A bovine whey protein extract can induce the generation of regulatory T cells and shows potential to alleviate asthma symptoms in a murine asthma model
Jiunn-Horng Chen1,2†, Po-Han Huang3†, Chen-Chen Lee3, 4 , Pin-Yu Chen5 and Hui-Chen Chen3*
1 Division of Rheumatology, Department of Medicine, China Medical University Hospital, Taichung, Taiwan
2 School of Medicine, China Medical University, Taichung, Taiwan
3 Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
4 Department of Microbiology and Immunology, School of Medicine, China Medical University, Taichung, Taiwan
5 Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada
† These authors contributed equally to this paper
*Corresponding author: Hui-Chen Chen
Address: No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan Tel: +886-4-2205-2121 ext. 7702
Fax: +886-4-22333641
E-mail: [email protected]
Running title: Whey protein extract and asthma
Key words: Bovine whey protein extract, Asthma, TGF-β, Regulatory T cells
glutathione; MCh: metacholine; MFI: mean fluorescence intensity; mLN: mediastinal lymph node; OVA: ovalbumin; PAS: periodic acid-Schiff; Treg: regulatory T cell; TH2: T helper cell type2; TGF-β: transforming growth factor-β; WPE: whey protein extract.
Abstract
The number of people with asthma has dramatically increased over the past few decades and the cost of care is more than $11.3 billion per year. The use of steroids is the major treatment to control asthma symptoms, but the side effects are often devastating. Seeking new drugs or new strategies to reduce the
dose of steroid taken has always been an important task. A bovine whey protein extract (WPE) that is enriched in transforming growth factor-β (TGF-β) has been demonstrated to have the potential for reducing symptoms associated with mild-to moderate T helper cell type1 (TH1) mediated psoriasis in human. However whether WPE also has potential for inhibiting T helper cell type2 (TH2)-mediated disease remains unclear. In this study, using a murine asthma model, we found that sensitized mice fed WPE daily, before they were challenged, resulted in reducing airway inflammation, serum OVA-specific IgE, TH2-related cytokine production and airway hyperresponsiveness. Increase in the regulatory T cell population in vitro and in vivo was observed when treated with WPE. According to the results from TGF-β blocking antibody study, we suggest that TGF-β is the main component that endows WPE with the potential to reduce the generation of regulatory T cells. Thus, our data suggest that WPE has the potential for alleviating the symptoms of asthma by inducing the generation of regulatory T cells. Therefore, regular administration of WPE might be potentially beneficial for patients with asthma.
Introduction
Asthma is a T cell-mediated allergic disease disorder that caused sensitized individuals to develop eosinophilic airway inflammation and mucus hypersecretion in response to inhaled aeroallergens. T helper cell type2 (TH2) cells play a central role in the disease progression through their production of the cytokines, particularly IL-4, IL-5, and IL-13, which contribute to IgE production by B cells, growth and differentiation
of eosinophils and mast cells, and the development of bronchial hyperactivity and goblet cell hyperplasia . Over the past decades, the prevalence of asthma has dramatically increased and cost of care is more than $11.3 billion per year. According to the records published by World Health Organization (WHO), asthma affects about 300 million people, and 255000 people died of asthma in 2005. The use of glucocorticosteroids is a major treatment for controlling symptoms of asthma. However, side effects of treatment, such as, high blood pressure, osteoporosis, immune system changes etc. are often inevitable.
Natural health products are generally used to support the treatment of many health problems. They often have fewer side effects and are used as reinforcement for regular medical treatments. Bovine whey protein extract (WPE), which is prepared from milk, is a source of bioactive proteins including lactoferrin, immunoglobulins, α-lactoglobulin, β-lactoglobulin, and growth factors. It has been scientifically demonstrated to possess many health benefits, including antioxidant, antitumor, hypolipidemic, anti-viral and anti-bacterial properties . Many studies have focused on the immune-booster effect of WPE . However,
Penttila et al. demonstrated that under some circumstances, WPE can also act as an immunosuppressive
agent .
The whey protein used in this study was produced from a bovine whey protein that is based on a patented process , and is rich in activated form of transforming growth factor-β (TGF-β), at concentrations of 5-50 μg/g powder. A previous study showed that this patented WPE inhibits the production of T helper 1 (TH1) related cytokines IFN-γ and IL-2 . Recently, an open-label clinical trial suggested that WPE has the potential to reduce psoriasis severity . There were no clinically significant side effects that were associated with the use of 2.5g of WPE twice daily for 112 days, suggesting that XP-828L may be a safe treatment . However the detailed mechanism regarding this inhibitory effect and whether WPE also has the potential to
inhibit TH2 mediated disease remains unclear. In this study, using an experimental asthma model, we found that the patented WPE was able to alleviate symptoms of asthma, possibly through the induction of regulatory T cell population in mice, which in turn inhibited immune responses.
Experimental methods
Animals
BALB/c mice were purchased from the National Animal Center, Taiwan. All of the mice were maintained and bred in an animal facility at China Medical University. For all of the animal experiments, female mice (8-12 week-old) were used and 6-8 mice were assigned to each group. All of the animal experiments were performed in accordance with the guidelines of Institutional Animal Care and Use Committee of China Medical University.
Reagents
WPE was obtained from EnBio-Life, the Asia exclusive agent of Advitech Solutions, Canada (Taichung, Taiwan). Mouse regulatory T cell staining kit, GATA-3-PE (TWAJ), CD3 (145.2C11) and anti-CD28 (37N.51) were purchased from eBioscience (San Diego, CA, USA). EasySep CD4+ T cell selection kit and EasySep B cell selection kit were purchased from STEMCELL Technologies Inc. (Vancouver, Canada). IL-4, IL-5, IL-13 and TGF-β Duo Set ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA). Alum (Imject Alum) was purchased from Pierce (Rockford, IL, USA). Ovalbumin (OVA) and mitomycin C were purchased from Sigma-Aldrich (St. Louis, MO, USA). TGF-β neutralizing antibody (1D11) was purchased from Genzyme Corporation (Framingham, MA, USA).
Experimental model of asthma and treatment of WPE
The mice were sensitized intraperitoneally by injection of 50 μg of OVA that had been emulsified in 4 mg of aluminum hydroxide in a total volume of 200 μL on days 0, 14 and 28, and then challenged with 100 μg OVA in a total volume of 50 μL by intranasal administration for 3 consecutive days and analyzed 1 day after they were challenged. Previous studies showed that psoriasis patients who receive orally a minimal dose of WPE of 8-10 mg/Kg twice daily for 56 days have improved physician’s global assessment (PGA) score and do not exhibit side effects . Based on the body surface area conversion of human to mice , the dose conversion from human to mice would be 200-1200 mg/Kg daily in mice. Therefore, for the treatment, 800 mg/Kg (20 mg/mice) WPE in 250 µL of PBS was administered to mice by gavage daily for 14 days (scheme shown in Fig. 1A).
Analysis of bronchoalveolar lavage
Mice were euthanized and cells in the bronchoalveolar lavage (BAL) cells were obtained as previously described . Briefly, BAL fluid was prepared by washing the lungs 3 times with 1 mL phosphate-buffered
saline (PBS). The cells from individual mice were centrifuged, resuspended and counted. Cytospin preparations were stained with Pappenheim staining. Differential cell counts were evaluated by counting at least 200 cells to determine the relative percentage of each cell type that was present in the BAL.
Lung histology
Paraffin-embedded lung sections were prepared as previously described and stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS). H&E images and PAS images were obtained at 100× and 200 × total magnification, respectively.
Re-stimulation of mediastinal lymph node cells from challenged mice
After sensitization and challenge, mediastinal LN cells were isolated. Single-cell suspensions were prepared and stimulated in vitro with 100 µg/mL OVA and syngeneic mitomycin C-treated B cells. The
concentrations of cytokines in culture supernatants were measured after 48 hours of culturing.
Isolation of CD4 T cell and B cell
CD4+ T cells or B cells were isolated from the spleens of mice by negative selection using EasySep CD4+ T cell selection kit or EasySep B cell selection kit, respectively. All the preparation procedures were carried out following the manufacturer’s instructions.
Serum antibody and TGF-β detection
Serum was obtained by cardiac puncturing for the measurement of OVA-specific IgE and TGF-β by ELISA.
Assay of airway hyperresponsiveness
The assays were performed as previously described . Briefly, airway resistance was assessed as the increase in pulmonary resistance after challenge with aerosolized methacholine (MCh) in anaesthetized mice. Mice were anaesthetized with 90–120 mg/kg ketamine along with 5–10 mg/kg xylazine. They were then
tracheotomized and mechanically ventilated at a rate of 150 breaths per min with a tidal volume of 0.3 mL and a positive end-expiratory pressure of 3–4 cm H2O using a computer-controlled small animal ventilator (flexiVent, SCIPEQ Scientific Respiratory Equipment INC.). An intravenous catheter tube was inserted into the trachea of each mouse to the level of the thorax and then coupled to a pressure transducer. Air flow was measured by electronic differentiation of the volume signal. Changes in pressure, flow, and volume were recorded. Pulmonary resistance was calculated using a software program (flexiVent version5.2). MCh aerosol was generated with an in-line nebulizer and administered directly through the ventilator.
Gene expression analysis by quantitative PCR
RNA was extracted using TRIzol Reagent (Invitrogen), and reverse transcription were carried out using oligo(dT) as a primer and MMTV Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol (Invitrogen). Quantitative PCR assays were performed using 20 μL of a reaction mixture that contained 8–10 ng of cDNA, SYBR Green, dNTP, primers, and Taq polymerase (ABI). The expression levels were normalized against HPRT.
Flow cytometry analysis
Cells were re-suspended in PBS/5% FCS containing 0.01% sodium azide, and incubated with anti-FcR (2.4G2) for 20 minutes on ice. The cells were then stained with the indicated antibodies for 30 minutes on ice. Intracellular Foxp3 and GATA-3 staining were conducted according to manufacturer’s protocol in the mouse regulatory T cell staining kit. FACSCalibur analyzer was used, and the data were analyzed with WinMDI.
Statistical analysis
The results were compared using Student’s t test by using the software program GraphPad Prism 5. The data are presented as mean ± SEM. Probability values (p) of less than 0.05 were considered statistically
significant.
Results
WPE reduces airway inflammation, hyperresponsiveness and TH2 cytokine production in a murine
asthma model
In order to test whether WPE has the potential to inhibit TH2-mediated disease such as asthma, a murine asthma model was used. The mice were sensitized and challenged to OVA. For the treatment, mice were orally administered 800 mg/Kg (20 mg/mice) of WPE daily for 14 consecutive days before they were challenged. During the period of treatment, the mice showed no signs of illness and weight loss.
To investigate the effect of WPE on airway inflammation, differential cell counts in BAL fluid were analyzed. In PBS-sensitized and challenged mice (negative control), no obvious increased inflammatory cells in BAL fluid was observed; mice sensitized and challenged with OVA (positive control) showed a typical TH2-type, eosinophilic BAL cell differentials. After treatment with WPE for 2 weeks before the challenge, less eosinophil infiltration was observed in the lungs (Fig. 1B). Histological examinations of the fixed lung tissue that were prepared from these mice revealed that peribronchiolar inflammation (indicated
by H&E staining) and mucus production (indicated by positive PAS staining) was present in the positive control. In contrast, the extent of cell infiltration and mucus secretion was lesser in the WPE-treated mice than in the positive control (Fig. 1C). Collectively, the results indicate that WPE treatment could inhibit airway inflammation.
We subsequently examined whether WPE treatment could suppress the development of airway hyperresponsiveness. As shown in Fig. 2, WPE-treated mice showed significant decreases in pulmonary resistance compared to the positive control.
Elevated levels of IgE in the patients of asthma are also an indicator of an allergy. Therefore we examined whether WPE treatment can reduce the level of specific IgE in serum. We found that OVA-specific IgE levels were significantly reduced after administration of WPE (Fig. 3A).
To further establish whether TH2 functions were inhibited by WPE treatment, we evaluated the ability of mediastinal lymph node (mLN) cells to produce TH2-type cytokines after they were challenged. Upon re-stimulation with OVA-pulsed B cells in vitro, mLN cells that were isolated from mice that had been sensitized and challenged with OVA produced TH2 cytokines such as IL-4, IL-5, and IL-13. However, the production of IL-4, IL-5, and IL-13 by the re-stimulated mLN cells from the WPE-treated mice was decreased (Fig. 3B).
WPE can induce the generation of regulatory T cells and reduce the CD4+GATA-3+ cell population in
vivo
The WPE that we used was prepared by acid precipitation according to a patented process , and is rich in rich in TGF-β (5–50 μg/g powder). TGF-β is a potent regulatory cytokines and has the ability to inhibit the development and progression of various immunopathological diseases . We therefore examined whether TGF-β levels could be elevated after oral administration of WPE. Indeed, elevated TGF-β levels in the
serum of the WPE-fed mice were observed (Fig. 4). TGF-β was found to be able to induce FoxP3 expression and the generation of regulatory cells (Treg) , which in turn block immune cell function. To explore the mechanisms by which WPE elicits its inhibitory effects, we first examined whether WPE can induce the generation of regulatory T cells. As shown in Fig. 5A, an increased proportion of Treg cells was observed in the blood and lungs from the mice that had been fed with WPE for 14 d before they were challenged. Regulatory T cells were found to be able to inhibit T cell proliferation and the expression of GATA-3, which is an important transcription factor that regulates the expression of the TH2 cytokines IL-4 and IL-5. Therefore, we next examined whether the TH2 cell (identified as being CD4+GATA-3+) population and/or GATA-3 expression were inhibited in the WPE-fed mice. We found that the TH2 cell population was increased in OVA-sensitized and challenged mice; however, it was reduced when the mice were fed with WPE before they were challenged (Fig. 5B). Interestingly, the expression levels of GATA-3 in the TH2 cells were similar among the groups (Fig. 5B), indicating that the decrease in TH2-related cytokine production observed in the WPE-fed mice was due to the decrease in TH2 cell numbers and not due to the decreased GATA-3 expression levels in the TH2 cells.
TGF-β is the active component in WPE that induces the generation of regulatory T cells
Finally, we aimed to investigate whether TGF-β is a major component in WPE that can induce the generation of regulatory T cells. We observed that WPE induced the generation of regulatory T cells when CD4 T cells were cultured in the presence of WPE (Fig. 6). However, this induction could be completely abolished when a TGF-β-blocking antibody (1D11) was used to neutralize the activity of TGF-β (Fig. 6). This finding indicates that without TGF-β, WPE lost its ability to induce the generation of regulatory T cells.
Discussion
Whether consumption of farm milk can prevent allergic diseases has always been controversial. Recently, the results of a large epidemiologic study (GABRIELA study) revealed that the consumption of unheated farm milk is inversely associated with childhood asthma and allergies . Elevated levels of TGF-β were found in unpasteurized cow milk . Further, milk TGF-β has been shown to help reduce the development of allergies in early childhood . All of the available evidence implicates the important role played by TGF-β in the prevention of allergic diseases in early life. Therefore, during manufacturing, enriching bioactive TGF-β may be useful for allergy prevention. In this study, we showed that the patented WPE, which is enriched in TGF-β, has the potential to alleviate the symptoms of asthma, including airway inflammation, serum IgE level, production of TH2-related cytokines (IL-4, IL-5, and IL-13), and airway hyperresponsiveness, which might be due to the induction of regulatory T cells in mice after ingestion of WPE.
Regulatory T cells has been shown to maintain immune homeostasis by suppressing T cell proliferation and the functional maturation of dendritic cells . It plays a central role in the prevention of extensive immune-mediated damage and autoimmune disease. Several Treg subsets have been described: one is the cells that are originated from thymus and called natural occurring Treg (nTreg) . Another is the subset that is
induced in the periphery and thus called inducible Treg (iTreg), which can be further divided into IL-10 secreting Treg and TGF-β secreting Treg. The subset that is induced by WPE could be TGF-β secreting Treg. The suppressive effect mediated by Treg is known to be not antigen-specific and involves cell-cell interaction or the inhibitory effects of TGF-β that is secreted by Treg .
Regulatory T cells from atopic individuals are defective in suppressing TH2 cell function compared with those from non-atopic individuals. Numerous experiments, carried out by either depleting regulatory T cells or adoptive transfer of antigen-specific regulatory cells, revealed that regulatory T cells can suppress allergic inflammation and hyperresponsiveness . Interestingly, our data show that WPE can induce the generation of Treg. WPE contains many bioactive proteins, including lactoferrin, immunoglobulins, α-lactoglobulin, β-α-lactoglobulin, and growth factors that have been demonstrated to regulate immune functions. However none of them have been shown to be able to induce the generation of regulatory T cells, except TGF-β that is enriched in this patented WPE . Research has demonstrated that orally administered TGF-β can still retain and exert its biological activity in the intestinal mucosa and can affect immune responses locally or systemically. Furthermore, the latent from of TGF-β, such as the TGF-β in whey-derived natural products, can be activated by gastric acid . Indeed, increased concentrations of TGF-β in the serum from the WPE-fed mice were observed in our study. We therefore hypothesize that the inhibitory effect that was elicited by WPE might have been due to the generation of regulatory T cells that were induced by the TGF-β enriched in the WPE.
TGF-β, produced by regulatory T cells, has diverse effects on various types of immune cells other that T cells, including dendritic cells (DCs), mast cells and granulocytes. DCs play a key role in linking innate and adaptive immune responses. Impaired DC functional maturation hampers the differentiation of TH2
cells. Evidence has shown that TGF-β negatively regulates DC maturation and its ability to present antigen to T cells . The mediators that are released by activated mast cells and eosinophils aggravate allergic inflammation. Cell activation depends on cross-linking of IgE to FcRI. Previous data have shown that TGF-β can inhibit the expression of FcRI . Taking the evidence together, the inhibitory effect of WPE during the disease progression of asthma might not be simply due to the inhibition of T cells function. The influence of WPE on other cells would be interesting to study.
Although we speculate that TGF-β serves as a key component that renders this patented WPE immunosuppressive effects, we cannot exclude the possibility that components other than TGF-β might also contribute to the alleviation of symptoms of asthma. For instance, whey protein is a source of cysteine-rich protein, may facilitate the synthesis of glutathione (GSH), which is a potent antioxidant found in the airways. It has been shown that GSH levels decrease in the lungs during the early stage of asthmatic reaction . GSH has also been shown to decrease allergen-induced contractions by relaxing airway smooth muscle and preventing histamine- and allergen-induced airway contractions . A whey-based glutathione-enhancing diet has also been shown to decrease allergen-induced airway contractions in a guinea-pig model of asthma. Therefore, the reduced airway hyperresponsiveness that we observed in the WPE-fed mice might be due to the increased GSH levels that reduced the oxidative stress in the airway. Lactoferrin, which is an iron-binding protein and has the ability to inhibit the potential causative agent of asthma, tryptase, was demonstrated to be able to abolish bronchoconstriction and airway hyperresponsiveness in an allergic sheep model . Furthermore, lactoferrin was shown to inhibit the migration of eosinophils. Currently, a phase II clinical trial using orally administrated recombinant human lactoferrin against asthma is underway. However, considering that the proportion of lactoferrin in this patent WPE is only 0-2%, lactoferrin might
not be the major component to alleviated symptoms of asthma in the current study.
Much research has focused on identifying nutrients with value more than nutrition itself but also with an ability to modulate the immune system. Undoubtedly, milk is an important one of interests. Our data further demonstrate that WPE can act as an immune modulator and that it might be potentially beneficial for patients with not only TH1- but also TH2-mediated immunopathological diseases.
Acknowledgements
This project was supported by China Medical University (CMU97-227 and CMU99-N1-20), Teh-Tzer Study Group for Human Medical Research Foundation (B991043) and EnBio-Life, the Asia exclusive agent of Advitech Solutions, Canada. J-H. C.and P-H. H. contributed equally to this paper. H-C. C, and J-H. C. designed the research protocol; P-H. H. conducted the research; H-C. C., J-H. C. and P-H. H. analyzed the data; P-H. H. prepared the figures and tables; H-C. C. wrote the paper; C-C. L. provided technical support and reagents that were used for AHR experiment. P-Y. C. was involved in revising the paper with respect to important intellectual content. All of the authors read and approved the final manuscript and declare no conflict of interest.
References
1. Renauld JC (2001) New insights into the role of cytokines in asthma. J Clin Pathol 54, 577-589.
2. Fallon PG, Jolin HE, Smith P et al. (2002) IL-4 induces characteristic Th2 responses even in the combined absence of IL-5, IL-9, and IL-13. Immunity 17, 7-17.
3. Krissansen GW (2007) Emerging health properties of whey proteins and their clinical implications. J Am
Coll Nutr 26, 713S-723S.
4. Rusu D, Drouin R, Pouliot Y et al. (2009) A bovine whey protein extract can enhance innate immunity by priming normal human blood neutrophils. J Nutr 139, 386-393.
5. Rusu D, Drouin R, Pouliot Y et al. (2010) A bovine whey protein extract stimulates human neutrophils to generate bioactive IL-1Ra through a NF-kappaB- and MAPK-dependent mechanism. J Nutr 140, 382-391. 6. Penttila IA, Zhang MF, Bates E et al. (2001) Immune modulation in suckling rat pups by a growth factor extract derived from milk whey. J Dairy Res 68, 587-599.
7. Juneau C DR, Moroni O (2007) Process for the manufacture of an anti-inflammatory composition. Patent No. 13431-15USPR.
8. Drouin R, Lamiot E, Cantin K et al. (2007) XP-828L (Dermylex), a new whey protein extract with potential benefit for mild to moderate psoriasis. Can J Physiol Pharmacol 85, 943-951.
9. Poulin Y, Bissonnette R, Juneau C et al. (2007) XP-828L in the treatment of mild to moderate psoriasis: randomized, double-blind, placebo-controlled study. Altern Med Rev 12, 352-359.
10. Drouin R, Moroni O, Cantin K et al. (2008) A double-blind, placebo-controlled, randomized trial of XP-828L (800 mg) on the quality of life and clinical symptoms of patients with mild-to-moderate psoriasis.
Altern Med Rev 13, 145-152.
11. Reagan-Shaw S, Nihal M & Ahmad N (2008) Dose translation from animal to human studies revisited.
FASEB J 22, 659-661.
12. Cohn L, Homer RJ, MacLeod H et al. (1999) Th2-induced airway mucus production is dependent on IL-4Ralpha, but not on eosinophils. J Immunol 162, 6178-6183.
13. Lee CC, Wang CN, Lai YT et al. (2010) Shikonin inhibits maturation of bone marrow-derived dendritic cells and suppresses allergic airway inflammation in a murine model of asthma. Br J Pharmacol 161, 1496-1511.
14. Li MO, Wan YY, Sanjabi S et al. (2006) Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol 24, 99-146.
15. Li MO, Sanjabi S & Flavell RA (2006) Transforming growth factor-beta controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms.
Immunity 25, 455-471.
16. Zhou L, Lopes JE, Chong MM et al. (2008) TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature 453, 236-240.
17. Lee HJ, Takemoto N, Kurata H et al. (2000) Gata-3 Induces T Helper Cell Type 2 (Th2) Cytokine Expression and Chromatin Remodeling in Committed Th1 Cells. The Journal of Experimental Medicine 192, 105-116.
18. Corthay A (2009) How do regulatory T cells work? Scand J Immunol 70, 326-336.
19. Jager A & Kuchroo VK (2010) Effector and regulatory T-cell subsets in autoimmunity and tissue inflammation. Scand J Immunol 72, 173-184.
Exp Allergy 41, 29-35.
21. Loss G, Apprich S, Waser M et al. (2011) The protective effect of farm milk consumption on childhood asthma and atopy: The GABRIELA study. J Allergy Clin Immunol.
22. Peroni DG, Piacentini GL, Bodini A et al. (2009) Transforming growth factor-beta is elevated in unpasteurized cow's milk. Pediatr Allergy Immunol 20, 42-44.
23. Oddy WH & Rosales F (2010) A systematic review of the importance of milk TGF-beta on immunological outcomes in the infant and young child. Pediatr Allergy Immunol 21, 47-59.
24. Serra P, Amrani A, Yamanouchi J et al. (2003) CD40 ligation releases immature dendritic cells from the control of regulatory CD4+CD25+ T cells. Immunity 19, 877-889.
25. Sakaguchi S, Sakaguchi N, Asano M et al. (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155, 1151-1164.
26. Thornton AM & Shevach EM (2000) Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J Immunol 164, 183-190.
27. Umetsu DT, Akbari O & Dekruyff RH (2003) Regulatory T cells control the development of allergic disease and asthma. J Allergy Clin Immunol 112, 480-487; quiz 488.
28. Strickland DH, Stumbles PA, Zosky GR et al. (2006) Reversal of airway hyperresponsiveness by induction of airway mucosal CD4+CD25+ regulatory T cells. J Exp Med 203, 2649-2660.
29. Lloyd CM & Hawrylowicz CM (2009) Regulatory T cells in asthma. Immunity 31, 438-449.
30. Bettelli E, Carrier Y, Gao W et al. (2006) Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235-238.
intestinal mucosa and enhances oral tolerance. J Allergy Clin Immunol 120, 916-923.
32. Nakamura Y, Miyata M, Ando T et al. (2009) The latent form of transforming growth factor-beta administered orally is activated by gastric acid in mice. J Nutr 139, 1463-1468.
33. Geissmann F, Revy P, Regnault A et al. (1999) TGF-beta 1 prevents the noncognate maturation of human dendritic Langerhans cells. J Immunol 162, 4567-4575.
34. Dong Y, Tang L, Letterio JJ et al. (2001) The Smad3 protein is involved in TGF-beta inhibition of class II transactivator and class II MHC expression. J Immunol 167, 311-319.
35. Gomez G, Ramirez CD, Rivera J et al. (2005) TGF-beta 1 inhibits mast cell Fc epsilon RI expression. J
Immunol 174, 5987-5993.
36. Kloek J, Mortaz E, van Ark I et al. (2010) Glutathione prevents the early asthmatic reaction and airway hyperresponsiveness in guinea pigs. J Physiol Pharmacol 61, 67-72.
37. Kloek J, van Ark I, Bloksma N et al. (2002) Glutathione and other low-molecular-weight thiols relax guinea pig trachea ex vivo: interactions with nitric oxide? Am J Physiol Lung Cell Mol Physiol 283, L403-408.
38. Kloek J, Mortaz E, Van Ark I et al. (2011) A whey-based glutathione-enhancing diet decreases allergen-induced airway contraction in a guinea-pig model of asthma. Br J Nutr 105, 1465-1470.
39. Elrod KC, Moore WR, Abraham WM et al. (1997) Lactoferrin, a potent tryptase inhibitor, abolishes late-phase airway responses in allergic sheep. Am J Respir Crit Care Med 156, 375-381.
40. Bournazou I, Mackenzie KJ, Duffin R et al. (2010) Inhibition of eosinophil migration by lactoferrin.
Immunol Cell Biol 88, 220-223.
Figures Legends:
animal sensitization, treatment and challenge that were carried out in this study. (B) Bronchoalveolar fluid (BALF) total cell number (left) and differential cell counts (right) that were assessed by Pappenheim staining. The numbers represent the mean± SEM of the cell number (n=6). (C) Representative lung sections stained with H&E (100×) or PAS (200×) from negative control (left), positive control (middle), or the mice that were treated with WPE before they were challenged (right). The arrows indicate areas of peribronchiolar cellular infiltrate (H&E) or positive mucus staining (PAS). ** p<0.01 compared with the positive control. Statistical significance was determined using Student’s t test. NC: negative control, mice that were sensitized and challenged with PBS. PC: positive control, mice that were sensitized and challenged with OVA without WPE treatment. WPE: the mice that were administered with WPE orally for 2 weeks before they were challenged.
Fig. 2. Airway hyperresponsiveness was inhibited by WPE. An airway resistance was measured by invasive body plethysmography. The data are expressed as the mean ± SEM of the pulmonary resistance (RL) (n=4). ** p<0.01, *** p<0.001 compared with the control. Statistical significance was determined using Student’s t test. NC: negative control, mice that were sensitized and challenged with PBS. PC: positive control, mice that were sensitized and challenged with OVA without WPE treatment. WPE: the mice that were administered with WPE orally for 2 weeks before they were challenged.
Fig. 3. Serum OVA-specific IgE and cytokine production by TH2 cells was reduced in the WPE-treated mice. (A) OVA-specific IgE in the serum was measured by ELISA. The numbers represent the mean± SEM of the O.D. (n=6). (B) Cytokines production by medistinal LN cells that were isolated from mice as indicated after re-stimulation with antigen presenting cells and OVA were analyzed by ELISA. In each experiment, mLN cells were pooled from six mice per group for culturing. * p<0.05, ** p<0.01 compared
with the positive control. Statistical significance was determined using Student’s t test.
Fig. 4. Concentrations of TGF-β in the blood. The concentrations of TGF-β in the serum samples were measured by ELISA. The numbers represent the mean± SEM of the concentrations (n=6). * p<0.05 compare with the positive control. Statistical significance was determined using Student’s t test. NC: negative control, mice that were sensitized and challenged with PBS. PC: positive control, mice that were sensitized and challenged with OVA without WPE treatment. WPE: the mice that were administered with WPE orally for 2 weeks before they were challenged.
Fig. 5. The generation of Treg cells and expression levels of GATA3 in vivo. (A) Treg population in the blood and lungs were identified as CD4+Foxp3+ cells using flow cytometry analysis. The numbers are expressed as the mean ± SEM of the Treg proportion in the blood (n=5). (B) CD4+GATA3+ cell population (left) and the mean fluorescence intensity (MFI) of GATA3 expression in the CD4+GATA3+ cells in the lungs were analyzed using flow cytometry analysis. The numbers are expressed as mean ± SEM of the CD4+GATA3+ cell proportion (left) or MFI (right) (n=4). * p<0.05, ** p<0.01 compared with the positive control. Statistical significance was determined using Student’s t test. NC: negative control, mice that were sensitized and challenged with PBS. PC: positive control, mice that were sensitized and challenged with OVA without WPE treatment. WPE: the mice that were administered with WPE orally for 2 weeks before they were challenged.
Fig. 6. Removal of TGF-β could abolish the effect of WPE on Treg differentiation. (A) Naïve CD4+ T cell were stimulated with anti-CD3 and anti-CD28 in the presence of indicated concentrations of WPE for 3 days. Treg was identified as CD4+Foxp3+ cells using flow cytometry analysis. The numbers represent the
mean ± SEM of the fold increase in Treg population compared with the cells without WPE treatment from 4 independent experiments. (B) Naïve CD4+ T cell were stimulated with anti-CD3 and anti-CD28 in the presence or absence of 250 µg/mL WPE with the indicated concentrations of anti-TGF-β antibodies for 3 days. Treg was identified as CD4+Foxp3+ cells using flow cytometry analysis. Numbers represent mean ± SEM of the Treg population in the culture (n=6). * p<0.05, ** p<0.01, *** p<0.001 compare with the one with WPE treatment but without the presence of anti-TGF-β antibodies. Statistical significance was determined using Student’s t test.
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