John R. Zibert

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.

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.

Halting angiogenesis by non-viral somatic gene therapy alleviates psoriasis and murine psoriasiform skin lesions

Dysregulated angiogenesis is a hallmark of chronic inflammatory diseases, including psoriasis, a common skin disorder that affects approximately 2% of the population. Studying both human psoriasis in 2 complementary xenotransplantation models and psoriasis-like skin lesions in transgenic mice with epidermal expression of human TGF-β1, we have demonstrated that antiangiogenic non-viral somatic gene therapy reduces the cutaneous microvasculature and alleviates chronic inflammatory skin disorders. Transient muscular expression of the recombinant disintegrin domain (RDD) of metargidin (also known as ADAM-15) by in vivo electroporation reduced cutaneous angiogenesis and vascularization in all 3 models. As demonstrated using red fluorescent protein-coupled RDD, the treatment resulted in muscular expression of the gene product and its deposition within the cutaneous hyperangiogenic connective tissue. High-resolution ultrasound revealed reduced cutaneous blood flow in vivo after electroporation with RDD but not with control plasmids. In addition, angiogenesis- and inflammation-related molecular markers, keratinocyte proliferation, epidermal thickness, and clinical disease scores were downregulated in all models. Thus, non-viral antiangiogenic gene therapy can alleviate psoriasis and may do so in other angiogenesis-related inflammatory skin disorders.

Ingenol Mebutate Signals via PKC/MEK/ERK in Keratinocytes and Induces Interleukin Decoy Receptors IL1R2 and IL13RA2

Squamous cell carcinoma (SCC) is the second most common human skin cancer and the second leading cause of skin cancer–related death. Recently, a new compound, ingenol mebutate, was approved for treatment of actinic keratosis, a precursor of SCC. As the mechanism of action is poorly understood, we have further investigated the mechanism of ingenol mebutate–induced cell death. We elucidate direct effects of ingenol mebutate on primary keratinocytes, patient-derived SCC cells, and a SCC cell line. Transcriptional profiling followed by pathway analysis was performed on ingenol mebutate–treated primary keratinocytes and patient-derived SCC cells to find key mediators and identify the mechanism of action. Activation of the resulting pathways was confirmed in cells and human skin explants and supported by a phosphorylation screen of treated primary cells. The necessity of these pathways was demonstrated by inhibition of certain pathway components. Ingenol mebutate inhibited viability and proliferation of all keratinocyte-derived cells in a biphasic manner. Transcriptional profiling identified the involvement of PKC/MEK/ERK signaling in the mechanism of action and inhibition of this signaling pathway rescued ingenol mebutate–induced cell death after treatment with 100 nmol/L ingenol mebutate, the optimal concentration for the first peak of response. We found the interleukin decoy receptors IL1R2 and IL13RA2 induced by ingenol mebutate in a PKC/MEK/ERK–dependent manner. Furthermore, siRNA knockdown of IL1R2 and IL13RA2 partially rescued ingenol mebutate–treated cells. In conclusion, we have shown that ingenol mebutate–induced cell death is mediated through the PKCδ/MEK/ERK pathway, and we have functionally linked the downstream induction of IL1R2 and IL13RA2 expression to the reduced viability of ingenol mebutate–treated cells. Mol Cancer Ther; 14(9); 2132–42. ©2015 AACR.

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.

Tumor-Preferential Induction of Immune Responses and Epidermal Cell Death in Actinic Keratoses by Ingenol Mebutate

The rapid and strong clinical efficacy of the first-in-class, ingenol mebutate, against actinic keratosis (AK) has resulted in its recent approval. We conducted the first comprehensive analysis of the cellular and molecular mode of action of topical ingenol mebutate 0.05% gel in both AK and uninvolved skin of 26 patients in a phase I, single-center, open-label, within-patient comparison. As early as 1 day after application, ingenol mebutate induced profound epidermal cell death, along with a strong infiltrate of CD4+ and CD8+ T-cells, neutrophils, and macrophages. Endothelial ICAM-1 activation became evident after 2 days. The reaction pattern was significantly more pronounced in AK compared with uninvolved skin, suggesting a tumor-preferential mode of action. Extensive molecular analyses and transcriptomic profiling of mRNAs and microRNAs demonstrated alterations in gene clusters functionally associated with epidermal development, inflammation, innate immunity, and response to wounding. Ingenol mebutate reveals a unique mode of action linking directly to anti-tumoral effects. Trial Registration: ClinicalTrials.gov NCT01387711

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).

Repeated treatments with ingenol mebutate for prophylaxis of UV-induced squamous cell carcinoma in hairless mice

BACKGROUND AND AIM The incidence of squamous cell carcinomas (SCC) is increasing, and effective chemopreventative strategies are needed. We hypothesized that repeated treatments with ingenol mebutate (IngMeb) would postpone development of SCC in hairless mice, and that application of a corticosteroid would reduce IngMeb-induced local skin responses (LSRs) without affecting tumor postponement. METHODS Hairless mice (n=150; 6 groups á 25 mice) were irradiated with solar simulated ultraviolet radiation (UVR) until SCC developed. During UV-irradiation and before tumor development, five single treatments (Tx) with IngMeb were given at four-week intervals (days 21, 49, 77, 105, 133). Clobetasol propionate (CP) was applied once daily for 5days prior to IngMeb, as well as 6h and 1day post treatment. Tumor formation was evaluated weekly for 52weeks. LSR (scale 0-24) were assessed at baseline, 1h, 6h, 1-, 2-, 3-, 4-, 5-, 6-, and 7days after each IngMeb treatment. RESULTS IngMeb significantly delayed tumor development compared to UVR alone (UVR day 168 vs. UVR+IngMeb day 189; p=0.025). LSR included erythema, flaking, crusting, bleeding, vesiculation, and ulceration. The composite LSR-scores were of moderate intensity in non-UV irradiated skin (max LSR IngMeb Tx 1-5: 1.5-2.5) and more pronounced in photodamaged skin (max LSR Tx 5; IngMeb 1.5 vs. UVR+IngMeb 1.8; p<0.001). LSR intensity correlated with tumor development by means of greater composite LSR-score resulted in longer tumor-free survival (r(2)=0.257, p<0.001). Contrary to our hypothesis, concurrent CP increased LSR (max LSR Tx 1-5: UVR+CP+IngMeb 3.2-4.9 vs. UVR+IngMeb 1.3-2.2, p<0.001) and postponed tumor development compared to IngMeb alone (UVR+CP+IngMeb day 217 vs. UVR+IngMeb day 189, p<0.001). CONCLUSION Repeated field-directed treatments with IngMeb delay development of UV-induced SCC in hairless mice, and increased IngMeb induced LSRs correlated with improved clinical outcomes. The findings highlight the potential of IngMeb as a prophylactic remedy for SCC in humans.

Repeated Treatments with Ingenol Mebutate Prevents Progression of UV-Induced Photodamage in Hairless Mice

Background and Aim Ingenol mebutate (IngMeb) is an effective treatment for actinic keratosis. In this study, we hypothesized that repeated treatments with IngMeb may prevent progression of UV-induced photodamage, and that concurrent application of a corticosteroid may reduce IngMeb-induced local skin responses (LSR). Methods Hairless mice (n = 60; 3 groups of 20 mice) were irradiated with solar simulated ultraviolet radiation (UVR) throughout the study. Five single treatments with IngMeb were given at 4-week intervals (Days 21, 49, 77, 105, and 133). Clobetasol propionate (CP) was applied once daily for 5 days prior to each IngMeb application, as well as 6 h and 1 day post treatment. One week after IngMeb treatment No. 1, 3, and 5 (Days 28, 84, and 140), biopsies from four mice in each group were collected for histological evaluation of UV-damage on a standardized UV-damage scale (0–12). LSR (0–24) were assessed once daily (Days 1–7) after each IngMeb treatment. Results IngMeb prevented progression of photodamage in terms of keratosis grade, epidermal hypertrophy, dysplasia, and dermal actinic damage with a lower composite UV-damage score on day 140 (UVR 10.25 vs. UVR+IngMeb 6.00, p = 0.002) compared to UVR alone. IngMeb induced LSR, including erythema, flaking, crusting, bleeding, vesiculation, and ulceration. Concurrent CP increased LSR (max LSR Tx 1–5: UVR+IngMeb+CP 3.6–5.5 vs. UVR+IngMeb 2.6–4.3) and provided better prevention of photodamage compared to IngMeb alone (Day 140: UVR+IngMeb 6.00 vs. UVR+IngMeb+CP 3.00 p < 0.001). Conclusion Repeated field-directed treatments with IngMeb prevent progression of cutaneous photodamage in hairless mice, while CP cannot be used to alleviate IngMeb-induced LSR. The findings suggest that IngMeb may potentially serve as a prophylactic treatment for UV-induced tumors.