Adjuvant Effect of Lactobacillus paracasei in Sublingual Immunotherapy of Asthmatic Mice

Abstract

Background: Sublingual immunotherapy (SLIT) has shown promise in mitigating allergic asthma symptoms; nevertheless, its high dose and prolonged duration of treatment raise safety concerns. This study explored the potential of Lactobacillus paracasei (L. paracasei) to enhance the effectiveness of SLIT in a mouse model of allergic asthma. Methods: Allergic asthma was induced in Balb/c mice following sensitization and challenge with a house dust mite (HDM) allergen. Subsequently, the mice were subjected to SLIT (66 and 132 µg) either alone or in combination with L. paracasei supplementation. Asthma-associated parameters, including rubbing frequency, IgE level, cytokine profiles, and histological changes, were evaluated to assess treatment efficacy. Results: mice that received SLIT 132 µg combined with the probiotic (combined 132) demonstrated a significant reduction in allergic symptoms (rubbing). This treatment strategy led to a marked IgE and eosinophil level decrease in serum; an increase in anti-inflammatory cytokines like IFN-γ and IL-10; and a reduction in pro-inflammatory cytokines IL-17 and TNF-α. The combination therapy also mitigated lung inflammation and supported the restoration of the structural integrity of the colon, promoting the recovery of goblet cells and mucus secretion. Probiotic treatment alone also effectively reduced IgE levels, increased IFN-γ, and decreased levels of IL-17 and TNF-α. Conclusions: The adjuvant effect of L. paracasei in enhancing SLIT represents a promising approach for improving asthma treatment efficacy.

Keywords: allergic asthma; house dust mite; probiotic; sublingual immunotherapy.

Figure 1

The effects of a probiotic, SLIT, and their combination on the frequency of nasal rubbing. Treatment with combined 132 demonstrated a significant decrease in the frequency of nasal rubbing on (A) day 19, (B) day 21, and (C) day 24 following an HDM challenge. * p < 0.05 vs. the control group; # p < 0.05 and ## p < 0.01 vs. the asthma group.

Figure 2

The effects of a probiotic, SLIT, and their combination on inflammatory cell count, IgE, and cytokine concentrations. Serum IgE and cytokines of asthmatic and treated mice of all groups were measured by ELISA. The concentrations of (A) pro-inflammatory cytokine IL-4, (B) Interferon gamma (IFN-γ), (C) HDM IgE, (D) IL-10, (E) IL-17, (F) tumor necrosis factor alpha (TNFα). The enumeration of inflammatory cells in blood, including (G) total blood cells, (H) eosinophils, and (I) neutrophils obtained from various groups. * p < 0.05, *** p < 0.001, and **** p < 0.0001 vs. the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001 vs. the asthma group.

Figure 3

A probiotic, SLIT, and their combination improved asthma-induced histopathological change in mice lungs. (A) H&E staining of mice lung tissues. Lung sections stained with H&E revealed infiltrations of inflammatory cells in HDM-sensitized and -challenged mice, contrasting with the control group where such aggregations were absent. Notably, treated mice exhibited distinctive features in this context. (B) Inflammation score. (C) PAS staining was applied to mouse lung sections following intranasal HDM exposure. (D) % PAS intensity of stained tissue. Scale bar = 200 μm; Magnification: 40×. *** p < 0.001 and **** p < 0.0001 vs. the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001 vs. the asthma group.

Figure 4

Microscopic examinations of colon tissues. (A) H&E staining of a transversely cut colon section; (B) colon sections stained with PAS; (C) the number of GCs/crypt; (D) % PAS-stained area of GCs and mucin; (E) colon sections stained with AB staining; (F) the number of GCs/crypt in AB-stained areas; and (G) % AB-stained areas were assessed in all groups. Magnification: 100×. **** p < 0.0001 vs. the control group; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001 vs. the asthma group.

Figure 5

Experimental protocol design of allergic asthma with HDM and L. paracasei treatment. Mice were intranasally sensitized to HDM Der p extract (25 µg Der p in 10 µL PBS) for 3 days. SLIT was administered for 5 consecutive days/week for 2 weeks, as either high dose (132 µg/day) or low dose (66 µg/day), followed by 6 days of intranasal challenge exposure (same dose with sensitization). Unlike SLIT HDM-treated groups, control and asthma and probiotic-treated mice received PBS for SLIT.