Induced apoptosis of Th2 lymphocytes and inhibition of
airway hyperresponsiveness and inflammation by
combined lactic acid bacteria treatment
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Wen-Hsin Lina, Chi-Rei Wub, Hong-Zin Leea, Yueh-Hsiung Kuoa, Hung-Shin Wena, Tze-Yi Linc,
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Chia-Ying Leed, Shi-Ying Huange*, Ching-Yuang Lind,f*
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a
School of Pharmacy, b Institute of Chinese Pharmaceutical Sciences, School of Pharmacy, China
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Medical University, Taichung, Taiwan
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c
Department of pathology, China Medical University Hospital, Taichung, Taiwan
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d
College of Medicine, China Medical University, Taichung, Taiwan
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e
Department of pediatrics, Armed Force Taoyuan General Hospital, Taoyuan, Taiwan
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f
Clinical Immunologic Center, Children’s Medical Center, China Medical University Hospital,
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Taichung, Taiwan
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Running Title:
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Ameliorate airway hyperresponsiveness by probiotics
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* Correspondence and equal correspondence: Ching-Yuang Lin and Shi-Ying Huang.
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Address for correspondence: Ching-Yuang Lin MD., Ph.D. Clinical Immunogical Center, China
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Medical University Hospital, 2 Yuh-Der Road, Taichung, 404, Taiwan
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Tel.: +886-4-22052121#1520; Fax: +886-4-22081079.
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E-mail address: cylin@mail.cmuh.org.tw
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Abstract
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Several lactic acid bacteria (LAB) demonstrably regulate the immune system and inhibit allergic
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disease. This study examined whether oral feeding of either Lactobacillus paracasei (L. paracasei)
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BB5 and/or L. rhamnosus BB1 suppress ovalbumin (OVA)-induced airway hyperresponsiveness
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(AHR) and inflammation in a murine model. OVA-specific immune responses, cell profile of
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bronchoalveolar lavage fluid (BALF), and airway AHR were assessed following OVA and
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methacholine challenge. We investigated whether LAB can enhance CD4+FoxP3+ and CD8+FoxP3+
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regulatory T (Treg) cells in splenic cells and apoptosis of CD4+IL-4+ T cells. Results found oral
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administration of combined LAB better than single L. paracasei or L. rhamnosus strain, improving
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Penh ratio after challenge with methacholine. High-dose combined LAB starkly decreased synthesis
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of OVA-specific IgE and IgG2a levels, as well as eosinophils infiltration in BALF. In addition,
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CD4+IL-4+ T cells decreased while CD4+FoxP3+ and CD8+FoxP3+ Treg cells increased significantly
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in splenic mononuclear cells of high-dose combined LAB group. Findings indicate allergen-induced
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AHR and airway allergic inflammation suppressed by enhances CD4+FoxP3+ and CD8+FoxP3+ Treg
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populations as well as Th1 cell response after treating with combined LAB. This study may provide a
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basis for developing a novel therapeutic or protective method for airway allergic disease.
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Key words: lactic acid bacteria, ovalbumin, airway hyperresponsiveness, regulatory T cells, apoptosis
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1. Introduction
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Asthma is a Th2-mediated chronic inflammatory disease releasing certain Th2 cytokines like
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interleukin (IL)-4 and IL-5 while increasing specific immunoglobin (Ig) E or accumulation of mucus
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and various inflammatory cells [1-4]. Epidemiologic studies indicate positive correlation between
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allergic diseases and changed microbial exposure patterns [5, 6]. These studies found more complex
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and intensive bacteria colonized in the intestinal tract of non-allergic versus allergic children [7-9]. In
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addition, major microbiota of non-allergic children intestine is Lactobacilli and Bifidobacteria genus,
9
but Staphylococcus genus is major in allergic children [9,10].
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Lactic acid bacteria (LAB) are regard as probiotics beneficial to human and animal. Studies
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suggest that yogurt containing live LAB potentiates a variety of intestinal immune responses and may
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yield clinical benefits against bacterial or viral infection [11-14]. Lactobacillus plantarum (L.
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plantarum) isolated from food is indicated to activate Th1 immune response and prevent influenza A
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viral infection by virus-specific neutralizing IgA in BALFs and sera [15]. Regular consumption of
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yogurt containing Lactobacillus bulgaricus (L. bulgaricus) and Streptococcus thermophilus
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demonstrably improves clinical scores among atopic patients or mice, which portends increased
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production of interferon -γ (IFN-γ) and decreased level of eosinophils [16, 17]. Also, some LAB
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strains have been reported as effectively and safely improving quality of life for patients suffering
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from allergic rhinitis, and shown to inhibit allergen-specific T-cell response [18].
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Human gastrointestinal tract microbiota has been estimated to consist of at least 400 different
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species and 1014 microorganisms [19, 20] that are major sources of microbial exposure for humans.
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These may influence immune homeostasis, depending on the strain. Probiotics are defined as living
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microorganisms beneficial to health. Evidence reveals strains of Lactobacilli and Bifidobacteria can
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influence immune function via diverse pathways, including effects on antigen-presenting cells (APC):
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e.g., circulating monocytes, local dendritic cells (DCs), regulatory T (Treg) cells, effector T and B
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1
cells or macrophage [21-24]. Studies also find cytokine profiles with obvious differences among
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strains of LAB co-cultured with human peripheral blood mononuclear cells (PBMC) or other APC
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[25-28]. Likewise, about 30 potential probiotic strains were isolated for prevention or treatment of
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immune imbalance disorders [29-31]; some strains of Bifidobacteria also enhanced Th1 responses or
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induced Treg cells. Other studies found few probiotics stimulating production of INF-γ and inhibiting
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secretion of allergy-related IL-4, IL-5 and specific-Immunoglobulin E (IgE) production. These
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indicate potential strains improving symptoms of allergy [32, 33]. Several clinical trials showed
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probiotics as reducing percentage and symptoms of atopic dermatitis or asthma in babies [34, 35].
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LAB was shown to promote local immune defenses and induce anti-inflammatory cytokines [23,
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36-38]. Treatment with screening probiotics demonstrated preventive effects against pathogenic- or
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chemical-induced colitis by gut barrier function and immunomodulatory efficacy [39-42].
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Lactobacillus paracasei (L. paracasei) treats human allergic rhinitis, and Bifidobacterium longum (B.
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longum) is proven to relieve clinical symptoms of Japanese cedar pollinosis [43, 44]. Induction of
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CD4+CD25+ Treg cells with increased IL-10 and transforming growth factor-β (TGF-β) secretion
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could maintain immune tolerance to allergen challenge [45-47]. CD4+CD25+ Treg cells could express
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toll-like receptor (TLR) in response to stimulation and might regulate allergic response [48, 49].
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Inducible CD8+FoxP3+ Treg cells and TGF-β production were identified recently to maintain
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homeostasis, promote immune tolerance, and regulate host defense against foreign pathogens or
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allergens [46, 47, 50]. CD8+FoxP3 Treg cells can suppress cellular proliferation of CD4+ naïve and
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effector T cell by cell-contact. CD8+CD25+ Treg cells directly induce CD4+CD45ROhigh cell
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apoptosis [51]. Our study tested whether oral administration of L. paracasei BB5, L. rhamnosus BB1
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or combination thereof effectively down-regulates serum IgE production, airway inflammation, Th2
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cytokines and airway hyperresponsiveness (AHR) for sensitized animals. We also tested whether
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LAB enhances CD4+FoxP3+ and CD8+FoxP3+ Treg population or helps suppress allergen-induced
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Th2 immune response. This study may elucidate a mechanism for novel immunotherapeutic
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treatment and prevention.
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2. Methods and materials
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2.1. Mice
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Five-week-old specific pathogen-free BALB/c male mice were purchased from the National
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Laboratory for Animal Breeding and Research Center at Taipei, then housed in six individual cages
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at 25°C and 60% relative humidity with a 12-h light cycle, eight mice per group. Basic feed was
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sterilized Laboratory Rodent Diet 5001 (PMI Nutrition International Inc., Richmond, IN), animal test
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and protocols approved by the China Medical University Hospital Animal Committee.
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2.2. Bacterial preparation
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LAB of L. paracasei BB5 and L. rhamnosus BB1 were isolated from infant feces and validated
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with effect for anti-allergy in our laboratory, both strains identified by API 50 CHL kit (Biomerieux,
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Marcy I’Etoile, France) and Lactobacillus identification system. Meanwhile, strains were confirmed
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by 16S rRNA gene sequence in the GenBank Database, LAB propagated under anaerobic conditions
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at 37°C in deMan-Rogosa-Sharpe (MRS) broth (Difco, Detroit, MI) until log phase of growth.
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Finally, cells of LAB were centrifuged and freeze-dried, viable count of powder adjusted to about
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1×1010 CFU/g.
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2.3. Study design and ovalbumin-induced asthma
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Experimental method was modified from Hong and others [52-54]. Six groups of BALB/c mice
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were designated to examine effects of LAB and their combination. Naïve control mice were neither
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sensitized with ovalbumin (OVA) nor given LAB treatment; other groups were challenged with OVA
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to induce allergic symptoms. To establish a murine model of asthma, these mice were immunized by
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peritoneal injection with 50 μg OVA (Sigma Chemical, St. Louis, MO) plus 1:1 mixture of
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incomplete Freund’s adjuvant (Sigma) on Days 14 and 21. Mice were exposed to OVA aerosol
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inhalation (2% OVA in normal saline solution) for 10 minutes on Days 27-28; hyperresponsiveness
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of airways was assessed on Day 29 after challenged with different concentration of methacholine
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(Sigma). During experiment (Days 0-28), mice of each group except naïve controls received orally
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administration of different samples daily: saline (OVA control group), 3.75 mg L. paracasei BB5
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(P-1×), 3.75 mg L. rhamnosus BB1 (R-1×), 3.75 mg (P+R,-1×) with 1.875 mg BB5 plus 1.875 mg
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BB1, and 11.25 mg (P+R,-3×) with three-fold dose of 3.75 mg P+R. Mice were sacrificed as of Day
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30 (Figure 1). Aerosol was generated by ultrasonic nebulizer in a chamber. Study was approved by
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our institutional Animal Care and Use Committee.
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2.4. Measurement of airway hyperresponsiveness
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AHR was assessed by whole-body plethymography (Buxco Electronics, Troy, NY) after the last
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OVA inhalation. Mice were placed in measured chamber and kept in a dark box. Aerosolized normal
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saline or methacholine in increasing concentration (6.25-25 mg/ml) were nebulized via chamber inlet
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for 3 min. Recordings and baselines were averaged for 3 min after each nebulization of variant
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methacholine concentration. AHR was expressed as mean enhanced pause (Penh = pause × peak
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expiratory box flow/peak inspiratory box flow), data expressed as ratio of PenhMCh and PenhNaCl.
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2.5. Analyzing cellular composition of BALF
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Mice were sacrificed by CO2 asphyxia after measuring AHR, their tracheas immediately lavaged
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three times via catheter with 1 ml normal saline. BALF was centrifuged (400 g) at 4°C for 5 min.
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After washing with normal saline, these cell pellets were resuspended in 1 ml normal saline. Total
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cell number was counted by standard haemocytometer, differential cell counts performed by staining
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with Liu’s stain solution and then counting at least 200 cells. Differentiating cell was judged by
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standard morphological criteria.
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2.6. Determination of serum antibody
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Serum samples collected from hearts after sacrificing were stored at -80°C until assay. Levels of
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serum antibodies were measured for IgE, IgA and IgG, using enzyme-linked immunosorbent assay
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(ELISA) kits (eBioscience, San Diego, CA). OVA-specific IgE, IgG1 and IgG2a were also measured.
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2.7. Preparations and cytokines expression of splenic mononuclear cells
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Spleens were removed aseptically and placed individually into 5 ml complete RPMI-1640
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medium. Single-cell suspensions were prepared by pipetting spleens into small pieces and single-cell
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suspensions with sterile syringe. Suspension was centrifuged at 180 g for 5 min. After discarding
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supernatant, the cell pellet was resuspended in lysis buffer (Tris-NH4Cl) to lyse erythrocytes; lysis
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buffer was washed twice in RPMI-1640 medium, cell viability and concentration determined by a
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hemocytometer after staining of trypan blue. Cell concentration was adjusted to 2×106 cells/ml in
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RPMI-1640 medium, and aliquots of 200 μl suspension added to each well of a 96-well plate and
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cultured in the presence of OVA (10 mg/ml; Sigma) for 48 h at 37°C. Cell-free supernatant fractions
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1
were harvested and stored at -80°C until assayed. Presence of IL-12, IFN-γ, IL-4 and IL-10 in culture
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supernatant fractions was determined by different commercial enzyme-linked immunosorbent assay
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(ELISA) kit, as per manufacturer’s instructions.
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2.8. Histologic examination
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After BAL, trachea and lungs from each animal were removed immediately and fixed by 10%
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buffered formalin. Sections of each lobe (five per animal) were stained with haematoxylin and eosin
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for microscopic examination to rate severity of inflammation. Two pathologists (blinded) assigned
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total inflammation score for each section of lung parenchyma: 0, normal; 1, inflammatory infiltration
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of eosinophils, lymphocytes, and neutrophils in <25% of the entire section; 2, inflammatory infiltrate
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in 25-50% of entire section; 3, inflammatory infiltrate >50% of entire section. Total inflammation
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score for each animal was calculated as mean score for five lung sections.
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2.9. Cellular staining procedure and cell apoptosis
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Splenic monocular cells were isolated from spleen and suspended in RPMI-1640 culture
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medium, 106 cells fixed with 4% paraformaldehyde for 30 min at 4°C, prior to staining for 5 min.
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Stained cells were washed with phosphate buffer saline (PBS, 0.2% BSA) and permeabilized for 5
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min on ice with 200 ul 70% ethanol. After washing, cells were washed three times with PBS
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containing 0.2% BSA buffer, then decanted and stained either with anti-CD4 or anti-CD8 fluorescein
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isothiocyanate (FITC)-conjugated monoclonal antibody (mAb) (eBioscience). To detect FoxP3
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expression of CD4+ or CD8+ T, cells were permeabilized and stained with anti-FoxP3 PE conjugated
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mAb, using standard protocol (eBioscience). Cells were analyzed by FACS scan flow-cytometer (FC
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500, Beckman Coulter, Miami, FL).
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Apoptosis of splenic mononuclear cells was studied with DNA strand breaks on the terminal
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deoxnucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) kit (Mebsttain, Immunotch,
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France). Briefly, 106 cells were fixed with 4% paraformaldehyde for 30 min at 4oC, washed (for 5
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min at 1500g) with phosphated buffer saline (PBS containing 0.2% BSA) and permeabilized for 5
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min on ice with 200 μl 70% ethanol. After washing, cells were decanted and resuspended in 30μl of
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TUNEL labeled as a negative control. After 60 min of incubation at 37oC in a humidified atmosphere,
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cells were washed three times with PBS containing 0.2% BSA buffer and stained with Flurescein
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isothiocyanate (FITC)-conjugated mAb for 30 min. For simultaneous detection of apoptosis, cytokine
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expression, and surface phenotype of a single cell level, anti-IL-4 phycoerythrin (PF) mAb, or
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corresponding isotype control mAbs (Coulter-Immunotech, Miami, FL) were labeled at the same
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time. After staining, cells were analyzed with a FACS scan flow cytometer (FC500, Beckman
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Coulter Inc., USA) acquiring 10,000 events.
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2.10. Statistical analysis
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Data were calculated with mean values, standard deviations (mean±SD) derived from triplicate
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trials. Significance was rated by one-way ANOVA (analysis of variance) and Duncan’s multiple range
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tests (P<0.05) using SPSS 10.0.7 software, figure drawn using Sigma Plot 10.0.
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3. Results
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3.1. Pulmonary function tests
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Mice treated with saline showed markedly higher Penh ratio after challenge with methacholine
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at 25 mg/ml when compared with the other groups (P<0.05). At dose of methacholine tested, Penh
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ratio was significantly lower in group of high-dose combined LAB (L. paracasei BB5 and L.
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rhamnosus BB1) than single strain of either L. paracasei BB5 or L. rhamnosus BB1 (P<0.05) (Fig.
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2). Meanwhile, Penh ratio was also significantly lower in high-dose combined LAB group than low
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dose of combined LAB group (P<0.05) (Fig. 2).
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3.2. Cell profile and cytokines of bronchoalveolar lavage fluid
In order to confirm inflammatory condition, cellular composition of BALF was analyzed, results
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shown in Figure 3. OVA-saline treated mice of positive control group induced a marked rise in cell
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numbers of eosinophil and macrophage (Fig. 3); naïve group had lowest total cell count. Eosinophil
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is main inflammatory cell in BALF; mice treated with probiotic LAB exhibited significantly reduced
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number of eosinophil and macrophage as compared with the OVA-saline treated group (P<0.05). For
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lymphocytes, percentages of treating with high-dose combined LAB group were higher than the other
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groups (P<0.05). For cytokines and total protein levels in BALF (Table 1). Naïve group had lowest
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IL-10, TGF-β, and total protein levels in BALF as compared to OVA-treated groups. Those treated
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with high-dose combined LAB mice had lower levels of TGF-β and higher levels of IL-10 than other
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LAB treated groups, and significantly different from OVA-saline treated group (P<0.05).
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3.3. Lung tissue histology
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Lung tissue and trachea of the OVA-saline group showed severe inflammation, characterized by
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hyperemia, interstitial edema, and inflammatory cell infiltration (Fig. 4a). Only mild hyperemia,
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interstitial edema, and inflammatory cell infiltration appeared in the lung tissues and airways of the
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high-dose combined LAB group (Fig. 4e). Inflammation was highest in the OVA-saline as compared
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to groups of P-1× (Fig. 4b), R-1× (Fig. 4c), low-dose of P+R,-1× (Fig. 4d), high-dose of P+R,-3×
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(Fig. 4d), and naïve control group (Fig. 4f). Mean inflammatory score of terminal bronchioles in the
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high-dose combined LAB group was lower than that of the OVA-saline group (1.2±0.3 vs. 2.2±0.4
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respectively, P<0.05). Mean inflammatory score of trachea in the high-dose combined LAB group
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was also lower than that of the OVA-saline group (1.0±0.2 vs. 2.1±0.4 respectively, P<0.05).
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3.4. Levels of serum antibody and OVA-specific antibody
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Figure 5 plots total serum immunoglobulins (Ig) including IgE, IgA and IgG. Levels of serum
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IgE treated with different LAB were lower than the OVA-saline group (Fig. 5a, P<0.05), as was level
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of IgG (Fig. 5b). Yet levels of IgA rose in mice treated with variant LAB (Fig. 5c); those treated with
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single strain of L. rhamnosus BB1 were higher than those treated with L. paracasei BB5 (P<0.05).
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Serum OVA-specific immunoglobulin levels including IgE, IgG1, and IgG2a were measured (Figure
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6). Figure 6a shows high-dose combined LAB-treatment with starkly lower OVA-specific IgE level
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compared with OVA-saline or other LAB-treated groups (P<0.05). Serum OVA-specific IgG1 level
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showed no significant differences among OVA-saline or other LAB-treated groups (Fig. 6b, P>0.05).
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While IgG2a serum levels related with Th1 cell pathway, results show LAB-treated groups sharply
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higher than the OVA-saline group (Fig. 6c, P<0.05). Combined LAB induced a stronger response of
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IgG2a. Hence either L. paracasei BB5 or L. rhamnosus BB1 strain could inhibit Th2 cell pathway to
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increase serum level of IgG2a and decrease serum level of specific IgE, with no effect on IgG1.
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3.5. Cytokines secretion from mouse splenic mononuclear cells
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To ascertain whether LAB influences secretion of IL-12, IFN-γ, IL-4 and IL-10, mice splenic
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mononuclear cells of each group were co-cultured with OVA for 48 h. Cell viabilities of prepared
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1
splenic cells before OVA treatment were above 95%. Figure 7 shows cytokine levels of IL-12 (Fig.
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7a) and IFN-γ (Fig. 7b); important Th1 cytokines increased significantly in mice treated with low- or
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high-dose combined LAB as compared with groups of naïve and OVA-saline treated mice (P<0.05).
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IL-4 production decreased significantly in single or combined LAB-treated mice than in those treated
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with OVA-saline (Fig. 7c, P<0.05); reduced effect of combined LAB was higher than the single LAB
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strain. IL-10 production of splenic mononuclear cells were increased significantly after treating with
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tested LAB as compared with naïve and OVA-saline treated mice (Fig. 7d, P<0.05).
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3.6. Induced apoptosis of Th2 lymphocytes
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Number of CD4+FoxP3+ T and CD8+FoxP3+ Treg cells significantly rose only in the high-dose
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combined LAB versus OVA-saline-treated group in splenic mononuclear cells (Figs. 8a-8b), with no
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difference emerging in CD4+FoxP3+ and CD8+FoxP3+ Treg cells between single-strain or low-dose
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combined strain groups. Ratio of CD4+IL-4+ TUNEL cells in splenic mononuclear cells rose
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significantly in the high-dose combined LAB-treated group, as compared to OVA-saline and other
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LAB-treated groups (Fig. 8c).
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4. Discussion
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Many studies attempted to devise some novel probiotic LAB to treat allergic disease [30, 31].
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The present study rated efficacy of single LAB and combined strains for inhibiting OVA-induced
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allergic AHR. Results proved combination of L. paracasei BB5 or L. rhamnosus BB1 with higher
25
efficacy than treatment with single strain; it also has dose-dependent effect. Serum OVA-specific IgE
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1
and splenic IL-4 levels were inhibited in the LAB-treated group; cytokine levels of IL-12, IL-10, and
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IFN-γ significantly increased after stimulation with OVA on splenic cells (Figs. 6-7). This study also
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found high-dose combined LAB had induced higher percentage of CD4+FoxP3+ and CD8+FoxP3+
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Treg cells as well as CD4+IL-4+ apoptotic cells than the other groups (Fig. 8). Results indicate a key
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role of combined LAB may attenuate a Th2 cell-mediated allergic immunity and induce CD4+ IL-4+
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cell apoptosis by enhancing expression of Th1 and Treg cells.
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Clinical study indicated supplementation of probiotics during pregnancy potentially enhancing
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immune parameters of IFN-γ and IgA, along with immunomodulatory factors of TGF-β in breast
9
milk [55, 56]. Mixture of probiotics and prebiotics also contributed a beneficial effect to modulated
10
infant’s intestinal microbiota successfully and IgE-associated atopic dermatitis [57-59]. Feleszko et al.
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demonstrated both Bifidobacterium lactis Bb-12 and Lactobacillus rhamnosus GG to reduce airway
12
hyperresponsiveness and percentage of inflammatory cells in BAL fluid [33]. Here we demonstrated
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screening L. paracasei BB5 and L. rhamnosus BB1 with potential effect to relieve allergic disease by
14
immunomodulation. Our prior studies and other reports had demonstrated how diverse LAB strains
15
produced different properties, as well as immune stimulation on antigen presenting cells or peripheral
16
blood mononuclear cells (PBMC) [28, 60-62]. Tested strains in this study induce different cytokine
17
profile when co-cultured with PBMC which Der p1 of house dust-challenged or naïve mice: e.g., L.
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paracasei BB5 induce high IFN-γ production in naïve mice, but L. rhamnosus BB1 induce high
19
IL-12 in Der p1-challenged mice. Results portend Gram-positive lactic acid bacteria producing
20
different peptidoglycans, lipoproteins, exopolysaccharide, enzyme, and cell wall. These productions
21
may induce different immune response via Toll-like receptors [63]. This study found better effect of
22
combined bacteria for regulating allergic immune response than single strain, possibly via synergistic
23
effect of different substance(s) from tested LAB strains. Other studies likewise found multistrains
24
more effective than monostrains [64, 65]. Gackowska et al. indicated LAB strains at least partially
25
dependent on their composition to regulate immune reactions. Low LAB mixture-induced IL-12 and
13
1
IFN- production and relatively high IL-10 and TNF-alpha expression may represent cellular activities
2
induced in vivo by combined action of bacterial antigens [66].
3
IL-10 plays a key role in maintenance of self-tolerance by induction of T cell with blockage of
4
CD28 T cell costimulation [45-47]. Our previous study results showed that specific-allergen induced
5
Th2 lymphocytes apoptosis in immunotherapy-treated asthmatic children or pollen-allergic patients
6
[67, 68]. In this study, increased CD4+FoxP3+ Treg cells with IL-10 production as well as IL-12 and
7
IFN-γ of Th1 cell-mediated cytokines suggest a possible mechanism of combined LAB-treatment
8
modulating Th2 to Th1 shift. Meanwhile, high-dose combined LAB treatment significantly induced
9
apoptosis of CD4+IL-4+ Th2 cells. Our prior study found that CD8+FoxP3+ Treg cells can suppress
10
CD4+ T cell proliferation by cell-contact inhibition [69]. TUNEL study revealed CD8+FoxP3 Treg
11
cells but not CD4+CD25+ Treg cells directly inducing CD4+CD45ROhigh apoptosis [51, 69]. In
12
addition, we found increased production of plasma IL-10 and TGF-β level significantly after one year
13
of immunotherapy-treating asthmatic children. During immunotherapy, both CD4+FoxP3+ and
14
CD8+FoxP3+ Treg cells increased significantly. Taken together, these in vivo human results suggest
15
increasing IL-10, TGF-β, and FoxP3+CD4/CD8+ Treg cells promotes immune tolerance. IL-10 is an
16
important cytokine to induce naïve T cell transform to Treg cells [51, 69].
17
Results indicate combination LAB treatment spawning immunomodulatory effect by inducing
18
both CD4+ and CD8+ Treg cells; it thus may be a good adjuvant for treating allergic asthma. This
19
study illustrated oral administration with high-dose combined L. paracasei BB5 and L. rhamnosus
20
BB1 ameliorating Th2 allergic AHR by boosting CD4+ Treg and CD8+ Treg cells function and
21
decreasing Th2 cytokines. Findings support the idea that combination of high-dose L. paracasei BB5
22
and L. rhamnosus BB1 as a candidate adjuvant for therapeutic intervention in allergic airway disease;
23
detailed mechanisms warrant study in the future.
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4
Acknowledgments
We thank the Armed Forces Taoyuan General Hospital and National Science Council of Taiwan
for supporting this study, People Diagnosis Project 9826 and NSC 98-2313-B-039-002-MY2.
5
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1
Figure legends
2
3
Fig. 1. Asthma induced protocol: Mice were fed orally with different samples daily from Day 0 to 28.
4
On Days 14 and 21, mice were immunized intraperitoneally (i.p) with 50 μg OVA. On Days
5
27-28, mice were challenged with OVA aerosol inhalation for 10 minutes; then the airway
6
hyper-responsiveness was measured after aerosolized methacholine challenge on Day 29.
7
Mice were sacrificed on Day 30.
8
9
Fig. 2. Effects on airway hyperresponsiveness after daily oral administration of single or combined
10
LAB from Days 0 to 28, airway Penh ratio estimated as lung function index after stimulation
11
with aerosolized methacholine on mice of OVA-induced allergic asthma, values expressed as
12
mean±SD (n=8). Letters represent significant difference (P<0.05).
13
14
15
Fig. 3. Cell profile of bronchoalveolar lavage fluid. Values are expressed as mean±SD (n=8). Letters
represent significantly different cells (P<0.05).
16
17
Fig. 4. Histopathology of airway inflammation. OVA-saline group showed severe inflammation of
18
lung tissue and trachea, characterized by hyperemia, interstitial edema and inflammatory cell
19
infiltration. Inflammation was highest in the OVA-saline group (4a) versus P-1 (4b), R-1 (4c),
20
P+R,-1× (4d), P+R,-3× (4e), and naïve control (4f). Hematoxylin and eosin stain 200×.
21
22
23
Fig. 5. Levels of nonspecific IgE (5a), IgG (5b) and IgA (5c) in mice sera, values expressed as mean
±SD (n=8). Different letters represents significantly difference (P<0.05).
24
25
Fig. 6. Levels of OVA-specific IgE (6a), IgG1 (6b) and IgG2a (6c) in mice sera, values expressed as
25
1
mean±SD (n=8). Different letters represent significant differences (P<0.05).
2
Fig. 7. Cytokine secretion of IL-12 (7a), IFN-γ (7b), IL-4 (7c) and IL-10 (7d) from mice spleen
3
mononuclear cells after treating with OVA, values expressed as mean±SD (n=8). Different
4
letters represent significant differences (P<0.05).
5
6
Fig. 8. Percentage of splenic monomclear cells with positive staining for CD4+FoxP3+ lymphocytes
7
(8a), CD8+FoxP3+ lymphocytes (8b) and CD4+IL-4+ TUNEL (8c), values expressed as mean±
8
SD (n=8). Different letters represents significantly difference (P<0.05).
9
26
1
2
Methacholine
challenge
3
OVA i.p
4
OVA i.p
1st & 2nd
inhalation
5
Sacrific
e
6
7
Day
0
8
14
21
Oral feeding different samples for 28 days
9
10
11
Fig. 1
12
27
27 28
29
30
1
2
6
Naive
OVA
P-1x
R-1x
P+R,-1x
P+R,-3x
Penh ratio
5
a
4
a
b
3
c
a
2
Saline
5
c
cd
b
c
c
c
c
1
6.25
d
d
12.5
Methacholine (mg/ml)
3
4
b
Fig. 2
6
28
c
c
d
25
1
2
60
a
Naive
OVA
P-1x
R-1x
P+R,-1x
P+R,-3x
Cell number (x104)
50
40
b
b
b
b
30
a
20
a
b
c
10
cc
d
bb
c
b
b
a a a aa
c
0
a
Macrophage Eosinophils Lymphocytes Basophils
3
4
5
Fig. 3
6
29
1
2
Fig. 4a
Fig. 4b
Fig. 4c
Fig. 4d
Fig. 4e
Fig. 4f
3
4
5
6
7
8
9
30
600
a
500
IgE (ng/ml)
400
300
b
200
b
b
b
100
c
0
1
2
Naive
OVA
P-1x
R-1x
P+R,-1x P+R,-3x
Fig. 5a
800
a
b
IgG (ng/ml)
600
b
b
b
b
400
200
0
3
4
Naive
OVA
P-1x
R-1x
P+R,-1x P+R,-3x
Fig. 5b
1600
a
1400
a
IgA (ng/ml)
1200
b
1000
800
600
a
c
d
400
200
0
5
6
Naive
OVA
P-1x
R-1x
P+R,-1x P+R,-3x
Fig. 5c
7
31
1
a
200
ab
bc
OVA-IgE (ng/ml)
c
150
d
100
50
ND
0
2
3
Naive
OVA
P-1x
a
a
OVA
P-1x
R-1x
P+R,-1x P+R,-3x
Fig. 6a
250
a
a
a
OVA-IgG1 (ng/ml)
200
150
100
50
ND
0
4
5
Naive
R-1x
P+R,-1x P+R,-3x
Fig. 6b
180
a
160
OVA-IgG2a (ng/ml)
140
b
120
c
c
100
80
d
60
40
20
ND
0
6
7
Naive
OVA
P-1x
R-1x
P+R,-1x P+R,-3x
Fig. 6c
8
32
1
600
a
200
b
150
IFN-(pg/ml)
IL-12 (pg/ml)
500
100
bc
50
a
400
b
300
c
200
d
c
c
100
c
e
f
0
0
Naive
2
3
OVA
P-1x
R-1x
Naive
P+R,-1x P+R,-3x
Fig. 7a
OVA
P-1x
R-1x
P+R,-1x P+R,-3x
Fig. 7b
40
30
a
a
25
b
c
cd
d
20
IL-10 (pg/ml)
IL-4 (pg/ml)
30
b
20
b
10
c
10
c
5
e
0
4
5
b
15
0
Naive
OVA
P-1x
R-1x
P+R,-1x P+R,-3x
Naive
Fig. 7c
Fig. 7d
6
33
OVA
P-1x
R-1x
P+R,-1x P+R,-3x
35
a
CD4+FoxP3+ (%)
30
25
bc
b
bc
20
c
15
10
d
5
0
1
2
Naive
OVA
P-1x
R-1x
P+R,-1x P+R,-3x
Fig. 8a
18
a
16
CD8+FoxP3+ (%)
14
12
bc
10
bc
b
c
8
6
4
d
2
0
3
4
Naive
OVA
P-1x
R-1x
P+R,-1x P+R,-3x
Fig. 8b
12
a
CD4+IL-4+ TUNEL (%)
10
b
8
d
d
Naive
OVA
c
c
P-1x
R-1x
6
4
2
0
5
6
P+R,-1x P+R,-3x
Fig. 8c
34
1
Table 1
2
IL-10, TGF-β, and total protein levels in bronchoalveolar lavage fluid
Naïve
OVA
P-1×
R-1×
P+R,-1×
P+R,-3×
IL-10 (pg/ml)
9.8±2.3 c
32.5±6.4 b
33.2±7.3 ab
38.1±5.7 ab
41.2±6.3 ab
46.5±5.4 a
TGF-β (pg/ml)
5.2±1.4 c
18.4±4.6 a
17.6±5.2 a
16.3±4.8 a
15.6±4.2 a
12.3±3.9 b
Protein (μg/ml) 96.5±20.4 b 312.4±62.3 a 321.2±58.4 a 343.5±51.4 a 351.2±53.4 a 381.3±55.8 a
3
Each value in the table represents mean value ± SD (n=8). Values in the same row followed by
4
different letters differ significantly (P<0.05) for treatment groups.
35