Although ozone induces exacerbations of asthma and COPD, and has also been implicated in accelerating cardiovascular disease, the precise cellular and molecular mechanisms are poorly described. Compelling evidence suggests that ozone induces airway inflammation manifested by increases in neutrophil number and levels of tumor necrosis factor-α and IL-1β. To date, the role of inflammation in mediating ozone-induced AHR remains controversial. Further, studies suggest uncoupling of airway inflammation and AHR after ozone exposure in humans 
The PCLS technique serves as a suitable model to measure ozone-induced AHR ex vivo
as demonstrated by an increase in airway sensitivity to a muscarinic agonist following ozone exposure (3 ppm; 2 h; T
+16 h: ), compared to air-matched controls. Increased levels of neutrophils were also evident. This was previously shown in a model of pulmonary resistance and was associated with an increase in trafficking cells in the BAL fluid, specifically neutrophils 
. Following exposures of the same concentration of ozone, but at a time where no infiltration cells were present (3 ppm; 2 h; T
0 h: ); no change in airway sensitivity compared to air-controls was observed. After mice were exposed to increased concentrations of ozone (6 ppm), at a time of increased neutrophils in the BAL fluid an increase in airway sensitive to carbachol was observed (6 ppm; 2 h; T
+16 h: ). Interestingly, when the time between ozone exposure and animal sacrifice was shortened from 16 to 0 h, AHR was still evident despite no increase in total cell number, neutrophils and KC levels, in the BAL fluid. Despite studies specifically examining neutrophil-dependent AHR 
, only a few in vivo
, and human studies 
have also noted an increase in airway sensitivity lacking a correlation with early airway inflammation. One study exposed neutrophil-depleted rats to ozone (1.0 ppm) for 8 h and concluded that neutrophils did not play a significant role in the acute ozone-induced early epithelial damage seen in non-neutrophil-depleted rats 
. One must therefore ask if an ozone-induced increase in IL-8 that attracts neutrophils into the lung is responsible for the latent AHR; what mechanism is causing the early phase AHR seen in our study? To determine whether the inhalation of ozone induced the release of a non-immunological mediator, or was directly modulating airway smooth muscle function rendering it more contractile; BAL fluid from mice exposed to ozone (6 ppm; 2 h; T
0 h) or filtered air was incubated overnight on lung slices from naive mice. Airways on naïve lung slices incubated with BAL fluid from ozone-exposed mice were more responsive and more sensitive to carbachol, as defined by an increase in the maximum contraction and a decrease in the log EC50
values respectively, than those incubated with BAL fluid from air-exposed mice. These data suggest that ozone promotes AHR via secretion of a soluble mediator from the airway into the BAL fluid.
Eicosinoids are increased in the lungs following ozone exposure 
. Alfaro et al.
reported that subjects with more sensitive airways had a greater amount of PGE2
in the exhaled breath condensate than subjects with less sensitive airways following an acute exposure to ozone 
. In the present study, BAL fluid from mice exposed to ozone (6 ppm, 2 h, T
0 h) was analyzed for quantities of oxidized lipids. Our targeted lipidomics approach for the analysis of BAL fluid revealed that 20-HETE levels were significantly increased upon exposure of mice to ozone. These data suggest that either CYP4A isoforms, responsible for ω-hydroxylation of AA, were up-regulated by ozone treatment 
, or that increases in AA substrate was generated leading to increases in 20-HETE, or that a combination of these mechanisms were in play. When the cytochrome P450 inhibitor ABT was administered 18 h prior to exposure to ozone, the levels of 20-HETE in BAL were reduced by 82%. This is similar to the 84% reduction of renal cortical 20-HETE formation observed in Sprague-Dawley rats treated with similar levels of ABT 
. The decreased concentrations of 20-HETE in BAL fluid coincided with the complete reversal of the ozone-induced increase in airway sensitivity, suggesting that 20-HETE production played an important role in the AHR when no infiltrating inflammatory cells or mediators were present. The lack of attenuation of the other HETEs suggests the specificity of ABT to 20-HETE in the murine model. Studies have suggested that 1-ABT is a non-selective inhibitor of CYP enzymes 
; however, the same concentration as was used in the present study demonstrated a fairly selective inhibition of the CYP4A/CYP4F catalyzed formation of 19-and 20-HETE in Sprague Dawley kidneys with no inhibition of epoxygenases 
. In the present study, no other HETEs were inhibited following 1-ABT administration suggesting it to be selective to the oxidized lipids that were measured. Future studies may warrant the use of the more selective 20-HETE synthesizing enzyme inhibitor HET0016 
were two of the other the most abundant eicosanoids detected in the BAL after ozone treatment () and are known to induce AHR; however, they were still present despite the ABT-induced decrease in airway sensitivity. The non-selective COX-inhibitor, indomethacin reduced the levels of PGE2
, and PGD2
levels by 100%, 100%, and 93%, respectively () but had no effect on the ozone-induced increase in airway sensitivity, suggesting that PGs do not play a significant role in this model of AHR. Indeed, other studies have shown no evidence of indomethacin inhibiting early phase ozone-induced airway hyper-responsiveness, and have suggested other arachidonic acid metabolites to be responsible for the increase in airway sensitivity 
. However, Nakando and colleagues demonstrated an increase in neutrophils immediately after ozone exposure, suggesting that neutrophil-dependent AHR was evident, that may be independent of AHR induced by increases in oxidized lipid products in their study. Hazucha and colleagues also demonstrated a lack of an effect with COX inhibition on an ozone-induced increase of airway resistance in human subjects, when PGE2
and thromboxane were reduced 
; however, like Nakando, but unlike the present study, neutrophils were also present. In addition, ozone exposure of mice deficient in PGE-synthase, which is needed for the generation of PGE2
, had little effect on AHR (unpublished data). Dexamethasone pre-treatment had no effect on the oxidized lipids in the BAL, or on the ozone-induced AHR. This could, in part, be due to steroids having little or no effect on models of oxidative stress 
. LOX inhibitors were not used as levels of LTs were not shown to be increased following the exposure of ozone, thus not thought to play a role in the AHR seen in the present study. Thromboxane levels were not detected following any condition.
Collectively, our data suggests that 20-HETE plays a significant role in the neutrophil-independent AHR because the inhibition of 20-HETE production by ABT attenuated ozone-induced increases in airway sensitivity, when other HETEs or PGs were not affected. Previous studies have suggested that 20-HETE modulates airway tone 
; however, our study failed to show evidence of the direct effect on airway contraction in murine airways. Incubation of 20-HETE with murine lung slices, however, increased its contractile sensitivity to carbachol as compared with diluent treated slices. Concentrations of 20-HETE used were comparable to those measured in the BAL fluid following ozone exposure. Concentrations 100 fold lower than those measured in the BAL fluid were also shown to increase sensitivity of airway contraction. The mechanism of 20-HETE-induced AHR is unknown at this time and warrants further investigation. Although the slices are exposed to ozone for 2 hours, airway functional assessment is not carried out until the next day. This is due to a limitation of the preparation in that although lung slices are prepared immediately, the removal of agarose and the allowance of time for the airway to revert back to basal tone following slicing is required. Within this time, the effects of the ozone exposure are still at play at the intra-cellular level. Therefore, to mimic this as close as possible, 20-HETE and BAL fluid was incubated over-night to time-match the ex vivo
To date, only CYP4A12a and 4A12b have been shown to generate 20-HETE from AA 
. In humans, CYP4F2 and CYP4F3B play a major role in generating 20-HETE. In mice, however, members of the CYP4F family modify the ω-, and ω-1 positions of AA to generate 18-HETE and 19-HETE. Therefore, though we determined the relative amounts of murine CYP4A family members and their relative change in response to ozone, we focused predominantly on the relative expression of the isoforms CYP4A12a and CYP4A12b in response to ozone. As shown in , CYP4A12a and CYP4A12b expression, as determined by qRT-PCR, were similar. Ozone had little effect on the relative expression of either gene product. Therefore the induction of 20-HETE must be regulated at a step proximal to CYP4A expression and is likely due to increases in substrate delivery by increased generation of AA via cPLA2
activity or at the re-acylation of AA into membrane phospholipids as previously described 
. Levels of AA were increased in murine BAL fluid following ozone exposure (), therefore providing increased levels of substrate for the CYP enzymes to convert to 20-HETE. Surprisingly, not all oxidized lipid mediators downstream from AA were increased such as the LTs, suggesting that CYP enzyme activity may be another mechanism modulating AHR following ozone exposure. Macrophages present in the lung tissue may also be a source of 20-HETE production.
It is worth emphasizing that the current study is taking a snap-shot immediately after ozone exposure, eliminating infiltrating inflammation by creating the slices, and hypothesizing that a CYP4A dependent pathway is (directly or indirectly) responsible for the AHR shown. A limitation of the current study is that we cannot rule out the exact mechanism as a time-course of the supernatants post-ozone post-slicing was not carried out; however, an advantage is that we can eliminate the role of infiltrating cells. This will lead us to our follow-up studies.
Although we recognize the concentration of ozone needed to achieve the phenomenon of neutrophil-independent AHR is greater than that defined by the EPA to be hazardous to humans 
, the fractional deposition of ozone in mice is only 25% compared to 100% for humans. The EPA also recognizes an exposure time of 8 h which is much longer than the 2 h duration in the present study. Our data suggests that 20-HETE generated by ozone may represent one of the earliest and most robust signals promoting ozone-induced AHR.
In summary, eicosinoids released into the airway increased following ozone that was not attenuated by dexamethasone or indomethacin. Only inhibition of 20-HETE production attenuated both the ozone-induced elevated levels 20-HETE and AHR. We postulate that the increased 20-HETE is derived from an increase in AA, as well as the possibility of increased CYP activity, but not by an increase expression of CYP enzymes. Modulation of the production of 20-HETE may serve as a new therapeutic target to prevent ozone-induced exacerbations of asthma and COPD or serve as an important biomarker of oxidative stress in the airways.