Interleukin-1α Mediates Ozone-Induced Myeloid Differentiation Factor-88-Dependent Epithelial Tissue Injury and Inflammation

Abstract

Air pollution associated with ozone exposure represents a major inducer of respiratory disease in man. In mice, a single ozone exposure causes lung injury with disruption of the respiratory barrier and inflammation. We investigated the role of interleukin-1 (IL-1)-associated cytokines upon a single ozone exposure (1 ppm for 1 h) using IL-1α-, IL-1β-, and IL-18-deficient mice or an anti-IL-1α neutralizing antibody underlying the rapid epithelial cell death. Here, we demonstrate the release of the alarmin IL-1α after ozone exposure and that the acute respiratory barrier injury and inflammation and airway hyperreactivity are IL-1α-dependent. IL-1α signaling via IL-1R1 depends on the adaptor protein myeloid differentiation factor-88 (MyD88). Importantly, epithelial cell signaling is critical, since deletion of MyD88 in lung type I alveolar epithelial cells reduced ozone-induced inflammation. In addition, intratracheal injection of recombinant rmIL-1α in MyD88^acid mice led to reduction of inflammation in comparison with wild type mice treated with rmIL-1α. Therefore, a major part of inflammation is mediated by IL-1α signaling in epithelial cells. In conclusion, the alarmin IL-1α released upon ozone-induced tissue damage and inflammation is mediated by MyD88 signaling in epithelial cells. Therefore, IL-1α may represent a therapeutic target to attenuate ozone-induced lung inflammation and hyperreactivity.

Keywords: epithelial cell; inflammation; interleukin-18; interleukin-1α; interleukin-1β; myeloid differentiation factor-88; ozone.

Figure 1

Interleukin (IL-1)α, IL-1β, and IL-18 expression in the lung 24 h after ozone exposure. Expression of IL-1α, IL-1β, and IL-18 mRNA (A) and protein (B) in lung of wild type mice, 24 h after single ozone exposure (1 ppm during 1 h). Representative data of two-independent experiments are shown. Results are expressed as mean ± SEM and are from one experiment with n = 4–6. Statistical test: Mann–Whitney, p value: *<0.05.

Figure 2

Inflammation in interleukin (IL-1)α^−/−, IL-1β^−/−, and IL-18^−/− mice 24 h after ozone exposure. Total cells (A), neutrophils and myeloperoxidase (B), protein and lipocalin-2 (C), tissue remodeling parameters, tissue inhibitor metalloproteinase 1, and matrix metalloproteinase 9 (D), histology, scale bar = 100 µm (F), in IL-1α^−/−, IL-1β^−/−, and IL-18^−/− mice and airway hyperreactivity in IL-1α^−/− mice (E), and microscopic analysis of the lung (F) at 24 h after ozone exposure. Representative data of two-independent experiments are shown. Results are expressed as mean ± SEM and are from one experiment, with n = 5–7. Statistical test: ordinary one-way ANOVA, with Bonferroni post test, p value: ****<0.0001, ***<0.001, **<0.01, *<0.05.

Figure 2

Inflammation in interleukin (IL-1)α^−/−, IL-1β^−/−, and IL-18^−/− mice 24 h after ozone exposure. Total cells (A), neutrophils and myeloperoxidase (B), protein and lipocalin-2 (C), tissue remodeling parameters, tissue inhibitor metalloproteinase 1, and matrix metalloproteinase 9 (D), histology, scale bar = 100 µm (F), in IL-1α^−/−, IL-1β^−/−, and IL-18^−/− mice and airway hyperreactivity in IL-1α^−/− mice (E), and microscopic analysis of the lung (F) at 24 h after ozone exposure. Representative data of two-independent experiments are shown. Results are expressed as mean ± SEM and are from one experiment, with n = 5–7. Statistical test: ordinary one-way ANOVA, with Bonferroni post test, p value: ****<0.0001, ***<0.001, **<0.01, *<0.05.

Figure 3

Interleukin-1 (IL-1)α antibody blockade reduces ozone-induced lung inflammation. A single administration of IL-1α neutralizing antibody 12 h before ozone exposure in wild type mice reduced inflammatory parameters. Total cell (A), neutrophils (count and myeloperoxidase) (B), total proteins and lipocalin-2 (C), matrix metalloproteinase 9, and tissue inhibitor metalloproteinase 1, (D) and histology with epithelial damage and inflammatory score (E), associated with histological picture (F), scale bar = 100 µm, 24 h after ozone exposure. Representative data of two-independent experiments are shown. Results are expressed as mean ± SEM and are from one experiment, with n = 5–6. Statistical test: ordinary one-way ANOVA, with Bonferroni post test, p value: ****<0.0001, ***<0.001, **<0.01, *<0.05.

Figure 4

The myeloid differentiation factor-88 (MyD88) pathway in epithelial cell is involved in inflammation induced by ozone. Total cell number (A), neutrophil counts and myeloperoxidase (B), protein and lipocalin-2 (C), tissue remodeling parameters, matrix metalloproteinase 9, and tissue inhibitor metalloproteinase 1 (D), and histology, scale bar = 100 µm (E) are shown in wild type, MyD88^−/− and MyD88^acid mice, 24 h after ozone exposure. Representative data from two-independent experiments are given. Results are expressed as mean ± SEM and are from one experiment, with n = 5–6. Statistical test: ordinary one-way ANOVA, with Bonferroni post test, p value: ****<0.0001, ***<0.001, **<0.01, *<0.05.

Figure 4

The myeloid differentiation factor-88 (MyD88) pathway in epithelial cell is involved in inflammation induced by ozone. Total cell number (A), neutrophil counts and myeloperoxidase (B), protein and lipocalin-2 (C), tissue remodeling parameters, matrix metalloproteinase 9, and tissue inhibitor metalloproteinase 1 (D), and histology, scale bar = 100 µm (E) are shown in wild type, MyD88^−/− and MyD88^acid mice, 24 h after ozone exposure. Representative data from two-independent experiments are given. Results are expressed as mean ± SEM and are from one experiment, with n = 5–6. Statistical test: ordinary one-way ANOVA, with Bonferroni post test, p value: ****<0.0001, ***<0.001, **<0.01, *<0.05.

Figure 5

The myeloid differentiation factor-88 (MyD88) pathway in epithelial cell partially though interleukin-1α signaling is involved in inflammation and cell damage induced by ozone. Total cell number (A), neutrophil counts and myeloperoxidase (B), protein and lipocalin-2 (C), tissue remodeling parameters, matrix metalloproteinase 9, and tissue inhibitor metalloproteinase 1 (D), histological scoring, epithelial cells desquamation (E), and histology, scale bar = 100 µm (F) are shown in wild type, MyD88^−/− and MyD88^acid mice, 24 h after ozone exposure. Results are expressed as mean ± SEM and are from one experiment, with n = 5–6. Statistical test: ordinary one-way ANOVA, with Bonferroni post test, p value: ****<0.0001, ***<0.001, **<0.01, *<0.05.