To the Editor
Ozone (O3) can injure or activate airway epithelial cells and macrophages and is the most common non-infectious environmental cause of asthma exacerbations. The typical airway response to O3 exposure includes neutrophilia, increased pro-inflammatory cytokine levels, and transient decrements in lung function. Although it was previously hypothesized that exacerbation of allergic inflammation mediated an allergic asthmatic’s (AA) increased susceptibility to O3, we have shown that sputum from AA displays evidence of an augmented innate immune response to O3 exposure compared to healthy volunteers (HV) (1, 2).
The IL-1 protein family members, including IL-1α and IL-1β, are important regulators of inflammatory responses. Both IL-1α and IL-1β signal through IL-1 receptor type 1 (IL-1R1) with downstream signaling leading to NFκB and AP-1-dependent transcription of pro-inflammatory genes, including genes for IL-8 and IL-6 (3). In the lung, IL-1β is produced by epithelial cells, macrophages, and mast cells and is notably upregulated in asthmatics (4, 5). O3 exposure of rat alveolar macrophages increases expression of IL-1α and IL-1β and promotes secretion of pro-inflammatory cytokines from epithelial cells (6). Similarly, mice lacking IL-1R1 have reduced expression of IL-6 and MIP-2 (a murine IL-8 homolog) after subacute O3 exposure (7), suggesting that signaling by either or both IL-1α and IL-1β are important regulators of the airway response to O3. IL-1 receptor antagonist (IL-1ra) is an anti-inflammatory protein released by monocytes and macrophages in response to IL-1β that blocks IL-1 signaling and mitigates a hyper-inflammatory state (8).
We previously demonstrated that, compared to HV, AA have elevated baseline and post-O3 sputum levels of IL-1β and elevated post-O3 sputum neutrophilia (1). Recently, we have shown that airway IL-1 responses to inhaled lipopolysaccharide (characterized by sputum neutrophilia and pro-inflammatory cytokine production) can be safely and effectively reduced by systemic administration of recombinant IL-1ra in HV (9). Due to similarities between airway inflammatory responses between O3 and LPS exposure, we hypothesized that the IL-1 axis is a key regulator of O3-induced airway inflammation. To explore this, we performed a retrospective correlation analysis of a subset of subjects from a previously published cohort of HV and AA before and 4 hours after the end of a 2 hour 0.4 ppm O3 exposure (1, 2). The subset of subjects was restricted to those with available sputum samples for further cytokine level analyses (HV, n=21; AA, n=18). Demographics and induced sputum cellularity for this subset are available in Table E1 of the online repository. We compared previously measured sputum neutrophilia, IL-1β, and IL-8 levels with newly measured sputum IL-1ra and IL-1α levels. IL-1α and IL-1ra levels were determined by ELISA (Mesoscale Discovery, Gaithersburg, MD, and R&D Systems, Minneapolis, MN, respectively). Spearman correlation analyses were performed to assess the association of O3-induced changes in IL-1α and IL-1β levels on the percentage of neutrophils of total sputum leukocytes (%Neutrophils) , Neutrophils/mg of sputum, and IL-8 and IL-1ra levels in each cohort in response to O3 exposure. Criterion for significance was taken to be p ≤ 0.05.
We first assessed the relationship of IL-1α and IL-1β with %Neutrophils and Neutrophils/mg in response to O3 exposure. We found that changes in IL-1β had a significant and positive correlation with changes in %Neutrophils in AA (Figure 1B), but not in HV (r=−0.06, p=0.8). There was no association between IL-1α and %Neutrophils or Neutrophils/mg in either HV or AA (Table 1), perhaps because there was no significant change in IL-1α levels after O3 exposure within each cohort, nor in comparison to each other (9.7 (−1356 to 2108) versus 75.37 (−1879 to 1218) pg/mL [Δ IL-1α, median (range)] , respectively). Because IL-8 is such a strong chemokine for neutrophils, and because IL-1 is known to upregulate IL-8 expression, we next assessed the relationship of IL-1α and IL-1β with IL-8 in response to O3 exposure. Whereas changes in IL-1α had no correlation with changes in IL-8 in either HV or AA, changes in IL-1β in response to O3 was significantly and positively correlated with changes in IL-8 in both HV and AA (Table 1). These data suggest that IL-1β is more strongly associated with pro-inflammatory signals in response to O3 than IL-1α. Furthermore, these data suggest that O3-induced IL-1β is a stronger pro-inflammatory signal in AA than in HV. Interestingly, O3-induced changes in IL-8 are significantly correlated with changes in %Neutrophils (Figure 1D) and Neutrophils/mg in AA (r=0.6, p=0.02) but not HV (r=0.1, p=0.68 for %Neutrophils; r=0.2, p=0.45 for Neutrophils/mg).
Figure 1.
Changes in IL-1β and IL-8 correlate with changes in %Neutrophils in sputum from AA. Linear regression with Spearman correlation analysis of O3-induced changes in sputum IL-1β and %Neutrophils in HV (A) and AA (B); correlations of O3-induced changes in sputum IL-8 and %Neutrophils in HV (C) and AA (D)
Table I.
Correlation of sputum IL-1α and IL-1β levels with PMNs/mg, IL-1ra, and IL-8
| Cohort | Independent analyte |
Δ Neutrophils/mg | Δ IL-8 | Δ IL-1ra | |||
|---|---|---|---|---|---|---|---|
| R | P | R | P | R | P | ||
| HV | Δ IL-1α | 0.3 | 0.2 | −0.01 | 0.97 | 0.39 | 0.08 |
| Δ IL-1β | −0.02 | 0.9 | 0.84 | <0.0001* | 0.01 | 0.96 | |
| AA | Δ IL-1α | 0.3 | 0.19 | 0.04 | 0.89 | 0.48 | 0.05 |
| Δ IL-1β | 0.4 | 0.09 | 0.71 | 0.001* | 0.42 | 0.08 | |
Spearman correlation analyses were performed to assess the relationship of sputum IL-1β & IL-1α levels with Neutrophils/mg of sputum, IL-1ra, and IL-8, and IL-6 levels in HV and AA subjects.
Given that IL-1ra is a natural antagonist to IL-1 signaling, we measured the levels of IL-1ra in the sputum after O3 exposure. There was no significant change in sputum IL-1ra levels after O3 exposure within either HV or AA cohorts, nor in comparison to each other (−10 (−70 to 219) versus −24 (−181 to 114) ng/mL [Δ IL-1ra, median (range)], respectively). Because IL-1β has been shown to increase IL-1ra secretion (8), we next investigated if there were any correlations between O3-induced changes in IL-1β and IL-1ra. There was no significant correlation between O3-induced changes in IL-1β or IL-1α with changes in IL-1ra in either HV or AA (Table 1). We found that O3-induced changes in IL-1α were positively correlated with IL-1ra in AA (p=0.05), but not in HV. IL-1α-induced expression of IL-1ra in airway cells is not well described, therefore the clinical relevance of this suggestive finding is unclear. In conclusion, our data support the notion that IL-1β is associated with airway neutrophil recruitment in AA after O3 exposure, which may be mediated through regulation of IL-8. O3-induced neutrophilia was uncoupled from both IL-1β and IL-8 responses in HV, but not in AA. This is consistent with our previous report, where we found that despite similarities in neutrophil and macrophage proportions in induced sputum samples after O3 exposure, gene array profiles from AA showed increased innate immune signaling involving the NFκB pathway (1, 2), while HV attempted to mitigate the airway response to O3.
A limitation to the interpretation of these data for asthmatics is the early 4 hour timepoint analyzed after O3 exposure; the kinetics of airway inflammation have been well studied in HV (E1) but not in asthmatics. Although airway neutrophilia peaks 6 hours after O3 exposure in HV, most asthma excerbations occur the day after exposure to O3. Others have found that asthmatics requiring inhaled corticosteroids had increased airway neutrophilia 18 hours after O3 and evidence of mast cell inflammation (E2), though the degree of airway inflammatory responses was not compared to earlier or later timepoints. It is noteworthy that even at the early 4 hour timepoint after O3 exposure, we saw augmentation of the innate immune response in AA. We suspect that the primed inflammatory state in AA airways with elevated baseline IL-1β levels was further exacerbated after O3 through the IL-1 pathway. Further investigation of IL-1β regulation and kinetics of airway inflammatory responses in AA airways will provide key insights into mechanisms of environmentally-induced asthma exacerbations that can guide the development of focused therapeutic targets for susceptible populations.
Supplementary Material
Acknowledgments
Funding Sources: MLH is supported by NIEHS K23-ES021745. This project was supported in part by grants R01ES012706 and P30ES010126 from the National Institute of Environmental Health Sciences, U19AI077437 from the National Institute for Allergy and Infectious Diseases. This work was also funded by CR 83346301 from the US Environmental Protection Agency.
Footnotes
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Disclosures: There are no conflicts of interests.
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