1. Immunostimulatory Endogenous Nucleic Acids Drive the Lesional Inflammation in Cutaneous Lupus Erythematosus 
Benedikt Scholtissek, Sabine Zahn, Judith Maier, Sophie Klaeschen, Christine Braegelmann, Michael Hoelzel, Thomas Bieber, Winfried Barchet, Joerg Wenzel 
Journal of Investigative Dermatology 
Volume 137, Issue 7, Pages (July 2017)
DOI: /j.jid
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2. Figure 1
Synthetic nucleic acids induce expression of IFN-regulated chemokines in human keratinocytes. (a, b) Effect of different synthetic immunostimulatory nucleic acids on the induction of CLE-typical cytokines in cultured NHEK cells (for 24 hours). (a) Cytokine protein expression in the supernatant was measured by cytometric bead array. ∗P < 0.05; Mann-Whitney U test: stimulation vs. medium control; n = 5 in each group; ± standard error of the mean. (b) Cytokine gene expression by PCR. (c, d) Effect of poly(I:C) and poly(dA:dT) on cytokine-expression in a three-dimensional epidermis model (epiCS). (c) Immunohistochemistry, representative micrographs. Specimens after 24 hours of stimulation, original magnification ×200, scale bar = 100 μm. (d) Histological scoring of the effect of poly(I:C) and poly(dA:dT) on the protein expression of MxA and CXCL10. Mean expression score ± standard error of the mean; ∗P < 0.05; n = 3 in each group). CLE, cutaneous lupus erythematosus; Ctrl, control; lipo, lipofectamine; NHEK, normal human epidermal keratinocyte.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
Endogenous nucleic acids induce expression of IFN-regulated chemokines in human keratinocytes. (a, b) Concentration of CXCL10 in the supernatant of HaCaT- and NHEK-cells stimulated with eNAs (endogenous nucleic acids) and controls (medium and lipofectamine), both measured with ELISA (n = 5 in each group). (c) Typical clinical picture of sun-induced CLE skin lesions. (d–h) Histological micrographs showing co-expression of different pathogenetic factors in active CLE skin lesions. Black arrows indicate co-localization of (d) MxA, (e) 8-hydroxyguanosine nucleic acids, and (f) LL37 in serial sections. Scale bar = 100 μm. (g, h) Grey arrows highlight cytosolic localization of different nucleic acid motifs: (g) 8-hydroxyguanosine nucleic acid, scale bar = 50 μm and (h) DNA-AC3010, scale bar = 20 μm in CDLE. (h) White arrow highlights nuclear expression in HC. (i, j) Amount of IFN-λ, CXCL10, and CXCL9 in the supernatant of NHEK cells depending on different stimuli measured with (i) ELISA (n = 5 in each group) and (j) PCR (representative data from one gel; white lines indicate the removal of complete lanes). (k) Concentration of CXCL10 in the supernatant of NHEK cells stimulated with different combinations of nucleic acids and transfectants measured with ELISA (n = 5 in each group). All values are means ± standard error of mean. ∗P < 0.05; Mann-Whitney U test. (i, k) Stimulus versus medium control. Patient photograph published with consent. 8-OHG-NA, 8-hydroxyguanosine nucleic acid motif; CDLE, chronic discoid lupus erythematosus; CLE, cutaneous lupus erythematosus; CTR, control; eDNA, RNase-digested eNA; eNA, endogenous nucleic acid; eRNA, DNase-digested eNA; HC, healthy controls; Lipo, lipofectamine; NHEK, normal human epidermal keratinocyte.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
UV stress causes cytosolic translocation of DNA motifs and enhances their immunostimulatory capacity. (a–d) Representative immunofluorescence micrographs of cultured HaCaT cells (c, d) 4 hours after UV stimulation (150 mJ/cm2) and (a, b) without treatment (control). Scale bar = 10 μm. DAPI binding is represented in blue color (recognizes double-stranded genomic DNA only); red staining represents DNA antibody AC3010 (recognizes all types of DNA, including single-stranded DNA and shorter immunostimulatory DNA fragments). (e) Mean CXCL10 protein expression in the supernatant of HaCaT cells stimulated with UV light (UV direct) and with different preparations of eNAs (with and without lipofectamine and after UVB stimulation with 150 mJ/cm2 [UVeNA]), measured by ELISA (± standard error of the mean; ∗P < 0.05, ∗∗P < 0.01; Mann-Whitney U test; n = 8 in each group). eNA, endogenous nucleic acid; Lipo, lipofectamine; ns, not significant.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
UV light induces LE-like skin lesions in mice lacking the cytosolic DNase TREX1. (a–f) Clinical, histological (H&E), and immunohistological (CD45) findings in TREX1–/– mice after UV stimulation (3 × 450 mJ/cm2) on day 12. Scale bar = 100 μm. (g) Mean score of the mouse-adapted CLE Disease Area and Severity Index (Mouse Cutaneous Lupus Erythematosus Disease Area and Severity Index) ± standard error of the mean in n = 5 mice per group (∗P < 0.05; Mann-Whitney U test). CLE, cutaneous lupus erythematosus; H&E, hematoxylin and eosin; LE, lupus erythematosus; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions