The Pattern Recognition Receptor NOD2 Mediates Staphylococcus aureus–Induced IL- 17C Expression in Keratinocytes  Sarah A. Roth, Maren Simanski, Franziska Rademacher, Lena Schröder, Jürgen Harder  Journal of Investigative Dermatology  Volume 134, Issue 2, Pages 374-380 (February 2014) DOI: 10.1038/jid.2013.313 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Activation of the IL-17C promoter through nucleotide-binding oligomerization domain–containing protein 2 (NOD2). HEK293 cells were co-transfected with an IL-17C firefly luciferase promoter plasmid and the phRG-TK Renilla luciferase plasmid for normalization and either the wild-type NOD2 expression plasmid or a NOD2-R702W expression plasmid or a NOD2-3020insC expression plasmid. Cells were cotreated with 1 μg of muramyl dipeptide (MDP) or MDP-LL (inactive isomer). IL-17C promoter activity was analyzed after 24 hours by measuring luciferase activity. Promoter activity was defined as the ratio between firefly and Renilla luciferase activities in each sample. Data are means±SD of one representative experiment of two, each performed in triplicate (*P<0.05, Student’s t-test). Journal of Investigative Dermatology 2014 134, 374-380DOI: (10.1038/jid.2013.313) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Role of NF-κB for the nucleotide-binding oligomerization domain–containing protein 2 (NOD2)–mediated IL-17C promoter activation. (a) HEK293 cells were co-transfected with a NOD2 expression plasmid, the phRG-TK Renilla luciferase plasmid for normalization, and either with an IL-17C firefly luciferase promoter plasmid or an IL-17C luciferase promoter plasmid containing three mutated NF-κB-binding sites (NF-κB-mut1+2+3). Cells were cotreated with 1 μg of muramyl dipeptide (MDP) or the inactive isomer MDP-LL. IL-17C promoter activity was determined after 24 hours by measuring luciferase activity. Promoter activity was defined as the ratio between firefly and Renilla luciferase activities in each sample. (b) HEK293 cells were treated as in a but with three different plasmids each containing a separate mutated NF-κB-binding site. Data are means±SD of triplicate stimulations. Journal of Investigative Dermatology 2014 134, 374-380DOI: (10.1038/jid.2013.313) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Living, but not heat-inactivated, Staphylococcus aureus bacteria induce nucleotide-binding oligomerization domain–containing protein 2 (NOD2) and IL-17C gene expression in primary keratinocytes. (a and b) Primary human keratinocytes were stimulated for a total of 6 hours with living S. aureus or with heat-inactivated S. aureus, and (a) NOD2 gene expression and (b) IL-17C gene expression were analyzed by real-time PCR. For time-course experiments, keratinocytes were treated for 2, 4, and 6 hours with living S. aureus, and relative gene induction of (c) NOD2 and (d) IL-17C was analyzed by real-time PCR. Data are means±SD of one representative experiment of two, each performed in triplicate samples (*P<0.05, Student’s t-test; NS, not significant). Journal of Investigative Dermatology 2014 134, 374-380DOI: (10.1038/jid.2013.313) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Relevance of nucleotide-binding oligomerization domain–containing protein 2 (NOD2) for IL-17C expression in keratinocytes upon Staphylococcus aureus stimulation. Primary keratinocytes were transfected with an empty pEF-6/V5-His vector (mock transfection), a NOD2 expression plasmid, or a NOD2-R702W expression plasmid. Cells were left unstimulated or were stimulated with S. aureus for a total of 6 hours and relative IL-17C (a) or IL-8 (b) gene induction was analyzed by real-time PCR. Data represent the percentage of fold gene induction relative to induction in NOD2-overexpressing cells, which was set as 100%. Data are means±SD of four independent experiments, each performed in triplicate (*P<0.05, Student’s t-test). Journal of Investigative Dermatology 2014 134, 374-380DOI: (10.1038/jid.2013.313) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Small interfering RNA (siRNA)-mediated downregulation of nucleotide-binding oligomerization domain–containing protein 2 (NOD2) reduces Staphylococcus aureus–mediated IL-17C induction in keratinocytes. (a) Keratinocytes were transfected with a non-silencing control siRNA or two different NOD2 siRNAs, respectively. Subsequently, cells were left unstimulated (-SA) or were stimulated with living S. aureus (+SA) for 6 hours, and IL-17C gene expression was analyzed by real-time PCR. Data are means±SD of one representative experiment of two, each performed in triplicate (*P<0.05, Student’s t-test). (b) Keratinocytes were transfected with a non-silencing control siRNA or a NOD2 siRNA, followed by transfection with an IL-17C luciferase promoter plasmid and the phRG-TK Renilla luciferase plasmid used for normalization. Cells were then stimulated with living S. aureus for 6 hours and IL-17C promoter activation was analyzed after 24 hours. Shown is the relative S. aureus–mediated IL-17C promoter induction. Data are means±SD of two independent experiments each performed in triplicate. Journal of Investigative Dermatology 2014 134, 374-380DOI: (10.1038/jid.2013.313) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Influence of nucleotide-binding oligomerization domain–containing protein 2 (NOD2) and IL-17C on the Staphylococcus aureus killing activity in keratinocytes. (a) Primary keratinocytes transfected with an empty pEF-6/V5-His plasmid (=control), or a NOD2 or NOD2-R702W expression plasmid were infected with S. aureus for 6 hours. Remaining colony-forming unit (CFU) in the cell lysates were determined and CFU of control cells set as 100%. (b and c) Keratinocytes treated with (b) control siRNA and NOD2 siRNA or (c) IL-17C siRNA were infected with S. aureus for 6 hours and remaining CFUs in the cell lysates were determined. CFUs in the cells treated with NOD2 or IL-17C siRNA were set as 100%. Data are means±SD of at least three infections representative of two independent experiments (*P<0.05, **P<0.01, Student’s t-test; NS, not significant). Journal of Investigative Dermatology 2014 134, 374-380DOI: (10.1038/jid.2013.313) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions