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.
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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biomolecular Sciences University of Urbino Carlo Bo, Urbino (PU), Italy. alessandra.fraternale@uniurb.it
authors.forename: Alessandra
authors.initials: A
authors.lastname: Fraternale
authors.forename: Rita
authors.initials: R
authors.lastname: Crinelli
authors.forename: Anna
authors.initials: A
authors.lastname: Casabianca
authors.forename: Maria Filomena
authors.initials: MF
authors.lastname: Paoletti
authors.forename: Chiara
authors.initials: C
authors.lastname: Orlandi
authors.forename: Elisa
authors.initials: E
authors.lastname: Carloni
authors.forename: Michaël
authors.initials: M
authors.lastname: Smietana
authors.forename: Anna Teresa
authors.initials: AT
authors.lastname: Palamara
authors.forename: Mauro
authors.initials: M
authors.lastname: Magnani
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D003543:Cysteamine / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D005260:Female; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D050856:Interferon Regulatory Factor-1 / Q000378:metabolism*; D053762:Interleukin-12 Subunit p40 / Q000096:biosynthesis* / Q000235:genetics; D064094:Interleukin-27 / Q000096:biosynthesis* / Q000235:genetics; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D050794:STAT1 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000737:chemistry
chemical_list: C436346:I-152 compound; D050856:Interferon Regulatory Factor-1; D053762:Interleukin-12 Subunit p40; D064094:Interleukin-27; D016328:NF-kappa B; D050794:STAT1 Transcription Factor; D013438:Sulfhydryl Compounds; C562006:glutathione-C4; D003543:Cysteamine; D005978:Glutathione; D000111:Acetylcysteine
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