RESUMO
In the interfollicular epidermis, keratinocyte stem cells (KSC) generate a short-lived population of transit amplifying (TA) cells that undergo terminal differentiation after several cell divisions. Recently, we isolated and characterized a highly proliferative keratinocyte cell population, named "early" TA (ETA) cell, representing the first KSC progenitor with exclusive features. This work aims to evaluate epidermis, with a focus on KSC and ETA cells, during transition from infancy to childhood. Reconstructed human epidermis (RHE) generated from infant keratinocytes is more damaged by UV irradiation, as compared to RHE from young children. Moreover, the expression of several differentiation and barrier genes increases with age, while the expression of genes related to stemness is reduced from infancy to childhood. The proliferation rate of KSC and ETA cells is higher in cells derived from infants' skin samples than of those derived from young children, as well as the capacity of forming colonies is more pronounced in KSC derived from infants than from young children's skin samples. Finally, infants-KSC show the greatest regenerative capacity in skin equivalents, while young children ETA cells express higher levels of differentiation markers, as compared to infants-ETA. KSC and ETA cells undergo substantial changes during transition from infancy to childhood. The study presents a novel insight into pediatric skin, and sheds light on the correlation between age and structural maturation of the skin.
Assuntos
Diferenciação Celular , Queratinócitos , Células-Tronco , Humanos , Lactente , Células-Tronco/citologia , Células-Tronco/metabolismo , Queratinócitos/metabolismo , Queratinócitos/citologia , Pré-Escolar , Proliferação de Células , Células Epidérmicas/metabolismo , Células Epidérmicas/citologia , Criança , Pele/citologia , Pele/metabolismo , Feminino , Masculino , Epiderme/metabolismo , Células CultivadasRESUMO
IL-1 plays a crucial role in triggering sterile inflammation following tissue injury. Although most studies associate IL-1 release by injured cells to the recruitment of neutrophils for tissue repair, the inflammatory cascade involves several molecular and cellular actors whose role remains to be specified. In the present study, we identified dermal fibroblasts among the IL-1R1-expressing skin cells as key sensors of IL-1 released by injured keratinocytes. After in vitro stimulation by recombinant cytokines or protein extracts of lysed keratinocytes containing high concentrations of IL-1, we show that dermal fibroblasts are by far the most IL-1-responsive cells compared to keratinocytes, melanocytes and endothelial cells. Fibroblasts have the property to respond to very low concentrations of IL-1 (from 10 fg/ml), even in the presence of 100-fold higher concentrations of IL-1RA, by increasing their expression of chemokines such as IL-8 for neutrophil recruitment. The capacity of IL-1-stimulated fibroblasts to attract neutrophils has been demonstrated both in vitro using cell migration assay and in vivo using a model of superficial epidermal lesion in IL-1R1-deficient mice which harbored reduced expression of inflammatory mediators and neutrophil skin infiltration. Together, our results shed a light on dermal fibroblasts as key relay cells in the chain of sterile inflammation induced after epidermal lesion.
Assuntos
Dermatite , Interleucina-1 , Camundongos , Animais , Interleucina-1/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-8/metabolismo , Células Endoteliais/metabolismo , Células Cultivadas , Queratinócitos/metabolismo , Dermatite/metabolismo , Fibroblastos/metabolismo , Inflamação/metabolismoRESUMO
Introduction: Although the presence of pathogens in skin wounds is known to delay the wound healing process, the mechanisms underlying this delay remain poorly understood. In the present study, we have investigated the regulatory role of proinflammatory cytokines on the healing kinetics of infected wounds. Methods: We have developed a mouse model of cutaneous wound healing, with or without wound inoculation with Staphylococcus aureus and Pseudomonas aeruginosa, two major pathogens involved in cutaneous wound bacterial infections. Results: Aseptic excision in C57BL/6 mouse skin induced early expression of IL-1ß, TNFα and Oncostatin M (OSM), without detectable expression of IL-22 and IL-17A/F. S. aureus and P. aeruginosa wound inoculation not only increased the expression of IL-1ß and OSM, but also induced a strong cutaneous expression of IL-22, IL-17A and IL-17F, along with an increased number of infiltrating IL-17A and/or IL-22-producing γδ T cells. The same cytokine expression pattern was observed in infected human skin wounds. When compared to uninfected wounds, mouse skin infection delayed the wound healing process. Injection of IL-1α, TNFα, OSM, IL-22 and IL-17 together in the wound edges induced delayed wound healing similar to that induced by the bacterial infection. Wound healing experiments in infected Rag2KO mice (deficient in lymphocytes) showed a wound healing kinetic similar to uninfected Rag2KO mice or WT mice. Rag2KO infected-skin lesions expressed lower levels of IL-17 and IL-22 than WT, suggesting that the expression of these cytokines is mainly dependent on γδ T cells in this model. Wound healing was not delayed in infected IL-17R/IL-22KO, comparable to uninfected control mice. Injection of recombinant IL-22 and IL-17 in infected wound edges of Rag2KO mice re-establish the delayed kinetic of wound healing, as in infected WT mice. Conclusion: These results demonstrate the synergistic and specific effects of IL-22 and IL-17 induced by bacterial infection delay the wound healing process, regardless of the presence of bacteria per se. Therefore, these cytokines play an unexpected role in delayed skin wound healing.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Pseudomonas aeruginosa , Camundongos , Humanos , Animais , Pseudomonas aeruginosa/metabolismo , Interleucina-17/metabolismo , Staphylococcus aureus/metabolismo , Fator de Necrose Tumoral alfa , Oncostatina M , Staphylococcus aureus Resistente à Meticilina/metabolismo , Camundongos Endogâmicos C57BL , Interleucina 22RESUMO
Filaggrin is an epidermal protein involved in skin barrier formation and hydration, whose expression is altered in canine atopic dermatitis (CAD). CAD patients also present an abnormal immune response with an altered expression of antimicrobial peptides (AMPs), such as ß-defensins and cathelicidins. Sphingolipids and glycosaminoglycans (GAGs) have been reported to improve the skin barrier in several animal species, including dogs. Our objective was to evaluate the in vitro effects of a sphingomyelin-rich lipid extract (LE), a hyaluronic acid-rich GAG matrix, and their combination, on the expression of filaggrin and human ß-defensin 2 (hBD-2). Filaggrin expression was quantified in a reconstructed human epidermis (RHE), and hBD-2 in normal human epidermal keratinocyte (NHEK) cultures. LE and GAGs were tested at 0.02 mg/mL, with or without adding a cytokine mix. A significant increase in mean hBD-2, compared to the control (99 pg/mL) was achieved with LE (138 pg/mL) and LE+GAGs (165 pg/mL). Filaggrin increased with GAGs (202% ± 83) and LE (193% ± 44) vs. the stimulated control, but this difference was statistically significant (p < 0.05) only with LE+GAGs (210% ± 39). In conclusion, the tested GAGs and LE enhance filaggrin and AMP expression in vitro, which might benefit CAD patients if applied in vivo.
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Loss of melanocytes is the pathological hallmark of vitiligo, a chronic inflammatory skin depigmenting disorder induced by exaggerated immune response, including autoreactive CD8 T cells producing high levels of type 1 cytokines. However, the interplay between this inflammatory response and melanocyte disappearance remains to be fully characterized. Here, we demonstrate that vitiligo skin contains a significant proportion of suprabasal melanocytes, associated with disruption of E-cadherin expression, a major protein involved in melanocyte adhesion. This phenomenon is also observed in lesional psoriatic skin. Importantly, apoptotic melanocytes were mainly observed once cells were detached from the basal layer of the epidermis, suggesting that additional mechanism(s) could be involved in melanocyte loss. The type 1 cytokines IFN-γ and TNF-α induce melanocyte detachment through E-cadherin disruption and the release of its soluble form, partly due to MMP-9. The levels of MMP-9 are increased in the skin and sera of patients with vitiligo, and MMP-9 is produced by keratinocytes in response to IFN-γ and TNF-α. Inhibition of MMP-9 or the JAK/STAT signaling pathway prevents melanocyte detachment in vitro and in vivo. Therefore, stabilization of melanocytes in the basal layer of the epidermis by preventing E-cadherin disruption appears promising for the prevention of depigmentation occurring in vitiligo and during chronic skin inflammation.
Assuntos
Caderinas/metabolismo , Interferon gama/metabolismo , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz/biossíntese , Melanócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vitiligo/metabolismo , Animais , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Melanócitos/patologia , CamundongosRESUMO
Cutaneous squamous cell carcinoma (cSCC) is the second most common keratinocyte malignancy and accounts for 20% of skin cancer deaths. Cancer is closely related to inflammation, but the contribution of the tumor microenvironment to cSCC development is poorly understood. We previously showed that oncostatin M (OSM), a cytokine belonging to the IL-6 family, promotes normal keratinocyte proliferation and migration, skin inflammation, and epidermal hyperplasia, both in vitro and in vivo. Here, we show that OSM is overexpressed in human cSCC and is associated with type 1 immune polarization. In vitro, OSM induced STAT-3 and ERK signaling, modified the expression of genes involved in cytokine signaling, proliferation, inhibition of apoptosis, and immune responses, and promoted proliferation and migration of malignant keratinocyte PDVC57 cells. PDVC57 cells grafted in the skin of mice led to rapid cSCC development, associated with OSM expression by tumor-infiltrating neutrophils. Finally, the absence of OSM (OSM-KO mice) led to a 30% reduction of tumor size and reduced M2 polarization in the tumor microenvironment. Globally, these results support a pro-tumoral role of OSM in cSCC development and suggest that a new therapeutic approach targeting this cytokine could be considered.
RESUMO
Pseudomonas aeruginosa, an opportunistic pathogen involved in skin and lung diseases, possesses numerous virulence factors, including type 2 and 3 secretion systems (T2SS and T3SS) and its flagellum, whose functions remain poorly known during cutaneous infection. Using isogenic mutants deleted from genes encoding each or all of these three virulence factors, we investigated their role in induction of inflammatory response and in tissue invasiveness in human primary keratinocytes and reconstructed epidermis. Our results showed that flagellum, but not T2SS and T3SS, is involved in induction of a large panel of cytokine, chemokine, and antimicrobial peptide (AMP) mRNA in the infected keratinocytes. Chemokine secretion and AMP tissular production were also dependent on the presence of the bacterial flagellum. This pro-inflammatory effect was significantly reduced in keratinocytes infected in presence of anti-toll-like receptor 5 (TLR5) neutralizing antibody. Bacterial invasion of human epidermis and persistence in a mouse model of sub-cutaneous infection were dependent on the P. aeruginosa flagellum. We demonstrated that flagellum constitutes the main virulence factor of P. aeruginosa involved not only in early induction of the epidermis inflammatory response but also in bacterial invasion and cutaneous persistence. P. aeruginosa is mainly sensed by TLR5 during the early innate immune response of human primary keratinocytes.
Assuntos
Epiderme/microbiologia , Flagelos/fisiologia , Inflamação/microbiologia , Queratinócitos/imunologia , Pseudomonas aeruginosa/patogenicidade , Animais , Anticorpos Neutralizantes/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Células Cultivadas , Quimiocinas/genética , Quimiocinas/imunologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Imunidade Inata , Queratinócitos/efeitos dos fármacos , Queratinócitos/microbiologia , Masculino , Camundongos , Mutação , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/ultraestrutura , Receptor 5 Toll-Like/imunologia , Fatores de Virulência/deficiência , Fatores de Virulência/genéticaRESUMO
The development of three-dimensional models of reconstituted mouse epidermis (RME) has been hampered by the difficulty to maintain murine primary keratinocyte cultures and to achieve a complete epidermal stratification. In this study, a new protocol is proposed for the rapid and convenient generation of RME, which reproduces accurately the architecture of a normal mouse epidermis. During RME morphogenesis, the expression of differentiation markers such as keratins, loricrin, filaggrin, E-cadherin and connexins was followed, showing that RME structure at day 5 was similar to those of a normal mouse epidermis, with the acquisition of the natural barrier function. It was also demonstrated that RME responded to skin-relevant proinflammatory cytokines by increasing the expression of antimicrobial peptides and chemokines, and inhibiting epidermal differentiation markers, as in the human system. This new model of RME is therefore suitable to investigate mouse epidermis physiology further and opens new perspectives to generate reconstituted epidermis from transgenic mice.
Assuntos
Citocinas/toxicidade , Epiderme/efeitos dos fármacos , Mediadores da Inflamação/toxicidade , Modelos Biológicos , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proteínas Filagrinas , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Camundongos Endogâmicos C57BL , Morfogênese/efeitos dos fármacos , Receptores de Citocinas/metabolismoRESUMO
BACKGROUND: Acute-serum Amyloid A (A-SAA), one of the major acute-phase proteins, is mainly produced in the liver but extra-hepatic synthesis involving the skin has been reported. Its expression is regulated by the transcription factors NF-κB, C/EBPß, STAT3 activated by proinflammatory cytokines. OBJECTIVES: We investigated A-SAA synthesis by resting and cytokine-activated Normal Human Epidermal Keratinocytes (NHEK), and their inflammatory response to A-SAA stimulation. A-SAA expression was also studied in mouse skin and liver in a model mimicking psoriasis and in the skin and sera of psoriatic and atopic dermatitis (AD) patients. METHODS: NHEK were stimulated by A-SAA or the cytokines IL-1α, IL-17A, IL-22, OSM, TNF-α alone or in combination, previously reported to reproduce features of psoriasis. Murine skins were treated by imiquimod cream. Human skins and sera were obtained from patients with psoriasis and AD. A-SAA mRNA was quantified by RT qPCR. A-SAA proteins were dosed by ELISA or immunonephelemetry assay. RESULTS: IL-1α, TNF-α and mainly IL-17A induced A-SAA expression by NHEK. A-SAA induced its own production and the synthesis of hBD2 and CCL20, both ligands for CCR6, a chemokine receptor involved in the trafficking of Th17 lymphocytes. A-SAA expression was increased in skins and livers from imiquimod-treated mice and in patient skins with psoriasis, but not significantly in those with AD. Correlations between A-SAA and psoriasis severity and duration were observed. CONCLUSION: Keratinocytes could contribute to psoriasis pathogenesis via A-SAA production, maintaining a cutaneous inflammatory environment, activating innate immunity and Th17 lymphocyte recruitment.
Assuntos
Dermatite Atópica/patologia , Interleucina-17/farmacologia , Psoríase/patologia , Proteína Amiloide A Sérica/metabolismo , Pele/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Adulto , Idoso , Aminoquinolinas/farmacologia , Animais , Células Cultivadas , Quimiocina CCL20/metabolismo , Quimiocina CCL20/farmacologia , Citocinas/genética , Citocinas/metabolismo , Dermatite Atópica/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imiquimode , Interleucina-17/genética , Interleucina-17/metabolismo , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Psoríase/metabolismo , Receptores CCR6/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteína Amiloide A Sérica/análise , Proteína Amiloide A Sérica/genética , Pele/metabolismo , Células Th17/citologia , Células Th17/metabolismoRESUMO
Macroautophagy is a degradative pathway whereby cells encapsulate and degrade cytoplasmic material within endogenously-built membranes. Previous studies have suggested that autophagosome membranes originate from lipid droplets. However, it was recently shown that rapamycin could induce autophagy in cells lacking these organelles. Here we show that lipid droplet-deprived cells are unable to perform autophagy in response to nitrogen-starvation because of an accelerated lipid synthesis that is not observed with rapamycin. Using cerulenin, a potent inhibitor of fatty acid synthase, and exogenous addition of palmitic acid we could restore nitrogen-starvation induced autophagy in the absence of lipid droplets.
Assuntos
Autofagia , Ácidos Graxos/biossíntese , Metabolismo dos Lipídeos , Nitrogênio/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Oncostatin M (OSM) has been reported to be overexpressed in psoriasis skin lesions and to exert proinflammatory effects in vitro on human keratinocytes. Here, we report the proinflammatory role of OSM in vivo in a mouse model of skin inflammation induced by intradermal injection of murine OSM-encoding adenovirus (AdOSM) and compare with that induced by IL-6 injection. Here, we show that OSM potently regulates the expression of genes involved in skin inflammation and epidermal differentiation in murine primary keratinocytes. In vivo, intradermal injection of AdOSM in mouse ears provoked robust skin inflammation with epidermal thickening and keratinocyte proliferation, while minimal effect was observed after AdIL-6 injection. OSM overexpression in the skin increased the expression of the S100A8/9 antimicrobial peptides, CXCL3, CCL2, CCL5, CCL20, and Th1/Th2 cytokines, in correlation with neutrophil and macrophage infiltration. In contrast, OSM downregulated the expression of epidermal differentiation genes, such as cytokeratin-10 or filaggrin. Collectively, these results support the proinflammatory role of OSM when it is overexpressed in the skin. However, OSM expression was not required in the murine model of psoriasis induced by topical application of imiquimod, as demonstrated by the inflammatory phenotype of OSM-deficient mice or wild-type mice treated with anti-OSM antibodies.
Assuntos
Aminoquinolinas/efeitos adversos , Expressão Gênica , Oncostatina M/genética , Psoríase/etiologia , Psoríase/metabolismo , Animais , Biomarcadores , Diferenciação Celular/genética , Proliferação de Células , Modelos Animais de Doenças , Epiderme/imunologia , Epiderme/metabolismo , Epiderme/patologia , Proteínas Filagrinas , Regulação da Expressão Gênica , Imiquimode , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Psoríase/patologia , Pele/imunologia , Pele/metabolismo , Pele/patologiaRESUMO
Severe burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. However, neither early complete healing nor quality of epithelialization is satisfactory. An alternative approach is to graft isolated keratinocytes. We evaluated paediatric foreskin and auricular skin as donor sources, autologous keratinocyte transplantation, and compared the graft efficiency to the in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. Keratinocytes were isolated from surgical samples by enzymatic digestion. Living cell recovery, in vitro proliferation and epidermal reconstruction capacities were evaluated. Differentiation status was analysed, using qRT-PCR and immunolabelling. Eleven children were grafted with foreskin-derived (boys) or auricular (girls) keratinocyte suspensions dripped onto deep severe burns. The aesthetic and functional quality of epithelialization was monitored in a standardized way. Foreskin keratinocyte graft in male children provides for the re-epithelialization of partial deep severe burns and accelerates wound healing, thus allowing successful wound closure, and improves the quality of scars. In accordance, in vitro studies have revealed a high yield of living keratinocyte recovery from foreskin and their potential in terms of regeneration and differentiation. We report a successful method for grafting paediatric males presenting large severe burns through direct spreading of autologous foreskin keratinocytes. This alternative method is easy to implement, improves the quality of skin and minimizes associated donor site morbidity. In vitro studies have highlighted the potential of foreskin tissue for graft applications and could help in tissue selection with the prospect of grafting burns for girls.
Assuntos
Separação Celular/métodos , Prepúcio do Pênis/citologia , Queratinócitos/citologia , Transplante de Pele , Biomarcadores/metabolismo , Queimaduras/patologia , Diferenciação Celular , Células Cultivadas , Criança , Pré-Escolar , Orelha , Células Epidérmicas , Humanos , Imuno-Histoquímica , Lactente , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração , Células-Tronco/citologia , Transplante Autólogo , CicatrizaçãoRESUMO
The pathogenesis of inflammatory skin diseases such as psoriasis involves the release of numerous proinflammatory cytokines, including members of the IL-1 family. Here we report overexpression of IL-1α, IL-1ß, and IL-1 receptor antagonist mRNA, associated to expression of IL-23p19, IL-17A, and IL-22 in skin cells, upon topical application of the TLR7 agonist imiquimod (IMQ) in C57BL/6J mice. IMQ-induced skin inflammation was partially reduced in mice deficient for both IL-1α/IL-1ß or for IL-1 receptor type 1 (IL-1R1), but not in IL-1α- or IL-1ß-deficient mice, demonstrating the redundant activity of IL-1α and IL-1ß for skin inflammation. NLRP3 or apoptosis-associated Speck-like protein containing a Caspase recruitment domain-deficient mice had no significant reduction of skin inflammation in response to IMQ treatment, mainly due to the redundancy of IL-1α. However, IMQ-induced skin inflammation was abolished in the absence of MyD88, the adaptor protein shared by IL-1R and TLR signaling pathways. These results are consistent with the TLR7 dependence of IMQ-induced skin inflammation. Thus, IL-1R1 contributes to the IMQ-induced skin inflammation, and disruption of MyD88 signaling completely abrogates this response.
Assuntos
Adjuvantes Imunológicos/efeitos adversos , Aminoquinolinas/efeitos adversos , Proteínas de Transporte/imunologia , Toxidermias/imunologia , Inflamassomos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Receptores Tipo I de Interleucina-1/imunologia , Transdução de Sinais/imunologia , Adjuvantes Imunológicos/farmacologia , Aminoquinolinas/farmacologia , Animais , Proteínas de Transporte/genética , Citocinas/genética , Citocinas/imunologia , Toxidermias/genética , Toxidermias/patologia , Imiquimode , Inflamassomos/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptores Tipo I de Interleucina-1/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Pele/imunologia , Pele/patologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologiaRESUMO
Androgens act through non-genomic and androgen receptor (AR)-dependent genomic mechanisms. AR is expressed in the sebaceous gland and the importance of androgens in the sebaceous function is well established. However, the in vitro models used to date have failed to evidence a clear genomic effect (e.g., modification of gene expression profile) of androgens on human sebocyte cells. In order to study the impact of active androgens in sebocytes, we constructed a stable human sebocyte cell line derived from SEBO662 [17] constitutively expressing a fully functional AR. In these SEBO662 AR+ cells, dihydrotestosterone (DHT) induced AR nuclear translocation and the strong modulation of a set of transcripts (RASD1, GREB1...) known to be androgen-sensitive in other androgenic cells and tissues. Moreover, we observed that DHT precociously down-regulated markers for immature follicular cells (KRT15, TNC) and for hair lineage (KRT75, FST) and up-regulated the expression of genes potentially related to sebocyte differentiation (MUC1/EMA, AQP3, FADS2). These effects were fully confirmed at the protein level. In addition, DHT-stimulated SEBO662 AR+, cultured in a low-calcium defined keratinocyte medium without serum or any complement, neosynthesize lipids, including sebum lipids, and store increased amounts of triglycerides in lipid droplets. DHT also induces morphological changes, increases cell size, and treatments over 7 days lead to a time-dependent increase in the population of apoptotic DNA-fragmented cells. Taken together, these results show for the first time that active androgens alone can engage immature sebocytes in a clear lipogenic differentiation process (Graphical abstract). These effects depend on the expression of a functional AR in these cells. This model should be of interest for revisiting the mechanisms of the sebaceous function in vitro and for the design of relevant pharmacological models for drug or compound testing.
Assuntos
Androgênios/metabolismo , Di-Hidrotestosterona/metabolismo , Folículo Piloso/citologia , Receptores Androgênicos/biossíntese , Glândulas Sebáceas/citologia , Transporte Ativo do Núcleo Celular , Apoptose , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Queratinócitos/citologia , Glândulas Sebáceas/metabolismoRESUMO
Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.
Assuntos
Glioblastoma/metabolismo , Interleucinas/metabolismo , Receptores de Interleucina/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Expressão Gênica , Glioblastoma/genética , Humanos , Subunidade beta de Receptor de Interleucina-10/genética , Subunidade beta de Receptor de Interleucina-10/metabolismo , Interleucinas/genética , Sistema de Sinalização das MAP Quinases , Células-Tronco Neoplásicas , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina/genética , Fator de Transcrição STAT3/metabolismo , Interleucina 22RESUMO
OBJECTIVE: Severe burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. An alternative approach is to graft isolated keratinocytes. We evaluated foreskin and other anatomic sites as donor sources for autologous keratinocyte graft in children. We studied in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. METHODS: Keratinocytes were isolated from foreskin, auricular skin, chest and abdominal skin by enzymatic digestion. Living cell recovery, in vitro proliferation, epidermal reconstruction capacities and differentiation status were analyzed. RESULTS: In vitro studies revealed the higher yield of living keratinocyte recovery from foreskin and higher potential in terms of proliferative capacity, regeneration and differentiation. Cultured keratinocytes from foreskin express lower amounts of differentiation markers than those isolated from trunk and ear. Histological analysis of reconstituted human epidermis derived from foreskin and inguinal keratinocytes showed a structured multilayered epithelium, whereas those obtained from ear pinna-derived keratinocytes were unstructured. CONCLUSION: Our studies highlight the potential of foreskin tissue for autograft applications in boys. A suitable alternative donor site for autologous cell transplantation in female paediatric burn patients remains an open question in our department. We tested the hypothesis that in vitro studies and RHE reconstructive capacities of cells from different body sites can be helpful to select an optimal site for keratinocyte isolation before considering graft protocols for girls.
Assuntos
Queimaduras/cirurgia , Técnicas de Cultura de Células/métodos , Pavilhão Auricular/citologia , Células Epidérmicas , Prepúcio do Pênis/citologia , Queratinócitos/transplante , Transplante de Pele/métodos , Tronco , Adolescente , Diferenciação Celular , Proliferação de Células , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Transplante AutólogoRESUMO
BACKGROUND: Treatment of burned patients is a tricky clinical problem not only because of the extent of the physiologic abnormalities but also because of the limited area of normal skin available. METHODS: Literature indexed in the National Center (PubMed) has been reviewed using combinations of key words (burns, children, skin graft, tissue engineering, and keratinocyte grafts). Articles investigating the association between burns and graft therapeutic modalities have been considered. Further literature has been obtained by analysis of references listed in reviewed articles. RESULTS: Severe burns are conventionally treated with split-thickness skin autografts. However, there are usually not enough skin donor sites. For years, the question of how covering the wound surface became one of the major challenges in clinical research area and several procedures were proposed. The microskin graft is one of the oldest methods to cover extensive burns. This technique of skin expansion is efficient, but results remain inconsistent. An alternative is to graft cultured human epidermal keratinocytes. However, because of several complications and labor-intensive process of preparing grafts, the initial optimism for cultured epithelial autograft has gradually declined. In an effort to solve these drawbacks, isolated epithelial cells from selecting donor site were introduced in skin transplantation. CONCLUSIONS: Cell suspensions transplanted directly to the wound is an attractive process, removing the need for attachment to a membrane before transfer and avoiding one potential source of inefficiency. Choosing an optimal donor site containing cells with high proliferative capacity is essential for graft success in burns.
RESUMO
Keratinocyte differentiation program leading to an organized epidermis plays a key role in maintaining the first line of defense of the skin. Epidermal integrity is regulated by a tight communication between keratinocytes and leucocytes, particularly under cytokine control. Imbalance of the cytokine network leads to inflammatory diseases such as psoriasis. Our attempt to model skin inflammation showed that the combination of IL-17A, IL-22, IL-1α, OSM and TNFα (Mix M5) synergistically increases chemokine and antimicrobial-peptide expression, recapitulating some features of psoriasis. Other characteristics of psoriasis are acanthosis and down-regulation of keratinocyte differentiation markers. Our aim was to characterize the specific roles of these cytokines on keratinocyte differentiation, and to compare with psoriatic lesion features. All cytokines decrease keratinocyte differentiation markers, but IL-22 and OSM were the most powerful, and the M5 strongly synergized the effects. In addition, IL-22 and OSM induced epidermal hyperplasia in vitro and M5 induced epidermal thickening and decreased differentiation marker expression in a mouse model, as observed in human psoriatic skin lesions. This study highlights the precise role of cytokines in the skin inflammatory response. IL-22 and OSM more specifically drive epidermal hyperplasia and differentiation loss while IL-1α, IL-17A and TNFα were more involved in the activation of innate immunity.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Citocinas/farmacologia , Queratinócitos/citologia , Animais , Biomarcadores/metabolismo , Células Epidérmicas , Humanos , Mediadores da Inflamação/farmacologia , Interleucina-17/farmacologia , Interleucina-1alfa/farmacologia , Interleucinas/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Oncostatina M/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Interleucina 22RESUMO
OBJECTIVES: Genome-wide association studies have identified variants at multiple loci associated with prostate cancer (PCa) risk. Some of these loci include candidate susceptibility genes, such as MSMB, HNF1ß, and C-terminal-binding protein (CtBP2). Except for MSMB, the clinicopathological significance of these genes has not been investigated. We therefore aimed to analyze their expression in PCa tissues, in relation with tumor progression and aggressiveness. METHODS AND MATERIALS: Protein expression was evaluated by immunohistochemistry on tissue microarrays containing samples from normal prostate (NL, n = 91), high-grade prostatic intraepithelial neoplasia (PIN, n = 61), clinically localized PCa (CLC, n = 434), PCa metastases (M, n = 28), and castration-resistant PCa (CRC, n = 49). Moreover, mRNA expression for each marker was assessed by quantitative real-time polymerase chain reaction, on 53 frozen samples of NL, CLC, and CRC. RESULTS: These genes were differentially expressed at the different stages of PCa natural history. MSMB expression decreased with disease development and progression. In contrast, nuclear HNF1ß and CtBP2 staining significantly increased in the CRC and M groups when compared with CLC, together with the transcripts levels. In patients with CLC, HNF1ß and CtBP2 nuclear expressions were strongly associated with cancer cell proliferation. After adjusting for the Gleason score and the pathological stage, none of the candidate genes was significantly predictive of recurrence after radical prostatectomy. In patients with CRC, CtBP2 nuclear staining was associated with shorter overall survival. CONCLUSIONS: The decrease of MSMB expression during tumor progression strongly supports its role as a tumor-suppressor gene. Although its functions remain to be clarified in PCa cells, HNF1ß and CtBP2 are associated with cancer cell proliferation, tumor progression, and castration-resistant disease.