Bacteria and mucosal immunity

Bacteria and mucosal immunity

Digestive and Liver Disease 38 Suppl. 2 (2006) S256–S260 www.elsevier.com/locate/dld Bacteria and mucosal immunity Giovanni Monteleone*, Ilaria Pelus...

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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 [1]. 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] (G. Monteleone).

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 [4]. Similarly,

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G. Monteleone et al. / Digestive and Liver Disease 38 Suppl. 2 (2006) S256–S260

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 [5]. 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 [6]. 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 [1]. 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 [7]. 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 [8]. 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 [7]. 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 [9]. In normal individuals, CD4+ T gut lamina propria lymphocytes (LPL) do not proliferate following co-culture with sonicates of the endogenous microbial flora [10]. 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 [1]. 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.

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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 [14]. 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 [1]. 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 [15]. 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 [16]. 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 [17]. 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. [27] 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 [28]. Additionally, Kelly et al. [29] 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 [30]. 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 [31]. 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. [34] 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 [34].

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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 [35]. 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 [36]. 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 [13]. 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

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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.

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