Nrf2 activation by sulforaphane restores the age-related decrease of TH1 immunity: Role of dendritic cells Hyon-Jeen Kim, PhD, Berenice Barajas, BS,

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Nrf2 activation by sulforaphane restores the age-related decrease of TH1 immunity: Role of dendritic cells Hyon-Jeen Kim, PhD, Berenice Barajas, BS, Meiying Wang, MD, Andre E. Nel, MD, PhD Journal of Allergy and Clinical Immunology Volume 121, Issue 5, Pages 1255-1261.e7 (May 2008) DOI: 10.1016/j.jaci.2008.01.016 Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 SFN reverses the age-related decrease in the CHS response. A, Ear-swelling response (mean ± SD). B, Hematoxylin and eosin staining of ear tissue. C and D, Real-time PCR for mRNA levels of genes in the ear (Fig 1, C) and liver (Fig 1, D). Results represent the fold increase (means ± SDs) compared with the CON-Young group (n = 6). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. CON, Vehicle-treated control; SFN, SFN treated; DFNB, DNFB sensitized/challenged. Journal of Allergy and Clinical Immunology 2008 121, 1255-1261.e7DOI: (10.1016/j.jaci.2008.01.016) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Nrf2 deficiency suppresses the CHS response. A, Ear-swelling response (means ± SDs). B, IFN-γ and IL-4 mRNA levels were measured by means of real-time PCR. Results represent the fold increase (means ± SDs) compared with the CON-Nrf2+/+ group (n = 6). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. CON, Vehicle-treated control; OXA, OXA sensitized/challenged. Journal of Allergy and Clinical Immunology 2008 121, 1255-1261.e7DOI: (10.1016/j.jaci.2008.01.016) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 DCs from old mice have lower thiol levels and phase II mRNA expression. A, Mean fluorescence intensity for MBB staining in CD11c+ splenocytes from young and old mice (means ± SDs). B, Phase II mRNA expression in CD11c+ splenocytes from young and old mice. Results represent the fold increase (means ± SDs) of old compared with young mice (n = 4). ∗P < .05. Journal of Allergy and Clinical Immunology 2008 121, 1255-1261.e7DOI: (10.1016/j.jaci.2008.01.016) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 DC redox disequilibrium interferes in the CHS response on adoptive transfer. The ear-swelling response in recipient mice (3 months old) receiving DNBS-pulsed DCs from young versus old mice (A) or nrf2+/+ versus nrf2−/− mice (B), followed by DNFB challenge, is shown (n = 6). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. CON, Vehicle-treated control; DNBS, DNBS pulsed. Journal of Allergy and Clinical Immunology 2008 121, 1255-1261.e7DOI: (10.1016/j.jaci.2008.01.016) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 NAC or SFN treatment of DCs reverses the age-related decrease in the CHS response on adoptive transfer. NAC-treated (A) or SFN-treated (B) DCs were exposed to DNBS ex vivo and adoptively transferred into mice that were challenged with DNFB (mean ± SD, n = 6). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. CON, Vehicle-treated control; DNBS, DNBS pulsed. Journal of Allergy and Clinical Immunology 2008 121, 1255-1261.e7DOI: (10.1016/j.jaci.2008.01.016) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Nrf2 deficiency suppresses the CHS response induced by DNFB Nrf2 deficiency suppresses the CHS response induced by DNFB. The ear-swelling response was expressed as mean ± SD (n = 4). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. CON, Vehicle-treated control; DFNB, DNFB sensitized/challenged. Journal of Allergy and Clinical Immunology 2008 121, 1255-1261.e7DOI: (10.1016/j.jaci.2008.01.016) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Thiol levels in the BM-DCs decreased by aging, Nrf2 deficiency, or both. Cultured BM-DCs were surface stained with phycoerythrin-labeled anti-CD11c, followed by MBB staining and flow cytometry. A, Mean fluorescence intensity for MBB staining in CD11c+ splenocytes from young and old mice (means ± SDs). B, Mean fluorescence intensity for MBB staining in CD11c+ splenocytes from old Nrf2+/+ and Nrf2−/− mice (mean ± SD, n = 4). ∗P < .05. MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology 2008 121, 1255-1261.e7DOI: (10.1016/j.jaci.2008.01.016) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Increased thiol levels in the BM-DCs by NAC treatment Increased thiol levels in the BM-DCs by NAC treatment. Cultured BM-DCs were incubated with or without NAC (20 mmol/L for 24 hours) and then washed. Cells were surface stained with phycoerythrin-labeled anti-CD11c, followed by MBB staining and flow cytometry. The graph shows mean fluorescence intensity for MBB staining in CD11c+ BM-DCs (means ± SDs, n = 4). ∗P < .05. CON, Control group. Journal of Allergy and Clinical Immunology 2008 121, 1255-1261.e7DOI: (10.1016/j.jaci.2008.01.016) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

SFN treatment upregulates p2E message and thiol levels SFN treatment upregulates p2E message and thiol levels. Cultured BM-DCs were incubated with or without SFN (5 μmol/L for 24 hours) and then washed. A, Total RNA was extracted from CD11c+ BM-DCs to perform real-time PCR for p2E. B, Cells were surface stained with phycoerythrin-labeled anti-CD11c, followed by MBB staining and flow cytometry. The graph shows mean fluorescence intensity for MBB staining in CD11c+ BM-DCs (means ± SDs, n = 4). ∗P < .05, ∗∗∗P < .001. CON, Control group; HO1, heme oxygenase 1. Journal of Allergy and Clinical Immunology 2008 121, 1255-1261.e7DOI: (10.1016/j.jaci.2008.01.016) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions