The Nrf2 activator tBHQ inhibits T cell activation of primary human CD4 T cells

Abstract

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates a battery of antioxidant, detoxification, and cell stress genes. It is activated by oxidative stress and a number of exogenous compounds, one of which is tert-butylhydroquinone (tBHQ), a widely used food preservative. Nrf2 modulates immune responses in numerous rodent models of inflammation, but its effects on human immune cells are not well characterized. The purpose of these studies was to evaluate the effects of the Nrf2 activator tBHQ on early events of T cell activation in primary human cells. Treatment with tBHQ induced mRNA expression of the Nrf2 target genes HMOX-1, GCLC, and NQO1, and also increased NRF2 mRNA expression, albeit to a lesser extent than the other target genes. tBHQ decreased production of the cytokines IL-2 and IFN-γ at both the protein and mRNA levels after stimulation with anti-CD3/anti-CD28 in human peripheral blood mononuclear cells and to an even greater extent in isolated CD4 T cells. Likewise, tBHQ decreased induction of CD25 and CD69 in peripheral blood mononuclear cells (PBMCs) and this decrease was even more marked in isolated CD4 T cells. In addition, tBHQ inhibited induction of NFκB DNA binding in anti-CD3/anti-CD28-activated PBMCs. Collectively, these data suggest that tBHQ inhibits activation of primary human CD4 T cells, which correlates with activation of Nrf2 and inhibition of NFκB DNA binding. Although these studies suggest the food additive tBHQ negatively impacts T cell activation, further studies will be needed to fully elucidate the effect of tBHQ on human immune responses.

Keywords: CD4 T Cells; Human PBMC; Nrf2; tBHQ.

Figure 1. tBHQ treatment induces expression of the Nrf2 target genes NQO1, GCLC, and HMOX as well as NRF2 expression in PBMCs

PBMCs were either left untreated (Bkg), or pretreated with vehicle (Veh, 0.01% ethanol) or tBHQ (1–5µM) and then activated 30 min later with anti-CD3/anti-CD28. Total RNA was isolated after 6h and mRNA expressions of (A) NQO1, (B) GCLC, (C) HMOX, and (D) NRF2 were quantified by quantitative RT-PCR. * indicates p<0.05 compared to the Veh group.

Figure 2. tBHQ treatment inhibits production of the early cytokine IL-2

(A-B) PBMCs or (C) CD4 cells were either left untreated (Bkg), or pretreated with vehicle (Veh, 0.01% ethanol) or tBHQ (0.1–5µM) and then activated 30 min later with anti-CD3/anti-CD28. (A, C) Supernatants were collected after 24h and analyzed for IL-2 by ELISA. (B) Total RNA was isolated from PBMCs after 6h and mRNA expression of IL-2 was quantified by quantitative RTPCR. * indicates p<0.05 compared to the Veh group, ND indicates samples below the limit of detection.

Figure 3. tBHQ treatment inhibits production of the cytokine IFNγ

A-B) PBMCs or (C) CD4 cells were either left untreated (Bkg), or pretreated with vehicle (Veh, 0.01% ethanol) or tBHQ (0.1–5µM) and then activated 30 min later with anti-CD3/anti-CD28. (A, C) Supernatants were collected after 24h and analyzed for IFNγ by ELISA. (B) Total RNA was isolated from PBMCs after 6h and mRNA expression of IFN-γ was quantified by quantitative RTPCR. * indicates p<0.05 compared to the Veh group, ND indicates samples below the limit of detection.

Figure 4. tBHQ treatment inhibits the expression of the early cell surface markers CD25 and CD69 in CD4+ T cells within the PBMC cell population

PBMCs were either left untreated (Bkg), or pretreated with vehicle (Veh, 0.01% ethanol) or tBHQ (0.1–5µM) and then activated 30 min later with anti-CD3/anti-CD28. Cells were collected after 24h and labeled with fluorochrome-conjugated antibodies against CD25 and CD69. CD4+ T cells were gated prior to analysis of CD25 and CD69 expression. A) Representative dot plots from a PBMC experiment, and quantification of (B) CD25 and (C) CD69 protein expression. Total RNA was isolated from PBMCs 6h after activation, and mRNA expressions of (D) CD25 and (E) CD69 were quantified by q-RT-PCR. * indicates p<0.05 compared to the Veh group.

Figure 5. tBHQ treatment inhibits the expression of the early cell surface markers CD25 and CD69 in isolated CD4+ T cells

CD4 T cells were either left untreated (Bkg), or pretreated with vehicle (Veh, 0.01% ethanol) or tBHQ (0.1–5µM) and then activated 30 min later with anti-CD3/anti-CD28. Cells were collected after 24h and labeled with fluorochrome-conjugated antibodies against CD25 and CD69. CD4+ T cells were gated prior to analysis of CD25 and CD69 expression. A) Representative dot plots and quantification of (B) CD25 and (C) CD69 protein expression. * indicates p<0.05 compared to the Veh group.

Figure 6. tBHQ treatment inhibits NFκB DNA binding in PBMCs

PBMCs were left untreated (Bkg), or pretreated with vehicle (Veh, 0.01% ethanol) or tBHQ (1µM), then activated 30 min later with anti-CD3/anti-CD28. 3h after activation, cells were collected and nuclear protein was isolated. p65 NFκB DNA binding activity was quantified spectrophotometrically by measuring absorbance at 450 nm.