Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor Fc gamma RIIB expression on B cells

Abstract

Background: Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA) on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period.

Results: Maternal immunization with OVA showed increased levels of Fc gamma RIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-gamma-secreting T cells and IL-4 and IL-12- secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specific IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced Fc gamma RIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers.

Conclusion: Maternal immunization upregulates the inhibitory Fc gamma RIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.

Figure 1

Influence of maternal immunization with OVA on the ex vivo expression of B cell-costimulatory molecules from nonimmunized offspring. BALB/c offspring from mothers immunized with OVA prior to conception were evaluated at 3 d-o (A) or 20 d-o (B) for CD80, CD86, CD40 and CD23 expression on splenic B cells (B220+IgM+). The data obtained by flow cytometry represent the mean ± SEM of 12 mice per group. * P ≤ 0.05 compared to offspring from nonimmunized mothers.

Figure 2

Maternal immunization with OVA upregulates FcγRIIb expression on B cells from offspring. BALB/c offspring from mothers immunized with OVA prior to conception or from control mothers (nonimmunized) were immunized (3 d-o) or not with OVA and evaluated at 3 d-o and 20 d-o for FcγRIIb expression on splenic B cells (B220+IgM+). The data obtained by flow cytometry represent the mean ± SEM of 12 mice per group. Histogram of B cell FcγRIIb expression depicting the cells from offspring from immunized (shaded histogram, Mean fluorescence intensity (MFI) in bold numbers) and nonimmunized mothers (white histogram, MFI in light numbers). * P ≤ 0.05 compared to offspring from nonimmunized mothers, # P ≤ 0.05 compared to nonimmunized offspring (20 d-o) from control mothers.

Figure 3

Effect of maternal immunization with OVA on the immune response of nonimmunized or immunized neonates. Neonate pups (3 d-o) from control or immune mothers were immunized or not with OVA and evaluated (20 d-o) for: (A) IgG1, IgG2a and IgM by ELISA; (B) anti-OVA IgE Ab levels by PCA reaction; (C) intracellular cytokines of splenic B cells (B220+) or (d) CD4+ T cells after 24 h incubation with 10 μg/mL brefeldin A by flow cytometry. The results represent the mean ± SEM of 12 mice per group. *P ≤ 0.05 compared to offspring from nonimmunized mothers, # P ≤ 0.05 compared to nonimmunized offspring from control mothers, • P ≤ 0.05 compared to control offspring from immune mothers.

Figure 4

Impaired B cell activation by BCR-crosslinking of offpring from immunized mothers. Neonate pups (3 d-o) were immunized with OVA and evaluated (20 d-o) for: (A) proliferative spleen cell response to OVA; (B) purified B-cell response for CpG oligodeoxynucleotide type B (5 μg/mL) stimuli; (C) purified B cell from nonimmunized mice incubated with F(ab')₂ anti-mouse IgM Ab (50 μg/mL) in the presence of IgG from from OVA-immunized unrelated adult mice (10, 50, 100 μg/mL), or monoclonal antibody anti-OVA (α OVA, 5 μg/mL) plus OVA (10 μg/mL), incubated for 96 h [data expressed in Stimulation Index (S.I.)]; (D) cytokine measurements in the supernatants of spleen cell culture after 72 h of stimulation with OVA by cytometric bead array. The results represent the mean ± SEM of 9 mice per group. *P ≤ 0.05 compared to offspring from nonimmunized mothers.

Figure 5

Effect of passive IgG transference to neonates on B and T cell responses. Neonate pups (3 d-o) from nonimmunized mothers injected with IgG from nonimmunized or immunized mothers and simultaneously immunized with OVA were evaluated (20 d-o) for: (A) anti-OVA IgE Ab levels by PCA reaction; (B) B cell FcγRIIb expression (B220+IgM+) and histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers); (C) intracellular cytokines of splenic B cells (B220+) or (D) CD4+ T cells after 24 h incubation with 10 μg/mL brefeldin A; data shown in B-D were obtained flow cytometry. The results represent the mean ± SEM of 9 mice per group. * P ≤ 0.05 compared to offspring from nonimmunized mothers.

Figure 6

Effect of passive IgG transference to pregnant mice on neonatal B cell FcγRIIb expression. Nonimmunized pregnant mice were injected with IgG from nonimmunized or immunized mothers. Nonimmunized neonates were evaluated (3 d-o) for: (A) CD80, CD86, CD40, and CD23 molecule expression on splenic B cells (B220+) and (B) B cell FcγRIIb expression (B220+IgM+) by flow cytometry. Histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers). The results represent the mean ± SEM of 6 mice per group. * P ≤ 0.05 compared to offspring from nonimmunized mothers.

Figure 7

Effect of passive IgG transference to pregnant mice on offspring's B and T cell responses. Nonimmunized pregnant mice were injected with IgG from nonimmunized or immunized mothers. Offspring immunized with OVA were evaluated (20 d-o) for: (a) anti-OVA IgE Ab levels by PCA reaction; (b) CD80, CD86, CD40 CD23 molecule expression on splenic B cells (B220+); (c) B cell FcγRIIb expression (B220+IgM+) by flow cytometry. Histogram of FcγRIIb expression on B cells of offspring from immunized (shaded histogram, MFI in bold numbers) or nonimmunized mothers (white histogram, MFI in light numbers); (d) intracellular cytokines of splenic B cells (B220+) and (e) CD4+ T cells after 424 h incubation with 10 μg/mL brefeldin A, all by flow cytometry. The results represent the mean ± SEM of 6 mice per group. *P ≤ 0.05 compared to offspring from nonimmunized mothers.