Nuclear factor erythroid 2-related factor 2 (Nrf2) is a cytoprotective transcription factor activated by cellular stressors such as reactive oxygen species, electrophilic xenobiotics, and other forms of oxidative stress that regulates the transcription of a number of antioxidant, detoxification, and cytoprotective genes. Commonly used Nrf2 activators include 1[2-Cyano-3,12-di oxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) and the food additive tert-butylhydroquinone (tBHQ). Nrf2 was initially characterized as a cytoprotective transcription factor, and has since been shown to have additional effects, including a role in the immune system. Nrf2-null mice are more sensitive to a number of inflammatory stimuli, and cell-type specific effects of Nrf2 have been observed, mainly in macrophages and dendritic cells. However, the role of Nrf2 in T cell function has not been well studied. Prior studies from our lab have shown that activation of Nrf2 skews murine CD4 T cell differentiation towards a Th2 (allergy-like) phenotype. However, the role of Nrf2 in T cell activation, which sets up T cells for differentiation, and in human T cells is unknown. The purpose of the present studies is to determine the role of Nrf2 in primary CD4 T cell activation to fill this gap. To do this, the Nrf2 activators tBHQ and CDDO-Im were used to determine effects on T cell activation in murine or human primary CD4 T cells. In mice, tBHQ inhibited production of the cytokines interleukin-2 (IL-2), interferon gamma (IFNγ), and granulocyte-macrophage colony stimulating factor (GM-CSF) in both wild-type and Nrf2-null splenocytes, and inhibited tumor necrosis factor alpha (TNFα) only in wild-type splenocytes. CDDO-Im inhibited IFNγ and TNFα production, and increased IL-2 production, in a largely Nrf2-dependent manner, and increased GM-CSF independently of Nrf2. Both tBHQ and CDDO-Im had little effect on expression of the cell surface proteins CD25 and CD69. Both tBHQ and CDDO-Im increased nuclear translocation of the transcription factor c-Jun in wild-type but not Nrf2-null splenocytes, and inhibited p65 NF-κB DNA binding in wild-type splenocytes. Overall, the data suggest that Nrf2 activation has differential effects on murine T cell activation. Nrf2 activation inhibits IFNγ and TNFα secretion and p65 NF-κB DNA binding, and increases production of IL-2 and translocation of c-Jun, with little effect on CD25 and CD69 expression. In primary human CD4 T cells, tBHQ inhibits events of T cell activation, including production of IL-2 and IFNγ, expression of CD25 and CD69, and p65 NF-κB DNA binding. CDDO-Im also inhibits IL-2 and IFNγ production, and CD25 and CD69 expression in primary human CD4 T cells. However, the role of Nrf2 in mediating these effects was undetermined, so a primary human CD4 T cell Nrf2 knockdown model was developed. Nucleofection with siRNA directed against Nrf2 knocked down Nrf2 protein expression by ~90% 12 h after transfection. Suppression of IL-2, IFNγ, TNFα, and GM-CSF production by tBHQ occurred in both the scrambled control and Nrf2-deficient CD4 T cells, as did inhibition of CD25 and CD69 expression. RNA-seq analysis was performed and identified a number of differentially expressed genes between the scrambled control and Nrf2-deficient CD4 T cells, as well as genes differentially expressed with tBHQ treatment, indicating potential areas in which Nrf2 may play a role in primary human CD4 T cell activation.
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