Cigarette smoke enhances Th-2 driven airway inflammation and delays inhalational tolerance

Abstract

Background: Active smoking increases asthma severity and is related to diminished treatment efficacy. Animal models in which inhalation of both allergen and mainstream cigarette smoke are combined can help us to understand the complex interaction between both agents. We have recently shown that, in allergic mice, the airway inflammation can be cleared by repeated allergen challenge, resulting in the establishment of a state of inhalational tolerance.

Methods: In this study, we assessed in vivo the impact of cigarette smoke on the efficacy and time course of this form of tolerance induction. We exposed sensitized mice to concurrent mainstream cigarette smoke and allergen (Ovalbumin- OVA) and measured the airway inflammation at different time points.

Results: We first confirmed that aerosolized OVA administered for a prolonged time period (4-8 weeks) resulted in the establishment of tolerance. Concurrent OVA and smoke exposure for 2 weeks showed that tobacco smoke enhanced the Th-2 driven airway inflammation in the acute phase. In addition, the induction of the tolerance by repeated inhalational OVA challenge was delayed significantly by the tobacco smoke, since 4 weeks of concurrent exposure resulted in a more persistent eosinophilic airway inflammation, paralleled by a more mature dendritic cell phenotype. However, smoke exposure could not prevent the establishment of tolerance after 8 weeks of antigen exposure as shown by both histopathology (disappearance of the Th-2 driven inflammation) and by in vivo functional experiments. In these tolerized mice, some of the inflammatory responses to the smoke were even attenuated.

Conclusion: Cigarette smoke enhances acute allergic inflammation and delays, but does not abrogate the development of tolerance due to prolonged challenge with inhaled antigen in experimental asthma.

Figures

Figure 1

Cigarette smoke and allergen exposure protocols. Allergen exposure protocols (Allergen Protocols): Allergen Protocol 1A: Exposure of sensitized C57BL/6 mice to PBS or OVA aerosols for 2 weeks. (n = 8 mice/group). Allergen Protocol 1B: Prolonged PBS or OVA aerosol exposure in sensitized mice (8 weeks). (n = 8 mice/group). Allergen Protocol 2: OVA sensitized mice were exposed to OVA for 8 weeks ("chronic"), followed by HEL/alum immunisation and short-term ("acute") HEL aerosol challenge (OVA/HEL group). Control mice were exposed to PBS for 8 weeks, immunized with HEL/alum and challenged with either PBS (PBS/PBS group) or with HEL (PBS/HEL group). (n = 8–12 mice/group). Smoke exposure protocol (Smoke Protocol): C57BL/6 mice are exposed to Smoke or Air for 8 weeks. (n = 6 mice/group). Combined protocols (Combination Protocols): Combination Protocol 1: similar to Allergen Protocol 1B, combined with Smoke exposures or Air. (n = 8 mice/group). Combination Protocol 2: similar to Allergen Protocol 2, combined with or without Smoke. (n = 8 mice/group). Combination Protocol 3A: similar to Allergen Protocol 1A, combined with Smoke exposures or Air. (n = 8 mice/group). Combination Protocol 3B: 4 weeks of OVA aerosol or PBS exposures in sensitized mice, combined with Smoke exposures or Air. (n = 8 mice/group). Vertical arrows = aerosol challenges (OVA, HEL or PBS). Stripped horizontal bars = period of Smoke or Air exposures.

Figure 2

BALF cell differentiation (A) and BALF cytokines (pg/ml) MCP-1 and KC (B) after 8 week exposure to Smoke or Air in C57BL/6 mice (Smoke Protocol). n = 6 mice/group. * p ≤ 0.05: Smoke vs Air (Mann-Whitney U test); Bars indicate mean ± SEM. ND = Not Detectable (detection limit = 1–2 pg/ml).

Figure 3

Histology (Congo Red) of the airways of the four groups of mice after 8 week exposure to OVA or PBS aerosols combined with Smoke or Air in sensitized C57BL/6 mice (Combination Protocol 1) (Magnification = 400×; A = PBS/Air, B = PBS/Smoke, C = OVA/Air, D = OVA/Smoke).

Figure 4

Results obtained from combining 8 weeks OVA or PBS aerosol exposure with Smoke or Air in sensitized C57BL/6 mice (Combination Protocol 1). (A.) BALF analysis of total cell counts and differentiation after prolonged exposure (8 weeks) to a combination of OVA or PBS aerosols and Smoke or Air. (B) Evaluation of the peribronchial inflammation via a semi-quantitative method (Mean score/airway). (C) Percentage of T – lymphocytes in lung that are CD4+CD69+ (as a percentage within the T cell population). (D) Percentage of dendritic cells in digested lung tissue (percentage of total lung cells). (E) Th-2 cytokine (IL-13 and TARC) profiles in BALF (pg/ml). (F) Cytokines MCP-1 and KC in BALF (pg/ml). n = 8 mice/group; bars indicate mean ± SEM. * p ≤ 0.05: all groups vs PBS/Air (Mann-Whitney U test); + p ≤ 0.05: PBS/Smoke vs OVA/Smoke (Mann-Whitney U test); # p ≤ 0.05: OVA/Smoke vs OVA/Air (Mann-Whitney U test); ND = Not Detectable (detection limit = 1–2 pg/ml)

Figure 5

Results obtained 8 weeks of OVA aerosols followed by re-sensitisation (i. p.) and re-challenge to HEL aerosols (Allergen Protocol 2) combined with concurrent Smoke or Air-exposures (Combination Protocol 2). (A) BALF cell differentiation. (B) Peribronchial inflammation (Mean score/airway). n = 8 mice/group; bars indicate mean ± SEM. * p ≤ 0.05: OVA/HEL/Smoke vs OVA/HEL/Air (Mann-Whitney U test);

Figure 6

BALF cell differentiation and cytokines (pg/ml) IL-13 and TARC after respectively 2 (Fig. 6A and 6B) and 4 (Fig. 6C and 6D) weeks of concurrent exposure to OVA/PBS and Smoke or Air in C57BL/6 mice (Combination Protocol 3A and 3B). n = 8 mice/group; bars indicate mean ± SEM. * p ≤ 0.05: all groups vs PBS/Air (Mann-Whitney U test); + p ≤ 0.05: OVA/Smoke vs. PBS/Smoke (Mann-Whitney U test); # p ≤ 0.05: OVA/Smoke vs. OVA/Air (Mann-Whitney U test); ND = Not Detectable (detection limit = 1–2 pg/ml)

Figure 7

The evolution in the levels of TARC in BALF (pg/ml) and the percentage of eosinophils in BALF over time after 2, 4 and 8 weeks of concurrent exposure to OVA/PBS and to Smoke or Air in C57BL/6 mice (Combination Protocol 1, 3A and 3B). # p ≤ 0.05: OVA/Smoke vs. OVA/Air (Mann-Whitney U test);