Molecules Altering the Intracellular Thiol Content Modulate NF-kB and STAT-1/IRF-1 Signalling Pathways and IL-12 p40 and IL-27 p28 Production in Murine Macrophages
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{"title"=>"Molecules Altering the Intracellular Thiol Content Modulate NF-kB and STAT-1/IRF-1 Signalling Pathways and IL-12 p40 and IL-27 p28 Production in Murine Macrophages", "type"=>"journal", "authors"=>[{"first_name"=>"Alessandra", "last_name"=>"Fraternale", "scopus_author_id"=>"6701752044"}, {"first_name"=>"Rita", "last_name"=>"Crinelli", "scopus_author_id"=>"6701568072"}, {"first_name"=>"Anna", "last_name"=>"Casabianca", "scopus_author_id"=>"7003567684"}, {"first_name"=>"Maria Filomena", "last_name"=>"Paoletti", "scopus_author_id"=>"14012893000"}, {"first_name"=>"Chiara", "last_name"=>"Orlandi", "scopus_author_id"=>"7007139850"}, {"first_name"=>"Elisa", "last_name"=>"Carloni", "scopus_author_id"=>"8873143500"}, {"first_name"=>"Michaël", "last_name"=>"Smietana", "scopus_author_id"=>"23095941500"}, {"first_name"=>"Anna Teresa", "last_name"=>"Palamara", "scopus_author_id"=>"7003579999"}, {"first_name"=>"Mauro", "last_name"=>"Magnani", "scopus_author_id"=>"7103023340"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84874881483", "doi"=>"10.1371/journal.pone.0057866", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"23536773", "issn"=>"19326203", "scopus"=>"2-s2.0-84874881483", "pui"=>"368506799"}, "id"=>"36cf6d13-8ec8-3989-b2f9-6b36e1514302", "abstract"=>"BACKGROUND: The aim of this study was to investigate the molecular mechanisms involved in the production of Th1 cytokines, namely IL-12 and IL-27, when the intra-macrophage redox state was altered by different chemical entities such as GSH-C4, which is reduced glutathione carrying an aliphatic chain, or I-152, a pro-drug of N-acetyl-cysteine (NAC) and beta-mercaptoethylamine. We had already demonstrated that GSH-C4 and I-152 could shift the immune response towards Th1 in Ovalbumin-immunized mice as well as enhance Th1 response in HIV-1 Tat-immunized mice.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: By a new high performance liquid chromatography method, we found that 20 mM GSH-C4 provided a number of thiol species in the form of GSH, while 20 mM I-152 decreased GSH and increased the thiols in the form of NAC and I-152. Under these experimental conditions, GSH-C4 and I-152 enhanced and suppressed respectively the mRNA expression levels of IL-12 p40 induced by LPS/IFN-γ as assessed by Real-Time PCR. The protein production of IL-12 p40 was increased by GSH-C4 and decreased by I-152 as determined by Enzyme-linked immunosorbent assay. Western immunoblot and electrophoretic mobility shift assays revealed that Nuclear Factor -kB (NF-kB) activation was inhibited by I-152 and prolonged by GSH-C4. Twenty mM I-152 stimulated IL-27 p28 gene expression and sustained Signal Transducer and Activator of Transcription (STAT)-mediated interferon regulator factor 1 (IRF-1) de novo synthesis. By contrast, 20 mM GSH-C4 did not exert any effect on IL-27 p28 gene expression.\\n\\nCONCLUSIONS AND SIGNIFICANCE: an increase in the intra-macrophage redox state by GSH-C4 and I-152 enhances Th1 cytokine production although the chemical structure and the intra-cellular metabolism influence differently signalling pathways involved in IL-27 or IL-12 production. GSH-C4 and I-152 may be used as Th1 immunomodulators in some pathologies and in ageing where GSH depletion may contribute to the Th1/Th2 imbalance, and in new immunization strategies.", "link"=>"http://www.mendeley.com/research/molecules-altering-intracellular-thiol-content-modulate-nfkb-stat1irf1-signalling-pathways-il12-p40", "reader_count"=>38, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>5, "Researcher"=>6, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Student > Master"=>4, "Student > Bachelor"=>6, "Professor"=>3}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>5, "Researcher"=>6, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>7, "Student > Postgraduate"=>1, "Student > Master"=>4, "Student > Bachelor"=>6, "Professor"=>3}, "reader_count_by_subject_area"=>{"Unspecified"=>5, "Biochemistry, Genetics and Molecular Biology"=>7, "Agricultural and Biological Sciences"=>14, "Medicine and Dentistry"=>5, "Neuroscience"=>1, "Sports and Recreations"=>1, "Chemistry"=>2, "Immunology and Microbiology"=>3}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>5}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>2}, "Sports and Recreations"=>{"Sports and Recreations"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>14}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}, "Unspecified"=>{"Unspecified"=>5}}, "reader_count_by_country"=>{"United States"=>1, "Japan"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/984383"], "description"=>"<p>(A) extracts of untreated murine peritoneal macropahges; (B) extracts of murine peritoneal macrophages treated with 20 mM GSH-C4 for 2 hours; (C) extracts of macrophages treated with 20 mM I-152 for 2 hours. Murine macrophages were obtained and processed as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057866#s4\" target=\"_blank\">Materials and Methods</a> section. Experimental details and chromatographic conditions are described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057866#s4\" target=\"_blank\">Materials and Methods</a> section.</p>", "links"=>[], "tags"=>["dtnb", "r-tnb", "reversed-phase"], "article_id"=>650029, "categories"=>["Immunology"], "users"=>["Alessandra Fraternale", "Rita Crinelli", "Anna Casabianca", "Maria Filomena Paoletti", "Chiara Orlandi", "Elisa Carloni", "Michaël Smietana", "Anna Teresa Palamara", "Mauro Magnani"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057866.g001", "stats"=>{"downloads"=>5, "page_views"=>50, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Separation_of_DTNB_and_R_TNB_by_reversed_phase_HPLC_/650029", "title"=>"Separation of DTNB and R-TNB by reversed-phase HPLC.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-12 09:23:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/984420"], "description"=>"<p>(A): NF-kB nuclear translocation and DNA binding activity was assessed by Electrophoretic Mobility Shift Assay (EMSA) of nuclear extracts obtained from macrophages untreated (Control) or pre-treated with either 20 mM GSH-C4 or 20 mM I-152 for 2 hours and then stimulated with LPS and IFN-γ after molecule removal, for different times as indicated. (B): IkB-α degradation and re-synthesis as monitored by Immunoblotting analysis of IkB-α protein levels in the cytosolic fraction of macrophages treated as above. Cytosolic proteins (15 µgs) were separated by SDS-PAGE onto 10% acrylamide gels, transferred to nitrocellulose membrane and immunoblotted with an anti-IkB-α antibody. Actin was stained as a loading control. IkB-α densitometric values, normalized to actin, are reported immediately below the IkB-α blot.</p>", "links"=>[], "tags"=>["activation", "murine", "peritoneal"], "article_id"=>650055, "categories"=>["Immunology"], "users"=>["Alessandra Fraternale", "Rita Crinelli", "Anna Casabianca", "Maria Filomena Paoletti", "Chiara Orlandi", "Elisa Carloni", "Michaël Smietana", "Anna Teresa Palamara", "Mauro Magnani"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057866.g005", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_NF_kB_activation_in_murine_peritoneal_macrophages_/650055", "title"=>"NF-kB activation in murine peritoneal macrophages.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-12 09:26:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/984458"], "description"=>"<p>The binding of ligand (IFN- γ) to the receptor results in receptor oligomerization and subsequent activation of receptor-associated JAK tyrosine kinases (JAK1 and JAK2). Activated JAKs phosphorylate specific tyrosine residues in the cytoplasmic domain of the receptor which in turn serves as the docking sites for the cytoplasmic transcription factors known as STAT-1. STAT-1 are therefore recruited to the phosphorylated receptor and subsequently phosphorylated by JAKs. The phosphorylated STAT-1 then dimerize, leave the receptor, and translocate to the nucleus where they activate the transcription of several genes, such as IRF-1. I-152, increasing intracellular thiol content, may positively influence JAK2’s catalytic activity which is known to be directly regulated by the redox state of the cell. I-152 may inhibit protein tyrosine phosphatases by glutathionylation. Y: tyrosine; GAS: IFN-γ activation site.</p>", "links"=>[], "tags"=>["activation", "transcription"], "article_id"=>650087, "categories"=>["Immunology"], "users"=>["Alessandra Fraternale", "Rita Crinelli", "Anna Casabianca", "Maria Filomena Paoletti", "Chiara Orlandi", "Elisa Carloni", "Michaël Smietana", "Anna Teresa Palamara", "Mauro Magnani"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057866.g008", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_STAT_mediated_activation_of_gene_transcription_by_IFN_947_/650087", "title"=>"STAT-mediated activation of gene transcription by IFN-γ.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-12 09:29:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/984415"], "description"=>"<p>The peritoneal macrophages were obtained as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057866#s4\" target=\"_blank\">Materials and Methods</a> section. They were left untreated or treated with either 20 mM GSH-C4 or 20 mM I-152 for 2 hours and further stimulated with LPS and IFN-γ for 3 and 6 hours as indicated in the figure. Total RNA extraction and reverse transcription as well as IL-12 p40 and IL-27 p28 mRNA quantification were performed as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057866#s4\" target=\"_blank\">Materials and Methods</a> section. The results are the mean±S.D. of 4 values. *p<0.05 vs. Control.</p>", "links"=>[], "tags"=>["il-12", "p40", "il-27", "p28", "mrna", "murine", "peritoneal"], "article_id"=>650050, "categories"=>["Immunology"], "users"=>["Alessandra Fraternale", "Rita Crinelli", "Anna Casabianca", "Maria Filomena Paoletti", "Chiara Orlandi", "Elisa Carloni", "Michaël Smietana", "Anna Teresa Palamara", "Mauro Magnani"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057866.g004", "stats"=>{"downloads"=>1, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_IL_12_p40_and_IL_27_p28_mRNA_in_murine_peritoneal_macrophages_/650050", "title"=>"Expression of IL-12 p40 and IL-27 p28 mRNA in murine peritoneal macrophages.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-12 09:25:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/984409"], "description"=>"<p>The peritoneal macrophages were obtained as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057866#s4\" target=\"_blank\">Materials and Methods</a> section. They were left untreated or treated with either 20 mM GSH-C4 or 20 mM I-152 for 2 hours and further stimulated with LPS and IFN-γ for 24 hours. The IL-12 p40 (A) and IL-27 p28 (B) levels secreted in the culture supernatants were measured by ELISA. Results are expressed as the percentage respect to untreated stimulated macrophages (Control) and are the mean±S.D. of al least 5 different experiments. *p<0.05 vs. Control.</p>", "links"=>[], "tags"=>["p40", "il-27", "p28", "induction", "murine", "peritoneal"], "article_id"=>650044, "categories"=>["Immunology"], "users"=>["Alessandra Fraternale", "Rita Crinelli", "Anna Casabianca", "Maria Filomena Paoletti", "Chiara Orlandi", "Elisa Carloni", "Michaël Smietana", "Anna Teresa Palamara", "Mauro Magnani"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057866.g003", "stats"=>{"downloads"=>0, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_IL_12_p40_and_IL_27_p28_induction_in_murine_peritoneal_macrophages_/650044", "title"=>"IL-12 p40 and IL-27 p28 induction in murine peritoneal macrophages.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-12 09:25:26"}
  • {"files"=>["https://ndownloader.figshare.com/files/984449"], "description"=>"<p>The binding of ligand to a receptor leads to the recruitment and activation of an IKK complex which phosphorylates IkB-α leading to degradation by the proteasome. NF-kB then translocates to the nucleus to activate target genes regulated by kB sites, such as that of IL-12 p40. I-152, reducing intracellular GSH, may inhibit the enzymatic activity of IKK complex by glutathionylation; as a consequence IkB-α degradation and NF-kB nuclear translocation are inhibited as well. GSH-C4, increasing intracellular GSH, may maintain in a reduced state the cysteine located in the DNA-binding loop so favouring and prolonging NF-kB DNA binding. SCF: SCF complex, Skp, Cullin, F-box containing complex: multi-protein E3 ubiquitin ligase complex.</p>", "links"=>[], "tags"=>["canonical", "nf-kb", "signalling"], "article_id"=>650082, "categories"=>["Immunology"], "users"=>["Alessandra Fraternale", "Rita Crinelli", "Anna Casabianca", "Maria Filomena Paoletti", "Chiara Orlandi", "Elisa Carloni", "Michaël Smietana", "Anna Teresa Palamara", "Mauro Magnani"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057866.g007", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_canonical_NF_kB_signalling_pathway_/650082", "title"=>"The canonical NF-kB signalling pathway.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-12 09:28:15"}
  • {"files"=>["https://ndownloader.figshare.com/files/984404"], "description"=>"<p>The analysis was performed in murine peritoneal macrophages (left) and RAW 264.7 cells (right) treated with different concentrations of GSH-C4 (A) and I-152 (B). The peritoneal macrophages were obtained and processed as described in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057866#s4\" target=\"_blank\">Materials and Methods</a> section. Values represent the mean±S.D. of 3 experiments. *p<0.05 vs. Control.</p>", "links"=>[], "tags"=>["thiol"], "article_id"=>650040, "categories"=>["Immunology"], "users"=>["Alessandra Fraternale", "Rita Crinelli", "Anna Casabianca", "Maria Filomena Paoletti", "Chiara Orlandi", "Elisa Carloni", "Michaël Smietana", "Anna Teresa Palamara", "Mauro Magnani"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057866.g002", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantification_of_thiol_species_by_HPLC_/650040", "title"=>"Quantification of thiol species by HPLC.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-12 09:25:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/984434"], "description"=>"<p>Immunoblotting analysis of phospho Tyr701 STAT-1 and IRF-1 levels in cells untreated (lanes 1–5) or pre-treated with 20 mM I-152 for 2 hours (lanes 6–9) and then stimulated with LPS and IFN-γ after molecule removal, for different times as indicated (lanes 2–5 and 6–9). Whole protein extracts (20 µgs) were resolved onto 8% acrylamide gels, transferred to a nitrocellulose membrane and stained with anti IRF-1 and anti phospho Tyr701 STAT-1 specific antibodies (pSTAT-1). NF-kB p65 (RelA) was stained as a loading control. pSTAT-1 and IRF-1 densitometric values, normalized to p65, are reported immediately below the corresponding blot.</p>", "links"=>[], "tags"=>["phosphorylation", "irf-1"], "article_id"=>650067, "categories"=>["Immunology"], "users"=>["Alessandra Fraternale", "Rita Crinelli", "Anna Casabianca", "Maria Filomena Paoletti", "Chiara Orlandi", "Elisa Carloni", "Michaël Smietana", "Anna Teresa Palamara", "Mauro Magnani"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0057866.g006", "stats"=>{"downloads"=>3, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_STAT_1_phosphorylation_and_IRF_1_levels_in_RAW_264_7_cells_/650067", "title"=>"STAT-1 phosphorylation and IRF-1 levels in RAW 264.7 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-12 09:27:16"}

PMC Usage Stats | Further Information

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Relative Metric

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