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• W3136383723 abstract "Article16 March 2021Open Access Source DataTransparent process Psoriatic skin inflammation is promoted by c-Jun/AP-1-dependent CCL2 and IL-23 expression in dendritic cells Philipp Novoszel orcid.org/0000-0002-1398-9140 Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Martin Holcmann Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Gabriel Stulnig Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Cristiano De Sa Fernandes Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Victoria Zyulina Division of Immunology and Pathophysiology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria Search for more papers by this author Izabela Borek Division of Immunology and Pathophysiology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria Search for more papers by this author Markus Linder Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Alexandra Bogusch Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Barbara Drobits orcid.org/0000-0002-1155-2869 Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Thomas Bauer Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Carmen Tam-Amersdorfer orcid.org/0000-0003-3123-6342 Division of Immunology and Pathophysiology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria Search for more papers by this author Patrick M Brunner Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria Search for more papers by this author Georg Stary Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria Search for more papers by this author Latifa Bakiri Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria Search for more papers by this author Erwin F Wagner orcid.org/0000-0001-7872-0196 Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria Search for more papers by this author Herbert Strobl Division of Immunology and Pathophysiology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria Search for more papers by this author Maria Sibilia Corresponding Author [email protected] orcid.org/0000-0001-6129-5613 Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Philipp Novoszel orcid.org/0000-0002-1398-9140 Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Martin Holcmann Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Gabriel Stulnig Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Cristiano De Sa Fernandes Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Victoria Zyulina Division of Immunology and Pathophysiology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria Search for more papers by this author Izabela Borek Division of Immunology and Pathophysiology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria Search for more papers by this author Markus Linder Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Alexandra Bogusch Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Barbara Drobits orcid.org/0000-0002-1155-2869 Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Thomas Bauer Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Carmen Tam-Amersdorfer orcid.org/0000-0003-3123-6342 Division of Immunology and Pathophysiology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria Search for more papers by this author Patrick M Brunner Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria Search for more papers by this author Georg Stary Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria Search for more papers by this author Latifa Bakiri Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria Search for more papers by this author Erwin F Wagner orcid.org/0000-0001-7872-0196 Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria Search for more papers by this author Herbert Strobl Division of Immunology and Pathophysiology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria Search for more papers by this author Maria Sibilia Corresponding Author [email protected] orcid.org/0000-0001-6129-5613 Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria Search for more papers by this author Author Information Philipp Novoszel1, Martin Holcmann1, Gabriel Stulnig1, Cristiano De Sa Fernandes1, Victoria Zyulina2, Izabela Borek2, Markus Linder1, Alexandra Bogusch1, Barbara Drobits1, Thomas Bauer1, Carmen Tam-Amersdorfer2, Patrick M Brunner3, Georg Stary3, Latifa Bakiri3,4, Erwin F Wagner3,4, Herbert Strobl2 and Maria Sibilia *,1 1Department of Medicine I, Comprehensive Cancer Center, Institute of Cancer Research, Medical University of Vienna, Vienna, Austria 2Division of Immunology and Pathophysiology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria 3Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria 4Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria *Corresponding author. Tel: +43 1 40160 57502; E-mail: [email protected] EMBO Mol Med (2021)13:e12409https://doi.org/10.15252/emmm.202012409 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Toll-like receptor (TLR) stimulation induces innate immune responses involved in many inflammatory disorders including psoriasis. Although activation of the AP-1 transcription factor complex is common in TLR signaling, the specific involvement and induced targets remain poorly understood. Here, we investigated the role of c-Jun/AP-1 protein in skin inflammation following TLR7 activation using human psoriatic skin, dendritic cells (DC), and genetically engineered mouse models. We show that c-Jun regulates CCL2 production in DCs leading to impaired recruitment of plasmacytoid DCs to inflamed skin after treatment with the TLR7/8 agonist Imiquimod. Furthermore, deletion of c-Jun in DCs or chemical blockade of JNK/c-Jun signaling ameliorates psoriasis-like skin inflammation by reducing IL-23 production in DCs. Importantly, the control of IL-23 and CCL2 by c-Jun is most pronounced in murine type-2 DCs. CCL2 and IL-23 expression co-localize with c-Jun in type-2/inflammatory DCs in human psoriatic skin and JNK-AP-1 inhibition reduces the expression of these targets in TLR7/8-stimulated human DCs. Therefore, c-Jun/AP-1 is a central driver of TLR7-induced immune responses by DCs and JNK/c-Jun a potential therapeutic target in psoriasis. Synopsis Based on genetically engineered mouse models (GEMMs) and human psoriasis biopsies, this study suggests that c-Jun in Dendritic Cells (DC) contributes to psoriasis by controlling CCL2 and IL-23 production, and further identifies the JNK/c-Jun axis as a druggable target. TLR7 (IMQ)-induced skin inflammation was attenuated in mice lacking c-Jun in DCs. TLR7/JNK/c-Jun signalling was required for CCL2 and IL-23 transcription in human and murine DCs. c-Jun was co-expressed with CCL2 and IL-23 in type-2/inflammatory DCs of human psoriatic skin. Treatment with JNK inhibitor alleviated skin inflammation in mouse models of psoriasis. The paper explained Problem The TLR7/8 signaling pathway is an antiviral, innate immune defense mechanism implicated in the pathogenesis of psoriasis, a common inflammatory skin disease. TLR stimulation culminates in the activation of a set of downstream effectors, among them c-Jun/AP-1 proteins, but their role in regulating the cellular response to TLR7 signaling remains poorly understood. Results Here, we show that c-Jun/AP-1 is an essential mediator of the TLR7 signaling pathway in DCs to promote skin inflammation in two clinically relevant mouse models of psoriasis. Mechanistically, c-Jun/AP-1 regulates the pDC recruiting chemokine CCL2 and the T-cell activating cytokine IL-23 in murine and human DCs via the JNK/TLR7 signaling pathway. Inhibition of JNK/c-Jun activity by pharmacological blockade alleviated skin inflammation in a chemically (IMQ) and genetically induced murine psoriasis model. In psoriasis, we show co-expression of c-Jun with CCL2 and IL-23 in type-2/inflammatory DCs. Impact Our results have uncovered a novel role of c-Jun in immune cells for the pathogenesis of psoriasis, identified the JNK/c-Jun signaling axis as a druggable target, and thereby provided a new therapeutic treatment strategy for patients with psoriasis. Introduction Toll-like receptors (TLRs) are pattern recognition receptors of the innate immune system. TLRs recognize conserved motifs present on pathogens. Expression and ligand specificity is unique for each TLR and enables the recognition of bacterial, viral, and fungal pathogens (Iwasaki & Medzhitov, 2004). However, host-derived ligands such as self-RNA/DNA can cause inappropriate activation of TLR7/9, triggering auto-immune diseases such as systemic lupus erythematosus (SLE) or psoriasis (Lande et al, 2007; Ganguly et al, 2009). Psoriasis is a multifactorial, chronic, inflammatory skin disease whose pathogenesis is dependent on predisposing genetic mutations and environmental triggers (Wagner et al, 2010). It has been demonstrated that initially, self-RNA/DNA released from stressed or dying keratinocytes stimulates intracellular TLRs in plasmacytoid DCs (pDCs) and cutaneous DCs to initiate a Th1- and Th17-dependent immune response culminating in the activation of additional immune and skin cells. This leads to immune cell-rich, thickened skin regions clinically characterized as lesional psoriasis (Boehncke & Schön, 2015). Some of these effects can be mimicked by small molecule immune response modifiers of the Imidazoquinoline family, including Imiquimod (IMQ) or Resiquimod (R848), which are potent immunostimulators of the antiviral TLR7/8 signaling pathway (Hemmi et al, 2002). In mice, IMQ application induces a skin inflammation characterized by epidermal thickening, pDC and neutrophil influx, and inflammatory cytokine production (van der Fits et al, 2009). Using genetically engineered mouse models (GEMMs), DCs (Tortola et al, 2012) and γδ-T cells (Pantelyushin et al, 2012) were identified to mediate the production of IL-23 (Wohn et al, 2013) and IL-17 A/F (Pantelyushin et al, 2012; Riol-Blanco et al, 2014), respectively, to induce the inflammatory phenotype. TLR7 and TLR8 are expressed in the endosomal compartment of pDCs, monocytes, and B cells. Signal transduction via the adaptor protein MyD88 results in the activation of transcription factors like NF-κB or IRF7 along with components of the AP-1 family. NF-κB induces transcription of various inflammatory cytokines, whereas IRF7 is critical for the production of Type-I interferons (Kaisho & Tanaka, 2008). However, the role of AP-1 proteins downstream of TLR7/8 has so far been barely investigated. AP-1 proteins are basic leucine zipper transcription factors that form homo-or heterodimers. They include members of the Jun family, such as c-Jun and JunB, which play a prominent role in the skin. In mice, epidermal deletion of JunB affects skin, bone, and the hematopoietic system (Meixner et al, 2008; Uluckan et al, 2016) and combined epidermal deletion of c-Jun and JunB in adult mice results in a skin phenotype resembling psoriasis (Zenz et al, 2005). Previous studies have also shown a role for c-Jun (Riera-Sans & Behrens, 2007) and JunB (Yamazaki et al, 2017) in immune cell differentiation and production of a variety of cytokines in response to TLR stimulation (Vanden Bush & Bishop, 2008; Lin et al, 2011), which activates AP-1 proteins through the TRAF-6/TAK-1/JNK-p38 signaling axis (O'Neill et al, 2013). Given the importance of Jun proteins in skin homeostasis and their activation downstream of TLRs, we set out to genetically dissect the role of c-Jun downstream of TLR7 signaling in different skin cells, immune cells, and DCs/pDCs, specifically, as these innate immune cells are crucial TLR7-responsive immune potentiators. We show that deletion of c-Jun in DCs results in an attenuated IMQ-induced skin inflammation. Mechanistically, c-Jun/AP-1 exerts these effects by directly controlling CCL2 and IL-23 expression in TLR7 activated DCs. Our findings have clinical implications for patients with cutaneous diseases such as psoriasis. Results IMQ-induced skin inflammation requires c-Jun in dendritic cells To dissect the role of c-Jun/AP-1 in skin inflammation, we genetically inactivated c-Jun using CD11c-Cre and K5-Cre-ERT2 mice, which target predominantly DCs or keratinocytes, respectively. Additionally, for a broader deletion of c-Jun, in particular in all hematopoietic cells, we employed the inducible Mx1-Cre line, where poly I:C treatment induces interferons to activate the Mx-1 promoter. The treatment scheme for c-Jun deletion in these mouse models and induction of skin inflammation by Imiquimod (IMQ) is illustrated in (Fig EV1A). Epidermal thickness was used as an initial read-out for skin inflammation. Click here to expand this figure. Figure EV1. c-Jun in DCs is required for IMQ-induced keratinocyte proliferation and differentiation, but is dispensable for skin DC development A. Graphical timeline of IMQ treatment for the different Cre-Lines. Mice received poly I:C (Mx1-Cre) or Tamoxifen (K5-Cre-ERT2) for cell-type specific deletion of c-Jun and were waxed 2 days before start of IMQ treatment. IMQ was applied daily on the back skin and mice were taken for analysis after 1.5, 3, or 7 days after the first IMQ application, which corresponds to an IMQ treatment of 1, 3 or 7 times, respectively. After 7 days of IMQ application, mice were left untreated for another 3 days to study the resolution of IMQ-induced skin inflammation. Controls (c-Junfl/fl) were treated equally to Mx-1 or K5-Cre-ERT2 mice in all experiments, which is indicated by a color code (bronze = poly I:C and green = Tamoxifen) here and for all Figures henceforth. B–D. Validation of the deletion efficiency of c-Jun in the Mx1-Cre, the CD11c-Cre and K5-Cre-ERT2 mouse model. Immunofluorescence of c-Jun (green), K5 (red) and CD45 (white) (B), of c-Jun (green) and CD11c (red) (C) and of c-Jun (green) and K5 (red) (D) was performed in back skin from the indicated genotypes. DAPI was used to counterstain. Arrows indicate CD45+ (B) or CD11c+ skin cells (C). Magnification: 25× (B), 40× (C), 40× (D). Scale Bar: 50 µm. E, F. Ratio of K5 to K10 positive areas (E), and number of Ki-67+ cells (F) in epidermis of indicated mice was analyzed by immunofluorescence performed as described in Appendix Fig S1A and B. Three randomly chosen fields per section were analyzed for each sample (n = 6–10 (E), n = 5–12 (F); 2–4 independent experiments) G–I. Flow cytometry of DC subsets in total skin of indicated mice after 2, 3 and 5d of daily IMQ treatment. CD11b+ DC cells were defined as MHCII+CD172+CD11b+CD64− (G), CD103+ DC as MHCII+XCR1+CD103+CD64− (H) and Langerhans cells as MHCII+CD172+CD207+ CD64− (I) among live, single, CD45+ cells. Graphs show DC subsets as % of live, single cells (n = 6–12 (G), n = 4–11 (H), n = 5–13 (I); 2–4 independent experiments). Data information: Data are shown as mean ± SEM. P-values were calculated by one-way ANOVA with Tukey (E, F) or Bonferroni multiple comparison test (G-I). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See Appendix Table S3 for exact P-values. Source data are available online for this figure. Download figure Download PowerPoint c-JunΔ/Δ Mx1-Cre mice had significantly less IMQ-induced skin inflammation compared to poly I:C-treated c-Junfl/fl controls (Figs 1A and EV1B). This attenuated inflammatory response to IMQ was recapitulated by DC-specific deletion of c-Jun (c-JunΔ/Δ CD11c-Cre) (Figs 1B and EV1C), while the keratinocyte-specific c-Jun deletion (c-JunΔ/Δ K5-Cre-ERT2 mice) responded to IMQ similar to Tamoxifen treated c-Junfl/fl controls (Figs 1C and EV1D). Given that c-Jun seems to be required downstream of TLR7 signaling to mediate the inflammatory response in DCs, we focused our in vivo analyses on c-JunΔ/Δ CD11c-Cre mice. Figure 1. IMQ-induced skin inflammation requires c-Jun in dendritic cells A–C. Epidermal thickness of the back skin was analyzed at the indicated time points in c-JunΔ/Δ Mx1-Cre (A), c-JunΔ/Δ CD11c-Cre (B) and c-JunΔ/Δ K5-Cre-ERT2 mice (C) on hematoxylin and eosin (H&E) stained sections (n = 5–21 (A), n = 6–29 (B), n = 5–18 (C); ≥ 2 independent experiments). D. H&E stained sections of back skin from indicated mice treated with IMQ for 2, 3, or 5 days. Bright-field images, Magnification: 20×, Scale bar: 100 µm. E. Layers of epidermis (left) and Trans-epidermal water loss (TEWL) (right) were analyzed in the back skin of indicated mice (Left: n = 8–14, right: n = 6–14; ≥ 2 independent experiments). F–H. Flow cytometry of total back skin after 2, 3, and 5 days of IMQ treatment. Analyzed were dermal γδ T cells (γδ TCRint+) (F), monocytes (CD11b+Ly6Chi) (G), and neutrophils (CD11b+Ly6G+) (H). Graphs show immune cells as % of live, single cells (n = 10–28 (F), n = 10–21 (G), n = 4–25 (H); ≥ 2 independent experiments). Data information: Data are shown as mean ± SEM. P-values were calculated by one-way ANOVA with Tukey (A-C, and E) or Bonferroni multiple comparison test (F- H). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See Appendix Table S3 for exact P-values. Source data are available online for this figure. Source Data for Figure 1 [emmm202012409-sup-0003-SDataFig1.zip] Download figure Download PowerPoint To better understand the role of c-Jun in the onset of skin inflammation, we analyzed the hallmarks of IMQ-induced skin inflammation, which are acanthosis (epidermal thickening), loss of barrier integrity, and infiltration of immune cells such as neutrophils and dermal γδ T cells, the major IL-17-producing immune cell in murine skin. c-JunΔ/Δ CD11c-Cre mice showed a marked reduction in acanthosis and were protected from IMQ-induced epidermal barrier breakdown as measured by trans-epithelial water loss (TEWL) (Fig 1D and E). In addition, we observed reduced keratinocyte proliferation (less Ki67+ nuclei, less proliferative K5+ to differentiated K10+ cells) in c-JunΔ/ΔCD11c-Cre mice compared to control mice (Fig EV1E and F and Appendix Fig S1A and B). Infiltration of dermal γδ T cells (γδ TCRint+), monocytes (CD11b+Ly6Chi), and neutrophils (CD11b+Ly6G+) following IMQ treatment was also significantly decreased in c-JunΔ/ΔCD11c-Cre mice (Fig 1F–H), while levels of the two major dermal DC subsets, CD11b+ DCs and CD103+ DCs, and of epidermal Langerhans cells remained unchanged (Fig EV1, EV2-EV5). These results suggest that c-Jun does not directly control skin DC development, but rather their effector function downstream of TLR7 signaling, which is required for IMQ-induced skin inflammation. Click here to expand this figure. Figure EV2. Control of cytokine expression in IMQ-treated skin and BMDCs by c-Jun Immunofluorescence of c-Jun (green), CD11c (red) and DAPI in mouse back skin of indicated mice treated with IMQ for 8 h. Insets give an enlarged view of the framed area. Scale bar: 10 µm, Magnification: 40×. qRT–PCR detection of chemokines Cxcl1, Ccl20, antimicrobial proteins S100a8, S100a9 and the pro-inflammatory cytokines Tnfa, Il22, Il6 and Il1b in total RNA isolated from the back skin of indicated mice treated with IMQ for 32 or 48 h (n = 9–20; 3–6 experiments). qRT–PCR mRNA expression analysis of indicated cytokines in BMDCs. Bar graph shows fold change of targets in IMQ (4 h) stimulated BMDCs relative to LAL. Asterisk shows significance between the genotypes in IMQ-treated samples (n = 3–7; 2–3 independent experiments). Immunofluorescence of CD11c (green), IL-23p19 (red) and DAPI in indicated BMDCs stimulated with IMQ for 5 h in the presence of Brefeldin A. Scale bar: 25 µm, Magnification: 60×. Gating strategy to sort DCs (CD45+CD11c+ MHCII+), T cells (CD3ε+), granulocytes (Gr-1+) and non-immune cells (CD45−) from back skin treated with IMQ for 8 h. qRT–PCR detection of Il23p19 mRNA expression levels in CD3ε +, Gr-1+ and CD45− cells sorted from back skin as described in (E) (n = 4–6, 3 independent experiments). IL-23p19 and IL-12p40 expression in BMDCs was analyzed by intracellular flow cytometry. BMDCs were stimulated with IMQ for 4 h in the presence of Brefeldin A. Representative plots shown are pregated on live, single, CD45+ and CD11c+ cells. Bar graph shows % of live, single, CD45+, CD11c+ cells (% DCs) expressing IL12-p40 (n = 7–10; 3 independent experiments). qRT–PCR detection of Il23p19 mRNA expression in BMDCs pretreated with MG-132 (NF-κBi, 20 µM) for 1 h and stimulated with IMQ for 4 h (n = 5–6; 2 independent experiments). Data information: Data are shown as mean ± SEM. P-values were calculated by unpaired, two-tailed t-test (B, C, and F) or one-way ANOVA with Tukey multiple comparison test (H, I). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See Appendix Table S3 for exact P-values. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Validation of the therapeutic potential of JNK inhibition in psoriasis-like mouse models A–C. Back skin of c-Junfl/fl, c-JunΔ/ΔCD11c-Cre, Tlr7−/− and Tlr7−/− c-JunΔ/ΔCD11c-Cre mice was treated with IMQ and JNKi or vehicle for 5 days. Shown are representative H&E stained sections (Bright-field images, Magnification: 20×, Scale bar: 100 µm) (A), immunofluorescence staining’s of Ki-67 (green) and DAPI (Scale bar: 100 µm, Magnification: 20×) (B) and of K5 (red), K10 (green) and DAPI (Scale bar: 100 µm, Magnification: 20×) (C) in murine back skin. For quantification 2–3 randomly chosen fields per section were counted. D–F. Flow cytometric analysis of cell suspension from back skin of indicated mice and treatment. t-SNE- plots (D) show the cutaneous immune cell phenotype. Populations defined by t-SNE algorithm were confirmed by conventional gating as described in Appendix Fig S7A and representative plots are shown in (E) for Monocytes and Neutrophils and in (F) for γδ T cells and dendritic epidermal T cells (DETC). Numbers adjacent to marked areas indicate percentage of cell population among CD45+ cells (D) and live, single cells (E, F). Representative plots shown are pregated on live, single, CD45+ (D) and CD11b+ (E) or CD11b− cells (F). G. Experimental design for blocking IL-23 signaling in the IMQ-induced skin inflammation model. Back skin of wild-type mice was treated daily with IMQ and anti-IL23R antibody (15 mg/kg, i.p.) or isotype control for 5 consecutive days. To compare JNK Inhibitor (15 mg/kg, i.p.) was given (n = 3) H. Trans-epidermal water loss (TEWL) was analyzed in the back skin at the end of treatment. I. Flow cytometry of total back skin after 5 days of IMQ treatment. Analyzed were dermal γδ T cells (γδ TCRint+), Monocytes (CD11b+Ly6Chi), and Neutrophils (CD11b+Ly6G+). Graphs show immune cells as % of live, single cells. Data information: Data are shown as mean ± SEM. P-values were calculated by Tukey multiple comparison test (H, I). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P-values. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Characterization of c-Jun co-expression with CCL2 and IL-23 in DCs of lesional psoriatic skin A. Immunofluorescence of c-Jun (green), CD11c (red) and DAPI in psoriatic lesions from two patients and in non-lesional skin from one patient. Insets give an enlarged view of the framed area. Arrows indicate CD11c+ skin cells. Scale bar: 10 µm, Magnification: 40x. B. Linear regression analysis between c-Jun and Ccl2 in healthy (n = 38), non-lesional (n = 27) and lesional (n = 28) skin. Expression values were obtained from the GEO Data Set [GSE121212]. A Pearson’s correlation test was performed and r2 and P values are indicated in the plot. C. Expression values of Tlr7, Tlr8, Jnk1, Jnk2, Jnk3, c-Jun, Ccl2 and Il23p19 in healthy (n = 38), non-lesional (n = 27) and lesional (n = 28) skin. Expression values were obtained from the GEO Data Set [GSE121212]. Box and whiskers plot: Central band shows median, box extends from the 25th to 75th percentiles and whiskers go down to the smallest (min) and up to the largest (max) value. D. Representative immunofluorescence of CCL2 or IL-23p19 (green) and DAPI in healthy and lesional skin. Arrows indicate IL-23p19+ (upper panels) or CCL2+ dermal cells (lower panels). Magnification: 40x. Scale bar: 50 µm (n = 2 patient samples). E, F. Representative immunofluorescence of CCL2 (E) or IL-23p19 (F) (white), c-Jun (green) and the DC markers CD1c or CD14 (red) was performed on lesional psoriatic skin. DAPI was used to counterstain. Arrows indicate triple-positive cells and an asterisk highlights a representative one that is shown enlarged in a white-framed inlet. Magnification: 40×. Scale bar: 50 µm (n = 2 patient samples). Data information: Data are shown as mean ± SEM. P-values were calculated by Pearson´s correlation test (B) and one-way ANOVA with Tukey multiple comparison test (C). Statistical significance: ns > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See Appendix Table S3 for exact P-values. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Role of JNK-AP-1 signaling in the maturation of human mo-DCs stimulated with R848 or LL-37-RNA-40 A–D. Human mo-DCs were pretreated with DMSO (1:1,000) or JNK Inhibitor (SP600125, 25 µM) (A, B) or AP-1 Inhibitor (T-5224, 20 µM) (C, D) for 1 h and stimulated with R848 for 24 h. Gated CD1a+ cells were analyzed for surface expression of CD80 and CD86 by flow cytometry. Shown are representative histograms (A, C) and mean fluorescence intensity (MFI) (B, D) (n = 4 (B); representative result from 2 independent experiments is shown, n = 7 (D); 2 independent experiments). E–G. Human mo-DCs were stimulated with LL-37-RNA-40 complex or LL-37 or RNA-40 for 24 h. Representative flow cytometry plots and histograms are shown in (E) and mean fluorescence intensity (MFI) of CD80 and C86 is shown in (F). Expression of CCL2 and IL-23 was analyzed by ELISA (G) (n = 7, 2 independent experiments). Data information: Data are shown as mean ± SEM. P-values were calculated by paired, tw" @default.
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• W3136383723 title "Psoriatic skin inflammation is promoted by c‐Jun/AP‐1‐dependent CCL2 and IL‐23 expression in dendritic cells" @default.
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