Asthma Prevention by Lactobacillus Rhamnosus in a Mouse Model is Associated With CD4⁺CD25⁺Foxp3⁺ T Cells

Mice

Female BALB/c mice weighing 20-25 g (4 weeks old) were purchased from Orient Bio (Orient Bio Inc., Seongnam, Korea) and treated in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at Asan Medical Center and Ulsan University College of Medicine.

Probiotic preparation

The Lcr35 used in this study was obtained from the Lyocentre® laboratory (Aurillac, France) and was cultured in sterile Lactobacillus MRS broth (Difco, Sparks, MD, USA) at 37℃ for 24 hours. After cultivation, cells were collected by centrifugation at 5,000×g for 10 minutes, washed twice with phosphate buffered saline (PBS), and lyophilized. The lyophilized powder was reconstituted with sterile saline before use.

Establishment of allergic asthma and probiotic treatment

Six-week-old female BALB/c mice (N=7 per group) were sensitized by intraperitoneal (i.p.) administration of an ovalbumin (OVA; 10 µg, grade V; Sigma Chemical Co., St. Louis, MO, USA) and alum (2.25 mg; Imject®, Pierce, Rockford, IL, USA) mixture. One week after the first sensitization, the mixture was administered a second time. Seven days later, the mice inhaled 1% OVA via an ultrasonic sprayer (Nescosonic UN-511; Alfresa, Osaka, Japan) for 30 minutes daily for three successive days (OVA challenge). The mice received Lcr35 (1×10⁹ colony-forming units/600 µL/mouse/day) orally from one week before primary sensitization to the endpoint of the study. Negative controls received only saline instead of OVA at both sensitizations and airway challenge. Positive controls received nothing more after OVA sensitization.

Clinical evaluations in vivo

Airway hyperresponsiveness (AHR) in response to inhaled methacholine (MeCh; Sigma Chemical Co.), administered 24 hours after OVA challenge, was measured in conscious, unrestrained mice using a barometric whole-body plethysmograph (Buxco; EMKA Technologies, Paris, France). Briefly, mice were placed in a whole-body chamber, and basal readings were obtained for 3 minutes and averaged. Aerosolized saline followed by 5-50 mg/mL MeCh were inhaled for 3 minutes after each MeCh inhalation.

Treg cells were depleted using anti-CD25 monoclonal antibody (mAb). Briefly, mice received 250 µg of rat anti-mouse CD25 mAb (clone PC61; eBioscience, San Diego, CA, USA) i.p. in 400 µL of normal saline one day before 1% OVA challenge. Control mice were injected with 250 µg of rat IgG1 (Sigma Chemical Co.).

Bronchoalveolar lavage (BAL) fluid analysis

After measurement of AHR, the mice were anesthetized by i.p. administration of ketamine-xylazine, and the trachea was immediately exposed. The airways were lavaged through a tracheal cannula, two times with 1-mL aliquots of pyrogen-free saline warmed to 37℃. The recovered lavage fluid was pooled, and the cells were collected by centrifugation (5,000 rpm, 4℃, 5 minutes) and resuspended in 100 mL of cold PBS. The cells were stained with trypan blue to determine viability, and total nucleated cells were counted using a hemocytometer.

For differential BAL cell counts, cytospin preparations were made and stained with Diff-Quik (Sysmex, Takatsukadai, Japan). After the samples were coded, all cytospin preparations were evaluated by one observer using an oil immersion microscope (magnification, ×1,000). At least 200 cells were counted per preparation, and the absolute number of each cell type was calculated.

Serum Ig analysis

Serum was obtained from blood taken during exsanguination of the mice after airway measurement, and 100 µL (1/10 dilution in carbonate-bicarbonate buffer) were added to each well of a 96-well plate. An IgE-specific enzyme linked immunosorbent assay (ELISA) was used to quantitate total IgE in the serum, using matching antibody pairs (eBioscience) according to the manufacturer's instructions. For the ELISA, 96-well plates were first coated overnight with rat anti-mouse IgE (10 µL in 100 µL of PBS; PharMingen, San Diego, CA, USA), rat anti-mouse IgG1 (20 µg in 100 µL of PBS; PharMingen), or rat anti-mouse IgG_2a (20 µg in 100 µL of PBS; PharMingen). The remaining binding sites were blocked, and the plates were incubated with 100 µL of diluted serum (1:5 for IgE, 1:10 for IgG1 or IgG_2a). After the plate was washed, each of the following was sequentially added, incubated, and removed by washing: OVA (1 µg/100 µL), peroxidase-labeled rabbit anti-OVA Ig (240 ng/100 µL, PharMingen), and 3,3,5,5-tetramethylbenzidine solution (Sigma Chemical Co.). The optical density was measured at 450 nm, and the Ig level was determined relative to that of a reference pool of serum from OVA-sensitized BALB/c mice (assigned a value of 100 experimental units/mL). Determinations were performed in duplicate.

Cytokine assays

Commercial preparations of paired antibodies and protein standards for measurements of mouse IL-4, IL-5, IL-13, and IFN-γ (eBioscience) in sera were used to develop ELISAs according to the manufacturer's instructions. Determinations were performed in duplicate.

Lymphocyte proliferation assay

After BAL, the mouse spleen was resected. Mouse splenocytes were separated on a Histopaque (Sigma Chemical Co.) gradient, and the collected cells were washed with PBS. RBCs were lysed by gently mixing the cells with 3.6 mL of 0.24% NaCl for 20 seconds, followed by the quick addition of 0.3 mL of 8.7% NaCl and further dilution with PBS. The pellet was suspended in Iscove's Modified Dulbecco's Medium (IMDM), and stored overnight at 4℃. The next morning, the cells were centrifuged at 4℃, suspended in cold PBS, stained with trypan blue, and counted using a hemocytometer.

Splenic T cells were cultured in IMDM supplemented with 25 mM HEPES, 10% (v/v) heat inactivated fetal bovine serum (FBS), 60 mg/L (100 U/mL) penicillin, 100 mg/L streptomycin, and 0.29 g/L L-glutamine. Splenic T cells were adjusted to 1×10⁵ cells/200 µL/well, transferred to 96-well plates, and incubated at 37℃ in a humidified 5% CO₂ incubator for 72 hours. The cells were stimulated with OVA treatment (100 µg/mL) for 72 hours. At 12 hours before the end of the incubation, 1 µCi of [³H]-thymidine was added to each well. The cells were harvested onto a glass microfiber filter (Simport, Beloeil, Canada), and radioactivity was measured in a liquid scintillation counter. The incorporation during the last 12 hours of culture (counts per minute) was used as an index of proliferation. All cultures were tested in triplicate.

Flow cytometry

Mouse Treg cells were collected from the spleen and analyzed for CD4⁺CD25⁺Foxp3⁺ expression using a mouse Treg cell staining kit containing FITC-labeled anti-CD4, APC-labeled anti-CD25, and PE-labeled anti-Foxp3 (eBioscience) according to the manufacturer's instructions. Briefly, prepared cells (1×10⁶) were washed by centrifugation with cold PBS, resuspended in 1 mL of fixation/permeabilization solution, and incubated in the dark at 4℃ for 30-60 minutes. The cells were washed once with 2 mL of permeabilization buffer, collected by centrifugation, resuspended in 20 mL of blocking agent with 2% (2 mL) normal rat serum in permeabilization buffer, and incubated at 4℃ for 15 minutes. Next, 20 mL of fluorochrome-conjugated antibody or isotype control in permeabilization buffer were added, followed by incubation in the dark at 4℃ for 30 minutes. Finally, the cells were washed with 2 mL of permeabilization buffer, resuspended in flow cytometry buffer (PBS with 2% FBS), and analyzed by flow cytometry using a FACSCalibur with CellQuest software (BD Biosciences, Mountain View, CA, USA). Determinations were performed in duplicate.

Lung histopathology

For the histological evaluation of lung tissue, the left lung of each mouse was embedded in paraffin, sectioned to a thickness of 5 µm, and stained with hematoxylin and eosin (H&E) to assess eosinophilic infiltration. Inflammation was scored by two independent, blinded investigators. The degree of peribronchial and perivascular inflammation was evaluated on a subjective scale of 0-3, as described elsewhere.13 Cellular infiltration in five randomly selected fields was assessed under a Zeiss Axiophot microscope (magnification, ×100; Carl Zeiss, Inc., Thornwood, NY, USA).

Statistical analysis

We analyzed the association between groups and positive controls using the Mann-Whitney test. Data are expressed as means±standard error. All statistical analyses were performed using SPSS ver. 18.0 for Windows (SPSS Inc., Chicago, IL, USA). A P value<0.05 indicated significance.

Effect of Lcr35 treatment on maximal Penh and airway eosinophilia

Lcr35 administration to allergic mice significantly suppressed AHR, to the level of the negative control group at the maximum MeCh dose (negative control: 4.72±2.19; Lcr35-treated: 4.75±1.56; positive control: 9.00±2.87; P<0.01) (Fig. 1A).

Fig. 1
Effect of oral Lcr35 treatment on allergic asthma in a mouse model. (A) Airway hyperresponsiveness; (B) Eosinophil proportion in bronchoalveolar lavage fluid; (C) Peribronchial and perivascular lung inflammation score; (D) Lung pathology. ^*P<0.05 and ^**P<0.01. Negative control: Saline (i.p.)/saline challenge by inhalation. Lcr35-treated: OVA (i.p.)/OVA challenge by inhalation+Lcr35 (oral administration). Positive control: OVA (i.p.)/OVA challenge by inhalation.

Lcr35, Lactobacillus rhamnosus; i.p., intraperitoneal; OVA, ovalbumin.

• Click for larger image

Lcr35-treated mice exhibited a significant suppression of BAL fluid total cell count compared with positive controls (Lcr35-treated: 4.77×10⁵ cells/mL; positive control: 8.29×10⁵ cells/mL; P<0.05). Oral administration of Lcr35 significantly reduced the number of eosinophils in BAL fluid compared with positive controls (Lcr35-treated: 3.79±0.29%; positive control: 13.60±3.00%; P<0.05) (Fig. 1B).

Lung inflammation after OVA inhalation was significantly reduced by oral administration of Lcr35 (Lcr35-treated: 2.16±0.55; positive control: 0.92±0.52; P<0.05) (Fig. 1C). Examination of the lung tissue of positive control mice revealed peribronchial and perivascular cellular infiltrates; Lcr35 treatment produced a marked decrease in both cellular infiltration and inflammatory changes, as determined by histopathology (Fig. 1D). Thus, Lcr35 treatment inhibited allergen-induced pulmonary inflammation, including the influx of eosinophils.

Mechanism of the effect of Lcr35 on allergic responses

The administration of Lcr35 significantly reduced the total IgE level compared with that of the positive control, but had no significant effect on OVA-specific IgE, IgG1, or IgG_2a (Table). Serum IL-4, -5, and -13 levels were significantly suppressed by orally administered Lcr35. The IFN-γ level was also decreased in Lcr35-treated mice, but the difference was not significant (Table). These results suggest that Lcr35 treatment suppresses Th2-dependent cytokines. OVA-induced cell proliferation was significantly lower in Lcr35-treated mice than in positive control mice (Fig. 2). Thus, Lcr35 effectively inhibited the OVA-specific splenic T cell response.

Fig. 2
Effect of oral Lcr35 treatment on the suppression of OVA-specific splenic T cell proliferation in a mouse model. ^*P<0.05 and ^**P<0.01. Negative control: Saline (i.p.)/saline challenge by inhalation. Lcr35-treated: OVA (i.p.)/OVA challenge by inhalation+Lcr35 (oral administration). Positive control: OVA (i.p.)/OVA challenge by inhalation.

Lcr35, Lactobacillus rhamnosus; OVA, ovalbumin; i.p., intraperitoneal.

• Click for larger image

Table
Systemic immune responses and cytokine levels of each group

• Click for larger image

Lcr35 treatment led to a significant increase in CD4⁺CD25⁺Foxp3⁺ Treg cells, compared with the percentage in positive control mice (Lcr35-treated: 17.60±0.49%; positive control: 15.20±0.44%; P<0.05) (Fig. 3B). This suggests that Lcr35-induced attenuation of allergic responses in the mouse model of asthma is associated with an increased CD4⁺CD25⁺Foxp3⁺ Treg cell population. To confirm the involvement of CD4⁺CD25⁺Foxp3⁺ Treg cells, Treg cell depletion was achieved by anti-CD25 mAb treatment. Anti-CD25 mAb treatment before OVA challenge significantly increased AHR at the maximum MeCh dose, compared with AHR in Lcr35-treated mice (anti-CD25-treated: 8.16±1.63; Lcr35-treated: 3.30±0.57; P<0.05) (Fig. 4A). The isotype control rat IgG₁ had no effect on AHR (data not shown). Anti-CD25 treatment also significantly decreased the population of CD4⁺CD25⁺Foxp3⁺ Treg cells in Lcr35-treated mice (anti-CD25 treated: 1.00±0.08%; Lcr35-treated: 15.53±0.10%; P<0.05) (Fig. 4B). These results suggest that CD4⁺CD25⁺Foxp3⁺ Treg cells are involved in the suppression of AHR mediated by oral administration of Lcr35.

Fig. 3
Effect of oral Lcr35 treatment on the CD4⁺CD25⁺Foxp3⁺ Treg population in a mouse model. (A) Flow cytometric analysis of T cells in the mouse spleen. Plots show cells expressing CD4, CD25, and intracellular Foxp3. Numbers inside the histograms indicate the percentage of cells; (B) Relative proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells among total splenic T cells after Lcr35 treatment in comparison with cells obtained from the positive control (Group 3). ^*P<0.05. Negative control: Saline (i.p.)/saline challenge by inhalation. Lcr35-treated: OVA (i.p.)/OVA challenge by inhalation+Lcr35 (oral administration). Positive control: OVA (i.p.)/OVA challenge by inhalation.

Lcr35, Lactobacillus rhamnosus; i.p., intraperitoneal; OVA, ovalbumin.

• Click for larger image

Fig. 4
Effect of anti-CD25 mAb treatment on allergic asthma in a mouse model after oral Lcr35-treatment. (A) Airway hyperresponsiveness; (B) Relative proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells among total splenic T cells; (C) Comparison of pulmonary inflammation scores; (D) Lung pathology (H&E stain; ×100). ^*P<0.05. Lcr35-treated: OVA (i.p.)/OVA challenge by inhalation+Lcr35 (oral administration). Anti-CD25: OVA (i.p.)/OVA challenge by inhalation+Lcr35 (oral administration)+anti-CD25 Ab (i.p.). Negative control: Saline (i.p.)/saline challenge by inhalation. Positive control: OVA (i.p.)/OVA challenge by inhalation.

Lcr35, Lactobacillus rhamnosus; OVA, ovalbumin; i.p., intraperitoneal.

• Click for larger image

The pulmonary inflammation score was also significantly higher in anti-CD25 mAb-treated Lcr35 mice compared with Lcr35-treated mice that did not receive anti-CD25 mAb (anti-CD25 treated: 2.28±0.19; Lcr35-treated 1.32±0.20; P<0.01) (Fig. 4C). The histopathology revealed enhanced eosinophilic inflammation in the lungs of anti-CD25-treated Lcr35 mice compared with anti-CD25 mAb-non-treated Lcr35 mice (Fig. 4D). The data indicate that administration of anti-CD25 mAb reverses the effect of oral Lcr35 treatment on lung inflammation and that Treg cells mediate the effect of Lcr35.