The Protective Effect Of Neonatal BCG Vaccination On Airway Inflammation And The Underlying Mechanisms In A Mouse Model Of Asthma

Allergic asthma has been defined as a disease of immunodysregulation, which associated with pulmonary eosinophilic inflammation and increased mucus production in the airways. An attractive explanation is offered by the "hygiene hypothesis", which suggests a decrease of or an altered exposure to microbes in the environment results in alteration of immune-regulation. BCG is considered as a strong inducer of T-helper type 1 immune response and modulates the development of asthma both in animal models and human beings. Importantly, our previous study has shown that neonatal BCG vaccination elicited long-term protection on allergic airway inflammation in young and aged mice, which provides a new insight for the prevention of asthma.BCG vaccination/infection stimulates the production of Th1 cytokines and decreases Th2 cytokine secretion in both BALF and the splenetic supernatants. However, the exact mechanism underlying the BCG vaccination preventing the development of asthma remains obscure. With the discovery and functional analysis of Th17 and Treg, we want to know whether and how these new subsets of T cells involve in the process of protective effects of BCG on asthma. It has been reported that IL-17 is required to induce optimum Th1 in some condition, while inhibits the Th1 immune response at other condition. Given the mutual effects of Th17 and Thl, we proposed that Th17 may be play a role in the prevention of BCG on asthma.BCG has been reported to induce immune-regulatory responses. However, the role of Treg in the effects of BCG on allergic inflammation has been little studied. M. vaccae regulates the allergic host immune responses through the induction of Treg (IL-10 and TGF-β), rather than affecting the Th1/Th2 balance. Indeed, BCG vaccinations ameliorate de novo local eosinophilic inflammation induced by allergen and increase the numbers of CD4+CD25+ Treg cells and Foxp3 expression. Collectively, these data suggest Treg may involve in the protection against allergic asthma. Based on the aforementioned studies, whether Th17 and Treg involve in the protective effect of BCG vaccination on asthma needs to further confirm.The most acceptable view is the enhanced Th1 immune response by BCG vaccination play a central role in the inhibition of asthma. Previous studies focused on the increased IFN-γlevel of BALF and/or cultured supernatant of spleen, yet the local enhanced IFN-γ+ CD4 T has been considered to play a critical role against allergic airway inflammation. The migration of FN-y-positive CD4+T and CD8+T cells from circulation and/or the rapid expansion of the resting memory CD4+ T and CD8+ T cells in the local lung contribute to the accumulation. It is more acceptable for the former, as effector T cell can migrate into non-lymph tissue in a low-frequency pattern in the stable condition, while they can rapidly influx into inflamed tissues. In fact, it is hard to establish a real memory immune in BCG vaccination or M.tb choronical infection as the substantial antigen. However, an unresolved issue in these studies is where the IFN-y-positive Thl cells induced by BCG vaccination stem and how these cells recruit into the flamed site of pulmonary after OVA challenge. Thus, we put forward to the hypothesis that "the increased Thl in the draining lymph node for the site of BCG vaccination contribute to enhanced Thl cells in lung after OVA challenge and yield to a protective effect on asthma".To confirm this hypothesis, we first detect the changes of Thl, Th17 and Treg in the lung after BCG vaccination both in stable-state and in OVA-challenged condition, we found BCG vaccination significantly increased Thl cells after OVA challenge, while other T subset have no differences. Furthermore, we found the increased Thl in the lung resulted from the ILN (not from spleen), and the ILN serves as a pool of Thl cells and migrated into the lung directly to alleviate the OVA-induced airway inflammation and AHR. These data support further that neonatal BCG vaccination elicits a protection of asthma, and provide a new insight of immune vaccination. Part 1:Neonatal BCG vaccination elicits a protection of airway inflammation and airway hyperresponsiveness via the recruitment of IFN-γ-expression CD4+T cells in the lung.Objective:To investigate whether neonatal BCG vaccination at the base of tail elicits the protective effects from the development of asthma and to examine the changes of Thl, Th17 and Treg in the lung in both resting and OVA challenge condition.Methods:①C57BL/6 neonates were vaccinated with BCG At age of 6-8 weeks, these mice were invided into three groups:negative control mice (saline/saline), asthma model mice (saline/OVA) and BCG-treated mice (BCG/OVA). The protocol for asthma model described as previous. After the last OVA challenge, AHR, total cell counts and cell differentiation were detected and the airway inflammation and mucus secretion were examined. Then mRNA level of IFN-γ, Foxp3 and IL-17A in the lung and IFN-γconcentration in BALF were determined by real-time PCR and ELISA respectively.②In a reparatory experiment, mice were divided into 4 groups:saline/saline for negative control, saline/OVA for asthma model control, BCG/saline and BCG/OVA. Within 24 h after the last OVA challenge, cell suspensions of lung were obtained and decteced the frequency of Th1, Th17 and Treg via FCM. Finally, cell suspensions were isolated from MLN, spleen and ILN and decteced the percentage of IFN-γ-expession CD4+ T and CD8+T via FCM.Results:①Neonatal BCG vaccination at the base of tail decreased AHR, inhibited eosinophils infiltration and mucus overproduction (P< 0.05).②In resting state, the IFN-y-positive CD4+T cells were significantly increased in the lung compared with that in saline/saline mice, while no differences were observed about IFN-γ-positive CD8+T, Th17, IL-17-positive CD8+T and Treg in the lung. Interestingly, the frequency of Thl cells was significantly increased in BCG/OVA mice in compared to that in BCG/saline(p< 0.001). Although IFN-y-positive CD8+T was also significantly enhanced in the lung of BCG/OVA mice (p=0.026), the major of IFN-γ-positive T cells was CD4+T cells. However, these changes were not observed for Th17, IL-17-expression CD8+T and Treg.③In resting condition, BCG vaccination have no effects on the changes of IFN-y-expression CD4+T and CD8+T in the MLN and spleen. In contrast, the frequency of Thl was significantly increased in ILN compared to that in saline/saline mice (p=0.002). To our surprise, OVA challenge enhanced significantly the frequency of Th1 in the spleen from BCG/OVA mice compared to BCG/saline mice, which was accompanied with the reduced frequency of IFN-γ-expression CD4+T in the ILN in BCG/OVA mice compared to BCG/saline mice (p=0.035). Although the differences were not significant, the frequency of IFN-γ-expression CD4+T and CD8+T in MLN were indeed reduced in BCG/OVA mice compared with those in BCG/saline mice.Conclusion:①Neonatal BCG vaccination elicits the protective effects on the airway inflammation and AHR in an experimental asthma model, which were corrected with the recruitment of large IFN-γ-expression CD4+T cells but not the Th17 and Treg cells into the lung.②The enhanced Thl cells in the lung after OVA challenge in mice vaccinated with BCG were result from ILN via spleen pathway. Part 2:phenotype and functional analysis of CD4+T cells in ILNObjective:To investigate whether BCG vaccination influences the phenotype and function of CD4+T cells in ILN for the site of BCG vaccination.Methods:C57BL/6 neonates were vaccinated with BCG as described in Part 1. We dynastically observed the cell counts of spleen and ILN at the age of 4W,12W and 30W. To confirm further IFN-γ-expression CD4+ T was indeed increased in ILN of mice vaccinated with BCG, we also dectected the frequency of IFN-γ-expression CD4+ T and CD8+T cells in ILN at the age of 18W and 30W. At the age of 12W, cell counts of MLN, MLN and thymus were also counted, and mRNA level of IFN-γ, IL-17A and Foxp3 in ILN were evaluated by real-time PCR. In addition, the frequencies of Th17, IL-17A-expression CD8+ T and Treg were dectected via FCM in spleen and ILN. Subsequently, cell cycle of ILN, activation and cell phenotype of CD4+T cells in ILN were also illusiated by FCM. The prolification capacity of lymphocytes and CD4+T cells were determined by MTT and CFSE dilution analysis. Finally, the secretary capacity of cytokine was examined by ELISA.Results:①Cell counts of ILN from BCG mice increased early at 4 W, and peaked at 12 W, then formed a plat until 30 W. In contrast, cell counts of spleen, thymus and non-draining Lymph node (LN) such as MLN and MLN were no difference between in BCG and saline mice.②Besides at the age of 12 W(as described in part 1), the enhanced Th1 cells were also observed in ILN in BCG mice compared to that in saline mice whether at age of 18W or at 30W(p=0.027 at age of 18W; p=0.04 at age of 30 W, respectively).However, the similar changes were not obseaved for IFN-γ-expression CD8+T cells.In contrast, the frequency of Thl and IFN-γ-expression CD8+T cells in spleen were similar whether BCG vaccination or not. The percentage of Th17, IL-17A-expression CD8+T and Foxp3+CD4+T in spleen and ILN were not changed between in BCG and saline mice. The mRNA level of IFN-γand the transcriptor T-bet were significanted increased in lung of BCG mice compared with that in saline mice, while the mRNA level of IL-17A and the transcriptor Foxp3 were similar regardless of BCG vaccination.③Cell prolification index of ILN, the CD69 and CD25 molecular expression on CD4+T cells in ILN in BCG mice were similar to that in saline mice in steady-state condition. In contrast, CD25 expression on CD4+T cells in ILN of BCG mice was significantly increased in the stimulator compared to that in saline mice. The percentage of Tef/em in ILN in BCG mice was significantly but slighter than that in saline mice. The proliferation of CD4+T cells in ILN of BCG mice measured by MTT and CFSE-dilution was enhanced significantly in contrast to that in saline mice, which was accompanied with the increased IFN-y concentration in cultured supenarant of lymphocyte from ILN of BCG mice.Conclusion:①BCG vaccination promotes the prolification of ILN but not spleen, and much Thl but IFN-γ-expression CD8+T, Th17, IL-17-expression CD8+T and Treg was enriched in ILN of mice vaccinated with BCG.②CD4+ T cells in ILN of mice vaccinated with BCG was in a resting state in steady-state condition, which have a potential capacity of activation, prolification and secretion. Part 3:Lymphocyte isolated from ILN of BCG mice elicits directly protective effects on airway inflammation and AHR via a protential of migration into lung after OVA challeng.Objiective:To investigate directly the lymphocyte enriched with Thl cells in ILN of BCG mice elicits protective effects on airway inflammation and AHR in a mouse model of asthma.Methods:For adoptive transfer test of ILN cell suspensions, mice were divided into three groups:BCG-ILN/OVA, saline-ILN/OVA and PBS/OVA. Single-cell suspensions were obtained from ILN, and transferred into recipient mice. After the consequent 3 days challenge, AHR were assayed and samples were collected for inflammation measurement.In addition, the IFN-γmRNA expression in the lung and IFN-γconcentration in BAL fluid were analyzed by real-time PCR and ELISA respectively. Together, we also performed the adoptive transfer test of spleen cell suspensions. In other experiment for directly detect the migration capacity of CD4+T cells of ILN, single-cell suspensions of ILN were stained with CFSE in vitro, and injected into recipient mice. The percentage of CFSE-positive cells and CD4 T cells were assayed using FACS analysis. Furthermore, we detected the mRNA expression of associated chemokine receptors of T cells in ILN and matched chemokine in Lung.Results:①Adoptive transfer of ILN cells from BCG mice inhibited AHR, attenuated total numbers of eosinophils in BALF, and decreased pulmonary inflammation and mucus secretion. The IFN-γmRNA expression in the lung was enhanced from BCG-ILN/OVA mice compared with that from saline-ILN/OVA mice, which accompanied with the enhanced IFN-γselection in BALF. However, adoptive transferring of spleen cells from BCG mice has no benefit.In support, CFSE-positive CD4+T cells in BCG-ILN/OVA were enhanced in lung compared to those in saline-ILN/OVA (p=0.045). As expected, the mRNA level of CXCR3 in ILN of BCG mice was higher significantly than that in saline mice. Intriguingly, the matched chemokine CXCL9,10 and 11 for CXCR3 in Lung challenged with OVA were also significantly increased compared to saline mice. Conclusion:Lymphocyte enriched with Thl cells in ILN of BCG mice elicits protective effects on airway inflammation and AHR in a mouse model of asthma. Moreover, the protective effect was associated with the potential migration of CD4+T into inflamed lung. Moreover, the high expression of CXCR3 in ILN contributes to the potential migration of CD4+T cells.