Prophylactic Supplementation of Bifidobacterium longum 51A Protects Mice from Ovariectomy-Induced Exacerbated Allergic Airway Inflammation and Airway Hyperresponsiveness

Abstract

Asthma is a chronic inflammatory disease that affects more females than males after puberty, and its symptoms and severity in women change during menstruation and menopause. Recently, evidence has demonstrated that interactions among the microbiota, female sex hormones, and immunity are associated with the development of autoimmune diseases. However, no studies have investigated if therapeutic gut microbiota modulation strategies could affect asthma exacerbation during menstruation and menopause. Here we aimed to examine the preventive effects of a probiotic, Bifidobacterium longum 5^1A, on airway inflammation exacerbation in allergic ovariectomized mice. We first evaluated the gut microbiota composition and diversity in mice 10 days after ovariectomy. Next, we examined whether re-exposure of ovariectomized allergic mice to antigen (ovalbumin) would lead to exacerbation of lung inflammation. Finally, we evaluated the preventive and treatment effect of B. longum 5^1A on lung inflammation and airway hyperresponsiveness. Our results showed that whereas ovariectomy caused no alterations in the gut microbiota composition and diversity in this animal model, 10 days after ovariectomy, preventive use administration of B. longum 5^1A, rather than its use after surgery was capable of attenuate the exacerbated lung inflammation and hyperresponsiveness in ovariectomized allergic mice. This prophylactic effect of B. longum 5^1A involves acetate production, which led to increased fecal acetate levels and, consequently, increased Treg cells in ovariectomized allergic mice.

Keywords: Bifidobacterium longum; airway inflammation; microbiota; ovariectomy; probiotic.

FIGURE 1

Bacterial community profiling of fecal samples from Balb/c before and after ovariectomy. (A) Rarefaction analysis indicating the average number of OTUs per sequence sampling depth for each of the groups. Error bars represent the standard error of the mean. (B) Multidimensional scaling of Euclidean distances between mouse fecal samples. (C) Heatmap and hierarchical clustering of mouse fecal samples using rarefied OTU abundances. (D) Mean relative abundances of the most abundant bacterial families identified in the samples. (E) Mean relative abundances of the least abundant bacterial families identified in the samples. Error bars represent the standard error of the mean and ^∗ indicates a p-value < 0.05.

FIGURE 2

Ovariectomy exacerbates the lung inflammation and airway hyperresponsiveness of re-challenged allergic mice. (A) Ten days before being ovariectomized (OVx) or not (Sham), the animals were sensitized and challenged. Ten days after ovariectomy, the animals were re-challenged. All parameters were analyzed 24 h after the last re-challenge. (B) Measurement of AHR, as assessed by Newtonian airway resistance to increasing doses of methacholine. (C) Quantification of the total number of cells in the bronchoalveolar lavage (BAL). (D) Concentration of IL-10, IL-5, and INF-γ in the BAL. (E) Representative periodic acid–Schiff (PAS)-stained lung tissue from mice in the SHAM, OVx, SHAM OVA, and OVx OVA groups. Scale bars, 200 μm. All results are representative of data generated in two different experiments and are expressed as the mean ± SEM (n = 5 in all groups). Statistical significance was determined by a one-way analysis of variance (ANOVA), except in (B), in which a two-way ANOVA test was used. Hash symbol: non-allergic (sham and OVx) groups vs. respective allergic group (sham Ova and OVx OVA), ^###P < 0.001; asterisk: Sham OVA vs. OVx OVA. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 3

Preventive oral probiotic supplementation decreased the allergic airway inflammation and airway hyperresponsiveness of re-challenged ovariectomized allergic mice. (A) An oral probiotic (B. longum 5^1A) was administered by gavage daily for 15 days before the first OVA sensitization of mice and continued until 4 h before the ovaries of the mice were removed. (B) AHR in mice supplemented with the probiotic or saline. (C) Differential cell counts in the BAL. Total number of cells infiltrating the airways in the BAL. (D) Concentration of INF-γ, IL-4, IL-5, and IL-10 expression levels in the BAL. (E) Representative periodic acid–Schiff (PAS)-stained lung tissue from mice on either a control or probiotic on day 16. Scale bars, 200 μm. All results are representative of data generated in two different experiments and are expressed as the mean ± SEM (n = 4 control mice, and n = 5 mice given the probiotic supplementation). Statistical significance was determined with Student’s t-test, except in (B), in which a two-way ANOVA test was used. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, ^∗∗∗∗P < 0.0001.

FIGURE 4

Preventive supplementation with B. longum 5^1A increases the production of short-chain fatty acids (SCFAs) and the number of lung Treg cells of re-challenged OVx allergic mice. (A) Sterile bacterial culture medium and B. longum in culture medium. Fecal SCFA concentrations from the mice depicted in (B). (C) An oral probiotic (B. longum 5^1A) was administered by gavage daily for 15 days before the first OVA sensitization of mice and continued until 24 h before the mouse ovaries were removed. After the last re-challenge, cells from the BALF were subjected to flow cytometry and stained for CD4-APC, CD25-PE, and FoxP3-FITC. The dot plot demonstrates the gate strategy of Treg (Isotype control, Fluorescence Minus One Control-FMO, OVA-OVx, and OVA-OVx-probiotic) and the percentage of positive Treg cells. The graph is representative of two independent experiments. All results are representative of data generated in two different experiments and are expressed as the mean ± SEM [n = 3 for all groups in (A), n = 4 in (B,C)]. Statistical significance was determined by a one-way ANOVA. ^∗P < 0.05, ^∗∗∗P < 0.001.

FIGURE 5

Oral probiotic supplementation after ovariectomy does not alter airway inflammation. An oral probiotic (B. longum 5^1A) was administered by gavage 24 h after ovariectomy until the last re-challenge (A). All parameters were analyzed 24 h after the last re-challenge. (B) Quantification of the total cells number in the BAL. All results are representative of data generated in one experiment and are expressed as the mean ± SEM (n = 4–5 mice in all groups).

FIGURE 6

Schematic representation of mechanism underlying the effect of probiotic intake on the asthma exacerbation after ovariectomy. Ovariectomy exacerbates the lung inflammation of re-challenge allergic mice (A). Preventive supplementation with B. longum 5^1A decreases exarcebation of lung inflammation and increases the production of acetate and Treg cells in re-challenge OVx allergic mice (B).