Mechanism of oral tolerance induction to therapeutic proteins

Abstract

Oral tolerance is defined as the specific suppression of humoral and/or cellular immune responses to an antigen by 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 that cause a variety of diseases or that complicate treatment of a disease. Many researchers have induced oral tolerance to efficiently treat autoimmune and inflammatory diseases in different animal models. However, clinical trials yielded limited success. Thus, understanding the mechanisms of oral tolerance induction to therapeutic proteins is critical for paving the way for clinical development of oral tolerance protocols. This review will summarize progress on understanding the major underlying tolerance mechanisms and contributors, including antigen presenting cells, regulatory T cells, cytokines, and signaling pathways. Potential applications, examples for therapeutic proteins and disease targets, and recent developments in delivery methods are discussed.

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 antigen from gut lumen; antigens 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 and crosstalking with different cell types: intestinal epithelial cells releasing TGF-β and retinoic acid; lamina propria macrophages producing large amount of IL-10 and inhibit DCs induction 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 occur simultaneously and overlap. Apoptotic T cells, macrophages and DCs that clean up the debris, express high level of TGF-β and suppress inflammatory cytokines production. Treg suppressed effector T cells responses including inhibition of T cell proliferation and blockade of inflammatory cytokines release. TGF-b and IL-10 are high in gut microenvironment, which is essential for Treg function and maintenance.

Fig. 2. Example of prevention of a systemic immune response by oral tolerance induction

Prevention of inhibitory antibody formation and of anaphylactic reactions against intravenous 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 are Peyer’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 the onset 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 a furin 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 6^th injection of hF.IX to prevent anaphylaxis. Modified from Proc Natl Acad Sci USA 107(15): 7101-6, 2010; © 2010 by The National Academy of Sciences of the USA.

Fig. 3. Mechanisms of Treg induction in oral tolerance

A. Low dose antigen feeding results in active induction of Treg, which involves cross talk between different cell types. For example, CD103⁺ gut DCs are specialized in inducing Treg via production of retinoic acid (RA), TGF-β, and expression of indoleamine-2,3-dioxygenase (IDO). B. High dose oral antigen induces T cell anergy. Anergic T cells produce cytokines including IL-4, IL-10, TGF-β, and act as suppressor cells to evoke tolerance. High dose antigen feeding also increases susceptibility to apoptosis. Macrophage and DC clean up the apoptotic cells and exhibit up-regulation of TGF-β and down regulation of inflammatory cytokines. Apoptotic cells can also secrete TGF-β, which is critical for inducing and maintaining Treg. These two mechanisms may occur simultaneously and overlap as they shared some of the same characteristics such as cytokines production profiles and generation of induced Treg (iTreg).

Fig. 4. Induction and multifunction of TGF-β in the gut

TGF-β is a key cytokine for oral tolerance. Abundant in the gut microenvironment, TGF-β has multiple functions, including epithelial cell differentiation, IgA class switching, and induction and maintenance of Foxp3⁺ Treg. Additionally, TGF-β decreases the differentiation of naïve T cells into effector Th1 and Th2 cells, drives the development of TCRαβ⁺CD8αα⁺ intestinal intraepithelial lymphocytes (IEL). TGF-β is secreted by a variety of cells including CD4⁺, CD8⁺ T cells, Th3 cells, macrophages, enterocytes, antigen-pulsed intestinal epithelial cells (IEC), and gut DCs. Gut DCs produce retinoic acid (RA), which enhance Foxp3⁺ Treg induction by TGF-β. In MLN DCs, interaction with Foxp3⁺ Treg increases expression of indoleamine-2,3-dioxygenase (IDO), which depletes tryptophan into kynurenines and suppresses effector T cell responses. Besides the secreted form, an active membrane-bound form (Latency-associated peptide, LAP) exists. Some Treg express LAP and TGF-β on their cell surface, which is associated with the suppressive activity of Treg. LAP⁺ Treg are expanded in oral tolerance.

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. Highly expressed in the intestine, IL-10 producing cells increased even more after antigen feeding, especially in Peyer’s patch (PP), lamina propria, and MLN. Specifically, one regulatory subset of CD4⁺ T cells mainly producing IL-10, named Tr1 cells, is induced in oral tolerance. Other regulatory T cells such as Th3. Foxp3⁺ Treg may also secrete IL-10 and are induced by antigen feeding. Macrophages, B cells, DCs and CD8⁺ T cells, epithelial cells, 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 macrophages. 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.