crossmark

Three Novel Candidate Probiotic Strains with Prophylactic Properties in a Murine Model of Cow’s Milk Allergy Elodie Neau,a,b Johanne Delannoy,a Candice Marion,a Charles-Henry Cottart,c,d Chantal Labellie,a Sophie Holowacz,b Marie-José Butel,a Nathalie Kapel,a,e Anne-Judith Waligora-Duprieta EA 4065, Ecosystème Intestinal, Probiotiques, Antibiotiques, Université Paris Descartes, DHU Risques et Grossesse, Paris, Francea; Groupe PiLeJe, Paris, Franceb; AP-HP, G. H. Necker-Enfants Malades, Service de Biochimie A, Paris, Francec; INSERM U1151, Paris, Franced; AP-HP, G. H. Pitié Salpetrière-Charles Foix, Service de Coprologie Fonctionnelle, Paris, Francee

Food allergies can have significant effects on morbidity and on quality of life. Therefore, the development of efficient approaches to reduce the risk of developing food allergies is of considerable interest. The aim of this study was to identify and select probiotic strains with preventive properties against allergies using a combination of in vitro and in vivo approaches. To that end, 31 strains of bifidobacteria and lactic acid bacteria were screened for their immunomodulatory properties in two cellular models, namely, human peripheral blood mononuclear cells (PBMCs) and T helper 2 (Th2)-skewed murine splenocytes. Six strains inducing a high interleukin-10 (IL-10)/IL-12p70 ratio and a low secretion of IL-4 on the two cellular models were selected, and their protective impact was tested in vivo in a murine model of food allergy to ␤-lactoglobulin. Three strains showed a protective impact on sensitization, with a decrease in allergen-specific IgE, and on allergy, with a decrease in mast cell degranulation. Analysis of the impact of these three strains on the T helper balance revealed different mechanisms of action. The Lactobacillus salivarius LA307 strain proved to block Th1 and Th2 responses, while the Bifidobacterium longum subsp. infantis LA308 strain induced a pro-Th1 profile and the Lactobacillus rhamnosus LA305 strain induced pro-Th1 and regulatory responses. These results demonstrate that a combination of in vitro and in vivo screening is effective in probiotic strain selection and allowed identification of three novel probiotic strains that are active against sensitization in mice.

F

ood allergies (FA) are an important public health problem that affects adults and children. They have dramatically and rapidly increased in prevalence in the last several decades, especially in areas with a westernized lifestyle. Despite the associated risk of severe allergic reactions and even death, there is currently no treatment for FA; thus, management of the disease continues to be based on patient education, ensuring strict allergen avoidance, and treatment of symptoms (1). Therefore, the development of new strategies for FA prevention appears to be essential to address this unmet medical need. The increase in the prevalence of allergic diseases over the past 20 to 30 years and the dichotomy in their rates between industrialized and developing countries are two lines of evidence suggesting that environmental changes are a major factor for the development of allergies. Intestinal commensal bacteria and their sequential establishment are known to play a crucial role in the maturation of the intestinal immune system, modulation of the T helper (Th) balance, acquisition of oral tolerance, and maintenance of gut wall epithelial integrity (2, 3). Yet, numerous clinical and experimental studies have reported intestinal dysbiosis in patients with allergic symptoms, with a low diversity and qualitative and/or quantitative differences in the composition of their intestinal microbiota compared to that of healthy individuals (4). One of the factors associated with this delay in microbiota implantation can be related to the hygiene hypothesis. According to the hygiene hypothesis, antibiotic use, widespread vaccination, and exaggerated hygiene delay gut microbiota establishment in infancy, promoting an inadequate stimulation of the immune system, which leads to unrestricted Th2 responses, major factors for the onset of allergies and other associated diseases (5). These relationships between the intestinal microbiota and allergy have contributed to the concept of modulating the intestinal microbiota in

1722

aem.asm.org

order to prevent or manage allergic diseases and support the use of probiotics as antiallergy therapy. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer health benefits on the host” (6). Some of these microorganisms have proved their efficacy in the management of diseases resulting from immune deviation, such as ulcerative colitis and pouchitis (7). One of the most studied groups of probiotics includes Bifidobacterium and lactic acid bacteria (LAB), which include the genera Lactobacillus, Streptococcus, and Lactococcus (4). Numerous preclinical and clinical trials have evaluated their effectiveness in the prevention or treatment of allergic diseases, but results are conflicting. Indeed, some studies showed beneficial effects in the prevention of FA (8), atopic dermatitis (9), and respiratory allergies (10), while others did not (11, 12). Thereby, currently available evidence failed to indicate that probiotic supplementation reduces the risk of developing allergy in children, and the European Food Safety Authority did not deliver favorable opinions following some requests. How-

Received 21 October 2015 Accepted 29 December 2015 Accepted manuscript posted online 4 January 2016 Citation Neau E, Delannoy J, Marion C, Cottart C-H, Labellie C, Holowacz S, Butel M-J, Kapel N, Waligora-Dupriet A-J. 2016. Three novel candidate probiotic strains with prophylactic properties in a murine model of cow’s milk allergy. Appl Environ Microbiol 82:1722–1733. doi:10.1128/AEM.03440-15. Editor: E. G. Dudley, Pennsylvania State University Address correspondence to Anne-Judith Waligora-Dupriet, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.03440-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Applied and Environmental Microbiology

March 2016 Volume 82 Number 6

Candidate Probiotic Strains against Food Allergy

ever, the World Allergy Organization stressed that there was a likely net benefit from using probiotics, resulting primarily from prevention of eczema during pregnancy and breastfeeding and in infants at high risk of developing allergy (8). The discrepancies between studies can be attributed to the differences in populations (stage and type of disease, environment, genetic background), to the variety of probiotics used (strains, dose, duration, and time of administration), and finally to the follow-up period. Moreover, it is to be noted that each strain has unique properties, so the probiotic effect of a specific strain cannot be extrapolated to other strains or to other diseases. Consequently, the selection of the appropriate probiotic strain is an indispensable prerequisite for a successful clinical trial and is critical for the efficacy of the treatment. However, the rationale for the choice of the probiotic strain is usually poorly documented in published studies (4, 13). From this perspective, the development of a simple and predictive screening method to determine the immunomodulatory properties of probiotic strains with antiallergy potential is of critical importance. This type of screening has already shown its benefit in vivo for the selection of a probiotic strain with a protective impact in colitis (14, 15) and in influenza virus infection (16), but it was scarcely applied to FA. In this context, our aim was to select and characterize a candidate probiotic strain with prophylactic properties for allergy from a panel of 31 strains. To this end, we analyzed the in vitro patterns of cytokine production (i) by human peripheral blood mononuclear cells (PBMCs) in order to estimate the type of T helper profile that strains can induce in humans and (ii) by Th2-skewed murine splenocytes after bacterial stimulation to assess the impact of these different strains within an allergic context. Strains with an anti-inflammatory profile (high interleukin-10 [IL-10]/IL-12p70 ratio and low gamma interferon [IFN-␥] secretion) were evaluated in vivo in a mouse model of allergy to ␤-lactoglobulin (BLG) to confirm their protective impact. MATERIALS AND METHODS Bacterial strains and culture conditions. Thirty-one strains, including 21 Lactobacillus species, 6 Bifidobacterium species, 2 Lactococcus species, and 2 Streptococcus species strains provided by PiLeJe Larena Health Group, were studied (Table 1). Lactobacillus and Bifidobacterium strains were cultured under anaerobic conditions (CO2-H2-N2, 10:10:80) at 37°C in de Man, Rogosa, and Sharpe (MRS) medium (Oxoid, Thermo Scientific, Illkirch, France) and in MRS medium supplemented with 0.05% (wt/vol) L-cysteine-hydrochloride monohydrate (Sigma-Aldrich, France), respectively. Lactococcus strains were cultured at 37°C in TGYH medium (tryptone peptone, 30 g liter⫺1; glucose, 5 g liter⫺1; yeast extract, 20 g liter⫺1; and hemin, 5 g liter⫺1). Streptococcus strains were cultured at 42°C in brain heart infusion (BHI) medium (Oxoid, Thermo Scientific, Illkirch, France). Bacterial growth was monitored by determining optical density (OD) at 600 nm. One hour after the beginning of the stationary phase, bacteria were pelleted, washed twice, resuspended in phosphate-buffered saline (PBS; Gibco, Thermo Scientific, Illkirch, France) containing 15% glycerol, and stored at ⫺80°C until used for assays. The number of live bacteria (CFU) was estimated by culture using a Whitley automated spiral plater (WASP; AES-Chemunex, Bruz, France). Three independent growth experiments were performed for each strain. Animals and housing conditions. Female BALB/cByJ mice were purchased at weaning age (21 ⫾ 2 days of life) from Charles River Laboratories (CRL; l’Arbresle, France). Animals were housed in groups of 10 per cage in adapted and enriched cages, including polycarbonate cottage and vegetal

March 2016 Volume 82 Number 6

TABLE 1 Studied strain species and their origins Strain designation

Bacterial species/subspecies

Origin/source

PI1 PI2 PI3 PI4 LA306 PI5 PI6 PI7 PI8 PI9 PI10 PI11 PI12 PI13 PI14 LA305 LA308 PI15 PI16 PI17 PI18 PI19 PI20 LA307 PI21 PI22 PI23 PI24 PI25 PI26 PI27

Lactobacillus acidophilus Lactobacillus salivarius Lactobacillus plantarum Lactobacillus acidophilus Bifidobacterium animalis subsp. lactis Lactobacillus helveticus Lactobacillus helveticus Bifidobacterium animalis subsp. lactis Lactobacillus casei Lactococcus lactis Bifidobacterium longum subsp. longum Lactobacillus acidophilus Lactobacillus casei Lactobacillus helveticus Lactobacillus reuteri Lactobacillus rhamnosus Bifidobacterium longum subsp. infantis Lactobacillus paracasei Lactobacillus rhamnosus Lactobacillus gasseri Lactobacillus paracasei Lactobacillus plantarum Lactobacillus casei Lactobacillus salivarius Streptococcus thermophilus Bifidobacterium bifidum Lactococcus lactis Lactobacillus brevis Bifidobacterium animalis subsp. lactis Lactobacillus casei subsp. paracasei Streptococcus thermophilus

Human Unknown Vegetable Human Human Dairy products Dairy products Dairy products Dairy products Dairy products Dairy products Human Human Dairy products Human Human Human Human Human Human Human Dairy products Human Animal Dairy products Human Undetermined Dairy products Human Human Dairy products

nest, under standard conditions in temperature- and humidity-controlled conventional colony rooms and under reversed 12 h-12 h light-dark cycle. Mice were offered ad libitum intake of tap water and standard rodent pellets that lacked cow’s milk proteins (A03; SAFE, Augy, France). All animal experiments were started after 1 week of acclimation. All procedures were carried out in accordance with European guidelines for the care and use of laboratory animals. The protocol was approved by the Regional Council of Ethics for animal experimentation (Ile de France-Paris Descartes, CEEA34.AJWD.062.12). Experiments were performed in the animal care facilities of CRP2-UMS 3612 CNRS-US25 INSERM-IRD at the Faculté de Pharmacie de Paris, Université Paris Descartes, Paris, France. Human PBMC stimulation. Human PBMCs were extracted from the qualified buffy coat cells of five healthy donors (four males and one female; median age, 36 years [range, 21 to 61 years]; negative for HIV and hepatitis A and B viruses), which were obtained from the Etablissement Français du Sang (agreement no. 12/COCHIN/184). PBMCs were isolated by density Ficoll gradient (Sigma-Aldrich, France) centrifugation (1,400 rpm for 30 min at 20°C, no brake) and were washed twice in Hanks’ balanced salt solution (HBSS; Sigma-Aldrich, France) and adjusted to 1.5 ⫻ 106 cells/ml in Iscove’s modified Dulbecco’s medium (IMDM; Sigma-Aldrich, France) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Scientific, Illkirch, France), 1% L-glutamine, 1% penicillin-streptomycin, and 0.1% gentamicin. The number of viable cells was determined using the trypan blue dye exclusion method and an automated cell counter (TC20; Bio-Rad, Marnes-la-Coquette, France). PBMCs were seeded in 48-well plates at a density of 6.75 ⫻ 105 cells/ well. Bacteria from three independent cultures and adjusted to 108 CFU/ml were added with a cell/bacterium ratio of 1:10 (vol/vol), and

Applied and Environmental Microbiology

aem.asm.org

1723

Neau et al.

FIG 1 Experimental design of the murine model of food allergy to ␤-lactoglobulin (BLG). Three-week-old BALB/cByJ mice were supplemented with a probiotic strain or PBS for 6 weeks. In parallel, mice were orally sensitized with whey protein and cholera toxin (sensitized mice) once a week from day 15 to day 43. Nonsensitized mice were treated with CT alone. One week after the last sensitization, all mice received an oral challenge with BLG. Analysis of impact of supplementation on biological markers of allergy and sensitization and on local and systemic markers was performed on day 50, after the BLG challenge.

plates were incubated for 48 h at 37°C in an atmosphere containing 5% CO2 and 95% air. Culture medium and nonspecific T-cell mitogen concanavalin A (ConA; Sigma-Aldrich, France) at 3 ␮g/ml were used as negative and positive controls, respectively. Plates were stored at ⫺80°C until cytokine analysis. Levels of IL-4, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IFN-␥, and IL-22 were quantified in culture supernatants by Bio-Plex (Bio-Plex Pro human cytokine group 8-plex and Bio-Plex Pro human Th17 cytokine IL-22 set; Bio-Rad, Marnes-la-Coquette, France) according to the manufacturer’s recommendations. Each candidate probiotic strain was thus characterized by the amount of secretion of Th1 (IL-12p70 and IFN-␥), Th2 (IL-4, IL-6, IL-9, and IL-23), Th17 (IL-17A and IL-22), and T regulatory (Treg) (IL-10) cytokines induced on PBMCs. The IL-10/IL-12p70 ratio allows us to distinguish strains with an antiinflammatory profile (potentially regulatory strains) and those with a proinflammatory profile (high versus low ratio, respectively) (14). OVA-primed murine splenocytes. Ovalbumin (OVA)-primed murine splenocytes were used as Th2-skewed T cells. Ten female BALB/cByJ mice (6 weeks old) were intraperitoneally sensitized with ovalbumin (50 ␮g/mouse; Sigma-Aldrich, France) and aluminum hydroxide (alum, 1 mg/mouse; Sigma-Aldrich, France) on days 1 and 8. Mice were euthanized on day 15 by intraperitoneal injection of sodium pentobarbital (Ceva Santé Animale, France). Spleens were removed, pooled by groups of two, and crushed on a 70-␮m nylon filter (Falcon, Dutscher, Brumath, France) to isolate spleen cells. Splenocytes were treated with red blood cell lysing buffer (Sigma-Aldrich, France) to remove erythrocytes. After being washed, extracted cells were resuspended in RPMI 1640 medium (Gibco, Thermo Scientific, Illkirch, France) supplemented with 10% FBS, 1% Lglutamine, 1% penicillin-streptomycin, 0.1% gentamicin, and 0.1% 2-mercaptoethanol. Splenocytes were coincubated for 5 days in 96-well plates (2 ⫻ 105 cells/well) with bacterial strains (4 ⫻ 105 bacteria/well) with a cell/bacterium ratio of 1:2 (vol/vol), either in the absence or in the presence of OVA at 1,000 ␮g/ml. Measurements of IFN-␥, IL-4, IL-10, and IL-12p70 levels in supernatants were done by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Mouse ELISA Ready-SET-Go! kits; eBioscience, San Diego, CA). Mouse model of FA to BLG. Our model was adapted from Li et al. (17). After a 1-week adaptation period, female BALB/cByJ mice were randomly assigned to the control or probiotic group and were housed 10 per cage. Each group was divided into the following 2 subgroups: sensitized (20 animals) and nonsensitized (10 animals). Sensitization was performed by intragastric administration on days 16, 23, 30, 37, and 44 with whey protein (WP; 15 mg/mouse; Lacprodan 80; Arla, Lyon, France) and cholera toxin (CT; 10 ␮g/mouse; List Biological, Campbell, CA) suspended in 100 ␮l of PBS. Nonsensitized mice received CT alone in 100 ␮l of PBS. On day 50, all mice were orally challenged with 60 mg of BLG (Sigma-Aldrich, France) (Fig. 1).

1724

aem.asm.org

Probiotic supplementation. Between days 7 and 48 after reception, mice intragastrically received 109 CFU of each lyophilized bacterial strain (Genibio, Lorp-Sentaraille, France), suspended in 200 ␮l of PBS, 4 times per week. Gavages were performed by two researchers who exchanged cages every 2 days. Each of the six candidate probiotic strains was studied in separate animal experiments. Animals from the control group received 200 ␮l of sterile PBS (Fig. 1). Measurements of plasmatic mouse mast cell protease-1, total IgE, total IgG1, total IgG2a, and BLG-specific antibodies. Plasma from each mouse was collected 45 min after challenge in K3-EDTA tubes and was centrifuged immediately (3,000 rpm for 10 min at 4°C); supernatants were stored at ⫺80°C until used. Plasmatic mouse mast cell protease-1 (mMCP-1)—a mast cell degranulation marker—was measured by ELISA according to the manufacturer’s recommendations (mouse MCPT-1 ELISA Ready-SET-Go!; eBioscience, San Diego, CA). Total IgE, IgG1, and IgG2a were quantified using mouse ELISA ReadySET-Go! kits (eBioscience, San Diego, CA) according to the manufacturer’s instructions. Optimal sample dilutions avoiding assay saturation were determined to be 1/100 for total IgE, 1/40,000 for total IgG1, and 1/1,000 for total IgG2a. Measurements of BLG-specific IgE levels were performed by capturing with rat anti-mouse IgE (Pharmingen, BD Biosciences, Le Pont-de-Claix, France) antibody and by detecting with biotinylated BLG (Pierce, Rockford, IL) and streptavidin-horseradish peroxidase (HRP) (CliniSciences, Nanterre, France) as previously described (18). Samples were diluted 20fold and measured in duplicate, and data were expressed in terms of OD at 450 nm. Levels of anti-BLG IgG1 and IgG2a were determined using BLG as the capture antigen, and goat anti-mouse IgG1 and IgG2a-HRP (Southern Biotech, Birmingham, AL) were labeled as detection antibodies. Samples were diluted 40,500-fold for IgG1 and 50-fold for IgG2a and measured in duplicate, and ODs were read at 450 nm. The IgG1/IgG2a ratio was calculated as a reflection of Th2/Th1 reactivity (19). Cytokine production by BLG-stimulated splenocytes and MLNs. On the day of sacrifice (day 50), spleens and mesenteric lymph nodes (MLNs) were removed and placed in RPMI 1640 medium for culture. Ex vivo culture of splenocytes was performed as described previously (18). MLNs were crushed and filtered through a 70-␮m nylon filter and were resuspended in RPMI 1640 complete tissue culture. Splenocytes and MLN cells were adjusted to 2 ⫻ 106 cells per well and were cultured in 24-well plates with and without 2.5 mg/ml BLG at 37°C in a 5% CO2 and 95% air atmosphere. Supernatants were collected after 48 h of culture and were stored at ⫺80°C until further analysis. Splenocyte and MLN culture supernatant levels of IFN-␥, IL-12p70, IL-4, IL-5, and IL-10 were quantified by Bio-Plex (Pro mouse group I cytokine; Bio-Rad, Marnes-la-Coquette, France). Quantification of Th1/Th2/Th17 and Treg gene expression in the ileum by reverse transcription-quantitative PCR. On the day of sacrifice (day 50), the ileum was removed. Total RNA was isolated from a 2-cm

Applied and Environmental Microbiology

March 2016 Volume 82 Number 6

Candidate Probiotic Strains against Food Allergy

segment devoid of Peyer’s patches using an RNeasy Plus universal minikit (Qiagen, Courtaboeuf, France), according to the manufacturer’s instructions, and was stored at ⫺20°C until used. RNA was treated with DNase I (Invitrogen, Thermo Scientific, Illkirch, France), and first-strand cDNA was synthesized from 500 ng of total RNA with SuperScript II and oligo(dT)12–18 primers (Invitrogen, Thermo Scientific, Illkirch, France). Quantitative real-time PCR (qRTPCR) was performed on an ABI Prism 7900HT sequence detection system (Applied Biosystems). QuantiTect SYBR green and QuantiTect primer assays (Qiagen, Courtaboeuf, France) were used to quantify IFN-␥, IL-10, IL-4, and transforming growth factor ␤ (TGF-␤). TaqMan gene expression assays with TaqMan universal master mix II (Applied Biosystems, Thermo Scientific, Illkirch, France) were used to quantify IL-17A, ROR␥T, Foxp3, T-bet, and Gata3. Dosages were performed in duplicate, and gene expression levels were calculated using the 2⫺⌬⌬CT method (20), where CT is the threshold cycle, with the TATA box (TaqMan) assay as the reference gene. A fold increase in expression was normalized to expression levels in the nonsensitized PBS group. Cecal-microbiota analysis. Total DNA was extracted from 125-mg frozen fecal samples. Samples were resuspended in 125 ␮l of 4 M guanidine thiocyanate (Sigma-Aldrich, France) and in 500 ␮l of 5% N-lauroylsarcosine (Sigma-Aldrich, France), and they were incubated at 70°C for 30 min. A total of 750 ␮l of 0.1-mm-diameter glass beads (Sigma-Aldrich, France) previously sterilized by autoclaving was added, and the tube was shaken at 25 Hz for 10 min in a TissueLyser II (Qiagen, Courtaboeuf, France). The tube was vortexed and centrifuged for 5 min at 13,000 rpm. After recovery of the supernatant, the pellet was washed three times with 200 ␮l of TENP (50 mM Tris, 20 mM EDTA, and 1% polyvinylpolypyrrolidone) and was centrifuged for 5 min at 13,000 rpm. Following phenolchloroform purification, total DNA was precipitated using isopropanol and sodium acetate. Dry pellets were suspended in 50 ␮l of sterile water after a 70% ethanol wash. TaqMan quantitative PCR (qPCR) was used to quantify the total bacterial population and the dominant (⬎1% of fecal bacterial population) bacterial groups and genera, including Clostridium cluster IV (Clostridium leptum group), the Bacteroides/Prevotella group, and Bifidobacterium. qPCR using SYBR green was performed to quantify the Lactobacillus/Leuconostoc/Pediococcus group, Clostridium cluster XIVa (Clostridium coccoides group), Clostridium cluster XI, Clostridium cluster I/II, Staphylococcus, Enterococcus, and Escherichia coli (primers and probes; see Table S1 in the supplemental material). Standard curves were obtained from serial dilutions of a known concentration of plasmid DNA containing a 16S rRNA gene insert from each species or group. The coefficients of correlation between log10 CFU and rRNA gene copy numbers for each species and group were obtained from rrnDB (https://rrndb.umms.med.umich.edu/), which allowed for the calculation of the number of CFU/g of feces. When species or targeted taxonomic groups were not detected, the arbitrary value of 1.5 log10 CFU/g of cecal content was used. Staphylococcus spp. were also quantified using culture on Chapman medium, as previously described in the literature (21). Statistical analysis. For in vitro experiments, results are expressed as the means ⫾ the standard error of the means (SEM). For in vivo experiments, results are expressed as the median (range) and contain a minimum of 10 per group. Results were analyzed using the nonparametric Mann-Whitney U test. Differences were considered to be statistically significant when the P value was ⬍0.05. Data were analyzed using SPSS 20 software (Statistical Package for the Social Sciences; IBM, France).

RESULTS

In vitro screening of bacterial strains. (i) Immunomodulatory capacities of probiotic bacterial strains on human PBMCs. Human PBMCs were used to identify potential immunomodulatory properties of candidate probiotic strains. Despite differences in levels of secretions between donors, the immunomodulatory profile of strains was preserved from one donor to another, and the

March 2016 Volume 82 Number 6

ranking of strains according to their induced secretions was highly reproducible regardless of the donor. Undetectable or very low levels of IL-4, IL-9, IL-17A, and IL-22 were produced by most cultures, whereas every sample produced significant levels of IL-6, IL-10, IL-12p70, and IFN-␥. Cytokine levels displayed a strain-specific pattern. All of the 31 bacterial strains induced IL-10 and IL-12p70 secretions, with concentrations ranging from 26 to 328 pg/ml and from 2 to 98 pg/ml, respectively. For IFN-␥, major variations were observed between strains, covering a large range of cytokine levels from 7 to 2,034 pg/ml. The strains belonging to the Streptococcus and Lactococcus genera were among the most immunostimulatory strains, with high secretions of IFN-␥, IL-10, and IL-12p70. The IL-10/IL-12p70 ratio allowed us to classify strains as having a strong or weak anti-inflammatory profile, with a high or low IL-10/IL-12p70 ratio, respectively (Fig. 2A). Among our panel of 31 candidate probiotic strains, nine strains—including all Lactococcus and Streptococcus strains— had no impact on the IL-10/IL-12p70 ratio. In contrast, 22 strains (all Bifidobacterium and 16 Lactobacillus strains) displayed an increase in the IL-10/IL-12p70 ratio (5- to 22-fold, depending on the strain) compared to that of the culture medium, which can be associated with a regulatory profile. IFN-␥ is considered an inflammatory mediator. Bacteria enhancing its production were considered to have proinflammatory properties and were not chosen for further characterization. Of the 31 candidate strains, 11 strains—five Lactobacillus and the six Bifidobacterium strains— did not induce a significant secretion of IFN-␥ compared to the culture medium, so they did not lead to an immune deviation toward a Th1 profile (Fig. 2B). All of these 11 strains belonged to the 22 strains with a high IL-10/IL-12p70 ratio, so they were selected to test their ability to block the production of IL-4, a cytokine allowing Th2 cell differentiation. (ii) Immunomodulatory capacities of candidate probiotic bacterial strains on Th2-skewed murine splenocytes. In order to evaluate the capacity of these 11 strains to normalize the Th1/Th2 balance, Th2-skewed murine splenocytes derived from OVA-sensitized BALB/c mice, which mimic cells from allergic patients, were cocultured with bacterial strains, and levels of IL-4, IL-10, IL-12p70, and IFN-␥ were determined. None of the 11 strains had a significant impact on the IL-10/ IL-12p70 ratio (data not shown). However, Lactobacillus rhamnosus LA305 significantly decreased IL-4 secretion (P ⬍ 0.05) compared to that in the culture medium, Lactobacillus salivarius LA307 significantly increased IFN-␥ secretion (P ⬍ 0.001) and led to a switch from a Th2 to a Th1 response, and Lactobacillus gasseri PI17 downregulated all cytokine secretions (Fig. 3). The eight other strains had no impact on the Th1/Th2 balance. At the end of the screening process, six strains were selected to validate their potential protective impact in vivo in a murine model of FA: LA307 (inductor of Th1 cytokines), LA305 and PI17 (repressors of Th2 cytokines), Bifidobacterium bifidum PI22, Bifidobacterium animalis subsp. lactis LA306, and Bifidobacterium longum subsp. infantis LA308. The last three strains had no impact on Th2-skewed cells but had a high IL-10/IL-12p70 ratio on PBMCs and a high stability for industrial purposes; i.e., the probiotic lyophilized powder can be handled under controlled conditions (20°C with low humidity) and stored for 2 years in packaging sealed to light and moisture without significant loss of viability (⬍50%) (data not shown).

Applied and Environmental Microbiology

aem.asm.org

1725

Neau et al.

FIG 2 Impact of 31 probiotic candidates on the cytokine production of human PBMCs from five independent donors. PBMCs were stimulated for 2 days with culture medium, with concanavalin A (ConA; 3 ␮g/ml), or with live bacteria (1 ⫻ 108 CFU/ml). Cytokine secretions were determined by Bio-Plex. Results are expressed as means ⫾ SEM. Strains are classified in increasing order according to the value of their IL-10/IL-12p70 ratio (A) and their IFN-␥-induced secretion (B). The six strains selected for the mouse model are represented with a thick border. NS, not significant (indicates no difference from results for the culture medium according to the Mann-Whitney U test).

In vivo effects of selected strains in a murine model of FA to BLG. (i) Impact of probiotic supplementation on mast cell degranulation and BLG-specific antibody responses. The protective impact of the six selected strains was investigated in a murine model of FA to BLG. In nonsensitized groups, plasmatic levels of mMCP-1 and total and BLG-specific IgE, IgG1, and IgG2a anti-

1726

aem.asm.org

bodies were similar to those from the PBS group regardless of the strain. Thus, only one nonsensitized group was represented, belonging to the PBS group. All WP-sensitized groups had significantly higher levels of mMCP-1 (P ⬍ 0.001) than the nonsensitized groups. Mice receiving strain LA306, PI17, or PI22 had the same levels of plasma

Applied and Environmental Microbiology

March 2016 Volume 82 Number 6

Candidate Probiotic Strains against Food Allergy

FIG 3 Impact of the 11 probiotic candidates on cytokine secretion by Th2-skewed murine splenocytes from five independent donors. Th2-skewed murine splenocytes were cultured for 5 days in the presence of ovalbumin (OVA; 1 mg/ml) with culture medium, with concanavalin A (ConA; 3 ␮g/ml), or with live bacteria (2 ⫻ 107 CFU/ml). Cytokine secretions were determined by ELISA. The six strains selected for the mouse model are represented with a thick border. Data are expressed as means ⫾ SEM and were compared to results for the culture medium using the Mann-Whitney U test (*, P ⬍ 0.05; ***, P ⬍ 0.001).

mMCP-1 as the PBS group (median, 770 ng/ml), whereas plasma mMCP-1 was significantly lower (P ⬍ 0.001) in the LA307, LA308, and LA305 groups (the median was reduced by a factor of 1.9, 1.6, and 4.5, respectively), despite a large interindividual variability of responses (Fig. 4). BLG administration significantly increased the plasmatic total and BLG-specific IgE and IgG1 responses (P ⬍ 0.001) in all of the WP-sensitized groups compared with those of the nonsensitized groups. These increases were reduced when mice were supplemented with LA305, LA307, or LA308, with a gradient in response from LA307 to LA305 (Fig. 4). With LA305, the BLG-specific IgE levels were divided by 2.4. Calculation of IgG1/IgG2a levels showed that the PI22, LA305, LA307, and LA308 strains were able to skew the response toward a Th1 response, with a lower IgG1/IgG2a ratio than that of the PBS group (Fig. 4). Furthermore, LA305, LA307, or LA308 administration reduced BLG-specific IgG1/IgG2a ratios to levels similar to those of the nonsensitized groups. This decrease in the three groups was due to a decrease in BLG-specific IgG1 levels and to an increase in BLG-specific IgG2a levels. In contrast, BALB/c mice supplemented with LA306 predominantly generated a Th2 response as revealed by a higher IgG1/IgG2a ratio. (ii) Impact of probiotic supplementation on ex vivo cytokine responses to BLG. LA305, LA307, and LA308 strains demonstrated a decrease of allergic responses in treated mice. We thus evaluated whether this protective impact can be associated with a suppressor effect or a T helper induction at local (MLN cells) and systemic (splenocytes) levels. T-cell cytokine responses induced by probiotic supplementation were evaluated in supernatants of MLN cells and splenocytes isolated from BLG-sensitized mice treated and restimulated ex vivo with or without BLG. Unstimulated MLN cells and splenocytes secreted small amounts of cytokines (data not shown). Secretion of IFN-␥, which is produced by Th1 cells and inhibits

March 2016 Volume 82 Number 6

type 2 effector functions (22), was increased in splenocyte supernatants from the groups administered LA305, LA306, or LA308 (P ⬍ 0.05) (Fig. 5A) but in MLN cell supernatants only from the group given LA306 (P ⬍ 0.001) (Fig. 5B). The same secretion profile was observed at systemic and local levels in the group administered LA306 for IL-12p70, which is the main Th1-inducing cytokine produced by activated macrophages and dendritic cells (22; data not shown). IL-4, which is the major inducer of B-cell switching to IgE production (22), was significantly decreased at systemic and local levels in the groups treated with PI22, LA305, LA307, or LA308 (P ⬍ 0.01) (Fig. 5A and B). The same profile was obtained with IL-5 (data not shown), which potently and specifically stimulates eosinophil production (22). Secretion of IL-10, which suppresses specific T-cell responses (22), was significantly increased at systemic and local levels in the LA305 and LA306 groups (P ⬍ 0.001) and only in splenocyte supernatants for the LA308 group (P ⬍ 0.01). (iii) Impact of probiotic supplementation on Th1/Th2/Th17/ Treg balance at the ileum level. We showed that mice treated with LA305, LA307, or LA308 and challenged with BLG on day 50 displayed lower immunoallergic responses than controls. We thus investigated whether the protective impact of these strains can be linked to a restored T-cell response in the terminal ileum. For that reason, the intestinal profile of the immune response induced by the administration of these three strains was evaluated by analyzing the mRNA expression of ifn-g, il-4, il-10, tgf-b, il-17a, and key regulators for polarization toward Th1 (t-bet), Th2 (gata3), Th17 (ror␥t), and Treg (foxp3) cells in the ileum of sensitized mice. Gene expression levels were normalized to expression levels in the nonsensitized PBS group at day 50 (Fig. 6). Relative levels of mRNA expression of the proinflammatory cytokine ifn-g and the transcription factor of Th1 cells t-bet were not affected by treatment with probiotic strains (data not shown).

Applied and Environmental Microbiology

aem.asm.org

1727

Neau et al.

FIG 4 Impact of probiotic treatment on mast cell degranulation and BLG-specific antibody responses after an oral challenge with ␤-lactoglobulin (BLG). Mice received by gavage a 6-week probiotic treatment or PBS and 5 exposures (1 per week) to CT (diamonds, nonsensitized mice; n ⫽ 10) or whey protein (WP) with CT (circles, sensitized mice; n ⫽ 20 for each group). After BLG challenge, the levels of mouse mast cell protease-1 (mMCP-1) and BLG-specific IgE, IgG1, and IgG2a in plasma were determined by ELISA. Each point represents a mouse, and the central horizontal line represents the median of each group. Data were compared to WP-sensitized mice treated with PBS using the Mann-Whitney U test (**, P ⬍ 0.01; ***, P ⬍ 0.001).

No elicitation of ileal T-cell response was observed in mice supplemented with LA305. Supplementation with the LA308 strain induced a downregulation of the allergen-induced Th2 responses at the ileum level as indicated by the significant 3.5-fold decrease (P ⬍ 0.05) of il-4 expression by comparison with that of the PBS group. This decrease was associated with a significant reduction (P ⬍ 0.01) in the expression of gata3, the transcription factor that controls the differentiation of CD4⫹ T cells into Th2 effector cells. Surprisingly, LA307 and LA308 strains also decreased il-10 expression, despite a significant increase in foxp3 gene expression. Finally, mRNA expression of il-17a was the only one to be modulated by the three probiotic treatments, with a significant 2-fold increase (P ⬍ 0.01) of the expression level compared to that of the PBS group (Fig. 6). This was the only impact of the LA305 strain at the ileum level. This increase was not associated with an increase of ror␥t expression. On the contrary, its expression was even decreased in the LA307 and LA308 groups (P ⬍ 0.01). (iv) Impact of probiotic supplementation on cecal microbiota. All probiotic strains except for the LA306 strain decreased bacterial load (see Fig. S1 in the supplemental material). However, no specific profile was associated with a beneficial impact on allergy for any of the evaluated strains. The LA307 and LA308 strains

1728

aem.asm.org

induced a large reduction in E. coli, Clostridium cluster I/II, and Clostridium cluster XI levels, associated with a drastic impact on enterococcal levels for LA307 compared to those of the other strains. In contrast, LA305 and LA306 strains induced an increase in Clostridium cluster IVa. Moreover, the LA305 strain also increased staphylococci, not only in DNA levels but also in diversity, with a mean of 3.3 ⫾ 0.33 types of colony in the LA305 group compared to 1.3 ⫾ 0.33 in the PBS group (P ⬍ 0.05). Staphylococci identified in the LA305 group included Staphylococcus aureus but also different colonies of coagulase-negative staphylococci. Bifidobacteria were scarcely detected, even in mice supplemented with Bifidobacterium strains (data not shown). DISCUSSION

In the present study, we described a combination of in vitro and in vivo screenings that allowed us to select three candidate probiotic strains (L. rhamnosus LA305, L. salivarius LA307, and B. longum subsp. infantis LA308) that were able to prevent sensitization and provide protection against cow’s milk allergy. We first ranked the 31 bacterial strains with reference to the IL-10/IL-12p70 cytokine ratio induced on human PBMCs after bacterial stimulation. This ratio is predictive of a protective anti-

Applied and Environmental Microbiology

March 2016 Volume 82 Number 6

Candidate Probiotic Strains against Food Allergy

FIG 5 Impact of probiotic treatment on ex vivo cytokine secretion of ␤-lactoglobulin (BLG)-stimulated splenocytes (A) and mesenteric lymph node (MLN) cells (B). Mice received by gavage a 6-week probiotic treatment or PBS and 5 exposures (1 per week) to whey protein (WP) with CT (n ⫽ 20 for each group). MLN cells and splenocytes from WP-sensitized mice were cultured for 2 days in the presence of BLG (2.5 mg/ml). Cytokines were measured in culture supernatants by Bio-Plex. Each point represents a mouse, and the central horizontal line represents the median of each group. Data were compared to WP-sensitized mice treated with PBS using the Mann-Whitney U test (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).

inflammatory effect, as previously described in a model of experimental colitis and in a model of influenza virus infection (14, 16). We confirmed a strong strain-specific impact on in vitro cytokine responses of human PBMCs to bacterial strains, as observed previously (14, 23–25), except for Bifidobacterium strains, which all displayed the same regulatory profile, with high IL-10 secretion and low IL-12 secretion levels. These results are in line with the

March 2016 Volume 82 Number 6

study of Latvala et al., which showed that IL-10/IL-12 ratios were higher with Bifidobacterium strains than with Lactobacillus strains (26). All of our 11 selected strains had a high IL-10/IL-12p70 ratio, so they may reduce the development of allergy by enhancing regulatory mechanisms and thus counteract allergic sensitization (27). The use of the Th2-skewed model confirmed the ability of three bacterial strains to interfere with Th2 cytokine production to

Applied and Environmental Microbiology

aem.asm.org

1729

Neau et al.

FIG 6 Impact of probiotic treatment on the local profile of the immune response. Ileal gene mRNA expression in WP-sensitized mice was quantified by qRT-PCR and was normalized to expression levels in the nonsensitized PBS group. The box plots show the median (central horizontal line), the 25th percentile (lower box border), and the 75th percentile (upper box border). The lower and upper horizontal lines show the 10th and 90th percentiles, respectively. Data were compared to those for WP-sensitized mice treated with PBS using the Mann-Whitney U test (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).

restore the T helper balance. Lactobacillus salivarius LA307 showed a potential ability to restore the T helper balance by stimulating Th1 cell development via IFN-␥ induction, while Lactobacillus rhamnosus LA305 and Lactobacillus gasseri PI17 were able to suppress Th2 responses. Such a selection process has been efficiently performed by Fujiwara et al., who showed that the oral administration of a strong IL-12-inducing strain on OVA-sensitized BALB/c splenocytes repressed IgE synthesis in the OVA-sensitized mouse model of allergy (28). The Th2-skewed PBMC model has also been successfully used for probiotic selection (19).

1730

aem.asm.org

These two in vitro models allowed us to select 3 Lactobacillus strains. Three Bifidobacterium strains from the PBMC selection were added to the in vivo screening because of their high IL-10/IL12p70 ratio, associated with a high industrial stability. The potential protective impact of the six selected strains was investigated in a BALB/c model of IgE-mediated hypersensitivity to cow’s milk. This model was chosen because it displays patterns of allergy similar to human patterns in terms of IgE reactivity and it induces homogenous sensitization to allergens (29). Three out of the six selected strains displayed a protective impact on sensiti-

Applied and Environmental Microbiology

March 2016 Volume 82 Number 6

Candidate Probiotic Strains against Food Allergy

zation (IgG1, IgE, IL-4, and IL-5) and on allergy (mMCP-1 release). Supplementation with L. rhamnosus LA305, L. salivarius LA307, and B. longum subsp. infantis LA308 strains led to a significant decrease in plasmatic IgE and IgG1 titers and in mMCP-1 upon challenge with BLG. In accordance with these results, we showed that LA305, LA307, and LA308 strains blocked the secretion of cytokines known to be involved in allergy pathogenesis, allowing a major inhibitory effect on IL-4 and IL-5 secretion. Furthermore, we demonstrated that the consumption of LA305 or LA308 allowed the induction of the tolerance-related biomarkers IgG2a, IFN-␥, and IL-10, which suggests a skewing of the immune response toward Th1 and the induction of a regulatory response. This skewing may in part explain the low levels of specific IgE and IgG1 and mMCP-1 in the plasma (30) and the inhibition of the development of Th2 cells and, therefore, play a protective role against allergies. In the literature, the use of probiotic strains as providers of pro-Th1 and Treg immune signals appeared to be a promising tool in antiallergy therapy. For example, the L. rhamnosus LGG strain, which induces IFN-␥ and IL-10 production, alleviated skin symptoms in IgE-sensitized infants with atopic dermatitis (31, 32). In contrast, the LA307 strain showed a discrete cytokine profile in MLNs and splenocytes, with a low production of Th1, Th2, and Treg cytokines. Holvoet et al. suggested that such low-key cytokine patterns induced by probiotics may be associated with a homeostatic effect and may be an alternative cytokine profile for clinical success (33). To elucidate the mechanisms of action of these three probiotic strains, we studied their impact on the T-cell balance in the terminal ileum of sensitized mice, which contains many lymphoid structures, allowing an active communication between probiotic strains and the immune system. Mice supplemented with LA307 or LA308 presented an anti-Th2 profile in accordance with the protective impact observed in vivo, with a significant decrease in expression of gata3, a key molecule regulating the balance in the ratio of Th1 cells to Th2 cells, which is thought to be indicative of the pathogenesis of allergic diseases (34). These two strains also induced an ileal expression of foxp3, a main regulator for the development and the function of Treg, as its absence in specific knockout mice is responsible for massive lymphoproliferation and for a severe autoimmune syndrome (35). Indeed, Treg cells are essential for the induction and maintenance of immune tolerance by preventing Th2 induction and cytokine release (36) through the release of IL-10 and TGF-␤. The average level of foxp3 gene expression is significantly lower in allergic children than in healthy children (37). Moreover, Foxp3 may have a suppressive role in the anaphylactic response, controlling the degranulation of mast cells (38). Our results therefore suggest a link between foxp3 gene expression in the ileum and protection against food allergy. In our study, we observed a decrease in il-10 and an increase in il-17a expression for the two strains. These results suggest the induction of IL-17⫹ Foxp3⫹ Treg cells, a subpopulation of CD4⫹ CD25⫹ Foxp3⫹ Treg cells of the lamina propria of the small intestine (39), which may play a critical role in antimicrobial defense while controlling autoimmunity and inflammation by producing IL-17A and IL-21 (40). The IL-17A cytokine has a controversial role, but recently, two populations of Th17 cells were defined: the steady-state Th17 cells, which contribute to tolerance and host defense, and the pathogenic Th17 cells, involved in the development of inflammation (41). In our

March 2016 Volume 82 Number 6

model, the increase of il-17 ileum expression in the LA305, LA307, and LA308 groups is associated with a protective impact in allergy and may be linked to a stimulation of the steadystate Th17 cells. This activation of the Th17 pathway in a proTh2 environment may also contribute to the restoration of the T helper balance. Epidemiological studies have illustrated that gut microbiota plays a crucial role in the maintenance of immune homeostasis and influences the occurrence of later diseases (microbial programming) (42). This association between dysbiosis and allergy led us to examine the impact of supplementation on cecal microbiota composition. The results regarding the microbiota have not allowed us to associate a given modulation with the benefit of a probiotic strain. The LA305 and LA306 strains increased the levels of Clostridium cluster IV, a group known for its anti-inflammatory impact (43). The LA305 strain increased Staphylococcus spp., a genus associated with a healthy status and whose colonization has been correlated with less severity in the allergic response in our previous study (18). Therefore, it may be linked to a protective effect against allergy or it may be an indicator of the preservation of the microbial diversity, which is reduced in the first week of life in children who will develop atopic eczema (44). LA307 and LA308 strains decreased the bacterial groups that are often associated with allergy, such as enterobacteria and Clostridium cluster XI (42). High-throughput sequencing might help identify specific elements in the microbiota associated with a beneficial impact. Thereby, probiotic action may be indirectly mediated by microbiota and, in particular, by the short-chain fatty acid (SCFA) production by clostridia and bifidobacteria. It has been described that SCFAs were able to induce Foxp3⫹ colonic regulatory T cell frequency and number, promoting colonic homeostasis (45). Moreover, clostridial colonization has been associated with IL-22 production by ROR␥t⫹ innate lymphoid cells (ILCs) and T cells in the intestinal lamina propria, reducing the uptake of orally administered dietary antigen into the systemic circulation and thus contributing to protection against sensitization (46). On the whole, PBMC-based assay and Th2-skewed murine splenocytes serve as useful primary indicators. The Th2-skewed model appears to be a complementary tool of the PBMC model for the selection of candidate probiotic strains, providing clinical relevance. Indeed, among the three strains with a significant impact in the Th2-biased model, two strains had their beneficial impact confirmed in vivo. However, these two cellular models are not sufficient to select the most promising candidate probiotic strain. The in vivo model still needs to be implemented, as it allows the study of mechanisms of action of probiotics, which are known to be multifactorial, including modulation of the host’s microbiota, modulation of the immune system, and also improvement of the barrier function of the gut mucosa. Indeed, probiotic strains may also act in parallel on intestinal permeability, which is increased in allergic children (47). Nebot-Vivinus et al. showed that the multispecies probiotic Lactibiane Tolerance increased occluding and ZO-1 expression to prevent the disruption of the epithelial barrier (48), which protects the interaction between enteric bacteria and metabolically associated components that can trigger an immune response (49). This mechanism of action remains to be studied for our three preventive strains. To conclude, 3 strains out of 31 were selected for their protective impact in our allergy model. L. salivarius LA307 was rather immunosuppressive, with no impact on IFN-␥ and IL-10 produc-

Applied and Environmental Microbiology

aem.asm.org

1731

Neau et al.

tion, and might induce Treg cells. B. longum subsp. infantis LA308 increased Th1 cytokines, which inhibit the proliferation of Th2 cells, while L. rhamnosus LA305 had a pro-Th1 and a regulatory response. The activation of these different pathways suggests a restoration of the T helper balance, which may explain the protective impact of these strains. ACKNOWLEDGMENT We thank Iharilalao Dubail and her team from Animal Platform CRP2, Faculté de Pharmacie de Paris, for technical support in animal experiments.

FUNDING INFORMATION PiLeJe Larena Health Group provided funding to Elodie Neau. This work was also cofunded by the Association Nationale de la Recherche et de la Technologie (ANRT) (grant number 2012/0551). Sophie Holowacz is an employee of PiLeJe Larena Health Group. The funder PiLeJe had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

REFERENCES 1. Dupont C. 2011. Food allergy: recent advances in pathophysiology and diagnosis. Ann Nutr Metab 59(Suppl):S8 –S18. 2. Chistiakov DA, Bobryshev YV, Kozarov E, Sobenin IA, Orekhov AN. 2015. Intestinal mucosal tolerance and impact of gut microbiota to mucosal tolerance. Front Microbiol 5:781. 3. Slack E, Hapfelmeier S, Stecher B, Velykoredko Y, Stoel M, Lawson MA, Geuking MB, Beutler B, Tedder TF, Hardt WD, Bercik P, Verdu EF, McCoy KD, Macpherson AJ. 2009. Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism. Science 325: 617– 620. http://dx.doi.org/10.1126/science.1172747. 4. Waligora-Dupriet AJ, Butel MJ. 2012. Microbiota and allergy: from dysbiosis to probiotics, p 413– 434. In Pereira C (ed), Allergic diseases— highlights in the clinic, mechanisms and treatment. InTech, Rijeka, Croatia. http: //www.intechopen.com/books/allergic-diseaseshighlights-in-the-clinic -mechanisms-and-treatment/microbiota-and-allergy-from-dysbiosis-to -probiotics. 5. Gordon BR. 2011. The allergic march: can we prevent allergies and asthma? Otolaryngol Clin North Am 44:765–777. http://dx.doi.org/10 .1016/j.otc.2011.03.006. 6. FAO/WHO Working Group. 2002. Guidelines for the evaluation of probiotics in food. FAO/WHO, London, Ontario, Canada. 7. Shen J, Zuo ZX, Mao AP. 2014. Effect of probiotics on inducing remission and maintaining therapy in ulcerative colitis, Crohn’s disease, and pouchitis: meta-analysis of randomized controlled trials. Inflamm Bowel Dis 20:21–35. http://dx.doi.org/10.1097/01.MIB .0000437495.30052.be. 8. Fiocchi A, Pawankar R, Cuello-Garcia C, Ahn K, Al-Hammadi S, Agarwal A, Beyer K, Burks W, Canonica GW, Ebisawa M, Gandhi S, Kamenwa R, Lee BW, Li H, Prescott S, Riva JJ, Rosenwasser L, Sampson H, Spigler M, Terracciano L, Vereda-Ortiz A, Waserman S, YepesNunez JJ, Brozek JL, Schunemann HJ. 2015. World Allergy Organization-McMaster University guidelines for allergic disease prevention (GLAD-P): probiotics. World Allergy Organ J 8:4. http://dx.doi.org/10 .1186/s40413-015-0055-2. 9. Kim HJ, Kim HY, Lee SY, Seo JH, Lee E, Hong SJ. 2013. Clinical efficacy and mechanism of probiotics in allergic diseases. Korean J Pediatr 56:369 – 376. http://dx.doi.org/10.3345/kjp.2013.56.9.369. 10. Kuitunen M. 2013. Probiotics and prebiotics in preventing food allergy and eczema. Curr Opin Allergy Clin Immunol 13:280 –286. 11. Folster-Holst R, Muller F, Schnopp N, Abeck D, Kreiselmaier I, Lenz T, von Rüden U, Schrezenmeir J, Christophers E, Weichenthal M. 2006. Prospective, randomized controlled trial on Lactobacillus rhamnosus in infants with moderate to severe atopic dermatitis. Br J Dermatol 155: 1256 –1261. http://dx.doi.org/10.1111/j.1365-2133.2006.07558.x. 12. Helin T, Haahtela S, Haahtela T. 2002. No effect of oral treatment with an intestinal bacterial strain, Lactobacillus rhamnosus (ATCC 53103), on birch-pollen allergy: a placebo-controlled double-blind study. Allergy 57: 243–246. http://dx.doi.org/10.1034/j.1398-9995.2002.1s3299.x.

1732

aem.asm.org

13. Castellazzi AM, Valsecchi C, Caimmi S, Licari A, Marseglia A, Leoni MC, Caimmi D, Miraglia del Giudice M, Leonardi S, La Rosa M, Marseglia GL. 2013. Probiotics and food allergy. Ital J Pediatr 39:47. http: //dx.doi.org/10.1186/1824-7288-39-47. 14. Foligne B, Nutten S, Grangette C, Dennin V, Goudercourt D, Poiret S, Dewulf J, Brassart D, Mercenier A, Pot B. 2007. Correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria. World J Gastroenterol 13:236 –243. http://dx.doi.org/10.3748/wjg.v13.i2 .236. 15. Santos Rocha C, Lakhdari O, Blottiere HM, Blugeon S, Sokol H, Bermudez-Humaran LG, Azevedo V, Miyoshi A, Dore J, Langella P, Maguin E, van de Guchte M. 2012. Anti-inflammatory properties of dairy lactobacilli. Inflamm Bowel Dis 18:657– 666. http://dx.doi.org/10 .1002/ibd.21834. 16. Kechaou N, Chain F, Gratadoux JJ, Blugeon S, Bertho N, Chevalier C, Le Goffic R, Courau S, Molimard P, Chatel JM, Langella P, BermudezHumaran LG. 2013. Identification of one novel candidate probiotic Lactobacillus plantarum strain active against influenza virus infection in mice by a large-scale screening. Appl Environ Microbiol 79:1491–1499. http: //dx.doi.org/10.1128/AEM.03075-12. 17. Li XM, Schofield BH, Huang CK, Kleiner GI, Sampson HA. 1999. A murine model of IgE-mediated cow’s milk hypersensitivity. J Allergy Clin Immunol 103:206 –214. http://dx.doi.org/10.1016/S0091-6749(99)70492-6. 18. Rodriguez B, Prioult G, Bibiloni R, Nicolis I, Mercenier A, Butel MJ, Waligora-Dupriet AJ. 2011. Germ-free status and altered caecal subdominant microbiota are associated with a high susceptibility to cow’s milk allergy in mice. FEMS Microbiol Ecol 76:133–144. http://dx.doi.org /10.1111/j.1574-6941.2010.01035.x. 19. Du C, Nilsson S, Lu H, Yin J, Jiang N, Wahlgren M, Chen Q. 2010. Immunogenicity of the Plasmodium falciparum Pf332-DBL domain in combination with different adjuvants. Vaccine 28:4977– 4983. http://dx .doi.org/10.1016/j.vaccine.2010.05.027. 20. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2⫺⌬⌬CT method. Methods 25: 402– 408. http://dx.doi.org/10.1006/meth.2001.1262. 21. Waligora-Dupriet AJ, Campeotto F, Nicolis I, Bonet A, Soulaines P, Dupont C, Butel MJ. 2007. Effect of oligofructose supplementation on gut microflora and well-being in young children attending a day care centre. Int J Food Microbiol 113:108 –113. http://dx.doi.org/10.1016/j .ijfoodmicro.2006.07.009. 22. Akdis M, Burgler S, Crameri R, Eiwegger T, Fujita H, Gomez E, Klunker S, Meyer N, O’Mahony L, Palomares O, Rhyner C, Ouaked N, Schaffartzik A, Van De Veen W, Zeller S, Zimmermann M, Akdis CA. 2011. Interleukins, from 1 to 37, and interferon-gamma: receptors, functions, and roles in diseases. J Allergy Clin Immunol 127:701–721. http://dx .doi.org/10.1016/j.jaci.2010.11.050. 23. Meijerink M, Wells JM, Taverne N, de Zeeuw Brouwer ML, Hilhorst B, Venema K, van Bilsen J. 2012. Immunomodulatory effects of potential probiotics in a mouse peanut sensitization model. FEMS Immunol Med Microbiol 65:488 – 496. http://dx.doi.org/10.1111/j.1574 -695X.2012.00981.x. 24. Niers LE, Timmerman HM, Rijkers GT, van Bleek GM, van Uden NO, Knol EF, Kapsenberg ML, Kimpen JL, Hoekstra MO. 2005. Identification of strong interleukin-10 inducing lactic acid bacteria which downregulate T helper type 2 cytokines. Clin Exp Allergy 35:1481–1489. http: //dx.doi.org/10.1111/j.1365-2222.2005.02375.x. 25. Menard O, Butel MJ, Gaboriau-Routhiau V, Waligora-Dupriet AJ. 2008. Gnotobiotic mouse immune response induced by Bifidobacterium sp. strains isolated from infants. Appl Environ Microbiol 74:660 – 666. http://dx.doi.org/10.1128/AEM.01261-07. 26. Latvala S, Miettinen M, Kekkonen RA, Korpela R, Julkunen I. 2011. Lactobacillus rhamnosus GG and Streptococcus thermophilus induce suppressor of cytokine signalling 3 (SOCS3) gene expression directly and indirectly via interleukin-10 in human primary macrophages. Clin Exp Immunol 165:94 –103. http://dx.doi.org/10.1111/j.1365-2249.2011.04408.x. 27. Kwon HK, Lee CG, So JS, Chae CS, Hwang JS, Sahoo A, Nam JH, Rhee JH, Hwang KC, Im SH. 2010. Generation of regulatory dendritic cells and CD4⫹Foxp3⫹ T cells by probiotics administration suppresses immune disorders. Proc Natl Acad Sci U S A 107:2159 –2164. http://dx.doi.org/10 .1073/pnas.0904055107. 28. Fujiwara D, Inoue S, Wakabayashi H, Fujii T. 2004. The anti-allergic effects of lactic acid bacteria are strain dependent and mediated by effects

Applied and Environmental Microbiology

March 2016 Volume 82 Number 6

Candidate Probiotic Strains against Food Allergy

29.

30. 31.

32. 33.

34.

35. 36. 37.

38.

39.

on both Th1/Th2 cytokine expression and balance. Int Arch Allergy Immunol 135:205–215. http://dx.doi.org/10.1159/000081305. Neau E, Butel MJ, Waligora-Dupriet AJ. 2015. Probiotics and allergic diseases— usefulness of animal models—from strains selection to mechanistic studies, p 125–151. In Pereira C (ed), Allergic diseases—new insights. InTech, Rijeka, Croatia. http://www.intechopen.com/books/allergic-diseases-new -insights/probiotics-and-allergic-diseases-usefulness-of-animal-models -from-strains-selection-to-mechanistic-s. Akdis CA, Akdis M. 2009. Mechanisms and treatment of allergic disease in the big picture of regulatory T cells. J Allergy Clin Immunol 123:735– 746. http://dx.doi.org/10.1016/j.jaci.2009.02.030. Pohjavuori E, Viljanen M, Korpela R, Kuitunen M, Tiittanen M, Vaarala O, Savilahti E. 2004. Lactobacillus GG effect in increasing IFNgamma production in infants with cow’s milk allergy. J Allergy Clin Immunol 114:131–136. http://dx.doi.org/10.1016/j.jaci.2004.03.036. Pessi T, Sutas Y, Hurme M, Isolauri E. 2000. Interleukin-10 generation in atopic children following oral Lactobacillus rhamnosus GG. Clin Exp Allergy 30:1804 –1808. http://dx.doi.org/10.1046/j.1365-2222.2000.00948.x. Holvoet S, Zuercher AW, Julien-Javaux F, Perrot M, Mercenier A. 2013. Characterization of candidate anti-allergic probiotic strains in a model of Th2-skewed human peripheral blood mononuclear cells. Int Arch Allergy Immunol 161:142–154. http://dx.doi.org/10.1159/000343703. Bae CJ, Lee JW, Shim SB, Jee SW, Lee SH, Woo JM, Lee CK, Hwang DY. 2011. GATA binding protein 3 overexpression and suppression significantly contribute to the regulation of allergic skin inflammation. Int J Mol Med 28:171–179. Pellerin L, Jenks JA, Begin P, Bacchetta R, Nadeau KC. 2014. Regulatory T cells and their roles in immune dysregulation and allergy. Immunol Res 58:358 –368. http://dx.doi.org/10.1007/s12026-014-8512-5. Cottrez F, Hurst SD, Coffman RL, Groux H. 2000. T regulatory cells 1 inhibit a Th2-specific response in vivo. J Immunol 165:4848 – 4853. http: //dx.doi.org/10.4049/jimmunol.165.9.4848. Krogulska A, Polakowska E, Wasowska-Krolikowska K, Malachowska B, Mlynarski W, Borowiec M. 2015. Decreased FOXP3 mRNA expression in children with atopic asthma and IgE-mediated food allergy. Ann Allergy Asthma Immunol 115:415– 421. http://dx.doi.org/10.1016/j.anai .2015.08.015. Gri G, Piconese S, Frossi B, Manfroi V, Merluzzi S, Tripodo C, Viola A, Odom S, Rivera J, Colombo MP, Pucillo CE. 2008. CD4⫹CD25⫹ regulatory T cells suppress mast cell degranulation and allergic responses through OX40-OX40L interaction. Immunity 29:771–781. http://dx.doi .org/10.1016/j.immuni.2008.08.018. Zhou L, Lopes JE, Chong MM, Ivanov II, Min R, Victora GD, Shen Y, Du J, Rubtsov YP, Rudensky AY, Ziegler SF, Littman DR. 2008. TGFbeta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature 453:236 –240. http://dx.doi.org/10.1038 /nature06878.

March 2016 Volume 82 Number 6

40. Voo KS, Wang YH, Santori FR, Boggiano C, Wang YH, Arima K, Bover L, Hanabuchi S, Khalili J, Marinova E, Zheng B, Littman DR, Liu YJ. 2009. Identification of IL-17-producing FOXP3⫹ regulatory T cells in humans. Proc Natl Acad Sci U S A 106:4793– 4798. http://dx.doi.org/10.1073 /pnas.0900408106. 41. Tanabe S. 2013. The effect of probiotics and gut microbiota on Th17 cells. Int Rev Immunol 32:511–525. http://dx.doi.org/10.3109/08830185.2013 .839665. 42. Melli LC, do Carmo-Rodrigues MS, Araújo-Filho HB, Sole D, de Morais MB. 15 May 2015. Intestinal microbiota and allergic diseases: a systematic review. Allergol Immunopathol (Madr) http://dx.doi.org/10 .1016/j.aller.2015.01.013. 43. Sokol H, Pigneur B, Watterlot L, Lakhdari O, Bermudez-Humaran LG, Gratadoux JJ, Blugeon S, Bridonneau C, Furet JP, Corthier G, Grangette C, Vasquez N, Pochart P, Trugnan G, Thomas G, Blottiere HM, Dore J, Marteau P, Seksik P, Langella P. 2008. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci U S A 105:16731–16736. http://dx.doi.org/10.1073/pnas.0804812105. 44. Ismail IH, Oppedisano F, Joseph SJ, Boyle RJ, Licciardi PV, RobinsBrowne RM, Tang ML. 2012. Reduced gut microbial diversity in early life is associated with later development of eczema but not atopy in high-risk infants. Pediatr Allergy Immunol 23:674 – 681. http://dx.doi.org/10.1111 /j.1399-3038.2012.01328.x. 45. Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly Y, Glickman JN, Garrett WS. 2013. The microbial metabolites, shortchain fatty acids, regulate colonic Treg cell homeostasis. Science 341:569 – 573. http://dx.doi.org/10.1126/science.1241165. 46. Stefka AT, Feehley T, Tripathi P, Qiu J, McCoy K, Mazmanian SK, Tjota MY, Seo GY, Cao S, Theriault BR, Antonopoulos DA, Zhou L, Chang EB, Fu YX, Nagler CR. 2014. Commensal bacteria protect against food allergen sensitization. Proc Natl Acad Sci U S A 111:13145–13150. http://dx.doi.org/10.1073/pnas.1412008111. 47. Kalach N, Kapel N, Waligora-Dupriet AJ, Castelain MC, Cousin MO, Sauvage C, Ba F, Nicolis I, Campeotto F, Butel MJ, Dupont C. 2013. Intestinal permeability and fecal eosinophil-derived neurotoxin are the best diagnosis tools for digestive non-IgE-mediated cow’s milk allergy in toddlers. Clin Chem Lab Med 51:351–361. 48. Nebot-Vivinus M, Harkat C, Bzioueche H, Cartier C, Plichon-Dainese R, Moussa L, Eutamene H, Pishvaie D, Holowacz S, Seyrig C, Piche T, Theodorou V. 2014. Multispecies probiotic protects gut barrier function in experimental models. World J Gastroenterol 20:6832– 6843. http://dx .doi.org/10.3748/wjg.v20.i22.6832. 49. Groschwitz KR, Hogan SP. 2009. Intestinal barrier function: molecular regulation and disease pathogenesis. J Allergy Clin Immunol 124:3–20. http://dx.doi.org/10.1016/j.jaci.2009.05.038.

Applied and Environmental Microbiology

aem.asm.org

1733