Glutathione-redox balance regulates c-rel-driven IL-12 production in macrophages: possible implications in antituberculosis immunotherapy

Abstract

The glutathione-redox balance, expressed as the ratio of intracellular reduced glutathione (GSH) and oxidized glutathione, plays an important role in regulating cellular immune responses. In the current study, we demonstrate that alteration of glutathione-redox balance in macrophages by GSH donors like cell-permeable glutathione ethyl ester reduced or N-acetyl-L-cysteine (NAC) can differentially regulate production of IL-12 cytokine in macrophages. A low concentration of NAC increased IL-12 p40/p70 production, whereas at high concentration, IL-12 production was inhibited due to increased calmodulin expression that binds and sequesters c-rel in the cytoplasm. Although NAC treatment increased the IκBα phosphorylation, it failed to increase TNF-α levels due to enhanced expression of suppressor of cytokine signaling 1, which specifically prevented nuclear translocation of p65 NF-κB. We demonstrate that NAC at 3 mM concentration could increase bacillus Calmette-Guerin–induced IFN-γ production by PBMCs from patients with active tuberculosis and shifts the anti–bacillus Calmette-Guerin immune response toward the protective Th1 type. Our results indicate that redox balance of glutathione plays a critical role in regulating IL-12 induction in native macrophages and NAC can be used in tailoring macrophages to induce enhanced Th1 response that may be helpful to control tuberculosis and other pathophysiological disorders. Glutathione is the most predominant intracellular low m.w. thiol and acts as a reducing agent and an antioxidant. Glutathione is implicated in many cellular functions, including synthesis and degradation of protein and DNA. The intracellular redox environment is mainly controlled by the glutathione-redox, which is defined as the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) (1) and plays critical roles in maintaining cellular homeostasis and various physiological functions (2). The cellular glutathione-redox dynamically regulates protein functions by reversible disulfide bond formation on variety of proteins, including phosphatases, kinases and transcription factors (3–5) and is known to protect against oxidative stress (6, 7). The glutathione-redox balance in macrophages is shown to be critical for mounting innate immune responses and has also been implicated in many of the pathological conditions (2, 8–10), indicating its possible potential use as a therapeutic agent. However, redox-based therapy is still in its early developmental phases. It has been observed that triggering of macrophage receptors by pathogen products could change the macrophage redox status (9) and modulate the cytokine milieu (11, 12). Various studies indicate that glutathione levels in APCs can influence the Th response (13, 14). It is well known that the Th1/Th2 lineage commitment depends to a great extent on the cytokine milieu induced during innate activation of macrophages (15, 16). For example, IL-12 and TNF-α activate the Th1 T cell, whereas IL-10 cytokine favors the Th2 T cell development. Therefore, understanding the signaling pathways modulated by the glutathione-redox in macrophages and thereby influencing induction of these cytokines is important to understand how the T cell microenvironment is controlled during various pathophysiological conditions. However, the molecular mechanisms by which glutathione-redox can directly control macrophage functions and cytokine induction profile are largely not understood, other than in a few cases like GSH-mediated regulation of NF-κB (6, 7). There are indications that Th1 immune response is downregulated in patients with active tuberculosis (TB) infection (17). Interestingly, studies also indicate that patients with TB have altered glutathione balance (18). However, it is not clear whether Mycobacterium tuberculosis modulates the T cell responses by altering the glutathione-redox balance in macrophages. In the current study, we demonstrate that IL-12 induction in native macrophages is controlled directly by the intracellular glutathione-redox through calmodulin (CaM) and c-rel and manipulation of the macrophage-glutathione-redox can influence in vitro cellular immune response of PBMCs obtained from patients with active tuberculosis to Bacillus Calmette-Guerin (BCG).