1. Immunology and Inflammation
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Dithranol targets keratinocytes, their crosstalk with neutrophils and inhibits the IL-36 inflammatory loop in psoriasis

  1. Theresa Benezeder
  2. Clemens Painsi
  3. VijayKumar Patra
  4. Saptaswa Dey
  5. Martin Holcmann
  6. Bernhard Lange-Asschenfeldt
  7. Maria Sibilia
  8. Peter Wolf  Is a corresponding author
  1. Department of Dermatology, Medical University of Graz, Austria
  2. State Hospital Klagenfurt, Austria
  3. Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Austria
Research Article
Cite this article as: eLife 2020;9:e56991 doi: 10.7554/eLife.56991
5 figures, 8 tables, 2 data sets and 5 additional files

Figures

Figure 1 with 2 supplements
Dithranol leads to a fast reduction of psoriatic skin lesions as determined by local psoriasis severity index (PSI) of marker lesions.

(A,B) 15 psoriasis patients were treated with dithranol. Skin biopsies were taken from marker lesions at multiple timepoints: 1a = lesional skin at baseline, 2 = lesional skin at day 6, 3 = lesional skin at end of treatment, week 2–3, 4 = lesional skin at follow-up, 4–6 weeks after end of treatment. In addition, non-lesional skin at baseline (1b) was sampled. (C) Representative images of lesional psoriatic skin and local psoriasis severity index (PSI; sum of erythema (0–4), induration (0–4) and scaling (0–4); 0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe) at different time points.

Figure 1—figure supplement 1
Psoriasis area and severity (PASI) score at baseline (day 0), early during treatment (day 6), at end of treatment (week 2–3) and at follow-up (4–6 weeks after end of treatment) for all 15 patients (A1–A15) treated with dithranol.
Figure 1—figure supplement 2
Lesional and perilesional erythema does not correlate with reduction in PASI score.

(A) Mean erythema score (0 = none, 1 = mild, 2 = moderate, 3 = severe 4 = very severe erythema on lesional skin) and B) Mean perilesional erythema score (0 = none, 1 = mild, 2 = moderate, 3 = severe erythema on perilesional skin) at baseline (day 0), early during dithranol treatment (day 6), at end of treatment (week 2–3) and at follow-up (4–6 weeks after end of treatment). Mean ± SD (n = 15) is shown. (C–D) Erythema score (C) and perilesional erythema score (D) at day six during dithranol treatment was compared with reduction in PASI score at end of treatment using Spearman correlation. (E–F) Erythema score (E) and perilesional erythema score (F) at end of treatment (week 2–3) was compared with reduction in PASI score at end of treatment using Spearman correlation. n.s. = not significant.

Histological and immunohistochemical analysis of lesional skin before (day 0), during (day 6), at end of treatment (week 2–3) and 4–6 weeks after ending treatment (follow-up).

(A) Representative H and E images, epidermal thickness and cellular infiltrate scoring. In treated psoriatic lesions, epidermal thickness was significantly decreased at week 2–3 and at follow-up. Infiltrate score (0 = none, 0.5 = none/low, 1 = low, 1.5 = low/moderate, 2 = moderate, 2.5 = moderate/high, 3 = high infiltration of immune cells) was significantly higher in untreated psoriatic lesions compared to non-lesional skin (NL) and healthy controls (HC). In treated psoriatic lesions, infiltrate score was significantly decreased at follow-up. (B) Ki67 staining in epidermis was significantly reduced at week 2–3 and follow-up. (C) CK16 staining in epidermis was significantly reduced at week 2–3 and follow-up. (D–I) Representative IHC images and mean cell counts for epidermis and dermis. Neutrophil cell counts were significantly reduced at week 2–3 and follow-up in epidermis and dermis (D). Langerhans cell (CD1a+) numbers in epidermis were significantly increased at follow-up (E). Epidermal FoxP3 (H) and CD8 (I) cell counts were significantly reduced at week 2–3. Dermal CD3 (F), CD4 (G), and FoxP3 (H) positive cell counts display significant reduction at follow-up. One-way ANOVA with Dunnet’s multiple comparisons test was used for statistics. Bars represent mean ± SD; *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; scale bar = 100 µm.

Figure 3 with 3 supplements
Topical application of dithranol ameliorates psoriasis-like skin lesions in c-Jun/JunB knockout mice.

(A) Schematic representation of experimental set-up. Red arrows indicate dithranol application in series of increasing concentrations. (Exp. 1 and 2 = experiment 1 and 2) (B) Macroscopic ear thickness on day 1 compared to day 7. Dithranol treatment led to a significant reduction in ear thickness. Controls (n = 11), dithranol group (n = 11); Paired t-test was used for statistics. (C) Representative H and E images of untreated and dithranol-treated ears. (D) Dithranol treatment led to a significant reduction in epidermis thickness. Controls (n = 10), dithranol group (n = 11); unpaired t-test was used for statistics. Data from the two experiments was pooled (D). Bars represent mean ± SD; n.s. = not significant; *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; scale bar = 100 µm.

Figure 3—figure supplement 1
Strong inducing effect of dithranol in the mouse-tail test.

(A) Representative H and E images of vehicle-treated and dithranol-treated mouse tail skin. (B) Dithranol treatment significantly increased orthokeratosis. (C) Representative images of erythema and typical brownish discoloration of skin caused by dithranol. Bars represent mean ± SD (n = 10). Mann-Whitney test was used for statistics. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; Scale bar = 50 µm.

Figure 3—figure supplement 2
Gene expression analysis by RT-PCR of keratinization markers (Flg, Ivl, Krt16, Lce3e, Serpinb3a), AMPs (Lcn2, S100a8, S100a9, Camp, Defb1, Defb3) and inflammatory markers (Il1b, Il17, Il22, Cxcl1, Cxcl5) of dithranol- and vehicle-treated murine tail skin.

Bars represent mean ± SD (n = 10) of relative expression values (deltaCT) normalized to Ubc. Unpaired t-test was used for statistics (n.s. = not significant) Flg, filaggrin; Ivl, involucrin, Krt16, keratin16; Lce3e, Late Cornified Envelope 3e; Camp, Cathelicidin Antimicrobial Peptide; Defb1, Beta-Defensin 1; Defb3, Beta-Defensin 3; Il1b, Interleukin one beta; Il17, Interleukin 17; Il22, Interleukin 22; Cxcl1, C-X-C Motif Chemokine Ligand 1; Cxcl5, C-X-C Motif Chemokine Ligand 5.

Figure 3—figure supplement 3
Dithranol causes exacerbation of psoriasis-like skin lesions in imiquimod mouse model.

(A) Representative images of untreated, IMQ-treated and IMQ and dithranol-treated dorsal skin of mice. (B) Macroscopic skin thickness (red arrows indicate concentration of dithranol), (C–D) epidermal thickness measurements with corresponding H and E images and E) cellular infiltrate score show exacerbation of psoriasis-like dermatitis in IMQ and Dithranol-treated mice. Infiltrate score (0 = none, 0.5 = none/low, 1 = low, 1.5 = low/moderate, 2 = moderate, 2.5 = moderate/high, 3 = high infiltration of immune cells); Tukey’s multiple comparisons test (B), One-way ANOVA with Tukey’s multiple comparisons test (D) and Kruskal Wallis test with Dunn’s multiple comparisons test (E) was used for statistics. Bars represent mean ± SD (n = 5); *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; Scale bar = 50 µm.

Figure 4 with 1 supplement
Venn diagram (created using InteractiVenn Heberle et al., 2015) showing comparison and overlap between differentially expressed genes (DEGs) in dithranol-treated human psoriatic skin at week 2–3 vs. day 0, DEGs in dithranol-treated human psoriatic skin at day 6 vs. day 0 and DEGs in dithranol-treated c-Jun/JunB psoriatic skin.
Figure 4—figure supplement 1
Verification of microarray gene expression analysis: Gene expression analysis by RT-PCR of selected genes based on microarray data in dithranol-treated c-Jun/JunB knockout mice vs. vehicle-treated controls.

Bars represent mean ± SD (n = 5) of relative expression values (deltaCT) normalized to Ywhaz. Unpaired t-test was used for statistics. Il1f5 (IL36RN), interleukin 1 family member 5 (IL-36 receptor antagonist); Ivl, Involucrin; Serpinb7, Serine peptidase inhibitor, clade B member 7; Serpinb13, Serine peptidase inhibitor, clade B member 13.

Proposed model of dithranol’s mechanism of action in psoriasis.

Dithranol decreases expression of keratinocyte differentiation regulators (involucrin and serpins), IL-36-related genes, keratinocyte-derived antimicrobial peptides (AMPs) (S100A7/A12 and ß-defensins) and chemotactic factors for neutrophils (CXCL5, CXCL8). This disrupts the crosstalk between keratinocytes and neutrophils and leads to diminished neutrophilic infiltration, thereby halting the inflammatory feedback loop in psoriasis. Together, this results in clearance of psoriatic lesions.

Tables

Table 1
Psoriasis area and severity index (PASI) and psoriasis severity index (PSI) from 15 psoriasis patients.
ParameterTime point
BaselineDay 6End of treatmentFollow-up
PASI
mean ± SD13.6 ± 10.39.0 ± 6.3
(p<0.0001)*
5.1 ± 3.8
(p=0.0001)*
5.7 ± 6.7
(p=0.0002)*
%reduction, mean ± SD (range)-32.9 ± 8.0
(16.7–44.3)
57.5 ± 9.5
(41.8–74.2)
56.1 ± 23.3
(3.1–81.0)
Local PSI
mean ± SD6.7 ± 1.13.3 ± 1.6
(p<0.0001)*
2.0 ± 1.5
(p<0.0001)*
1.6 ± 1.3
(p<0.0001)*
%reduction, mean ± SD (range)-52.6 ± 21.9
(14.3–100)
69.0 ± 23.9
(16.7–100)
76.3 ± 18.3
(37.5–100)
  1. *P value was determined using Wilcoxon test comparing indicated value to baseline.

Table 2
Top 45 differentially regulated genes (p-value<0.05, fold change >1.5) in dithranol-treated lesional skin after 6 days compared to baseline from 15 patients with psoriasis.
Probe set IDGene symbolGene titleDay 6 vs. baseline
(fold change)
16693318FLG2Filaggrin family member 22.89
16693303HRNRHornerin2.64
16903552NEBNebulin2.18
16765017KRT2Keratin 22.04
16693308FLGFilaggrin1.97
16761221CLEC2AC-type lectin domain family 2, member A1.92
16730782ELMOD1ELMO/CED-12 domain containing 11.91
16735288OVCH2Ovochymase 21.83
16990787SPINK7Serine peptidase inhibitor, Kazal type 7 (putative)1.78
16852824SERPINB12Serpin peptidase inhibitor, clade B (ovalbumin), member 121.74
16693341LCE1CLate cornified envelope 1C1.71
16693249THEM5Thioesterase superfamily member 51.70
16921644MIRLET7CMicroRNA let-7c1.69
16670681ANXA9Annexin A91.68
16821186CLEC3AC-type lectin domain family 3, member A1.68
16859090CASP14Caspase 141.68
16765005KRT73Keratin 731.66
16945497COL6A5Collagen, type VI, alpha 51.66
16976615SULT1E1Sulfotransferase family 1E, estrogen-preferring, member 11.65
16898858CD207CD207 molecule, langerin1.63
16861126UPK1AUroplakin 1A1.60
16704475FAM35DPFamily with sequence similarity 35, member A pseudogene1.60
17085015FRMPD1FERM and PDZ domain containing 11.59
16817034CHP2Calcineurin-like EF-hand protein 21.56
16671082LCE1ALate cornified envelope 1A1.55
16671037LCE2DLate cornified envelope 2D1.55
16748835PIK3C2GPhosphatidylinositol-4-phosphate 3-kinase, catalytic subunit1.54
17039517LY6G6CLymphocyte antigen six complex, locus G6C1.53
16673713FMO2Flavin containing monooxygenase 2 (non-functional)1.52
16812344BCL2A1BCL2-related protein A1−1.52
16968213ANXA3Annexin A3−1.54
17104519RNY4P23RNA, Ro-associated Y4 pseudogene 23−1.55
16948835MIR1224MicroRNA 1224−1.55
16815310TNFRSF12ATumor necrosis factor receptor superfamily, member 12A−1.60
17015084SERPINB1Serpin peptidase inhibitor, clade B (ovalbumin), member 1−1.67
17106398SLC6A14Solute carrier family 6 (amino acid transporter), member 14−1.69
16693375SPRR2FSmall proline-rich protein 2F−1.69
17065458DEFB4ADefensin, beta 4A−1.75
17074361DEFB4BDefensin, beta 4B−1.77
16698947RNU5A-8PRNA, U5A small nuclear 8, pseudogene−1.78
16976827CXCL5Chemokine (C-X-C motif) ligand 5−1.81
16976821PPBPPro-platelet basic protein (chemokine (C-X-C motif) ligand 7)−1.82
17019056TREM1Triggering receptor expressed on myeloid cells 1−1.99
17050251SLC26A4Solute carrier family 26 (anion exchanger), member 4−2.19
16967771CXCL8Chemokine (C-X-C motif) ligand 8−3.36
Table 3
Top 45 differentially regulated genes in dithranol-treated lesional skin at end of treatment compared to baseline from 15 patients with psoriasis (p<0.05, fold change >1.5).
Probe set IDGene symbolGene titleEnd of treatment vs. baseline (fold change)
16947045AADACArylacetamide deacetylase3.07
16765005KRT73Keratin 732.31
16976868BTCBetacellulin2.27
16924792CLDN8Claudin 82.11
17125092CNTNAP3Contactin associated protein-like 32.09
17097358SLC46A2Solute carrier family 46, member 22.07
16780133SLITRK6SLIT and NTRK-like family, member 62.00
16834436RAMP2Receptor (G protein-coupled) activity modifying protein 21.96
17123970ZDHHC11BZinc finger, DHHC-type containing 11B1.88
17104313ARAndrogen receptor1.85
16951140MIR548I2MicroRNA 548i-21.85
17063366ATP6V0A4ATPase, H+ transporting, lysosomal V0 subunit a41.84
16974224MIR548I2MicroRNA 548i-21.83
16687914CYP2J2Cytochrome P450, family 2, subfamily J, polypeptide 21.82
17125106CNTNAP3BContactin associated protein-like 3B1.80
16903552NEBNebulin1.80
17125094CNTNAP3BContactin associated protein-like 3B1.80
16770284TMEM116Transmembrane protein 1161.79
16765041KRT77Keratin 771.79
17123972ZDHHC11BZinc finger, DHHC-type containing 11B1.78
16688210MIR3671MicroRNA 36711.78
17125218CNTNAP3Contactin associated protein-like 31.74
16764923KRT6AKeratin 6A−3.69
16767261IL22Interleukin 22−3.87
17065453DEFB103ADefensin, beta 103A−3.94
17074366DEFB103ADefensin, beta 103A−3.94
16671027LCE3CLate cornified envelope 3C−4.14
16743751MMP12Matrix metallopeptidase 12 (macrophage elastase)−4.42
16979444TNIP3TNFAIP3 interacting protein 3−4.48
17106398SLC6A14Solute carrier family 6 (amino acid transporter), member 14−4.60
16686734CYP4Z2PCytochrome P450, family 4, subfamily Z, polypeptide 2−4.89
16671144S100A7AS100 calcium binding protein A7A−5.00
16764907KRT6CKeratin 6C−5.01
16730157HEPHL1Hephaestin-like 1−5.15
16967831EPGNEpithelial mitogen−5.31
16842517NOS2Nitric oxide synthase 2, inducible−5.38
16693409S100A12S100 calcium binding protein A12−6.12
16813112RHCGRh family, C glycoprotein−6.18
16738803TCN1Transcobalamin I (vitamin B12 binding protein, R binder family)−6.45
16924785CLDN17Claudin 17−8.09
16693365SPRR2CSmall proline-rich protein 2C (pseudogene)−8.72
16967771CXCL8Chemokine (C-X-C motif) ligand 8−9.14
17074361DEFB4BDefensin, beta 4B−9.39
16693375SPRR2FSmall proline-rich protein 2F−10.12
16884602IL36AInterleukin 36, alpha−10.50
Table 4
Top 20 significantly enriched pathways as determined by Gene Ontology (GO) enrichment analysis in dithranol-treated lesional skin after 6 days compared to baseline from 15 patients with psoriasis.

(GO was done using Cytoscape software [Bindea et al., 2009; Shannon et al., 2003]; a.o. = among others).

GOIDGO termP-Value% Associated GenesAssociated genes found
GO:0001533Cornified envelope3.8E-1210.45FLG, HRNR, KRT2, LCE1A, LCE1C, LCE2D, SPRR2F
GO:0031424Keratinization7.7E-123.93CASP14, FLG, HRNR, KRT2, KRT73, LCE1A, LCE1C, LCE2D, SPRR2F
GO:0030216Keratinocyte differentiation79.0E-123.02CASP14, FLG, HRNR, KRT2, KRT73, LCE1A, LCE1C, LCE2D, SPRR2F
GO:0018149Peptide cross-linking300.0E-129.84FLG, KRT2, LCE1A, LCE1C, LCE2D, SPRR2F
GO:0070268Cornification12.0E-95.31CASP14, FLG, KRT2, KRT73, LCE1A, SPRR2F
GO:0004168Receptor CXCR2 binds ligands CXCL1 to 71.0E-633.33CXCL5, CXCL8, PPBP
GO:0042379Chemokine receptor binding5.4E-66.25CXCL5, CXCL8, DEFB4A, PPBP
GO:0045236CXCR chemokine receptor binding6.0E-618.75CXCL5, CXCL8, PPBP
GO:0061436Establishment of skin barrier18.0E-613.04FLG, FLG2, HRNR
GO:0033561Regulation of water loss via skin21.0E-612.00FLG, FLG2, HRNR
GO:0004657IL-17 signaling pathway21.0E-64.30CXCL5, CXCL8, DEFB4A, DEFB4B
GO:0007874Keratin filament formation21.0E-64.17FLG, KRT2, KRT73, SPRR2F
GO:0030593Neutrophil chemotaxis21.0E-64.17CXCL5, CXCL8, PPBP, TREM1
GO:1990266Neutrophil migration27.0E-63.85CXCL5, CXCL8, PPBP, TREM1
GO:0071621Granulocyte chemotaxis38.0E-63.31CXCL5, CXCL8, PPBP, TREM1
GO:1902622Regulation of neutrophil migration45.0E-67.50CXCL5, CXCL8, PPBP
GO:0071622Regulation of granulocyte chemotaxis71.0E-65.66CXCL5, CXCL8, PPBP
GO:0030104Water homeostasis130.0E-64.00FLG, FLG2, HRNR
GO:0002690Positive regulation of leukocyte chemotaxis120.0E-63.30CXCL5, CXCL8, PPBP
GO:0004867Serine-type endopeptidase inhibitor activity79.0E-63.00SERPINB1, SERPINB12, SPINK7
Table 5
Top 20 significantly enriched pathways as determined by Gene Ontology (GO) enrichment analysis in dithranol-treated lesional skin at end of treatment compared to baseline from 15 patients with psoriasis.

(GO was done using Cytoscape software; Bindea et al., 2009; Shannon et al., 2003 a.o. = among others).

GOIDGO termP-Value% Associated GenesAssociated genes found
GO:0006954Inflammatory response17.0E-186.93IL17A, IL1B, IL20, IL22, IL36A, IL36G, IL36RN, a.o.
GO:0006952Defense response52.0E-184.60CXCL8, CXCL9, DEFB103A, DEFB4A, IFNG, IL17A, IL1B, IL20, IL22, IL36A, IL36G, IL36RN, IL4R, IRAK2, IRF1, KRT16, a.o.
GO:0051707Response to other organism56.0E-186.02CCL2, CCL20, CCL22, CD24, CD80, COTL1, CXCL13, CXCL16, CXCL8, CXCL9, DDX21, DEFB103A, DEFB4A, a.o.
GO:0050663Cytokine secretion480.0E-1813.78IFNG, IL17A, IL1B, IL26, IL36RN, IL4R, LYN, MMP12, NOS2, PAEP, PANX1, PNP, S100A12, S100A8, S100A9, a.o.
GO:0001816Cytokine production680.0E-186.76IDO1, IFNG, IL17A, IL1B, IL26, IL36A, IL36RN, IL4R, IRF1, LTF, LYN, MB21D1, MMP12, NOS2, a.o.
GO:0012501Programmed cell death1.0E-154.05CASP5, CASP7, CCL2, CCL21, CD24, CD274, CD38, IFNG, IL17A, IL1B, IRF1, IVL, KLK13, KRT16, KRT17, KRT31, KRT6A, KRT6C, KRT73, KRT74, KRT77, a.o.
GO:0051240Positive regulation of multicellular organismal process1.5E-154.57CXCL17, CXCL8, FBN2, FERMT1, GBP5, GPR68, HPSE, HRH2, IDO1, IFNG, IL17A, IL1B, IL20, IL26, IL36A, S100A8, S100A9, SERPINB3, SERPINB7, a.o.
GO:0001817Regulation of cytokine production2.0E-157.04CCL2, CCL20, CD24, CD274, CD80, CD83, CXCL17, IFNG, IL17A, IL1B, IL26, IL36A, IL36RN, IL4R, IRF1, TNFRSF9, WNT5A, a.o.
GO:0002237Response to molecule of bacterial origin15.0E-159.22S100A8, S100A9, SELE, SOD2, TIMP4, TNFRSF9, TNIP3, WNT5A, ZC3H12A, a.o.
GO:0070268Cornification100.0E-1517.70DSC2, DSG3, IVL, KLK13, KRT16, KRT17, KRT31, KRT6A, KRT6C, KRT73, KRT74, KRT77, PI3, SPRR2A, SPRR2B, SPRR2D, a.o.
GO:0043588Skin development190.0E-158.17IL20, IVL, KLK13, KRT16, KRT17, KRT31, KRT6A, KRT6C, KRT73, KRT74, KRT77, LCE3A, LCE3C, LCE3E, a.o.
GO:0008544Epidermis development220.0E-157.63DSC2, DSG3, EPHA2, FERMT1, FOXE1, FURIN, HPSE, IL20, IVL, KLK13, KRT16, KRT17, KRT31, KRT6A, KRT6C, KRT73, KRT74, KRT77, a.o.
GO:0009617Response to bacterium240.0E-156.63CCL2, CCL20, CD24, CD80, CXCL13, CXCL16, CXCL8, CXCL9, DEFB103A, DEFB4A, S100A12, S100A8, S100A9, TREM1, a.o.
GO:0001819Positive regulation of cytokine production1.4E-127.89CCL2, CCL20, CD274, CD80, CD83, CXCL17, FERMT1, GBP5, HPSE, IDO1, IFNG, IL17A, IL1B, IL26, IL36A, a.o.
GO:0050707Regulation of cytokine secretion2.4E-1213.10AIM2, CASP5, CD274, FERMT1, GBP1, IFNG, IL17A, IL1B, IL26, IL36RN, IL4R, LYN, MMP12, PAEP, PANX1, a.o.
GO:0005125Cytokine activity4.4E-1210.64CCL20, CCL21, CCL22, CCL4L2, CXCL13, CXCL16, CXCL8, CXCL9, FAM3D, IFNG, IL17A, IL1B, IL20, IL22, IL26, a.o.
GO:0031424Keratinization21.0E-1210.48IVL, KLK13, KRT16, KRT17, KRT31, KRT6A, KRT6C, KRT73, KRT74, KRT77, LCE3A, LCE3C, LCE3E, PI3, SPRR2A, SPRR2B, a.o.
GO:0002790Peptide secretion97.0E-126.25CD274, CD38, DOC2B, FAM3D, FERMT1, GBP1, GBP5, GLUL, GPR68, IFNG, IL17A, IL1B, IL26, IL36RN, IL4R, LYN, MMP12, NOS2, TREM1, WNT5A, a.o.
GO:0032940Secretion by cell140.0E-124.04AIM2, AMPD3, CASP5, IL36RN, IL4R, LCN2, LRG1, LTF, LYN, MMP12, NOS2, NR1D1, NR4A3, OLR1, PAEP, PANX1, PLA2G3, a.o.
GO:0009913Epidermal cell differentiation190.0E-127.91DSC2, DSG3, EPHA2, FURIN, IL20, IVL, KLK13, KRT16, KRT17, KRT31, KRT6A, KRT6C, KRT73, LCE3E, PI3, SLITRK6, SPRR2A, SPRR2B, SPRR2D, SPRR2E, a.o.
Table 6
Top 45 differentially regulated genes in dithranol-treated psoriatic skin of c-Jun/JunB knockout mice compared to that of vehicle-treated controls (p<0.05, fold change (FC) >1.5).
Probe set IDGene symbolGene titleFold change
TC0Y00000006.mm.2Eif2s3yEukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked10,37
TC0Y00000233.mm.2UtyUbiquitously transcribed tetratricopeptide repeat gene, Y chromosome6,38
TC0Y00000235.mm.2Ddx3yDEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked5,40
TC0900000047.mm.2Mmp3Matrix metallopeptidase 33,64
TC0Y00000079.mm.2Ssty2Spermiogenesis specific transcript on the Y 23,31
TC0100001632.mm.2Ifi209Interferon activated gene 2093,23
TC0500002755.mm.2Cxcl9Chemokine (C-X-C motif) ligand 92,85
TC0300003133.mm.2Ifi44Interferon-induced protein 442,69
TC0100003591.mm.2Ifi213Interferon activated gene 2132,53
TC0100001634.mm.2Ifi208Interferon activated gene 2082,49
TC1900000217.mm.2Ms4a4cMembrane-spanning 4-domains, subfamily A, member 4C2,45
TC0300002684.mm.2Chil3Chitinase-like 32,39
TC0100003550.mm.2Slamf7SLAM family member 72,33
TC0500000922.mm.2Cxcl13Chemokine (C-X-C motif) ligand 132,26
TC1900000500.mm.2Ifit2Interferon-induced protein with tetratricopeptide repeats 22,20
TC0500002840.mm.2Plac8Placenta-specific 82,20
TC0700001630.mm.2AdmAdrenomedullin2,15
TC1900000501.mm.2Ifit3Interferon-induced protein with tetratricopeptide repeats 32,13
TC1800000610.mm.2Iigp1Interferon inducible GTPase 12,07
TC0300001446.mm.2Gbp3Guanylate binding protein 32,07
TC0300003134.mm.2Ifi44lInterferon-induced protein 44 like2,05
TC1900000502.mm.2Ifit3bInterferon-induced protein with tetratricopeptide repeats 3B2,02
TC0400003296.mm.2Skint5Selection and upkeep of intraepithelial T cells 5−2,37
TC0300000482.mm.2AadacArylacetamide deacetylase−2,37
TC1100000469.mm.2Fndc9Fibronectin type III domain containing 9−2,39
TC0300002399.mm.2Lce6aLate cornified envelope 6A−2,42
TC0300002409.mm.2Lce1iLate cornified envelope 1I−2,44
TC0300002412.mm.2KprpKeratinocyte expressed, proline-rich−2,44
TC0300000846.mm.2HrnrHornerin−2,48
TC0300002407.mm.2Lce1gLate cornified envelope 1G−2,49
TC1600000324.mm.2FetubFetuin beta−2,53
TC1300001377.mm.2Akr1c18Aldo-keto reductase family 1, member C18−2,56
TC0300002406.mm.2Lce1fLate cornified envelope 1F−2,57
TC1500001714.mm.2Slurp1Secreted Ly6/Plaur domain containing 1−2,57
TC1500002344.mm.2Gsdmc2Gasdermin C2−2,59
TC1000000145.mm.2Il20raInterleukin 20 receptor, alpha−2,66
TC0700000443.mm.2Cyp2b19Cytochrome P450, family 2, subfamily b, polypeptide 19−2,70
TC0100000103.mm.2Ly96Lymphocyte antigen 96−2,89
TC0700000778.mm.2Klk5Kallikrein related-peptidase 5−3,15
TC0300003252.mm.2Clca3a2Chloride channel accessory 3A2−3,20
TC0900002312.mm.2Elmod1ELMO/CED-12 domain containing 1−3,20
TC0300002401.mm.2Lce1a1Late cornified envelope 1A1−3,26
TC0700000484.mm.2FcgbpFc fragment of IgG binding protein−3,83
TC0300002405.mm.2Lce1eLate cornified envelope 1E−4,16
TC1500002274.mm.2Krt2Keratin 2−5,95
Table 7
Top 20 significantly enriched pathways as determined by Gene Ontology (GO) enrichment analysis in dithranol-treated psoriatic skin of c-Jun/JunB knockout mice compared to that of vehicle-treated controls.

(GO was done using Cytoscape software Bindea et al., 2009; Shannon et al., 2003; a.o. = among others).

Go idGO termP-Value% Associated GenesAssociated genes found
GO:0043588Skin development14,0E-1811,11Casp14, Cldn1, Flg2, Gjb3, Hrnr, Ivl, Krt2, Lce1a1, Lce1a2, Lce1l, Lce1m, Lor, Pou2f3, Ptgs1, Scel, Tfap2b, a.o.
GO:0008544Epidermis development73,0E-159,18Acer1, Alox8, Casp14, Cnfn, Cst6, Dnase1l2, Hrnr, Ivl, Krt2, Lce1c, Lce1d, Lce1e, Lce1f, Lce1g, Lce1h, Lce1i, Lce1j, a.o.
GO:0071345Cellular response to cytokine stimulus1,7E-126,26Ccdc3, Ccl2, Ccl5, Ccr9, Cxcl13, Cxcl9, Edn1, Il18, Il1f5, Il1f8, Il1rl1, Il20ra, Stat2, a.o.
GO:0034097Response to cytokine7,1E-125,62Cd38, Chad, Cldn1, Csf3, Edn1, Gbp2, Gbp3, Gbp4, Gbp7, Gbp8, Gbp9, Gm4951, Ifi203, Ifi204, Ifi209, Ifit1, Ifit2, Ifit3, Ifit3b, Xaf1, a.o.
GO:0035456Response to interferon-beta12,0E-1225,00Gbp2, Gbp3, Gbp4, Gm4951, Ifi203, Ifi204, Ifi209, Ifit1, Ifit3, Iigp1, Irgm1, Xaf1
GO:0006952Defense response63,0E-124,11Ccl2, Ccl5, Cd180, Cd59a, Cxcl13, Cxcl9, Cybb, Defb6, Drd1, Herc6, Hp, Il18, Il1f5, Il1f8, Il1rl1, Irgm1, Kalrn, Klk5, a.o.
GO:0030855Epithelial cell differentiation430,0E-125,51Casp14, Cdkn1a, Cldn1, Cnfn, Dlx3, Dnase1l2, Gsdmc2, Gstk1, Hrnr, Ivl, Klf15, Krt2, Lce1a1, Lor, Pou2f3, a.o.
GO:0020005Symbiont-containing vacuole membrane1,4E-966,67Gbp2, Gbp3, Gbp4, Gbp7, Gbp9, Iigp1
GO:0044216Other organism cell5,6E-930,77C4b, Gbp2, Gbp3, Gbp4, Gbp7, Gbp9, Iigp1, Tap1
GO:0044406Adhesion of symbiont to host120,0E-937,50Ace2, Gbp2, Gbp3, Gbp4, Gbp7, Gbp9
GO:0045087Innate immune response390,0E-94,35Ccl2, Ccl5, Cd180, Cfb, Cldn1, Ifit3, Iigp1, Il1f5, Il1f8, Irgm1, Lbp, Ly96, Mx1, Parp9, Sla, Slamf6, Slamf7, Stat2, Tlr7, Trim62, a.o.
GO:0034341Response to interferon-gamma1,0E-610,58Ccl2, Ccl5, Cldn1, Edn1, Gbp2, Gbp3, Gbp4, Gbp7, Gbp8, Gbp9, Parp9
GO:0006954Inflammatory response5,9E-64,29C3, C4b, Ccl2, Ccl5, Cd180, Cd59a, Chil3, Crip2, Ctla2a, Cxcl13, Cxcl9, Cybb, Hp, Il18, Il1f5, Il1f8, Il1rl1, Lbp, Ly96, a.o.
GO:0042832Defense response to protozoan9,4E-619,35Gbp2, Gbp3, Gbp4, Gbp7, Gbp9, Iigp1
GO:0035457Cellular response to interferon-alpha21,0E-636,36Ifit1, Ifit2, Ifit3, Ifit3b
GO:0030414Peptidase inhibitor activity22,0E-66,19C3, C4b, Cst6, Ctla2b, Fetub, R3hdml, Serpinb12, Serpinb13, Serpinb2, Serpinb7, Spink14, Tfap2b, Wfdc12, Wfdc5
GO:0098542Defense response to other organism34,0E-64,05Adm, Ccl5, Cxcl13, Cxcl9, Defb6, Gbp2, Gbp3, Hp, Ifit1, Ifit2, Ifit3, Ifit3b, Iigp1, Il1f5, Klk5, Lbp, Mx1, Oas1f, Plac8, Stat2, Tlr7, Wfdc12, a.o.
GO:0031424Keratinization43,0E-615,00Casp14, Cnfn, Hrnr, Ivl, Krt2, Lor
GO:0044403Symbiosis, encompassing mutualism through parasitism55,0E-64,11Ace2, Acta2, Atg16l2, Ccl5, Cxcl9, Gbp2, Gbp3, Gbp4, Gbp7, Gbp9, Ifit1, Ifit2, Ifit3, Ifit3b, Lbp, Mx1, Oas1f, Pou2f3, Rab9, Stat2, Tap1, Tlr7, Trim62
Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional information
Antibodyanti-CD1a; mouse monoclonalImmunotech, Beckman CoulterClone: O10
RRID:AB_10547704
(undiluted)
Antibodyanti-CD3; mouse monoclonalNovocastra, Leica BiosystemsClone: PS1
RRID:AB_2847892
(1:100)
Antibodyanti-CD4; mouse monoclonalNovocastra, Leica BiosystemsClone: 1F6
RRID:AB_563559
(1:30)
Antibodyanti-CD8; mouse monoclonalDako, AgilentClone: C8/144b
RRID:AB_2075537
(1:25)
Antibodyanti-CK16; rabbit monoclonalAbcamClone: EPR13504
RRID:AB_2847885
(1:1000)
Antibodyanti-FoxP3; mouse monoclonalBio-RadClone: 236A/E7
RRID:AB_2262813
(1:100)
Antibodyanti-Ki-67; mouse monoclonalDako, AgilentClone: MIB-1
RRID:AB_2631211
(1:50)
Antibodyanti-MPO; mouse monoclonalDako, AgilentClone: MPO-7
RRID:AB_578599
(1:100)
Strain, strain background
Mus musculus
BALB/c, wild-typeCharles River LaboratoriesRRID:IMSR_CRL:028
Charles River Strain code#: 028
Strain, strain background
Mus musculus
JunBf/f c-Junf/f K5-Cre-ERTPMID:16163348Obtained from the laboratory of Maria Sibilia (Medical University of Vienna)
Commercial assay or kitmiRNeasy Mini KitQiagenCat #: 217004
Commercial assay or kitiScript Reverse Transcription SupermixBio-RadCat #: 1708841
Commercial assay or kitGoTag qPCR Master MixPromegaCat #: A6001
Commercial assay or kitHuman GeneChip2.0 ST arraysAffymetrix, ThermoFisher ScientificCat #:902113
Commercial assay or kitmouse Clariom S AssayAffymetrix, ThermoFisher ScientificCat #:902919
Commercial assay or kitGeneChip WT PLUS Reagent KitThermoFisher ScientificCat #: 902280
Commercial assay or kitGeneChip WT Terminal Labeling KitThermoFisher
Scientific
Cat #: 900671
Commercial assay or kitGeneChip Hybridization, Wash and Stain KitThermoFisher
Scientific
Cat #: 900720
Commercial assay or kitnCounter GX Custom codesetNanoString TechnologiesCustom codeset (80 target genes, four reference genes
Chemical compound, drugAldara (Imiquimod)5% creamMEDA PharmaceuticalsCat #: 111981
Chemical compound, drugTamoxifenSigma-AldrichCat #: T5648
Chemical compound, drugDithranol (1,8-Dihydroxy-9(10H)-anthracenone)Gatt-Koller GmbH PharmaceuticalsCat #: 8069994Dissolved in vaseline and provided by the pharmacy of the Medical University of Graz, Austria
Software, algorithmGraphPad Prism version 8GraphPadRRID:SCR_002798
https://www.graphpad.com/scientific-software/prism/
Software, algorithmInteracti VennPMID:25994840http://www.interactivenn.net/
Software, algorithmCytoscapePMID:19237447RRID:SCR_003032
https://cytoscape.org/
Software, algorithmTranscriptome Analysis Console (TAC) 4.0.2ThermoFisher
Scientific
https://www.thermofisher.com/at/en/home/life-science/microarray-analysis/microarray-analysis-instruments-software-services/microarray-analysis-software/affymetrix-transcriptome-analysis-console-software.html
Software, algorithmPartek Genomics Suite version 6.6Partek IncRRID:SCR_011860
https://www.partek.com/partek-genomics-suite/
Software, algorithmR Version 3.5.1The R Project for Statistical
Computing
RRID:SCR_001905
https://www.r-project.org/
Software, algorithmnSolver 2.5 SoftwareNanoString Technologieshttps://www.nanostring.com/products/analysis-software/nsolver

Data availability

All microarray data has been deposited at the public repository Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSE145126 and GSE145127.

The following data sets were generated
    1. Benezeder T
    2. Painsi C
    3. Patra V
    4. Dey S
    5. Holcmann M
    6. Lange-Asschenfeldt B
    7. Sibilia M
    8. Wolf P
    (2020) NCBI Gene Expression Omnibus
    ID GSE145126. Microarray analysis of c-Jun/JunB knockout mice treated with dithranol.
    1. Benezeder T
    2. Painsi C
    3. Patra V
    4. Dey S
    5. Holcmann M
    6. Lange-Asschenfeldt B
    7. Sibilia M
    8. Wolf P
    (2020) NCBI Gene Expression Omnibus
    ID GSE145127. Microarray analysis of dithranol-treated psoriasis.

Additional files

Supplementary file 1

Top 45 upregulated genes in lesional psoriatic skin compared to non-lesional skin at baseline from 15 patients with psoriasis (p<0.05, fold change >1.5).

https://cdn.elifesciences.org/articles/56991/elife-56991-supp1-v1.docx
Supplementary file 2

Verification of microarray results of dithranol-treated skin samples of psoriasis patients with Nanostring analysis (nCounter GX Custom codeset) of 80 target genes and four reference genes.

FC, fold change

https://cdn.elifesciences.org/articles/56991/elife-56991-supp2-v1.docx
Supplementary file 3

Top 20 significantly enriched pathways as determined by Gene Ontology (GO) enrichment analysis in histological responders compared to non-responders in the psoriasis patient cohort (GO was done using Cytoscape software; [Bindea et al., 2009; Shannon et al., 2003] a.o. = among others).

https://cdn.elifesciences.org/articles/56991/elife-56991-supp3-v1.docx
Supplementary file 4

qPCR Primer sequences and corresponding annealing temperatures.

https://cdn.elifesciences.org/articles/56991/elife-56991-supp4-v1.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/56991/elife-56991-transrepform-v1.pdf

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