Balance between early life tolerance and sensitization in allergy: dependence on the timing and intensity of prenatal and postnatal allergen exposure of the mother

Abstract

Allergens can be maternally transferred to the fetus or neonate, though it is uncertain how this initial allergen exposure may impact the development of allergy responses. To evaluate the roles of timing and level of maternal allergen exposure in the early life sensitization of progeny, female BALB/c mice were given ovalbumin (OVA) orally during pregnancy, lactation or weekly at each stage to investigate the immunoglobulin E (IgE) antibody production and cellular responsiveness of their offspring. Exposure to OVA during pregnancy was also evaluated in OVA-specific T-cell receptor (TCR) transgenic (DO11.10) mice. The effect of prenatal antigen exposure on offspring sensitization was dependent on antigen intake, with low-dose OVA inducing tolerance followed by neonatal immunization that was sustained even when pups were immunized when 3 weeks old. These offspring received high levels of transforming growth factor-beta via breastfeeding. High-dose exposure during the first week of pregnancy or perinatal period induced transient inhibition of IgE production following neonatal immunization; although for later immunization IgE production was enhanced in these offspring. Postnatal maternal antigen exposure provided OVA transference via breastfeeding, which consequently induced increased offspring susceptibility to IgE antibody production according to week post-birth. The effect of low-dose maternal exposure during pregnancy was further evaluated using OVA transgenic TCR dams as a model. These progeny presented pronounced entry of CD4(+) T cells into the S phase of the cell cycle with a skewed T helper type 2 response early in life, revealing the occurrence of allergen priming in utero. The balance between tolerance and sensitization depended on the amount and timing of maternal allergen intake during pregnancy.

Figure 1

Determination of ovalbumin (OVA) levels in milk after postnatal maternal antigen exposure. Female BALB/c mice were given OVA orally during the first week post-birth (1·5, 3·0 or 9·0 mg), divided into three doses (Protocol II). Breast milk samples were obtained from the stomach of 5-day-old newborn mice. OVA in milk samples was assessed by enzyme-linked immunosorbent assay. Horizontal line represents means. *P≤ 0·05 when compared with offspring from non-exposed mothers.

Figure 2

Effect of maternal allergen exposure on the development of the immunoglobulin E (IgE) response of offspring. Three-day-old (a) or 25-day-old BALB/c (b) offspring from mothers exposed to ovalbumin (OVA; 1·5 or 9·0 mg) during the prenatal or postnatal periods, described as Protocol I and II, respectively, in the Materials and methods section, were immunized with OVA. Anti-OVA IgE antibody was evaluated by means of a passive cutaneous anaphylaxis reaction. Horizontal line represents means. *P≤ 0·05 when compared with offspring from non-exposed mothers.

Figure 3

Effect of timing of ovalbumin (OVA) exposure in the prenatal or postnatal period on the neonatal immunoglobulin E (IgE) development. Female BALB/c mice were mated and orally administered 9·0 mg of OVA prenatally, divided into three doses in the first or second, or third week of the pregnancy or in the postnatal period, in the first or second, or third week of suckling (Protocol III). Three-day-old offspring were immunized with OVA, and IgE antibody production was evaluated by passive cutaneous anaphylaxis reaction. Horizontal line represents means. *P≤ 0·05 when compared with offspring from non-exposed mothers.

Figure 4

Maternal low-dose exposure during pregnancy induces inhibition of offspring cellular responsiveness. Three-day-old BALB/c mice from mothers exposed to ovalbumin (OVA; dotted bar) during prenatal period (1·5 mg, Protocol I) or from non-exposed mothers (open bar) were immunized with OVA or hen egg lysozyme (HEL). Proliferative spleen cells from OVA-immunized offspring (20 days old) in response to OVA are represented as the stimulation index (SI) (a); interferon-γ (IFN-γ) (b), interleukin-4 (IL-4) (c), and IL-10 (d) measurements in the supernatants of splenic cell culture after 24 hr of stimulation with anti-CD3 monoclonal antibody or 72 hr with OVA were detected by enzyme-linked immunosorbent assay (ELISA). Total IgE antibody (e) and anti-OVA IgG1 (f) and IgG2a antibody (g) with their respective total concentration levels were evaluated by means of ELISA. Proliferative spleen cell response of offspring immunized with HEL from non-exposed (vertical hatched bar) or exposed (horizontal hatched bar) mothers to anti-CD3 stimuli is represented as SI (hr); IFN-γ (i), IL-4 (j), and IL-10 (l) measurements in the supernatants of splenic cell culture after 24 hr of stimulation with anti-CD3 monoclonal antibody by ELISA. Representative results from five to eight mice/per group are expressed as mean ± SEM. *P≤ 0·05 when compared with offspring from non-exposed mothers.

Figure 5

Transforming growth factor-β (TGF-β) levels in milk of mothers exposed to antigen during the prenatal or postnatal period. Female BALB/c mice received ovalbumin (OVA) by the oral route during gestation (1·5 mg, Protocol I, dotted bar) or in the first week of suckling (9·0 mg, Protocol II, solid bar). TGF-β₁ in individual milk samples (n= 5) (a) or total immunoglobulin A (IgA) levels (b) were assessed by enzyme-linked immunosorbent assay. Results are expressed as mean ± SEM. *P≤ 0·05 when compared with offspring from non-exposed mothers (open bar).

Figure 6

Prenatal ovalbumin (OVA) exposure induces activation of OVA-specific CD4⁺ T cells in the offspring of transgenic (Tg) DO11.10 mice. Female DO11.10 mice were mated and given OVA orally (1·5 mg) during the pregnancy (Protocol I). Splenic cells of offspring (20 days old) were pooled and cultured with OVA to evaluate the CD4⁺ cell cycle phase through bromodeoxyuridine (BrdU) incorporation. Spleen cells were stained with fluorescein isothiocyanate-labelled anti-BrdU monoclonal antibody, phycoerythrin-labelled anti-CD4 monoclonal antibody, and 7-amino-actinomycin D (7-AAD) for flow cytometry analysis. The percentage of gated CD4⁺ cells in the G0/G1, S, G2/M, and Ap (Apoptotic) stages of the cell cycle, represented by bars, were determined for offspring from non-exposed (a) or exposed (b) mothers and are representative of two experiments. A representative fluorescence-activated cell sorting analysis showing the percentage of stimulated cells following culture with OVA for 72 hr is shown. The percentages of cells in each phase of the cell cycle are shown in parentheses.

Figure 7

Cytokine production from the offspring of transgenic (Tg) DO11.10 mice subsequent to prenatal ovalbumin (OVA) exposure. Splenic cells from Tg offspring (20 days old) from mothers that were exposed (solid bar) to antigen during pregnancy (1·5 mg, Protocol I) or from non-exposed mothers (open bar) were stimulated with OVA (400 μg/ml). Cytokine measurements in the supernatant after 48 and 72 hr of stimulation were assessed by enzyme-linked immunosorbent assay (ELISA) (a); splenic B220⁺ IgM⁺ and B220⁺ CD23⁺ cells were evaluated by flow cytometry (b), and anti-OVA immunoglobulin G (IgG) antibody serum levels by ELISA (c). Results represent values from four to six mice/per group expressed as mean ± SEM. *P≤ 0·05 when compared with offspring from non-exposed mothers.