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. 2014 Nov 18;9(11):e113180.
doi: 10.1371/journal.pone.0113180. eCollection 2014.

IL-17A and serum amyloid A are elevated in a cigarette smoke cessation model associated with the persistence of pigmented macrophages, neutrophils and activated NK cells

Michelle J Hansen  1 , Sheau Pyng J Chan  1 , Shenna Y Langenbach  1 , Lovisa F Dousha  1 , Jessica E Jones  1 , Selcuk Yatmaz  1 , Huei Jiunn Seow  1 , Ross Vlahos  1 , Gary P Anderson  1 , Steven Bozinovski  1
Affiliations

IL-17A and serum amyloid A are elevated in a cigarette smoke cessation model associated with the persistence of pigmented macrophages, neutrophils and activated NK cells

Michelle J Hansen et al. PLoS One. .

Abstract

While global success in cessation advocacy has seen smoking rates fall in many developed countries, persistent lung inflammation in ex-smokers is an increasingly important clinical problem whose mechanistic basis remains poorly understood. In this study, candidate effector mechanisms were assessed in mice exposed to cigarette smoke (CS) for 4 months following cessation from long term CS exposure. BALF neutrophils, CD4+ and CD8+ T cells and lung innate NK cells remained significantly elevated following smoking cessation. Analysis of neutrophil mobilization markers showed a transition from acute mediators (MIP-2α, KC and G-CSF) to sustained drivers of neutrophil and macrophage recruitment and activation (IL-17A and Serum Amyoid A (SAA)). Follicle-like lymphoid aggregates formed with CS exposure and persisted with cessation, where they were in close anatomical proximity to pigmented macrophages, whose number actually increased 3-fold following CS cessation. This was associated with the elastolytic protease, MMP-12 (macrophage metallo-elastase) which remained significantly elevated post-cessation. Both GM-CSF and CSF-1 were significantly increased in the CS cessation group relative to the control group. In conclusion, we show that smoking cessation mediates a transition to accumulation of pigmented macrophages, which may contribute to the expanded macrophage population observed in COPD. These macrophages together with IL-17A, SAA and innate NK cells are identified here as candidate persistence determinants and, we suggest, may represent specific targets for therapies directed towards the amelioration of chronic airway inflammation.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. Smoke-induced weight loss was reversed 12 weeks after CS cessation.
Male BALB/c mice were either exposed to 6 cigarettes/day, 6 days/week (▪) or sham handled (▴) for 16 weeks. After smoke exposure, groups of mice were then exposed to room air without cigarette smoke for either 4 weeks (∇) or 12 weeks (♦). For all groups body weight was determined weekly. Data are shown as mean ± SE for n = 14–18 per treatment group.
Figure 2
Figure 2. Effect of sub-chronic smoke exposure and 12 weeks of CS cessation on BALF cellularity and lung neutrophilia.
Male BALB/c mice were either exposed to 6 cigarettes/day, 6 days/week (▪) or sham handled (□) for 16 weeks. After smoke exposure a group of mice was then exposed to room air without cigarette smoke for 12 weeks. Total cells (A), macrophages (B) and neutrophils (C) were determined in BALF. Data are shown as mean ± SE for n = 8–11 per treatment group. (D) Single cell suspension of the lungs was used to determine neutrophil numbers in the lung tissue by flow cytometry. Data were analysed by two-way ANOVA and when significance was achieved a post hoc Bonferroni test was performed. #P<0.05 significant post hoc effect of CS compared to sham animals at the same recovery time-point.
Figure 3
Figure 3. Effect of sub-chronic smoke exposure and 12 weeks of CS cessation on BALF and lung CD4+ and activated CD8+ lymphocytes.
Male BALB/c mice were either exposed to 6 cigarettes/day, 6 days/week (▪) or sham handled (□) for 16 weeks. After smoke exposure a group of mice was then exposed to room air without cigarette smoke for 12 weeks. CD4+CD3+ lymphocyte number was determined in individual lung single cell suspensions (A) and BALF cells (B) using FACS analysis. Activated CD8+CD69+ lymphocyte number was determined in individual lung single cell suspensions (C) and BALF cells (D) using FACS analysis. In addition, activated NK cell numbers were quantified in individual lung single cell suspensions (E) and BALF cells (F). Data are shown as mean ± SE for n = 7–8 per treatment group. Data were analysed by two-way ANOVA and when significance was achieved a post hoc Bonferroni test was performed. #P<0.05 significant post hoc effect of CS compared to sham animals at the same recovery time-point.
Figure 4
Figure 4. Sub-chronic smoke exposure resulted in the prolonged presence of pigmented macrophages.
Representative histological staining of hematoxylin and eosin sections from sham and CS exposed mice and after 12 weeks of recovery (A). Magnification, x100 and x1000. The histological sections were scored for the number of lymphoid aggregates (B). The histological sections were scored for the presence of pigmented macrophages (C). Gene expression of the macrophage survival cytokines GM-CSF (D) and CSF-1 (E) was determined by Q-PCR, normalized to 18S rRNA and expressed as a fold change relative to the Sham no recovery group. Data are shown as mean ± SE for n = 7–8 per treatment group for QPCR and n = 4–6 for immunhistochemistry. Data were analysed by two-way ANOVA and when significance was achieved a post hoc Bonferroni test was performed. #P<0.05 significant post hoc effect.
Figure 5
Figure 5. Effect of sub-chronic smoke exposure and 12 weeks of CS cessation on alternative macrophage marker mRNA expression in lung tissue.
Male BALB/c mice were either exposed to 6 cigarettes/day, 6 days/week (▪) or sham handled (□) for 16 weeks. After smoke exposure a group of mice was then exposed to room air without cigarette smoke for 12 weeks. Gene expression of the alternative macrophage markers, MMP-12 (A) and IL-10 (B) was determined by Q-PCR, normalized to 18S rRNA and expressed as a fold change relative to the Sham 0 weeks recovery group. Data are shown as mean ± SE for n = 7–8 per treatment group. Data were analysed by two-way ANOVA and when significance was achieved a post hoc Bonferroni test was performed. #P<0.05 significant post hoc effect.
Figure 6
Figure 6. Effect of sub-chronic smoke exposure and 12 weeks of CS cessation on classic neutrophil mobilization mediators.
Male BALB/c mice were either exposed to 6 cigarettes/day, 6 days/week (▪) or sham handled (□) for 16 weeks. After smoke exposure a group of mice was then exposed to room air without cigarette smoke for 12 weeks. Gene expression of MIP-2α (A), KC (B) and G-CSF (C) was determined by Q-PCR, normalized to 18S rRNA and expressed as a fold change relative to the Sham group. Data are shown as mean ± SE for n = 7–8 per treatment group. Data were analysed by two-way ANOVA and when significance was achieved a post hoc Bonferroni test was performed. #P<0.05 significant post hoc effect.
Figure 7
Figure 7. Effect of sub-chronic smoke exposure and 12 weeks of CS cessation on IL-17A and SAA expression.
Male BALB/c mice were either exposed to 6 cigarettes/day, 6 days/week (▪) or sham handled (□) for 16 weeks. After smoke exposure a group of mice was then exposed to room air without cigarette smoke for 12 weeks. Gene expression of IL-17A (A), SAA (B), IL-6 (C) and IL23 (D) was determined by Q-PCR, normalized to 18S rRNA and expressed as a fold change relative to the Sham group. Data are shown as mean ± SE for n = 6–8 per treatment group. Data were analyzed by two-way ANOVA and when significance was achieved a post hoc Bonferroni test was performed.
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This work was supported by NHMRC (National Health and Medical Research Council) Australia (www.NHMRC.gov.au), project grant number 628492. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.