Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1

Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology

Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin

<p>Abstract</p> <p>Background</p> <p>Psoriasis is a chronic inflammatory skin disorder that shows as erythematous and scaly lesions. The pathogenesis of psoriasis is driven by a dysregulation of the immune system which leads to an altered cytokine production. Proinflammatory cytokines that are up-regulated in psoriasis include tumor necrosis factor alpha (TNFα), interleukin-12 (IL-12), and IL-23 for which monoclonal antibodies have already been approved for clinical use. We have previously documented the therapeutic applicability of targeting TNFα mRNA for RNA interference-mediated down-regulation by anti-TNFα small hairpin RNAs (shRNAs) delivered by lentiviral vectors to xenografted psoriatic skin. The present report aims at targeting mRNA encoding the shared p40 subunit (IL-12B) of IL-12 and IL-23 by cellular transduction with lentiviral vectors encoding anti-IL12B shRNAs.</p> <p>Methods</p> <p>Effective anti-IL12B shRNAs are identified among a panel of shRNAs by potency measurements in cultured cells. The efficiency and persistency of lentiviral gene delivery to xenografted human skin are investigated by bioluminescence analysis of skin treated with lentiviral vectors encoding the luciferase gene. shRNA-expressing lentiviral vectors are intradermally injected in xenografted psoriatic skin and the effects of the treatment evaluated by clinical psoriasis scoring, by measurements of epidermal thickness, and IL-12B mRNA levels.</p> <p>Results</p> <p>Potent and persistent transgene expression following a single intradermal injection of lentiviral vectors in xenografted human skin is reported. Stable IL-12B mRNA knockdown and reduced epidermal thickness are achieved three weeks after treatment of xenografted psoriatic skin with lentivirus-encoded anti-IL12B shRNAs. These findings mimick the results obtained with anti-TNFα shRNAs but, in contrast to anti-TNFα treatment, anti-IL12B shRNAs do not ameliorate the psoriatic phenotype as evaluated by semi-quantitative clinical scoring and by immunohistological examination.</p> <p>Conclusions</p> <p>Our studies consolidate the properties of lentiviral vectors as a tool for potent gene delivery and for evaluation of mRNA targets for anti-inflammatory therapy. However, in contrast to local anti-TNFα treatment, the therapeutic potential of targeting IL-12B at the RNA level in psoriasis is questioned.</p

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

Exploring valrubicin’s effect on Propionibacterium acnes-induced skin inflammation in vitro and in vivo

Acne is a common skin disease involving colonization with <em>Propionibacterium</em> <em>acnes</em> (<em>P</em>. <em>acnes</em>), hyperproliferation of the follicular epithelium and inflammatory events. Valrubicin is a second-generation anthracycline, non-toxic upon contact, and available in a topical formulation. Valrubicin’s predecessor doxorubicin possesses antibacterial effects and previously we demonstrated that valrubicin inhibits keratinocyte proliferation and skin inflammation suggesting beneficial topical treatment of acne with valrubicin. This study aims to investigate valrubicin’s possible use in acne treatment by testing valrubicin’s antibacterial effects against <em>P</em>. <em>acnes</em> and <em>P.</em> <em>acnes</em>-induced skin inflammation <em>in</em> <em>vitro</em> and <em>in</em> <em>vivo</em>. Valrubicin was demonstrated not to possess antibacterial effects against <em>P.</em> <em>acnes</em>. Additionally, valrubicin was demonstrated not to reduce mRNA and protein expression levels of the inflammatory markers interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α <em>in</em> <em>vitro</em> in human keratinocytes co-cultured with <em>P</em>. <em>acnes</em>. Moreover, <em>in</em> <em>vivo</em>, valrubicin, applied both topically and intra-dermally, was not able to reduce signs of inflammation in mouse ears intra-dermally injected with <em>P</em>. <em>acnes</em>. Taken together, this study does not support beneficial antibacterial and anti inflammatory effects of topical valrubicin treatment of acne

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

Exploring valrubicin’s effect on Propionibacterium acnes-induced skin inflammation in vitro and in vivo

Acne is a common skin disease involving colonization with Propionibacterium acnes (P. acnes), hyperproliferation of the follicular epithelium and inflammatory events. Valrubicin is a second-generation anthracycline, non-toxic upon contact, and available in a topical formulation. Valrubicin’s predecessor doxorubicin possesses antibacterial effects and previously we demonstrated that valrubicin inhibits keratinocyte proliferation and skin inflammation suggesting beneficial topical treatment of acne with valrubicin. This study aims to investigate valrubicin’s possible use in acne treatment by testing valrubicin’s antibacterial effects against P. acnes and P. acnes-induced skin inflammation in vitro and in vivo. Valrubicin was demonstrated not to possess antibacterial effects against P. acnes. Additionally, valrubicin was demonstrated not to reduce mRNA and protein expression levels of the inflammatory markers interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α in vitro in human keratinocytes co-cultured with P. acnes. Moreover, in vivo, valrubicin, applied both topically and intra-dermally, was not able to reduce signs of inflammation in mouse ears intra-dermally injected with P. acnes. Taken together, this study does not support beneficial antibacterial and anti inflammatory effects of topical valrubicin treatment of acne

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

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