Transcription profiling of responses to TNF-alpha, IL-1beta, peptidoglycan or lipopolysaccharide (TLR2/4 agonists) evaluated at 4 hrs in peripheral blood monocytes (PBM) from the patient bearing the IRAK4 Q293X mutation vs PBM from 5 healthy individuals
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Naran Hirani
PROVIDER: E-HGMP-1 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:IL-1R associated kinase-4 (IRAK4) is a key enzyme required for activation of the common Toll-like Receptor (TLR) signaling cascade, which results in the transcription of inflammatory and immunity genes. IRAK-4 deficiency has recently been described as a rare form of innate immunodeficiency. Patients present with pyogenic bacterial infections and bacteraemia, particularly with Gram+ Streptococcus pneumoniae, and isolated PBMC from these patients fail to produce inflammatory cytokines in response to TLR agonists. We embarked on this study for several purposes: (1) to identify defective gene response resulting from IRAK4-deficiency that are responsible for the patients' susceptibility to infection by particular bacteria (2) to identify genetic responses that confer relatively normal immunity in these patients despite having a defect in such a critical component of the innate immune system (3) to gain understanding of the transcriptional regulation of inflammatory genes (4) to gain insight into TLR signal transduction pathways, in particular, the role of IRAK4 in the TLR2 and TLR4 pathways initiated by Gram+ and Gram- bacterial components respectively. Transcriptional responses to TNF-alpha, IL-1beta, peptidoglycan or lipopolysaccharide (TLR2/4 agonists) were evaluated at 4 hrs in peripheral blood monocytes (PBM) from the patient bearing the IRAK4 Q293X mutation compared to PBM from 5 healthy individuals.
2010-05-19 | E-FPMI-7 | biostudies-arrayexpress
Project description:BackgroundToll like receptor 2 (TLR2) signaling can regulate the pathogenesis of otitis media (OM). However, the precise role of TLR2 signaling in OM has not been clarified due to the lack of an optimal animal model. Peptidoglycan-polysaccharide (PGPS) of the bacterial cell wall can induce inflammation by activating the TLR2 signaling. This study aimed at examining the pathogenic characteristics of OM induced by PGPS in Tlr2(-/-) mice, and the potential therapeutic effect of sodium aescinate (SA) in this model.MethodsWild-type (WT) and Tlr2(-/-) mice were inoculated with streptococcal PGPS into their middle ears (MEs) and treated intravenously with vehicle or SA daily beginning at 3days prior to PGPS for 6 consecutive days. The pathologic changes of individual mice were evaluated longitudinally.ResultsIn comparison with WT mice, Tlr2(-/-) mice were susceptible to PGPS-induced OM. Tlr2(-/-) mice displayed greater hearing loss, tympanic membrane damage, ME mucosal thickening, longer inflammation state, cilia and goblet cell loss. SA-treatment decreased neutrophil infiltration, modulated TLR2-related gene expression and improved ciliary organization.ConclusionsPGPS induced a relatively stable OM in Tlr2(-/-) mice, providing a new model for OM research. Treatment with SA mitigated the pathogenic damage in the ME and may be valuable for intervention of OM.
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Project description:Melanoma is the most lethal form of skin cancer. Clinical efforts to combat melanoma include immune therapies whose benefit depends on antitumor T-cells, to target and to clear melanoma. However, most tumors lack significant immune infiltration prior to therapy, and some immune therapies are hindered by a persistent lack of immune-cell infiltration. Chemokines can promote T-cell migration into tumors; therefore, agents that induce T-cell attracting chemokines in the tumor microenvironment could potentially improve the clinical activity of current immune therapies for melanoma. CXCL10 has been implicated as a critical chemokine supporting T-cell infiltration into the tumor microenvironment. Here we report that combination treatment of human melanoma cell lines with Toll-like receptor (TLR) 2/6 agonists MALP-2 or FSL-1 +IFNlambda synergize to induce production of immune-cell attracting chemokines CCL3 and CXCL10 by melanoma cells. We find that TLR2 and TLR6 are widely expressed on human melanoma cells, and that stimulation of fresh patient melanoma specimens with TLR2/6 agonists+IFNlambda induces CXCL10 production from melanoma cells, endothelial and immune-cells. Furthermore, ex vivo migration assays demonstrate that stimulation of melanoma cells with TLR2/6 agonists+IFNlambda increases CD4+ and CD8+ T-cell migration toward melanoma. Collectively, these data identify a novel synergy of TLR2/6 agonists+IFNlambda for inducing CXCL10 production by melanoma cells and suggest that intralesional administration of TLR2/6 agonists+IFNlambda may improve immune signatures in melanoma metastases and have value in combination with other immune therapies, by supporting better T-cell migration to melanoma.
2015-08-13 | GSE49632 | GEO
Project description:Clinical approaches to treat advanced melanoma include immune therapies, whose benefits depend on tumor-reactive T-cells to infiltrate metastases. However, most tumors lack significant immune infiltration prior to therapy, and some immune therapies are hindered by a persistent lack of immune cell infiltration. CXCL10 has been implicated as a critical chemokine supporting T-cell migration into tumors; thus agents that induce CXCL10 in tumors may improve patient responses to systemic immune therapy. We find that melanoma cells treated with TLR2/6 agonists (MALP-2 or FSL-1) and interferon-gamma (IFNgamma) upregulate CXCL10 production, when compared to IFNgamma treatment alone or no treatment. Gene profiling of melanoma cells lines treated with TLR2/6 agonists and IFNgamma demonstrate that a selective profile of genes are induced which may be favorable for promoting immune cell infiltration of tumors. TLR2 and TLR6 are widely expressed on human melanoma cells, and treatment of melanoma cells with TLR2/6 agonists and IFNgamma does not hinder melanoma cell apoptosis or promote proliferation. Furthermore, melanoma cells from surgically resected patient tumors upregulate CXCL10 production after treatment with TLR2/6 agonists and IFNgamma when compared to treatment with either agent alone. Collectively, these data identify TLR2/6 agonists and IFNgamma as a novel target for promoting CXCL10 production directly from melanoma cells.
2015-11-22 | GSE64506 | GEO
Project description:The presence of a reservoir of latently infected cells in HIV-infected patients is a major barrier towards finding a cure. One active cure strategy is to find latency-reversing agents that induce viral reactivation, thus leading to immune cell recognition and elimination of latently infected cells, known as the shock-and-kill strategy. Therefore, the identification of molecules that reactivate latent HIV and increase immune activation has the potential to further these strategies into the clinic. Here, we characterized synthetic molecules composed of a TLR2 and a TLR7 agonist (dual TLR2/7 agonists) as latency-reversing agents and compared their activity with that of the TLR2 agonist Pam2CSK4 and the TLR7 agonist GS-9620. We found that these dual TLR2/7 agonists reactivate latency by 2 complementary mechanisms. The TLR2 component reactivates HIV by inducing NF-κB activation in memory CD4+ T cells, while the TLR7 component induces the secretion of TNF-α by monocytes and plasmacytoid dendritic cells, promoting viral reactivation in CD4+ T cells. Furthermore, the TLR2 component induces the secretion of IL-22, which promotes an antiviral state and blocks HIV infection in CD4+ T cells. Our study provides insight into the use of these agonists as a multipronged approach targeting eradication of latent HIV.
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Project description:Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1beta and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1beta activity, involving the upregulation of both IL-1beta and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1beta was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-kappaB sites, whereas the cathelicidin promoter had three VDREs and no NF-kappaB sites. Transfection of NF-kappaB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1beta in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.
| S-EPMC2686169 | biostudies-literature
Project description:Toll-like receptor (TLR) agonists activate both the innate and the adaptive immune systems. These TLR agonists have been exploited as potent vaccine adjuvants and antitumor agents. We describe the identification and characterization of a small molecule, N-methyl-4-nitro-2-(4-(4-(trifluoromethyl)phenyl)-1 H-imidazol-1-yl)aniline (CU-T12-9), that directly targets TLR1/2 to initiate downstream signaling. CU-T12-9 specifically induces TLR1/2 activation, which can be blocked by either the anti-hTLR1 or the anti-hTLR2 antibody, but not the anti-hTLR6 antibody. Using a variety of different biophysical assays, we have demonstrated the binding mode of CU-T12-9. By binding to both TLR1 and TLR2, CU-T12-9 facilitates the TLR1/2 heterodimeric complex formation, which in turn activates the downstream signaling. Fluorescence anisotropy assays revealed competitive binding to the TLR1/2 complex between CU-T12-9 and Pam3CSK4 with a half-maximal inhibitory concentration (IC50) of 54.4 nM. Finally, we showed that CU-T12-9 signals through nuclear factor κB (NF-κB) and invokes an elevation of the downstream effectors tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and inducible nitric oxide synthase (iNOS). Thus, our studies not only provide compelling new insights into the regulation of TLR1/2 signaling transduction but also may facilitate future therapeutic developments.
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Project description:Clinical approaches to treat advanced melanoma include immune therapies, whose benefits depend on tumor-reactive T-cells to infiltrate metastases. However, most tumors lack significant immune infiltration prior to therapy, and some immune therapies are hindered by a persistent lack of immune cell infiltration. CXCL10 has been implicated as a critical chemokine supporting T-cell migration into tumors; thus agents that induce CXCL10 in tumors may improve patient responses to systemic immune therapy. We find that melanoma cells treated with TLR2/6 agonists (MALP-2 or FSL-1) and interferon-gamma (IFNgamma) upregulate CXCL10 production, when compared to IFNgamma treatment alone or no treatment. Gene profiling of melanoma cells lines treated with TLR2/6 agonists and IFNgamma demonstrate that a selective profile of genes are induced which may be favorable for promoting immune cell infiltration of tumors. TLR2 and TLR6 are widely expressed on human melanoma cells, and treatment of melanoma cells with TLR2/6 agonists and IFNgamma does not hinder melanoma cell apoptosis or promote proliferation. Furthermore, melanoma cells from surgically resected patient tumors upregulate CXCL10 production after treatment with TLR2/6 agonists and IFNgamma when compared to treatment with either agent alone. Collectively, these data identify TLR2/6 agonists and IFNgamma as a novel target for promoting CXCL10 production directly from melanoma cells. Samples from four human melanoma cell lines, VMM1 (n=6), DM13 (n=6), DM93 (n=6) and VMM39 (n=6), were treated with media alone, MALP-2 (TLR2/6 agonist), FSL-1 (TLR2/6 agonist), IFNgamma alone, MALP-2 and IFNgamma, or FSL-1 and IFNgamma.
2015-11-22 | E-GEOD-64506 | biostudies-arrayexpress
Project description:IL-1beta produced by phagocytes is important for protection against the mucosal pathogen Staphylococcus aureus. Processing and maturation of this cytokine requires activation of the multiprotein inflammasome complex. We observed that the bacterial cell wall component peptidoglycan (PGN) must be particulate and internalized via phagocytosis to activate NLRP3 inflammasomes and IL-1beta secretion. In the context of S. aureus infection of macrophages, we find that phagocytosis and lysozyme-based bacterial cell wall degradation are necessary to induce IL-1beta secretion. Further, an S. aureus enzyme, PGN O-acetyltransferase A, previously demonstrated to make cell wall PGN resistant to lysozyme, strongly suppresses inflammasome activation and inflammation in vitro and in vivo. These observations demonstrate that phagocytosis and lysozyme-based cell wall degradation of S. aureus are functionally coupled to inflammasome activation and IL-1beta secretion and illustrate a case whereby a bacterium specifically subverts IL-1beta secretion through chemical modification of its cell wall PGN.
| S-EPMC2818986 | biostudies-literature
Project description:Background: Infection/inflammation is an important causal factor in spontaneous preterm birth (sPTB). Most mechanistic studies have concentrated on the role of bacteria, with limited focus on the role of viruses in sPTB. Murine studies support a potential multi-pathogen aetiology in which a double or sequential hit of both viral and bacterial pathogens leads to a higher risk preterm labour. This study aimed to determine the effect of viral priming on bacterial induced inflammation in human in vitro models of ascending and haematogenous infection. Methods: Vaginal epithelial cells, and primary amnion epithelial cells and myocytes were used to represent cell targets of ascending infection while interactions between peripheral blood mononuclear cells (PBMCs) and placental explants were used to model systemic infection. To model the effect of viral priming upon the subsequent response to bacterial stimuli, each cell type was stimulated first with a TLR3 viral agonist, and then with either a TLR2 or TLR2/6 agonist, and responses compared to those of each agonist alone. Immunoblotting was used to detect cellular NF-κB, AP-1, and IRF-3 activation. Cellular TLR3, TLR2, and TLR6 mRNA was quantified by RT-qPCR. Immunoassays were used to measure supernatant cytokine, chemokine and PGE2 concentrations. Results: TLR3 ("viral") priming prior to TLR2/6 agonist ("bacterial") exposure augmented the pro-inflammatory, pro-labour response in VECs, AECs, myocytes and PBMCs when compared to the effects of agonists alone. In contrast, enhanced anti-inflammatory cytokine production (IL-10) was observed in placental explants. Culturing placental explants in conditioned media derived from PBMCs primed with a TLR3 agonist enhanced TLR2/6 agonist stimulated production of IL-6 and IL-8, suggesting a differential response by the placenta to systemic inflammation compared to direct infection as a result of haematogenous spread. TLR3 agonism generally caused increased mRNA expression of TLR3 and TLR2 but not TLR6. Conclusion: This study provides human in vitro evidence that viral infection may increase the susceptibility of women to bacterial-induced sPTB. Improved understanding of interactions between viral and bacterial components of the maternal microbiome and host immune response may offer new therapeutic options, such as antivirals for the prevention of PTB.
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