Anti-Inflammatory Action of Keratinocyte-Derived Vaspin Anja Saalbach, Jenny Tremel, Diana Herbert, Katharina Schwede, Elke Wandel, Christine Schirmer, Ulf Anderegg, Annette G. Beck- Sickinger, John T. Heiker, Stephan Schultz, Thomas Magin, Jan C. Simon  The American Journal of Pathology  Volume 186, Issue 3, Pages 639-651 (March 2016) DOI: 10.1016/j.ajpath.2015.10.030 Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Figure 1 Differentiation-dependent expression of vaspin in human keratinocytes (KCs). A: Keratinocytes were seeded onto a fibroblast monolayer. Two days before and 6, 8, 10, 12, and 14 days after raising cells to air-liquid interphase, RNA was isolated and vaspin and cytokeratin 10 (CK10) expression was detected by quantitative RT-PCR (RT-qPCR). B: Vaspin gene expression was determined in confluent and proliferating, subconfluent keratinocytes. C and D: Keratinocytes were stimulated with 20 ng/mL epidermal growth factor (EGF) for 24 and 72 hours. C: Vaspin gene expression was detected by RT-qPCR. mRNA values were normalized to the housekeeping gene RPS26. Arbitrary units (AUs) of untreated cells were set to 1. D: Vaspin detection in the supernatant by enzyme-linked immunosorbent assay. E: Vaspin (red), and CK10 and CK14 (green), expression in healthy skin by immunofluorescence staining. Isotype control antibody (ctr) was used as negative control. Nuclei were stained by DAPI (blue). Images represent one representative example of three different skin biopsy specimens. Values are given as means ± SD (A–D). n = 4 (B and C); n = 5 (D). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bar = 100 μm (E). The American Journal of Pathology 2016 186, 639-651DOI: (10.1016/j.ajpath.2015.10.030) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Figure 2 Vaspin expression is down-regulated in psoriatic skin inflammation. A: Vaspin expression was quantified in healthy and psoriatic skin biopsy specimens by quantitative RT-PCR (RT-qPCR). mRNA values were normalized to the housekeeping gene RPS26. B: Vaspin (red) expression in healthy, nonlesional, and lesional psoriatic skin was detected by immunofluorescence staining. Isotype control antibody (Ctr) was used as negative control. One representative experiment is shown. Vaspin expression was quantified by fluorescence intensity across the epidermis of 11 different samples from nonlesional psoriatic, lesional psoriatic, and healthy skin. C and D: BALB/c mice were treated for 4 consecutive days (d) with imiquimod ointment (Aldara; Meda Pharma GmbH, Bad Homurg, Germany; 62.5 mg) on the shaved back. After 1, 2, 3, and 4 days, mice were analyzed. C: Tissue sections were stained with hematoxylin. Proliferating cells (Ki-67), myeloid cells (anti-CD11b), and vaspin were detected by immunofluorescence labeling. Isotype ctr was used as a negative control. Images represent one representative example of three different mice. D: Vaspin expression was quantified by RT-qPCR. mRNA values were normalized to the unregulated housekeeping gene RS36B4. Untreated mice were set to 1, and x-fold change is shown. E–G: Human keratinocytes were stimulated with indicated cytokines for 24 and 72 hours (h). E and F: Vaspin expression was quantified by RT-qPCR. mRNA values were normalized to the unregulated housekeeping gene RPS26. Untreated keratinocytes were set to 1 (dashed line), and x-fold change is shown. G: Vaspin secretion into the supernatant was detected by enzyme-linked immunosorbent assay. Values are given as means ± SD (A, B, and D–G). n = 10 (A); n = 11 (B); n = 4 (D, untreated and imiquimod-treated mice); n = 5 (E–G). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bar = 100 μm (B and C). AU, arbitrary unit; TNF-α, tumor necrosis factor-α. The American Journal of Pathology 2016 186, 639-651DOI: (10.1016/j.ajpath.2015.10.030) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Figure 3 Action of vaspin on keratinocytes (KCs). A–C: Human primary keratinocytes isolated from three different skin donors were transfected with vaspin siRNA or a scrambled siRNA (scr) for 24 hours. Medium was replaced and cells were cultured for 3 days, followed by stimulation with 10 ng/mL tumor necrosis factor (TNF)-α and 20 ng/mL IL-17. A: Vaspin secretion was detected by enzyme-linked immunosorbent assay. B and C: After 24 hours, RNA was isolated and genome-wide expression analysis was performed. Expression in scr-transfected cells was set to 1, and x-fold change is shown. Values are given as means ± SD (A–C). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OAS, 2′,5′-oligoadenylate synthetase. The American Journal of Pathology 2016 186, 639-651DOI: (10.1016/j.ajpath.2015.10.030) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Figure 4 Keratinocyte (KC)-derived vaspin has anti-inflammatory potential. A–C: HaCaTs were transfected with an expression vector encoding for human vaspin or a control vector (Ctr vec). A: Vaspin secretion in the supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA). B and C: A total of 400,000 dendritic cells (DCs), Langerhans cells (LCs), monocytes, macrophages, and neutrophils were cultured with HaCaT-vaspin or HaCaT-Ctr vec in DC-medium. After 6 hours of co-culture, cells were stimulated with 100 ng/mL lipopolysaccharide (LPS). Supernatants were collected after 24 hours for the detection of the indicated cytokines by ELISA. Data are given as percentage of control (co-culture on HaCat-ctr vec = 100%, dashed line). D: Human primary keratinocytes transfected with vaspin siRNA or a scrambled siRNA (scr) were co-cultured with 400,000 monocytes separated by a 0.4-μm Transwell insert. LPS was added after 6 hours. As control, monocytes were stimulated with 10 ng/mL vaspin. Cytokine secretion was detected by ELISA after 24 hours. Data are given as percentage of control (scr-siRNA). E and F: KCs were co-cultured with 400,000 monocytes separated by a 0.4-μm Transwell insert. LPS was added after 6 hours. RNA from either monocytes or KCs after co-culture was isolated. In parallel, RNAs from KCs and monocytes cultured separately were isolated. Tumor necrosis factor (TNF)-α and IL-1β gene expression was detected by real-time quantitative PCR analysis. mRNA values were normalized to the unregulated housekeeping gene RPS26. E: Myeloid cells were detected by staining with aCD11b, neutrophils by aGr-1, and macrophages by aF4/80. Isotype control antibody (ctr) was used as a negative control. F: The number of labeled cells was calculated using a BZ-9000E analyzer (Keyence). G and H: BALB/c mice were treated daily with imiquimod to induce psoriasis-like skin inflammation, and 12.5 μg/kg recombinant vaspin or vehicle control was applied daily. After 5 days, skin was analyzed by immunofluorescence staining. Data represent means ± SD (A–F and H). n = 4 independent experiments (A–D); n = 3 (E and F); n = 6 mice (H). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bar = 100 μm (G). AU, arbitrary unit; MCP, monocyte chemoattractant protein; ROS, reactive oxygen species. The American Journal of Pathology 2016 186, 639-651DOI: (10.1016/j.ajpath.2015.10.030) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Figure 5 Action of vaspin in skin. Vaspin expression in human epidermis is associated with the differentiation of keratinocytes (KCs). Proliferating KCs do not express vaspin, whereas differentiated KCs express high levels of vaspin. Complex changes in KCs by the expression of vaspin result in a modulation of the communication between KCs and immune cells. Expression of vaspin supports the differentiation of KCs and suppresses the expression of inflammatory mediators. Consequently, vaspin-expressing KCs down-regulate the secretion of inflammatory mediators from a range of immune cells. Vaspin itself had no immune-modulatory activity on immune cells. Moreover, vaspin attenuated the infiltration of myeloid cells into the skin during inflammation. Thus, a paucity of the anti-inflammatory vaspin in psoriatic epidermis might contribute to the vicious circle of chronic skin inflammation in psoriasis. In conclusion, vaspin represents a novel link between KC differentiation/activation and the control of cutaneous immune responses in the chronic inflammatory skin disease psoriasis. DC, dendritic cell; MCP, monocyte chemoattractant protein; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-α. The American Journal of Pathology 2016 186, 639-651DOI: (10.1016/j.ajpath.2015.10.030) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions