Seasonal influenza poses a significant threat to the public due to its ability to cause illness that ranges in severity and can lead to hospitalization and death. Research to identify potential contributing factors to the susceptibility and severity of influenza infection is necessary to decrease the burden of disease. Natural killer (NK) cells provide early protection during infection by providing an early source of cytokines and cytotoxicity of virally infected cells. Recently, an immunomodulatory role for the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), has been shown in several immune cell types and is activated by oxidative stress and various exogenous compounds, such as the food additive, tert-butylhydroquinone (tBHQ). However, the role of Nrf2 and tBHQ in NK cells remains largely unknown. The overall goal of these studies is to understand the impact of tBHQ and Nrf2 on NK cell activity and to determine the effect of tBHQ on NK cell response to influenza. To assess this, we determined the effects of tBHQ on NK cell activation and effector function and whether these effects were dependent on Nrf2. Activation of NK cells was significantly decreased as shown by a reduction in early activation markers, CD25 and CD69, with 1 and 5 ¹̐ƯM tBHQ compared to the control vehicle group (VEH). Additionally, tBHQ induced maturation as demonstrated by a decrease in the percentage of intermediate stage NK cells, CD27+CD11b+, and an increase in terminally differentiated NK cells (CD27-CD11b+). tBHQ also inhibited production of the antiviral cytokine, IFNÎđ, at 1 and 5 ÎơM tBHQ compared to the VEH control. Furthermore, cytotoxic molecules, perforin and granzyme B, were also reduced with tBHQ in a concentration-dependent manner. To investigate the role of Nrf2, Nrf2-deficient mice were utilized to assess whether these effects were mediated by Nrf2. Maturation of NK cells had a pronounced genotype and tBHQ effect with a significant increase in the percentage of immature NK cells (CD27-CD11b-) in activated splenocytes from Nrf2-null mice. In addition, tBHQ caused a Nrf2-dependent decrease in the expression of the activation markers, CD25 and CD69, in PMA/ionomycin-activated NK cells. Likewise, tBHQ decreased expression of fas ligand in IL-12/IL-18-activated NK cells in a Nrf2-dependent manner. In contrast, inhibition of IFNÎđ induction by tBHQ was only partially Nrf2-dependent. These findings demonstrate tBHQ negatively impacts NK cell activation and effector function in vitro. However, little is known regarding the effect of dietary tBHQ on NK cells in vivo, specifically in response to influenza infection. Mice were fed a diet containing either 0.0014% tBHQ or control diet 10 days prior to being infected with influenza A/PR/8/34 (H1N1). NK cell responses were assessed at days two and three post-infection. Mice exhibited similar weight loss throughout infection regardless of their respective diet. At day three, tBHQ modestly decreased the percentage of NK cells in the lung, and NK cells adopted a more immature phenotype. tBHQ significantly reduced expression of fas ligand and production of IFNÎđ in NK cells compared to those on a control diet at day two. NK cell cytotoxicity decreased with tBHQ at day three, as demonstrated by reduced CD107a expression. Induction of effector genes was decreased in the lungs of mice on a tBHQ diet at days two and three of infection. Collectively, these are the first studies show that the food additive, tBHQ, negatively impacts NK cell activation, effector function and early responses to influenza infection.
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