Protective role of interleukin-10 in ozone-induced pulmonary inflammation

Abstract

Background: The mechanisms underlying ozone (O₃)-induced pulmonary inflammation remain unclear. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is known to inhibit inflammatory mediators.

Objectives: We investigated the molecular mechanisms underlying interleuken-10 (IL-10)-mediated attenuation of O₃-induced pulmonary inflammation in mice.

Methods: Il10-deficient (Il10(-/-)) and wild-type (Il10(+/+)) mice were exposed to 0.3 ppm O₃ or filtered air for 24, 48, or 72 hr. Immediately after exposure, differential cell counts and total protein (a marker of lung permeability) were assessed from bronchoalveolar lavage fluid (BALF). mRNA and protein levels of cellular mediators were determined from lung homogenates. We also used global mRNA expression analyses of lung tissue with Ingenuity Pathway Analysis to identify patterns of gene expression through which IL-10 modifies O₃-induced inflammation.

Results: Mean numbers of BALF polymorphonuclear leukocytes (PMNs) were significantly greater in Il10(-/-) mice than in Il10(+/+) mice after exposure to O₃ at all time points tested. O₃-enhanced nuclear NF-κB translocation was elevated in the lungs of Il10(-/-) compared with Il10(+/+) mice. Gene expression analyses revealed several IL-10-dependent and O₃-dependent mediators, including macrophage inflammatory protein 2, cathepsin E, and serum amyloid A3.

Conclusions: Results indicate that IL-10 protects against O₃-induced pulmonary neutrophilic inflammation and cell proliferation. Moreover, gene expression analyses identified three response pathways and several genetic targets through which IL-10 may modulate the innate and adaptive immune response. These novel mechanisms of protection against the pathogenesis of O₃-induced pulmonary inflammation may also provide potential therapeutic targets to protect susceptible individuals.

Figure 1

Changes in number of PMNs (A) and total protein concentration (B) recovered in BALF from Il10^+/+ and Il10^−/− mice in response to air or 0.3 ppm O₃ (means ± SE; n = 8/group). *p < 0.05, air versus 0.3 ppm O₃; **p < 0.05, Il10^+/+ versus Il10^−/−. (C) H&E staining of 5-μm lung sections from Il10^+/+ and Il10^−/− mice after air or 0.3 ppm O₃ (72 hr). Arrows illustrate areas of greatest neutrophilic inflammation, particularly around terminal bronchioles (TB) and blood vessels (V). Sections are representative of mice from each treatment group. (D) Ki67 immunostaining of 5-μm lung sections from Il10^+/+ and Il10^−/− mice after air or 0.3 ppm O₃ (72 hr). Arrows indicate foci of cellular proliferation (arrows indicate the arrowheads shown in the inset). Sections are representative of mice from each treatment group.

Figure 2

(A) MIP-2 protein levels in BALF recovered from Il10^+/+ and Il10^−/− mice exposed to air and 0.3 ppm O₃ (means ± SE; n = 4 per group). *p < 0.05, air versus O₃; **p < 0.05, Il10^+/+ versus Il10^−/−. (B) Socs3 mRNA expression in Il10^+/+ and Il10^−/− mice in response to air or 0.3 ppm O₃. We determined the ratio of Socs3 to 18S RNA from whole-lung homogenates from PCR (means ± SE; n = 3 per group). *p < 0.05, air versus 0.3 ppm O₃. (C) Lung CD86 protein expression in Il10^+/+ and Il10^−/− mice exposed to air and 0.3 ppm O₃ (means ± SE; n = 3–5 per group). We detected CD86 expression from whole-lung homogenates by Western blot analysis and reprobed blots with actin for normalization. +p < 0.05.

Figure 3

(A) EMSA to determine DNA binding activity of total NF-κB (top left) and specific p50 κB (top right) after 0.3 ppm O₃ in Il10^+/+ and Il10^−/− mice. Each lane represents nuclear protein pooled from three representative animals of each treatment group and was repeated three times. SB, shifted band; SSB, supershifted band; FP, free probe. Quantified p65 κB determined by ELISA is presented below (means ± SE; n = 3 per group). *p < 0.05, air versus 0.3 ppm O₃; **p < 0.05, Il10^+/+ versus Il10^−/−. (B) Phosphorylated STAT3 (p-STAT3) in Il10^+/+ and Il10^−/− mice in response to air or 0.3 ppm O₃. We determined the ratio of p-STAT3 to total STAT3 from whole-lung homogenates by Western blot analysis (means ± SE; n = 3 per group). *p < 0.05, air versus 0.3 ppm O₃.

Figure 4

IPA illustrates the three most highly associated networks (A–C) of IL-10–dependent genes in response to O₃. Red symbols, genes whose profiles were derived from Supplemental Material Figure 5A [see Supplemental Material, Figure 5, Table 2 (doi:10.1289/ehp.1002182)]; yellow symbols, genes with profiles that followed the pattern illustrated in Supplemental Material Figure 5B (see Supplemental Material, Figure 5, Table 2); green symbols, genes with profiles that differ between Il10^+/+ and Il10^−/− mice [see Supplemental Material, Figure 5D, Table 2 (doi:10.1289/ehp.1002182)]. Gene abbreviations are identified in Supplemental Material, Table 2. GPCR, G-protein–coupled receptor.