Myeloid Differentiation Primary Response Gene Research Articles

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.

Contribution of TLR4 and MyD88 for adjuvant monophosphoryl lipid A (MPLA) activity in a DNA prime–protein boost HIV-1 vaccine

Recombinant protein vaccines are commonly formulated with an immune-stimulatory compound, or adjuvant, to boost immune responses to a particular antigen. Recent studies have shown that, through recognition of molecular motifs, receptors of the innate immune system are involved in the functions of adjuvants to generate and direct adaptive immune responses. However, it is not clear to which degree those receptors are also important when the adjuvant is used as part of a novel heterologous prime-boost immunization process in which the priming and boosting components are not the same type of vaccines. In the current study, we compared the immune responses elicited by a pentavalent HIV-1 DNA prime–protein boost vaccine in mice deficient in either Toll-like receptor 4 (TLR4) or myeloid differentiation primary response gene 88 (MyD88) to wildtype mice. HIV gp120 protein administered in the boost phase was formulated with either monophosphoryl lipid A (MPLA), QS-21, or Al(OH)3. Endpoint antibody titer, serum cytokine response and T-cell memory response were assessed. Neither TLR4 nor MyD88 deficiency had a significant effect on the immune response of mice given vaccine formulated with QS-21 or Al(OH)3. However, TLR4- and MyD88-deficiency decreased both the antibody and T-cell responses in mice administered HIV gp120 formulated with MPLA. These results further our understanding of the activation of TLR4 and MyD88 by MPLA in the context of a DNA prime/protein boost immunization strategy.

Leptospiral lipopolysaccharide stimulates the expression of toll‐like receptor 2 and cytokines in pig fibroblasts

Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L-LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L-LPS. Here, we stimulated pig fibroblasts by L-LPS and then quantitatively measured gene and protein expression levels of two toll-like receptors (TLRs), TLR2 and TLR4, by real-time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up-regulated within 24 h after L-LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL-6) and IL-8 gene expressions were markedly up-regulated by L-LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L-LPS stimulation, thereby inducing expression of cytokines.

H2S Protecting against Lung Injury following Limb Ischemia-reperfusion by Alleviating Inflammation and Water Transport Abnormality in Rats

ObjectiveTo investigate the effect of H2S on lower limb ischemia-reperfusion (LIR) induced lung injury and explore the underlying mechanism. MethodsWistar rats were randomly divided into control group, IR group, IR+ Sodium Hydrosulphide (NaHS) group and IR+ DL-propargylglycine (PPG) group. IR group as lung injury model induced by LIR were given 4 h reperfusion following 4 h ischemia of bilateral hindlimbs with rubber bands. NaHS (0.78 mg/kg) as exogenous H2S donor and PPG (60 mg/kg) which can suppress endogenous H2S production were administrated before LIR, respectively. The lungs were removed for histologic analysis, the determination of wet-to-dry weight ratios and the measurement of mRNA and protein levels of aquaporin-1 (AQP1), aquaporin-5 (AQP5) as indexes of water transport abnormality, and mRNA and protein levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary-response gene 88 (MyD88) and p-NF-κB as indexes of inflammation. ResultsLIR induced lung injury was accompanied with upregulation of TLR4-Myd88-NF-κB pathway and downregulation of AQP1/AQP5. NaHS pre-treatment reduced lung injury with increasing AQP1/AQP5 expression and inhibition of TLR4-Myd88-NF-κB pathway, but PPG adjusted AQP1/AQP5 and TLR4 pathway to the opposite side and exacerbated lung injury. ConclusionEndogenous H2S, TLR4-Myd88-NF-κB pathway and AQP1/AQP5 were involved in LIR induced lung injury. Increased H2S would alleviate lung injury and the effect is at least partially depend on the adjustment of TLR4-Myd88-NF-κB pathway and AQP1/AQP5 expression to reduce inflammatory reaction and lessen pulmonary edema.

Effect of acute beer ingestion on the liver: studies in female mice.

The aim of the present study was to assess whether the effects of acute consumption of stout or pilsner beer on the liver differ from those of plain ethanol in a mouse model. Seven-week-old female C57BL/6J mice received either ethanol, stout or pilsner beer (ethanol content: 6g/kg body weight) or isocaloric maltodextrin solution. Plasma alanine transaminase, markers of steatosis, lipogenesis, activation of the toll-like receptor-4 signaling cascade as well as lipid peroxidation and fibrogenesis in the liver were measured 12h after acute ethanol or beer intake. Acute alcohol ingestion caused a marked ~11-fold increase in hepatic triglyceride accumulation in comparison to controls, whereas in mice exposed to stout and pilsner beer, hepatic triglyceride levels were increased only by ~6.5- and ~4-fold, respectively. mRNA expression of sterol regulatory element-binding protein 1c and fatty acid synthase in the liver did not differ between alcohol and beer groups. In contrast, expression of myeloid differentiation primary response gene 88, inducible nitric oxide synthases, but also the concentrations of 4-hydroxynonenal protein adducts, nuclear factor κB and plasminogen activator inhibitor-1 were induced in livers of ethanol treated mice but not in those exposed to the two beers. Taken together, our results suggest that acute ingestion of beer and herein especially of pilsner beer is less harmful to the liver than the ingestion of plain ethanol.

Steroids inhibit vascular endothelial growth factor expression via TLR4/Akt/NF-κB pathway in chronic rhinosinusitis with nasal polyp.

Vascular endothelial growth factor (VEGF) is elevated in chronic rhinosinusitis with nasal polyps. Steroids have anti-inflammatory properties and are ideal candidates for treating chronic inflammatory airways. The aims of this study were to identify the inhibitory effects and mechanisms of steroids on lipopolysaccharide (LPS)-induced VEGF expression in nasal polyps. Nasal polyp-derived fibroblasts (NPDFs) were stimulated with LPS alone or with both LPS and steroids were used to determine the expression levels of toll-like receptor (TLR)-4, myeloid differentiation primary response gene 88 (MyD88), and VEGF by using reverse transcription-polymerase chain reaction (RT-PCR). VEGF protein level was analyzed by immunocytochemical staining and enzyme-linked immunosorbent assay (ELISA). Small interfering RNA (siRNA) for TLR4 was transfected to down-regulate TLR4 expression. Activation of Akt and nuclear factor κB (NF-κB) pathway on VEGF expression was determined by Western blot analysis, immunocytochemical staining, and ELISA. Nasal polyp organ cultures were stimulated with LPS alone or in conjunction with steroids or LPS-RS (TLR4 inhibitor) and accessed the expression of VEGF. Steroids decreased the expressions of TLR4, MyD88, and VEGF mRNA and VEGF protein in LPS-stimulated NPDFs. Steroids inhibited LPS-induced VEGF expression levels in dose-dependent manner. The suppression of TLR4 transcription by siRNA treatment reduced LPS-induced expression of both TLR4 and VEGF in NPDFs. Furthermore, steroids inhibited the production of VEGF by blocking Akt and NF-κB activation and preventing with NF-κB translocation. Also, steroid and TLR4 inhibitor decreased VEGF expression in nasal polyp organ cultures. These results indicate that steroids inhibit LPS-induced VEGF expression through the TLR4/Akt/NF-κB signaling pathway in chronic rhinosinusitis with nasal polyp.

Toll-like receptor 2/4 heterodimer mediates inflammatory injury in intracerebral hemorrhage.

Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced secondary brain injury. However, the upstream events that initiate inflammatory responses following ICH remain elusive. Our previous studies suggested that Toll-like receptor 4 (TLR4) may be the upstream signal that triggers inflammatory injury in ICH. In addition, recent clinical findings indicated that both TLR2 and TLR4 may participate in ICH-induced brain injury. However, it is unclear how TLR2 functions in ICH-induced inflammatory injury and how TLR2 interacts with TLR4. The role of TLR2 and TLR2/TLR4 heterodimerization in ICH-induced inflammatory injury was investigated in both in vivo and in vitro models of ICH. TLR2 mediated ICH-induced inflammatory injury, which forms a heterodimer with TLR4 in both in vivo and in vitro models of ICH. Hemoglobin (Hb), but not other blood components, triggered inflammatory injury in ICH via assembly of TLR2/TLR4 heterodimers. MyD88 (myeloid differentiation primary response gene 88), but not TRIF (Toll/IR-1 domain-containing adaptor protein inducing interferon-beta), was required for ICH-induced TLR2/TLR4 heterodimerization. Mutation of MyD88 Arg196 abolished the TLR2/TLR4 heterodimerization. Our results suggest that a novel TLR2/TLR4 heterodimer induced by Hb initiates inflammatory injury in ICH. Interfering with the assembly of the TLR2/TLR4 heterodimer may be a novel target for developing effective treatment of ICH.

Protective effect of resveratrol against acute lung injury induced by lipopolysaccharide via inhibiting the myd88-dependent Toll-like receptor 4 signaling pathway.

Resveratrol, a polyphenol mainly present in grapes and red wine, demonstrated varied pharmacological activities. The protective effects of resveratrol on acute lung injury(ALI) induced by lipopolysaccharide(LPS) have remained elusive. The present study investigated whether the protective effect of resveratrol on ALI induced by LPS was via inhibiting the myeloid differentiation primary response gene(myd88)‑dependent toll‑like receptor(TLR)4 signaling pathway. Mice were pretreated with 5 or 45mg/kg resveratrol for threedays prior to bronchial perfusion with 25mg/kg LPS. At 12h after surgery, the ratio of the wet to the dry (w/d) lung was calculated to assess tissue edema. Histological changes of the lungs were detected using hematoxylin and eosin staining and the protein expression levels of TLR4, myd88 and nuclear factor kappa‑light‑chain‑enhancer of activatedB cells (NF‑κB) were measured by western blot analysis. The concentration of interleukin (IL)‑6 and cyclooxygenase (COX)‑2 in the bronchoalveolar lavage fluid were detected by ELISA. The results showed that resveratrol can suppress the edema, inflammatory cell infiltration and alveolar structure damage of lungs in mice with ALI and decrease the lung w/d ratio. Additionally, resveratrol markedly decreased the expression of TLR4, myd88 and NF‑κB and decreased the concentration of inflammatory cytokines, including IL‑6 and COX‑2. Therefore, it can be concluded that resveratrol has a protective effect against ALI induced by LPS, at least in part by inhibiting the myd88‑dependent TLR4 signaling pathway. In conclusion, resveratrol pretreatment has a protective effect against LPS‑induced ALI in mice, which indicates that resveratrol may serve as a potential therapeutic agent for treating ALI in the clinic.

The NLRP3/ASC inflammasome promotes T-cell-dependent immune complex glomerulonephritis by canonical and noncanonical mechanisms

Interleukin (IL)-1β contributes to renal injury in immune complex glomerulonephritis. However, production of mature IL-1β depends on activation of the inflammasome that cleaves pro-IL-1β into its secretable form. A functional role of the NLRP3-containing inflammasome, which responds to various endogenous danger signals, was found in tubulointerstitial nephropathies, but its function in glomerular disease has not been established. To determine whether NLRP3 and its adapter molecule ASC contribute to glomerulonephritis, we induced T-cell-dependent autologous nephrotoxic serum nephritis in Nlrp3- and Asc-deficient mice. Renal expression of NLRP3/ASC inflammasome components and pro-IL-1β increased during nephrotoxic serum nephritis and was abundant in renal dendritic cells. This was associated with renal production of mature IL-1β, indicating inflammasome activation. Nlrp3 and Asc deficiency significantly attenuated glomerular injury, renal leukocyte infiltration, and T-cell activation. Production of mature IL-1β was abrogated in Asc-deficient mice, consistent with a loss of inflammasome-dependent IL-1β activation. Surprisingly, renal IL-1β secretion remained intact in Nlrp3-deficient mice, indicating noncanonical pro-inflammatory effects of NLRP3 in autologous nephrotoxic serum nephritis. This may include NLRP3-mediated glomerular release of pro-inflammatory high-mobility group box 1 protein as a noncanonical function of NLRP3/ASC in glomerulonephritis. Thus, therapeutic blockade of the NLRP3/ASC/IL-1β axis may be beneficial in glomerulonephritis.

Toll-Like Receptor 4 Signaling Contributes to Paclitaxel-Induced Peripheral Neuropathy

This paper tests the contribution of the toll-like receptors, TLR4 in particular, in the initiation and maintenance of paclitaxel-related chemotherapy-induced peripheral neuropathy. TLR4 and its immediate downstream signaling molecules—myeloid differentiation primary response gene 88 (MyD88) and toll/interleukin 1 receptor domain–containing adapter-inducing interferon-β (TRIF)—were found to be increased in the dorsal root ganglion (DRG) using Western blot by day 7 of paclitaxel treatment. The behavioral phenotype, the increase of both TLR4 and MyD88, was blocked by cotreatment with the TLR4 antagonist lipopolysaccharide–Rhodobacter sphaeroides during chemotherapy. A similar, but less robust, behavioral effect was observed using intrathecal treatment of MyD88 homodimerization inhibitory peptide. DRG levels of TLR4 and MyD88 reduced over the next 2 weeks, whereas these levels remained increased in spinal cord through day 21 following chemotherapy. Immunohistochemical analysis revealed TLR4 expression in both calcitonin gene-related peptide–positive and isolectin B4–positive small DRG neurons. MyD88 was only found in calcitonin gene-related peptide–positive neurons, and TRIF was found in both calcitonin gene-related peptide–positive and isolectin B4–positive small DRG neurons as well as in medium- and large-size DRG neurons. In the spinal cord, TLR4 was only found colocalized to astrocytes but not with either microglia or neurons. Intrathecal treatment with the TLR4 antagonist lipopolysaccharide–R. sphaeroides transiently reversed preestablished chemotherapy-induced peripheral neuropathy mechanical hypersensitivity. These results strongly implicate TLR4 signaling in the DRG and the spinal cord in the induction and maintenance of paclitaxel-related chemotherapy-induced peripheral neuropathy. PerspectiveThe toll-like receptor TLR4 and MyD88 signaling pathway could be a new potential therapeutic target in paclitaxel-induced painful neuropathy.

Both bone marrow‐derived and non‐bone marrow‐derived cells contribute to AIM2 and NLRP3 inflammasome activation in a MyD88‐dependent manner in dietary steatohepatitis

Inflammation promotes the progression of non-alcoholic steatohepatitis (NASH). Toll-like receptor 4 (TLR4) and TLR9 activation through myeloid differentiation primary response gene 88 (MyD88) and production of mature interleukin-1β (IL-1β) via inflammasome activation contribute to steatohepatitis. Here, we investigated the inter-relationship between TLR signalling and inflammasome activation in dietary steatohepatitis. Wild type (WT), TLR4- and MyD88-deficient (KO) mice received methionine-choline-deficient (MCD) or -supplemented (MCS) diets for 5 weeks and a subset was challenged with TLR9 ligand CpG-DNA. TLR4, TLR9, AIM2 (absent in melanoma 2) and NLRP3 (NLR family pyrin domain containing 3) inflammasome mRNA, and mature IL-1β protein levels were increased in MCD diet-induced steatohepatitis compared to MCS controls. TLR9 stimulation resulted in greater up-regulation of the DNA-sensing AIM2 expression and IL-1β production in livers of MCD compared to MCS diet-fed mice. High mobility group box 1 (HMGB1), a TLR9-activating danger molecule and phospho-HMGB1 protein levels were also increased in livers of MCD diet-fed mice. MyD88- but not TLR4-deficiency prevented up-regulation of AIM2, NLRP3 mRNA and IL-1β protein production in dietary steatohepatitis. Selective MyD88 deficiency either in bone marrow (BM)-derived or non-BM-derived cells attenuated hepatic up-regulation of inflammasome mRNA, caspase-1 activation and IL-1β protein production, but only BM-derived cell-specific MyD88-deficiency attenuated liver injury. Our data demonstrate that both bone marrow-derived and non-BM-derived cells contribute to inflammasome activation in a MyD88-dependent manner in dietary steatohepatitis. We show that AIM2 inflammasome expression and activation are further augmented by TLR9 ligands in dietary steatohepatitis.

Dispensable role of myeloid differentiation primary response gene 88 (MyD88) and MyD88-dependent toll-like receptors (TLRs) in a murine model of osteoarthritis

ObjectivesThe aim of our study was to evaluate the role of cell-membrane expressed TLRs and the signaling molecule MyD88 in a murine model of OA induced by knee menisectomy (surgical partial removal of the medial meniscus [MNX]). MethodsOA was induced in 8–10weeks old C57Bl/6 wild-type (WT) female (n=7) mice and in knockout (KO) TLR-1 (n=7), -2 (n=8), -4 (n=9) -6 (n=5), MyD88 (n=8) mice by medial menisectomy, using the sham-operated contralateral knee as a control. Cartilage destruction and synovial inflammation were evaluated by knee joint histology using the OARSI scoring method. Apoptotic chondrocytes and cartilage metabolism (collagen II synthesis and MMP-mediated aggrecan degradation) were analyzed using immunohistochemistry. ResultsOperated knees exhibited OA features at 8weeks post-surgery compared to sham-operated ones. In menisectomized TLR-1, -2, -4, and -6 deficient mice, cartilage lesions, synovial inflammation and cartilage metabolism were similar to that in operated WT mice. Accordingly, using the same approach, we found no significant protection in MyD88-deficient mice in terms of OA progression as compared to WT littermates. ConclusionsDeficiency of TLRs or their signalling molecule MyD88 did not impact on the severity of experimental OA. Our results demonstrate that MyD88-dependent TLRs are not involved in this murine OA model. Moreover, the dispensable role of MyD88, which is also an adaptor for IL-1 receptor signaling, suggests that IL-1 is not a key mediator in the development of OA. This latter hypothesis is strengthened by the lack of efficiency of IL-1β antagonist in the treatment of OA.

Toll-like Receptor 4-mediated Endoplasmic Reticulum Stress in Intestinal Crypts Induces Necrotizing Enterocolitis

The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4(ΔIEC-OVER) mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4(ΔIEC) mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.

Aloin protects against chronic alcoholic liver injury via attenuating lipid accumulation, oxidative stress and inflammation in mice

The present study was designed to investigate the protective effect of aloin against alcoholic liver disease in a chronic alcohol feeding mouse model. Mice were given alcohol twice a day by intragastric administration for 11 weeks (4.0, 4.7, 5.5 g/kg bw/day for the first 3 weeks respectively, 6.3 g/kg bw/day for the following 8 weeks). Aloin (10, 30 mg/kg bw) or vehicle was given by gavage to mice after each alcohol administration. Alcohol elevated the serum transaminases alanine aminotransferase, aspartate aminotransferase, total cholesterol and triglyceride levels which were significantly attenuated by the co-administration of aloin (p < 0.05). Histopathological observations were consistent with these indices. Co-administration of aloin significantly suppressed the alcohol-dependent induction of sterol regulatory element-binding protein-1c expression (p < 0.01) and remarkably up-regulated the mRNA levels of AMP-activated protein kinase-α2 (p < 0.001). Furthermore, aloin supplementation significantly inhibited the alcohol-dependent elevation of malondialdehyde and cytochrome P4502E1 expression (p < 0.05), and significantly elevated superoxide dismutase activity (p < 0.01). The up-regulation of serum lipopolysaccharide (LPS), hepatic nitric oxide, tumor necrosis factor α, toll-like receptor-4, and myeloid differentiation primary response gene 88 were also markedly suppressed by the co-administration of aloin (p < 0.05) in alcohol-treated mice. These results suggest that aloin may represent a novel, protective strategy against chronic alcoholic liver injury by attenuating lipid accumulation, oxidative stress and LPS-induced inflammatory response.

Graphene Oxide Triggers Toll‐Like Receptors/Autophagy Responses In Vitro and Inhibits Tumor Growth In Vivo

Graphene oxide (GO) is a nanomaterial with burgeoning bioapplications, while autophagy is implicated in cancer therapy. Although induction of autophagy by nanomaterials is reported, the underlying signaling mechanism in cancer cells and how this implicates the potential of GO in cancer therapy remain obscure. Here, it is shown that GO itself can induce the toll-like receptors (TLRs) responses and autophagy in cancer cells and confer antitumor effects in mice. GO can be phagocytosed by CT26 colon cancer cells, simultaneously triggering autophagy as well as TLR-4 and TLR-9 signaling cascades. By dissecting the crosstalk between the TLRs and autophagy pathways, it is uncovered that the GO-activated autophagy is regulated through the myeloid differentiation primary response gene 88 (MyD88)- and TNF receptor-associated factor 6 (TRAF6)-associated TLR-4/9 signaling pathways. Injection of GO alone into immunocompetent mice bearing the CT26 colon tumors not only suppresses the tumor progression but also enhances cell death, autophagy, and immune responses within the tumor bed. These data altogether implicate the potential of GO as an effective nanomaterial for autophagy induction and cancer therapy.

The metastasis-associated protein S100A4 promotes the inflammatory response of mononuclear cells via the TLR4 signalling pathway in rheumatoid arthritis

S100A4 has been implicated in cancer and several inflammatory diseases, including RA. The aim of the present study was to determine whether S100A4 can stimulate proinflammatory cytokine production in mononuclear cells. Peripheral blood mononuclear cells (PBMCs) isolated from patients with RA were stimulated with S100A4, S100A8, S100A9 and S100A12. The production of IL-1β, IL-6 and TNF-α was measured by ELISA. Receptor for advanced glycation end products (RAGEs) and Toll-like receptor 4 (TLR4) signalling were examined. For signalling pathway blocking studies, inhibitors of myeloid differentiation primary response gene 88 (MyD88), nuclear factor kappa B (NF-κB) and the mitogen activated protein (MAP) kinases p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) were used. MAP kinase activation was evaluated by western blotting. Stimulation of PBMCs with S100A4 significantly up-regulated IL-1β, IL-6 and TNF-α production compared with unstimulated cells (P < 0.001). Importantly, the production of these cytokines was markedly enhanced in response to S100A4 compared with S100A8 and S100A12; however, it was less pronounced compared with S100A9. Furthermore, enhanced production of proinflammatory cytokines in S100A4-stimulated PMBCs was at least partly mediated via TLR4, but not RAGEs, and by activation of the transcription factor NF-κB and the MAP kinases p38 and ERK1/2. This is the first study to demonstrate that S100A4 can induce an inflammatory response mediated by TLR4 and by the activation of NF-κB and the kinases p38 and ERK1/2 in mononuclear cells from patients with RA. Therefore S100A4 may be a potential therapeutic target for immune-mediated diseases.

Baicalin inhibits TLR7/MYD88 signaling pathway activation to suppress lung inflammation in mice infected with influenza A virus.

The present study aimed to investigate the protective effects and underlying mechanisms of baicalin on imprinting control region mice infected with influenza A/FM/1/47 (H1N1) virus. Oral administration of baicalin into mice infected with H1N1 prevented death, increased the mean time to death and inhibited lung index and lung consolidation. Subsequently, fluorescence quantitative polymerase chain reaction was used to assess the mRNA expression of toll-like receptor 7 (TLR7) and myeloid differentiation primary response gene 88 (MYD88), and western blot analysis was used to determine the expression of phosphorylated nuclear factor κB (NF-κB)-P65 and c-jun/activator protein 1 (AP-1). An enzyme-linked immunosorbent assay was applied to test for the inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β and IL-6, in the lung tissue. The findings indicated that baicalin downregulated the mRNA expression of TLR7 and MYD88, significantly downregulated the protein expression of NF-κB-P65 and AP-1 and also inhibited the secretion of TNF-α, IL-1β and IL-6. In conclusion, baicalin effectively reduced the pathological damage and inflammation of the lungs by downregulating the TLR7/MYD88-mediated signaling pathway.

Human Metapneumovirus M2-2 Protein Inhibits Innate Immune Response in Monocyte-Derived Dendritic Cells

Human metapneumovirus (hMPV) is a leading cause of lower respiratory infection in young children, the elderly and immunocompromised patients. Repeated hMPV infections occur throughout life. However, immune evasion mechanisms of hMPV infection are largely unknown. Recently, our group has demonstrated that hMPV M2-2 protein, an important virulence factor, contributes to immune evasion in airway epithelial cells by targeting the mitochondrial antiviral-signaling protein (MAVS). Whether M2-2 regulates the innate immunity in human dendritic cells (DC), an important family of immune cells controlling antigen presenting, is currently unknown. We found that human DC infected with a virus lacking M2-2 protein expression (rhMPV-ΔM2-2) produced higher levels of cytokines, chemokines and IFNs, compared to cells infected with wild-type virus (rhMPV-WT), suggesting that M2-2 protein inhibits innate immunity in human DC. In parallel, we found that myeloid differentiation primary response gene 88 (MyD88), an essential adaptor for Toll-like receptors (TLRs), plays a critical role in inducing immune response of human DC, as downregulation of MyD88 by siRNA blocked the induction of immune regulatory molecules by hMPV. Since M2-2 is a cytoplasmic protein, we investigated whether M2-2 interferes with MyD88-mediated antiviral signaling. We found that indeed M2-2 protein associated with MyD88 and inhibited MyD88-dependent gene transcription. In this study, we also identified the domains of M2-2 responsible for its immune inhibitory function in human DC. In summary, our results demonstrate that M2-2 contributes to hMPV immune evasion by inhibiting MyD88-dependent cellular responses in human DC.

Lipopolysaccharide differentially modulates expression of cytokines and cyclooxygenases in dorsal root ganglion cells via Toll-like receptor-4 dependent pathways

We have examined the functional expression of Toll-like receptor 4 (TLR4) in adult male rat dorsal root ganglion (DRG) cells in culture by studying changes in pro-inflammatory cytokines and cyclooxygenase (COX)-dependent prostanoid production. In the mixed population of DRG neurons and glial cells, only DRG neurons expressed cell surface TLR4 along with MD-2 and CD14. This classical TLR4 signaling complex on DRG neurons responded to lipopolysaccharide (LPS) with a TLR4-dependent and time-dependent increase in interleukin-1β and tumor necrosis factor-α mRNA expression which was entirely dependent on NF-κB activity. In contrast, after 2-h incubation with DRG cells, LPS-stimulated COX-2 was regulated by both NF-κB and transactivation of epidermal growth factor receptor (EGFR) with potential downstream activation of ERK1/2 and p38 kinase. In contrast to this evidence for myeloid differentiation primary response gene-88 (MyD88)-dependent signaling, no evidence was obtained for TIR-domain-containing adaptor-inducing interferon-ß (TRIF)-dependent signaling from TLR4 in DRG neurons. LPS surprisingly produced a time-dependent decrease in COX-1 protein which likely facilitates the COX-2-dependent production of prostaglandin E2 and prostacyclin. Our study is the first to demonstrate the activation of TLR4-dependent production of prostaglandin E2 and prostacyclin in DRG cell cultures. Our findings support the concept that the activation of TLR4 on primary sensory neurons by endogenous ligands may underlie neuropathic and inflammatory pain states.

A Wnt/β-catenin negative feedback loop represses TLR-triggered inflammatory responses in alveolar epithelial cells

Increasing evidence has demonstrated that the epithelial cells in the lung play crucial roles in regulating certain inflammatory responses by modulating Wnt signaling during microbial infection. However, the anti-microbial functions of Wnt signaling in alveolar epithelial cells remain elusive. In this report, we show that Wnt/β-catenin signaling is repressed in A549 alveolar epithelial cells during a Toll-like receptor ligand stimulation with Mycobacterium bovis Bacillus Calmette-Guerin (BCG) or lipopolysaccharide (LPS). In addition to activating TLR signaling, a stimulation of BCG or LPS led to the up-regulation of a Wnt receptor Frizzled-1, cytosolic GSK3β and Axin, and the down-regulation of nuclear β-catenin, lymphoid enhancer factor 1 and transcription factor 4. While an enhancement of β-catenin activity suppressed the TLR signal response, and substantially led to alleviate the TLR ligand-induced pro-inflammatory responses. Importantly, gain and loss of function studies by overexpressing or silencing of TLR signaling adaptor, myeloid differentiation primary response gene 88 (MyD88) further demonstrated an inverse relationship between TLR signaling and canonical Wnt signaling in A549 cells. These data imply that Wnt/β-catenin signaling acts as a negative feedback loop to suppress inflammation in alveolar epithelial cells, and averts cell injury from excessive inflammatory reactions. This study thus reveals a novel immunoregulatory mechanism in alveolar epithelial cells in response to bacterial infection.