Impact And Partial Pathogenesis Of Recombinant Bacillus Calmette-guerin Secreting IL-12 Neonatal Vaccination On OVA-induced Murine Model Of Asthma Post RSV Infection

Background and Objective Asthma is one of the most common chronic inflammation diseases of airway. So far, prevention and treatment of asthma have not met WHO's demands of primary prevention. Balillus Calmette-Guerin(BCG) is an attenuated live vaccine which has been broadly vaccinated in developing countries including China. Animal experiments have showed BCG can induce Th1 immunity response and inhibit the development of asthma, but that has not been proved in human being. Our previous researches have showed that neonatal BCG could attenutate airway inflammation and AHR in OVA-induced murine model of asthma. However, RSV infection could reverse the anti-asthma effects of BCG vaccination.These years mycobacterium has been recombinated via molecular biology techniques and it can express IL-12. It is worth to study to learn if recombinant BCG vaccination can induce Th1 immunity response and interrupt reversed anti-asthma effect of RSV infection.Th17 cell is a new Th cells subset recently discovered, producing IL-17. Increased lever of IL-17 has been found both in human asthmatics and in animal models of asthma, which suggest IL-17 plays an important role in the pathogenesis of asthma. It is meaningful to know whether neonatal BCG vaccination can reduce the lever of IL-17 induced by OVA and whether RSV infection interrupts anti-asthma effect of BCG via IL-17.Our purpose is to know the role of IL-17 in the pathogenesis of experimental asthma and in the reversed anti-asthma effect of RSV infection and whether neonatal recombinant BCG vaccination can interrupt reversed anti-asthma effect of RSV infection so that anti-asthma. This is very helpful to understand the mechanism of reversed anti-asthma effect of RSV infection and is the basis of developing new anti-asthma vaccination.Part I. Impact of IL-17 on anti-asthma effect of RSV infection in OVA-induced murine model of asthma of neonatal Bacillus Calmette-Guerin vaccinationObjective To explore the role of IL-17 in the pathogenesis of experimental asthma and impact of IL-17 on anti-asthma effect of RSV infection in OVA-induced murine model of asthma of neonatal Bacillus Calmette-Guerin vaccinationMethods Neonatal BALB/c mice were divided into five groups: Control, OVA, BCG/OVA, RSV/OVA, BCG/RSV/OVA groups. Neonatal mice were vaccinated with BCG in BCG/OVA and BCG/RSV/OVA groups. Mice of RSV/OVA, BCG/RSV/OVA groups were infected RSV by intranasal inoculation of RSV at the third week after birth. Except control group, mice were sensitized and undergone OVA challenge in the other four groups. Inflammatory cell numbers and morphological identification of leucocytes in bronchoalveolar lavage fluid (BALF) were measured by light microscopy. Inflammatory characteristics of lungs were scored by staining with hematoxylin and eosin. Cytokine IFN-γ, IL-5, IL-10 and IL-17 levels in BALF and OVA-specific IgE in serum were measured by ELISA.Results There was a significantly larger number of total cells, lymphocytes, eosinophils and neutrophils in BALF of all asthmatic groups compared with control group(P<0.01 or P<0.05). The total cells and the percentage of lymphocytes in BCG/RSV/OVA group were statistically increased compared with BCG/OVA group(P<0.01 or P<0.05). Histological score of peribronchiolifis,perivasculitis and alveolifis in all OVA-induced groups was significantly higher than that in control group(P<0.01). All groups infected RSV and OVA group had severer perivasculitis and alveolifis than BCG/OVA group(P<0.01 or P<0.05), while there was no significant difference among themselves. The level of IFN-γin BALF was significantly lower in all asthmatic groups compared with control group (P<0.01). The level of IL-5 in BALF was significantly higher in all asthmatic groups compared with control group (P<0.01 or P<0.05). The level of IFN-γand IL-5 in BALF among all asthmatic groups was no significant difference. The level of IL-17 was significantly higher in all asthmatic groups except BCG/OVA group compared with control groups (P<0.05). The level of IL-17 in BCG/RSV/OVA group was statistically increased compared with BCG/OVA group. The level of IL-10 and IL-12 in BALF among all groups was respectively no statistical difference. All OVA-induced groups showed significantly higher serum OVA-specific IgE than control group (P<0.01), while no significant difference between all OVA-induced groups.Conclusions IL-17 played the role in the pathogenesis of asthma. Treatment of neonatal BALB/c mice with BCG can reduce the level of IL-17 in BALF and can attenuate lung inflammation, but this effect can be reversed by RSV infection. It may be led to following increasing IL-17 of airway on RSV infection, and the exact mechanism need to be further studied.Part II. Combined effects of neonatal recombinant Bacillus Calmette-Guerin vaccination secreting IL-12 and RSV infection on OVA-induced murine model of asthmaObjective To explore whether neonatal IL-12 recombinant BCG vaccination can interrupt reversed anti-asthma effect of RSV infection or not.Methods Neonatal BALB/c mice were divided into five groups: Control, OVA, rBCG/OVA, RSV/OVA, rBCG/RSV/OVA groups. Neonatal mice were vaccinated with rBCG only in rBCG/OVA and rBCG/RSV/OVA groups. Mice of RSV/OVA, rBCG/RSV/OVA groups were infected RSV by intranasal inoculation of RSV at the third week after birth. Except control group, mice were sensitized and undergone OVA challenge in the other four groups. Inflammatory cell numbers and morphological identification of leucocytes in bronchoalveolar lavage fluid (BALF) were measured by light microscopy. Inflammatory characteristics of lungs were scored by staining with hematoxylin and eosin. Cytokine IFN-γ, IL-5, IL-10 and IL-17 levels in BALF and OVA-specific IgE in serum were measured by ELISA.Results There was a significantly larger number of total cells, lymphocytes, eosinophils and neutrophils in BALF of all asthmatic groups compared with control group (P<0.01 or P<0.05). The total cells and the percentage of lymphocytes in rBCG/RSV/OVA group were statistically increased compared with rBCG/OVA group ((P<0.01). Histological score of peribronchiolifis,perivasculitis and alveolifis in all OVA-induced groups was significantly higher than that in control group(P<0.01). rBCG/RSV/OVA group had severer perivasculitis and alveolifis than rBCG/OVA group (P<0.01 or P<0.05). The level of IFN-γin BALF was significantly lower in all asthmatic groups compared with control group(P<0.01). The level of IL-5 in BALF was significantly higher in all asthmatic groups compared with control group (P<0.01 or P<0.05). The level of IFN-γand IL-5 in BALF among all asthmatic groups was no significant difference. The level of IL-12 was significantly higher in rBCG/OVA group compared with other groups (P<0.01). The ratio of IFN-γ/IL-5 in in rBCG/OVA group was statistically increased compared with other asthmatic group (P<0.01 or P<0.05). The level of IL-10 in BALF among all groups was no statistical difference. All OVA-induced groups showed significantly higher serum OVA-specific IgE than control group (P<0.01),while no significant difference among all OVA-induced groups.Conclusions Neonatal recombinant BCG vaccination secreting IL-12 can improve the lever of IL-12 in the lung of asthmatic mice. However, IL-12 can be inhibited and infiltration of inflammatory cells and pathological changes of lung are significant on RSV infection. Neonatal recombinant BCG vaccination secreting IL-12 can not interrupt reversed anti-asthma effect of RSV infection. It is still needed to study whether reversed anti-asthma effect of RSV infection is associated with IL-17 and this is helpful to develop more effective vaccination of anti-asthma.