Glutathione peroxidase-1 primes pro-inflammatory cytokine production after LPS challenge in vivo

Abstract

Reactive oxygen species produced during the innate immune response to LPS are important agents of anti-pathogen defence but may also cause oxidative lung damage. Glutathione peroxidase-1 (gpx-1) is an anti-oxidant enzyme that may protect lungs from such damage. We assessed the in vivo importance of gpx-1 in LPS-induced lung inflammation. Male wild-type (WT) or gpx-1 deficient (gpx-1(-/-)) mice were treated intranasally with PBS or 10 µg LPS and killed 3 and 24 h post LPS. Lungs were lavaged with PBS and then harvested for inflammatory marker expression. LPS caused an intense neutrophilia in WT BALF evident 3 and 24 h post challenge that was reduced in gpx-1(-/-) mice. In addition, LPS-treated gpx-1(-/-) mice had significantly fewer macrophages than LPS-treated WT mice. To understand the basis for this paradoxical reduction we assessed inflammatory cytokines and proteases at protein and transcript levels. MMP-9 expression and net gelatinase activity in BALF of gpx-1(-/-) mice treated with LPS for 3 and 24 h was no different to that found in LPS-treated WT mice. BALF from LPS-treated gpx-1(-/-) mice (3 h) had less TNF-α, MIP-2 and GM-CSF protein than LPS-treated WT mice. In contrast, LPS-induced increases in TNF-α, MIP-2 and GM-CSF mRNA expression in WT mice were similar to those observed in gpx-1(-/-) mice. These attenuated protein levels were unexpectedly not mirrored by reduced mRNA transcripts but were associated with increased 20S proteasome expression. Thus, these data suggest that gpx-1 primes pro-inflammatory cytokine production after LPS challenge in vivo.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Effect of LPS on BALF cellularity in WT and gpx-1−/− mice.

Mice were treated with PBS or LPS (10 µg/mouse) for 3 h (Panels A–C) and 24 h (Panels D–E) and the number of total cells, neutrophils and macrophages counted. Data are shown as mean ± SEM for 6 mice per treatment group. Clear bars represent PBS-treated mice and black bars represent LPS-treated mice. *P<0.001 vs PBS (ANOVA and Bonferroni's post hoc test), †P<0.001 vs WT (for total cells and neutrophils, ANOVA followed by Bonferroni's post hoc test), †P<0.05 vs WT (for macrophages, ANOVA followed by Bonferroni's post hoc test).

Figure 2. Effect of LPS treatment on MMP-9 expression in BALF of WT and gpx-1−/− mice.

WT and gpx-1−/− mice were treated with LPS (10 µg/mouse) for 3 h (Panel A) and 24 h (Panel C) and MMP-9 expression in BALF of individual mice measured by zymogarphy. Panels A & C show MMP-9 assayed by gelatin zymography under reducing conditions and Panels B & D are the respective densitometric measurements. Densitometry data are shown as mean ± SEM for 5–6 mice per treatment group. Clear bars represent PBS-treated mice and black bars represent LPS-treated mice. *P<0.05 vs respective PBS-treated mice (ANOVA and Bonferroni's post hoc test).

Figure 3. LPS-induced increases in net gelatinase activity is not affected by gpx-1 deletion.

Panels A & B show net free gelatinase activity in neat BALF from individual mice treated with LPS for 3 and 24 h, respectively. Data are shown as mean ± SEM for 6 mice per treatment group. Clear bars represent PBS-treated mice and black bars represent LPS-treated mice. *P<0.05 vs respective PBS-treated mice (ANOVA and Bonferroni's post hoc test), **P<0.01 vs respective PBS-treated mice (ANOVA and Bonferroni's post hoc test).

Figure 4. LPS-induced increases in BALF TNF-α, MIP-2 and GM-CSF protein are reduced in gpx-1−/− mice.

Data are shown as mean ± SEM for 6 mice per treatment group. Clear bars represent PBS-treated mice and black bars represent LPS-treated mice. *P<0.001 vs respective PBS-treated mice (ANOVA and Bonferroni's post hoc test), †P<0.05 vs WT LPS-treated mice (ANOVA followed by Bonferroni's post hoc test).

Figure 5. Effect of LPS on whole lung TNF-α, MIP-2, GM-CSF and MMP-9 mRNA expression in WT and gpx-1−/− mice.

mRNA expression for all genes was measured simultaneously under identical conditions using quantitative real-time PCR. Responses are shown as fold change relative to 18S from 6 individual mice. Clear bars represent WT mice and black bars represent gpx-1−/− mice. *P<0.001 vs PBS (ANOVA and Bonferroni's post hoc test), †P<0.05 vs WT (ANOVA followed by Bonferroni's post hoc test).

Figure 6. Effect of LPS on 20S proteasome concentrations in BALF of WT and gpx-1−/− mice.

WT and gpx-1−/− mice were treated with LPS (10 µg/mouse) for 3 h and 20S proteasome concentrations measured in BALF. Data are shown as the mean for BALF pooled from 5–6 individual mice per treatment group. Clear bars represent PBS-treated mice and black bars represent LPS-treated mice.