ARTICLE
Oral Immunotherapy for Allergic Conjunctivitis Waka Ishida, MS,* Ken Fukuda, MD, PhD,* Yosuke Harada, MD, PhD,* Hideo Yagita, PhD,† and Atsuki Fukushima, MD, PhD*
Abstract: Antigen-specific immunotherapy is expected to be a desirable treatment for allergic diseases. Currently, antigenspecific immunotherapy is performed by administering diseasecausing antigens subcutaneously or sublingually. These approaches induce long-term remission in patients with allergic rhinitis or asthma. The oral route is an alternative to subcutaneous and sublingual routes, and can also induce long-term remission, a phenomenon known as “oral tolerance.” The effectiveness of oral tolerance has been reported in the context of autoimmune diseases, food allergies, asthma, atopic dermatitis, and allergic rhinitis in both human patients and animal models. However, few studies have examined its efficacy in animal models of allergic conjunctivitis. Previously, we showed that ovalbumin feeding suppressed ovalbumin-induced experimental allergic conjunctivitis, indicating the induction of oral tolerance is effective in treating experimental allergic conjunctivitis. In recent years, transgenic rice has been developed that can induce oral tolerance and reduce the severity of anaphylaxis. The major Japanese cedar pollen antigens in transgenic rice, Cryptomeria japonica 1 and C. japonica 2, were deconstructed by molecular shuffling, fragmentation, and changes in the oligomeric structure. Thus, transgenic rice may be an effective treatment for allergic conjunctivitis. Key Words: allergic conjunctivitis, oral tolerance, transgenic rice (Cornea 2014;33(Suppl):S32–S36)
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pproximately 30% of the population in industrialized countries has allergic diseases such as atopic dermatitis, asthma, rhinitis, or conjunctivitis.1 Moreover, the prevalence of these diseases has increased in recent decades and is still rising. Treatment is centered on medicines such as antihistamines and corticosteroids; however, although these medicines can relieve symptoms, they are not curative.1 Allergic symptoms are elicited by environmental antigens such as pollens or house dust mites. Allergic conjunctivitis caused by pollen antigens is an immunoglobulin E (IgE)-mediated type I allergic reaction regulated by T cells.2 Therefore, the main aim of antigen-specific immunotherapy (antigen-SIT) is the From the *Department of Ophthalmology, Kochi Medical School, Kochi, Japan; and †Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan. A. Fukushima has received payment as a board member for Kyoto Cornea Club. The other authors have no funding or conflicts of interest to disclose. Reprints: Atsuki Fukushima, MD, PhD, Department of Ophthalmology, Kochi Medical School, Kohasu, Oko-cho, Nankoku City, Kochi 783-8505, Japan (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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induction of tolerance against antigen-specific T cells. Recent studies have clearly demonstrated the impact of antigen-SIT on autoimmune and allergic inflammatory diseases.3–25 Here, we discuss past, present, and future directions of antigen-SIT for several diseases, with a special focus on oral immunotherapy for allergic conjunctivitis.
ANTIGEN-SPECIFIC IMMUNOTHERAPY Subcutaneous and Sublingual Immunotherapy Antigen-SIT has been performed for almost a century and remains one of the few disease-modifying treatments for allergic diseases.3 Antigen-SIT was first reported in the early 1900s by Leonard Noon, who studied the efficacy of antigen immunotherapy in individuals with seasonal allergic rhinitis. He injected patients with increasing doses of pollen extract and then challenged them with eye drops containing the antigen. He observed that pollen-specific injection immunotherapy suppressed ocular symptoms.4 Antigen-SIT is performed by repeatedly administering disease-causing antigens either subcutaneously or sublingually. Subcutaneous immunotherapy (SCIT) consists of repeated subcutaneous injection of antigen extracts, known as “allergy shots.” Sublingual immunotherapy involves administering antigen-containing drops or tablets into the sublingual area. SCIT prevents the development of asthma in patients with allergic rhinoconjunctivitis.5 Sublingual immunotherapy has also been used successfully to treat rhinoconjunctivitis.6 However, the disadvantages of conventional antigen-SIT include systemic allergic side effects such as anaphylaxis and the need for long-term treatment. Moreover, SCIT is accompanied by pain and discomfort associated with repeated injections. Therefore, more convenient and safer treatment methods are needed.
Oral Immunotherapy Administration of immunotherapy through the oral route is an alternative method of curative treatment and induces a phenomenon known as “oral tolerance.”7 Several animal studies have demonstrated the efficacy of orally administered antigens for the treatment of autoimmune and inflammatory diseases.8 Oral tolerance suppresses immune responses in animal models of experimental autoimmune encephalomyelitis (EAE),9–11 uveitis,12,13 and asthma.14,15 Moreover, oral tolerance has been studied extensively in the context of food allergies,16 asthma,17 and atopic dermatitis18 in human studies. Cornea Volume 33, Number 11, Supplement, November 2014
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THERAPEUTIC APPLICATION OF ORAL TOLERANCE IN HUMANS AND ANIMAL MODELS OF AUTOIMMUNE AND ALLERGIC INFLAMMATORY DISEASES EAE and Multiple Sclerosis EAE is an animal model of a T-cell-mediated disease that mimics the human demyelinating disease, multiple sclerosis (MS). Javed et al9 found that EAE was suppressed by myelin basic protein (MBP) administered orally at high doses and that the underlying mechanism involved immunosuppression through T-cell anergy. Another study obtained similar results, but this time, oral administration of low-dose MBP or its fragments was effective.10 Khoury et al11 reported that EAE suppression by lowdose MBP was mediated through the upregulation of antiinflammatory cytokines such as transforming growth factor-b (TGF-b). A human study of MS showed that the number of MBPand proteolipid protein-specific TGF-b-secreting T cells in the peripheral blood of MS patients receiving oral MBP was higher than in untreated patients.19 This suggested that autoantigen-specific TGF-b-secreting T cells were generated by oral administration of human autoantigens.
Experimental Autoimmune Uveitis and Uveitis Experimental autoimmune uveitis (EAU) is widely used as a model for forms of human uveitis such as Vogt–Koyanagi– Harada disease and Behçet disease. Studies show that EAU is suppressed by oral administration of a retinal autoantigen, S-antigen (S-Ag), or by oral administration of S-Ag-derived peptides. Indeed, Nussenblatt et al12 demonstrated that S-Aginduced-EAU was prevented, or that symptoms were markedly reduced, by orally administered S-Ag. Torseth et al13 reported that oral administration of bovine S-Ag-derived peptides only inhibited mild disease when feeding was delayed until after immunization, and that relatively high-dose feeding was required. A phase I/II trial of retinal S-Ag and a mixture of soluble retinal antigens in uveitis patients showed positive results with oral S-Ag, but not with the antigen mixture.20
Experimental Models of Asthma Asthma is a common chronic inflammatory disease characterized by intermittent and reversible airway obstruction and airway hyperresponsiveness. Russo et al14 demonstrated that oral administration of ovalbumin (OVA) suppressed OVAinduced airway inflammation and type 2 T-helper (Th2) cytokine production in BALB/c and BP2 mice. Chung et al showed that oral administration of OVA suppressed airway hyperresponsiveness, antigen-specific IgE production, and the generation of Th2-derived cytokines in a murine model of asthma. In addition, feeding mice multiple high doses of OVA suppressed disease progression and mild asthma. However, high-dose feeding did not suppress airway reactivity or eosinophilia in the bronchoalveolar lavage fluid in mice with severe asthma.15
Experimental Allergic Rhinitis Allergic rhinitis is characterized by inflammation of the nasal membranes, sneezing, nasal congestion, and rhinorrhea. Ó 2014 Lippincott Williams & Wilkins
Oral Immunotherapy for Allergic Conjunctivitis
Kim et al investigated airway remodeling and the preventive effects of different doses of orally administered OVA in a mouse model of allergic rhinitis. Groups of mice were fed either highor low-dose OVA. The results showed that the allergic behavior scores, OVA-specific Ig levels, and inflammatory cytokine levels in the bronchoalveolar lavage fluids were suppressed in both groups compared with the control group.21
Experimental Allergic Conjunctivitis Allergic conjunctivitis is ocular-surface inflammation mediated by a type I allergic reaction.2 Clinical symptoms of allergic conjunctivitis consist of redness, tearing, itching, and conjunctival edema. These acute reactions are associated with type I hypersensitivity, an early-phase reaction, and can occur within minutes of antigen exposure.26 The early response is followed by a more sustained inflammation, termed latephase reaction. The late response involves the recruitment and activation of effector cells, notably Th2 lymphocytes, eosinophils, and basophils.26 Regarding allergic inflammatory diseases as described above, the effectiveness of oral immunotherapy has been examined extensively. In contrast, only 2 studies have examined the efficacy of oral tolerance induction in animal models of allergic conjunctivitis.22,23 Koizumi et al induced oral tolerance in experimental allergic conjunctivitis (EAC)-induced rats through oral administration of OVA. The OVA-fed group showed significant suppression of the IgE-mediated early-phase reaction.22 Zemann et al induced oral tolerance in an IgE highresponder dog model by feeding OVA dissolved in cow’s milk. They found that allergic conjunctivitis, hyperemia, and edema were significantly inhibited by feeding OVA.23 Neither of these studies examined the mechanisms underlying the induction of oral tolerance by antigen feeding. Furthermore, only a single dose of antigen was examined in each study. Therefore, we examined the mechanisms underlying oral tolerance induction by different doses of antigen in a murine model of EAC.
HYPOTHETICAL MECHANISMS UNDERLYING ORAL TOLERANCE Oral tolerance is thought to be mediated by more than 1 mechanism, and is dose dependent.27,28 High doses of antigen favor the induction of clonal anergy29 or clonal deletion,30 whereas low doses favor the induction of active suppression by regulatory T cells. Active suppression describes a mechanism by which regulatory T cells suppress immune responses by secretion of soluble antiinflammatory cytokines such as interleukin-10 (IL-10) and TGF-b,31,32 or by cell-to-cell contact (Fig. 1).33
PROPHYLACTIC HIGH- AND LOW-DOSE OVA FEEDING SUPPRESSES EAC DEVELOPMENT High-Dose OVA Feeding Photographs and micrographs of conjunctiva from mice fed high-dose OVA (100 mg) showed an apparent reduction in clinical appearance (Fig. 2A, top row) and conjunctival eosinophil infiltration (Fig. 2A, bottom row), respectively. www.corneajrnl.com |
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high-dose OVA feeding led to a reduction in serum IgE levels and cytokine production by antigen-specific Th1 and Th2 cells.24 Taken together, these data suggested that antigenspecific T cells or B cells were either depleted or nonfunctional. Thus, high-dose OVA feeding may induce clonal anergy or clonal deletion in this model.
Low-Dose OVA Feeding
FIGURE 1. Hypothetical mechanisms underlying oral tolerance induction. Generation of an immune response requires ligation of a T-cell receptor (TCR) by a peptide-major histocompatibility complex. An active immune response is then generated if the appropriate costimulatory molecules and cytokines are expressed. Three hypothetical mechanisms underlie the induction of oral tolerance. First, TCR cross-linking by antigens can occur in the absence of costimulation, which leads to “clonal anergy.” Second, TCR cross-linking by antigens can occur in the presence of Fas and Fas ligand, leading to apoptosis and “clonal deletion.” Third, an antigen can activate regulatory T cells, which suppress immune responses by secreting soluble antiinflammatory cytokines such as IL-10 and TGF-b, or by direct cell-to-cell contact, a phenomenon known as active suppression.
Furthermore, there was a significant reduction in conjunctival inflammation, as measured by clinical scores and conjunctival eosinophil numbers, in high-dose OVA-fed mice compared with phosphate-buffered saline (PBS)-fed mice.24 In addition,
When mice were fed low-dose OVA (2.5 mg), the clinical appearance was similar between the control and low-dose OVA-fed groups (Fig. 2B, top row). However, micrographs of conjunctiva showed an apparent reduction in conjunctival eosinophil infiltration in the low-dose OVA-fed group (Fig. 2B, bottom row). Although lower numbers of conjunctival eosinophils were observed in the low-dose OVA-fed group, serum IgE levels and antigen-specific cytokine production were not suppressed.24 As stated earlier, active suppression by regulatory T cells is mediated by antiinflammatory cytokines or cell-to-cell contact. Therefore, we examined the possible contribution of each mechanism. To investigate the involvement of antiinflammatory cytokines, mice were treated every other day with neutralizing antibodies against IL-10 or TGF-b. However, neither treatment increased the number of conjunctival eosinophils.24 Therefore, IL-10 and TGF-b are unlikely to play a role in the induction of oral tolerance by feeding mice OVA in this experimental setting. Next, we examined the role of cell-to-cell contact. OVA-fed immunocompetent mice (which contain regulatory T cells) showed a significant reduction in the number of conjunctival eosinophils compared with PBS-fed mice.24 In contrast to PBS-fed mice, OVA-fed mice depleted of regulatory T cells also showed reduced conjunctival eosinophil numbers, although the difference between the 2 groups was not significant.24
FIGURE 2. Effects of OVA feeding on allergic inflammation in EAC. Clinical appearance was observed after administration of OVA eye drops in OVA-sensitized mice fed high-dose (A, top row) or low-dose (B, top row) OVA. Images were obtained 20 minutes after OVA eye drops were administered. Giemsa-stained paraffin sections from OVA-sensitized mice conjunctivas were harvested 24 hours after OVA eye drops were administered to high-dose (A, bottom row) or low-dose (B, bottom row) OVA-fed mice. Arrows indicate eosinophils. Scale bars = 50 mm.
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Thus, it seems that the regulatory T cells may play a role, at least in part, in OVA-induced oral tolerance. Taken together, the data from the above experiments suggest that high-dose OVA feeding induces oral tolerance by the induction of clonal anergy or clonal deletion, whereas low-dose OVA feeding induces oral tolerance by regulatory T-cell-mediated active suppression. Thus, the induction of oral tolerance suppresses EAC in mice. Furthermore, the mechanisms underlying immune suppression differ depending on the dose of orally administered antigen.24 This study identified the optimal antigen-feeding dose regimens that induce oral tolerance and showed that different mechanisms are involved depending on the dose of antigen. These findings provide valuable information that will assist future studies.
FUTURE DIRECTIONS In recent years, staple foods (eg, rice) have been “designed” to act as antigen sources that can reduce severe adverse reactions such as anaphylactic shock. Crude antigens must be modified before they can be used for desensitization. Such antigens must show reduced IgE binding activity without any loss of T-cell epitopes. For example, “transgenic rice” has been “designed” to express the deconstructed major Japanese cedar pollen antigens, Cryptomeria japonica 1 (Cry j-1) and C. japonica 2 (Cry j-2).25 Cry j-1 and Cry j-2 were deconstructed by fragmentation and molecular shuffling, respectively. The pollen antigens in transgenic rice do not bind to Cry j-specific IgE antibodies but still express the full range of T-cell epitopes. Furthermore, they are resistant to digestive enzymes in the gastrointestinal tract; thus, transgenic rice can reduce the risk of anaphylaxis. Indeed, Wakasa et al25 showed that oral administration of transgenic rice seeds inhibited nasal symptoms and inflammation in a mouse model.
CONCLUSIONS Over the past several decades, antigen-SIT has shown promising results in both autoimmune and allergic inflammatory disease human patients and animal models. Recent studies have elucidated the mechanisms underlying oral tolerance. Immune suppression is mediated by clonal anergy, clonal deletion, or regulatory T-cell-mediated active suppression. However, regarding allergic conjunctivitis, there is insufficient evidence for the efficacy and safety of this therapeutic approach. We demonstrated that oral immunotherapy could suppress the development of allergic conjunctivitis in mice. However, further investigations are needed before oral immunotherapy can be provided to patients in clinical practice. Our future work will aim to explore the effectiveness of transgenic rice, a staple food, for the treatment of allergic conjunctivitis in humans. REFERENCES 1. Bielory L. Update on ocular allergy treatment. Expert Opin Pharmacother. 2002;3:541–553. 2. Friedlaender MH. Immunologic aspects of diseases of the eye. JAMA. 1992;268:2869–2873.
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3. Valenta R, Campana R, Marth K, et al. Allergen-specific immunotherapy: from therapeutic vaccines to prophylactic approaches. J Intern Med. 2012;272:144–157. 4. Noon L. Prophylactic inoculation against hay fever. Lancet. 1911;1: 1572–1573. 5. Jacobsen L, Niggemann B, Dreborg S, et al. Specific immunotherapy has long-term preventive effect of seasonal and perennial asthma: 10-year follow-up on the PAT study. Allergy. 2007;62:943–948. 6. Calderon MA, Simons FE, Malling HJ, et al. Sublingual allergen immunotherapy: mode of action and its relationship with the safety profile. Allergy. 2012;67:302–311. 7. Weiner HL. Oral tolerance. Proc Natl Acad Sci U S A. 1994;91: 10762–10765. 8. Faria AM, Weiner HL. Oral tolerance. Immunol Rev. 2005;206:232–259. 9. Javed NH, Gienapp IE, Cox KL, et al. Exquisite peptide specificity of oral tolerance in experimental autoimmune encephalomyelitis. J Immunol. 1995;155:1599–1605. 10. Higgins PJ, Weiner HL. Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein and its fragments. J Immunol. 1988;140:440–445. 11. Khoury SJ, Hancock WW, Weiner HL. Oral tolerance to myelin basic protein and natural recovery from experimental autoimmune encephalomyelitis are associated with downregulation of inflammatory cytokines and differential upregulation of transforming growth factor beta, interleukin 4, and prostaglandin E expression in the brain. J Exp Med. 1992; 176:1355–1364. 12. Nussenblatt RB, Caspi RR, Mahdi R, et al. Inhibition of S-antigen induced experimental autoimmune uveoretinitis by oral induction of tolerance with S-antigen. J Immunol. 1990;144:1689–1695. 13. Torseth JW, Gregerson DS. Oral tolerance in experimental autoimmune uveoretinitis: feeding after disease induction is less protective than prefeeding. Clin Immunol Immunopathol. 1998;88:297–304. 14. Russo M, Nahori MA, Lefort J, et al. Suppression of asthma-like responses in different mouse strains by oral tolerance. Am J Respir Cell Mol Biol. 2001;24:518–526. 15. Chung Y, Cho J, Chang YS, et al. Preventive and therapeutic effects of oral tolerance in a murine model of asthma. Immunobiology. 2002;206: 408–423. 16. Brozek JL, Terracciano L, Hsu J, et al. Oral immunotherapy for IgE-mediated cow’s milk allergy: a systematic review and meta-analysis. Clin Exp Allergy. 2012;42:363–374. 17. Nelson HS. Advances in upper airway diseases and allergen immunotherapy. J Allergy Clin Immunol. 2006;117:1047–1053. 18. Pajno GB, Finegold I. SIT beyond respiratory diseases. Ann Allergy Asthma Immunol. 2011;107:395–400. 19. Fukaura H, Kent SC, Pietrusewicz MJ, et al. Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-beta1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. J Clin Invest. 1996;98:70–77. 20. Nussenblatt RB, Gery I, Weiner HL, et al. Treatment of uveitis by oral administration of retinal antigens: results of a phase I/II randomized masked trial. Am J Ophthalmol. 1997;123:583–592. 21. Kim SJ, Shin JH, Kim SC, et al. Preventive effects of oral tolerance on allergic inflammation and airway remodeling in a murine model. Am J Rhinol Allergy. 2013;27:e11–e16. 22. Koizumi T, Abe T. Induction of oral tolerance to experimental allergic conjunctivitis in rats. Nihon Ganka Gakkai Zasshi. 1995;99:515–520. 23. Zemann B, Schwaerzler C, Griot-Wenk M, et al. Oral administration of specific antigens to allergy-prone infant dogs induces IL-10 and TGF-beta expression and prevents allergy in adult life. J Allergy Clin Immunol. 2003;111:1069–1075. 24. Ishida W, Fukuda K, Harada Y, et al. Oral administration of Ag suppresses Ag-induced allergic conjunctivitis in mice: critical timing and dose of Ag. Br J Ophthalmol. 2013;97:492–497. 25. Wakasa Y, Takagi H, Hirose S, et al. Oral immunotherapy with transgenic rice seed containing destructed Japanese cedar pollen allergens, Cry j 1 and Cry j 2, against Japanese cedar pollinosis. Plant Biotechnol J. 2013;11:66–76. 26. Galli SJ, Tsai M, Piliponsky AM. The development of allergic inflammation. Nature. 2008;454:445–454. 27. Vickery BP, Burks AW. Immunotherapy in the treatment of food allergy: focus on oral tolerance. Curr Opin Allergy Clin Immunol. 2009;9:364–370.
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28. Mayer L, Shao L. Therapeutic potential of oral tolerance. Nat Rev Immunol. 2004;4:407–419. 29. Whitacre CC, Gienapp IE, Orosz CG, et al. Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy. J Immunol. 1991;147:2155–2163. 30. Chen Y, Inobe J, Marks R, et al. Peripheral deletion of antigen-reactive T cells in oral tolerance. Nature. 1995;376:177–180. 31. Faria AM, Maron R, Ficker SM, et al. Oral tolerance induced by continuous feeding: enhanced up-regulation of transforming growth
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factor-beta/interleukin-10 and suppression of experimental autoimmune encephalomyelitis. J Autoimmun. 2003;20:135–145. 32. Min SY, Hwang SY, Park KS, et al. Induction of IL-10-producing CD4+CD25+ T cells in animal model of collagen-induced arthritis by oral administration of type II collagen. Arthritis Res Ther. 2004;6: R213–R219. 33. Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor beta. J Exp Med. 2001;194:629–644.
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Oral Immunotherapy for Allergic Conjunctivitis Waka Ishida, MS,* Ken Fukuda, MD, PhD,* Yosuke Harada, MD, PhD,* Hideo Yagita, PhD,† and Atsuki Fukushima, MD, PhD*
Abstract: Antigen-specific immunotherapy is expected to be a desirable treatment for allergic diseases. Currently, antigenspecific immunotherapy is performed by administering diseasecausing antigens subcutaneously or sublingually. These approaches induce long-term remission in patients with allergic rhinitis or asthma. The oral route is an alternative to subcutaneous and sublingual routes, and can also induce long-term remission, a phenomenon known as “oral tolerance.” The effectiveness of oral tolerance has been reported in the context of autoimmune diseases, food allergies, asthma, atopic dermatitis, and allergic rhinitis in both human patients and animal models. However, few studies have examined its efficacy in animal models of allergic conjunctivitis. Previously, we showed that ovalbumin feeding suppressed ovalbumin-induced experimental allergic conjunctivitis, indicating the induction of oral tolerance is effective in treating experimental allergic conjunctivitis. In recent years, transgenic rice has been developed that can induce oral tolerance and reduce the severity of anaphylaxis. The major Japanese cedar pollen antigens in transgenic rice, Cryptomeria japonica 1 and C. japonica 2, were deconstructed by molecular shuffling, fragmentation, and changes in the oligomeric structure. Thus, transgenic rice may be an effective treatment for allergic conjunctivitis. Key Words: allergic conjunctivitis, oral tolerance, transgenic rice (Cornea 2014;33(Suppl):S32–S36)
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pproximately 30% of the population in industrialized countries has allergic diseases such as atopic dermatitis, asthma, rhinitis, or conjunctivitis.1 Moreover, the prevalence of these diseases has increased in recent decades and is still rising. Treatment is centered on medicines such as antihistamines and corticosteroids; however, although these medicines can relieve symptoms, they are not curative.1 Allergic symptoms are elicited by environmental antigens such as pollens or house dust mites. Allergic conjunctivitis caused by pollen antigens is an immunoglobulin E (IgE)-mediated type I allergic reaction regulated by T cells.2 Therefore, the main aim of antigen-specific immunotherapy (antigen-SIT) is the From the *Department of Ophthalmology, Kochi Medical School, Kochi, Japan; and †Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan. A. Fukushima has received payment as a board member for Kyoto Cornea Club. The other authors have no funding or conflicts of interest to disclose. Reprints: Atsuki Fukushima, MD, PhD, Department of Ophthalmology, Kochi Medical School, Kohasu, Oko-cho, Nankoku City, Kochi 783-8505, Japan (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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induction of tolerance against antigen-specific T cells. Recent studies have clearly demonstrated the impact of antigen-SIT on autoimmune and allergic inflammatory diseases.3–25 Here, we discuss past, present, and future directions of antigen-SIT for several diseases, with a special focus on oral immunotherapy for allergic conjunctivitis.
ANTIGEN-SPECIFIC IMMUNOTHERAPY Subcutaneous and Sublingual Immunotherapy Antigen-SIT has been performed for almost a century and remains one of the few disease-modifying treatments for allergic diseases.3 Antigen-SIT was first reported in the early 1900s by Leonard Noon, who studied the efficacy of antigen immunotherapy in individuals with seasonal allergic rhinitis. He injected patients with increasing doses of pollen extract and then challenged them with eye drops containing the antigen. He observed that pollen-specific injection immunotherapy suppressed ocular symptoms.4 Antigen-SIT is performed by repeatedly administering disease-causing antigens either subcutaneously or sublingually. Subcutaneous immunotherapy (SCIT) consists of repeated subcutaneous injection of antigen extracts, known as “allergy shots.” Sublingual immunotherapy involves administering antigen-containing drops or tablets into the sublingual area. SCIT prevents the development of asthma in patients with allergic rhinoconjunctivitis.5 Sublingual immunotherapy has also been used successfully to treat rhinoconjunctivitis.6 However, the disadvantages of conventional antigen-SIT include systemic allergic side effects such as anaphylaxis and the need for long-term treatment. Moreover, SCIT is accompanied by pain and discomfort associated with repeated injections. Therefore, more convenient and safer treatment methods are needed.
Oral Immunotherapy Administration of immunotherapy through the oral route is an alternative method of curative treatment and induces a phenomenon known as “oral tolerance.”7 Several animal studies have demonstrated the efficacy of orally administered antigens for the treatment of autoimmune and inflammatory diseases.8 Oral tolerance suppresses immune responses in animal models of experimental autoimmune encephalomyelitis (EAE),9–11 uveitis,12,13 and asthma.14,15 Moreover, oral tolerance has been studied extensively in the context of food allergies,16 asthma,17 and atopic dermatitis18 in human studies. Cornea Volume 33, Number 11, Supplement, November 2014
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THERAPEUTIC APPLICATION OF ORAL TOLERANCE IN HUMANS AND ANIMAL MODELS OF AUTOIMMUNE AND ALLERGIC INFLAMMATORY DISEASES EAE and Multiple Sclerosis EAE is an animal model of a T-cell-mediated disease that mimics the human demyelinating disease, multiple sclerosis (MS). Javed et al9 found that EAE was suppressed by myelin basic protein (MBP) administered orally at high doses and that the underlying mechanism involved immunosuppression through T-cell anergy. Another study obtained similar results, but this time, oral administration of low-dose MBP or its fragments was effective.10 Khoury et al11 reported that EAE suppression by lowdose MBP was mediated through the upregulation of antiinflammatory cytokines such as transforming growth factor-b (TGF-b). A human study of MS showed that the number of MBPand proteolipid protein-specific TGF-b-secreting T cells in the peripheral blood of MS patients receiving oral MBP was higher than in untreated patients.19 This suggested that autoantigen-specific TGF-b-secreting T cells were generated by oral administration of human autoantigens.
Experimental Autoimmune Uveitis and Uveitis Experimental autoimmune uveitis (EAU) is widely used as a model for forms of human uveitis such as Vogt–Koyanagi– Harada disease and Behçet disease. Studies show that EAU is suppressed by oral administration of a retinal autoantigen, S-antigen (S-Ag), or by oral administration of S-Ag-derived peptides. Indeed, Nussenblatt et al12 demonstrated that S-Aginduced-EAU was prevented, or that symptoms were markedly reduced, by orally administered S-Ag. Torseth et al13 reported that oral administration of bovine S-Ag-derived peptides only inhibited mild disease when feeding was delayed until after immunization, and that relatively high-dose feeding was required. A phase I/II trial of retinal S-Ag and a mixture of soluble retinal antigens in uveitis patients showed positive results with oral S-Ag, but not with the antigen mixture.20
Experimental Models of Asthma Asthma is a common chronic inflammatory disease characterized by intermittent and reversible airway obstruction and airway hyperresponsiveness. Russo et al14 demonstrated that oral administration of ovalbumin (OVA) suppressed OVAinduced airway inflammation and type 2 T-helper (Th2) cytokine production in BALB/c and BP2 mice. Chung et al showed that oral administration of OVA suppressed airway hyperresponsiveness, antigen-specific IgE production, and the generation of Th2-derived cytokines in a murine model of asthma. In addition, feeding mice multiple high doses of OVA suppressed disease progression and mild asthma. However, high-dose feeding did not suppress airway reactivity or eosinophilia in the bronchoalveolar lavage fluid in mice with severe asthma.15
Experimental Allergic Rhinitis Allergic rhinitis is characterized by inflammation of the nasal membranes, sneezing, nasal congestion, and rhinorrhea. Ó 2014 Lippincott Williams & Wilkins
Oral Immunotherapy for Allergic Conjunctivitis
Kim et al investigated airway remodeling and the preventive effects of different doses of orally administered OVA in a mouse model of allergic rhinitis. Groups of mice were fed either highor low-dose OVA. The results showed that the allergic behavior scores, OVA-specific Ig levels, and inflammatory cytokine levels in the bronchoalveolar lavage fluids were suppressed in both groups compared with the control group.21
Experimental Allergic Conjunctivitis Allergic conjunctivitis is ocular-surface inflammation mediated by a type I allergic reaction.2 Clinical symptoms of allergic conjunctivitis consist of redness, tearing, itching, and conjunctival edema. These acute reactions are associated with type I hypersensitivity, an early-phase reaction, and can occur within minutes of antigen exposure.26 The early response is followed by a more sustained inflammation, termed latephase reaction. The late response involves the recruitment and activation of effector cells, notably Th2 lymphocytes, eosinophils, and basophils.26 Regarding allergic inflammatory diseases as described above, the effectiveness of oral immunotherapy has been examined extensively. In contrast, only 2 studies have examined the efficacy of oral tolerance induction in animal models of allergic conjunctivitis.22,23 Koizumi et al induced oral tolerance in experimental allergic conjunctivitis (EAC)-induced rats through oral administration of OVA. The OVA-fed group showed significant suppression of the IgE-mediated early-phase reaction.22 Zemann et al induced oral tolerance in an IgE highresponder dog model by feeding OVA dissolved in cow’s milk. They found that allergic conjunctivitis, hyperemia, and edema were significantly inhibited by feeding OVA.23 Neither of these studies examined the mechanisms underlying the induction of oral tolerance by antigen feeding. Furthermore, only a single dose of antigen was examined in each study. Therefore, we examined the mechanisms underlying oral tolerance induction by different doses of antigen in a murine model of EAC.
HYPOTHETICAL MECHANISMS UNDERLYING ORAL TOLERANCE Oral tolerance is thought to be mediated by more than 1 mechanism, and is dose dependent.27,28 High doses of antigen favor the induction of clonal anergy29 or clonal deletion,30 whereas low doses favor the induction of active suppression by regulatory T cells. Active suppression describes a mechanism by which regulatory T cells suppress immune responses by secretion of soluble antiinflammatory cytokines such as interleukin-10 (IL-10) and TGF-b,31,32 or by cell-to-cell contact (Fig. 1).33
PROPHYLACTIC HIGH- AND LOW-DOSE OVA FEEDING SUPPRESSES EAC DEVELOPMENT High-Dose OVA Feeding Photographs and micrographs of conjunctiva from mice fed high-dose OVA (100 mg) showed an apparent reduction in clinical appearance (Fig. 2A, top row) and conjunctival eosinophil infiltration (Fig. 2A, bottom row), respectively. www.corneajrnl.com |
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high-dose OVA feeding led to a reduction in serum IgE levels and cytokine production by antigen-specific Th1 and Th2 cells.24 Taken together, these data suggested that antigenspecific T cells or B cells were either depleted or nonfunctional. Thus, high-dose OVA feeding may induce clonal anergy or clonal deletion in this model.
Low-Dose OVA Feeding
FIGURE 1. Hypothetical mechanisms underlying oral tolerance induction. Generation of an immune response requires ligation of a T-cell receptor (TCR) by a peptide-major histocompatibility complex. An active immune response is then generated if the appropriate costimulatory molecules and cytokines are expressed. Three hypothetical mechanisms underlie the induction of oral tolerance. First, TCR cross-linking by antigens can occur in the absence of costimulation, which leads to “clonal anergy.” Second, TCR cross-linking by antigens can occur in the presence of Fas and Fas ligand, leading to apoptosis and “clonal deletion.” Third, an antigen can activate regulatory T cells, which suppress immune responses by secreting soluble antiinflammatory cytokines such as IL-10 and TGF-b, or by direct cell-to-cell contact, a phenomenon known as active suppression.
Furthermore, there was a significant reduction in conjunctival inflammation, as measured by clinical scores and conjunctival eosinophil numbers, in high-dose OVA-fed mice compared with phosphate-buffered saline (PBS)-fed mice.24 In addition,
When mice were fed low-dose OVA (2.5 mg), the clinical appearance was similar between the control and low-dose OVA-fed groups (Fig. 2B, top row). However, micrographs of conjunctiva showed an apparent reduction in conjunctival eosinophil infiltration in the low-dose OVA-fed group (Fig. 2B, bottom row). Although lower numbers of conjunctival eosinophils were observed in the low-dose OVA-fed group, serum IgE levels and antigen-specific cytokine production were not suppressed.24 As stated earlier, active suppression by regulatory T cells is mediated by antiinflammatory cytokines or cell-to-cell contact. Therefore, we examined the possible contribution of each mechanism. To investigate the involvement of antiinflammatory cytokines, mice were treated every other day with neutralizing antibodies against IL-10 or TGF-b. However, neither treatment increased the number of conjunctival eosinophils.24 Therefore, IL-10 and TGF-b are unlikely to play a role in the induction of oral tolerance by feeding mice OVA in this experimental setting. Next, we examined the role of cell-to-cell contact. OVA-fed immunocompetent mice (which contain regulatory T cells) showed a significant reduction in the number of conjunctival eosinophils compared with PBS-fed mice.24 In contrast to PBS-fed mice, OVA-fed mice depleted of regulatory T cells also showed reduced conjunctival eosinophil numbers, although the difference between the 2 groups was not significant.24
FIGURE 2. Effects of OVA feeding on allergic inflammation in EAC. Clinical appearance was observed after administration of OVA eye drops in OVA-sensitized mice fed high-dose (A, top row) or low-dose (B, top row) OVA. Images were obtained 20 minutes after OVA eye drops were administered. Giemsa-stained paraffin sections from OVA-sensitized mice conjunctivas were harvested 24 hours after OVA eye drops were administered to high-dose (A, bottom row) or low-dose (B, bottom row) OVA-fed mice. Arrows indicate eosinophils. Scale bars = 50 mm.
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Thus, it seems that the regulatory T cells may play a role, at least in part, in OVA-induced oral tolerance. Taken together, the data from the above experiments suggest that high-dose OVA feeding induces oral tolerance by the induction of clonal anergy or clonal deletion, whereas low-dose OVA feeding induces oral tolerance by regulatory T-cell-mediated active suppression. Thus, the induction of oral tolerance suppresses EAC in mice. Furthermore, the mechanisms underlying immune suppression differ depending on the dose of orally administered antigen.24 This study identified the optimal antigen-feeding dose regimens that induce oral tolerance and showed that different mechanisms are involved depending on the dose of antigen. These findings provide valuable information that will assist future studies.
FUTURE DIRECTIONS In recent years, staple foods (eg, rice) have been “designed” to act as antigen sources that can reduce severe adverse reactions such as anaphylactic shock. Crude antigens must be modified before they can be used for desensitization. Such antigens must show reduced IgE binding activity without any loss of T-cell epitopes. For example, “transgenic rice” has been “designed” to express the deconstructed major Japanese cedar pollen antigens, Cryptomeria japonica 1 (Cry j-1) and C. japonica 2 (Cry j-2).25 Cry j-1 and Cry j-2 were deconstructed by fragmentation and molecular shuffling, respectively. The pollen antigens in transgenic rice do not bind to Cry j-specific IgE antibodies but still express the full range of T-cell epitopes. Furthermore, they are resistant to digestive enzymes in the gastrointestinal tract; thus, transgenic rice can reduce the risk of anaphylaxis. Indeed, Wakasa et al25 showed that oral administration of transgenic rice seeds inhibited nasal symptoms and inflammation in a mouse model.
CONCLUSIONS Over the past several decades, antigen-SIT has shown promising results in both autoimmune and allergic inflammatory disease human patients and animal models. Recent studies have elucidated the mechanisms underlying oral tolerance. Immune suppression is mediated by clonal anergy, clonal deletion, or regulatory T-cell-mediated active suppression. However, regarding allergic conjunctivitis, there is insufficient evidence for the efficacy and safety of this therapeutic approach. We demonstrated that oral immunotherapy could suppress the development of allergic conjunctivitis in mice. However, further investigations are needed before oral immunotherapy can be provided to patients in clinical practice. Our future work will aim to explore the effectiveness of transgenic rice, a staple food, for the treatment of allergic conjunctivitis in humans. REFERENCES 1. Bielory L. Update on ocular allergy treatment. Expert Opin Pharmacother. 2002;3:541–553. 2. Friedlaender MH. Immunologic aspects of diseases of the eye. JAMA. 1992;268:2869–2873.
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Oral Immunotherapy for Allergic Conjunctivitis
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factor-beta/interleukin-10 and suppression of experimental autoimmune encephalomyelitis. J Autoimmun. 2003;20:135–145. 32. Min SY, Hwang SY, Park KS, et al. Induction of IL-10-producing CD4+CD25+ T cells in animal model of collagen-induced arthritis by oral administration of type II collagen. Arthritis Res Ther. 2004;6: R213–R219. 33. Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor beta. J Exp Med. 2001;194:629–644.
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