Glutathione Peroxidase-1 Primes Pro-Inflammatory Cytokine Production after LPS Challenge In Vivo

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

Non-essential role for TLR2 and its signaling adaptor Mal/TIRAP in preserving normal lung architecture in mice

Myeloid differentiation factor 88 (MyD88) and MyD88-adaptor like (Mal)/Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) play a critical role in transducing signals downstream of the Toll-like receptor (TLR) family. While genetic ablation of the TLR4/MyD88 signaling axis in mice leads to pulmonary cell death and oxidative stress culminating in emphysema, the involvement of Mal, as well as TLR2 which like TLR4 also signals via MyD88 and Mal, in the pathogenesis of emphysema has not been studied. By employing an in vivo genetic approach, we reveal here that unlike the spontaneous pulmonary emphysema which developed in Tlr42/2 mice by 6 months of age, the lungs of Tlr22/2 mice showed no physiological or morphological signs of emphysema. A more detailed comparative analysis of the lungs from these mice confirmed that elevated oxidative protein carbonylation levels and increased numbers of alveolar cell apoptosis were only detected in Tlr42/2 mice, along with up-regulation of NADPH oxidase 3 (Nox3) mRNA expression. With respect to Mal, the architecture of the lungs of Mal2/2 mice was normal. However, despite normal oxidative protein carbonylation levels in the lungs of emphysema-free Mal2/2 mice, these mice displayed increased levels of apoptosis comparable to those observed in emphysematous Tlr42/2 mice. In conclusion, our data provide in vivo evidence for the non-essential role for TLR2, unlike the related TLR4, in maintaining the normal architecture of the lung. In addition, we reveal that Mal differentially facilitates the anti-apoptotic, but not oxidant suppressive, activities of TLR4 in the lung, both of which appear to be essential for TLR4 to prevent the onset of emphysema

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

<p>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. *<i>P</i><0.001 vs PBS (ANOVA and Bonferroni's <i>post hoc</i> test), ^†<i>P</i><0.05 vs WT (ANOVA followed by Bonferroni's <i>post hoc</i> test).</p

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

<p>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. *<i>P</i><0.05 vs respective PBS-treated mice (ANOVA and Bonferroni's <i>post hoc</i> test).</p

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

<p>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. *<i>P</i><0.001 vs PBS (ANOVA and Bonferroni's <i>post hoc</i> test), ^†<i>P</i><0.001 vs WT (for total cells and neutrophils, ANOVA followed by Bonferroni's <i>post hoc</i> test), ^†<i>P</i><0.05 vs WT (for macrophages, ANOVA followed by Bonferroni's <i>post hoc</i> test).</p

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

<p>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.</p

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

<p>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. *<i>P</i><0.001 vs respective PBS-treated mice (ANOVA and Bonferroni's <i>post hoc</i> test), ^†<i>P</i><0.05 vs WT LPS-treated mice (ANOVA followed by Bonferroni's <i>post hoc</i> test).</p

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

<p>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. *<i>P</i><0.05 vs respective PBS-treated mice (ANOVA and Bonferroni's <i>post hoc</i> test), **<i>P</i><0.01 vs respective PBS-treated mice (ANOVA and Bonferroni's <i>post hoc</i> test).</p

Intranasal and epicutaneous administration of Toll-like receptor 7 (TLR7) agonists provides protection against influenza A virus-induced morbidity in mice

Abstract Toll-like receptor 7 (TLR7) is a pattern recognition receptor that recognizes viral RNA following endocytosis of the virus and initiates a powerful immune response characterized by Type I IFN production and pro-inflammatory cytokine production. Despite this immune response, the virus causes very significant pathology, which may be inflammation-dependent. In the present study, we examined the effect of intranasal delivery of the TLR7 agonist, imiquimod or its topical formulation Aldara, on the inflammation and pathogenesis caused by IAV infection. In mice, daily intranasal delivery of imiquimod prevented peak viral replication, bodyweight loss, airway and pulmonary inflammation, and lung neutrophils. Imiquimod treatment also resulted in a significant reduction in pro-inflammatory neutrophil chemotactic cytokines and prevented the increase in viral-induced lung dysfunction. Various antibody isotypes (IgG1, IgG2a, total IgG, IgE and IgM), which were increased in the BALF following influenza A virus infection, were further increased with imiquimod. While epicutaneous application of Aldara had a significant effect on body weight, it did not reduce neutrophil and eosinophil airway infiltration; indicating less effective drug delivery for this formulation. We concluded that intranasal imiquimod facilitates a more effective immune response, which can limit the pathology associated with influenza A virus infection