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. 2015 May;172(10):2588-603.
doi: 10.1111/bph.13080. Epub 2015 Mar 24.

LPS exacerbates functional and inflammatory responses to ovalbumin and decreases sensitivity to inhaled fluticasone propionate in a guinea pig model of asthma

A P P Lowe  1 , R S Thomas, A T Nials, E J Kidd, K J Broadley, W R Ford
Affiliations

LPS exacerbates functional and inflammatory responses to ovalbumin and decreases sensitivity to inhaled fluticasone propionate in a guinea pig model of asthma

A P P Lowe et al. Br J Pharmacol. 2015 May.

Abstract

Background and purpose: Asthma exacerbations contribute to corticosteroid insensitivity. LPS is ubiquitous in the environment. It causes bronchoconstriction and airway inflammation and may therefore exacerbate allergen responses. This study examined whether LPS and ovalbumin co-administration could exacerbate the airway inflammatory and functional responses to ovalbumin in conscious guinea pigs and whether these exacerbated responses were insensitive to inhaled corticosteroid treatment with fluticasone propionate (FP).

Experimental approach: Guinea pigs were sensitized and challenged with ovalbumin and airway function recorded as specific airway conductance by whole body plethysmography. Airway inflammation was measured from lung histology and bronchoalveolar lavage. Airway hyper-reactivity (AHR) to inhaled histamine was examined 24 h after ovalbumin. LPS was inhaled alone or 24 or 48 h before ovalbumin and combined with ovalbumin. FP (0.05-1 mg·mL(-1) ) or vehicle was nebulized for 15 min twice daily for 6 days before ovalbumin or LPS exposure.

Key results: Ovalbumin inhalation caused early (EAR) and late asthmatic response (LAR), airway hyper-reactivity to histamine and influx of inflammatory cells into the lungs. LPS 48 h before and co-administered with ovalbumin exacerbated the response with increased length of the EAR, prolonged response to histamine and elevated inflammatory cells. FP 0.5 and 1 mg·mL(-1) reduced the LAR, AHR and cell influx with ovalbumin alone, but was ineffective when guinea pigs were exposed to LPS before and with ovalbumin.

Conclusions and implications: LPS exposure exacerbates airway inflammatory and functional responses to allergen inhalation and decreases corticosteroid sensitivity. Its widespread presence in the environment could contribute to asthma exacerbations and corticosteroid insensitivity in humans.

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Figures

Figure 1
Figure 1
Ova and LPS protocols used in the present study. (A) Pre-Ova challenge LPS administration. (B) LPS and Ova co-administration. (C) LPS without Ova protocol.
Figure 2
Figure 2
The mean time courses for changes in sGaw following Ova challenge of Ova-sensitized guinea pigs. Guinea pigs were treated with either saline or 30 μg·mL−1 LPS for 1 h, (A) 72 h and 24 h pre-Ova challenge, or (B) 48 h before Ova challenge and co-administered with Ova. Histograms represent the maximum bronchoconstriction values recorded during the EAR (0–6 h) and LAR (6–12 h). (C) Analysis of the time taken for EAR to recover to 50% of peak bronchoconstriction values. (D) Mean time courses for changes in sGaw following the second of two LPS or saline exposures in non-sensitized guinea pigs. Mean changes in sGaw are expressed as mean ± SEM percentage change from baseline prior to Ova challenge. n = 5–10. *Significantly different from respective naïve or Ova challenge and double-saline treatment group P < 0.05, **P < 0.01, ***P < 0.001; performed with one-way anova followed by Bonferroni post-test.
Figure 3
Figure 3
Response of the airways to nebulized histamine (0.3 mM, 2 min) before and 24 h after Ova challenge of Ova-sensitized guinea pigs. Guinea pigs were treated 72 and 24 h pre-Ova with: (A) saline twice, (B) saline and LPS (30 μg·mL−1), (C) LPS twice, or treated 48 h pre-Ova and with Ova challenge with (D) saline twice, (E) saline and LPS (F) LPS twice. Mean changes in sGaw are expressed as mean ± SEM percentage change from baseline. A negative value represents a bronchoconstriction. n = 5–10. *Significantly different from pre-Ova challenge values P < 0.05, **P < 0.01, ***P < 0.001; performed with a two-tailed t-test.
Figure 4
Figure 4
The total cell (A), macrophage (B), eosinophil (C), lymphocyte (D) and neutrophil (E) counts in bronchoalveolar fluid and IL-13 (F), IL-8 (G) IL-17 (H) in homogenized lung and lavage fluid protein levels (I) of Ova-sensitized guinea pigs determined 24 h after Ova challenge. Guinea pigs were treated with either saline or 30 μg·mL−1 LPS for 1 h either 72 and 24 h pre-Ova or 48 h before Ova challenge and co-administered with Ova. Naïve (unsensitized and challenged), Ova sensitized and challenged double-saline and double-LPS challenged guinea pigs are also shown. IL-8 was undetectable in Ova-sensitized and -challenged guinea pigs and was therefore analysed by Kruskal–Wallis non-parametric anova. Results are expressed as mean ± SEM; n = 5–10. *Significantly different from respective Ova challenge and double-saline treatment group (saline/saline/Ova) P < 0.05, **P < 0.01, ***P < 0.001; performed with one-way anova followed by Bonferroni post-test. ^^Significantly different from naive animals P < 0.01, ^^^P < 0.001. ###Significantly different from saline ×2 P < 0.001.
Figure 5
Figure 5
Bronchiolar changes and goblet cell staining in guinea pigs: naïve (A and E), double-LPS challenged (B and F), Ova sensitized and challenged treated with either saline (C and G) or LPS 48 h pre-Ova and co-administered with Ova (D and H). ASM, airway smooth muscle; E, epithelium; IC, inflammatory cell; G, goblet cell; L, lumen. A–D were stained with haematoxylin and eosin, original magnification 100× (bar = 25 μm). E–H with alcian blue/periodic acid Schiff, original magnification 400× (bar = 100 μm). (I) The mean pathology score of the PB area. (J) The number of mucin-associated goblet cells per 10 000 epithelial pixels. Results are expressed as mean ± SEM; n = 5–10. *Significantly different from respective naïve P < 0.05, **P < 0.01, ***P < 0.001 performed with anova followed by Tukey's post-test. ^Significantly different from saline control (saline/saline+Ova) P < 0.01.
Figure 6
Figure 6
The mean time courses for changes in sGaw following (A) Ova challenge in sensitized guinea pig (B) treatment with LPS (30 μg·mL−1) 48 h pre-Ova and co-administered with Ova challenge in sensitized guinea pigs treated with either inhaled vehicle or FP (0.5 or 1 mg·mL−1), split into twice daily doses. The histogram represents the maximum bronchoconstriction values recorded during the EAR (0–6 h) and LAR (6–12 h). (C) AUC analysis of sGaw values for 0–12, 0–6 and 6–12 h post-Ova challenge. (D) Analysis of the time taken for EARs to recover to 50% of peak bronchoconstriction values after Ova and LPS co-administration. Mean changes in sGaw are expressed as mean ± SEM percentage change from baseline prior to Ova challenge. n = 5–10. *Significantly different from vehicle treatment P < 0.05, performed with anova followed by Dunnett's post-test.
Figure 7
Figure 7
Response of the airways to nebulized histamine delivered in a plethysmograph (0.3 mM, 10% duty cycles and 0.5 L·s–1/chamber over 2 min) in guinea pigs (A) sensitized and challenged with Ova and treated with inhaled vehicle or (B) FP (1 mg·mL−1) split into twice daily doses or (C) sensitized and challenged with Ova treated with LPS (30 μg·mL−1) 48 h pre-Ova and co-administered with Ova and treated with inhaled vehicle or (D) FP (1 mg·mL−1) split into twice daily doses or (E) treated with LPS (30 μg·mL−1) twice and treated with inhaled vehicle or (F) FP (1 mg·mL−1), split into twice daily doses. The histogram represents the maximum bronchoconstriction values recorded during pre- and post-challenge histamine challenge. Values were recorded pre- and 24 h post-Ova challenge. Mean changes in sGaw are expressed as mean ± SEM percentage change from baseline. A negative value represents a bronchoconstriction. n = 5–10. *Significantly different from pre-Ova challenge values P < 0.05, **P < 0.01, ***P < 0.001; performed with a two-tailed t-test.
Figure 8
Figure 8
The total cell (A), macrophage (B), eosinophil (C), lymphocyte (D) and neutrophil (E) counts in the bronchoalveolar fluid and homogenized lung (F) IL-13, (G) IL-8 (H) IL-17 and lavage fluid protein levels (I) in guinea pigs sensitized and challenged with Ova, sensitized and challenged with Ova and treated with LPS (30 μg·mL−1) 48 h pre-Ova and co-administered with Ova or treated with LPS (30 μg·mL−1) twice. Guinea pigs were treated with either inhaled vehicle or FP (0.5 or 1 mg·mL−1) split into twice daily doses. Naïve (unsensitized and challenged) guinea pigs are also shown for comparative purposes only. Results are expressed as mean ± SEM; n = 5–10. *Significantly different from respective vehicle P < 0.05, **P < 0.01, ***P < 0.001; ∧∧∧Significantly different from naïve; performed with anova followed by Dunnett's post-test or by Kruskal–Wallis non-parametric test in the case of IL-8.
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