1. Toll-Like Receptor-Mediated Upregulation of CXCL16 in Psoriasis Orchestrates Neutrophil Activation 
Sabine Steffen, Susanne Abraham, Maik Herbig, Franziska Schmidt, Kristin Blau, Susann Meisterfeld, Stefan Beissert, Jochen Guck, Claudia Günther 
Journal of Investigative Dermatology 
Volume 138, Issue 2, Pages (February 2018)
DOI: /j.jid
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2. Figure 1
CXCL16 expression and secretion by monocytes and slanDCs is regulated by TLR stimulation. (a) Flow cytometric analysis of CXCL16 expression on freshly blood-isolated antigen-presenting cells. Mean ± standard deviation (SD) values of six psoriasis patients and six healthy controls are shown; unpaired Student t test. (b−f) Expression of CXCL16 after 24 hours culture in medium compared to freshly isolated cells (w/o) (b) and compared to TLR stimulation on CD14+CD16low (c), CD14lowCD16+ monocytes (d), and slanDCs (e). Concentration of CXCL16 in PBMC supernatants measured by ELISA (f). (g) CXCL16 expression and production of PBMCs from psoriasis patients compared to healthy controls (24 hours). Mean ± SD values of at least five independent donors are shown; paired Student t test (b), one-way analysis of variance with Bonferroni’s multiple comparison test (c−f), one-tailed unpaired Student t test (g). CXCL, CXC chemokine ligand; DC, dendritic cell; LPS, lipopolysaccharide; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; slan, 6-sulfo LacNAc; TLR, Toll-like receptor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
TNF-α blockade reduces CXCL16 expression in vitro and in the skin of psoriasis patients. (a−c) PBMCs were cultivated in medium with TNF-α, Pam2, or R837 upon treatment with etanercept or adalimumab for 24 hours. CXCL16 expression was analyzed by flow cytometry (a, c) and CXCL16 production by ELISA (b, c). Three individual experiments (a) performed in duplicates (b) and 6 (mean fluorescence intensity CXCL16) or at least five (concentration CXCL16) individual experiments (c) are shown; one-tailed Mann-Whitney U test (a) and one-way analysis of variance with Bonferroni’s multiple comparison test (b, c). (d) Immunofluorescence staining of CXCL16 (green) in psoriatic skin before treatment with adalimumab and at week 4 (n = 3). The basement membrane zone is marked by the solid line and the epidermis is indicated by “e.” Scale bar = 50 μm. CXCL, CXC chemokine ligand; LPS, lipopolysaccharide; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; TNF-α, tumor necrosis factor−α.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
CXCL16 expression and secretion by primary keratinocytes of psoriasis patients upon TLR and TNF-α stimulation (24 hours). (a) Expression and (b) secretion of CXCL16 with and without poly(IC) stimulation compared to healthy controls analyzed by flow cytometry and ELISA. (c) CXCL16/β-actin protein expression ratio of in medium cultured psoriatic keratinocytes (P1−P3) compared to healthy controls (H1−H3) detected by western blot. C = chemokine domain. (d) CXCL16 expression and (e) secretion with and without TLR stimulation evaluated by flow cytometry and ELISA. CXCL16 secretion upon TNF-α stimulation is inhibited by etanercept (f). Three individual experiments (a, c, d) performed in duplicate (b, e, f) are shown; Friedman test (d), Mann-Whitney U test (a, c), one-way analysis of variance with Bonferroni’s multiple comparison test (e, f), and unpaired Student t test (d). CXCL, CXC chemokine ligand; poly(IC), polyinosinic-polycytidylic acid; TLR, Toll-like receptor; TNF-α, tumor necrosis factor−α.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
CXCL16 expression and secretion is upregulated by type I IFN in psoriasis patients (24 hours). (a, b) Type I IFN concentrations in supernatant of R837-stimulated PBMCs (a) and poly(IC)-stimulated keratinocytes (b) analyzed by IFN reporter assay. Mean ± standard deviation (SD) values of three individual experiments of three donors are shown; paired Student t test. (c, d) CXCL16 expression on CD14lowCD16+ monocytes and slanDCs (c) and CXCL16 secretion by keratinocytes upon IFN-beta stimulation compared to healthy controls (d) detected by flow cytometry and ELISA. Data are given as mean ± SD of three independent donors (c) performed in duplicates (d); one-tailed Mann-Whitney U test (c), paired and unpaired Student t test (d). CXCL, CXC chemokine ligand; DC, dendritic cell; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; poly(IC), polyinosinic-polycytidylic acid; slan, 6-sulfo LacNAc.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
CXCL16 mediates chemotaxis of CXCL16 expressing neutrophils from psoriasis patients. (a) Migrating neutrophils in response to CXCL16 and/or IL-8 analyzed by transwell migration assay. Mean ± standard deviation (SD) of at least five independent donors are shown; one-way analysis of variance with Bonferroni’s multiple comparison test. (b, c) Immunofluorescence staining of CXCL16 (green) by neutrophils (CD66b, red; overview: scale bar = 30 μm, detail marked by square: scale bar = 10 μm; b) and in IL-8-expressing dermal vessels (red; scale bar = 10 μm; c) in psoriasis vulgaris. (d) Western blot of CXCL16 protein expression. (e) CXCL16 secretion after stimulation with IL-8 (50 ng/ml) for 24 hours examined by ELISA. Mean ± SD values of three individual experiments performed in duplicate are shown; paired Student t test. C, chemokine domain; CXCL, CXC chemokine ligand; PBMC, peripheral blood mononuclear cell; PMN, polymorphonuclear neutrophils; T, transmembrane full-length form; S, soluble form.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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7. Figure 6
Morphological and mechanical properties of neutrophils analyzed by real-time deformability cytometry. Representative scatter and contour plots (a) and statistical analysis (b) of area, area ratio, and deformation of neutrophils from healthy controls and patients with psoriasis after stimulation with or without CXCL16 or IL-8 for 1 hour (area ratio 1.0:1.2; 0.04 μl/s flow rate). (c) Deformation of round, inactivated neutrophils (area ratio 1.0:1.05) from healthy donors. Results of three independent donors are shown; linear mixed-effects models. CXCL, CXC chemokine ligand.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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