Disruption of the transcription factor Nrf2 promotes pro-oxidative dendritic cells that stimulate Th2-like immunoresponsiveness upon activation by ambient particulate matter

Abstract

Oxidative stress is important in dendritic cell (DC) activation. Environmental particulate matter (PM) directs pro-oxidant activities that may alter DC function. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates expression of antioxidant and detoxification genes. Oxidative stress and defective antioxidant responses may contribute to the exacerbations of asthma. We hypothesized that PM would impart differential responses by Nrf2 wild-type DCs as compared with Nrf2(-/-) DCs. We found that the deletion of Nrf2 affected important constitutive functions of both bone marrow-derived and highly purified myeloid lung DCs such as the secretion of inflammatory cytokines and their ability to take up exogenous Ag. Stimulation of Nrf2(-/-) DCs with PM augmented oxidative stress and cytokine production as compared with resting or Nrf2(+/+) DCs. This was associated with the enhanced induction of Nrf2-regulated antioxidant genes. In contrast to Nrf2(+/+) DCs, coincubation of Nrf2(-/-) DCs with PM and the antioxidant N-acetyl cysteine attenuated PM-induced up-regulation of CD80 and CD86. Our studies indicate a previously underappreciated role of Nrf2 in innate immunity and suggest that deficiency in Nrf2-dependent pathways may be involved in susceptibility to the adverse health effects of air pollution in part by promoting Th2 cytokine responses in the absence of functional Nrf2. Moreover, our studies have uncovered a hierarchal response to oxidative stress in terms of costimulatory molecule expression and cytokine secretion in DCs and suggest an important role of heightened oxidative stress in proallergic Th2-mediated immune responses orchestrated by DCs.

FIGURE 1

Determination of the cell surface markers that characterize bone marrow-derived DCs (BM-DC) in liquid static culture (A) for the experiments described herein and the highly purified rare myeloid conventional DCs subpopulation enriched from the pooled lungs of Nrf2^+/+ (B) and Nrf2^−/− (C) mice. Cell surface expressions, shown here as flow cytofluorographic histograms, were typical of several enrichments conducted in our laboratory and were determined by real-time flow cytometry (FACScalibur and CellQuest software). For bone-marrow-derived DCs, the myeloid DC phenotype (A) was confirmed by high expression of an CD11c-allophycocyanin conjugate and coexpression of CD11b-PE, with low-moderate expression of MHC class II-FITC typical of resting state immature DCs. For purified lung DCs, the myeloid DC phenotype was confirmed by expression of CD11c-PE, absence of the plasmacytoid DC marker PDCA-1-FITC, and low to very low expression of B220-PE in both Nrf2^+/+ DCs and Nrf2^−/− DCs. Data are described as MFI.

FIGURE 2

Flow cytometric determination of cell surface expression of function-associated molecules by bone marrow-derived DCs and purified lung DCs. A and B, Bone marrow-derived DCs from Nrf2^+/+ and Nrf2^−/− mice were stimulated in vitro with particulate matter at 10 μg/ml for 48 h and surface expressions of CD40 and MHC class II (A) CD80 and CD86 (B) were determined by multiparameter flow cytometry. C, In addition, flow histograms of those cell surface markers present on bone marrow-derived DCs of Nrf2^+/+ and Nrf2^−/− mice from one of the experiments is shown. D, Expressions of CD11c, CD40, CD80, CD86, and MHC class II were found on highly purified lung DCs of Nrf2^+/+ and Nrf2^−/− mice. Data are described as geometric mean ± SD of data collected as MFI from n = 6 age- and sex-matched mice per group. Data inserted in the representative flow histograms is also defined as geometric MFI units. Absolute levels of significance (p values) are shown in the figures.

FIGURE 3

Flow cytometric quantitation of function-associated molecule expression by DCs showing the effects of NAC on PM-induced alterations in cell surface expression of CD40 (A), CD80 (B), CD86 (C), and MHC class II (D) on bone marrow-derived DCs from Nrf2^+/+ and Nrf2^−/− mice. Data shown are mean ± SD of geometric MFI from n = 6 age- and sex-matched mice per group. Levels of tests of significance between treatments are shown and are as described in the text.

FIGURE 4

Quantitation of inflammatory cytokine production by PM-exposed DCs from Nrf2^+/+ and Nrf2^−/− mice. The secretion of immunoreactive IL-12p70 and IL-6 (A), IL-10 and TNF (B), as well as IL-18 (C) and VEGF (D) is shown. Bone marrow-derived DCs from the indicated strains were incubated with or without (Resting) particulate matter for 48 h, followed by analysis of cell-free supernatants by commercially available ELISA. Data are expressed as picograms per million cells and are the product of the mean ± SD of n = 6 sex- and age-matched mice per group.

FIGURE 5

An analysis of the time-dependent secretion of inflammatory cytokines of resting (unstimulated) lung DCs from Nrf2^+/+ and Nrf2^−/− mice as compared with PM-exposed DCs. The secretion of TNF-α (A) and KC (B) was assessed by commercial ELISA protocols at 24, 48, and 72 h following the exposure of DCs to PM. Data are described as mean picograms per million DCs ± 1 SD. Levels of significance as indicated (***) are p < 0.01.

FIGURE 6

Determination of Th1-type (IL-12p70 and IFN-γ) vs Th2-type (IL-13 and IL-5) cytokines by OVA-pulsed (50 μg/ml) lung DCs from Nrf2^+/+ and Nrf2^−/− mice and coculture with naive CD4⁺CD45RA⁺ allogeneic OT-II T cells at a stimulator (DC) to responder T cell ratio of 1:5 (see Materials and Methods). The secretion of IL-13 and IL-5 (A) and the secretion IL-12p70 and IFN-γ (B) are shown. In addition, a ratiometric analysis of the secretion of IL-13 produced by PM-stimulated Nrf2^+/+ and Nrf2^−/− DCs relative to either IFN-γ or IL-12p70 secretion is shown (C). Data are described as picograms of cytokine per milliliter produced in the coculture.

FIGURE 7

Flow cytometric quantitation of the endocytic uptake of FITC-DX (40 kDa) by resting (A) or PM-exposed (B) DCs from Nrf2^+/+ (WT, wild type) and Nrf2^−/− (Nrf2 ko (knockout)) mice. The time-dependent endocytosis of FITC-dextran is shown and levels of tests of significance between Nrf2^+/+ as compared with the Nrf2^−/− DCs are also described for comparison. Data are described as geometric MFI.

FIGURE 8

Flow cytometric quantitation of H₂O₂ production and accumulation of resting DCs as compared with PM-exposed DCs. Data are geometric MFI ± SD as a function of DCF fluorescence (oxidized DCFHDA). The respiratory burst of DCs derived from Nrf2^+/+ and Nrf2^−/− mice is shown following stimulation with PM (A) and as compared with the negative control carbon black (CB) particulates (B), bacterial LPS/endotoxin (C), and trimeric CD40L (D). ko, Knockout.

FIGURE 9

The effect of PM and the induction of enhanced oxidative stress in Nrf2^−/− DCs as compared with Nrf2^+/+ DCs. In this assay, real-time H₂O₂ formation was measured by a peroxidase luminol chemiluminescence (CL) method. The CL response was initiated by adding 5 μM luminol and 10 μg/ml HRP and continuously monitored at 37°C for 1 h. Data are mean ± SD of three independent experiments. Levels of significance between resting and PM-treated DCs are described as absolute values on the figure; ** represents enhanced production of H₂O by Nrf2^−/− DCs as compared with their wt counterparts at p < 0.001. ko, Knockout.

FIGURE 10

Increased transcriptional induction of antioxidant genes in Nrf2^+/+ DCs as compared with Nrf2^−/− DC. Quantitative real-time RTPCR analysis showed increased levels of mRNA for genes such as γGCLc (A), γ GCLm (B), and HO-1 (C) in PM-exposed wt Nrf2^+/+ DCs as compared with Nrf2^−/− DCs. Results are mean ± SD of three independent experiments. Levels of statistical significance (**, p ≤ 0.05) are for Nrf2^+/+ DCs as compared with Nrf2^−/− DCs. For comparative purposes, the inductions of γGCLc (A), γ GCLm (B), and HO-1 (C) in response to both LPS and trimeric CD40L are shown. In each case, PM was more effective at inducing antioxidant gene expression than either LPS or CD40L in this model. Ct, Cycle threshold.