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1.
PLoS Pathog ; 16(9): e1008886, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32931525

RESUMO

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus. Meiwa kumquat (Fortunella crassifolia) has shown a durable resistance against Xcc. Here, we aimed to characterize the mechanisms responsible for such a durable resistance by characterizing the transcriptional and physiological responses of Meiwa kumquat to Xcc. Inoculation of Meiwa kumquat with Xcc promoted immune responses such as upregulation of PR genes, accumulation of salicylic acid, hypersensitive response (HR)-like cell death and early leaf abscission. Hypertrophy and hyperplasia symptoms, which are known to be caused by Xcc-induction of the canker susceptibility gene LOB1 through the transcription activator-like effector (TALE) PthA4, always appear prior to the development of cell death. Mutation of pthA4 in Xcc abolished the induction of LOB1, canker symptoms, cell death, and leaf abscission and reduced the expression of PR genes in inoculated kumquat leaves without reducing Xcc titers in planta. Transcriptome analysis demonstrated that PthA4 promotes plant biotic and abiotic stress responses and the biosynthesis of abscisic acid. Transcriptional induction of LOB1 homologs in Meiwa kumquat by Xcc pthA4 mutant strains carrying a repertoire of designer TALEs promoted the elicitation of HR-like phenotype and leaf abscission, suggesting that kumquat response to Xcc is associated with upregulation of LOB1. Our study suggests a novel mechanism of plant resistance to Xanthomonas via elicitation of immune responses by upregulation of a host susceptibility gene.


Assuntos
Citrus , Genes de Plantas/imunologia , Doenças das Plantas , Imunidade Vegetal , Proteínas de Plantas , Transativadores , Xanthomonas/imunologia , Citrus/genética , Citrus/imunologia , Citrus/microbiologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Transativadores/genética , Transativadores/imunologia
2.
Int J Cancer ; 146(8): 2182-2193, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31904872

RESUMO

Most genome-wide association studies (GWASs) identify genetic variants for breast cancer occurrence. In contrast, few are for recurrence and mortality. We conducted a GWAS on breast cancer survival after diagnosis in estrogen receptor-positive patients, including 953 Taiwanese patients with 159 events. Through Cox proportional hazard models estimation, we identified 24 risk SNPs with p < 1 × 10-5 . Based on imputation and integrated analysis, one SNP, rs1024176 (located in 1q24.2, p = 2.43 × 10-5 ) was found to be a functional variant associated with breast cancer survival and XCL1 gene expression. A series of experimental approaches, including cell-based analyses and CRISPR/Cas9 genome-editing system, were then used and identified the transcription factor MYBL2 was able to discriminately bind to the A allele of rs1024176, the protective variant for breast cancer survival, which promoted XCL1 expression, but not to the G allele of rs1024176. The chemokine XCL1 attracts type 1 dendritic cells (DC1s) to the tumor microenvironment. In breast cancer tissues, we applied a two-step Mendelian randomization analysis, using expression quantitative trait loci as instrumental variables, to confirm higher XCL1 expression was correlated with higher DC1 signatures and favorable disease progression, through the causal effect of rs1024176-A allele. Our study supports the genetic effect on preventing breast cancer survival through XCL1-induced DC1 recruitment in tumor microenvironment.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Quimiocinas C/genética , Quimiocinas C/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Quimiocinas C/biossíntese , Células Dendríticas/imunologia , Células Dendríticas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Locos de Características Quantitativas , Transativadores/genética , Transativadores/imunologia , Adulto Jovem
3.
J Microbiol ; 58(1): 54-60, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31898253

RESUMO

We previously reported that human cytomegalovirus (HCMV) 86 kDa immediate-early 2 gene product (IE86) promotes proteasome-dependent degradation of STING. In the present study, we determined the specific residues of IE86 responsible for STING degradation using a STING-firefly luciferase fusion protein expression system for quantitative meas-urement of STING protein levels. IE86 amino acids (aa) 136-289 were sufficient to promote STING degradation and further induced down-regulation of 2'3'-cyclic GMP-AMP (cGAMP)-mediated IFN-ß promoter activation. Interestingly, transactivation domains (TAD) of the IE86 protein located at the N- and C-termini were required for down-regulation of Toll/interleukin-1 receptor (TIR) domain-containing adaptor-inducing interferon ß (IFN-ß) (TRIF)-mediated IFN-ß-and p65/RelA-induced NF-κB-dependent promoter activation while amino acids (aa) 136-289 had no significant effects. Our collective data suggest that the IE86 protein utilizes the aa 136-289 region to promote STING degradation and inhibit the cGAS-STING pathway.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Proteínas Imediatamente Precoces , Proteínas de Membrana/imunologia , Nucleotidiltransferases/imunologia , Transativadores , Proteínas Virais , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/imunologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Ligação Proteica , Transativadores/imunologia , Transativadores/metabolismo , Proteínas Virais/imunologia , Proteínas Virais/metabolismo
4.
J Immunol ; 204(5): 1201-1213, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31932499

RESUMO

Vitamin D can modulate the innate and adaptive immune system. Vitamin D deficiency has been associated with various autoimmune diseases. Th9 cells are implicated in the pathogenesis of numerous autoimmune diseases. Thus, we investigated the role of calcitriol (active metabolite of vitamin D) in the regulation of Th9 cell differentiation. In this study, we have unraveled the molecular mechanisms of calcitriol-mediated regulation of Th9 cell differentiation. Calcitriol significantly diminished IL-9 secretion from murine Th9 cells associated with downregulated expression of the Th9-associated transcription factor, PU.1. Ectopic expression of VDR in Th9 cells attenuated the percentage of IL-9-secreting cells. VDR associated with PU.1 in Th9 cells. Using a series of mutations, we were able to dissect the VDR domain involved in the regulation of the Il9 gene. The VDR-PU.1 interaction prevented the accessibility of PU.1 to the Il9 gene promoter, thereby restricting its expression. However, the expression of Foxp3, regulatory T cell-specific transcription factor, was enhanced in the presence of calcitriol in Th9 cells. When Th9 cells are treated with both calcitriol and trichostatin A (histone deacetylase inhibitor), the level of IL-9 reached to the level of wild-type untreated Th9 cells. Calcitriol attenuated specific histone acetylation at the Il9 gene. In contrast, calcitriol enhanced the recruitment of the histone modifier HDAC1 at the Il9 gene promoter. In summary, we have identified that calcitriol blocked the access of PU.1 to the Il9 gene by reducing its expression and associating with it as well as regulated the chromatin of the Il9 gene to regulate expression.


Assuntos
Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/imunologia , Interleucina-9/imunologia , Proteínas Proto-Oncogênicas/imunologia , Linfócitos T Reguladores/imunologia , Transativadores/imunologia , Acetilação/efeitos dos fármacos , Animais , Diferenciação Celular/imunologia , Feminino , Regulação da Expressão Gênica/imunologia , Histonas/imunologia , Camundongos , Regiões Promotoras Genéticas/imunologia , Receptores de Calcitriol/imunologia , Linfócitos T Reguladores/citologia
5.
Microb Pathog ; 139: 103865, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31715318

RESUMO

Brucella spp. are facultative intracellular pathogens and zoonotic agents which pose a huge threat to human health and animal husbandry. The B. melitensis, B. abortus, and B. suis cause undulant fever and influenza-like symptoms in humans. However, the effects of B. canis have not been extensively studied. The quorum sensing-dependent transcriptional regulator VjbR influences the Brucella virulence in smooth type Brucella strains, such as B. melitensis, B. abortus and rough type Brucella ovis. However, the function of VjbR in the rough-type B. canis is unknown. In the present study, we discovered that deletion of this regulator significantly affected Brucella virulence in macrophage and mice infection models. The expression levels of virB operon and the ftcR gene were significantly altered in the vjbR mutant strain. We further investigated the protective effect of different doses of the vjbR mutant in mice and the results indicated that VjbR conferred protection against the virulent B. canis strain. This study presents the first evidence that the transcriptional regulator VjbR has important function in B. canis. In addition, according to its reduced virulence and the protective immunity it induces in mice, it can be a potential live attenuated vaccine against B. canis.


Assuntos
Proteínas de Bactérias/genética , Brucella canis/fisiologia , Brucelose/microbiologia , Regulação Bacteriana da Expressão Gênica , Mutação , Proteínas Repressoras/genética , Transativadores/genética , Sistemas de Secreção Tipo IV/fisiologia , Animais , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas/imunologia , Brucelose/imunologia , Brucelose/prevenção & controle , Linhagem Celular , Deleção de Genes , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Percepção de Quorum/genética , Células RAW 264.7 , Proteínas Repressoras/imunologia , Proteínas Repressoras/metabolismo , Transativadores/imunologia , Transativadores/metabolismo , Virulência , Fatores de Virulência/genética
6.
Retrovirology ; 16(1): 34, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783769

RESUMO

BACKGROUND: Immunity against pathogens evolved through complex mechanisms that only for sake of simplicity are defined as innate immunity and adaptive immunity. Indeed innate and adaptive immunity are strongly intertwined each other during evolution. The complexity is further increased by intrinsic mechanisms of immunity that rely on the action of intracellular molecules defined as restriction factors (RFs) that, particularly in virus infections, counteract the action of pathogen gene products acting at different steps of virus life cycle. MAIN BODY AND CONCLUSION: Here we provide an overview on the nature and the mode of action of restriction factors involved in retrovirus infection, particularly Human T Leukemia/Lymphoma Virus 1 (HTLV-1) infection. As it has been extensively studied by our group, special emphasis is given to the involvement of the MHC class II transactivator CIITA discovered in our laboratory as regulator of adaptive immunity and subsequently as restriction factor against HIV-1 and HTLV-1, a unique example of dual function linking adaptive and intrinsic immunity during evolution. We describe the multiple molecular mechanisms through which CIITA exerts its restriction on retroviruses. Of relevance, we review the unprecedented findings pointing to a concerted action of several restriction factors such as CIITA, TRIM22 and TRIM19/PML in synergizing against retroviral replication. Finally, as CIITA profoundly affects HTLV-1 replication by interacting and inhibiting the function of HTLV-1 Tax-1 molecule, the major viral product associated to the virus oncogenicity, we also put forward the hypothesis of CIITA as counteractor of HTLV-1-mediated cancer initiation.


Assuntos
Imunidade Adaptativa , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Proteínas Nucleares/imunologia , Transativadores/imunologia , Replicação Viral , Animais , Humanos , Leucemia/virologia , Linfoma/virologia , Proteínas Repressoras/genética , Infecções por Retroviridae/complicações , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/virologia , Fatores de Transcrição/genética
7.
PLoS Pathog ; 15(11): e1008122, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31765434

RESUMO

The T cell receptor (TCR) repertoire is an essential component of the CD8 T-cell immune response. Here, we seek to investigate factors that drive selection of TCR repertoires specific to the HLA-A2-restricted immunodominant epitope BRLF1109-117 (YVLDHLIVV) over the course of primary Epstein Barr virus (EBV) infection. Using single-cell paired TCRαß sequencing of tetramer sorted CD8 T cells ex vivo, we show at the clonal level that recognition of the HLA-A2-restricted BRLF1 (YVL-BR, BRLF-1109) epitope is mainly driven by the TCRα chain. For the first time, we identify a CDR3α (complementarity determining region 3 α) motif, KDTDKL, resulting from an obligate AV8.1-AJ34 pairing that was shared by all four individuals studied. This observation coupled with the fact that this public AV8.1-KDTDKL-AJ34 TCR pairs with multiple different TCRß chains within the same donor (median 4; range: 1-9), suggests that there are some unique structural features of the interaction between the YVL-BR/MHC and the AV8.1-KDTDKL-AJ34 TCR that leads to this high level of selection. Newly developed TCR motif algorithms identified a lysine at position 1 of the CDR3α motif that is highly conserved and likely important for antigen recognition. Crystal structure analysis of the YVL-BR/HLA-A2 complex revealed that the MHC-bound peptide bulges at position 4, exposing a negatively charged aspartic acid that may interact with the positively charged lysine of CDR3α. TCR cloning and site-directed mutagenesis of the CDR3α lysine ablated YVL-BR-tetramer staining and substantially reduced CD69 upregulation on TCR mutant-transduced cells following antigen-specific stimulation. Reduced activation of T cells expressing this CDR3 motif was also observed following exposure to mutated (D4A) peptide. In summary, we show that a highly public TCR repertoire to an immunodominant epitope of a common human virus is almost completely selected on the basis of CDR3α and provide a likely structural basis for the selection. These studies emphasize the importance of examining TCRα, as well as TCRß, in understanding the CD8 T cell receptor repertoire.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Proteínas Imediatamente Precoces/imunologia , Epitopos Imunodominantes/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T Citotóxicos/imunologia , Transativadores/imunologia , Sequência de Aminoácidos , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Epitopos de Linfócito T/imunologia , Infecções por Vírus Epstein-Barr/virologia , Antígeno HLA-A2/imunologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transativadores/genética , Transativadores/metabolismo
8.
Sci Rep ; 9(1): 16660, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31723204

RESUMO

Peptides presented by Human leukocyte antigen (HLA) class-I molecules are generally 8-10 amino acids in length. However, the predominant pool of peptide fragments generated by proteasomes is less than 8 amino acids in length. Using the Epstein - Barr virus (EBV) Rta-epitope (ATIGTAMYK, residues 134-142) restricted by HLA-A*11:01 which generates a strong immunodominant response, we investigated the minimum length of a viral peptide that can constitute a viral epitope recognition by CD8 T cells. The results showed that Peripheral blood mononuclear cells (PBMCs) from healthy donors can be stimulated by a viral peptide fragment as short as 4-mer (AMYK), together with a 5-mer (ATIGT) to recapitulate the full length EBV Rta epitope. This was confirmed by generating crystals of the tetra-complex (2 peptides, HLA and ß2-microglobulin). The solved crystal structure of HLA-A*11:01 in complex with these two short peptides revealed that they can bind in the same orientation similar to parental peptide (9-mer) and the free ends of two short peptides acquires a bulged conformation that is directed towards the T cell receptor. Our data shows that suboptimal length of 4-mer and 5-mer peptides can complement each other to form a stable peptide-MHC (pMHC) complex.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Antígenos HLA-A/química , Herpesvirus Humano 4/imunologia , Proteínas Imediatamente Precoces/química , Leucócitos Mononucleares/imunologia , Fragmentos de Peptídeos/química , Transativadores/química , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Infecções por Vírus Epstein-Barr/virologia , Antígenos HLA-A/imunologia , Humanos , Proteínas Imediatamente Precoces/imunologia , Leucócitos Mononucleares/virologia , Fragmentos de Peptídeos/imunologia , Conformação Proteica , Linfócitos T Citotóxicos/imunologia , Transativadores/imunologia
9.
Front Immunol ; 10: 1912, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474989

RESUMO

Neutrophils are implicated in almost every stage of oncogenesis and paradoxically display anti- and pro-tumor properties. Accumulating evidence indicates that neutrophils display diversity in their phenotype resulting from functional plasticity and/or changes to granulopoiesis. In cancer, neutrophils at a range of maturation stages can be identified in the blood and tissues (i.e., outside of their developmental niche). The functional capacity of neutrophils at different states of maturation is poorly understood resulting from challenges in their isolation, identification, and investigation. Thus, the impact of neutrophil maturity on cancer progression and therapy remains enigmatic. In this review, we discuss the identification, prevalence, and function of immature and mature neutrophils in cancer and the potential impact of this on tumor progression and cancer therapy.


Assuntos
Diferenciação Celular/imunologia , Células-Tronco Hematopoéticas/imunologia , Leucopoese/imunologia , Neoplasias/imunologia , Neutrófilos/imunologia , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/imunologia , Fator de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucopoese/genética , Neoplasias/genética , Neoplasias/terapia , Neutrófilos/citologia , Neutrófilos/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/genética , Transativadores/imunologia , Transativadores/metabolismo
10.
JCI Insight ; 4(20)2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31527312

RESUMO

Tissue engineering may address organ shortages currently limiting clinical transplantation. Off-the-shelf engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony-forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells can express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. CRISPR/Cas9-mediated dual ablation of ß2-microglobulin and class II transactivator (CIITA) in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4+ effector memory T cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.


Assuntos
Aloenxertos/imunologia , Células Endoteliais/imunologia , Rejeição de Enxerto/prevenção & controle , Proteínas Nucleares/genética , Engenharia Tecidual/métodos , Transativadores/genética , Microglobulina beta-2/genética , Aloenxertos/irrigação sanguínea , Aloenxertos/provisão & distribução , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Progenitoras Endoteliais , Feminino , Sangue Fetal/citologia , Técnicas de Inativação de Genes , Rejeição de Enxerto/sangue , Rejeição de Enxerto/imunologia , Voluntários Saudáveis , Humanos , Isoanticorpos/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/genética , Camundongos , Microvasos/citologia , Microvasos/imunologia , Microvasos/transplante , Proteínas Nucleares/imunologia , Transplante de Órgãos/efeitos adversos , Transplante de Órgãos/métodos , Cultura Primária de Células , Transativadores/imunologia , Microglobulina beta-2/imunologia
11.
Front Immunol ; 10: 1670, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379861

RESUMO

Pseudomonas aeruginosa is the most prevalent opportunistic pathogen in the airways of cystic fibrosis (CF) patients. The pulmonary disorder is characterized by recurrent microbial infections and an exaggerated host inflammatory immune response led primarily by influx of neutrophils. Under these conditions, chronic colonization with P. aeruginosa is associated with diminished pulmonary function and increased morbidity and mortality. P. aeruginosa has a wide array of genetic mechanisms that facilitate its persistent colonization of the airway despite extensive innate host immune responses. Loss of function mutations in the quorum sensing regulatory gene lasR have been shown to confer survival advantage and a more pathogenic character to P. aeruginosa in CF patients. However, the strategies used by LasR-deficient P. aeruginosa to modulate neutrophil-mediated bactericidal functions are unknown. We sought to understand the role of LasR in P. aeruginosa-mediated neutrophil extracellular trap (NET) formation, an important anti-microbial mechanism deployed by neutrophils, the first-line responder in the infected airway. We observe mechanistic and phenotypic differences between NETs triggered by LasR-sufficient and LasR-deficient P. aeruginosa strains. We uncover that LasR-deficient P. aeruginosa strains fail to induce robust NET formation in both human and murine neutrophils, independently of bacterial motility or LPS expression. LasR does not mediate NET release via downstream quorum sensing signaling pathways but rather via transcriptional regulation of virulence factors, including, but not restricted to, LasB elastase and LasA protease. Finally, our studies uncover the differential requirements for NADPH oxidase in NET formation triggered by different P. aeruginosa strains.


Assuntos
Proteínas de Bactérias/imunologia , Armadilhas Extracelulares/imunologia , Pseudomonas aeruginosa/imunologia , Transativadores/imunologia , Fatores de Virulência/imunologia , Virulência/imunologia , Animais , Humanos , Camundongos , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Virulência/genética , Fatores de Virulência/genética
12.
DNA Cell Biol ; 38(10): 1040-1047, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31414895

RESUMO

The helper T cell 9 (Thelper-9, Th9), as a functional subgroup of CD4+T cells, was first discovered in 2008. Th9 cells expressed transcription factor PU.1 and cytokine interleukin-9 (IL-9) characteristically. Recent researches have shown that the differentiation of Th9 cells was coregulated by cytokine transforming growth factor ß, IL-4, and various transcription factors. Th9 cells, as a new player, played an important role in various immune-related diseases, including tumors, inflammatory diseases, parasite infection, and other diseases. In this article, we summarize the related research progress and discuss the possible prospect.


Assuntos
Interleucina-9/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Neoplasias/imunologia , Proteínas Proto-Oncogênicas/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Transativadores/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Diferenciação Celular , Fator de Transcrição GATA3/deficiência , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/imunologia , Regulação da Expressão Gênica , Humanos , Inflamação , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-9/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/patologia , Proteínas Proto-Oncogênicas/genética , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/imunologia , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/patologia , Transativadores/genética , Fator de Crescimento Transformador beta/genética
13.
Front Immunol ; 10: 1806, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31417570

RESUMO

Construction of an optimal vaccine against tumors relies on the availability of appropriate tumor-specific antigens capable to stimulate CD4+ T helper cells (TH) and CD8+ cytolytic T cells (CTL). CTL are considered the major effectors of the anti-tumor adaptive immune response as they recognize antigens presented on MHC class I (MHC-I) molecules usually expressed in all cells and thus also in tumors. However, attempts to translate in clinics vaccination protocols based only on tumor-specific MHC-I-bound peptides have resulted in very limited, if any, success. We believe failure was mostly due to inadequate triggering of the TH arm of adaptive immunity, as TH cells are necessary to trigger and maintain the proliferation of all the immune effector cells required to eliminate tumor cells. In this review, we focus on a novel strategy of anti-tumor vaccination established in our laboratory and based on the persistent expression of MHC class II (MHC-II) molecules in tumor cells. MHC-II are the restricting elements of TH recognition. They are usually not expressed in solid tumors. By genetically modifying tumor cells of distinct histological origin with the MHC-II transactivator CIITA, the physiological controller of MHC-II gene expression discovered in our laboratory, stable expression of all MHC class II genes was obtained. This resulted in tumor rejection or strong retardation of tumor growth in vivo in mice, mediated primarily by tumor-specific TH cells as assessed by both depletion and adoptive cell transfer experiments. Importantly these findings led us to apply this methodology to human settings for the purification of MHC-II-bound tumor specific peptides directly from tumor cells, specifically from hepatocarcinomas, and the construction of a multi-peptide (MHC-II and MHC-I specific) immunotherapeutic vaccine. Additionally, our approach unveiled a noticeable exception to the dogma that dendritic cells are the sole professional antigen presenting cells (APC) capable to prime naïve TH cells, because CIITA-dependent MHC-II expressing tumor cells could also perform this function. Thus, our approach has served not only to select the most appropriate tumor specific peptides to activate the key lymphocytes triggering the anti-tumor effector functions but also to increase our knowledge of intimate mechanisms governing basic immunological processes.


Assuntos
Células Apresentadoras de Antígenos , Vacinas Anticâncer , Regulação Neoplásica da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II , Proteínas de Neoplasias , Neoplasias , Proteínas Nucleares , Transativadores , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologia , Transativadores/genética , Transativadores/imunologia
14.
Nucleic Acids Res ; 47(16): 8838-8859, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31329944

RESUMO

Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem-loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem-loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem-loops, thus licensing Regnase-1 to proceed with RNA degradation. Following translation, Regnase-1 physically associates with UPF1 using two distinct points of interaction: The Regnase-1 RNase domain binds to SMG1-phosphorylated residue T28 in UPF1; in addition, an intrinsically disordered segment in Regnase-1 binds to the UPF1 RecA domain, enhancing the helicase activity of UPF1. The SMG1-UPF1-Regnase-1 axis targets pioneer rounds of translation and is critical for rapid resolution of inflammation through restriction of the number of proteins translated by a given mRNA. Furthermore, small-molecule inhibition of SMG1 prevents RNA unwinding in dendritic cells, allowing post-transcriptional control of innate immune responses.


Assuntos
Macrófagos Peritoneais/imunologia , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Ribonucleases/genética , Transativadores/genética , Animais , Fibroblastos/citologia , Fibroblastos/imunologia , Células HEK293 , Células HeLa , Homeostase/genética , Homeostase/imunologia , Humanos , Imunidade Inata , Inflamação , Sequências Repetidas Invertidas , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Knockout , Mutação , Cultura Primária de Células , Ligação Proteica , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/imunologia , RNA Mensageiro/metabolismo , Ribonucleases/deficiência , Ribonucleases/imunologia , Imagem Individual de Molécula , Transativadores/imunologia
15.
J Virol ; 93(16)2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31167911

RESUMO

The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the host DNA damage-binding protein 1 (DDB1) E3 ubiquitin ligase to target Smc5/6 for degradation. HBx is an attractive therapeutic target for the treatment of chronic hepatitis B (CHB), but it is challenging to study this important viral protein in the context of natural infection due to the lack of a highly specific and sensitive HBx antibody. In this study, we developed a novel monoclonal antibody that enables detection of HBx protein in HBV-infected primary human hepatocytes (PHH) by Western blotting and immunofluorescence. Confocal imaging studies with this antibody demonstrated that HBx is predominantly located in the nucleus of HBV-infected PHH, where it exhibits a diffuse staining pattern. In contrast, a DDB1-binding-deficient HBx mutant was detected in both the cytoplasm and nucleus, suggesting that the DDB1 interaction plays an important role in the nuclear localization of HBx. Our study also revealed that HBx is expressed early after infection and has a short half-life (∼3 h) in HBV-infected PHH. In addition, we found that treatment with small interfering RNAs (siRNAs) that target DDB1 or HBx mRNA decreased HBx protein levels and led to the reappearance of Smc6 in the nuclei of HBV-infected PHH. Collectively, these studies provide the first spatiotemporal analysis of HBx in a natural infection system and also suggest that HBV transcriptional silencing by Smc5/6 can be restored by therapeutic targeting of HBx.IMPORTANCE Hepatitis B virus X protein (HBx) is a promising drug target since it promotes the degradation of the host structural maintenance of chromosomes 5/6 complex (Smc5/6) that inhibits HBV transcription. To date, it has not been possible to study HBx in physiologically relevant cell culture systems due to the lack of a highly specific and selective HBx antibody. In this study, we developed a novel monoclonal HBx antibody and performed a spatiotemporal analysis of HBx in a natural infection system. This revealed that HBx localizes to the nucleus of infected cells, is expressed shortly after infection, and has a short half-life. In addition, we demonstrated that inhibiting HBx expression or function promotes the reappearance of Smc6 in the nucleus of infected cells. These data provide new insights into HBx and underscore its potential as a novel target for the treatment of chronic HBV infection.


Assuntos
Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Hepatócitos/virologia , Transativadores/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Proteínas de Ligação a DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Expressão Gênica , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica , Transporte Proteico , Transativadores/química , Transativadores/genética , Transativadores/imunologia , Proteínas Virais Reguladoras e Acessórias
16.
Gastroenterology ; 157(1): 227-241.e7, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30930022

RESUMO

BACKGROUND & AIMS: One strategy to treat chronic hepatitis B virus (HBV) infection could be to increase the functions of virus-specific T cells. We performed a multicenter phase 2 study to evaluate the safety and efficacy of GS-4774, a yeast-based therapeutic vaccine engineered to express HBV antigens, given with tenofovir disoproxil fumarate (TDF) to untreated patients with chronic HBV infection. METHODS: We performed an open-label study at 34 sites in Canada, Italy, New Zealand, Romania, South Korea, and United States from July 2014 to August 2016. Adults who were positive for HB surface antigen (HBsAg) > 6 months and levels of HBV DNA ≥2000 IU/mL who had not received antiviral treatment for HBV within 3 months of screening were randomly assigned (1:2:2:2) to groups given oral TDF 300 mg daily alone (n = 27; controls) or with 2, 10, or 40 yeast units GS-4774 (n = 168), administered subcutaneously every 4 weeks until week 20 for a total of 6 doses. Blood samples were collected and analyzed and patients received regular physical examinations. Efficacy was measured by decrease in HBsAg from baseline to week 24. Specific responses to HBV (production of interferon gamma [IFNG], tumor necrosis factor [TNF], interleukin 2 [IL2], and degranulation) were measured in T cells derived from 12 HBeAg-negative patients with genotype D infections, after overnight or 10 days of stimulation of peripheral blood mononuclear cells with peptides from the entire HBV proteome. T-regulatory cells were analyzed for frequency and phenotype. Data from studies of immune cells were compared with data on reductions in HBsAg, HBV DNA, and alanine aminotransferase in blood samples from patients. RESULTS: GS-4774 was safe and well tolerated but did not produce significant decreases in levels of HBsAg. Production of IFNG, TNF, and IL2 increased significantly at weeks 24 and 48, compared with baseline, in HBV-specific CD8+ T cells from patients given GS-4774 but not from controls. GS-4774 had greater effects on CD8+ than CD4+ T cells, which were not affected at all or very weakly by TDF with or without GS-4774. GS-4774 did not affect responses of T cells to other viruses tested. HBV core peptides induced the greatest production of IFNG by T cells following overnight stimulation, whereas HBV envelope antigens did not induce a response. Following 10 days of stimulation, production of IFNG and TNF increased with time of exposure to GS-4774; the greatest levels of responses were to HBV envelope antigens followed by core and polymerase peptides. We observed a correlation in patients given GS-4774 between increased T-cell functions and reductions in numbers of T-regulatory cells. CONCLUSIONS: In a phase 2 study of patients with chronic HBV infection given TDF with or without GS-4774, we found that vaccination can increase production of IFNG, TNF, and IL2 by CD8+ T cells exposed to antigenic peptides, with little effect on CD4+ T cells. Although GS-4774 did not reduce levels of HBsAg in patients, its strong immune stimulatory effect on CD8+ T cells might be used in combination with other antiviral agents to boost the antivirus immune response. Clinicaltrials.gov no: NCT02174276.


Assuntos
Antivirais/uso terapêutico , Vacinas contra Hepatite B/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , Tenofovir/uso terapêutico , Adolescente , Adulto , Idoso , Linfócitos T CD8-Positivos/imunologia , DNA Viral , Quimioterapia Combinada , Feminino , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Humanos , Tolerância Imunológica/imunologia , Interferon gama/imunologia , Interleucina-2/imunologia , Masculino , Pessoa de Meia-Idade , Transativadores/imunologia , Fator de Necrose Tumoral alfa/imunologia , Carga Viral , Proteínas Virais Reguladoras e Acessórias , Adulto Jovem
17.
Proc Natl Acad Sci U S A ; 116(19): 9511-9520, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31000603

RESUMO

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.


Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Fatores Reguladores de Interferon/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Proteínas Proto-Oncogênicas/imunologia , Transativadores/imunologia , Animais , Linfócitos B/citologia , Centro Germinativo/citologia , Switching de Imunoglobulina/imunologia , Fatores Reguladores de Interferon/genética , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Transativadores/genética
18.
Nat Immunol ; 20(5): 546-558, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30911105

RESUMO

Neutrophils are essential first-line defense cells against invading pathogens, yet when inappropriately activated, their strong immune response can cause collateral tissue damage and contributes to immunological diseases. However, whether neutrophils can intrinsically titrate their immune response remains unknown. Here we conditionally deleted the Spi1 gene, which encodes the myeloid transcription factor PU.1, from neutrophils of mice undergoing fungal infection and then performed comprehensive epigenomic profiling. We found that as well as providing the transcriptional prerequisite for eradicating pathogens, the predominant function of PU.1 was to restrain the neutrophil defense by broadly inhibiting the accessibility of enhancers via the recruitment of histone deacetylase 1. Such epigenetic modifications impeded the immunostimulatory AP-1 transcription factor JUNB from entering chromatin and activating its targets. Thus, neutrophils rely on a PU.1-installed inhibitor program to safeguard their epigenome from undergoing uncontrolled activation, protecting the host against an exorbitant innate immune response.


Assuntos
Epigênese Genética/imunologia , Epigenômica/métodos , Neutrófilos/imunologia , Proteínas Proto-Oncogênicas/imunologia , Transativadores/imunologia , Animais , Candida albicans/imunologia , Candida albicans/fisiologia , Candidíase/genética , Candidíase/imunologia , Candidíase/microbiologia , Resistência à Doença/genética , Resistência à Doença/imunologia , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Análise de Sobrevida , Transativadores/deficiência , Transativadores/genética , Transcriptoma/genética , Transcriptoma/imunologia
19.
J Cell Mol Med ; 23(5): 3737-3746, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30895711

RESUMO

Adipose-derived stem cells (ASCs) are highly attractive for cell-based therapies in tissue repair and regeneration because they have multilineage differentiation capacity and are immunosuppressive. However, the detailed epigenetic mechanisms of their immunoregulatory capacity are not fully defined. In this study, we found that Mysm1 was induced in ASCs treated with inflammatory cytokines. Adipose-derived stem cells with Mysm1 knockdown exhibited attenuated immunosuppressive capacity, evidenced by less inhibition of T cell proliferation, more pro-inflammatory factor secretion and less nitric oxide (NO) production in vitro. Mysm1-deficient ASCs exacerbated inflammatory bowel diseases but inhibited tumour growth in vivo. Mysm1-deficient ASCs also showed depressed miR-150 expression. When transduced with Mysm1 overexpression lentivirus, ASCs exhibited enhanced miR-150 expression. Furthermore, Mysm1-deficient cells transduced with lentivirus containing miR-150 mimics produced less pro-inflammatory factors and more NO. Our study reveals a new role of Mysm1 in regulating the immunomodulatory activities of ASCs by targeting miR-150. These novel insights into the mechanisms through which ASCs regulate immune reactions may lead to better clinical utility of these cells.


Assuntos
Tecido Adiposo/citologia , Epigênese Genética/imunologia , MicroRNAs/imunologia , Células-Tronco/imunologia , Transativadores/imunologia , Proteases Específicas de Ubiquitina/imunologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Interferon gama/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Óxido Nítrico/imunologia , Óxido Nítrico/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Transativadores/genética , Transativadores/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo
20.
Front Immunol ; 10: 2923, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31969878

RESUMO

Macrophages have a major role in infectious and inflammatory diseases, and the available data suggest that Helicobacter pylori persistence can be explained in part by the failure of the bacterium to be killed by professional phagocytes. Macrophages are cells ready to kill the engulfed pathogen, through oxygen-dependent and -independent mechanisms; however, their killing potential can be further augmented by the intervention of T helper (Th) cells upon the specific recognition of human leukocyte antigen (HLA)-II-peptide complexes on the surface of the phagocytic cells. As it pertains to H. pylori, the bacterium is engulfed by macrophages, but it interferes with the phagosome maturation process leading to phagosomes with an altered degradative capacity, and to megasomes, wherein H. pylori resists killing. We recently showed that macrophages infected with H. pylori strongly reduce the expression of HLA-II molecules on the plasma membrane and this compromises the bacterial antigen presentation to Th lymphocytes. In this work, we demonstrate that H. pylori hampers HLA-II expression in macrophages, activated or non-activated by IFN-γ, by down-regulating the expression of the class II major histocompatibility complex transactivator (CIITA), the "master control factor" for the expression of HLA class II genes. We provided evidence that this effect relies on the up-regulation of let-7f-5p, let-7i-5p, miR-146b-5p, and -185-5p targeting CIITA. MiRNA expression analysis performed on biopsies from H. pylori-infected patients confirmed the up-regulation of let-7i-5p, miR-146b-5p, and -185-5p in gastritis, in pre-invasive lesions, and in gastric cancer. Taken together, our results suggest that specific miRNAs may be directly involved in the H. pylori infection persistence and may contribute to confer the risk of developing gastric neoplasia in infected patients.


Assuntos
Antígenos HLA/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Macrófagos/imunologia , MicroRNAs/imunologia , Proteínas Nucleares/imunologia , Transativadores/imunologia , Regulação para Cima/imunologia , Idoso , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/imunologia
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