DCs are thought to contribute to allergic diseases by initiating and propagating T-cell–dependent immune responses and thus provide a link between environmental exposures and allergen-specific immunity. DCs also secrete cytokines that act on many other cell types and cause tissue inflammation. Despite their important role as environmental sensors, relatively little is known about how allergens activate DCs. Pollen allergens including ragweed have been shown to possess intrinsic oxidase activity (13
), and oxidative stress has been reported to play a critical role in DC maturation and cytokine secretion. In the present study, we show that a commonly used extract of short ragweed Ambrosia artemisiifolia
affected DC surface molecule expression, fluid-phase endocytosis, and cytokine secretion in an oxidant-dependent manner and resulted in an unusual phenotype of DC activation. RWE-dependent DC activation was significantly enhanced in DCs derived from mice deficient in Nrf2, the master antioxidant transcription factor, in association with increased oxidative stress and reduced expression of three key Nrf2-regulated antioxidant genes. Taken together, these data suggest that ragweed is sensed as a “danger signal” by dendritic cells in an oxidative stress- and Nrf2-dependent manner.
During DC maturation, DCs lose their ability to capture and process antigens, increase their expression of MHC II and costimulatory molecules, and up-regulate their production of cytokines (33
). Recent studies have revealed that ROS play a critical role in DC activation and maturation (15
). Boldogh and colleagues demonstrated the critical role of ROS generated by the intrinsic NADPH oxidase activity of RWE in enhancing antigen-induced allergic airway inflammation in mice, possibly by activating airway epithelial cells (13
). We used the same extract in our comprehensive in vitro
analyses and show that DCs are one key cell type targeted by this common aeroallergen.
RWE significantly induced oxidative stress in DCs, and this was augmented in Nrf2−/−
DCs compared with Nrf2+/+
DCs. RWE-induced oxidative stress was lost upon heat inactivation, suggesting that a protein component harbors the oxidative capacity, in keeping with the studies of Boldogh and colleagues (13
). We also found that RWE-induced oxidative stress was accompanied by strong up-regulation of CD80, CD86, and HLA-DR cell surface molecules, similar to a recent study by Allakhverdi and colleagues (38
) using pollen grain–treated human monocyte–derived DCs. Immature Nrf2−/−
DCs expressed higher levels of CD80, CD86, and MHCII when compared with resting Nrf2+/+
DCs at baseline, and this was augmented further by RWE. The antioxidant NAC significantly inhibited RWE-induced up-regulation of each of these molecules in Nrf2+/+
DCs but only CD86 in Nrf2−/−
DCs. Taken together, these data suggest that RWE induces surface molecule expression in DCs in an oxidative stress-dependent manner and that the ability of antioxidants to inhibit this process is compromised in the absence of Nrf2.
Dendritic cell maturation is not an all-or-none phenomenon, and subtle differences during this process can affect subsequent immune responses due to soluble and contact-dependent signals that influence T-cell differentiation. In contrast to Th1-promoting DCs, which secrete large amounts of IL-12 and related cytokines, Th2-promoting DCs are generally IL-12lo
and express combinations of other cytokines and cell surface receptors (39
). We found that RWE induced several features consistent with DC maturation (e.g., enhanced MHCII and costimulatory molecule expression) but inhibited basal IL-12 production in wild-type DCs (). Similarly, work by Allakhverdi and colleagues (38
) showed that pollen allergen inhibits IL-12 secretion by human monocyte–derived DCs. How exposure to RWE and other allergens influence DC activation and Th2 immune responses is an active area of research. A recent report found that birch pollen inhibited IL-12 production by DCs (41
), similar to our findings with RWE. This was attributed to pollen-associated phytoprostanes (41
), which, to our knowledge, have not been associated with RWE. We also found that the effects of RWE on BM-DC cytokine production were dependent on Nrf2. In addition to IL-12, RWE induced expression of significantly more TNF-α and IL-6 in BM-DC and primary lung DCs from Nrf2−/−
mice compared with their wild-type counterparts. Taken together with the recent observation that Nrf2-deficient mice produced enhanced levels of IL-12, IL-6, and TNF-α after exposure to LPS (29
), this suggests that, by keeping proinflammatory cytokine gene expression in check, Nrf2 may play a critical role in regulating activation of innate immune cells in different models. Future studies are needed to determine the precise mechanisms by which Nrf2 regulates cytokine production in dendritic cells and whether Nrf2 deficiency is a risk factor for allergen sensitization in human subjects.
We found the inhibition of IL-12 secretion by RWE in Nrf2+/+
DCs was not restored by the antioxidant NAC and that RWE-induced secretion of IL-12 was enhanced by NAC in Nrf2−/−
online supplement). The effects of NAC on IL-12 secretion have been shown to be stimulus- and concentration dependent (17
), and exactly how Nrf2 deficiency modulates the actions of NAC requires further study. Other products produced by DCs that affect Th2 differentiation include VEGF and IL-18, and we found that secretion of both of these was affected by RWE. Ragweed increased the secretion of VEGF but inhibited IL-18 production by DCs. VEGF has been reported to inhibit IL-12 production and Th1 differentiation by LPS-activated DCs (43
), and studies using VEGF overexpressing transgenic mice indicated that VEGF stimulated airway inflammation and remodeling in association with expansion of Th2-promoting lung DCs (44
). NAC did not inhibit VEGF secretion in Nrf2+/+
DCs, suggesting that this cytokine may be relatively resistant to the effects of antioxidants. IL-18 was the only cytokine we examined that was produced in higher amounts by resting Nrf2+/+
DCs compared with Nrf2−/−
DCs and was inhibited by RWE in DCs of both genotypes. The role of IL-18 has been investigated in murine models of allergic airway inflammation with complex results. In at least one mouse model, IL-18 (together with IL-12) inhibited Th2-driven responses (45
). Taken together, our findings suggest that ragweed may promote Th2-domininant allergic immune responses at least in part by inducing IL-12lo
DCs. However, we did not directly investigate the ability of RWE-exposed DCs to stimulate T cells in coculture assays, which requires further study. Future studies should investigate how Nrf2 and antioxidant defenses affect DC maturation and cytokine profiles after RWE exposure in vivo
We recently reported that Nrf2-deficient BM-DCs were strikingly susceptible to activation by ambient outdoor particulate matter in an oxidative stress-dependent manner (46
). Taken together with the data in this report, we conclude that two highly relevant but structurally distinct environmental exposures converge on a common mechanism of DC activation. It will be interesting to study the role of oxidative stress and Nrf2 in innate immune response to other allergens in future studies. Our study revealed increased basal level expression of Nrf2-regulated antioxidant genes GCLc and HO-1 in resting Nrf2+/+
DCs compared with Nrf2−/−
). Exposure to RWE further augmented the expression of GCLc and GCLm, the two key enzymes involved in glutathione synthesis. Ragweed extract also significantly induced the expression of HO-1, another Nrf2-regulated antioxidant gene, in Nrf2+/+
DCs when compared with Nrf2−/−
DCs. HO-1 is a rate-limiting intracellular enzyme that degrades heme to biliverdin, free divalent iron, and CO (48
). HO-1 is a stress-responsive gene whose expression has been shown to have a cytoprotective effect against oxidative injury and inflammation (48
). In human and rat DCs, induction of HO-1 expression with cobalt protoporphyrin inhibited LPS-induced phenotypic maturation and secretion of proinflammatory cytokines IL-12 p40, TNF-α, and IL-6 (49
), suggesting the importance of antioxidant genes in the modulation of DC maturation and cytokine secretion. Thus, decreased expression of HO-1 likely contributes to enhanced activation of Nrf2
-deficient BM-DCs in response to RWE.
In summary, our study adds to a growing body of literature indicating that the oxidant activity of allergens is an important determinant of allergic inflammation and suggests a novel link between allergen-induced oxidative stress and activation of DCs. We demonstrate the importance of Nrf2-mediated antioxidant signaling mechanisms in DC maturation, cytokine secretion, and endocytic function and conclude that the inability to combat oxidative stress by host factors such as Nrf2 may enhance the “innate allergic” immune response. Future studies investigating these pathways in human subjects may enhance our understanding of the susceptibility to allergic inflammation and asthma.