Background
Bronchial asthma is a highly prevalent chronic airway disease affecting nearly 300 million people worldwide [
1] and characterized by airway inflammation and airway hyperresponsiveness (AHR). Acute exacerbation of asthma is characterized by severe airflow obstruction, due to the enhanced airway inflammation, hypercontractility of airway smooth muscle and airway wall edema
. Asthma exacerbations are often triggered by environmental allergens, virus infections and air pollutions [
2]. As one of the air pollutants, ozone (O
3) is a ubiquitous photochemical oxidant and has potential adverse impacts on human health, especially the respiratory system [
3]. Exposure to O
3 not only increases the burden of oxidative stress in lungs [
4,
5], but also exerts detrimental effects on respiratory mechanics [
6,
7].
In asthmatic patients, O
3 exposure was found to partially contributes to their exacerbations. Several studies have reported that the elevation of local atmosphere O
3 level is associated with the average visits of asthmatic patients to emergency departments, implying a causative role for O
3 in triggering the exacerbation of asthma [
8,
9]. Particularly, continuous exposure to O
3 is very harmful to the asthma patients. However, it is by far not clear how O
3 influences asthma patients. Understanding the action of O
3 on asthma exacerbation may offer asthmatic patients with more inclusive advices and potential therapeutic options.
Though numerous animal studies have explored the influences of O
3 on the airways with acute allergic inflammation, most of them applied O
3 exposure before or after the challenge process. However, studies have shown that O
3 interfered with the immune responses during the challenge process of allergy establishment. For examples, Depuydt et al. have proved that O
3 does not affect the sensitization process but does affect the challenge process [
10]. In fact, in a scenario that the exacerbations of asthmatic patients are triggered by ambient O
3, the challenge process will be subject to the O
3’s influence. Therefore, under such scenario both the immune response process and the subsequent allergic airway inflammation of these patients are vulnerable to the ozonic effects. For the best of our knowledge, so far there is no comprehensive animal studies to investigate the effects of O
3 on the pathophysiological features of an allergic asthma model during the challenge process.
To date, the underlying mechanisms for in vivo ozonic effects on exacerbation of asthma remain elusive. Studies have shown that p38 mitogen-activated protein kinas (MAPK) might be involved in this process. For example, Williams et al. reported that p38 MAPK contributes to the O
3-induced airway hyperresponsiveness (AHR) [
11], while Li et al. later demonstrated that p38 MAPK activation in the airway smooth muscle further activated heat shock protein (HSP) 27 and subsequently contributed to the O
3-increased contractility [
12]. On the other hand, other researchers speculated that oxidative stress could be the major player in the action of O
3, based on the fact that O
3 exposure elevates the oxidative stress level in lung tissues and airway lumen in both humans [
13] and rodents [
14]. It is by far not known whether the activation of p38 MAPK and the oxidative stress are involved in the ozonic effects during the challenge process, triggering the asthma exacerbations.
In current study, we exposed an OVA-sensitized asthmatic mouse model to O3 during the OVA challenge process to mimic O3-induced asthma exacerbation. To further illustrate the underlying mechanisms of ozonic effects on this model, we investigated the biological function of p38 MAPK and oxidative stress using their corresponding inhibitors. This study revealed specific ozonic effects on an allergic asthma model involved p38 MPAK and oxidative stress. Additionally, it led to a possible strategy to attenuate the O3-elicited detrimental effects on asthma exacerbation and in other oxidative stress-related inflammatory airway diseases, like chronic obstructive pulmonary diseases.
Discussion
In this study, we investigated how O3 exposure during the OVA challenge affects the asthma exacerbation in an OVA-allergic mouse asthma model. We found that O3 exposure during the OVA challenge process increased asthmatic inflammation in the airway and lungs, particularly promoting AHR and the airway resistance synergistically. We further demonstrated that p38 MAPK and oxidative stress play important roles in the observed ozonic effects on the asthma exacerbation.
Although there have been several similar mouse studies looking at the O
3 effects on the asthmatic inflammation [
10,
18,
22‐
24], our work is unique in the animal protocol to mimic the real situation of O
3-induced asthma exacerbation in human. It is worth to note that the concentration of O
3 used in this study (1.0 ppm) is relative higher than the atmosphere O
3 concentration (~0.01 ppm) for inducing a measurable biological response. The major difference between our model and others was that the O
3 exposure was applied during a different stage of immune establishment. Some groups conducted the exposure right after the OVA sensitization instead of during the challenge process [
22], some introduced O
3 exposure after the OVA challenge process was completed [
18,
23,
24]. Though these studies did contribute to our understanding of the different perspectives of O
3 effects in allergic asthma model, they are less relevant to the real-life situation of asthmatic patients undergoing O
3 triggered exacerbation. It has been well-documented that the susceptibility to antigen challenge of asthmatic patients can be enhanced by the exposure to the ambient O
3 [
25‐
27]. Thus, applying the O
3 exposure in the antigen challenge process, would better mimic the patient conditions of the ambient O
3 induced asthma exacerbation. To date, there is only one study using a similar animal protocol of ours (i.e. O
3 exposure during the process of antigen challenge), demonstrating that O
3 promoted the eosinophilic airway and lung inflammation in the OVA-allergic mice [
10]. Our study took one step forward to further address the specific ozonic effects on the AHR and lung mechanics (airway resistance Raw and lung compliance CL); more importantly, we defined the possible underlying mechanisms of these specific effects, suggesting that p38 MAPK and oxidative stress were critically involved in the process.
It has been reported that p38 MAPK pathway is involved in the O
3-induced AHR and pulmonary inflammation in normal mice [
11]. O
3 exposure can activate p38 MAPK in the airway smooth muscle (ASM) of normal mice, and the phosphorylation of p38 MAPK will result in the phosphorylation of HSP27 (known as the p38 MAPK-HSP27 cascade), which eventually increases the contractility of ASM by enhancing its sensitivity to agonists, such as acetylcholine [
12] and carbachol [
28]. Our study further demonstrated that p38 MAPK-HSP27 pathway was also involved in the O
3 induced asthma exacerbation as the phosphorylation of p38 MAPK and HSP27 was elevated in tracheal tissues in the OVA-O
3 mouse model (Fig.
3). In addition, we also observed the profound increase in the AHR and Raw in the O
3-exposed OVA-allergic asthma model (Fig.
2a). Note that the O
3 exposure during OVA challenge could even cause a synergistic effect on p38 phosphorylation. Furthermore, inhibition of p38 MAPK with specific chemical inhibitor leads to the decrease in the AHR and Raw (Fig.
5a). Taken together, these data suggest that p38 MAPK-HSP27 cascade play an important role in the O
3-induced elevation of the AHR and Raw in the allergic asthma model.
In addition to the p38 MAPK-HSP27 cascade, we also found that the oxidative stress is another key factor mediating the O3-enhanced AHR and Raw in the current OVA-O3 mouse model. First, the oxidative stress level in the lung tissues of O3-exposed normal mice was elevated as reflected by the MDA content and the activity of GSH-Px. Second, such increase, particularly in the GSH-Px activity, was further boosted by the synergistic effect from the O3 exposure and OVA challenge together. Third, the mitigation of oxidative stress by a ROS scavenger, α-tocopherol, led to the reduction of O3-enhanced AHR and Raw. All these evidences indicated that oxidative stress indeed contributed to the profound increase in AHR and Raw in mice of OVA-O3 model.
It has been shown that hyaluronans (low molecular weight, LMW) in the BALF of mice play essential roles in O
3-induced enhancements in both AHR and the mucus production [
18,
29]. In current study, we found that the hyaluronan in the BALF was synergistically increased by OVA challenge and O
3 exposure; this effect could be inhibited by α-tocopherol alone but not the p38 inhibitor SB239063 (Fig.
5d), suggesting that oxidative stress is associated with the production of hyaluronan in the airway lumen. Consistently with this observation, previous study has demonstrated that the production of LMW-hyaluronan from the depolymerization of HMW-hyaluronan can be promoted significantly by ROS [
30]. Furthermore, it has been reported that the mRNA expression of hyaluronan synthases (HAS1 and HAS2) was upregulated in a murine model of asthma [
31], and the production of ROS can be detected within 2 h after the stimulation of ozone on airway epithelium [
32]. Therefore, ozone exposure on asthma model could produce more LMW-hyaluronan than normal subjects, which offers an explanation for the synergistic effects of ozone and OVA on the change of BAL hyaluronan. Interestingly, in our previous study where the O
3 was applied after the OVA challenge process was completed, we did not observe the synergistic effect of OVA and O
3 on the hyaluronan production in BALF [
18]. This is most likely because different protocols of O
3 exposure were used. This also provides evidence for the importance of O
3 exposure during the OVA challenge process.
Notably, we observed that the inhibition on oxidative stress in the OVA-O
3 model could decrease the phosphorylation of p38 MAPK, but the inhibition on the p38 MAPK had no effect on the oxidative stress. Such inhibition by α-tocopherol on the p38 phosphorylation was much less than those caused by SB239063. In addition, α-tocopherol was incapable of inhibiting the downstream HSP27 phosphorylation as well as decreasing the Raw in the OVA-O
3 mice. Together, these observations suggest that the oxidative stress pathway may probably be one of many upstream mediators of p38 MAPK activation in the OVA-O
3 model. Though the underlying molecular mechanisms for oxidative stress induced p38 MAPK activation remain elusive, it is suggested by Williams, A. S. et al. that the Toll-like receptor (mainly TLR4 and TLR2) signaling pathways may be involved, as the p38 MAPK-mediated ozone-induced airway hyperresponsiveness was blocked by genetic inhibitions of TLR4 and TLR2 in mice [
33]. Nevertheless, more detailed studies need to be conducted in the future to better understand this phenomenon.
In addition, our studies indicated that oxidative stress, but not p38 MAPK, contributed to the aggravation of allergic inflammation and immune responses by O
3 exposure for the following reasons. First, we found that O
3 exposure barely affected the inflammation in peribronchial area (Fig.
2c), thus, the peribronchial inflammation is mainly allergic in OVA-O
3 model. We have shown that p38 MAPK inhibition did not reduce the peribronchial lung inflammation, however, the oxidative stress inhibition did (Fig.
5c), suggesting that the oxidative stress was involved in the production of O
3-enhanced allergic peribronchial inflammation. Actually, this phenomenon has been previously described by Cook-Mills et al.
, finding that α-tocopherol decreased the allergic lung inflammation in mice induced by the house dust mite [
34]. Secondly, inhibition of oxidative stress was also found to inhibit the accumulation of eosinophils and the local production of IL-13 and IL-5 in the airway lumen of mice in OVA-O
3 model. Similar findings were reported using rat model [
35]. Mabalirajan, U. et al. found that the antioxidant treatment improved AHR and Th2 inflammatory response in an OVA-established asthma mouse model [
17]. These findings suggested that the oxidative stress, but not the p38 MAPK, mediates the O
3-enhanced allergic immune response.
Different from the eosinophils-mediated pulmonary allergic inflammation that is exclusively influenced by the oxidative stress in the current OVA-O
3 model, the neutrophilic airway inflammation is attributed to both p38 MAPK activation and oxidative stress. Simultaneous inhibition of these two pathways exhibited synergistic effects on reducing the neutrophils infiltration to the lung (Fig.
5b). Consistently, lung mRNA expression of CXCL-1 (a main chemokine for neutrophil migration) was exclusively inhibited by the combined inhibition of both pathways. These results indicate a collaborative role of p38 MAPK and oxidative stress pathways in the O
3-induced accumulation of neutrophils in the airway lumen of the OVA-allergic asthma model.