Abstract
Certain types of regulatory T cells are preferentially induced at mucosal surfaces to maintain tolerance. However, the mechanisms involved in their induction are unclear. New evidence suggests DCs orchestrate this mucosal tolerogenic immune response.

The immune system provides surveillance of both the internal and external environments and must do so without causing harm to the host. This is accomplished by “immunological tolerance”, a concept that has undergone revision in recent years as more is learned about the workings of the immune system. It is now recognized that tolerance does not involve simple deletion or absence of autoreactive cells, but involves ignorance, anergy, receptor down-regulation, and active cellular regulation. Although active cellular regulation (previously called “suppression”) was, for a time, out-of-favor in the immunological community, it is now clear that suppressor or regulatory cells play an integral role in the immune homeostasis and that there is more that one type of regulatory cell (refs 1-3). Certain types of regulatory cells are preferentially induced at mucosal surfaces, although the mechanisms involved in their induction are not well characterized. Recent studies have shown that dendritic cells may be a key element in the differentiation of such regulatory cells at mucosal sites.
Mucosal surfaces are continuously exposed to environmental antigens. This exposure is of critical immunological importance because antigens that gain priviledged access to the internal milieu via the mucosa must be dealt with in a manner that does not injure the host. This indeed is the case; since 1911 ithas been known that such exposure induces oral tolerance (ref. 5). A well developed mucosal immune system exists both in the gut and respiratory tract and active immunological events occur when antigen contacts the mucosal surfaces; different mechanism of tolerance are induced depending on the antigen, dose and route of administration. Thus, understanding the immunological milieu in which an antigen is processed by the immune system is crucial not only for understanding mucosal tolerance, but for understanding mechanisms of tolerance in general.
The central role of DCs in initiating immune responses is well known (ref. 6) and DCs of different phenotypes may polarize T cells in different directions. DC1 cells, for example, selectively enhance naive cells so that they differentiate into Th1 cells, whereas the DC2 phenotype enhances Th2 induction. Regarding mucosal surfaces, the primary antigen-presenting cell at surfaces is the DC. It has been shown that resting DCs in the respiratory tract preferentially stimulate Th2 responses and when transfered to naive mice can, under certain circumstances, induce Th2 type responses that contribute to allergic asthma. Because the mature DC phenotype drives Th1 responses, they require obligatory cytokine signals for the induction of Th1 immunity (ref. 8).
Regarding the gut, specifically activating DCs in vivo by administering Flt-3 ligand enhances oral tolerance, which demonstrates the important role DCs play in the induction of mucosal tolerance (ref. 12). In addition, others have shown that freshly isolated DCs from Peyer’s patches, but not spleen, produce IL-10 and induce the differentiation of Th2 type cells (ref. 9).
Akbari et al also showed that DC are the heart of immunological tolerance when the respiratory system is exposed to allergen and tolerance is dependent on IL-10 (ref 4). Pulmonary dendritic cells (DCs) that were isolated after respiratory exposure to antigen produced IL-10 and induced CD4+ T regulator type (Tr1) cells. DCs isolated from mesenteric lymph nodes after mice had been fed with OVA expressed amounts of tranforming growth factor-beta(TGF-b) and enhanced production of TGF-b by CD4+ cells. This may explain why antigen administered via the gut preferentially induces T helper type 3 (Th3) regulatory cells (ref. 5). TGF-b is also a switch factor for immunoglobulin A (IgA) and Peyer’s patch-DC mixtures preferentially induce IgA (ref. 10,11); this is consistent with the observation that gut DCs produce this cytokine. Interestingly, in the system described by Akbari et al, pulmonary DCs from nasally could transfer tolerance in adoptive transfer experiments even though they expressed a mature phenotype and elevated amounts of CD80, CD86 and CD40. Thus, after intranasal administration of OVA, a mature DC acquires antigen, differentiates in the respiratory mucosa and becomes tolerogenic. These mature pulmonary DCs from tolerant mice induce regulatory CD4+ T cells in a process that requires co-stimulation via the inducible co-stimulator (ICOS-ICOS ligand pathway) (ref. 16).
However, in the context of maintaining tolerance, mucosal surfaces must also deal with pathogens and, thus, may respond by stimulating a Th1 responses via DC1 cells; these responses may be induced in the gut by pathogen-derived molecules such as CpG motifs from bacterial DNA.
There are obvious advantages of using the nasal route for tolerance induction. Smaller doses of antigen can be administered compared with orally, perhaps because there is less degradation of the proteins before they reach the intestinal mucosal surface. As for the mechanisms triggered, similarities and differences can be observed between orally- and nasally-induced tolerance.
Th3 cells that produce TGF-b are generated after feeding in many reported models of oral tolerance (ref. 5). We found recently that CD4+CD25+ T cells that are induced after feeding secrete IL-10 and TGF-b. Thymic generated CD4+CD25+ cells that are involved in tolerance to autocomponents seem to mediate suppression in a cytokine independent fashion (ref. 17). Adoptive transfer of CD4+CD25+ cells from fed OVA TCR transgenic mice suppress DTH responses in BALB/c mice suggesting that these cells participate in oral tolerance induction to specific antigens (ref. 13). TGF-b has also been associated with the prevention of experimental colitis in mice. The ability of CD4+CD45RBlo T cells to protect against disease induced by transfer of inflammatory CD4+CD45RBhi T cells into scid mice is inhibited by anti-TGF-b antibodies (ref. 18), as is the protective effect of feeding antigen on the colitis induced by local administration of contact sensitising agents to normal animals (ref. 19). CD4+CD25- T cells expressing LAP, the latent protein associated with the precursor of TGF-b, also inhibit colitis in a TGF-b-dependent fashion (ref. 20).
On the other hand, IL-10 appears, in most of the described models, as the key regulatory cytokine for nasal tolerance. Few reports show that TGF-b production can be also detected in spleen cells from nasally tolerized animals in some systems (ref. 21-24).
Of note, nasal tolerance may also generate bystander suppression to unrelated but co-localized antigens in experimental models of atherosclerosis in LDL-R deficient mice (ref. 25), Alzheimer’s disease in PD-APP mice (ref. 26) and stroke (ref. 21, 27). Production of IL-10 but not TGF-b is up-regulated in these models. It is not know what is the importance of DCs and IL-10 in the bystander effect observed but they are likely to play a role.
A striking observation in several reports is the selective inability of nasally-induced tolerance to suppress specific IgG1 production (ref. 16, 25, 28). All other immune responses seem to be sensitive to nasal tolerante as they are to oral tolerance. Th1- and Th2-mediated inflammatory conditions can be inhibited as well as IFN-g, IL-4, IL-13 and specific IgE production. It is also plausible that differences in antigen presentation at different mucosal sites may determine distinct inhibitory patterns after nasal versus oral antigen administration.
It appears, therefore, that the mucosal immune system has a unique immunological milieu that is based on two tolerance-inducing cytokines – IL-10 and TGF-b – and the milieu acts, in part, via the DCs to induce different phenotypes of regulatory cells. Many unanswered questions exist regarding the mechanisms by which the mucosal milieu conditions DCs. Does the milieu act to irreversibly differentiate DCs or are the DCs tolerogenic only the mucosal environment? Is there a mature tolerizing DC that differs from DC1s and DC2s? What does the conditioning represent in molecular terms for DCs and how does that translate into the induction of different types of immune responses?
As mentioned above, one form of T cell regulation is mediated by secretion of the solube IL-10 (Tr1 cells) and/or TGF-b (Th3 cells) after antigen-specific triggering. These two types are in some way related, although their relationship is not clearly understood. For example, Tr1 cells, which suppress colitis and are induced by IL-10, mediate regulation, in part, by the secretion of TGF-b (ref. 1). A relationship between Th3 and Tr1 cells is not unexpected if both are induced via the mucosal route, although via different compartments. IL-10 and TGF-b are important regulatory cytokines because TGF-b-deficient animals have widespread inflammation and IL-10-deficient animals develop colitis. Another key regulatory cell develops in the thymus and is associated with protection from a number of autoimmune processes, although its mechanism of action is dependent on cell-to-cell contact and thus is distinct form the antigen-driven cytokine-mediated suppression of Th3 and Tr1 cells. It is worth noting that CD4+CD25+ regulatory cells may be induced after oral antigen. Interestingly, there are suggestions that the biology of CD4+CD25+ cells may in some way invove IL-10 and TGF-b, and thus there may be common links between the different regulatory cells, even though they come from different compartments and function differently.
An attractive feature of mucosally administratered antigen is the possibility that it could be used therapeutically in situations where immune responses are detrimental to the host. Models of allergic lung disease have been suppressed by oral antigen – Th2 type responses were suppressed by TGF-b (ref. 14, 29, 30) – and there have been positive reports in several human studies of mucosal tolerance for asthma and allergy (ref. 5, 15). The results presented by Akbari et al present the possibility that defective production of IL-10 by DCs in the respiratory tract contributes to asthma, which provides a new immune strategy for this condition. From the perspective of immunological tolerance, it is increasingly clear that local microenvironments play a key role in maintaining tolerogenic cytokines such as IL-10 and TGF-b, as well as by DC polarization. For example, TGF-b appears crucial in immune tolerance associated with the anterior chamber of the eye. Thus, understanding what creates the milieu of tolerogenic immune microenvironments will shed light both on mechanisms of tolerance and strategies of immune manipulation that are beneficial for the host.


Howard L. Weiner, M.D.
Robert L. Kroc Professor of Neurology
Harvard Medical School

Director, Multiple Sclerosis Center
Brigham & Women's and Massachusetts General Hospital

Co-Director, Center for Neurological Diseases
Brigham & Women's Hospital
Address:
Harvard Medical School, Center for Neurologic Diseases,
Brigham & Women’s Hospital
77 Ave Louis Pasteur, HIM 720
Boston, MA 02115
USA
Hweiner@rics.bwh.harvard.edu

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