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Epicutaneous immunization with protein antigen TNP-Ig and NOD2 ligand muramyl dipeptide (MDP) reverses skin-induced suppression of contact hypersensitivity
Pharmacological Reports 66 (2014) 137–142
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Epicutaneous immunization with protein antigen TNP-Ig and NOD2 ligand muramyl dipeptide (MDP) reverses skin-induced suppression of contact hypersensitivity Monika Majewska-Szczepanik, Iwona Doroz˙yn´ska, Anna Strze˛pa, Marian Szczepanik * Department of Medical Biology, Jagiellonian University College of Medicine, Krako´w, Poland
A R T I C L E I N F O
Article history: Received 27 February 2013 Accepted 14 June 2013 Available online 1 February 2014 Keywords: Epicutaneous immunization NOD-like receptors Reversal of suppression Contrasuppression
A B S T R A C T
Background: Epicutaneous (EC) immunization offers a new method of a needle-free and selfadministrable immunization by using a topically applied patch to deliver vaccine. We have previously shown that EC immunization with hapten-conjugated protein antigen TNP-Ig prior to hapten sensitization inhibits Th1-mediated contact hypersensitivity (CHS) in mice. Our further work showed that EC immunization with TNP-Ig and Toll-like receptor (TLR) ligands prior to hapten sensitization reverses skin-induced suppression. Methods: Animal model of contact hypersensitivity was used to study reversal of skin-induced suppression. Results: Current work showed that EC immunization with protein antigen TNP-Ig and MDP NOD2 agonist – muramyldipeptide (L isoform) reverses skin-induced suppression of CHS. On the other hand L18-MDP NOD2 agonist – muramyldipeptide with a C18 fatty acid chain and MDP control – negative control for MDP – muramyldipeptide (D isoform, inactive) did not reverse skin-induced suppression. ‘‘Transfer in’’ experiment showed that reversal of skin-induced suppression can be adoptively transferred with lymphoid cells isolated from donors EC treated with TNP-Ig and MDP NOD2 agonist. Moreover, experiment employing two non-cross-reacting antigens TNP-Ig and OX-Ig proved that reversal of skin-induced suppression is antigen specific. Additionally, lymph node cells isolated from mice EC immunized with TNP-Ig and MDP NOD2 agonist produced increased level of IFN-g suggesting that this cytokine might be involved in reversal of skin-induced suppression. Conclusion: This work shows that EC immunization with protein antigen plus NOD2 ligand MDP may be a potential tool to increase the immunogenicity of weekly immunogenic antigens. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction The skin forms an effective barrier between the host and the environment preventing invasion of pathogens, protecting from chemical and physical aggression. The concept that the skin is a unique and important immune organ is certainly not new, and the Abbreviations: ALNC, axillary and inguinal lymph node cells; CFA, Freund’s adjuvant; CHS, contact hypersensitivity; CIA, collagen induced arthritis; EAE, experimental autoimmune encephalomyelitis; EC, epicutaneous; FCS, fetal calf serum; IFN-g, interferon gamma; L18-MDP, muramyldipeptide with a C18 fatty acid chain; MBP, myelin basic protein; MDP, muramyldipeptide; MHC, class II major histocompatibility complex class II; NF-kB, nuclear factor kappa-light-chainenhancer of activated B cells; NK, natural killer; NOD2, nucleotide-binding oligomerization domain containing 2; OX, oxazolone; PAMPS, pathogen-associated molecular patterns; PBS, phosphate buffered saline; PRR, pathogen recognition receptors; TLR, Toll-like receptor; TNBS, trinitrobenzene sulfonic acids; TNP-Cl, trinitrophenyl chloride. * Corresponding author. E-mail address: [email protected] (M. Szczepanik).
cellular components that communicate and form a network have been investigated extensively. Recently skin was found as an attractive place of vaccination. Epicutaneous (EC) immunization offers a new method of a needlefree and self-administrable immunization by using a topically applied patch to deliver vaccine [24]. Our previous work showed that EC immunization of mice with different protein antigens (Ag) applied on the skin in the form of a patch or cream emulsion induces a state of subsequent tolerance that inhibits both Th1 and Tc1 mediated contact hypersensitivity (CHS) [17,23,25,29]. Maneuver of EC immunization with protein antigen also suppressed NK dependent CHS [16]. Similar results were found in animal model of multiple sclerosis (EAE) and collagen induced arthritis (CIA) where EC immunization with myelin basic protein (MBP) or collagen respectively reduced disease severity and decreased disease incidence [26–28]. Our further work employing allogeneic skin graft showed that EC immunization with a protein antigen delays graft rejection [14].
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.06.008
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Finally, we showed that EC immunization with protein antigen TNP-Ig alleviates TNBS-induced colitis in mice [15]. Thus our animal studies suggest that method of EC immunization might be sufficient strategy to treat various inflammatory diseases. Indeed, maneuver of EC immunization seems to bear fruit as we found that myelin peptides applied EC to multiple sclerosis (MS) patients generates type 1, IL-10 producing regulatory T cells causing suppression of specific autoreactive proliferative responses and suppression of IFN-g and TGF-b production. Moreover, we demonstrated for the first time that EC immunization with myelin peptides generates tolerogenic responses in vivo and attenuates autoimmunity in MS patients [9]. More recently we showed that EC immunization with protein antigen TNP-Ig and Toll-like receptor (TLR) ligands reversed skin induced suppression [13,21,22]. This finding suggests that EC immunization with an antigen together with TLR ligands that represent pathogen-associated molecular patterns (PAMPS) might play an important role in immunopotentiation. Potentially such maneuver may be effective for new vaccines and anti-cancer therapy. The aim of current work was to determine whether ligands that stimulate other pathogen recognition receptors (PRR) than TLR could also reverse skin induced suppression and become potential adjuvant then. Materials and methods Mice Male CBA/J mice 6–8 weeks old were from the breeding unit of the Department of Medical Biology, Jagiellonian University, College of Medicine. Mice were fed autoclaved food and water. All experiments were conducted according to guidelines of the Jagiellonian University College of Medicine. Reagents Trinitrophenyl chloride (TNP-Cl) (Chemica Alta, Edmonton, Canada); MDP NOD2 agonist – muramyldipeptide (L isoform), L18MDP NOD2 agonist – muramyldipeptide with a C18 fatty acid chain and MDP control – negative control for MDP – muramyldipeptide (D isoform, inactive) were from Inviogen (San Diego, CA, USA). Mouse immunoglobulins (Ig) were prepared from CBA/J mouse sera and conjugated with TNP hapten [12,19]. A single preparation with the level of substitution of 40 TNP per Ig molecule (TNP40-Ig) was used throughout. Mouse Ig were also conjugated with oxazolone (OX) (OX20-Ig) as described by Askenase and Asherson [1]. To measure the levels of TNF-a, IL-4, IL-6, IL-10, IL-12p70, IL17A, IFN-g and TGF-b ELISA kits; BD OptEIA Set (BD Bioscience, San Diego, CA, USA) were used. Additionally horseradish peroxidase streptavidin (Vector Laboratories, Burlingame, CA), and o-phenylenediamine, hydrogen peroxide (Sigma, St. Louis, MO) were used.
Active sensitization and measurement of contact hypersensitivity (CHS) in vivo Mice were actively sensitized by topical application of 0.15 ml of 5% TNP-Cl in acetone-ethanol mixture (1:3) to the shaved abdomen, and hind feet. Control mice were shaved and painted with acetone–ethanol mixture alone as a sham sensitization. Four days later, mice were challenged on both sides of the ears with 10 ml of 0.4% TNP-Cl in olive oil-acetone mixture (1:1) correspondingly onto both sides of the ears. The positive control group consisted of animals which were only skin-sensitized. The subsequent increase in ear thickness was measured 24 h later with an engineer’s micrometer (Mitutoyo, Tokyo, Japan) and expressed in mm SE [2]. Background increase in ear thickness of litter-mate sham sensitized animals that were similarly challenged, was evaluated. Each experimental and control group consisted of 6–8 mice. Transfer of regulatory cells into EC tolerized mice (‘‘transfer in’’ protocol) To test whether skin-induced suppression could be reversed by adoptive transfer of regulatory cells and to determine their antigen specificity, we performed a ‘‘transfer in’’ protocol [22]. On day ‘‘0’’ recipients received iv injection of 4 107 axillary and inguinal lymph node cells (ALNC) isolated from donors patched with TNP-Ig plus MDP, OX-Ig plus MDP or MDP alone. Two hours after cell transfer animals underwent EC immunization with TNP-Ig for one week (days ‘‘0’’ and ‘‘+4’’) to induce suppression (Fig. 2A). Animals that did not receive any cell transfer but were EC immunized with TNP-Ig served as a suppression control. On day ‘‘+7’’ patches were removed and mice were sensitized with 5% TNP-Cl. Also mice in positive control group (patched with PBS) were sensitized with TNP-Cl. Four days later mice were challenged and tested for CHS. Background increase in ear thickness of litter-mate sham sensitized animals that were similarly challenged, was evaluated. Tissue culture and cytokine immunoassays Lymph node cells (3 106) from mice EC treated with PBS (control group), TNP-Ig, or TNP-Ig +MDP, or MDP alone, were cultured in 1 ml RPMI 1640 medium supplemented with 5% FCS in the presence of 100 mg/ml TNP40-Ig. Cells were distributed in triplicate wells in flat 24-well Falcon plates. After 48 h culture supernatants were harvested and then tested for cytokine concentration [22]. Culture supernatants were tested for TNF-a, IL-6, IL-10, IL12p70, IL-17A, IFN-g and TGF-b by ELISA with the use of BD OptEIA Set (BD Bioscience, San Diego, CA, USA). Statistics Data in graphs are shown as the mean SE. ANOVA followed by Student’s t-test was used for multiple comparisons. Statistical significance was set at p < 0.05.
Epicutaneous immunization with TNP-Ig and NOD2 ligands Results EC immunization was performed by applying to the shaved skin of the mouse dorsum a 1 cm2 gauze patch soaked with a solution containing 100 mg TNP-Ig alone or TNP-Ig and 100 mg of MDP or L18-MDP or MDP control in a volume of 100 ml PBS on day 0. The patch was secured by adhesive tape wrapped around the midsection [22]. In the positive control mice were patched with PBS alone. The patch was left in place from day 0 until day 4, when it was replaced by a fresh patch. On day 7 patches were removed and mice were actively sensitized with TNP-Cl (Fig. 1A).
Skin-induced suppression is reversed by NOD2 ligand MDP Our previous work showed that EC immunization with protein antigen e.g. TNP-Ig induces antigen non-specific suppression of CHS sensitivity [16,17,23,25,29]. The aim of this experiment was to determine whether EC immunization with hapten-conjugated protein antigen TNP-Ig in the presence of NOD2 ligand muramyl dipeptide (MDP) could
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Fig. 1. EC immunization with TNP-Ig and NOD2 ligand MDP reverses skin induced tolerance. (A) Reversal of skin-induced suppression. CBA/J mice were EC immunized with 100 mg of TNP-Ig alone or TNP-Ig plus NOD2 ligand applied in a gauze patch on days ‘‘0’’ and ‘‘+4’’. On day ‘‘+7’’ patches were removed and animals were contact sensitized by abdominal skin painting with 5% TNP-Cl. Four days later (day ‘‘+11’’) mice were challenged by painting of both ears with 10 ml of 0.4% TNP-Cl in vehicle and 24 h later (day ‘‘+12’’) animals were tested for CHS. (B) EC treatment with antigen and MDP overcomes skin-induced suppression. Mice were patched with TNP-Ig alone (group C) or TNP-Ig plus MDP (L isoform) (group D) or TNP-Ig plus L18-MDP (group E) or TNP-Ig plus MDP control (D isoform) (group F). In control group mice were EC exposed to PBS alone (group B). On day ‘‘+7’’ patches were removed, mice (groups B–F) were sensitized with 5% TNP-Cl and then tested for CHS. Mice that were not sensitized but challenged were used as a negative control. Ear swelling was expressed in mm SE. n = 12; ***p 0.001; ns = non-significant.
reverse skin induced suppression. Here we show that indeed EC application of TNP-Ig plus MDP (L isoform) reverses skin-induced suppression (Fig. 1B, group D vs. group C). On the other hand EC immunization with TNP plus L18-MDP or MDP control prior to TNP-Cl sensitization does not reverse skin-induced suppression (Fig. 1B, groups E and F vs. group C). Reversal of skin-induced suppression is transferable and antigen specific Our previous work on skin-induced suppression showed that this phenomenon is antigen nonspecific [25,29]. On the other hand reversal of skin-induced suppression via stimulation of TLR turned out to be antigen-specific [13,21,22]. To determine whether EC induced reversal of skin-induced suppression via stimulation of NOD2 receptor is transferable and antigen specific we performed a ‘‘transfer in’’ experiment. Data presented in Fig. 2B show that reversal of skin-induced suppression is transferable with ALNC
(group D vs. group C) and this phenomenon is antigen specific (groups E and F vs. group C). Epicutaneous immunization with TNP-Ig and MDP increases release of proinflammatory cytokines in vitro To investigate whether maneuver of EC immunization with TNP-Ig and MDP affects cytokine production by lymph node cells, the following cytokines: IL-4, IL-6, IL-10, IL-12p70, IL-17 A, TNF-a, IFN-g and TGF-b were measured in culture supernatants. Data presented in Fig. 3 show that EC immunization with TNP-Ig plus MDP results in increased production of TNF-a (Fig. 3A; group C vs. B), IL-6 (Fig. 3B; group C vs. B), IL-17A (Fig. 3C; group C vs. B) when compared to immunization with TNP-Ig alone. However, similar increase in cytokine production was observed after patching with MDP alone (Fig. 3A–C; group D vs. B) when compared to immunization with TNP-Ig alone. There was no significant difference in TNF-a, IL-6 and IL-17A production between groups
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Fig. 2. Reversal of skin-induced suppression is transferable. (A) ‘‘Transfer in’’ protocol. On day ‘‘0’’ mice were iv injected with 4 107 axillary and inguinal lymph node cells (ALNC) isolated from donors patched with TNP-Ig plus MDP, OX-Ig plus MDP, MDP alone for one week. Two hours later recipient mice underwent EC immunization with TNPIg for one week (days ‘‘0’’ and ‘‘+4’’) to induce suppression. Animals that did not receive any cell transfer but were EC immunized with TNP-Ig served as a suppression control. On day ‘‘+7’’ patches were removed and mice were sensitized with 5% TNP-Cl. Also mice in positive control group (patched with PBS) were sensitized with TNP-Cl. Four days later (day ‘‘+11") mice were challenged and tested for CHS 24 h later (day ‘‘+12"). (B) Reversal of skin-induced suppression is transferable and is antigen specific. EC tolerized mice that were transferred with ALNC from donors patched TNP-Ig plus MDP (group D), OX-Ig plus MDP (group E), MDP alone (group F) or did not receive any cells (group C) were sensitized with TNP-Cl and tested for CHS. Positive control contained mice patched with PBS and then TNP-Cl sensitized (group B). Mice in negative control consisted of sham sensitized animals (group A). Ear swelling was expressed in mm SE. n = 12; **p 0.01; ***p 0.001.
patched with TNP-Ig plus MDP or MDP alone (Fig. 3A–C; group D vs. C). Additionally, EC immunization with TNP-Ig plus MDP results in significant production of IFN-g when compared to mice EC treated with TNP-Ig alone or MDP alone (Fig. 3D; group C vs. B and D). Data presented in Fig. 3E show that EC immunization with TNPIg induces higher but non-significant production of IL-10 when compared to PBS or TNP-Ig plus MDP or MDP alone treated mice (group B vs. A, C and D). We did not detect any production of IL-4 and TGF-b in tested supernatants (data not shown). Discussion In early 1970s, muramyl dipeptide (MDP) was discovered to be the smallest motif required for the efficacy of Freund’s adjuvant (CFA), one of the most potent and commonly used adjuvants in
animal studies [6]. Muramyl dipeptide (MDP) is a minimal bioactive peptidoglycan pattern present in all bacteria composed of N-acetylmuramic acid linked by its lactic acid moiety to the Nterminus of an L-alanine D-isoglutamine dipeptide [8]. It has been shown that MDP is recognized by NOD2 (NLRC2) cytoplasmic sensor of PAMPS, but not TLR2, TLR2/1 or TLR2/6 dimers [7]. It is believed that potent adjuvant activity of MDP may also be linked to an activation of NALP3/Cryopyrin inflammasome [18]. Recognition of MDP by NOD2 triggers activation of NF-kB which results in the production of proinflammatory cytokines and chemokines such as IL-6, IL-12, TNF-a, IL-8 and MCP-1 [10]. Additionally, it was shown that NOD2-induced autophagy directs the adaptive immune response because it provides antigen to MHC class II loading compartments in antigen presenting cells what is required for the development of CD4 T cell-mediated immune responses [5].
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Fig. 3. EC immunization with TNP-Ig plus MDP induces IFN-g production. Mice were EC immunized with TNP-Ig (group B) or TNP-Ig plus MDP (L isoform) (group C) or MDP alone (group D) for 7 days. Control mice were patched with PBS alone (group A). On day ‘‘+7’’ patches were removed and mice were sacrificed, ALN collected and processed under aseptic, conditions and then 3 106 ALNC were cultured in the presence of 100 mg/ml TNP-Ig. Cells were distributed in triplicate wells in flat 24-well Falcon plates. After 48 h culture supernatants were collected
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A variety of MDP analogs and derivatives have been chemically synthesized, and the relationship between their structure and immuno-pharmacological activity has been described. It has been reported that MDP and its derivatives stimulated host resistance against Klebsiella pneumonia infection in mice [4]. Additionally, it has been shown that several MDP derivatives have hoststimulating activities against bacterial infections in experimental models [4]. It is also well accepted that nonspecific host stimulation with adjuvants is a key factor for the treatment of immunosuppressed cancer patients. Indeed MDP derivatives were used to boost immune responses as a form of cancer therapy [3]. There are reports showing that macrophages activated by a liposome-encapsulated immunomodulator such as MDP derivative result in tumoricidal activity [20]. Additionally, it was found that Paclitaxel (Taxol) conjugated to MDP has not only anti-tumor activity but also immunostimulatory effects [11]. Our previous work showed that EC immunization with protein antigens induces an antigen non-specific suppression of CHS responses to subsequent active sensitization [16,17,23,25,29], whereas EC treatment with protein antigen in the presence of TLR ligands reverses skin-induced tolerance [13,21,22]. In this paper we attempted to determine whether NOD2 ligands could reverse skin-induced suppression. For that purpose we have selected MDP – muramyldipeptide (L isoform) and L18-MDP – muramyldipeptide with a C18 fatty acid chain. As a control MDP control – muramyldipeptide (D isoform, inactive) were used. Data presented in Fig. 1B show that EC immunization with TNP-Ig and MDP prior to TNP-Cl sensitization reverses skin-induced suppression (group D vs. group C). Neither L18-MDP nor MDP control has such ability (groups E and F vs. group B). Both MDP and L18-MDP are the minimal bioactive motifs common to all bacteria, the essential structures required for adjuvant activity in vaccines. The difference between MDP and L18-MDP in efficacy to reverse skininduced suppression can be related to the ability of MDP to stimulate not only NOD2 receptor but also NALP3/Cryopyrin inflammasome [18]. Thus, it is possible that simultaneous activation of NOD2 receptor and NALP3/Cryopyrin inflammasome provides more powerful adjuvant activity than stimulation of NOD2 receptor alone by L18 MDP. Then, ‘‘transfer in’’ experiment showed that reversal of skininduced suppression can be transferred with axillary and inguinal lymph node cells (ALNC) isolated from mice EC treated with TNPIg and MDP (Fig. 2B group D vs. group C). Transferring ALNC from donors patched with TNP-Ig plus MDP or OX-Ig plus MDP or MDP alone to TNP-tolerized recipients we showed that investigated reversal of suppression is antigen-specific (groups E and F vs. group D). To determine whether cytokines are involved in the reversal of skin-induced tolerance, we measured concentration of pro- and anti-inflammatory cytokines. This experiment showed that EC immunization with TNP-Ig plus MDP results in increased production of TNF-a, IL-6 and IL-17A when compared to immunization with TNP-Ig alone (Fig. 3A–C, group C vs. B). However, patching with MDP alone caused similar increase of TNF-a, IL-6 and IL-17A (Fig. 3A–C; group D vs. B), but there was no significant difference in TNF-a, IL-6 and IL-17A secretion between groups patched with TNP-Ig plus MDP or MDP alone (Fig. 3A–C; group D vs. C). These data may suggest that these three proinflammatory cytokines do not play a crucial role in reversal of skin-induced suppression as patching with antigen plus MDP and MDP alone caused similar increase in cytokine synthesis. These findings rather suggest nonspecific stimulation of cytokine production what is not a case in and then tested for concentration of the following cytokines TNF-a (A), IL-6 (B), IL17A (C), IFN-g (D) and IL-10 (group E). Results are presented as the mean SE. n = 4. **p 0.01;***p 0.001; ns = non-significant.
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antigen-specific response. On the other hand EC immunization with TNP-Ig plus MDP results in increased production of IFN-g when compared to mice patched with TNP-Ig alone or MDP alone (Fig. 3D; group C vs. B and D). This finding shows that upregulated production of IFN-g requires both stimuli, an antigen e.g. TNP-Ig and MDP, which supports our finding by proving that observed reversal of skin-induced suppression is antigen-specific. Involvement of IFN-g in the reversal of skin-induced suppression is in line with our previous findings showing that this cytokine plays an important role in contrasuppression induced via EC immunization with TNP-Ig and TLR4 ligand LPS [22]. In summary, this finding suggests that EC immunization with an antigen together with MDP may play an important role in immunopotentiation and that such a maneuver may be beneficial to enhance the immune response to weekly immunogenic antigens. Conflict of interest No conflict of interest. Funding This work was supported by grants from Ministry of Science and Higher Education N N401 545940 and National Center of Science UMO-2012/05/B/NZ6/00997 to Marian Szczepanik, and UMO2011/03/B/NZ6/00821 to Monika Majewska-Szczepanik. References [1] Askenase PW, Asherson GL. Contact sensitivity to oxazolonein the mouse. VIII: demonstration of several classes of antibody in the sera of contact sensitized and immunized mice by a simplified antiglobulin assay. Immunology 1972;26:206–14. [2] Askenase PW, Itakura A, Leite-de-Moraes MC, Lisbonne M, Roongapinun S, Goldstein DR, et al. TLR-dependent IL-4 production by invariant Valpha14+Jalpha18+NKT cells to initiate contact sensitivity in vivo. J Immunol 2005;175:6390–401. [3] Azuma I, Seya T. Development of immunoadjuvants for immunotherapy of cancer. Int Immunopharmacol 2001;1:1249–59. [4] Chedid L, Prant M, Lefrancier P, Choay J, Lederer L. Enhancement of nonspecific immunity to Klebsiella pneumoniae infection by synthetic immunoadjuvant (N-acetyl-muramyl-L-alanyl-D-isoglutamine) and several analogs. Proc Natl Acad Sci U S A 1997;74:2089–93. [5] Cooney R, Baker J, Brain O, Danis B, Pichulik T, Allan P, et al. Nod2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation. Nat Med 2010;16:90–7. [6] Ellouz F, Adam A, Ciorbaru R, Lederer E. Minimal structural requirements for adjuvant activity of bacterial peptidoglycan derivatives. Biochem Biophys Res Commun 1974;59:1317–25. [7] Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 2003;278:8869–72. [8] Inohara N, Ogura Y, Fontalba A, Gutierrez O, Pons F, Crespo J, et al. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J Biol Chem 2003;278:5509–12. [9] Jurynczyk M, Walczak A, Jurewicz A, Jesionek-Kupnicka D, Szczepanik M, Selmaj K. Immune regulation of multiple sclerosis by transdermally applied myelin peptides. Ann Neurol 2010;68:593–601.
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Epicutaneous immunization with protein antigen TNP-Ig and NOD2 ligand muramyl dipeptide (MDP) reverses skin-induced suppression of contact hypersensitivity Monika Majewska-Szczepanik, Iwona Doroz˙yn´ska, Anna Strze˛pa, Marian Szczepanik * Department of Medical Biology, Jagiellonian University College of Medicine, Krako´w, Poland
A R T I C L E I N F O
Article history: Received 27 February 2013 Accepted 14 June 2013 Available online 1 February 2014 Keywords: Epicutaneous immunization NOD-like receptors Reversal of suppression Contrasuppression
A B S T R A C T
Background: Epicutaneous (EC) immunization offers a new method of a needle-free and selfadministrable immunization by using a topically applied patch to deliver vaccine. We have previously shown that EC immunization with hapten-conjugated protein antigen TNP-Ig prior to hapten sensitization inhibits Th1-mediated contact hypersensitivity (CHS) in mice. Our further work showed that EC immunization with TNP-Ig and Toll-like receptor (TLR) ligands prior to hapten sensitization reverses skin-induced suppression. Methods: Animal model of contact hypersensitivity was used to study reversal of skin-induced suppression. Results: Current work showed that EC immunization with protein antigen TNP-Ig and MDP NOD2 agonist – muramyldipeptide (L isoform) reverses skin-induced suppression of CHS. On the other hand L18-MDP NOD2 agonist – muramyldipeptide with a C18 fatty acid chain and MDP control – negative control for MDP – muramyldipeptide (D isoform, inactive) did not reverse skin-induced suppression. ‘‘Transfer in’’ experiment showed that reversal of skin-induced suppression can be adoptively transferred with lymphoid cells isolated from donors EC treated with TNP-Ig and MDP NOD2 agonist. Moreover, experiment employing two non-cross-reacting antigens TNP-Ig and OX-Ig proved that reversal of skin-induced suppression is antigen specific. Additionally, lymph node cells isolated from mice EC immunized with TNP-Ig and MDP NOD2 agonist produced increased level of IFN-g suggesting that this cytokine might be involved in reversal of skin-induced suppression. Conclusion: This work shows that EC immunization with protein antigen plus NOD2 ligand MDP may be a potential tool to increase the immunogenicity of weekly immunogenic antigens. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction The skin forms an effective barrier between the host and the environment preventing invasion of pathogens, protecting from chemical and physical aggression. The concept that the skin is a unique and important immune organ is certainly not new, and the Abbreviations: ALNC, axillary and inguinal lymph node cells; CFA, Freund’s adjuvant; CHS, contact hypersensitivity; CIA, collagen induced arthritis; EAE, experimental autoimmune encephalomyelitis; EC, epicutaneous; FCS, fetal calf serum; IFN-g, interferon gamma; L18-MDP, muramyldipeptide with a C18 fatty acid chain; MBP, myelin basic protein; MDP, muramyldipeptide; MHC, class II major histocompatibility complex class II; NF-kB, nuclear factor kappa-light-chainenhancer of activated B cells; NK, natural killer; NOD2, nucleotide-binding oligomerization domain containing 2; OX, oxazolone; PAMPS, pathogen-associated molecular patterns; PBS, phosphate buffered saline; PRR, pathogen recognition receptors; TLR, Toll-like receptor; TNBS, trinitrobenzene sulfonic acids; TNP-Cl, trinitrophenyl chloride. * Corresponding author. E-mail address: [email protected] (M. Szczepanik).
cellular components that communicate and form a network have been investigated extensively. Recently skin was found as an attractive place of vaccination. Epicutaneous (EC) immunization offers a new method of a needlefree and self-administrable immunization by using a topically applied patch to deliver vaccine [24]. Our previous work showed that EC immunization of mice with different protein antigens (Ag) applied on the skin in the form of a patch or cream emulsion induces a state of subsequent tolerance that inhibits both Th1 and Tc1 mediated contact hypersensitivity (CHS) [17,23,25,29]. Maneuver of EC immunization with protein antigen also suppressed NK dependent CHS [16]. Similar results were found in animal model of multiple sclerosis (EAE) and collagen induced arthritis (CIA) where EC immunization with myelin basic protein (MBP) or collagen respectively reduced disease severity and decreased disease incidence [26–28]. Our further work employing allogeneic skin graft showed that EC immunization with a protein antigen delays graft rejection [14].
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.06.008
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Finally, we showed that EC immunization with protein antigen TNP-Ig alleviates TNBS-induced colitis in mice [15]. Thus our animal studies suggest that method of EC immunization might be sufficient strategy to treat various inflammatory diseases. Indeed, maneuver of EC immunization seems to bear fruit as we found that myelin peptides applied EC to multiple sclerosis (MS) patients generates type 1, IL-10 producing regulatory T cells causing suppression of specific autoreactive proliferative responses and suppression of IFN-g and TGF-b production. Moreover, we demonstrated for the first time that EC immunization with myelin peptides generates tolerogenic responses in vivo and attenuates autoimmunity in MS patients [9]. More recently we showed that EC immunization with protein antigen TNP-Ig and Toll-like receptor (TLR) ligands reversed skin induced suppression [13,21,22]. This finding suggests that EC immunization with an antigen together with TLR ligands that represent pathogen-associated molecular patterns (PAMPS) might play an important role in immunopotentiation. Potentially such maneuver may be effective for new vaccines and anti-cancer therapy. The aim of current work was to determine whether ligands that stimulate other pathogen recognition receptors (PRR) than TLR could also reverse skin induced suppression and become potential adjuvant then. Materials and methods Mice Male CBA/J mice 6–8 weeks old were from the breeding unit of the Department of Medical Biology, Jagiellonian University, College of Medicine. Mice were fed autoclaved food and water. All experiments were conducted according to guidelines of the Jagiellonian University College of Medicine. Reagents Trinitrophenyl chloride (TNP-Cl) (Chemica Alta, Edmonton, Canada); MDP NOD2 agonist – muramyldipeptide (L isoform), L18MDP NOD2 agonist – muramyldipeptide with a C18 fatty acid chain and MDP control – negative control for MDP – muramyldipeptide (D isoform, inactive) were from Inviogen (San Diego, CA, USA). Mouse immunoglobulins (Ig) were prepared from CBA/J mouse sera and conjugated with TNP hapten [12,19]. A single preparation with the level of substitution of 40 TNP per Ig molecule (TNP40-Ig) was used throughout. Mouse Ig were also conjugated with oxazolone (OX) (OX20-Ig) as described by Askenase and Asherson [1]. To measure the levels of TNF-a, IL-4, IL-6, IL-10, IL-12p70, IL17A, IFN-g and TGF-b ELISA kits; BD OptEIA Set (BD Bioscience, San Diego, CA, USA) were used. Additionally horseradish peroxidase streptavidin (Vector Laboratories, Burlingame, CA), and o-phenylenediamine, hydrogen peroxide (Sigma, St. Louis, MO) were used.
Active sensitization and measurement of contact hypersensitivity (CHS) in vivo Mice were actively sensitized by topical application of 0.15 ml of 5% TNP-Cl in acetone-ethanol mixture (1:3) to the shaved abdomen, and hind feet. Control mice were shaved and painted with acetone–ethanol mixture alone as a sham sensitization. Four days later, mice were challenged on both sides of the ears with 10 ml of 0.4% TNP-Cl in olive oil-acetone mixture (1:1) correspondingly onto both sides of the ears. The positive control group consisted of animals which were only skin-sensitized. The subsequent increase in ear thickness was measured 24 h later with an engineer’s micrometer (Mitutoyo, Tokyo, Japan) and expressed in mm SE [2]. Background increase in ear thickness of litter-mate sham sensitized animals that were similarly challenged, was evaluated. Each experimental and control group consisted of 6–8 mice. Transfer of regulatory cells into EC tolerized mice (‘‘transfer in’’ protocol) To test whether skin-induced suppression could be reversed by adoptive transfer of regulatory cells and to determine their antigen specificity, we performed a ‘‘transfer in’’ protocol [22]. On day ‘‘0’’ recipients received iv injection of 4 107 axillary and inguinal lymph node cells (ALNC) isolated from donors patched with TNP-Ig plus MDP, OX-Ig plus MDP or MDP alone. Two hours after cell transfer animals underwent EC immunization with TNP-Ig for one week (days ‘‘0’’ and ‘‘+4’’) to induce suppression (Fig. 2A). Animals that did not receive any cell transfer but were EC immunized with TNP-Ig served as a suppression control. On day ‘‘+7’’ patches were removed and mice were sensitized with 5% TNP-Cl. Also mice in positive control group (patched with PBS) were sensitized with TNP-Cl. Four days later mice were challenged and tested for CHS. Background increase in ear thickness of litter-mate sham sensitized animals that were similarly challenged, was evaluated. Tissue culture and cytokine immunoassays Lymph node cells (3 106) from mice EC treated with PBS (control group), TNP-Ig, or TNP-Ig +MDP, or MDP alone, were cultured in 1 ml RPMI 1640 medium supplemented with 5% FCS in the presence of 100 mg/ml TNP40-Ig. Cells were distributed in triplicate wells in flat 24-well Falcon plates. After 48 h culture supernatants were harvested and then tested for cytokine concentration [22]. Culture supernatants were tested for TNF-a, IL-6, IL-10, IL12p70, IL-17A, IFN-g and TGF-b by ELISA with the use of BD OptEIA Set (BD Bioscience, San Diego, CA, USA). Statistics Data in graphs are shown as the mean SE. ANOVA followed by Student’s t-test was used for multiple comparisons. Statistical significance was set at p < 0.05.
Epicutaneous immunization with TNP-Ig and NOD2 ligands Results EC immunization was performed by applying to the shaved skin of the mouse dorsum a 1 cm2 gauze patch soaked with a solution containing 100 mg TNP-Ig alone or TNP-Ig and 100 mg of MDP or L18-MDP or MDP control in a volume of 100 ml PBS on day 0. The patch was secured by adhesive tape wrapped around the midsection [22]. In the positive control mice were patched with PBS alone. The patch was left in place from day 0 until day 4, when it was replaced by a fresh patch. On day 7 patches were removed and mice were actively sensitized with TNP-Cl (Fig. 1A).
Skin-induced suppression is reversed by NOD2 ligand MDP Our previous work showed that EC immunization with protein antigen e.g. TNP-Ig induces antigen non-specific suppression of CHS sensitivity [16,17,23,25,29]. The aim of this experiment was to determine whether EC immunization with hapten-conjugated protein antigen TNP-Ig in the presence of NOD2 ligand muramyl dipeptide (MDP) could
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Fig. 1. EC immunization with TNP-Ig and NOD2 ligand MDP reverses skin induced tolerance. (A) Reversal of skin-induced suppression. CBA/J mice were EC immunized with 100 mg of TNP-Ig alone or TNP-Ig plus NOD2 ligand applied in a gauze patch on days ‘‘0’’ and ‘‘+4’’. On day ‘‘+7’’ patches were removed and animals were contact sensitized by abdominal skin painting with 5% TNP-Cl. Four days later (day ‘‘+11’’) mice were challenged by painting of both ears with 10 ml of 0.4% TNP-Cl in vehicle and 24 h later (day ‘‘+12’’) animals were tested for CHS. (B) EC treatment with antigen and MDP overcomes skin-induced suppression. Mice were patched with TNP-Ig alone (group C) or TNP-Ig plus MDP (L isoform) (group D) or TNP-Ig plus L18-MDP (group E) or TNP-Ig plus MDP control (D isoform) (group F). In control group mice were EC exposed to PBS alone (group B). On day ‘‘+7’’ patches were removed, mice (groups B–F) were sensitized with 5% TNP-Cl and then tested for CHS. Mice that were not sensitized but challenged were used as a negative control. Ear swelling was expressed in mm SE. n = 12; ***p 0.001; ns = non-significant.
reverse skin induced suppression. Here we show that indeed EC application of TNP-Ig plus MDP (L isoform) reverses skin-induced suppression (Fig. 1B, group D vs. group C). On the other hand EC immunization with TNP plus L18-MDP or MDP control prior to TNP-Cl sensitization does not reverse skin-induced suppression (Fig. 1B, groups E and F vs. group C). Reversal of skin-induced suppression is transferable and antigen specific Our previous work on skin-induced suppression showed that this phenomenon is antigen nonspecific [25,29]. On the other hand reversal of skin-induced suppression via stimulation of TLR turned out to be antigen-specific [13,21,22]. To determine whether EC induced reversal of skin-induced suppression via stimulation of NOD2 receptor is transferable and antigen specific we performed a ‘‘transfer in’’ experiment. Data presented in Fig. 2B show that reversal of skin-induced suppression is transferable with ALNC
(group D vs. group C) and this phenomenon is antigen specific (groups E and F vs. group C). Epicutaneous immunization with TNP-Ig and MDP increases release of proinflammatory cytokines in vitro To investigate whether maneuver of EC immunization with TNP-Ig and MDP affects cytokine production by lymph node cells, the following cytokines: IL-4, IL-6, IL-10, IL-12p70, IL-17 A, TNF-a, IFN-g and TGF-b were measured in culture supernatants. Data presented in Fig. 3 show that EC immunization with TNP-Ig plus MDP results in increased production of TNF-a (Fig. 3A; group C vs. B), IL-6 (Fig. 3B; group C vs. B), IL-17A (Fig. 3C; group C vs. B) when compared to immunization with TNP-Ig alone. However, similar increase in cytokine production was observed after patching with MDP alone (Fig. 3A–C; group D vs. B) when compared to immunization with TNP-Ig alone. There was no significant difference in TNF-a, IL-6 and IL-17A production between groups
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Fig. 2. Reversal of skin-induced suppression is transferable. (A) ‘‘Transfer in’’ protocol. On day ‘‘0’’ mice were iv injected with 4 107 axillary and inguinal lymph node cells (ALNC) isolated from donors patched with TNP-Ig plus MDP, OX-Ig plus MDP, MDP alone for one week. Two hours later recipient mice underwent EC immunization with TNPIg for one week (days ‘‘0’’ and ‘‘+4’’) to induce suppression. Animals that did not receive any cell transfer but were EC immunized with TNP-Ig served as a suppression control. On day ‘‘+7’’ patches were removed and mice were sensitized with 5% TNP-Cl. Also mice in positive control group (patched with PBS) were sensitized with TNP-Cl. Four days later (day ‘‘+11") mice were challenged and tested for CHS 24 h later (day ‘‘+12"). (B) Reversal of skin-induced suppression is transferable and is antigen specific. EC tolerized mice that were transferred with ALNC from donors patched TNP-Ig plus MDP (group D), OX-Ig plus MDP (group E), MDP alone (group F) or did not receive any cells (group C) were sensitized with TNP-Cl and tested for CHS. Positive control contained mice patched with PBS and then TNP-Cl sensitized (group B). Mice in negative control consisted of sham sensitized animals (group A). Ear swelling was expressed in mm SE. n = 12; **p 0.01; ***p 0.001.
patched with TNP-Ig plus MDP or MDP alone (Fig. 3A–C; group D vs. C). Additionally, EC immunization with TNP-Ig plus MDP results in significant production of IFN-g when compared to mice EC treated with TNP-Ig alone or MDP alone (Fig. 3D; group C vs. B and D). Data presented in Fig. 3E show that EC immunization with TNPIg induces higher but non-significant production of IL-10 when compared to PBS or TNP-Ig plus MDP or MDP alone treated mice (group B vs. A, C and D). We did not detect any production of IL-4 and TGF-b in tested supernatants (data not shown). Discussion In early 1970s, muramyl dipeptide (MDP) was discovered to be the smallest motif required for the efficacy of Freund’s adjuvant (CFA), one of the most potent and commonly used adjuvants in
animal studies [6]. Muramyl dipeptide (MDP) is a minimal bioactive peptidoglycan pattern present in all bacteria composed of N-acetylmuramic acid linked by its lactic acid moiety to the Nterminus of an L-alanine D-isoglutamine dipeptide [8]. It has been shown that MDP is recognized by NOD2 (NLRC2) cytoplasmic sensor of PAMPS, but not TLR2, TLR2/1 or TLR2/6 dimers [7]. It is believed that potent adjuvant activity of MDP may also be linked to an activation of NALP3/Cryopyrin inflammasome [18]. Recognition of MDP by NOD2 triggers activation of NF-kB which results in the production of proinflammatory cytokines and chemokines such as IL-6, IL-12, TNF-a, IL-8 and MCP-1 [10]. Additionally, it was shown that NOD2-induced autophagy directs the adaptive immune response because it provides antigen to MHC class II loading compartments in antigen presenting cells what is required for the development of CD4 T cell-mediated immune responses [5].
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Fig. 3. EC immunization with TNP-Ig plus MDP induces IFN-g production. Mice were EC immunized with TNP-Ig (group B) or TNP-Ig plus MDP (L isoform) (group C) or MDP alone (group D) for 7 days. Control mice were patched with PBS alone (group A). On day ‘‘+7’’ patches were removed and mice were sacrificed, ALN collected and processed under aseptic, conditions and then 3 106 ALNC were cultured in the presence of 100 mg/ml TNP-Ig. Cells were distributed in triplicate wells in flat 24-well Falcon plates. After 48 h culture supernatants were collected
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A variety of MDP analogs and derivatives have been chemically synthesized, and the relationship between their structure and immuno-pharmacological activity has been described. It has been reported that MDP and its derivatives stimulated host resistance against Klebsiella pneumonia infection in mice [4]. Additionally, it has been shown that several MDP derivatives have hoststimulating activities against bacterial infections in experimental models [4]. It is also well accepted that nonspecific host stimulation with adjuvants is a key factor for the treatment of immunosuppressed cancer patients. Indeed MDP derivatives were used to boost immune responses as a form of cancer therapy [3]. There are reports showing that macrophages activated by a liposome-encapsulated immunomodulator such as MDP derivative result in tumoricidal activity [20]. Additionally, it was found that Paclitaxel (Taxol) conjugated to MDP has not only anti-tumor activity but also immunostimulatory effects [11]. Our previous work showed that EC immunization with protein antigens induces an antigen non-specific suppression of CHS responses to subsequent active sensitization [16,17,23,25,29], whereas EC treatment with protein antigen in the presence of TLR ligands reverses skin-induced tolerance [13,21,22]. In this paper we attempted to determine whether NOD2 ligands could reverse skin-induced suppression. For that purpose we have selected MDP – muramyldipeptide (L isoform) and L18-MDP – muramyldipeptide with a C18 fatty acid chain. As a control MDP control – muramyldipeptide (D isoform, inactive) were used. Data presented in Fig. 1B show that EC immunization with TNP-Ig and MDP prior to TNP-Cl sensitization reverses skin-induced suppression (group D vs. group C). Neither L18-MDP nor MDP control has such ability (groups E and F vs. group B). Both MDP and L18-MDP are the minimal bioactive motifs common to all bacteria, the essential structures required for adjuvant activity in vaccines. The difference between MDP and L18-MDP in efficacy to reverse skininduced suppression can be related to the ability of MDP to stimulate not only NOD2 receptor but also NALP3/Cryopyrin inflammasome [18]. Thus, it is possible that simultaneous activation of NOD2 receptor and NALP3/Cryopyrin inflammasome provides more powerful adjuvant activity than stimulation of NOD2 receptor alone by L18 MDP. Then, ‘‘transfer in’’ experiment showed that reversal of skininduced suppression can be transferred with axillary and inguinal lymph node cells (ALNC) isolated from mice EC treated with TNPIg and MDP (Fig. 2B group D vs. group C). Transferring ALNC from donors patched with TNP-Ig plus MDP or OX-Ig plus MDP or MDP alone to TNP-tolerized recipients we showed that investigated reversal of suppression is antigen-specific (groups E and F vs. group D). To determine whether cytokines are involved in the reversal of skin-induced tolerance, we measured concentration of pro- and anti-inflammatory cytokines. This experiment showed that EC immunization with TNP-Ig plus MDP results in increased production of TNF-a, IL-6 and IL-17A when compared to immunization with TNP-Ig alone (Fig. 3A–C, group C vs. B). However, patching with MDP alone caused similar increase of TNF-a, IL-6 and IL-17A (Fig. 3A–C; group D vs. B), but there was no significant difference in TNF-a, IL-6 and IL-17A secretion between groups patched with TNP-Ig plus MDP or MDP alone (Fig. 3A–C; group D vs. C). These data may suggest that these three proinflammatory cytokines do not play a crucial role in reversal of skin-induced suppression as patching with antigen plus MDP and MDP alone caused similar increase in cytokine synthesis. These findings rather suggest nonspecific stimulation of cytokine production what is not a case in and then tested for concentration of the following cytokines TNF-a (A), IL-6 (B), IL17A (C), IFN-g (D) and IL-10 (group E). Results are presented as the mean SE. n = 4. **p 0.01;***p 0.001; ns = non-significant.
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antigen-specific response. On the other hand EC immunization with TNP-Ig plus MDP results in increased production of IFN-g when compared to mice patched with TNP-Ig alone or MDP alone (Fig. 3D; group C vs. B and D). This finding shows that upregulated production of IFN-g requires both stimuli, an antigen e.g. TNP-Ig and MDP, which supports our finding by proving that observed reversal of skin-induced suppression is antigen-specific. Involvement of IFN-g in the reversal of skin-induced suppression is in line with our previous findings showing that this cytokine plays an important role in contrasuppression induced via EC immunization with TNP-Ig and TLR4 ligand LPS [22]. In summary, this finding suggests that EC immunization with an antigen together with MDP may play an important role in immunopotentiation and that such a maneuver may be beneficial to enhance the immune response to weekly immunogenic antigens. Conflict of interest No conflict of interest. Funding This work was supported by grants from Ministry of Science and Higher Education N N401 545940 and National Center of Science UMO-2012/05/B/NZ6/00997 to Marian Szczepanik, and UMO2011/03/B/NZ6/00821 to Monika Majewska-Szczepanik. References [1] Askenase PW, Asherson GL. Contact sensitivity to oxazolonein the mouse. VIII: demonstration of several classes of antibody in the sera of contact sensitized and immunized mice by a simplified antiglobulin assay. Immunology 1972;26:206–14. [2] Askenase PW, Itakura A, Leite-de-Moraes MC, Lisbonne M, Roongapinun S, Goldstein DR, et al. TLR-dependent IL-4 production by invariant Valpha14+Jalpha18+NKT cells to initiate contact sensitivity in vivo. J Immunol 2005;175:6390–401. [3] Azuma I, Seya T. Development of immunoadjuvants for immunotherapy of cancer. Int Immunopharmacol 2001;1:1249–59. [4] Chedid L, Prant M, Lefrancier P, Choay J, Lederer L. Enhancement of nonspecific immunity to Klebsiella pneumoniae infection by synthetic immunoadjuvant (N-acetyl-muramyl-L-alanyl-D-isoglutamine) and several analogs. Proc Natl Acad Sci U S A 1997;74:2089–93. [5] Cooney R, Baker J, Brain O, Danis B, Pichulik T, Allan P, et al. Nod2 stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation. Nat Med 2010;16:90–7. [6] Ellouz F, Adam A, Ciorbaru R, Lederer E. Minimal structural requirements for adjuvant activity of bacterial peptidoglycan derivatives. Biochem Biophys Res Commun 1974;59:1317–25. [7] Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 2003;278:8869–72. [8] Inohara N, Ogura Y, Fontalba A, Gutierrez O, Pons F, Crespo J, et al. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J Biol Chem 2003;278:5509–12. [9] Jurynczyk M, Walczak A, Jurewicz A, Jesionek-Kupnicka D, Szczepanik M, Selmaj K. Immune regulation of multiple sclerosis by transdermally applied myelin peptides. Ann Neurol 2010;68:593–601.
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