RESUMO
Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.
Assuntos
Células Dendríticas , Homeostase , Megacariócitos , Trombopoese , Animais , Feminino , Humanos , Masculino , Camundongos , Apoptose , Plaquetas/citologia , Medula Óssea , Linhagem da Célula , Proliferação de Células , Células Dendríticas/imunologia , Células Dendríticas/citologia , Retroalimentação Fisiológica , Imunidade Inata , Microscopia Intravital , Megacariócitos/citologia , Megacariócitos/imunologia , Camundongos Endogâmicos C57BL , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/fisiopatologia , COVID-19/virologiaRESUMO
Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17â¼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.
Assuntos
Colite/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Repressoras/imunologia , Serina-Treonina Quinases TOR/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Diferenciação Celular , Colite/genética , Colite/patologia , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/imunologia , Regulação da Expressão Gênica , Centro Germinativo/imunologia , Centro Germinativo/patologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/imunologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/imunologia , Fosfatidilinositol 3-Quinases/genética , Cultura Primária de Células , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Transdução de Sinais , Baço/imunologia , Baço/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Serina-Treonina Quinases TOR/genética , Células Th17/imunologia , Células Th17/patologia , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
CD8+ T cells are crucial for the clearance of viral infections. During the acute phase, proinflammatory conditions increase the amount of circulating phosphatidylserine+ (PS) extracellular vesicles (EVs). These EVs interact especially with CD8+ T cells; however, it remains unclear whether they can actively modulate CD8+ T cell responses. In this study, we have developed a method to analyze cell-bound PS+ EVs and their target cells in vivo. We show that EV+ cell abundance increases during viral infection and that EVs preferentially bind to activated, but not naive, CD8+ T cells. Superresolution imaging revealed that PS+ EVs attach to clusters of CD8 molecules on the T cell surface. Furthermore, EV-binding induces antigen (Ag)-specific TCR signaling and increased nuclear translocation of the transcription factor Nuclear factor of activated T-cells (NFATc1) in vivo. EV-decorated but not EV-free CD8+ T cells are enriched for gene signatures associated with T-cell receptor signaling, early effector differentiation, and proliferation. Our data thus demonstrate that PS+ EVs provide Ag-specific adjuvant effects to activated CD8+ T cells in vivo.
Assuntos
Vesículas Extracelulares , Viroses , Humanos , Linfócitos T CD8-Positivos , Fosfatidilserinas/metabolismo , Vesículas Extracelulares/metabolismo , Viroses/metabolismo , Diferenciação CelularRESUMO
Platelets are not only the first responders in thrombosis and hemostasis but also central players in inflammation. Compared with platelets recruited to thrombi, immune-responsive platelets use distinct effector functions including actin-related protein complex 2/3-dependent migration along adhesive substrate gradients (haptotaxis), which prevents inflammatory bleeding and contributes to host defense. How platelet migration in this context is regulated on a cellular level is incompletely understood. Here, we use time-resolved morphodynamic profiling of individual platelets to show that migration, in contrast to clot retraction, requires anisotropic myosin IIa-activity at the platelet rear which is preceded by polarized actin polymerization at the front to initiate and maintain migration. Integrin GPIIb-dependent outside-in signaling via Gα13 coordinates polarization of migrating platelets to trigger tyrosine kinase c-Src/14-3-3ζ-dependent lamellipodium formation and functions independent of soluble agonists or chemotactic signals. Inhibitors of this signaling cascade, including the clinically used ABL/c-Src inhibitor dasatinib, interfere predominantly with the migratory capacity of platelets, without major impairment of classical platelet functions. In murine inflammation models, this translates to reduced migration of platelets visualized by 4D intravital microscopy, resulting in increased inflammation-associated hemorrhage in acute lung injury. Finally, platelets isolated from patients with leukemia treated with dasatinib who are prone to clinically relevant hemorrhage exhibit prominent migration defects, whereas other platelet functions are only partially affected. In summary, we define a distinct signaling pathway essential for migration and provide novel mechanistic insights explaining dasatinib-related platelet dysfunction and bleeding.
Assuntos
Plaquetas , Trombose , Humanos , Camundongos , Animais , Plaquetas/metabolismo , Proteínas 14-3-3/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Dasatinibe , Actinas/metabolismo , Trombose/metabolismo , Inflamação/metabolismoRESUMO
Impairment of vascular integrity is a hallmark of inflammatory diseases. We recently reported that single immune-responsive platelets migrate and reposition themselves to sites of vascular injury to prevent bleeding. However, it remains unclear how single platelets preserve vascular integrity once encountering endothelial breaches. Here we demonstrate by intravital microscopy combined with genetic mouse models that procoagulant activation (PA) of single platelets and subsequent recruitment of the coagulation cascade are crucial for the prevention of inflammatory bleeding. Using a novel lactadherin-based compound, we detect phosphatidylserine (PS)-positive procoagulant platelets in the inflamed vasculature. We identify exposed collagen as the central trigger arresting platelets and initiating subsequent PA in a CypD- and TMEM16F-dependent manner both in vivo and in vitro. Platelet PA promotes binding of the prothrombinase complex to the platelet membrane, greatly enhancing thrombin activity and resulting in fibrin formation. PA of migrating platelets is initiated by costimulation via integrin αIIbß3 (GPIIBIIIA)/Gα13-mediated outside-in signaling and glycoprotein VI signaling, leading to an above-threshold intracellular calcium release. This effectively targets the coagulation cascade to breaches of vascular integrity identified by patrolling platelets. Platelet-specific genetic loss of either CypD or TMEM16F as well as combined blockade of platelet GPIIBIIIA and glycoprotein VI reduce platelet PA in vivo and aggravate pulmonary inflammatory hemorrhage. Our findings illustrate a novel role of procoagulant platelets in the prevention of inflammatory bleeding and provide evidence that PA of patrolling platelet sentinels effectively targets and confines activation of coagulation to breaches of vascular integrity.
Assuntos
Plaquetas , Glicoproteínas da Membrana de Plaquetas , Animais , Plaquetas/metabolismo , Hemorragia/metabolismo , Camundongos , Ativação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismoRESUMO
Development of dendritic cells (DCs) commences in the bone marrow, from where pre-DCs migrate to peripheral organs to differentiate into mature DCs in situ. However, the factors that regulate organ-specific differentiation to give rise to tissue-specific DC subsets remain unclear. Here we show that the Ras-PI3Kγ-Akt-mTOR signaling axis acted downstream of FLT3L signaling and was required for development of lung CD103(+) DCs and, to a smaller extent, for lung CD11b(+) DCs, but not related DC populations in other non-lymphoid organs. Furthermore, we show that in lymphoid organs such as the spleen, DCs depended on a similar signaling network to respond to FLT3 ligand with overlapping and partially redundant roles for kinases PI3Kγ and PI3Kδ. Thus we identified PI3Kγ as an essential organ-specific regulator of lung DC development and discovered a signaling network regulating tissue-specific DC development mediated by FLT3.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/fisiologia , Células Dendríticas/citologia , Pulmão/imunologia , Transdução de Sinais/fisiologia , Tirosina Quinase 3 Semelhante a fms/fisiologia , Animais , Apoptose , Diferenciação Celular/fisiologia , Classe Ib de Fosfatidilinositol 3-Quinase/deficiência , Células Dendríticas/classificação , Compostos Heterocíclicos com 3 Anéis/farmacologia , Homeostase/imunologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Pulmão/citologia , Pulmão/enzimologia , Tecido Linfoide/citologia , Tecido Linfoide/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/fisiologia , Especificidade de Órgãos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Quimera por Radiação , Proteínas Recombinantes/farmacologia , Serina-Treonina Quinases TOR/fisiologiaRESUMO
Plasmacytoid dendritic cells (pDCs) display an increased abundance in visceral adipose tissue (VAT) of humans with obesity. In the current study, we set out to decipher the molecular mechanisms of their recruitment to VAT and the functional relevance of this process. We observed increased pDC numbers in murine blood, liver, spleen, and VAT after feeding a high-fat diet (HFD) for 3 wk when compared with a standard diet. pDCs were enriched in fat-associated lymphoid clusters representing highly specific lymphoid regions within VAT. HFD led to an enlargement of fat-associated lymphoid clusters with an increased density and migratory speed of pDCs as shown by intravital multiphoton microscopy. For their recruitment into VAT, pDCs employed P-selectin with E-selectin and L-selectin being only critical in response to HFD, indicating that the molecular cues underlying pDC trafficking were dependent on the nutritional state. Subsequent recruitment steps required α4ß1 and α4ß7 integrins and engagement of CCR7. Application of fingolimod (FTY720) abrogated egress of pDCs from VAT, indicating the involvement of sphingosine-1-phosphate in this process. Furthermore, HFD altered pDC functions by promoting their activation and type 1 IFN expression. Blocking pDC infiltration into VAT prevented weight gain and improved glucose tolerance during HFD. In summary, a HFD fundamentally alters pDC biology by promoting their trafficking, retention, and activation in VAT, which in turn seems to regulate metabolism.
Assuntos
Dieta Hiperlipídica , Gordura Intra-Abdominal , Tecido Adiposo , Animais , Células Dendríticas , Gordura Intra-Abdominal/metabolismo , Camundongos , FenótipoRESUMO
Colorectal cancer is one of the most common cancers and a major cause of mortality. Proinflammatory and antitumor immune responses play critical roles in colitis-associated colon cancer. CCL17, a chemokine of the C-C family and ligand for CCR4, is expressed by intestinal dendritic cells in the steady state and is upregulated during colitis in mouse models and inflammatory bowel disease patients. In this study, we investigated the expression pattern and functional relevance of CCL17 for colitis-associated colon tumor development using CCL17-enhanced GFP-knockin mice. CCL17 was highly expressed by dendritic cells but also upregulated in macrophages and intermediary monocytes in colon tumors induced by exposure to azoxymethane and dextran sodium sulfate. Despite a similar degree of inflammation in the colon, CCL17-deficient mice developed fewer tumors than did CCL17-competent mice. This protective effect was abrogated by cohousing, indicating a dependency on the microbiota. Changes in microbiota diversity and composition were detected in separately housed CCL17-deficient mice, and these mice were more susceptible to azoxymethane-induced early apoptosis in the colon affecting tumor initiation. Immune cell infiltration in colitis-induced colon tumors was not affected by the lack of CCL17. Taken together, our results indicate that CCL17 promotes colitis-associated tumorigenesis by influencing the composition of the intestinal microbiome and reducing apoptosis during tumor initiation.
Assuntos
Colite , Neoplasias do Colo , Microbioma Gastrointestinal , Camundongos , Animais , Carcinogênese , Transformação Celular Neoplásica , Azoximetano/toxicidade , Neoplasias do Colo/patologia , Quimiocina CCL17RESUMO
Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DCs) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute illness to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage-HLADR+ cells lacking DC markers. Increased frequency of CD163+ CD14+ cells within the recently discovered DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of programmed death-ligand 1 (PD-L1) in conventional DCs (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.
Assuntos
COVID-19/imunologia , Células Dendríticas/imunologia , Regeneração/imunologia , SARS-CoV-2/imunologia , Adulto , Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , COVID-19/patologia , Células Dendríticas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/patologia , Receptor de Morte Celular Programada 1/imunologiaRESUMO
High-content imaging and single-cell genomics are two of the most prominent high-throughput technologies for studying cellular properties and functions at scale. Recent studies have demonstrated that information in large imaging datasets can be used to estimate gene mutations and to predict the cell-cycle state and the cellular decision making directly from cellular morphology. Thus, high-throughput imaging methodologies, such as imaging flow cytometry can potentially aim beyond simple sorting of cell-populations. We introduce IFC-seq, a machine learning methodology for predicting the expression profile of every cell in an imaging flow cytometry experiment. Since it is to-date unfeasible to observe single-cell gene expression and morphology in flow, we integrate uncoupled imaging data with an independent transcriptomics dataset by leveraging common surface markers. We demonstrate that IFC-seq successfully models gene expression of a moderate number of key gene-markers for two independent imaging flow cytometry datasets: (i) human blood mononuclear cells and (ii) mouse myeloid progenitor cells. In the case of mouse myeloid progenitor cells IFC-seq can predict gene expression directly from brightfield images in a label-free manner, using a convolutional neural network. The proposed method promises to add gene expression information to existing and new imaging flow cytometry datasets, at no additional cost.
Assuntos
Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/métodos , Processamento de Imagem Assistida por Computador/métodos , Aprendizado de Máquina , Análise de Célula Única/métodos , Animais , Biologia Computacional/métodos , Bases de Dados Genéticas , Feminino , Sangue Fetal/metabolismo , Regulação da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Células Progenitoras Mieloides/metabolismo , Redes Neurais de Computação , TranscriptomaRESUMO
Loss of appetite (anorexia) is a typical behavioral response to infectious diseases that often reduces body weight. Also, anorexia can be observed in cancer and trauma patients, causing poor quality of life and reduced prospects of positive therapeutic outcomes. Although anorexia is an acute symptom, its initiation and endocrine regulation during antiviral immune responses are poorly understood. During viral infections, plasmacytoid dendritic cells (pDCs) produce abundant type I interferon (IFN-I) to initiate first-line defense mechanisms. Here, by targeted ablation of pDCs and various in vitro and in vivo mouse models of viral infection and inflammation, we identified that IFN-I is a significant driver of somatostatin (SST). Consequently, SST suppressed the hunger hormone ghrelin that led to severe metabolic changes, anorexia, and rapid body weight loss. Furthermore, during vaccination with Modified Vaccinia Ankara virus (MVA), the SST-mediated suppression of ghrelin was critical to viral immune response, as ghrelin restrained the production of early cytokines by natural killer (NK) cells and pDCs, and impaired the clonal expansion of CD8+ T cells. Thus, the hormonal modulation of ghrelin through SST and the cytokine IFN-I is fundamental for optimal antiviral immunity, which comes at the expense of calorie intake.
Assuntos
Apetite , Grelina , Interferon Tipo I/imunologia , Somatostatina/imunologia , Viroses/imunologia , Animais , Linfócitos T CD8-Positivos , Células Dendríticas , Imunidade Inata , Camundongos , Qualidade de VidaRESUMO
The host-cell restriction factor SERINC5 potently suppresses the infectivity of HIV, type 1 (HIV-1) particles, and is counteracted by the viral pathogenesis factor Nef. However, the molecular mechanism by which SERINC5 restricts HIV-1 particle infectivity is still unclear. Because SERINC proteins have been suggested to facilitate the incorporation of serine during the biosynthesis of membrane lipids and because lipid composition of HIV particles is a major determinant of the infectious potential of the particles, we tested whether SERINC5-mediated restriction of HIV particle infectivity involves alterations of membrane lipid composition. We produced and purified HIV-1 particles from SERINC5293T cells with very low endogenous SERINC5 levels under conditions in which ectopically expressed SERINC5 restricts HIV-1 infectivity and is antagonized by Nef and analyzed both virions and producer cells with quantitative lipid MS. SERINC5 restriction and Nef antagonism were not associated with significant alterations in steady-state lipid composition of producer cells and HIV particles. Sphingosine metabolism kinetics were also unaltered by SERINC5 expression. Moreover, the levels of phosphatidylserine on the surface of HIV-1 particles, which may trigger uptake into non-productive internalization pathways in target cells, did not change upon expression of SERINC5 or Nef. Finally, saturating the phosphatidylserine-binding sites on HIV target cells did not affect SERINC5 restriction or Nef antagonism. These results demonstrate that the restriction of HIV-1 particle infectivity by SERINC5 does not depend on alterations in lipid composition and organization of HIV-1 particles and suggest that channeling serine into lipid biosynthesis may not be a cardinal cellular function of SERINC5.
Assuntos
HIV-1/patogenicidade , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Vírion/patogenicidade , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Ligação Competitiva , Linhagem Celular Tumoral , Deleção de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , HIV-1/química , HIV-1/fisiologia , Humanos , Cinética , Lipossomos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfatidilserinas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Esfingosina/metabolismo , Propriedades de Superfície , Vírion/química , Vírion/fisiologia , Montagem de Vírus , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
The costimulatory molecule CD40 is a major driver of atherosclerosis. It is expressed on a wide variety of cell types, including mature dendritic cells (DCs), and is required for optimal T-cell activation and expansion. It remains undetermined whether and how CD40 on DCs impacts the pathogenesis of atherosclerosis. Here, the effects of constitutively active CD40 in DCs on atherosclerosis were examined using low-density lipoprotein-deficient (Ldlr-/-) bone marrow chimeras that express a transgene containing an engineered latent membrane protein 1 (LMP)/CD40 fusion protein conferring constitutive CD40 signaling under control of the DC-specific CD11c promoter (DC-LMP1/CD40). As expected, DC-LMP1/CD40/Ldlr-/- chimeras (DC-LMP1/CD40) showed increased antigen-presenting capacity of DCs and increased T-cell numbers. However, the mice developed extensive neutrophilia compared to CD40wt/Ldlr-/- (CD40wt) chimeras. Despite overt T-cell expansion and neutrophilia, a reduction in conventional DC frequency and a dramatic (approximately 80%) reduction in atherosclerosis was observed. Further analyses revealed that cholesterol and triglyceride levels had decreased by 37% and 60%, respectively, in DC-LMP1/CD40 chimeras. Moreover, DC-LMP1/CD40 chimeras developed inflammatory bowel disease characterized by massive transmural influx of leukocytes and lymphocytes, resulting in villous degeneration and lipid malabsorption. Constitutive activation of CD40 in DCs results in inflammation of the gastrointestinal tract, thereby impairing lipid uptake, which consequently results in attenuated atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Antígenos CD40/metabolismo , Colesterol/metabolismo , Células Dendríticas/metabolismo , Transdução de Sinais/fisiologia , Animais , Aterosclerose/imunologia , Células Dendríticas/imunologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Ativação Linfocitária/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos MutantesRESUMO
Dendritic cells (DCs) are considered the most potent antigen-presenting cells (APCs), which directly prime or cross-prime MHC I-restricted cytotoxic T cells (CTLs). However, recent evidence suggests the existence of other, as-yet unidentified APCs also able to prime T cells. To identify those APCs, we used adenoviral (rAd) vectors, which do not infect DCs but selectively accumulate in CD169(+) macrophages (MPs). In mice that lack DCs, infection of CD169(+) MPs was sufficient to prime CTLs specific for all epitopes tested. In contrast, CTL responses relying exclusively on cross-presenting DCs were biased to selected strong MHC I-binding peptides only. When both DCs and MPs were absent, no CTL responses could be elicited. Therefore, CD169(+) MPs can be considered APCs that significantly contribute to CTL responses.
Assuntos
Apresentação de Antígeno/fisiologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Lectinas Tipo C/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Reações Cruzadas/imunologia , Células Dendríticas/citologia , CamundongosRESUMO
Immature dendritic cells (DCs) sample tissue-specific antigens (TSAs) and process them for "crosspresentation" via major histocompatibility complex (MHC) class I and II molecules. Findings with adoptively transferred T cell receptor (TCR)-transgenic CD8+ T cells in transgenic mice expressing model TSA indicate that this process contributes to tolerance induction of CD8+ T cells, a phenomenon termed "crosstolerance." However, up to now it has been unknown whether "crosstolerance" can also control autoimmune T cells specific for physiological nontransgenic TSA. Here, we showed that a DC-specific deficiency in uptake of apoptotic material inhibits crosspresentation in vivo. This defect allowed the accumulation of fully functional autoreactive CD8+ T cells that could be activated for autoimmune attack in peripheral lymphoid organs. Thus, our data demonstrate the importance of crosstolerance induction by DCs as a vital instrument for controlling self-reactive T cells from the peripheral repertoire and preventing autoimmune disease.
Assuntos
Antígenos/imunologia , Antígenos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Apresentação Cruzada/imunologia , Tolerância Imunológica , Animais , Apoptose/genética , Apoptose/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/transplante , Linhagem Celular , Apresentação Cruzada/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Tolerância Imunológica/genética , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Tecido Linfoide/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Ovalbumina/metabolismo , Proteínas rac1 de Ligação ao GTP/biossíntese , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Tissues accommodate defined numbers of dendritic cells (DCs) in highly specific niches where different intrinsic and environmental stimuli control DC life span and numbers. DC homeostasis in tissues is important, because experimental changes in DC numbers influence immunity and tolerance toward various immune catastrophes and inflammation. However, the precise molecular mechanisms regulating DC life span and homeostasis are unclear. We report that the GTPase RhoA controls homeostatic proliferation, cytokinesis, survival, and turnover of cDCs. Deletion of RhoA strongly decreased the numbers of CD11b(-)CD8(+) and CD11b(+)Esam(hi) DC subsets, whereas CD11b(+)Esam(lo) DCs were not affected in conditional RhoA-deficient mice. Proteome analyses revealed a defective prosurvival pathway via PI3K/protein kinase B (Akt1)/Bcl-2-associated death promoter in the absence of RhoA. Taken together, our findings identify RhoA as a central regulator of DC homeostasis, and its deletion decreases DC numbers below critical thresholds for immune protection and homeostasis, causing aberrant compensatory DC proliferation.
Assuntos
Apoptose/imunologia , Células Dendríticas/imunologia , Homeostase/imunologia , Proteína rhoA de Ligação ao GTP/imunologia , Animais , Apoptose/genética , Western Blotting , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Proliferação de Células/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Citocinese/genética , Citocinese/imunologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Homeostase/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Baço/citologia , Baço/imunologia , Baço/metabolismo , Proteína de Morte Celular Associada a bcl/imunologia , Proteína de Morte Celular Associada a bcl/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The PI3K/Akt/mTOR pathway has emerged as a critical regulator of dendritic cell (DC) development and function. The kinase mTOR is found in 2 distinct complexes, mTORC1 and mTORC2. In this study, we show that mTORC1 but not mTORC2 is required for epidermal Langerhans cell (LC) homeostasis. Although the initial seeding of the epidermis with LCs is not affected, the lack of mTORC1 activity in DCs by conditional deletion of Raptor leads to a progressive loss of LCs in the skin of mice. Ablation of mTORC2 function by deletion of Rictor results in a modest reduction of LCs in skin draining lymph nodes. In young mice Raptor-deficient LCs show an increased tendency to leave the skin, leading to a higher frequency of migratory DCs in skin draining lymph nodes, indicating that the loss of LCs results from enhanced migration. LCs lacking Raptor are smaller and display reduced expression of Langerin, E-cadherin, ß-catenin, and CCR7 but unchanged levels of MHC-II, ruling out enhanced spontaneous maturation. Ki-67 and annexin V stainings revealed a faster turnover rate and increased apoptosis of Raptor-deficient LCs, which might additionally affect the preservation of the LC network. Taken together our results show that the homeostasis of LCs strictly depends on mTORC1.
Assuntos
Homeostase , Células de Langerhans/fisiologia , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Western Blotting , Citometria de Fluxo , Imuno-Histoquímica , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos TransgênicosRESUMO
Epidermal Langerhans cells (LCs) of the skin represent the prototype migratory dendritic cell (DC) subtype. In the skin, they take up Ag, migrate to the draining lymph nodes, and contribute to Ag transport and immunity. Different depletion models for LCs have revealed contrasting roles and contributions of this cell type. To target the migratory properties of DCs, we generated mice lacking the Rho-family GTPase Cdc42 specifically in DCs. In these animals, the initial seeding of the epidermis with LCs is functional, resulting in slightly reduced Langerhans cell numbers. However, Cdc42-deficient LCs fail to leave the skin in steady state as well as upon stimulation, as they do not enter the skin-draining afferent lymph vessels. Similarly, also other Cdc42-deficient migratory DC subsets fail to home properly to the corresponding draining lymph nodes. We used this novel mouse model, in which LCs are locked out, to demonstrate that these cells contribute substantially to priming of Ag-specific CD4 and CD8 T cell responses upon epicutaneous immunization, but could not detect a role in the induction of contact hypersensitivity to various doses of hapten.
Assuntos
Inibição de Migração Celular/imunologia , Movimento Celular/imunologia , Células de Langerhans/imunologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Animais , Inibição de Migração Celular/genética , Movimento Celular/genética , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Dermatite de Contato/genética , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Modelos Animais de Doenças , Epiderme/enzimologia , Epiderme/imunologia , Epiderme/patologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Células de Langerhans/enzimologia , Células de Langerhans/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Quimera por Radiação/genética , Quimera por Radiação/imunologia , Proteína cdc42 de Ligação ao GTP/deficiência , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Chronic viral infections lead to CD8(+) T-cell exhaustion, characterized by impaired cytokine secretion and loss of proliferative capacity. While viral load and T-cell dysfunction correlate, it is currently unclear whether the quality of a cell type presenting antigen determines the degree of T-cell exhaustion or if the overall amount of antigen recognized by T cells promotes exhaustion. We found that chronic lymphocytic chorio-meningitis virus infection led to decreased CD8(+) T-cell exhaustion in DC-MHC class I (MHCI) mice, in which CD8(+) T cells can only recognize antigen on DCs. However, this increase in CD8(+) T-cell function came at the expense of fatal immunopathology. Additional antigen recognition on nonhematopoietic cells in DC-MHCI mice promoted T-cell exhaustion and avoidance of immunopathology. Likewise, increased numbers of antigen-expressing hematopoietic cells, as well as a selective elevation of the number of DCs as the only cell type presenting antigen in DC-MHCI mice, resulted in compromised T-cell function. These results favor a scenario in which the overall amount of antigen exposure, rather than the type of cell engaging with virus-specific CD8(+) T cells, is responsible for their functional exhaustion. Furthermore, exhaustion of virus-specific CD8(+) T cells leads to avoidance of life-threatening immunopathology.