Leslie van der Fits

Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice Is Mediated via the IL-23/IL-17 Axis

Topical application of imiquimod (IMQ), a TLR7/8 ligand and potent immune activator, can induce and exacerbate psoriasis, a chronic inflammatory skin disorder. Recently, a crucial role was proposed for the IL-23/IL-17 axis in psoriasis. We hypothesized that IMQ-induced dermatitis in mice can serve as a model for the analysis of pathogenic mechanisms in psoriasis-like dermatitis and assessed its IL-23/IL-17 axis dependency. Daily application of IMQ on mouse back skin induced inflamed scaly skin lesions resembling plaque type psoriasis. These lesions showed increased epidermal proliferation, abnormal differentiation, epidermal accumulation of neutrophils in microabcesses, neoangiogenesis, and infiltrates consisting of CD4+ T cells, CD11c+ dendritic cells, and plasmacytoid dendritic cells. IMQ induced epidermal expression of IL-23, IL-17A, and IL-17F, as well as an increase in splenic Th17 cells. IMQ-induced dermatitis was partially dependent on the presence of T cells, whereas disease development was almost completely blocked in mice deficient for IL-23 or the IL-17 receptor, demonstrating a pivotal role of the IL-23/IL-17 axis. In conclusion, the sole application of the innate TLR7/8 ligand IMQ rapidly induces a dermatitis closely resembling human psoriasis, critically dependent on the IL-23/IL-17 axis. This rapid and convenient model allows further elucidation of pathogenic mechanisms and evaluation of new therapies in psoriasis.

Effective Treatment of Psoriasis with Narrow-Band UVB Phototherapy Is Linked to Suppression of the IFN and Th17 Pathways

Narrow-band ultraviolet-B (NB-UVB) phototherapy is an effective treatment for psoriasis. The molecular mechanisms underlying its efficacy are incompletely understood. To identify NB-UVB-induced molecular pathways that may account for its anti-inflammatory efficacy, gene expression profiling was performed using epidermal RNA from lesional and nonlesional skin from patients with psoriasis undergoing NB-UVB therapy. Downregulation of Th17 signaling pathway was observed during NB-UVB therapy in psoriatic epidermis. Strong inhibition of the Th17 pathway by UVB was confirmed in an ex vivo organ culture system by demonstrating reduced signal transducer and activator of transcription 3 (STAT3) phosphorylation and β-defensin-2 production. These results were further substantiated by demonstrating that NB-UVB inhibited the Th17-dependent psoriasis-like dermatitis in mice. Other pathways affected by NB-UVB therapy include the IFN signaling pathway, epidermal differentiation, and other well-known therapeutic targets in psoriasis, such as the glucocorticoid, vitamin D, peroxisome proliferator-activated receptor, and IL-4 signaling pathways. In conclusion, clinical improvement of psoriasis by NB-UVB is linked to suppression of Th17 and type I and type II IFN signaling pathways, which are critical in the pathogenesis of the disease. Our results show that clinically effective NB-UVB therapy is based on suppression of a broad range of important molecular pathways in psoriatic skin.

MicroRNA-21 Expression in CD4+ T Cells Is Regulated by STAT3 and Is Pathologically Involved in Sézary Syndrome

MicroRNAs (miRNAs) are small RNAs that control gene expression, and are involved in the regulation of fundamental biological processes including development, cell differentiation, proliferation, and apoptosis. miRNAs regulate gene expression in normal hematopoiesis, and aberrant miRNA expression might contribute to leukomogenesis. Specifically, miR-21 is abundantly expressed in various tumors including leukemia and lymphoma, and is functionally involved in oncogenic processes. We investigated a role for miR-21 in Sézary Syndrome (SS), a cutaneous T-cell lymphoma (CTCL) with CD4+ tumor cells (Sézary cells) present in the skin, lymph nodes, and peripheral blood. It was shown previously that SS is characterized by constitutively activated signal transducer and activator of transcription 3 (STAT3) signaling. In this study we show by chromatin immunoprecipitation that miR-21 is a direct STAT3 target in Sézary cells. Stimulation of Sézary cells or healthy CD4+ T cells with the common-γ chain cytokine IL-21 results in a strong activation of STAT3, and subsequent upregulation of miR-21 expression. Both pri- and mature miR-21 expression are increased in Sézary cells when compared with CD4+ T cells from healthy donors. Silencing of miR-21 in Sézary cells results in increased apoptosis, suggesting a functional role for miR-21 in the leukomogenic process. Consequently, miR-21 might represent a therapeutic target for the treatment of SS.

Autocrine IL-21 Stimulation Is Involved in the Maintenance of Constitutive STAT3 Activation in Sézary Syndrome

Sézary syndrome (SS) is a cutaneous T-cell lymphoma (CTCL) with malignant CD4+ T cells (SS cells) in skin, lymph nodes, and blood. Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in SS cells, whereas this activation is lost upon in vitro culturing, indicating that STAT3 activation observed in vivo is the result of activating factors in the micromilieu of the malignant cells. We investigated which factors are involved in STAT3 activation in SS, focusing on cytokines of the common γ-chain family because of their crucial role in T-cell activation. Furthermore, downstream effects of STAT3 signaling in SS cells were assayed. In SS cells, STAT3 was strongly activated by IL-21, and increased expression of IL-21 and its receptor chains was observed in peripheral blood SS cells. IL-21 and IL-21R protein expression was detectable on neoplastic cells in SS skin biopsies. Using short-term culturing experiments, we demonstrate that IL-21 itself and the α-chain of the IL-2 receptor are STAT3 target genes in SS cells, thereby rendering cells more sensitive to stimulation with the T-cell proliferation and activating cytokine IL-2. Combined, our data point toward a pivotal role for an autocrine positive feedback loop involving IL-21 and consequent persistent STAT3 activation in the pathogenesis of SS, thereby indicating IL-21 and IL-21R as new therapeutical targets.

Caspase-14-Deficient Mice Are More Prone to the Development of Parakeratosis

Caspase-14 is an important protease in the proper formation of a fully functional skin barrier. Newborn mice that are deficient in caspase-14 exhibit increased transepidermal water loss and are highly sensitive to UVB-induced photodamage. Decreased caspase-14 expression and incomplete caspase-14 processing in lesional psoriatic parakeratotic stratum corneum has been reported previously. In this study, we show that caspase-14-deficient skin frequently displays incompletely cornified cells in the transitional zone between the granular and the cornified layers, pointing to a delay in cornification. We also demonstrate that after challenge of epidermal permeability barrier function by repetitive acetone treatment, a higher incidence of large parakeratotic plaques was observed in caspase-14-deficient skin. Furthermore, caspase-14-deficient mice are more prone than control mice to the development of parakeratosis upon induction of psoriasis-like dermatitis by imiquimod treatment. These results show that lack of caspase-14 expression predisposes to the development of parakeratosis and that caspase-14 has an important role in keratinocyte terminal differentiation and the maintenance of normal stratum corneum, especially in conditions causing epidermal hyperproliferation.

Psoriatic lesional skin exhibits an aberrant expression pattern of interferon regulatory factor-2 (IRF-2).

Psoriasis is a T‐cell‐mediated inflammatory skin disease. A Th1 cytokine profile with increased levels of interferon‐gamma (IFN‐γ) is predominant in skin and peripheral blood mononuclear cells (PBMCs) from psoriasis patients. Furthermore, psoriatic keratinocytes exhibit an aberrant sensitivity and response to IFN‐γ. The transcriptional activator interferon regulatory factor‐1 (IRF‐1) plays a crucial role in the activation of IFN‐γ‐induced gene expression. Recently it was shown that mice deficient in IRF‐2, a transcriptional repressor of IFN signalling and thereby acting as an IRF‐1 antagonist, display psoriasis‐like skin abnormalities. It was therefore hypothesized that a dysbalance between IRF‐1 and IRF‐2, the activator and repressor of IFN responses, respectively, contributes to the altered IFN‐γ signalling observed in patients with psoriasis. In the epidermis of patients with psoriasis and healthy controls, similar IRF‐1 and IRF‐2 mRNA expression levels were observed. Furthermore, it was not possible to detect any differences in IRF‐1 and IRF‐2 protein levels in nuclear extracts from the epidermis of controls and psoriasis patients by electrophoretic mobility shift assay and western blot analysis. Using double immunofluorescence labelling, it was observed that in normal skin IRF‐1 was expressed in keratinocytes throughout the epidermis, whereas IRF‐2 was restricted to the basal cell layer. In psoriatic skin, IRF‐1 expression was comparable to normal skin, whereas IRF‐2 was expressed in both basal and suprabasal cell layers. This altered IRF‐2 expression in suprabasal cell layers may therefore result in a dysbalance between the activator and repressor of IFN responses in these cell layers, putatively contributing to aberrant responses to IFN‐γ and eventually to the psoriatic skin phenotype. Copyright

Exploring the IL-21-STAT3 Axis as Therapeutic Target for Sezary Syndrome

Sézary syndrome is an aggressive cutaneous T-cell lymphoma. The malignant cells (Sézary cells) are present in skin, lymph nodes, and blood, and express constitutively activated signal transducer and activator of transcription (STAT)3. STAT3 can be activated by IL-21 in vitro and the IL-21 gene itself is a STAT3 target gene, thereby creating an autocrine positive feedback loop that might serve as a therapeutic target. Sézary cells underwent apoptosis when incubated with Stattic, a selective STAT3 inhibitor. STAT3 activation in Sézary cells did not affect expression of the supposed anti-apoptotic STAT3 target genes BCL2, BCL-xL, and SURVIVIN, whereas expression of (proto)oncogenes miR-21, TWIST1, MYC, and PIM1 was significantly increased. CD3/CD28-mediated activation of Sézary cells induced IL-21 expression, accompanied by STAT3 activation and increased proliferation. Blocking IL-21 in CD3/CD28-activated cells had no effects, whereas Stattic abrogated IL-21 expression and cell proliferation. Thus, specific inhibition of STAT3 is highly efficient in the induction of apoptosis of Sézary cells, likely mediated via the regulation of (proto)oncogenes. In contrast, blocking IL-21 alone seems insufficient to affect STAT3 activation, cell proliferation, or apoptosis. These data provide further insights into the pathogenic role of STAT3 in Sézary syndrome and strengthen the notion that STAT3 represents a promising therapeutic target in this disease.

NOTCH1 signaling as a therapeutic target in Sézary syndrome.

NOTCH signaling is important for development and tissue homeostasis and is activated in many human cancers. We investigated a role for NOTCH1 signaling in Sézary syndrome (SS), a cutaneous T-cell lymphoma in which CD4+ tumor cells (Sézary cells) are present in the skin, lymph nodes, and peripheral blood. We show consistent expression of activated NOTCH1 by Sézary cells isolated from peripheral blood of SS patients, as well as the SS-derived cell lines SeAx and HuT78. In addition, immunohistochemical stainings of skin biopsies from SS patients showed consistent expression of nuclear NOTCH1 and its downstream target hairy/enhancer of split-1 (HES1) by Sézary cells. We demonstrate that this persistent NOTCH1 activation is not caused by mutations in the coding regions of NOTCH1 and F-box and WD40 domain protein 7 (FBWX7) genes. Inhibition of NOTCH1 signaling by gamma secretase inhibitors decreased cellular viability and induced apoptosis of Sézary cells. These observations argue that NOTCH1 signaling is functionally involved in the pathogenesis of SS, and inhibition of NOTCH1 signaling represents a new therapeutic target for the treatment of SS.

A novel mouse model for Sézary Syndrome using xenotransplantation of Sézary cells into immunodeficient RAG2−/− γc−/− mice

Sézary syndrome (SS) is an aggressive cutaneous T‐cell lymphoma with CD4+ tumor cells localized in the skin, lymph nodes and peripheral blood. Characteristic molecular aberrancies in SS have been identified; however, paucity of functional models severely hampered the translation of these observations into pathogenic mechanisms, and subsequent validation of novel therapeutic targets. We therefore developed a mouse model for SS using intrahepatic injection of SS cells in newborn immunodeficient RAG2−/− γc−/− mice that are completely devoid of T‐, B‐ and NK‐cell activity. Injection of the SS cell line SeAx led to long‐term and reproducible systemic repopulation of the mice. Injection of mice with the SS cell line HuT‐78 led to the death of the mice owing to massive growth of internal tumors. Four weeks after injection of primary SS cells, human CD3+ T cells could be tracked back in the liver, peripheral blood, lymph nodes, spleen and skin of the mice, although the engraftment rate varied when using cells from different patients. In conclusion, we demonstrate that injection of SS cell lines or primary cells in newborn RAG2−/− γc−/− mice results in long‐term systemic repopulation of the mice, thereby providing a novel mouse model for Sézary syndrome.

Mutation in PIGA Results in a CD52-Negative Escape Variant in a Sézary Syndrome Patient during Alemtuzumab Treatment

ted skin on UVR-blood flow doseresponse (Young et al., 1985; Flanagan et al., 2001). In view of an encouraging report, we also explored the possibility of using a* (‘redness’), a hemoglobin (Hb) index, and Hb oxygen (HbO2) saturation gained with a standard spectrophotometer (CM-600D; Konica Minolta) to describe erythemal responses (Kollias et al., 1994). However, we found that they by no means reached the level of reliability of the MFD in assessing minimal sunburn in pigmented skin (Supplementary Figure 1a–d online). MFD permits objective assessment of UVR-vasodilatation response, which is transferable between laboratories. Although our data suggest that 30% flux increase is a good threshold for MFD, this should be verified in a larger study with more extensive MED and MFD assessments. Laser speckle contrast imaging appears to be more sensitive and more reliable compared with other available objective methods, such as determining oxyhemoglobin levels from reflectance spectra. Hence, we find 785 nm laser speckle contrast imaging excellently suited to assess robustly a minimal sunburn reaction (by vasodilatation) even in heavily pigmented skin. Influence of skin pigmentation on UVR responses is an area of substantial current interest and controversy (Brenner and Hearing, 2008; Bogh et al., 2010; Farrar et al., 2011), and this technique will assist its further exploration.