Effect of neonatal BCG vaccination on phenotype and function of splenic dendritic cells of BALB/c mice / 中华儿科杂志

<p><b>OBJECTIVE</b>The impact of dendritic cells (DCs) and regulatory T cells (Tr) on the pathogenesis of asthma have been investigated over the past decades. As the professional antigen presenting cells, DCs not only prime immune response directing Th0 cells toward different T subtypes but also induce immune tolerance. As an immunoregulator, Bacillus Calmette-Guerin (BCG) has potential to be applied in allergic diseases such as asthma for prevention. Previous study showed that neonatal BCG vaccination could induce Th1/Tr1 development in mice in vivo. To further identify the mechanism of neonatal BCG vaccination on T cell subsets differentiation, the present study was designed to investigate the impact of BCG vaccination on splenic DCs development in neonatal mice.</p><p><b>METHODS</b>Neonatal specific pathogen free (SPF) BALB/c mice (2-3 days) were divided into intraperitoneal BCG-treated group, subcutaneous BCG-treated group and control group; simultaneously adult SPF BALB/c mice (6-8 weeks) were divided into intraperitoneal BCG-treated and control group. The BCG-treated mice were inoculated with 1 x 10(5) CFU BCG, the mice in control group were not inoculated with any vaccine. Four weeks post BCG vaccination, spleen cells were isolated. With flow cytometry, subtype and maturity of splenic DCs were analysed. Moreover, cells were further separated into mononuclear cell by Ficoll solution. The mononuclear cells were stimulated by 1 microg/ml lipopolysaccharide (LPS) for 18 or 10(5) CFU /ml BCG for 48 hours at 37 degrees C in a humidified atmosphere containing 5% CO2 and cytokines concentration was detected by ELISA.</p><p><b>RESULTS</b>(1) CD11c(+) CD8alpha(+) and CD11c(+) CD8alpha(-) DCs were found in spleen cells of the BALB/c mice. In comparison with the control group, the percent of CD11c(+) CD8alpha(-) DCs in intraperitoneal BCG group significantly declined (P < 0.01) and that of CD11c(+) CD8alpha(+) DCs significantly increased (P < 0.01), there were no significant difference in DC subtypes between intraperitoneal and subcutaneous BCG-vaccinated mice. In contrast, the percent of CD11c(+) CD8alpha(-)DCs markedly increased (P < 0.01) and that of CD11c(+) CD8alpha(+)DCs noticeably reduced (P < 0.01) in adult BCG-vaccinated mice. The percent of CD11c(+)CD8alpha(-)DC was significantly higher and that of CD11c(+) CD8alpha(+)DC was significantly lower in adult-vaccinated BALB/c mice than that of neonatal-vaccinated ones. (2) The expression of costimulatory molecules CD40 on CD11c(+) CD8alpha(-) DCs and CD86 on CD11c(+) CD8alpha(+) DCs in neonatal BCG-treated BALB/c mice was higher than the controls. There were no significant difference in expression of costimulatory molecules on DC between neonatal BCG-vaccinated mice. Compared with the control group, expression of CD40 and MHC-II molecules on CD11c(+) CD8alpha(-) and CD11c(+) CD8alpha(+)DC was significantly higher and that of CD86 was significantly lower in adult BCG-vaccinated mice. The expression of costimulatory molecules on DC had no significant difference between neonatal and adult BCG vaccinated BALB/c mice. (3) As compared with the control mice, concentration of IL-12p70 induced by LPS and IL-10 induced by BCG in vitro from spleen cells culture supernatant was noticeably elevated (P < 0.05) in neonatal BCG-treated BALB/c mice, but that of IL-6 did not change by LPS or BCG stimulation.</p><p><b>CONCLUSION</b>(1) By up-regulating splenic CD8alpha(+)DCs and inducing IL-12p70 and IL-10 production in BALB/c mice, neonatal BCG vaccination promoted Th1/Tr1 development. (2) The effect of BCG vaccination on DC was different between neonates and adult BALB/c mice.</p>

Allergic airway response associated with the intestinal microflora disruption induced by antibiotic therapy / 中华儿科杂志

<p><b>OBJECTIVE</b>Over the past several decades, there has been a significant increase in allergy and asthma in the world, which correlates with alterations in microflora and widespread use of antibiotics. The authors have developed a mouse model of antibiotics-induced microbiota disruption. In that model, mice were challenged by intranasal exposure to Aspergillus fumigatus allergens to explore the relation of allergic airway response and intestinal microflora disruption.</p><p><b>METHODS</b>Sixty female BALB/c mice were divided at random into 6 groups with 10 mice in each. (1) First antibiotic therapy group: the mice were given oral cefoperazone for 7 days, on day 7, mice were inoculated with Candida albicans (10(9)/ml, 50 microl) orally. (2) First control group: the mice were treated as first antibiotic therapy group, but cefoperazone and Candida albicans were replaced by saline. The mice in groups (1) and (2) were sacrificed on day 8, and cecal contents were collected for quantitative analysis of the intestinal bacterial flora. (3) Antibiotic therapy and challenge group: the mice were treated as the first antibiotic therapy group, then challenged (day 9 and 16) by intranasal exposure to Aspergillus fumigatus allergen. (4) Second antibiotic therapy group: the mice were treated as the first antibiotic therapy group, then challenged (day 9 and 16) by intranasal exposure to saline. (5) Challenge group: the mice were treated as the first control group, then challenged (day 9 and 16) by intranasal exposure to Aspergillus fumigatus allergen. (6) Second control group: the mice were treated as the first control group, then challenged (day 9 and 16) by intranasal exposure to saline. The mice in (3) - (6) group were killed for analysis of allergic airway response on day 19.</p><p><b>RESULTS</b>The quantity of Enterobacteriaceae, Enterococcus, Bifidobacterium and Lactobacillus in first antibiotic therapy group was significantly lower than that in the first control group, the quantity of Candida albicans increased in the first antibiotic therapy group as compared with the first control group. Mice intestinal microflora were disrupted with weight reduction and increased moisture in feces. After challenging with Aspergillus fumigatus allergens via intranasal inhalation, the total cell count, eosinophils, lymphocytes and neutrophils increased in BALF, especially in bronchoalveolar lavage fluid (BALF) from the mice in antibiotic therapy and challenge groups. IL-4 level in BALF from antibiotic therapy and challenge group (45.35 +/- 2.36) pg/ml was higher than that in the second control group (35.32 +/- 2.53) pg/ml. The expression of GATA-3 mRNA in the mice lung tissue (0.569 +/- 0.023) was higher than that in the second control group (0.410 +/- 0.020), and the ratios of T-bet/GATA-3 (0.578 +/- 0.021) decreased as compared with that in the second control group (0.804 +/- 0.035). IFN-gamma level in BALF from any group was not significantly different. In the absence of antibiotics, mice exposed to Aspergillus fumigatus allergen did not develop an allergic response in the airways.</p><p><b>CONCLUSIONS</b>The allergic (Th2) immune response can be induced by airway challenge with Aspergillus fumigatus allergen in the mice in which the intestinal microflora disruption resulted from antibiotic therapy, this result suggests that the intestinal microflora disruption resulted from antibiotic therapy is a risk factor for allergy and asthma.</p>