TLR4 signaling is involved in the protective effect of propofol in BV2 microglia against OGD/reoxygenation

Propofol exhibits neuroprotective effects against hypoxic–ischemic brain injury, but the underlying mechanisms are still not clear. Toll-like receptor 4 (TLR4) plays a considerable role in the induction of innate immune and inflammatory responses. The purposes of this study are to investigate the effect of propofol on the oxygen and glucose deprivation (OGD)/reoxygenation (OGD/R) BV2 microglia and to explore the role of TLR4/myeloid differentiation protein 88 (MyD88)/nuclear factor-kappa B (NF-κB) pathway in the neuroprotective effects of propofol. BV2 microglia were placed into an airtight chamber and in glucose-free medium for OGD/reoxygenation. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. TLR4 and its downstream signaling molecules, MyD88 and NF-κB expressions were detected by Western blotting. Level of tumor necrosis factor alpha (TNF-α) in culture medium was determined with enzyme-linked immunosorbent assay. BV2 microglia apoptosis was determined by flow cytometry. We found that pretreatment with propofol significantly alleviated the hypoxic injury in BV2 microglia. Propofol inhibited upregulation of TLR4, MyD88, and NF-κB expressions in BV2 microglia exposed to OGD/reoxygenation. Propofol pretreatment also significantly reduced the production of TNF-α and apoptosis in OGD/reoxygenation BV2 microglia. The results indicated that TLR4 and its downstream MyD88-dependent signaling pathway contributed to neuroprotection of propofol to microglia exposed to OGD/reoxygenation.     

Eupatilin alleviates inflammatory response after subarachnoid hemorrhage by inhibition of TLR4/MyD88/NF-κB axis

Early brain injury (EBI) is associated with the adverse prognosis of subarachnoid hemorrhage (SAH) patients. The key bioactive component of the Chinese herbal medicine Artemisia asiatica Nakai (Asteraceae) is eupatilin. Recent research reports that eupatilin suppresses inflammatory responses induced by intracranial hemorrhage. This work is performed to validate whether eupatilin can attenuate EBI and deciphers its mechanism. A SAH rat model was established by intravascular perforation in vivo. At 6 h after SAH in rats, 10 mg/kg eupatilin was injected into the rats via the caudal vein. A Sham group was set as the control. In vitro, BV2 microglia was treated with 10 μM Oxyhemoglobin (OxyHb) for 24 h, followed by 50 μM eupatilin treatment for 24 h. The SAH grade, brain water content, neurological score, and blood-brain barrier (BBB) permeability of the rats were measured 24 h later. The content of proinflammatory factors was detected via enzyme-linked immunosorbent assay. Western blot analysis was conducted to analyze the expression levels of TLR4/MyD88/NF-κB pathway-associated proteins. In vivo, eupatilin administration alleviated neurological injury, and decreased brain edema and BBB injury after SAH in rats. Eupatilin markedly reduced the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), and suppressed the expression levels of MyD88, TLR4, and p-NF-κB p65 in the SAH rats' cerebral tissues. Eupatilin treatment also reduced the levels of IL-1β, IL-6, and TNF-α, and repressed the expression levels of MyD88, TLR4, and p-NF-κB p65 in OxyHb-induced BV2 microglia. Additionally, pyrrolidine dithiocarbamate or resatorvid enhanced the suppressive effects of eupatilin on OxyHb-induced inflammatory responses in BV2 microglia. Eupatilin ameliorates SAH-induced EBI via modulating the TLR4/MyD88/NF-κB pathway in rat model.     

TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation caused by proteinuria

Proteinuria is an important risk factor for chronic kidney diseases (CKD). Several studies have suggested that proteinuria initiates tubulointerstitial inflammation, while the mechanisms have not been fully understood. In this study, we hypothesized whether the activation of the TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation induced by proteinuria. We observed expression of TLR2, MyD88, NF-κB, as well as TNF-α and IL-6 detected by immunohistostaining, Western blotting and real-time PCR in albumin-overloaded (AO) nephropathy rats. In vitro, we observed these markers in HK-2 cells stimulated by albumin. We used TLR2 siRNA or the NF-κB inhibitor BAY 11-7082 to observe the influence of TNF-α and IL-6 expression caused by albumin overload. Finally, we studied these markers in non-IgA mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria. It was demonstrated that expression of TLR2, MyD88 and NF-κB were significantly increased in AO rats and in non-IgA MsPGN patients with high levels of proteinuria, and TNF-α and IL-6 expressions were increased after NF-κB activation. Furthermore, TNF-α and IL-6 expression was positively correlated with the level of proteinuria. Albumin-overload induced TNF-α and IL-6 secretions by the TLR2–MyD88–NF-κB pathway activation, which could be attenuated by the TLR2 siRNA or BAY 11-7082 in HK-2 cells. In summary, we demonstrated that proteinuria may exhibit an endogenous danger-associated molecular pattern (DAMP) that induces tubulointerstitial inflammation via the TLR2–MyD88–NF-κB pathway activation.     

Histone Deacetylase 2 Inhibitor CAY10683 Alleviates Lipopolysaccharide Induced Neuroinflammation Through Attenuating TLR4/NF-κB Signaling Pathway

Neuroinflammation involves in the progression of many central nervous system diseases. Several studies have shown that histone deacetylase (HDAC) inhibitors modulated inflammatory responses in lipopolysaccharide (LPS) stimulated microglia. While, the mechanism is still unclear. The aim of present study was to investigate the effect of HDAC2 inhibitor CAY10683 on inflammatory responses and TLR4/NF-κB signaling pathways in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. The effect of CAY10683 on cell viability of BV2 microglial cells was detected by CCK-8 assay. The expressions of inflammatory cytokines were analyzed by western blotting and RT-PCR respectively. The TLR4 protein expression was measured by western blotting, immunofluorescence, immunohistochemistry respectively. The protein expressions of MYD88, phospho-NF-κB p65, NF-κB-p65, acetyl-H3 (AH3), H3, and HDAC2 were analyzed by western blotting. We found that CAY10683 could inhibit expression levels of inflammatory cytokine TNF-α and IL-1β in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. It could induce TLR4, MYD88, phospho-NF-κB p65, and HDAC2 expressions. Moreover, CAY10683 increased the acetylation of histones H3 in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. Taken together, our findings suggested that HDAC2 inhibitor CAY10683 could suppress neuroinflammatory responses and TLR4/NF-κB signaling pathways by acetylation after LPS stimulation.     

Activation of BV2 microglia by lipopolysaccharide triggers an inflammatory reaction in PC12 cell apoptosis through a toll-like receptor 4-dependent pathway

Microglia play an important role in neuronal protection and damage. However, the molecular and cellular relationship between microglia and neurons is unclear. We carried out a prospective study to detect that activation of BV2 microglia induced PC12 cell apoptosis in vitro through the TLR4/adapter protein myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. BV2 microglia were treated with different concentrations of LPS for 24 h. Western blot was utilized to detect the expression of TLR4 and the downstream signaling pathway. The level of inflammatory mediator was quantified using a specific ELISA kit. The supernatant of 10 μg/ml LPS-treated BV2 cells was used as conditioned medium (CM). PC12 cells were co-culture with CM for 24 h. Cell viability was determined by MTT assay and cell apoptosis was tested by flow cytometry. BV2 microglia were treated with 10, 20, or 30 μg/ml LPS for 24 h. The expression of TLR4, MyD88, and NF-κB significantly increased. When PC12 cells were co-cultured with CM for 24 h, cell viability decreased. CM up-regulated the Bax level and down-regulated the Bcl-2 protein level in PC12 cells. PC12 cells pretreated with interleukin-1 receptor antagonist (IL-1RA) for 30 min, significantly alleviated CM-induced PC12 cell apoptosis. These results suggest that BV2 microglia activated by LPS triggered TLR4/MyD88/NF-κB signaling pathway that induced the release of IL-1β and could participate in the PC12 cells injury.     

Bevacizumab cured age-related macular degeneration (AMD) via down-regulate TLR2 pathway

AMD is the main cause of visual impairment in people over 50 years of age and the most common cause of blindness. In recent years, the use of bevacizumab to treat neovascular AMD has become a preferred treatment in the United States. However, whether bevacozumab is available for RPE or AMD patients is unknown. We firstly indicate that Pam3CSK4 (P3C) activates TLR2 pathway during ARPE-19 apoptosis as determined by western blotting. And then, the expression of MyD88, NF-κB, p-IKK in primary RPE cells from AMD patients is significantly down-regulated after treatment with 50 µg L^?1 Bevacizumab. Therefore, our data shows that MyD88 is involved in the TLR2 pathway in ARPE-19 cell apoptosis resulting from Pam3CSK4 (P3C). And more importantly, our findings suggested that Bevacizumab cured age-related macular degeneration (AMD) via down-regulate Toll—like receptor 2 (TLR2) pathway in RPE from AMD patients.     

Clostridium butyricum activates TLR2-mediated MyD88-independent signaling pathway in HT-29 cells

Oral administration of Clostridium butyricum as probiotic is increasingly gaining importance in the treatment of diarrhea and the improvement of animal performance. However, the mechanisms of host cell receptor recognition of C. butyricum and the downstream immune signaling pathways leading to these benefits remain unclear. The objective of this study was to analyze the mechanisms involved in C. butyricum induction of the toll-like receptor (TLR) signaling. Knockdown of myeloid differentiation primary response protein 88 (MyD88) expression using small interfering RNA in this manner did not affect C. butyricum-induced elevated levels of nuclear factor κB (NF-κB), interleukin-8 (IL-8), IL-6, and tumor necrosis factor alpha (TNF-α), suggesting a MyD88-independent route to TLR signaling transduction. However, a significant reduction in the levels of NF-κB, IL-8, IL-6, and TNF-α was evident in the absence of TLR2 expression, implying the need for TLR2 in C. butyricum recognition. Hence, C. butyricum activates TLR2-mediated MyD88-independent signaling pathway in human epithelial cells, which adds to our understanding of the molecular mechanisms of this probiotic action on gut epithelium.     

TLR2/MyD88/NF-κB pathway, reactive oxygen species, potassium efflux activates NLRP3/ASC inflammasome during respiratory syncytial virus infection

Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although "controlled" inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1β (IL-1β) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1β is synthesized as an immature pro-IL-1β form. It is cleaved by activated caspase-1 to yield mature IL-1β that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1β release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1β secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1β production during RSV infection. Further studies illustrated that prior to inflammasome formation; the "first signal" constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-κB pathway. TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection. Following expression of these genes, two "second signals" are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K(+)) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-κB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1β release during RSV infection.     

Salvianolic acid A alleviated inflammatory response mediated by microglia through inhibiting the activation of TLR2/4 in acute cerebral ischemia-reperfusion

BackgroundToll-like receptor 2 and Toll-like receptor 4 (TLR2/4) on microglia have been found as important regulators in the inflammatory response during cerebral ischemia/reperfusion (I/R). In China, traditional Chinese medicine Salvia miltiorrhiza (danshen) and its some components are considered to be effective in rescuing cerebral I/R injury through clinical practice.Hypothesis/PurposeHere we examined the effect of Salvianolic acid A (SAA), a monomer compound in the water extract of Salvia miltiorrhiza, on TLR2/4 of microglia and its mediated inflammatory injury during cerebral I/R in vivo and in vitro.Study DesignFor exploring the effect of SAA on cerebral I/R and TLR2/4, classic middle cerebral artery occlusion (MCAO) model and oxygen glucose deprivation / reoxygenation (OGD/R) model of co-culture with primary hippocampal neurons and microglia in vitro were used. Signal pathway research and gene knockout have been applied to further explain its mechanism.MethodsThe evaluation indexes of I/R injury included infarct size, edema degree and pathology as well as primary hippocampal neurons and microglia culture, ELISA, western, RT-PCR, HE staining, immunofluorescence, flow cytometry, siRNA gene knockout were also employed.ResultsSAA significantly improved the degree of brain edema and ischemic area in I/R rats accompanied by decreases in levels of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α). Pathological staining revealed that SAA could reduce inflammatory cell infiltration and mcirogila activation after reperfusion. Both protein and gene expression of TLR2 and TLR4 in ischemic hemisphere were obviously inhibited by SAA treatment while changes were not found in the non-ischemic hemisphere. In order to further study its mechanism, OGD/R model was used to mimic inflammatory damage of ischemic tissue by co-culturing primary rat hippocampal neurons and microglial cells. It was found that SAA also inhibited the protein and gene expression of TLR2 and TLR4 after OGD/R injury in microglia. After TLR2/4 knockout, the inhibitory effect of SAA on IL-1β and TNF-α levels in cell supernatant and neuron apoptosis were significantly weakened in each dose group. Moreover, expression levels of myeloid differentiation factor 88 (MyD88), NFκB, IL-1β and IL-6 in TLR2/4 mediated inflammatory pathway were reduced with SAA treatment.ConclusionSAA could significantly reduce the inflammatory response and injury in cerebral ischemia-reperfusion in vivo and in vitro, and its mechanism may be through the inhibition of TLR2/4 and its related signal pathway.     

The role of intermediary domain of MyD88 in cell activation and therapeutic inhibition of TLRs

Adaptor MyD88 has a pivotal role in TLR and IL-1R signaling and is involved in mediating excessive inflammation. MyD88 is composed of a death domain and a Toll/IL-1R domain connected by an intermediary domain (INT). The alternatively spliced form of MyD88 lacking the INT prevents signaling through MyD88-dependent TLRs. We designed a peptide from the INT and showed that it inhibits TLR4 activation by LPS when linked to a cell-penetrating peptide. As a new approach for the delivery of signaling-inhibitory peptides, INT peptide acylation also provided efficient cell translocation and inhibition of activation. We determined that INT peptide targets IL-1R-associated kinase 4. Furthermore, MyD88 mutant and molecular modeling refines the MyD88- IL-1R-associated kinase 4 interaction model based on the Myddosome structure. In addition to TLR4, INT peptide also inhibited TLR5, TLR2, TLR9, and IL-1R signaling but not TLR3, which uses Toll/IL-1R domain-containing adapter inducing IFN-β signaling adaptor. Inhibition of signaling in murine and human cells was observed by decreased NF-κB activation, cytokine mRNA synthesis, and phosphorylation of downstream kinases. In the endotoxemic mouse model, INT peptide suppressed production of inflammatory cytokines and improved survival, supporting therapeutic application of INT peptides for the suppression of inflammatory conditions mediated by MyD88.     

Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-β (IFN-β) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-β. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-κB and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88) MyD88 expression and NF-κB activation, confirming that hypoxia suppressed the LPS-induced inflammatory response by affecting TLR4 signaling. In conclusion, our results demonstrated that hypoxia attenuated the host immune and inflammatory response against Acanthamoeba infection by suppressing TLR4 signaling, indicating that hypoxia might impair the host cell's ability to eliminate the Acanthamoeba invasion and that hypoxia could enhance cell susceptibility to Acanthamoeba infection. These results may explain why contact lens use is one of the most prominent risk factors for AK.     

Forsythiaside A Exhibits Anti-inflammatory Effects in LPS-Stimulated BV2 Microglia Cells Through Activation of Nrf2/HO-1 Signaling Pathway

Inflammation and oxidative stress have been reported to play critical roles in the pathogenesis of neurodegenerative disease. Forsythiaside A, a phenylethanoside product isolated from air-dried fruits of Forsythia suspensa, has been reported to have anti-inflammatory and antioxidant effects. In this study, the anti-inflammatory effects of forsythiaside A on LPS-stimulated BV2 microglia cells and primary microglia cells were investigated. The production of inflammatory mediators TNF-α, IL-1β, NO and PGE₂ were detected in this study. NF-κB, nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) expression were detected by western blot analysis. Our results showed that forsythiaside A significantly inhibited LPS-induced inflammatory mediators TNF-α, IL-1β, NO and PGE₂ production. LPS-induced NF-κB activation was suppressed by forsythiaside A. Furthermore, forsythiaside A was found to up-regulate the expression of Nrf2 and HO-1. In conclusion, this study demonstrates that forsythiaside A inhibits LPS-induced inflammatory responses in BV2 microglia cells and primary microglia cells through inhibition of NF-κB activation and activation of Nrf2/HO-1 signaling pathway.     

Interleukin-10 activates Toll-like receptor 4 and requires MyD88 for cardiomyocyte survival

Toll-like receptors (TLRs) are important in a variety of inflammatory diseases including acute cardiac disorders. TLR4 innate signaling regulates the synthesis of anti-inflammatory cytokine, interleukin-10 (IL-10) upon TLR4 agonists’ re-stimulation. Anti-apoptotic action of IL-10 in cardiac dysfunction is generally accepted but its protective mechanism through TLR4 is not yet understood. We studied the effect of IL-10 in the activation of TLR4 downstream signals leading to cardiomyocytes survival. IL-10 caused a significant increase in the expression of CD14, MyD88 and TLR4. TLR4 activation led to the translocation of the interferon regulatory factor 3 (IRF3) into the nucleus. Phosphorylation of IRF3 enhanced mRNA synthesis for IL-1β but not TNF-α and was elevated even after removal of IL-10 stimulation. Furthermore, degradation of inhibitory kappa B (IκB) kinase (Ikk) suggested that IκBβ was the main activating kinase for IRF3-regulated NF-κB activation and phosphorylation of p65. Phosphorylated NF-κB p65 was translocated into the nucleus. Concomitantly, an increase in Bcl-xL activity inhibited Bax and the proteolytic activity of caspase 3 as well as a decrease in PARP cleavage. An inhibition of MyD88, modulated the above listed responses to IL-10 as there was a decrease in TLR4 and IRF3 and an increase in TNF-α mRNA. This was associated with a decrease in NF-κB p65, Bcl-xL mRNA and protein levels as well as there was an activation of Bax and PARP cleavage independent of caspase 3 activation. These data in cardiomyocytes suggest that IL-10 induced anti-apoptotic signaling involves upregulation of TLR4 through MyD88 activation.     

Induction of COX-2/PGE2/IL-6 is crucial for cigarette smoke extract-induced airway inflammation: Role of TLR4-dependent NADPH oxidase activation

Exposure to cigarette smoke extract (CSE) leads to airway and lung inflammation through an oxidant-antioxidant imbalance. Cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂) have been shown to play critical roles in respiratory inflammation. Here, we show that COX-2/PGE₂/IL-6 induction is dependent on Toll-like receptor 4 (TLR4)/NADPH oxidase signaling in human tracheal smooth muscle cells (HTSMCs). CSE induced COX-2 expression in vitro in HTSMCs and in vivo in the airways of mice. CSE also directly caused an increase in TLR4. Moreover, CSE-regulated COX-2, PGE₂, and IL-6 generation was inhibited by pretreatment with TLR4 Ab; inhibitors of c-Src (PP1), NADPH oxidase (diphenylene iodonium chloride and apocynin), p38 MAPK (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-κB (helenalin); a ROS scavenger (N-acetyl-l-cysteine); and transfection with siRNA of TLR4, MyD88, TRAF6, Src, p47^phox, p38, p42, JNK2, or p65. CSE-induced leukocyte numbers in BAL fluid were also reduced by pretreatment with these inhibitors. Furthermore, CSE induced p47^phox translocation and TLR4/MyD88/TRAF6 and c-Src/p47^phox complex formation. We found that PGE₂ enhanced IL-6 production in HTSMCs and leukocyte count in BAL fluid. In addition, treatment with nicotine could induce COX-2, PGE₂, and IL-6 generation in in vivo and in vitro studies. These results demonstrate that CSE-induced ROS generation was mediated through the TLR4/MyD88/TRAF6/c-Src/NADPH oxidase pathway, in turn initiated the activation of MAPKs and NF-κB, and ultimately induced COX-2/PGE₂/IL-6-dependent airway inflammation.     

CpG oligonucleotides induce an immune response of odontoblasts through the TLR9, MyD88 and NF-κB pathways

Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-α expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-κB or IκBα markedly inhibited CpG ODN-induced IL-6 and TNF-α expression. Moreover, the CpG ODN-mediated increase of κB-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9, MyD88 and IκBα, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-κB are involved in this process.     

TLR2 hypersensitivity of astrocytes as functional consequence of previous inflammatory episodes

Precedent inflammatory episodes may drastically modify the function and reactivity of cells. We investigated whether priming of astrocytes by microglia-derived cytokines alters their subsequent reaction to pathogen-associated danger signals not recognized in the quiescent state. Resting primary murine astrocytes expressed little TLR2, and neither the TLR2/6 ligand fibroblast-stimulating lipopeptide-1 (FSL1) nor the TLR1/2 ligand Pam(3)CysSK(4) (P3C) triggered NF-κB translocation or IL-6 release. We made use of single-cell detection of NF-κB translocation as easily detectable and sharply regulated upstream indicator of an inflammatory response or of c-Jun phosphorylation to measure restimulation events in astrocytes under varying conditions. Cells prestimulated with IL-1β, with a TLR3 ligand, with a complete cytokine mix consisting of TNF-α, IL-1β, and IFN-γ, or with media conditioned by activated microglia responded strongly to FSL1 or P3C stimulation, whereas the sensitivity of the NF-κB response to other pattern recognition receptors was unchanged. This sensitization to TLR2 ligands was associated with an initial upregulation of TLR2, displayed a "memory" window of several days, and was largely independent of the length of prestimulation. The altered signaling led to altered function, as FSL1 or P3C triggered the release of IL-6, CCL-20, and CXCL-2 in primed cells, but not in resting astrocytes. These data confirmed the hypothesis that astrocytes exposed to activated microglia assume a different functional phenotype involving longer term TLR2 responsiveness, even after the initial stimulation by inflammatory mediators has ended.     

Membrane-tethered MUC1 mucin is phosphorylated by epidermal growth factor receptor in airway epithelial cells and associates with TLR5 to inhibit recruitment of MyD88

MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surface of mucosal epithelial cells. Previous in vivo and in vitro studies established that MUC1 counterregulates airway inflammation by suppressing TLR signaling. In this article, we elucidate the mechanism by which MUC1 inhibits TLR5 signaling. Overexpression of MUC1 in HEK293 cells dramatically reduced Pseudomonas aeruginosa-stimulated IL-8 expression and decreased the activation of NF-κB and MAPK compared with cells not expressing MUC1. However, overexpression of MUC1 in HEK293 cells did not affect NF-κB or MAPK activation in response to TNF-α. Overexpression of MyD88 abrogated the ability of MUC1 to inhibit NF-κB activation, and MUC1 overexpression inhibited flagellin-induced association of TLR5/MyD88 compared with controls. The MUC1 cytoplasmic tail associated with TLR5 in all cells tested, including HEK293T cells, human lung adenocarcinoma cell line A549 cells, and human and mouse primary airway epithelial cells. Activation of epidermal growth factor receptor tyrosine kinase with TGF-α induced phosphorylation of the MUC1 cytoplasmic tail at the Y46EKV sequence and increased association of MUC1/TLR5. Finally, in vivo experiments demonstrated increased immunofluorescence colocalization of Muc1/TLR5 and Muc1/phosphotyrosine staining patterns in mouse airway epithelium and increased Muc1 tyrosine phosphorylation in mouse lung homogenates following P. aeruginosa infection. In conclusion, epidermal growth factor receptor tyrosine phosphorylates MUC1, leading to an increase in its association with TLR5, thereby competitively and reversibly inhibiting recruitment of MyD88 to TLR5 and downstream signaling events. This unique ability of MUC1 to control TLR5 signaling suggests its potential role in the pathogenesis of chronic inflammatory lung diseases.     

Curcumin Attenuates gp120-Induced Microglial Inflammation by Inhibiting Autophagy via the PI3K Pathway

Microglial inflammation plays an essential role in the pathogenesis of HIV-associated neurocognitive disorders. A previous study indicated that curcumin relieved microglial inflammatory responses. However, the mechanism of this process remained unclear. Autophagy is a lysosome-mediated cell content-dependent degradation pathway, and uncontrolled autophagy leads to enhanced inflammation. The role of autophagy in curcumin-attenuating BV2 cell inflammation caused by gp120 was investigated with or without pretreatment with the autophagy inhibitor 3-MA and blockers of NF-κB, IKK, AKT, and PI3K, and we then detected the production of the inflammatory mediators monocyte chemoattractant protein-1 (MCP-1) and IL17 using ELISA, and autophagy markers ATG5 and LC3 II by Western Blot. The autophagic flux was observed by transuding mRFP-GFP-LC3 adenovirus. The effect of the blockers on gp120-induced BV2 cells was examined by the expression of p-AKT, p-IKK, NF-κB, and p65 in the nuclei and LC3 II and ATG5. gp120 promoted the expression of MCP-1 and IL-17, enhanced autophagic flux, and up-regulated the expression of LC3 II and ATG5, while the autophagy inhibitor 3-MA down-regulated the phenomena above. Curcumin has similar effects with 3-MA, in which curcumin inhibited NF-κB by preventing the translocation of NF-κB p65. Curcumin also inhibited the phosphorylation of p-PI3K, p-AKT, and p-IKK, which leads to down-regulation of NF-κB. Curcumin reduced autophagy via PI3K/AKT/IKK/NF-κB, thereby reducing BV2 cellular inflammation induced by gp120.