Inflammation modulates intercellular adhesion and mechanotransduction in human epidermis via ROCK2.

Aberrant mechanotransduction and compromised epithelial barrier function are associated with numerous human pathologies including inflammatory skin disorders. However, the cytoskeletal mechanisms regulating inflammatory responses in the epidermis are not well understood. Here we addressed this question by inducing a psoriatic phenotype in human keratinocytes and reconstructed human epidermis using a cytokine stimulation model. We show that the inflammation upregulates the Rho-myosin II pathway and destabilizes adherens junctions (AJs) promoting YAP nuclear entry. The integrity of cell-cell adhesion but not the myosin II contractility per se is the determinative factor for the YAP regulation in epidermal keratinocytes. The inflammation-induced disruption of AJs, increased paracellular permeability, and YAP nuclear translocation are regulated by ROCK2, independently from myosin II activation. Using a specific inhibitor KD025, we show that ROCK2 executes its effects via cytoskeletal and transcription-dependent mechanisms to shape the inflammatory response in the epidermis.

The Impact of JWH-133 on Articular Cartilage Regeneration in Osteoarthritis Via Metalloproteinase 13-Dependent Mechanism.

Objective: Osteoarthritis (OA) is common degenerative joint disease, mostly characterized by gradual cartilage breakdown. Currently there are no disease-modifying drugs available, therefore, there is an increasing need for basic research to focus on cartilage function in OA. Changes in cannabinoid receptor 2 (CB2) expression were observed in the OA-affected joints, although its action on cartilage chondrocytes remain unclear. We studied the action of dimethylbutyl-deoxy-delta-8-THC (JWH-133), selective CB2 agonist, on chondrocytes metabolism using both in vitro and in vivo studies. Design: Intraarticular (i.a.) injection of monoiodoacetate (MIA) was used to induce OA in rats. OA-related pain symptoms were assessed by pressure application measurements (PAMs). Primary human chondrocytes treated with MIA were used to investigate action of JWH-133 on chondrocytes viability, proliferation, and motility. Cannabinoid system components, inflammatory cytokines and metalloproteinases (MMPs) expression was measured on messenger RNA and protein levels in chondrocytes and animal cartilage. Results: Repeated, i.a. administration of JWH-133 showed antinociceptive potential in PAM, as well as decreased levels of MMPs, which suggests that CB2 agonism may modify degradation of cartilage. JWH-133 administration partially reduced toxicity, increased proliferation, and chondrocytes' migration. Moreover, our data suggest that CB2 agonism leads to alleviation of MMPs expression both in vitro and in vivo. Conclusions: In this study, we demonstrate modifying effect of JWH-133 local administration on cartilage metabolism and MMP13 expression that was shown to be involved in cartilage degradation. CB2 receptors' activation may be of benefit for chondrocytes' proliferation, therefore delaying disease progression. Our results propose direction of studies on OA-modifying treatment that can benefit in management of human OA.

IL-38 orchestrates proliferation and differentiation in human keratinocytes.

Interleukin (IL)-38 is a member of the IL-1 cytokine family with reported anti-inflammatory activity. The highest constitutive IL-38 expression is detected in the skin, where it is mainly produced by differentiating keratinocytes. However, little data are available regarding its biological functions. In this study, we investigated the role of IL-38 in skin physiology. We demonstrate here that dermal fibroblasts and epithelial cells of skin appendages, such as eccrine sweat glands and sebaceous glands, also express IL-38. Next, using two- and three-dimensional cell cultures, we show that endogenous expression of IL-38 correlates with keratinocyte differentiation and its ectopic overexpression inhibits keratinocyte proliferation and enhances differentiation. Accordingly, immunohistochemical analysis revealed downregulation of IL-38 in skin pathologies characterized by keratinocyte hyperproliferation, such as psoriasis and basal or squamous cell carcinoma. Finally, intracellular IL-38 can shuttle between the nucleus and the cytoplasm and its overexpression modulates the activity of the transcription regulators YAP and ID1. Our results indicate that IL-38 can act independently from immune system activation and suggest that it may affect the epidermis directly by decreasing proliferation and promoting differentiation of keratinocytes. These data suggest an important role of keratinocyte-derived IL-38 in skin homeostasis and pathologies characterized by epidermal alterations.

IL-25 participates in keratinocyte-driven dermal matrix turnover and is reduced in systemic sclerosis epidermis.

OBJECTIVES: Evidence shows that dysfunctional SSc keratinocytes contribute to fibrosis by altering dermal homeostasis. Whether IL-25, an IL-17 family member regulating many epidermal functions, takes part in skin fibrosis is unknown. Here we address the role of IL-25 in skin fibrosis. METHODS: The expression of IL-25 was evaluated by immunofluorescence and in situ hybridization in 10 SSc and seven healthy donor (HD) skin biopsies. Epidermal equivalents (EE) reconstituted by primary HD keratinocytes were used as a model to study transcriptomic changes induced by IL-25 in the epidermis. RNA expression profile in EEs was characterized by RNAseq. The conditioned medium (CM) from primary SSc and HD keratinocytes primed with IL-25 was used to stimulate fibroblasts. IL-6, IL-8, MMP-1, type-I collagen (Col-I), and fibronectin production by fibroblasts was assessed by ELISA. RESULTS: SSc epidermis expressed lower levels of IL-25 compared with HDs. In EEs, IL-25 regulated several molecular pathways related to wound healing and extracellular matrix remodelling. Compared with control CM, the CM from IL-25-primed keratinocytes enhanced the fibroblast production of MMP-1, IL-6 and IL-8, but not of Col-I nor fibronectin. However, IL-25 significantly reduced the production of Col-I when applied directly to fibroblasts. The activation of keratinocytes by IL-25 was receptor-dependent and evident after a very short incubation time (10 min), largely mediated by IL-1, suggesting enhanced and specific release of preformed mediators. CONCLUSIONS: These results show that IL-25 participates in skin homeostasis, and its decreased expression in SSc may contribute to skin fibrosis by favouring extracellular matrix deposition over degradation.

A Methylome and Transcriptome Analysis of Normal Human Scar Cells Reveals a Role for FOXF2 in Scar Maintenance.

Scars are maintained for life and increase in size during periods of growth such as puberty. Epigenetic changes in fibroblasts after injury may underpin the maintenance and growth of scars. In this study, we combined methylome and transcriptome data from normotrophic mature scar and contralateral uninjured normal skin fibroblasts to identify potential regulators of scar maintenance. In total, 219 significantly differentially expressed and 1,199 significantly differentially methylated promoters were identified, of which there were 12 genes both significantly differentially methylated and expressed. Of these, the two transcription factors, FOXF2 and MKX, were selected for further analysis. Immunocytochemistry and qPCR suggested that FOXF2 but not MKX had elevated expression in scar fibroblasts. Using RNA sequencing, FOXF2 knockdown was shown to significantly reduce the expression of extracellular matrix‒related genes, whereas MKX did not appear to affect similar pathways. Finally, FOXF2 knockdown was also shown to significantly decrease collagen I production in scar and keloid fibroblasts. This study provides insights into the maintenance of normotrophic scar, suggesting that FOXF2 is an important regulator of this process. Targeting genes responsible for maintenance of scar phenotype may ameliorate scar appearance and improve patient outcomes in the future.

Dysfunctional Keratinocytes Increase Dermal Inflammation in Systemic Sclerosis: Results From Studies Using Tissue-Engineered Scleroderma Epidermis.

OBJECTIVE: Evidence suggests that keratinocyte-fibroblast interactions are abnormal in systemic sclerosis (SSc). The present study was undertaken to investigate potential epidermal dysfunction in SSc and its effects on dermal homeostasis. METHODS: Epidermal equivalents (EEs) were generated using keratinocytes from 6 healthy donors and 4 individuals with SSc. Skin and EE expression of markers of proliferation, differentiation, and activation was evaluated by immunohistochemistry. The transcriptomic profile of SSc EEs and healthy donor EEs was identified by RNA sequencing. EE conditioned medium (CM) was used to stimulate fibroblasts, and their production of interleukin-6 (IL-6), IL-8, matrix metalloproteinase 1 (MMP-1), type I collagen, and fibronectin was assessed by enzyme-linked immunosorbent assay. RESULTS: Compared to healthy donor EEs, SSc EEs exhibited aberrant differentiation, enhanced expression of activation markers, and a lower rate of basal keratinocyte mitosis, reproducing most of the abnormalities observed in SSc epidermis. RNA sequencing analysis revealed that, compared to healthy donor EEs, SSc EEs were characterized by lower expression of homeobox gene family members and by enhanced metabolic and oxidative stress molecular pathways. EE CM enhanced fibroblast production of IL-6, IL-8, MMP-1, type I collagen, and fibronectin (P < 0.05). Except for type I collagen and fibronectin, this effect was 2-fold higher in the presence of CM generated form SSc EEs. IL-1 was responsible, at least in part, for keratinocyte-dependent fibroblast activation. CONCLUSION: SSc EEs recapitulate the in vivo characteristics of SSc epidermis, demonstrating that SSc keratinocytes have an intrinsically altered differentiation program, possibly due to the dysregulation of genes from the homeobox family. The increased metabolic and oxidative stress associated with SSc epidermis may contribute to chronic inflammation and fibrosis of the dermis.

IL-25 (IL-17E) in epithelial immunology and pathophysiology.

IL-25, also known as IL-17E, is a unique cytokine of the IL-17 family. Indeed, IL-25 exclusively was shown to strongly induce expression of the cytokines associated with type 2 immunity. Although produced by several types of immune cells, such as T cells, dendritic cells, or group 2 innate lymphoid cells, a vast amount of IL-25 derives from epithelial cells. The functions of IL-25 have been actively studied in the context of physiology and pathology of various organs including skin, airways and lungs, gastrointestinal tract, and thymus. Accumulating evidence suggests that IL-25 is a "barrier surface" cytokine whose expression depends on extrinsic environmental factors and when upregulated may lead to inflammatory disorders such as atopic dermatitis, psoriasis, or asthma. This review summarizes the progress of the recent years regarding the effects of IL-25 on the regulation of immune response and the balance between its homeostatic and pathogenic role in various epithelia. We revisit IL-25's general and tissue-specific mechanisms of action, mediated signaling pathways, and transcription factors activated in immune and resident cells. Finally, we discuss perspectives of the IL-25-based therapies for inflammatory disorders and compare them with the mainstream ones that target IL-17A.

IL-17E (IL-25) and IL-17A Differentially Affect the Functions of Human Keratinocytes.

Our group has recently shown that keratinocyte-derived IL-17E (IL-25), one of six members of the IL-17 family, is overexpressed in lesional psoriatic skin and is involved in its pathophysiology. We show here that IL-22 enhances IL-17E production in human keratinocytes and that these cells display a complete IL-17E receptor at their surface, the expression of which is further induced by IL-17A, indicating a potential autocrine effect of IL-17E. Therefore, we addressed the impact of IL-17E on the function of human primary keratinocytes. IL-17E promoted the proliferation of keratinocytes in two-dimensional and three-dimensional cultures and caused the concomitant upregulation of differentiation-associated gene transcripts (e.g., keratin 10), whereas their expression was either inhibited or not changed by IL-17A. Contrary to IL-17A, IL-17E was not involved in the induction of antimicrobial proteins. Time-lapse analysis of cell movement showed that IL-17E influences cell motility, increasing both cell speed and displacement. This was associated with specific changes in the actin cytoskeleton organization and the cell-substrate adhesion. No such effects were observed upon IL-17A stimulation. In summary, we identified effects of IL-17E clearly distinct from IL-17A, pointing toward an important role of IL-17E in the physiology and pathophysiology of the epidermis.

Keratinocyte-specific ablation of Mcpip1 impairs skin integrity and promotes local and systemic inflammation.

MCPIP1 (Regnase-1, encoded by the ZC3H12A gene) regulates the mRNA stability of several inflammatory cytokines. Due to the critical role of this RNA endonuclease in the suppression of inflammation, Mcpip1 deficiency in mice leads to the development of postnatal multiorgan inflammation and premature death. Here, we generated mice with conditional deletion of Mcpip1 in the epidermis (Mcpip1EKO). Mcpip1 loss in keratinocytes resulted in the upregulated expression of transcripts encoding factors related to inflammation and keratinocyte differentiation, such as IL-36α/γ cytokines, S100a8/a9 antibacterial peptides, and Sprr2d/2h proteins. Upon aging, the Mcpip1EKO mice showed impaired skin integrity that led to the progressive development of spontaneous skin pathology and systemic inflammation. Furthermore, we found that the lack of epidermal Mcpip1 expression impaired the balance of keratinocyte proliferation and differentiation. Overall, we provide evidence that keratinocyte-specific Mcpip1 activity is crucial for the maintenance of skin integrity as well as for the prevention of excessive local and systemic inflammation. KEY MESSAGES: Loss of murine epidermal Mcpip1 upregulates transcripts related to inflammation and keratinocyte differentiation. Keratinocyte Mcpip1 function is essential to maintain the integrity of skin in adult mice. Ablation of Mcpip1 in mouse epidermis leads to the development of local and systemic inflammation.

Interleukin-38 interacts with destrin/actin-depolymerizing factor in human keratinocytes.

Interleukin (IL)-38 is a member of the IL-1 family of cytokines, which was proposed to exert anti-inflammatory effects. IL-38 is constitutively expressed in the skin, where keratinocytes are the main producing cells. Little information is currently available concerning IL-38 biology. Here, we investigated the subcellular localization and interaction partners of the IL-38 protein in human keratinocytes. IL-38 expression was reduced in primary keratinocytes grown in monolayer (2D) cultures. We thus used IL-38 overexpressing immortalized normal human keratinocytes (NHK/38) to study this cytokine in cell monolayers. In parallel, differentiation of primary human keratinocytes in an in vitro reconstructed human epidermis (RHE) 3D model allowed us to restore endogenous IL-38 expression. In NHK/38 cells and in RHE, IL-38 was mainly cell-associated, rather than released into culture supernatants. Intracellular IL-38 was preferentially, although not exclusively, cytoplasmic. Similarly, in normal human skin sections, IL-38 was predominantly cytoplasmic in the epidermis and essentially excluded from keratinocyte nuclei. A yeast two-hybrid screen identified destrin/actin-depolymerizing factor (DSTN) as a potential IL-38-interacting molecule. Co-immunoprecipitation and proximity ligation assay confirmed this interaction. We further observed partial co-localization of IL-38 and DSTN in NHK/38 cells. Endogenous IL-38 and DSTN were also co-expressed in all epidermal layers in RHE and in normal human skin. Finally, IL-38 partially co-localized with F-actin in NHK/38 cells, in particular along the cortical actin network and in filopodia. In conclusion, IL-38 is found predominantly in the cytoplasm of human keratinocytes, where it interacts with DSTN. The functional relevance of this interaction remains to be investigated.

Fenofibrate enhances barrier function of endothelial continuum within the metastatic niche of prostate cancer cells.

OBJECTIVE: Extravasation of circulating cancer cells is an important step of the metastatic cascade and a potential target for anti-cancer strategies based on vasoprotective drugs. Reports on anti-cancer effects of fenofibrate (FF) prompted us to analyze its influence on the endothelial barrier function during prostate cancer cell diapedesis. RESEARCH DESIGN AND METHODS: In vitro co-cultures of endothelial cells with cancer cells imitate the 'metastatic niche' in vivo. We qualitatively and quantitatively estimated the effect of 25 µM FF on the events which accompany prostate carcinoma cell diapedesis, with the special emphasis on endothelial cell mobilization. RESULTS: Fenofibrate attenuated cancer cell diapedesis via augmenting endothelial cell adhesion to the substratum rather than through the effect on intercellular communication networks within the metastatic niche. The inhibition of endothelial cell motility was accompanied by the activation of PPARα-dependent and PPARα-independent reactive oxygen species signaling, Akt and focal adhesion kinase (FAK) phosphorylation, in the absence of cytotoxic effects in endothelial cells. CONCLUSIONS: Fenofibrate reduces endothelial cell susceptibility to the paracrine signals received from prostate carcinoma cells, thus inhibiting endothelial cell mobilization and reducing paracellular permeability of endothelium in the metastatic niche. Our data provide a mechanistic rationale for extending the clinical use of FF and for the combination of this well tolerated vasoactive drug with the existing multidrug regimens used in prostate cancer therapy.

Poly(L-lactide-co-glycolide) thin films can act as autologous cell carriers for skin tissue engineering.

Degradable aliphatic polyesters such as polylactides, polyglycolides and their copolymers are used in several biomedical and pharmaceutical applications. We analyzed the influence of poly(L-lactide-co-glycolide) (PLGA) thin films on the adhesion, proliferation, motility and differentiation of primary human skin keratinocytes and fibroblasts in the context of their potential use as cell carriers for skin tissue engineering. We did not observe visible differences in the morphology, focal contact appearance, or actin cytoskeleton organization of skin cells cultured on PLGA films compared to those cultured under control conditions. Moreover, we did not detect biologically significant differences in proliferative activity, migration parameters, level of differentiation, or expression of vinculin when the cells were cultured on PLGA films and tissue culture polystyrene. Our results indicate that PLGA films do not affect the basic functions of primary human skin keratinocytes and fibroblasts and thus show acceptable biocompatibility in vitro, paving the way for their use as biomaterials for skin tissue engineering.

Microcystin-LR affects properties of human epidermal skin cells crucial for regenerative processes.

The occurrence of cyanobacterial toxic peptides, including microcystins (MCs), is an emerging health issue due to the eutrophication of water bodies. MCs have a strong influence on human cells, predominantly hepatocytes, however, toxicity was also observed in kidney, lung and dermal skin cells. Skin as the most external barrier of the human body is responsible for the maintenance of homeostasis of the whole organism. Simultaneously, skin cells may be the most exposed to MCs during recreational activity. The aim of this study was to examine the impact of MC-LR on processes indispensable for normal skin function and regeneration, namely, viability, migration and actin cytoskeleton organization of human keratinocytes. The results showed that short exposure to MC-LR does not affect proliferation of human skin keratinocytes but it is toxic after longer incubation in dose-dependent manner. Total disruption of the actin cytoskeleton was observed under the same MC-LR concentration. Furthermore, keratinocyte migration was inhibited at MC-LR concentrations of 50 µM after incubation for only 4 h. Some of the negative impacts of MC-LR on the examined cell processes may be partly reversible. The observed effects, regarding the possible high exposition of keratinocytes to toxins including MCs, are severe and may cause diverse health problems.

Effects triggered by platinum nanoparticles on primary keratinocytes.

The platinum (Pt)-group elements (PGEs) represent a new kind of environmental pollutant and a new hazard for human health. Since their introduction as vehicle-exhaust catalysts, their emissions into the environment have grown considerably compared with their low natural concentration in the earth crust. PGE emissions from vehicle catalysts can be also in the form of nanometer-sized particles (Pt nanoparticles [PtNPs]). These elements, both in their metallic form or as ions solubilized in biological media, are now recognized as potent allergens and sensitizers. Human skin is always exposed to toxic particles; therefore, in the present study we addressed the question of whether polyvinylpyrrolidone-coated PtNPs may have any negative effects on skin cells, including predominantly epidermal keratinocytes. In this study, PtNPs of two sizes were used: 5.8 nm and 57 nm, in concentrations of 6.25, 12.5, and 25 µg/mL. Both types of NPs were protected with polyvinylpyrrolidone. Primary keratinocytes were treated for 24 and 48 hours, then cytotoxicity, genotoxicity, morphology, metabolic activity, and changes in the activation of signaling pathways were investigated in PtNP-treated cells. We found that PtNPs trigger toxic effects on primary keratinocytes, decreasing cell metabolism, but these changes have no effects on cell viability or migration. Moreover, smaller NPs exhibited more deleterious effect on DNA stability than the big ones. Analyzing activation of caspases, we found changes in activity of caspase 9 and caspase 3/7 triggered mainly by smaller NPs. Changes were not so significant in the case of larger nanoparticles. Importantly, we found that PtNPs have antibacterial properties, as is the case with silver NPs (AgNPs). In comparison to our previous study regarding the effects of AgNPs on cell biology, we found that PtNPs do not exhibit such deleterious effects on primary keratinocytes as AgNPs and that they also can be used as potential antibacterial agents, especially in the treatment of Escherichia coli, representing a group of Gram-negative species.

Chemerin is an antimicrobial agent in human epidermis.

Chemerin, a chemoattractant ligand for chemokine-like receptor 1 (CMKLR1) is predicted to share similar tertiary structure with antibacterial cathelicidins. Recombinant chemerin has antimicrobial activity. Here we show that endogenous chemerin is abundant in human epidermis, and that inhibition of bacteria growth by exudates from organ cultures of primary human skin keratinocytes is largely chemerin-dependent. Using a panel of overlapping chemerin-derived synthetic peptides, we demonstrate that the antibacterial activity of chemerin is primarily mediated by Val(66)-Pro(85), which causes direct bacterial lysis. Therefore, chemerin is an antimicrobial agent in human skin.

Effect of silver nanoparticles on human primary keratinocytes.

Silver nanoparticles (AgNPs) have many biological applications in biomedicine, biotechnology and other life sciences. Depending on the size, shape and the type of carrier, AgNPs demonstrate different physical and chemical properties. AgNPs have strong antimicrobial, antiviral and antifungal activity, thus they are used extensively in a range of medical settings, particularly in wound dressings but also in cosmetics. This study was undertaken to examine the potential toxic effects of 15 nm polyvinylpyrrolidone-coated AgNPs on primary normal human epidermal keratinocytes (NHEK). Cells were treated with different concentrations of AgNPs and then cell viability, metabolic activity and other biological and biochemical aspects of keratinocytes functioning were studied. We observed that AgNPs decrease keratinocyte viability, metabolism and also proliferatory and migratory potential of these cells. Moreover, longer exposure resulted in activation of caspase 3/7 and DNA damage. Our studies show for the first time, that AgNPs may present possible danger for primary keratinocytes, concerning activation of genotoxic and cytotoxic processes depending on the concentration.

[Metabolic changes in bone marrow in patients submitted to immunosuppressive treatment]. / Zmiany metaboliczne w tkance kostnej u pacjentów poddanych leczeniu immunosupresyjnemu.

A historical outline of the immunosuppressive treatment and related developments in transplantology is presented here. It is accompanied by a description of the essential knowledge of the bone marrow histology as well as of the role of mesenchymal stem cells (MSCs) in the regenerative processes occurring in the organism following tissue damage. We have reviewed contemporary knowledge of negative (quantitative and qualitative) changes in bone tissues caused by immunosuppressive treatment after an organ transplantation and mentioned prophylactic measures which may be used in such cases. We also describe basic immunosuppressive medications taking into account their side effects on the organism of the patient and, in particular, on the bone marrow metabolism as well as the mechanisms of their action at the cellular level.