PRESENTATION

Probiotics and the Immune Response

Karen Madsen, PhD

Abstract: Benecial eects exerted by probiotic bacteria in the treatment of human disease may be broadly classied as those eects which arise due to activity in the large intestine and are related to colonization or inhibition of pathogen growth; and those eects which arise in both the small and large intestine, and are related to enhancement of the host immune response and intestinal barrier function. In a strain dependent fashion, probiotic bacteria can enhance intestinal barrier function and modulate signal transduction pathways and gene expression in epithelial and immune cells. Oral administration of live probiotics and bacterial structural components can also dierentially modulate dendritic cells resulting in an increased production of IL-10 and regulatory T cells. Both innate and adaptive immune responses can be modulated by probiotic bacteria. Key words: Lactobacillus, Bidobacteria, epithelium, dendritic cells, Treg cells (J Clin Gastroenterol 2006;40:232234)

robiotic bacteria interact with and inuence all cells within the gut. Mechanisms of probiotic actions include eects on luminal microbial ecology,1 modulation of immune function,2,3 and enhancement of epithelial barrier function.4,5 Probiotic bacteria exhibit host-specic and strain-specic dierences in their actions, and also in their resistance to acid and bile, ability to colonize the gastrointestinal tract, and clinical ecacy. Furthermore, the activity of individual strains of bacteria may be enhanced, or alternatively, eliminated, by the presence of other strains in vivo. Also, eects of probiotic bacteria in vitro may not always translate into similar in vivo behavior.6

PROBIOTICS AND EPITHELIAL CELLS

Selected probiotic strains are able to modulate cytokine release from a variety of cell types, thus inuencing both mucosal and systemic innate and
Received for Publication September 23, 2005; accepted November 16, 2005. From the University of Alberta, 6146 Dentistry Pharmacy Building, Edmonton, AL, Canada T6G 2C2. Supported by Alberta Heritage Foundation for Medical Research, Canadian Institutes for Health Research, Crohns and Colitis Foundation of Canada. Reprints: Dr Karen Madsen, PhD, University of Alberta, 6146 Dentistry Pharmacy Building, Edmonton, AL, Canada T6G 2C2 (e-mail: karen.madsen@ualberta.ca). Copyright r 2006 by Lippincott Williams & Wilkins

adaptive immune function. Probiotic bacteria can interact with epithelial cells and alter cytokine production through modulation of cellular signal transduction pathways.79 Epithelial cells recognize and respond to whole bacteria and bacterial components in a dierential manner, releasing proinammatory cytokines such as interleukin (IL)-8 in response to pathogenic bacteria, although showing no response to probiotic strains.10 Certain probiotic strains can elicit anti-inammatory responses and/or inhibit the nuclear factor-kB pathway in epithelial cells through various mechanisms, including blocking inhibitor kB degradation by inhibiting ubiquination11; by inhibiting proteasome function12; and by regulating nuclear-cytoplasmic movement of RelA through a peroxisome proliferator activated receptorgamma dependent pathway.13 The probiotic Lactobacillus rhamnosus GG can prevent cytokine-induced apoptosis in colon cells by activation of the antiapoptotic Akt and protein kinase B, and inactivation of the propaptoptic p38 mitogen-activated protein kinase signalling pathway.14 Two secreted proteins elicited the same activity as did the live bacteria.14 However, anti-inammatory eects of probiotics are not limited to live bacteria or secreted protein products. Bacterial DNA is also recognized in a dierential manner by epithelial cells; with pathogenic strains evoking a phosphorylation of the extracellular signal-regulated kinase pathway and activation of activator protein-1,15 and probiotic strains modulating the nuclear factor-kB pathway.12 Probiotic bacteria DNA can also suppress systemic inammatory responses to pathogenic bacterial DNA.12 Studies to date have shown a requirement for Toll-like receptor 9 (TLR9) for cellular recognition and response to bacterial DNA.16 Intestinal epithelial cells express several antigenpresenting molecules and costimulatory molecules and play a role in the activation and expansion of both CD4+and CD8+ regulatory T-cell (Treg) subsets.17 Proteasomes have a key role in the degradation of endogenous and exogenous proteins for antigen presentation by both major histocompatability complex class 1 and 2 molecules.17 Probiotic bacteria modulate epithelial cell proteasomal activity,12,18 thus providing a potential mechanism for probiotic-induced alterations in epithelialdriven T-cell activation in the gut. Probiotics can modulate epithelial barrier function,4 possibly through interactions with TLR2.19 TLR2 recognizes bacterial lipoproteins, lipoteichoic acid, and zymosan. Studies have shown that the synthetic bacterial lipopeptide PamcCysSK4, a peptidoglycan, can induce
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activation of protein kinase C and a tightening and sealing of tight junction associated zonula-occludins-1 (ZO-1).20

EFFECTS OF PROBIOTICS ON DENDRITIC CELLS

Dendritic cells (DC) are antigen-presenting cells found throughout the gut that continually sample enteric antigens and present them to naive T cells, resulting in T-cell activation and dierentiation. DC are able to discriminate between dierent microbial strains through the expression of pattern-recognition receptors that recognize specic pathogen-associated molecular patterns.21 Microbes will stimulate DC to acquire signals to drive the development of either TH1, TH2, or Treg cell responses.22 Some probiotic bacteria induce a pattern of maturation of DC characterized by the release of small amounts of tumor necrotic factor a and IL-12, with increased levels of IL-10, and inhibit the generation of proinammatory TH1 cells.23 Bidobacteria in particular induce an upregulation of IL-10 production by DC and decrease expression of the costimulatory molecules, CD80 and CD40. This increase in IL-10 production may act both by having direct anti-inammatory eects and by enhancing the generation of Treg cells. In contrast to the eects of Bidobacteria, some Lactobacilli strains tend to generate a phenotype of DC characterized by increased costimulatory marker expression but low production of proinammatory cytokines.24 Interestingly, the ratio of bacteria/DC seems to aect DC response, suggesting that dierent intracellular signaling pathways are activated when bacteria are present at high doses.25 Overall, probiotic bacteria interactions with DC leads to an immunoregulatory state, rather than aggressive immune responses.

homeostasis within the gut lumen. Further, both innate and adaptive immune responses can be modulated by probiotic bacteria in a strain-dependent and dosedependent fashion through interactions with mucosal DC and intestinal epithelial cells.

REFERENCES
1. Backhed F, Ley RE, Sonnenburg JL, et al. Host-bacterial mutualism in the human intestine. Science. 2005;307:19151920. 2. Pickard KM, Bremner AR, Gordon JN, et al. Microbial-gut interactions in health and disease. Immune responses. Best Pract Res Clin Gastroenterol. 2004;18:271285. 3. Kalliomaki MA, Isolauri E. Probiotics and down-regulation of the allergic response. Immunol Allergy Clin North Am. 2004; 24: 739752,viii. 4. Madsen K, Cornish A, Soper P, et al. Probiotic bacteria enhance murine and human intestinal epithelial barrier function. [comment]. Gastroenterology. 2001;121:580591. 5. Isolauri E, Salminen S. Probiotics, gut inammation and barrier function. Gastroenterol Clin North Am. 2005;34:437450, viii. 6. Ibnou-Zekri N, Blum S, Schirin EJ, et al. Divergent patterns of colonization and immune response elicited from two intestinal Lactobacillus strains that display similar properties in vitro. Infect Immun. 2003; 71:428436. 7. Neish AS. Bacterial inhibition of eukaryotic pro-inammatory pathways. Immunol Res. 2004; 29:175186. 8. Otte JM, Podolsky DK. Functional modulation of enterocytes by gram-positive and gram-negative microorganisms. Am J Physiol Gastrointest Liver Physiol. 2004; 286:G613G626. 9. Ruiz PA, Homann M, Szcesny S, et al. Innate mechanisms for Bidobacterium lactis to activate transient pro-inammatory host responses in intestinal epithelial cells after the colonization of germfree rats. Immunology. 2005;115:441450. 10. Lammers KM, Helwig U, Swennen E, et al. Eect of probiotic strains on interleukin 8 production by HT29/19A cells. Am J Gastroenterol. 2002;97:11821186. 11. Neish AS, Gewirtz AT, Zeng H, et al. Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination. Science. 2000;289:15601563. 12. Jijon H, Backer J, Diaz H, et al. DNA from probiotic bacteria modulates murine and human epithelial and immune function. Gastroenterology. 2004;126:13581373. 13. Kelly D, Campbell JI, King TP, et al. Commensal anaerobic gut bacteria attenuate inammation by regulating nuclear-cytoplasmic shuttling of PPAR-gamma and RelA. Nat Immunol. 2004;5: 104112. 14. Yan F, Polk DB. Probiotic bacterium prevents cytokine-induced apoptosis in intestinal epithelial cells. J Biol Chem. 2002;277: 5095950965. 15. Akhtar M, Watson JL, Nazli A, et al. Bacterial DNA evokes epithelial IL-8 production by a MAPK-dependent, NFkappaBindependent pathway. FASEB J. 2003;17:13971321. 16. Watson JL, McKay DM. The immunophysiological impact of bacterial CpG DNA on the gut. Clin Chim Acta. 2006;364:111. 17. Mukherjee P, Dani A, Bhatia S, et al. Ecient presentation of both cytosolic and endogenous transmembrane protein antigens on MHC class II is dependent on cytoplasmic proteolysis. J Immunol. 2001; 167:26322641. 18. Petrof EO, Kojima K, Ropeleski MJ, et al. Probiotics inhibit nuclear factor-kappaB and induce heat shock proteins in colonic epithelial cells through proteasome inhibition. Gastroenterology. 2004;127:14741487. 19. Kalliomaki MA, Walker WA. Physiologic and pathologic interactions of bacteria with gastrointestinal epithelium. Gastroenterol Clin North Am. 2005;34:383399, vii. 20. Cario E, Gerken G, Podolsky DK. Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C. Gastroenterology. 2004;127:224238.

PROBIOTICS AND TREG CELLS

Treg cells seem to function in the suppression or regulation of eector T-cell function through production of cytokines such as IL-10 and/or transforming growth factor b, or through receptor-ligand interactions such as cytotoxic T-lymphocyte-associated protein-4 (CTLA-4).26 To date, several CD4+ T regulatory subsets have been identied, including Tr1, which produces high levels of IL-1; TH3, which produces transforming growth factor b; and CD4+CD25+ T cells that mediate suppression in a contact-dependent manner through cytotoxic T cellassociated antigen 4.26 Induction of Treg cells by certain probiotic bacterial strains may explain how probiotics given by the subcutaneous route may be eective not only against inammation in the gut, but also in the treatment of arthritis.27

CONCLUSIONS
The commensal bacteria in the gastrointestinal tract are the primary stimulus for the intestinal mucosal immune system and are necessary for normal immune development. Interactions between microbial species and dendritic and epithelial cells are key to the maintenance of
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21. Hart AL, Lammers K, Brigidi P, et al. Modulation of human dendritic cell phenotype and function by probiotic bacteria. Gut. 2004;53:16021609. 22. Kalinski P, Hilkens CM, Wierenga EA et al. T-cell priming by type1 and type-2 polarized dendritic cells: the concept of a third signal. Immunol Today. 1999;20:561567. 23. Drakes M, Blanchard T, Czinn S. Bacterial probiotic modulation of dendritic cells. Infect Immun. 2004;72:32993309. 24. Christensen HR, Frokiaer H, Pestka JJ. Lactobacilli dierentially modulate expression of cytokines and maturation surface markers in murine dendritic cells. J Immunol. 2002;168:171178.

25. Smits HH, Engering A, van der Kleij D, et al. Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specic intercellular adhesion molecule 3-grabbing nonintegrin. J Allergy Clin Immunol. 2005;115:12601267. 26. Paust S, Cantor H. Regulatory T cells and autoimmune disease. Immunol Rev. 2005;204:195207. 27. Sheil B, McCarthy J, OMahony L, et al. Is the mucosal route of administration essential for probiotic function? Subcutaneous administration is associated with attenuation of murine colitis and arthritis. Gut. 2004;53:694700.

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