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Open Access

Peer-reviewed

Research Article

Oral administration of Limosilactobacillus reuteri VHProbi® M07 alleviates ovalbumin-induced allergic asthma in mice

Abstract

Aims

Asthma is characterized by chronic airway inflammation, persistent cough, wheezing, and dyspnea. This study aimed to evaluate the efficacy of Limosilactobacillus reuteri VHProbi® M07 (M07) administration in alleviate the asthma severity in a mice model.

Methods and results

In vitro studies confirmed that M07 can survive and proliferate within the gastrointestinal tract. BALB/c mice were administered M07 both before and after ovalbumin (OVA) challenge. Serum levels of OVA-specific immunoglobulin (Ig) E and IgG1, inflammatory cells and cytokines in bronchoalveolar lavage fluid were assessed, along with histopathological examination of lung tissue. Compared to the placebo (PLA) group, mice treated with M07 exhibited significantly lower levels of OVA-specific IgE and IgG1 (P < 0.01). The counts of eosinophils and neutrophils were also significantly reduced in both the pretreated (PRE) group and post-treated (POS) group compared with the PLA group (P < 0.01). Histological analysis of lung tissues verified the protective effects of M07 against inflammation, demonstrating reduced infiltration of inflammatory cells. Additionally, mice in the PRE and POS groups showed significantly increased levels of IL-10 (P < 0.01), and significantly decreased levels of IL-5, IL-13, MCP-1, eotaxin, and tumor necrosis factor-α (P < 0.01).

Conclusions

Oral administration of M07 mitigated key features of inflammatory responses in the OVA-induced mice asthma model. These findings suggest that M07 holds therapeutic potential for the treatment of allergic asthma.

Citation: Meng G, Cui H, Feng C, Guo C, Song L, Duan Z (2025) Oral administration of Limosilactobacillus reuteri VHProbi® M07 alleviates ovalbumin-induced allergic asthma in mice. PLoS ONE 20(1): e0317587. https://doi.org/10.1371/journal.pone.0317587

Editor: Misbahuddin Rafeeq, King Abdulaziz University Faculty of Medicine, SAUDI ARABIA

Received: November 7, 2024; Accepted: January 1, 2025; Published: January 16, 2025

Copyright: © 2025 Meng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: l relevant data are within the manuscript and its Supporting information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Asthma is characterised by recurrent airway limitations and respiratory symptoms such as wheezing, coughing, breathlessness, and chest tightness. It is one of the most common chronic, non-communicable diseases globally [1]. Despite its relatively low mortality rate, asthma imposes a significant burden on healthcare systems and adversely affects patients and their families [2, 3]. Estimates suggest that over 334 million individuals worldwide are afflicted by asthma, with projections this number may rise to 400 million by 2025 [4].

The prevalence of asthma is highest in developed countries, where it has stabilized or is decreasing, and lowest in many developing countries, where it is increasing rapidly due to modernized lifestyles [1, 5]. Generally, chronic inflammation, airway obstruction, and hyperresponsiveness are the primary mechanisms involved in the pathogenesis of asthma [6]. Specifically, chronic airway inflammation is a hallmark feature of asthma regardless of disease duration and severity [7].

From an immunological perspective, three main processes are involved in the development of asthma: sensitization, challenge, and inflammation [8–11]. Elevated levels of antigen-specific immunoglobulins and proinflammatory cytokines, such as interleukin (IL)-4, IL-9 and IL-13, are observed in the serum accompanied by eosinophil infiltration as evidenced by histological findings [12–15]. Commonly, pharmacological treatments, including inhaled corticosteroids (ICS), short-acting and long-acting β2 agonists (SABA/LABA) are recommended for asthma management [16–18]. However, given the limitations of these therapies—such as poor adherence, over-reliance, and an increased risk of exacerbation and mortality—developing new treatments that target the underlying immunological pathways to enhance efficacy and improve asthma control is a rational approach [19–22].

In recent decades, the potential impact of intestinal microbiota dysbiosis on the pathogenesis of respiratory illnesses and associated disease symptoms has garnered increasing attention [23–25]. The maturation of host’s immune system and its susceptibility to diseases such as respiratory infections and asthma are closely linked to the composition and diversity of gut microbiota [26]. Consequently, restoring gastrointestinal microflora to promote host health through nonpharmacological interventions, including probiotics, represents a promising approach.

Probiotics have been defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host [27]. Studies have suggested that probiotics can be utilized to prevent and/or alleviate asthma due to their anti-inflammatory and immunomodulatory properties [28]. For instance, Zhang et al. demonstrated that oral administration of Clostridium butyrium can prevent and treat airway inflammation in mice with ovalbumin (OVA)-induced asthma by exerting immunoregulatory effects [29]. Drago et al. conducted a clinical trial using a mixture of probiotics to ameliorate asthma severity, showing that certain probiotic strains can serve as adjuvant therapies [2]. Given the high degree of heterogeneity among probiotic strains, including their immunological characteristics and genetic backgrounds, identifying additional microorganisms with potential probiotic properties to enhance efficacy is of significant importance [30].

Previously, we isolated and characterized the probiotic strain Limosilactobacillus reuteri VHProbi® M07 (hereafter referred as to “M07”) based on its phenotypic and genotypic properties [31]. In this study, we investigated whether M07 could prevent and/or mitigate asthma using an OVA-induced asthma model in mice.

Methods and materials

Culture conditions

M07 was inoculated in the de Man-Rogosa-Sharpe (MRS) broth medium (Beijing Land Bridge, China) and incubated anaerobically overnight at 37°C. This experiment was conducted from May 1, 2020 to May 30 2020 and was approved by the Ethics Committee of Qingdao Vland Biotech Inc. with approval number VL-HC-20200329. Fermented cultures with a live bacterial count of 1×10^9 CFU/mL were directly used for subsequent experiments [32].

Tolerance of M07 to simulated gastrointestinal (GI) juice

The protocol was developed based on the method described by Millette [33]. Briefly, porcine pepsin (Sigma) and pancreatin (Sigma) were added to simulated gastric juice and intestinal fluid, respectively, and incubated in a water bath at 37°C for 1 h before use. To simulate gastrointestinal transit, 1 mL of fermented supernatants were mixed with 9 mL of simulated gastric juice and incubated for 2 h at 37°C. Subsequently, 1 mL of this mixture was co-incubated with 24 mL of simulated intestinal fluid for 3 h at 37°C. Enumeration of live bacteria was conducted before and after the simulated digestion to evaluate M07’s resistance to gastric acid and bile salts. The assay was repeated twice.

Adhesion of M07 to Caco-2 cells

The adhesive property of M07 to the intestinal epithelial cells was examined according to the method described by Berenet [34]. Briefly, Caco-2 cells were seeded on glass coverslips and washed twice with phosphate-buffered saline (PBS, Sigma). Culture medium (1mL) was added to achieve a final concentration of 2 × 10^5 cells/well. 200 μL of bacterial supernatant (1 × 10^9 CFU/mL) were added to the 96-well plates, and the mixture was incubated in a CO2 incubator at 37°C for 2 h. The monolayers were then washed three times with PBS, fixed and stained for microscopic examination. Subsequently, the monolayers were co-incubated with trypsin, and the number of live bacteria was counted using the conventional plate count method. The test was performed in duplicate. The Adhesion Index (CFU/cell) was calculated as the total bacterial count per well divided by the total cell counts per well.

Animal experiments

Twenty-four specific pathogen-free (SPF) BALB/c mice, aged 8–9 weeks, were obtained from the Pengyue Experimental Animal Centre (animal permit number SCXK(Lu)20140007; Jinan, Shandong). The mice were provided with standard pellet feed and water ad libitum. They were housed in an environment maintained at 20–26°C, with relatively humidity of 40–70%, and exposed to a 12-h light-dark cycle [35]. Housing conditions, ventilation, air quality, caging, bedding, and quarantine met the requirements of SPF mice. The animal experiment was conducted under the supervision and approval of the Laboratory Animal Ethic Committee of Nanjing BIOGENE Biotech Co., Ltd (approval number:20230710105501). The study adheres to the ARRIVE guidelines.

Mice were adaptively fed for 7 days. Subsequently, the mice were randomly divided into four groups (n = 6 per group): normal (NOR), placebo (PLA), pre-treated probiotics (PRE), and post-treated probiotics (POS). All mice were provided with a regular diet throughout the study period. Mice in the PRE group received 200 μL of M07 suspension (1 × 10^9 CFU/mL) by gavage once daily for 10 days prior to the first sensitization until the study ended. Mice in the POS group were administered the same volume of M07 suspension from day 0 to day 27. Mice in the NOR and PLA groups received 200 μL of sterile saline solution (0.85%) daily from day 0 to day 27.

To establish an asthma model, mice in all groups except the NOR group were intraperitoneally injected with 200 μL of an allergen solution containing ovalbumin (OVA, 50 μg; Shanghai Yuanye Bio-Technology, Shanghai, China) and aluminium hydroxide (800 μg) dissolved in saline on days 0 and 6. The allergen solution was filtered and sterilized prior to use to ensure contamination-free conditions. Mice were also exposed to aerosolized OVA (2% in saline) using an air pressure atomizer (OMRON® C28S) for 30 min on days 12, 14, 17, 20, 23, and 27 to exacerbate asthma symptoms. The dose of the allergen administered via aerosol was based on the duration of exposure. Meanwhile, mice in the NOR were treated with the same volume of PBS during sensitization and challenge. All mice were euthanized using pentobarbital sodium (2%, 0.045 mL/g body weight) on day 28. The asthma model was established as shown schematically in Fig 1, with slight modifications from the method described by Qian‑Lin Yu [36].

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Fig 1. Schematic of the OVA-induced asthma mouse model protocol.

NOR, normal group; PLA, placebo group; PRE, pre-treated group; POS, post-treated group; OVA, ovalbumin; i.p. intraperitoneal; Dn, Day n.

https://doi.org/10.1371/journal.pone.0317587.g001

Detection of OVA-specific immunoglobulin (Ig)E and IgG1.

Blood samples were collected from the orbital venous plexus of all mice after the mice immediately after euthanasia on day 28 and stored at 4°C overnight. The next day, blood samples were centrifuged at 3000 rpm for 15 min, and serum was obtained for OVA-specific IgE and IgG1 assays using enzyme- linked immunosorbent assay (ELISA) kits (Beyotime) according to the manufacturer’s instructions [37].

Determination of cytokines and inflammatory cells in bronchoalveolar lavage fluid.

Tracheotomy was performed immediately after euthanasia. Bronchoalveolar lavage fluids (BALF) was obtained by flushing the lungs three times with 800 mL of PBS. The BALF samples were centrifuged at 1200 rpm for 10 min at 4°C. The pelleted cells were resuspended in 1 mL of PBS and stained with Trypan blue (Yuanye, CN) to count viable cells. Cell smears were prepared using a cytospin centrifuge (ELITechGroup), stained with methvlem blue and Eostm Y (Sigma), and examined under an Olympus® microscope to differentiate cell types based on cytochemical and morphological characteristics. The supernatants were collected to determine cytokine levels using ELISA kits such as interleukin-5 (IL-5), IL-10, IL-13, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha(TNF-α), and eotaxin [38].

Histology of lung tissue.

Lung tissues were fixed in 10% formalin for 24h. Paraffin blocks were prepared, and sections (5μm thick) were obtained. The tissue sections were stained with hematoxylin and eosin (HE, Promega), and examined under a light microscope at×400 magnification (LEICA, Germany) [39].

Statistical analysis

Data are presented as means ± standard deviations. Differences in the mean values among groups were analyzed using one-way analysis of variances (ANOVA) followed by Duncan’s multiple range test. P value < 0.05 and < 0.01 was defined as statistically significant and extremely significant differences, respectively. Statistical analysis was conducted using SPSS 20.0 (SPSS Inc, USA). GraphPad Prism 8 (GraphPad Software Inc, USA) was used to generate plots.

Results

Tolerance to artificial GI juice

In vitro studies demonstrated that M07 exhibits excellent tolerance to simulated GI fluid. The enumeration of live bacteria is shown in Table 1. The initial count was 9.86 Log CFU/mL, which decreased to 9.33 Log CFU/mL after co-incubation with simulated gastric juice. The final surviving bacterial count was 9.02 Log CFU/mL, representing a reduction of 0.84 Log compared to the initial count. Therefore, M07 has the potential to survive in the gastrointestinal tract.

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Table 1. Viability of M07 after co-incubation with GI fluid (Log CFU/mL).

https://doi.org/10.1371/journal.pone.0317587.t001

Adherence to the Caco-2 cells

Microscopy results showed that M07 was capable of adhering to the surface of Caco-2 cells (Fig 2). The Adhesion Index value was 23.18 ± 1.52. This indicates that M07 has the potential to colonize human intestinal epithelial cells.

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Fig 2. Microscopic examination of M07 adhesion to Caco-2 cells (400x).

Yellow arrows indicate bacterial cells adhered to Caco-2 cell after washing.

https://doi.org/10.1371/journal.pone.0317587.g002

Levels of OVA-specific IgE and IgG1 in serum

Serum was collected to determine the levels of OVA-specific IgE and IgG1. Compared with the NOR group, the levels of OVA-specific IgE and IgG1 were significantly higher in the PLA group (P < 0.01) (Fig 3), indicating that OVA-sensitized mice underwent allergic reactions. In the PRE group, the level of OVA-specific IgE decreased by 32.0% (P < 0.01) and the level of OVA-specific IgG1 decreased by 23.2% (P < 0.01) compared with the PLA group. The levels of OVA-specific IgE and IgG1 in the POS group were also significantly lower than those in the PLA group (P < 0.01), but higher than those in the PRE group.

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Fig 3. Levels of OVA-specific IgE and IgG1 in serum from different groups of mice.

NOR, normal group; PLA, placebo group; PRE, pre-treated group; and POS, post-treated group. OVA, ovalbumin; IgE, immunoglobulin E; IgG1, immunoglobulin G1; ##P < 0.01 compared with NOR; **P < 0.01 compared with PLA.

https://doi.org/10.1371/journal.pone.0317587.g003

Enumeration of differential leukocytes in BALF

The total numbers of inflammatory cells and eosinophils in the BALF were significantly increased in the PLA group compared with those in the NOR group (P < 0.01) (Table 2). The lymphocyte count in the PLA group was 4.67-fold higher than that in the NOR group, which was significantly different (P < 0.01). These data indicate that the OVA-induced asthma mouse model has been successfully established.

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Table 2. Differential counts of leukocytes in mouse BALF.

https://doi.org/10.1371/journal.pone.0317587.t002

Mice in the PRE and POS groups that received M07 gavage showed a significant reduction in total cell numbers and eosinophil count compared with mice in the PLA group (P < 0.01). Specifically, the total cell counts in the PRE and POS groups decreased by 58.4% and 42.7%, respectively, compared with the PLA group. The eosinophil counts in the PRE and POS groups decreased by 79.6% and 67.4%, respectively, compared with PLA group. There were no significant differences in lymphocyte or macrophage counts among the four groups.

Concentration of different cytokines in BALF

Detailed changes in cytokine levels are depicted in Fig 4. After establishing the asthma model, the levels of IL-5, IL-13, MCP-1, TNF-α, and eotaxin significantly increased in the PLA group compared with those in the NOR group (P < 0.01), while the IL-10 level significantly decreased by 42.0% (P < 0.01). Mice that underwent gavage with M07 in the PRE and POS groups showed a significantly lower level of IL-5 compared with the PLA group (P < 0.05); specifically, the IL-5 level decreased by 26.8% in the PRE group and 18.7% in the POS group.

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Fig 4. Levels of pro- and anti-inflammatory cytokines in BALF.

NOR, normal group; PLA, placebo group; PRE, pre-treated group; and POS, post-treated group. IL, interleukin; MCP-1, monocyte chemotactic protein-1; TNF-α, tumor necrosis factor- alpha. ##P < 0.01 compared with NOR; * P <0.05, ** P <0.01.

https://doi.org/10.1371/journal.pone.0317587.g004

The level of the anti-inflammatory cytokine IL-10 in the PRE group was significantly higher than that in the PLA group (P < 0.01), increasing by 20.8%. There was no significant difference in IL-10 levels between the PLA and POS groups (P > 0.05). The levels of IL-13 and MCP-1 in the POS group were significantly lower than those in the PLA group (P < 0.05), decreasing by 38.0% and 17.2%, respectively. The TNF-α level in both the PRE and POS groups was significantly lower than that in the PLA group (P < 0.01), reducing by 43.0% and 59.4%, respectively.

Meanwhile, the level of eotaxin exhibited a similar trend to that of IL-5, decreasing by 27.6% in the PRE group and 31.9% in the POS group, with significant differences compared with the PLA group (P < 0.01).

Histopathological examination of the lung tissue

The lung tissues from each mouse were dissected and stained with HE. Changes in the structure of pulmonary alveoli, bronchi, and the infiltration of inflammatory cells were qualitatively analyzed. Mice in the NOR group exhibited normal respiratory epithelium within branching bronchioles (Fig 5). Pulmonary alveoli primarily consisted of type I and type II alveolar cells. There was no infiltration of inflammatory cells into peribranchial, perivascular, or intra-alveolar spaces.

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Fig 5. Representative sections of HE-stained lung tissue from different groups under light microscopy(400X).

NOR, normal group; PLA, placebo group; PRE, pre-treated group; and POS, post-treated group. The yellow triangle indicates vessels; the red rhombus indicates bronchioles; the black arrow indicates infiltration of inflammatory cells; the green circle indicates inflammatory hyperplasia.

https://doi.org/10.1371/journal.pone.0317587.g005

Mice in the PLA group showed inflammatory hyperplasia around terminal bronchioles, which extended into larger bronchi. The number of macrophages in the pulmonary alveoli increased significantly. In the PRE group, mice had an increased number of macrophages in the pulmonary alveoli but no other significant inflammatory changes. Mice in the POS group had vasodilated vessels in lung tissues, along with significant infiltration of inflammatory cells and an increased number of macrophages.

Discussion

In recent decades, various studies have demonstrated the existence of bidirectional communication between the respiratory system and gastrointestinal tract, known as the gut-lung axis [26, 40, 41]. Dysbiosis of gut commensal microflora appears to play a crucial role in the initiation and progression of allergic diseases. For instance, germ-free mice tend to develop immune system disorders when exposed to allergens due to the absence of gut microbiota [42, 43]. In recent years, a prevailing hypothesis has emerged, suggesting that allergic disease, including asthma, atopic dermatitis, and rheumatoid arthritis, are closely related to lifestyle factors that influence the diversity and abundance of microbiome, such as diet, antibiotic use, and hygiene practices [44–47].

Manipulating the human gut microflora to enhance immune functions, particularly through probiotic supplementation, has increasingly drawn global attention [48–50]. For example, Moura et al. conducted a small clinical study and demonstrated that supplementation with L. reuteri as an adjuvant therapy could mitigate the severity of asthma in children and adults [51]. Forsythe et al. showed that oral administration of L. reuteri improved the major characteristics of OVA- sensitized asthma in mice, including cytokine levels, eosinophilia, and airway hyperresponsiveness [52]. Raftis et al. found that Bifidobacterium breve MRx0004 could protect against inflammatory cell infiltration in a severe asthma model by reducing neutrophil and eosinophil counts [38]. Therefore, developing non-pharmacological strategies, such as novel probiotic strains for the clinical management of asthma, brings substantial benefits to patients and society.

The gastric juice and intestinal fluids pose a primary challenge for probiotics to survive in the harsh gut environment [53]. Probiotics with favourable tolerance to GI fluids are more likely to colonize in the gut tract, thereby conferring beneficial effects on the host. We demonstrated that the loss of M07 was less than 1 Log cycle after successive co-incubation with simulated gastric and intestinal juice, indicating its robust survival capability. The ability to hostile conditions is a fundamental characteristic of effective probiotic dietary supplements. Adherence to the epithelial cells is a prerequisite for probiotics to confer benefits in the GI tract, including competing for binding sites and nutrients with harmful gut microbiota and communicating with the host immune system [54–56].

Caco-2 cell lines derived from human colon adenocarcinoma are commonly used as an in vitro model to assess the adhesion properties of probiotics [57]. Our results showed that M07 could adhere to Caco-2 cells, as evidenced by microscopic examination and cell counts. This finding aligns with studies on extensively researched probiotics such as Lacticaseibacillus rhamnosus GG, which exhibits strong affinity for intestinal mucosal cells and resistance to gastric acids and bile salts [58]. Similarly, Lactobacillus acidophilus NCFM has demonstrated the capacity to adhere to Caco-2 cells and resist gastrointestinal fluids [59, 60]. These consistent findings suggest that M07 has the potential to colonize in the human gut tract.

From the perspective of pulmonary immunology, asthma has been considered a hallmark of T-help type 2 (Th2) disorders in the airways [12]. Elevated levels of IgE and IgG1 are indicative of a Th2-type adaptive immune response [61]. Mice in the PLA group exhibited significantly increased levels of OVA-specific IgE and IgG1 in serum, indicating a Th2 immune response. Mice treated with M07 displayed significantly reduced levels of OVA-specific IgE and IgG1 compared with those in the PLA group. Similar results have also been observed in various OVA-induced mice model experiments.

For example, Hougee et al. conducted a comparative study and demonstrated that oral treatment with B. breve M-16V or Lactiplantibacillus plantarum NumRes8 reduced the levels of OVA-specific IgE and IgG1 in OVA-sensitized mice [61]. Lan and colleagues investigated the efficacy of L. plantarum CQPC11 in attenuating airway inflammation in OVA-induced mice and documented reduced serum levels of OVA-specific IgE and OVA-specific IgG1 [62]. Furthermore, Hagner et al. illustrated that intranasal administration of Staphylococcus sciuri W620 in an OVA-exposed mouse model led to significantly reduced serum levels of OVA-IgG1 and IgE compared with untreated mice [63]. Taken together, these data suggest that consumption of M07 could relieve the severity of asthma by suppressing serum levels of both OVA-specific IgE and IgG1.

Airway inflammation is a pathogenic factor in asthma and involves leukocytes such as eosinophils, neutrophils, lymphocytes, and macrophages [64, 65]. Restoring the dysfunction of immune cells plays a crucial role in alleviating allergic diseases and associated inflammation [66]. In mice that received M07 gavage, the counts of total inflammatory cells and eosinophils were significantly reduced compared with those in the PLA group and approached levels seen in the NOR group. These reductions were consistent with declines in eotaxin levels. There were no significant differences in the counts of lymphocytes and macrophages among mice in PLA, PRE and POR groups.

Raftis et al. discovered that severe asthma mice treated with B.breve MRx0004 showed therapeutic effects by suppressing neutrophil and eosinophil counts in lung tissues [38]. Forsythe and coworkers demonstrated that oral treatment with live L. reuteri ATCC 23272 could assist in treating allergic airway responses in mice by reducing eosinophil infiltration into the airway lumen and parenchyma [52]. Spacova et al. found that intranasal administration of L. rhamnosus could prevent birch pollen-induced allergic asthma in a murine model, with significantly lower eosinophil counts in BALF [67].

Our results are consistent with these studies, which describe reductions in pulmonary eosinophil numbers in asthma mouse model using probiotics. Oral administration of M07 could attenuate asthma severity by suppressing the number of total leukocytes and eosinophils.

IL-5 and IL-13 are pivotal in the development of an allergen-sensitized asthma model. IL-5 is responsible for the maturation and differentiation of eosinophils [1]. Delivering IL-13 to the airways induces asthma symptoms such as eosinophil infiltration and a vigorous response in the airway, while inhibition of IL-13 levels could attenuate these effects [11, 68]. IL-10 is an immunosuppressive cytokine, and clinical studies have revealed that serum IL-10 level can reflect the severity of asthma [69].

Analysis of cytokine levels in BALF revealed elevated levels of pro-inflammatory cytokines (IL-5, IL-13, MCP-1, and TNF-α) in the PLA group, while a decreased level of the anti-inflammatory cytokine of IL-10 was observed. Mice treated with M07 via gavage displayed diametrically opposite trends in cytokine levels compared with those in the PLA group. Mice pretreated with M07 in the PRE group showed greater recovery of cytokine levels compared with those in the POS group, although no significant difference was observed between these two groups.

Zhang et al. found that administration of L. rhamnosus GG led to reductions in IL-5, IL-13, and MCP-1 levels and an increase in IL-10 levels in OVA-sensitized mice [70]. Oral administration of L. reuteri ATCC 23272 also resulted in immunoregulatory effects, with elevated IL-10 levels and reduced levels of IL-5, IL-13, MCP-1, and TNF-α in OVA-induced asthma mice [52]. One could speculate that oral administration of M07 suppress asthma development by regulating pro-inflammation and anti-inflammation cytokines.

M07 also confers enhanced effects on lung histopathology, as illustrated by the significantly reduced infiltration of inflammatory cells into perivascular and peribronchiolar areas. The improvements in lung tissue pathology were associated with prolonged M07 administration. The reduced infiltration of inflammatory cells was consistent with the decreased counts of eosinophils and neutrophils.

We acknowledge that the present study has certain limitations. Although M07 confer benefits in relieving OVA-induced asthma in mice, the mechanisms of action of M07 need to be investigated in greater detail. For instance, further research is required to understand how the airway responsiveness changes during asthma development. Additionally, the effect of M07 on the abundance and diversity of gut microflora should be explored. Moreover, it is crucial to accumulate positive evidence of the beneficial effects of M07 by conducting human clinical trials.

In conclusion, this study clearly showed that M07 can tolerate and survive in the harsh gastrointestinal environment. More importantly, it alleviates OVA-induced asthma in mice by reducing the Th2-type immune response, including decreased infiltration of inflammatory cells and specific antibody levels, as well as regulating of pro- and anti-inflammatory cytokines, thereby restoring immunological balance. Moreover, the efficacy of M07 in ameliorating asthma symptoms was greater in the PRE group compared to the POS group. Taken together, our findings provide a promising probiotic strain, M07, for managing allergic respiratory diseases and support the concept that oral administration of probiotics can benefit distal sites such as respiratory tract.

Acknowledgments

We sincerely appreciate all the staff who participated in this study.

References

  1. 1. Papi A, Brightling C, Pedersen SE, Reddel HK. Asthma. Lancet. 2018;391: 783–800. pmid:29273246
  2. 2. Drago L, Cioffi L, Giuliano M, Pane M, Amoruso A, Schiavetti I, et al. The Probiotics in Pediatric Asthma Management (PROPAM) Study in the Primary Care Setting: A Randomized, Controlled, Double-Blind Trial with Ligilactobacillus salivarius LS01 (DSM 22775) and Bifidobacterium breve B632 (DSM 24706). J Immunol Res. 2022;2022: 1–7. pmid:35083341
  3. 3. Asher I, Pearce N. Global burden of asthma among children. Int J Tuberc lung Dis. 2014;18: 1269–1278. pmid:25299857
  4. 4. Liu A, Ma T, Xu N, Jin H, Zhao F, Kwok L-Y, et al. Adjunctive Probiotics Alleviates Asthmatic Symptoms via Modulating the Gut Microbiome and Serum Metabolome. Microbiol Spectr. 2021;9: 1–25. pmid:34612663
  5. 5. To T, Stanojevic S, Moores G, Gershon AS, Bateman ED, Cruz AA, et al. Global asthma prevalence in adults: findings from the cross-sectional world health survey. BMC Public Health. 2012;12: 1–8.
  6. 6. Baarnes CB, Hansen AV, Ulrik CS. Enrolment in an asthma management program during pregnancy and adherence with inhaled corticosteroids: the ‘Management of Asthma during Pregnancy’program. Respiration. 2016;92: 9–15. pmid:27348313
  7. 7. Ma J, Zhang J, Li Q, Shi Z, Wu H, Zhang H, et al. Oral administration of a mixture of probiotics protects against food allergy via induction of CD103+ dendritic cells and modulates the intestinal microbiota. J Funct Foods. 2019;55: 65–75.
  8. 8. Lee J, Kim TH, Murray F, Li X, Choi SS, Broide DH, et al. Cyclic AMP concentrations in dendritic cells induce and regulate Th2 immunity and allergic asthma. Proc Natl Acad Sci. 2015;112: 1529–1534. pmid:25605931
  9. 9. Vroman H, Hendriks RW, Kool M. Dendritic cell subsets in asthma: impaired tolerance or exaggerated inflammation? Front Immunol. 2017;8: 941. pmid:28848549
  10. 10. Eisenbarth SC, Piggott DA, Bottomly K. The master regulators of allergic inflammation: dendritic cells in Th2 sensitization. Curr Opin Immunol. 2003;15: 620–626. pmid:14630194
  11. 11. Eisenbarth SC, Cassel S, Bottomly K. Understanding asthma pathogenesis: linking innate and adaptive immunity. Curr Opin Pediatr. 2004;16: 659–666. pmid:15548929
  12. 12. Lambrecht BN, Hammad H. The immunology of asthma. Nat Immunol. 2015;16: 45–56. pmid:25521684
  13. 13. Raymond M, Van VQ, Wakahara K, Rubio M, Sarfati M. Lung dendritic cells induce TH17 cells that produce TH2 cytokines, express GATA-3, and promote airway inflammation. J Allergy Clin Immunol. 2011;128: 192–201. pmid:21601259
  14. 14. Lambrecht BN, De Veerman M, Coyle AJ, Gutierrez-Ramos J-C, Thielemans K, Pauwels RA. Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation. J Clin Invest. 2000;106: 551–559. pmid:10953030
  15. 15. Hammad H, Plantinga M, Deswarte K, Pouliot P, Willart MAM, Kool M, et al. Inflammatory dendritic cells—not basophils—are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen. J Exp Med. 2010;207: 2097–2111. pmid:20819925
  16. 16. Parsons JP, Hallstrand TS, Mastronarde JG, Kaminsky DA, Rundell KW, Hull JH, et al. An official American Thoracic Society clinical practice guideline: exercise-induced bronchoconstriction. Am J Respir Crit Care Med. 2013;187: 1016–1027. pmid:23634861
  17. 17. Boulet L-P, Vervloet D, Magar Y, Foster JM. Adherence: the goal to control asthma. Clin Chest Med. 2012;33: 405–417. pmid:22929091
  18. 18. Reddel HK, Busse WW, Pedersen S, Tan WC, Chen Y-Z, Jorup C, et al. Should recommendations about starting inhaled corticosteroid treatment for mild asthma be based on symptom frequency: a post-hoc efficacy analysis of the START study. Lancet. 2017;389: 157–166. pmid:27912982
  19. 19. Papi A, Corradi M, Pigeon-Francisco C, Baronio R, Siergiejko Z, Petruzzelli S, et al. Beclometasone–formoterol as maintenance and reliever treatment in patients with asthma: a double-blind, randomised controlled trial. Lancet Respir Med. 2013;1: 23–31. pmid:24321801
  20. 20. Gibeon D, Heaney LG, Brightling CE, Niven R, Mansur AH, Chaudhuri R, et al. Dedicated severe asthma services improve health-care use and quality of life. Chest. 2015;148: 870–876. pmid:25789861
  21. 21. Kew KM, Dahri K. Long‐acting muscarinic antagonists (LAMA) added to combination long‐acting beta 2‐agonists and inhaled corticosteroids (LABA/ICS) versus LABA/ICS for adults with asthma. Cochrane Database Syst Rev. 2016.
  22. 22. Bisgaard H, Le Roux P, Bjåmer D, Dymek A, Vermeulen JH, Hultquist C. Budesonide/formoterol maintenance plus reliever therapy: a new strategy in pediatric asthma. Chest. 2006;130: 1733–1743. pmid:17166990
  23. 23. Haahtela T. A biodiversity hypothesis. Allergy. 2019;74: 1445–1456. pmid:30835837
  24. 24. Lukacs NW, Huang YJ. Microbiota–immune interactions in asthma pathogenesis and phenotype. Curr Opin Immunol. 2020;66: 22–26. pmid:32320899
  25. 25. Barcik W, Boutin RCT, Sokolowska M, Finlay BB. The role of lung and gut microbiota in the pathology of asthma. Immunity. 2020;52: 241–255. pmid:32075727
  26. 26. Dang AT, Marsland BJ. Microbes, metabolites, and the gut–lung axis. Mucosal Immunol. 2019;12: 843–850. pmid:30976087
  27. 27. Hill C. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol. 2014;11. pmid:24912386
  28. 28. Maldonado Galdeano C, Cazorla SI, Lemme Dumit JM, Vélez E, Perdigón G. Beneficial effects of probiotic consumption on the immune system. Ann Nutr Metab. 2019;74: 115–124. pmid:30673668
  29. 29. ZHANG J, WEI C, LIU M, HUANG J, WU H, ZHENG Y, et al. Inhibitory effect of Clostridium butyrium on the inflammatory response in asthmatic mice. J Shanxi Med Univ. 2015;46: 1083–1807.
  30. 30. Lin J, Zhang Y, He C, Dai J. Probiotics supplementation in children with asthma: a systematic review and meta‐analysis. J Paediatr Child Health. 2018;54: 953–961. pmid:30051941
  31. 31. Cui H, Wu S, Duan Z. Complete Genome Sequence of Limosilactobacillus reuteri Strain VHProbi M07, Isolated from Breast Milk. Microbiol Resour Announc. 2022;11: 10–11. pmid:36287064
  32. 32. Cui H, Wang Q, Feng C, Guo C, Zhang J, Bu X, et al. Positive effect of Bifidobacterium animalis subsp. lactis VHProbi YB11 in improving gastrointestinal movement of mice having constipation. Front Microbiol. 2022;13. pmid:36532450
  33. 33. Millette M, Nguyen A, Amine KM, Lacroix M. Gastrointestinal survival of bacteria in commercial probiotic products. Int J Probiotics Prebiotics. 2013;8: 149–156.
  34. 34. Bernet MF, Brassart D, Neeser JR, Servin AL. Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions. Appl Environ Microbiol. 1993;59: 4121–4128. pmid:8285709
  35. 35. Van Hove CL, Maes T, Cataldo DD, Guéders MM, Palmans E, Joos GF, et al. Comparison of acute inflammatory and chronic structural asthma-like responses between C57BL/6 and BALB/c mice. Int Arch Allergy Immunol. 2009;149: 195–207. pmid:19218812
  36. 36. Yu QL, Chen Z. Establishment of different experimental asthma models in mice. Exp Ther Med. 2018;15: 2492–2498. pmid:29456654
  37. 37. Abril-Gil M, Massot-Cladera M, Pérez-Cano FJ, Castellote C, Franch À, Castell M. A diet enriched with cocoa prevents IgE synthesis in a rat allergy model. Pharmacol Res. 2012;65: 603–608. pmid:22342543
  38. 38. Raftis EJ, Delday MI, Cowie P, McCluskey SM, Singh MD, Ettorre A, et al. Bifidobacterium breve MRx0004 protects against airway inflammation in a severe asthma model by suppressing both neutrophil and eosinophil lung infiltration. Sci Rep. 2018;8: 1–13.
  39. 39. Choi S, Kim JH, Kim K, Kim M, Choi HJ, Kim YM, et al. Primary hepatic extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue. J Pathol Transl Med. 2020;54: 340. pmid:32702946
  40. 40. Enaud R, Prevel R, Ciarlo E, Beaufils F, Wieërs G, Guery B, et al. The gut-lung axis in health and respiratory diseases: a place for inter-organ and inter-kingdom crosstalks. Front Cell Infect Microbiol. 2020;10: 9. pmid:32140452
  41. 41. Marsland BJ, Trompette A, Gollwitzer ES. The gut–lung axis in respiratory disease. Ann Am Thorac Soc. 2015;12: S150–S156. pmid:26595731
  42. 42. Herbst T, Sichelstiel A, Schär C, Yadava K, Bürki K, Cahenzli J, et al. Dysregulation of allergic airway inflammation in the absence of microbial colonization. Am J Respir Crit Care Med. 2011;184: 198–205. pmid:21471101
  43. 43. He Y, Wen Q, Yao F, Xu D, Huang Y, Wang J. Gut–lung axis: the microbial contributions and clinical implications. Crit Rev Microbiol. 2017;43: 81–95. pmid:27781554
  44. 44. Oliver JE, Silman AJ. Risk factors for the development of rheumatoid arthritis. Scand J Rheumatol. 2006;35: 169–174. pmid:16766362
  45. 45. Solomon I, Ilie MA, Draghici C, Voiculescu VM, Căruntu C, Boda D, et al. The impact of lifestyle factors on evolution of atopic dermatitis: An alternative approach. Exp Ther Med. 2019;17: 1078–1084. pmid:30679977
  46. 46. Huovinen E, Kaprio J, Koskenvuo M. Factors associated to lifestyle and risk of adult onset asthma. Respir Med. 2003;97: 273–280. pmid:12645835
  47. 47. Lambrecht BN, Hammad H. The immunology of the allergy epidemic and the hygiene hypothesis. Nat Immunol. 2017;18: 1076–1083. pmid:28926539
  48. 48. Riazi-Rad F, Behrouzi A, Mazaheri H, Katebi A, Ajdary S. Impact of gut microbiota on immune system. Acta Microbiol Immunol Hung. 2021;68: 135–144. pmid:34375301
  49. 49. Bengmark S. Gut microbiota, immune development and function. Pharmacol Res. 2013;69: 87–113. pmid:22989504
  50. 50. Kosiewicz MM, Zirnheld AL, Alard P. Gut microbiota, immunity, and disease: a complex relationship. Front Microbiol. 2011;2: 180. pmid:21922015
  51. 51. Moura JCV, Moura ICG, Gaspar GR, Mendes GMS, Faria BAV, Jentzsch NS, et al. The use of probiotics as a supplementary therapy in the treatment of patients with asthma: A pilot study and implications. Clinics. 2019;74: 1–6. pmid:31411278
  52. 52. Forsythe P, Inman MD, Bienenstock J. Oral treatment with live Lactobacillus reuteri inhibits the allergic airway response in mice. Am J Respir Crit Care Med. 2007;175: 561–569. pmid:17204726
  53. 53. Guo X, Li D, Lu W, Piao X, Chen X. Screening of Bacillus strains as potential probiotics and subsequent confirmation of the in vivo effectiveness of Bacillus subtilis MA139 in pigs. Antonie van Leeuwenhoek, Int J Gen Mol Microbiol. 2006;90: 139–146. pmid:16820971
  54. 54. Westerdahl A, Olsson JC, Kjelleberg S, Conway PL. Isolation and characterization of turbot (Scophtalmus maximus)-associated bacteria with inhibitory effects against Vibrio anguillarum. Appl Environ Microbiol. 1991;57: 2223–2228. pmid:1768092
  55. 55. Schiffrin EJ, Brassart D, Servin AL, Rochat F, Donnet-Hughes A. Immune modulation of blood leukocytes in humans by lactic acid bacteria: criteria for strain selection. Am J Clin Nutr. 1997;66: 515S–520S. pmid:9250141
  56. 56. Salminen S, Bouley C, Boutron M-C, Cummings JH, Franck A, Gibson GR, et al. Functional food science and gastrointestinal physiology and function. Br J Nutr. 1998;80: S147–S171. pmid:9849357
  57. 57. Tuomola EM, Salminen SJ. Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures. Int J Food Microbiol. 1998;41: 45–51. pmid:9631336
  58. 58. Walter J. Ecological role of lactobacilli in the gastrointestinal tract: implications for fundamental and biomedical research. Appl Environ Microbiol. 2008;74: 4985–4996. pmid:18539818
  59. 59. Klindt-Toldam S, Larsen SK, Saaby L, Olsen LR, Svenstrup G, Müllertz A, et al. Survival of Lactobacillus acidophilus NCFM® and Bifidobacterium lactis HN019 encapsulated in chocolate during in vitro simulated passage of the upper gastrointestinal tract. LWT. 2016;74: 404–410.
  60. 60. Wang R-M, Li N, Zheng K, Hao J-F. Enhancing acid tolerance of the probiotic bacterium Lactobacillus acidophilus NCFM with trehalose. FEMS Microbiol Lett. 2018;365: fny217. pmid:30184196
  61. 61. Hougee S, Vriesema AJM, Wijering SC, Knippels LMJ, Folkerts G, Nijkamp FP, et al. Oral treatment with probiotics reduces allergic symptoms in ovalbumin-sensitized mice: A bacterial strain comparative study. Int Arch Allergy Immunol. 2010;151: 107–117. pmid:19752564
  62. 62. Lan H, Gui Z, Zeng Z, Li D, Qian B, Qin L, et al. Oral administration of Lactobacillus plantarum CQPC11 attenuated the airway inflammation in an ovalbumin (OVA)‐induced Balb/c mouse model of asthma. J Food Biochem. 2022;46: e14036. pmid:34981513
  63. 63. Hagner S, Harb H, Zhao M, Stein K, Holst O, Ege MJ, et al. Farm-derived Gram-positive bacterium Staphylococcus sciuri W620 prevents asthma phenotype in HDM- and OVA-exposed mice. Allergy. 2013;68: 322–329. pmid:23369007
  64. 64. Busse WW. Inflammation in asthma: the cornerstone of the disease and target of therapy. J Allergy Clin Immunol. 1998;102: S17–22. pmid:9798720
  65. 65. Lane SJ, Lee TH. Mast cell effector mechanisms. J Allergy Clin Immunol. 1996;98: S67–71; discussion S71-2. pmid:8939179
  66. 66. Jutel M, Agache I, Bonini S, Burks AW, Calderon M, Canonica W, et al. International consensus on allergy immunotherapy. J Allergy Clin Immunol. 2015;136: 556–568. pmid:26162571
  67. 67. Spacova I, Petrova MI, Fremau A, Pollaris L, Vanoirbeek J, Ceuppens JL, et al. Intranasal administration of probiotic Lactobacillus rhamnosus GG prevents birch pollen-induced allergic asthma in a murine model. Allergy Eur J Allergy Clin Immunol. 2019;74: 100–110. pmid:29888398
  68. 68. Kuperman DA, Huang X, Koth LL, Chang GH, Dolganov GM, Zhu Z, et al. Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat Med. 2002;8: 885–889. pmid:12091879
  69. 69. Kearley J, Barker JE, Robinson DS, Lloyd CM. Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4+ CD25+ regulatory T cells is interleukin 10 dependent. J Exp Med. 2005;202: 1539–1547. pmid:16314435
  70. 70. Zhang J, Ma J yi, Li Q hong, Su H, Sun X. Lactobacillus rhamnosus GG induced protective effect on allergic airway inflammation is associated with gut microbiota. Cell Immunol. 2018;332: 77–84. pmid:30097177