Digestive and Liver Disease 38 Suppl. 2 (2006) S256–S260 www.elsevier.com/locate/dld
Bacteria and mucosal immunity Giovanni Monteleone*, Ilaria Peluso, Daniele Fina, Roberta Caruso, Fabio Andrei, Claudio Tosti, Francesco Pallone Department of Internal Medicine, Gastroenterology Unit, Tor Vergata University, Rome, Italy
Abstract In normal individuals, the intestine is a site of intense immunological activity due to the continuous stimulation by luminal antigens mostly derived from the normal bacterial ﬂora. This is reﬂected in the huge amount of IgA produced in the gut and the abundant T cells in the lamina propria and epithelium. It is also becoming clear that products of the normal ﬂora may regulate the cytokine environment within the inductive sites of the mucosal immune responses, such as the Peyer’s patches of the small bowel. Thus normal ﬂora could either negatively or positively regulate speciﬁc immune responses by dictating the proﬁle of locally released cytokines. For example, it is known that in Crohn’s disease the antigens that drive the strongly polarized Th1 tissue-damaging response are derived from the normal bacteria ﬂora. Emerging evidence also indicates that gut microﬂora can contribute to maintain the mucosal homeostasis by promoting the generation and/or expansion of counter-regulatory mechanisms. © 2006 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Bacterial ﬂora; Crohn’s disease; IgA; Mucosal immune response; T-cells
1. Introduction Human beings harbour a complex and abundant bacterial ﬂora. Most of these microbes reside in the intestine. Indeed, the distal ileum and colon are host to over 400 species of bacteria comprising 1014 organisms, that feed on the food we ingest and multiply. Gut ﬂora has many effects on host gene expression, and these are likely to represent the net effect of the outcomes of multiple interactions between the host cells and the different bacterial species. Crosstalk occurs not only between microbial ﬂora and epithelium but also between bacteria and immune system. Indeed, luminal bacteria represent a major stimulus for the development of the host’s gut mucosal immune system. This is reﬂected in the huge amount of IgA produced in the gut and the abundant T cells in the lamina propria and epithelium . No studies to date have however shown that IgA is involved in protecting against the normal ﬂora, and systemic infections with gut bacteria are not seen in IgA-deﬁcient animals or humans. Similarly, mice without T or B cells survive with a normal ﬂora as long as they are kept in a speciﬁc pathogen-free environment, and there is little evidence of excessive penetration and persistence of normal ﬂora in systemic tissues. However, lymphocytesdeﬁcient mice succumb rapidly when exposed to even low* Corresponding author. Giovanni Monteleone. Dipartimento di Medicina Interna, Universit`a Tor Vergata, Via Montpellier, 1, 00133 Rome, Italy. Tel.: +39 06 72596158; fax: +39 06 72596391. E-mail address: [email protected]
grade pathogens, thus supporting the role of gut bacterial commensals in sustaining lymphocyte-mediated immunity against pathogens.
2. Normal bacteria ﬂora elicits the development of the mucosal immune system That commensal bacteria interact with and inﬂuence the mucosal immune system was originally supposed by observations made with germ-free mice. Investigators realized that these animals differed in many aspects of the mucosal immune system from their conventional counterparts with bacteria ﬂora. One of the most striking differences was seen in the Peyer’s patches (PP) and in the lamina propria. Those tissues in conventional animals are prominent and contain high numbers of activated/ memory mononuclear cells, whereas in germ-free animals PP are very small with no germinal centers, and very low numbers of IgA-making plasma cells are seen in the lamina propria. Germ-free mice also have markedly fewer lamina propria CD4+ T cells and intraepithelial abTCR CD8+ cells than normal mice [1–3]. Germ-free mice can be colonized by one or more species of bacteria, thus becoming ‘gnotobiotes’. This approach has revealed that members of the autochthonous ﬂora can inﬂuence speciﬁc immune responses. For example, colonization of germ-free mice with the commensal organism Morganella morganii leads to a prompt increase in IgA production . Similarly,
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introduction of Bacteroides thetaiotaomicron, a member of the murine and human intestinal ﬂora, in germ-free mice again results in an inﬂux of IgA-making plasma cells into the lamina propria along with enhanced expression of the polymeric immunoglobulin receptor . Association of germ-free mice with an unculturable segmented ﬁlamentous bacterium (SFB) has been shown to cause an up-regulation of MHC class II molecules on intestinal cells, and to increase the number of intraepithelial lymphocytes . In line with these ﬁndings, studies in human babies at birth, when we are essentially germ-free, have shown that the lymphoid tissue in the gut is large, but there are no IgA plasma cells and only primary B-cell follicles. However, expansion of Ig-secreting plasma cells and germinal centers occurs within a few weeks, likely as a result of the active interaction between bacterial ﬂora and immune system . Studies in both human and murine systems also indicate that some commensal gut bacteria stimulate the appearance of IgA plasma cells in gut lamina propria of formerly germfree mice better than others. For example, the differentiation of IgA-secreting plasma cells is more sustained in germfree mice mono-associated with Listeria monocytogenes actA in comparison to animals colonized with Morganella morganii or Helicobacter muridarum . Consistent with these observations, infants colonized with Bacillus fragilis were found to have more IgA-and IgM-secreting cells in peripheral blood in comparison to controls . Finally, studies in gnotobiotic mice have shown that mixtures of isolated commensals are more effective in inducing the development of IgA plasma cells than single gut bacteria . There is considerably less information concerning the role of gut microﬂora in the development and activation of the elements of the cellular immune responses. Colonization of germ-free mice with SFB or E. coli results in a marked increase in Natural Killer cells in the intraepithelial (IEL) spaces. Similarly, after colonization of germ-free mice with gut commensal bacteria, the proportion of a/b TCR+, Thy-1+, and a/b CD8+ T cells in the IEL compartment increases, even if it still unknown how bacterial colonizers promote the accumulation, survival and activation of these cells . In normal individuals, CD4+ T gut lamina propria lymphocytes (LPL) do not proliferate following co-culture with sonicates of the endogenous microbial ﬂora . However, studies in germ-free mice colonized with SFB have documented a shift of CD45RBhigh cells, indicative of na¨ıve or unprimed T cells, towards the CD45RBlow phenotype, suggesting a nonspeciﬁc role for bacterial ﬂora in activating CD4+ T cells . Consistently studies in patients with inﬂammatory bowel disease (IBD) and animal models of IBD have documented commensal bacterial product-driven proliferation and cytokine production by gut CD4+ LPL [11–13]. In general, these T-cell responses appear to be dependent on MHC class II molecules and can be inhibited in vitro with speciﬁc anti-class II antibody.
3. The inﬂuence of normal ﬂora on the activity of immune cells Immune cells express receptors for bacterial products/ components, the so-called Toll-like receptors (TLRs). TLRs are characterized by multiple leucine-rich repeats, a membrane-spanning domain, and a cytosolic domain. TLRs associate with the adaptor protein MyD88 (myeloid differentiation protein) which triggers a signaling pathway that leads to the activation of the transcription factor NF-úB . Interaction between commensal bacteria and gut immune cells can occur at 3 distinct sites (Fig. 1). First, this crosstalk occurs at the PP level, where a specialized epithelial cell in the follicle-associated epithelium (FAE), called M cell, accomplishes the translocation of bacterial antigens from the gut lumen to the tissue beneath the FAE. At this level, immature myeloid dendritic cells (DC) encounter and process the antigen, becoming mature DC capable of driving a T-cell response. DC may present antigen locally to T cells, migrate to T-cell zones or to mesenteric lymp nodes, or interact with memory B cells . Luminal bacterial antigens can also enter into the mucosal tissue through an alternative M cell-independent pathway that is mediated by lamina propria DC, that are able to sample the luminal contents by extending intraepithelial dendrites . Lamina propria DC sampling involves both pathogenic and nonpathogenic bacteria, and requires the expression of the chemokine receptor CX3CR1, as DC from mice lacking this receptor are normally recruited to the gut but are unable to produce dendrites . Another inductive site on the mucosal immune responses is represented by the isolated lymphoid follicle (ILF), which contains a single B-cell follicle, as well as DC and small numbers of T cells. In contrast to PP, ILFs do not develop during fetal life but form after birth, when intestine is colonized by bacteria . Expression of TLRs on DC at each of these inductive sites of gut immune response has not yet been reported, but studies in other systems indicate that distinct subsets of DC express particular repertoires of TLRs. For example, myeloid DC, that are contained in PP, express TLR2 and TL4 and produce IL-12 in response to TLR signaling. In contrast, plasmacytoid DC, that are also present in PP, express TLR9 and produce IFN-a [18–20]. These ﬁndings clearly suggest that in response to bacteria stimulation immune cells can produce cytokines, which then inﬂuence the type and duration of the immune response. Molecular analysis of the cytokine proﬁle in PP has provided conﬂicting data between humans and mice, and this may be related to the intestinal ﬂora, which is markedly different between the two species. While a predominant production of IL-10 and TGF-b1 has been documented in mouse PP, isolation of human PP T cells and analysis of their cytokine pattern has revealed the predominance of IFN-g [21–24].
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Fig. 1. Diagrammatic illustration of sites where normal ﬂora can interact with gut immune cells. Bacteria can cross-talk with both epithelial cells (1) or immune cells. This latter phenomenon can occur at: (2) the Peyer’s patches of the small bowel; (3) the lamina propria where dendritic cells (DC) sample the luminal content by extending intraepithelial dendrites; and (4) the isolated lymphoid follicles.
Consistently, high production of IL-12, the major Th1inducing factor, and constitutive activation of the Th1associated transcription factors, Stat4 and T-bet, have been documented in human PP [25,26]. Luminal bacteria can also use TLR-independent pathways to activate resident immune cells. Recently, Mazmanian et al.  have shown that mono-colonization of germfree mice with Bacteroides fragilis (BF), an ubiquitous Gram-negative anaerobe that colonizes the mammalian lower gastrointestinal tract, results in CD4+ T-cell expansion. The immunomodulatory effects of BF require the presence of PSA, a capsular polysaccharide, that is taken up by CD11c+ DC. This leads to the production of IL-12 and expansion of Th1 cells. The exact mechanisms by which DC handle PSA and activate T cells remains however unknown.
4. Effects of gut microﬂora on immune tolerance Evidence has been accumulating that gut commensal bacteria may help maintain the mucosal homeostasis. This can rely on the ability of microﬂora to inﬂuence the activity of both immune and epithelial cells. In this context, it was recently shown that an avirulent Salmonella strain inhibits the in vitro synthesis of inﬂammatory cytokines in human intestinal epithelial cells. This has been linked to the ability of Salmonella to prevent the degradation of IkBa, and to block the activation of NF-úB and the expression of NF-úB-dependent inﬂammatory genes . Additionally, Kelly et al.  showed that Bacteroides thetaiotaomicron
can restrict NF-úB activity in intestinal epithelial cell lines by accelerating the nuclear export of the transcription factor. Studies in mice have also suggested that gut microﬂora may inﬂuence the generation and expansion of counterregulatory mechanisms. In particular, studies in models of IBD induced by selective transfer of naive T cells into SCID recipients have shown that bacterial commensals are crucial in promoting the development and/or maintenance of regulatory cells that limit the ongoing mucosal inﬂammation . Similarly, feeding probiotics in mice has been shown to be effective in inhibiting acute ﬂare-ups induced by repeated rectal administrations of the haptenating reagent, 2,4,6-trinitrobenzene sulfonic acid. The protective effect of probiotics is associated with enhanced expansion of IL-10-producing cells, and neutralization of IL-10 activity prevents the beneﬁcial effect of probiotics . Commensals acquired during the early postnatal period could also be necessary for the development of tolerance to other luminal antigens, including dietary antigens. For example, it was shown that LPS-responder mice, such as C3H/HeN-strain mice, develop a prolonged and sustained oral tolerance to sheep erythrocytes or ovalbumin, while LPS-nonresponder mice, such as C3H/HeJ mice, do not [32,33]. Additionally, Sudo et al.  reported that T helper 2-mediated immune responses to ovalbumin were not susceptible to oral tolerance induction in germ-free mice, but susceptibility was restored after the introduction of a single component of the microﬂora .
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5. Contributions of the commensal ﬂora to immune-mediated gut diseases The functional and tightly regulated dialogue between indigenous gut microbes and immune cells can be perturbed by changes in dietary habits, by ingestion of toxic compounds, or by infections with pathogens, all of which can result in aberrant local immune response and pathogenicity. A classical example is represented by the tissue-damaging inﬂammatory response occurring in patients with IBD. Indeed, the “inappropriate” activation of the mucosal immune system in this condition has been linked to a loss of tolerance to gut commensals. At least four strands of evidence have contributed to illustrate this point. First, Crohn’s disease patients respond to treatment with broad-spectrum antibiotics . Second, a multitude of animal models of IBD has shown that the lesion is dependent on the presence of a normal ﬂora, given that colitis is abrogated when animals are raised under germ-free conditions . Third, it has been demonstrated that T cells from the lamina propria of Crohn’s disease patients respond in vitro to the antigens of their own ﬂora with a marked production of inﬂammatory Th1 cytokines . Finally, evidence comes from patients in whom diversion of the fecal stream after surgery prevents the recurrence of Crohn’s disease, and exposing the bowel to fecal contents results in inﬂammation [37,38]. In this context, it is also noteworthy that many strains of colitic mice have an exaggerated Th1 immune response directed towards commensal bacterial ﬂagellins, and as well as in Crohn’s disease patients, serum IgG1 levels against ﬂagellins are elevated [39,40].
6. Intestinal commensals as therapeutic agents The ability of commensals to profoundly inﬂuence the activity of mucosal immune cells has provided a rationale for using these organisms as therapeutic agents in immunemediated diseases. In its simplest expression, components of the normal ﬂora are given as live biological supplements (probiotics) that confer some host beneﬁt. For example, probiotics can attenuate colitis both in IBD patients and in animals [41–43]. The exact mechanism underlying the beneﬁcial effect of probiotic interventions is not yet fully understood. As speciﬁed above, probiotics can enhance the production of counter-regulatory molecules such as IL-10 and TGF-b1 or directly inhibit inﬂammatory pathways which sustains the mucosal inﬂammation, such as NF-úB. Another possibility is that probiotics can alter the proﬁle of Th1/Th2 cytokines released by resident T cells. Indeed, Gram-positive bacteria, including some lactobacilli, are potent inducers of IL-12, IL-18, and Th1 cell responses [44–46]. It is thus tempting to speculate that giving patients large amounts of lactobacilli might boost mucosal Th1 responses, which is probably not a good choice in Crohn’s disease. However, the same approach may
be useful in limiting Th2-associated immune responses, such as those occurring in patients with allergic diseases, or particularly advantageous in vaccination strategy.
7. Conclusion Our gastrointestinal tract is colonized by a vast community of commensals that have important effects on mucosal immune functions. These commensals normally do not cause any harm, and are supposed to play a decisive role in the development and activity of the gut immune system. The use of modern genetic techniques has contributed to clarify where and how bacteria interact with immune cells, thus providing novel clues on the essential nature on these interactions. There is no doubt that commensal bacteria prevent colonization of enteric pathogens. Additionally, it is known that some bacteria (i.e. Clostridium difﬁcile) may become enteropathogens and cause tissue damage if allowed to proliferate as a result of a marked reduction of competing normal ﬂora by antibiotics. However, tolerance against intestinal bacteria is broken in patients with IBD, thereby leading to an inappropriate activation of the mucosal immune system and tissue destruction. In these patients, using genetically engineered commensals or live biological supplements could be a promising strategy to restore the mucosal homeostasis.
References  Pickard KM, Bremner AR, Gordon JN, MacDonald TT. Microbialgut interactions in health and disease. Immune responses. Best Pract Res Clin Gastroenterol 2004;18:271−85.  Macpherson AJ, Hunziker L, McCoy K, Lamarre A. IgA responses in the intestinal mucosa against pathogenic and non-pathogenic microorganisms. Microbes Infect 2001;3:1021−35.  Moreau MC, Ducluzeau R, Guy-Grand D, Muller MC. Increase in the population of duodenal immunoglobulin A plasmocytes in axenic mice associated with different living or dead bacterial strains of intestinal origin. Infect Immun 1978;21:532−9.  Logan AC, Chow KP, George A, Weinstein PD, Cebra JJ. Use of Peyer’s patch and lymph node fragment cultures to compare local immune responses to Morganella morganii. Infect Immun 1991;59: 1024−31.  Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science 2001;291:881−4.  Umesaki Y, Setoyama H, Matsumoto S, Imaoka A, Itoh K. Differential roles of segmented ﬁlamentous bacteria and clostridia in development of the intestinal immune system. Infect Immun 1999;67:3504−11.  Bos NA, Jiang HQ, Cebra JJ. T cell control of the gut IgA response against commensal bacteria. Gut 2001;48:762−4.  Gronlund MM, Arvilommi H, Kero P, Lehtonen OP, Isolauri E. Importance of intestinal colonisation in the maturation of humoral immunity in early infancy: a prospective follow up study of healthy infants aged 0−6 months. Arch Dis Child Fetal Neonatal Ed. 2000; 83:F186−92.  Cebra J J, Jiang H-Q, Boiko N, Tlaskalova-Hogenova H. The role of mucosal microbiota in the development, maintenance, and pathologies of the mucosal immune system. In: Mestecky J, Lamm ME, Strober W,
    
G. Monteleone et al. / Digestive and Liver Disease 38 Suppl. 2 (2006) S256–S260 Bienenstock J, McGhee JR, Mayer L (editors), Mucosal Immunology. 2005; Academic Press, Vol I, pp. 335−68. Duchmann R, May E, Heike M, Knolle P, Neurath M, Meyer zum Buschenfelde KH. T cell speciﬁcity and cross reactivity towards enterobacteria, bacteroides, biﬁdobacterium, and antigens from resident intestinal ﬂora in humans. Gut 1999;44:812−8. Brimnes J, Reimann J, Nissen M, Claesson M. Enteric bacterial antigens activate CD4(+) T cells from scid mice with inﬂammatory bowel disease. Eur J Immunol 2001;31:23−31. Cong Y, Brandwein SL, McCabe RP, Lazenby A, Birkenmeier EH, Sundberg JP, Elson CO. CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell type 1 response and ability to transfer disease. J Exp Med 1998;187:855−64. Duchmann R, Kaiser I, Hermann E, Mayet W, Ewe K, Meyer zum Buschenfelde KH. Tolerance exists towards resident intestinal ﬂora but is broken in active inﬂammatory bowel disease (IBD). Clin Exp Immunol 1995;102:448−55. Takeuchi O, Akira S. Toll-like receptors; their physiological role and signal transduction system. Int Immunopharmacol 2001;1:625−35. Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio R, Granucci F, Kraehenbuhl JP, Ricciardi-Castagnoli P. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2001;2:361−7. Niess JH, Brand S, Gu X, Landsman L, Jung S, McCormick BA, Vyas JM, Boes M, Ploegh HL, Fox JG, Littman DR, Reinecker HC. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science 2005;307:254−8. Eberl G. Inducible lymphoid tissues in the adult gut: recapitulation of a fetal developmental pathway? Nat Rev Immunol 2005;5:413−20. Pasare C, Medzhitov R. Toll-like receptors: linking innate and adaptive immunity. Adv Exp Med Biol 2005;560:11−8. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004;5:987−95. Kaisho T, Akira S. Regulation of dendritic cell function through toll-like receptors. Curr Mol Med 2003;3:759−71. Kellermann SA, McEvoy LM. The Peyer’s patch microenvironment suppresses T cell responses to chemokines and other stimuli. J Immunol 2001;167:682−90. Iwasaki A, Kelsall BL. Freshly isolated Peyer’s patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells. J Exp Med 1999;190:229−39. Hauer AC, Bajaj-Elliott M, Williams CB, Walker-Smith JA, MacDonald TT. An analysis of interferon gamma, IL-4, IL-5 and IL-10 production by ELISPOT and quantitative reverse transcriptasePCR in human Peyer’s patches. Cytokine 1998;10:627−34. Nagata S, McKenzie C, Pender SL, Bajaj-Elliott M, Fairclough PD, Walker-Smith JA, Monteleone G, MacDonald TT. Human Peyer’s patch T cells are sensitized to dietary antigen and display a Th cell type 1 cytokine proﬁle. J Immunol 2000;165:5315−21. Monteleone G, Holloway J, Salvati VM, Pender SL, Fairclough PD, Croft N, MacDonald TT. Activated STAT4 and a functional role for IL-12 in human Peyer’s patches. J Immunol. 2003;170:300−7. MacDonald TT, Monteleone G. IL-12 and Th1 immune responses in human Peyer’s patches. Trends Immunol 2001;22:244−7. Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 2005;122:107−18. Neish AS, Gewirtz AT, Zeng H, Young AN, Hobert ME, Karmali V, Rao AS, Madara JL. Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination. Science 2000;289: 1560−3.
 Kelly D, Campbell JI, King TP, Grant G, Jansson EA, Coutts AG, Pettersson S, Conway S. Commensal anaerobic gut bacteria attenuate inﬂammation by regulating nuclear-cytoplasmic shuttling of PPARgamma and RelA. Nat Immunol 2004;5:104−12.  Strauch UG, Obermeier F, Grunwald N, Gurster S, Dunger N, Schultz M, Griese DP, Mahler M, Scholmerich J, Rath HC. Inﬂuence of intestinal bacteria on induction of regulatory T cells: lessons from a transfer model of colitis. Gut 2005;54:1546−52.  Di Giacinto C, Marinaro M, Sanchez M, Strober W, Boirivant M. Probiotics ameliorate recurrent Th1-mediated murine colitis by inducing IL-10 and IL-10 dependent TGF-beta-bearing regulatory cells. J Immunol 2005;174:3237−46.  Kiyono H, McGhee, JR, Wannemuehler J, Michalek SM. Lack of oral tolerance in C3H/HeJ mice. J Exp Med 1982;155:605−10.  Moreau M C, Corthier G. Effect of the gastrointestinal microﬂora on induction and maintenance of oral tolerance to ovalbumin in C3H/HeJ mice. Infect Immun 1988;56:2766−8.  Sudo N, Sawamura S, Tanaka K, Aiba Y, Kubo C, Koga Y. The requirement of intestinal bacterial ﬂora for the development of an IgE production system fully susceptible to oral tolerance induction. J Immunol 1997;159:1739−45.  Thukral C, Travassos WJ, Peppercorn MA. The Role of Antibiotics in Inﬂammatory Bowel Disease. Curr Treat Options Gastroenterol 2005;8:223−8.  Strober W, Fuss IJ, Blumberg RS. The immunology of mucosal models of inﬂammation. Annu Rev Immunol 2002;20:495–549.  Rutgeerts P, Goboes K, Peeters M, Hiele M, Penninckx F, Aerts R, Kerremans R, Vantrappen G. Effect of faecal stream diversion on recurrence of Crohn’s disease in the neoterminal ileum. Lancet 1991; 338:771−4.  D’Haens GR, Geboes K, Peeters M, Baert F, Penninckx F, Rutgeerts P. Early lesions of recurrent Crohn’s disease caused by infusion of intestinal contents in excluded ileum. Gastroenterology 1998;114: 262−7.  Lodes MJ, Cong Y, Elson CO, Mohamath R, Landers CJ, Targan SR, Fort M, Hershberg RM. Bacterial ﬂagellin is a dominant antigen in Crohn disease. J Clin Invest 2004;113:1296–1306.  Sitaraman SV, Klapproth JM, Moore DA 3rd, Landers C, Targan S, Williams IR, Gewirtz AT. Elevated ﬂagellin-speciﬁc immunoglobulins in Crohn’s disease. Am J Physiol Gastrointest Liver Physiol 2005; 288:G403−6.  Bibiloni R, Fedorak RN, Tannock GW, Madsen KL, Gionchetti P, Campieri M, De Simone C, Sartor RB. VSL#3 probiotic-mixture induces remission in patients with active ulcerative colitis. Am J Gastroenterol 2005;100:1539−46.  Sartor RB. Therapeutic manipulation of the enteric microﬂora in inﬂammatory bowel diseases: antibiotics, probiotics, and prebiotics. Gastroenterology 2004;126:1620−33.  Shanahan F. Probiotics and inﬂammatory bowel disease: is there a scientiﬁc rationale? Inﬂamm Bowel Dis 2000;6:107−15.  Maassen CB, van Holten-Neelen C, Balk F, den Bak-Glashouwer MJ, Leer RJ, Laman JD, Boersma WJ, Claassen E. Strain-dependent induction of cytokine proﬁles in the gut by orally administered Lactobacillus strains. Vaccine 2000;18:2613−23.  Mohamadzadeh M, Olson S, Kalina WV, Ruthel G, Demmin GL, Warﬁeld KL, Bavari S, Klaenhammer TR. Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization. Proc Natl Acad Sci USA 2005;102:2880−85.  Hessle C, Hanson LA, Wold AE. Lactobacilli from human gastrointestinal mucosa are strong stimulators of IL-12 production. Clin Exp Immunol 1999;116:276−82.