Experimental immunology
Effect of Lactobacillus rhamnosus GG encapsulation on interleukin 10 release in vitro

Introduction

Using functional food products serves as a preventive measure in development of several diseases, including immune system disorders. Currently functional food is very popular due to consumers demand and consciousness. Probiotic bacteria belong to the components which give the food products unique properties. Several studies proved that probiotics can have immunomodulating effect by increasing the synthesis of some antibodies, T and B lymphocytes, macrophages and NK cells in intestinal tract [1]. It was determined that Lactobacillus GG can decrease the expression of symptoms of alimentary allergy and atopic dermatitis development by stimulation of Th3 lymphocytes, that are able to release IL-10 and TGF- [2-4]. This is caused by colonization of the intestines by Lactobacillus GG interacting with immune system mainly through cell membrane structures and exopolysaccharide. In available literature there are no data concerning the effect of only Lactobacillus GG metabolites on immune system, but there are reports showing the possibility of causing bacteriemia in people with extremely reduced immunity. Probiotic bacteria are considered as safe, but in case of extreme low immunity, as any other ‘safe’ microorganism, they can become the cause of infection. It should be stressed that the frequency of infections caused by lactic acid bacteria is incomparably lower than infections caused by other Gram-positive and Gram-negative comensal microflora colonizing human organism. Infections caused by Lactobacillus strains were reported e.g. after liver transplants, chirurgical surgeries, long-term antibiotic therapies or chemotherapy [5, 6].

Taking the above aspects into consideration it seemed justified to enclose the bacteria in alginate microcapsules. The lack of direct contact with bacteria can, on one hand, protect immunosuppressed consumer from the infection and, on the other hand, can reduce the possibility of bacterial phagocytosis, hence facilitating longer survival of probiotic bacteria in the organism.

Previous reports on the effect of alginate on immunity, which suggested its immunomodulating activity [7] had significant influence on the materials selected for this study. The aim of this work was to determine the effect of Lactobacillus GG metabolites on IL-10 release. Therefore the concentrations of IL-10 from lymphocyte cultures stimulated by alginate, Lactobacillus GG metabolites and Lactobacillus GG immobilized in alginate microcapsules were compared in vitro.

Material and methods

Bacterial cell cultures



For the analyses Lactobacillus GG ATCC 53103 lyophilisate was used from London ATCC cultures collection. Lactobacillus GG lyophilisate was suspended in MRS broth (according to De Man Rogosa Sharpe) (Oxoid CM0359). The bacteria were cultured for 24 h at 36.6°C until the medium was turbid.



Microcapsules preparation



The cultured bacteria were immobilized in alginate microcapsules obtained by precipitation of polymers from sodium alginate solution through calcium cations instillation. In this study water soluble 1.5% sodium alginate was used, obtained from Natura-Sweet Kelco Company (USA), known commercially as Keltone HV, with viscous average molecular mass Mn-440 000. Prepared 1.5% sodium alginate in physiological salt solution was filtered with sterile Millex GP filtration sets. Filters contained regenerated cellulose membranes with pore size 0.22 μm. Alginate solution was combined with previously cultured bacteria. Obtained 0.7% solution (bacteria + alginate) was instilled into 1% CaCl2 in physiological salt solution during 20 minutes. During microcapsule formation CaCl2 in physiological salt solution was mixed with magnetic stirrer. Rapid jellification took place due to the presence of calcium ions. A syringe pump SEP 11S & SEP21S, SEP11 Model (ASCOR S.A.) was used in order to obtain microcapsules of repeatable shapes. Microcapsules were rinsed three times with sterile physiological salt solution to accurately rinse out the solvent and Lactobacillus GG cells adhering to the outer surface of the capsules. Obtained microcapsules had spherical shapes.

The factors which had the direct influence on formation of microcapsules of desired spherical shape were: rotation speed of magnetic stirrer – 500 rpm, the distance of needle tip from the surface of CaCl2 solution (in physiological salt solution) – ca. 3 cm, and application of appropriate instillation pressure – 40 ml/h. Obtained microcapsules were subjected to lyophilization.



Assessment of microcapsules tightness



Lyophilized microcapsules were again suspended in MRS broth. During 14 days samples were collected and preparations were observed by scanning microscope Joel JSM 6100 under a magnification of 10 000× (Fig. 1).

The purpose of cell culture establishment was to examine the immunomodulating effects of Lactobacillus GG metabolites. Firstly 5 ml of venous blood was obtained from 10 adult volunteers. The blood was poured into test tubes containing 150 units of heparin (Sigma) in 0.5 ml of 0.9% NaCl.

Afterwards the media for lymphocytes cultures were prepared.

Composition of 250 ml medium:

• 20% of inactivated calf’s serum (Biomed, Lublin),

• 80% of RPMI 1640 medium with L-glutamine and NaHCO3 (Biomed, Lublin),

• 5 μg of phytohemagglutinin PHA-P (Sigma),

• 100 μg of streptomycin in 2.5 ml of 0.9% NaCl,

• 100 U of penicillin in 2.5 ml of 0.9% NaCl.

The complete medium of pH 7.2-7.4 was filtered on sterile Millex GP sets. Five ml of culture medium was poured into 50 sterile test tubes (5 ml each) adding 0.1 ml of the whole peripheral blood. The test tubes were divided into five groups.

Group I (control): consisted only of lymphocytes in culture medium.

Group II: lymphocytes + the addition of 0.005 mg of empty alginate capsules.

Group III: lymphocytes + 0.5 ml of supernatant with metabolites from the 24 h culture of Lactobacillus GG in MRS (7 in McFarland scale).

Group IV: lymphocytes + contained the addition of 0.5 ml liquid MRS medium.

Group V: lymphocytes + 0.005 mg of lyophilized microcapsules containing Lactobacillus GG.

The cultures were incubated at 36.6°C for 5 days, slightly shaken daily. After 5 days the test tubes were centrifuged, 2000 rpm, for 5 minutes at 4°C. Supernatants were collected from the cultures, frozen at –20°C and stored for further study to determine cytokines.

ELISA analyzes were performed using OptEIA kit (Becton Dickinson and Company USA (cat. No 550613). The kit included polystyrene plate coated with monoclonal antibodies against IL-10 and reagents. 100 μl of serum or standards from 7.8 to 500 pg/ml for IL-10 were added to the wells and plates were incubated for 2 hours at room temperature. Then the wells were washed five times with automatic washer EL50 (BIO-TEK). Next, monoclonal antibodies against IL-10 coupled with horseradish peroxidase were added and incubated for 1 h at room temperature. After washing each well seven times tetramethylbenzidine was added and samples were incubated for 30 min in the dark. Colour formation reaction was stopped by adding the STOP solution. The absorbance was read at 450 nm wavelength by multichannel reader ELISA-ELx808 (BIO-TEK). The accuracy of the method was > 2 pg/ml. The analyses were performed in duplicates.



Statistical analysis



Analyses were performed using Statistica 7.1 (Statsoft). To determine the statistically significant differences of concentrations between the analyzed groups, non-parametric Wilcoxon match-pairs ranks test was used [8, 9], which is a non-parametric alternative of t-test for correlated samples.

Results and discussion

The concentrations of IL-10 in cell cultures in vitro were analyzed using ELISA and the resulting arithmetical means are presented in Table 1. The highest values (188.39 pg/ml) of IL-10 concentration were obtained for Group V, where lymphocytes were stimulated with lyophilized microcapsules containing Lactobacillus GG. It was observed that MRS broth (IV Group) also stimulated IL-10 release at the concentration of 62.68 pg/ml. The addition of supernatant culture stimulated the release of cytokine in concentration of 46.33 pg/ml. In Group II, where empty alginate capsules were used, the this value amounted to 23.98 pg/ml, as compared to the control group of non-stimulated lymphocytes culture – 26.58 pg/ml.

Using non-parametric Wilcoxon match-pairs ranks test the statistically significant differences in IL-10 concentrations between the groups V and I, II, II, IV and between group II and IV were determined (Fig. 2, solid lines). The lack of statistically significant differences, however, was observed between the groups I and II (Fig. 2, dashed line).

Therefore, the statistically significant increase of IL-10 release was determined under the influence of lyophilized in alginate capsules bacterial cells of Lactobacillus GG, as compared to the control and other groups. Enclosing the bacteria in microcapsules limited the presentation of cell membrane antigens (peptidoglycan) of Lactobacillus GG to immune response cells. The latter were mainly macro­phages and lymphocytes present in cell cultures. Thus the potential stimulants were exopolysaccharide released by bacteria and bacterial metabolites including nitrogen oxide and hydrogen peroxide, supporting the effect of lactic acid [10-13].

It is known that orally admitted Lactobacillus GG in people activates Th3 lymphocytes to the synthesis of the cytokines TGF- and IL-10, which keep the Th1/Th2 balance. Regulating Th3 lymphocytes inhibit pro-inflammatory and allergic response [1, 14]. The studies of Pessi et al. and Pochard and Pohjavuori confirmed the favourable influence of this strain on the expression of atopic dermatitis and food allergy symptoms because Lactobacillus GG activated IL-10 release [15-17], which in turn inhibited the synthesis of pro-inflammatory cytokines (IL-2 and IL-3). On the other hand it was proved that inappropriate IL-10 concentration can lead to numerous disorders – too high concentration can increase susceptibility to viral infections (e.g. HIV), but too low can intensify inflammatory reactions [18]. Interleukin 10 released in high concentrations exhibits immunomodulating effect to a large extent [19-23]. Moon et al. showed significant favourable influence of Lactobacillus GG in chronic colitis treatment [24]. The results of many studies confirm synergistic effect of sodium alginate through the increase of T lymphocytes and macrophages proliferation, thus the use of alginate as a stimulant of immune response seemed to be promising in clinical use [25-29].

In our study and with reference to increased IL-10 release in vitro the use of microcapsules containing Lactobacillus GG can be considered as a components of special food for people with immune disorders. The results justify further studies on the possibilities of the use of Lactobacillus GG metabolites in vivo as an immunomodulator for localized treatment, bringing the balance between the Th1 and Th2 lymphocytes and safe for humans.

Conclusions

1. In cultures of lymphocytes from whole blood stimulated with metabolites released by Lactobacillus GG immobilized in alginate microcapsules a high, statistically significant increase of IL-10 concentration was observed.

2. Lactobacillus GG enclosed in alginate capsules did not form biofilm on the outer surface of the membrane during 14 days of observations.

3. No influence of sodium alginate on the concentration of released IL-10 in lymphocyte cultures was observed.

References

 1. Madaliński K, Szajewska H (2004): Probiotics: mechanism of action, immunomodulation and potential use in alimentary tract diseases. Zakażenia 5: 43-48 [article in Polish].

 2. Kirjavainen PV, Apostolou E, Salminen SJ, Isolauri E (2001): New aspect of probiotics – a novel approach in the management of food allergy. Allergy 6: 1-6.

 3. Nowak K, Śliżewska K, Libudzisz Z (2010): Probiotics – history and mechanisms of action. ŻYWNOŚĆ. Nauka. Technologia. Jakość 4: 5-19 [article in Polish].

 4. Nowak K, Śliżewska K, Libudzisz Z, Socha J (2010): Probiotics – health effects. ŻYWNOŚĆ. Nauka. Technologia. Jakość 4: 20-36 [article in Polish].

 5. De Groote MA, Frank DN, Dowell E, et al. (2005): Lactobacillus rhamnosus GG bacteremia associated with probiotic use in a child with short gut syndrome. Pediatr Infect Dis J 24: 278-280.

 6. Land MH, Rouster-Stevens K, Woods CR, et al. (2005): Lactobacillus sepsis associated with probiotic therapy. Pediatrics 115: 178-181.

 7. Stahl B, M’Rabet L, Vos Arjan P, et al. (2004): Immunomodulating oligosaccharides. 25.10.2004 Nr pat. 06111971.5 PL/EP 1733730 [in Polish].

 8. Dobosz M (2001): Computer aided statistical analysis of research data. Akademicka Oficyna Wydawnicza EXIT, Warszawa [in Polish].

 9. Stanisz A (1998): Accessible course in statistics based on STATISTICA software exemplified with medical data. Tom I. StatSoft INc, Kraków [in Polish].

10. Avonts L, Van Uytven E, De Vuyst L (2004): Cell growth and bacteriocin production of probiotic Lactobacillus strains in differnet media. Int Dairy J 14: 947-955.

11. Martirosian G (1993): Activity of probiotics on selected anaerobic bacteria of food tract. Postępy w Medycynie Zakażeń 11: 741-743 [article in Polish].

12. Salminen S, Wright A (1993): Lactic acid bacteria. Dekker, New York.

13. Ciszek-Lenda M, Nowak B, Śróttek M, et al. (2011): Immunoregulatory potential of exsopolysaccharide from Lactobacillus rhamnosus KL37. Effects on the production of inflammatory mediators by mouse macrophages. Centr Eur

J Immunol 36: 51-55.

14. Cukrowska B, Czarnowska E (2007): Effect of probiotics on immune system. Zakażenia 1: 59-64 [article in Polish].

15. Pessi T, Sütas Y, Hurme M, Isolauri E (2000): Interleukin-10 generation in atopic children following oral Lactobacillus rhamnosus GG. Clin Exp Allergy 30: 1804-1808.

16. Pochard P, Gosset P, Grangette C, et al. (2002): Lactic acid bacteria inhibit TH2 cytokine production by mononuclear cells from allergic patients. J Allergy Clin Immunol 32: 563-570.

17. Pohjavuori E, Viljanen M, Korpela R, et al. (2004): Lactobacillus GG effect in increasing IFN-gamma production in infants with cow’s milk allergy. J Allergy Clin Immunol 114: 131-136.

18. Chung EY, Liu J, Homma Y (2007): Interleukin-10 expression in macrophages during phagocytosis of apoptotic cells is mediated by homeodomain proteins Pbx1 and Prep-1. Immunity 27: 952-964.

19. Drakes M, Blanchard T, Czinn S (2004): Bacterial probiotic modulation of dendritic cells. Infect Immun 72: 3299-3309.

20. Hermann C, Spreitzer I, Schröder NW, et al. (2002): Cytokine induction by purified lipoteichoic acids from various bacterial species – role of LBP, sCD14, CD14 and failure to increase IL-12 and subsequent IFN-gamma release. Eur J Immunol 32: 541-551.

21. Howard M, O’Garra A (1992): Biological properties of interleukin 10. Immunol Today 13: 198-200.

22. Marcinkiewicz J (2005): Effect of probiotics on immune system. Zakażenia 3: 37-40 [article in Polish].

23. Matsuguchi T, Takagi A, Matsuzaki T, et al. (2003): Lipoteichoic acids from Lactobacillus strains elicit strong tumor necrosis factor alpha –inducing activities in macrophages through toll-like receptor 2. Clin Diagn Lab Immunol 3: 259-266.

24. Moon G, Myung SJ, Jeong JY, et al. (2004): Prophylactic effect of Lactobacillus GG in animal colitis and its effect on cytokine secretion and mucin gene expressions. Korean J Gastroenterol 43: 234-245.

25. Arya V, Gupta VK (2011): A review on marine immunomodulators. Int J Pharm Life Sci 5: 751-758.

26. Harikrishnan R, Kim MC, Kim JS, et al. (2011): Immunomodulatory effect of sodium alginate enriched diet in kelp grouper Epinephelus brneus against Streptococcus iniae. Fish Shellfish Immunol 30: 543-549.

27. Huang R, Lee H (2005): Immunological properties of the marine brown alga endarachne binghamiae (Phaeophyceae). Int J Applied Sci Engineering 3: 167-173.

28. Otterlei M, Ostgaard K, Skja°k-Braek G, et al. (1991): Induction of cytokine production from human monocytes stimulated with alginate. J Immunother 10: 286-291.

29. Shan BE, Yoshida Y, Kuroda E, Yamashita U (1999): Immunomodulating activity of seaweed extract on human lymphocytes in vitro. Int J Immunopharmacol 21: 59-70.