Resveratrol Ameliorates Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice

<div><p>Background</p><p>The polyphenol resveratrol has anti-inflammatory effects in various cells, tissues, animals and human settings of low-grade inflammation. Psoriasis is a disease of both localized and systemic low-grade inflammation. The Sirtuin1 enzyme thought to mediate the effects of resveratrol is present in skin and resveratrol is known to down regulate NF-κB; an important contributor in the development of psoriasis. Consequently we investigated whether resveratrol has an effect on an Imiquimod induced psoriasis-like skin inflammation in mice and sought to identify candidate genes, pathways and interleukins mediating the effects.</p><p>Methods</p><p>The study consisted of three treatment groups: A control group, an Imiquimod group and an Imiquimod+resveratrol group. Psoriasis severity was assessed using elements of the Psoriasis Area Severity Index, skin thickness measurements, and histological examination. We performed an RNA microarray from lesional skin and afterwards Ingenuity pathway analysis to identify affected signalling pathways. Our microarray was compared to a previously deposited microarray to determine if gene changes were psoriasis-like, and to a human microarray to determine if findings could be relevant in a human setting.</p><p>Results</p><p>Imiquimod treatment induced a psoriasis-like skin inflammation. Resveratrol significantly diminished the severity of the psoriasis-like skin inflammation. The RNA microarray revealed a psoriasis-like gene expression-profile in the Imiquimod treated group, and highlighted several resveratrol dependent changes in relevant genes, such as increased expression of genes associated with retinoic acid stimulation and reduced expression of genes involved in IL-17 dependent pathways. Quantitative PCR confirmed a resveratrol dependent decrease in mRNA levels of IL-17A and IL-19; both central in developing psoriasis.</p><p>Conclusions</p><p>Resveratrol ameliorates psoriasis, and changes expression of retinoic acid stimulated genes, IL-17 signalling pathways, IL-17A and IL-19 mRNA levels in a beneficial manner, which suggests resveratrol, might have a role in the treatment of psoriasis and should be explored further in a human setting.</p></div

Epidermal thickness measured in skin sections, presentation of the mouse phenotype and HE sectioned skin.

<p>(a) Epidermal thickness; Means of epidermal thickness was calculated based on 15–20 random site measurements. (b-d) Presentation of phenotype of mice from control, IMQ and IMQ-RSV groups, respectively. Photograph is taken after 5 days of treatment. (e-g) HE-stained skin sections from the backs of the mice. Sections were used for evaluation of epidermal thickness. In the lower right corner of photos the white box = 100μm. Columns in a) are group means ±SEM (n = 7, n = 5, n = 5 for controls, IMQ, IMQ-RSV respectively). Clamped bar with * above indicates the pair of column means are significantly different (p<0.05). (Symbols: Striped fill = control, black fill = IMQ, grey fill = IMQ-RSV).</p

Significantly RSV changed pathway.

<p>List of RSV dependent pathway changes reaching statistical significance. Probe sets with fold change> 1.5 were analysed and a p-value < 0.05 was considered statistically significant in the analysis. The list is ranked by p-value from lowest to highest. Pathway analysis was performed using Ingenuity Pathway Analysis software.</p><p>Significantly RSV changed pathway.</p

Calliper measurement of skin thickness.

<p>The right ear fold and the skinfold on the backs of the mice were measured to quantify the thickening of the skin caused by Imiquimod treatment. (a) Skinfold thickness on the backs of the mice. (b) Right ear fold thickness. Columns represent group means ±SEM of skin/ear fold measurements day 7 ((n = 8, n = 10, n = 10 for controls, IMQ, IMQ-RSV respectively). Clamped bar with * above indicates the pair of column means are significantly different (p<0.05).</p

RSV effects on IL-17A, IL-19 and IL-23p19 gene expression.

<p>Quantitative PCR of IL-17A, IL-19 and IL-23p19 gene expression was determined to quantify effects of RSV on IL-17A, IL-19 and IL-23p19 gene expression. The mRNA levels of IL-17A, IL-19 and IL-23p19 were quantified using MYO18B as reference gene. Clamped bar with * above indicates the pair of column means are significantly different (p<0.05). Striped fill = control, black fill = IMQ, grey fill = IMQ-RSV.</p

Erythema and scales score of the skin on the backs BALB/c mice.

<p>Scoring was performed on days 0, 2, 4 and 7 using the erythema and scales elements of the Psoriasis Area Severity Index (PASI) to assign a score of 0–4 to each animal and thereby assess the effects of daily treatment with Imiquimod cream and vehicle cream. (a) Erythema score: Data points are presented as group means ±SEM (n = 8, n = 10, n = 10 for controls, IMQ and IMQ-RSV respectively) (X = control group, O = IMQ-RSV group, □ = IMQ group). (b) Scales score: Data points are presented as group means ±SEM (n = 8, n = 10, n = 10 for controls, IMQ, IMQ-RSV respectively) (X = control group, O = IMQ-RSV group, □ = IMQ group).</p

Quantitative PCR of microarray genes.

<p>Selected qPCR of genes that were 1.5 fold or more changed by RSV treatment in the microarray (RSV treated compared with the IMQ group). The mRNA levels were quantified using MYO18B as reference gene. (a) Phosphoenolpyruvate Carboxykinase 1 (PCK1). (b) Tripartite Motif Containing 63, E3 Ubiquitin Protein Ligase (TRIM63). (c) Protein Phosphatase 1, Regulatory (inhibitor) Subunit 3C (PPP1R3C). Columns in (a-c) are group means ±SEM (n = 8, n = 10, n = 10 for controls, IMQ, IMQ-RSV respectively). Clamped bar with * above indicates the pair of column means are significantly different (p<0.05).(Symbols: Striped fill = control, black fill = IMQ, grey fill = IMQ-RSV).</p

Keratin23 (KRT23) Knockdown Decreases Proliferation and Affects the DNA Damage Response of Colon Cancer Cells

<div><p>Keratin 23 (KRT23) is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression <i>in vitro.</i> Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed that KRT23 depletion affected molecules of the cell cycle and DNA replication, recombination and repair. <i>In vitro</i> analyses confirmed that KRT23 depletion significantly decreased the cellular proliferation of SW948 and LS1034 cells and markedly decreased the expression of genes involved in DNA damage response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced proliferation of the KRT23 depleted cells compared to irradiated control cells.</p></div

Knockdown of KRT23.

<p><b>A</b>) Western Blot of freshly made SW948-ctrl and SW948-sh1506 cells extracts with 20 µg extract per lane using a monospecific anti-K23 antibody in a 1∶150 dilution, Marker Biorad All Blue. Stable knockdown of K23 in the SW948-sh1506 cells resulted in >80% reduced K23 protein expression compared to the SW948-ctrl cells. Beta-actin was used as loading control. B) Immunofluorescence analysis confirmed a decreased K23 expression in SW948-sh1506 cells; anti-K23 antibody 1∶500, detection with Alexa 488, nuclear stain Hoechst, magnification 630x C) Visual inspection indicated a lower cell density for SW948-sh1506 cells 48 h post seeding. D) SW948-sh1506 cells showed less nuclear expression of the proliferation marker KI67 (green); anti-KI67 1:100 E) SW948-ctrl and SW948-sh1506 cells were seeded on 96-well plates with 4000 cells per well (n = 12) and proliferation was analyzed post-seeding at five time-points. Proliferation of SW948-sh1506 cells was significantly (Fisher’s exact t-test, p<0.0001) decreased at 96 hours and 120 hours post-seeding. F) The MTT assay was repeated by seeding 16000 cells per well (n = 11) on a 96 well plate and proliferation/viability was analyzed at 48 h post-seeding and percentage viability was measured. The proliferation of KRT23 depleted cells was significantly (p = 2.6E-06) decreased by about 30%. G) SW948-ctrl and SW948-sh1506 cells were seeded on 96-well RTCA-plates with 8000 cells/well (n = 3). Values are shown as medians and standard deviations for each group at selected time points for a representative experiment.</p

Colon cancer cells depleted from KRT23 compared to control cells.

*<p>number of molecules differentially expressed to number of molecules in given pathway.</p