The Human Skin–Associated Autoantigen α-NAC Activates Monocytes and Dendritic Cells via TLR-2 and Primes an IL-12-Dependent Th1 Response  Susanne Hradetzky, Hari Balaji, Lennart M. Roesner, Annice Heratizadeh, Irene Mittermann, Rudolf Valenta, Thomas Werfel  Journal of Investigative Dermatology  Volume 133, Issue 9, Pages 2289-2292 (September 2013) DOI: 10.1038/jid.2013.161 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 α-NAC (the alpha-chain of the nascent polypeptide–associated complex) binds to and activates monocytes. Peripheral blood mononuclear cells (PBMCs) were incubated for 30minutes with α-NAC and then stained with biotin-coupled rabbit α-NAC antibodies, detected by streptavidin-allophycocyanin (APC), and FITC-coupled mouse antibodies against CD14, CD4, CD8, and CD19. (a) Depicts representative dot plots from one of eight donors, comparing the percentage of α-NAC+ CD14+ cells of gated monocytes without and with α-NAC incubation. (b–e) PBMCs and isolated monocytes from the same healthy donors were stimulated with α-NAC (2.5μg/ml), IL-2 (50U/ml), or a combination of both for 72hours (b, d) or 8hours (c, e). Supernatants were measured for levels of cytokines by ELISA (b, d). Relative levels of mRNA were determined by reverse transcriptase quantitative PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (c, e). In d and e, PBMCs are represented by black dots and monocytes by open dots. Medians are shown. *P<0.05, **P<0.01. (f) Isolated monocytes were stimulated with α-NAC (2.5μg/ml), IL-2 (50U/ml), or a combination of both for 30minutes. Western blots for Phospho-p44/42 or Phospho-NF-κB and GAPDH were performed. One representative experiment of 10 independent experiments is shown. Journal of Investigative Dermatology 2013 133, 2289-2292DOI: (10.1038/jid.2013.161) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 α-NAC (the alpha-chain of the nascent polypeptide–associated complex)-induced IL-12 is required for IFN-γ secretion in peripheral blood mononuclear cells (PBMCs). (a) Human PBMCs from eight different donors were preincubated with a blocking antibody against IL-12 p40 (10μg/ml) or the respective isotype control and then stimulated with α-NAC (2.5μg/ml), IL-2 (50U/ml), or a combination of both for 2 days. Medians are shown. (b) Human PBMCs from 11 different donors and purified CD4+ T cells from the same donor were stimulated with α-NAC (2.5μg/ml) and IL-2 (50U/ml) for 3 days. Supernatants from both a and b were collected, and IFN-γ secretion was detected using a sandwich ELISA. **P<0.01. PBMCs (c) or monocytes (d) were preincubated with 8μg/ml TLR-2-blocking antibody or the respective isotype control for 30minutes and then stimulated with α-NAC (2.5μg/ml), IL-2 (50U/ml), or a combination of both for 72hours. Supernatants were measured for levels of IL-12 p40, IFN-γ, and IL-6 by ELISA. Isolated monocytes were treated for 3 days with 1μM small interfering RNA (siRNA; smart-Pool TLR-2 or nontargeting pool as control) and later stimulated with α-NAC (2.5μg/ml) for 3 more days. At day 6, cells were analyzed by FACS for surface expression of CD14 and TLR-2 (e). Supernatants were taken and measured for levels of IL-12 p40 (f). Medians are shown. *P<0.05, **P<0.01, ***P<0.001. Journal of Investigative Dermatology 2013 133, 2289-2292DOI: (10.1038/jid.2013.161) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions