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DTT 2024; 3(2): 159-168
Published online September 30, 2024
https://doi.org/10.58502/DTT.24.0006
Copyright © The Pharmaceutical Society of Korea.
Tae Il Park*, Jin Yong Song* 
, Ji-Yun Lee
Correspondence to:Ji-Yun Lee, jylee98@cau.ac.kr
*These authors contributed equally to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Asthma, a prevalent chronic inflammatory lung disease that affects over 330 million people worldwide, manifests through symptoms such as wheezing, coughing, dyspnea, and chest tightness. Probiotics, such as Bacillus subtilis (B. subtilis) and Lactobacillus rhamnosus (L. rhamnosus), provide protection against allergic airway inflammation. This is achieved by the induction of regulatory immune responses and restoration of cytokine balance in activated immune cells. This study aimed to investigate the protective effects of probiotics against inflammation in pulmonary diseases by assessing their impact on airway resistance, immune cell infiltration, and allergy-related chemokine levels in an ovalbumin (OVA)-induced asthmatic mouse model. Oral administration of live L. rhamnosus and B. subtilis significantly decreased airway resistance and reduced infiltration of immune cells like eosinophils and macrophages. Histological assessment demonstrated the anti-inflammatory effects of B. subtilis and L. rhamnosus. Furthermore, both probiotics could decrease the production of allergic factors, such as interleukin-13 (IL-13) and immunoglobulin (Ig) E and IgG1. IL-13 reduction by B. subtilus and L. rhamnosus also led to decreasing mucus production. This study demonstrated that oral administration of probiotics from Korean fermented foods may induce potentially preventive effects against asthmatic lung inflammation.
Keywordsallergy, asthma, Bacillus subtilis, Lactobacillus rhamnosus, probiotics, mucin
Asthma is a common chronic inflammatory lung disease that affects over 330 million people worldwide. It is characterized by respiratory symptoms, such as wheezing, coughing, dyspnea, and chest tightness (Paoletti et al. 2022). Moreover, patients with asthma suffer from a low quality of life (Agache et al. 2021). A recent study found that people with asthma had a higher rate of mortality related to coronavirus disease 2019. (Bloom et al. 2021). Previous studies have shown that Asian sand dust blowing into Korea from China and Mongolia in the spring worsens Ovalbumin (OVA)-induced asthma (Kim et al. 2022). The European Respiratory Society/American Thoracic Society guidelines recommend the use of anti-interleukin (IL)-5, anti-immunoglobulin E (IgE), and anti-IL-4/IL-13 therapeutic agents for severe asthma cases (Holguin et al. 2020). Type 2 helper T (Th2) cytokines, such as IL-4, IL-5, and IL-13, play a central role in allergic diseases including asthma and atopic dermatitis (Webb et al. 2000; Kim 2022). IL-4 mediates allergic responses by enhancing the proliferation and survival of Th2 cells, as well as the synthesis of IgE (Gour and Wills-Karp 2015). IL-5 induces differentiation, infiltration, and degranulation of eosinophils (Pelaia et al. 2019). IL-13 induces goblet cell hyperplasia, mucus hyperproduction, and subepithelial airway fibrosis (Zhu et al. 1999). IgE sensitizes mast cells by detecting high-affinity IgE receptors, which increase cytokine secretion and histamine release (Yamaguchi et al. 1997).
Several studies have demonstrated that probiotics regulate the body systems including immune system and central nervous system (Penders et al. 2007; Guan et al. 2024). Probiotics can mitigate the symptoms of chronic diseases via their immune-modulatory effects in the host (Forsythe 2011) and their capacity to recalibrate the immunological balance from allergic disease-associated Th2 to Th1 (Rigaux et al. 2009). Additionally, probiotics have been demonstrated to induce the expression of regulatory T cells by increasing the expression of forkhead box P3 in murine models of allergic diseases (Feleszko et al. 2007). Thus, probiotics can be used to restore the cytokine balance by inhibiting activated immune cells, such as eosinophils, B cells, and mast cells (Kalliomäki and Isolauri 2003). Based on these findings, probiotics are considered potent therapeutic agents for chronic diseases like allergic asthma (Kalliomäki et al. 2010).
Traditional Korean foods, particularly “kimchi” and “cheonggukjang”, consist of large amounts of probiotics, including Lactobacillus (Park et al. 2014) and Bacillus strains (Na et al. 2020). The recent rise in health concerns has led to an increase in the number of studies on probiotics. One prominent area of such investigation is the relationship between probiotics and the immune system. For example, a strain of Lactiplantibacillus plantarum isolated from kimchi has been demonstrated to modulate innate immunity and protect against influenza virus infection (Park et al. 2013). Moreover, kimchi intake has been associated with a reduced risk of asthma, as indicated by a low asthma occurrence rate among Koreans who consume kimchi (Kim et al. 2014). Similarly, cheonggukjang has been demonstrated to exert an inhibitory effect on allergic asthma in a murine model (Bae et al. 2014). Furthermore, preclinical studies have shown that administering L. rhamnosus can mitigate virus-induced pulmonary inflammation and allergic airway inflammation in a mouse model. Also, intranasal administration of L. rhamnosus prior to influenza infection has been demonstrated to induce an early increase in the transcription of type I interferon (IFN), resulting in enhanced resistance to infection-related mortality (Kumova et al. 2019). Furthermore, intranasal administration of L. rhamnosus has been demonstrated to improve the survival rates of influenza virus-infected mice by increasing the mRNA expressions of IL-1β, tumor necrosis factor, and monocyte chemoattractant protein-1 (Harata et al. 2010). In addition, long-term oral administration of L. rhamnosus has been demonstrated to provide protection against allergic airway inflammation in OVA-induced inflammation model by regulating the gut microbiota (Zhang et al. 2018).
Exopolysaccharide (EPS) from B. subtilis is known to alleviate asthmatic airway inflammation. When administered orally, EPS (B. subtilis) has been demonstrated to reduce the number of inflammatory cells and alleviate reactive oxygen species-induced inflammation in an OVA-induced inflammation model. This was achieved by attenuating Th2 cell-mediated inflammation via the nuclear factor-kappa B and signal transducer and activator of transcription 6 pathways in the airways (Zhang and Yi 2022). Moreover, oral administration of B. subtilis spores could protect the airways of house dust mite-induced allergic lung models against eosinophilic infiltration (Monroy Del Toro et al. 2023). Intranasal administration of B. subtilis in piglets has been shown to increase the number of immune cells in the nasal mucosa and tonsils by inducing IL-6 production (Yang et al. 2018).
Despite these important findings, the therapeutic efficacies of B. subtilis and L. rhamnosus have not been comparatively assessed to date. There is yet to be a study that has investigated the use of probiotics in an asthma mouse model. Therefore, in this study, the protective effects of probiotics on allergic asthma were investigated by evaluating and comparing the cytokine and antibody production induced by B. subtilis and L. rhamnosus. The analysis comprised assessments of lung functions, biochemical parameters in serum and bronchoalveolar lavage fluid (BALF), as well as histological characteristics.
Specific-pathogen-free, 6-week-old female BALB/c mice (6 mice per group) were purchased from Youngbio (Seongnam, Kyunggi-do, Republic of Korea) and maintained under standard conditions (22-25°C; 45-55% relative humidity; 12 h light/12 h dark cycle). The animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Chung-Ang University (IACUC approval no.: 202100124).
Lactobacillus rhamnosus 53103TM and Bacillus subtilis 6633TM strains used in this experiment were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). L. rhamnosus was grown in an ATCC medium with 416 Lactobacilli MRS broth at 37°C under 5% CO2. B. subtilis was grown in an ATCC medium with 44 brain–heart infusion broth at 30°C under anaerobic conditions. The probiotics were harvested during the stationary phase. The bacterial count was obtained via serial plate counting, and the optical density was determined using absorption spectrophotometry. The mice were orally administered with L. rhamnosus or B. subtilis (1 × 109 CFU in 0.2 mL phosphate-buffered saline) or 0.2 mL PBS per mouse three times a week, approximately one week before sensitization.
The sensitization was performed on Days 0 and 7. The mice were sensitized via intraperitoneal injections of a mixture containing 0.1 mL alum (Imject Alum, Pierce Biotechnology, Rockford, IL, USA) and 10 µg OVA (Sigma-Aldrich, St. Louis, MO, USA), absorbed onto 2.25 mg of alum. The control mice were intraperitoneally injected with 0.1 mL of Dulbecco’s PBS (DPBS) instead of OVA-alum mixture. From Days 14 to 28, the mice were subjected to an aerosolized OVA challenge (1% OVA in DPBS) for 30 min thrice weekly; the control mice were not challenged with OVA. The methacholine test was performed 29 days after the last OVA challenge, and the mice were sacrificed on the same day. All mice were divided randomly into five groups each containing six mice: (1) control (CON) group; (2) OVA group, which comprised mice sensitized with OVA; (3) OVA + B. subtilis group, which comprised mice pretreated with B. subtilis for 4 weeks and sensitized with OVA; (4) OVA + L. rhamnosus group, which comprised mice pretreated with L. rhamnosus for 4 weeks and sensitized with OVA; and (5) OVA + dexamethasone (DEXA) group, which comprised mice treated with DEXA and sensitized with OVA (Fig. 1).
Figure 1.Experimental schedule and measurement of AHR. (A) The experimental protocol for asthmatic murine model. The (B) sRaw and (C) TV values were measured using barometric plethysmography in a double chamber (n = 6). *p < 0.05 and **p < 0.01 indicate significant differences compared to the OVA group. ##p < 0.01 denotes significant differences compared to the CON group. AHR, airway hyperresponsiveness; sRAW, specific airway resistance; TV, tidal volume; p.o., oral administration; OVA, ovalbumin; CON, control; DEXA, dexamethasone.
The methacholine test was performed on Day 29 to assess tidal volume (TV) and specific airway resistance (sRaw). First, 0.5 mL of methacholine solution (6.25 mg/mL) was nebulized to conscious mice for 1 min. TV and sRaw were then measured for 3 min using barometric double-chambered plethysmography (BuxcoVR FinePointe Non-Invasive Airway Mechanics, DSITM, MN, USA). Sequentially, 12.5, 25, and 50 mg/mL methacholine solutions were nebulized, and the measurements were obtained in the same manner. sRaw (mmHg × s) was used as the main index of airway hyperresponsiveness.
On Day 29, the mice were euthanized with 5 mg/kg xylazine and 40 mg/kg zoletil. The trachea was cannulated using a syringe, and the lung was flushed with 0.7 mL DPBS twice to obtain BALF. The total and differential cell counts in BALF were determined using hemocytometry and cytospin preparation, which was stained using Kwik-DiffTM staining kits (Thermo ScientificTM, Waltham, MA, USA). The numbers of eosinophils, macrophages, neutrophils, and lymphocytes were determined by microscopy.
After the mice were euthanized on Day 29, serum samples were obtained from their inferior vena cava using a 1-mL syringe with 30 μL 3.2% sodium citrate (anticoagulant). The obtained serum samples were centrifuged at 1,500 g for 10 min at 4 °C. The supernatants of the centrifuged sera were used for analysis. The OVA-specific IgG1 and IgE enzyme-linked immunosorbent assay (ELISA) kits (Cayman Chemical, Ann Arbor, MI, USA) were used for analysis. The serum samples were stored at –80°C until analysis.
To obtain BALF supernatants, the BALF samples were centrifuged at 1,500 g for 10 min at 4°C. Next, IL-4, IL-5, IL-13, and IL-17 concentrations in the BALF supernatants were measured using ELISA kits (Quantikine ELISA, R&D Systems, MN, USA). The BALF supernatants were stored at –80°C for subsequent analysis.
The unflushed lungs were used for histopathological analysis. These samples were fixed with 10% formalin for 1 week and embedded in a paraffin block using Tissue-Tek® (Sakura Finetek®, Torrance, CA, USA). The blocks were sectioned using Leica Microtome 820 (Leica Microsystems, Wetzlar, Germany) at a slice thickness of 4 µm and stained with hematoxylin and eosin (H&E) to assess the degree of lung inflammation. The inflammation degree was scored as 0 for no inflammation, 1 for occasional cuffing with inflammatory cells, 2 when most bronchi or vessels were surrounded by a thin layer (1-5 cells) of inflammatory cells, and 3 when most bronchi or vessels were surrounded by a thick layer (>5 cells) of inflammatory cells. Furthermore, the lung tissues were stained with a periodic acid–Schiff (PAS) stain kit (Abcam, Cambridge, UK). The mucin production in the alveoli was quantified using ImageJ (NIH Image, Bethesda, MD, USA). Both H&E- and PAS-stained tissue sections were evaluated under a microscope (Leica Microsystems), and the images were captured using Leica DM 480 Camera (Leica Microsystems).
The lung tissues were homogenized using Miccra D8 homogenizer (MICCRA GmbH, Heitersheim, Germany) and incubated in radioimmunoprecipitation assay buffer (Thermo ScientificTM, Waltham, MA, USA) to extract the lung proteins. The protein concentrations were determined using Pierce Bicinchoninic Acid Protein Assay Kit (Thermo ScientificTM, Waltham, MA, USA). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer, the membranes were blocked with 5% skim milk and treated with anti-MUC5AC and anti-GAPDH antibodies (ABclonal, MA, USA) overnight at 4°C. Next, the membranes were treated with peroxidase-conjugated secondary antibodies at 22°C for 1 h. The immunoreactive bands were visualized using Fusion Solo X (Vilber, Paris, France). Whole membrane is shown in supplementary materials (Supplementary Fig. 1 and 2).
The acquired data were analyzed using one-way analysis of variance (ANOVA), two-way ANOVA, and student’s t-test. Statistical analyses were performed using GraphPad Prism version 7 (GraphPad Software Inc., San Diego, CA, USA) and Microsoft Excel (Microsoft Corporation, Redmond, WA, USA).
sRaw and TV were determined based on the methacholine test. The sRaw values at 50 mg/mL of methacholine showed statistically significant differences: 165.3 ± 15.2% (p < 0.01), 149.9 ± 5.9% (p < 0.05), 146.0 ± 10.2% (p < 0.05), 136.2 ± 10.0% (p < 0.01), and 130.5 ± 10.2% in the OVA, OVA + B. subtilis, OVA + L. rhamnosus, OVA + DEXA, and CON groups, respectively. The OVA group showed the highest sRaw value. The OVA + B. subtilis, OVA + L. rhamnosus, and OVA + DEXA groups showed significantly decreased values. No significant difference was observed between the OVA + B. subtilis and OVA + L. rhamnosus groups (Fig. 1B). Moreover, no significant intergroup difference was observed for TV values (Fig. 1C).
The macrophages, lymphocytes, neutrophils, and eosinophils in BALF were stained by Kwik-DiffTM staining kits; a representative image is displayed in Fig. 2A. These cells were counted to determine the total and differentiated immune cells in the lung. The number of immune cells in BALF in the OVA group was significantly increased compared to the CON group. The cell counts were as follows: 54.5 ± 29.2 × 103, 690.9 ± 314.7 × 103 (p < 0.001), 433.3 ± 90.55 × 103 (p < 0.05), 390.9 ± 121.3 × 103 (p < 0.05), and 235 ± 128.3 × 103 cells (p < 0.01) in the CON, OVA, OVA + B. subtilis, OVA + L. rhamnosus, and OVA + DEXA groups, respectively. The number of total immune cells in BALF was significantly decreased in the OVA + B. subtilis, OVA + L. rhamnosus, and OVA + DEXA groups (Fig. 2B). However, no significant difference was observed between the OVA + B. subtilis and OVA + L. rhamnosus groups. The differentiated immune cells were also analyzed (Fig. 2C). The number of macrophages and eosinophils showed a similar trend to that of total immune cells, while the neutrophil and lymphocyte counts did not show significant differences (data not shown).
Figure 2.Immune cells in BALF. (A) Optical microscopy images of immune cells stained with Kwik-DiffTM. Magnification, ×100. Scale bars, 100 µm. The numbers of (B) total immune cells and (C) differentiated immune cells were quantified using a hemocytometer (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences compared to the OVA group. ###p < 0.01 indicate significant differences compared to the CON group. BALF, bronchoalveolar lavage fluid; OVA, ovalbumin; CON, control; DEXA, dexamethasone.
IL-13 concentration was measured using ELISA to determine the relationship between Th2 cytokines and asthmatic lung inflammation. The OVA group showed a significantly increased IL-13 concentration (23.8 ± 5.9 pg/mL; p < 0.01) compared to the CON group (7.3 ± 0.2 pg/mL). The OVA + B. subtilis (14.5 ± 6.9 pg/mL; p < 0.05) and OVA + L. rhamnosus groups (15.3 ± 2.8 pg/mL; p < 0.05) exhibited significantly decreased concentrations compared to the OVA group. The OVA + DEXA group (8.2 ± 0.6 pg/mL; p < 0.01), which was the positive control group, also showed a significantly decreased concentration. Moreover, no significant difference was observed between the OVA + L. rhamnosus and OVA + B. subtilis groups (Fig. 3). The IL-4, IL-5, and IL-17 levels were also measured, but no significant intergroup difference was observed for any of these cytokines.
Figure 3.IL-13 level in BALF and serum levels of OVA-specific IgE and OVA-specific IgG1. (A) IL-13 levels in BALF. (B) Serum levels of OVA-specific IgE and OVA-specific IgG1. These levels were determined using ELISA (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences compared to the OVA group. ##p < 0.01 and ###p < 0.001 indicate significant differences compared to the CON group. BALF, bronchoalveolar lavage fluid; IL, interleukin; Ig, immunoglobulin; OVA, ovalbumin; CON, control; DEXA, dexamethasone.
Next, the OVA-specific IgE and IgG1 concentrations were measured using ELISA to determine the relationship between systemic allergy and asthmatic lung inflammation (Fig. 3B). The OVA-specific IgE concentration significantly increased in the OVA group (55.1 ± 7.4 ng/mL; p < 0.001) compared to the CON group (0.7 ± 1.1 ng/mL). The OVA + L. rhamnosus (14.4 ± 10.9 ng/mL; p < 0.01) and OVA + B. subtilis (14.7 ± 10.8 ng/mL; p < 0.01) groups exhibited significantly decreased concentrations compared to the OVA group. The OVA + DEXA (10.3 ± 5.7 ng/mL; p < 0.01) group also showed a significantly decreased concentration. In terms of OVA-specific IgG1, the OVA group showed a significantly increased concentration (108.4 ± 22.7 ng/mL; p < 0.001) compared to the CON group (2.6 ± 3.3 ng/mL). The OVA + B. subtilis (77.7 ± 23.9 ng/mL; p < 0.01) and OVA + L. rhamnosus (56.9 ± 13.2 ng/mL; p < 0.001) groups exhibited significantly decreased concentrations compared to the OVA group. The OVA + DEXA group also showed a significantly decreased concentration (15.1 ± 9.1 ng/mL; p < 0.001). On the other hand, no significant difference was observed between the OVA + B. subtilis and OVA + L. rhamnosus groups.
The histopathological changes in the bronchial tubes and pulmonary alveoli were assessed by H&E staining (Fig. 4A). The challenge with OVA significantly increased the inflammatory cell infiltration. Notably, the treatment with L. rhamnosus or B. subtilis could reduce such infiltration and the thickness of the lung epithelial membranes. The width of the alveolar wall was thicker in the OVA group (3.1 ± 0.9; p < 0.01) than in the CON group (0.3 ± 0.5). The OVA+ L. rhamnosus (1.1 ± 0.7; p < 0.01) and OVA+ B. subtilis groups (1.3 ± 0.9) exhibited less severe thickness than the OVA group (Fig. 4B). No significant differences were observed between the OVA + L. rhamnosus and the OVA + B. subtilis groups.
Figure 4.Histopathological images of the lung tissues stained with H&E. (A) H&E staining of the alveoli. Magnification, ×100. Scale bars, 100 µm. (B) Inflammation scores were determined using the histological grading criteria (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 indicates significant differences compared to the OVA group. ###p < 0.001 indicates significant differences compared to the CON group. OVA, ovalbumin; CON, control; DEXA, dexamethasone.
PAS staining was performed to assess the mucin production levels (Fig. 5A). The level was highest in the OVA group (20.0 ± 3.4%; p < 0.01), while the OVA + L. rhamnosus (4.75 ± 1.3%; p < 0.01) and OVA + B. subtilis groups (5.5 ± 1.3%; p < 0.01) exhibited similar levels (Fig. 5B). Both the B. subtilis and L. rhamnosus groups showed decreased MUC5AC production levels, but the B. subtilis group exhibited a greater reduction than the L. rhamnosus group (Fig. 5C).
Figure 5.Mucin production in the lung. (A) PAS staining was performed to assess the mucin production levels at magnifications of 100x and 400x. The black scale bars are 100 µm at ×100 magnification, and 25 µm at ×400 magnification. (B) The mucin production level was highest in the OVA group (20.0 ± 3.4%; p < 0.01). (C) The OVA + L. rhamnosus (4.75 ± 1.3%) and OVA + B. subtilis (5.5 ± 1.3%) groups showed similar levels (p < 0.01) (n = 6). Western blot analysis was performed to assess the MUC5AC levels; the band intensity of MUC5AC was highest in the OVA group. **p < 0.01 indicates significant differences compared to the OVA group. ##p < 0.01 indicates significant differences compared to the CON group. PAS, Periodic Acid-Schiff; OVA, ovalbumin; CON, control; DEXA, dexamethasone; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
It has been prominently demonstrated that Lactobacillus strains can alleviate allergic diseases like atopic dermatitis (Kim et al. 2020), allergic rhinitis (AR) (Kim et al. 2019), and allergic airway disease (Li et al. 2010). Furthermore, probiotics administration has been associated with immune regulation and changes in the composition of gut microbiota, with evident mitigation of atopic dermatitis. Similarly, it has been demonstrated that probiotics can suppress the differentiation of splenic T cells into Th2 cells while mediating their differentiation into regulatory T cells in vitro to alleviate the symptoms of AR and disturbances in the gut microbiota (Kim et al. 2019). Furthermore, probiotics can modulate the immune system (Li et al. 2010).
Traditional Korean foods, particularly kimchi, have been demonstrated to alleviate the symptoms of allergic diseases. A previous study (Kim et al. 2016) reported that kimchi is beneficial for treating allergic diseases like AR and asthma. Furthermore, although focused on the effects of vegetables, lactic acid, vitamins (ascorbic acid), and probiotics, another previous study (Kang et al. 2009) indicated that bacteria in kimchi may mitigate allergic diseases like rhinitis and asthma, as investigated using a murine model. It was revealed that Leuconostoc citreum HJ-P4 (KACC 91035), which was isolated from kimchi, improved the immune system by decreasing the serum levels of total IgE and IgG1 and enhancing the secretion of antigen-specific IFN-γ. Similarly, Leuconostoc citreum EFEL2061, a bacterium also isolated from kimchi, induced cytokine production and decreased the serum level of IgE in an allergic mouse model. This effect was attributed to the enhanced production of innate immune cells and decreased activation of bystander B-cells (Kang et al. 2016).
In addition, cheonggukjang (Wei et al. 2015) has also been prominently demonstrated for its antiviral effects against the influenza A virus. Ethyl acetate extracts of cheonggukjang have shown inhibitory effects against neuraminidase in vitro. Furthermore, studies have reported the potential anti-inflammatory and antiviral properties of the chemical constituents of soybeans and their fermentative metabolites (Kwon et al. 2019).
In this study, we investigated the protective effects of orally administered probiotics (Lactobacillus rhamnosus 53103TM and Bacillus subtilis 6633TM) against OVA-induced asthmatic lung inflammation in mice.
OVA allergen stimulation by sensitization and challenge increased the levels of specific airway resistance, immune cell infiltration into the lung, cytokine levels (IL-13, OVA-specific IgE, and OVA-specific IgG1), and mucin production, which collectively indicated the successful establishment of a murine model of asthmatic lung inflammation.
IL-13 activates eosinophils, increases airway resistance, promotes mucin production, and mediates metaplasia and hyperplasia of goblet cells that produce the mucus (Ingram and Kraft 2012). The level of IL-13 can be increased by activated T lymphocytes (Zurawski and de Vries 1994). Also, IL-13 increases mucus production by inducing mucus metaplasia, MUC5AC expression, and differentiation of goblet cells (Kanoh et al. 2011). Moreover, elevated eosinophil counts in asthma are caused by Th2 cell activation (Boonpiyathad et al. 2019). In this study, the OVA group showed an increased IL-13 level, eosinophil count, and mucus production. These trends indicated that IL-13 and eosinophils play important roles in allergic diseases (Kita 2011).
Importantly, L. rhamnosus and B. subtilis provided preventive effects against allergic asthma. In the OVA + B. subtilis and OVA + L. rhamnosus groups, airway resistance and infiltration of total immune cells – including macrophages and eosinophils but excluding neutrophils and lymphocytes – decreased significantly. These groups also showed decreased IL-13 levels and mucin production. Notably, the B. subtilis group showed a more significant reduction in the production of MUC5AC production compared to the L. rhamnosus group. MUC5AC, which is highly expressed in the bronchial epithelium and submucosal glands, is the major mucin released from goblet cells. Controlling excessive mucus secretion is very important in asthma, as excessive mucus secretion can worsen the disease progression (Bonser and Erle 2017). Therefore, it is expected that the intake of B. subtilis will have a clinically beneficial effect on asthma. This study has limitations in that it did not observe changes in gut microbacteria species and metabolites such as short chain fatty acids. In further studies, it will be possible to explain changes in the expression level of Muc5AC through direct comparison of gut microbacteria species and metabolites in L. rhamnosus and B. subtilis administration groups.
This study compared two types of probiotics to demonstrate their preventive effects against asthma. Although additional studies are needed to clarify the underlying mechanisms, this study demonstrated that oral administration of B. subtilis and L. rhamnosus can mitigate the symptoms of asthma by reducing mucin production. Collectively, our findings indicate the therapeutic potential of probiotics against asthma.
Supplementary materials can be found via https://doi.org/10.58502/DTT.24.0006.
dtt-3-2-159-supple.pdfThe authors declare that they have no conflict of interest.
This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2022R1F1A1076528).
DTT 2024; 3(2): 159-168
Published online September 30, 2024 https://doi.org/10.58502/DTT.24.0006
Copyright © The Pharmaceutical Society of Korea.
Tae Il Park*, Jin Yong Song* , Ji-Yun Lee
College of Pharmacy, Chung-Ang University, Seoul, Korea
Correspondence to:Ji-Yun Lee, jylee98@cau.ac.kr
*These authors contributed equally to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Asthma, a prevalent chronic inflammatory lung disease that affects over 330 million people worldwide, manifests through symptoms such as wheezing, coughing, dyspnea, and chest tightness. Probiotics, such as Bacillus subtilis (B. subtilis) and Lactobacillus rhamnosus (L. rhamnosus), provide protection against allergic airway inflammation. This is achieved by the induction of regulatory immune responses and restoration of cytokine balance in activated immune cells. This study aimed to investigate the protective effects of probiotics against inflammation in pulmonary diseases by assessing their impact on airway resistance, immune cell infiltration, and allergy-related chemokine levels in an ovalbumin (OVA)-induced asthmatic mouse model. Oral administration of live L. rhamnosus and B. subtilis significantly decreased airway resistance and reduced infiltration of immune cells like eosinophils and macrophages. Histological assessment demonstrated the anti-inflammatory effects of B. subtilis and L. rhamnosus. Furthermore, both probiotics could decrease the production of allergic factors, such as interleukin-13 (IL-13) and immunoglobulin (Ig) E and IgG1. IL-13 reduction by B. subtilus and L. rhamnosus also led to decreasing mucus production. This study demonstrated that oral administration of probiotics from Korean fermented foods may induce potentially preventive effects against asthmatic lung inflammation.
Keywords: allergy, asthma, Bacillus subtilis, Lactobacillus rhamnosus, probiotics, mucin
Asthma is a common chronic inflammatory lung disease that affects over 330 million people worldwide. It is characterized by respiratory symptoms, such as wheezing, coughing, dyspnea, and chest tightness (Paoletti et al. 2022). Moreover, patients with asthma suffer from a low quality of life (Agache et al. 2021). A recent study found that people with asthma had a higher rate of mortality related to coronavirus disease 2019. (Bloom et al. 2021). Previous studies have shown that Asian sand dust blowing into Korea from China and Mongolia in the spring worsens Ovalbumin (OVA)-induced asthma (Kim et al. 2022). The European Respiratory Society/American Thoracic Society guidelines recommend the use of anti-interleukin (IL)-5, anti-immunoglobulin E (IgE), and anti-IL-4/IL-13 therapeutic agents for severe asthma cases (Holguin et al. 2020). Type 2 helper T (Th2) cytokines, such as IL-4, IL-5, and IL-13, play a central role in allergic diseases including asthma and atopic dermatitis (Webb et al. 2000; Kim 2022). IL-4 mediates allergic responses by enhancing the proliferation and survival of Th2 cells, as well as the synthesis of IgE (Gour and Wills-Karp 2015). IL-5 induces differentiation, infiltration, and degranulation of eosinophils (Pelaia et al. 2019). IL-13 induces goblet cell hyperplasia, mucus hyperproduction, and subepithelial airway fibrosis (Zhu et al. 1999). IgE sensitizes mast cells by detecting high-affinity IgE receptors, which increase cytokine secretion and histamine release (Yamaguchi et al. 1997).
Several studies have demonstrated that probiotics regulate the body systems including immune system and central nervous system (Penders et al. 2007; Guan et al. 2024). Probiotics can mitigate the symptoms of chronic diseases via their immune-modulatory effects in the host (Forsythe 2011) and their capacity to recalibrate the immunological balance from allergic disease-associated Th2 to Th1 (Rigaux et al. 2009). Additionally, probiotics have been demonstrated to induce the expression of regulatory T cells by increasing the expression of forkhead box P3 in murine models of allergic diseases (Feleszko et al. 2007). Thus, probiotics can be used to restore the cytokine balance by inhibiting activated immune cells, such as eosinophils, B cells, and mast cells (Kalliomäki and Isolauri 2003). Based on these findings, probiotics are considered potent therapeutic agents for chronic diseases like allergic asthma (Kalliomäki et al. 2010).
Traditional Korean foods, particularly “kimchi” and “cheonggukjang”, consist of large amounts of probiotics, including Lactobacillus (Park et al. 2014) and Bacillus strains (Na et al. 2020). The recent rise in health concerns has led to an increase in the number of studies on probiotics. One prominent area of such investigation is the relationship between probiotics and the immune system. For example, a strain of Lactiplantibacillus plantarum isolated from kimchi has been demonstrated to modulate innate immunity and protect against influenza virus infection (Park et al. 2013). Moreover, kimchi intake has been associated with a reduced risk of asthma, as indicated by a low asthma occurrence rate among Koreans who consume kimchi (Kim et al. 2014). Similarly, cheonggukjang has been demonstrated to exert an inhibitory effect on allergic asthma in a murine model (Bae et al. 2014). Furthermore, preclinical studies have shown that administering L. rhamnosus can mitigate virus-induced pulmonary inflammation and allergic airway inflammation in a mouse model. Also, intranasal administration of L. rhamnosus prior to influenza infection has been demonstrated to induce an early increase in the transcription of type I interferon (IFN), resulting in enhanced resistance to infection-related mortality (Kumova et al. 2019). Furthermore, intranasal administration of L. rhamnosus has been demonstrated to improve the survival rates of influenza virus-infected mice by increasing the mRNA expressions of IL-1β, tumor necrosis factor, and monocyte chemoattractant protein-1 (Harata et al. 2010). In addition, long-term oral administration of L. rhamnosus has been demonstrated to provide protection against allergic airway inflammation in OVA-induced inflammation model by regulating the gut microbiota (Zhang et al. 2018).
Exopolysaccharide (EPS) from B. subtilis is known to alleviate asthmatic airway inflammation. When administered orally, EPS (B. subtilis) has been demonstrated to reduce the number of inflammatory cells and alleviate reactive oxygen species-induced inflammation in an OVA-induced inflammation model. This was achieved by attenuating Th2 cell-mediated inflammation via the nuclear factor-kappa B and signal transducer and activator of transcription 6 pathways in the airways (Zhang and Yi 2022). Moreover, oral administration of B. subtilis spores could protect the airways of house dust mite-induced allergic lung models against eosinophilic infiltration (Monroy Del Toro et al. 2023). Intranasal administration of B. subtilis in piglets has been shown to increase the number of immune cells in the nasal mucosa and tonsils by inducing IL-6 production (Yang et al. 2018).
Despite these important findings, the therapeutic efficacies of B. subtilis and L. rhamnosus have not been comparatively assessed to date. There is yet to be a study that has investigated the use of probiotics in an asthma mouse model. Therefore, in this study, the protective effects of probiotics on allergic asthma were investigated by evaluating and comparing the cytokine and antibody production induced by B. subtilis and L. rhamnosus. The analysis comprised assessments of lung functions, biochemical parameters in serum and bronchoalveolar lavage fluid (BALF), as well as histological characteristics.
Specific-pathogen-free, 6-week-old female BALB/c mice (6 mice per group) were purchased from Youngbio (Seongnam, Kyunggi-do, Republic of Korea) and maintained under standard conditions (22-25°C; 45-55% relative humidity; 12 h light/12 h dark cycle). The animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Chung-Ang University (IACUC approval no.: 202100124).
Lactobacillus rhamnosus 53103TM and Bacillus subtilis 6633TM strains used in this experiment were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). L. rhamnosus was grown in an ATCC medium with 416 Lactobacilli MRS broth at 37°C under 5% CO2. B. subtilis was grown in an ATCC medium with 44 brain–heart infusion broth at 30°C under anaerobic conditions. The probiotics were harvested during the stationary phase. The bacterial count was obtained via serial plate counting, and the optical density was determined using absorption spectrophotometry. The mice were orally administered with L. rhamnosus or B. subtilis (1 × 109 CFU in 0.2 mL phosphate-buffered saline) or 0.2 mL PBS per mouse three times a week, approximately one week before sensitization.
The sensitization was performed on Days 0 and 7. The mice were sensitized via intraperitoneal injections of a mixture containing 0.1 mL alum (Imject Alum, Pierce Biotechnology, Rockford, IL, USA) and 10 µg OVA (Sigma-Aldrich, St. Louis, MO, USA), absorbed onto 2.25 mg of alum. The control mice were intraperitoneally injected with 0.1 mL of Dulbecco’s PBS (DPBS) instead of OVA-alum mixture. From Days 14 to 28, the mice were subjected to an aerosolized OVA challenge (1% OVA in DPBS) for 30 min thrice weekly; the control mice were not challenged with OVA. The methacholine test was performed 29 days after the last OVA challenge, and the mice were sacrificed on the same day. All mice were divided randomly into five groups each containing six mice: (1) control (CON) group; (2) OVA group, which comprised mice sensitized with OVA; (3) OVA + B. subtilis group, which comprised mice pretreated with B. subtilis for 4 weeks and sensitized with OVA; (4) OVA + L. rhamnosus group, which comprised mice pretreated with L. rhamnosus for 4 weeks and sensitized with OVA; and (5) OVA + dexamethasone (DEXA) group, which comprised mice treated with DEXA and sensitized with OVA (Fig. 1).
Figure 1. Experimental schedule and measurement of AHR. (A) The experimental protocol for asthmatic murine model. The (B) sRaw and (C) TV values were measured using barometric plethysmography in a double chamber (n = 6). *p < 0.05 and **p < 0.01 indicate significant differences compared to the OVA group. ##p < 0.01 denotes significant differences compared to the CON group. AHR, airway hyperresponsiveness; sRAW, specific airway resistance; TV, tidal volume; p.o., oral administration; OVA, ovalbumin; CON, control; DEXA, dexamethasone.
The methacholine test was performed on Day 29 to assess tidal volume (TV) and specific airway resistance (sRaw). First, 0.5 mL of methacholine solution (6.25 mg/mL) was nebulized to conscious mice for 1 min. TV and sRaw were then measured for 3 min using barometric double-chambered plethysmography (BuxcoVR FinePointe Non-Invasive Airway Mechanics, DSITM, MN, USA). Sequentially, 12.5, 25, and 50 mg/mL methacholine solutions were nebulized, and the measurements were obtained in the same manner. sRaw (mmHg × s) was used as the main index of airway hyperresponsiveness.
On Day 29, the mice were euthanized with 5 mg/kg xylazine and 40 mg/kg zoletil. The trachea was cannulated using a syringe, and the lung was flushed with 0.7 mL DPBS twice to obtain BALF. The total and differential cell counts in BALF were determined using hemocytometry and cytospin preparation, which was stained using Kwik-DiffTM staining kits (Thermo ScientificTM, Waltham, MA, USA). The numbers of eosinophils, macrophages, neutrophils, and lymphocytes were determined by microscopy.
After the mice were euthanized on Day 29, serum samples were obtained from their inferior vena cava using a 1-mL syringe with 30 μL 3.2% sodium citrate (anticoagulant). The obtained serum samples were centrifuged at 1,500 g for 10 min at 4 °C. The supernatants of the centrifuged sera were used for analysis. The OVA-specific IgG1 and IgE enzyme-linked immunosorbent assay (ELISA) kits (Cayman Chemical, Ann Arbor, MI, USA) were used for analysis. The serum samples were stored at –80°C until analysis.
To obtain BALF supernatants, the BALF samples were centrifuged at 1,500 g for 10 min at 4°C. Next, IL-4, IL-5, IL-13, and IL-17 concentrations in the BALF supernatants were measured using ELISA kits (Quantikine ELISA, R&D Systems, MN, USA). The BALF supernatants were stored at –80°C for subsequent analysis.
The unflushed lungs were used for histopathological analysis. These samples were fixed with 10% formalin for 1 week and embedded in a paraffin block using Tissue-Tek® (Sakura Finetek®, Torrance, CA, USA). The blocks were sectioned using Leica Microtome 820 (Leica Microsystems, Wetzlar, Germany) at a slice thickness of 4 µm and stained with hematoxylin and eosin (H&E) to assess the degree of lung inflammation. The inflammation degree was scored as 0 for no inflammation, 1 for occasional cuffing with inflammatory cells, 2 when most bronchi or vessels were surrounded by a thin layer (1-5 cells) of inflammatory cells, and 3 when most bronchi or vessels were surrounded by a thick layer (>5 cells) of inflammatory cells. Furthermore, the lung tissues were stained with a periodic acid–Schiff (PAS) stain kit (Abcam, Cambridge, UK). The mucin production in the alveoli was quantified using ImageJ (NIH Image, Bethesda, MD, USA). Both H&E- and PAS-stained tissue sections were evaluated under a microscope (Leica Microsystems), and the images were captured using Leica DM 480 Camera (Leica Microsystems).
The lung tissues were homogenized using Miccra D8 homogenizer (MICCRA GmbH, Heitersheim, Germany) and incubated in radioimmunoprecipitation assay buffer (Thermo ScientificTM, Waltham, MA, USA) to extract the lung proteins. The protein concentrations were determined using Pierce Bicinchoninic Acid Protein Assay Kit (Thermo ScientificTM, Waltham, MA, USA). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer, the membranes were blocked with 5% skim milk and treated with anti-MUC5AC and anti-GAPDH antibodies (ABclonal, MA, USA) overnight at 4°C. Next, the membranes were treated with peroxidase-conjugated secondary antibodies at 22°C for 1 h. The immunoreactive bands were visualized using Fusion Solo X (Vilber, Paris, France). Whole membrane is shown in supplementary materials (Supplementary Fig. 1 and 2).
The acquired data were analyzed using one-way analysis of variance (ANOVA), two-way ANOVA, and student’s t-test. Statistical analyses were performed using GraphPad Prism version 7 (GraphPad Software Inc., San Diego, CA, USA) and Microsoft Excel (Microsoft Corporation, Redmond, WA, USA).
sRaw and TV were determined based on the methacholine test. The sRaw values at 50 mg/mL of methacholine showed statistically significant differences: 165.3 ± 15.2% (p < 0.01), 149.9 ± 5.9% (p < 0.05), 146.0 ± 10.2% (p < 0.05), 136.2 ± 10.0% (p < 0.01), and 130.5 ± 10.2% in the OVA, OVA + B. subtilis, OVA + L. rhamnosus, OVA + DEXA, and CON groups, respectively. The OVA group showed the highest sRaw value. The OVA + B. subtilis, OVA + L. rhamnosus, and OVA + DEXA groups showed significantly decreased values. No significant difference was observed between the OVA + B. subtilis and OVA + L. rhamnosus groups (Fig. 1B). Moreover, no significant intergroup difference was observed for TV values (Fig. 1C).
The macrophages, lymphocytes, neutrophils, and eosinophils in BALF were stained by Kwik-DiffTM staining kits; a representative image is displayed in Fig. 2A. These cells were counted to determine the total and differentiated immune cells in the lung. The number of immune cells in BALF in the OVA group was significantly increased compared to the CON group. The cell counts were as follows: 54.5 ± 29.2 × 103, 690.9 ± 314.7 × 103 (p < 0.001), 433.3 ± 90.55 × 103 (p < 0.05), 390.9 ± 121.3 × 103 (p < 0.05), and 235 ± 128.3 × 103 cells (p < 0.01) in the CON, OVA, OVA + B. subtilis, OVA + L. rhamnosus, and OVA + DEXA groups, respectively. The number of total immune cells in BALF was significantly decreased in the OVA + B. subtilis, OVA + L. rhamnosus, and OVA + DEXA groups (Fig. 2B). However, no significant difference was observed between the OVA + B. subtilis and OVA + L. rhamnosus groups. The differentiated immune cells were also analyzed (Fig. 2C). The number of macrophages and eosinophils showed a similar trend to that of total immune cells, while the neutrophil and lymphocyte counts did not show significant differences (data not shown).
Figure 2. Immune cells in BALF. (A) Optical microscopy images of immune cells stained with Kwik-DiffTM. Magnification, ×100. Scale bars, 100 µm. The numbers of (B) total immune cells and (C) differentiated immune cells were quantified using a hemocytometer (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences compared to the OVA group. ###p < 0.01 indicate significant differences compared to the CON group. BALF, bronchoalveolar lavage fluid; OVA, ovalbumin; CON, control; DEXA, dexamethasone.
IL-13 concentration was measured using ELISA to determine the relationship between Th2 cytokines and asthmatic lung inflammation. The OVA group showed a significantly increased IL-13 concentration (23.8 ± 5.9 pg/mL; p < 0.01) compared to the CON group (7.3 ± 0.2 pg/mL). The OVA + B. subtilis (14.5 ± 6.9 pg/mL; p < 0.05) and OVA + L. rhamnosus groups (15.3 ± 2.8 pg/mL; p < 0.05) exhibited significantly decreased concentrations compared to the OVA group. The OVA + DEXA group (8.2 ± 0.6 pg/mL; p < 0.01), which was the positive control group, also showed a significantly decreased concentration. Moreover, no significant difference was observed between the OVA + L. rhamnosus and OVA + B. subtilis groups (Fig. 3). The IL-4, IL-5, and IL-17 levels were also measured, but no significant intergroup difference was observed for any of these cytokines.
Figure 3. IL-13 level in BALF and serum levels of OVA-specific IgE and OVA-specific IgG1. (A) IL-13 levels in BALF. (B) Serum levels of OVA-specific IgE and OVA-specific IgG1. These levels were determined using ELISA (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences compared to the OVA group. ##p < 0.01 and ###p < 0.001 indicate significant differences compared to the CON group. BALF, bronchoalveolar lavage fluid; IL, interleukin; Ig, immunoglobulin; OVA, ovalbumin; CON, control; DEXA, dexamethasone.
Next, the OVA-specific IgE and IgG1 concentrations were measured using ELISA to determine the relationship between systemic allergy and asthmatic lung inflammation (Fig. 3B). The OVA-specific IgE concentration significantly increased in the OVA group (55.1 ± 7.4 ng/mL; p < 0.001) compared to the CON group (0.7 ± 1.1 ng/mL). The OVA + L. rhamnosus (14.4 ± 10.9 ng/mL; p < 0.01) and OVA + B. subtilis (14.7 ± 10.8 ng/mL; p < 0.01) groups exhibited significantly decreased concentrations compared to the OVA group. The OVA + DEXA (10.3 ± 5.7 ng/mL; p < 0.01) group also showed a significantly decreased concentration. In terms of OVA-specific IgG1, the OVA group showed a significantly increased concentration (108.4 ± 22.7 ng/mL; p < 0.001) compared to the CON group (2.6 ± 3.3 ng/mL). The OVA + B. subtilis (77.7 ± 23.9 ng/mL; p < 0.01) and OVA + L. rhamnosus (56.9 ± 13.2 ng/mL; p < 0.001) groups exhibited significantly decreased concentrations compared to the OVA group. The OVA + DEXA group also showed a significantly decreased concentration (15.1 ± 9.1 ng/mL; p < 0.001). On the other hand, no significant difference was observed between the OVA + B. subtilis and OVA + L. rhamnosus groups.
The histopathological changes in the bronchial tubes and pulmonary alveoli were assessed by H&E staining (Fig. 4A). The challenge with OVA significantly increased the inflammatory cell infiltration. Notably, the treatment with L. rhamnosus or B. subtilis could reduce such infiltration and the thickness of the lung epithelial membranes. The width of the alveolar wall was thicker in the OVA group (3.1 ± 0.9; p < 0.01) than in the CON group (0.3 ± 0.5). The OVA+ L. rhamnosus (1.1 ± 0.7; p < 0.01) and OVA+ B. subtilis groups (1.3 ± 0.9) exhibited less severe thickness than the OVA group (Fig. 4B). No significant differences were observed between the OVA + L. rhamnosus and the OVA + B. subtilis groups.
Figure 4. Histopathological images of the lung tissues stained with H&E. (A) H&E staining of the alveoli. Magnification, ×100. Scale bars, 100 µm. (B) Inflammation scores were determined using the histological grading criteria (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 indicates significant differences compared to the OVA group. ###p < 0.001 indicates significant differences compared to the CON group. OVA, ovalbumin; CON, control; DEXA, dexamethasone.
PAS staining was performed to assess the mucin production levels (Fig. 5A). The level was highest in the OVA group (20.0 ± 3.4%; p < 0.01), while the OVA + L. rhamnosus (4.75 ± 1.3%; p < 0.01) and OVA + B. subtilis groups (5.5 ± 1.3%; p < 0.01) exhibited similar levels (Fig. 5B). Both the B. subtilis and L. rhamnosus groups showed decreased MUC5AC production levels, but the B. subtilis group exhibited a greater reduction than the L. rhamnosus group (Fig. 5C).
Figure 5. Mucin production in the lung. (A) PAS staining was performed to assess the mucin production levels at magnifications of 100x and 400x. The black scale bars are 100 µm at ×100 magnification, and 25 µm at ×400 magnification. (B) The mucin production level was highest in the OVA group (20.0 ± 3.4%; p < 0.01). (C) The OVA + L. rhamnosus (4.75 ± 1.3%) and OVA + B. subtilis (5.5 ± 1.3%) groups showed similar levels (p < 0.01) (n = 6). Western blot analysis was performed to assess the MUC5AC levels; the band intensity of MUC5AC was highest in the OVA group. **p < 0.01 indicates significant differences compared to the OVA group. ##p < 0.01 indicates significant differences compared to the CON group. PAS, Periodic Acid-Schiff; OVA, ovalbumin; CON, control; DEXA, dexamethasone; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
It has been prominently demonstrated that Lactobacillus strains can alleviate allergic diseases like atopic dermatitis (Kim et al. 2020), allergic rhinitis (AR) (Kim et al. 2019), and allergic airway disease (Li et al. 2010). Furthermore, probiotics administration has been associated with immune regulation and changes in the composition of gut microbiota, with evident mitigation of atopic dermatitis. Similarly, it has been demonstrated that probiotics can suppress the differentiation of splenic T cells into Th2 cells while mediating their differentiation into regulatory T cells in vitro to alleviate the symptoms of AR and disturbances in the gut microbiota (Kim et al. 2019). Furthermore, probiotics can modulate the immune system (Li et al. 2010).
Traditional Korean foods, particularly kimchi, have been demonstrated to alleviate the symptoms of allergic diseases. A previous study (Kim et al. 2016) reported that kimchi is beneficial for treating allergic diseases like AR and asthma. Furthermore, although focused on the effects of vegetables, lactic acid, vitamins (ascorbic acid), and probiotics, another previous study (Kang et al. 2009) indicated that bacteria in kimchi may mitigate allergic diseases like rhinitis and asthma, as investigated using a murine model. It was revealed that Leuconostoc citreum HJ-P4 (KACC 91035), which was isolated from kimchi, improved the immune system by decreasing the serum levels of total IgE and IgG1 and enhancing the secretion of antigen-specific IFN-γ. Similarly, Leuconostoc citreum EFEL2061, a bacterium also isolated from kimchi, induced cytokine production and decreased the serum level of IgE in an allergic mouse model. This effect was attributed to the enhanced production of innate immune cells and decreased activation of bystander B-cells (Kang et al. 2016).
In addition, cheonggukjang (Wei et al. 2015) has also been prominently demonstrated for its antiviral effects against the influenza A virus. Ethyl acetate extracts of cheonggukjang have shown inhibitory effects against neuraminidase in vitro. Furthermore, studies have reported the potential anti-inflammatory and antiviral properties of the chemical constituents of soybeans and their fermentative metabolites (Kwon et al. 2019).
In this study, we investigated the protective effects of orally administered probiotics (Lactobacillus rhamnosus 53103TM and Bacillus subtilis 6633TM) against OVA-induced asthmatic lung inflammation in mice.
OVA allergen stimulation by sensitization and challenge increased the levels of specific airway resistance, immune cell infiltration into the lung, cytokine levels (IL-13, OVA-specific IgE, and OVA-specific IgG1), and mucin production, which collectively indicated the successful establishment of a murine model of asthmatic lung inflammation.
IL-13 activates eosinophils, increases airway resistance, promotes mucin production, and mediates metaplasia and hyperplasia of goblet cells that produce the mucus (Ingram and Kraft 2012). The level of IL-13 can be increased by activated T lymphocytes (Zurawski and de Vries 1994). Also, IL-13 increases mucus production by inducing mucus metaplasia, MUC5AC expression, and differentiation of goblet cells (Kanoh et al. 2011). Moreover, elevated eosinophil counts in asthma are caused by Th2 cell activation (Boonpiyathad et al. 2019). In this study, the OVA group showed an increased IL-13 level, eosinophil count, and mucus production. These trends indicated that IL-13 and eosinophils play important roles in allergic diseases (Kita 2011).
Importantly, L. rhamnosus and B. subtilis provided preventive effects against allergic asthma. In the OVA + B. subtilis and OVA + L. rhamnosus groups, airway resistance and infiltration of total immune cells – including macrophages and eosinophils but excluding neutrophils and lymphocytes – decreased significantly. These groups also showed decreased IL-13 levels and mucin production. Notably, the B. subtilis group showed a more significant reduction in the production of MUC5AC production compared to the L. rhamnosus group. MUC5AC, which is highly expressed in the bronchial epithelium and submucosal glands, is the major mucin released from goblet cells. Controlling excessive mucus secretion is very important in asthma, as excessive mucus secretion can worsen the disease progression (Bonser and Erle 2017). Therefore, it is expected that the intake of B. subtilis will have a clinically beneficial effect on asthma. This study has limitations in that it did not observe changes in gut microbacteria species and metabolites such as short chain fatty acids. In further studies, it will be possible to explain changes in the expression level of Muc5AC through direct comparison of gut microbacteria species and metabolites in L. rhamnosus and B. subtilis administration groups.
This study compared two types of probiotics to demonstrate their preventive effects against asthma. Although additional studies are needed to clarify the underlying mechanisms, this study demonstrated that oral administration of B. subtilis and L. rhamnosus can mitigate the symptoms of asthma by reducing mucin production. Collectively, our findings indicate the therapeutic potential of probiotics against asthma.
Supplementary materials can be found via https://doi.org/10.58502/DTT.24.0006.
dtt-3-2-159-supple.pdfThe authors declare that they have no conflict of interest.
This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2022R1F1A1076528).
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