The Clinical And Experimental Study On The Prevention And Treatment Of Asthma At The T Cell Subsets Level

Background and Objective Asthma is one of the most common chronic diseases characterized by airway lymphocyte and eosinocyte et al infiltration and airway hyperresponsiveness(AHR)in childhood.Over the past three decades,there has been a significant increase incidence in allergic disease such as asthma in the world.The host genetics can be an important factor to influence allergy and asthma development, but the genetic backgrounds of human being can not be changed in a short course. Therefore this marked increase of asthma incidence is almost certainly due to changes in environmental factors, this idea is called as the"hygiene hypothesis".Th1-Th2 model is the initially widely accepted to explain the mechanisms of the hygiene hypothesis.Recently it has been emphasize that a deregulated Th1 and Th2 balance is not the major factor for the development of allergic disease,and the regulatory T cell play the important role.Th1/Th2/Treg model may be well to explain the pathogenesis of asthma.It is great significance that regulate the development of T cell subsets,in particular,to promote the development of Th1/Tr cell subsets in the development of the immune system in children by some certain drugs or microbial agents.In our previous study,we find that the percentage of CD4~+CD25~+Tr cells and the expression of Foxp3 mRNA in asthmatic children was significantly decrease;Neonatal BCG vaccination could alleviated airway inflammation and AHR in OVA-sensitizated/challenged asthma mouse model by induce more Th1/Tr cells ,but RSV infection could reverse the anti-asthma effect of neonatal BCG vaccination.Here,we hypothesized that 1)Glucocorticoids as a choice drug for asthma could regulate the CD4~+Foxp3~+Tr cells by some cytokines and its signal passway.2)It can enhance the anti-asthma effect of neonatal BCG vaccination by Poly I:C and BCG co-vaccination or constructing a recombinant BCG secreting IL-12.To test the hypothesis mentioned above,we conducted clinic and animal experiments in three parts.Part I. Effects of glucocorticoids on Foxp3~+ regulatory T cells and interleukin-2/ interleukin-6 in pediatric asthmaObject:To investigate the effects of inhaled fluticasone propionate(FP) on Foxp3~+regulatory T cells,interleukin-2/interleukin-6 and STAT5 expression of childhood in vivo and the effects of dexamethasone(Dex)on its in vitro Methods:Totally 15 patients with acute asthma exacerbation,15 children with asthma remitted by inhaled FP and 10 children for health examination and without atopic disorders or history of allergic diseases or respiratory infections within a month as controls were recruited in this study from Oct.2006 to Mar.2007. The each PBMC of other six health children were assigned into four groups(blank,Dex,PHA,PHA plus Dex)in the experiment in vitro,which to observe the effects of Dex on above-mentioned factors.The percentage of CD4~+Foxp3~+ Tr cells were detected by flow cytometry.The levels of IL-2,IL-6 in plasma and supernatant were assayed by ELISA.The levels of STAT5/p-STAT5 of PBMC were detected by Western blot.Results:The percentage of CD4~+Foxp3~+Tr cells in exacerbation asthmatic children was significantly lower than of the control group both pre-(P<0.05)and post-stimulation with PHA(P<0.01).The percentage of CD4~+Foxp3~+Tr cells in FP remitted asthmatic children was significantly higher than in exacerbation asthmatic children both pre- and post-stimulation.The percentage of CD4~+Foxp3~+Tr cells of all groups was significantly higher after PHA stimulation 24 hours,the percentage of remission and control children were increase to 1.89,2.01 times respectively, and the exacerbation children only to 1.56 times.The level of IL-6 in plasma of asthmatic children was higher than in control and FP remitted children, while there was no significant difference in the level of IL-2 in plasma of three groups.The percentage of CD4~+Foxp3~+Tr cells,the level of IL-2 and IL-6 in PBMC of health children were significantly higher after PHA,stimulation,and decreased after Dex administration;The pSTAT5 expression level in exacerbation asthmatic children was significantly lower than in control and FP remitted children,and the STAT5 expression have no significant differences after PHA-stimulation.Dex can inhibit activation effects of STAT5 signaling pathway of PHA.Conclusions:Inhaled fluticasone propionate may effectively increase the percentage of CD4~+Foxp3~+Tr cells in exacerbation asthmatic children by decrease the levels of IL-6 and activate STAT5 signaling pathway.Dex can increase the level of IL-6 in supernatant of Dex-stimulated PBMC of health children in vitro,and inhibit the up-regulation effects on the percentage of CD4~+Foxp3~+Tr cells and activation of STAT5 signaling pathway of PHA;different glucocorticoids have divergent effects on Foxp3 by different mechanism.Part II. Effects of neonatal BCG and Poly I:C co-vaccination on development of T cell subsets and airway inflammation in OVA-induced murine model of asthma.Objective:To explore the effects of neonatal BCG and Poly I:C co-vaccination on the development of spleen T cell subsets and airway inflammation of OVA-induced murine model of asthma. Methods:(1)Thirty-two neonatal BALB/c mices were divided 4 groups:control,BCG,Poly I:C and BCG+Poly I:C groups,which inoculated with BCG and/or Poly I:C intraperiotoneally within 2～3days after birth. Four weeks later, spleen cells of mice were isolated and the percentage of Tc1/Th1/Tc2/Th2/Treg cells respectively were detected by flow cytometry at single cells level.(2)Forty neonatal BALB/c mices were divided 5 groups: control,OVA,BCG+OVA,Poly I:C+OVA and BCG+Poly I:C+OVA groups, which inoculated with BCG and/or Poly I:C intraperiotoneally within 2～3 days after birth.Four weeks later,all groups except control group,undergone OVA sensization and challenge.Bronchoalveolar lavage was performed after last challenge.Cells in BALF were counted.Cytokines in BALF and serum OVA-specific IgE were detected by ELISA and inflammatory characteristics of lungs was scored by staining with hematoxylin and eosin.Results:(1)The percentages of Th1,Tc1 cells of BCG-vaccinated mice, Poly I:C-vaccinated mice and BCG+Poly I:C-vaccinated mice were significant higher than control mice(P<0.05 or P<0.01),and there was no difference among the three vaccinated groups.The ratio of Th1/Th2 and total IFN-γ/IL-4 of three vaccinated groups were higher than control group,but not the ratio of Tc1/Tc2.The Th1/Th2 ratio of BCG+Poly I:C-vaccinated group was higher than BCG-vaccinated group(P<0.05).The percentages of CD4~+Foxp3~+Tr cells were no difference among the four groups.(P>0.05)(2)OVA-sensitizated/challenged mice showed obvious asthmatic manifestation;The numbers of total white cells,lymphocytes, neutrophils,and eosinophils in the BALF from all OVA-sensitized/challenged groups(OVA,BCG+OVA,Poly I:C+OVA,and BCG+Poly I:C+OVA group)were all significantly greater than those in control(P<0.01).BCG+OVA group,BCG+Poly I:C+OVA group had significantly lower total white cells, lymphocytes and eosinophils in BALF as compared with OVA group or Poly I:C+OVA group,there are no difference between BCG+OVA group and BCG+Poly I:C+OVA group;All OVA-sensitized/challenged groups had significantly lower IFN-γ(P<0.05), higher IL-4(P<0.05),lower IL-10(P<0.01)level in BALF,higher IgE (P<0.01)level in serum as compared with control group.BCG+OVA group and BCG+PolyI:C+OVA group had significantly higer IFN-γlevel in BALF(P<0.05 or P<0.01),lower IgE(P<0.05)level in serum as compared with OVA group or PolyI:C+OVA group respectively.There was no significant difference in IFN-γ,IL-4,IgE level between BCG+OVA group and BCG+PolyI:C+OVA group or OVA group or PolyI:C+OVA group. There was no significant difference in IL-4,IL-10 level between all experimental groups;Histological score of peribronchiolitis, perivasculitis, and alveolitis in all OVA-sensitized/challenged groups was significantly higher than that in control.BCG+OVA group and BCG+PolyI:C+OVA group had significantly lighter peribronchiolitis,perivasculitis,and alveolitis than OVA,Poly I:C+OVA group(P<0.05).However, there was no difference between BCG+OVA and BCG+PolyI:C+OVA group or OVA and PolyI:C+OVA group.Conclusions:Neonatal Poly I:C vaccination can induce Th1/Tc1 immune response like BCG,but can't decrease the IgE level of serum and alleviate the airway inflammation in OVA-induced murine model of asthma as BCG.,and has no a synergistic effect of BCG vaccination.It's suggest that Th1/Tc1 immune response induced by virus and BCG respectively have the divergent effect on asthma.Part III. Effects of neonatal recombinant Balillus Calmette-Guerin secreting IL-12 vaccination on development of T cell subsets and airway inflammation of OVA-induced murine model of asthma.Objective:To explore the effects of neonatal recombinant BCG secreting IL-12 vaccination on the development of spleen T cell subsets and airway inflammation in OVA-induced murine model of asthma.Methods:(1)Thirty-two neonatal BALB/c mices were divided 4 groups:control,BCG,r-BCGand M12(a shuttler Plasmid secreting IL-12) groups,which inoculated with PBS,BCG,r-BCG,M12 respectively intraperiotoneally within 2～3days after birth. Four weeks later,spleen cells of mice were isolated and the percentage of Tc1/Th1/Tc2/Th2/Treg cells respectively were detected by flow cytometry at single cells level.(2)Forty neonatal BALB/c mices were divided 5 groups:control,OVA, BCG+OVA,r-BCG+OVA and M12+OVA groups,which inoculated with PBS,PBS,BCG,r-BCG,M12 respectively intraperiotoneally within 2～3days after birth.Four weeks later,all groups except control group,undergone OVA sensization and challenge.Bronchoalveolar lavage was performed after challenge.Cell in BALF were counted.Cytokines in BALF and serum OVA-specific IgE were detected by ELISA and inflammatory characteristics of lungs was scored by staining with hematoxylin and eosin.Results:(1)The percentages of Th1,Tc1 cell,the ratio of Th1/Th2and total IFN-γ/IL-4,and index of spleen of BCG-vaccinated mice and r-BCG-vaccinated mice were significant higher than control mice and M12-vaccinated mice respectively(P<0.05or P<0.01),and there was no difference between BCG and r-BCG-vaccinated group.The percentages of Th2,Tc2,Tr cells were no difference among the four groups.(P>0.05)(2) OVA-sensitizated/challenged mice showed obvious asthmatic manifestation; The numbers of total white cells,lymphocytes, neutrophils,and eosinophils in the BALF from all OVA-sensitized/challenged groups(OVA, BCG+OVA,r-BCG+OVA,and M12+OVA group)were all significantly greater than those in control(P<0.01).BCG+OVA group,r-BCG+OVA group had significantly lower total white cells,lymphocytes and eosinophils in BALF as compared with OVA group or M12+OVA group,there are no difference between BCG+OVA group and r-BCG+OVA group;All OVA-sensitized/challenged groups had significantly lower IFN-γ(P<0.05), higher IL-4(P<0.05),lower IL-10(P<0.01)level in BALF,higher IgE(P<0.01) level in serum as compared with control group.BCG+OVA and r-BCG+OVA group had significantly higer IFN-γlevel in BALF(P<0.05 or P<0.01),lower IgE level in serum(P<0.05)as compared with OVA group or M12+OVA group respectively.There was no significant difference in IFN-γ,IL-4,IgE level between BCG+OVA and r-BCG+OVA group or M12 and OVA group.There was no significant difference in IL-4,IL-10 level between all OVA-sensitized/challenged groups;Histological score of peribronchiolitis,perivasculitis,and alveolitis in all OVA-sensitized/ challenged groups was significantly higher than that in control.BCG+OVA and r-BCG+OVA group had significantly lighter peribronchiolitis, perivasculitis,and alveolitis than OVA and M12+OVA group respectively (P<0.05).Histological score of peribronchiolitis and alveolitis of r-BCG group was significantly lighter than BCG group(P<0.05).Conclusions:Neonatal recombinant BCG secreting IL-12 vaccination can significantly decrease the IgE level of serum and alleviate the airway inflammation in OVA-induced murine model of asthma.r-BCG vaccination is better than BCG to alleviate the airway inflammation not only by develop Tc1 and Th1 cell subsets and elevate Th1/Th2 ratio but other mechanisms.