| Background and Objective Bronchial asthma is the most common chronic airway inflammatory disease in childhood, which has become the public health problem in worldwide. Allergic asthma has been defined as a disease of immunodysregulation, where the pathology is a direct consequence of excessive T-helper type 2 immune responses (Th2) to allergens in the lungs. Disease is associated with increased secretion of type-2 cytokines such as interleukin (IL)-4, IL-5 and IL-13 which, among other things, promotes enhanced IgE levels, induces pulmonary eosinophilic inflammation and increased mucus production in the airways.Th17 cells are a new subset of helper T cell characterized by the production of IL-17A, IL-17F, IL-6, IL-21 and IL-22. The proinflammatory cytokine IL-17 has previously been forwarded as a link between activated T-lymphocytes and the recruitment and activation of neutrophils in various types of airway inflammation. It has been shown that the concentration of IL-17 is increased in BALF, sputum, and blood from patients with asthma. Recently, it has been suggested that at least two inflammatory subtypes of asthma exist: the eosinophilic and the non-eosinophilic type. Neutrophils are specially prominent in acute, severe exacerbations of asthma and potentially contribute to airway gland hypersecretion, bronchial hyper-reactivity and to airway wall remodelling. Given that Th17 cells play a crucial role in the pathogenesis of neutrophilic inflammation, Th17 might be a predominant player in non-eosinophilic Asthma. However, the exact role of Th17 in asthma is not well known. In a series of mouse model of allergic airway inflammation, blockade of IL-17 exhibited different results in the airway. Both Th1 and Th17 cells co-localize within the region of inflammation and they may require each other for recruitment and/or entry to this region. In the lung, however, the reverse entry sequence has been reported in mice receiving a post-vaccination challenge of Mycobacteriumtuberculosis; Th17 cells were recruited to the lung prior to the appearance of IFN-γ-producing memory cells and IL-17-induced chemokine expression was essential for the rapid accumulation of these Th1 effectors.In our preliminary data, RSV reversed the anti-asthma effect of neonatal BCG vaccination in BALB/c mice. IL-12~+ -recombined BCG inhibited the inflammatory response by decreasing IL-17 and regulating the imbalance between IL-17/IFN-γ.The aim of this study is to investigate the effect of neonatal BCG/ IL-12~+ -recombined BCG vaccination on development of Th cells and Th17/Th1 balance, determine the effect and mechanism of neonatal BCG vaccination on murine asthma model through IL-17/IFN-γbalance and Th17/Th1 balance, elucidate the effect and mechanism of neonatal IL-12~+ -recombined BCG vaccination on murine asthma model through IL-17/IFN-γbalance and Th17/Th1 balance.To test the hypothesis mentioned above, we conducted experiments in animal model in three parts.Part one Effect of neonatal BCG and IL-12~+ -recombined BCG vaccination on development and function of Th cellsObjective: To explore the effects of neonatal BCG and IL-12~+ -recombined BCG vaccination on the development and function of lung and spleen Th cell subsets .Methods: (1) Neonatal BALB/c mice were divided into 3 groups: control, BCG, and rBCG groups, which inoculated with BCG and rBCG subcutaneously within 2～3 days after birth. Four weeks later, lung cells and spleen cells of mice were isolated and the percentage of four groups of Th cells respectively were detected by flow cytometry at single cells level. (2) RNA was extracted from lung and spleen tissue, and then reverse transcribed into cDNA. Real time PCR were performed to identify the gene expression of transcription factors including RORγt, T-bet, GATA3 and Foxp3. (3) Single-cell suspensions of lung and spleen were prepared , plated in 12 well plates (2×106 cells/mL) in duplicate, and cultured with medium containing RSV/OVA/LPS at 37°C in 5% CO2 for 72 h. The supernatants were then collected and stored at -20°C until analysis. IFN-γand IL-17 were assayed by ELISA kits.Results: (1) In comparison with mice in the BCG and control group , RORγt and T-bet mRNA and the percentages of CD3~+IL-17~+ cells and Th17 cells in the lung of mice in the rBCG group were increased. Compared with mice in the control group, T-bet mRNA in the lung of mice in the rBCG group was increased. The cultures of lung cells from mice in the BCG and rBCG group contained significantly higher levels of IL-17 , IFN-γand IL-17/IFN-γratio compared with the mice in the control group. (2) The mice in the BCG and rBCG group had a higher mRNA expression of T-bet, Foxp3 and lower mRNA expression of RORγt than those in the control group. In comparison with mice in the BCG and control group , the percentages of CD3~+IFN-γ~+ cells, Th1 cells, CD4~+CD25~+Foxp3~+ cells and ratio of Th1/Th17 in the lung of mice in the rBCG group were increased.Conclusion: (1) Neonatal BCG and rBCG vaccination have impact on the development of Th cells, but the target tissue itself participates actively in creating the site-specific milieu and contribute to the development of Th cells. (2) Neonatal BCG rBCG vaccination promotes enhanced IL-17/Th17 development in lung and induced Th1 cell, Tr in spleen. These data suggest IL-12 play an important role in the development of Th1, Th17 and Tr.Part two Effect and mechanism of neonatal BCG vaccination on murine asthma model through IL-17/IFN-γbalance Objective: To determine the effect and mechanism of neonatal BCG vaccination on OVA sensitized mice and OVA sensitized and challenged mice, and investigate the role of Th17/Th1 balance in the pathogenesis of asthma.Methods: (1) Neonatal BALB/c mices were divided into 3 groups: control, OVA, BCG~+OVA groups. Mice in BCG~+OVA group inoculated with BCG subcutaneously within 2～3 days after birth. Then Using the intraperitoneal OVA with alum sensitization protocol at 4-week and 6-week as mice in OVA group. Mice which were used to perform asthma model were challenged by an aerosol of OVA at 7- week for one week. (2) Bronchoalveolar lavage was performed before challenge. Cells in BALF were counted. Cytokines in BALF and serum OVA-specific IgE were detected by ELISA and Real time PCR were performed to identify the gene expression of transcription factors including RORγt, T-bet, GATA3 and Foxp3. (3) Inflammatory characteristics of lungs was scored by staining with hematoxylin and eosin, and airway hyperresponsiveness to methacholine in conscious, spontaneously breathing animals was measured by enhanced pause.Results: (1) After sensitization, none of the mice in the three groups showed obvious asthmatic manifestation. Difference was not found in peribronchiolitis, perivasculitis and alveolitis among the mice in the three groups. The numbers of total white cells,lymphocytes, neutrophils in the BALF of the mice from all OVA-sensitized groups (OVA, BCG~+OVA) were significantly greater than those in the control group. In comparison with OVA group, the numbers of total white cells, lymphocytes, neutrophils in the BALF were decreased in the BCG~+OVA group. Mice in the BCG~+OVA group had lower IL-17 concentration and higher IL-10, IL-12, IFN-γconcentration in BALF than mice in the OVA group. Ratio of IL-17/IFN-γwas reduced in mice of the BCG~+OVA group. (2) OVA-sensitizated/challenged mice showed obvious asthmatic manifestation. The numbers of total white cells and percentages of lymphocytes, neutrophils and eosinophils in the BALF of the mice from all OVA-sensitized/challenged groups (OVA, BCG~+OVA) were significantly greater than those in the control group. Mice in BCG~+OVA group had significantly lower total white cells and percentages of lymphocytes, neutrophils and eosinophils in BALF and airway hyperresponsiveness than those in the OVA group. Mice in the BCG~+OVA group had lower concentration of IL-17 and higher concentration of IFN-γin BALF than those in the OVA group. Ratio of IL-17/IFN-γwas downregulated in mice of BCG~+OVA group.Conclusion: (1) After OVA sensitization airway inflammation was not found in murine model, but inflammatory cells were recruited, increased OVA-specific IgE level and airway hyperreactivity were exist. (2) Enhanced Th2 response was showed after OVA sensitization, which induced increased OVA-specific IgE level and IL-5, IL-10 level in BALF. (3) After OVA sensitization inflammatory cells recruitment and airway hyperreactivity were mediated by IL-17. (4) Neonatal BCG vaccination reduce airway inflammatory cells recruitment and airway hyperreactivity through the regulation of the balance of IL-17/IFN-γ, but have not impact on enhanced Th2 response and increased OVA-specific IgE level. (5) After challenge, airway inflammatory cells recruitment and airway hyperreactivity were reduced in the neonatal BCG vaccinated mice by .the regulation of the balance of IL-17/IFN-γ.Part three Effect and mechanism of neonatal IL-12~+ -recombined BCG vaccination on murine asthma modelObjective: To investigate Effect and mechanism of neonatal IL-12~+ -recombined BCG vaccination on murine asthma model.Methods: (1) Neonatal BALB/c mice were divided three groups: control, OVA and rBCG~+OVA groups, which inoculated with rBCG subcutaneously within 2～3 days after birth. Then Using the intraperitoneal OVA with alum sensitization protocol at 4-week and 6-week as mice in OVA group. Mice which were used to perform asthma model were challenged by an aerosol of OVA at 7- week for one week. (2) Bronchoalveolar lavage was performed before challenge. Cells in BALF were counted. Cytokines in BALF and serum OVA-specific IgE were detected by ELISA and Real time PCR were performed to identify the gene expression of transcription factors including RORγt, T-bet, GATA3 and Foxp3. (3) Inflammatory characteristics of lungs was scored by staining with hematoxylin and eosin, and airway hyperresponsiveness to methacholine in conscious, spontaneously breathing animals was measured by enhanced pause.Results: (1) After sensitization, in comparison with OVA group inflammatory cell recruitment and airway hyperresponsiveness were decreased in the rBCG~+OVA group. T-bet mRNA was increased and GATA3, Foxp3 mRNA were decreased in the lung of mice in the rBCG group compared with mice in the OVA group. (2) Mice in the BCG~+OVA group had significantly lower total white cells and percentages of lymphocytes, neutrophils and eosinophils in BALF and airway hyperresponsiveness, OVA specific IgE than those in the OVA group. Mice in the BCG~+OVA group had higher percentages of Th1 cell and Tr cell than those in the OVA group. Ratio of IL-17/IFN-γwas downregulated in mice of BCG~+OVA group by increased level of IFN-γin BALF.Conclusions: (1) After sensitization or challenge, airway inflammatory cells recruitment and airway hyperreactivity were reduced in the neonatal BCG vaccinated mice. (2) Neonatal rBCG vaccination elicited protection in murine asthma model by downregulation of IL-17/IFN-γratio, which is different from BCG. (3) Neonatal rBCG vaccination induced Tr recruitment to mediate protection in murine asthma model.(4) Diversity neonatal inmmune stimulation had different impact on the prognosis of asthma. |
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