Psoriasiform Dermatitis Model Research Articles

An integrated bioinformatics approach for unravelling the molecular insights into psoriasis pathology and therapeutics

Psoriasis is a chronic relapsing inflammatory disease of the skin that affects almost 2–3 % population worldwide. The current treatment strategies include palliative therapeutics targeting the inflammatory pathways that do not completely cure the lesioned skin. The molecular cues in the hyper-proliferative and aberrantly differentiated keratinocytes of the psoriatic lesioned skin remain unknown. Through an integrative in-silico approach, we have analyzed human psoriatic skin samples from 3 RNA-Seq and 3 microarray datasets to identify 340 differentially expressed genes (DEGs). Further, these DEGs were analyzed using gene ontology enrichment, KEGG pathways, and protein-protein interaction networks for their role in disease pathology and the identification of hub genes. The expression of the hub genes was validated in a preclinical murine model of psoriasiform dermatitis. Finally, the ten hub genes were assessed for their drugability, which revealed 74 drugs targeting 7 hub genes (CCNA2, TOP2A, BIRC5, RRM2, CDK1, AURKA, and CCNB1) that can be repurposed for psoriasis treatment. This study provides an understanding of psoriasis pathophysiology and suggests key molecular biomarkers as therapeutic targets for effective mitigation of the disease.

Mitochondrial DNA variants and microbiota: An experimental strategy to identify novel therapeutic potential in chronic inflammatory diseases

We previously demonstrated that mice carrying natural mtDNA variants of the FVB/NJ strain (m.7778 G>T in the mt-Atp8 gene in mitochondrial complex V), namely C57BL/6 J-mtFVB/NJ (B6-mtFVB), exhibited (i) partial protection from experimental skin inflammatory diseases in an anti-murine type VII collagen antibody-induced skin inflammation model and psoriasiform dermatitis model; (ii) significantly altered metabolites, including short-chain fatty acids, according to targeted metabolomics of liver, skin and lymph node samples; and (iii) a differential composition of the gut microbiota according to bacterial 16 S rRNA gene sequencing of stool samples compared to wild-type C57BL/6 J (B6) mice. To further dissect these disease-contributing factors, we induced an experimental antibody-induced skin inflammatory disease in gnotobiotic mice. We performed shotgun metagenomic sequencing of caecum contents and untargeted metabolomics of liver, CD4+ T cell, and caecum content samples from conventional B6-mtFVB and B6 mice. We identified D-glucosamine as a candidate mediator that ameliorated disease severity in experimental antibody-induced skin inflammation by modulating immune cell function in T cells, neutrophils and macrophages. Because mice carrying mtDNA variants of the FVB/NJ strain show differential disease susceptibility to a wide range of experimental diseases, including diet-induced atherosclerosis in low-density lipoprotein receptor knockout mice and collagen antibody-induced arthritis in DBA/1 J mice, this experimental approach is valuable for identifying novel therapeutic options for skin inflammatory conditions and other chronic inflammatory diseases to which mice carrying specific mtDNA variants show differential susceptibility.

Keratinocyte-to-macrophage communication exacerbate psoriasiform dermatitis via LRG1-enriched extracellular vesicles.

Rationale: Macrophage-associated inflammation and keratinocytes excessive proliferation and inflammatory cytokines secretion induced by stimulation play an important role in the progression of psoriasiform dermatitis. However, how these two types of cells communicate remains obscure. Methods: We induced a mouse model with experimental psoriasiform dermatitis by Imiquimod (IMQ). To investigate whether damaged keratinocytes promote macrophage polarization and accelerate skin lesions by releasing extracellular vesicle (EV), purified EV were isolated from the primary epidermis of 5-day IMQ-induced psoriasiform dermatitis model mice, and then fluorescence-labeled the EV with PKH67. The EV was injected into the skin of mice treated with IMQ or vehicle 2 days in situ. In addition, we established a co-culture system of the human monocytic cell line (THP-1) and HaCaT, and THP-1/HaCaT conditioned media culture model in vitro respectively. Subsequently, we evaluated the effect of Leucine-rich α-2-glycoprotein 1 (LRG1)-enriched EV on macrophage activation. Results: We demonstrated macrophages can significantly promote keratinocyte inflammation and macrophage polarization may be mediated by intercellular communication with keratinocytes. Interestingly, IMQ-induced 5-day, keratinocyte-derived EV recruited macrophage and enhanced the progression of skin lesions. Similar to results in vivo, EV released from M5-treated HaCaT significantly promotes Interleukin 1β (IL-1β) and Tumor necrosis factor α (TNF-α) expression of THP-1 cells. Importantly, we found that LRG1-enriched EV regulates macrophages via TGF beta Receptor 1 (TGFβR1) dependent process. Conclusion: Our findings indicated a novel mechanism for promoting psoriasiform dermatitis, which could be a potential therapeutic target.

Attenuated effects of topical vinpocetine in an imiquimod-induced mouse model of psoriasis

Psoriasis is an uncontrolled, long-lasting inflammatory dermatosis distinguished by thickened, erythematous, and flaky skin lesions. Massive amounts of inflammatory cytokines are produced when immune system imbalances are driven by genetic and environmental triggers. Vinpocetine (VNP), a man-made analogue of the compound vincamine found in the dwarf periwinkle herb, has robust anti-inflammatory, immunomodulatory, and anti-oxidative effects; alleviates the epidermal penetration of immune cells, such as eosinophils and neutrophils; and abolishes the generation of pro-inflammatory molecules. ObjectiveThis study was aimed at exploring the effects of long-term topical VNP, both alone and co-administered with clobetasol propionate, in an imiquimod-induced mouse model of psoriasiform dermatitis. MethodsThe study protocol consisted of 48 Swiss albino mice, randomly divided into six groups of eight mice each. In group I, petroleum jelly was administered daily for 8 days. In group II, imiquimod was administered topically at 62.5 mg daily for 8 days. In groups III, VI, V, and VI, 0.05% clobetasol propionate, 1% VNP, 3% VNP, and 3% VNP plus 0.05% clobetasol were administered topically for an additional 8 days after the induction, thus resulting in a total trial length of 16 days. ResultsTopical VNP at various doses alleviated the severity of imiquimod-induced psoriatic lesions—including erythema, silvery-white scaling, and thickening—and reversed the histopathological abnormalities. Moreover, imiquimod-exposed animals treated with VNP showed markedly diminished concentrations of inflammatory biomarkers, including tumour necrosis factor-α, interleukin (IL)-8, IL-17A, IL-23, IL-37, nuclear factor-kappa B (NF-κB), and transforming growth factor-β1. ConclusionThis research provides new evidence that VNP, alone and in combination with clobetasol, may serve as a potential adjuvant for long-term management of autoimmune and autoinflammatory skin diseases, particularly psoriasis, by attenuating psoriatic lesion severity, suppressing cytokine generation, and limiting NF-κB-mediated inflammation.

Importance of PLD2 in an IL-23 driven psoriasiform dermatitis model and potential link to human psoriasis.

Phospholipase D2 (PLD2), a major isoform of the PLD family, has been reported to regulate inflammatory responses. Thus far, the relevance of PLD2 in psoriasis, an inflammatory skin disease, has not been explored. In the current study, we examined PLD2 expression in the skin of psoriasis patients and the role of PLD2 in an interleukin (IL)-23-induced mouse model of psoriasiform dermatitis. Both in situ hybridization and bulk RNA sequencing showed PLD2 gene expression is significantly higher in lesional relative to non-lesional skin of psoriasis patients or the skin of healthy subjects. PLD2 expression is also enriched in residual lesions from patients on biologic therapies. Murine in vivo studies showed that PLD2 deficiency significantly reduced psoriasiform inflammation in IL-23-injected ears, as reflected by decreases in ear thickness, expression of defensin beta 4A and the S100 calcium binding protein A7A, macrophage infiltrate, and expression of CXCL10 and IL-6. However, the expression of type 17 cytokines, IL-17A and IL-17F, were not reduced. Dual knockout of PLD1 and PLD2 offered little additional protection compared to PLD2 knockout alone in the IL-23 model. In addition, pharmacological inhibition with a pan-PLD1/PLD2 inhibitor also suppressed IL-23-induced psoriasiform dermatitis. Bone-marrow-derived macrophages from wild type (WT) and PLD2 knockout (KO) mice exhibited little difference in viability and sensitivity to lipopolysaccharide and/or interferon gamma, or resiquimod (R848). PLD2 deficiency did not alter the differentiation and function of Th17 cells in an ex vivo study with splenocytes isolated from WT and PLD2 KO mice. Overall, these data suggest that PLD2 may play a role in the pathophysiology of psoriasis. Reducing macrophage infiltrate and cytokine/chemokine production might contribute to an anti-inflammatory effect observed in PLD2 knockout mice. Further studies are required to better understand the mechanisms by which PLD2 contributes to skin lesions in psoriasis patients and psoriasiform dermatitis models.

IL-17A exacerbates psoriasis in a STAT3 overexpressing mouse model.

Psoriasis is an autoimmune skin disease characterized by immunocyte activation, excessive proliferation, and abnormal differentiation of keratinocytes. Signal transducers and activators of transcription 3 (STAT3) play a crucial role in linking activated keratinocytes and immunocytes during psoriasis development. T helper (Th) 17 cells and secreted interleukin (IL)-17A contribute to its pathogenesis. IL-17A treated STAT3 overexpressing mouse model might serve as an animal model for psoriasis. In this study, we established a mouse model of psoriasiform dermatitis by intradermal IL-17A injection in STAT3 overexpressing mice. Transcriptome analyses were performed on the skin of wild type (WT), STAT3, and IL-17A treated STAT3 mice. Bioinformatics-based functional enrichment analysis was conducted to predict biological pathways. Meanwhile, the morphological and pathological features of skin lesions were observed, and the DEGs were verified by qPCR. IL-17A treated STAT3 mice skin lesions displayed the pathological features of hyperkeratosis and parakeratosis. The DEGs between IL-17A treated STAT3 mice and WT mice were highly consistent with those observed in psoriasis patients, including S100A8, S100A9, Sprr2, and LCE. Gene ontology (GO) analysis of the core DEGs revealed a robust immune response, chemotaxis, and cornified envelope, et al. The major KEGG enrichment pathways included IL-17 and Toll-like receptor signaling pathways. IL-17A exacerbates psoriasis dermatitis in a STAT3 overexpressing mouse.

A Human STAT3 Gain of Function Variant Drives Th17 Expansion and IL-22-dependent Skin Inflammation in a Model of Psoriasiform Dermatitis

Patients with germline gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3) present with early-onset polyautoimmunity, including psoriasis. STAT3 is a pleiotropic transcription factor that plays a role in the proliferation, survival, and function of many different cell types. In T cells, STAT3 is required for the polarization and function of Th17 cells and can inhibit development of peripheral Tregs. We hypothesized that STAT3 GOF variants enhance Th17 responses following an inflammatory stimulus, leading to autoimmune disease. To examine this, we generated a mouse model of STAT3 GOF using an amino acid substitution identified in patients (p.G421R). Aged STAT3 GOF mice develop spontaneous skin inflammation of the ears associated with increased neutrophil and T cell infiltration into the skin. The frequency of Th17 in the aged skin was also increased compared to age-matched WT littermates. In adult STAT3 GOF mice, Th17 polarization of naïve T cells was enhanced in vitro, indicating a potential for CD4 skewing toward Th17. To test if this would drive disease susceptibility in vivo, WT and STAT3 GOF mice were treated with topical imiquimod (IMQ). Following treatment, STAT3 GOF mice demonstrated increased ear swelling and an increased Th17 response in the skin and periphery. Adaptive lymphocytes mediated this response; however, CD8T cells and γδ T cells are not required. Analysis of T cell clonality by scRNA-seq revealed an increased frequency of expanded clones in untreated and IMQ-treated CD4 and CD8T cells of effector and exhausted subsets. Among the expanded clones, Il22 transcript was upregulated in STAT3 GOF IMQ-treated CD4s. IL-22 was then found to partially mediate the response to IMQ in STAT3 GOF. Using bone marrow chimeras, we identified a role for both radiosensitive and resistant cells in this inflammatory response. We also observed a competitive advantage for STAT3 GOF Th17 cells in the skin. Collectively, these data demonstrate that STAT3 GOF results in a cell-intrinsic skewing of T cells with a Th17 phenotype, suggesting a potential mechanism for the development of autoimmunity and a target for therapeutics in patients with STAT3 GOF syndrome.

1114 Study of long-term (15-day) imiquimod treatment and drug intervention in localized psoriasiform dermatitis model

1114 Study of long-term (15-day) imiquimod treatment and drug intervention in localized psoriasiform dermatitis model

486 Glibenclamide ameliorates skin inflammation in a TRPM4 gain-of-function murine model of imiquimod-mediated psoriasiform dermatitis

A heterozygous (het) gain-of-function (GoF) mutation (I1033M or I1040T) of TRPM4, a calcium-activated nonselective monovalent cation channel, has been reported as the cause of progressive symmetric erythrokeratodermia. This genetic disease is characterized by red, scaly plaques in periorificial and acral regions that are reminiscent of human psoriasis. To determine whether the TRPM4 mutation alone is sufficient to lead to skin disease, we created a mouse (TRPM4I1029M, genetically equivalent to the human I1033M mutation) using CRISPR-Cas9 methods. Unperturbed homo and het TRPM4I1029M mice showed no apparent skin changes compared to wild type (WT) littermates. We compared the response of TRPM4I1029M and WT littermates to topical imiquimod (IMQ) application, a model of psoriasiform dermatitis (PsD). Het TRPM4I1029M showed enhanced skin inflammation with greater clinical severity scores (200% vs WT), thicker epidermis, more infiltration of CCR6+ γδ low T cells, higher expression of p-STAT3 protein, and higher mRNA levels of Il17a (1.5-fold) compared to WT mice. In vivo dendritic cell (DC) migration assay showed enhanced DC migration in het TRPM4I1029M. Cell cycle analysis showed that keratinocytes (KCs) from het TRPM4I1029M were in proliferative cell cycle status compared to KCs from WT mice. However, TRPM4I1029M did not show exaggerated responses in other in vivo models such as croton oil-induced irritant dermatitis and DNFB-induced contact dermatitis models. Glibenclamide, a classic diabetes drug, is a known TRPM4 inhibitor and ameliorated IMQ-PsD both in TRPM4I1029M and WT mice with milder clinical scores (50% vs vehicle), thinner epidermis, less infiltration of CCR6+ γδ low T cells, and lower mRNA levels of Il17a (30% vs vehicle). Our results suggest a possible therapeutic application of TRPM4 inhibitors in psoriasis.

Bile Acids Improve Psoriasiform Dermatitis through Inhibition of IL-17A Expression and CCL20-CCR6–Mediated Trafficking of T Cells

Bile acids (BAs), produced in the liver and further transformed in the gut, are cholesterol-derived molecules involved in essential physiological processes. Recent studies suggest that BAs regulate T helper 17 cell function, but the underlying mechanism of this action and their therapeutic value in disease models remains unclear. Using an IL-23 minicircle DNA-based murine model of psoriasiform dermatitis, we showed that oral administration of secondary BAs, including lithocholic acid (LCA), deoxycholic acid, and 3-oxoLCA, significantly improved psoriasiform dermatitis without inducing apparent hepatotoxicity. Of the BAs tested, LCA possessed the greatest potency in treating psoriasiform dermatitis. Intravenous administration of LCA at a much lower dosage (compared with oral treatment) showed a comparable antipsoriatic effect and markedly suppressed the IL-17A response. Exvivo experiments revealed that LCA reduced IL-17A production in IL-23-stimulated murine T cells in the absence of BA receptors TGR5 or FXR. Strikingly, BAs inhibited CCL20 expression in keratinocytes, which led to reduced migration of CCR6-expressing Jurkat cells cultured in the conditioned medium of stimulated keratinocytes. Thus, BAs improve psoriasiform dermatitis with minimal toxicity via direct inhibition of IL-17A production and blockade of CCL20-mediated trafficking, supporting the potential use of BAs in psoriasis.

Capsaicin attenuates imiquimod-induced epidermal hyperplasia and cutaneous inflammation in a murine model of psoriasis

Psoriasis is one of the most common chronic inflammatory diseases that is characterized by well-defined erythematous plaques, with typical histopathological findings of lymphocytic infiltration and epidermal hyperplasia. Topical treatments of psoriasis are either associated with limited response or with side effects. Up to date, topicals targeting neuroimmune axis in psoriasis or psoriasiform dermatitis have not been explored. Here, we investigated whether percutaneous delivery of capsaicin could attenuate the pathological change of psoriasiform inflammation. Imiquimod-induced psoriasis-like murine model was used to evaluate therapeutic effects from topical application of capsaicin. An additional model of psoriasiform dermatitis induced by direct IL-23 injection was used to identify the level of action from capsaicin in this neuroimmune axis. Cutaneous inflammation was assessed by erythema level and ear thickness change. Key cytokines, infiltrating cells in the skin, and draining lymph node cells were investigated. The results showed that capsaicin administration obstructed the activation of IL-23/IL-17 pathway induced by imiquimod, presenting with significantly reduced psoriasiform dermatitis both in gross appearance and microscopic features. Tissue gene expression of psoriatic core cytokines induced by imiquimod (including IL-23, IL-17A, IL-22, TNF-α, and IL-6) were greatly decreased by capsaicin application. This protective effect from capsaicin could be hampered by direct intradermal injection of IL-23. ConclusionEpicutaneous delivery of capsaicin on imiquimod-treated murine skin could significantly decrease expression of multiple inflammatory cytokines and the severity of prototypic change of psoriasiform inflammation. The beneficial effect imposed by capsaicin reinforces the neuroimmune contribution towards psoriasiform inflammation and provides a potential non-steroidal therapeutic alternative for topical treatment of psoriasiform dermatitis.

ACT1 Is Required for Murine IL-23–Induced Psoriasiform Inflammation Potentially Independent of E3 Ligase Activity

Psoriasis is a debilitating skin disease characterized by epidermal thickening, abnormal keratinocyte differentiation, and proinflammatory immune cell infiltrate into the affected skin. IL-17A plays a critical role in the etiology of psoriasis. ACT1, an intracellular adaptor protein and a putative ubiquitin E3 ligase, is essential for signal transduction downstream of the IL-17A receptor. Thus, IL-17A signaling in general, and ACT1 specifically, represent attractive targets for the treatment of psoriasis. We generated Act1 knockout and Act1 L286G knockin (ligase domain) mice to investigate the potential therapeutic effects of targeting ACT1 and its U-box domain, respectively. Act1 knockout, but not Act1 L286G knockin, mice were resistant to increases in CXCL1 plasma levels induced by subcutaneous injection of recombinant IL-17A. Moreover, in a mouse model of psoriasiform dermatitis induced by intradermal IL-23 injection, Act1 knockout, but not Act1 L286G knockin, was protective against increases in ear thickness, keratinocyte hyperproliferation, expression of genes for antimicrobial peptides and chemokines, and infiltration of monocytes and macrophages. Our studies highlight the critical contribution of ACT1 to proinflammatory skin changes mediated by the IL-23/IL-17 signaling axis and illustrate the need for further insight into ACT1 E3 ligase activity.

Potential Role of Cytosolic RNA Sensor MDA5 as an Inhibitor for Keratinocyte Differentiation in the Pathogenesis of Psoriasis.

BackgroundPsoriasis is a chronic inflammatory skin disease. The etiology of psoriasis is not fully understood, but the genetic background is considered to be the most important factor. To date, many psoriasis-related genes have been discovered, but the role of many important genes has not been well understood.ObjectiveThe purpose of this study is to uncover possible roles of MDA5 in psoriasis.MethodsExpression of MDA5 was investigated using immunohistochemistry. Then, MDA5 was overexpressed in keratinocytes using a recombinant adenovirus.ResultsAs a result of immunohistochemical staining, the expression of MDA5 was significantly increased in the epidermis of psoriasis compared to normal skin. Similarly, the expression of MDA5 was increased in imiquimod-induced psoriasiform dermatitis model. In cultured keratinocytes, toll-like receptor 3 agonist poly(I:C) induced expression of MDA5 at both mRNA and protein levels. When MDA5 was overexpressed using a recombinant adenovirus, poly(I:C)-induced cytokine expression was significantly increased. Finally, MDA5 overexpression significantly inhibited calcium-induced differentiation of keratinocytes.ConclusionThese results suggest that MDA5 increases in psoriasis and negatively regulates keratinocyte differentiation.

Immunological Memory in Imiquimod-Induced Murine Model of Psoriasiform Dermatitis.

Psoriasis is a common chronic inflammatory skin condition manifested by T cell responses and characterized by preferential recurrence at previously inflamed sites upon withdrawal of treatment. The site-specific disease memory in psoriasis has been linked to CD8+CD103+ tissue-resident memory T cells (Trm) in the epidermis which were previously thought to only provide “frontline” protection against pathogens and immunosurveillance during cancer development. In this study, we correlated the presence of a subset of the Trm cells which are also CD49a+ with disease severity in human psoriatic lesions with acute and chronic disease. Using an imiquimod (IMQ)-induced murine model of psoriasiform dermatitis, we also investigated the level of CD49a+ Trm cells in acute, chronic and resolved psoriatic lesions. Investigation of clinical human samples showed that patient disease severity highly correlated with the numbers of epidermal CD49a+ Trm cells. Additionally, this subset of Trm cells was shown to persist in resolved lesions of murine psoriasiform dermatitis once clinical disease features had subsided. Importantly, these CD49a+ Trm cells showed significantly higher levels of granzyme B (GzmB) production compared to acute disease, suggesting a potential role of CD49a+ Trm cells for psoriatic re-occurrence in resolved patients. Better understanding of epidermal CD49a+ Trm cell activity is necessary for development of advanced treatment strategies for psoriasis to permit long-term, continuous disease control.

A monocyte-keratinocyte-derived co-culture assay accurately identifies efficacies of BET inhibitors as therapeutic candidates for psoriasiform dermatitis

BackgroundBromodomain and extra-terminal (BET) proteins perform key roles in epigenetic control of gene expression that is involved in inflammatory conditions, including psoriasiform dermatitis (PsD). Predicting which (of many potential available BET inhibitors) will be effective in vivo is challenging. ObjectiveWe determine if a novel in vitro assay that includes two critical cell types involved in human psoriasis can predict the therapeutic potential of specific BET inhibitors in vivo. MethodsAn in vitro model consisting of U-937 and HaCaT cell co-culture was created to screen small molecule BET antagonists for inhibition of cutaneous inflammatory genes. Efficacious BET inhibitors were tested in a mouse imiquimod (IMQ)-induced PsD model. ResultsIn the co-culture system, HaCaT cells exhibited a marked increase in the secretion of a characteristic set of proinflammatory and Th17-associated cytokines. Of the ten commercially-available small molecules targeting BET proteins assayed, most compounds exhibited inhibitory functions at 1 μM against inflammatory activation, but responded variably at lower concentrations. OTX015, a typical representative for most of the compounds, barely inhibited the inflammatory reactions at 0.1 μM. By contrast, ABBV075 was effective in concentrations as low as 0.01 μM. While oral administration OTX015 in IMQ-treated mice reduced disease severity, ABBV075 equally decreased the symptoms and molecular and cellular severity markers at one-tenth of the minimal dosing required for OTX015. ConclusionIn vitro screening system combined with an in vivo animal model, can serve as a convenient pre-clinical screening tool for the selection of BET inhibitors (and possibly other drugs) that may have clinical potential in psoriasis therapy.

A Localized Aldara (5% Imiquimod)-Induced Psoriasiform Dermatitis Model in Mice Using Finn Chambers.

The expanding number of research studies utilizing the imiquimod-induced psoriasiform dermatitis model attests to the usefulness of this procedure. Advantages of this model include rapid development of the skin response and cost-effectiveness. A major limitation is that application of imiquimod cream over large areas of skin, as well as licking and ingestion of the cream, may lead to severe systemic inflammation, which can cause a general decline in health, splenomegaly, and death. In this protocol, Finn chambers are used to localize the imiquimod cream to a small area of the skin. This results in production of severe and reproducible psoriatic skin reactions with significantly less imiquimod, greatly reducing the possibility of untoward systemic effects. Moreover, having psoriasiform and control skin areas on the same mice decreases inter-animal differences. The protocol can be readily adapted for other skin disease models involving topical application of test agents. This article also details functional measurements performed during assays, including skin thickness, blood perfusion, semiquantitative histopathological evaluation, determination of scaling score to monitor psoriatic symptoms, and collection of spleen and body weight data to identify systemic effects. © 2020 The Authors. Basic Protocol: Use of Finn chambers to induce psoriasiform skin reactions with imiquimod Support Protocol 1: Measurement of double-fold dorsal skin thickness Support Protocol 2: Measurement of blood perfusion Support Protocol 3: Determination of scaling score Support Protocol 4: Semiquantitative histopathological scoring Support Protocol 5: Assessment of systemic side effects in response to imiquimod application.

Validation of a chronic imiquimod-induced psoriasiform dermatitis model in Balb/c mice

Abstract We developed a modified imiquimod (IMQ)-induced dermatitis protocol (Horvath et al, Scientific Reports 2019) using Finn chambers to minimize the unwanted systemic side effects of this widely used psoriasis animal model. It enables to perform prolonged imiquimod treatment in a self-control way as well. In the present study we investigate the long-term application of IMQ in this localized model. 20 mg Aldara cream (5% IMQ) or 20 mg vaseline were applied in two Finn chambers on the dorsal skin of Balb/c mice during the first 4 days of the experiment and every second or third day from day 5 to day 15, in separate groups. Vaseline or corticosteroid cream were applied on the intermediate days. Skin thickness, blood perfusion, body weight and skin scaling were measured daily. Histopathological alterations were evaluated on H&E stained sections, inflammatory cytokine concentrations (IL-1β, TNF-α, IL-17, IL-22, IL-23) were measured with Luminex technology. Skin edema increased to 80–120% in the every 2nd or 3rd days treated group. Blood perfusion reached maximal response on day 4 and then gradually decreased until the end of the experiment. Moderate body weight loss was observed on the first 5 days of the experiment. Inflammatory cytokine concentrations in the skin were elevated during the onset of the symptoms (1–5 days) but dropped in the chronic phase (day 10 or 15). Topical corticosteroid treatment significantly reduced skin edema from day 8, but it had no effect on blood perfusion. Our results proved that long-term Aldara application in Finn chambers enables the study of chronic psoriatic skin inflammation. Furthermore, our chronic model may be used to investigate topically applied anti-inflammatory drug candidates in psoriasiform dermatitis.

434 Development of Aldara-induced chronic (15-day) psoriasiform dermatitis model

434 Development of Aldara-induced chronic (15-day) psoriasiform dermatitis model

Reinforcing involvement of NK cells in psoriasiform dermatitis animal model.

Psoriasis (Ps) is a chronic inflammatory immune-mediated disease with skin and joint manifestations, characterized by abnormal and rapid proliferation of keratinocytes and infiltration of psoriatic lesions with immune cells. Extensive literature suggests that Ps is a T-cell mediated disease its pathogenesis being highly related to innate and adaptative immune cells. Although natural killer (NK) cells are involved in the inflammatory process of Ps through pro-inflammatory cytokine secretion (tumor necrosis factor α, interferon γ), their role in this pathology is not yet fully elucidated. In order to study the involvement of NK subpopulations in the pathogenesis of Ps we used the imiquimod-based mouse model of psoriasiform dermatitis and NK cells complex phenotype patterns from peripheral blood (PB) and spleen were investigated. Skin inflammation and the disease severity were assessed using in vivo measurements (erythema, desquamation and induration parameters, PASI modified score), splenomegaly assessment and histopathological evaluation. Phenotypic characterization of NK cells in imiquimod (IMQ)-treated mice was performed by flow cytometry, for both PB and spleen cell suspension. A large panel of surface markers was used: maturation and activation markers [cluster of differentiation (CD)49b, CD11b, CD43, CD27, KLRG1, CD335, CD69, CD28, gp49R, CD45R, CD11c] and markers for cytokine receptors (CD25, CD122, CD132). Our experimental data showed important differences in IMQ-treated mouse NK cell phenotype as compared to control group. The maturation markers (CD11b, CD43, CD27, KLRG1) were found increased on NK cells, in periphery and spleen, while CD49b+NK1.1+ was significantly lower, and the alterations correlated with the severity of the disease. Our findings reflect the immune engagement toward activatory profile of NK cells and draw attention to evaluating Ps intensity correlated with the mature profile of circulating NK cells.

036 CXCL10 expression is regulated by keratinocyte STAT3 signaling and inhibits skin inflammation

The role of STAT3 signaling in psoriasis is not entirely clear as transgenic mice that overexpress STAT3 in keratinocytes (K14-stat3c) spontaneously develop psoriasiform dermatitis whereas STAT3 is also thought to promote IL-17A/F production by T cells. Therefore, in this study, the relative contribution of STAT3 in keratinocytes versus T cells was evaluated in the imiquimod mouse model of psoriasiform dermatitis by evaluating cre/lox mice with either keratinocyte inducible deletion of STAT3 (K5-creERT2×STAT3fl/fl [K5-STAT3]) or specific deletion of STAT3 in T-cells (Lck-cre×STAT3 fl/fl [Lck-STAT3]). Unexpectedly, psoriasiform skin inflammation was diminished in K5-STAT3 mice whereas Lck-STAT3 mice developed similar skin inflammation as WT mice. In addition, K5-STAT3 mice had increased IFN-γ+ T cells but less IL-17+ T cells compared to wt mice, indicating that loss of STAT3 signaling in keratinocytes dampened inflammation by inhibiting IL-17 responses while promoting IFN-γ responses. Interestingly, mRNA and histologic expression of the interferon response gene, CXCL10, inversely correlated with the skin inflammation in deletion or overexpression of STAT3 in the K5-STAT3 or K14-stat3c mice, respectively. Additionally, we discovered that neutralizing CXCL10 signaling enhanced imiquimod-induced skin inflammation, suggesting that CXCL10 acts to inhibit skin inflammation in this model. Taken together, these findings define a novel mechanism by which keratinocyte but not T cell-intrinsic STAT3 signaling induces psoriasiform-like skin inflammation via regulation of CXCL10 expression, proinflammatory IL-17 and anti-inflammatory IFN-γ T cell responses.