oral tolerance wang2013

Advanced Drug Delivery Reviews xxx (2012) xxx–xxx

ADR-12419; No of Pages 15

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Advanced Drug Delivery Reviews

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Mechanism of oral tolerance induction to therapeutic proteins☆

Xiaomei Wang a, Alexandra Sherman a, Gongxian Liao b, Kam W. Leong c, Henry Daniell d,Cox Terhorst b, Roland W. Herzog a,⁎a Dept. of Pediatrics, University of Florida, Gainesville, FL 32610, USAb Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USAc Department of Biomedical Engineering, Duke University, Durham, NC 27708, USAd Dept. of Molecular Biology and Microbiology, University of Central Florida, Orlando, FL 32816, USA

☆ This review is part of the Advanced Drug Delivery Re⁎ Corresponding author at: University of Florida, Cance

E-mail addresses: [email protected] (X. Wang), a(H. Daniell), [email protected] (C. Terhorst), rhe

0169-409X/$ – see front matter © 2012 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.addr.2012.10.013

Please cite this article as: X. Wang, et al., Mdx.doi.org/10.1016/j.addr.2012.10.013

a b s t r a c t
a r t i c l e i n f o
Article history:Received 8 August 2012Accepted 24 October 2012Available online xxxx

Keywords:Oral toleranceDendritic cellsTregIL-10TGF-βTr1Th3NanoparticlesTransgenic plantsOral deliveryProtein antigen

Oral tolerance is defined as the specific suppression of humoral and/or cellular immune responses to an antigenby administration of the same antigen through the oral route. Due to its absence of toxicity, easy administration,and antigen specificity, oral tolerance is a very attractive approach to prevent unwanted immune responses thatcause a variety of diseases or that complicate treatment of a disease. Many researchers have induced oral toler-ance to efficiently treat autoimmune and inflammatory diseases in different animal models. However, clinicaltrials yielded limited success. Thus, understanding the mechanisms of oral tolerance induction to therapeuticproteins is critical for paving the way for clinical development of oral tolerance protocols. This review willsummarize progress on understanding the major underlying tolerance mechanisms and contributors, includingantigen presenting cells, regulatory T cells, cytokines, and signaling pathways. Potential applications, examplesfor therapeutic proteins and disease targets, and recent developments in delivery methods are discussed.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.1. GALT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2. APC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.2.1. M cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2.2. DC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.3. T cell responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.4. Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.4.1. Transforming growth factor-β (TGF-β) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.4.2. Interleukin-10 (IL-10) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.5. Other immune regulatory pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.5.1. Retinoic acid (RA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.5.2. Treg expressing transcription factor Foxp3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3. Therapeutic proteins and disease targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04. Delivery methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

views theme issue on “Nanoparticle- and biomaterials-mediated oral delivery for drug, gene, and immunotherapy”.r and Genetics Research Center, 2033 Mowry Road, Gainesville, FL 32610, USA. Tel.: +1 352 273 8149; fax: +1 352 273 [email protected] (A. Sherman), [email protected] (G. Liao), [email protected] (K.W. Leong), [email protected]@ufl.edu (R.W. Herzog).

l rights reserved.

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2 X. Wang et al. / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx

1. Introduction

The human mucosal surfaces of the gastrointestinal (GI) tract,with an area of around 300 m2, are constantly in contact with alarge variety of antigens, including dietary proteins and constituentsof commensal bacteria [1]. Approximately 30 kg of food proteins peryear reaches the gut, and 1012 bacteria per g of stool colonize thehuman intestinal mucosa [2,3]. Under such a high antigen pressure,it is natural for the gut to develop a mechanism to abrogate potential-ly injurious inflammatory responses and favor a tolerogenic environ-ment. However, most pathogens also enter the human body throughthe GI tract [4]. The gut immune system has evolved a complicatedand tightly regulated mechanism to suppress unwanted inflammato-ry responses while at the same time protecting the body from patho-genic organisms. Generally, there are at least three types of responsesto oral antigen administration— a local secretory IgA antibody responsewhich protects mucus layer by forming a barrier capable of neutralizingthe pathogen before it get to the cells; local and systemic suppression ofthe activation of damaging immunological responses (this type ofresponse is termed as oral tolerance); systemic immune responsesincluding generating serum antibodies such as IgG and cell-mediatedimmunity such as by cytotoxic T lymphocytes (CTL) [5].

Particularly, oral tolerance is defined as the specific suppression ofhumoral and/or cellular immune responses, such as antibody formationof different isotypes, production of inflammatory cytokines, and cellularimmune responses including lymphocyte proliferation, delayed type-hypersensitivity reactions, etc. to an antigen by prior administration ofthe same antigen through the oral route [6]. Tolerance should not berestricted to the local intestinal tissue but also include the systemic im-mune system [6]. It is an active adaptive immune response rather thanpassive unresponsiveness or blindness of the immune system to exoge-nous antigens. It is “anymechanismbywhich a potentially injurious im-mune response is prevented, suppressed, or shifted to a non-injuriousclass of immune responses [7].”

Currently, intravenous infusion of recombinant protein is a routinetreatment for genetic deficiencies such as hemophilia and lysosomalstorage diseases. However, immune responses develop in a certain per-centage of patients receiving the therapeutic protein, which representsa big hurdle for therapy [8–10]. All these urgently call for an efficientimmune tolerance protocol. Due to the absence of toxicity, easy admin-istration, and targeting of a specific antigen, oral tolerance is a very at-tractive candidate. Indeed, people have successfully lowered antibodytiters and cell mediated immunitywith oral tolerance therapy in animalmodels of autoimmune diseases and inflammatory diseases, includingexperimental autoimmune encephalomyelitis (EAE), uveitis, thyroid-itis, myasthenia gravis, arthritis, diabetes, experimental colitis, as wellas graft-versus host disease, allergy, anti-phospholipid syndrome, asth-ma, stroke, and atherosclerosis [11]. Several clinical trials have also beenconducted inmultiple sclerosis, uveitis, thyroid disease, Crohn's disease,rheumatoid arthritis, hepatitis, and diabetes [7,12]. The results fromhuman studies have yielded limited success, possibly due to inappropri-ate selection of dosage and delivery methods, indicating that there isstill a long way to go in understanding the mechanisms of oral toler-ance. The present reviewwill focus on themolecular and cellularmech-anisms currently known to direct oral tolerance, emphasizing clinicalapplications of oral tolerance in protein therapy, and discussing severalemerging and promising delivery methods for oral tolerance therapy.

2. Mechanisms

2.1. GALT

The intestine has the most abundant populations of immune cells,with every meter of human small intestine housing 1012 lymphoidcells [13]. These immune cells are located in three compartments:scattered throughout the epithelium; in the lamina propria of the

Please cite this article as: X. Wang, et al., Mechanism of oral tolerance indx.doi.org/10.1016/j.addr.2012.10.013

mucosa; or residing in the gut-associated lymphoid tissue (GALT),which are organized lymphoid aggregates along the submucosa of theentire small intestine [14]. Consisting of three parts — Peyer's patches,appendix, and isolated lymphoid follicles, the GALT contains approxi-mately 5×1010 lymphocytes [4]. Its structure is similar to lymphnodes in that they both have follicular B cell zones, inter-follicularT cell zones, and antigen-presenting cells like dendritic cells (DCs)and macrophages [15]. However, the GALT is not encapsulated andcontains no lymphatic vessels, acquiring antigens directly from theintestinal mucosal surface [15]. GALT and the mesenteric lymphnode (MLN), the largest lymph node in the body, are consideredthe primary inductive sites of adaptive immune responses, whereasthe lamina propria and epithelium of mucosa have effector andmem-ory functions (Fig. 1). For example, B cell differentiate into plasmacells and generating antibodies in lamina propria with helper T cellsgetting signals from local antigen presenting DC [14]. Intraepitheliallymphocytes (IELs) regulate innate and adaptive immune responses.About one IEL can be found per 10 intestinal villous epithelial cells[16]. The majority of IELs are CD8+ T cells and express αβ or γδ Tcell receptors, which is required for oral tolerance. Depletion of γδIEL impaired oral tolerance induction and maintenance [17].

The development of the GALT requires stimulation from commensalmicrobiota and food antigens. Newborns develop germinal centers, andIgA positive plasmablasts after 1 month [18]. Germ free mice have un-derdeveloped lymphoid follicles and low IgA production, which nor-malized after 1-month exposure to conventional gut microbiota [19].Similarly, mice on a balanced protein free diet showed immatureGALT with reduced IgA [20]. Microbiota play a role in tolerance induc-tion, as numbers of regulatory T cells (Treg), one of the key cell typesin tolerance, were lower in germ-free mice than in normal microbiotamice [21]. Colonization of germ-free mice studies demonstrated thatClostridium species, but not Lactobacillus, segmented filamentous bacte-ria (SFB), or Bacteroides, specifically promoted intestinal epithelial cellsto produce transforming growth factor-β (TGF-β), which leads to Tregdifferentiation and survival [22].

Interestingly, MLN but not GALT was demonstrated to be essentialand sufficient for oral tolerance (Fig. 1). Clostridium can induce Treg ac-cumulation in mice with deficient Peyer's patches and lymphoid folli-cles [22]. Oral tolerance can be induced in the absence of Peyer'spatches andMcells [23], though oral tolerance can be induced in Peyer'spatches as well [2]. Functional MLN are essential for oral tolerance in-duction as lymphotoxin α deficient mice, which is a model for Peyer'spatches and lymph node deficiency, lost oral tolerance induction;whereas reconstitution of MLN with anti-lymphotoxin β receptorantibody was able to restore oral tolerance [24]. This is additionallysupported by a studywhere surgical ablation of theMLN resulted in de-creased oral tolerance [25]. Similarly, surgical removal of nose-draininglymph nodes abolished nasal tolerance, which can be restored by trans-plantation of nose-draining lymph nodes but not peripheral lymphnodes [26].

2.2. APC

Orally administered antigens are sampled by different mechanismsand cell types. Microfold cells (M cells), DCs, and enterocytes were allreported to actively take up antigens [27]. In addition, some protein an-tigens can directly cross the epithelial layer and get into circulation. Theroute and mechanism of antigen uptake may be dependent on the nat-ural characteristics of that antigen [28]. Peyer's patches might be in-volved more with bacteria antigens [29], whereas some haptensmight reach the liver via portal vein, which is believed to play a prom-inent role in oral tolerance induction [30]. Antigen presenting cells(APC) in the gut are particularly prone to induce antigen-specific Treg,which migrate and suppress damaging immune responses and secretantigen non specific cytokines such as TGF-β, contributing to bystandersuppression (Fig. 1) [27].

duction to therapeutic proteins, Adv. Drug Deliv. Rev. (2012), http://



Fig 1. Overview of mechanism of oral tolerance induction. Oral antigens are sampled by different ways: M cells transfer antigen to DCs by transcytosis; DCs directly acquire antigenfrom gut lumen; antigens are endotyosized by enterocytes and DCs or macrophages engulf enterocytes debris containing antigens; antigens directly cross the epithelial layer.Plasmacytoid or CD11c+ DCs, CD103+ DCs, and CD11b+ DCs are specialized in inducing oral tolerance. DCs get educated in local microenvironment through local factors andcrosstalking with different cell types: intestinal epithelial cells releasing TGF-β and retinoic acid; lamina propria macrophages produce large amount of IL-10 and inhibit DC induc-tion of Th-17 cells; oral immunized memory T cells secrete IL-4 and IL-10 to educate DCs and induce naïve T cells to produce the same cytokines. DCs are critical in gut hemostasis:DCs from Peyer's patches and MLN induced B cells to transform into plasma cells and produce IgA; DCs abrogate a large fraction of antigen-specific CD8+ T cells in liver and MLNs;activated DCs migrate to MLNs and induce Treg. Generally, high dose of oral antigens induce T cell deletion/anergy and low dose antigens induce Treg. These two mechanisms occursimultaneously and overlap. Apoptotic T cells, macrophages and DCs that clean up the debris, express high level of TGF-β and suppress inflammatory cytokine production. Tregsuppress effector T cell responses including inhibition of T cell proliferation and blockade of inflammatory cytokines release. TGF-β and IL-10 are high in the gut microenvironment,which is essential for Treg function and maintenance.

3X. Wang et al. / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx

2.2.1. M cellsM cells, with broad microfolds instead of microvilli on their apical

surface, are mainly located in the follicle associated epithelium thatlines Peyer's patches and isolated lymphoid follicles in the small intes-tine (Table 1). M cells take up antigens in gut lumen by endocytosisand effectively transport them to professional antigen presenting cellsresiding in the subepithelial dome region of the follicles. Antigen loadedAPCs further present the antigens to naïve T cells in the GALT ormigrateto gut-drainingMLNs [29]. TargetingM cells using a recombinant reovi-rus protein sigma 1(pσ1) conjugated with ovalbumin (OVA), enhancedoral tolerance as antibody titers and CD4+ T cell responses againstOVA were greatly suppressed compared with controls. CD4+CD25+

forkhead box protein 3 (Foxp3+) T cells from the spleen, MLNs, andPeyer's patches showed dramatically increased expression of TGF-βand IL-10 (Fig. 1) [31]. In one example, fed antigen predominantly local-ized to the interphase between M cells and CD11c+ DC in Peyer'spatches, consistent with themodel that M cells take up antigen and de-liver to DC by transcytosis. Two to five hours after oral gavage, antigencan be found in the liver and plasma (Fig. 2) [32]. This transcellular-Mpathway was also identified as one of the mechanisms in nanoparticleoral delivery system by fluorescent image studies [33]. Particles with adiameter of less than 5 μm were able to be taken up by M cells, with adiameter around 100 nm having higher efficiency compared with big-ger nanoparticle sizes [34,35].

2.2.2. DCIntestinal DCs, the major APCs in the gut, are located in the lamina

propria, Peyer's patches, and MLN [36]. DCs constitutively engulf


apoptotic enterocytes, which sampled intestine luminal antigens. Ad-ditionally, DCs under the epithelial of lamina propria are able to openthe tight junctions between adjacent epithelial cells and extend theirdendrites to reach luminal content to acquire antigen directly fromintestine lumen (Fig. 1) [37,38].

It was suggested that resident DCs in the gut have intrinsicnon-inflammatory characteristics (Table 1). Compared to splenicDCs, DCs from Peyer's patches induced tolerance rather than primedT cells to an effector phenotype [39]. DCs isolated from MLN andPeyer's patches induced B cell production of IgA, and T cell expressionof gut-homing receptors CCR9 and α4β7 [40,41], which are criticallyrequired for oral tolerance [11]. In vivo expansion of DCs using flt3 li-gand enhanced oral tolerance [36]. Although intestinal antigens wereshown to reach the circulation and peripheral lymph nodes [42], evi-dence indicated that it is not sufficient to induce significant responses.Oral tolerance relies on the gut DCs' uptake of antigens and subsequentmigration to MLN on a CCR7-dependent manner [25].

Besides inducing Treg, the gut's DCs are able to initiate effector T cellsin response to invasive pathogens, depending on subsets of DCs involvedand surface receptors engaged [43]. Plasmacytoid or conventionalCD11c+ DCs, CD103+ DCs, and CD11b+ DCs may be specialized for in-ducing oral tolerance. In an asthma model, transferring splenic CD11c+

plasmacytoid DCs (pDCs) isolated from OVA-fed mice transferred toler-ance [44]. In a delayed type hypersensitivity model, in vivo depletion ofpDCs diminished oral tolerance [45]. Adoptively transferring pDCsfrom liver and MLN but not spleen of antigen fed animals transferredtolerance. A study showed that pDCs contributed to around 70–80%anergy/deletion of antigen-specific cells [45]. In a contact dermatitis




Table 1Key cells, cytokines, and pathways involved in oral tolerance induction.

Characteristics Function Findings in oral tolerance Reference

Cells M cells Broad microfolds. Located in Peyer's patches and lymphofollicles in small intestine

Take up antigens and effectively transport to antigenpresenting cells

Targeting M cells using pσ1 conjugation with antigenenhanced oral tolerance.

[29,31,32]

Dendritic cells CD11c+ DCs, CD103+ DCs, and CD11b+ DCs specialize ininducing oral tolerance. Gut DCs are mainly located inlamina propria, Peyer's patches, MLN.

Antigen presentation to T cells. Gut DCs may induce Treg,increase T cells expression of gut homing receptors, andinduce B cells production of IgA.

In vivo expansion of DCs enhances oral tolerance whereasdepletion of DCs diminished oral tolerance. Adoptivetransfer of MLN pDCs transferred tolerance.

[36,40,41,44,45]

Tr1 CD4+ T cells with low proliferative capacity, high levels ofIL-10, and low levels of IL-2, no IL-4.

Suppress T cells responses through IL-10. Induce Foxp3+ Treg. Involved in low dose oral tolerance [74,80,110]

Th3 CD4+ T cells producing TGF-β with various amounts of IL-4and IL-10. Dependent on IL-4 rather than IL-2 for growth.First isolated from MLN of orally tolerized mice.

Suppress T cells responses. Induce Foxp3+ Treg. Population increased in orally tolerized mice andperipheral blood of humans fed MBP.

[6,79,99]

Th17 Secrete high amounts of IL-17 and IL-22. Induced by IL-23or IL-6.

Pro-inflammation. Important in autoimmune diseases.Decrease Treg.

Oral tolerance decreased Th17 cells [49,195]

Foxp3+ Treg CD4+ CD25+ Foxp3+ regulatory T cells Suppressing an effector T cell response such as T cell pro-liferation and inflammatory cytokine productions.

Oral antigen administration induced Foxp3+ Treg, whichtransferred tolerance to naïve recipients.

[76,77]

LAP+ Treg CD4+ LAP+ regulatory T cells in both CD25+ and CD25− Suppressing an effector T cell response Population increased in oral tolerance. Anti-TGF-β anti-body reversed the suppressive effects.

[81–83]

Cytokines TGF-β Abundant in the gut microenvironment. Secreted by CD4+

CD8+ T cells, macrophages, enterocytes, and epithelial cells.Pivotal in epithelial cell differentiation, IgA classswitching, and strong immunosuppressive effects onlymphocytes. Induce Treg.

Up-regulated in oral tolerance. Neutralized TGF-β impairedoral tolerance. TGF-β knockoutmice exhibited no tolerance inlow dose feeding and partial tolerance in high dose feeding.

[90,91,97,98,100,101,103]

IL-10 Highly expressed in the intestine. Secreted by certainCD4+ T cells (Th2 cells, Tr1 cells, Th3 cells, Foxp3+ Treg),epithelial cells, macrophages, NK cells, B cells, DCs andsome CD8+ T cells.

Broad anti-inflammatory cytokine. Inhibits Th1 responses,down-regulates MHC class II and co-stimulatory molecules.Important for Treg induction and function.

Antigen feeding induced IL-10 production and Tr1 cells.IL-10 deficient mice failed in oral tolerance induction.However, some reported oral tolerance could be inducedwith anti-IL-10 antibody administration.

[80,109,113,115–118]

IFN-Υ Th1 type cytokine Regulates the expression of adhesion molecules essentialfor Treg migration.

Controversial. Some reported that oral tolerance cannot beinduced or adoptive transferred in IFN-Υ deficient mice.Others found it did not affect oral tolerance induction ormaintenance.

[121–124]

Pathways Cox-2 Inducible enzyme in arachidonic acid metabolism Induces Foxp3 expression Selective Cox-2 inhibition led to loss of oral tolerance [126,127]Retinoic acid Metabolite of vitamin A. Produced by mucosal DCs,

epithelial cells, MLN stromal cells, and macrophages.Critical for mucosal DCs function. Regulates Th17 cellresponses. Boost Treg induction.

CD103+ DCs in MLN expressed high level of Aldhla2,which made their own RA.

[51–53,135–139]

Foxp3 Mainly expressed in CD4+ T cells. Marker of Treg. Transcriptional factor. Pivotal for Treg functions. Mutation caused severe autoimmune disease. Retroviralgene transfer turned naïve T cells to Treg-like phenotype andfunction.

[142–148,150,151]

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Fig. 2. Example of prevention of a systemic immune response by oral tolerance induction: Prevention of inhibitory antibody formation and of anaphylactic reactions against intra-venous human F.IX (hF.IX) by oral administration of CTB-hF.IX chloroplast transgenic plant material in hemophilia B mice. A. Delivery of hF.IX antigen to the GALT. Shown arePeyer's patch and villi of ileum of a fed mouse stained for hF.IX (red), M cells (UEA-1, green), and CD11c (blue). B. Survival of mice fed with wild-type (WT, n=10 mice at theonset of protein therapy), CTB-FIX (n=17), or CTB-FFIX (n=15) plant material as a function of the number of intravenous injections of hF.IX protein (CTB-FFIX contained afurin cleavage site between the CTB and hF.IX portions of the fusion protein). C. Inhibitor titers (in BU/ml) at 3-month time point mice (i.e. after 8 weekly IV injections of hF.IX)in unfed, WT fed, CTB-FIX fed, and CTB-FFIX fed hemophilia B mice. αhis/PAF: titers in unfed mice that received anti-histamine/anti-PAF prior to a 6th injection of hF.IX to preventanaphylaxis.Modified from Proc Natl Acad Sci USA 107(15): 7101-6, 2010; © 2010 by The National Academy of Sciences of the USA.


model, pDCs first abrogated a large fraction of antigen-specific CD8+

T cells in liver and MLNs, followed by induction of suppressive Treg[30]. CD103+ DCs produced retinoic acid, which is important in

Fig. 3. Mechanisms of Treg induction in oral tolerance: A. Low dose antigen feeding resultsexample, CD103+ gut DCs are specialized in inducing Treg via production of retinoic acid (Rtigen induces T cell anergy. Anergic T cells produce cytokines including IL-4, IL-10, TGF-β, asusceptibility to apoptosis. Macrophage and DC clean up the apoptotic cells and exhibit up-ralso secrete TGF-β, which is critical for inducing and maintaining Treg. These two mechanisistics such as cytokine production profiles and generation of induced Treg (iTreg).


boosting Foxp3 Treg [46]. CD103+ DCs also expressed indoleamine-2,3-dioxygenase (IDO), required for the induction of Foxp3 Treg anddevelopment of oral tolerance. TGF-β as well as prostaglandin E2, was

in active induction of Treg, which involves cross talk between different cell types. ForA), TGF-β, and expression of indoleamine-2,3-dioxygenase (IDO). B. High dose oral an-nd act as suppressor cells to evoke tolerance. High dose antigen feeding also increasesegulation of TGF-β and down regulation of inflammatory cytokines. Apoptotic cells canms may occur simultaneously and overlap as they shared some of the same character-





involved in inducing IDO expression in CD103+ DCs [47]. CD11b+ DCs,which produced IL-10 and IL-27, increased dramatically during oral tol-erance. IL-27 enhanced IL-10 production by Treg [48]. CD11b deficientanimals were unable to induce oral tolerance [49].

Gut mucosal environment is important in educating DCs (Fig. 1). Astheymigrate from the lamina propria to theMLNs, DCs are influenced bylocal factors and crosstalkingwith different cell types. Intestinal epithe-lial cells are in close contact with lamina propria DCs and have beenshown to condition DCs into “tolerogenic cells” by releasing TGF-β,retinoic acid, and thymic stromal lymphopoietin [50,51]. Other innatecells may play roles, such as lamina propria macrophages producinglarge amount of IL-10 and inhibiting DCs induction of TH-17 cells [52].MLN stromal cells are essential for DC induced CCR9 expression ofT cells and express high level of retinoic acid-producing enzymes [53].Orally immunized memory CD4+ T cells secrete IL-4 and IL-10 to edu-cate DCs, which further induce naïve T cells to produce the same cyto-kines that the immunized T cells are producing [54].

2.3. T cell responses

T cell responses play a central role in immunity. Oral tolerance is or-chestrated by distinct mechanisms (Fig. 3). Generally, when given ahigh dose oral antigen, it induces T cell deletion/anergy with IgA pro-duction; when adding certain adjuvants, the response can be convertedto systemic activation such as CTLs and IgG.When given lowdoses, it re-sults in active suppressionwith IgA secretion and induction of Treg pro-ducing IL-4, IL-10 and TGF-β [6]. These two mechanisms may occursimultaneously and overlap instead of being mutually exclusive, asthey share some of the same characteristics such as similar cytokineproduction, and anergic T cells perform regulatory function identicalto Treg [55], which will be discussed further below. Suppression of spe-cific effector T cell responses by oral antigen administration has a differ-ent susceptibility. For example, Th1 type inflammation can be easilysuppressed long-term with lower doses of antigen [56]. Th2 responsesneed higher amounts of antigen or increased feeding frequency [57], ex-cept for IL-4 dependent IgE response, which is highly susceptible to oraltolerance induction [58].

The concept of suppression mediated by T cells was first de-scribed in the 1970s [59]. More than 1000 scientific papers werepublished on that topic, mainly referring to CD8+ (Lyt-2) T cells[59,60]. However, due to lack of specific markers and poor character-ization, suppressor T cells were nearly abandoned by the end of the1980s [61]. In the mid-1990s, a new subset of suppressive CD4+ Tcells was characterized as regulatory T cells (or “Treg”). Besideshigh level of IL-2 receptor (IL-2Rα or CD25) expression, othermarkers were also reported such as transcriptional factor Foxp3,cytotoxic T lymphocyte antigen 4 (CTLA-4), and glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR)[59]. Commonly, CD4+ T cells now are divided into two distinct line-ages, conventional T helper (Th) cells and Treg [59]. Conventional Thcells regulate the adaptive immunity by activating other effector cellsincluding CD8+ CTLs, B cells, andmacrophages in an antigen dependentmanner. Treg are defined as T cells diminishing potentially harmful ac-tivity of Th cells [62]. Treg exhibit regulatory functions to suppress aneffector T cell response including inhibition of T cell proliferation andblockade of inflammatory cytokines release. Treg can transfer toleranceto naïve recipients [59]. Theymay secrete high levels of IL-10 and TGF-βand require TGF-β for their development [62].

It is well accepted that oral antigen administration activates or in-duces Treg, though themechanisms are not completely clear, most like-ly involving cross talk betweenmacrophages, DCs and T cells (Fig. 3). Asmentioned above, gut DCs have the intrinsic characteristic to inducetolerogenic T cell response rather than prime effector T cells. An inter-esting link between apoptosis and generation of Treg has been de-scribed in high dose feeding. Feeding high doses of antigen in miceseems to increase the susceptibility of lymphocytes to apoptosis [63].


Similarly, human lamina propria T cells show increased susceptibilityto Fas-mediated apoptosis upon CD2 pathway stimulation [64]. Macro-phages,which cleanup the apoptotic cells throughαvβ3 integrin, exhib-it up-regulation of TGF-β and down-regulation of pro-inflammatorycytokine production [65]. In addition, a subset of gut DCs was discov-ered to take up apoptotic enterocytes, and has relatively low stimulato-ry activity for T cells [66]. Apoptotic T cells also secrete TGF-β, as a resultfrom existing cytokine redistribution following loss of mitochondrialmembrane potential [67]. TGF-β released from macrophages, DCs andapoptotic T cells, is a critical cytokine in the differentiation and survivalof Treg. Another explanation for high dose oral tolerance is anergy ofspecific T cells. Anergy is the basis of peripheral tolerance toself-antigens. It also plays a role in oral tolerance, first indicated that Tcell tolerance can be reversed by exogenous IL-2 in vitro [68]. AnergicT cells are not just passive in the tolerance they evoke. They produce cy-tokines including IL-4, IL-10, TGF-β and act as suppressor cells in vivoand in vitro [69,70]. Anergic CD4+ T cells frommice tolerized by feedingcasein canmediate active suppression when transferred to severe com-bined immunodeficiency (SCID) mice [71].

Various subsets of Treg in oral tolerance have been reported, such asthymus derived Treg or natural Treg, induced Foxp3+ Treg, Tr1 cells,and Th3 cells (Table 1). Thymus derived CD4+CD25+Foxp3+ Treg ornatural Treg play critical role in abrogation of auto-immunity andmain-tain self-tolerance [72]. In addition, they involve in suppressing im-mune responses toward commensal bacteria in the gut [73]. They arereported to be unregulated in oral/mucosal tolerance [11]. However,in a mice model containing a monoclonal population of CD4+ T cellsspecific for OVA crossed on a RAG-1 deficient background, it was dem-onstrated that oral tolerance to OVA was effectively induced withoutthymus-derived Treg [74].

Peripherally induced or adaptive Treg are essential for oral tolerance[75]. They develop outside the thymus within specific microenviron-ment. For example, CD4+CD25+Foxp3+ Treg were markedly inducedin Peyer's patches of OVA TCR transgenic mice fed high doses of OVA.They have suppressive properties in vitro and mediate tolerance trans-fer to naive animals [76]. CD4+CD25+CTLA4+ Treg were upregulatedas long as 4 weeks after OVA feeding. They expressed high level ofTGF-β and IL-10 and mediate adoptive tolerance transfer in BALB/cmice [77]. In a high dose OVA feeding model, induced CD4+CD25+

Treg expressed TGF-β and were anergic T cells. Their oral tolerancewas IL-18 dependent [78]. One subset of CD4+ T cells producingTGF-β is termed Th3 cells, which was initially isolated and clonedfrom the lymphoid tissues of mice tolerated by low dose antigen feed-ing (Table 1) [79]. They appear to be dependent on IL-4 rather thanIL-2 for growth and may produce variable amounts of Th2 cytokineslike IL-4 and IL-10 [6]. Another subset of IL-10 producing type 1 regula-tory T cells, named Tr1 cells, mediate suppression through IL-10 and in-duce Foxp3+ Treg, is reported to be involved in lose dose oral tolerance(Table 1) [74,80]. A membrane-bound form of TGF-β containinglatency-associated peptide (LAP) has been identified [81]. LAP+ Tregoccur in both CD25+ or CD25− cells (Table 1) [82]. All these subsetsare not mutually exclusive as they produce similar cytokines. For in-stance, Foxp3+ Treg, Th3 cells, and Tr1 cells, all secrete IL-10. As a resultof different experimental settings and lack of exclusivemarkers, one canhardly determinewhat percentage a certain cell type contributes in oraltolerance. Moreover, various subsets might act in synergy andconnected to each other in performing their functions. As such, Th3and Tr1 cells induce Foxp3+ Treg through TGF-β and IL-10 [83].

Additionally, a subpopulation of CD8+ lymphocytes might be alsoinvolved in tolerance induction. Intestinal epithelial cells (IEC) acti-vated CD8+ T cells with a regulatory function [84]. Feeding a MHCclass I immunodominant peptide of OVA induced CD8+ T cells withregulatory function. These cells inhibited Th1/Th17 responses butnot Th2 responses [85]. Defects in CD8+ T cells were observed in in-flammatory bowel disease patients and correlated with a failure toinduce oral tolerance in these patients [86]. However, anti-CD8





antibody studies demonstrated that CD8+ cells are not essential for oraltolerance induction and maintenance of systemic tolerance [87,88].

2.4. Cytokines

2.4.1. Transforming growth factor-β (TGF-β)TGF-β is a multifunctional polypeptide involved in various cellular

processes, such as extracellular matrix production, angiogenesis, dif-ferentiation, proliferation, apoptosis, and immunomodulation [89].Three isoforms exist in mammals including TGF-β1, TGF-β2, andTGF-β3. TGF-β1 is the foremost in leukocyte populations [89]. TGF-βhas emerged as a key regulator of both innate and adaptive immuneresponses, in a context dependent manner [89]. Abundant in the gutmicroenvironment, TGF-β is pivotal in epithelial cell differentiation,IgA class switching, and has strong immunosuppressive effects onlymphocytes [90]. TGF-β mediates immune tolerance via inductionand maintenance of Foxp3+ Treg. In vitro studies revealed thatTGF-β activates naïve T cells into Foxp3+ expressing T cells with sup-pressive capacity [91]. Neutralization of TGF-β during induction oforal tolerance diminished Foxp3 expression in MLN and spleen [74].TGF-β inhibits T cell proliferation with no exogenous IL-2, and de-creases the differentiation of naïve T cells into effector Th1 and Th2cells via the downregulation of T-bet and GATA3 [92–94]. TGF-β is se-creted as an inactivated form. Latent TGF-β can be activated by gutDCs and IECs through integrins αvβ8 and α4β6 [95,96]. Mucosal DCsexpressed more αvβ8 than splenic DCs and release more bioactiveTGF-β, which might in part explain why mucosal DC induce moreFoxp3+ Treg [95].

Involvement of TGF-β in oral tolerancewas extensively documented(Fig. 4). TGF-β production was unregulated in Peyer's patches, laminapropria and draining lymphoid tissues after induction of oral tolerance[97,98]. TGF-β producing Th3, other CD4+ cells, and CD8+ T cellswere isolated and cloned from the Peyer's path and MLN of orallytolerized mice, and peripheral blood of humans fed MBP [6,99]. In theexperimental models of EAE and IBD, oral antigen feeding reduced dis-eases dependent on TGF-β productions, as neutralized TGF-βwith anti-bodies impaired the development of oral tolerance [100,101]. TGF-βdeficient mice die soon after birth from wide spread inflammation[102]. Using an anti-LFA-1 antibody to inhibit inflammation in TGF-βdeficient mice, it was shown that low doses and high dose feeding ofOVA lymphocytes had reduced proliferation [103]. However, some

Fig. 4. Induction and multifunction of TGF-β in the gut: TGF-β is a key cytokine for oral toleepithelial cell differentiation, IgA class switching, and induction and maintenance of Foxp3+

and Th2 cells, drives the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocmacrophages, enterocytes, antigen-pulsed intestinal epithelial cells (IEC), and gut DCs. GutMLN DCs, interaction with Foxp3+ Treg increases expression of indoleamine-2,3-dioxygenresponses. Besides the secreted form, an active membrane-bound form (latency-associatedassociated with the suppressive activity of Treg. LAP+ Treg are expanded in oral tolerance.


mice in low dose feeding exhibited no tolerance or even priming sys-temic proliferation responses; and the degree of tolerancewas only par-tial for all systemic responses compared with wild type mice [103].Thus, TGF-β plays a critical role in oral tolerance, although it may notbe the exclusive mechanism involved.

The source of TGF-β in gut is not only from CD4+ or CD8+ T cells.Many other cell types such as macrophages and enterocytes can alsoproduce this cytokine [104]. One study even indicated that antigen-pulsed epithelial cells might inhibit T cell activation through secretionof TGF-β [105].

In addition to the secreted form of TGF-β, an active membrane-bound form was also described. Latency-associated peptide (LAP) isthe amino-terminal domain of the TGF-β precursor peptide. Aftercleavage, LAP forms the latent complex with TGF-β via noncovalentassociation [82]. Certain Treg express LAP and TGF-β on their cell sur-face, which is associated with the suppressive activity of Treg [81,82].LAP+ Treg are expanded in oral tolerance, both CD4+CD25+ andCD4+CD25− [82]. Anti-TGF-β antibody can reverse the suppressiveeffects of LAP+ Treg [83].

2.4.2. Interleukin-10 (IL-10)IL-10 initially has been described as a Th2 type cytokine to sup-

press Th1 type responses, including cytokines IL-2 and IFN-γ produc-tion and cell proliferation in oral tolerance, which is still widely usedin measurement of oral tolerance induction by many researchers[54,106]. Low dose antigen feeding induced IL-10 and IL-4 in tolerat-ed mice [107]. However, oral tolerance also inhibits Th2 type re-sponses although the susceptibility is different [57]. The role of Th2cells in mediating oral tolerance is further questioned since neutraliz-ing IL-4 with antibody or IL-4 deficient mice, which are models forTh2 cell deficiency, still develops oral tolerance [108].

IL-10 is now regarded as a broad anti-inflammatory cytokine(Fig. 5). It not only inhibits Th1 responses, but also down-regulateMHC class II and costimulatory molecule expression [109]. BesidesTh2 cells, IL-10 can be secreted from a variety of cells. Specifically,one regulatory subset of CD4+ T cells mainly producing IL-10, isnamed Tr1 cells [110]. Other populations of CD4+ T cells are capableof secreting IL-10, such as Th3 cells, Th17 cells, and Foxp3+ Treg[109]. Epithelial cells, macrophages, NK cells, B cells, DCs and CD8+

T cells, all have been reported to produce IL-10 [109]. An interestinglinking has been revealed between IL-10 producing mucosal APC

rance. Abundant in the gut microenvironment, TGF-β has multiple functions, includingTreg. Additionally, TGF-β decreases the differentiation of naïve T cells into effector Th1ytes (IEL). TGF-β is secreted by a variety of cells including CD4+, CD8+ T cells, Th3 cells,DCs produce retinoic acid (RA), which enhances Foxp3+ Treg induction by TGF-β. Inase (IDO), which depletes tryptophan into kynurenines and suppresses effector T cellpeptide, LAP) exists. Some Treg express LAP and TGF-β on their cell surface, which is


ThanhLuan

Highlight



Fig. 5. IL-10 expression and function in the gut: IL-10 is a cytokine with broad anti-inflammatory properties and constitutes another key component in oral tolerance. Highlyexpressed in the intestine, IL-10 producing cells increase even more after antigen feeding, especially in Peyer's patch (PP), lamina propria, and MLN. Specifically, one regulatorysubset of CD4+ T cells mainly producing IL-10, named Tr1 cells, is induced in oral tolerance. Other regulatory T cells such as Th3 or Foxp3+ Treg may also secrete IL-10 and areinduced by antigen feeding. Macrophages, B cells, DCs and CD8+ T cells, epithelial cells, and NK cells all have been reported to produce IL-10. IL-10 can be produced by Th2,Th17, and Th1 cells in certain situations, which serves as a negative feedback loop to limit effector T cell responses, possibly via regulation of antigen presenting DCs and macro-phages. IL-10 is critical for induction of certain subsets of regulatory T cells and/or their function, which provides a positive regulatory loop for establishment of oral tolerance.


and IL-10 producing T cells, as plasmacytoid DCs from MLN, CD11b+

DCs from PP, and CD11b+F4/80+ lamina propria macrophages can in-duce IL-10 producing T cells through their own secretion of IL-10 [52].

Highly expressed in the intestine, IL-10 is critical in establishingtolerance to intestinal commensal bacteria. IL-10 deficient mice de-velop spontaneous colitis associated with abnormal T cell activation[111]. Exogenous IL-10 was able to restore tolerance in the colitismice [109]. It has been reported that all IL-10 producing T cells inthe colonic lamina propria express Foxp3, but not in the small intes-tine [112]. Selective deletion of IL-10 in Foxp3+ T cells in the colonleads to spontaneous colitis, indicating that IL-10 is important inFoxp3+ Treg suppressive function in the colon [113]. In addition,IL-10 producing macrophages in lamina propria are crucial insupporting the Foxp3+ Treg function as well [114].

The role of IL-10 in oral tolerance is controversial. Lots of IL-10producing T cells exist in the gut microenvironment [115]. Antigenfeeding increases IL-10 production even more, especially in PP,MLN, also in spleen and serum [80]. Tr1 cells are induced in oral tol-erance [116]. However, oral tolerance can still be induced in micetreated with anti-IL-10 antibodies and anti-IL-10 antibodies do notabrogate established tolerance in vivo [117]. But in one study withIL-10 deficient mice treated recombinant IL-2, multiple feeding of an-tigen failed to induce tolerance [80]. Another IL-4 and IL-10 knockoutmouse study demonstrated that both cytokines are required for lowdose oral tolerance [118].

Cytokines are essential for oral tolerance induction. Cross-talkingbetween different cytokine producing cells occurs in oral tolerance.TGF-β may induce the differentiation of IL-10 producing cells[112,119]. IL-10 may be involved in TGF-β production as anti-IL-10treatment decreased TGF-β levels [120].

Besides the twomajor suppressive cytokines TGF-β and IL-10, othercytokines may also be involved. IFN-γ regulates the expression level ofadhesionmolecules essential for Tregmigration [121]. It is reported thatoral tolerance cannot be induced in IFN-γ deficient mice or adoptivetransfer of TCR transgenic T cells with an IFN-γ deficient background[121,122]. However, other reports described that deficiency of IFN-γdoes not affect oral tolerance induction or maintenance [123,124].Some anti-tolerance cytokines have also been described. IL-6 andIL-17 treatment can decrease the Treg cell population and impair toler-ance [125]. Oral tolerance suppresses Th17 cells, which secrete highamounts of IL-17 and IL-22 [49].


2.5. Other immune regulatory pathways

Many signal pathways are important in regulating immune re-sponses for oral tolerance (Table 1). Cyclooxyenase-2 (COX-2) is theinducible enzyme in arachidonic acid metabolism. PGE2, the productof COX-2 was shown to directly induce Foxp3 expression in T cells[126,127]. Selective COX-2 inhibition led to loss of tolerance to oralantigen administration [126]. NF-κB and MAPK pathways are essen-tial for Treg development and Akt has a negative effect on Treg[128]. Retinoic acid and Foxp3 are two pathways which were inten-sively studied, and will be discussed further below.

2.5.1. Retinoic acid (RA)Retinoic acid (RA) is the metabolite of vitamin A by alcohol dehy-

drogenase (ADH) and retinal dehydrogenase (RALDH) [129]. It is crit-ical for proper function of mucosal DCs in oral tolerance. Mucosal DCinduction of gut homing receptors includingαEβ7 and CCR9 in T and Bcells is dependent on the ability of DCs to convert vitamin A into RA[130,131]. RA regulates Th17 cell responses, contingent on the localcytokine environment [132,133]. TGF-β mediated Foxp3+ Treg in-duction can be boosted by RA [134,135]. It is well known that mucosalDCs have a greater capacity to induce Foxp3 expression in T cells thannon-mucosal DCs, which is associated with the DCs ability to convertvitamin A into RA [134]. CD103+ DCs in MLN express high level ofAldhla2, the gene encoding RALDH2 [46]. So that CD103+ DCs butnot CD103− DCs can induce Foxp3+ Treg in the absence of exogenousRA and TGF-β. CD103− DCs have weaker induction of Foxp3+ Tregcompared with CD103+ DCs even in the presence of exogenousTGF-β [46].

Multiple pathways might mediate the mechanisms of RA en-hancement of Foxp3+ Treg induction by TGF-β. First, RA inhibits theproduction of some cytokines in effector and memory T cells, whichsuppress naïve T cell differentiation into Foxp3+ Treg [136,137]. Fur-thermore, RA suppresses the effects of pro-inflammatory cytokineson naïve T cells, such as down-regulation of IL-6 receptor levels[138]. Vitamin A deficiency results in increased inflammation [129].In addition, RA increases TGF-β signaling by raising the expressionof Smad3 [138]. RA up-regulates Foxp3 expression directly via bind-ing to the promoter region with RA receptor α [139]. Moreover, RAattenuates the co-stimulation from interfering Foxp3+ Treg differen-tiation [135]. However, there is a report that RA's effects can be





independent on inhibitory cytokines and RA induction of Foxp3 ex-pression is largely Smad3 independent [140]. Thus, the effects of RAare most likely integrative instead of just relying on a single pathway.

Besides mucosal DCs, other cell types in the gut are able to convertvitamin A into RA, such as intestinal epithelial cells, MLN stromal cells,and lamina propria macrophages [51–53]. The epithelial cells and stro-mal cells generated RA might be transferred to DC as a mechanism oflocal education of DCs. DCs were reported to take RA from outside,store it, and acquire tolerogenic characteristics [141]. Macrophagesmight function in a similar way with DCs in that macrophages induceFoxp3+ Treg dependent on RA and TGF-β [52]. The difference is macro-phage require IL-10 in inducing Treg besides RA and TGF-β [52].

2.5.2. Treg expressing transcription factor Foxp3Foxp3 belongs to the forkhead/winged-helix family of transcription-

al factors and is highly conserved in humans [142,143]. Acting as the ac-tivator or suppressor, Foxp3 binds to the promoter region of 700–1100genes, most of the genes associated with TCR signaling [142,143]. Theexpression of Foxp3 is largely in T cells, mainly in CD4+ T cells, someCD8+ T cells also express it, very low or undetectable in B cells, naturalkiller cells, macrophages and DCs [142,143]. To date, Foxp3 is consid-ered one of the most reliable Treg markers, although some reportsfound Foxp3 also exist in non-Treg [144]. There is an overlap of expres-sion between Foxp3 and CD25, the traditional Treg marker. In lymphnodes and spleen, most CD4+CD25+ cells express Foxp3 [144,145]. Al-though Foxp3 can be positive in CD4+CD25+ and CD4+CD25− cells, itis more abundant in CD4+CD25+ cells in mice [145].

Foxp3 is a pivotal factor for Treg function. Foxp3 mutationscause X-linked autoimmunity allergic dysregulation syndromeand immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, which are severe autoimmune diseases in humans[146–148]. Scurfy mice, the mice orthologue of Foxp3 deficiency, de-velop lymphoproliferative disorder and die one month after birth[149]. T cell targeted deficiency of Foxp3 lead to lymphoproliferativedisease [144]. Retroviral-mediated Foxp3 expression in naïve T cellsresulted in those T cells acquiring Treg-like phenotype and functions[150,151]. Foxp3 transgenic mice show markedly increased numberof CD4+CD25+ Treg, and CD8+ T cells expressing Foxp3 also demon-strate immunosuppressive activity [152]. Continuous Foxp3 expres-sion is associated with maintenance of the Treg in the periphery[153]. Expression of Foxp3 is stabilized by demethylation, and thisepigenetic modification is essential for the development of a perma-nent Treg cell lineage [154].

Foxp3+ Treg can convert to conventional T cells. In vitro TGF-β in-duced Treg with partial promoter demethylation lost Foxp3 expressionand suppressive activity upon restimulation in the absence of TGF-β[154]. A large fraction of labeled Foxp3+ Treg became Foxp3− after a4 week transfer into T cell deficient mice. Some of them convertedto Th1 cells, Th2 cells, and Th17 cells in lymph nodes and spleen[155,156]. Some converted to Tfh cells that function in assisting IgA pro-duction by B cells in Peyer's patches [157]. The mechanism of this con-version is not clear, possibly involving inflammatory cytokines. IL-6 candecrease Foxp3 expression and reprogram Treg into Th17 cells [158]. Inthe presence of inflammatory signals, Treg differentiated into Th17 cellsin vivo [159].

Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR; also called TNFRSF18 or CD357) is constitutive-ly expressed on Treg cells and is inducible upon activation on naïveCD4+ T cells [160–162]. Although the number of Foxp3+ Treg is nor-mal under steady state, it is significantly induced in wild-type miceafter administration of soluble Fc-GITR-L fusion protein, which hasbeen shown to transiently induce tolerance to the coagulation factorIX expression in a murine model of hemophilia B [163]. TransgenichCD19-GITR-L mice also showed increased numbers of Foxp3+ Tregand were more resistant to the induction of experimental autoim-mune encephalitis (EAE) [164]. In addition to regulating the balance


between Teff and Treg cells, the GITR-L/GITR interaction also controlsthe development of DC, macrophages and/or monocytes. Therefore,the numbers of tolerogenic CD103+ DCs and pDCs are reduced inGITR deficient mice, thereby causing the expansion of Th1 cells andinhibiting the development of Foxp3+ Treg after their exposure to thelarge amount of food antigens as well as to commensal bacteria[165]. GITR-L/GITR engagement may also potentially influence theestablishment of oral tolerance by a reverse signaling through GITR-Lin pDC. For example, GITR-L engagement by GITR::Fc fusion protein in-duced expression of IDO in pDC, a pathway thatmay also aid in inducingoral tolerance [166].

3. Therapeutic proteins and disease targets

Hemophilia A and B are X-linked bleeding diseases resulting fromdeficiency of coagulation factor VIII (F.VIII) or factor IX (F.IX), respec-tively. Current clinical treatment is based on periodical intravenousadministration of the deficient coagulation factor. The most problem-atic complication associated with therapeutic protein replacement isthe development of neutralizing antibodies (inhibitors), which occursin treatment of 25–30% of hemophilia A and of 2–4% of hemophilia Bpatients. Some hemophilia B patients with inhibitors are also at riskfor severe allergic/anaphylactic reactions to F.IX, contributing to ahigher morbidity and mortality rate. Inhibitors develop due to the in-duction of adaptive immune responses against the therapeutic pro-teins, which are perceived as non-self antigens. High-level inhibitorsdecrease the therapeutic effects of coagulation factors and require ei-ther bypass therapy or immune tolerance induction (ITI). However,several disadvantages limited these applications: high cost; inabilityto induce tolerance in some patients; and requirement for frequentmonitoring [167,168].

Pompe disease, which is an autosomal recessive lysosomal storagedisorder with a deficiency of acid α-glucosidase (GAA), causes deathearly in childhood due to hypertrophic cardiomyopathy and respiratoryfailure. Similarly, enzyme replacement therapy (ERT) based on intrave-nous infusion of recombinant human GAA, is the standard therapy,which prolongs the life of affected infants. However, anti-GAA antibodyresponses develop and compromise the beneficial effects. Only recently,immune tolerance protocols to prevent anti-GAA antibody formation inpatients with infantile onset disease are being developed to main-tain the efficacy of ERT [10]. However, these heavily rely on immunesuppression. One study addressed oral tolerance as an alternative(Table 2). Oral recombinant human GAA administration successfullylowered anti-GAA antibody titers in C57BL/6 and BALB/c mice. GAAspecific IgE titers in post-immune serum were lowered too [169].

Oral tolerance for inherited protein deficiencies offers many advan-tages in suppressing adaptive immune responses: low costs; low ad-verse effects; easy delivery; and higher efficacy in achieving antigenspecific peripheral tolerance. Several studies have been performed inhemophilia (Table 2). F.VIII orally administered with sodium bicarbon-ate at a dose of 1250 IU once daily for 3 months successfully decreasedF.VIII inhibitors titers in two of three patients, lasting for 6 months aftertermination of the treatment. The other patient showed no decrease ofinhibitors but still had a decreased in vivo proliferation rate of mononu-cleated cells and reduced cytokine production, including IL-6, IL-1β andTNF-α [170]. Another study reported that orally administered F.VIII atintervals ranging from 1 day to 1 week, and dosage increasing from20 to 70 units/kg, markedly diminished the inhibitor from 42 BU/mlto 11 BU/ml in one patient. However, the inhibitor relapsed whenhigh dosage of prothrombin complex concentrates (which containsF.VIII light chain fragment) was administered for control of bleeding[171]. Mouse studies showed that F.VIII-C2 oral and nasal applicationssignificantly suppressed antibody and inhibitor formation to this do-main but not to the entire F.VIII protein. Tolerance was associatedwith increased IL-10 production and could be adoptively transferred.However, tolerance was not sustained after additional F.VIII-C2




Table 2Studies on oral administration of therapeutic protein to induce tolerance in treatment of disease.

Therapeuticprotein

Disease Species Feeding regimen Results Reference

F.VIII Hemophilia A Human 1250 IU F.VIII with sodium bicarbonate once daily

for 3 months

2 of 3 patients had decreased inhibitor titer lasted for 6 months;the other patients had decreased in vivo proliferation rate ofmononucleated cells and reduced inflammatory cytokine productions.

[170]

F.VIII Hemophilia A Human 20 to 70 U/kg F.VIII orally administered at intervalsfrom 1 day to 1 week

Diminished inhibitors titers from 42 BU/ml to 11 BU/ml in onepatient; Inhibitors come back when high dose of prothrombincomplex concentrates was used.

[171]

F.VIII C2 Hemophilia A Mice Oral or nasal 1–50 μg F.VIII C2 for 5 consecutive days Decreased the titer of anti-F.VIII C2 inhibitors and antibodies; tolerancewas associated with increased IL-10 and can be adoptively transferred.

[172]

F.VIII Hemophilia A Mice 1 to 2 μg F.VIII oral gavage neonatal mice every 2 to3 days for 9 total feedings

No protection against inhibitors formation developed after F.VIII ivinjection.

[196]

F.IX Hemophilia B Mice Tobacco leaves contain CTB-F.IX or CTB-FF.IX twice perweek for 2 months; equivalent to 5–80 μg/kgrecombinant F.IX

Eliminated fatal anaphylactic reactions; blocked formation of inhibitoryantibodies undetectable or up to 100-fold less than controls; controlledinhibitor formation and anaphylaxis long-term, up to 7 months, about40% life span of this mouse strain.

[32]

GAA Pompedisease

Mice 16 mg recombinant human GAA 5 times every otherday for 6 days

Significantly lower anti-GAA antibody titers; reduced specific IgEagainst GAA titers. Not effective with 1 mg and 10 mg oral GAA 3times every other day.

[169]

Insulin Diabetes Human 2.5 or 7.5 mg oral insulin or placebo daily for 1 year inautoantibody positive patients

No differences were seen in the time course or titers of antibodiesto insulin, glutamic acid decarboxylase or islet antigen 2. Similarinsulin requirements, hemoglobin A1c concentrations, and C peptideconcentrations.

[197]

Insulin Diabetes Human 1 or 10 mg insulin tolerance trial in newly diagnoseddiabetic patients with cytoplasmic islet cell autoantibodies

Improved plasma C-peptide responses in patients diagnosed atages greater than 20 years, best seen at low (1 mg/daily) over high(10 mg/day) dose. There were no adverse effects.

[198]

Insulin Diabetes Human Oral insulin or placebo treatment of type 1 diabetes for1 year

In oral insulin group, significantly higher TGF-β; markedly reducedIFN-γ; similar levels of IL-4 and IL-5 in challenged lymphocytes;significantly lower circulating levels of IgG1 and IgG3 against insulin.

[199]

Insulin Diabetes Mice Tobacco or lettuce leaves contain CTB-proinsulin(about 14 μg of fusion protein) or control once a weekfor 7 weeks

Decreased infiltration of cells characteristics of lymphocytes;insulin producing β cells were preserved; increased expression ofimmunosuppressive cytokines

[173]

Insulin Diabetes Mice Silkworm produced GFP–CTB–insulin (about 50 μg offusion protein) every other day for 5 weeks

Induced special tolerance; delayed the development of diabeticsymptoms; increased the numbers of CD4+CD25+Foxp3+ Treg.

[200]

Insulin Diabetes Mice Potato expressed CTB–insulin, CTB, or insulin(20–30 μg) once per week for 5 weeks

Only CTB–insulin group showed a reduction in insulitis and a delayin the diabetes progression, but not insulin or CTB alone group.

[201]

Insulin Diabetes Mice Glutamic acid decarboxylase expressing transgenicplant as a dietary supplement

Inhibits the development of diabetes in the non-obese diabeticmouse

[202]

Insulin Diabetes Mice 1 mg porcine insulin orally twice a week for 5 weeks,then weekly until 1 year.

Reduced lymphocytic infiltration of pancreatic islets; splenic Tcells from orally treated animal adoptively transfer protectionagainst diabetes.

[178]

Insulin Diabetes Mice 0.8 mg human insulin in PBS 3 times a week for2–4 weeks

Induced insulin B-chain reactive regulatory T cells to blockcytokine secretion and migration of diabetogenic effector T cells

[203]

Insulin Diabetes Mice 600 μg insulin B chain or 250 μg peptide 5 consecutivedays, once a week thereafter

Slowed diabetes development in a co-transfer model of diabetes inNODmouse; a decrease in Th1 cytokine and increase in Th2 cytokines.

[204]

Insulin Diabetes Mice 1 mg equine insulin or ovalbumin twice a week for5 weeks

Considerably less insulitis in insulin fed NOD mice; residualmononuclear cells expressing IL-4, IL-10, PGE, TGF-β, and anabsence of IL-2, IFN-γ or TNF-α.

[205]

Insulin Diabetes Rat 0.5 or 1 mg insulin 3 times weekly for 90 days No protection effect was observed in diabetes prone or diabetesresistant BB rats

[206]

Egg white Egg allergy Human 2 g egg white powder per day for 10 months; 10 g eggwhite power till 22 months

55% in oral-immunotherapy group passed the oral food challengeafter 10 months therapy; 75% of children were desensitized in theoral-immunotherapy group at 24 months.

[207]


injections [172]. Oral delivery of human F.IX bioencapsulated in chloro-plast transgenic plant cells was successful in controlling inhibitor for-mation to factor replacement therapy in hemophilia B mice with F9gene deletion long-term [32]. Moreover, this treatment also preventedIgE formation and the associated life-threatening anaphylactic reactionsthat normally occur in response to repeated intravenous administrationof human F.IX in these mice (Fig. 2).

In contrast to the still limited data in genetic disorders caused by sin-gle gene deficiencies, oral tolerance has initially and most intensivelybeen studied in autoimmune disease. These investigations includewell-established animal models and clinical trial trials, particularly inmultiple sclerosis (MS), rheumatoid arthritis (RA), and diabetes(Table 2). For example, oral insulin has been used to slow or prevent di-abetes in the non-obese diabetic (NOD) and in a viral induced diabeticmouse models, accompanied by decreased IFN-γ and increased IL-4,IL-10 and TGF-β responses to the insulin antigen. In otherwork, oral an-tigens tolerized mice showed markedly reduced disease severity incollagen-induced arthritis model, adjuvant arthritis model, pristine-


induced arthritis model, and silicone-induced arthritis [11,12]. Studiesfrom different groups demonstrated that oral myelin basic protein(MBP) significantly suppressed experimental autoimmune encephalitis(EAE), which is a murinemodel for MS [6]. Human trials have also beenperformed in autoimmune diseases, MS, RA, and diabetes [11,12]. Al-though clinical efficacy has been limited, and approval of an oral toler-ance drug is still a distance away from reality, these trials continue toyield valuable information. For example, no systemic toxicity and exac-erbation of diseases have been observed. Several small studies showedpositive effects but also indicate a need for further optimization of dos-age, scheduling of the regimen, and delivery methodologies [11,12].

4. Delivery methods

The majority of the animal experiments and human trials wereconducted using protein concentrates or solutions. Although the sup-pressions of immune responses from animal studies were dramaticand sufficient to protect from diseases, clinical trials in patients





were generally of limited success. One possible explanation is that thedoses of antigens applied to humans were low compared with that ofmice, due to the large absorptive intestinal mucosal area [7,173]. Thusone critical question to address in oral tolerance clinical applicationsis to enhance the efficacy or to obtain sufficient amounts of antigenfor repeated oral administration.

Transgenic plants are an attractive system for the production of oralantigens. Plants are easy to produce for large scale-up at low cost withno need of expensive culture media. They are highly amenable to oralapplication without extensive protein purification. They are stable andeasy to store. The protein and peptide synthesized in plants can bestructurally and functionally identical to their native counterparts, andare protected by bioencapsulation. Indeed, many publications havereported the production of pharmaceutical proteins such as antibodies,cytokines, enzymes, vaccine antigens, hormones etc. in plant cells. Someof them have entered clinical development stage. Transgenic plants in-cluding tobacco, rice, potato, lettuce, and tomato have all been created,either as a stable transgenic like nuclear system or transient expressionusing plant virus vectors [174]. Several interesting studies have triedplant material in oral tolerance therapy in animal models of allergies,rheumatoid arthritis, diabetes and hemophilia B. One study, usingtransgenic rice with mite allergen encapsulated in endoplasmicreticulum derived protein bodies in mice, markedly suppressedallergen-specific IgE and IgG production, allergen-induced CD4+ T cellproliferation and production of Th2 cytokines, infiltration of eosino-phils, neutrophils, andmononuclear cells into the airways [175]. Anoth-er group generated transgenic rice with T cell epitopes of cedar pollenfused with cholera toxin B (CTB), suppressed allergen specific IgE re-sponses and pollen induced clinical asthma symptoms [176]. Oral appli-cation of CTB-fused proinsulin fusion protein expressed in lettuce andtobacoo chloroplasts, significantly inhibited insulitis in NODmice. Insu-lin producing β cells were greatly preserved; blood and urine glucoselevels were reduced; and IL-4 and IL-10 were increased [173]. FeedingDBA/1 mice, which are models of rheumatoid arthritis, with transgenicrice seeds of collagen type II peptides, lowered serum specific IgG2a re-sponses against subsequent and repeated intraperitoneal injection oftype II collagen [177]. Our studies utilized oral delivery of chloroplasttransgenic (“transplastomic”) leaf material containing CTB-fused F.IXto control inhibitor formation and anaphylaxis to human F.IX in hemo-philia B mice in a highly effective manner for at least 7 months, about40% of the life span for the mouse strain used [32]. From these earlyplant studies, it appears that the effective dosage required for plant de-livery system is considerably lower than for conventional protein orpeptide concentrates, especially when the plant expressed protein orpeptide was fused with a mucosal carrier like CTB. For example, 1 mgof porcine insulin twice a week for 5 weeks, and then weekly for1 year was used to effectively suppress diabetes in NOD mice [178];whereas only about 14 μg of the CTB-proinsulin fusion proteinexpressed in tobacco once a week for 7 weeks markedly suppressedinsulitis in NOD mice [173]. In the hemophilia A mouse studies,500 μg of F.VIII C2 peptide protein concentrations was used for oral ad-ministration to induce partial immune tolerance in hemophilia A micemodel; whereas we used only 0.14 μg to 2 μg F.IX with CTB fusionexpressed in tobacco induced complete oral tolerance in the hemophiliaB mouse model [32,172]. 100 μg collagen type II (250–270) syntheticpeptide every other day for 20 days (total 1 mg) vs 25 μg same peptideexpressed in rice seeds per day for 2 weeks (total 350 μg) successfullysuppressed immune responses against type II collagen in DBA/1 mice[177,179].

CTB, the nontoxic cholera toxin B subunit, is a potent antigen-adjuvant. It binds to GM1 ganglioside expressed on the live intestinalepithelial cells, and facilitates uptake via an actin- and ATP-dependentprocesses [173,180]. The binding of CTB does not alter the organizationof the plasma membrane and has minimal effect on the diffusion ofother molecules [180]. CTB-coupled antigens have been shown to sup-press DC activation and promote induction of Foxp3+ Treg [181–183].


One of the reasons for requirement of lower dosage of proteinbioencapsulated in plant cells is protection of antigens from degrada-tion in the stomach by acids and enzymes [184]. Also, transplastomicplants are capable of very high levels of expression of therapeutic pro-teins, up to 70% of the total leaf protein [185]. In addition, multi-geneengineering to express several antigens in a single transformationevent is a major advantage [185–187]. Plant cells can be lyophilizedand stored at room temperature for several months or years withoutdegradation of antigens or autoantigens [188]. Most importantly, har-vest of leaves before emergence of reproductive structures and ma-ternal inheritance of transformed chloroplast genomes containinggenes coding for therapeutic proteins offer several layers of biologicalcontainment of transgenes [189]; such containment addresses one ofthe major environmental concerns in using genetically modifiedplants. Therefore, plant delivery system has several unique advan-tages in delivering autoantigens to confer oral tolerance.

Nanometer-sized particles like proteins and viruses are activelyinvolved in diverse cellular activities. Recently, synthetic nano-particles were developed to transport drugs and proteins of interestinto target cells. Oral formulation of nanoparticles encapsulatesdrugs or proteins, protects and releases them in a temporally or spa-tially controlled manner. Particle size, surface charge, and surfacechemistry all influence delivery efficacy. The particle surface can bemodified or coated to enhancing specific targeting. For example, coat-ing insulin-nanoparticles with pH sensitive material or conjugatingwith vitamin B-12 or derivatives can greatly enhance insulin absorp-tion and bioavailability. To date, the well recognized oral nanoparticleformulation is complex, multilayered, mucoadhesive, biodegradable,biocompatible, and acid-protected [190]. A few oral tolerance studieshave used nanoparticle as a delivery method. Polylactic-co-glycolicacid (PLGA) nanoparticles delivered collagen type II protected exper-imental arthritis in mice [191]. Another study used poly-ethylene gly-col (PEP) conjugated collagen peptide suppressed collagen-inducedarthritis [192]. Besides protein, peptide or drugs, cDNA can be effec-tively delivered using chitosan, which is a cationic polymer that inter-acts with cDNA via electorstatic interactions, protects entrappedcDNA from digestion and facilitates gut delivery. Using chitosan-based nanoparticles, F.VIII DNA was successfully delivered into hemo-philia A knockout mice with functional F.VIII protein detected in plas-ma at a peak level of 2–4% F.VIII activity. 13 out of 30 mice showed aphenotypic correction in bleeding challenge [193]. Another study,using canine F.VIII cDNA formulated in chitosan, provided transientF.VIII activity that was however sustainable upon re-administration.No neutralizing F.VIII antibody was detected, suggesting oral toler-ance involvement [194].

In conclusion, elucidation of the mechanisms of oral tolerance willfurther facilitate the success of oral tolerance as a means of control-ling or preventing immune responses to therapeutic proteins. Furtherinvestigations are needed in several areas such as the characterizationof therapeutic antigens, modes of antigen processing and presenta-tion, co-stimulatory requirements, induction of cytokine responses,and others. In this context, murine studies may be supplementedwith translational studies in large animal models of disease. As weunderstand more, we will get closer to effective clinical developmentof oral tolerance in treatment of human diseases. For example,targeting pivotal antigen presentation cells, adding specific adjuvants,co-administration of cytokines such as IL-10 or TGF-β, optimization oforal antigen delivery, its dosage and feeding frequency, all seempromising aspects that would greatly improve efficacy of oral toler-ance therapy for human disease.

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