Effects of lysed Enterococcus faecalis FK-23 on experimental allergic rhinitis in a murine model

Abstract

In the current study, we sought to investigate whether lysed Enterococcus faecalis FK-23 (LFK), a heat-killed probiotic preparation, attenuated eosinophil influx into the upper airway and had immunomodulatory activity in a murine allergic rhinitis model. Eighteen BALB/c mice were divided into three groups; the ovalbumin (OVA)-sensitized/challenged group, which received saline orally for 6 weeks (OVA group), the OVA-sensitized/challenged group, which received LFK orally for 6 weeks (LFK-fed group), and the non-sensitized group, which received saline for 6 weeks (saline control group). Nasal rubbing and sneezing were monitored during the study. After the final challenge, interleukin (IL)-4, interferon (IFN)-γ, and OVA-specific IgE levels in the sera and splenocyte culture supernatants were determined, eosinophilic infiltrate into the upper airway was quantified, and splenic CD4+CD25+ regulatory T cells (Tregs) were examined by flow cytometry. We found that nasal rubbing was significantly reduced in LFK-fed mice compared to the OVA group on d 27 and 35, and sneezing was significantly inhibited by LFK administration for 35 d. LFK-fed mice had significantly less eosinophil influx into the nasal mucosa than the OVA group. There were no significant differences between the LFK-fed group and OVA group in the serum and splenocyte culture supernatant levels of IL-4, IFN-γ, and OVA-specific IgE. Interestingly, the LFK-fed mice had a significantly greater percentage of splenic CD4+CD25+ Tregs than OVA group. Our results indicate that oral administration of LFK may alleviate nasal symptoms, reduce nasal eosinophilia, and increase the percentage of CD4+CD25+ Tregs in experimental allergic rhinitis.

Keywords: Enterococcus faecalis; allergic rhinitis; cytokines; eosinophils; mice; probiotics; regulatory T-lymphocytes.

Fig. 1. Sensitization and challenge protocols for the experimental murine model.

BALB/c mice were divided into three groups: ovalbumin (OVA)-sensitized/challenged and orally administered saline (OVA group), OVA-sensitized/challenged and orally administered LFK (LFK-fed group), and non-sensitized and orally administered saline (saline control group). Mice in the OVA group and the LFK group were sensitized by the intraperitoneal (i.p.) injection of 0.1 mg OVA and 2 mg Al(OH)₃ in a volume of 0.2 mL on d 0, 7, and 14 and were subsequently challenged intranasally (i.n.) daily with 20 µL OVA solution (50 mg/mL) on d 21 to 27 and on alternate days from d 29 to 41. The LFK-fed mice were fed 60 mg LFK, and the mice in the control group were orally administered 0.5 mL saline during the period of sensitization and challenge.

Fig. 2. Effects of 35 consecutive days of LFK administration on nasal rubbing.

Episodes of nasal rubbing was counted for 10 min after nasal ovalbumin (OVA) challenge on d 21 (A), d 27 (B), and d 35 (C). Each column and vertical bar shows the mean±SEM (n=5 each group). *P < 0.05; **P < 0.01.

Fig. 3. Effects of 35 consecutive days of LFK administration on sneezing.

Nasal sneezes were counted for 10 min after nasal ovalbumin (OVA) challenge on d 21 (A), d 27 (B), and d 35 (C). Each column and vertical bar shows the mean±SEM (n = 5 each group). *P < 0.05; **P < 0.01.

Fig. 4. Representative H&E stained sections of the nasal turbinate tissue of the ovalbumin (OVA) group (A), LFK-fed group (B), and saline control group (C) (magnification, ×200).

Nasal mucosa samples were anatomically separated on d 41, fixed with 10% formalin for 24 h, decalcified in EDTA for 1 week, dehydrated, and embedded in paraffin. Increased eosinophilic infiltration in the nasal submucosa and lamina propria was apparent in the nasal turbinate tissue of the OVA control mice compared to that of the saline control mice, whereas the eosinophilic cellular infiltrate was decreased in the LFK-fed group compared to the OVA group. D: The statistical analysis of the number of eosinophils in three groups (n = 4). Each column and vertical bar shows mean±SEM. *P < 0.05.

Fig. 5. Effects of orally administered LFK on the percentage of splenic CD4⁺CD25⁺ Tregs.

BALB/c mice were injected intraperitoneally on d 0, 7, and 14 with 0.1 mg ovalbumin (OVA) and 2 mg Al(OH)₃ in a total volume of 0.2 mL. The mice were orally administered LFK (60 mg/0.5 mL) daily for 41 d. Spleens were removed within 24 h after the final nasal challenge on d 41, splenocyte single-cell suspensions were prepared, and the percentages of splenic CD4⁺CD25⁺ Tregs in OVA group (A), LFK-fed group (B), and saline control group (C) were measured by flow cytometry. D: The data shown are representative of three independent experiments. Each column and vertical bar shows the mean±SEM (n = 3). *P < 0.05.