Marianne B. Løvendorf

Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL)

Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that the most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) miRNAs distinguish CTCL from benign skin diseases with > 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Quantitative (q)RT-PCR analysis of 103 patients with CTCL and benign skin disorders validates differential expression of 4 of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A qRT-PCR-based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity and sensitivity, and with a classification accuracy of 95%, indicating that miRNAs have a high diagnostic potential in CTCL.

MicroRNAs and potential target interactions in psoriasis.

BACKGROUND Psoriasis is a chronic inflammatory skin disease often seen in patients with a genetic susceptibility. MicroRNAs (miRNA) are endogenous, short RNA molecules that can bind to parts of mRNA target genes, thus inhibiting their translation and causing accelerated turnover or transcript degradation. MicroRNAs are important in the pathogenesis of human diseases such as immunological disorders, as they regulate a broad range of biological processes. OBJECTIVE We investigated miRNA-mRNA interactions in involved (PP) and non-involved (PN) psoriatic skin compared with healthy skin (NN). METHODS Biopsies were obtained from PP, PN and NN, the miRNA and mRNA expression was analyzed by microarray techniques and a subset of miRNAs and mRNAs were validated by q-RT-PCR. Novel target interactions in psoriasis were found using PubMed, miRBase and RNAhybrid. In addition, TIMP3 protein expression was studied in PP, PN and NN. Finally, the miR-221/2-TIMP3 target interaction was studied in primary human keratinocytes by endogenous overexpression of the miRNAs. RESULTS We identified 42 upregulated miRNAs and 5 downregulated miRNAs in PP compared with NN, and only few deregulated miRNAs in PN compared with NN. Based on the miRNA and mRNA profiles miR-21, -205, -221 and -222 were found to have the following potential mRNA targets in psoriatic skin: PDCD4, TPM1, P57, C-KIT, RTN4, SHIP2, TIMP3, RECK and NFIB. The identified target mRNAs were likely to be involved in cellular growth, proliferation, apoptosis and degradation of the extracellular matrix. Finally we found that TIMP3 is downregulated in psoriatic skin. In vitro overexpression of miR-221 and miR-222 lead to degradation of TIMP3 resulting in decreased TIMP3 protein level. CONCLUSION Our data indicate several novel important associations for miRNAs in psoriasis and in particular the miR-221/2-TIMP3 target interaction could among others play a role in the psoriasis pathogenesis.

MicroRNA-223 and miR-143 are important systemic biomarkers for disease activity in psoriasis.

BACKGROUND Psoriasis is a systemic inflammatory skin disease. MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that recently have been found in the blood to be relevant as disease biomarkers. OBJECTIVE We aimed to explore miRNAs potential as blood biomarkers for psoriasis. METHODS Using microarray and quantitative real-time PCR we measured the global miRNA expression in whole blood, plasma and peripheral blood mononuclear cells (PBMCs) from patients with psoriasis and healthy controls. RESULTS We identified several deregulated miRNAs in the blood from patients with psoriasis including miR-223 and miR-143 which were found to be significantly upregulated in the PBMCs from patients with psoriasis compared with healthy controls (FCH=1.63, P<0.01; FCH=2.18, P<0.01, respectively). In addition, miR-223 and miR-143 significantly correlated with the PASIscore (r=0.46, P<0.05; r=0.55, P<0.02, respectively). Receiver-operating characteristic analysis (ROC) showed that miR-223 and -143 have the potential to distinguish between psoriasis and healthy controls (miR-223: area under the curve (AUC)=0.80, miR-143: AUC=0.75). Interestingly, after 3-5 weeks of treatment with methotrexate following a significant decrease in psoriasis severity, miR-223 and miR-143 were significantly downregulated in the PBMCs from patients with psoriasis. CONCLUSION We suggest that changes in the miR-223 and miR-143 expressions in PBMCs from patients with psoriasis may serve as novel biomarkers for disease activity in psoriasis; however, further investigations are warranted to clarify their specific roles.

Laser capture microdissection followed by next‐generation sequencing identifies disease‐related microRNAs in psoriatic skin that reflect systemic microRNA changes in psoriasis

Psoriasis is a systemic disease with cutaneous manifestations. MicroRNAs (miRNAs) are small non‐coding RNA molecules that are differentially expressed in psoriatic skin; however, only few cell‐ and region‐specific miRNAs have been identified in psoriatic lesions. We used laser capture microdissection (LCM) and next‐generation sequencing (NGS) to study the specific miRNA expression profiles in the epidermis (Epi) and dermal inflammatory infiltrates (RD) of psoriatic skin (N = 6). We identified 24 deregulated miRNAs in the Epi and 37 deregulated miRNAs in the RD of psoriatic plaque compared with normal psoriatic skin (FCH > 2, FDR < 0.05). Interestingly, 9 of the 37 miRNAs in RD, including miR‐193b and miR‐223, were recently described as deregulated in circulating peripheral blood mononuclear cells (PBMCs) from patients with psoriasis. Using flow cytometry and qRT‐PCR, we found that miR‐193b and miR‐223 were expressed in Th17 cells. In conclusion, we demonstrate that LCM combined with NGS provides a robust approach to explore the global miRNA expression in the epidermal and dermal compartments of psoriatic skin. Furthermore, our results indicate that the altered local miRNA changes seen in the RD are reflected in the circulating immune cells, suggesting that miRNAs may contribute to the pathogenesis of psoriasis.

The Vitamin D Analogue Calcipotriol Reduces the Frequency of CD8+IL-17+ T Cells in Psoriasis Lesions

The vitamin D analogue calcipotriol is an immunomodulatory drug widely used to treat psoriasis; however, how calcipotriol affects the immune cells in psoriasis lesions is not fully understood. The aim of this atudy was to investigate the effect of calcipotriol on the frequency of CD4+ and CD8+ T cells and innate lymphoid cells (ILC) and their production of IL‐17A, IFN‐γ and IL‐22 in psoriasis lesions in patients with chronic plaque psoriasis. Eighteen patients with psoriasis were included, and two similar psoriasis lesions were chosen for each patient. One lesion was treated with calcipotriol (50 μg/g) and the other with vehicle twice a day for 14 days. The clinical effect was measured by degree of erythema, scaling and induration in each lesion (SUM score). Skin biopsies were collected for histological and immunohistochemical analyses. Skin‐derived cells were isolated and analysed by flow cytometry. After 14 days of treatment with calcipotriol, a significant clinical and histological effect was seen; however, we found no differences in the frequency of CD4+ and CD8+ T cells or ILC between calcipotriol‐ and vehicle‐treated skin. The main finding was a significant decrease in CD8+IL‐17+ T cells in skin‐derived cells from calcipotriol‐treated skin, which was further supported by the absence of CD8+IL‐17+ T cells in immunohistochemical staining of calcipotriol‐treated skin. No changes in the frequency of IL‐22+ or IFN‐γ+ cells were observed. Our findings show that the vitamin D analogue calcipotriol reduces the frequency of CD8+IL‐17+ T cells in psoriasis lesions concomitant with clinical improvement.

Comparison of microRNA expression using different preservation methods of matched psoriatic skin samples

Abstract:  MicroRNAs are non‐coding RNA molecules modulating gene expression post‐transcriptionally. Formalin‐fixed, paraffin‐embedding (FFPE) is a standard preservation method often used in clinical practices, but induces RNA degradation. Extracting high‐quality RNA from human skin can be challenging as skin contains high levels of RNases. As microRNAs are 19‐23 nucleotides long and lack a poly‐A tail, they may be less prone to RNA degradation than mRNAs. We investigated whether microRNAs in psoriatic (FFPE) samples reliably reflect microRNA expression in samples less prone to RNA degradation such as fresh‐frozen (FS) and Tissue‐Tek‐embedding (OCT). We found a strong correlation of the microRNA expression levels between all preservation methods of matched psoriatic skin samples (rs ranging from 0.91 to 0.95 (P < 0.001)). These observations were further confirmed with qRT‐PCR. Our results demonstrate that microRNA detection in human skin is robust irrespective of preservation method; thus, microRNAs offer an appropriate and flexible approach in clinical practices and for diagnostic purposes in skin disorders.

miRNAs in inflammatory skin diseases and their clinical implications

miRNAs are a class of non-coding RNA molecules that modulate gene expression post-transcriptionally. They have a major impact on several physiological and pathological cellular processes including modulation of the innate and the adaptive immune system. The role of miRNAs in skin biology is still incomplete; however, it is known that miRNAs are implicated in various cellular processes of both normal and diseased skin. Some miRNAs appear to be consistently deregulated in several different inflammatory skin diseases, including psoriasis and atopic dermatitis, indicating a common role in fundamental biological processes. The clinical implications of miRNAs are intriguing, both from a diagnostic and a therapeutic perspective. Accordingly, there is emerging evidence for the clinical potential of miRNAs as both biomarkers and possible therapeutic targets in skin diseases. Future studies will hopefully establish the biological significance of miRNAs in skin biology, paving the way for new miRNA-based diagnostic and therapeutic applications in dermatology.

Unique microRNAs appear at different times during the course of a delayed-type hypersensitivity reaction in human skin

Diphencyprone (DPCP) is a hapten that induces delayed‐type hypersensitivity (DTH) reactions. MicroRNAs (miRNAs) are short non‐coding RNAs that negatively regulate gene expression and have been implicated in various inflammatory skin diseases, but their role in DTH reactions is not well understood. We generated global miRNA expression profiles (using next‐generation sequencing) of DPCP reactions in skin of seven healthy volunteers at 3, 14 and 120 days after challenge. Compared to placebo‐treated sites, DPCP‐challenged skin at 3 days (peak inflammation) had 127 miRNAs significantly deregulated. At 14 days (during resolution of inflammation), 43 miRNAs were deregulated and, at 120 days (when inflammation had completely resolved), six miRNAs were upregulated. While some miRNAs have been observed in psoriasis or atopic dermatitis, most of the deregulated miRNAs have not yet been studied in the context of skin biology or immunology. Across the three time points studied, many but not all miRNAs were uniquely expressed. As various miRNAs may influence T cell activation, this may indicate that the miRNAs exclusively expressed at different time points function to promote or resolve skin inflammation, and therefore, may inform on the paradoxical ability of DPCP to treat both autoimmune conditions (alopecia areata) and conditions of ineffective immunity (melanoma).

MicroRNAs in Skin Diseases

MicroRNAs (miRNAs) comprise a class of small noncoding RNA molecules that are involved in the epigenetic modulation of gene expression. miRNAs act primarily within the cell; however, recent evidence has also shown the presence of miRNAs in cell-free environments, including serum and plasma. The role of miRNAs in skin biology is still incomplete; however, it is now evident that miRNAs indeed are involved in various cellular processes in both normal and diseased skin. Only a few skin-specific miRNAs have been described, e.g., miR-203. Until now the majority of studies have characterized specific patterns of miRNA expression levels in lesional diseased skin relative to nonlesional and healthy skin. Thus, both benign inflammatory skin diseases such as psoriasis, atopic dermatitis (AD), and allergic contact dermatitis (ACD) and malignant skin diseases including basal cell carcinoma and melanoma have been associated with specific miRNA signatures. Some miRNAs appear to be consistently deregulated across several skin diseases indicating a common role in fundamental cellular processes in these pathologies. For instance, aberrant expression of miR-21, -142-3p, and -155 has been reported for psoriasis, AD, and ACD. In this chapter, we summarize the current knowledge on the aberrant expression and biological role of miRNAs in skin diseases. In addition, we present the most recent progress within the clinical utility of miRNAs as biomarkers for various skin diseases.

Allergic contact dermatitis to nickel is characterized by a specific micro-RNA signature.

To the Editor: Allergic contact dermatitis (ACD) is a type IV delayed type hypersensitivity response to allergens that come in contact with the skin. Micro RNAs (miRNAs) are noncoding RNA molecules that modulate gene expression posttranscriptionally. Here, we investigated the miRNA expression in skin after challenge to nickel (Ni). Using global miRNA expression analysis on skin obtained from the challenge reaction (Ni and vehicle) from 3 subjects with a patch test positive to Ni (PP) and 3 healthy subjects with no history of dermatitis (NN) (Table S1, http://links.lww.com/DER/A4), we identified 159 miRNAs that were differentially expressed (P G 0.05; Table S2, http://links.lww.com/DER/A4). Unsupervised hierarchical clustering based on the 159 deregulated miRNAs showed a clear separation related to the elicitation reaction (Fig. 1A). Micro RNA-193a-3p was the only miRNA that was previously reported as deregulated in another allergeninduced ACD (diphenylcyclopropenone (DPCP)-challenged skin) indicating that the miRNA expression profiles in ACD may be allergen specific. In an ACD reaction, immune cells migrate from the blood into the skin. To determine whether the disease-related miRNAs could be confined specifically to the immune cells in the dermal inflammatory compartment, we next investigated the global miRNA expression in peripheral blood mononuclear cells (PBMCs) exposed to Ni or vehicle. We found 19 miRNAs that were differentially expressed between PBMCs from subjects with allergy to Ni stimulated with Ni compared with vehicle (P G 0.05; Table S3, http://links.lww.com/DER/A4). Among those, miR-155 that previously has been shown to be increased in the dermal infiltrating immune cells in lesions from patients with psoriasis and atopic dermatitis, altogether suggesting a common inflammatory role of miR-155 in skin diseases. A comparison between deregulated miRNAs in Nichallenged skin and PBMCs from subjects with allergy to Ni showed only a minimal overlap (Fig. 1B). An explanation for the limited overlap could be the fact that the identified miRNA signature from the PBMCs may represent a systemic component rather than local miRNA changes in the skin. Finally, our sample population was small, and the adjusted P values were high increasing the risk of detecting false-positive miRNAs. Nonetheless, unsupervised hierarchical clustering of the PP skin samples and the PBMCs support that the changes in the miRNA expressions are related to biological variation. Because ACD clinically can resemble irritant contact dermatitis (ICD), we finally sought to investigate whether miRNA expression profiles can be used to distinguish ACD