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Epicutaneous and Oral Low-Zone Tolerance Protects from Colitis in Mice
Accepted Manuscript Epicutaneous and oral low zone tolerance protects from colitis in mice Talkea Schmidt, Nadine Lorenz, Verena Raker, Sonja Reißig, Ari Waisman, Benno Weigmann, Kerstin Steinbrink PII:
S0022-202X(16)31248-9
DOI:
10.1016/j.jid.2016.04.037
Reference:
JID 350
To appear in:
The Journal of Investigative Dermatology
Received Date: 9 October 2015 Revised Date:
7 March 2016
Accepted Date: 11 April 2016
Please cite this article as: Schmidt T, Lorenz N, Raker V, Reißig S, Waisman A, Weigmann B, Steinbrink K, Epicutaneous and oral low zone tolerance protects from colitis in mice, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.04.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Epicutaneous and oral low zone tolerance protects from colitis in mice Talkea Schmidt1, Nadine Lorenz1, Verena Raker1, Sonja Reißig2, Ari Waisman2, Benno Weigmann3, and Kerstin Steinbrink1,4 1
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Address correspondence to: Kerstin Steinbrink Department of Dermatology, University Medical Center University of Mainz Langenbeckstrasse 1 55131 Mainz, Germany phone: +49-6131-17-3792 fax: + 49-6131-17-5527 [email protected]
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Department of Dermatology, University Medical Center, Johannes Gutenberg-University of Mainz, Germany, 2 Institute for Molecular Medicine, University Medical Center, Johannes Gutenberg-University of Mainz, Germany 3 Department of Gastroenterology, University of Erlangen, Germany 4 To whom correspondence should be addressed
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Short title: Low zone tolerance protects from colitis
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CHS, contact hypersensitivity; DNBS, 2,4-dinitrobenzenesulfonic acid; DNFB, 1-Fluro-2,4dinitrobenzene; DT, diptheria toxin; LZT, low zone tolerance; TNCB, 2,4,6-trinitro-1chlorobenzene; TNBS; picrylsulfonic acid, 2,4,6-trinitro-benzenesulfonic acid; Tregs, regulatory T cells
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ABSTRACT Tolerance to environmental antigens which encounter the organism at interfaces like skin or gut prevents deleterious systemic immune responses. The aim of this study was to analyze whether
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and how low doses of haptens by entry through the skin or gastrointestinal tract affect the outcome of the predominantly Th1/Th17-mediated TNBS-induced colitis, which mimics an autoimmune bowl disease in man. Epicutaneous and oral applications of low doses of the allergen
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resulted in the induction of low zone tolerance (LZT) and protected from colitis development, demonstrated by a significantly reduced inflammatory response of the gut in vivo. In line with
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this observation, we found a significantly diminished Th1/Th17-mediated T cell response and reduced T cell proliferation after both tolerance regimes, indicating that epicutaneous LZT is just as well efficient as oral tolerance in prevention of a gut-associated inflammatory immune response. Use of a second, unrelated hapten for LZT induction revealed an antigen-specific
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tolerance mechanism. Intriguingly, in the absence of hapten-activated CD4+CD25+Foxp3+ Tregs and IL-10, epicutaneous and oral LZT failed to abrogate the development of the intestinal inflammation. In conclusion, this study highlights in particular epicutaneous immunotherapies in
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form of LZT through activation of CD4+CD25+Foxp3+ Tregs as treatment strategies for
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inflammatory, allergic or autoimmune diseases.
Key words:
Low zone tolerance; epicutaneous tolerance; oral tolerance; regulatory T cells; contact allergen; hapten; colitis; IL-10
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INTRODUCTION Antigens elicit qualitatively distinct immune responses based on their portal of entry, their dose and their specific properties, thereby either inducing activated immune responses or leading
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to tolerance. Exposure of the skin to high doses of contact allergens (haptens) results in the development of an allergic contact dermatitis, a CD8+ Tc1-mediated cutaneous inflammation and one of the most frequent occupational skin diseases in man (Fyhrquist et al., 2014). Cutaneous
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antigen-presenting cells are activated by contact allergens and migrate to the skin-draining lymph nodes where they stimulate antigen-specific CD8+ effector T cells (Kaplan et al., 2012, Martin,
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2015). In contrast, we have shown previously that epicutaneous applications of small amounts of haptens did not induce the generation of an allergic contact dermatitis but resulted in a haptenspecific tolerance reaction which is termed low zone tolerance (LZT). LZT significantly inhibited the development of a contact hypersensitivity (CHS), the murine model of the allergic contact
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dermatitis in man (Luckey et al., 2011, Luckey et al., 2012, Steinbrink et al., 1996). Epicutaneous LZT was mediated by CD8+ suppressor T cells which activated TNF-producing killer dendritic cells leading to apoptosis of CD8+ effector T cells of CHS, and thereby, to
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prevention of the allergic contact dermatitis (Luckey et al., 2011, Luckey et al., 2012). One well known physiological mechanism of peripheral tolerance is the induction of oral
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tolerance that prevents the development of local and systemic T cell-mediated inflammatory responses to self and exogenous dietary and environmental antigens (Cassani et al., 2011, Mowat, 2003). Low doses favor the activation of regulatory CD4+ T cells, whereas high doses result in anergy and deletion of antigen-specific T cells (Cassani et al., 2011, Mowat, 2003). Numerous studies in rodents have documented that oral tolerance to protein antigens is an efficient mean to inhibit autoimmune diseases in experimental models such as experimental arthritis, uveitis or
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diabetes (Higgins and Weiner, 1988, Nussenblatt, 1990, Thompson and Staines, 1986, Zhang et al., 1991). Understanding of the immune system´s ability to balance between tolerance and immunity
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remains one of the key challenges in immunology and may result in the development of innovative therapeutic strategies. Therefore, in the context of LZT we have addressed two questions: First, whether low doses of antigens in form of allergens/haptens may affect the
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outcome of a predominantly CD4+ Th1/Th17-mediated intestinal inflammation by use of the TNBS-induced model of colitis which resembles Crohn´s disease as an inflammatory
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autoimmune disorder in man (Wirtz et al., 2007, Wirtz and Neurath, 2007). Second, whether in particular the epicutaneous (as well as the oral) route of LZT induction may be efficient in the control of systemic T cell-mediated immune responses. Our data demonstrate that irrespective of the route of application, LZT significantly inhibited the development of colitis by activation of
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CD4+CD25+Foxp3+ Treg- and IL-10-controlled immune reactions. Thus, this study indicates that epicutaneously and orally applied low doses of haptens induce a tolerance reaction that protects from a systemic inflammatory disease and supported the concept of epicutaneous
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immunotherapies for allergic and autoimmune diseases.
RESULTS
Epicutaneous and oral induction of low zone tolerance to contact allergens prevents colitis development. As previously demonstrated (Luckey et al., 2011, Luckey et al., 2012, Seidel-Guyenot et al., 2006, Steinbrink et al., 1996), LZT to haptens abolished the contact hypersensitivity (CHS) 4
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reaction, a cutaneous CD8+ Tc1-mediated immune response. In this study, we addressed the question whether low allergen doses do affect the course of a predominantly Th1/Th17-mediated immune response mimicking an autoimmune disease in man (Crohn´s disease) and whether
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epicutaneous and oral LZT to haptens can inhibit an inflammatory immune response of an organ system which is related (gut) or unrelated (skin) to the site of tolerance induction.
Repeated epicutaneous painting or oral feeding of low, subimmunogenic doses of the
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hapten TNCB (or the water-soluble form TNBS for oral application) (Fig. 1a) resulted in LZT induction and in a significantly impaired colitis as observed by markedly reduced inflammation
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of the gut compared with control animals (Fig. 1b-f). The reduced intestinal inflammation was determined by a decreased immune response in the intestine according to the endoscopic score by high resolution mini-endoscopy (Fig.S1a, b)(Fig. 1b, total score [left panel], single parameters [right panel]). A significantly reduction of stool consistency, granularity of the mucosal surface,
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changes of the vascular pattern, visibility of fibrin and translucency of the colon was demonstrated, indicating that oral as well as epicutaneous LZT induction prevented colitis development (Fig. 1b, c). Histological analysis of rectum tissue confirmed the clinical
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observations by demonstrating a preserved architecture of the gut with very low levels of cellular infiltrate after epicutaneous and oral tolerization (Fig. 1d). In contrast, mice suffering from colitis
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exhibited an extensive transmural leukocyte infiltration and thickening and destruction of the colon wall with loss of goblet cells. Furthermore, analysis of the hapten-specific T cell response revealed a diminished T cell
proliferation (Fig. 1e) and significantly reduced production of IFN-γ, IL-2 and IL-17 in T cells of tolerized mice as compared to control animals (Fig. 1f), indicating an abrogation of the colitisrelated Th1/Th17-mediated immune response after LZT induction. Intriguingly, we did not
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observe any significant differences between the oral and epicutaneous route of tolerization in terms of colitis protection (Fig. 1b-f).
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Abrogation of colitis through LZT induction is allergen-specific.
Next, we tested whether colitis prevention by LZT is antigen-specific. Hereby, a second TNCB-unrelated hapten, DNFB (and the soluble form DNBS for oral applications) was used for
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tolerance induction, followed by sensitization and challenge with TNCB for colitis initiation. Applications of low amounts of the second hapten DNFB/DNBS for tolerization did not result in
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LZT induction as we observed an unrestricted TNBS-induced colitis reaction after both oral (Fig. 2a) or epicutaneous (Fig. 2c) administrations that was comparable to control colitis mice (Fig. 2a, c). In line with these data, the experiments revealed an unaffected hapten-specific T cell response after oral (Fig. 2b) and epicutaneous (Fig. 2d) tolerization with DNFB/DNBS, similar to colitis
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mice. Thus, these data indicate an antigen-specific mechanisms of LZT induction in colitis
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Epicutaneous and oral LZT results in activation of CD4+CD25+Foxp3+ Tregs. CD4+CD25+Foxp3+ Tregs play pivotal roles in tolerance processes to prevent and control
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immune reactions (McMurchy et al., 2010, Shevach, 2009, Wing and Sakaguchi, 2010). Therefore, we evaluated their potential function in epicutaneous and oral LZT induction. For this purpose, we first analyzed the frequency of CD4+CD25+Foxp3+ Tregs in mesenteric and skindraining lymph nodes after oral and epicutaneous tolerance induction. In these experiments, we did not find any differences in the percentages of Tregs in gastrointestinal and cutaneous compartments compared between control and tolerized mice, respectively (Fig. 3a, gating strategy Fig. S2a.). Nevertheless, the role of Tregs in colitis prevention may be due to their 6
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activation-induced efficient suppressive activity rather than their increased ratios in LZT. To identify an activated subpopulation of Tregs induced by the LZT protocol, we analyzed the expression of a panel of molecules considered to be involved in Treg activation, function and
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antigen experience (CD25, CD44, CD62L, CD69, CD103, CCR7, CTLA-4, ICOS, PD-1) (Wing and Sakaguchi, 2010). We did not observe any changes in the expression of CD44, CD62L, CD69, and CTLA-4 on Tregs after tolerization (data not shown). However, the experiments
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revealed significantly enhanced numbers of CD103, ICOS and PD-1 positive CD4+CD25+Foxp3+ Tregs and an increased surface expression of CCR7 on CD4+CD25+Foxp3+ Tregs after
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epicutaneous and oral tolerization compared to control animals (Figure 3b-e, Fig. S2b), indicating an activated phenotype of Treg after hapten-specific tolerance induction. Furthermore, we observed stimulated CCR7, CD103, ICOS and PD-1 positive CD4+CD25+Foxp3+ Tregs in both compartments, skin-draining and mesenteric lymph nodes, regardless of the route of tolerization
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(Figure 3b-e, Fig. S2b).
CD4+CD25+Foxp3+ Tregs are critical for prevention of colitis by oral LZT.
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For functional analysis, Tregs were depleted in the induction phase of LZT and in solventtreated control mice using an anti-CD25 mAb (PC61) (Fig. 4a). Solvent-painted and tolerized
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mice without Treg depletion served as controls. Subsequently, all experimental groups were sensitized and challenged for colitis induction. The efficacy of depletion during tolerization and recovery of Tregs prior to sensitization were assessed by flow cytometry (Fig. S3a, b) to exclude a direct effect of Treg depletion on the intestinal inflammation. In contrast to orally tolerized mice, development of LZT was completely abrogated in Treg-depleted as observed by a pronounced intestinal inflammation (Fig. 4b, total score [upper panel], score of single parameters [lower panel], Fig. 4c). Histological analysis demonstrated a 7
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pronounced cellular infiltration in the gut, accompanied by a destruction of the mucosal architecture in absence of Tregs (Fig. 4d). Consistently, analysis of the T cell response showed an enhanced hapten-specifc T cell proliferation (Fig. 4e) and IFN-γ and IL-2 production (Fig. 4f)
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after Treg depletion.
Colitis prevention by epicutaneous LZT is mediated via CD4+CD25+Foxp3+ Tregs.
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In contrast to epicutaneous LZT, in the model of oral tolerance the gut is target organ of both, tolerance induction and gastrointestinal inflammation (colitis). Therefore, we addressed the
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question whether the epicutaneous route of tolerization also resulted in Treg activation as mechanism of colitis prevention. Here again, mice were epicutaneously tolerized (LZT Treg depleted) or solvent treated (Colitis Treg depleted) after Treg depletion by injections of antiCD25-mAb (PC61) (Fig. 5a, Fig. S3a, b). In the absence of Tregs, epicutaneous applications of
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low hapten doses failed to induce tolerance and resulted in an elevated gut inflammation after colitis induction (Fig. 5b-d), determined by a significantly increased colitis score (Fig. 5b), clinical symptoms (Fig. 5c) and loss of histo-architecture and pronounced inflammatory
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infiltration of the colon (Fig. 5d). As shown for oral tolerization, Treg depletion resulted in a strong hapten-specific T cell response, with a vigorous T cell proliferation (Fig. 5e) and cytokine
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production after hapten-specific restimulation (Fig. 5f). Considering that CD25 is also upregulated on activated effector T cells and that Tregs are
also compromised by a small fraction of a CD25-negative subset, DEREG mice were used to investigate the role of Foxp3+ Tregs (Lahl et al., 2007). DEREG mice express the diphtheria toxin (DT) receptor under the control of the Foxp3 promoter. Therefore, injection of DT led to depletion of Foxp3+ cells as confirmed by flow cytometry analysis (Fig. S4a, b). Similar to antibody-induced depletion, absence of Foxp3+ Tregs in DEREG mice during LZT induction 8
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resulted in a significantly enhanced inflammation of the colon and pronounced destruction of the gut (as demonstrated by clinical symptoms and histology) compared to tolerized mice (Fig. S5ac).
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These data demonstrated a pivotal role of CD4+CD25+Foxp3+ Tregs in colitis prevention by orally as well as epicutaneously induced LZT and indicated that Treg function was not
IL-10 is required for LZT-mediated colitis prevention.
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exclusively controlled by an organ-specific T cell imprinting during LZT induction.
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The immunosuppressive cytokine IL-10 critically contributes to Treg activation and their suppressive activities, in particular in prevention of intestinal inflammation (Asseman et al., 1999, Groux et al., 1997). To unravel the function of IL-10 for CD4+CD25+Foxp3+ Tregcontrolled colitis protection by LZT, we used IL-10 deficient mice. To exclude a development of
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a spontaneous colitis we used animals on the C57BL/6 background at the age of 8-10 weeks which, in contrast to IL-10-/- animals on BALB/c background, develop a spontaneous colitis at the earliest after 6-7 months (Im et al., 2014). Intriguingly, IL-10 deficiency resulted in the
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complete abrogation of oral and epicutaneous LZT as shown by the development of a strong colitis reaction (by endoscopic score, Fig. 6a, b), inflammatory reaction in the gut (by histology,
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Fig. 6c) and pronounced T cell response (Fig. 6d). The extent of the immune responses of tolerzied IL10-/- mice was comparable to control colitis mice, indicating a critical role of IL-10 in colitis prevention by oral and epicutaneous LZT.
DISCUSSION
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In our study, we demonstrated that oral and epicutaneous applications of low doses of haptens resulted in the induction of LZT which significantly abolished the development of the predominantly Th1/Th17-mediated TNBS-induced colitis. In addition to CD4+ T cells, in the
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laminia propria of TNBS-colitis mice CD8+ Tc1 T cells were found which may also exhibit a pathogenic role in the intestinal inflammation (Nitta et al., 2003). Previously, we figured out that epicutaneous LZT did as well protect from the CHS reaction, a CD8+ Tc1-mediated skin
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inflammatory disease, which mimics the allergic contact dermatitis in man (Seidel-Guyenot et al., 2006). Thus, these results identify LZT to haptens as an efficient regime for modulation of
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systemic inflammatory immune responses, irrespective of the tolerization site. The phenomenon of oral tolerance has been known for over a century. However, in the vast majority of studies, protein antigens have been used for immuno-modulation. Here, we administered the hapten TNCB for tolerance induction, a small chemical compound which has to
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bind to (self) proteins for recognition by the immune system. Therefore, the immune mechanisms of hapten-specific LZT are not necessarily transferable to protein-induced models of tolerance/inflammation. Several studies have illustrated that immune responses induced by
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haptens or proteins, respectively, can differ in the underlying mechanisms. For example, organized Peyer's patches are required for oral tolerance to proteins, whereas orally applied
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haptens elicit systemic unresponsiveness even in Peyer´s patches deficient mice (Fujihashi et al., 2001). Former studies reported that oral application of high doses of TNCB (in the range of mg vs µg in the present study) abrogated the intestinal inflammatory response of a TNBS-mediated colitis or that feeding of high doses of hapten abrogated cutaneous inflammatory reactions (Dubois et al., 2003, Dubois et al., 2009, Elson et al., 1996, Gautam and Battisto, 1985, Neurath et al., 1996), emphasizing the phenomenon of LZT in our model.
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Two general mechanisms of oral tolerance have been proposed: feeding of high doses of antigen typically results in T cell deletion or anergy, while low doses of oral antigen administration lead to active immunosuppression by induction of Tregs (Cassani et al., 2011).
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Accordingly, depletion experiments clearly demonstrated that CD4+CD25+Foxp3+ Tregs were critical required for colitis protection by oral as well as epicutaneous LZT. These results are in line with our observation of enhanced percentages of activated CD4+CD25+Foxp3+ Tregs in skin-
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draining and mesenteric lymph nodes after epicutaneous and oral tolerance induction. We found an increased expression of CCR7 on CD4+CD25+Foxp3+ Tregs, a chemokine receptor which is
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known to be critically involved in the suppressor function of Tregs in vivo, in particular in cutaneous skin inflammation, and in Treg migration (Förster et al., 1999, Luckey et al., 2012, Schneider et al., 2007). Previous studies revealed that in contrast to control Tregs the adaptive transfer of CCR7 deficient Tregs after epicutaneous LZT induction failed to inhibit the
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development of CHS in recipient animals, emphasizing the relevance of CCR7 Tregs in LZT (Luckey et al., 2012). In addition, higher percentages of CD103, ICOS and PD-1 expressing CD4+CD25+Foxp3+ were assessed after epicutaneous as well as after oral induction of LZT.
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These data are in line with previous reports demonstrating that ICOS is essential for proper Treg cell function and that ICOS expressing antigen-specific Foxp3+ Tregs control the generation of
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CD8+ effector T cells in CHS (Burmeister et al., 2008, Dong and Nurieva, 2003, Ito et al., 2008, Vocanson et al., 2010). ICOS expression was also associated with the control of IL-10 production by Tregs, supporting our data that LZT requires Tregs and IL-10 for colitis prevention (Coquerelle et al., 2009, Redpath et al., 2013). In addition, several studies pointed out that CD103 and PD-1 expression was found on activated and antigen-experienced Tregs (Mouly et al., 2010, Riella et al., 2012, Sather et al., 2007, Suffia et al., 2005).
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Moreover, our study demonstrated that IL-10 crucially contributed to oral and epicutaneous LZT in colitis protection as demonstrated in the current study and in CHS prevention as previously described (Luckey et al., 2011, Luckey et al., 2012). IL-10 exhibits a
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multitude of functions in peripheral tolerance and in suppression of intestinal inflammation, in particular, in the interaction with Tregs. In this context, it was reported that protection from colitis by feeding of haptenated colonic protein was due to IL-10-dependent Treg development
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(Fuss et al., 2002, Neurath et al., 1996). In addition, IL-10 was required for the maintenance of Foxp3 expression and the suppressive function of Tregs resulting in protection from colitis and
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inhibition of Th17-mediated gut inflammation (Asseman et al., 1999, Chaudhry et al., 2011, Murai et al., 2009). These results are in line with our observations that IL-10 and Tregs critically contributed to LZT resulting in colitis prevention. In previous studies of oral high zone tolerance resulting in prevention of skin inflammatory immune reactions (CHS), a functional role of IL-10
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was excluded but activated pDC had been shown as key players of oral tolerance (Dubois et al., 2009, Goubier et al., 2008). In contrast in CHS prevention by LZT, CD8+ suppressor T cells have been identified that stimulate TNF producing killer CD8+ DC in the effector phase of tolerance
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(Luckey et al., 2011, Luckey et al., 2012), indicating significant differences in the underlying immune responses between low and high dose tolerance to haptens. Furthermore, these results
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suggest that in addition to the critical role of CD4+CD25+Foxp3+ Tregs, CD8+ suppressor T cells may also play a role in hapten-specific LZT protecting from colitis development. Intriguingly, we figured out that the epicutaneous LZT abrogated the colitis development
as efficiently as the cutaneous CHS reaction. Several previous studies suggested a crucial role for the tissue microenvironment in the instruction of T cells for tissue-selective homing (Dudda et al., 2004, Mora and von Andrian, 2006, Sather et al., 2007). In mice, skin-tropic T cell express Eand P-selection ligands in combination with CCR4 and CCR10, whereas gut-tropic T cells 12
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express high levels of the integrin α4β7 and CCR9 (Mora and von Andrian, 2006). Oral tolerance which resulted in prevention of a skin-related immune responses was abolished in the absence of gut-homing molecules CCR9 and α4β7 (Cassani et al., 2011), demonstrating that a tissue-
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specific education during the priming of the immune responses is required. However, deficiency of the skin-tropic CCR4 led to protection from skin inflammation after oral sensitization but cutaneous challenge (Oyoshi et al., 2011), indicating that cutaneous re-exposure to allergens can
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re-program gut-tropic effector/regulatory T cells to express homing receptors for the skin. These observations are consistent with our present study and our former results regarding CHS control
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by oral LZT that, regardless of the tolerization route, the challenge site (skin or gut) controls the organ-specific immune responses. In this context, epicutaneous application of the hapten may also lead to a distribution of the allergen into non-regional lymphatic organs. However, this question has not been addressed in detail und controversial results have been reported: Macher et 14
C-labelled DNCB
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al. did not find radioactivity in the mesenteric lymph nodes after use of
(Macher and Chase, 1969), whereas application of FITC onto ear skin resulted in detection of the allergen in mesenteric lymph nodes but to significantly lower degrees compared to skin-draining
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retroauricular lymph nodes (Pior et al., 1999).
Thus, in the current study, an unexpected link between cutaneous/oral tolerization and
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control of gut- (and skin)-related T cell-mediated immune responses had been demonstrated. In addition to oral LZT, the epicutaneous route of tolerance induction efficaciously protected from intestinal inflammatory immune responses. The therapeutic potential of oral tolerance was efficiently used in mouse models of autoimmunity and in some studies of food allergies in humans (Kulis and Burks, 2013, Mayer and Shao, 2004). However, our findings may attract notice also to an epicutaneous immunotherapy with intention of tolerance induction. In this field, some placebo-controlled phase I/IIb trails of epicutaneous immunotherapy (EPIT) for IgE13
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mediated type I allergies have been conducted efficiently by application of high amounts of allergens but T cell-mediated disorders like contact allergies or autoimmune diseases have not
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been addressed so far (Senti et al., 2015, Senti et al., 2014).
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MATERIAL AND METHODS
Reagents
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TNBS (Picrylsulfonic acid, 2,4,6-trinitro-benzenesulfonic acid), DNFB (1-Fluro-2,4dinitrobenzene), DNCB (2,4-dinitrobenzenesulfonic acid) was purchased by Sigma-Aldrich (Hamburg; Germany) and TNCB (Picrylchloride, 2,3,6-trinitro-1-chlorobenzene) by VeZwerf Laborsynthesen (Idar-Oberstein, Germany). In contrast to the haptens TNCB and DNFB which
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are applied in vivo for epicutaneous sensitization and tolerization, TNBS and DNBS are the water-soluble corresponding sulfonic acids and therefore are used for oral applications, and TNBS also for in vitro experiments. Diphtheria toxin (DT) was obtained from Calbiochem (San
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Diego, CA, USA).
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Colitis induction and elicitation
For induction of colitis mice were epicutaneously sensitized with 3000µg TNBS in 150µl
acetone/ olive oil solvent (AOO: v/v 4:1) onto a shaved area of the back. 5 days later, rectal challenge was performed by inserting a catheter (Certofix-Mono, Melsungen, Germany) into the colon of an anesthetized mouse administering 100µl of a 2500µg TNBS (25% absolute ethanol/ PBS) solution.
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Tolerance induction For epicutaneous tolerization, mice were painted 8-10 times every other day with
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tolerizing doses of 4.5µg TNCB or 0.01% DNFB dissolved in 15µl AOO (v/v 3:1) or AOO alone as control onto shaved areas of the body. Oral tolerance induction was achieved by oral gavage of the water-soluble forms of the contact allergens 4.5µg TNBS or 0.01% DNBS in PBS or PBS
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alone, respectively, 8-10 times every other day.
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Animal studies were approved by the Animal Care Committee of Rhineland-Palatinate and
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supervised by the Animal Protection Representatives of the University Medical Center Mainz.
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Conflict of interest
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The authors disclose no conflict of interest.
Acknowledgements
This work was supported by the German Research Foundation (STE791/6-1, STE791/9-1, CRC
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1066/B6, TR156/A04/C05), by the German Cancer Aid and by intramural grants to KS.
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Neurath MF, Fuss I, Kelsall BL, Presky DH, Waegell W, and Strober W (1996) Experimental granulomatous colitis in mice is abrogated by induction of TGF-beta-mediated oral tolerance. J Exp Med 183(6):2605-16
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Nitta T, Iwata H, Mori Y, Takagi H, Hirota T, Kanetake K et al. (2003) Specific CTL activity of CD8+ TCR Vβ14+ T cell in mouse 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis. Dig Dis Sci 48(10):2095-103 Nussenblatt RB (1990) The natural history of uveitis. Int Ophthalmol 14(5-6):303-8 Oyoshi MK, Elkhal A, Scott JE, Wurbel MA, Hornick JL, Campbell JJ et al. (2011) Epicutaneous challenge of orally immunized mice redirects antigen-specific gut-homing T cells to the skin. J Clin Invest 121(6):2210-20 Pior J, Vogl T, Sorg C, and Macher E (1999) Free hapten molecules are dispersed by way of the bloodstream during contact sensitization to fluorescein isothiocyanate. J Invest Dermatol 113(6):888-93 19
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Redpath SA, van der Werf N, Cervera AM, MacDonald AS, Gray D, Maizels RM et al. (2013) ICOS controls Foxp3+ regulatory T‐cell expansion, maintenance and IL‐10 production during helminth infection. Eur J Immunol 43(3):705-15
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Wirtz S, and Neurath MF (2007) Mouse models of inflammatory bowel disease. Adv Drug Delivery Rev 59(11):1073-83
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FIGURE LEGENDS
Epicutaneous and oral low zone tolerance protects from colitis.
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Figure 1 a-f
(a) Protocol of LZT and colitis induction. (b) Results were presented as pooled data (mean ± SD) of 3 independent experiments of the total endoscopic score (left panel) and of single
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parameters (right panel). Representative images of the mini-endoscopy (c) and of histology (d) of the rectum tissue are displayed. Scale bar = 150 µm. (e, f) Three days after TNBS enema, lymph
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nodes were obtained for T cell analysis. (e) After hapten-specific restimulation, T cell proliferation was depicted (mean value of triplicates in cpm). One representative out of 5 independent experiments is shown. (f) Pooled data of T cell cytokine production (IFN-γ, IL-2 and IL-17) (mean ± SEM, n = 3-5) are demonstrated. *p<0.05, **p<0.01, ***p<0.001, n.s. not
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Figure 2 a-d
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significant
Immune mechanisms of epicutaneous and oral tolerance in colitis protection are hapten-
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specific.
In addition to TNCB, DNFB was used as a second, unrelated hapten to test the allergen-
specificity of the LZT. In addition to the epicutaneous painting, for oral applications the water soluble forms of TNCB and DNFB, TNBS or DNBS, respectively, were used. Mice were (a, b) orally or (c, d) epicutaneously tolerized with TNBS/TNCB or the second unrelated hapten DNBS/DNFB, respectively. Subsequently, the sensitization and challenge for the TNBS-induced colitis was performed. Pooled data of the endoscopic score of (a) oral and (c) epicutaneous 22
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tolerance of two independent experiments are shown (mean ± SD). Hapten-specific T cell proliferation of (b) oral and (d) epicutaneous LZT is demonstrated. **p<0.01, ***p<0.001, n.s.
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not significant.
Figure 3a-e
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Increased numbers of activated CD4+CD25+Foxp3+ Tregs after epicutanoeus and oral tolerization.
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Mice have been epicutaneously or orally tolerized as described in Materials and Methods. Skin draining and mesenteric lymph nodes were obtained for Treg analysis of tolerized or control mice, respectively. (a) Percentages of CD25+Foxp3+ Tregs, gated on CD4+ T cells are shown. Pooled data of 3 independent experiments are depicted (mean ± SEM, 15 mice per group). (b)
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Expression of CCR7 (mean) and percentage of (c) CD103, (d) ICOS and (e) PD-1 positive CD4+CD25+Foxp3+ Tregs are depicted. Pooled data of 3 independent experiments are demonstrated (mean ± SEM, 15 mice per group). *p<0.05, **p<0.01, ***p<0.001, n.s. not
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significant.
Figure 4 a-f
CD4+CD25+Foxp3+ Treg cells are crucial for colitis prevention by oral LZT. (a) Protocol of Treg depletion. TNBS-colitis was induced after Treg recurrence (Fig. S2).
Solvent-treated mice +/-Treg depletion served as controls (colitis / colitis Treg depleted). (b) Inflammatory colitis symptoms according to the total endoscopic score (upper panel) and single parameters (lower panel). Pooled data of 2 experiments are shown (mean ± SD). (c) Images of 23
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mini-endoscopy representing the average endoscopic score and (d) of histology of rectum tissue sections are shown. Scale bar = 150 µm. (e) Hapten-specific T cell proliferation in vitro was assessed (mean value of triplicates in cpm). One representative out of 2 independent experiments
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with similar results is shown. (f) Representative data of hapten-specific T cell cytokine production (IFN-γ, IL-2) of 2 independent experiments are depicted (mean value of duplicates ±
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SD). **p<0.01, ***p<0.001, n.s. not significant.
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Figure 5 a-f
Epicutaneous LZT requires CD4+CD25+Foxp3+ Treg cells for colitis inhibition. (a) Protocol of Treg depletion. After Treg repopulation (Fig. S2) colitis was induced by sensitization and rectal challenge. Solvent-treated mice +/-Treg depletion served as controls
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(colitis / colitis Treg depleted). (b) Total endoscopic score (upper panel) and single parameter of the score were given as pooled data of 3 experiments (mean ± SD). (c) Images of the miniendoscopy representing the average endoscopic scores and (d) of histology of rectum tissue are
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shown. Scale bar = 150 µm. (e) Hapten-specific T cell proliferation was depicted (mean value of triplicates in cpm) after hapten-specific restimulation in vitro. One representative out of 3
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independent experiments with similar results is shown. (f) Representative data of T cell cytokine production (IFN-γ, IL-2) out of 3 independent experiments are demonstrated (mean value of duplicates ± SD). ***p<0.001, n.s. not significant.
Figure 6 a-d IL-10 is critical for oral and epicutaneous tolerance resulting in colitis prevention. 24
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WT and IL-10 deficient mice were epicutaneously or orally tolerized, respectively. Subsequently, colitis induction was performed. Pooled data of (a) the total endoscopic score of 3 experiments are depicted (mean ± SD). Representative images of clinical score of mini-
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endoscopy (b) and of histology (c) of the gut tissue are shown. Scale bar = 150 µm. (d) The hapten-specific T cell proliferation in vitro was depicted (mean value of triplicates in cpm). One representative out of 3 independent experiments with similar results is shown. *p<0.05,
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***p<0.001, n.s. not significant.
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S0022-202X(16)31248-9
DOI:
10.1016/j.jid.2016.04.037
Reference:
JID 350
To appear in:
The Journal of Investigative Dermatology
Received Date: 9 October 2015 Revised Date:
7 March 2016
Accepted Date: 11 April 2016
Please cite this article as: Schmidt T, Lorenz N, Raker V, Reißig S, Waisman A, Weigmann B, Steinbrink K, Epicutaneous and oral low zone tolerance protects from colitis in mice, The Journal of Investigative Dermatology (2016), doi: 10.1016/j.jid.2016.04.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Epicutaneous and oral low zone tolerance protects from colitis in mice Talkea Schmidt1, Nadine Lorenz1, Verena Raker1, Sonja Reißig2, Ari Waisman2, Benno Weigmann3, and Kerstin Steinbrink1,4 1
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Address correspondence to: Kerstin Steinbrink Department of Dermatology, University Medical Center University of Mainz Langenbeckstrasse 1 55131 Mainz, Germany phone: +49-6131-17-3792 fax: + 49-6131-17-5527 [email protected]
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Department of Dermatology, University Medical Center, Johannes Gutenberg-University of Mainz, Germany, 2 Institute for Molecular Medicine, University Medical Center, Johannes Gutenberg-University of Mainz, Germany 3 Department of Gastroenterology, University of Erlangen, Germany 4 To whom correspondence should be addressed
Abbreviations:
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Short title: Low zone tolerance protects from colitis
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CHS, contact hypersensitivity; DNBS, 2,4-dinitrobenzenesulfonic acid; DNFB, 1-Fluro-2,4dinitrobenzene; DT, diptheria toxin; LZT, low zone tolerance; TNCB, 2,4,6-trinitro-1chlorobenzene; TNBS; picrylsulfonic acid, 2,4,6-trinitro-benzenesulfonic acid; Tregs, regulatory T cells
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ABSTRACT Tolerance to environmental antigens which encounter the organism at interfaces like skin or gut prevents deleterious systemic immune responses. The aim of this study was to analyze whether
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and how low doses of haptens by entry through the skin or gastrointestinal tract affect the outcome of the predominantly Th1/Th17-mediated TNBS-induced colitis, which mimics an autoimmune bowl disease in man. Epicutaneous and oral applications of low doses of the allergen
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resulted in the induction of low zone tolerance (LZT) and protected from colitis development, demonstrated by a significantly reduced inflammatory response of the gut in vivo. In line with
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this observation, we found a significantly diminished Th1/Th17-mediated T cell response and reduced T cell proliferation after both tolerance regimes, indicating that epicutaneous LZT is just as well efficient as oral tolerance in prevention of a gut-associated inflammatory immune response. Use of a second, unrelated hapten for LZT induction revealed an antigen-specific
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tolerance mechanism. Intriguingly, in the absence of hapten-activated CD4+CD25+Foxp3+ Tregs and IL-10, epicutaneous and oral LZT failed to abrogate the development of the intestinal inflammation. In conclusion, this study highlights in particular epicutaneous immunotherapies in
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form of LZT through activation of CD4+CD25+Foxp3+ Tregs as treatment strategies for
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inflammatory, allergic or autoimmune diseases.
Key words:
Low zone tolerance; epicutaneous tolerance; oral tolerance; regulatory T cells; contact allergen; hapten; colitis; IL-10
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INTRODUCTION Antigens elicit qualitatively distinct immune responses based on their portal of entry, their dose and their specific properties, thereby either inducing activated immune responses or leading
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to tolerance. Exposure of the skin to high doses of contact allergens (haptens) results in the development of an allergic contact dermatitis, a CD8+ Tc1-mediated cutaneous inflammation and one of the most frequent occupational skin diseases in man (Fyhrquist et al., 2014). Cutaneous
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antigen-presenting cells are activated by contact allergens and migrate to the skin-draining lymph nodes where they stimulate antigen-specific CD8+ effector T cells (Kaplan et al., 2012, Martin,
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2015). In contrast, we have shown previously that epicutaneous applications of small amounts of haptens did not induce the generation of an allergic contact dermatitis but resulted in a haptenspecific tolerance reaction which is termed low zone tolerance (LZT). LZT significantly inhibited the development of a contact hypersensitivity (CHS), the murine model of the allergic contact
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dermatitis in man (Luckey et al., 2011, Luckey et al., 2012, Steinbrink et al., 1996). Epicutaneous LZT was mediated by CD8+ suppressor T cells which activated TNF-producing killer dendritic cells leading to apoptosis of CD8+ effector T cells of CHS, and thereby, to
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prevention of the allergic contact dermatitis (Luckey et al., 2011, Luckey et al., 2012). One well known physiological mechanism of peripheral tolerance is the induction of oral
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tolerance that prevents the development of local and systemic T cell-mediated inflammatory responses to self and exogenous dietary and environmental antigens (Cassani et al., 2011, Mowat, 2003). Low doses favor the activation of regulatory CD4+ T cells, whereas high doses result in anergy and deletion of antigen-specific T cells (Cassani et al., 2011, Mowat, 2003). Numerous studies in rodents have documented that oral tolerance to protein antigens is an efficient mean to inhibit autoimmune diseases in experimental models such as experimental arthritis, uveitis or
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diabetes (Higgins and Weiner, 1988, Nussenblatt, 1990, Thompson and Staines, 1986, Zhang et al., 1991). Understanding of the immune system´s ability to balance between tolerance and immunity
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remains one of the key challenges in immunology and may result in the development of innovative therapeutic strategies. Therefore, in the context of LZT we have addressed two questions: First, whether low doses of antigens in form of allergens/haptens may affect the
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outcome of a predominantly CD4+ Th1/Th17-mediated intestinal inflammation by use of the TNBS-induced model of colitis which resembles Crohn´s disease as an inflammatory
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autoimmune disorder in man (Wirtz et al., 2007, Wirtz and Neurath, 2007). Second, whether in particular the epicutaneous (as well as the oral) route of LZT induction may be efficient in the control of systemic T cell-mediated immune responses. Our data demonstrate that irrespective of the route of application, LZT significantly inhibited the development of colitis by activation of
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CD4+CD25+Foxp3+ Treg- and IL-10-controlled immune reactions. Thus, this study indicates that epicutaneously and orally applied low doses of haptens induce a tolerance reaction that protects from a systemic inflammatory disease and supported the concept of epicutaneous
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immunotherapies for allergic and autoimmune diseases.
RESULTS
Epicutaneous and oral induction of low zone tolerance to contact allergens prevents colitis development. As previously demonstrated (Luckey et al., 2011, Luckey et al., 2012, Seidel-Guyenot et al., 2006, Steinbrink et al., 1996), LZT to haptens abolished the contact hypersensitivity (CHS) 4
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reaction, a cutaneous CD8+ Tc1-mediated immune response. In this study, we addressed the question whether low allergen doses do affect the course of a predominantly Th1/Th17-mediated immune response mimicking an autoimmune disease in man (Crohn´s disease) and whether
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epicutaneous and oral LZT to haptens can inhibit an inflammatory immune response of an organ system which is related (gut) or unrelated (skin) to the site of tolerance induction.
Repeated epicutaneous painting or oral feeding of low, subimmunogenic doses of the
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hapten TNCB (or the water-soluble form TNBS for oral application) (Fig. 1a) resulted in LZT induction and in a significantly impaired colitis as observed by markedly reduced inflammation
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of the gut compared with control animals (Fig. 1b-f). The reduced intestinal inflammation was determined by a decreased immune response in the intestine according to the endoscopic score by high resolution mini-endoscopy (Fig.S1a, b)(Fig. 1b, total score [left panel], single parameters [right panel]). A significantly reduction of stool consistency, granularity of the mucosal surface,
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changes of the vascular pattern, visibility of fibrin and translucency of the colon was demonstrated, indicating that oral as well as epicutaneous LZT induction prevented colitis development (Fig. 1b, c). Histological analysis of rectum tissue confirmed the clinical
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observations by demonstrating a preserved architecture of the gut with very low levels of cellular infiltrate after epicutaneous and oral tolerization (Fig. 1d). In contrast, mice suffering from colitis
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exhibited an extensive transmural leukocyte infiltration and thickening and destruction of the colon wall with loss of goblet cells. Furthermore, analysis of the hapten-specific T cell response revealed a diminished T cell
proliferation (Fig. 1e) and significantly reduced production of IFN-γ, IL-2 and IL-17 in T cells of tolerized mice as compared to control animals (Fig. 1f), indicating an abrogation of the colitisrelated Th1/Th17-mediated immune response after LZT induction. Intriguingly, we did not
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observe any significant differences between the oral and epicutaneous route of tolerization in terms of colitis protection (Fig. 1b-f).
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Abrogation of colitis through LZT induction is allergen-specific.
Next, we tested whether colitis prevention by LZT is antigen-specific. Hereby, a second TNCB-unrelated hapten, DNFB (and the soluble form DNBS for oral applications) was used for
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tolerance induction, followed by sensitization and challenge with TNCB for colitis initiation. Applications of low amounts of the second hapten DNFB/DNBS for tolerization did not result in
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LZT induction as we observed an unrestricted TNBS-induced colitis reaction after both oral (Fig. 2a) or epicutaneous (Fig. 2c) administrations that was comparable to control colitis mice (Fig. 2a, c). In line with these data, the experiments revealed an unaffected hapten-specific T cell response after oral (Fig. 2b) and epicutaneous (Fig. 2d) tolerization with DNFB/DNBS, similar to colitis
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mice. Thus, these data indicate an antigen-specific mechanisms of LZT induction in colitis
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Epicutaneous and oral LZT results in activation of CD4+CD25+Foxp3+ Tregs. CD4+CD25+Foxp3+ Tregs play pivotal roles in tolerance processes to prevent and control
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immune reactions (McMurchy et al., 2010, Shevach, 2009, Wing and Sakaguchi, 2010). Therefore, we evaluated their potential function in epicutaneous and oral LZT induction. For this purpose, we first analyzed the frequency of CD4+CD25+Foxp3+ Tregs in mesenteric and skindraining lymph nodes after oral and epicutaneous tolerance induction. In these experiments, we did not find any differences in the percentages of Tregs in gastrointestinal and cutaneous compartments compared between control and tolerized mice, respectively (Fig. 3a, gating strategy Fig. S2a.). Nevertheless, the role of Tregs in colitis prevention may be due to their 6
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activation-induced efficient suppressive activity rather than their increased ratios in LZT. To identify an activated subpopulation of Tregs induced by the LZT protocol, we analyzed the expression of a panel of molecules considered to be involved in Treg activation, function and
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antigen experience (CD25, CD44, CD62L, CD69, CD103, CCR7, CTLA-4, ICOS, PD-1) (Wing and Sakaguchi, 2010). We did not observe any changes in the expression of CD44, CD62L, CD69, and CTLA-4 on Tregs after tolerization (data not shown). However, the experiments
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revealed significantly enhanced numbers of CD103, ICOS and PD-1 positive CD4+CD25+Foxp3+ Tregs and an increased surface expression of CCR7 on CD4+CD25+Foxp3+ Tregs after
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epicutaneous and oral tolerization compared to control animals (Figure 3b-e, Fig. S2b), indicating an activated phenotype of Treg after hapten-specific tolerance induction. Furthermore, we observed stimulated CCR7, CD103, ICOS and PD-1 positive CD4+CD25+Foxp3+ Tregs in both compartments, skin-draining and mesenteric lymph nodes, regardless of the route of tolerization
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(Figure 3b-e, Fig. S2b).
CD4+CD25+Foxp3+ Tregs are critical for prevention of colitis by oral LZT.
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For functional analysis, Tregs were depleted in the induction phase of LZT and in solventtreated control mice using an anti-CD25 mAb (PC61) (Fig. 4a). Solvent-painted and tolerized
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mice without Treg depletion served as controls. Subsequently, all experimental groups were sensitized and challenged for colitis induction. The efficacy of depletion during tolerization and recovery of Tregs prior to sensitization were assessed by flow cytometry (Fig. S3a, b) to exclude a direct effect of Treg depletion on the intestinal inflammation. In contrast to orally tolerized mice, development of LZT was completely abrogated in Treg-depleted as observed by a pronounced intestinal inflammation (Fig. 4b, total score [upper panel], score of single parameters [lower panel], Fig. 4c). Histological analysis demonstrated a 7
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pronounced cellular infiltration in the gut, accompanied by a destruction of the mucosal architecture in absence of Tregs (Fig. 4d). Consistently, analysis of the T cell response showed an enhanced hapten-specifc T cell proliferation (Fig. 4e) and IFN-γ and IL-2 production (Fig. 4f)
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after Treg depletion.
Colitis prevention by epicutaneous LZT is mediated via CD4+CD25+Foxp3+ Tregs.
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In contrast to epicutaneous LZT, in the model of oral tolerance the gut is target organ of both, tolerance induction and gastrointestinal inflammation (colitis). Therefore, we addressed the
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question whether the epicutaneous route of tolerization also resulted in Treg activation as mechanism of colitis prevention. Here again, mice were epicutaneously tolerized (LZT Treg depleted) or solvent treated (Colitis Treg depleted) after Treg depletion by injections of antiCD25-mAb (PC61) (Fig. 5a, Fig. S3a, b). In the absence of Tregs, epicutaneous applications of
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low hapten doses failed to induce tolerance and resulted in an elevated gut inflammation after colitis induction (Fig. 5b-d), determined by a significantly increased colitis score (Fig. 5b), clinical symptoms (Fig. 5c) and loss of histo-architecture and pronounced inflammatory
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infiltration of the colon (Fig. 5d). As shown for oral tolerization, Treg depletion resulted in a strong hapten-specific T cell response, with a vigorous T cell proliferation (Fig. 5e) and cytokine
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production after hapten-specific restimulation (Fig. 5f). Considering that CD25 is also upregulated on activated effector T cells and that Tregs are
also compromised by a small fraction of a CD25-negative subset, DEREG mice were used to investigate the role of Foxp3+ Tregs (Lahl et al., 2007). DEREG mice express the diphtheria toxin (DT) receptor under the control of the Foxp3 promoter. Therefore, injection of DT led to depletion of Foxp3+ cells as confirmed by flow cytometry analysis (Fig. S4a, b). Similar to antibody-induced depletion, absence of Foxp3+ Tregs in DEREG mice during LZT induction 8
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resulted in a significantly enhanced inflammation of the colon and pronounced destruction of the gut (as demonstrated by clinical symptoms and histology) compared to tolerized mice (Fig. S5ac).
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These data demonstrated a pivotal role of CD4+CD25+Foxp3+ Tregs in colitis prevention by orally as well as epicutaneously induced LZT and indicated that Treg function was not
IL-10 is required for LZT-mediated colitis prevention.
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exclusively controlled by an organ-specific T cell imprinting during LZT induction.
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The immunosuppressive cytokine IL-10 critically contributes to Treg activation and their suppressive activities, in particular in prevention of intestinal inflammation (Asseman et al., 1999, Groux et al., 1997). To unravel the function of IL-10 for CD4+CD25+Foxp3+ Tregcontrolled colitis protection by LZT, we used IL-10 deficient mice. To exclude a development of
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a spontaneous colitis we used animals on the C57BL/6 background at the age of 8-10 weeks which, in contrast to IL-10-/- animals on BALB/c background, develop a spontaneous colitis at the earliest after 6-7 months (Im et al., 2014). Intriguingly, IL-10 deficiency resulted in the
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complete abrogation of oral and epicutaneous LZT as shown by the development of a strong colitis reaction (by endoscopic score, Fig. 6a, b), inflammatory reaction in the gut (by histology,
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Fig. 6c) and pronounced T cell response (Fig. 6d). The extent of the immune responses of tolerzied IL10-/- mice was comparable to control colitis mice, indicating a critical role of IL-10 in colitis prevention by oral and epicutaneous LZT.
DISCUSSION
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In our study, we demonstrated that oral and epicutaneous applications of low doses of haptens resulted in the induction of LZT which significantly abolished the development of the predominantly Th1/Th17-mediated TNBS-induced colitis. In addition to CD4+ T cells, in the
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laminia propria of TNBS-colitis mice CD8+ Tc1 T cells were found which may also exhibit a pathogenic role in the intestinal inflammation (Nitta et al., 2003). Previously, we figured out that epicutaneous LZT did as well protect from the CHS reaction, a CD8+ Tc1-mediated skin
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inflammatory disease, which mimics the allergic contact dermatitis in man (Seidel-Guyenot et al., 2006). Thus, these results identify LZT to haptens as an efficient regime for modulation of
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systemic inflammatory immune responses, irrespective of the tolerization site. The phenomenon of oral tolerance has been known for over a century. However, in the vast majority of studies, protein antigens have been used for immuno-modulation. Here, we administered the hapten TNCB for tolerance induction, a small chemical compound which has to
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bind to (self) proteins for recognition by the immune system. Therefore, the immune mechanisms of hapten-specific LZT are not necessarily transferable to protein-induced models of tolerance/inflammation. Several studies have illustrated that immune responses induced by
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haptens or proteins, respectively, can differ in the underlying mechanisms. For example, organized Peyer's patches are required for oral tolerance to proteins, whereas orally applied
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haptens elicit systemic unresponsiveness even in Peyer´s patches deficient mice (Fujihashi et al., 2001). Former studies reported that oral application of high doses of TNCB (in the range of mg vs µg in the present study) abrogated the intestinal inflammatory response of a TNBS-mediated colitis or that feeding of high doses of hapten abrogated cutaneous inflammatory reactions (Dubois et al., 2003, Dubois et al., 2009, Elson et al., 1996, Gautam and Battisto, 1985, Neurath et al., 1996), emphasizing the phenomenon of LZT in our model.
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Two general mechanisms of oral tolerance have been proposed: feeding of high doses of antigen typically results in T cell deletion or anergy, while low doses of oral antigen administration lead to active immunosuppression by induction of Tregs (Cassani et al., 2011).
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Accordingly, depletion experiments clearly demonstrated that CD4+CD25+Foxp3+ Tregs were critical required for colitis protection by oral as well as epicutaneous LZT. These results are in line with our observation of enhanced percentages of activated CD4+CD25+Foxp3+ Tregs in skin-
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draining and mesenteric lymph nodes after epicutaneous and oral tolerance induction. We found an increased expression of CCR7 on CD4+CD25+Foxp3+ Tregs, a chemokine receptor which is
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known to be critically involved in the suppressor function of Tregs in vivo, in particular in cutaneous skin inflammation, and in Treg migration (Förster et al., 1999, Luckey et al., 2012, Schneider et al., 2007). Previous studies revealed that in contrast to control Tregs the adaptive transfer of CCR7 deficient Tregs after epicutaneous LZT induction failed to inhibit the
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development of CHS in recipient animals, emphasizing the relevance of CCR7 Tregs in LZT (Luckey et al., 2012). In addition, higher percentages of CD103, ICOS and PD-1 expressing CD4+CD25+Foxp3+ were assessed after epicutaneous as well as after oral induction of LZT.
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These data are in line with previous reports demonstrating that ICOS is essential for proper Treg cell function and that ICOS expressing antigen-specific Foxp3+ Tregs control the generation of
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CD8+ effector T cells in CHS (Burmeister et al., 2008, Dong and Nurieva, 2003, Ito et al., 2008, Vocanson et al., 2010). ICOS expression was also associated with the control of IL-10 production by Tregs, supporting our data that LZT requires Tregs and IL-10 for colitis prevention (Coquerelle et al., 2009, Redpath et al., 2013). In addition, several studies pointed out that CD103 and PD-1 expression was found on activated and antigen-experienced Tregs (Mouly et al., 2010, Riella et al., 2012, Sather et al., 2007, Suffia et al., 2005).
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Moreover, our study demonstrated that IL-10 crucially contributed to oral and epicutaneous LZT in colitis protection as demonstrated in the current study and in CHS prevention as previously described (Luckey et al., 2011, Luckey et al., 2012). IL-10 exhibits a
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multitude of functions in peripheral tolerance and in suppression of intestinal inflammation, in particular, in the interaction with Tregs. In this context, it was reported that protection from colitis by feeding of haptenated colonic protein was due to IL-10-dependent Treg development
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(Fuss et al., 2002, Neurath et al., 1996). In addition, IL-10 was required for the maintenance of Foxp3 expression and the suppressive function of Tregs resulting in protection from colitis and
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inhibition of Th17-mediated gut inflammation (Asseman et al., 1999, Chaudhry et al., 2011, Murai et al., 2009). These results are in line with our observations that IL-10 and Tregs critically contributed to LZT resulting in colitis prevention. In previous studies of oral high zone tolerance resulting in prevention of skin inflammatory immune reactions (CHS), a functional role of IL-10
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was excluded but activated pDC had been shown as key players of oral tolerance (Dubois et al., 2009, Goubier et al., 2008). In contrast in CHS prevention by LZT, CD8+ suppressor T cells have been identified that stimulate TNF producing killer CD8+ DC in the effector phase of tolerance
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(Luckey et al., 2011, Luckey et al., 2012), indicating significant differences in the underlying immune responses between low and high dose tolerance to haptens. Furthermore, these results
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suggest that in addition to the critical role of CD4+CD25+Foxp3+ Tregs, CD8+ suppressor T cells may also play a role in hapten-specific LZT protecting from colitis development. Intriguingly, we figured out that the epicutaneous LZT abrogated the colitis development
as efficiently as the cutaneous CHS reaction. Several previous studies suggested a crucial role for the tissue microenvironment in the instruction of T cells for tissue-selective homing (Dudda et al., 2004, Mora and von Andrian, 2006, Sather et al., 2007). In mice, skin-tropic T cell express Eand P-selection ligands in combination with CCR4 and CCR10, whereas gut-tropic T cells 12
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express high levels of the integrin α4β7 and CCR9 (Mora and von Andrian, 2006). Oral tolerance which resulted in prevention of a skin-related immune responses was abolished in the absence of gut-homing molecules CCR9 and α4β7 (Cassani et al., 2011), demonstrating that a tissue-
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specific education during the priming of the immune responses is required. However, deficiency of the skin-tropic CCR4 led to protection from skin inflammation after oral sensitization but cutaneous challenge (Oyoshi et al., 2011), indicating that cutaneous re-exposure to allergens can
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re-program gut-tropic effector/regulatory T cells to express homing receptors for the skin. These observations are consistent with our present study and our former results regarding CHS control
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by oral LZT that, regardless of the tolerization route, the challenge site (skin or gut) controls the organ-specific immune responses. In this context, epicutaneous application of the hapten may also lead to a distribution of the allergen into non-regional lymphatic organs. However, this question has not been addressed in detail und controversial results have been reported: Macher et 14
C-labelled DNCB
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al. did not find radioactivity in the mesenteric lymph nodes after use of
(Macher and Chase, 1969), whereas application of FITC onto ear skin resulted in detection of the allergen in mesenteric lymph nodes but to significantly lower degrees compared to skin-draining
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retroauricular lymph nodes (Pior et al., 1999).
Thus, in the current study, an unexpected link between cutaneous/oral tolerization and
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control of gut- (and skin)-related T cell-mediated immune responses had been demonstrated. In addition to oral LZT, the epicutaneous route of tolerance induction efficaciously protected from intestinal inflammatory immune responses. The therapeutic potential of oral tolerance was efficiently used in mouse models of autoimmunity and in some studies of food allergies in humans (Kulis and Burks, 2013, Mayer and Shao, 2004). However, our findings may attract notice also to an epicutaneous immunotherapy with intention of tolerance induction. In this field, some placebo-controlled phase I/IIb trails of epicutaneous immunotherapy (EPIT) for IgE13
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mediated type I allergies have been conducted efficiently by application of high amounts of allergens but T cell-mediated disorders like contact allergies or autoimmune diseases have not
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been addressed so far (Senti et al., 2015, Senti et al., 2014).
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MATERIAL AND METHODS
Reagents
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TNBS (Picrylsulfonic acid, 2,4,6-trinitro-benzenesulfonic acid), DNFB (1-Fluro-2,4dinitrobenzene), DNCB (2,4-dinitrobenzenesulfonic acid) was purchased by Sigma-Aldrich (Hamburg; Germany) and TNCB (Picrylchloride, 2,3,6-trinitro-1-chlorobenzene) by VeZwerf Laborsynthesen (Idar-Oberstein, Germany). In contrast to the haptens TNCB and DNFB which
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are applied in vivo for epicutaneous sensitization and tolerization, TNBS and DNBS are the water-soluble corresponding sulfonic acids and therefore are used for oral applications, and TNBS also for in vitro experiments. Diphtheria toxin (DT) was obtained from Calbiochem (San
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Diego, CA, USA).
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Colitis induction and elicitation
For induction of colitis mice were epicutaneously sensitized with 3000µg TNBS in 150µl
acetone/ olive oil solvent (AOO: v/v 4:1) onto a shaved area of the back. 5 days later, rectal challenge was performed by inserting a catheter (Certofix-Mono, Melsungen, Germany) into the colon of an anesthetized mouse administering 100µl of a 2500µg TNBS (25% absolute ethanol/ PBS) solution.
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Tolerance induction For epicutaneous tolerization, mice were painted 8-10 times every other day with
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tolerizing doses of 4.5µg TNCB or 0.01% DNFB dissolved in 15µl AOO (v/v 3:1) or AOO alone as control onto shaved areas of the body. Oral tolerance induction was achieved by oral gavage of the water-soluble forms of the contact allergens 4.5µg TNBS or 0.01% DNBS in PBS or PBS
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alone, respectively, 8-10 times every other day.
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Animal studies were approved by the Animal Care Committee of Rhineland-Palatinate and
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supervised by the Animal Protection Representatives of the University Medical Center Mainz.
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Conflict of interest
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The authors disclose no conflict of interest.
Acknowledgements
This work was supported by the German Research Foundation (STE791/6-1, STE791/9-1, CRC
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1066/B6, TR156/A04/C05), by the German Cancer Aid and by intramural grants to KS.
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FIGURE LEGENDS
Epicutaneous and oral low zone tolerance protects from colitis.
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Figure 1 a-f
(a) Protocol of LZT and colitis induction. (b) Results were presented as pooled data (mean ± SD) of 3 independent experiments of the total endoscopic score (left panel) and of single
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parameters (right panel). Representative images of the mini-endoscopy (c) and of histology (d) of the rectum tissue are displayed. Scale bar = 150 µm. (e, f) Three days after TNBS enema, lymph
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nodes were obtained for T cell analysis. (e) After hapten-specific restimulation, T cell proliferation was depicted (mean value of triplicates in cpm). One representative out of 5 independent experiments is shown. (f) Pooled data of T cell cytokine production (IFN-γ, IL-2 and IL-17) (mean ± SEM, n = 3-5) are demonstrated. *p<0.05, **p<0.01, ***p<0.001, n.s. not
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Figure 2 a-d
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significant
Immune mechanisms of epicutaneous and oral tolerance in colitis protection are hapten-
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specific.
In addition to TNCB, DNFB was used as a second, unrelated hapten to test the allergen-
specificity of the LZT. In addition to the epicutaneous painting, for oral applications the water soluble forms of TNCB and DNFB, TNBS or DNBS, respectively, were used. Mice were (a, b) orally or (c, d) epicutaneously tolerized with TNBS/TNCB or the second unrelated hapten DNBS/DNFB, respectively. Subsequently, the sensitization and challenge for the TNBS-induced colitis was performed. Pooled data of the endoscopic score of (a) oral and (c) epicutaneous 22
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tolerance of two independent experiments are shown (mean ± SD). Hapten-specific T cell proliferation of (b) oral and (d) epicutaneous LZT is demonstrated. **p<0.01, ***p<0.001, n.s.
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not significant.
Figure 3a-e
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Increased numbers of activated CD4+CD25+Foxp3+ Tregs after epicutanoeus and oral tolerization.
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Mice have been epicutaneously or orally tolerized as described in Materials and Methods. Skin draining and mesenteric lymph nodes were obtained for Treg analysis of tolerized or control mice, respectively. (a) Percentages of CD25+Foxp3+ Tregs, gated on CD4+ T cells are shown. Pooled data of 3 independent experiments are depicted (mean ± SEM, 15 mice per group). (b)
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Expression of CCR7 (mean) and percentage of (c) CD103, (d) ICOS and (e) PD-1 positive CD4+CD25+Foxp3+ Tregs are depicted. Pooled data of 3 independent experiments are demonstrated (mean ± SEM, 15 mice per group). *p<0.05, **p<0.01, ***p<0.001, n.s. not
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significant.
Figure 4 a-f
CD4+CD25+Foxp3+ Treg cells are crucial for colitis prevention by oral LZT. (a) Protocol of Treg depletion. TNBS-colitis was induced after Treg recurrence (Fig. S2).
Solvent-treated mice +/-Treg depletion served as controls (colitis / colitis Treg depleted). (b) Inflammatory colitis symptoms according to the total endoscopic score (upper panel) and single parameters (lower panel). Pooled data of 2 experiments are shown (mean ± SD). (c) Images of 23
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mini-endoscopy representing the average endoscopic score and (d) of histology of rectum tissue sections are shown. Scale bar = 150 µm. (e) Hapten-specific T cell proliferation in vitro was assessed (mean value of triplicates in cpm). One representative out of 2 independent experiments
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with similar results is shown. (f) Representative data of hapten-specific T cell cytokine production (IFN-γ, IL-2) of 2 independent experiments are depicted (mean value of duplicates ±
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SD). **p<0.01, ***p<0.001, n.s. not significant.
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Figure 5 a-f
Epicutaneous LZT requires CD4+CD25+Foxp3+ Treg cells for colitis inhibition. (a) Protocol of Treg depletion. After Treg repopulation (Fig. S2) colitis was induced by sensitization and rectal challenge. Solvent-treated mice +/-Treg depletion served as controls
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(colitis / colitis Treg depleted). (b) Total endoscopic score (upper panel) and single parameter of the score were given as pooled data of 3 experiments (mean ± SD). (c) Images of the miniendoscopy representing the average endoscopic scores and (d) of histology of rectum tissue are
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shown. Scale bar = 150 µm. (e) Hapten-specific T cell proliferation was depicted (mean value of triplicates in cpm) after hapten-specific restimulation in vitro. One representative out of 3
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independent experiments with similar results is shown. (f) Representative data of T cell cytokine production (IFN-γ, IL-2) out of 3 independent experiments are demonstrated (mean value of duplicates ± SD). ***p<0.001, n.s. not significant.
Figure 6 a-d IL-10 is critical for oral and epicutaneous tolerance resulting in colitis prevention. 24
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WT and IL-10 deficient mice were epicutaneously or orally tolerized, respectively. Subsequently, colitis induction was performed. Pooled data of (a) the total endoscopic score of 3 experiments are depicted (mean ± SD). Representative images of clinical score of mini-
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endoscopy (b) and of histology (c) of the gut tissue are shown. Scale bar = 150 µm. (d) The hapten-specific T cell proliferation in vitro was depicted (mean value of triplicates in cpm). One representative out of 3 independent experiments with similar results is shown. *p<0.05,
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***p<0.001, n.s. not significant.
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