Intranasal coadministration of live lactococci producing interleukin-12 and a major cow's milk allergen inhibits allergic reaction in mice

Abstract

The Th1/Th2 balance deregulation toward a Th2 immune response plays a central role in allergy. We previously demonstrated that administration of recombinant Lactococcus lactis strains expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, partially prevents mice from sensitization. In the present study, we aimed to improve this preventive effect by coadministration of L. lactis BLG and a second recombinant L. lactis strain producing biologically active interleukin-12 (IL-12). This L. lactis strain producing IL-12 was previously used to enhance the Th1 immune response in a tumoral murine model (L. G. Bermúdez-Humarán et al., J. Immunol. 175:7297-7302, 2005). A comparison of the administration of either BLG alone or BLG in the presence of IL-12 was conducted. A BLG-specific primary Th1 immune response was observed only after intranasal coadministration of both L. lactis BLG and IL-12-producing L. lactis, as demonstrated by the induction of serum-specific immunoglobulin G2a (IgG2a) concomitant with gamma interferon secretion by splenocytes, confirming the adjuvanticity of IL-12-producing L. lactis. Immunized mice were further sensitized by intraperitoneal administration of purified BLG, and the allergic reaction was elicited by intranasal challenge with purified BLG. Mice pretreated with BLG in either the presence or the absence of IL-12 were rendered completely tolerant to further allergic sensitization and elicitation. Pretreatment with either L. lactis BLG or L. lactis BLG and IL-12-producing L. lactis induces specific anti-BLG IgG2a production in serum and bronchoalveolar lavage (BAL) fluid. Although specific serum IgE was not affected by these pretreatments, the levels of eosinophilia and IL-5 secretion in BAL fluid were significantly reduced after BLG challenge in the groups pretreated with L. lactis BLG and L. lactis BLG-IL-12-producing L. lactis, demonstrating a decreased allergic reaction. Our data demonstrate for the first time (i) the induction of a protective Th1 response by the association of L. lactis BLG and IL-12-producing L. lactis which inhibits the elicitation of the allergic reaction to BLG in mice and (ii) the efficiency of intranasal administration of BLG for the induction of tolerance.

FIG. 1.

Experimental protocol. Groups of mice (n = 12) were intranasally immunized on days 1 to 3, 14 to 16, and 28 to 30 with a live recombinant L. lactis strain producing BLG, L. lactis BLG plus IL-12-producing recombinant L. lactis, nonrecombinant L. lactis, purified protein BLG alone, or BLG in combination with IL-12. Control animals were immunized with saline. On days 37 and 50 the mice were intraperitoneally sensitized with 5 μg of BLG emulsified in incomplete Freund's adjuvant. Allergen challenge was performed on days 55 and 56 by intranasal administration of 20 μg BLG. Naïve mice (n = 5) were left untreated.

FIG. 2.

Intranasal treatment of mice with L. lactis BLG (L[BLG]) plus L. lactis IL-12 (L[IL-12]) induces a BLG-specific Th1 immune response. Immunized mice (n = 5; Fig. 1) were killed on day 42. Serum samples and spleen cells restimulated in vitro with BLG were tested for IgG2a (A) and IFN-γ (B) production, respectively. Bars represent the means ± standard deviations. The P values obtained with JMP software after comparison of the treated group and the saline (control) group are shown, and a P value of <0.05 indicated statistical significance when the results for the different groups are compared. L−, L. lactis strain containing an empty vector.

FIG. 3.

Pretreatment of recombinant lactococci did not affect BLG-specific IgE antibody production. Five days after the second sensitization (day 55), the sera of mice (n = 7) were collected and tested for BLG-specific IgE (A), IgG1 (B), and IgG2a (C) antibodies. Bars represent the means ± standard deviations. The P values obtained with JMP software after comparison of treated group and the saline (control) are shown. L[BLG], L. lactis BLG; L[IL-12], L. lactis IL-12; L−, L. lactis strain containing an empty vector.

FIG. 4.

Pretreatment with L. lactis BLG (L[BLG]) and L. lactis BLG plus L. lactis IL-12 (L[IL-12]) allows the production of IgG2a in BAL fluid. On day 57, sensitized and challenged mice (n = 7) were killed and BAL fluids were collected as described in Materials and Methods. BLG-specific IgG1 (A) and IgG2a (B) antibody concentrations were determined by quantitative immunoassays. Bars represent the means ± standard deviations. The P values obtained with JMP software after comparison of treated group and the saline (control) are shown. L−, L. lactis strain containing an empty vector.

FIG. 5.

Intranasal pretreatment with L. lactis BLG (L[BLG]) and L. lactis BLG plus L. lactis IL-12 (L[IL-12]) reduced IL-5 and IL-4 levels and decreased the level of airway eosinophilia. On day 57, sensitized and challenged mice were killed and BAL fluids were collected. IL-4 (A) and IL-5 (B) cytokines were measured by ELISA, and eosinophil influx (C) was quantified by differential cellular counts under May-Grunwald and Giemsa staining. Bars represent the means ± standard deviations. The P values obtained with JMP software after comparison of treated group and the saline (control) are shown. L−, L. lactis strain containing an empty vector.