Human Keratinocytes Express Functional Toll-Like Receptor 3, 4, 5, and 9  Maria C. Lebre, Angelic M.G. van der Aar, Lisa van Baarsen, Toni M.M. van Capel, Joost H.N. Schuitemaker, Martien L. Kapsenberg, Esther C. de Jong  Journal of Investigative Dermatology  Volume 127, Issue 2, Pages 331-341 (February 2007) DOI: 10.1038/sj.jid.5700530 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Detection of TLR mRNA by RT-PCR in cultured human keratinocytes. Human keratinocytes express constitutively TLR1, 2, 3, 4, 5, 6, 9, and 10, but not TLR7 and 8. (a) For RT-PCR, cDNAs were amplified for 45 cycles and were separated on a 1% agarose gel containing ethidium bromide. Plasmacytoid dendritic cells (pDC) express TLR7 and monocyte-derived DC (moDC) express TLR8. (b) For iCycler RT-PCR (TLR7 and 8), cDNAs were amplified for 40 cycles and were separated on a 1% agarose gel containing ethidium bromide. The data shown are representative of four independent experiments. Journal of Investigative Dermatology 2007 127, 331-341DOI: (10.1038/sj.jid.5700530) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Kinetics of chemokine and cytokine production by keratinocytes stimulated with different concentrations of TLR3, 4, 5, and 9 ligands. (a) Keratinocytes were cultured with poly-I:C (1, 10, and 100μg/ml), LPS (1, 10, and 100μg/ml), flagellin (1, 10, and 100pg/ml), or CpG-ODN 2006 or 2216 (1 and 10μM) for 4, 8, 24, 48, and 72hours, and the concentrations of CXCL8 and TNF-α in the cell-free supernatants were measured with specific ELISA. Results, expressed as mean±SD of triplicate cultures are from one experiment representative of five with different donors. (b) Keratinocytes were cultured with non-CpG controls, CpG 2216c and 2006c (both 10μM), or poly-I:C (10μg/ml) for 48hours, and the concentrations of CXCL8 and TNF-α in the cell-free supernatants were measured with specific ELISA. Data were analyzed for statistical significance using analysis of variance followed by Dunnett's multiple comparisons test, *P<0.05, **P<0.01. (c) TNF-α, IFN-α, and IFN-α expression by human keratinocytes. Keratinocytes were cultured for 4hours with poly-I:C (10μg/ml), or CpG-ODN 2006 (10μM) or 2216 (10μM), and the cells lyzed for RT-PCR analysis. The data shown are representative of four experiments. Journal of Investigative Dermatology 2007 127, 331-341DOI: (10.1038/sj.jid.5700530) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Kinetics of CCL2, CCL20, and CCL27 production by keratinocytes stimulated with different concentrations of TLR3, 4, 5, and 9 ligands. Keratinocytes were cultured with poly-I:C, LPS, flagellin, or CpG-ODN 2006 or 2216, as stated in Materials and Methods, and the concentrations of chemokines in the cell-free supernatants were measured with specific ELISA. Results, expressed as mean±SD of triplicate cultures, are from one experiment representative of five. Journal of Investigative Dermatology 2007 127, 331-341DOI: (10.1038/sj.jid.5700530) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Kinetics of the Th1-attracting chemokines CXCL9 and CXCL10 production by keratinocytes stimulated with different concentrations of TLR3, 4, 5, and 9 ligands. Keratinocytes were cultured with poly-I:C, LPS, flagellin, or CpG-ODN 2006 or 2216, as stated above, and the concentrations of chemokines in the cell-free supernatants were measured with specific ELISA. Results, expressed as mean±SD of triplicate cultures, are from one experiment representative of five. Journal of Investigative Dermatology 2007 127, 331-341DOI: (10.1038/sj.jid.5700530) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Keratinocytes modulate the expression of cell surface molecules in response to different TLR ligands. Keratinocytes were stained with antibodies to ICAM-1, HLA-DR, HLA-ABC, FasR, or CD40 48hours after exposure to the indicated TLR ligands (poly-I:C, 10μg/ml; LPS, 100μg/ml; flagellin, 100ng/ml; CpG-ODN 2006, 10μM; CpG-ODN 2216, 10μM) or to medium alone, and analyzed by FACS. Results are expressed as mean of duplicate cultures. ΔMean fluorescence intensity represents the difference between the various stainings and the isotype control. Data are representative of four experiments with different donors with similar results. Journal of Investigative Dermatology 2007 127, 331-341DOI: (10.1038/sj.jid.5700530) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Phosphorylation of IκBα by TLR3-, 4-, 5-, or 9-triggered human keratinocytes. Keratinocytes were stimulated with different concentrations of poly-I:C (1 and 10μg/ml), LPS (10 and 100μg/ml), CpG-ODN 2006, or CpG-ODN 2216 (1 and 10μM) for 4hours. Cell lysates were fractionated by SDS-PAGE and then analyzed by Western blotting using antibodies against phospho-IκBα. A single representative experiment is shown from three different experiments. Journal of Investigative Dermatology 2007 127, 331-341DOI: (10.1038/sj.jid.5700530) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Detection of nucleus translocation of the NF-κB subunit p65 expression in TLR3, 4, 5, and 9 ligand-stimulated keratinocytes. Detection by (a) confocal laser scanning microscopy (original magnification, × 64, bar=20μm) or (b–q) fluorescence microscopy (original magnification, × 40, bar=20μm). (a) The upper panel shows the 4,6-diamidino-2-phenylindole staining (blue) indicating the nucleus; the middle panel shows the NF-κB staining (Alexa-488, green); and the lower panel the overlay. NF-κBp65 present in the nucleus is indicated by arrows. The data shown are representative of four experiments. (b–q) As in (a) NF-κBp65 present in the nucleus is indicated by arrows. (b) Unstimulated; (c) LPS (1ng/ml); (d) LPS (10ng/ml); (e) LPS (100ng/ml); (f) LPS (1μg/ml); (g) LPS (10μg/ml); (h) LPS (100μg/ml); (i) poly-I:C (10μg/ml); (j) flagellin (100ng/ml); (k) CpG 2006 (1μM); (l) CpG 2006 (10μM); (m) control-CpG 2006 (10μM); (n) CpG 2216 (1μM); (o) CpG 2216 (10μM); (p) control-CpG 2216 (10μM); and (q) control Ig. Journal of Investigative Dermatology 2007 127, 331-341DOI: (10.1038/sj.jid.5700530) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions