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

BackgroundThe 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.Methodology/Principal FindingsBy 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 and Significancean 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.

Glutathione Depletion Is Linked with Th2 Polarization in Mice with a Retrovirus-Induced Immunodeficiency Syndrome, Murine AIDS: Role of Proglutathione Molecules as Immunotherapeutics

“…Determination of the amount of thiol in peritoneal macrophages was performed as previously described (47). The removed macrophages were immediately lysed with 100 l of lysis buffer (0.1% Triton X-100, 0.1 M Na 2 HPO 4 , 5 mM EDTA, pH 7.5).…”

“…Peritoneal exudate cells of B6 mice were obtained by peritoneal lavage as previously described (47). Macrophages were incubated for 24 h in 35-mm-diameter dishes with RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (EuroClone), 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin (Sigma) and then processed differently according to the determinations that were to be made.…”

“…The IL-12 p40 mRNA in murine peritoneal macrophages infected in vitro was quantified as previously described (47). Briefly, total RNA was isolated from about 2.5 ϫ 10 5 macrophages using an RNeasy minikit (Qiagen), and 0.8 g was used for cDNA synthesis (RT 2 first-strand cDNA synthesis kit; Qiagen) according to the manufacturer's instructions.…”

Interleukin-27 inhibits foam cell formation by promoting macrophage ABCA1 expression through JAK2/STAT3 pathway

“…Recent studies have shown that IL-27 is a member of IL-12 family cytokines that is composed of IL-27p28 and Epstein-Barr virus induced gene 3 (EBI3) [1,[5][6][7]. The IL-27 receptor that consists of WSX-1 and gp130 and is required for all established signaling pathways initiated IL-27 [2,5].…”

Analise de componentes do metabolismo lipídico na resposta inflamatória induzida por ligadura e punção cecal em ratos diabéticos

“…As células T helper tipo 1 (Th1), secretam citocinas pró-inflamatórias, como por exemplo, TNF-α e IL-1β, e ao contrário, as células T helper tipo 2 (Th2) secretam interleucinas anti-inflamatórias (IL-4, IL-10), promovendo com isso um feedback positivo. 39,45,46,47,48 Outro fator importante é a fagocitose de células apoptóticas por macrófagos e células dendríticas e com isto, estas células estimulam a tolerância imune ao induzir a liberação de citocinas anti-inflamatórias, incluindo a IL-10 e TGF-β, o qual suprimi a secreção de citocinas pró-inflamatórias. Esta potente ligação entre a liberação de IL-10 pela apoptose das células e a imunossupressão na sepse é preditiva de um desfecho fatal em pacientes críticos.…”