GSH levels in APC play a central role in determining whether Th1 or Th2 cytokine response patterns predominate in immune responses. GSH depletion leads to a shift away from the typical Th1 cytokine profile toward Th2 response patterns 
Th1 cytokine production is positively or negatively polarized by augmenting or depleting, respectively, the intracellular content of GSH in APC 
. In previous papers we reported that administration of GSH-C4 and I-152 could shift the immune response towards Th1 both in mice immunized with Ova in which a prevalent Th2 response is developed and in mice immunized with HIV-1 Tat in which Th1 response is predominant 
. The pro-GSH molecules used in these studies were an aliphatic derivative of GSH, GSH-C4 
and a NAC/MEA conjugate compound, I-152 
both designed to facilitate cell entry. In this paper, we investigate the molecular mechanisms whereby GSH-C4 and I-152 can influence the production of Th1 cytokines; in particular, we studied the LPS/IFN-γ-induced expression of both IL-12 p40 and IL-27 p28 in macrophages pre-treated with the molecules. The results obtained show that the two chemical entities exert different effects on the production of the above cytokines, probably due to their different intracellular metabolism. The developed HPLC method used in this paper allowed the distinction of the different thiol species generated intracellularly by GSH-C4 and I-152, while the spectrophotometric methods previously used 
had provided total thiol content 
. This is a very simple method allowing an accurate study of the metabolism of the molecules of interest containing thiol groups. GSH-C4 provided a number of thiol species in the form of GSH, while high concentrations of I-152 reduced the intracellular GSH content and provided a high concentration of NAC and I-152. Although both molecules increased thiol content about 2–3 fold over the basal level, they displayed a very different effect on immune signalling pathway responsible for IL 12 p40 and IL-27 p28 expression, i.e. the NF-kB and the STAT-1/IRF-1 signalling pathways, suggesting that several investigations based on the sole determination of intracellular thiols are not adequate to understand the role of the cell redox state on Th1/Th2 polarization. It is known that the canonical signalling transduction pathway of NF-kB activation, which involves IkB phosphorylation and degradation, is sensitive to oxidant/antioxidant homeostasis, especially to the thiol/disulphide balance 
. Data presented demonstrate that the thiol increase in the form of GSH, induced by GSH-C4 treatment, does not alter NF-kB activation in response to LPS/IFN-γ; in fact, IkB-α degradation and NF-kB nuclear translocation occurred to a similar extent and with the same kinetic as in Control cells. GSH-C4 rather interferes with the regulatory mechanism(s) that determine NF-kB inactivation. In particular, prolonged NF-kB activation was observed in GSH-C4-treated cells compared to stimulated control cells, an observation which could explain the accumulation of IL-12 p40 mRNA and protein in GSH-C4-treated cells. The best known mechanism for termination of NF-kB response involves re-synthesis of IkB proteins induced by activated NF-kB. However, IkB-α protein was re-synthesized in GSH-C4-treated cells to the same extent and with the same kinetic observed in untreated control macrophages (). In recent years, additional inhibitory mechanisms that function later in the pathway and directly affect the active DNA-bound NF-kB have been identified. One of these mechanisms involves oxidation of redox-sensitive cysteine located in the DNA-binding loop; a cysteine residue in a well-conserved motif in all Rel/kB proteins is critical and must be maintained in a reduced state to allow DNA binding 
. Thus, it could be speculated that GSH-C4, by increasing the intracellular GSH content, may control NF-kB DNA binding activity favouring and prolonging its association with IL-12 p40 promoter sequence.
Contrarily to GSH-C4, I-152 inhibited IkB-α degradation and as a consequence NF-kB nuclear translocation. There is increasing interest in new possible mechanisms in the regulation of NF-kB activation, and data already reported in literature show that multiple points of redox regulation exist, including S-glutathionylation of the IkB kinase IKK-β and the S-glutathionylation of IkB-α which has functional consequences on catalytic activity and protein–protein interactions 
. In agreement with these findings, it has been shown that chemicals able to decrease intracellular GSH content inhibit IkB-α degradation 
. Thus, it could be hypothesized that I-152, by decreasing intracellular GSH, may induce IKK-β and/or IkB-α glutathionylation, resulting in the inhibition of IkB-α degradation and consequently NF-kB nuclear entry (). On the other hand, I-152 determines an increase in thiol content in the form of NAC, a molecule which is known to interfere with IkB degradation and NF-kB activation 
The canonical NF-kB signalling pathway.
High concentrations of I-152 significantly increased IL-27 p28 production while GSH-C4 had no significant effects. The study of IRF-1 expression revealed that I-152 maintains the STAT-1/IRF-1 signalling pathway active for periods longer than controls. The persistence of STAT-1/IRF-1 signalling may explain the higher levels of IL-27 p28 found in macrophages treated with I-152. A possible mechanism by which I-152 could prolong activation of the STAT-1/IRF-1 signalling involves redox regulation of JAK2 through thiols provided by I-152 (in the form of NAC and I-152), since cysteine residues within JAK2 catalytic domain are crucial for enzymatic activity which is inhibited by oxidation and restored via reduction to the thiol state 
(). Another mechanism may involve inhibition of tyrosine phosphatases since oxidation/reduction of conserved cysteinyl residues in the catalytic domains is an important factor in the regulation of their activity and a variety of redox-active metabolites can oxidatively inactivate protein tyrosine phosphatases with potentially profound implications for signal transduction 
. Glutathionylation, as suggested above for inhibition of IkB degradation, can be proposed as possible mechanism for inhibition of tyrosine phosphatase by I-152. Having established that GSH-C4 and I-152 differentially modulate signalling pathway involved in IL-12 p40 and IL-27 p28 expression, further studies will be necessary to establish which step(s) of the NF-kB and STAT-1/IRF-1 signalling cascades may be selectively affected by GSH-C4 or I-152 treatment. It would also be interesting to study the contribution of GSH-C4 and I-152 to functional activity and expression of TLR and IFNGR as well as their influence on the intracellular molecules identified as essential for TLR-mediated signalling. In particular, by altering the intracellular thiol content, these molecules may influence the post-translational modification required for TLR function 
STAT-mediated activation of gene transcription by IFN-γ.
On the whole, these results suggest that it is possible to use different molecules to change the redox state of macrophages with the aim to induce Th1 cytokine production. In vivo we previously found that both GSH-C4 and I-152 increased circulating IL-12 levels and shifted the immune response towards Th1 in Ova- and Tat-immunized mice. The results reported here explain the possible mechanism through which GSH-C4 leads to an increase in IL-12 production while I-152 seems to have different effects in vivo and in vitro depending on the intracellular redox status. In fact, in animals immunized by an antigen, i.e. Ova, the content of GSH is reduced in APC because employed in the processing of antigens with disulfide bonds 
. Thus, it could be hypothesized that I-152 is metabolized to replenish/increase GSH levels favoring NF-kB activation and IL-12 production. On the other hand, in vitro experiments here reported show that I-152 is able to enhance the production of the Th1 cytokine IL-27, favoring polarization towards Th1 profile. These results also suggest that increased IL-12 production is not the common effect of molecules that alter the redox state of APC but is the effect of GSH.
AIDS, cancer, allergic disorders, as well as aging, are characterized by a limited ability of organisms to mount a Th1 immune response against an infectious agent or a vaccine antigen. Of interest, GSH depletion in APC correlates with defective antigen processing and reduced secretion of Th1 cytokines, favoring polarization from the typical Th1 cytokine profile towards Th2 response patterns. In view of therapeutic interventions, one can envisage the application of GSH-C4 and I-152 both to interfere with viral infections, and to replenish intracellular GSH in APC favouring an efficient antiviral response and an efficient Th1 immune response.