RESUMO
In the skin, the lack of markers permitting the unambiguous identification of macrophages and of conventional and monocyte-derived dendritic cells (DCs) complicates understanding of their contribution to skin integrity and to immune responses. By combining CD64 and CCR2 staining, we successfully identified each of these cell types and studied their origin, transcriptomic signatures, and migratory and T cell stimulatory properties. We also analyzed the impact of microbiota on their development and their contribution to skin inflammation during contact hypersensitivity. Dermal macrophages had a unique scavenging role and were unable to migrate and activate T cells. Conventional dermal DCs excelled both at migrating and activating T cells. In the steady-state dermis, monocyte-derived DCs are continuously generated by extravasated Ly-6C(hi) monocytes. Their T cell stimulatory capacity combined with their poor migratory ability made them particularly suited to activate skin-tropic T cells. Therefore, a high degree of functional specialization occurs among the mononuclear phagocytes of the skin.
Assuntos
Células Dendríticas/citologia , Macrófagos/citologia , Pele/citologia , Animais , Antígenos de Diferenciação/análise , Antígeno CD11b/análise , Linhagem da Célula , Quimiotaxia de Leucócito , Cromatografia em Gel , Células Dendríticas/imunologia , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Derme/citologia , Regulação da Expressão Gênica no Desenvolvimento , Imunofenotipagem/métodos , Células de Langerhans/citologia , Células de Langerhans/imunologia , Cooperação Linfocítica , Macrófagos/fisiologia , Camundongos , Microbiota/imunologia , Monócitos/citologia , Análise de Componente Principal , Quimera por Radiação , Receptores CCR2/análise , Receptores de IgG/análise , Pele/imunologia , Pele/microbiologia , Organismos Livres de Patógenos Específicos , Coloração e Rotulagem/métodos , TranscriptomaRESUMO
The development of vaccines inducing efficient CD8(+) T cell responses is the focus of intense research. Dendritic cells (DCs) expressing the XCR1 chemokine receptor, also known as CD103(+) or CD8α(+) DCs, excel in the presentation of extracellular Ags to CD8(+) T cells. Because of its high numbers of DCs, including XCR1(+) DCs, the skin dermis is an attractive site for vaccine administration. By creating laser-generated micropores through the epidermis, we targeted a model protein Ag fused to XCL1, the ligand of XCR1, to dermal XCR1(+) DCs and induced Ag-specific CD8(+) and CD4(+) T cell responses. Efficient immunization required the emigration of XCR1(+) dermal DCs to draining lymph nodes and occurred irrespective of TLR signaling. Moreover, a single intradermal immunization protected mice against melanoma tumor growth in prophylactic and therapeutic settings, in the absence of exogenous adjuvant. The mild inflammatory milieu created in the dermis by skin laser microporation itself most likely favored the development of potent T cell responses in the absence of exogenous adjuvants. The existence of functionally equivalent XCR1(+) dermal DCs in humans should permit the translation of laser-assisted intradermal delivery of a tumor-specific vaccine targeting XCR1(+) DCs to human cancer immunotherapy. Moreover, considering that the use of adjuvants in vaccines is often associated with safety issues, the possibility of inducing protective responses against melanoma tumor growth independently of the administration of exogenous adjuvants should facilitate the development of safer vaccines.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Neoplasias/imunologia , Receptores de Quimiocinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Vacinas Anticâncer/administração & dosagem , Quimiocinas C/genética , Quimiocinas C/metabolismo , Modelos Animais de Doenças , Injeções Intradérmicas , Melanoma Experimental , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Ovalbumina/genética , Ovalbumina/imunologia , Ligação Proteica , Receptores de Quimiocinas/genética , Subpopulações de Linfócitos T/imunologia , Carga Tumoral/imunologiaRESUMO
Psoriasis is a chronic inflammatory skin disease of unknown etiology. Previous studies showed that short-term, 5-7 d-long application of imiquimod (IMQ), a TLR7 agonist, to the skin of mice triggers a psoriasis-like inflammation. In the current study, by applying IMQ for 14 consecutive d, we established an improved mouse psoriasis-like model in that it recapitulated many of the clinical and cellular hallmarks observed in human patients during both the early-onset and the late-stable phase of psoriasis. Although macrophages and dendritic cells (DCs) have been proposed to drive the psoriatic cascade, their largely overlapping phenotype hampered studying their respective role. Based on our ability to discriminate Langerhans cells (LCs), conventional DCs, monocytes, monocyte-derived DCs, macrophages, and plasmacytoid DCs in the skin, we addressed their dynamics during both phases of our biphasic psoriasis-like model. Plasmacytoid DCs were not detectable during the whole course of IMQ treatment. During the early phase, neutrophils infiltrated the epidermis, whereas monocytes and monocyte-derived DCs were predominant in the dermis. During the late phase, LCs and macrophage numbers transiently increased in the epidermis and dermis, respectively. LC expansion resulted from local proliferation, a conclusion supported by global transcriptional analysis. Genetic depletion of LCs permitted to evaluate their function during both phases of the biphasic psoriasis-like model and demonstrated that their absence resulted in a late phase that is associated with enhanced neutrophil infiltration. Therefore, our data support an anti-inflammatory role of LCs during the course of psoriasis-like inflammation.
Assuntos
Aminoquinolinas/efeitos adversos , Células Dendríticas/imunologia , Macrófagos/imunologia , Psoríase/induzido quimicamente , Psoríase/imunologia , Pele/imunologia , Transcriptoma/efeitos dos fármacos , Aminoquinolinas/farmacologia , Animais , Células Dendríticas/patologia , Modelos Animais de Doenças , Humanos , Imiquimode , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Psoríase/patologia , Pele/patologiaRESUMO
Targeting antigens to cross-presenting dendritic cells (DCs) is a promising method for enhancing CD8(+) T-cell responses. However, expression patterns of surface receptors often vary between species, making it difficult to relate observations in mice to other animals. Recent studies have indicated that the chemokine receptor Xcr1 is selectively expressed on cross-presenting murine CD8α(+) DCs, and that the expression is conserved on homologous DC subsets in humans (CD141(+) DCs), sheep (CD26(+) DCs), and macaques (CADM1(+) DCs). We therefore tested if targeting antigens to Xcr1 on cross-presenting DCs using antigen fused to Xcl1, the only known ligand for Xcr1, could enhance immune responses. Bivalent Xcl1 fused to model antigens specifically bound CD8α(+) DCs and increased proliferation of antigen-specific T cells. DNA vaccines encoding dimeric Xcl1-hemagglutinin (HA) fusion proteins induced cytotoxic CD8(+) T-cell responses, and mediated full protection against a lethal challenge with influenza A virus. In addition to enhanced CD8(+) T-cell responses, targeting of antigen to Xcr1 induced CD4(+) Th1 responses and highly selective production of IgG2a antibodies. In conclusion, targeting of dimeric fusion vaccine molecules to CD8α(+) DCs using Xcl1 represents a novel and promising method for induction of protective CD8(+) T-cell responses.
Assuntos
Anticorpos Antivirais/biossíntese , Células Dendríticas/efeitos dos fármacos , Imunoglobulina G/biossíntese , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Receptores de Quimiocinas/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Proliferação de Células , Apresentação Cruzada , Células Dendríticas/imunologia , Feminino , Expressão Gênica , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunidade Celular , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Receptores de Quimiocinas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologiaRESUMO
BACKGROUND AND OBJECTIVES: Cutaneous squamous cell carcinoma (SCC) is known for its capacity to metastasize via lymphatic vessels. In recent studies, the level of lymphangiogenesis has been reported as a potential prognostic factor for several skin tumors. The aim of this study was to quantify lymphangiogenesis in SCC using either computer-assisted image analysis or the Chalkley count technique. Vascular parameters were evaluated and compared with respect to their predictive power for tumor metastasis. PATIENT AND METHODS: In this case-control study, clinical and histological data of 15 metastatic and 15 nonmetastatic SCC patients were retrospectively analyzed. SCC samples were immunostained for the lymphatic endothelial marker D2-40 and the panvascular marker CD31, and analyzed using computer-assisted morphometric image analyses within hot spots as well as the digitalized Chalkley counting method. RESULTS: Lymphatic vessel density, relative lymphatic vessel area, and lymphatic Chalkley count were significantly elevated in metastatic SCC. Tumor thickness was significantly higher in metastatic SCC, and had the highest predictive power for metastatic disease. Tumor thickness was a significant predictor of lymphangiogenic parameters. CONCLUSIONS: Lymphangiogenesis is elevated in metastatic SCC but its extent is influenced by tumor thickness. Tumor thickness remains the most reliable predictive factor for metastatic disease.
Assuntos
Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/secundário , Densitometria/métodos , Linfangiogênese , Metástase Linfática/patologia , Vasos Linfáticos/patologia , Neoplasias Cutâneas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Densitometria/estatística & dados numéricos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Prognóstico , Reprodutibilidade dos Testes , Fatores de Risco , Sensibilidade e EspecificidadeRESUMO
HINTERGRUND UND ZIELE: Kutane Plattenepithelkarzinome (SCC) sind bekannt für ihre Fähigkeit, über Lymphgefäße zu metastasieren. In neueren Studien wird das Ausmaß der Lymphangiogenese als möglicher prognostischer Faktor bei einigen Hauttumoren genannt. Ziel dieser Studie war die Quantifizierung der Lymphangiogenese bei SCC entweder durch computergestützte Bildanalyse oder mithilfe der Zählmethode nach Chalkley. Gefäßparameter wurden im Hinblick auf ihre Vorhersagekraft für die Bildung von Tumormetastasen beurteilt und verglichen. PATIENTEN UND METHODEN: In dieser Fallkontrollstudie wurden die klinischen und histologischen Daten von jeweils 15 SCC-Patienten mit bzw. ohne Metastasen retrospektiv analysiert. In den SCC-Proben wurde der für das Lymphendothel spezifische Marker D2-40 und der pan-vaskuläre Marker CD31 immunhistochemisch angefärbt und durch computergestützte morphometrische Bildanalyse in Hotspots sowie mithilfe der digitalisierten Zählmethode nach Chalkley analysiert. ERGEBNISSE: Die Dichte von Lymphgefäßen, die relative Lymphgefäßfläche und die mit der Chalkley-Methode ermittelte Zahl an Lymphgefäßen (Chalkley-Count) waren bei metastasierten SCC signifikant erhöht. Die Tumordicke war bei metastasierten SCC signifikant höher und besaß die höchste Vorhersagekraft für eine Metastasierung. Die Tumordicke war ein signifikanter Prädiktor für Lymphangiogeneseparameter. SCHLUSSFOLGERUNGEN: Die Lymphangiogenese ist bei metastasierten SCC erhöht, doch ihr Ausmaß wird von der Tumordicke beeinflusst. Die Tumordicke bildet weiterhin den zuverlässigsten prädiktiven Faktor für die Metastasierung.
RESUMO
BACKGROUND AND OBJECTIVES: Erythrodermic cutaneous T-cell lymphomas are aggressive diseases posing diagnostic and therapeutic challenges. Numerous indicators for confirming diagnosis and disease-monitoring have been proposed. CD26-negativity of peripheral CD4+ T-cells has been reported to have these properties. Our aim was to test, if the CD4(+) T-cell count, fraction of CD26- or CD7-negative CD4+ T-cells during the course of disease are valuable markers to predict therapeutic efficacy or disease progression in relation to changes in skin status. PATIENTS AND METHODS: Retrospective cohort analysis of eleven patients treated at a tertiary referral centre. Statistics were done by linear regression analysis and logrank test. RESULTS: Five patients displayed response to therapy in the skin, nine in the blood. Patients with cutaneous response showed a decrease of CD4+ T-cells, preceding the clinical response in most patients, whereas the percentage of CD26-negative T-cells changed first during clinical improvement. The calculated positive predictive values for response or progression were low for both CD4-count and CD26-expression. CONCLUSIONS: CD26 is not a reliable marker of either response or progression. As cutaneous response was always associated with a response in blood and not vice versa, we conclude, that the clinical status represents the most important parameter for guiding therapeutic decisions.
Assuntos
Biomarcadores Tumorais/sangue , Linfoma Cutâneo de Células T/sangue , Linfoma Cutâneo de Células T/tratamento farmacológico , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/tratamento farmacológico , Idoso , Antígenos CD4/sangue , Contagem de Linfócito CD4/métodos , Estudos de Coortes , Dipeptidil Peptidase 4/sangue , Progressão da Doença , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Linfoma Cutâneo de Células T/diagnóstico , Masculino , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Neoplasias Cutâneas/diagnóstico , Resultado do TratamentoRESUMO
BACKGROUND: Interferon-alfa is used in the treatment of primary cutaneous B-cell lymphoma (PCBCL). Therapy with interferon-alfa has thus far been reported solely in case reports and small case series, mostly describing intralesional use. OBJECTIVE: We sought to evaluate efficacy, response rate, time to response, duration of response, and safety of subcutaneously administered interferon-alfa for the treatment of cutaneous B-cell lymphoma. METHODS: We conducted a retrospective chart analysis of patients given the diagnosis of PCBCL and treated with interferon-alfa subcutaneously at a tertiary referral center. RESULTS: Fifteen patients with indolent subtypes of PCBCL were identified. The overall response rate was 66.7%; all responding patients went into complete remission. Response was not significantly associated with the maximum tolerated dose. Within the median follow-up time of 40 months, 90% of the responders experienced a relapse; median duration of response was 15.5 months. Adverse events were predominantly mild and in no case led to cessation of therapy. LIMITATIONS: Retrospective nature of the analysis and small number of patients because of scarcity of the disease are limitations. CONCLUSION: Treatment of indolent PCBCL with subcutaneously injected interferon-alfa demonstrated good response rates and tolerability. Response was not dose dependent. Relapses were observed in nearly all responding patients raising the question of interferon-alfa maintenance therapy in PCBCL.
Assuntos
Interferon-alfa/uso terapêutico , Linfoma de Células B/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Neoplasias Cutâneas/tratamento farmacológico , Adulto , Idoso , Biópsia por Agulha , Estudos de Coortes , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Seguimentos , França , Humanos , Imuno-Histoquímica , Injeções Subcutâneas , Interferon-alfa/efeitos adversos , Estimativa de Kaplan-Meier , Linfoma de Células B/mortalidade , Linfoma de Células B/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/mortalidade , Estadiamento de Neoplasias , Estudos Retrospectivos , Medição de Risco , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Estatísticas não Paramétricas , Taxa de Sobrevida , Resultado do TratamentoAssuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Urticária/tratamento farmacológico , Adulto , Anticorpos Monoclonais Humanizados/efeitos adversos , Asma/tratamento farmacológico , Doença Crônica , Esquema de Medicação , Eosinofilia/tratamento farmacológico , Feminino , Volume Expiratório Forçado/efeitos dos fármacos , Humanos , Doenças do Complexo Imune/induzido quimicamente , Resultado do TratamentoRESUMO
The dynamic process of wound healing is routinely evaluated by clinical or histological evaluation. Recently, a number of non-invasive imaging techniques have been evaluated for their clinical applicability in dermatology. Among them, reflectance confocal microscopy (RCM) represents a non-invasive imaging technique that allows the in vivo characterization of the skin at near-histological resolution. The aim of this study was to monitor epidermal wound repair using RCM in a model of tissue damage induced by cryosurgery. For this purpose, contact cryosurgery was performed at -32 °C for 10 seconds on the volar forearm of five healthy volunteers. Clinical and RCM evaluations were performed at nine consecutive time points. RCM allowed the visualization of edema formation and blood vessel dilatation immediately after cryosurgery, as well as morphologic features of wound repair, including the formation of finger-like protusions of keratinocytes into the wound bed, the appearance of hairpin-like vessels, and inflammatory cells. This pilot study illustrates that RCM represents a promising technique for quasi-real-time monitoring the kinetics of wound repair non-invasively and over time, thus offering new insights into in vivo processes of cutaneous wound repair and angiogenesis, as well as potential effects of topically applied drugs on the process of tissue repair.
Assuntos
Microscopia Confocal , Pele/patologia , Cicatrização , Adulto , Criocirurgia , Derme/patologia , Dermoscopia , Epiderme/patologia , Feminino , Humanos , Masculino , Pele/lesõesRESUMO
The family of toll-like receptors (TLRs) plays a central role in the cutaneous immune defense system. To date, different TLRs have been found on several major cell populations of the skin, such as keratinocytes, fibroblasts, antigen-presenting cells, and melanocytes. Activation of TLRs leads, via different intracellular signaling pathways, to the production of pro-inflammatory stimuli, and is considered a danger signal that should transform the skin in to the functional state of defense. However, TLRs have also been implicated in tissue homeostasis and renewal. Within the group of TLRs, two types have been identified: surface-expressed TLRs, which are predominantly active against bacterial cell wall compounds; and intracellular receptors, which preferentially recognize virus-associated pattern molecules. In addition, surface-expressed receptors trigger phagocytotic and maturation signals, while the intracellular TLRs lead to the induction of antiviral genes. Our review aims to outline the importance of TLRs in the pathogenesis of numerous skin diseases and the potential of TLR agonists as a treatment option for various skin diseases.
Assuntos
Dermatopatias/imunologia , Pele/imunologia , Receptores Toll-Like/imunologia , Acne Vulgar/imunologia , Células Apresentadoras de Antígenos/imunologia , Doenças Autoimunes/imunologia , Biomarcadores/metabolismo , Infecções por Borrelia/imunologia , Dermatite Atópica/imunologia , Fármacos Dermatológicos/uso terapêutico , Fibroblastos/imunologia , Humanos , Queratinócitos/imunologia , Hanseníase/imunologia , Melanócitos/imunologia , Psoríase/imunologia , Transdução de Sinais/imunologia , Pele/metabolismo , Dermatopatias/tratamento farmacológico , Dermatopatias/metabolismo , Sífilis/imunologia , Receptores Toll-Like/agonistasAssuntos
Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/etiologia , Biópsia , Criança , Doenças Endêmicas , Granuloma/diagnóstico , Humanos , Leishmania donovani/metabolismo , Leishmaniose Cutânea/tratamento farmacológico , Masculino , Paromomicina/uso terapêutico , Pele/patologia , Viagem , Resultado do TratamentoRESUMO
The efficacious delivery of antigens to antigen-presenting cells (APCs), in particular, to dendritic cells (DCs), and their subsequent activation remains a significant challenge in the development of effective vaccines. This study highlights the potential of dissolving microneedle (MN) arrays laden with nanoencapsulated antigen to increase vaccine immunogenicity by targeting antigen specifically to contiguous DC networks within the skin. Following in situ uptake, skin-resident DCs were able to deliver antigen-encapsulated poly-d,l-lactide-co-glycolide (PGLA) nanoparticles to cutaneous draining lymph nodes where they subsequently induced significant expansion of antigen-specific T cells. Moreover, we show that antigen-encapsulated nanoparticle vaccination via microneedles generated robust antigen-specific cellular immune responses in mice. This approach provided complete protection in vivo against both the development of antigen-expressing B16 melanoma tumors and a murine model of para-influenza, through the activation of antigen-specific cytotoxic CD8(+) T cells that resulted in efficient clearance of tumors and virus, respectively. In addition, we show promising findings that nanoencapsulation facilitates antigen retention into skin layers and provides antigen stability in microneedles. Therefore, the use of biodegradable polymeric nanoparticles for selective targeting of antigen to skin DC subsets through dissolvable MNs provides a promising technology for improved vaccination efficacy, compliance, and coverage.
Assuntos
Antígenos/administração & dosagem , Células Dendríticas/imunologia , Nanocápsulas/administração & dosagem , Imunidade Adaptativa , Animais , Peptídeos Catiônicos Antimicrobianos/química , Sistemas de Liberação de Medicamentos , Feminino , Injeções Intradérmicas , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/prevenção & controle , Camundongos , Camundongos Transgênicos , Nanocápsulas/química , Nanotecnologia , Agulhas , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/prevenção & controle , Vírus Sendai/imunologia , Pele/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas/administração & dosagemRESUMO
There is a high demand for noninvasive imaging techniques for wound assessment. In vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical technique for noninvasive evaluation of normal and diseased skin in vivo at near cellular resolution. This study was designed to test the feasibility of CLSM for noninvasive analysis of cutaneous wound healing in 15 patients (7 male/8 female), including acute and chronic, superficial and deep dermal skin wounds. A commercially available CLSM system was used for the assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. CLSM was able to visualize features of cutaneous wound repair in epidermal and superficial dermal wounds, including aspects of inflammation, neovascularisation, and tissue remodelling in vivo. Limitations include the lack of mechanic fixation of the optical system on moist surfaces restricting the analysis of chronic skin wounds to the wound margins, as well as a limited optical resolution in areas of significant slough formation. By describing CLSM features of cutaneous inflammation, vascularisation, and epithelialisation, the findings of this study support the role of CLSM in modern wound research and management.