RESUMO
BACKGROUND: Langerhans cells (LCs) are skin-resident dendritic cells (DCs) that orchestrate skin immunity. CCCTC-binding factor (CTCF) is a highly conserved DNA-binding protein that regulates higher-order chromatin organization and is involved in various gene regulation processes. OBJECTIVE: We sought to clarify a possible role for CTCF in LC homeostasis and function in vivo. METHODS: We used a conditional gene deletion mouse system to generate DC- and LC-specific CTCF-ablated mice. Short hairpin RNA-mediated RNA interference was used to silence CTCF expression in human monocyte-derived Langerhans cells. DC populations were assessed by using flow cytometry and immunofluorescence. Gene expression arrays were performed to identify genes regulated by CTCF in LCs. Contact hypersensitivity and epicutaneous sensitization responses were measured to examine the functional significance of CTCF ablation. RESULTS: DC-specific CTCF deletion led to a reduced pool of systemic DCs, with LCs most severely affected. Decreases in epidermal LC numbers were specifically associated with self-turnover defects. Interestingly, CTCF-deficient LCs demonstrated impaired migration out of the epidermis. Whole-transcriptome analyses revealed that genes that promoted cell adhesion were highly expressed, but CCR7 was downregulated in CTCF-depleted LCs. Hapten-induced contact hypersensitivity responses were more sustained in LC-specific CTCF-deficient mice, whereas epicutaneous sensitization to protein antigen was attenuated, indicating that CTCF-dependent LC homeostasis is required for optimal immune function of LCs in a context-dependent manner. CONCLUSION: Our results show that CTCF positively regulates the homeostatic pool and the efficient emigration of LCs, which are required for modulating the functional immune network of the skin.
Assuntos
Dermatite de Contato/genética , Homeostase/genética , Células de Langerhans/metabolismo , Proteínas Repressoras/genética , Animais , Fator de Ligação a CCCTC , Adesão Celular , Movimento Celular/genética , Movimento Celular/imunologia , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Epiderme/imunologia , Epiderme/metabolismo , Epiderme/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Haptenos , Homeostase/imunologia , Humanos , Células de Langerhans/imunologia , Células de Langerhans/patologia , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Receptores CCR7/genética , Receptores CCR7/imunologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/deficiência , Proteínas Repressoras/imunologia , Transdução de SinaisRESUMO
In contact hypersensitivity (CHS), multiple cells, inflammatory mediators and cytokines are known to be involved in the regulation of the immune response. Previously, we revealed the reactive oxygen species generation by 2, 4, 6-trinitrobenzene sulphonic acid (TNBS) in vivo, followed by heat shock protein 70 (Hsp70) carbonylation and the exogenous antioxidant role of cell-permeable Hsp70. Here, we demonstrate the role of Hsp70 using cell-permeable Hsp70 in the mouse CHS model. Pretreatment of cell-permeable Hsp70: (i) suppressed ear swelling; (ii) down-regulated phosphorylated p38, but up-regulated phosphorylated extracellular signal-regulated kinase; (iii) increased population of CD4(+) CD25(+) Foxp3(+) T cells; (iv) decreased secretion of tumor necrosis factor-α (TNF-α), IL-12, interferon-γ and IL-2 and (v) but up-regulated IL-4 and transforming growth factor beta (TGF-ß) in the lymph nodes. In conclusion, cell-permeable Hsp70 attenuates CHS through modulation of MAPK pathway and regulation of Th1, Th2 and regulatory T cells.
Assuntos
Dermatite de Contato/metabolismo , Dermatite de Contato/terapia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Transdução de Sinais/fisiologia , Administração Tópica , Animais , Modelos Animais de Doenças , Feminino , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transdução Genética/métodosRESUMO
Psoriasis is a chronic skin inflammation caused by a dysfunctional immune system, which causes systemic inflammation in various organs and tissues. Due to the risk of systemic inflammation and recurrence of psoriasis, it is important to identify the critical targets in the pathogenesis of psoriasis and develop targeted therapeutics. Dimerized translationally controlled tumor protein (dTCTP) promotes immune cell activation as a pro-inflammatory cytokine and plays a role in developing allergic diseases such as asthma and rhinitis. Here, we sought to explore whether dTCTP and its inhibition contributed to the development and control of imiquimod (IMQ)-induced psoriasis. Topical application of IMQ inflamed the skin of the back and ear, increased inflammatory cytokines, and decreased regulatory T cell markers. Interestingly, TCTP was significantly increased in inflamed skin and immune cells such as T cells, B cells, and macrophages after IMQ treatment and was secreted into the serum to undergo dimerization. Extracellular dTCTP treatment selectively suppressed regulatory T (Treg) cells, not other effector T helper (Th) cells, and increased M1 macrophages. Moreover, dTCTP-binding peptide 2 (dTBP2), a dTCTP inhibitor peptide, effectively attenuated the systemic inflammatory responses, including Th17 cell response, and alleviated psoriatic skin inflammation. dTBP2 blocked dTCTP-mediated Treg suppression and stimulated the expression of Treg cell markers in the spleen and inflammatory skin lesions. These results suggest that dTCTP dysregulated immune balance through Treg suppression in psoriatic inflammation and that functional inhibition of dTCTP by dTBP2 maintained immune homeostasis and attenuated inflammatory skin diseases by expanding Treg cells.
Assuntos
Psoríase , Linfócitos T Reguladores , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Imiquimode/farmacologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/farmacologia , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico , Pele , Linfócitos T Reguladores/metabolismo , Células Th17 , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
ROS are produced in dendritic cells (DCs) during antigen presentation in contact hypersensitivity (CHS). As a result, ROS cause a number of nonenzymatic protein modifications, including carbonylation, which is the most widely used marker of oxidative stress. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) is a well-characterized contact allergen that results in the formation of ROS. However, proteins that are carbonylated in DCs in response to TNBS have not been identified. To study ROS-dependent protein carbonylation in response to TNBS, we used the well-established mouse DC line, XS-106. We focused on the effects of TNBS on oxidation by examining selected oxidative markers. We identified TNBS-induced ROS and myeloperoxidase (MPO) proteins and demonstrated that the increase in ROS resulted in IL-12 production. The increase in oxidation was further confirmed by an oxidation-dependent increase in protein modifications, such as carbonylation. In fact, TNBS strongly induced carbonylation of mitochondrial adenosine triphosphate (ATP) synthase in XS-106 DCs, as determined by MALDI-TOF analysis and 2-D Western blotting. ROS production and protein carbonylation were confirmed in human monocyte-derived DCs (Mo-DCs). Furthermore, glutathione (GSH) decreased ROS and protein carbonylation in Mo-DCs. Carbonylation of ATP synthase in DCs may contribute to the pathophysiology of CHS.
Assuntos
Células Dendríticas/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Ácido Trinitrobenzenossulfônico/farmacologia , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células , Linhagem Celular , Meios de Cultivo Condicionados , Células Dendríticas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Camundongos , Monócitos/citologia , Peroxidase/genética , Peroxidase/metabolismo , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Pele/citologiaRESUMO
4-Hydroxyhexenal (HHE) is known to affect redox balance during aging, included are vascular dysfunctions. To better understand vascular abnormality through the molecular alterations resulting from HHE accumulation in aging processes, we set out to determine whether up-regulation of mitogen-activated protein kinase (MAPK) by HHE is mediated through nuclear factor kappa B (NF-kappaB) activation in endothelial cells. HHE induced NF-kappaB activation by inhibitor of kappaB (IkappaB) phosphorylation via the IkappaB kinase (IKK)/NF-kappaB inducing kinase (NIK) pathway. HHE increased the activity of p38 MAPK and extracellular signal regulated kinase (ERK), but not c-jun NH(2)-terminal kinase, indicating that p38 MAPK and ERK are closely involved in HHE-induced NF-kappaB transactivation. Pretreatment with ERK inhibitor PD98059, and p38 MAPK inhibitor SB203580, attenuated the induction of p65 translocation, IkappaB phosphorylation, and NF-kappaB luciferase activity. These findings strongly suggest that HHE induces NF-kappaB activation through IKK/NIK pathway and/or p38 MAPK and ERK activation associated with oxidative stress in endothelial cells.
Assuntos
Aldeídos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Transporte Biológico , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Quinase I-kappa B , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases , Masculino , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Ácido Peroxinitroso/biossíntese , Próstata/citologia , Piridinas/farmacologia , Ratos , Fator de Transcrição RelA , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno , Quinase Induzida por NF-kappaBRESUMO
Mitochondrial adenine nucleotide translocator (ANT) plays important roles in the regulation of mitochondrial permeability transition and cell bioenergetics. The mouse has three ANT isoforms (1, 2 and 4) showing tissue-specific expression patterns. Although ANT1 is known to have a pro-apoptotic property, the specific functions of ANT2 have not been well determined. In the present study, ANT2 expression was significantly lower in the aged rat liver and in a liver fibrosis model. To explore the protective role of ANT2 in the liver, we established a hepa1c1c7 cell line overexpressing ANT2. Overexpression of ANT2 caused hepa1c1c7 cells to be more resistant to oxidative stress, and mitochondrial membrane potential (MMP, ∆Ψm) was relatively intact in ANT2-overexpressing cells under oxidative stress. In addition, ANT2 was found to increase ATP production by influencing mitochondrial bioenergetics. These results imply that the hepatoprotective effect of ANT2 is due to the stabilization of MMP and enhanced ATP production, and thus, maintaining ANT2 levels in the liver might be important to enhance resistance to aging and oxidative stress.
Assuntos
Translocador 2 do Nucleotídeo Adenina/metabolismo , Envelhecimento/metabolismo , Fígado/metabolismo , Estresse Oxidativo/fisiologia , Translocador 2 do Nucleotídeo Adenina/biossíntese , Translocador 2 do Nucleotídeo Adenina/genética , Animais , Modelos Animais de Doenças , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Cirrose Hepática/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Organismos Livres de Patógenos Específicos , TransfecçãoRESUMO
PURPOSE: Dendritic cell (DC) vaccination for melanoma was introduced because melanoma carries distinct tumor-associated antigens. The purpose of this study was to investigate the efficacy and safety of DC vaccination for melanoma in Korea. MATERIALS AND METHODS: Five patients with stage IV and one with stage II were enrolled. Autologous monocyte-derived DCs (MoDCs) were cultured and pulsed with tumor-lysate, keyhole limpet hemocyanin, and cytokine cocktail for mature antigen-loaded DC. DC vaccination was repeated four times at 2-week intervals and 2-4×107 DC were injected each time. RESULTS: Reduced tumor volume was observed by PET-CT in three patients after DC vaccination. Delayed type hypersensitivity responses against tumor antigen were induced in five patients. Tumor antigen-specific IFN-γ-producing peripheral blood mononuclear cells were detected with enzyme-linked immunosorbent spot in two patients. However, the overall clinical outcome showed disease progression in all patients. CONCLUSION: In this study, DC vaccination using tumor antigen-loaded, mature MoDCs led to tumor regression in individual melanoma patients. Further standardization of DC vaccination protocol is required to determine which parameters lead to better anti-tumor responses and clinical outcomes.
Assuntos
Células Dendríticas/citologia , Imunoterapia/métodos , Melanoma/terapia , Monócitos/citologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Resultado do TratamentoRESUMO
BACKGROUND: Although reactive oxygen species (ROS) have been produced in both mouse bone marrow-derived dendritic cells (DCs) and XS-106 DCs by contact sensitizers and irritants in previous studies, the generation of ROS in human monocyte-derived DCs (MoDCs) and their role in contact hypersensitivity (CHS) has yet to be elucidated. OBJECTIVE: The purpose of this study was to determine whether contact allergens and irritants induce ROS in MoDCs and, if so, to evaluate the role of contact allergen and irritant induced-ROS in MoDCs in CHS. METHODS: Production of ROS was measured by 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA) assay. Surface CD86 and HLA-DR molecules were detected by flow cytometry. Protein carbonylation was detected by Western blotting. RESULTS: ROS were produced by contact allergens such as dinitrochlorobenzene (DNCB) and thimerosal and the irritant benzalkonium chloride (BKC). DNCB-induced, but not BKC-induced, ROS increased surface CD86 and HLA-DR molecules on MoDCs and induced protein carbonylation. These changes were reduced in the presence of antioxidant N-acetyl cysteine. CONCLUSION: Our results suggest that DNCB-induced ROS may be different from those induced by irritant BKC. The DNCB-induced ROS may be associated with the CHS response, because they activate surface molecules on DCs that are important for generating immune reactions.
RESUMO
Lipid peroxidation and its products such as 4-hydroxy-2-nonenal (HNE) and 4-hydroxyhexenal (HHE) are known to affect redox balance during aging and various degenerative processes, including vascular dysfunction. Deterioration of the endothelial cells that line the vascular wall is known to be an underlying cause of vascular dysfunction. At present, little is known about the mechanism by which HHE induces endothelial cell death (i.e. apoptosis), although HNE-induced apoptotic cell death has been reported. The aim of this study was to determine whether apoptosis induced by HHE in endothelial cells involves peroxynitrite (ONOO(-)). Our results show that in endothelial cells HHE triggers apoptotic cell death by inducing apoptotic Bax coupled with a decrease in anti-apoptotic Bcl-2. Results show that HHE induces reactive oxygen species (ROS), nitric oxide, and ONOO(-) generation, leading to redox imbalance. Furthermore, the antioxidant N-acetyl cysteine, ROS scavenger, and penicillamine, an ONOO(-) scavenger, were found to block HHE-mediated apoptosis. We used confocal laser microscopy to estimate the ability of these inhibitors to attenuate HHE-induced intracellular ONOO(-) levels thus confirming the oxidative mediation of apoptosis in endothelial cells. These findings strongly suggest that accumulated HHE triggers reactive species-mediated endothelial apoptosis, leading to vascular dysfunction as well as vascular aging. During aging, increased lipid peroxidation and its associated production of HHE may exacerbate the weakened redox balance, leading to various chronic degenerative processes including vascular dysfunction.