Early Postnatal Exposure to Cigarette Smoke Leads to Later Airway Inflammation in Asthmatic Mice

Abstract

Background and objective: Asthma is one of the most common airway inflammatory diseases. In most cases, asthma development is related to ubiquitous harmful environmental exposure factors in early-life. Previous studies have indicated that smoking can promote asthma development and increase the difficulty of asthma control. The aim of this study was to determine the effects of early-life CS exposure on ovalbumin (OVA)-sensitized asthmatic mice.

Methods: Pathological and immunological functions were analyzed in an adult asthma mice model in which mice were sensitized with OVA combined with early-life CS exposure.

Results: Mice exposed to CS for only 5 weeks demonstrated significantly reduced pulmonary compliance, increased airway inflammation, and augmented cellular and humoral immune responses. In addition, CS inhalation was sufficient to facilitate OVA sensitization and challenge asthmatic development. Meanwhile, CS exposure amplified regulatory T cell-mediated immunity inhibition, but still did not offset the increased effector T cell-mediated inflammatory response.

Conclusion: Early-life CS exposure is significantly associated with later pulmonary injury and aggravation of T-cell immunologic derangement in asthmatic mice.

Fig 1. Schematic of the mouse model of early-life expose to CS and OVA sensitization.

The processes of CS exposure and OVA sensitization in BALB/c mice are illustrated. The number in grey box indicates the number of weeks post-delivery. The numbers above the line indicate the time in postnatal days. The arrow represents the time of each treatment. The treatment method and time are described above or below the arrow.

Fig 2. Early-life CS exposure aggravated asthmatic airway inflammation.

(A) Pulmonary resistance was analyzed with the flexiVent forced oscillation technique (SCIREQ). Pulmonary resistance was measured in response to methacholine (MCh) at the indicated concentration in the air exposure with PBS sensitization (air-PBS), CS exposure with PBS sensitization (CS-PBS), air exposure with OVA sensitization (air-OVA), and CS exposure with OVA sensitization (CS-OVA) groups. The percentage rates of the baseline were normalized to 100. (B) The numbers of eosinophils, neutrophils, lymphocytes, macrophages, and total cells were counted in the BALF of the air-PBS, CS-PBS, air-OVA, and CS-OVA groups. (C) HE staining of lung sections from the air-PBS (upper left), CS-PBS (upper right), air-OVA (lower left), and CS-OVA (lower right)-treated mice. (D) The degree of inflammation of the groups in (C) was analyzed by scoring the cells around the airways (0, no cells; 1, a few cells; 2, a ring of cells 1 cell layer deep; 3, a ring of cells 2–4 cell layers deep; and 4 with a ring of cells more than 4 cell layers deep) in the four groups. All values are the means ±SD of the results from 6 mice per group. ^#P<0.05 vs. Air-PBS group; *P<0.05 vs. Air-OVA group.

Fig 3. Early-life CS exposure induced Treg-mediated immune suppressive activity.

(A-C) The population of CD4⁺CD25⁺ Tregs of lung tissue was analyzed by flow cytometry. The Treg frequency (A) and number (B) were determined from the data shown in (C). Foxp3 expression of lung tissue was estimated by RT-PCR (D) and western blotting (E-F). The proliferation of lung T cells at different ratios of CD4⁺CD25^- to CD4⁺CD25⁺ were assayed by the [³H]-thymidine incorporation method. The fresh Tregs and the induced Tregs were isolated from the lung of untreated mice and CS-PBS mice, respectively. (G) The values are the means ±SD of the results from 6 mice per group. ^#P<0.05 vs. Air-PBS group; *P<0.05 vs. Air-OVA group in (A, B, D and F). ^#P<0.05 vs. Fresh Tregs in (G).

Fig 4. Early-life CS exposure suppressed cellular immunity.

(A) The percentage of (CD4⁺CD25⁺) Tregs was analyzed in lungs of the air-PBS, CS-PBS, air-OVA, and CS-OVA-treated mice. The cytokines INF-γ (B), IL-4, -5, and -14 (C), IL-9 (D), and IL-17, -21, -23 (E) were detected in BALF samples from the air-PBS, CS-PBS, air-OVA, and CS-OVA groups. The ratio of Treg/effector T cells (%) in the lungs were calculated in F. (G) Serum antibody concentrations were determined by ELISA in the same groups as above. All data were obtained by flow cytometric analysis, and the values are the means ±SD of the results from 6 mice per group. ^#P<0.05 vs. Air-PBS group; *P<0. 05 vs. Air-OVA group.