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.     

Outer membrane protein A (OmpA) of Shigella flexneri 2a links innate and adaptive immunity in a TLR2-dependent manner and involvement of IL-12 and nitric oxide

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.     

Characterization of sparstolonin B, a Chinese herb-derived compound, as a selective Toll-like receptor antagonist with potent anti-inflammatory properties

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.     

Sialyl residues modulate LPS-mediated signaling through the Toll-like receptor 4 complex

We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFκB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation.     

The molecular mechanisms of the effect of Dexamethasone and Cyclosporin A on TLR4 /NF-κB signaling pathway activation in oral lichen planus

Toll-like receptors (TLRs) and the nuclear factor-kappa B (NF-κB) signaling transduction pathway play important roles in the pathogenesis of several chronic inflammatory diseases, but its function in oral lichen planus (OLP) remains unclear. In this study, we examined the expression of TLR4 and NF-κB-p65 and inflammatory cytokines TNF-α and IL-1β by immunohistochemistry in OLP tissues, and found that TLR4 and NF-κB-p65 were significantly upregulated in OLP compared to normal oral mucosa (P<0.05). We used keratinocytes HaCaT stimulated with lipopolysaccharide (LPS) to simulate the local OLP immune environment to some extent. RT-PCR and immunoblotting analyses showed significant activation of TLR4 and NF-κB-p65 in the circumstance of LPS-induced inflammatory response. The high expression of TLR4 and NF-κB-p65 are correlated with expression of cytokines TNF-α and IL-1β (P<0.05). We further showed that NF-κB could act as an anti-apoptotic molecule in OLP. We conclude that TLR4 and the NF-κB signaling pathway may interact with the perpetuation of OLP. Steroids and cyclosporine are effective in the treatment of symptomatic OLP. However, there was some weak evidence for the mechanism over Dexamethasone (DeX) and Cyclosporine A (CsA) for the palliation of symptomatic OLP. In the present study, we found that Dexamethasone and Cyclosporine A negatively regulated NF-κB signaling pathway under LPS simulation in HaCaT cells by inhibiting TLR4 expression, on the other hand, Cyclosporine A could inhibit HaCaT cell proliferation by the induction of the apoptosis of HaCaT cells to protect OLP from the destruction of epidermal cells effectively.     

Mas-related G protein-coupled receptor D participates in inflammatory pain by promoting NF-κB activation through interaction with TAK1 and IKK complex

Mas-related G protein-coupled receptor D (MrgprD) is mainly expressed in small-diameter sensory neurons of the dorsal root ganglion (DRG). Results from previous studies suggest that MrgprD participates in mechanical hyperalgesia and nerve injury-induced neuropathic pain. However, it remains elusive whether and how MrgprD is involved in inflammatory pain. Here, we used a mouse model of chronic inflammatory pain established by intraperitoneal administration of lipopolysaccharide (LPS). The LPS injection induced an evident peripheral neuroinflammation and mechanical hyperalgesia in the mice and increased MrgprD expression in the DRG. The LPS administration also augmented the proportion of MrgprD-expressing neurons in the lumbar 4 DRG. Behaviorally, the LPS-induced hypersensitivities to mechanical and cold stimuli, but not to a heat stimulus, were substantially attenuated in Mrgprd-knockout mice compared with wildtype littermates. Mrgprd deletion in DRGs suppressed the LPS-triggered activation of the NF-κB signaling pathway and attenuated LPS-induced up-regulation of pro-inflammatory factors. Moreover, ectopic overexpression of MrgprD in HEK293 cells stably expressing mouse toll-like receptor 4 (TLR4) markedly promoted the LPS-induced NF-κB activation and enhanced NF-κB's DNA-binding activity. Furthermore, MrgprD physically interacted with TGF-β-activated kinase 1 (TAK1) and I-kappa-B-kinase (IKK) complexes, but not with mitogen-activated protein kinases (MAPKs) in mouse DRGs. In macrophage-like RAW 264.7 cells, MrgprD also interacted with TAK1 and IKK complex, and the treatment of MrgprD agonist elicited the activation of NF-κB signaling, but not of mitogen-activated protein kinases (MAPKs) signaling pathway. Our findings indicate that MrgprD facilitates the development of LPS-triggered persistent inflammatory hyperalgesia by promoting canonical NF-κB activation, highlighting the important roles of MrgprD in NF-κB-mediated inflammation and chronic pain.     

Heterotrimeric Gi/Go proteins modulate endothelial TLR signaling independent of the MyD88-dependent pathway

The innate immune recognition of bacterial lipopolysaccharide (LPS) is mediated by Toll-like receptor 4 (TLR4) and results in activation of proinflammatory signaling including NF-κB and MAPK pathways. Heterotrimeric G proteins have been previously implicated in LPS signaling in macrophages and monocytes. In the present study, we show that pertussis toxin sensitive heterotrimeric G proteins (Gα(i/o)) are involved in the activation of MAPK and Akt downstream of TLR2, TLR3, and TLR4 in endothelial cells. Gα(i/o) are also required for full activation of interferon signaling downstream of TLR3 and TLR4 but are not required for the activation of NF-κB. We find that Gα(i/o)-mediated activation of the MAPK is independent of the canonical MyD88, interleukin-1 receptor-associated kinase, and tumor necrosis factor receptor-associated factor 6 signaling cascade in LPS-stimulated cells. Taken together, the data presented here suggest that heterotrimeric G proteins are widely involved in TLR pathways along a signaling cascade that is distinct from MyD88-TRAF6.     

Spontaneously diabetic Ins2(+/Akita):apoE-deficient mice exhibit exaggerated hypercholesterolemia and atherosclerosis

Type 2 diabetes (T2DM) is characterized by hyperglycemia, dyslipidemia, and increased inflammation. Previously, we showed that high glucose (HG) induces Toll-like receptor (TLR) expression, activity, and inflammation via NF-κB followed by cytokine release in vitro and in vivo. Here, we determined how HG-induced inflammation is affected by free fatty acids (FFA) in human monocytes. THP-1 monocytic cells, CD14(+) human monocytes, and transiently transfected HEK293 cells were exposed to various FFA (0-500 μM) and glucose (5-20 mM) for evaluation of TLR2, TLR4, NF-κB, IL-1β, monocyte chemoattractant protein-1 (MCP-1), and superoxide release. In THP-1 cells, palmitate increased cellular TLR2 and TLR4 expression, generated reactive oxygen species (ROS), and increased NF-κB activity, IL-1β, and MCP-1 release in a dose- and time-dependent manner. Similar data were observed with stearate and FFA mixture but not with oleate. Conversely, NADPH oxidase inhibitor treatment repressed glucose- and palmitate-stimulated ROS generation and NF-κB activity and decreased IL-1β and MCP-1 expression. Silencing TLR2, TLR4, and p47phox with small inhibitory RNAs (siRNAs) significantly reduced superoxide release, NF-κB activity, IL-1β, and MCP-1 secretion in HG and palmitate-treated THP-1 cells. Moreover, data from transient transfection experiments suggest that TLR6 is required for TLR2 and MD2 for TLR4 to augment inflammation in FFA- and glucose-exposed cells. These findings were confirmed with human monocytes. We conclude that FFA exacerbates HG-induced TLR expression and activity in monocytic cells with excess superoxide release, enhanced NF-κB activity, and induced proinflammatory factor release.     

Essential role of Toll-like receptor 2 in macrophage activation by glycogen

We prepared enzymatically synthesized glycogen (ESG) with the same characteristics as natural glycogen and investigated whether the macrophage-stimulating activity of glycogen was related to Toll-like receptors (TLRs), which are important receptors for innate immunity. ESG induced no nuclear factor-kappa B (NF-κB) activity in TLR4/MD-2/CD14-expressed human embryonic kidney 293 (HEK293) reporter cells, whereas this polysaccharide did activate peritoneal exude cells (PECs) derived from TLR4-deficient mice at the same level as those from wild-type (WT) mice. Similarly, ESG did not activate HEK293 cells expressing TLR3, 5, 7, 8 or 9, suggesting that these TLRs were irrelevant to the activity of ESG. In contrast, ESG enhanced the NF-κB activity of TLR2-expressed HEK293 reporter cells in a concentration-dependent manner. Furthermore, the cell-stimulating activity of ESG was remarkably lower for PECs from TLR2-deficient mice compared with those from WT mice. The activity of ESG completely disappeared after treatment with a glycogen-degrading enzyme, indicating that the activity derived from ESG itself and not from contamination with canonical TLR2 ligands such as bacterial lipopeptides. Moreover, it was clarified by ELISA that ESG was directly bound to TLR2. Taken together, these results demonstrated that TLR2 directly recognizes glycogen and that the recognition activates immunocytes such as macrophages to enhance the production of nitric oxide and inflammatory cytokines. In addition, it was suggested that TLR2 could be involved in the glycogen activity in vivo. We propose that glycogen act as an activator to potentiate the host defense through TLR2 on the macrophage.     

Toll-like receptor 2 mediates invasion via activating NF-κB in MDA-MB-231 breast cancer cells

MDA-MB-231 breast cancer cells have a high invasive potential, yet the mechanisms involved are not known. This study showed that Toll-like receptor 2 (TLR2) was highly expressed in MDA-MB-231 cells and played a critical role in cell invasion. Compared with the poorly invasive MCF-7 cells, MDA-MB-231 cells expressed 10.5-fold more TLR2. Using TLR2 agonist pg-LPS and TLR2 neutralizing antibody, we found that TLR2 activation significantly promoted MDA-MB-231 invasion, whereas TLR2 blockade diminished this capacity. TLR2 activation enhanced the activity of NF-κB and induced phosphorylation of TAK1 and IκBα in the TLR2/NF-κB signaling pathway in MDA-MB-231, but not in MCF-7 cells. TLR2 activation increased IL-6, TGF-β, VEGF and MMP9 secretion, which are associated with TLR2-NF-κB signaling. We demonstrated that TLR2 is a critical receptor responsible for NF-κB signaling activity and highly invasive capacity of MDA-MB-231 cells.     

Herbal melanin activates TLR4/NF-κB signaling pathway

Expression of many pro-inflammatory cytokines is controlled by the NF-κB signaling pathway. NF-κB is induced by LPS through activation of TLR4. Melanins extracted from fungal, plant and human sources modulate cytokine production and activate NF-κB pathway. We showed that a herbal melanin (HM) from Nigella sativa L. modulates cytokine production and suggested it as a ligand for TLR4. In this study we investigated the possibility that the HM-induced cytokine production is via an NF-κB signaling pathway. We found that HM induced the degradation of IκBα, a key step in the activation of NF-κB. Moreover, addition of IκB kinase (IKK) specific inhibitors effectively inhibited the observed HM-induced production of IL-8 and IL-6 by TLR4-transfected HEK293 cells and THP-1 cells. Our results have also shown that HM induced cleavage of caspase 8, and that this cleavage was partially abrogated by IKK inhibitors. We suggest that HM can modulate the inflammatory response by inducing IL-8 and IL-6 production via TLR4-dependent activation of the NF-κB signaling pathway.