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  Journal of Investigative Dermatology  Volume 136, Issue 9, Pages 1831-1839 (September 2016) DOI: 10.1016/j.jid.2016.04.037 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Epicutaneous and oral low-zone tolerance (LZT) protects from colitis. (a) Protocol of LZT and colitis induction. (b) Results were presented as pooled data (mean ± SD) of three independent experiments of the total endoscopic score (left panel) and of single 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 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 five 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 significant. cpm, counts per minute, TNBS, picrylsulfonic acid, 2,4,6-trinitro-benzenesulfonic acid; SD, standard deviation; SEM, standard error of the mean. Journal of Investigative Dermatology 2016 136, 1831-1839DOI: (10.1016/j.jid.2016.04.037) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Immune mechanisms of epicutaneous and oral tolerance in colitis protection are hapten-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 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. not significant. DNBS, 2,4-dinitrobenzenesulfonic acid; DNFB, 1-fluro-2,4-dinitrobenzene; LZT, low-zone tolerance; SD, standard deviation; TNBS, 2,4,6-trinitro-benzenesulfonic acid; TNCB, 2,4,6-trinitro-1-chlorobenzene. Journal of Investigative Dermatology 2016 136, 1831-1839DOI: (10.1016/j.jid.2016.04.037) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Increased numbers of activated CD4+CD25+Foxp3+ Tregs after epicutanoeus and oral tolerization. Mice have been epicutaneously or orally tolerized as described in the Materials and Methods section. 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 three independent experiments are depicted (mean ± SEM, 15 mice per group). (b) 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 three independent experiments are demonstrated (mean ± SEM, 15 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant. LZT, low-zone tolerance; SEM, standard error of the mean; Tregs, regulatory T cells. Journal of Investigative Dermatology 2016 136, 1831-1839DOI: (10.1016/j.jid.2016.04.037) Copyright © 2016 The Authors Terms and Conditions

Figure 4 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 (Supplementary Figure 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 two experiments are shown (mean ± SD). (c) Images of 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 two independent experiments with similar results is shown. (f) Representative data of hapten-specific T cell cytokine production (IFN-γ, IL-2) of two independent experiments are depicted (mean value of duplicates ± SD). **P < 0.01, ***P < 0.001, n.s. not significant. cpm, counts per minute; LZT, low-zone tolerance; SD, standard deviation; TNBS, 2,4,6-trinitro-benzenesulfonic acid; Tregs, regulatory T cells. Journal of Investigative Dermatology 2016 136, 1831-1839DOI: (10.1016/j.jid.2016.04.037) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Epicutaneous LZT requires CD4+CD25+Foxp3+ Treg cells for colitis inhibition. (a) Protocol of Treg depletion. After Treg repopulation (Supplementary Figure S2) colitis was induced by sensitization and rectal challenge. Solvent-treated mice ± Treg depletion served as controls (colitis/colitis Treg depleted). (b) Total endoscopic score (upper panel) and single parameter of the score were given as pooled data of three experiments (mean ± SD). (c) Images of the mini-endoscopy representing the average endoscopic scores and (d) of histology of rectum tissue are 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 three independent experiments with similar results is shown. (f) Representative data of T cell cytokine production (IFN-γ, IL-2) out of three independent experiments are demonstrated (mean value of duplicates ± SD). ***P < 0.001, n.s. not significant. cpm, counts per minute; LZT, low-zone tolerance; SD, standard deviation; Tregs, regulatory T cells. Journal of Investigative Dermatology 2016 136, 1831-1839DOI: (10.1016/j.jid.2016.04.037) Copyright © 2016 The Authors Terms and Conditions

Figure 6 IL-10 is critical for oral and epicutaneous tolerance resulting in colitis prevention. 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 three experiments are depicted (mean ± SD). Representative images of clinical score of mini-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 counts per minute). One representative out of three independent experiments with similar results is shown. *P < 0.05, ***P < 0.001, n.s. not significant. cpm, counts per minute; LZT, low-zone tolerance; SD, standard deviation; WT, wild type. Journal of Investigative Dermatology 2016 136, 1831-1839DOI: (10.1016/j.jid.2016.04.037) Copyright © 2016 The Authors Terms and Conditions