, the off-target effects of ST2825 should be considered. Further investigation is required to assess the potential of MYD88 inhibitors as novel molecular-targeted drugs against lymphoma and leukemia. DisclosuresNo relevant conflicts of interest to declare.

MyD 88 Polymorphisms in Children Diagnosed with Sepsis

Myeloid differentiation primary response gene 88 (MyD 88) is an intracellular adapter protein that mediates the early immune response to pathogens. Toll-like receptors (except TLR-3) induce the immune response through a MyD 88-dependent signal pathway. We aimed to investigate the MyD 88 polymorphisms that play important roles in the immune response in septic children and to evaluate whether or not they were risk factors in the development of sepsis. Case-control study. Sixty-five patients diagnosed with sepsis in the Pediatric Intensive Care Unit during the period from April 2010 to January 2012 were included as the study group. Sixty-five children without sepsis were included as controls. After DNA was obtained from blood samples in the study and control groups, MyD 88 polymorphisms were analyzed. According to the genotype and allele frequencies, the distributions of MyD 88 polymorphisms [Single nucleotide polymorphism (SNP) - 938 C/A (rs4988453), MyD 88 SNP 1944 C/G (rs4988457)] were analyzed in both the study and control groups. The C/C genotype of MyD 88 SNP -938 was significantly more common than the C/A genotype in the patient group (p=0.002). No statistically significant difference in the frequency of the MyD 88 SNP 1944 genotype was found between the study and control groups (p=0.272). Gene polymorphism studies could elucidate our understanding of sepsis in terms of prevalence and the management of treatment. It was shown in this study that children with the MyD 88 SNP -938 C/C genotype had a greater tendency toward sepsis. However, additional studies should be performed.

A comparison of the immunological potency of Burkholderia lipopolysaccharides in endotoxemic BALB/c mice.

Lipopolysaccharide is one of the virulence factors of the soil-borne pathogens Burkholderia pseudomallei, B. thailandensis, B. cenocepacia and B. multivorans, which cause septic melioidosis (often in B. pseudomallei infections but rarely in B. thailandensis infections) or cepacia syndromes (commonly in B. cenocepacia infections but rarely in B. multivorans infections). The inflammatory responses in Burkholderia LPS-induced endotoxemia were evaluated in this study. Prior to induction, the conserved structures and functions of each purified LPS were determined using electrophoretic phenotypes, the ratios of 3-hydroxytetradecanoic to 3-hydroxyhexadecanoic acid and endotoxin units. In an in vitro assay, cytokine expression of myeloid differentiation primary response gene 88 and Toll/IL-1 receptor domain containing adapter-inducing INF-β-dependent signaling-dependent signaling differed when stimulated by different LPS. Endotoxemia was induced in mice by s.c. injection as evidenced by increasing serum concentrations of 3-hydroxytetradecanoic acid and the septic prognostic markers CD62E and ICAM-1. During endotoxemia, splenic CD11b+ I-A+ , CD11b+ CD80+ , CD11b+ CD86+ and CD11b+ CD11c+ subpopulations increased. After induction with B. pseudomallei LPS, there were significant increases in splenic CD49b NK cells and CD14 macrophages. The inflamed CD11b+ CCR2+ , CD11b+ CD31+ , CD11b+ CD14+ , resident CD11b+ CX3 CR1+ and progenitor CD11b+ CD34+ cells showed delayed increases in bone marrow. B. multivorans LPS was the most potent inducer of serum cytokines and chemokines, whereas B. cenocepacia LPS induced relatively low concentrations of the chemokines MIP-1α and MIP-1β. Endotoxin activities did not correlate with the virulence of Burkholderia strains. Thus factors other than LPS and/or other mechanisms of low activity LPS must mediate the pathogenicity of highly virulent Burkholderia strains.

Redox sensitivity of the MyD88 immune signaling adapter

The transcription factor nuclear factor-κB (NF-κB) mediates expression of key genes involved in innate immunity and inflammation. NF-κB activation has been repeatedly reported to be modulated by hydrogen peroxide (H2O2). Here, we show that the NF-κB-activating signaling adapter myeloid differentiation primary response gene 88 (MyD88) is highly sensitive to oxidation by H2O2 and may be redox-regulated in its function, thus facilitating an influence of H2O2 on the NF-κB signaling pathway. Upon oxidation, MyD88 forms distinct disulfide-linked conjugates which are reduced by the MyD88-interacting oxidoreductase nucleoredoxin (Nrx). MyD88 cysteine residues functionally modulate MyD88-dependent NF-κB activation, suggesting a link between MyD88 thiol oxidation state and immune signaling.

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

BackgroundPolyunsaturated 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.MethodsForty 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)-1β, 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.ResultsThe mildest macroscopic/microscopic esophagitis was found in the n-3 group (P < 0.05). The expression of IL-1β, 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).Conclusionn-3 PUFAs may reduce acute RE in rats, which may be due to inhibition of the MyD88-NF-kB pathway and limit oxidative damage.

In vivo modeling of diffuse large B cell lymphoma (DLBCL) with the myeloid differentiation primary response gene 88 (MYD88) L265P mutation

In vivo modeling of diffuse large B cell lymphoma (DLBCL) with the myeloid differentiation primary response gene 88 (MYD88) L265P mutation

Hops (Humulus lupulus) Content in Beer Modulates Effects of Beer on the Liver After Acute Ingestion in Female Mice.

Using a binge-drinking mouse model, we aimed to determine whether hops (Humulus lupulus) in beer is involved in the less damaging effects of acute beer consumption on the liver in comparison with ethanol. Female C57BL/6 J mice were either fed one iso-alcoholic and iso-caloric bolus dose of ethanol, beer, beer without hops (6 g ethanol/kg body weight) or an iso-caloric bolus of maltodextrin control solution. Markers of steatosis, intestinal barrier function, activation of toll-like receptor 4 signaling cascades, lipid peroxidation and lipogenesis were determined in liver, small intestine and plasma 2 h and 12 h after acute alcohol ingestion. Alcohol-induced hepatic fat accumulation was significantly attenuated in mice fed beer whereas in those fed beer without hops, hepatic fat accumulation was similar to that found in ethanol-fed mice. While markers of intestinal barrier function e.g. portal endotoxin levels and lipogenesis only differed slightly between groups, hepatic concentrations of myeloid differentiation primary response gene 88, inducible nitric oxide synthase (iNOS) and plasminogen-activator inhibitor 1 protein as well as of 4-hydroxynonenal and 3-nitrotyrosine protein adducts were similarly elevated in livers of mice fed ethanol or beer without hops when compared with controls. Induction of these markers was markedly attenuated in mice fed hops-containing beer. Taken together, our data suggest that hops in beer markedly attenuated acute alcohol-induced liver steatosis in female mice through mechanisms involving a suppression of iNOS induction in the liver.

Rosmarinic Acid Methyl Ester Inhibits LPS-Induced NO Production via Suppression of MyD88- Dependent and -Independent Pathways and Induction of HO-1 in RAW 264.7 Cells.

In this study, we investigated the anti-inflammatory effect of rosmarinic acid methyl ester (RAME) isolated from a mutant cultivar of Perilla frutescens (L.) Britton. We found that RAME inhibits lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with an IC50 of 14.25 µM, in RAW 264.7 cells. RAME inhibited the LPS-induced expression of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, monocyte chemoattractant protein-1, interferon-β, and inducible nitric oxide synthase (iNOS). Moreover, RAME suppressed the activation of nuclear factor kappa B. These results suggest that the downregulation of iNOS expression by RAME was due to myeloid differentiation primary response gene 88 (MyD88)-dependent and -independent pathways. Furthermore, RAME induced the expression of heme oxygenase-1 (HO-1) through activation of nuclear factor-erythroid 2-related factor 2. Treatment with tin protoporphyrin, an inhibitor of HO-1, reversed the RAME-induced suppression of NO production. Taken together, RAME isolated from P. frutescens inhibited NO production in LPS-treated RAW 264.7 cells through simultaneous induction of HO-1 and inhibition of MyD88-dependent and -independent pathways.

Ginkgo biloba L. extract protects against chronic cerebral hypoperfusion by modulating neuroinflammation and the cholinergic system

BackgroundGinkgo biloba extract (GBE)—a widely used nutraceutical—is reported to have diverse functions, including positive effects on memory and vasodilatory properties. Although numerous studies have assessed the neuroprotective properties of GBE in ischemia, only a few studies have investigated the neuro-pharmacological mechanisms of action of GBE in chronic cerebral hypoperfusion (CCH). PurposeIn the present study, we sought to determine the effects of GBE on CCH-induced neuroinflammation and cholinergic dysfunction in a rat model of bilateral common carotid artery occlusion (BCCAo). MethodsChronic BCCAo was induced in adult male Wistar rats to reflect the CCH conditions. On day 21 after BCCAo, the animals were treated orally with saline or GBE (5, 10, 20, and 40mg/kg) daily for 42 days. After the final treatment, brain tissues were isolated for the immunohistochemical analysis of glial markers and choline acetyltransferase (ChAT), as well as for the western blot analysis of proinflammatory cytokines, toll-like receptor (TLR)-related pathway, receptor for advanced glycation end products (RAGE), angiotensin-II (Ang-II), and phosphorylated mitogen-activated protein kinases (MAPKs). ResultsBCCAo increased glial proliferation in the hippocampus and white matter, whereas proliferation was significantly attenuated by GBE treatment. GBE also attenuated the BCCAo-related increases in the hippocampal expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6), TLR4, myeloid differentiation primary response gene 88, RAGE, Ang-II, and phosphorylated MAPKs (ERK, p38, and JNK). Furthermore, GBE treatment restored the ChAT expression in the basal forebrain following BCCAo. ConclusionsThese findings suggest that GBE has specific neuroprotective effects that may be useful for the treatment of CCH. The pharmacological mechanism of GBE partly involves the modulation of inflammatory mediators and the cholinergic system.

Sam68 is a regulator of Toll-like receptor signaling

Recognition of pathogens by Toll-like receptors (TLR) activate multiple signaling cascades and expression of genes tailored to mount a primary immune response, inflammation, cell survival and apoptosis. Although TLR-induced activation of pathways, such as nuclear factor kappaB (NF-κB) and mitogen-activated protein kinases (MAPK), has been well studied, molecular entities controlling quantitative regulation of these pathways during an immune response remain poorly defined. We identified Sam68 as a novel regulator of TLR-induced NF-κB and MAPK activation. We found that TLR2 and TLR3 are totally dependent, whereas TLR4 is only partially dependent on Sam68 to induce the activation of NF-κB c-Rel. Absence of Sam68 greatly decreased TLR2- and TLR3-induced NF-κB p65 activation, whereas TLR4-induced p65 activation in a Sam68-independent manner. In contrast, Sam68 appeared to be a negative regulator of MAPK pathways because absence of Sam68 enhanced TLR2-induced activation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK). Interestingly, TLR2- and TLR3-induced gene expression showed a differential requirement of Sam68. Absence of Sam68 impaired TLR2-induced gene expression, suggesting that Sam68 has a critical role in myeloid differentiation primary response gene 88-dependent TLR2 signaling. TLR3-induced gene expression that utilize Toll/Interleukin-1 receptor-domain-containing adapter-inducing beta interferon pathway, depend only partially on Sam68. Our findings suggest that Sam68 may function as an immune rheostat that balances the activation of NF-κB p65 and c-Rel, as well as MAPK, revealing a potential novel target to manipulate TLR signaling.

Modulation of Macrophage Inflammatory Nuclear Factor κB (NF-κB) Signaling by Intracellular Cryptococcus neoformans

Cryptococcus neoformans (Cn) is a common facultative intracellular pathogen that can cause life-threatening fungal meningitis in immunocompromised individuals. Shortly after infection, Cn is detectable as both extra- and intracellular yeast particles, with Cn being capable of establishing long-lasting latent infections within host macrophages. Although recent studies have shown that shed capsular polysaccharides and intact extracellular Cn can compromise macrophage function through modulation of NF-κB signaling, it is currently unclear whether intracellular Cn also affects NF-κB signaling. Utilizing live cell imaging and computational modeling, we find that extra- and intracellular Cn support distinct modes of NF-κB signaling in cultured murine macrophages. Specifically, in RAW 264.7 murine macrophages treated with extracellular glucuronoxylomannan (GXM), the major Cn capsular polysaccharide, LPS-induced nuclear translocation of p65 is inhibited, whereas in cells with intracellular Cn, LPS-induced nuclear translocation of p65 is both amplified and sustained. Mathematical simulations and quantification of nascent protein expression indicate that this is a possible consequence of Cn-induced "translational interference," impeding IκBα resynthesis. We also show that long term Cn infection induces stable nuclear localization of p65 and IκBα proteins in the absence of additional pro-inflammatory stimuli. In this case, nuclear localization of p65 is not accompanied by TNFα or inducible NOS (iNOS) expression. These results demonstrate that capsular polysaccharides and intact intracellular yeast manipulate NF-κB via multiple distinct mechanisms and provide new insights into how Cn might modulate cellular signaling at different stages of an infection.

TLR2, TLR4 AND MyD88 Mediate Allergic Airway Disease (AAD) and Streptococcus pneumoniae-Induced Suppression of AAD.

BackgroundExposure to non-pathogenic Streptococcus pneumoniae and vaccination are inversely associated with asthma. Studies in animal models demonstrate that airway administration of S. pneumoniae (live or killed), or its vaccines or components, suppresses the characteristic features of asthma in mouse models of allergic airway disease (AAD). These components could be developed into immunoregulatory therapies. S. pneumoniae components are recognized by Toll-like receptors (TLR) 2 and TLR4, and both induce inflammatory cell responses through the adaptor protein myeloid differentiation primary response gene 88 (MyD88). The involvement of TLR2, TLR4 and MyD88 in the pathogenesis of AAD and asthma is incompletely understood, and has not been studied in S. pneumoniae-mediated suppression of AAD. We investigated the role of TLR2, TLR4 and MyD88 in the development of AAD and S. pneumoniae-mediated suppression of AAD.Methods and FindingsOVA-induced AAD and killed S. pneumoniae-mediated suppression of AAD were assessed in wild-type, TLR2-/-, TLR4-/-, TLR2/4-/- and MyD88-/- BALB/c mice. During OVA-induced AAD, TLR2, TLR4 and MyD88 were variously involved in promoting eosinophil accumulation in bronchoalveolar lavage fluid and blood, and T-helper type (Th)2 cytokine release from mediastinal lymph node T cells and splenocytes. However, all were required for the induction of airways hyperresponsiveness (AHR). In S. pneumoniae-mediated suppression of AAD, TLR2, TLR4 and MyD88 were variously involved in the suppression of eosinophilic and splenocyte Th2 responses but all were required for the reduction in AHR.ConclusionsThese results highlight important but complex roles for TLR2, TLR4 and MyD88 in promoting the development of OVA-induced AAD, but conversely in the S. pneumoniae-mediated suppression of AAD, with consistent and major contributions in both the induction and suppression of AHR. Thus, TLR signaling is likely required for both the development of asthma and the suppression of asthma by S. pneumoniae, and potentially other immunoregulatory therapies.

Hypoxia-inducible factor-1α mediates the toll-like receptor 4 signaling pathway leading to anti-tumor effects in human hepatocellular carcinoma cells under hypoxic conditions.

Hypoxia-inducible factor-1α (HIF-1α) and toll-like receptor 4 (TLR4) are involved in numerous mechanisms of cancer biology, including cell proliferation and survival; however the interaction of the two factors under hypoxic conditions remains unclear. The present study investigated the in vitro mechanism that results in the suppression of tumor cell growth and cellular functions when HIF-1α is silenced. In the present study, the human hepatocellular carcinoma HepG2 cell line was transfected with short hairpin RNA (shRNA) against HIF-1α and cultured under hypoxic conditions (1% O2 for 24 h). The expression of HIF-1α and various growth factors, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), were examined using quantitative polymerase chain reaction and immunoblotting. Tumor growth was measured using a Cell Counting Kit-8 assay and tumor activity was measured using tumor cell invasion and migration assays. Lipopolysaccharide and TAK-242 were used to activate and inhibit TLR4, respectively, to observe the role of TLR4 in the HIF-1α silenced tumor cells. The expression of TLR4 signaling pathway associates, including myeloid differentiation primary response gene 88 (MyD88), apoptosis signal-regulating kinase 1 (ASK1), p38 mitogen-activated protein kinases and HIF-1α, were analyzed by western blot assay. Under hypoxic conditions, silencing of HIF-1α expression suppressed tumor cell growth and regulated the expression of tumor growth-associated genes, including EGF, HGF, VEGF and FG2. Suppression of tumor cell invasion and migration was also observed in the HIF-1α silenced HepG2 cell line. In addition, TLR4 was identified to be involved in HIF-1α and MyD88 accumulation, and activation of ASK1 and p38 were demonstrated to be critical for TLR4-mediated HIF-1α pathway. In conclusion, silencing of HIF-1α expression may induce anti-tumor effects under hypoxic conditions in HepG2 cells via the TLR4 mediated pathway, suggesting that the HIF-1α/TLR4 signaling cohort may act as a novel therapeutic target for the treatment of hepatocellular cancer.

Toll-Like Receptor 4 Deficiency Improves Short-term Renal Function but not Long-term Graft Survival in a Fully MHC-Mismatched Murine Model of Renal Allograft Transplantation.

We have previously demonstrated that absence of myeloid differentiation primary response gene 88 (MyD88) induced donor-specific kidney allograft tolerance. The upstream pathways of MyD88 that mediate this process, however, remain unclear. Toll-like receptor 4 (TLR4) is an innate immune receptor that is dependent upon MyD88 for activity of its dominant signaling pathway. Here, we investigated the role of TLR4 in kidney allograft rejection using a fully major histocompatibility complex-mismatched, life-sustaining, murine model of renal allograft rejection. Donor (BALB/c) and recipient (C57BL/6) mice either both deficient or sufficient for TLR4 underwent heterotopic renal allograft transplantation, with an additional group of mice receiving renal isografts as controls. Survival was assessed up to 100 days posttransplantation. Animals were also sacrificed 14 days posttransplantation for assessment of the acute allograft rejection response. Both wild-type (WT) and TLR4 allografts showed inferior survival compared to isografts, with no difference in survival between the allograft groups. Serum creatinine was lower in TLR4 allografts at day 14 posttransplantation compared with WT allografts, but this was not sustained by day 100. At day 14 posttransplant, increased CD11c dendritic cell accumulation, expression of IL-2 and indoleamine 2,3-dioxygenase were evident in TLR4 compared with WT allografts, whereas expression of inducible nitric oxide synthase was decreased. Acute kidney allograft rejection was modestly attenuated in TLR4 mice; however, long-term allograft survival and function were not affected in our model. Protection against acute rejection may involve increased accumulation of CD11c cells and indoleamine 2,3-dioxygenase expression.