Fig 1.
TNF-α and IL-17A drive psoriasis transcript expression in ex vivo skin organ cultures.
To validate the use of cytokine-stimulated normal human skin biopsies as a model of skin inflammation, we treated biopsies with a combination of 10 ng/ml TNF-α and 20 ng/ml IL-17A for 12, 24, 48, and 72 h and monitored expression of DEFB4, PI3, LCN2, S100A7, and IL36G transcripts by qRT-PCR. Values are mean ± SD of results in triplicate from 3 donors. Statistical significance indicated by *p<0.05, **p<0.01, as compared with untreated cultures at each time point.
Fig 2.
Rhodomyrtone decreases expression of inflammatory transcripts by TNF-α+IL-17A stimulated in skin organ cultures.
Skin biopsies were pre-treated with 400 ng/ml rhodomyrtone 6 h before stimulation with 10 ng/ml TNF-α and 20 ng/ml IL-17A. Rhodomyrtone treatment decreases production of DEFB4, IL1B, IL17C, IL36G, LCN2, PI3, S100A7, and S100A8 transcripts as measured by qRT-PCR at 72 h. Statistical significance determined by one-way ANOVA and Dunn’s multiple comparison test, indicated as *p<0.05, **p<0.01, ****p<0.0001 versus TNF-α+IL-17A treatment.
Fig 3.
Rhodomyrtone inhibits TNF-α+IL-17A-induced inflammatory responses in skin organ cultures.
Cultures were pre-treated with 400 ng/ml rhodomyrtone for 6 h before stimulation with 10 ng/ml TNF-α and 20 ng/ml IL-17A for 72 h. Immunohistochemical staining for HBD-2, IL-36γ, and S100A7 revealed prominent induction of all three proteins by TNF+IL17A treatment (a). ELISA of conditioned media at 72 h confirmed the induction of HBD-2, IL-36γ, and S100A7 by IL17A+TNF (b). Staining from 1 representative donor of 5 shown, staining from 4 others shown in S2 Fig. Bars indicate mean ± SD, n = 5. Statistical significance determined by one-way ANOVA and Dunn’s multiple comparison test, indicated as*p<0.05 versus TNF-α and IL-17A treatment alone. Scale bar, 100 μm.
Fig 4.
Rhodomyrtone preferentially down-regulates transcripts involved in epidermal responses, myeloid leukocyte chemotaxis and inflammatory responses.
Global transcriptome sequencing (RNA-seq) was performed on NHK treated with IL-17A+TNF for 24 h with or without 1 h pre-treatment with 400 ng/ml rhodomyrtone. Rhodomyrtone inhibited the expression of 579 of 1066 IL-17A/TNF-induced genes by NHK (2-fold change, TREAT p<0.01 [16]) and increased the expression of 145/521 IL-17A/TNF-suppressed genes (a). Gene ontology (GO) [19] analysis of these DEGs revealed suppression of epidermal responses, myeloid leukocyte chemotaxis and inflammatory response pathways (e). KEGG pathway analysis of the DEGs revealed that while the cytokine vs control (c) and cytokine+rhodomyrone vs control (d) comparisons shared many of the same up-regulated gene functions, there was an overall reduction in DEG expression levels and/or number of DEGs per KEGG function (e.g. CXCL1) in the IL-17 pathway when cytokine exposed cells were pretreated with rhodomyrtone. These shifts in gene expression were highlighted when cytokine+rhodomyrtone transcripts were compared against cytokine-only transcripts (e). Node color and font size is the sum of the log2 fold-change of either the up-regulated or down-regulated DEGs. The line color and thickness is the sum of averaged DEG read counts across the 3 samples of either the numerator condition (up-regulated) or denominator condition (down-regulated). Gene set enrichment analysis (GSEA) [17] showed a decrease in the expression of IL-17/TNF induced transcripts area under curve = 0.46 (f) vs. 0.30 (h) and a decrease in genes overlapping with a plaque psoriasis gene signature, area under curve = 0.21 (g) vs. 0.07 (i).
Fig 5.
Rhodomyrtone dose dependently decreases production of CXCL1, IL-36G, IL-36RN, MMP-13, S100A7, S100A8, and S100A12 transcripts by IL-17A/TNF-α-stimulated primary human keratinocytes.
Keratinocytes were pretreated with rhodomyrtone (50, 100, 200, 400, and 600 ng/ml) before stimulation with 20ng/ml IL-17A+10ng/ml TNF-α. The mRNA expression levels were measured at 24 h by qRT-PCR. Values are mean±SD of results from three independent experiments in triplicate. Statistical significance determined using one-way ANOVA and Dunnett’s multiple comparison test and noted ****p<0.0001, as compared with IL-17A/TNF-α treatment.
Fig 6.
Rhodomyrtone inhibits IL-17A/TNF-α-induced inflammatory responses in keratinocytes.
Primary human keratinocytes were pretreated with rhodomyrtone (50, 100, 200, 400, and 600 ng/ml) for 1 h before stimulation with 20 ng/ml IL-17A+10 ng/ml TNF-α. HBD-2 (a), and CXCL-1 (b), were assayed in conditioned media at 24 h by ELISA and found to be reduced in a dose-dependent manner in the presence of rhodomyrtone. Values are mean ± SD of results from three independent experiments in triplicate. Statistical significance determined using one-way ANOVA and Dunnett’s multiple comparison test and noted ****p<0.0001, as compared with TNF-α treatment alone.
Fig 7.
Rhodomyrtone suppressed the TNF-α induced phosphorylation of MAPKs and NF-κBp65 in primary human keratinocytes.
Keratinocytes were pre-incubated with 600 ng/ ml rhodomyrtone for 24 h and stimulated with TNF-α for 0, 10, 20, 30, and 60 min. The ratio of p-ERK/ERK (a), p-p38/p38 (b), p-JNK/JNK (c), and p-p65/p65 (d) was analyzed by western blot analysis, and protein expression was quantitated using Image Studio Lite v5.2.Ink software and normalized to β-actin. Values are mean ± SD of results from three independent experiments in triplicate. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, as compared with TNF-α at each time point).
Fig 8.
Rhodomyrtone attenuates imiquimod (IMQ)-induced skin inflammation in mice.
Mice were treated for 6 consecutive days with IMQ on the shaved back. Betamethasone (0.0155 mg/cm2), rhodomyrtone (0.181 mg/cm2), rhodomyrtone (0.364 mg/cm2), IMQ, and vehicle were topically administered for 15 days. A skin lesion biopsy was removed and fixed in 10% (v/v) paraformaldehyde for 3 (■), 6 (), and 9 (□) days. Representative H&E-stained sections of skin after 15 days of treatment (a) and epidermal thickness of the back skin was measured in 10 subsequent areas in μm and the mean was calculated (b). n = 9 each group, ***p<0.001, as a significant compared with vehicle-only.