RESUMO
In recent decades, the demand for biomedical imaging tools has grown very rapidly as a key feature for biomedical research and diagnostic applications. Particularly, fluorescence imaging has gained increased attention as a non-invasive, inexpensive technique that allows real-time imaging. However, tissue auto-fluorescence under external illumination, together with a weak tissue penetration of low wavelength excitation light, largely restricts the application of the technique. Accordingly, new types of fluorescent labels are currently being investigated and, in this search, phosphorescent nanoparticles promise great potential, as they combine the interesting size-dependent properties of nanoscale materials with a long-lasting phosphorescence-type emission that allows optical imaging well after excitation (so avoiding autofluorescence). In this work, core-shell structures consisting of SrAlO:Eu,Dy luminescent cores encapsulated within a biocompatible silica shell were prepared, showing a green persistent phosphorescence with an afterglow time of more than 1000 s. A high-energy ball milling procedure was used to reduce the size of the starting phosphors to a size suitable for cellular uptake, while the silica coating was produced by a reverse micelle methodology that eventually allows the excitation and emission light to pass efficiently through the shell. Confocal fluorescence microscopy using HeLa cancer cells confirmed the potential of the all-ceramic composites produced as feasible labels for in vitro optical imaging.
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Metais Terras Raras , Nanopartículas , Humanos , Luminescência , Nanopartículas/química , Imagem Óptica , Dióxido de Silício , EstrôncioRESUMO
Lymphocyte migration, which is essential for effective immune responses, belongs to the so-called amoeboid migration. The lymphocyte migration is up to 100 times faster than between mesenchymal and epithelial cell types. Migrating lymphocytes are highly polarized in three well-defined structural and functional zones: uropod, medial zone, and leading edge (LE). The actiomyosin-dependent driving force moves forward the uropod, whereas massive actin rearrangements protruding the cell membrane are observed at the LE. These actin rearrangements resemble those observed at the immunological synapse driven by clathrin, a protein normally involved in endocytic processes. Here, we used cell lines as well as primary lymphocytes to demonstrate that clathrin and clathrin adaptors colocalize with actin at the LE of migrating lymphocytes, but not in other cellular zones that accumulate both clathrin and actin. Moreover, clathrin and clathrin adaptors, including Hrs, the clathrin adaptor for multivesicular bodies, drive local actin accumulation at the LE. Clathrin recruitment at the LE resulted necessary for a complete cell polarization and further lymphocyte migration in both 2D and 3D migration models. Therefore, clathrin, including the clathrin population associated to internal vesicles, controls lymphocyte migration by regulating actin rearrangements occurring at the LE.
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Actinas/metabolismo , Movimento Celular , Clatrina/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fosfoproteínas/metabolismo , Linfócitos T/fisiologia , Movimento Celular/genética , Polaridade Celular , Clatrina/genética , Humanos , Sinapses Imunológicas , Células Jurkat , Transporte Proteico , RNA Interferente Pequeno/genética , Vesículas Transportadoras/metabolismoRESUMO
We have investigated the expression and role of galectin-1 and other galectins in psoriasis and in the Th1/Th17 effector and dendritic cell responses associated with this chronic inflammatory skin condition. To determine differences between psoriasis patients and healthy donors, expression of galectins was analysed by RT-PCR in skin samples and on epidermal and peripheral blood dendritic cells by immunofluorescence and flow cytometry. In the skin of healthy donors, galectin-1, -3 and -9 were expressed in a high proportion of Langerhans cells. Also, galectins were differentially expressed in peripheral blood dendritic cell subsets; galectin-1 and galectin-9 were highly expressed in peripheral myeloid dendritic cells compared with plasmacytoid dendritic cells. We found that non-lesional as well as lesional skin samples from psoriasis patients had low levels of galectin-1 at the mRNA and protein levels, in parallel with low levels of IL-10 mRNA compared with skin from healthy patients. However, only lesional skin samples expressed high levels of Th1/Th17 cytokines. The analysis of galectin-1 expression showed that this protein was down-regulated in Langerhans cells and dermal dendritic cells as well as in peripheral blood CD11c(+) DCs from psoriasis patients. Expression of galectin-1 correlated with IL-17 and IL-10 expression and with the psoriasis area and index activity. Addition of galectin-1 to co-cultures of human monocyte-derived dendritic cells with autologous T lymphocytes from psoriasis patients attenuated the Th1 response. Conversely, blockade of galectin binding increased IFNγ production and inhibited IL-10 secretion in co-cultures of monocyte-derived dendritic cells with CD4(+) T cells. Our results suggest a model in which galectin-1 down-regulation contributes to the exacerbation of the Th1/Th17 effector response in psoriasis patients.
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Galectinas/genética , Células de Langerhans/imunologia , Psoríase/imunologia , Pele/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas de Cocultura , Regulação para Baixo , Feminino , Citometria de Fluxo , Galectina 1/genética , Galectina 1/metabolismo , Galectina 1/farmacologia , Galectina 3/genética , Galectina 3/metabolismo , Galectinas/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-10/antagonistas & inibidores , Interleucina-10/metabolismo , Células de Langerhans/efeitos dos fármacos , Células de Langerhans/metabolismo , Células de Langerhans/patologia , Masculino , Pessoa de Meia-Idade , Psoríase/genética , Psoríase/patologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/metabolismo , Pele/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Group 3 innate lymphoid cells (ILC3s) are vital for defending tissue barriers from invading pathogens. Hypoxia influences the production of intestinal ILC3-derived cytokines by activating HIF. Yet, the mechanisms governing HIF-1α in ILC3s and other innate RORγt+ cells during in vivo infections are poorly understood. In our study, transgenic mice with specific Hif-1a gene inactivation in innate RORγt+ cells (RAG1KO HIF-1αâµRorc) exhibit more severe colitis following Citrobacter rodentium infection, primarily due to the inability to upregulate IL-22. We find that HIF-1αâµRorc mice have impaired IL-22 production in ILC3s, while non-ILC3 innate RORγt+ cells, also capable of producing IL-22, remain unaffected. Furthermore, we show that IL-18, induced by Toll-like receptor 2, selectively triggers IL-22 in ILC3s by transcriptionally upregulating HIF-1α, revealing an oxygen-independent regulatory pathway. Our results highlight that, during late-stage C. rodentium infection, IL-18 induction in the colon promotes IL-22 through HIF-1α in ILC3s, which is crucial for protection against this pathogen.
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Colite , Interleucinas , Camundongos , Animais , Interleucinas/genética , Interleucinas/metabolismo , Imunidade Inata , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Linfócitos/metabolismo , Interleucina-18 , Inflamação , Camundongos Transgênicos , Camundongos Endogâmicos C57BLRESUMO
Introduction: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. Methods: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. Results: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. Conclusion: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.
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Colite Ulcerativa , Doenças Inflamatórias Intestinais , Humanos , Colite Ulcerativa/patologia , Células CACO-2 , Tapsigargina , Estresse do Retículo Endoplasmático/genética , Doenças Inflamatórias Intestinais/metabolismo , Células Epiteliais/metabolismo , Hidroximetilglutaril-CoA SintaseRESUMO
Cancer is a multifactorial chronic illness caused by a combination of genetic and environmental factors. A tumor is more than just a collection of cancer cells, it also contains infiltrating and resident host cells that are constantly interacting with it. Innate lymphoid cells (ILCs) have been recently found to be within the tumor and its microenvironment in close relationship with cancer cells. Although ILCs lack an antigen-specific receptor, they can respond to environmental stress signals, aiding in the fast orchestration of an early immune response. They are tissue resident cells mostly located in mucosa and first barrier organs that have been mainly studied in the defense against pathogens, lymphoid development, and tissue repair, however, current research has begun to elucidate their involvement in carcinogenesis. Nevertheless, among all ILCs, ILC3s have been found to be the most controversial in terms of tumor immunity. It has been found that they enhance anti-tumor immunity by detecting cancerous cells and helping lymphocytes infiltrate tumors. However, some recent studies have revealed that IL-23 stimulating ILC3s may promote tumor growth. In this review, we have incorporated the most recent studies on the involvement of ILC3s in cancer development to offer an overview of the role of ILC3s in cancer emphasis on their particular activity in several organs primarily in the mucosa, but also in breast, pancreas, liver, and skin, realizing that their role likely depends on the tissue microenvironment and the subtype of ILC3s.
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Linfócitos , Neoplasias , Humanos , Imunidade Inata , Fígado , Microambiente TumoralRESUMO
BACKGROUND: Experimental autoimmune myocarditis (EAM), a mouse model of post-infectious cardiomyopathy, reflects mechanisms of inflammatory cardiomyopathy in humans. EAM is characterized by an infiltration of inflammatory cells into the myocardium that can be followed by myocyte fibrosis, edema, and necrosis, leading to ventricular wall dysfunction and heart failure. Different data indicate that CD69 exerts an important immunoregulatory effect in vivo. However, the possible role of CD69 in autoimmune myocarditis has not been studied. METHODS AND RESULTS: We have explored the role of the leukocyte regulatory molecule CD69 in the inflammation that leads to cardiac dysfunction after myocardial injury in EAM. We have found that after induction of EAM, the draining lymph nodes from CD69-deficient mice developed an exacerbated Th17 inflammatory response, resulting in increases in the numbers of infiltrating leukocytes in the myocardium. In the chronic phase of EAM, transthoracic echocardiography revealed a significantly reduced left ventricular fractional shortening and a decreased ejection fraction in CD69-deficient mice, indicative of an impaired cardiac contractility. This condition was accompanied by a greater extent of myocardial fibrosis, an elevated number of sinus pauses on ECG, and an enhanced ratio of heart weight to body weight in CD69-/- mice. Moreover, both bone marrow transplantation and adoptive transfer of Th17 cells isolated from immunized CD69-/- mice with EAM into naive wild-type recipients reproduced the severity of the disease, demonstrating that CD69 exerts its function within the lymphocyte compartment. CONCLUSION: Our findings indicate that CD69 negatively regulates heart-specific Th17 responses, cardiac inflammation, and heart failure progression in EAM.
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Doenças Autoimunes/fisiopatologia , Lectinas Tipo C/deficiência , Miocardite/fisiopatologia , Animais , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Cruzamentos Genéticos , Fibrose Endomiocárdica/imunologia , Fibrose Endomiocárdica/fisiopatologia , Feminino , Citometria de Fluxo , Humanos , Lectinas Tipo C/genética , Linfonodos/citologia , Linfonodos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Miocardite/genética , Miocardite/imunologia , Cadeias Pesadas de Miosina/imunologia , Fragmentos de Peptídeos/imunologia , Índice de Gravidade de Doença , Baço/citologia , Baço/fisiologiaRESUMO
BACKGROUND: Allergic diseases have a major health care impact in industrialized countries. The development of these diseases is influenced by exposure to allergen and to immunological and genetic factors. However, the molecular mechanisms underlying the inflammatory response that triggers allergy are not well defined. OBJECTIVE: We have investigated the role of the leukocyte activation antigen CD69 in the regulation of two allergic diseases, asthma and contact dermatitis. METHODS: Analysis of two models of allergic diseases in CD69 knockout and wild-type mice: ovalbumin-induced allergic airway inflammation (BALB/c genetic background) and contact hypersensitivity to oxazolone (C57BL/6J genetic background). RESULTS: CD69 deficiency dramatically enhanced the inflammatory response in the ovalbumin-induced asthma model of antigen-induced airway allergy. CD69 knockout mice showed exacerbated pulmonary eosinophil recruitment, high vascular cell adhesion molecule 1 expression levels in lung vasculature, and enhanced T(H)2 and T(H)17 cytokines in the bronchoalveolar space and lung tissue. In the hapten-induced cutaneous contact hypersensitivity model, both CD69 deficiency and treatment with anti-CD69 mAb increased inflammation. Treatment with contact allergens induced enhanced T(H)1 and T(H)17 responses in CD69 deficient mice, and neutralizing anti-IL-17 antibodies reduced skin inflammation. In both experimental systems, adoptive transfer of lymph node cells from CD69 knockout mice increased the inflammatory response in recipient mice. CONCLUSION: These results demonstrate that the early activation receptor CD69 is an intrinsic modulator of immune allergic processes through the negative regulation of allergen-induced T-cell effector responses.
Assuntos
Asma/imunologia , Dermatite Alérgica de Contato/imunologia , Eosinófilos/imunologia , Células Th1/imunologia , Células Th2/imunologia , Transferência Adotiva , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Antígenos CD , Antígenos de Diferenciação de Linfócitos T , Asma/genética , Asma/patologia , Dermatite Alérgica de Contato/genética , Dermatite Alérgica de Contato/patologia , Modelos Animais de Doenças , Eosinófilos/patologia , Feminino , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-17/antagonistas & inibidores , Interleucina-17/genética , Interleucina-17/imunologia , Lectinas Tipo C , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células Th1/patologia , Células Th2/patologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
Type 3 Innate lymphoid cells (ILC3s) have been described as tissue-resident cells and characterized throughout the body, especially in mucosal sites and classical first barrier organs such as skin, gut and lungs, among others. A significant part of the research has focused on their role in combating pathogens, mainly extracellular pathogens, with the gut as the principal organ. However, some recent discoveries in the field have unveiled their activity in other organs, combating intracellular pathogens and as part of the response to viruses. In this review we have compiled the latest studies on the role of ILC3s and the molecular mechanisms involved in defending against different microbes at the mucosal surface, most of these studies have made use of conditional transgenic mice. The present review therefore attempts to provide an overview of the function of ILC3s in infections throughout the body, focusing on their specific activity in different organs.
Assuntos
Interações Hospedeiro-Patógeno , Linfócitos/imunologia , Animais , Trato Gastrointestinal/imunologia , Humanos , Imunidade Inata , Infecções/imunologia , Pulmão/citologia , Pulmão/imunologia , Mucosa Bucal/citologia , Mucosa Bucal/imunologia , Pele/citologia , Pele/imunologiaRESUMO
Since their discovery, innate lymphoid cells (ILCs) have gradually been gaining greater relevance in the field of immunology due to their multiple functions in the innate immune response. They can mainly be found in mucosal and barrier organs like skin, gut, and lungs, and have been classified into five main types (NKs, ILC1s, ILC2s, ILC3s, and Lti cells) according to their function and development. They all play major roles in functions such as tissue homeostasis, early pathogen defense, regulation of inflammation, or tissue remodeling. ILCs are mostly tissue-resident cells tightly bound to the tissue structure, a fact that requires long and complex protocols that do not always provide sufficient yield for analysis. This suggests the need for optimized approaches aimed at ensuring that enriched and viable ILC samples are obtained, in order to furnish quality results. Herein a detailed protocol is established for obtaining a single-cell suspension highly enriched in lymphoid cells from mouse gut in order to identify the different subsets of ILCs by means of flow cytometry. The cell marker panel and flow cytometry gating strategies for identification and quantification of all the different ILC populations are provided for simultaneous analysis. Moreover, the protocol described includes a procedure for studying the different cytokines produced by ILC3s involved in maintaining the integrity of the gut barrier and defending against extracellular pathogens. As a result, herein an efficient method is presented for studying mouse ILCs within the lamina propria of the small intestine and colon; this can constitute a useful tool for future investigations in the field.
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Separação Celular/métodos , Colo/imunologia , Imunidade Inata , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Linfócitos/imunologia , Animais , Citocinas/metabolismo , Citometria de Fluxo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The original version of this Article contained an error in the spelling of the author José María González-Granado, which was incorrectly given as José María Gozález-Granado. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMO
Bacterial phagocytosis and antigen cross-presentation to activate CD8+ T cells are principal functions of professional antigen presenting cells. However, conventional CD4+ T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8+ T cells, which proliferate and become cytotoxic in response. CD4+ T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8+ T cells with low PD-1 expression. Moreover, transphagocytic CD4+ T cells induce protective anti-tumour immune responses by priming CD8+ T cells, highlighting the potential of CD4+ T cells as a tool for cancer immunotherapy.
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Apresentação de Antígeno/imunologia , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Animais , Células Cultivadas , Apresentação Cruzada/imunologia , Citotoxicidade Imunológica/imunologia , Memória Imunológica/imunologia , Sinapses Imunológicas/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagocitose/imunologia , Receptor de Morte Celular Programada 1/imunologiaRESUMO
During infections, the first reaction of the host against microbial pathogens is carried out by innate immune cells, which recognize conserved structures on pathogens, called pathogen-associated molecular patterns. Afterward, some of these innate cells can phagocytose and destroy the pathogens, secreting cytokines that would modulate the immune response to the challenge. This rapid response is normally followed by the adaptive immunity, more specific and essential for a complete pathogen clearance in many cases. Some innate immune cells, usually named antigen-presenting cells, such as macrophages or dendritic cells, are able to process internalized invaders and present their antigens to lymphocytes, triggering the adaptive immune response. Nevertheless, the traditional boundary of separated roles between innate and adaptive immunity has been blurred by several studies, showing that very specialized populations of lymphocytes (cells of the adaptive immunity) behave similarly to cells of the innate immunity. These "innate-like" lymphocytes include γδ T cells, invariant NKT cells, B-1 cells, mucosal-associated invariant T cells, marginal zone B cells, and innate response activator cells, and together with the newly described innate lymphoid cells are able to rapidly respond to bacterial infections. Strikingly, our recent data suggest that conventional CD4+ T cells, the paradigm of cells of the adaptive immunity, also present innate-like behavior, capturing bacteria in a process called transinfection. Transinfected CD4+ T cells digest internalized bacteria like professional phagocytes and secrete large amounts of proinflammatory cytokines, protecting for further bacterial challenges. In the present review, we will focus on the data showing such innate-like behavior of lymphocytes following bacteria encounter.
RESUMO
Recently, we have shown, contrary to what is described, that CD4(+) T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4(+) T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.
Assuntos
Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Animais , Apresentação de Antígeno , Bactérias/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Dendritic cells (DCs) phagocytose, process, and present bacterial antigens to T lymphocytes to trigger adaptive immunity. In vivo, bacteria can also be found inside T lymphocytes. However, T cells are refractory to direct bacterial infection, leaving the mechanisms by which bacteria invade T cells unclear. We show that T cells take up bacteria from infected DCs by the process of transinfection, which requires direct contact between the two cells and is enhanced by antigen recognition. Prior to transfer, bacteria localize to the immunological synapse, an intimate DC/T cell contact structure that activates T cells. Strikingly, T cells efficiently eliminate the transinfecting bacteria within the first hours after infection. Transinfected T cells produced high levels of proinflammatory cytokines and were able to protect mice from bacterial challenge following adoptive transfer. Thus, T lymphocytes can capture and kill bacteria in a manner reminiscent of innate immunity.
Assuntos
Infecções Bacterianas/microbiologia , Células Dendríticas/imunologia , Listeria monocytogenes/imunologia , Salmonella enterica/imunologia , Staphylococcus aureus/imunologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Citocinas/imunologia , Células Dendríticas/microbiologia , Feminino , Humanos , Imunidade Inata , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose , Linfócitos T/microbiologiaRESUMO
CD69 is involved in immune cell homeostasis, regulating the T cell-mediated immune response through the control of Th17 cell differentiation. However, natural ligands for CD69 have not yet been described. Using recombinant fusion proteins containing the extracellular domain of CD69, we have detected the presence of a ligand(s) for CD69 on human dendritic cells (DCs). Pulldown followed by mass spectrometry analyses of CD69-binding moieties on DCs identified galectin-1 as a CD69 counterreceptor. Surface plasmon resonance and anti-CD69 blocking analyses demonstrated a direct and specific interaction between CD69 and galectin-1 that was carbohydrate dependent. Functional assays with both human and mouse T cells demonstrated the role of CD69 in the negative effect of galectin-1 on Th17 differentiation. Our findings identify CD69 and galectin-1 to be a novel regulatory receptor-ligand pair that modulates Th17 effector cell differentiation and function.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Galectina 1/imunologia , Lectinas Tipo C/imunologia , Células Th17/imunologia , Animais , Adesão Celular/imunologia , Humanos , Inflamação/imunologia , Interferon gama/biossíntese , Interleucina-17/biossíntese , Células Jurkat , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/imunologia , Receptores Fc/imunologia , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69-/- DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69-/- DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P(1)) by CD69-/- DCs, whereas S1P(3) expression levels were similar in wild-type and CD69-/- DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P(1) function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery.
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Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/fisiologia , Movimento Celular , Células de Langerhans/fisiologia , Lectinas Tipo C/fisiologia , Lisofosfolipídeos/fisiologia , Esfingosina/análogos & derivados , Animais , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Diferenciação Celular , Células de Langerhans/citologia , Lectinas Tipo C/análise , Camundongos , Camundongos Endogâmicos C57BL , Microscopia , Esfingosina/fisiologiaRESUMO
The leukocyte activation marker CD69 functions as a negative regulator of the immune response, both in NK-dependent tumor rejection and in the inflammation associated with lymphocyte-dependent collagen-induced arthritis. In contrast, it has been reported that CD69-deficient mice are refractory to the neutrophil-dependent acute inflammatory response associated with anti-type II collagen antibody-induced arthritis (CAIA), suggesting a positive regulatory role for CD69 in neutrophil function during arthritis induction. To clarify this discrepancy, the CAIA response was independently analyzed in our CD69-deficient mice. In these experiments, the inflammatory response was unaffected by CD69 deficiency. Additionally, the in vivo down-regulation of CD69 expression by treatment of wild-type mice with the anti-CD69 mAb 2.2, which mimics the CD69-deficient phenotype, did not affect the course of arthritis in this model. Moreover, down-regulation of CD69 expression increased expression in arthritic joints of key inflammatory mediators, including IL-1beta, IL-6 and the chemokine MCP-1. Neutrophil accumulation in zymosan-treated air pouches and in thioglycolate-treated peritoneal cavities was also unaffected in CD69-deficient mice. In addition, CD69 expression was absent in activated neutrophils. Taken together, these results rule out a significant stimulatory role for CD69 in acute inflammatory responses mediated by neutrophils.
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Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Artrite Experimental/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Quimiocina CCL2/biossíntese , Regulação para Baixo , Feminino , Citometria de Fluxo , Inflamação/imunologia , Interleucina-1beta/biossíntese , Interleucina-6/biossíntese , Lectinas Tipo C , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Rho GTPases control many facets of cell polarity and migration; namely, the reorganization of the cellular cytoskeleton to extracellular stimuli. Rho GTPases are activated by GTP exchange factors (GEFs), which induce guanosine diphosphate (GDP) release and the stabilization of the nucleotide-free state. Thus, the role of GEFs in the regulation of the cellular response to extracellular cues during cell migration is a critical step of this process. In this report, we have analyzed the activation and subcellular localization of the hematopoietic GEF Vav in human peripheral blood lymphocytes stimulated with the chemokine stromal cell-derived factor-1 (SDF-1alpha). We show a robust activation of Vav and its redistribution to motility-associated subcellular structures, and we provide biochemical evidence of the recruitment of Vav to the membrane of SDF-1alpha-activated human lymphocytes, where it transiently interacts with the SDF-1alpha receptor CXCR4. Overexpression of a dominant negative form of Vav abolished lymphocyte polarization, actin polymerization, and migration. SDF-1alpha-mediated cell polarization and migration also were impaired by overexpression of an active, oncogenic Vav, although the mechanism appears to be different. Together, our data postulate a pivotal role for Vav in the transmission of the migratory signal through the chemokine receptor CXCR4.