Dataset Information

Nuclear factor kappaB (NF-kappaB) activation primes cells to a pro-inflammatory polarized response to a Toll-like receptor 7 (TLR7) agonist.

ABSTRACT: TLR7 (Toll-like receptor 7) mediates anti-viral immunity by recognizing ssRNA (single-stranded RNA) viruses. Small-molecular-mass TLR7 agonists have been approved, or are being evaluated, for treatment of cancers or infectious diseases. Although TLR7 is predominantly expressed in a restricted set of immune cell types, including pDCs (plasmacytoid dendritic cells), it is also expressed in non-native expressing cells (e.g. hepatocytes) under certain circumstances. To elucidate the molecular basis of TLR7 induction by pro-inflammatory stimulation and the subsequent cellular responses in these non-native TLR7-expressing cell types, we first cloned and characterized the 5'-promoter region of TLR7. The proximal region of this promoter drives the transcription of the TLR7 gene. Pro-inflammatory stimuli activated TLR 7 transcription via a NF-kappaB (nuclear factor kappaB)-binding motif in this region, and this activation could be blocked by mutation of the NF-kappaB binding site or addition of NF-kappaB inhibitors. Further studies showed that pretreatment of the Hep3B hepatocytes with TNF-alpha (tumour necrosis factor-alpha) or IL-1 (interleukin-1) rendered them responsive to TLR7 activation by a TLR7 agonist. However, distinct from TLR7 activation in pDCs, which respond to stimulation with Th1 polarized cytokine production, TLR7 induction by pro-inflammatory signals in hepatocytes reconstitutes the NF-kappaB-dependent cascade but not the IRF7 (interferon regulatory factor 7)-dependent cascade, resulting in a pro-inflammatory polarized response rather than a Th1 polarized response. These results indicate that inflammatory stimulation is capable of priming cells to respond to TLR7 agonist with an immune response that differs from that in native TLR7-expressing cells.

SUBMITTER: Lee J

PROVIDER: S-EPMC2746977 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Nuclear factor kappaB (NF-kappaB) activation primes cells to a pro-inflammatory polarized response to a Toll-like receptor 7 (TLR7) agonist.

Lee Jongdae J Hayashi Masaaki M Lo Jeng-Fan JF Fearns Colleen C Chu Wen-Ming WM Luo Yunping Y Xiang Rong R Chuang Tsung-Hsien TH

The Biochemical journal 20090626 2

TLR7 (Toll-like receptor 7) mediates anti-viral immunity by recognizing ssRNA (single-stranded RNA) viruses. Small-molecular-mass TLR7 agonists have been approved, or are being evaluated, for treatment of cancers or infectious diseases. Although TLR7 is predominantly expressed in a restricted set of immune cell types, including pDCs (plasmacytoid dendritic cells), it is also expressed in non-native expressing cells (e.g. hepatocytes) under certain circumstances. To elucidate the molecular basis ...[more]

PMID: 19426145

Similar Datasets

Project description:The ability of the transcription factor NF-kappaB to upregulate anti-apoptotic proteins has been linked to the chemoresistance of solid tumors to standard chemotherapy. In contrast, recent studies have proposed that, in response to doxorubicin, NF-kappaB can be pro-apoptotic through repression of anti-apoptotic target genes. However, there is little evidence analyzing the outcome of NF-kappaB inhibition on the cytotoxicity of doxorubicin in studies describing pro-apoptotic NF-kappaB activity. In this study, we further characterize the activation of NF-kappaB in response to doxorubicin and evaluate its role in chemotherapy-induced cell death in sarcoma cells where NF-kappaB is reported to be pro-apoptotic. Doxorubicin treatment in U2OS cells induced canonical NF-kappaB activity as evidenced by increased nuclear accumulation of phosphorylated p65 at serine 536 and increased DNA-binding activity. Co-treatment with a small molecule IKKbeta inhibitor, Compound A, abrogated this response. RT-PCR evaluation of anti-apoptotic gene expression revealed that doxorubicin-induced transcription of cIAP2 was inhibited by Compound A, while doxorubicin-induced repression of other anti-apoptotic genes was unaffected by Compound A or siRNA to p65. Furthermore, the combination of doxorubicin and canonical NF-kappaB inhibition with Compound A or siRNA to p65 resulted in decreased cell viability measured by trypan blue staining and MTS assay and increased apoptosis measured by cleaved poly (ADP-ribose) polymerase and cleaved caspase 3 when compared to doxorubicin alone. Our results demonstrate that doxorubicin-induced canonical NF-kappaB activity associated with phosphorylated p65 is anti-apoptotic in its function and that doxorubicin-induced repression of anti-apoptotic genes occurs independent of p65. Therefore, combination therapies incorporating NF-kappaB inhibitors together with standard chemotherapies remains a viable method to improve the clinical outcomes in patients with advanced stage malignancies.

Project description:Picroliv, an iridoid glycoside derived from the plant Picrorhiza kurroa, is used traditionally to treat fever, asthma, hepatitis, and other inflammatory conditions. However, the exact mechanism of its therapeutic action is still unknown. Because nuclear factor-kappaB (NF-kappaB) activation plays a major role in inflammation and carcinogenesis, we postulated that picroliv must interfere with this pathway by inhibiting the activation of NF-kappaB-mediated signal cascade. Electrophoretic mobility shift assay showed that pretreatment with picroliv abrogated tumor necrosis factor (TNF)-induced activation of NF-kappaB. The glycoside also inhibited NF-kappaB activated by carcinogenic and inflammatory agents, such as cigarette smoke condensate, phorbol 12-myristate 13-acetate, okadaic acid, hydrogen peroxide, lipopolysaccharide, and epidermal growth factor. When examined for the mechanism of action, we found that picroliv inhibited activation of IkappaBalpha kinase, leading to inhibition of phosphorylation and degradation of IkappaBalpha. It also inhibited phosphorylation and nuclear translocation of p65. Further studies revealed that picroliv directly inhibits the binding of p65 to DNA, which was reversed by the treatment with reducing agents, suggesting a role for a cysteine residue in interaction with picroliv. Mutation of Cys(38) in p65 to serine abolished this effect of picroliv. NF-kappaB inhibition by picroliv leads to suppression of NF-kappaB-regulated proteins, including those linked with cell survival (inhibitor of apoptosis protein 1, Bcl-2, Bcl-xL, survivin, and TNF receptor-associated factor 2), proliferation (cyclin D1 and cyclooxygenase-2), angiogenesis (vascular endothelial growth factor), and invasion (intercellular adhesion molecule-1 and matrix metalloproteinase-9). Suppression of these proteins enhanced apoptosis induced by TNF. Overall, our results show that picroliv inhibits the NF-kappaB activation pathway, which may explain its anti-inflammatory and anticarcinogenic effects.

Project description:BackgroundMicroglia, the resident macrophage-like cells in the brain, regulate innate immune responses in the CNS to protect neurons. However, excessive activation of microglia contributes to neurodegenerative diseases. Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Here, the coordinated activities of GR and MR on the modulation of the nuclear factor-?B (NF-?B) pathway in murine BV-2 microglial cells were studied.MethodsBV-2 cells were treated with different corticosteroids in the presence or absence of MR and GR antagonists. The impact of the glucocorticoid-activating enzyme 11?-hydroxysteroid dehydrogenase type 1 (11?-HSD1) was determined by incubating cells with 11-dehydrocorticosterone, with or without selective inhibitors. Expression of interleukin-6 (IL-6), tumor necrosis factor receptor 2 (TNFR2), and 11?-HSD1 mRNA was analyzed by RT-PCR and IL-6 protein expression by ELISA. NF-?B activation and translocation upon treatment with various corticosteroids were visualized by western blotting, immunofluorescence microscopy, and translocation assays.ResultsGR and MR differentially regulate NF-?B activation and neuroinflammatory parameters in BV-2 cells. By converting inactive 11-dehydrocorticosterone to active corticosterone, 11?-HSD1 essentially modulates the coordinated action of GR and MR. Biphasic effects were observed for 11-dehydrocorticosterone and corticosterone, with an MR-dependent potentiation of IL-6 and tumor necrosis factor-? (TNF-?) expression and NF-?B activation at low/moderate concentrations and a GR-dependent suppression at high concentrations. The respective effects were confirmed using the MR ligand aldosterone and the antagonist spironolactone as well as the GR ligand dexamethasone and the antagonist RU-486. NF-?B activation could be blocked by spironolactone and the inhibitor of NF-?B translocation Cay-10512. Moreover, an increased expression of TNFR2 was observed upon treatment with 11-dehydrocorticosterone and aldosterone, which was reversed by 11?-HSD1 inhibitors and/or spironolactone and Cay-10512.ConclusionsA tightly coordinated GR and MR activity regulates the NF-?B pathway and the control of inflammatory mediators in microglia cells. The balance of GR and MR activity is locally modulated by the action of 11?-HSD1, which is upregulated by pro-inflammatory mediators and may represent an important feedback mechanism involved in resolution of inflammation.

Project description:Fractalkine (also known as CX3CL1), a CX3C chemokine, activates and attracts monocytes/macrophages to the site of injury/inflammation. It binds to CX3C receptor 1 (CX3CR1), a pertussis toxin-sensitive G-protein-coupled receptor. In smooth muscle cells (SMCs), fractalkine is induced by proinflammatory cytokines [tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)], which may mediate monocyte adhesion to SMCs. However, the mechanisms underlying its induction are unknown. In addition, it is unlear whether SMCs express CX3CR1. TNF-alpha activated nuclear factor kappaB (NF-kappaB) and induced fractalkine and CX3CR1 expression in a time-dependent manner in rat aortic SMCs. Transient transfections with dominant-negative (dn) inhibitory kappaB (IkappaB)-alpha, dnIkappaB-beta, dnIkappaB kinase (IKK)-gamma, kinase-dead (kd) NF-kappaB-inducing kinase (NIK) and kdIKK-beta, or pretreatment with wortmannin, Akt inhibitor, pyrrolidinecarbodithioc acid ammonium salt ('PDTC') or MG-132, significantly attenuated TNF-alpha-induced fractalkine and CX3CR1 expression. Furthermore, expression of dn TNF-alpha-receptor-associated factor 2 (TRAF2), but not dnTRAF6, inhibited TNF-alpha signal transduction. Pretreatment with pertussis toxin or neutralizing anti-CX3CR1 antibodies attenuated TNF-alpha-induced fractalkine expression, indicating that fractalkine autoregulation plays a role in TNF-alpha-induced sustained fractalkine expression. Fractalkine induced its own expression, via pertussis toxin-sensitive G-proteins, phosphoinositide 3-kinase (PI 3-kinase), phosphoinositide-dependent kinase 1 (PDK1), Akt, NIK, IKK and NF-kappaB activation, and induced SMC cell-cell adhesion and cellular proliferation. Taken together, our results demonstrate that TNF-alpha induces the expression of fractalkine and CX3CR1 in rat aortic SMCs and that this induction is mediated by NF-kappaB activation. We also show that fractalkine induces its own expression, which is mediated by the PI 3-kinase/PDK1/Akt/NIK/IKK/NF-kappaB signalling pathway. More importantly, fractalkine increased cell-cell adhesion and aortic SMC proliferation, indicating a role in initiation and progression of atherosclerotic vascular disease.

Dataset Information

Nuclear factor kappaB (NF-kappaB) activation primes cells to a pro-inflammatory polarized response to a Toll-like receptor 7 (TLR7) agonist.

Publications

Nuclear factor kappaB (NF-kappaB) activation primes cells to a pro-inflammatory polarized response to a Toll-like receptor 7 (TLR7) agonist.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure