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1.
Nature ; 574(7777): 249-253, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31578523

RESUMO

The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells1. The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality2-5. Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6-/- skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.


Assuntos
Diferenciação Celular , Células Epidérmicas/citologia , Epiderme/fisiologia , Fatores Reguladores de Interferon/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Anormalidades Múltiplas/genética , Animais , Fenda Labial/genética , Fissura Palatina/genética , Cistos/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Células Epidérmicas/metabolismo , Epiderme/embriologia , Anormalidades do Olho/genética , Feminino , Dedos/anormalidades , Regulação da Expressão Gênica , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Joelho/anormalidades , Articulação do Joelho/anormalidades , Lábio/anormalidades , Metabolismo dos Lipídeos/genética , Deformidades Congênitas das Extremidades Inferiores/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/genética , Sindactilia/genética , Anormalidades Urogenitais/genética
2.
Nat Commun ; 10(1): 3974, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481662

RESUMO

Tumor-associated macrophages (TAMs) usually express an M2 phenotype, which enables them to perform immunosuppressive and tumor-promoting functions. Reprogramming these TAMs toward an M1 phenotype could thwart their pro-cancer activities and unleash anti-tumor immunity, but efforts to accomplish this are nonspecific and elicit systemic inflammation. Here we describe a targeted nanocarrier that can deliver in vitro-transcribed mRNA encoding M1-polarizing transcription factors to reprogram TAMs without causing systemic toxicity. We demonstrate in models of ovarian cancer, melanoma, and glioblastoma that infusions of nanoparticles formulated with mRNAs encoding interferon regulatory factor 5 in combination with its activating kinase IKKß reverse the immunosuppressive, tumor-supporting state of TAMs and reprogram them to a phenotype that induces anti-tumor immunity and promotes tumor regression. We further establish that these nanoreagents are safe for repeated dosing. Implemented in the clinic, this immunotherapy could enable physicians to obviate suppressive tumors while avoiding systemic treatments that disrupt immune homeostasis.


Assuntos
Macrófagos/imunologia , Nanopartículas , Neoplasias/imunologia , RNA Mensageiro/administração & dosagem , Animais , Linhagem Celular Tumoral , Reprogramação Celular , Feminino , Glioblastoma/imunologia , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Imunossupressão , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Ativação de Macrófagos , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Metástase Neoplásica , Neoplasias/genética , Neoplasias/patologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , RNA Mensageiro/genética , Linfócitos T/imunologia , Fatores de Transcrição/genética , Transfecção
3.
Nat Immunol ; 20(9): 1174-1185, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406377

RESUMO

Classical type 1 dendritic cells (cDC1s) are required for antiviral and antitumor immunity, which necessitates an understanding of their development. Development of the cDC1 progenitor requires an E-protein-dependent enhancer located 41 kilobases downstream of the transcription start site of the transcription factor Irf8 (+41-kb Irf8 enhancer), but its maturation instead requires the Batf3-dependent +32-kb Irf8 enhancer. To understand this switch, we performed single-cell RNA sequencing of the common dendritic cell progenitor (CDP) and identified a cluster of cells that expressed transcription factors that influence cDC1 development, such as Nfil3, Id2 and Zeb2. Genetic epistasis among these factors revealed that Nfil3 expression is required for the transition from Zeb2hi and Id2lo CDPs to Zeb2lo and Id2hi CDPs, which represent the earliest committed cDC1 progenitors. This genetic circuit blocks E-protein activity to exclude plasmacytoid dendritic cell potential and explains the switch in Irf8 enhancer usage during cDC1 development.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células Dendríticas/citologia , Elementos Facilitadores Genéticos/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Fatores Reguladores de Interferon/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Repressoras/metabolismo , Células-Tronco/citologia
4.
Nat Immunol ; 20(9): 1161-1173, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406378

RESUMO

Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification.


Assuntos
Células Dendríticas/citologia , Elementos Facilitadores Genéticos/genética , Fatores Reguladores de Interferon/metabolismo , Macrófagos/citologia , Monócitos/citologia , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Linhagem da Célula , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Fatores Reguladores de Interferon/genética , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Células-Tronco/citologia , Células Tumorais Cultivadas
6.
Nat Commun ; 10(1): 3203, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324791

RESUMO

Viral control of mitochondrial quality and content has emerged as an important mechanism for counteracting the host response to virus infection. Despite the knowledge of this crucial function of some viruses, little is known about how herpesviruses regulate mitochondrial homeostasis during infection. Human herpesvirus 8 (HHV-8) is an oncogenic virus causally related to AIDS-associated malignancies. Here, we show that HHV-8-encoded viral interferon regulatory factor 1 (vIRF-1) promotes mitochondrial clearance by activating mitophagy to support virus replication. Genetic interference with vIRF-1 expression or targeting to the mitochondria inhibits HHV-8 replication-induced mitophagy and leads to an accumulation of mitochondria. Moreover, vIRF-1 binds directly to a mitophagy receptor, NIX, on the mitochondria and activates NIX-mediated mitophagy to promote mitochondrial clearance. Genetic and pharmacological interruption of vIRF-1/NIX-activated mitophagy inhibits HHV-8 productive replication. Our findings uncover an essential role of vIRF-1 in mitophagy activation and promotion of HHV-8 lytic replication via this mechanism.


Assuntos
Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8/genética , Fatores Reguladores de Interferon/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Virais/metabolismo , Antivirais/farmacologia , Apoptose , Linhagem Celular Tumoral , Herpesvirus Humano 8/efeitos dos fármacos , Herpesvirus Humano 8/patogenicidade , Homeostase , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/virologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
7.
J Immunol Res ; 2019: 7059680, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31321244

RESUMO

Background: Adipose-derived mesenchymal stem cells (ADMSCs) can promote healing and inhibit inflammation/immune response in local tissues, while the detailed mechanism remains unknown. Results: ADMSCs and peritoneal macrophages were collected from C57BL/6 mice. The culture medium (CM) from ADMSCs (24 hours cultured) was collected. The CM was added to the Mφ culture system with lipopolysaccharide (LPS) or IL-4/IL-13 or blank. And those Mφ cultures without adding CM were used as controls. A series of classification markers and signaling pathways for Mφ polarization were detected by using flow cytometry, RT-PCR, and western blotting. Furthermore, the cell viability of all the groups was detected by CCK8 assay. After CM induction in different groups, M1-Mφ markers and M2a-Mφ were decreased; however, M2b/c-Mφ markers increased. STAT3/SOCS3 and STAT6/IRF4 were suppressed in all 3 CM-treated groups. Moreover, the cell viability of all 3 groups which were induced by CM significantly increased as compared to that of the control groups without adding CM. Conclusion: ADMSCs can induce nonactivated macrophage and M1-Mφ into M2b/c-Mφ. Downregulation of the STAT3 and STAT6 pathway may involve in this process. This data shows that the anti-inflammatory role of ADMSC in local tissues may be partly due to their effect on Mφ to M2b/c-Mφ.


Assuntos
Tecido Adiposo/citologia , Macrófagos Peritoneais/imunologia , Células-Tronco Mesenquimais/imunologia , Animais , Diferenciação Celular/imunologia , Sobrevivência Celular , Inflamação , Fatores Reguladores de Interferon/metabolismo , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo
8.
Chem Biol Interact ; 311: 108773, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31351048

RESUMO

Hemangioma (HA) is tumor formed by hyper-proliferation of vascular endothelial cells. However, the potential effects of mono-(2-ethylhexyl) phthalate (MEHP) on the progression of HA are not well illustrated. Our present study revealed that MEHP exposure can significantly increase the in vitro proliferation of hemangioma-derived endothelial cells (HemECs). MEHP treatment can activate yes-associated protein (YAP), a key effector of Hippo pathway, by inhibiting its phosphorylation. The dephosphorylation of YAP induced by MEHP can promote the nuclear accumulation of YAP. Knockdown of YAP or its inhibitor can block MEHP triggered cell proliferation. MEHP can increase the levels of precursor and mature mRNA of YAP in HemECs. As well, MEHP extended the half-life of YAP protein. Mechanistically, MEHP can decrease the phosphorylation of YAP via suppressing the activity of large tumor suppressor kinase 1/2 (LATS1/2) to inhibit it induced degradation of YAP. Further, MEHP increased the expression of interferon regulatory factor 1 (IRF1), which can bind to the promoter of YAP to initiate its transcription. Collectively, we revealed that Hippo-YAP signal is involved in MEHP-induced proliferation of HA cells.


Assuntos
Proliferação de Células/efeitos dos fármacos , Dietilexilftalato/análogos & derivados , Hemangioma/patologia , Proteínas Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular , Células Cultivadas , Dietilexilftalato/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Hemangioma/metabolismo , Humanos , Fatores Reguladores de Interferon/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Estabilidade Proteica/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição Genética/efeitos dos fármacos
9.
Hematol Oncol ; 37 Suppl 1: 53-61, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31187530

RESUMO

Pediatric-type follicular lymphoma (PTFL), pediatric nodal marginal zone lymphoma (pnMZL), and large B-cell lymphoma (LBCL) with IRF4 rearrangement have been introduced into the current World Health Organization (WHO) classification. They account for 5% to 10% of mature B-cell lymphomas in children and adolescents. Both PTFL and pnMZL predominantly affect male adolescents and usually present with localized lymphadenopathy in the head and neck region. The cells within the follicles of PTFL typically show high-grade cytology, IGH monoclonality and lack the t(14;18) chromosomal alteration. In contrast, pnMZL is characterized by progressive transformation of germinal center (PTGC)-like features and interfollicular proliferation of the cells with expansion of the marginal zones with diffuse areas. Watch and wait after complete resection seems an adequate therapy with chemotherapy restricted to incompletely resected disease. All children with PTFL and pnMZL reported, so far, survived. B-cell lymphomas presenting in the Waldeyer's ring are characterized by the expression of IRF4/MUM1 and often associated with IRF4 rearrangements. Because of the frequent diffuse component, treatment often follows current protocols for mature B-NHL. The prognosis is excellent.


Assuntos
Linfoma de Células B/diagnóstico , Adolescente , Fatores Etários , Biópsia , Criança , Terapia Combinada , Humanos , Imuno-Histoquímica , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Linfoma de Células B/epidemiologia , Linfoma de Células B/etiologia , Linfoma de Células B/terapia , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Linfoma de Zona Marginal Tipo Células B/epidemiologia , Linfoma de Zona Marginal Tipo Células B/terapia , Linfoma Folicular/diagnóstico , Linfoma Folicular/epidemiologia , Linfoma Folicular/terapia , Técnicas de Diagnóstico Molecular , Gradação de Tumores , Estadiamento de Neoplasias , Resultado do Tratamento
10.
Nat Neurosci ; 22(6): 1021-1035, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061494

RESUMO

While the roles of parenchymal microglia in brain homeostasis and disease are fairly clear, other brain-resident myeloid cells remain less well understood. By dissecting border regions and combining single-cell RNA-sequencing with high-dimensional cytometry, bulk RNA-sequencing, fate-mapping and microscopy, we reveal the diversity of non-parenchymal brain macrophages. Border-associated macrophages (BAMs) residing in the dura mater, subdural meninges and choroid plexus consisted of distinct subsets with tissue-specific transcriptional signatures, and their cellular composition changed during postnatal development. BAMs exhibited a mixed ontogeny, and subsets displayed distinct self-renewal capacity following depletion and repopulation. Single-cell and fate-mapping analysis both suggested that there is a unique microglial subset residing on the apical surface of the choroid plexus epithelium. Finally, gene network analysis and conditional deletion revealed IRF8 as a master regulator that drives the maturation and diversity of brain macrophages. Our results provide a framework for understanding host-macrophage interactions in both the healthy and diseased brain.


Assuntos
Encéfalo/citologia , Fatores Reguladores de Interferon/metabolismo , Macrófagos/citologia , Macrófagos/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia
11.
PLoS Pathog ; 15(5): e1007743, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31059555

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) belongs to the subfamily of Gammaherpesvirinae and is the etiological agent of Kaposi's sarcoma as well as of two lymphoproliferative diseases: primary effusion lymphoma and multicentric Castleman disease. The KSHV life cycle is divided into a latent and a lytic phase and is highly regulated by viral immunomodulatory proteins which control the host antiviral immune response. Among them is a group of proteins with homology to cellular interferon regulatory factors, the viral interferon regulatory factors 1-4. The KSHV vIRFs are known as inhibitors of cellular interferon signaling and are involved in different oncogenic pathways. Here we characterized the role of the second vIRF protein, vIRF2, during the KSHV life cycle. We found the vIRF2 protein to be expressed in different KSHV positive cells with early lytic kinetics. Importantly, we observed that vIRF2 suppresses the expression of viral early lytic genes in both newly infected and reactivated persistently infected endothelial cells. This vIRF2-dependent regulation of the KSHV life cycle might involve the increased expression of cellular interferon-induced genes such as the IFIT proteins 1, 2 and 3, which antagonize the expression of early KSHV lytic proteins. Our findings suggest a model in which the viral protein vIRF2 allows KSHV to harness an IFN-dependent pathway to regulate KSHV early gene expression.


Assuntos
Endotélio Vascular/virologia , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Fatores Reguladores de Interferon/metabolismo , Interferons/metabolismo , Sarcoma de Kaposi/virologia , Proteínas Virais/metabolismo , Ativação Viral , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Humanos , Proteínas Imediatamente Precoces/genética , Fatores Reguladores de Interferon/genética , Interferons/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Proteínas Virais/genética , Latência Viral
12.
Tuberculosis (Edinb) ; 116S: S131-S137, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31085128

RESUMO

Mycobacterium tuberculosis (Mtb) is a facultative intracellular pathogen that infects macrophages where it avoids elimination by interfering with host defense mechanisms, including phago-lysosome fusion. Endosomal Toll-like receptors (TLRs) generate Type I Interferons (IFNs), which are associated with active tuberculosis (TB). We aimed to explore if DNA from different Mtb lineages lead to differences in the inflammatory response of human monocytic/macrophage cells. THP-1 cells which express two inducible reporter constructs for interferons (IFNs) as well as for NF-κB, were stimulated via endosomal delivery of Mtb DNA as a nanocomplex with PEI. DNA from different Mtb phylogenetic lineages elicited differential inflammatory responses in human macrophages. An initial relatively weak IRF-mediated response to DNA from HN878 and H37Rv increased if the cells were pre-treated with Vitamin D (Vit D) for 72 h. RNAseq of THP-1 under different transformation conditions showed that pre-treatment with Vit D upregulated several TLR9 variants, as well as genes involved in inflammatory immune response to infection, immune cell activation, Type I IFN regulation, and regulation of inflammation. Vit D appears to be important in increasing low IRF responses to DNA from certain lineages of Mtb. Variations in the IRF-mediated response to DNA derived from different Mtb genotypes are potentially important in the pathogenesis of tuberculosis since Type I IFN responses are associated with active disease. The role of Vit D in these responses could also translate into future therapeutic approaches.


Assuntos
Calcitriol/farmacologia , DNA Bacteriano/metabolismo , Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Interações Hospedeiro-Patógeno , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interferon gama/farmacologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Células THP-1 , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo
13.
Nat Commun ; 10(1): 2201, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101814

RESUMO

Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease. It is thought that many common variant gene loci of weak effect act additively to predispose to common autoimmune diseases, while the contribution of rare variants remains unclear. Here we describe that rare coding variants in lupus-risk genes are present in most SLE patients and healthy controls. We demonstrate the functional consequences of rare and low frequency missense variants in the interacting proteins BLK and BANK1, which are present alone, or in combination, in a substantial proportion of lupus patients. The rare variants found in patients, but not those found exclusively in controls, impair suppression of IRF5 and type-I IFN in human B cell lines and increase pathogenic lymphocytes in lupus-prone mice. Thus, rare gene variants are common in SLE and likely contribute to genetic risk.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico/genética , Proteínas de Membrana/genética , Quinases da Família src/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Adulto , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Criança , Modelos Animais de Doenças , Feminino , Frequência do Gene , Células HEK293 , Voluntários Saudáveis , Humanos , Fatores Reguladores de Interferon/imunologia , Fatores Reguladores de Interferon/metabolismo , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto , Sequenciamento Completo do Exoma , Quinases da Família src/metabolismo
14.
Nat Commun ; 10(1): 1903, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015418

RESUMO

Single-cell transcriptomic assays have enabled the de novo reconstruction of lineage differentiation trajectories, along with the characterization of cellular heterogeneity and state transitions. Several methods have been developed for reconstructing developmental trajectories from single-cell transcriptomic data, but efforts on analyzing single-cell epigenomic data and on trajectory visualization remain limited. Here we present STREAM, an interactive pipeline capable of disentangling and visualizing complex branching trajectories from both single-cell transcriptomic and epigenomic data. We have tested STREAM on several synthetic and real datasets generated with different single-cell technologies. We further demonstrate its utility for understanding myoblast differentiation and disentangling known heterogeneity in hematopoiesis for different organisms. STREAM is an open-source software package.


Assuntos
Algoritmos , Linhagem da Célula/genética , Genômica/métodos , Células-Tronco Hematopoéticas/metabolismo , Análise de Célula Única/estatística & dados numéricos , Transcriptoma , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Eritroides/citologia , Células Eritroides/metabolismo , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos , Redução Dimensional com Múltiplos Fatores , Células Mieloides/citologia , Células Mieloides/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
J Neuroinflammation ; 16(1): 82, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975169

RESUMO

BACKGROUND: Neuropathic pain is caused by sensory nerve injury, but effective treatments are currently lacking. Microglia are activated in the spinal dorsal horn after sensory nerve injury and contribute to neuropathic pain. Accordingly, molecules expressed by these cells are considered potential targets for therapeutic strategies. Our previous gene screening study using a mouse model of motor nerve injury showed that the G-protein-coupled receptor 34 gene (GPR34) is induced by nerve injury. Because GPR34 is now considered a microglia-enriched gene, we explored the possibility that it might be involved in microglial activation in the dorsal horn in a mouse model of neuropathic pain. METHODS: mRNA expression of GPR34 and pro-inflammatory molecules was determined by quantitative real-time PCR in wild-type and GPR34-deficient mice with L4 spinal nerve injury. In situ hybridization was used to identify GPR34 expression in microglia, and immunohistochemistry with the microglial marker Iba1 was performed to examine microglial numbers and morphology. Mechanical sensitivity was evaluated by the von Frey hair test. Liquid chromatography-tandem mass spectrometry quantified expression of the ligand for GPR34, lysophosphatidylserine (LysoPS), in the dorsal horn, and a GPR34 antagonist was intrathecally administrated to examine the effect of inhibiting LysoPS-GPR34 signaling on mechanical sensitivity. RESULTS: GPR34 was predominantly expressed by microglia in the dorsal horn after L4 nerve injury. There were no histological differences in microglial numbers or morphology between WT and GPR34-deficient mice. However, nerve injury-induced pro-inflammatory cytokine expression levels in microglia and pain behaviors were significantly attenuated in GPR34-deficient mice. Furthermore, the intrathecal administration of the GPR34 antagonist reduced neuropathic pain. CONCLUSIONS: Inhibition of GPR34-mediated signal by GPR34 gene deletion reduced nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia without affecting their morphology. Therefore, the suppression of GPR34 activity may have therapeutic potential for alleviating neuropathic pain.


Assuntos
Microglia/metabolismo , Neuralgia/metabolismo , Neuralgia/patologia , Receptores de Lisofosfolipídeos/metabolismo , Medula Espinal/patologia , Análise de Variância , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Fatores Reguladores de Interferon/metabolismo , Lisofosfolipídeos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Neuralgia/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Medição da Dor , Limiar da Dor/fisiologia , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Receptores de Lisofosfolipídeos/antagonistas & inibidores , Receptores de Lisofosfolipídeos/genética , Fatores de Tempo
16.
BMC Cancer ; 19(1): 273, 2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30917792

RESUMO

BACKGROUND: Programmed cell death receptor 1 ligand 1 (PD-L1) expression in various tumors, including hematologic malignancies, has recently become a research topic of great interest. We performed a meta-analysis to evaluate the prognostic and clinicopathological value of PD-L1 expressed in tumor cells of patients with diffuse large B-cell lymphoma (DLBCL). METHODS: Relevant studies were identified from PubMed, EMBASE, Web of Science and the Cochrane Library. The hazard ratio (HR) and 95% confidence interval (95% CI) were used for analyzing survival outcomes, and the odds ratio (OR) was used for analyzing clinicopathological parameters. RESULTS: Pooled results showed that tumor cell PD-L1 expression is associated with poor overall survival (OS) (HR = 2.128, 95% CI: 1.341-3.378, P = 0.001), the non-germinal center B-cell-like subtype (OR = 2.891, 95% CI: 2.087-4.003, P < 0.000), high international prognostic index score (3-5) (OR = 1.552, 95% CI: 1.111-2.169, P = 0.010), B symptoms (OR = 1.495, 95% Cl: 1.109-2.015, P = 0.008), positive MUM1 expression (OR = 3.365, 95% Cl: 1.578-7.175, P = 0.002) and negative BCL6 expression (OR = 0.414, 95% Cl: 0.217-0.792, P = 0.008). Sensitivity analysis showed that there was no publication bias among these studies. CONCLUSIONS: Our meta-analysis supported the idea that tumor cell PD-L1 expression may represent a promising biomarker for predicting poor prognosis and is associated with adverse clinicopathologic features in DLBCL patients.


Assuntos
Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores Reguladores de Interferon/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Razão de Chances , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Análise de Sobrevida
17.
Int J Mol Sci ; 20(6)2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30901970

RESUMO

Interferons (IFNs) are very powerful cytokines, which play a key role in combatting pathogen infections by controlling inflammation and immune response by directly inducing anti-pathogen molecular countermeasures. There are three classes of IFNs: type I, type II and type III. While type II IFN is specific for immune cells, type I and III IFNs are expressed by both immune and tissue specific cells. Unlike type I IFNs, type III IFNs have a unique tropism where their signaling and functions are mostly restricted to epithelial cells. As such, this class of IFN has recently emerged as a key player in mucosal immunity. Since the discovery of type III IFNs, the last 15 years of research in the IFN field has focused on understanding whether the induction, the signaling and the function of these powerful cytokines are regulated differently compared to type I IFN-mediated immune response. This review will cover the current state of the knowledge of the similarities and differences in the signaling pathways emanating from type I and type III IFN stimulation.


Assuntos
Interferon Tipo I/metabolismo , Interferons/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Ativação Enzimática , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Janus Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo
18.
Nat Commun ; 10(1): 1349, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30902986

RESUMO

During infection, transcription factor interferon regulatory factor 5 (IRF5) is essential for the control of host defense. Here we show that the microtubule-associated guanine nucleotide exchange factor (GEF)-H1, is required for the phosphorylation of IRF5 by microbial muramyl-dipeptides (MDP), the minimal structural motif of peptidoglycan of both Gram-positive and Gram-negative bacteria. Specifically, GEF-H1 functions in a microtubule based recognition system for microbial peptidoglycans that mediates the activation of IKKε which we identify as a new upstream IKKα/ß and IRF5 kinase. The deletion of GEF-H1 or dominant-negative variants of GEF-H1 prevent activation of IKKε and phosphorylation of IRF5. The GEF-H1-IKKε-IRF5 signaling axis functions independent of NOD-like receptors and is critically required for the recognition of intracellular peptidoglycans and host defenses against Listeria monocytogenes.


Assuntos
Quinase I-kappa B/metabolismo , Fatores Reguladores de Interferon/metabolismo , Listeria monocytogenes/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Quinases Associadas a rho/metabolismo
19.
Diabetologia ; 62(6): 993-999, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30830262

RESUMO

AIMS/HYPOTHESIS: Mild islet inflammation has been suggested as a contributing factor to beta cell failure in type 2 diabetes. Macrophage levels are elevated in the islets of humans and mice with type 2 diabetes, but their effects on beta cells are not understood. Our goal was to examine the gene expression changes in islet-associated macrophages in obesity models with opposing disposition to diabetes development and to assess their potential contribution to beta cell (mal)adaptation. METHODS: Islets were isolated from lean control mice, obese diabetes-prone db/db mice and obese diabetes-resistant ob/ob mice. Macrophages were sorted using flow cytometry. Islets were treated ex vivo with clodronate-containing liposomes to deplete macrophages. Gene expression was assessed by real-time RT-PCR. RESULTS: Macrophage levels were increased in islets from db/db mice but not in islets from ob/ob mice compared with lean control mice. Macrophages from db/db and ob/ob islets displayed distinct changes in gene expression compared with control islet macrophages, suggesting differential shifts in functional state. Macrophages from db/db islets displayed increased expression of interferon regulatory factor 5 (Irf5), IL-1 receptor antagonist (Il1rn) and mannose receptor C-type 1 (Mrc1), whereas macrophages from ob/ob islets showed elevated levels of transforming growth factor beta 1 (Tgfb1) and reduced IL-1ß (Il1b). Clodronate-liposome treatment of islets depleted macrophages, as evidenced by reduced mRNA expression of Cd11b (also known as Itgam) and F4/80 (also known as Adgre1) compared with PBS-liposome-treated islets. The depletion of macrophages in db/db islets increased the expression of genes related to beta cell identity. The mRNA levels of islet-associated transcription factors (Mafa and Pdx1), glucose transporter (Glut2 [also known as Slc2a2]), ATP-sensitive K+ channel (Kcnj11), incretin receptor (Gipr) and adaptive unfolded protein response (UPR) genes (Xbp1, Hspa5, Pdia4 and Fkbp11) were increased in db/db islets after macrophage depletion, whereas the mRNA levels of the deleterious UPR effector, Ddit3, were reduced. In contrast, depletion of macrophages in islets of ob/ob mice did not affect beta cell identity gene expression. CONCLUSIONS/INTERPRETATION: The findings of this study suggest that distinct alterations in islet macrophages of obese mice are critically important for the disruption of beta cell gene expression in diabetes.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Animais , Citometria de Fluxo , Humanos , Fatores Reguladores de Interferon/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Ilhotas Pancreáticas/citologia , Lipossomos/metabolismo , Camundongos , Camundongos Obesos , Reação em Cadeia da Polimerase em Tempo Real , Resposta a Proteínas não Dobradas/fisiologia
20.
Immunol Res ; 67(1): 58-69, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30820875

RESUMO

Alternatively activated macrophages (M2) exert anti-inflammatory effects and are crucial for keeping balance between protective and destructive cell-mediated immunity in healing phase of inflammation. Two members of the interferon regulatory factors family, IRF5 and IRF4, are known to promote M1 or M2 phenotype, respectively. Our study aimed to analyse the effectiveness of the M2 differentiation process in vitro (achieved by IL-4 stimulation) and its relationship to the stage of type 1 diabetes mellitus (DM1) in juvenile patients. To identify the basic changes in M2 phenotype, we examined the expression of the surface CD206, CD14, CD86 molecules, intracellular IRF4 and IRF5 transcription factors as well as IL-10 and TNFα intracellular production. Ten newly diagnosed (ND-DM1) and ten long-standing (LS-DM1) patients were enrolled into the study. The control group consisted of six children. We observed a significantly higher number of unpolarised CD206+CD14+ cells in the M2 cultures of DM1 subjects when compared to healthy ones. Examined cells presented common features with M1 macrophages (high levels of the CD14/CD86/IRF5 markers); however, they were weak TNFα producers in ND-DM1 patients. For the first time, we have revealed dysregulated IRF4/IRF5 axis in the analysed subpopulation derived from diabetic patients. Additionally, monocytes of ND-DM1 children were still able to differentiate into regulatory IL-10+ M2 macrophages, while this process was highly limited in LS-DM1 patients. Summarising, we suggest that the M2 polarisation process is less effective in DM1 patients than in healthy subjects and it may vary depending on the stage of disease. It can be concluded that in vitro differentiated M2 macrophages may be used in the future as inflammatory inhibitors for adoptive therapy experiments in ND-DM1 subjects.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Interleucina-10/metabolismo , Macrófagos/imunologia , Adolescente , Diferenciação Celular , Células Cultivadas , Criança , Feminino , Humanos , Fatores Reguladores de Interferon/metabolismo , Interleucina-4/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Lipopolissacarídeos , Masculino , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Células Th2/imunologia
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