Consumption of Galactooligosaccharides together with Probiotics Stimulates the In Vitro Peripheral Blood Mononuclear Cell Proliferation and IFNγ Production in Healthy Men

1 Pharmacology, Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 Helsinki, Finland 2 Valio Ltd., R&D, P.O. Box 30, FIN-00039 Helsinki, Finland 3 STAT Consulting, Vahverokatu 6, FIN-37130 Nokia, Finland 4 Immune Response Unit, Department of Vaccination and Immune Protection, National Institute for Health and Welfare, P.O. Box 104, FIN-00251 Helsinki, Finland 5 Folkhälsan Research Center, University of Helsinki, Biomedicum, P.O. Box 63, FIN-00014 Helsinki, Finland


Introduction
The intestine has a dual task to develop tolerance to the dietary antigens and commensal microbiota and to develop immunity against potential pathogens [1].The gut-associated lymphoid tissue consists of organized tissue (Peyer's patches, mesenteric lymph nodes) and diffused tissue (immunoglobulin A-producing plasma cells, mature T cells) and contains the largest mass of immune cells in the hu-man body [1,2].The tolerance and defense systems are not accomplished by intestinal immune system alone, but demand the cooperation between epithelial barrier and gut microbiota [1][2][3].Intestinal microbiota modulates immune system either through pathogen-associated molecular patterns on the surface of microbes, such as lipopolysaccharides and peptidoglycans [2], or through microbial products, such as short-chain fatty acids, interacting with both immune cells and enterocytes [4,5].
Probiotics and prebiotics are potentially important regulators of immune response and have been used in the prevention and treatment of the immune-mediated disorders such as allergies.The criteria of probiotics, (live microorganisms which when administered in adequate amounts confer a health benefit on the host) [6], is fulfilled most of all by several species of lactobacilli and bifidobacteria [7].Prebiotics are defined as "nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or limited number of bacteria in the colon, and thus improve host health" [8].Prebiotics most widely used belong to two chemical groups, galactooligosaccharides (GOSs) and inulin-type fructans [9].Probiotics may affect immune system directly [10] or by modifying microbial environment or the processing of antigens in the gut [11].It is clear that there are differences in immunological effects between probiotic species and strains [12].Prebiotics may modulate the immune system directly through ligation of carbohydrate or pattern recognition receptors on immune and epithelial cells [5,9].More likely, however, prebiotics affect the immune response indirectly by enhancing intestinal fermentation and growth of certain microbes [5,9].
The aim of the present study was to investigate in healthy men the immunomodulatory potential of the four-strain probiotic combination (Lactobacillus rhamnosus GG, L. rhamnosus LC705, Propionibacterium freudenreichii ssp.shermanii JS, and Bifidobacterium breve Bb99) alone and together with GOSs, which stimulate the growth of bifidobacteria and, to a smaller extent, lactobacilli in the colon [13].

2.1.
Participants.This immunological study was performed as a part of an intervention study evaluating the effects of probiotics and prebiotics on serum enterolactone concentration [14].Eighteen healthy Finnish men aged 30-60 years (mean 45 years) volunteered to participate in this study.Gender is known to affect the outcome of the immune response [15], and therefore only men were included.Before entering the study, the participants were interviewed for illness, medication, diet, and smoking.Exclusion criteria included antibiotic treatment for four weeks before the intervention, chronic gastrointestinal diseases, infections of urinary tract with antibiotic treatment, and the use of chemotherapeutics.Two participants used medication for high blood pressure, one for heart disease, one for asthma, and five of the participants smoked.All participants consumed omnivorous diet.The study protocol had been approved by the Ethics Committee of the Foundation for Nutrition Research, Helsinki, Finland, and was carefully explained to the participants, who then gave their written informed consent.

2.2.
Intervention.This immunological study consisted of a 3week pretreatment period and a 4-week intervention period.Intervention consisted of two 2-week periods given in a fixed order: (1) juice with probiotic bacteria (65 mL/day), (2) juice with probiotic bacteria (65 mL/day) and GOS (3.8 g/day).During the study, the subjects were not allowed to eat products containing probiotic bacteria and were instructed not to change their ordinary diet.The probiotic juice (Valio Ltd., Helsinki, Finland) contained two Lactobacillus rhamnosus strains, GG (ATCC 53103) and LC705 (DSM7061), one bifidobacterium strain, Bifidobacterium breve Bb99 (DSM 13692), and one propionibacterium strain, Propionibacterium freudenreichii ssp shermanii JS (DSM7067), total amount 2×10 10 viable bacteria daily.After the probiotic juice period, 10.3 g GOS-syrup (Valio Ltd) was added to probiotic juice.It contained 37% of pure GOS resulting in a daily amount of 3.8 g GOS per subject.

Data Collection.
Heparinized blood samples for this immunological study were collected at the end of the pretreatment period, probiotics period, and probiotics+GOS period.The sampling was performed in the morning and always on the same day of the week.Peripheral blood mononuclear cells (PBMCs) were separated from heparinized blood by Ficoll centrifugation and plasma was collected and stored at −20 • C until analysis.Plasma concentrations of interleukins IL-2, IL-4, IL-5 and IL-10 as well as tumour necrosis factor-α (TNFα) and interferon-γ (IFNγ) were analyzed at baseline as well as at the end of probiotics and probiotics+GOS periods by using a bead-based immunoassay (BD, San Jose, CA, USA).Secretion of IL-4, IL-5, transforming growth factor-β1 (TGFβ1) and IFNγ by PBMC and their proliferation were assessed at baseline and at the end of the probiotic+GOS period, but not at the end of probiotics period.For the cytokine secretion assay, PBMC were stimulated with either phytohemagglutinin (PHA) 5 μg/mL or medium alone in U-bottomed wells (Nunc, Roskilde, Denmark) at the density of 200, 000 cells in 200 μL of RPMI medium (Gibco, Paisley, UK) containing 5% inactivated AB + serum (Finnish Red Cross, Helsinki, Finland) and L-glutamine.The medium was collected after 48 h and the cytokines IL-4, IL-5, TGFβ1, and IFNγ were assessed by ELISA.Commercial kits were used when analyzing IL-4 (PeliKine Compact, CLB, Amsterdam, the Netherlands) and TGFβ1 (Quantikine, R&D Systems, Minneapolis, Minn, USA).IL-5 and IFNγ were analyzed as previously described [16,17].For the proliferation assay, PBMC were cultured at the density 100 000 cells per 200 μL medium either with or without PHA 5 μg/mL for 72 h, the cells were harvested 16 h after adding radioactive thymidine (Amersham, Aylesbury, UK), and the incorporation of radioactive thymidine into DNA (cpm) was measured as previously described [18].

Statistical
Analysis.The Cochran's Q test was used to compare the percentages of positive (above the detection limits) serum cytokines during pretreatment period, probiotic period and probiotic+GOS periods.In case of positivity of 50% or more, the mean concentrations were compared using the ANOVA of repeated measures.The values below the detection limit were first replaced by "detection limit/2."The distributions of serum cytokines were skew to the right and were logarithmically transformed before the analysis.Multiple comparisons were not performed as the global tests were nonsignificant for all cytokines.The Wilcoxon-signed ranks test was used to compare probiotic+GOS to the pretreatment period with respect to spontaneous and PHAstimulated proliferation of PBMC and cytokine production in vitro.The data were analyzed using PASW version 18.0 (SPSS Inc.Chicago, Ill, USA).

Results
All the participants completed the study and none of them used antibiotics during the study.
The proliferation of spontaneous as well as PHA-stimulated PBMC increased during the consumption of pro-biotics+GOS when compared to the baseline (Figure 1).The percentage increase was 133% for the spontaneous proliferation and 104% for the PHA-stimulated.The secretion of IFNγ by PHA-stimulated PBMC increased during the same period (Table 1).The secretions of IL-4, IL-5, and TGFβ1 by PBMC were not significantly affected.Plasma concentrations of IL-2, IL-4, IL-5, IL-10, TNFα, and IFNγ were analyzed at baseline and during the probiotics period and the probiotics+GOS period.No significant chan-ges were found in percentages of positive (above the detection limit) serum cytokines or in mean concentrations of IL-4, IL-5, and IFNγ during the study (Table 2).The concentration of IL-2 was below detection limit in more than 50% of the participants during each period and of IL-10 and TNFα during two periods, and therefore the mean values are not presented.

Discussion
In the present study, proliferation response of PBMC, both spontaneous and mitogen-stimulated, as well as their mitogen-stimulated IFNγ secretion significantly increased during consumption of the probiotic mixture together with GOS in healthy men.The secretion of other cytokines measured was not significantly affected nor were the serum concentrations of any cytokines measured, which indicates a selective response.IFNγ is not only antiviral and antimicrobial agent with a broad spectrum, but also an important regulator of overall inflammatory responses [19].Defective IFNγ production in the infancy has been associated with the development of IgE-mediated allergy, and IFNγ, and other Th1 lymphocytes, may counteract the Th2 responses of allergy [20].In fact, the increase of IFNγ secretion by stimulated PBMC has been correlated with clinical improvement in children with atopic dermatitis or cow's milk allergy [21].This kind of effect could explain the reduced eczema and increased resistance to respiratory infections in infants with high risk for allergy by the same probiotic mixture administered together with GOS as used in the present study [22,23].
The same probiotic mixture as used in the present study (without GOS) did not have a significant effect on IFNγ secretion in stimulated PBMC in earlier studies in infants with atopic dermatitis and cow's milk allergy [17].Similarly, a single-strain consumption of L. rhamnosus GG or Propionibacterium freudenreichii ssp.shermanii did not affect serum IFNγ concentration or IFNγ production by stimulated PBMC in healthy adults [24].In children with atopic dermatitis or cow's milk allergy, L. rhamnosus GG consumption increased IFNγ secretion by stimulated PBMC [17], but it did not affect lymphocyte proliferation in infants [25] nor proliferative responses of stimulated PBMC in supplemented mothers or in their neonates [26].Therefore, the effects on IFNγ production in the present study do not seem to be due to L. rhamnosus GG alone.It is possible that GOSs are needed to enhance the colonization of the probiotic bacteria or the immunomodulatory effects of prebiotics and probiotics are additive, as there is data suggesting they enhance the effects of probiotics on the modulation of intestinal environment [14,27].GOS supplementation alone was reported recently to reduce the days of cold or flu and stress-induced gastrointestinal dysfunction in healthy young adults [28].On the other hand, another prebiotic, a mixture of raftilose and raftiline, did not have any effect on IFNγ production, or other immune responses, in the elderly [29].
There are numerous studies on the effects of different probiotics and their combinations on the immune system in different ages and diseases, and the results are very different from study to study [12].The potentially beneficial effects  of several probiotics on immune system, such as increased IFNγ secretion by stimulated PBMC, have been reported in allergic children [17,21,30,31], whereas their effects in healthy adults are not as often shown: L. casei strain Shirota, L. casei DN-114001 nor B. animalis ssp.lactis Bb12 together with L. paracasei ssp.paracasei CRL-431, have not significantly affected production of IFNγ [32][33][34][35].In a small study in rhinopathic patients, the combination of L. acidophilus and a bifidobacterium strain did not affect PBMC proliferation [36].However, L. acidophilus LAFTI L10 consumption slightly increased IFNγ secretion from T cells in fatigued athletes and IFN-γ concentration in saliva in healthy athletes [37].Our results are supported by a previous study where a consumption of prebiotic inulin enriched with oligofructose in combination with the probiotics L. rhamnosus GG and B. lactis Bb12 increased the capacity of stimulated PBMC to selectively produce IFNγ in resected colon cancer patients [38].The present study design with sequential interventions has disadvantages.The randomized controlled trial (RCT) such as parallel groups with three groups or four groups (i.e., factorial design) or crossover study with randomized order of treatments were not possible for practical reasons.Thus, in the present study, the time-dependent covariates and carryover effects are possible sources of bias and may reduce the differences between treatments.

Conclusions
The consumption of this specific four-strain probiotic combination together with GOS stimulated the in vitro PBMC proliferation and IFNγ production.These effects may be beneficial in states where IFNγ production is insufficient, such as in allergies.

Figure 1 :
Figure 1: Box-blot figures for spontaneous (a) and PHA-stimulated (b) proliferation of PBMC during a pretreatment (run-in) period and during 2 weeks of treatment with the probiotic juice with GOS.n = 18.Wilcoxon signed ranks test.The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile.Whiskers above and below the box indicate the 90th and 10th percentiles.The outliers are given as circles.

Table 1 :
Cytokine production in vitro by PBMC with or without stimulation by PHA during a pretreatment period and 2 weeks of treatment with probiotic juice with GOS.= 18 for all variables except IFNγ spont n = 14.a Wilcoxon signed ranks test.

Table 2 :
Serum cytokines during a pretreatment period, 2 weeks of treatment with probiotic juice, and 2 weeks of treatment with probiotic juice with GOS.
pg/mL.n = 18. a Geometric means are given if at least half of observations are above the detection limit.Values below the detection limit were replaced by "detection limit/2".b Cochran's Q test.c ANOVA for repeated measures using logarithmically (ln) transformed values.