Intranasal immunization with a colloid-formulated bacterial extract induces an acute inflammatory response in the lungs and elicits specific immune responses

Abstract

Nonspecific stimulation of lung defenses by repeated oral administration of immunomodulators, such as bacterial extracts, has shown potential for the prevention of respiratory tract infections. Here, we show that intranasal (i.n.) immunization with a bacterial extract formulated as a colloid induces an acute inflammatory response in the lungs characterized by increased production of CCL and CXCL chemokines and a major influx of dendritic cells (DCs) and neutrophils, with a higher proportion of DCs showing an activated phenotype (high CD80/CD86 expression). Cytokine levels measured in bronchoalveolar-lavage samples showed a small increase in the production of tumor necrosis factor alpha and similar levels of the other cytokines measured (interleukin 10 [IL-10], IL-12, and gamma interferon [IFN-gamma]) in immunized mice compared with control mice. However, the recall response of primed animals after antigenic challenge induced increased expression of IL-12 and IFN-gamma mRNAs in lung homogenates. Overall, all these effects were not due to the lipopolysaccharide content in the bacterial extract. Furthermore, we found that three i.n. doses administered 2 to 3 weeks apart were enough to elicit long-lasting specific serum immunoglobulin G (IgG) and secretory IgA antibody responses. Assessment of IgG subclasses showed a balanced pattern of IgG1-IgG2a responses. The serum total IgE concentrations were also elevated in immunized mice 2 weeks after the third dose, but they significantly decreased soon afterwards. Our results suggest that simple formulations of bacterial extracts administered i.n. are highly immunogenic, eliciting local and systemic immune responses, and may serve as the basis for cost-effective immunotherapies for the prevention and treatment of respiratory infections.

FIG. 1.

Pulmonary DCs from immunized and control mice on days 1 and 7 after the last dose. The percentages of DCs in total lung cells were calculated by FC analysis of mice that received three i.n. doses of either CBE (immunized) or PBS (control) administered 1 day apart and were sacrificed 1 and 7 days later. For FC analysis, 10⁶ cells from five mice per group were immunostained with Cychrome 5-CD11c and PE-MHC class II, and DCs were defined as described in Materials and Methods. The data were collected on a FACSCalibur. The results are expressed as mean ± standard deviation. *, P < 0.05 compared to the control group (two-tailed Mann-Whitney test), corresponding to five mice per group analyzed individually.

FIG. 2.

Identification of pulmonary DC population. Lung single-cell suspensions prepared from mice that received three i.n. doses of CBE, PBS, or 40 pg of LPS administered 1 day apart or a single LPS dose of 5 μg and that were sacrificed 24 h later. Cells (10⁶) from a pool of five mice per group were immunostained with Cychrome 5-CD11c, PE-MHC class II, and FITC-CD80 and -CD86. The data were collected on a FACSCalibur. DCs are represented in R2 (CD11c⁺ MHC class II⁺ cells).

FIG. 3.

Chemokine and proinflammatory-cytokine mRNA expression in lungs from mice that received three i.n. doses of CBE or PBS administered 1 day apart and were sacrificed 4 and 24 h later. Total lung RNA was extracted, and the expression of chemokines and cytokines was evaluated by RT-PCR. The amplification products were separated by electrophoresis in acrylamide gels and silver stained. The results are representative of three independent experiments.

FIG. 4.

Cytokine levels in BAL samples from naïve mice and mice that received three i.n. doses of CBE (Immunized) or PBS (Control) and were sacrificed 1 day later. IL-10 (A), IL-12 (B), and IFN-γ (C) concentrations were determined by ELISA. (D) For TNF-α, an additional group that received three i.n. doses of LPS (40 pg) was also analyzed. The results are expressed as the mean of duplicate optical-density values for individual mice and the mean values for the groups (horizontal bars).

FIG. 5.

Production of IL-12 and IFN-γ mRNAs in lungs after antigen challenge. Two groups of mice received three i.n. doses of CBE administered 1 day apart. Seven days later, one group received an additional dose of CBE, and the other group served as controls (Not challenged). All mice were sacrificed 24 h later, total lung RNA was extracted, and the mRNA expression of IL-12 and IFN-γ was evaluated by RT-PCR. The amplification products were separated by electrophoresis in acrylamide gels and silver stained. The results are representative of three independent experiments.

FIG. 6.

Total IgG antibody response against CBE, as detected by ELISA analysis of sera taken on days 0, 54, 110, and 120 after priming. The results are expressed as the mean of duplicate optical-density (OD) values for individual mice immunized with CBE as described in Materials and Methods and the mean values for the groups of five mice (horizontal bars). *, P < 0.05 compared to day 0. (Inset) IgG antibody response against recombinant pneumolysin analyzed at the same time points. Data are the means for five mice per group ± standard deviation.

FIG. 7.

Serum IgG1 (A) and IgG2a (B) against CBE, as detected by ELISA analysis of sera taken on days 0, 54, 110, and 120 after priming. The results are expressed as the mean of duplicate optical-density (OD) values for individual mice immunized with CBE as described in Materials and Methods and the mean values for the groups of five mice (horizontal bars).

FIG. 8.

IgA response against CBE as detected by ELISA analysis of sera taken on days 0 and 54 after priming. The results are expressed as the mean of duplicate optical-density (OD) values for individual mice immunized with CBE as described in Materials and Methods and the mean values for the groups of five mice (horizontal bars). P < 0.05 compared to day 0 (two-tailed Student t test).

FIG. 9.

Total IgE antibody concentration in sera taken on days 0, 54, 110, and 120 after priming from mice immunized with CBE. Each value is the average for duplicate ELISA determinations for individual mice, and the horizontal bars represent the mean values for the groups.

FIG. 10.

Total IgA and IgE antibody concentrations in BAL samples from naïve and immunized mice 2 weeks after the last booster (day 120 after priming). The results are expressed as mean concentrations fromduplicate ELISA determinations for individual mice.