Skin Commensals Amplify the Innate Immune Response to Pathogens by Activation of Distinct Signaling Pathways  Ines Wanke, Heiko Steffen, Christina Christ, Bernhard Krismer, Friedrich Götz, Andreas Peschel, Martin Schaller, Birgit Schittek  Journal of Investigative Dermatology  Volume 131, Issue 2, Pages 382-390 (January 2011) DOI: 10.1038/jid.2010.328 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Differential induction of antimicrobial peptides/proteins (AMPs) by staphylococcal preparations in differentiated human primary keratinocytes and influence on bacterial viability. (a–e) RNA expression of human β-defensin (HBD)-1, HBD-2, HBD-3, and RNase 7 compared with the unstimulated control set as 1. (a, b) Ca2+-differentiated primary keratinocytes were stimulated by live (a) Staphylococcus aureus 113 Δspa or (b) S. epidermidis 1457 for 6, 12, 18, or 24hours. (c–e) Differentiated human primary keratinocytes were infected by either (c) live or (d) heat-killed bacteria, or with (e) bacterial-conditioned medium (BCM) of S. aureus 113 Δspa or S. epidermidis 1457. (f) Percentages of counted colony-forming units (CFUs) normalized to untreated control cells of an antimicrobial test with lysates from human primary keratinocytes challenged with BCM from S. epidermidis 1457 or S. aureus 113 Δspa with the same strains. Journal of Investigative Dermatology 2011 131, 382-390DOI: (10.1038/jid.2010.328) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Human β-defensin-3 (HBD-3) and RNase 7 expression is induced in differentiated primary keratinocytes by several skin colonizing strains of Staphylococcus aureus. (a–e) Relative RNA expression of the indicated antimicrobial peptides/proteins (AMPs) compared with the unstimulated control set as 1. Expression of (a) HBD-3 or (b) RNase7 in differentiated primary human keratinocytes after 24hours of stimulation with bacterial-conditioned medium (BCM) of 12 different strains and isolates of S. epidermidis, 12 unidentified commensals from people with healthy skin, and 30 strains and isolates of S. aureus. Basal AMP expression in Ca2+-differentiated compared with undifferentiated primary human keratinocytes. Successful differentiation was verified by expression of involucrin (inlet). (c) Comparison of Ca2+-differentiated and undifferentiated primary human keratinocytes and HaCaT after 24hours of challenge with BCM of (d) S. aureus 113 Δspa or (e) S. epidermidis 1457. Journal of Investigative Dermatology 2011 131, 382-390DOI: (10.1038/jid.2010.328) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Staphylococcus epidermidis amplifies the innate immune response of keratinocytes. (a) Relative RNA expression of indicated antimicrobial peptides/proteins (AMPs) in differentiated primary keratinocytes after stimulation with bacterial-conditioned medium (BCM) of S. epidermidis 1457, S. aureus 113 Δspa, or both together for 24hours or after sequential stimulation as indicated compared with the unstimulated control cells set as 1. (b) Human β-defensin-3 (HBD-3) protein levels in cell culture supernatants after 24hours of prechallenge with the indicated BCM following 4hours of secretion into fresh medium are shown. Journal of Investigative Dermatology 2011 131, 382-390DOI: (10.1038/jid.2010.328) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Toll-like receptor-2 (TLR-2), NF-κB, and EGFR signaling is involved in the induction of human β-defensin-3 (HBD-3) and RNase7 expression by Staphylococcus epidermidis bacterial-conditioned medium (BCM), but not by S. aureus BCM. (a) Relative RNA expression compared with uninduced cells set as 1 after stimulation with BCM of either S. aureus 113 Δspa or S. epidermidis 1457 for 24hours. (b) Differentiated primary keratinocytes were stimulated with (b–d) Pam2Cys, (b, e, f) transforming growth factor-α (TGFα), (b) EGF, or (c–f) BCM of S. epidermidis 1457 or S. aureus 113 Δspa alone or both together. Cells were incubated 2hours before stimulation with a (c) TLR-2 or (e) an EGFR blocking antibody or simultaneously with (d) celastrol or (f) gefitinib and the values normalized to challenged cells without inhibitor set as 100%. Note that S. epidermidis induces much lower antimicrobial peptide/protein (AMP) levels than S. aureus. Journal of Investigative Dermatology 2011 131, 382-390DOI: (10.1038/jid.2010.328) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 NF-κB signaling is involved in induction of human β-defensin-3 (HBD-3) and RNase7 expression by secreted factors of Staphylococcus epidermidis, but not of S. aureus. (a, b) Ca2+-differentiated primary keratinocytes were incubated with the NF-κB inhibitors celastrol or JSH-23 and simultaneously stimulated for 24hours with living bacteria or bacterial-conditioned medium (BCM) from (a) S. aureus 113 Δspa or (b) S. epidermidis 1457. Indicated are the percentages of antimicrobial peptide/protein (AMP) RNA expression levels normalized to induced cells without the appropriate inhibitor (set as 100%). Note that S. epidermidis induces much lower HBD-3 and RNase7 levels than S. aureus. Journal of Investigative Dermatology 2011 131, 382-390DOI: (10.1038/jid.2010.328) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Staphylococcus aureus activates the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathways and suppresses NF-κB activation in primary human keratinocytes. (a) Western blot analyses of lysates of differentiated primary human keratinocytes at 15 and 30minutes and at 1 and 4hours after stimulation with bacterial-conditioned medium (BCM) of S. aureus 113 Δspa or S. epidermidis 1457 alone or both together or the unstimulated control cells. (b) Activation of the NF-κB signaling pathway was analyzed using differentiated primary human keratinocytes transfected with an NF-κB-luciferase reporter plasmid. Shown is the x-fold induction of the firefly/Renilla luciferase activity ratio after 24-hour stimulation of the cells with either tumor necrosis factor (TNF)/IL-1β or BCM of S. epidermidis, S. aureus, or both together. Journal of Investigative Dermatology 2011 131, 382-390DOI: (10.1038/jid.2010.328) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions