Negative regulation of dendritic cell activation in psoriasis mediated via CD100–plexin-B2

Psoriasis is a chronic inflammatory skin disease in which dendritic cells (DCs) play a pivotal role by inducing Th1/Th17 immune responses; however, the regulation of DC activation in psoriasis remains largely unknown. Previously we found that the level of soluble CD100 was increased in sera of psoriasis patients, and CD100 promoted the activation of inflammasome in keratinocytes. In the present study, CD100 knockout mice were utilized for generation of imiquimod (IMQ)-induced psoriatic dermatitis, with the result that skin inflammation in the early, but not late, phase of the psoriatic dermatitis was significantly exacerbated compared to that in wild-type controls. This was attributed mainly to the deficiency of CD100 in hematopoietic cells. Bone marrow-derived DCs, but not T cells or keratinocytes, from CD100 knockout mice produced significantly increased levels of IL-1β, IL-36, and IL-23 upon stimulation with IMQ in a plexin-B2-dependent manner. Moreover, the surface level of plexin-B2 on DCs of psoriasis patients was lower than that of healthy individuals, and CD100 attenuated IMQ-induced production of IL-1β and IL-36 from monocyte-derived DCs of psoriasis patients. Our results uncovered a negative regulatory mechanism for DCs activation in psoriasis, which was mediated via CD100–plexin-B2 in a cell type- and receptor-specific manner. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Inhibitory effect of Paeonia lactiflora Pallas extract (PE) on poly (I:C)-induced immune response of epidermal keratinocytes

Epidermal keratinocytes provide protective role against external stimuli by barrier formation. In addition, kertinocytes exerts their role as the defense cells via activation of innate immunity. Disturbance of keratinocyte functions is related with skin disorders. Psoriasis is a common skin disease related with inflammatory reaction in epidermal cells. We attempted to find therapeutics for psoriasis, and found that Paeonia lactiflora Pallas extract (PE) has an inhibitory potential on poly (I:C)-induced inflammation of keratinocytes. PE significantly inhibited poly (I:C)-induced expression of crucial psoriatic cytokines, such as IL-6, IL-8, CCL20 and TNF-α, via down-regulation of NF-κB signaling pathway in human keratinocytes. In addition, PE significantly inhibited poly (I:C)-induced inflammasome activation, in terms of IL-1β and caspase-1 secretion. Finally, PE markedly inhibited poly (I:C)-increased NLRP3, an important component of inflammasome. These results indicate that PE has an inhibitory effect on poly (I:C)-induced inflammatory reaction of keratinocytes, suggesting that PE can be developed for the treatment of psoriasis.     

IL-1β and IFN-γ induce the regenerative epidermal phenotype of psoriasis in the transwell skin organ culture system. IFN-γ up-regulates the expression of keratin 17 and keratinocyte transglutaminase via endogenous IL-1 production

Skin biopsies from healthy human skin and non-lesional skin from patients with psoriasis were cultured for 24h and stimulated with interleukin-1β(IL-1β) and interferon-γ (IFN-γ) in a skin organ culture model and the induction of the psoriasiform regenerative epidermal phenotype was analysed using immunostaining. In the presence of IL-1β, the psoriasiform regenerative epidermal phenotype was clearly induced. This involved strong up-regulation of the expression of keratin 16, keratin 17, and keratinocyte transglutaminase (TGk) in the suprabasal layers, strong up-regulation and a shift of the expression of keratin 5 and integrin β₁from the basal to suprabasal keratinocytes, and induction of the expression of ICAM-1 and HLA-DR on basal keratinocytes. The effects of IL-1β in the organ cultures of normal skin could be completely neutralized by anti-IL-1 polyclonal antibodies. The effects of IFN-γ in healthy and non-lesional psoriatic skin were qualitatively similar to those of IL-1β. The IFN-γ-induced epidermal expression of keratin 17 and TGk could be completely blocked by culturing the biopsies in the presence of IL-1ra or anti-IL-1 antibodies, while the induction of HLA-DR and ICAM-1 was not inhibited. The induction of the psoriasiform regenerative epidermal phenotype by IFN-γ is partially mediated via endogenous epidermal IL-1. Copyright © 1999 John Wiley & Sons, Ltd.     

Revisiting the Koebner phenomenon: role of NGF and its receptor system in the pathogenesis of psoriasis

Nerve growth factor (NGF) influences the key pathological events of psoriasis: keratinocyte proliferation, angiogenesis, and T-cell activation. We have systematically examined the kinetics of NGF expression, keratinocyte proliferation, and migration of T lymphocytes in the epidermis in Koebner-induced developing psoriatic plaques. In skin traumatized by the tape-stripping method (n = 12), a marked up-regulation of NGF in Koebner-positive lesions (n = 7) was observed 24 hours after trauma. Synthesis of NGF reached its maximum level in the 2nd week. Furthermore, cultured keratinocytes from nonlesional skin of psoriasis patients produced 10 times higher levels of NGF compared with keratinocytes from healthy individuals. To substantiate the in vivo effect of NGF secreted by keratinocytes in psoriatic plaques, we studied psoriatic plaques and normal human skin in a SCID-human skin xenograft model. The transplanted psoriatic plaques demonstrated marked proliferation of NGF-R (p75)-positive nerve fibers compared with only a few nerves in the transplanted normal human skin. Our results demonstrate that 1) in a developing psoriatic lesion, up-regulation of NGF together with keratinocyte proliferation are early events and precede epidermotropism of T lymphocytes; 2) keratinocytes in patients with psoriasis are primed to produce elevated levels of NGF; and 3) NGF synthesized by these keratinocytes is functionally active.     

The antimicrobial protein REG3A regulates keratinocyte proliferation and differentiation after skin injury

Epithelial keratinocyte proliferation is an essential element of wound repair, and abnormal epithelial proliferation is an intrinsic element in the skin disorder psoriasis. The factors that trigger epithelial proliferation in these inflammatory processes are incompletely understood. Here we have shown that regenerating islet-derived protein 3-alpha (REG3A) is highly expressed in keratinocytes during psoriasis and wound repair and in imiquimod-induced psoriatic skin lesions. The expression of REG3A by keratinocytes is induced by interleukin-17 (IL-17) via activation of keratinocyte-encoded IL-17 receptor A (IL-17RA) and feeds back on keratinocytes to inhibit terminal differentiation and increase cell proliferation by binding to exostosin-like 3 (EXTL3) followed by activation of phosphatidylinositol 3 kinase (PI3K) and the kinase AKT. These findings reveal that REG3A, a secreted intestinal antimicrobial protein, can promote skin keratinocyte proliferation and can be induced by IL-17. This observation suggests that REG3A may mediate the epidermal hyperproliferation observed in normal wound repair and in psoriasis.     

Increased expression of interleukin-4 receptors on psoriatic epidermal cells.

Abnormal keratinocyte proliferation and differentiation, the main characteristics of psoriasis, may be induced and maintained by cytokines produced by activated resident and recruited inflammatory cells in lesional skin. As the epidermal cytokine profile is clearly altered in psoriasis and because increased expression of interleukin-4 receptor (IL-4R) has been reported in some epithelial proliferative diseases, we investigated the expression of IL-4R on psoriatic epidermal cells (ECs). The expression of IL-4R on freshly isolated ECs from healthy skin and untreated psoriatic lesions was studied by immunostaining using an IL-4R-specific antibody and by examining their capacity to bind biotinylated recombinant human IL-4 using flow cytometry. The number of IL-4R+ ECs and the number of binding sites per cell were significantly increased on psoriatic ECs as compared with healthy control ECs. Immunostaining confirmed these results, whereby staining was mainly observed in the lower epidermal layers. In addition, an increased IL-4R mRNA expression was also observed in psoriatic epidermis using a digoxigenin-labeled IL-4R RNA probe. In short-term in vitro cultures, lipopolysaccharide/phorbol-myristate-acetate-stimulated and unstimulated psoriatic ECs did not produce any immunoreactive IL-4. The results of this study together with the reported increased expression of IL-4R in epithelial neoplasias suggest an association between overexpression of IL-4R and abnormal keratinocyte activation and proliferation.     

Proinflammatory effect of high‐mobility group protein B1 on keratinocytes: an autocrine mechanism underlying psoriasis development

Psoriasis is an autoimmune skin disease, in which keratinocytes play a crucial pathogenic role. High‐mobility group protein B1 (HMGB1) is an inflammatory factor that can be released from keratinocyte nuclei in psoriatic lesions. We aimed to investigate the proinflammatory effect of HMGB1 on keratinocytes and the contribution of HMGB1 to psoriasis development. Normal human keratinocytes were treated with recombinant human HMGB1, and the production of inflammatory factors and the intermediary signalling pathways were examined. Furthermore, the imiquimod‐induced psoriasis‐like mouse model was used to investigate the role of HMGB1 in psoriasis development in vivo. A total of 11 inflammatory factors were shown to be upregulated by HMGB1 in keratinocytes, among which interleukin (IL)‐18 showed the greatest change. We then found that activation of the nuclear factor‐κB signalling pathway and inflammasomes accounted for HMGB1‐induced IL‐18 expression and secretion. Moreover, HMGB1 and downstream IL‐18 contributed to the development of psoriasiform dermatitis in the imiquimod‐treated mice. In addition, T‐helper 17 immune response in the psoriasis‐like mouse model could be inhibited by both HMGB1 and IL‐18 blockade. Our findings indicate that HMGB1 secreted from keratinocytes can facilitate the production and secretion of inflammatory factors such as IL‐18 in keratinocytes in an autocrine way, thus promoting the development of psoriasis. Blocking the proinflammatory function of the HMGB1–IL‐18 axis may be useful for psoriasis treatment in the future. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Interleukin-18 expression and the response to treatment in patients with psoriasis

Introduction

The aim of the study was to demonstrate Interleukin-18 (IL-18) expression in keratinocytes from psoriatic lesions in comparison to keratinocytes from uninvolved skin and to study the change of expression after therapeutic interventions.

Material and methods

This study included 16 patients of different clinical subtypes of psoriasis. IL-18 gene expression analysis was performed using real-time quantitative PCR. Three biopsies were obtained from each patient. Two were taken from the lesional psoriatic skin and from uninvolved skin before starting treatment. A third lesional skin biopsy was taken at the end of two months'' treatment course. The treatment was in the form of topical steroids or oral systemic methotrexate.

Results

Of all 16 studied patients significantly increased IL-18 expression was noted in keratinocytes from psoriatic lesions before and after treatment when compared to keratinocytes from uninvolved skin (P = 0.001 and 0.002 respectively). The IL-18 expression in the skin lesions after treatment was significantly lower than lesional skin before treatment (P = 0.023). In psoriatic skin lesions of all studied patients IL-18 expression was significantly correlated with disease duration (r = 0.40 and P = 0.01) and clinical severity of psoriasis (r = 0.72 and P = 0.001).

Conclusions

Increased IL-18 expression in keratinocytes from psoriatic lesions of our patients and its correlation with disease duration and severity supported the concept which views psoriasis as a T-cell-mediated autoimmune disease. This could establish therapeutic and preventive approaches for psoriasis that ultimately lead to improved outcomes for patients.     

Interleukin-18 expression and the response to treatment in patients with psoriasis

Introduction

The aim of the study was to demonstrate interleukin-18 (IL-18) expression in keratinocytes from psoriatic lesions in comparison to keratinocytes from uninvolved skin and to study the change of expression after therapeutic interventions.

Material and methods

This study included 16 patients of different clinical subtypes of psoriasis. Interleukin-18 gene expression analysis was performed using real time quantitative PCR. Three biopsies were obtained from each patient. Two were taken from the lesional psoriatic skin and from uninvolved skin before starting treatment. A third lesional skin biopsy was taken at the end of 2 months of treatment. The treatment was in the form of topical steroids or oral systemic methotrexate.

Results

Of all 16 studied patients, significantly increased IL-18 expression was noted in keratinocytes from psoriatic lesions before and after treatment when compared to keratinocytes from uninvolved skin (p = 0.001 and p = 0.002 respectively). The IL-18 expression in the skin lesions after treatment was significantly lower than lesional skin before treatment (p = 0.023). In psoriatic skin lesions of all studied patients IL-18 expression was significantly correlated with disease duration (r = 0.40 and p = 0.01) and clinical severity of psoriasis (r = 0.72 and p = 0.001).

Conclusions

Increased IL-18 expression in keratinocytes from psoriatic lesions of our patients and its correlation with disease duration and severity supported the concept of psoriasis as a T cell mediated autoimmune disease. This could establish therapeutic and preventive approaches for psoriasis that ultimately lead to improved outcomes for patients.     

Role of NLRP3 and CARD8 in the regulation of TNF-α induced IL-1β release in vascular smooth muscle cells

Interleukin (IL)-1β is known to be activated by the inflammasome. Inflammasome activities depend on a plethora of moieties including NLRP3 and CARD8, which have been reported to be associated with several inflammatory diseases. Aortic smooth muscle cells (AOSMCs) were transfected with siRNA targeting the NLRP3 and CARD8 genes, followed by tumor necrosis factor-α (TNF-α) treatment. We found that TNF-α induces IL-1β, IL-1Ra and NLRP3 genes but not CARD8. Silencing of the NLRP3 gene significantly decreased IL-1β expression and release, the IL-1Ra expression showed a borderline non-significant increment, while CARD8 knockdown did not affect the IL-1β and IL-1Ra mRNA expression or IL-1β protein release. Our results suggest that mainly NLRP3 plays a role in the regulation of IL-1β expression and release in AOSMC and could be a potential future target for the treatment of atherosclerosis and other inflammatory diseases.     

Pathological crosstalk in vitro between T lymphocytes and lesional keratinocytes in psoriasis: necessity of direct cell-to-cell contact

Psoriasis, a chronic autoimmune-related skin disease, involves both immune and non-immune cells like T cells and keratinocytes. This study investigates the regulatory role of T cells-keratinocyte interactions during psoriasis on immune factors production. Cytokines and chemokines were evaluated by multiplex and ELISA assays in an in vitro model of co-culture of keratinocytes with T lymphocytes. Keratinocytes were from psoriatic skin lesions or healthy skin. T lymphocytes were from healthy volunteers. Psoriatic keratinocytes (PKs) alone generated concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-8, monocyte chemotactic protein (MCP)-1, interferon-γ-induced protein 10 kDa (IP-10) and vascular endothelial growth factor (VEGF) higher than those produced by healthy keratinocytes (HKs). In contrast, IL-1α and IL-Ra production was reduced in PKs. Normal T cells, which had no effect on HKs, increased the production of TNF-α, IL-6, GM-CSF, IL-8, MCP-1 and IP-10 by PKs, but did not influence PK production of IL-1β, IL-1α, IL-Ra and VEGF. The most striking effects were obtained with PK- and IL-2-stimulated T lymphocytes: most of the above cytokines and chemokines were greatly upregulated, except IL-1β and VEGF that were decreased or unchanged, respectively. In addition, fractalkine was overproduced in this latter condition only. Our results indicate (1) a functional interaction between keratinocytes and T lymphocytes that requires a direct cellular contact, and (2) a reciprocal influence that depends on cytokine and chemokine types. In conclusion, lesional keratinocytes from psoriasis vulgaris alter functions of normal T lymphocytes that conversely modulate these keratinocytes.     

Biological therapy downregulates the heterodimer S100A8/A9 (calprotectin) expression in psoriatic patients

The pathophysiology of psoriasis is very complex and involves an interplay between immune cells and keratinocytes. The keratinocyte production of calprotectin (S100A8/A9), induced by the inflammatory psoriatic milieu, may be involved in initiating immune cell invasion, as well as in propagating inflammation. However, the exact role of calprotectin in psoriasis remains unclear. Therapeutic approaches utilizing adalimumab, etanercept and ustekinumab are widely used in psoriatic treatment, but their anti-inflammatory mechanisms are not fully understood. The aim of this study was to investigate, by immunohistochemical analysis, the expression of the heterocomplex S100A8/A9 in lesional skin from psoriatic patients undergoing biological therapy with adalimumab, etanercept or ustekinumab. Our results showed that S100A8/A9, absent or present at very low level in skin biopsies from healthy subjects, is dramatically upregulated in each epidermal layer from psoriatic patients. Interestingly, calprotectin was mainly localized in keratinocyte nuclei from psoriatic patients, suggesting a role of S100A8/A9 in keratinocyte nuclear function. Furthermore, we have shown that the biological treatment induced a drastic reduction of S100A8/A9 expression in skin biopsies from treated patients, correlating with PASI reduction. Our results suggest that calprotectin may play a crucial role as a significant marker of inflammation in psoriasis, and that its reduction of expression may be considered a favourable prognostic marker in psoriasis.     

Discordant expression of Bcl-x and Bcl-2 by keratinocytes in vitro and psoriatic keratinocytes in vivo.

Apoptosis is a required event in maintaining kinetic homeostasis within continually renewing tissues such as skin. However, no systematic study of the apoptotic process in epidermal keratinocytes of the skin has been performed. In this report, we examined the expression of proteins associated with promoting (Fas) or preventing (Bcl-2, Bcl-x, CD40) apoptosis in the normal, psoriatic, and malignant keratinocyte. Immunohistochemical staining and flow cytometry analysis revealed that normal cultured keratinocytes express low levels of Fas, CD40, and Bcl-x that was enhanced by cytokines including gamma-interferon (IFN-gamma) and a phorbol ester tumor promoter, TPA. Only faint Bcl-2 staining was detected in cultured keratinocytes exposed to IFN-gamma and TPA compared with the prominent expression of Bcl-x. Biopsies of normal skin, psoriatic plaques, and basal cell carcinomas were examined to extend the in vitro observations. Immunohistochemical staining revealed that while keratinocytes in normal epithelium express low to absent levels of Fas and Bcl-x, psoriatic keratinocytes expressed significantly higher levels of Fas and Bcl-x. In contrast, malignant keratinocytes in basal cell carcinomas expressed high levels of Bcl-2, but minimal Bcl-x, and no Fas. Immunoblot analysis revealed that the long form of Bcl-x (Bcl-xI), which prevents apoptosis in lymphocytes, is expressed by cultured keratinocytes and psoriatic plaque keratinocytes. We conclude that normal cytokine-activated keratinocytes can express an apoptotic (Fas) and an anti-apoptotic protein (Bcl-x). The overexpression of Bcl-x in psoriasis, or Bcl-2 in basal cell carcinomas, may contribute to the longevity of these cells by blocking the normal apoptotic process involved in the terminal differentiation program of epidermal keratinocytes.