Effects of Lactobacillus rhamnosus GG supplementation on cow's milk allergy in a mouse model

Abstract

Background: Cow's milk allergy (CMA) is one of the most prevalent human food-borne allergies, particularly in infants and young children from developed countries. Our study aims to evaluate the effects of Lactobacillus rhamnosus GG (LGG) administration on CMA development using whole cow's milk proteins (CMP) sensitized Balb/C mice by two different sensitization methods.

Methods: LGG supplemented mice were either sensitized orally with CMP and cholera toxin B-subunit (CTB) as adjuvant, or intraperitoneally (IP) with CMP but without the adjuvant. Mice were then orally challenged with CMP and allergic responses were accessed by monitoring hypersensitivity scores, measuring the levels of CMP-specific immunoglobulins (IgG1, IgG2a and IgG) and total IgE from sera, and cytokines (IL-4 and IFN-γ) from spleen lysates.

Results: Sensitization to CMP was successful only in IP sensitized mice, but not in orally sensitized mice with CMP and CTB. Interestingly, LGG supplementation appeared to have reduced cow's milk allergy (CMA) in the IP group of mice, as indicated by lowered allergic responses.

Conclusions: Adjuvant-free IP sensitization with CMP was successful in inducing CMA in the Balb/C mice model. LGG supplementation favourably modulated immune reactions by shifting Th2-dominated trends toward Th1-dominated responses in CMP sensitized mice. Our results also suggest that oral sensitization by the co-administration of CMP and CTB, as adjuvant, might not be appropriate to induce CMA in mice.

Figure 1

Schematic overview of CMP sensitization and challenge protocol in Balb/C mice. (a) Intragastrically sensitized (gavage) group. Mice were sub-divided into 5 treatments and sensitized intragastrically for 4 weeks as follows (n = 6); CTL-: PBS; CTB: PBS+ CTB (0.25 μg/g BW); CTL+: CMP (1 mg/g BW) + CTB; LGG1: CTL+ mice supplemented with LGG for 5 weeks and LGG2: CTL+ mice supplemented with LGG for 4 weeks. At d 35, all sensitized mice were intragastrically challenged two times with CMP (30 mg/mouse) at 30 min apart. Two hours after final challenge, mice were euthanized with CO2 inhalation and blood and spleen samples were collected. (b) Intraperitoneally sensitized (IP) groups. Mice were sub-divided into 4 treatments and sensitized intraperitoneally for 2 weeks as followed (n = 6); CTL-: PBS; CTL+: CMP (10 mg/mouse); LGG1: CTL+ mice supplemented with LGG for 5 weeks and LGG2: CTL+ mice supplemented with LGG for 4 weeks. At d 35, all sensitized mice were intragastrically challenged two times with CMP (30 mg/mouse) at 30 min apart. Two hours after final challenge, mice were euthanized with CO2 inhalation and blood and spleen samples were collected.

Figure 2

Hypersensitivity scores of intraperitoneally sensitized (IP) mice. Hypersensitivity symptoms were scored one hour after last challenge with CMP. Each point represents an individual mouse. Values are means, n = 6 per treatment. CTL-: PBS, control mice; CTL+: CMP sensitized mice; LGG1: CTL+ mice supplemented with LGG for 5 weeks and LGG2: CTL+ mice supplemented with LGG for 4 weeks. Means with different letters differ, P ≤ 0.05.

Figure 3

CMP-specific serum IgG1 and IgG2a titres of IP mice. CTL-: PBS; CTL+: CMP (10 mg/mouse); LGG1: CTL+ mice supplemented with LGG for 5 weeks and LGG2: CTL+ mice supplemented with LGG for 4 weeks. Values are presented as Mean ± standard deviation. Values with different letters in the same column differ, P ≤ 0.05.

Figure 4

Lactobacillus rhamnosus GG (LGG) analysis from fecal genomic DNA using LGG-specific primers [24]and subsequent agarose gel electrophoresis. Lane 1, DNA extracted from supplemented LGG (ATCC53103); lanes 2 and 3, CTL + mice; lanes 4 and 5, LGG1 mice; lanes 6 and 7, LGG2 mice.