Gastroenterology
Volume 133, Issue 2 , Pages 706-709, August 2007

Teaching Tolerance With a Probiotic Antigen Delivery System

  • Michel H. Maillard
    • ,
  • Scott B. Snapper

      Affiliations

    • Corresponding Author InformationAddress requests for reprints to: Scott B. Snapper, MD, Department of Medicine, Massachusetts General Hospital, 55 Blossom Street, Boston, Massachusetts 02114. fax: (617) 726-2373.

Gastrointestinal Unit and the Center for the Study of Inflammatory Bowel Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts

Article Outline

 

See “Induction of ovalbumin-specific tolerance by oral administration of Lactococcus lactis secreting ovalbumin” by Huibregtse IL, Snoeck V, de Creus A, et al on page 517.

The gastrointestinal (GI) tract is constantly exposed to a diverse set of antigens of dietary, microbial, and host origin. These luminal antigens influence the host innate and adaptive immune systems through interactions with intestinal epithelial cells and immune cells residing in the lamina propria. Although these dietary and microbial antigens are “foreign,” the human immune system has developed exquisite regulatory mechanisms to prevent unwanted and potentially pathogenic immune responses to these innocuous molecules—leading to a state of unresponsiveness or “tolerance.” Indeed, a breakdown of tolerance can lead to various disorders, including food allergies and inflammatory bowel diseases (IBD).1, 2, 3 Recently, investigators have attempted to develop strategies to induce such tolerogenic mechanisms to prevent a hyperactive immune system associated with a variety of diseases.

Induction of suppression of immune responses to an antigen by its prior oral administration is defined as “oral tolerance.”4 There are 2 main mechanisms by which oral tolerance is achieved: (1) deletion/anergy of pathogenic antigen-specific T-cell clones; and (2) induction of regulatory/suppressor T cells (Tregs), cells with the unique ability to actively control effector immune responses. Although controversial, a contributing factor implicated in determining which mechanism occurs after oral administration of an antigen is antigen dosage. Low doses of antigen favor the development of regulatory T cells, whereas high doses result preferentially in deletion/anergy of reactive T-cell clones.5, 6, 7 However, it seems likely that both regulatory mechanisms (deletion/anergy of specific T-cell clones and regulatory T-cell induction) may occur simultaneously in vivo. Although deletion/anergy of specific T-cell clones and regulatory T-cell induction require antigen specificity, regulatory T cells stimulated in an antigen-specific manner can actively suppress other T cells in an antigen-independent manner (ie, bystander suppression).8

The first description of the induction of a regulatory T-cell population with tolerogenic properties following prior oral feeding of a specific antigen was reported in the experimental autoimmune encephalomyelitis model. These regulatory cells, defined as Th3 cells, secrete transforming growth factor (TGF)-β, possess suppressive properties in vitro, and are capable of transferring tolerance to non-fed animals.9, 10, 11 Th3 cells are part of a broader family of adaptive regulatory T cells (aTregs) that are peripherally activated, and whose suppressive activity is, at least in part, cytokine dependent.12 This family also includes interleukin (IL)-10–secreting cells or Tr1 cells that were originally described as T-cell clones arising in vitro after prolonged stimulation with IL-10.13 Tr1 cells were capable of protecting against colitis induced by transfer of CD4+CD45RBhi T cells into lymphopenic hosts.13 Interestingly, the gut flora, and more specifically Helicobacter hepaticus, seem to modulate the presence of Tr1 cells in the GI tract.14

It has become clear over the past few decades that a fraction of peripheral T cells that maintain tolerance to self-antigens exist, in contrast with aTregs, under normal physiologic conditions. These cells are defined as naturally occurring regulatory T cells (nTregs). nTregs are generated in the thymus and express the forkhead-box transcription factor Foxp3, a molecule involved both in nTreg cell development and function.15 Other associated surface markers expressed by nTregs include the IL-2Rα chain (CD25), GITR, and CTLA-4. nTregs have been shown to play a nonredundant role in preventing autoimmune disease, preventing graft-versus-host disease, suppressing antitumor immunity, and modulating immune responses to infectious pathogens.16 In mice, transfer of nTreg cell-depleted CD4+ cells into lymphopenic hosts leads to severe chronic colitis.17 Furthermore, genetic targeting of key molecules in nTreg cell function/homeostasis (ie, CD25, IL-2, Foxp3) results in colitis in mice.18, 19, 20 Thus, nTregs appear to play a central role in regulating gut immune homeostasis; however, the extent by which nTregs contribute to oral tolerance is still unknown. In this regard, Thorstenson et al21 have reported the appearance of an ovalbumin (OVA)-specific CD4+CD25+ cell population after oral administration of low doses of OVA to WT mice adoptively transferred with OVA-specific T cells and have shown that these cells are suppressive ex vivo in a TGF-β– and IL-10–independent manner.21 Nonetheless, it remains unclear whether these cells are bona fide thymic-derived nTregs.

A third category of regulatory T cells has been described that acquire Foxp3 expression upon TGF-β stimulation. These so-called inducible regulatory T cells (iTregs) may be related to Th17 cells that arise in vitro in the presence of both IL-6 and TGF-β.22 Inducible Treg cells have regulatory functions both in vitro23 and in vivo.24 Interestingly, maintenance of these cells in culture with TGF-β induces TGF-β secretion.23 Thus, TGF-β–dependent regulation is central to both Th3 and iTreg development/function and suggests that some overlap may exist between these 2 cell types in vivo.

Although oral tolerance has been effectively achieved in mice using various systems, clinical attempts in humans have thus far been unsuccessful.4 In this issue of Gastroenterology, Huibregtse et al25 developed a novel approach of oral antigen delivery in mice that employs the probiotic bacterial strain Lactococcus lactis as an antigen delivery system. This approach has several features that may make it an attractive antigen delivery system for the use in modulating immune responses in humans.

L lactis is a Gram-positive, noncolonizing, nonpathogenic, noninvasive bacterium that is not normally present in the human or mouse gut flora and is commonly used as a fermenting agent in the food industry. Previous work by Steidler et al26 showed that a strain of L lactis modified to express IL-10 was effective at preventing colitis in IL-10 knockout mice and after oral administration of dextran sodium sulfate. L lactis has also been used to stimulate IL-10 secretion in the GI tract when expressing the low-calcium-response V antigen (LcrV) protein from Yersinia pseudotuberculosis.27 Oral administration of LcrV-secreting L lactis was effective at protecting mice from experimental colitis.28 Recently, a Phase I trial was conducted in humans with moderate to severe active Crohn’s disease that demonstrated that IL-10–secreting L lactis was safe and may be effective for the treatment of Crohn’s disease.29

Probiotics can be defined as live microorganisms that have a beneficial effect to the health or well-being of the host.30 L lactis is a member of the lactic acid bacteria family, which are among the most commonly used probiotic agents. These bacteria have the ability to induce Th1-cytokine production by splenocytes in vitro31 and inhibit Th2 cytokine production by mononuclear cells from allergic patients.32 Moreover, L lactis has an adjuvant effect when co-administered with antigen in murine models of birch pollen or food allergies.31, 33 These models are associated with Th2 responses and one of the postulated mechanisms of action for L lactis is the stimulation of counter-regulatory Th1 responses.

In the current report, Huibregtse et al25 show decreased delayed-type hypersensitivity in OVA-specific T-cell receptor (TCR)-transgenic mice that were fed L lactis engineered to express OVA (LL-OVA) compared with those fed high- or low-dose soluble OVA. This response was accompanied by an up-regulation of IL-10 in the gut-associated lymphoid tissue (GALT) and spleen of the tolerized animals in an antigen-specific manner. Of note, although this response was more pronounced after LL-OVA administration, mice receiving L lactis alone, without OVA expression, also partially responded. Although IL-10 induction was specifically up-regulated after LL-OVA administration, partial suppression of splenocyte proliferation and interferon-γ secretion was achieved with L lactis alone.

An important advance of this study is assessing the role of the mode of antigen delivery in tolerance induction. The precise mechanism by which L lactis enhances tolerogenic signals remains unclear. L lactis may directly modulate antigen processing, antigen presentation, and/or expression of co-stimulatory molecules on dendritic cells (DCs) through stimulation, at least in part, of innate immune molecules.34, 35 Most of the luminal OVA found after oral gavage was in the cecum and colon, and most of the mucosal OVA was in the terminal ileum. It is unknown which site is most important for tolerance induction. DCs residing throughout the GI tract have been shown to directly sample luminal antigens through the epithelial cell layer.36, 37 Through an association with the intestinal epithelium, L lactis may permit more efficient antigen uptake than is available through oral administration of OVA. Alternatively, the authors report mucosal associated OVA in the terminal ileum and cecum in LL-OVA–fed mice. A bacterial-delivered antigen may provide a more effective dose of antigen to the intestine than obtained with soluble oral antigen alone. The authors have not evaluated whether similar suppressive effects occur in the presence of L lactis plus soluble antigen, which would point to whether bacterial expression of antigen is critical. Furthermore, whether LL-OVA antigen delivery modulates anergy/deletion of a specific T-cell clones was not assessed.

As mentioned previously, TGF-β regulates both the function of Th3 cells and development of iTregs. In this issue of Gastroenterology, Huibregtse et al25 demonstrate that oral administration of LL-OVA leads to the appearance of a suppressive T-cell subset whose activity was almost completely dependent on TGF-β—a classic property of Th3 cells. Through adoptive transfer experiments, they further refine the suppressive activity to a CD4+CD25− T-cell subset—a subset that does not typically include nTregs. Interestingly, these cells expressed Foxp3 and CTLA-4, a phenotype consistent with iTregs. As noted, IL-10 was up-regulated in the GALT and spleen of LL-OVA treated mice, and although not required for suppression, this cytokine may be important for the generation/differentiation of the CD4+CD25−Foxp3+ cell population. A similar cross-talk between IL-10 and TGF-β has previously been reported in experimental colitis.38 Taken together, these results highlight how different Treg populations can overlap in function and phenotype and hint at the complexity of the regulatory pathways involved in oral tolerance.

Several immune-mediated diseases (such as food allergies) are triggered by well-defined antigens; therefore, tolerance protocols aimed at targeting these antigens are clearly warranted. In the current report, Huibregtse et al25 successfully induced antigen-specific immune tolerance in TCR-transgenic mice. Whether antigen-specific immune suppression can be achieved in a normal host bearing a broader TCR repertoire remains unknown. Regardless, nonspecific bystander suppression of immune responses may be beneficial in the context of a disease driven by multiple antigens or where specific antigen triggers have remained elusive (eg, IBD). Oral administration of OVA using L lactis may trigger both antigen-specific as well as antigen-nonspecific regulatory pathways.

In conclusion, this is the first report demonstrating that L lactis can be used to efficiently deliver antigen to the intestinal mucosa for the induction of antigen-specific peripheral tolerance (Figure 1). This mode of administration induces much more efficient responses than with purified antigen and alleviates the need for large-scale protein purification. Adequate biological containment strategies and efficient antigen delivery make L lactis an attractive candidate for tolerance induction to known antigens. This approach gives hope for novel therapeutic interventions in antigen-driven diseases such as allergies or some autoimmune diseases.

  • View full-size image.
  • Figure 1. 

    Mechanisms for induction of oral tolerance. Oral administration of antigen leads to either deletion/anergy of antigen-specific T-cell clones (high dose) or induction of TGF-β-secreting regulatory T cells (Th3) (low dose). L lactis engineered to produce OVA (LL-OVA) may deliver antigen to dendritic cells: (1) via direct sampling in the intestinal lumen; or (2) through contact with DCs following penetration to the lamina propria. Oral administration of LL-OVA results in the appearance of CD4+CD25-Foxp3+CTLA-4+ regulatory T cells that may control local and systemic antigen-specific immune responses in a TGF-β-dependent manner.

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References 

  1. Iweala OI, Nagler CR. Immune privilege in the gut: the establishment and maintenance of non-responsiveness to dietary antigens and commensal flora. Immunol Rev. 2006;213:82–100
  2. Strobel S, Mowat AM. Oral tolerance and allergic responses to food proteins. Curr Opin Allergy Clin Immunol. 2006;6:207–213
  3. Strober W, Fuss I, Mannon P. The fundamental basis of inflammatory bowel disease. J Clin Invest. 2007;117:514–521
  4. Faria AM, Weiner HL. Oral tolerance. Immunol Rev. 2005;206:232–259
  5. Friedman A, Weiner HL. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc Natl Acad Sci U S A. 1994;91:6688–6692
  6. Chen Y, Inobe J, Marks R, et al. Peripheral deletion of antigen-reactive T cells in oral tolerance. Nature. 1995;376:177–180
  7. Garside P, Steel M, Worthey EA, et al. Lymphocytes from orally tolerized mice display enhanced susceptibility to death by apoptosis when cultured in the absence of antigen in vitro. Am J Pathol. 1996;149:1971–1979
  8. Miyara M, Sakaguchi S. Natural regulatory T cells: mechanisms of suppression. Trends Mol Med. 2007;13:108–116
  9. Lider O, Santos LM, Lee CS, et al. Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein (II. Suppression of disease and in vitro immune responses is mediated by antigen-specific CD8+ T lymphocytes). J Immunol. 1989;142:748–752
  10. Santos LM, al-Sabbagh A, Londono A, et al. Oral tolerance to myelin basic protein induces regulatory TGF-beta-secreting T cells in Peyer’s patches of SJL mice. Cell Immunol. 1994;157:439–447
  11. Miller A, Lider O, Roberts AB, et al. Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor beta after antigen-specific triggering. Proc Natl Acad Sci U S A. 1992;89:421–425
  12. Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells. Nat Rev Immunol. 2003;3:253–257
  13. Groux H, O’Garra A, Bigler M, et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature. 1997;389:737–742
  14. Kullberg MC, Jankovic D, Gorelick PL, et al. Bacteria-triggered CD4(+) T regulatory cells suppress Helicobacter hepaticus-induced colitis. J Exp Med. 2002;196:505–515
  15. Zheng Y, Rudensky AY. Foxp3 in control of the regulatory T cell lineage. Nat Immunol. 2007;8:457–462
  16. Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol. 2005;6:345–352
  17. Powrie F, Leach MW, Mauze S, et al. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C (B-17 scid mice). Int Immunol. 1993;5:1461–1471
  18. Lyon MF, Peters J, Glenister PH, et al. The scurfy mouse mutant has previously unrecognized hematological abnormalities and resembles Wiskott-Aldrich syndrome. Proc Natl Acad Sci U S A. 1990;87:2433–2437
  19. Willerford DM, Chen J, Ferry JA, et al. Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity. 1995;3:521–530
  20. Sadlack B, Merz H, Schorle H, et al. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell. 1993;75:253–261
  21. Thorstenson KM, Khoruts A. Generation of anergic and potentially immunoregulatory CD25+CD4 T cells in vivo after induction of peripheral tolerance with intravenous or oral antigen. J Immunol. 2001;167:188–195
  22. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235–238
  23. Fantini MC, Becker C, Monteleone G, et al. Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol. 2004;172:5149–5153
  24. Chen W, Jin W, Hardegen N, et al. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med. 2003;198:1875–1886
  25. Huibregtse IL, Snoeck V, de Creus A, et al. Induction of ovalbumin-specific tolerance by oral administration of Lactococcus lactis secreting ovalbumin. Gastroenterology. 2007;133:517–528
  26. Steidler L, Hans W, Schotte L, et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science. 2000;289:1352–1355
  27. Sing A, Rost D, Tvardovskaia N, et al. Yersinia V-antigen exploits toll-like receptor 2 and CD14 for interleukin 10-mediated immunosuppression. J Exp Med. 2002;196:1017–1024
  28. Foligne B, Dessein R, Marceau M, et al. Prevention and treatment of colitis with Lactococcus lactis secreting the immunomodulatory Yersinia LcrV protein. Gastroenterology. 2007;133:(in press)
  29. Braat H, Rottiers P, Hommes DW, et al. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease. Clin Gastroenterol Hepatol. 2006;4:754–759
  30. Broekaert IJ, Walker WA. Probiotics and chronic disease. J Clin Gastroenterol. 2006;40:270–274
  31. Repa A, Grangette C, Daniel C, et al. Mucosal co-application of lactic acid bacteria and allergen induces counter-regulatory immune responses in a murine model of birch pollen allergy. Vaccine. 2003;22:87–95
  32. Pochard P, Gosset P, Grangette C, et al. Lactic acid bacteria inhibit TH2 cytokine production by mononuclear cells from allergic patients. J Allergy Clin Immunol. 2002;110:617–623
  33. Adel-Patient K, Ah-Leung S, Creminon C, et al. Oral administration of recombinant Lactococcus lactis expressing bovine beta-lactoglobulin partially prevents mice from sensitization. Clin Exp Allergy. 2005;35:539–546
  34. Yarovinsky F, Kanzler H, Hieny S, et al. Toll-like receptor recognition regulates immunodominance in an antimicrobial CD4+ T cell response. Immunity. 2006;25:655–664
  35. Blander JM, Medzhitov R. Toll-dependent selection of microbial antigens for presentation by dendritic cells. Nature. 2006;440:808–812
  36. Rescigno M, Urbano M, Valzasina B, et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol. 2001;2:361–367
  37. Niess JH, Brand S, Gu X, et al. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science. 2005;307:254–258
  38. Fuss IJ, Boirivant M, Lacy B, et al. The interrelated roles of TGF-beta and IL-10 in the regulation of experimental colitis. J Immunol. 2002;168:900–908

PII: S0016-5085(07)01287-5

doi:10.1053/j.gastro.2007.06.055

Refers to article:

  • Induction of Ovalbumin-Specific Tolerance by Oral Administration of Lactococcus lactis Secreting Ovalbumin , 04 May 2007

    Inge L. Huibregtse, Veerle Snoeck, An de Creus, Henri Braat, Ester C. de Jong, Sander J.H. van Deventer, Pieter Rottiers
    Gastroenterology August 2007 (Vol. 133, Issue 2, Pages 517-528)

Gastroenterology
Volume 133, Issue 2 , Pages 706-709, August 2007

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