The Effect Of Neonatal Multiple BCG Vaccination On The Airway Remodeling Of Asthma And The Underlying Immunological Mechanisms

Asthma is a chronic airway inflammatory disease, which is characterized by the presence of increased numbers of eosinophils and a predominantly Th2 response. A persistent Th2 response leads to airway remodeling, characterized by increased globet cell numbers and consequent mucus production, smooth muscle hypertrophy and hyperplasia, collagen and elastic fiber deposition, and increased angiogenesis.Current asthma management strategies principally involve the use of inhaled corticosteroids to control symptoms, but it has little effect on inhibiting the disease progression and the development of airway remodeling. Therefore, preventative interventions may be a better approach to protect individuals from asthma. BCG as a strong inducer for Thl-type immune response, is considered as a potential candidate. Lately a mata-analysis for 23 studies suggested a protective effect of BCG exposure on childhood asthma occurrence, supports the hypothesis that exposure to the BCG vaccine in early life prevents asthma. Several factors may affect the efficacy of BCG vaccination, including the route and time of delivery. Our group has demonstrated previously that neonatal BCG vaccination has a long-term effect on inhibiting AHR and eosinophilia in mouse asthma. However, the potential of neonatal multiple BCG vaccination in preventing the development of airway remodeling associated with repeated allergen exposure remains to be addressed.Concerning the underlying mechanisms of BCG vaccination against asthma, most previous studies demonstrated that BCG inhibited Th2 response by inducing a strong Thl-type immune response. But lately some studies also suggested that BCG or killed Mycobacterium vaccae confer protection against airway inflammation by giving rise to allergen-specific regulatory T cells. Th17 is the newest member of the Th cell family. It's function in asthma, especially in chronic and severe asthma, is receiving attention. How neonatal BCG vaccination eliciting an immunological response in an airway remodeling model remains to be addressed.In this study, using a chronic asthma model of OVA-sensitized and repeated challenged mice, we further determined if neonatal multiple BCG vaccination is capable of reducing the airway remodeling. Then we used Flow Cytometric and Real-time PCR analysis to examine the differentiation of pulmonary T cell populations and to determine the underlying mechanisms of neonatal BCG vaccination in inhibiting asthma.Part I The establishment of murine model of airway remodeling and the effect of neonatal multiple BCG vaccination on the airway remodeling of asthmaObjective:To investigate whether neonatal multiple vaccination with BCG could decrease the airway remodeling of asthma.Methods:BALB/c mice were injected intradermally with 25μl Freeze-dried living BCG suspension [105 infection units (IU)] at birth, and boosted two times on days 7,14 with the same dose of BCG. At 5 weeks of age, mice were immunized i.p. on days 0,14 with 20μg of OVA emulsified in 100μl Imject Alum. Groups of mice that had been sensitized with OVA were challenged with an aerosolized OVA thrice a week for 8 weeks. Age-and sex-matched mice that had been sensitized and challenged with saline at each time point were used as negative controls.24h after the final OVA challenge, airway hyperresponsiveness (AHR) to inhaled methacholine was measured. Mice were sacrificed 48h after the final OVA challenge and bronchoalveolar lavage fluid (BALF) and lungs were analyzed. Lungs were processed for either histologic staining (HE, PAS and Masson trichrome) or immunostaining (a-SMA). Lung inflammation, smooth muscle layer thickness, peribronchial fibrosis, airway mucus expression were analyzed.Results:OVA sensitive and repeated challenge induced airway remodeling including increased mucus production, smooth muscle hyperplasia, increased collagen deposition and airway hyperresponsiveness. Multiple BCG vaccination decreased levels of BALF total cells (30.27±3.06×104 vs 41.72±3.11×104 BCG+OVA vs OVA; p<0.05) and eosinophils (1.22±0.28×104 vs 8.46±1.34×104; BCG+OVA vs OVA; p<0.01). BCG vaccination also inhibited airway responsiveness, reduced the levels of peribronchial a-smooth muscle actin stained area (0.93±0.08 vs 1.45±0.12μm2/μm; BCG+OVA vs OVA; p< 0.05) and peribronchial collagen deposition (0.64±0.06 vs 0.88±0.09μm2/μm; BCG+OVA vs OVA;p<0.05). The mucus overproduction also decreased.Conclusion:Allergen sensitive and repeated aerosolized allergen challenge established murine model of airway remodeling. Multiple neonatal BCG vaccination reduced levels of allergen-induced airway remodeling and airway hyperresponsiveness in mice.Part II The possible mechanisms of BCG vaccination inhibiting the airway remodeling of miceObjective:To investigate the pulmonary T cell response following neonatal BCG vaccination and the possible mechanisms of BCG vaccination inhibiting the airway remodeling.Methods:48h after the final OVA challenge, mice were sacrificed and BALF was collected. Levels of selected cytokines and chemokines in BALF (IFN-y, IL-5, IL-17, IL-13, eotaxin-1, TGF-β1) were assayed using ELISA kits. Lung lymphocytes were isolated and labeled with T cells surface Abs (CD3, CD4, CD8 and CD25) and intracellular Abs (IFN-γ, IL-4, IL-17A, IL-10, Foxp3). Data were collected and analyzed using a FACScan for three-color flow cytometry. Lung tissue RNA was extracted with Trizol. Real-time PCR quantitative analyses were performed to detect the level of IFN-y, IL-17A, Foxp3mRNA. Lung tissues paraffin were also immunostained with TGF-β1 Ab, and peribronchial TGF-β1+cells were counted.Results:Neonatal BCG vaccination induced significantly increased level of BALF IFN-y and decreased the levels of BALF IL-5 and BALF eotaxin-1 compared with non-vaccinted and chronic OVA challenge alone. The level of IL-13 also decreased but had not statistical significance. Flow cytometric detection of T cell subpopulations in the lungs showed that BCG vaccination increased almost 6-fold CD3+CD8- IFN-γ+T cells (5.01±0.66 vs 0.83±0.18% of CD3+T cells) in the lungs as compared with the non-vaccinated mice. No difference was observed in the frequency of CD3+CD8- IL-4+T cells, CD3+CD8- IL-17+T cells and CD4+Foxp3+T cells(Treg) in the lungs of BCG vaccinated and non-BCG vaccinated mice, but IL-4+T cells and IL-17+T cells of OVA and BCG+OVA group mice significantly increased comparing with control mice. The results of IFN-y, IL-17A, Foxp3mRNA level in lung tissue were similar with flow cytometric detection of T cell subpopulations. Exposure of mice to chronic OVA challenge induced a significant increase in the level of TGF-β1 in BALF and peribronchial TGF-β1+cells. Multiple BCG vaccination decreased the level of TGF-β1 in BALF and peribronchial TGF-β1+cells.Conclusion:Our data suggested that neonatal multiple BCG vaccination primarily increased the level of IFN-y and IFN-y producing T cells in lungs. But has little effect on IL-17 producing T cells and pulmonary regulatory T cells. BCG vaccination decreased the level of IL-5 and Eotaxin-1 in BALF, also reduced levels of TGF-β1 in BALF and peribronchial TGF-β1+cells which may contribute to the abatement of airway remodeling. Our previous study showed that compared to other studies with single dose of BCG immunization, neonatal multiple BCG vaccination elicited longer-term protection by inhibiting allergic airway inflammation. In the present study, we further investigated the effect of multiple vaccination with BCG at newborn on the airway remodeling of chronic asthma and determined the possible immunological mechanisms. Conclusions obtained from our data were listed as following:1. We established a stable murine model of chronic asthma with typical airway remodeling features.2. Multiple neonatal BCG vaccination reduced levels of allergen-induced airway remodeling and airway hyperresponsiveness, as well as the airway inflammation.3. Multiple neonatal BCG vaccination decreased the levels of IL-5 and TGF-β1 in lungs, which may contribute to the inhibition of airway remodeling.4. Multiple BCG vaccination increased the frequency of Thl cells and IFN-y level, but not Treg cells, which suggested that the inhibition of asthma by BCG vaccination was not associated with an increased number of pulmonary regulatory T cells, but was positively correlated with the increase of IFN-y producing T cells.5. Thl 7 cells and IL-17 contribute to the development of airway remodeling. Neonatal BCG vaccination did not change the frequency of Th17 cells and IL-17 level.