This study explored the effects of a recently identified component of airborne PM formed during processes of combustion, namely, the EPFRs. The EPFR-containing ultrafine particle used in this study was created by combusting 1,2-monochlorophenol in the presence of a metal catalyst and silica substrate (i.e., MCP230). In particular, this study explored the role of MCP230 in altering pulmonary immunologic homeostasis in normal mice or mice with allergic airway inflammation (i.e., mice with asthma). Our data demonstrated that MCP230 directly induced the maturation of pulmonary DCs and enhanced OVA-induced allergic airway inflammation. This did not correlate with eosinophilic inflammation, as expected of a traditional model of OVA-induced asthma, but rather with pulmonary neutrophilia. The observed neutrophilia appeared to be directly related to MCP230 exposure. In addition, MCP230 enhanced the Th17 immune response observed after OVA sensitization and challenge. Neutrophilic inflammation and Th17 immune responses are predominantly associated with persistent and difficult-to-control asthma (i.e., with poor responses to corticosteroids) (11
Exposure to MCP230 induced the maturation of lung DCs in vivo
, and stimulated the expression of the costimulatory molecules CD80 and CD86 in BMDCs in vitro
, suggesting that MCP230 directly stimulates the maturation of DCs. Comparable effects on the maturation of DCs were observed after exposure of human blood-derived monocytes or BMDCs to freshly collected air pollution particles and ultrafine carbon black particles (17
The mechanism by which ambient PM induces the maturation of DCs remains unclear. However, it seems logical to assume that particle uptake, by either specialized phagocytic or endocytic mechanisms, is necessary to promote the maturation of DCs. Indeed, our data demonstrate that particle uptake is necessary for increasing the expression of the costimulatory molecules CD86 and CD80.
Oxidative stress is also thought to play an important role in inducing the maturation of DCs (19
). GSH is an important tripeptidethiol antioxidant, and its intracellular concentration and a decreased GSH/GSSG ratio are indicators of oxidative stress (9
). In this study, exposure to MCP230 induced oxidative stress in BMDCs, as evidenced by decreased GSH and GSH/GSSG ratio. The antioxidant PBN was able to decrease the MCP230-induced oxidative stress of BMDCs, and also inhibited their maturation (i.e., decreased the expression of CD86 and CD80). Because amorphous silica particles, which do not contain an EPFR, failed to induce the maturation of BMDCs, these data cumulatively suggest that the pro-oxidant property of MCP230 is responsible for inducing the maturation of DCs.
We further examined the effects of pulmonary exposure to MCP230 on pulmonary DCs in the presence of an antigen (OVA). As expected, MCP230 enhanced the expression of costimulatory molecules on DCs in the presence of the antigen. Furthermore, these more mature DCs were capable of inducing stronger CD4+ T-cell proliferation and activation responses. These data are compatible with the enhanced inflammation in the lung and enlarged MLNs in the MCP230-exposed and OVA-challenged mice. These findings suggest that MCP230 amplifies the functional activation of DCs by inducing the elevated expression of costimulatory molecules, thus exerting a durable effect on the antigen-presenting ability of DCs. In mice with asthma, MCP230 appeared to synergize with allergen, to exacerbate pulmonary pathophysiology.
In this study, exposure to MCP230 elicited an enhanced Th17 immune response in a mouse model of asthma. This result is contrary to previous reports of Th responses after many other exposures, including PM and DEPs (22
). Many studies with DEPs, for example, demonstrated that the generation of oxidative stress by DEPs favors a Th2 skewing of the immune response, while suppressing Th1 differentiation (24
). This Th2 skew correlated with significant eosinophilic inflammation in the lung and an OVA–IgG1 response in already sensitized animals (28
). In this study, the Th2 immune responses of OVA + MCP230–exposed animals were not statistically different from those of OVA–sensitized or OVA–challenged animals, and the Th17 responses were significantly greater. This result is compatible with the demonstration in our model of increased IL-17A gene expression in the lung and increased neutrophil concentrations in the BALF of mice exposed to MCP230.
These differences in immune responses are most likely related to the differences in chemical composition of various PMs and DEPs and MCP230. For example, carbonaceous particles and a quinone in DEPs were shown to promote Th1 immune responses, whereas chemical components extracted with benzene–ethanol initiated Th2 responses (22
). Th17 and associated cytokines were implicated in the development and promotion of steroid-resistant asthma and airway inflammation and hyperresponsiveness in humans and mice (12
). The mechanism by which MCP230 induces Th17 but not Th2 immune responses is not clear. But in total, our data suggest that exposure to MCP230 may be a significant and unrealized risk factor in the development of steroid-resistant asthma.
In conclusion, our data demonstrate that EPFR-containing PM formed during processes of combustion directly induced the maturation of DCs in an uptake-dependent and oxidative stress–dependent manner. Alterations in the maturation of DCs were accompanied by alterations in Th cell subsets in the lung. The local Th17-biased phenotype (increased Th17 cells and cytokines) was accompanied by significant pulmonary neutrophilia. Pulmonary neutrophilic inflammation, the presence of Th17 cytokines, and elevated numbers of Th17 cells in the lung are reminiscent of severe, steroid-insensitive asthma in humans. Thus, exposure to EPFR-containing PM may constitute an important and unrecognized risk factor in the exacerbation and development of a severe and steroid-resistant asthma phenotype in humans.