Natural killer (NK) cells are essential for immune protection against tumors and viruses. Disease environments impose oxidative stress and impair immune cell functions. Glutathione (GSH) is a major cellular antioxidant and is critical for the immune response, but how it modulates NK cell function remains largely unknown. Using a mouse model with a specific deletion of the catalytic subunit of glutamate-cysteine ligase (Gclc) in NK cells, we demonstrate that GSH supports interleukin-15 (IL-15)-driven activation of NK cells. Gclc deficiency causes an intracellular accumulation of reactive oxygen species (ROS), which impairs the metabolism of NK cells. This is accompanied by defective proliferation and cytokine production concurrent with subverted mTOR and STAT5 activation. During acute lymphocytic choriomeningitis virus (LCMV) infection, Gclc-deficient NK cells are unable to suppress the antiviral T cell response. Remarkably, Gclc deficiency impairs NK cell-mediated protection against tumor lung metastases. Our findings highlight an essential role of GSH in maintaining NK cell functionality.
Reactive oxygen species (ROS) are generated by various mechanisms, with mitochondria being a major source. Excessive accumulation of ROS alters protein structure, impairing cellular function. Thus, ROS scavenging by antioxidants is crucial for cellular homeostasis. Elevated levels of free radicals have been shown to regulate phosphatases and the mitogen-activated protein kinase (MAPK)/ERK pathway to control immune cell activation.
Glutathione (GSH) is the most abundant antioxidant in mammalian cells. It is a critical mediator for immune homeostasis and metabolism of B cells and T cells. In conventional CD4+ and CD8+ T cells, the absence of GSH impairs priming of antigen-specific T cells. This results in a defective antiviral response and an attenuated disease pathology in a murine model of multiple sclerosis. In the context of an intestinal bacterial infection model, GSH-deficient Th17 cells in the lamina propria were not able to produce interleukin-22 (IL-22), culminating in severe intestinal damage and mortality. A redox imbalance impairs metabolic regulators, such as mTOR, cMyc, and NFAT signaling, undermining metabolic activation required for T cell effector functions.
Similar to T cells, natural killer (NK) cells critically rely on mTOR-dependent metabolic reprogramming upon IL-15-induced activation. While OXPHOS is critical for receptor-stimulated NK cells, high-dose IL-15 stimulation overcomes OXPHOS inhibition. IL-15- and IL-12-dependent activation of NK cells drives mTORC1-dependent upregulation of glycolysis, which is essential for the production of effector molecules, such as interferon γ (IFN-γ) and granzyme B. It has been shown that IL-15 drives mTOR and STAT5 activity, which is critical for NK cell development and function. IL-15 also sustains the antitumor response mediated by NK cells. In addition, IL-2 and IL-12 activate MYC and SREBP to sustain OXPHOS and glycolysis. These findings highlight that NK cell function depends on distinct metabolic rewiring in response to a wide array of activation signals similar to those observed in T cells.
The shared metabolic requirement of T cells and IL-15-activated NK cells demonstrates the need to investigate the effect of oxidative stress on NK cell metabolism and function. Extracellular ROS, generated by monocytes and granulocytes, has been demonstrated to suppress NK cell cytotoxic functions. Depletion of myeloid-derived suppressor cells, important sources of ROS during viral infection, enhances NK cell expansion and activation. In addition, the major ROS-producing enzyme NADPH oxidase (NOX) was also observed to suppress IFN-γ+ NK cell expansion. Deprivation of L-cystine and glutamine, essential substrates for GSH synthesis, also significantly suppress NK cell proliferation. Paradoxically, antioxidant treatment inhibited nuclear factor κB (NF-κB) activity and the cytotoxic potential of NK cells, highlighting the importance of ROS during NK cell activation.
In vivo, elevated expression of surface thiols by the IL-15-mTOR signaling cascade also enhances tumor infiltration by NK cells. A cytotoxic response against Mycobacterium tuberculosis is induced by culturing NK cells with the antioxidants GSH or N-acetyl-L-cysteine (NAC). Interestingly, patients with human immunodeficiency virus (HIV) have lower levels of GSH in NK cells, which is often accompanied by opportunistic infections with M. tuberculosis. These observations imply that the antioxidant response critically regulates NK cell effector functions.
Here, we explored the significance of GSH in NK cell function. We found that NK cell-specific ablation of GSH production leads to defective NK cell expansion and cytokine production in response to IL-15 stimulation. This was coupled with metabolic dysfunction and decreased activity of mTOR and STAT5 signaling. The absence of GSH impaired NK cell effector functions, resulting in defective regulation of antiviral immunity and impaired protection against tumor metastasis. Our data implicate GSH as an important regulator of NK cell function.
To investigate the redox balance coupled with IL-15-driven NK cell activation, we stimulated splenic NK cells derived from C57BL/6J wild-type (WT) mice for 24 h and measured the changes in intracellular levels of ROS by a fluorescent probe, DCF-DA. When compared to their unstimulated counterparts, IL-15-stimulated NK cells showed significantly decreased levels of cytosolic ROS as well as reduced accumulation of ROS in the mitochondria, measured by MitoSOX. ROS are critical for cell activation and can modulate NK cell cytotoxic capacity. NK cells primed with IL-15 increased the production of intracellular thiols, as shown by higher monobromobimane (MBB) fluorescence. Consistently, GSH levels were increased in NK cells upon IL-15 stimulation. Thus, we hypothesized that GSH modulates IL-15-mediated NK cell activation.
Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step of GSH synthesis. To examine the role of GSH in NK cell homeostasis and function, we used Gclc fl/fl mice, in which the catalytic subunit of the glutamine cysteine ligase (Gclc) locus is flanked by loxP elements. These mice were crossed with Ncr1-Cre + mice, where the Cre recombinase gene is expressed under control of the Ncr1 promoter, a gene largely restricted to NK cells. The progeny (Gclc fl/fl Ncr1-Cre +) produces NK cells that are unable to generate GSH. Gclc fl/fl Ncr1-Cre + mice displayed a significantly decreased frequency of splenic NK cells, suggesting involvement of GSH in NK cell homeostasis. Terminal maturation and differentiation of NK cells is characterized by sequential loss of CD27, which is paralleled by acquisition of CD11b from immature (CD11b− CD27+) to intermediate (CD11b+ CD27+) and then to terminally mature (CD11b+ CD27−) NK cell subsets. The maturation of splenic Gclc-deficient NK cells was comparable to Gclc fl/fl (control) controls. However, in line with reduced NK cell frequency in the spleen, the abundance of splenic NK cells, in particular that of CD11b+ CD27− NK cells, was reduced in Gclc fl/fl Ncr1-Cre + mice. T cells in naive Gclc-deficient mice remained unaffected.
In summary, these results suggest that GSH regulates redox balance in IL-15-activated NK cells and maintains NK cell homeostasis in the periphery.
Despite the lower levels of intracellular thiols, Gclc-deficient splenic NK cells at the steady state did not present altered cytosolic and mitochondrial ROS levels compared with control NK cells.
