Cytokine Effects Induced by the Human Autoallergen α-NAC Susanne Hradetzky, Lennart M. Roesner, Hari Balaji, Annice Heratizadeh, Irene Mittermann, Rudolf Valenta, Thomas Werfel  Journal of Investigative Dermatology  Volume 134, Issue 6, Pages 1570-1578 (June 2014) DOI: 10.1038/jid.2014.25 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Alpha-chain of the nascent polypeptide-associated complex (α-NAC) induces secretion and transcription of T-cell cytokines. Peripheral blood mononuclear cells (PBMCs) from atopic dermatitis (AD) patients and control donors were stimulated with α-NAC (2.5 μg ml–1), IL-2 (50 U ml–1), or a combination of both for 72 hours (a) or 8 hours (b). Supernatants were measured for levels of IFN-γ, IL-17, and IL-22 by ELISA (a) and relative levels of mRNA were determined by reverse-transcriptase–quantitative PCR (RT–qPCR) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (b). Medians are shown. *P<0.05, **P<0.01, ***P<0.001. Journal of Investigative Dermatology 2014 134, 1570-1578DOI: (10.1038/jid.2014.25) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Monocytes are important for induction of T-cell cytokines by the alpha-chain of the nascent polypeptide-associated complex (α-NAC). Peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from the same donors were stimulated with α-NAC (2.5 μg ml–1), IL-2 (50 U ml–1), or a combination of both for 72 hours (a). Isolated monocytes were stimulated with α-NAC (2.5 μg ml–1), IL-2 (50 U ml–1), or a combination of both for 72 hours. Isolated CD4+ T cells were incubated with these supernatants for 72 hours. For comparison, PBMCs and isolated CD4+ T cells from the same donors were stimulated with α-NAC (2.5 μg ml–1), IL-2 (50 U ml–1), or a combination of both for 72 hours (b). Supernatants were measured for levels of IL-17 and IL-22 by ELISA. Medians are shown. **P<0.01. SN, supernatant. Journal of Investigative Dermatology 2014 134, 1570-1578DOI: (10.1038/jid.2014.25) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Monocyte-derived IL-23 is important for the alpha-chain of the nascent polypeptide-associated complex (α-NAC)-induced IL-17 secretion. Peripheral blood mononuclear cells (PBMCs) were stimulated with α-NAC (2.5 μg ml–1) for 4 hours and relative levels of mRNA were determined by reverse-transcriptase–quantitative PCR (RT-qPCR) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (a). PBMCs were stimulated with α-NAC (2.5 μg ml–1), IL-2 (50 U ml–1), or a combination of both for 72 hours. IL-23 levels were measured by ELISA (b). PBMCs were preincubated with anti-IL-23 p19 antibody or the isotype control, followed by stimulation with α-NAC (2.5 μg ml–1), IL-2 (50 U ml–1), or a combination of both for 72 hours. IL-17 and IL-22 were measured by ELISA (c). Medians are shown. *P<0.05, **P<0.01. Journal of Investigative Dermatology 2014 134, 1570-1578DOI: (10.1038/jid.2014.25) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Stimulation with the alpha-chain of the nascent polypeptide-associated complex (α-NAC) leads to signal transducer and activator of transcription 3 (STAT3) phosphorylation in peripheral blood mononuclear cells (PBMCs). PBMCs from 26 donors were stimulated with α-NAC (2.5 μg ml–1), IL-2 (50 U ml–1), or a combination of both for 30 minutes. Western blots for phospho-STAT3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed. One representative experiment is shown (a). Integrated density values (IDV) of phospho-STAT3 bands were normalized to the corresponding GAPDH bands. Medians are shown (b). *P<0.05, **P<0.01. Journal of Investigative Dermatology 2014 134, 1570-1578DOI: (10.1038/jid.2014.25) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Signal transducer and activator of transcription 3 (STAT3) is essential for the alpha-chain of the nascent polypeptide-associated complex (α-NAC)-induced cytokine secretion. Peripheral blood mononuclear cells (PBMCs) were preincubated with 20 μM STAT3-inhibitor (Stattic) or the solvent DMSO alone and then stimulated with α-NAC (2.5 μg ml–1), IL-2 (50 U ml–1), or a combination of both for 30 minutes and blotted as described above. One representative experiment of 10 independent experiments from different donors is shown in (a) and integrated density values (IDVs) of phospho-STAT3 normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown in (b). PBMCs were preincubated with 5 μM Stattic or DMSO for 30 minutes and then stimulated with α-NAC (2.5 μg ml–1), IL-2 (50 U ml–1), or a combination of both for 72 hours. Supernatants were measured for levels of IFN-γ (n=16), IL-17 (n=13), and IL-22 (n=15) by ELISA (c) and 7-AAD-staining was performed (n=6) (d). PBMCs were pre-treated with Stattic as described above and stimulated with Staphylococcus enterotoxin B. Supernatants were analyzed by ELISA (e). Medians are shown. *P<0.05, **P<0.01, ***P<0.001. 7-AAD, 7-aminoactinomycin D. Journal of Investigative Dermatology 2014 134, 1570-1578DOI: (10.1038/jid.2014.25) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Peripheral blood mononuclear cells (PBMCs) of the alpha-chain of the nascent polypeptide-associated complex (α-NAC)-sensitized atopic dermatitis (AD) patients fail to secrete IL-10 in response to α-NAC. PBMCs from AD patients and non-allergic donors were stimulated with α-NAC (2.5 μg ml–1), IL-2 (50 U ml–1), or a combination of both for 72 hours. AD, α-NAC-sensitized: open boxes (n=11), controls: grey boxes (n=18), AD, not α-NAC-sensitized: striped boxes (n=12) (a). PBMCs, monocytes, and CD4+ T cells from non-sensitized donors were stimulated with α-NAC (2.5 μg ml–1), IL-2 (50 U ml–1), or a combination of both for 72 h (b). Supernatants were analyzed for levels of IL-10 by ELISA. Medians are shown. Groups were compared by Mann–Whitney test. *P<0.05, **P<0.01. ns, not significant. Journal of Investigative Dermatology 2014 134, 1570-1578DOI: (10.1038/jid.2014.25) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions