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1、精品论文Immunomodulatory effects of Lactobacillus casei Zhang in a murine model of peanut allergyMengsha Hu, Qiuxiang Zhang, Xiaoming Liu, Gang Wang, Hao Zhang, Wei Chen5(School of Food Science and Technology, Jiangnan University, JiangSu WuXi 214122) Abstract: Probiotics can modulate systemic and mucos
2、al immune systems, as well as induce immune tolerance in allergic diseases. The aim of this study was to evaluate the immunomodulatory potential of Lactobacillus casei Zhang on prophylactic and therapeutic effects in a mouse model of peanut allergy. Intragastrical administration with L. casei Zhang,
3、 prior or after allergic sensitization, led to increased10levels of serum allergen-specific IgG2a and mucosal IgA antibodies. Additionally, systemic anaphylactic symptoms and Th2 responses (IgE antibody, histamine release and IL-4 production) wereattenuated. L. casei Zhang had less influence on the
4、serum IgG1 antibody and other cytokines (IL-10, IL-12 and IFN-). Successful immunomodulation by pre and post-treatment were further demonstrated by an augment of the IFN-/IL-4 ratio in vitro and enrichment of CD4+ Foxp3+ Tregs in spleen and15mesenteric lymph nodes. These results suggested that L.cas
5、ei Zhang may play a role in preventing and treating peanut allergy.Keywords: Food Science; Lactobacillus casei Zhang; peanut allergy; Th1/Th2 balance; regulatory Tcells200IntroductionFood allergy has become a major health concern in developed as well as developing countries nowadays. Eight kinds of
6、food are responsible for more than 90% of food allergies: crustaceans (shrimp, lobster, crab), egg, fish, milk, peanuts, soybeans, tree nuts (including almond), and wheat1, 2. Among them, peanut allergy deserves special attention because of its fatal outcomes. It25tends to present early in life, and
7、 typically lifelong with only 20% of young children can outgrown their allergy3, 4. In most people with peanut allergy, symptoms develop after ingesting substantially less than 1 peanut, and in highly allergic people trace quantities can induce an allergic reaction2. The anaphylaxis provoked by pean
8、uts is an IgE-mediated type I hypersensitivity reaction. In such reactions, when peanut allergens penetrate mucosal barriers,30they crosslink with the specific IgE antibodies on mast cells and basophils, which results in degranulation of preformed allergic mediators and subsequent cell activation. T
9、hen these cells may secrete various cytokines and chemokines, which recruit other inflammatory cells and contribute to the IgE-mediated allergic response2, 5. The symptoms of peanut allergy include increasing in vascular permeability and smooth muscle contraction that can cause acute urticaria35(hiv
10、es), swelling of the mouth, face, tongue and pharynx, diarrhea, asthma5, 6.Yet to date, strict avoidance is the only effective means to prevent peanut allergy, which can be difficult due to erroneous food labeling, cross-contamination during food processing7, 8. As a result, accidental ingestion of
11、peanut-containing food is not rare and accounts for many deaths each year9. There has been growing recognition of the contributing role of the “hygiene40hypothesis” over decades. According to the “hygiene hypothesis”, an evident decline in microbialFoundations: the National Natural Science Foundatio
12、n of China (No. 31200691), the National Science Fund of Distinguished Young Scholars (No. 31125021), the National High Technology Research and Development Program of China (863 Program No. 2011AA100901, 2011AA100902), the Key program of National Natural Science Foundation of China (No. 20836003), th
13、e National Basic Research Program of China (973 Program No.2012CB720802), the Key projects in the national science & technology pillar program during the twelfth five-yearplan period (No. 2012BAD12B08, 2012BAD28B07), the National Science and Technology Pillar Program (No.2010C0070311), the 111 proje
14、ct B07029, and Fundamental Research Funds for the Central Universities (JUSRP111A31).Brief author introduction:Mengsha Hu, (1988-), female, master, food science and technology. Correspondance author: Wei Chen, (1966-), male, professor, food science and technology. E-mail: - 13 -exposure during early
15、 childhood is one of the most plausible causes of the increasing rates of allergic disease10. The human intestinal microflora represents the most important microbial exposure for the developing infants11. Furthermore, the indigenous microflora of the gastrointestinal tract is required to maintain im
16、mune homeostasis and promoted the maturation of45the hosts immune system12. Consequently, a variety of clinical studies investigated thedifferences of the intestinal microbial composition between allergic and healthy infants. Atopic infants had been shown to be less colonized with bifidobacteria or
17、lactobacilli, particularly during the period preceding the occurrence of atopic disease13, 14. Along these lines, human trials using specific lactic acid bacteria (LAB) strains for oral interventions have pointed out a potential for50prevention of allergic diseases and inspired further research15.LA
18、B are non-pathogenic，non-invasive microorganisms with GRAS (generally regarded as safe) status and have been used in food industry for centuries. Probioticmediated immunomodulation represents an interesting option in the management of food allergy and it was shown that both systemic and mucosal immu
19、ne systems can be modulated by orally delivered55bacteria16, 17. Lactobacillus rhamnosus GG, an extensively studied LAB strain, has been reported to alleviate the symptoms of gastrointestinal inflammation and atopic dermatitis with food allergy18, 19. Moreover, LAB have the capacity to induce immune
20、 tolerance in allergic diseases. Prioult et al. reported that L. paracasei can induce oral tolerance response to bovine -lactoglobulin (BLG) in mice20. Nevertheless, not all candidate probiotics have been proven60equally efficient due to the differences in survival and persistence of the strain in t
21、he gastro-intestinal tract, and strain-specific interactions of the probiotic with the host immune system20, 21. According to the research of de Jonge et al., L. casei Shirota administration shifted the Th1/Th2 ratio towards Th2 dominance in Brown Norway rats sensitized to peanut extract, which enha
22、nced allergic reactions22. Therefore, its particularly important to select a successful65protective strain to reduce allergic reactions. In the present study, the immunomodulatory effects of L. casei Zhang were investigated in a peanut allergy mouse model.1Material and methods1.1AnimalsFour-week-old
23、 female Balb/c mice, specific pathogen-free, were purchased from Slaccas70Laboratory (Shanghai, China). In our experimental animal center, mice were acclimated for 1 week before experiments. All mice were kept in well-controlled animal house, at meantemperature of 23 2, a relative humidity of at lea
24、st 40% and not exceeding 60% and a 12 hlight/dark cycle. The Animal Experimentation Ethics Committee of the Jiangnan University approved all experiments.751.2Bacterial strain and culture conditionL. casei Zhang was kindly provided by Professor Heping Zhang (Inner Mongolia Agricultural University). T
25、he bacterium was cultured in MRS (Man-Rogasa Sharpe) broth at 37 for 1824 h. Cellular pellets were then harvested by centrifugation (6000 g, 10 min at 4) and washed twice with PBS. Then, the pellets were resuspended in saline to a final concentration of 2109 CFU in80200 l (inoculum) and were immedia
26、tely administered to mice (see below).1.3Preparation of crude peanut extract (CPE)The preparation of CPE was performed as previously described16, 23 with some modifications.859095100105110115Peanut were ground into small granules and protein extract was made by blending 100 g peanut with 250 mL 20 m
27、M Tris buffer (pH 7.2). After 2 h stirring at intervals of 20 minutes for a minuteat room temperature, the aqueous fraction was collected by centrifugation (3000 g, 30 min at 4).To remove residual traces of fat and insoluble particles, the aqueous phase was subsequently centrifuged (10000 g, 30 min
28、at 4). Protein concentrations were measured using a Pierce BCA Protein Assay kit (Thermo Scientific, USA) with BSA as a standard. CPE contained 28 mg/mlprotein and was stored at -20. SDS-PAGE of the peanut protein extraction showed protein bands between 14 and approximately 120 kDa (data not shown).
29、1.4Mouse immunization protocolThe immunization protocol was described in Fig. 1. Briefly, 24 5-week-old female Balb/c mice were randomly divided into four groups, named Group 1, 2, 3 and 4. Group 1 was the negative control group, i.e. mice in this group received PBS only during the sensitization and
30、 treatment phases. Group 2 was the positive control group. In this group, oral sensitization with CPE was performed by intragastric dosing of 6 mg of CPE mixed with 10 g of Cholera Toxin (CT) on days 17, 22, 28, 34, 40 and 46. Additionally, mice were received PBS instead of L. casei Zhang in treatme
31、nt phase. Group 3 was the prophylactic group. Prior to the sensitization phase(day 1), mice (5 weeks of age) were intragastrically (i.g.) administered 2109 CFU of L. caseiZhang strain (diluted in 200 L PBS), 4 consecutive days per week. The therapeutic group was Group 4. Treatment with L. casei Zhan
32、g was initiated after the sensitization phase (day 53) in this group. Finally, all mice were orally challenged with 12 mg of CPE alone on day 70.Fig. 1 The immunization protocol. Balb/c mice were randomly divided into 4 groups (n=6). Group 1:unsensitization group; Group 2: CPE-sensitization group, m
33、ice were sensitized i.g. with CPE together with CT over an 4-week period; Group 3: prophylactic group, i.e. L. casei Zhang administration prior to sensitization phase; Group 4: therapeutic group, i.e. L. casei Zhang administration after sensitizations phase. All mice were orally challenged with 12 m
34、g of CPE alone on day 70.1.5Assessment of hypersensitivity reactionsAnaphylactic symptoms were evaluated 30 to 60 minutes after the oral challenge, and the scoring system was established as previously described24: 0, no symptoms; 1, scratching and rubbing around the head and nose; 2, diarrhea, pilar
35、 erect, puffiness around the mouth and eyes, reduced activity and/or decreased activity with increasing respiratory rate; 3, wheezing, labored respiration, cyanosis around the mouth; 4, no activity after prodding, or tremor and convulsion; and 5, death. Scoring of symptoms was executed in a blinded
36、manner.1.6Measurement of serum total IgE and IgG antibodiesAfter evaluation of the anaphylactic reactions, mice were sacrificed immediately, and sera120125130135140145150155were obtained. After clotting and centrifugation, serum samples were collected and stored at 70 until determined. Total IgE and
37、 IgG antibodies were determined using enzyme immunoassay kits (Bethyl Laboratories Montgomery, USA) as described by the manufacturer.1.7Measurement of CPE-specific IgE, IgG1 and IgG2a antibodiesELISA assays were performed as described above. 96-well microtiter plates (Maxisorp Nunc, Roskilde, Denmar
38、k) were coated with 100 l/well CPE (10 g/ml) diluted in 0.05 M sodiumcarbonate buffer (pH 9.5) overnight at 4. After washing with PBS containing 0.05% Tween 20(PBST), plates were blocked 2 h at 37 with 1% BSA diluted in PBST. Serum samples (100l/well) were added in duplicate at the appropriate dilut
39、ion (diluted 1:20 for IgE measurement,1:103 for IgG2a and 1:105 for IgG1) as primary antibodies. After incubation at 37 for 1 h, the plates were washed three times using PBST. For detection of CPE-specific IgE，IgG1 and IgG2a,100 l of the HPR-conjugated goat anti-mouse IgE，IgG1 or IgG2a antibodies (1
40、:6000 dilution; Southern Biotechnologies Associates, USA) were added separately for a further 1 h reaction at 37. Subsequently, plates were extensively washed and solid-phase bound HPR activity wasdetermined by addition of 100 l/well of TMB (3, 3, 5, 5-tetramethylbenzidine) as substrate. Absorbance
41、was then measured at 450 nm.1.8Histamine assay and faecal IgA measurementFaeces were collected during the anaphylactic reaction (3060 min after challenge) on day 70, then pooled and immediately extracted. Briefly, fresh faecal pellets (0.1 g) were suspended in 1 ml PBS. After completely shaking, all
42、 solid material was disrupted and then centrifuged at 7500 g for5 min at 4. The supernatant was stored at -70 for further analysis. According to themanufacturers instructions, histamine and IgA levels were assayed in faecal samples by ELISAkits (MyBioSource, USA for histamine; Bethyl Laboratories, U
43、SA for IgA).1.9Cell culture and cytokine measurementSpleen and mesenteric lymph nodes (MLN) were collected from each group of mice, which had been sacrificed immediately following evaluation of the anaphylactic reactions. Single cell suspensions were prepared and resuspended in RPMI-1640 medium (The
44、rmo Scientific HyClone, USA) containing 10% fetal bovine serum (Gibco, USA), 1% penicillin/streptomycin, and 1%glutamine. They were cultured in 96-well plates (5106 cells/well) in the presence or absence ofCPE (200 g/ml) or lipopolysaccharide (1 g/ml, positive control) for 72 h under 5% CO2 at 37. S
45、upernatants were then collected and aliquots were stored at -70 until analyzed. IL-4, IL-10,IL-12 (p70) and IFN- levels were determined by ELISA kits according to the manufacturers instructions (BD Biosciences Pharmingen, USA). According to the description of Shahid et al.25, the ratio of IFN-/IL-4
46、was used to further assessing the Th1/Th2 balance.1.10 Quantification of CD4+ Foxp3+ Tregs by flow cytometryFresh single cell suspensions from spleen and MLN (1 106 cells) was stained with extracellular CD4 and intracellular Foxp3. Cells were primarily stained with fluoresceinisothiocyanate (FITC)-labeled anti-mouse CD4 antibody (eBioscience, USA) for 30 min at 4 inthe dark condition. Then the samples were fixed, permeabilized, and stained for intracellular antigen. Intracellular staining of Foxp3 was performed using the phycoerythrin (PE)-conjugated Foxp3 antibody