Michał J. Kowalczyk

Expression of selected human endogenous retroviral sequences in skin and peripheral blood mononuclear cells in morphea.

Introduction Morphea or localized scleroderma is a relatively rare disease whose main symptom is excessive skin fibrosis. Here we focus on the involvement of human endogenous retroviruses (HERVs) in morphea. The HERVs are a vast and intensely growing field in genomics. HERVs are of special interest as far as autoimmune disorders are concerned, yet little effort has been made until now to assess the possible changes of their expression in morphea. Material and methods Six sequences of particular interest were chosen for this study. Real-time polymerase chain reaction was performed on samples derived from peripheral blood mononuclear cells (PBMCs) and skin biopsies. The results were normalized to the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcription. Results In PBMCs we found a statistically significant decrease of transcription of HERV-E pol, while HERV-K env, HERV-R pol-env, and HERV-W env were found to be up-regulated. In skin biopsies HERV-K env was strongly up-regulated. On the other hand, we noted a decrease of transcription of HERV-H env 62, HERV-K10 gag, HERV-R pol-env, and HERV-W env. In PBMCs we found a statistically significant decrease of transcription of HERV-E pol (–81.8%, p < 0.001), while HERV-K env (+94.1%, p = 0.010), HERV-R pol-env (+140.0%, p < 0.001), and HERV-W env (+97.7%, p < 0.001) were found to be up-regulated. In skin biopsies HERV-K env was strongly up-regulated (+713.0%, p = 0.003). On the other hand, we noted a decrease of transcription of HERV-H env 62 (–83.5%, p < 0.001, HERV-K10 gag (-33.7%, p = 0.044), HERV-R pol-env (–71.3%, p < 0.001), and HERV-W env (–59.3%, p = 0.029). Conclusions The studied HERV sequences generally show an increase of transcription in PBMCs of morphea patients, while being down-regulated in their skin, with some exceptions for both types of tissue.

The role of altered cutaneous immune responses in the induction and persistence of rosacea.

Rosacea is a chronic inflammatory skin condition that predominantly affects the skin of the face and the eyes. Several factors are associated with the onset and persistence of the condition, including an altered immune response in the skin and elevated levels of Demodex mites. Alterations in the immune response include elevated levels of LL-37 in rosacea skin, increased expression of TLR-2 and increased amounts of vitamin D3 in epidermal tissue. The combined effect of these changes may make the skin more sensitive to external and internal stimuli. External stimuli that may trigger or sustain rosacea inflammation include exposure to ultraviolet light, while internal factors may include the presence of elevated numbers of Demodex mites. These mites may directly stimulate an immune response or release bacteria within the pilosebaceous unit that act as a trigger for inflammation. This review will highlight the changes that occur in the immune response of the skin and describe how Demodex mites and associated bacteria may activate this response and lead to the characteristics of rosacea.

Transforming growth factor-β1 in plaque morphea

Introduction Morphea (localized scleroderma) is a rare cutaneous disease characterized by skin fibrosis of unknown pathogenesis. Transforming growth factor-β (TGF-β) is a potent profibrotic factor. The role of TGF-β in morphea remains unclear. Aim The goal of this study was to estimate the expression level of TGF-β1 in skin and peripheral blood mononuclear cells as well as the plasma levels of TGF-β1 in plaque morphea (MEP). Material and methods The study involved 20 MEP patients. Three control groups were involved: 1 – plasma: 36 healthy volunteers; 2 – PBMC: 47 healthy volunteers; 3 – skin biopsies: 13 samples collected during mastectomy (breast cancer was not skin involved). The analysis of TGF-β1 plasma levels was performed with the use an adequate ELISA kit, while real-time polymerase chain reaction was employed for the expression of TGF-β1 in peripheral blood mononuclear cells (PBMC) and skin. Results In our study we have not detected differences in TGF-β 1 expression in PBMC, skin, nor in plasma levels of TGF-β1 between MEP patients and healthy controls, regardless of disease activity and its duration. Conclusions The results of our study contradict the claim of the substantial role of TGF-β1 in the most common morphea subtype – MEP.

The role of dendritic cells and regulatory T cells in the pathogenesis of morphea.

Morphea is one of diseases characterised by fibrosis of the skin and subcutaneous tissue. It is a chronic disease that does not shorten the life of the patient, yet significantly affects its quality. The group of factors responsible for its pathogenesis is thought to include disturbed functioning of endothelial cells as well as immune disturbances leading to chronic inflammatory conditions, accompanied by increased production of collagen and of other extracellular matrix components. Dendritic cells (DC) are a type of professional antigen-presenting cells and can be found in almost all body tissues. Individual investigations have demonstrated high numbers of plasmacytoid DC (pDC) in morphoeic skin lesions, within deeper dermal layers, around blood vessels, and around collagen fibres in subcutaneous tissue. It appears that DC has a more pronounced role in the development of inflammation and T cell activation in morphea, as compared to systemic sclerosis (SSc). Regulatory T (Treg) cells represent a subpopulation of T cells with immunosuppressive properties. Recent studies have drawn attention to the important role played by Treg in the process of autoimmunisation. Just a few studies have demonstrated a decrease in the number and activity of Treg in patients with SSc, and only such studies involve morphea. This article reviews recent studies on the role of DC and regulatory T cells in the pathogenesis of morphea. Moreover, mechanisms of phototherapy and potential therapeutic targets in the treatment of morphea are discussed in this context.

Human endogenous retroviruses and chosen disease parameters in morphea

Introduction Morphea (localized scleroderma) is a relatively rare disease characterized by excessive skin fibrosis. Human endogenous retroviruses (HERV) are largely distributed within the human genome with hundreds of thousands of elements. The HERV have been widely studied in autoimmune disorders, yet hardly ever assessed in diseases with a good prognosis such as morphea. Aim In this study we focus on the possible relations between the expression of chosen HERV and factors influencing the pathomechanism of the disease, such as age, sex, titres of anti-nuclear antibodies, as well as duration, activity, and severity of the disease (LoSSI index). Material and methods Real-time polymerase chain reaction (PCR) targeting six HERV sequences of interest were performed on samples derived from peripheral blood mononuclear cells (PBMC) and skin biopsies. Results In PBMC we found a statistically significant negative correlation between HERV-W env expression and LoSSI index (p = 0.01). Additionally, HERV-W env was downregulated in patients with the active form of morphea. In all other cases we found no correlation whatsoever nor statistically significant differences below the p = 0.05 threshold. Conclusions Morphea seems to be an autoimmune disease where the impact of HERV is not so apparent. It seems that probing many patients for the expression of just a few sequences is not as effective as previously expected. For initial studies of HERV in other diseases we recommend high throughput techniques such as HERV-dedicated DNA microarrays or massive parallel sequencing.

Expression of selected genes of dendritic and Treg cells in blood and skin of morphea patients treated with UVA1 phototherapy

Introduction Morphea is a chronic autoimmune disease characterized by fibrosis of the skin. Dendritic cells (DC) and regulatory T cells (Tregs) play a significant role in development of autoimmune and tolerance mechanisms. The aim of the study was to establish the expression of selected genes of plasmacytoid and myeloid DC, Treg cells, and the microenvironment of cytokines (interleukin-17A (IL-17A), transforming growth factor β (TGF-β)) in blood and skin of morphea patients. In addition, the effect of UVA1 phototherapy on expression of the aforementioned genes was evaluated. Material and methods The study was performed on 15 blood and 10 skin samples from patients with morphea. The evaluation included expression of CLEC4C (C-type lectin domain family 4, member C receptor), Lymphocyte antigen 75 (LY75), Forkhead box p3 (foxp3) transcription factor, IL-17A and TGF-β genes in peripheral blood mononuclear cells (PBMC) and in skin samples both before and after UVA1 phototherapy using real-time polymerase chain reaction. Results The study revealed lower expression of CLEC4C before (p = 0.010) and after (p = 0.009) phototherapy and lower expression of IL-17A before (p = 0.038) phototherapy in PBMC of patients with morphea vs. the control group. Expression of CLEC4C in PBMC correlated negatively (rho = –0.90; p = 0.001) with activity of disease after phototherapy. No significant differences were found between expression of analysed genes before and after UVA1 therapy in PBMC and skin of morphea patients. Conclusions The results do not confirm the involvement of analysed subsets of DC and Tregs in UVA1 phototherapy in morphea, but point to CLEC4C as a possible biomarker associated with the disease activity.

Effects of UVA1 Phototherapy on Expression of Human Endogenous Retroviral Sequence (HERV)-K10 gag in Morphea: A Preliminary Study.

Background Morphea, also known as localized scleroderma, is a rare autoimmune connective tissue disease characterized by skin fibrosis. UVA1 phototherapy is an important asset in the reduction of clinical manifestations in morphea. There are studies claiming that UV light modulates the expression of some human endogenous retroviral sequences. The aim of this study was to determine if the expression of HERV-K10 gag element is lowered by UVA1 phototherapy in morphea, a disease in which such irradiation has a soothing effect. Material/Methods The expression levels of the HERV-K10 gag were assessed by real-time PCR (polymerase chain reaction) in peripheral blood mononuclear cells (PBMC) and skin-punch biopsies of healthy volunteers and 9 morphea patients before and after phototherapy. Additionally, correlations between the HERV-K10 gag expression and age, disease duration, the Localized Scleroderma Skin Severity Index (LoSSI), and antinuclear antibody (ANA) titers were assessed. Results In PBMC, HERV-K10 gag mRNA was significantly elevated after UVA1 phototherapy compared to healthy controls. Most of the patients responded with an increased expression level of this sequence. However, we found no statistical evidence at this point that phototherapy indeed has an effect on the HERV-K10 gag expression (there were no statistical differences in PBMC of morphea patients before and after phototherapy). Similarly, there was no statistically relevant effect of the UVA1 on the expression of HERV-K10 gag in skin. Conclusions At this point, the effect of UVA1 phototherapy on the expression of HERV-K10 gag cannot be statistically confirmed.