Effect of Vitamin D on Peripheral Blood Mononuclear Cells from Patients with Psoriasis Vulgaris and Psoriatic Arthritis

BackgroundPsoriasis, a chronic skin disease with or without joint inflammation, has increased circulating proinflammatory cytokine levels. Vitamin D is involved in calcium homeostasis, bone formation, osteoclastogenesis and osteoclast activity, as well as regulation of immune response. We aimed to study osteoclast differentiation and cytokine secretion of peripheral blood mononuclear cells (PBMCs) from patients with psoriasis vulgaris and psoriatic arthritis, in response to 1,25(OH)₂D₃.MethodsSerum levels of bone turnover markers were measured by ELISA in patients with psoriasis vulgaris and psoriatic arthritis, and healthy controls. PBMCs were isolated and cultured with or without RANKL/M-CSF and 1,25(OH)₂D₃. Osteoclast differentiation and cytokine secretion were assessed.ResultsPsoriatic arthritis patients had lower osteocalcin, as well as higher C-telopeptide of type I collagen and cathepsin K serum levels compared with psoriasis vulgaris patients and controls. RANKL/M-CSF-stimulated PBMCs from psoriatic arthritis patients produced higher proinflammatory cytokine levels and had a differential secretion profile in response to 1,25(OH)₂D₃, compared with psoriasis vulgaris and control PBMCs.ConclusionsOur data confirmed altered bone turnover in psoriatic arthritis patients, and demonstrated increased osteoclastogenic potential and proinflammatory cytokine secretion capacity of these PBMCs compared with psoriasis vulgaris and controls. 1,25(OH)₂D₃ abrogated these effects.     

Transforming growth factor-beta 1 localization in normal and psoriatic epidermal keratinocytes in situ.

Transforming growth factor-beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell proliferation and its effects on growth and differentiation have been extensively characterized in cultured keratinocytes. We used two TGF beta 1-specific polyclonal antibodies (anti-LC and anti-CC) to determine the presence of TGF beta 1 peptide in keratinocytes in sections of normal human skin in situ and in both plaque and nonplaque skin from individuals with psoriasis. In contrast to the differentiation phenotype expressed by keratinocytes in normal epidermis, keratinocytes in the psoriatic plaque exhibit a hyperproliferative/regenerative differentiation phenotype. Anti-TGF beta 1 staining was observed primarily in the epidermis. Anti-LC TGF beta 1 antibody stained nonproliferating, differentiated suprabasal keratinocytes intracellularly in normal skin but did not stain psoriatic plaques from five of seven patients. In contrast, anti-CC TGF beta 1 antibody stained suprabasal keratinocytes extracellularly in psoriatic plaques, but did not stain normal skin. Both anti-LC and anti-CC stained suprabasal keratinocytes intracellularly in nonplaque psoriatic skin. Thus, the conformation or structure of TGF beta 1 and its localization vary in keratinocytes with distinct differentiation phenotypes suggesting that TGF beta 1 is a potential modulator of keratinocyte differentiation in vivo. Selective association of TGF beta 1 with nonproliferating keratinocytes in the suprabasal layers of the epidermis and its exclusion from the proliferating keratinocytes in the basal layer suggest that it may be a physiological regulator of keratinocyte proliferation. In addition, the intracellular localization of TGF beta 1 peptide in both normal and psoriatic keratinocytes suggests that it is constitutively synthesized by epidermal keratinocytes in vivo.     

Reduced TRPC channel expression in psoriatic keratinocytes is associated with impaired differentiation and enhanced proliferation

Psoriasis is a characteristic inflammatory and scaly skin condition with typical histopathological features including increased proliferation and hampered differentiation of keratinocytes. The activation of innate and adaptive inflammatory cellular immune responses is considered to be the main trigger factor of the epidermal changes in psoriatic skin. However, the molecular players that are involved in enhanced proliferation and impaired differentiation of psoriatic keratinocytes are only partly understood. One important factor that regulates differentiation on the cellular level is Ca(2+). In normal epidermis, a Ca(2+) gradient exists that is disturbed in psoriatic plaques, favoring impaired keratinocyte proliferation. Several TRPC channels such as TRPC1, TRPC4, or TRPC6 are key proteins in the regulation of high [Ca(2+)](ex) induced differentiation. Here, we investigated if TRPC channel function is impaired in psoriasis using calcium imaging, RT-PCR, western blot analysis and immunohistochemical staining of skin biopsies. We demonstrated substantial defects in Ca(2+) influx in psoriatic keratinocytes in response to high extracellular Ca(2+) levels, associated with a downregulation of all TRPC channels investigated, including TRPC6 channels. As TRPC6 channel activation can partially overcome this Ca(2+) entry defect, specific TRPC channel activators may be potential new drug candidates for the topical treatment of psoriasis.     

RAS-association domain family 1A regulates the abnormal cell proliferation in psoriasis via inhibition of Yes-associated protein

Psoriasis is a chronic, inflammatory skin disease with a high incidence and recurrence; however, its exact pathogenesis and aetiology remain unclear. This study aimed to analyse the effect of the upstream negative regulator RAS-association domain family 1A (RASSF1A) on Yes-associated protein (YAP) in psoriasis. Skin lesions of 22 patients with psoriasis and 19 healthy controls were used. Human epidermal keratinocytes stimulated by M5 (IL-1α, IL-17, IL-22, TNF-α and oncostatin M) were used to establish a psoriatic cell model. BALB/c mice treated with topical imiquimod were used to establish a psoriatic mouse model. As the methylation level of RASSF1A increased, its expression in psoriatic patients and mice model decreased. Addition of the methylation inhibitor 5-Aza-CdR or RASSF1A-overexpressing lentivirus vector increased RASSF1A and reduced YAP expression; meanwhile improved skin lesions, reduced cell proliferation, induced cell cycle arrest in the G0/G1 phase, increased apoptosis, reduced inflammatory cytokines and activities of ERK, STAT3 and NF-κB signalling pathways. The results indicated that RASSF1A could play a role in the treatment of psoriasis by inhibiting YAP expression. Based on these findings, targeted drugs that can inhibit the methylation or increase the expression of RASSF1A may be useful for treating psoriasis.     

Psoriasis alters HDL composition and cholesterol efflux capacity

Psoriasis, a chronic inflammatory skin disease, has been linked to increased myocardial infarction and stroke. Functional impairment of HDL may contribute to the excess cardiovascular mortality of psoriatic patients. However, data available regarding the impact of psoriasis on HDL composition and function are limited. HDL from psoriasis patients and healthy controls was isolated by ultracentrifugation and shotgun proteomics, and biochemical methods were used to monitor changed HDL composition. We observed a significant reduction in apoA-I levels of HDL from psoriatic patients, whereas levels of apoA-II and proteins involved in acute-phase response, immune response, and endopeptidase/protease inhibition were increased. Psoriatic HDL contained reduced phospholipid and cholesterol. With regard to function, these compositional alterations impaired the ability of psoriatic HDL to promote cholesterol efflux from macrophages. Importantly, HDL-cholesterol efflux capability negatively correlated with psoriasis area and severity index. We observed that control HDL, as well as psoriatic HDL, inhibited dihydrorhodamine (DHR) oxidation to a similar extent, suggesting that the anti-oxidative activity of psoriatic HDL is not significantly altered. Our observations suggest that the compositional alterations observed in psoriatic HDL reflect a shift to a pro-inflammatory profile that impairs cholesterol efflux capacity of HDL and may provide a link between psoriasis and cardiovascular disease.     

MiR-20a-3p regulates TGF-β1/Survivin pathway to affect keratinocytes proliferation and apoptosis by targeting SFMBT1 in vitro

Psoriasis is a common immune-mediated chronic inflammatory skin disease characterized by abnormal keratinocyte proliferation, differentiation and apoptosis. However, the exact etiology and pathogenesis are still unclear. Evidence is rapidly accumulating for the role of microRNAs in psoriasis. It has been demonstrated that Interleukin-22 (IL-22) plays vital role in T cell-mediated immune response by interacting with keratinocytes in the pathogenesis of psoriasis. The aim of our study was to explore the possible functional role of miR-20a-3p in psoriasis and in IL-22 induced keratinocyte proliferation. Here, we found that miR-20a-3p was down-regulated in psoriatic lesions and in HaCaT cells (human keratinocyte cell line) treated by IL-22 stimulation. Functional experiments showed that overexpression of miR-20a-3p in HaCaT cells suppressed proliferation and induced apoptosis while its knockdown promoted cell proliferation and reduces cell apoptosis. Mechanistically, SFMBT1 was identified as the direct target of miR-20a-3p by dual luciferase reporter assay. SFMBT1 knockdown was demonstrated to inhibit cell growth and induced apoptosis, which was consistent with the function of miR-20a-3p upregulation in HaCaT cells. In addition, results of western blot analysis showed that miR-20a-3p upregulation or SFMBT1 knockdown changed the protein expression levels of TGF-β1 and survivin. Our findings suggest that miR-20a-3p play roles through targeting SFMBT1 and TGF-β1/Survivin pathway in HaCaT cells, and loss of miR-20a-3p in psoriasis may contribute to hyperproliferation and aberrant apoptosis of keratinocytes.     

The role of LncRNA MALAT-1 and MiRNA-9 in Psoriasis

BackgroundPsoriasis is a chronic skin disorder manifested by recurrent episodes of scaly, red, itchy skin patches that occur within apparently normal skin.ObjectivesThis study was performed to detect the expression of serum and tissue (lesion and non-lesion) LncRNA MALAT-1 and MiRNA-9 that might be used as biomarkers for psoriasis.MethodsBlood samples were obtained from 60 psoriasis patients and 40 controls, as well as 4 mm punch biopsy from lesional and non lesional skin of psoriatic patient and normal skin of healthy controls. Expression of LncRNA MALAT-1 and miRNNA-9 in serum and tissues was detected by real time qRT-PCR.Resultsa statistically significant increase in the expression of MALAT-1 in lesional and non-lesional skin and serum of psoriatic patients in comparison to controls were detected. Moreover, there was statistically significant increase in serum MiRNA-9 in patients in comparison to controls, while its tissue level was significantly lower in patients.ConclusionThis study highlights the dysregulation of LncRNA MALAT-1 and miRNA-9 in psoriasis. Elevated expression of MALAT-1 in lesional skin of psoriatic patients compared to non-lesional skin may possibly contribute to the development of psoriatic plaques.     

Potential role of chemerin in recruitment of plasmacytoid dendritic cells to diseased skin

Interferon α-producing plasmacytoid dendritic cells (pDC) are crucial contributors to pro-inflammatory or tolerogenic immune responses and are important in autoimmune diseases such as psoriasis. pDC accumulate in the lesional skin of psoriasis patients, but are rarely found in the affected skin of patients with atopic dermatitis (AD). While homeostatic chemokine CXCL12 and inducible pro-inflammatory CXCR3 chemokine ligands may regulate pDC influx to psoriatic skin, the mechanism responsible for selective pDC recruitment in psoriasis vs. AD remains unknown. Circulating pDC from normal donors express a limited number of chemoattractant receptors, including CXCR3 and CMKLR1 (chemokine-like receptor 1). In this work, we demonstrate that circulating pDC from normal donors as well as psoriasis and AD patients express similar levels of CXCR3 and responded similarly in functional migration assays to CXCL10. We next found that blood pDC from normal, AD, and psoriasis patients express functional CMKLR1. In contrast to normal skin, however, lesional skin from psoriasis patients contains the active form of the CMKLR1 ligand chemerin. Furthermore, in affected skin from psoriatic patients the level of active chemerin was generally higher than in AD skin. Taken together, these results indicate that local generation of active chemerin may contribute to pDC recruitment to psoriatic skin.     

Regulation of skin inflammation and angiogenesis by EC-SOD via HIF-1α and NF-κB pathways

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that breaks down superoxide anion into oxygen and hydrogen peroxide in extracellular spaces and plays key roles in controlling pulmonary and vascular diseases in response to oxidative stresses. We aimed to investigate the role of EC-SOD in angiogenesis and inflammation in chronic inflammatory skin disorders such as psoriasis. Overexpressed EC-SOD reduced expression of angiogenic factors and proinflammatory mediators in hypoxia-induced keratinocytes and in ultraviolet B-irradiated mice, whereas the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase-1 and anti-inflammatory cytokine interleukin-10 were increased. EC-SOD decreased new vessel formation, epidermal edema, and inflammatory cell infiltration in UVB-irradiated transgenic mice. Moreover, cells treated with recombinant human EC-SOD showed inhibited endothelial tube formation and cell proliferation. Overall, the antiangiogenic and anti-inflammatory effects of EC-SOD might be due to suppression of hypoxia-inducible factor-1α, protein kinase C, and nuclear factor-κB expression. Furthermore, EC-SOD expression in tissue from psoriasis patients was markedly decreased in psoriatic lesional and nonlesional skins from psoriasis patients in comparison to normal skin from healthy volunteers. Together, these results suggest that EC-SOD may provide a novel therapeutic approach to treating angiogenic and inflammatory skin diseases such as psoriasis.     

S100A7: A rAMPing up AMP molecule in psoriasis

S100A7 (psoriasin), an EF-hand type calcium binding protein localized in epithelial cells, regulates cell proliferation and differentiation. An S100A7 overexpression may occur in response to inflammatory stimuli, such in psoriasis, a chronic inflammatory autoimmune-mediated skin disease. Increasing evidence suggests that S100A7 plays critical roles in amplifying the inflammatory process in psoriatic skin, perpetuating the disease phenotype. This review will discuss the interactions between S100A7 and cytokines in psoriatic skin. Furthermore, we will focus our discussion on regulation and functions of S100A7 in psoriasis. Finally, we will discuss the possible use of S100A7 as therapeutic target in psoriasis.     

Secretory leukocyte proteinase inhibitor-competent DNA deposits are potent stimulators of plasmacytoid dendritic cells: implication for psoriasis

Secretory leukocyte proteinase inhibitor (SLPI) is a well-established inhibitor of serine proteases such as human neutrophil elastase (HNE) and a NF-κB regulatory agent in immune cells. In this paper, we report that SLPI plays a previously uncharacterized role in regulating activation of plasmacytoid dendritic cells (pDCs). As the main source of IFN type I (IFNI), pDCs are crucial contributors to inflammatory and likely wound-healing responses associated with psoriasis. The mechanisms responsible for activation of pDCs in psoriatic skin are therefore of substantial interest. We demonstrate that in lesional skin of psoriasis patients, SLPI together with its enzymatic target HNE and DNA, is a component of neutrophil extracellular traps (NETs). Whereas SLPI(+) neutrophils and NETs were found to colocalize with pDCs in psoriatic skin, a mixture of SLPI with neutrophil DNA and HNE induced a marked production of IFNI by pDCs. IFNI synthesis by stimulated pDCs was dependent on intracellular DNA receptor TLR9. Thus, SLPI may contribute to psoriasis by enabling pDCs to sense extracellular DNA and produce IFNI.     

Identification of extra- and intracellular alanyl aminopeptidases as new targets to modulate keratinocyte growth and differentiation

Aminopeptidase inhibitors strongly affect proliferation, differentiation, and function of immune cells and show therapeutic potential in inflammatory disorders. In psoriatic lesions, keratinocytes display increased cellular turnover and disturbed differentiation, leading to epidermal hyperplasia accompanied by the loss of stratum granulosum. Here, we report in the HaCaT hyperproliferative keratinocyte cell line as well as in two primary keratinocyte strains in vitro a molecular and biochemical analysis of the expression of both membrane and cytosol alanyl aminopeptidase (cAAP) on the mRNA, protein, and enzymatic activity level. We found a clear dose-dependent suppression of DNA synthesis in vitro in the presence of the inhibitors actinonin, bestatin, and the cAAP-specific inhibitor PAC-22 correlating well with the simultaneous decrease in enzyme activity. In vivo, actinonin dose-dependently restored the stratum granulosum and ameliorated the impaired keratinocyte differentiation in the mouse tail model of psoriasis. Taken together, these data suggest that targeting alanyl aminopeptidases may be beneficial for psoriasis and other inflammatory skin disorders.     

Biological action of docosahexaenoic acid in a 3D tissue-engineered psoriatic skin model: Focus on the PPAR signaling pathway

N-3 polyunsaturated fatty acids (n-3 PUFAs), and in particular docosahexaenoic acid (DHA), have many beneficial metabolic effects, including reducing epidermal thickness in patients with psoriasis. The positive impacts of DHA in psoriasis could be mediated by its interactions with the PPAR signaling pathway, as well as by its secretion of anti-inflammatory bioactive metabolites, but the detailed metabolism is still not understood. In the present study, we evaluated the influence of DHA on the main features of psoriasis and its effects on the PPAR signaling pathway, in a psoriatic in vitro skin model. Healthy and psoriatic skin substitutes were produced according to the tissue-engineered self-assembly method, using culture media supplemented with 10 μM of DHA. The presence of DHA led to a reduction in the abnormal cell differentiation of psoriatic keratinocytes, seen in the increased expression of filaggrin and keratin 10. DHA was incorporated into the membrane phospholipids of the epidermis and transformed principally into eicosapentaenoic acid (EPA). Furthermore, the addition of DHA into the culture medium led to a decrease in the levels of lipid mediators derived from n-6 PUFAs, mainly prostaglandin E₂ (PGE₂) and 12-hydroxyeicosatetraenoic acid (12-HETE). Finally, DHA supplementation rebalanced the expression of PPAR receptors and caused a decrease in the secretion of TNF-α. Altogether, our results show that DHA possesses the ability to attenuate the psoriatic characteristics of psoriatic skin substitutes, mostly by restoring epidermal cell differentiation and proliferation, as well as by reducing inflammation.     

Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells

Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and play an important role in development and in many cellular processes. We have found that BMP-2, BMP-6, and BMP-9 induce the most potent osteogenic differentiation of mesenchymal stem cells. Expression profiling analysis has revealed that the Inhibitors of DNA binding/differentiation (Id)-1, Id-2, and Id-3 are among the most significantly up-regulated genes upon BMP-2, BMP-6, or BMP-9 stimulation. Here, we sought to determine the functional role of these Id proteins in BMP-induced osteoblast differentiation. We demonstrated that the expression of Id-1, Id-2, and Id-3 genes was significantly induced at the early stage of BMP-9 stimulation and returned to basal levels at 3 days after stimulation. RNA interference-mediated knockdown of Id expression significantly diminished the BMP-9-induced osteogenic differentiation of mesenchymal progenitor cells. Surprisingly, a constitutive overexpression of these Id genes also inhibited osteoblast differentiation initiated by BMP-9. Furthermore, we demonstrated that BMP-9-regulated Id expression is Smad4-dependent. Overexpression of the three Id genes was shown to promote cell proliferation that was coupled with an inhibition of osteogenic differentiation. Thus, our findings suggest that the Id helix-loop-helix proteins may play an important role in promoting the proliferation of early osteoblast progenitor cells and that Id expression must be down-regulated during the terminal differentiation of committed osteoblasts, suggesting that a balanced regulation of Id expression may be critical to BMP-induced osteoblast lineage-specific differentiation of mesenchymal stem cells.     

Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation

The balance between regulatory and effector functions is important for maintaining efficient immune responses, while avoiding autoimmunity. The inflammatory skin disease psoriasis is sustained by the ongoing activation of pathogenic effector T cells. We found that a CD4(+) T lymphocyte subpopulation in peripheral blood, phenotypically CD25(high), CTLA-4(+), Foxp3(high) (regulatory T (Treg) cells), is deficient in its suppressor activity in psoriasis. This was associated with accelerated proliferation of CD4(+) responder T cells in psoriasis, the majority of which expressed CXCR3. Nevertheless, criss-cross experiments isolated the defect to psoriatic Treg cells. To examine Treg cells in a nonlymphoid tissue of a human T cell-mediated disease, Treg cells were also analyzed and isolated from the site of inflammation, psoriatic lesional skin. At the regulatory vs effector T cells ratios calculated to be present in skin, however, the psoriatic Treg cell population demonstrated decreased suppression of effector T cells. Thus, dysfunctional blood and target tissue CD4(+)CD25(high) Treg cell activity may lead to reduced restraint and consequent hyperproliferation of psoriatic pathogenic T cells in vivo. These findings represent a critical component of human organ-specific autoimmune disease and may have important implications with regard to the possible therapeutic manipulation of Treg cells in vivo.