Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress activated transcription factor, which is activated by reactive oxygen species, reactive electrophiles, and other xenobiotics. Under basal conditions, Nrf2 is tethered in the cytosol by its canonical repressor, Kelch-like ECH-associated protein 1 (Keap1). Keap1 acts as an adapter protein to an E3 ubiquitin ligase and facilitates the continual polyubiquitination and degradation of Nrf2 by the 26s proteasome, in unstressed conditions. In the presence of stressful stimulus, such as electrophilic compounds, key cysteines on Keap1 are modified, disrupting the ability of Keap1 to facilitate Nrf2 turnover, resulting in Nrf2 nuclear accumulation. In the nucleus, Nrf2 heterodimerizes with small Maf proteins and binds its consensus regulatory element, the antioxidant response element (ARE). To date, Nrf2 has been shown to drive the transcription of well over 100 genes, which are largely cytoprotective in function. Recently, Nrf2 has been shown to have a role beyond the antioxidant response. Studies have shown that Nrf2 plays a pivotal role in modulating immune response and inflammatory disease, ranging from autoimmunity to sepsis. Additionally, our lab demonstrated that activation of Nrf2 by the electrophilic food preservative tBHQ skews CD4 T cell fate toward a Th2 phenotype, in primary mouse CD4 T cells, which may increase susceptibility to allergy. In the present studies, we aimed to determine the role of the Nrf2/Keap1 system in human T cell activation, using the human Jurkat E6-1 cell line. In my first study, treatment of Jurkat T cells with the Nrf2 activator, tBHQ, resulted in a dramatic reduction in the early cytokine IL-2 at 24h. Although not as marked as the suppression of IL-2, tBHQ treatment also significantly decreased CD25 expression in these cells but had no effect on CD69 expression, indicating T cell activation was not suppressed as a whole. In correlation with the decrease in IL-2 induction, tBHQ also inhibited the transcriptional activity of NFB, an important transcription factor for IL-2 gene regulation. These were the first studies to demonstrate that the Nrf2 activator, tBHQ, suppresses the early events of T cell activation, using a human model; but it did not address whether the effects observed were due to the activation of Nrf2 or rather to off-target effects of tBHQ. Toward this end, we used CRISPR/Cas9 to generate Nrf2-null Jurkat cells. Treatment of wild-type and Nrf2-null Jurkat T cells with tBHQ demonstrated that the suppression of IL-2 and NFĸB by tBHQ was largely independent of Nrf2, whereas tBHQ-mediated inhibition of CD25 induction was largely Nrf2 dependent. To further characterize the role of Nrf2 in this model, 1[2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), a more potent and selective Nrf2 activator, was used. Interestingly, treatment of Jurkat cells with CDDO-Im resulted in a Nrf2-dependent suppression in IL-2 secretion. These were the first studies to demonstrate that activation of Nrf2 by tBHQ/CDDO-Im modulates immune function in a human model, but did not address basal genotype differences in the absence of xenobiotic. To address this, I utilized CRISPR/Cas9 to knock out the repressor of Nrf2, Keap1, resulting in Jurkat T cells with constitutive Nrf2 activity. Analysis of early endpoints of T cell activation in wild-type, Nrf2-null, and Keap1-null cells show a marked increase in IL-2 induction in Nrf2-null cells, which is consistent with the Nrf2-dependent suppression of IL-2 by CDDO-Im. Furthermore, Keap1-null cells, which have constitutive Nrf2 activity, show marked suppression of IL-2 secretion. Interestingly, Keap1-null cells also displayed a substantial increase in apoptosis, which may be the mechanism by which IL-2 is suppressed in these cells. In addition, modulation of nuclear translocation of c-jun and NFkB was observed in both Nrf2-null and Keap1-null genotypes. These are the first studies to demonstrate that the Nrf2/Keap1 system significantly modulates a number of early immune endpoints of T cell activation, including cytokine production, cell surface protein expression, and cell viability. These are the first studies, using a human model, to demonstrate a role for the Nrf2/Keap1 pathway in modulation of the early events of T cell activation.
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