RESUMO
The antiviral state, an initial line of defense against viral infection, is established by a set of IFN-stimulated genes (ISGs) encoding antiviral effector proteins. The effector ISGs are transcriptionally regulated by type I IFNs mainly via activation of IFN-stimulated gene factor 3 (ISGF3). In this study, the regulatory elements of effector ISGs were characterized to determine the (epi)genetic features that enable their robust induction by type I IFNs in multiple cell types. We determined the location of regulatory elements, the DNA motifs, the occupancy of ISGF3 subunits (IRF9, STAT1, and STAT2) and other transcription factors, and the chromatin accessibility of 37 effector ISGs in murine dendritic cells. The IFN-stimulated response element (ISRE) and its tripartite version occurred most frequently in the regulatory elements of effector ISGs than in any other tested ISG subsets. Chromatin accessibility at their promoter regions was similar to most other ISGs but higher than at the promoters of inflammation-related cytokines, which were used as a reference gene set. Most effector ISGs (81.1%) had at least one ISGF3 binding region proximal to the transcription start site (TSS), and only a subset of effector ISGs (24.3%) was associated with three or more ISGF3 binding regions. The IRF9 signals were typically higher, and ISRE motifs were "stronger" (more similar to the canonical sequence) in TSS-proximal versus TSS-distal regulatory regions. Moreover, most TSS-proximal regulatory regions were accessible before stimulation in multiple cell types. Our results indicate that "strong" ISRE motifs and universally accessible promoter regions that permit robust, widespread induction are characteristic features of effector ISGs.
Assuntos
Fatores de Restrição Antivirais , Cromatina , Animais , Camundongos , Cromatina/genética , Motivos de Nucleotídeos , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Interferons/metabolismoRESUMO
Dendritic cell (DC) activation and cytokine production is tightly regulated. In this study, we found that Zbtb10 expression is activation dependent and it is essential for the immunogenic function of cDC1. Zbtb10 knockdown (KD) significantly reduced the expression of co-stimulatory genes CD80 and CD86 along with cytokines including IL-12, IL-6, and IL-10, in activated cDC1 Mutu-DC line. Consequently, the clonal expansion of CD44+ effector T cells in co-cultured CD4+ T cells was drastically reduced owing to significantly reduced IL-2. At the same time, these CD44+ effector T cells were unable to differentiate toward Tbet+ IFNγ+ Th1 subtype. Instead, an increased frequency of Th2 cells expressing GATA3+ and IL-13+ was observed. Interestingly, in Zbtb10 KD condition the co-cultured T cells depicted increased expression of PD1 and LAG3, the T-cell anergic markers. Moreover, the global transcriptome analysis identified that Zbtb10 is pertinent for DC activation and its depletion in cDC1 completely shuts down their immune responses. Mechanistic analysis revealed that Zbtb10 KD enhanced the expression of NKRF (NF-κB repressing factor) leading to drastic suppression of NF-κB related genes. Zbtb10 KD abrogated p65 and RelB nuclear translocation, thereby controlling the activation and maturation of cDC1 and the ensuing adaptive T cell responses.
Assuntos
Citocinas/biossíntese , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Biomarcadores , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ativação Linfocitária/imunologia , Camundongos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Plasmacytoid dendritic cells (DCs) are reported to induce robust type-I interferon (IFN) response, whereas cDC1 DCs develop moderate type-I IFN response upon TLR9 stimulation. It is very interesting to understand how this signaling under TLR9 is tightly regulated for the induction of type-I IFNs. Here, we report co-repressor protein NCoR1 as the major factor fine-tuning the signaling pathways regulating IFN-ß expression under TLR9 in cDC1 DCs. We found that NCoR1 knockdown induced a robust IFN-ß-mediated antiviral response upon TLR9 activation in cDC1 DCs. At the molecular level, we showed that NCoR1 directly repressed MyD88-IRF7 signaling axis in cDC1 cells. Therefore, NCoR1 depletion enhanced pIRF7 levels, IFN-ß secretion, and downstream pSTAT1-pSTAT2 signaling, leading to sustained induction of IFN stimulatory genes. Integrative genomic analysis depicted strong enrichment of an antiviral gene-module in CpG-activated NCoR1 knockdown DCs upon TLR9 activation. Moreover, we confirmed our findings in primary DCs derived from splenocytes of WT and NCoR1 DC-/- animals, which showed protection from Sendai and Vesicular Stomatitis viruses upon CpG activation. Ultimately, we identified that NCoR1-HDAC3 complex is involved in repressing the type-I IFN response in cDC1 DCs.
Assuntos
Células Dendríticas/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/fisiologiaRESUMO
Identification of disease-associated autoantibodies is of high importance. Their assessment could complement current diagnostic modalities and assist the clinical management of patients. We aimed at developing and validating high-throughput protein microarrays able to screen patients' sera to determine disease-specific autoantibody-signatures for pancreatic cancer (PDAC), chronic pancreatitis (CP), autoimmune pancreatitis and their subtypes (AIP-1 and AIP-2). In-house manufactured microarrays were used for autoantibody-profiling of IgG-enriched preoperative sera from PDAC-, CP-, AIP-1-, AIP-2-, other gastrointestinal disease (GID) patients and healthy controls. As a top-down strategy, three different fluorescence detection-based protein-microarrays were used: large with 6400, intermediate with 345, and small with 36 full-length human recombinant proteins. Large-scale analysis revealed 89 PDAC, 98 CP and 104 AIP immunogenic antigens. Narrowing the selection to 29 autoantigens using pooled sera first and individual sera afterwards allowed a discrimination of CP and AIP from PDAC. For validation, predictive models based on the identified antigens were generated which enabled discrimination between PDAC and AIP-1 or AIP-2 yielded high AUC values of 0.940 and 0.925, respectively. A new repertoire of autoantigens was identified and their assembly as a multiplex test will provide a fast and cost-effective tool for differential diagnosis of pancreatic diseases with high clinical relevance.
Assuntos
Autoanticorpos/sangue , Pancreatite Autoimune/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Análise Serial de Proteínas/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/imunologia , Pancreatite Autoimune/imunologia , Diagnóstico Diferencial , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/imunologia , Pancreatite Crônica/diagnóstico , Pancreatite Crônica/imunologia , Pacientes , Neoplasias PancreáticasRESUMO
Th17 cells are often associated with autoimmunity and been shown to be increased in CD11b-/- mice. Here, we examined the role of CD11b in murine collagen-induced arthritis (CIA). C57BL/6 and CD11b-/- resistant mice were immunized with type II collagen. CD11b-/- mice developed arthritis with early onset, high incidence, and sustained severity compared with C57BL/6 mice. We observed a marked leukocyte infiltration, and histological examinations of the arthritic paws from CD11b-/- mice revealed that the cartilage was destroyed in association with strong lymphocytic infiltration. The CD11b deficiency led to enhanced Th17-cell differentiation. CD11b-/- dendritic cells (DCs) induced much stronger IL-6 production and hence Th17-cell differentiation than wild-type DCs. Treatment of CD11b-/- mice after establishment of the Treg/Th17 balance with an anti-IL-6 receptor mAb significantly suppressed the induction of Th17 cells and reduced arthritis severity. Finally, the severe phenotype of arthritis in CD11b-/- mice was rescued by adoptive transfer of CD11b+ DCs. Taken together, our results indicate that the resistance to CIA in C57BL/6 mice is regulated by CD11b via suppression of IL-6 production leading to reduced Th17-cell differentiation. Therefore, CD11b may represent a susceptibility factor for autoimmunity and could be a target for future therapy.
Assuntos
Artrite Experimental/imunologia , Antígeno CD11b/metabolismo , Cartilagem/imunologia , Células Dendríticas/imunologia , Interleucina-6/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Transferência Adotiva , Animais , Anticorpos Bloqueadores/farmacologia , Antígeno CD11b/genética , Diferenciação Celular , Células Cultivadas , Colágeno Tipo II/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-6/imunologiaRESUMO
Experimental autoimmune myocarditis (EAM) is a CD4(+) T-cell-mediated model of human inflammatory dilated cardiomyopathies. Heart-specific CD4(+) T-cell activation is dependent on autoantigens presented by MHC class II (MHCII) molecules expressed on professional APCs. In this study, we addressed the role of inflammation-induced MHCII expression by cardiac nonhematopoietic cells on EAM development. EAM was induced in susceptible mice lacking inducible expression of MHCII molecules on all nonhematopoietic cells (pIV-/- K14 class II transactivator (CIITA) transgenic (Tg) mice) by immunization with α-myosin heavy chain peptide in CFA. Lack of inducible nonhematopoietic MHCII expression in pIV-/- K14 CIITA Tg mice conferred EAM resistance. In contrast, cardiac pathology was induced in WT and heterozygous mice, and correlated with elevated cardiac endothelial MHCII expression. Control mice with myocarditis displayed an increase in infiltrating CD4(+) T cells and in expression of IFN-γ, which is the major driver of nonhematopoietic MHCII expression. Mechanistically, IFN-γ neutralization in WT mice shortly before disease onset resulted in reduced cardiac MHCII expression and pathology. These findings reveal a previously overlooked contribution of IFN-γ to induce endothelial MHCII expression in the heart and to progress cardiac pathology during myocarditis.
Assuntos
Doenças Autoimunes/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Miocardite/imunologia , Animais , Autoantígenos , Linfócitos T CD4-Positivos , Modelos Animais de Doenças , Endotélio/imunologia , Inflamação , Interferon gama/imunologia , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Miocárdio/ultraestrutura , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Transativadores/genéticaRESUMO
The ability of pathogens to influence host cell survival is a crucial virulence factor. Listeria monocytogenes (Lm) infection is known to be associated with severe apoptosis of hepatocytes and spleen cells. This impairs host defense mechanisms and thereby facilitates the spread of intracellular pathogens. The general mechanisms of apoptosis elicited by Lm infection are understood, however, the roles of BH3-only proteins during primary Lm infection have not been examined. To explore the roles of BH3-only proteins in Lm-induced apoptosis, we studied Listeria infections in mice deficient in Bim, Bid, Noxa or double deficient in BimBid or BimNoxa. We found that BimNoxa double knockout mice were highly resistant to high-dose challenge with Listeria. Decreased bacterial burden and decreased host cell apoptosis were found in the spleens of these mice. The ability of the BH3-deficient mice to clear bacterial infection more efficiently than WT was correlated with increased concentrations of ROS, neutrophil extracellular DNA trap release and downregulation of TNF-α. Our data show a novel pathway of infection-induced apoptosis that enhances our understanding of the mechanism by which BH3-only proteins control apoptotic host cell death during Listeria infection.
Assuntos
Apoptose , Listeria monocytogenes , Listeriose/etiologia , Listeriose/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/deficiência , Proteína 11 Semelhante a Bcl-2/deficiência , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Resistência à Doença/genética , Resistência à Doença/imunologia , Suscetibilidade a Doenças , Armadilhas Extracelulares/imunologia , Feminino , Expressão Gênica , Listeriose/mortalidade , Listeriose/patologia , Masculino , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Espécies Reativas de Oxigênio/metabolismo , Baço/imunologia , Baço/metabolismo , Baço/patologia , Taxa de SobrevidaRESUMO
BACKGROUND: Discriminating between autoimmune pancreatitis (AIP), chronic pancreatitis (CP), and pancreatic ductal adenocarcinoma (PDAC) can be challenging. In this retrospective study, levels of serum and tissue cytokines were analyzed as part of the clinical strategy for the preoperative differentiation between AIP and PDAC. The identification of differential cytokine profiles may help to prevent unnecessary surgical resection and allow optimal treatment of these pathologies. METHODS: To compare the cytokine profiles of AIP, CP, and PDAC patients, serum and pancreatic tissue homogenates were subjected to multiplex analysis of 17 inflammatory mediators. In total, serum from 73 patients, composed of 29 AIP (14 AIP-1 and 15 AIP-2), 17 CP, and 27 PDAC, and pancreatic tissue from 36 patients, including 12 AIP (six AIP-1 and six AIP-2), 12 CP, and 12 PDAC, were analyzed. RESULTS: Comparing AIP and PDAC patients' serum, significantly higher concentrations were found in AIP for interleukins IL-1ß, IL-7, IL-13, and granulocyte colony-stimulating factor (G-CSF). G-CSF also allowed discrimination of AIP from CP. Furthermore, once AIP was divided into subtypes, significantly higher serum levels for IL-7 and G-CSF were measured in both subtypes of AIP and in AIP-2 for IL-1ß when compared to PDAC. G-CSF and TNF-α were also significantly differentially expressed in tissue homogenates between AIP-2 and PDAC. CONCLUSIONS: The cytokines IL-1ß, IL-7, and G-CSF can be routinely measured in patients' serum, providing an elegant and non-invasive approach for differential diagnosis. G-CSF is a good candidate to supplement the currently known serum markers in predictive tests for AIP and represents a basis for a combined blood test to differentiate AIP and particularly AIP-2 from PDAC, enhancing the possibility of appropriate treatment.
Assuntos
Adenocarcinoma/diagnóstico , Doenças Autoimunes/diagnóstico , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/diagnóstico , Citocinas/sangue , Neoplasias Pancreáticas/diagnóstico , Pancreatite Crônica/sangue , Pancreatite Crônica/diagnóstico , Adenocarcinoma/sangue , Adenocarcinoma/fisiopatologia , Adulto , Doenças Autoimunes/sangue , Doenças Autoimunes/fisiopatologia , Carcinoma Ductal Pancreático/fisiopatologia , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Pancreatite Crônica/fisiopatologia , Curva ROC , Neoplasias PancreáticasRESUMO
Because of their unique capacity to cross-present Ags to CD8(+) T cells, mouse lymphoid tissue-resident CD8(+) dendritic cells (DCs) and their migratory counterparts are critical for priming antiviral T cell responses. High expression of the dsRNA sensor TLR3 is a distinctive feature of these cross-presenting DC subsets. TLR3 engagement in CD8(+) DCs promotes cross-presentation and the acquisition of effector functions required for driving antiviral T cell responses. In this study, we performed a comprehensive analysis of the TLR3-induced antiviral program and cell-autonomous immunity in CD8(+) DC lines and primary CD8(+) DCs. We found that TLR3-ligand polyinosinic-polycytidylic acid and human rhinovirus infection induced a potent antiviral protection against Sendai and vesicular stomatitis virus in a TLR3 and type I IFN receptor-dependent manner. Polyinosinic-polycytidylic acid-induced antiviral genes were identified by mass spectrometry-based proteomics and transcriptomics in the CD8(+) DC line. Nanostring nCounter experiments confirmed that these antiviral genes were induced by TLR3 engagement in primary CD8(+) DCs, and indicated that many are secondary TLR3-response genes requiring autocrine IFN-ß stimulation. TLR3-activation thus establishes a type I IFN-dependent antiviral program in a DC subtype playing crucial roles in priming adaptive antiviral immune responses. This mechanism is likely to shield the priming of antiviral responses against inhibition or abrogation by the viral infection. It could be particularly relevant for viruses detected mainly by TLR3, which may not trigger type I IFN production by DCs that lack TLR3, such as plasmacytoid DCs or CD8(-) DCs.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Interferon beta/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Apresentação Cruzada/imunologia , Humanos , Interferon beta/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/virologia , Poli I-C/imunologia , Receptor de Interferon alfa e beta/imunologia , Rhinovirus/imunologia , Vírus Sendai/imunologia , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
Beta-catenin signaling has recently been tied to the emergence of tolerogenic dendritic cells (DCs). In this article, we demonstrate a novel role for beta-catenin in directing DC subset development through IFN regulatory factor 8 (IRF8) activation. We found that splenic DC precursors express beta-catenin, and DCs from mice with CD11c-specific constitutive beta-catenin activation upregulated IRF8 through targeting of the Irf8 promoter, leading to in vivo expansion of IRF8-dependent CD8a+, plasmacytoid, and CD103+ CD11b2 DCs. beta-cateninstabilized CD8a+ DCs secreted elevated IL-12 upon in vitro microbial stimulation, and pharmacological beta-catenin inhibition blocked this response in wild-type cells. Upon infections with Toxoplasma gondii and vaccinia virus, mice with stabilized DC beta-catenin displayed abnormally high Th1 and CD8+ T lymphocyte responses, respectively. Collectively, these results reveal a novel and unexpected function for beta-catenin in programming DC differentiation toward subsets that orchestrate proinflammatory immunity to infection.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Inflamação/imunologia , Fatores Reguladores de Interferon/genética , beta Catenina/imunologia , Animais , Antígenos CD/imunologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Antígeno CD11c/imunologia , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Ativação Enzimática , Feminino , Cadeias alfa de Integrinas/imunologia , Fatores Reguladores de Interferon/imunologia , Interleucina-12/biossíntese , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Carga Parasitária , Regiões Promotoras Genéticas , Pirimidinonas/farmacologia , Receptores de Superfície Celular/genética , Transdução de Sinais/imunologia , Baço/citologia , Baço/imunologia , Células Th1/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Vacínia/imunologia , Vaccinia virus/imunologia , beta Catenina/antagonistas & inibidores , beta Catenina/biossínteseRESUMO
Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1ß stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucina-1beta/imunologia , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Transdução de Sinais/imunologia , Animais , Apresentação de Antígeno/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Celular/imunologia , Interleucina-1beta/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The activation, or maturation, of dendritic cells (DCs) is crucial for the initiation of adaptive T-cell mediated immune responses. Research on the molecular mechanisms implicated in DC maturation has focused primarily on inducible gene-expression events promoting the acquisition of new functions, such as cytokine production and enhanced T-cell-stimulatory capacity. In contrast, mechanisms that modulate DC function by inducing widespread gene-silencing remain poorly understood. Yet the termination of key functions is known to be critical for the function of activated DCs. Genome-wide analysis of activation-induced histone deacetylation, combined with genome-wide quantification of activation-induced silencing of nascent transcription, led us to identify a novel inducible transcriptional-repression pathway that makes major contributions to the DC-maturation process. This silencing response is a rapid primary event distinct from repression mechanisms known to operate at later stages of DC maturation. The repressed genes function in pivotal processes--including antigen-presentation, extracellular signal detection, intracellular signal transduction and lipid-mediator biosynthesis--underscoring the central contribution of the silencing mechanism to rapid reshaping of DC function. Interestingly, promoters of the repressed genes exhibit a surprisingly high frequency of PU.1-occupied sites, suggesting a novel role for this lineage-specific transcription factor in marking genes poised for inducible repression.
Assuntos
Células Dendríticas/metabolismo , Inativação Gênica , Proteínas Nucleares/genética , Transativadores/genética , Transcrição Gênica , Animais , Humanos , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismoRESUMO
Leishmania major infection induces self-healing cutaneous lesions in C57BL/6 mice. Both IL-12 and IFN-γ are essential for the control of infection. We infected Jun dimerization protein p21SNFT (Batf3(-/-) ) mice (C57BL/6 background) that lack the major IL-12 producing and cross-presenting CD8α(+) and CD103(+) DC subsets. Batf3(-/-) mice displayed enhanced susceptibility with larger lesions and higher parasite burden. Additionally, cells from draining lymph nodes of infected Batf3(-/-) mice secreted less IFN-γ, but more Th2- and Th17-type cytokines, mirrored by increased serum IgE and Leishmania-specific immunoglobulin 1 (Th2 indicating). Importantly, CD8α(+) DCs isolated from lymph nodes of L. major-infected mice induced significantly more IFN-γ secretion by L. major-stimulated immune T cells than CD103(+) DCs. We next developed CD11c-diptheria toxin receptor: Batf3(-/-) mixed bone marrow chimeras to determine when the DCs are important for the control of infection. Mice depleted of Batf-3-dependent DCs from day 17 or wild-type mice depleted of cross-presenting DCs from 17-19 days after infection maintained significantly larger lesions similar to mice whose Batf-3-dependent DCs were depleted from the onset of infection. Thus, we have identified a crucial role for Batf-3-dependent DCs in protection against L. major.
Assuntos
Apresentação de Antígeno , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Apresentação Cruzada , Células Dendríticas/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Proteínas Repressoras/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/genética , Anticorpos Antiprotozoários/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Apresentação Cruzada/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Imunoglobulina E/sangue , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Interferon gama , Leishmania major/metabolismo , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/patologiaRESUMO
Fungal pathogens are a frequent cause of opportunistic infections. They live as commensals in healthy individuals but can cause disease when the immune status of the host is altered. T lymphocytes play a critical role in pathogen control. However, specific Ags determining the activation and function of antifungal T cells remain largely unknown. By using an immunoproteomic approach, we have identified for the first time, to our knowledge, a natural T cell epitope from Candida albicans. Isolation and sequencing of MHC class II-bound ligands from infected dendritic cells revealed a peptide that was recognized by a major population of all Candida-specific Th cells isolated from infected mice. Importantly, human Th cells also responded to stimulation with the peptide in an HLA-dependent manner but without restriction to any particular HLA class II allele. Immunization of mice with the peptide resulted in a population of epitope-specific Th cells that reacted not only with C. albicans but also with other clinically highly relevant species of Candida including the distantly related Candida glabrata. The extent of the reaction to different Candida species correlated with their degree of phylogenetic relationship to C. albicans. Finally, we show that the newly identified peptide acts as an efficient vaccine when used in combination with an adjuvant inducing IL-17A secretion from peptide-specific T cells. Immunized mice were protected from fatal candidiasis. Together, these results uncover a new immune determinant of the host response against Candida ssp. that could be exploited for the development of antifungal vaccines and immunotherapies.
Assuntos
Candida albicans/imunologia , Candidíase/prevenção & controle , Epitopos de Linfócito T/imunologia , Vacinas Fúngicas/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Candidíase/imunologia , Linhagem Celular , Vacinas Fúngicas/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T Auxiliares-Indutores/microbiologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
OBJECTIVE: In gout, monosodium urate crystals are taken up by macrophages, triggering the activation of the NLRP3 inflammasome and the maturation of IL-1ß. This study aimed to investigate the role of integrin CD11b in inflammasome activation in macrophages stimulated by MSU. METHODS: BMDM from WT and CD11b KO mice were stimulated in vitro with MSU crystals. Cellular supernatants were collected to assess the expression of the inflammatory cytokines by enzyme-linked immunosorbent assay and western blot methods. The role of integrin CD11b in MSU-induced gouty arthritis in vivo was investigated by intra-articular injection of MSU crystals. Real-time extracellular acidification rate and oxygen consumption rate of BMDMs were measured by Seahorse Extracellular Flux Analyzer. RESULTS: We demonstrate that CD11b-deficient mice developed exacerbated gouty arthritis with increased recruitment of leukocytes in the joint and higher IL-1ß levels in the sera. In macrophages, genetic deletion of CD11b induced a shift of macrophage metabolism from oxidative phosphorylation to glycolysis, thus decreasing the overall generation of intracellular ATP. Upon MSU stimulation, CD11b-deficient macrophages showed an exacerbated secretion of IL-1ß. Treating wild-type macrophages with a CD11b agonist, LA1, inhibited MSU-induced release of IL-1ß in vitro and attenuated the severity of experimental gouty arthritis. Importantly, LA1, was also effective in human cells as it inhibited MSU-induced release of IL-1ß by peripheral blood mononuclear cells from healthy donors. CONCLUSION: Our data identified the CD11b integrin as a principal cell membrane receptor that modulates NLRP3 inflammasome activation by MSU crystal in macrophages, which could be a potential therapeutic target to treat gouty arthritis in human patients.
Assuntos
Artrite Gotosa , Antígeno CD11b , Inflamassomos , Macrófagos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ácido Úrico , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Macrófagos/metabolismo , Antígeno CD11b/metabolismo , Inflamassomos/metabolismo , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/metabolismo , Camundongos , MasculinoRESUMO
MicroRNAs (miRNAs) are small, noncoding RNAs that regulate target mRNAs by binding to their 3' untranslated regions. There is growing evidence that microRNA-155 (miR155) modulates gene expression in various cell types of the immune system and is a prominent player in the regulation of innate and adaptive immune responses. To define the role of miR155 in dendritic cells (DCs) we performed a detailed analysis of its expression and function in human and mouse DCs. A strong increase in miR155 expression was found to be a general and evolutionarily conserved feature associated with the activation of DCs by diverse maturation stimuli in all DC subtypes tested. Analysis of miR155-deficient DCs demonstrated that miR155 induction is required for efficient DC maturation and is critical for the ability of DCs to promote antigen-specific T-cell activation. Expression-profiling studies performed with miR155(-/-) DCs and DCs overexpressing miR155, combined with functional assays, revealed that the mRNA encoding the transcription factor c-Fos is a direct target of miR155. Finally, all of the phenotypic and functional defects exhibited by miR155(-/-) DCs could be reproduced by deregulated c-Fos expression. These results indicate that silencing of c-Fos expression by miR155 is a conserved process that is required for DC maturation and function.
Assuntos
Células Dendríticas/fisiologia , Inativação Gênica/imunologia , MicroRNAs/imunologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Células Dendríticas/citologia , Evolução Molecular , Humanos , Camundongos , Camundongos Mutantes , MicroRNAs/genética , Monócitos/citologia , RNA Mensageiro/genética , RNA Mensageiro/imunologiaRESUMO
Cystatin C (CstC) is a cysteine protease inhibitor of major clinical importance. Low concentration of serum CstC is linked to atherosclerosis. CstC can prevent formation of amyloid ß associated with Alzheimer's disease and can itself form toxic aggregates. CstC regulates NO secretion by macrophages and is a TGF-ß antagonist. Finally, the serum concentration of CstC is an indicator of kidney function. Yet, little is known about the regulation of CstC expression in vivo. In this study, we demonstrate that the transcription factor IFN regulatory factor 8 (IRF-8) is critical for CstC expression in primary dendritic cells. Only those cells with IRF-8 bound to the CstC gene promoter expressed high levels of the inhibitor. Secretion of IL-10 in response to inflammatory stimuli downregulated IRF-8 expression and consequently CstC synthesis in vivo. Furthermore, the serum concentration of CstC decreased in an IL-10-dependent manner in mice treated with the TLR9 agonist CpG. CstC synthesis is therefore more tightly regulated than hitherto recognized. The mechanisms involved in this regulation might be targeted to alter CstC production, with potential therapeutic value. Our results also indicate that caution should be exerted when using the concentration of serum CstC as an indicator of kidney function in conditions in which inflammation may alter CstC production.
Assuntos
Cistatina C/biossíntese , Cistatina C/sangue , Regulação para Baixo/imunologia , Mediadores da Inflamação/fisiologia , Fatores Reguladores de Interferon/biossíntese , Interleucina-10/fisiologia , Animais , Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/patologia , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Cistatina C/deficiência , Células Dendríticas/classificação , Células Dendríticas/imunologia , Células Dendríticas/patologia , Regulação para Baixo/genética , Mediadores da Inflamação/antagonistas & inibidores , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/fisiologia , Interleucina-10/metabolismo , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
Plasma cells represent the end stage of B-cell development and play a key role in providing an efficient antibody response, but they are also involved in numerous pathologies. Here we show that CD93, a receptor expressed during early B-cell development, is reinduced during plasma-cell differentiation. High CD93/CD138 expression was restricted to antibody-secreting cells both in T-dependent and T-independent responses as naive, memory, and germinal-center B cells remained CD93-negative. CD93 was expressed on (pre)plasmablasts/plasma cells, including long-lived plasma cells that showed decreased cell cycle activity, high levels of isotype-switched Ig secretion, and modification of the transcriptional network. T-independent and T-dependent stimuli led to re-expression of CD93 via 2 pathways, either before or after CD138 or Blimp-1 expression. Strikingly, while humoral immune responses initially proceeded normally, CD93-deficient mice were unable to maintain antibody secretion and bone-marrow plasma-cell numbers, demonstrating that CD93 is important for the maintenance of plasma cells in bone marrow niches.
Assuntos
Formação de Anticorpos/imunologia , Medula Óssea/imunologia , Glicoproteínas de Membrana/imunologia , Plasmócitos/imunologia , Receptores de Complemento/imunologia , Animais , Diferenciação Celular , Galinhas , Fator de Transcrição Ikaros , Imunização , Vírus do Tumor Mamário do Camundongo/imunologia , Camundongos , Plasmócitos/citologia , Fator 1 de Ligação ao Domínio I Regulador Positivo , Infecções por Retroviridae/imunologia , Sindecana-1/imunologia , Linfócitos T/imunologia , Transativadores/imunologia , Fatores de Transcrição/imunologia , gama-Globulinas/imunologiaRESUMO
Dendritic cell (DC) fine-tunes inflammatory versus tolerogenic responses to protect from immune-pathology. However, the role of co-regulators in maintaining this balance is unexplored. NCoR1-mediated repression of DC immune-tolerance has been recently reported. Here we found that depletion of NCoR1 paralog SMRT (NCoR2) enhanced cDC1 activation and expression of IL-6, IL-12 and IL-23 while concomitantly decreasing IL-10 expression/secretion. Consequently, co-cultured CD4+ and CD8+ T-cells depicted enhanced Th1/Th17 frequency and cytotoxicity, respectively. Comparative genomic and transcriptomic analysis demonstrated differential regulation of IL-10 by SMRT and NCoR1. SMRT depletion represses mTOR-STAT3-IL10 signaling in cDC1 by down-regulating NR4A1. Besides, Nfkbia and Socs3 were down-regulated in Ncor2 (Smrt) depleted cDC1, supporting increased production of inflammatory cytokines. Moreover, studies in mice showed, adoptive transfer of SMRT depleted cDC1 in OVA-DTH induced footpad inflammation led to increased Th1/Th17 and reduced tumor burden after B16 melanoma injection by enhancing oncolytic CD8+ T-cell frequency, respectively. We also depicted decreased Ncor2 expression in Rheumatoid Arthritis, a Th1/Th17 disease.
Assuntos
Interleucina-10 , Interleucina-6 , Animais , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Células Dendríticas/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Interleucina-6/metabolismo , Camundongos , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear , Fator de Transcrição STAT3 , Serina-Treonina Quinases TOR/metabolismoRESUMO
B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors--transmembrane activator and calcium signal--modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)--that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific "receptor." This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRIL's basic sequence. Moreover, syndecan-1-positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.