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1.
Med Sci Monit ; 25: 7795-7807, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31622282

RESUMO

BACKGROUND The methylation status of RUNX3 promoter region, its impact on RUNX3 gene expression, and Th1/Th2 imbalance are unknown in bronchiolitis. This study aimed to explore the predictors of bronchiolitis developing into asthma. MATERIAL AND METHODS The methylation status of RUNX3 promoter was assessed using Illumina HiSeq platform method. The relative RUNX3 mRNA levels in PBMCs were measured by qRT-PCR. Serum IL-4 and IFN-γ concentrations were measured by ELISA. RESULTS A series of sites with significantly higher levels of methylation as compared to their corresponding controls were identified, including 24 sites in group Ba vs. group Cn, 13 sites in group Ba vs. group Ca, 7 sites in group Ba vs. group Bn, 16 sites in group Bn vs. group Cn, 11 sites in group Ca vs. group Cn, and 23 sites in group B vs. group C; P<0.05. The relative mRNA levels in group Ba were significantly lower than those in groups Cn, Ca, Bn; P<0.05. The serum IL-4 concentrations in group Ba were significantly higher than those in group Cn; P<0.05. The serum IFN-γ concentrations in group Ba were significantly lower than those in groups Cn, Ca, Bn; P<0.05. Correlation analysis showed that differentially methylated RUNX3 promoter region sites were significantly negatively correlated with levels of relative RUNX3 mRNA and IFN-γ, and were significantly positively correlated with IL-4 levels. CONCLUSIONS The methylation status of RUNX3 promoter region plays a role in Th1/Th2 imbalance by silencing RUNX3 gene expression, which can serve as predictive marker for the development of bronchiolitis into asthma.


Assuntos
Bronquiolite/genética , Bronquiolite/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/imunologia , Metilação de DNA , Células Th1/imunologia , Células Th2/imunologia , Asma/genética , Asma/imunologia , Biomarcadores/análise , Estudos de Casos e Controles , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Prognóstico , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Células Th1/metabolismo , Células Th2/metabolismo , Transcriptoma
2.
J Neuroinflammation ; 16(1): 167, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31416452

RESUMO

BACKGROUND: Although effective in reducing relapse rate and delaying progression, current therapies for multiple sclerosis (MS) do not completely halt disease progression. T cell autoimmunity to myelin antigens is considered one of the main mechanisms driving MS. It is characterized by autoreactivity to disease-initiating myelin antigen epitope(s), followed by a cascade of epitope spreading, which are both strongly patient-dependent. Targeting a variety of MS-associated antigens by myelin antigen-presenting tolerogenic dendritic cells (tolDC) is a promising treatment strategy to re-establish tolerance in MS. Electroporation with mRNA encoding myelin proteins is an innovative technique to load tolDC with the full spectrum of naturally processed myelin-derived epitopes. METHODS: In this study, we generated murine tolDC presenting myelin oligodendrocyte glycoprotein (MOG) using mRNA electroporation and we assessed the efficacy of MOG mRNA-electroporated tolDC to dampen pathogenic T cell responses in experimental autoimmune encephalomyelitis (EAE). For this, MOG35-55-immunized C57BL/6 mice were injected intravenously at days 13, 17, and 21 post-disease induction with 1α,25-dihydroxyvitamin D3-treated tolDC electroporated with MOG-encoding mRNA. Mice were scored daily for signs of paralysis. At day 25, myelin reactivity was evaluated following restimulation of splenocytes with myelin-derived epitopes. Ex vivo magnetic resonance imaging (MRI) was performed to assess spinal cord inflammatory lesion load. RESULTS: Treatment of MOG35-55-immunized C57BL/6 mice with MOG mRNA-electroporated or MOG35-55-pulsed tolDC led to a stabilization of the EAE clinical score from the first administration onwards, whereas it worsened in mice treated with non-antigen-loaded tolDC or with vehicle only. In addition, MOG35-55-specific pro-inflammatory pathogenic T cell responses and myelin antigen epitope spreading were inhibited in the peripheral immune system of tolDC-treated mice. Finally, magnetic resonance imaging analysis of hyperintense spots along the spinal cord was in line with the clinical score. CONCLUSIONS: Electroporation with mRNA is an efficient and versatile tool to generate myelin-presenting tolDC that are capable to stabilize the clinical score in EAE. These results pave the way for further research into mRNA-electroporated tolDC treatment as a patient-tailored therapy for MS.


Assuntos
Células Dendríticas/metabolismo , Eletroporação/métodos , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/terapia , Glicoproteína Mielina-Oligodendrócito/metabolismo , RNA Mensageiro/metabolismo , Animais , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Tolerância Imunológica/fisiologia , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Glicoproteína Mielina-Oligodendrócito/imunologia , RNA Mensageiro/administração & dosagem , RNA Mensageiro/imunologia
3.
Pediatr Hematol Oncol ; 36(4): 198-210, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31287345

RESUMO

The pathogenesis of aplastic anemia (AA) in children is not clear. This study was conducted to investigate the changes in the proportion and function of regulatory T cells (Tregs) in pediatric AA. The proportion of Tregs, mRNA levels of transcription factors, and concentrations of cytokines were measured by flow cytometry, reverse transcription-PCR, and enzyme-linked immunosorbent assay, respectively. Tregs were co-cultured with effector T cells (Teff) to evaluate the function of Tregs. The proportion of Tregs after immunosuppressive therapy (IST) in pediatric AA was monitored dynamically. Compared to the control, the proportions of Tregs in peripheral blood and bone marrow lymphocytes of the untreated AA group were lower (1.31% ± 0.73% vs. 3.16% ± 0.92%, 1.49% ± 0.81% vs. 3.06% ± 0.82%, respectively, p < 0.001). The mRNA levels of FOXP3 and STAT3 in the AA group were lower (p = 0.014; p < 0.001). However, the mRNA levels of T-BET did not significantly differ between groups. The concentration of interferon-γ and interleukin-17 in the AA group were higher (p = 0.004; p = 0.003), whereas the concentration of TGF-ß decreased (p = 0.044). The immunosuppressive function of Tregs was impaired in the AA group. After IST, the proportion of Tregs was significantly lower than that in the control. The proportion of Tregs at the time of diagnosis in the nonresponsive group was lower than that in the responsive group, but the difference was not significant. Treg levels were significantly decreased and were functionally impaired at the time of diagnosis of pediatric AA. However, there was no significant change in Tregs at the resolution of AA.


Assuntos
Anemia Aplástica/imunologia , Células da Medula Óssea/imunologia , Linfócitos T Reguladores/imunologia , Anemia Aplástica/patologia , Células da Medula Óssea/patologia , Criança , Pré-Escolar , Feminino , Fatores de Transcrição Forkhead/imunologia , Humanos , Masculino , Proteínas de Neoplasias/imunologia , RNA Mensageiro/imunologia , Fator de Transcrição STAT3/imunologia , Proteínas com Domínio T/imunologia , Linfócitos T Reguladores/patologia
4.
Drug Metab Pharmacokinet ; 34(4): 280-286, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31262603

RESUMO

UDP-Glucuronosyltransferase (UGT) 2A3 belongs to a UGT superfamily of phase II drug-metabolizing enzymes that catalyzes the glucuronidation of many endobiotics and xenobiotics. Previous studies have demonstrated that UGT2A3 is expressed in the human liver, small intestine, and kidney at the mRNA level; however, its protein expression has not been determined. Evaluation of the protein expression of UGT2A3 would be useful to determine its role at the tissue level. In this study, we prepared a specific antibody against human UGT2A3 and evaluated the relative expression of UGT2A3 in the human liver, small intestine, and kidney. Western blot analysis indicated that this antibody is specific to UGT2A3 because it did not cross-react with other human UGT isoforms or rodent UGTs. UGT2A3 expression in the human small intestine was higher than that in the liver and kidney. Via treatment with endoglycosidase, it was clearly demonstrated that UGT2A3 was N-glycosylated. UGT2A3 protein levels were significantly correlated with UGT2A3 mRNA levels in a panel of 28 human liver samples (r = 0.64, p < 0.001). In conclusion, we successfully prepared a specific antibody against UGT2A3. This antibody would be useful to evaluate the physiological, pharmacological, and toxicological roles of UGT2A3 in human tissues.


Assuntos
Anticorpos/imunologia , Glucuronosiltransferase/imunologia , Reações Antígeno-Anticorpo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Microssomos/imunologia , Microssomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas
5.
Gene Ther ; 26(9): 363-372, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31300730

RESUMO

Self-amplifying RNA (saRNA) is a promising biotherapeutic tool that has been used as a vaccine against both infectious diseases and cancer. saRNA has been shown to induce protein expression for up to 60 days and elicit immune responses with lower dosing than messenger RNA (mRNA). Because saRNA is a large (~9500 nt), negatively charged molecule, it requires a delivery vehicle for efficient cellular uptake and degradation protection. Lipid nanoparticles (LNPs) have been widely used for RNA formulations, where the prevailing paradigm is to encapsulate RNA within the particle, including the first FDA-approved small-interfering siRNA therapy. Here, we compared LNP formulations with cationic and ionizable lipids with saRNA either on the interior or exterior of the particle. We show that LNPs formulated with cationic lipids protect saRNA from RNAse degradation, even when it is adsorbed to the surface. Furthermore, cationic LNPs deliver saRNA equivalently to particles formulated with saRNA encapsulated in an ionizable lipid particle, both in vitro and in vivo. Finally, we show that cationic and ionizable LNP formulations induce equivalent antibodies against HIV-1 Env gp140 as a model antigen. These studies establish formulating saRNA on the surface of cationic LNPs as an alternative to the paradigm of encapsulating RNA.


Assuntos
Nanopartículas , RNA Mensageiro/administração & dosagem , Animais , Cátions , Ácidos Graxos Monoinsaturados/química , Feminino , Células HEK293 , Anticorpos Anti-HIV/biossíntese , Humanos , Lipídeos/administração & dosagem , Lipídeos/química , Luciferases de Vaga-Lume/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/química , Tamanho da Partícula , Compostos de Amônio Quaternário/química , Estabilidade de RNA , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Transfecção , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
6.
J Immunol ; 203(2): 400-407, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31189570

RESUMO

Immune responses are tightly controlled by T cell costimulatory and coinhibitory molecules. In this study, we identify Skint8 as a new member of the T cell coinhibitory group, whose extracellular domains share significant homology with existing B7 family members. Skint8 mRNA is expressed in resting and activated B cells, monocytes, and CD4 T cells. The Skint8 putative receptor is expressed on activated CD4 and CD8 T cells, B cells, monocytes and dendritic cells. Recombinant Skint8-IgG Fc fusion protein inhibits T cell proliferation, activation, and cytokine production in vitro. In vivo administration of Skint8-IgG Fc reduces T cell activation and alleviates experimental autoimmune encephalomyelitis in mice. The findings broaden our understanding of the regulation of immune responses and may have implications for treating immune-related diseases.


Assuntos
Antígeno B7-1/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Animais , Linfócitos B/imunologia , Proliferação de Células/fisiologia , Citocinas/imunologia , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Feminino , Imunoglobulina G/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , RNA Mensageiro/imunologia
7.
Curr Med Sci ; 39(3): 356-362, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31209803

RESUMO

Poly(ADP-ribose) polymerase 1 (PARP1) plays important roles in the regulation of transcription factors. Mounting evidence has shown that inhibition of PARP1 influences the expression of genes associated with inflammatory response. Interferon regulatory factor 1 (IRF1) is a critical transcription factor for the development of both the innate and adaptive immune responses against infections. However, the molecular mechanism through which PARP1 mediates the effects has not been clearly demonstrated. Jurkat cells were exposed to dexamethasone (Dex) or PARP1 inhibitor PJ34. The expression levels of IL-12, LMP2, OAS1 and PKR were detected using real-time RT-PCR. The interactions between PARP1 and IRF1 were examined by co-immunoprecipitation (co-IP) assays. We further explored the mechanism of PARP1 suppressing IRF1 by assessing the activities of interferon stimulated response element (ISRE). The mRNA expression of IL-12, LMP2, OAS1 and PKR was obviously suppressed by Dex in Jurkat cells, which could be rescued by PJ34 treatment. Luciferase study revealed that poly(ADP-ribosyl)- ation suppressed IRF1-mediated transcription through preventing the binding of IRF1 to ISREs. PARP1 inhibited IRF1-mediated transcription in Jurkat cells by preventing IRF1 binding to ISREs in the promoters of target genes. It is suggested that PARP1 is a crucial regulator of IRF1-mediated immune response. This study provides experimental evidence for the possible application of PARP1 inhibitors in the treatment of IRF1-related immune anergy.


Assuntos
Dexametasona/farmacologia , Regulação Leucêmica da Expressão Gênica , Fator Regulador 1 de Interferon/genética , Poli(ADP-Ribose) Polimerase-1/genética , Transcrição Genética/efeitos dos fármacos , 2',5'-Oligoadenilato Sintetase/antagonistas & inibidores , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/imunologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Humanos , Fator Regulador 1 de Interferon/imunologia , Interleucina-12/antagonistas & inibidores , Interleucina-12/genética , Interleucina-12/imunologia , Células Jurkat , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/imunologia , Poli Adenosina Difosfato Ribose/imunologia , Poli Adenosina Difosfato Ribose/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Elementos de Resposta , Transdução de Sinais , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética , eIF-2 Quinase/imunologia
8.
PLoS One ; 14(6): e0215031, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31163034

RESUMO

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction.


Assuntos
Bromovirus/metabolismo , Proteínas do Capsídeo/genética , Células Dendríticas/imunologia , RNA Mensageiro/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Bromovirus/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Cricetinae , Células Dendríticas/virologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Camundongos , RNA Mensageiro/imunologia , Análise de Célula Única , Montagem de Vírus
9.
Mol Immunol ; 111: 136-144, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31054407

RESUMO

Production of antimicrobial peptides cathelicidins, interferons and cytokines is an important feature in airway epithelial host defense. The innate immune response to alpha-herpesvirus infection at the sites of primary replication has not been fully studied. Thus, the aim of this study was to determine the expression of innate immune components, cathelicidins, IFNß, TNFα and TNF receptors (TNFRI and TNFRII) during acute infection and reactivation of bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) in the respiratory tract and lymphoid tissue of their natural host. We found that BoHV infection modulates mainly the expression of BMAP28, a key cathelicidin in cattle. It was downregulated by both viruses in retropharyngeal lymph nodes of acutely infected-calves, and it was accompanied by a lower expression of IFNß, TNFα and TNFRI. BoHV-5 showed a pronounced role in the downregulation of BMAP28, even in nasal mucosa and lung. However, during reactivation, BoHV-5 upregulated both BMAP28 and IFNß in retropharyngeal lymph nodes. Acute replication induced also TNFα mRNA and protein synthesis, and expression of TNFRI and II was positively regulated during both acute infection and reactivation, particularly in the trachea. Moreover, BMAP27 was detected during BoHV-1 reactivation suggesting a potential role at this stage. Thus, cathelicidins are implicated in alpha-herpesvirus infections of the bovine respiratory system and the response is distinct during BoHV-1 and BoHV-5 acute infection and reactivation. This demonstrates that these viruses modulate differentially the components of innate immune response, possibly influencing their pathogenesis. This study provides an initial pilot analysis of factors that might be implicated in alpha-herpesvirus infection of the bovine respiratory system.


Assuntos
Catelicidinas/imunologia , Doenças dos Bovinos/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Bovino 1/imunologia , Interferon beta/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Bovinos , Citocinas/imunologia , Infecções por Herpesviridae/veterinária , Imunidade Inata/imunologia , Projetos Piloto , RNA Mensageiro/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Sistema Respiratório/imunologia , Sistema Respiratório/virologia , Regulação para Cima/imunologia
10.
Fish Shellfish Immunol ; 91: 333-342, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31129189

RESUMO

In recent years, microRNAs (miRNAs) have been shown to play important roles in immunity. Analyses of the functions of miRNAs and their targets are useful in understanding the regulation of the immune response. To understand the relationships between miRNAs and their targets during infection, we used zebrafish as an infection model in which to characterize the miRNA and mRNA transcriptomes of zebrafish larvae infected with Vibrio parahaemolyticus. We identified the differentially expressed miRNAs and mRNAs. Overall, 37 known zebrafish miRNAs were differentially expressed in the infection group and 107 predicted target genes of 26 miRNAs were differentially expressed in the mRNA transcriptome. These targets with specific Gene Ontology (GO) terms, such as peripheral nervous system neuron axonogenesis, organophosphate metabolic process, heme binding, protein binding, tetrapyrrole binding, protein dimerization activity, and aromatase activity, which regulate nerve conduction, energy metabolism, hematopoiesis, and protein synthesis. They were also associated with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways such as phototransduction, tryptophan metabolism, notch signaling, and purine metabolism. Our findings indicate that miRNAs regulate the innate immune response via complex networks, and zebrafish (Danio rerio, dre)-miR-205-3p, dre-miR-141-5p, dre-miR-200a-5p, dre-miR-92a-2-5p, dre-miR-192, and dre-miR-1788 may play important roles in the innate immune response by regulating target genes.


Assuntos
Doenças dos Peixes/imunologia , Imunidade Inata/genética , MicroRNAs/genética , RNA Mensageiro/genética , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Animais , Doenças dos Peixes/microbiologia , Perfilação da Expressão Gênica/veterinária , MicroRNAs/imunologia , RNA Mensageiro/imunologia , Vibrioses/imunologia , Vibrioses/microbiologia , Vibrioses/veterinária , Vibrio parahaemolyticus/fisiologia
11.
Mol Immunol ; 111: 162-171, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31063937

RESUMO

B cells have been reported to have a suppressive function in autoimmune diseases, which appears to require an increase of CD11b expression on B cells. However, little is known how CD11b is induced in B cells to play the function. In this study, we found that the high expression of CD11b in B cells occurred not only in the mucosal immune organs, but also in systemically immune organs such as the spleen during dextran sulfate sodium (DSS)-induced colitis. Since the inflammatory lesions in mouse models of inflammatory bowel disease (IBD) were revealed to be significantly hypoxic or even anoxic, the B cells from colitic mice Peyer's patches (PP) were investigated to express higher levels of hypoxia-inducible factor-1α (HIF-1α) than naïve B cells from wildtype (WT) mice. HIF-1α siRNA transfection or HIF-1α protein inhibition led to decreased CD11b expression at both the mRNA and protein levels in vitro. B cells with HIF-1α specific knockdown were then adoptively transferred to Rag-1-/- mice. The result displayed that CD11b expression was decreased in B cells and an exacerbated colitis occurred. The bio-informatics promoter analysis and ChIP assay showed that HIF-1α was the critical transcription factor for CD11b and cooperatively formed a complex with the p-STAT3 homodimers to bind onto hypoxia-responsive element (HRE) regions, which was guaranteed by MEK/ERK pathway activation and IL-10 secretion. In conclusion, our study demonstrated the key function of the hypoxia-associated transcription factor HIF-1α together with p-STAT3 in driving CD11b transcription in B cells and controlling B cell's protective activity in experimental inflammatory bowel disease (IBD).


Assuntos
Linfócitos B/imunologia , Antígeno CD11b/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Doenças Inflamatórias Intestinais/imunologia , Fator de Transcrição STAT3/imunologia , Transdução de Sinais/imunologia , Animais , Colite/imunologia , Colo/imunologia , Regulação da Expressão Gênica/imunologia , Proteínas de Homeodomínio/imunologia , Imunossupressão/métodos , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/imunologia , RNA Mensageiro/imunologia , Baço/imunologia , Fatores de Transcrição/imunologia
12.
Nat Commun ; 10(1): 1898, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015515

RESUMO

N6-methyladenosine (m6A) modification plays important roles in various cellular responses by regulating mRNA biology. However, how m6A modification is involved in innate immunity via affecting the translation of immune transcripts remains to be further investigated. Here we report that RNA methyltransferase Mettl3-mediated mRNA m6A methylation promotes dendritic cell (DC) activation and function. Specific depletion of Mettl3 in DC resulted in impaired phenotypic and functional maturation of DC, with decreased expression of co-stimulatory molecules CD40, CD80 and cytokine IL-12, and reduced ability to stimulate T cell responses both in vitro and in vivo. Mechanistically, Mettl3-mediated m6A of CD40, CD80 and TLR4 signaling adaptor Tirap transcripts enhanced their translation in DC for stimulating T cell activation, and strengthening TLR4/NF-κB signaling-induced cytokine production. Our findings identify a new role for Mettl3-mediated m6A modification in increasing translation of certain immune transcripts for physiological promotion of DC activation and DC-based T cell response.


Assuntos
Adenosina/análogos & derivados , Células Dendríticas/imunologia , Epigênese Genética , Metiltransferases/genética , RNA Mensageiro/genética , Linfócitos T/imunologia , Adenosina/imunologia , Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Antígenos/farmacologia , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Antígenos CD40/genética , Antígenos CD40/imunologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Interleucina-12/genética , Interleucina-12/imunologia , Ativação Linfocitária , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Metilação , Metiltransferases/imunologia , Camundongos , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/imunologia , Peptídeos/imunologia , Peptídeos/farmacologia , Cultura Primária de Células , Biossíntese de Proteínas , RNA Mensageiro/imunologia , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia
13.
Nat Commun ; 10(1): 1364, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30910999

RESUMO

The mechanisms linking muscle injury and regeneration are not fully understood. Here we report an unexpected role for ZEB1 regulating inflammatory and repair responses in dystrophic and acutely injured muscles. ZEB1 is upregulated in the undamaged and regenerating myofibers of injured muscles. Compared to wild-type counterparts, Zeb1-deficient injured muscles exhibit enhanced damage that corresponds with a retarded p38-MAPK-dependent transition of their macrophages towards an anti-inflammatory phenotype. Zeb1-deficient injured muscles also display a delayed and poorer regeneration that is accounted by the retarded anti-inflammatory macrophage transition and their intrinsically deficient muscle satellite cells (MuSCs). Macrophages in Zeb1-deficient injured muscles show lower phosphorylation of p38 and its forced activation reverts the enhanced muscle damage and poorer regeneration. MuSCs require ZEB1 to maintain their quiescence, prevent their premature activation following injury, and drive efficient regeneration in dystrophic muscles. These data indicate that ZEB1 protects muscle from damage and is required for its regeneration.


Assuntos
Músculo Esquelético/metabolismo , Distrofias Musculares/genética , RNA Mensageiro/genética , Regeneração/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Cromonas/farmacologia , Modelos Animais de Doenças , Flavonoides/farmacologia , Regulação da Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/imunologia , Laminina/genética , Laminina/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Morfolinas/farmacologia , Músculo Esquelético/imunologia , Músculo Esquelético/lesões , Distrofias Musculares/imunologia , Distrofias Musculares/patologia , Fenótipo , Fosforilação , RNA Mensageiro/imunologia , Regeneração/imunologia , Células Satélites de Músculo Esquelético/imunologia , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/patologia , Transdução de Sinais , Homeobox 1 de Ligação a E-box em Dedo de Zinco/deficiência , Homeobox 1 de Ligação a E-box em Dedo de Zinco/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia
14.
Mol Cancer ; 18(1): 24, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30744655

RESUMO

Axl receptor tyrosine kinase is involved in the growth and metastasis and is an indicator of poor prognosis in several cancers including lung cancers. Although a mitogen-activated protein kinase (MAPK) pathway and an epithelial-to-mesenchymal transition (EMT) program are critical, molecular mechanisms underlying the Axl-driven cancer progression have not been fully elucidated. We aimed to identify molecules up-regulated by Axl kinase in lung adenocarcinomas. Through the global gene expression analysis and the functional annotation clustering, we found that AXL expression positively correlated with mRNA expressions of immune checkpoint molecules and chemokine receptors in non-small-cell lung cancers. Validation cohorts including our biobank confirmed that the AXL expression significantly correlated with expression of genes encoding programmed death-ligand1 (PD-L1) and CXC chemokine receptor 6 (CXCR6) in lung adenocarcinoma, especially in epidermal growth factor receptor (EGFR) mutation-positive adenocarcinoma. Pharmacological inhibition of Axl kinase activity decreased mRNA expressions of PD-L1 and CXCR6 in EGFR mutation-positive cell lines. Our data indicates the novel role of Axl kinase as a driver of immune checkpoint molecules and chemokine signalling pathways in the progression of lung adenocarcinomas. This study also highlights the necessity of clinical trials in order to test the efficacy of Axl kinase inhibition in the Axl-highly expressing subset of lung adenocarcinomas. .


Assuntos
Adenocarcinoma de Pulmão/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Antineoplásicos/farmacologia , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Benzocicloeptenos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Estudos de Coortes , Progressão da Doença , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/imunologia , Receptores ErbB/genética , Receptores ErbB/imunologia , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Proteína Tirosina Quinases/imunologia , Receptores CXCR6/genética , Receptores CXCR6/imunologia , Transdução de Sinais/imunologia , Triazóis/farmacologia
15.
Cancer Immunol Immunother ; 68(5): 799-812, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30770959

RESUMO

CV9201 is an RNActive®-based cancer immunotherapy encoding five non-small cell lung cancer-antigens: New York esophageal squamous cell carcinoma-1, melanoma antigen family C1/C2, survivin, and trophoblast glycoprotein. In a phase I/IIa dose-escalation trial, 46 patients with locally advanced (n = 7) or metastatic (n = 39) NSCLC and at least stable disease after first-line treatment received five intradermal CV9201 injections (400-1600 µg of mRNA). The primary objective of the trial was to assess safety. Secondary objectives included assessment of antibody and ex vivo T cell responses against the five antigens, and changes in immune cell populations. All CV9201 dose levels were well-tolerated and the recommended dose for phase IIa was 1600 µg. Most AEs were mild-to-moderate injection site reactions and flu-like symptoms. Three (7%) patients had grade 3 related AEs. No related grade 4/5 or related serious AEs occurred. In phase IIa, antigen-specific immune responses against ≥ 1 antigen were detected in 63% of evaluable patients after treatment. The frequency of activated IgD+CD38hi B cells increased > twofold in 18/30 (60%) evaluable patients. 9/29 (31%) evaluable patients in phase IIa had stable disease and 20/29 (69%) had progressive disease. Median progression-free and overall survival were 5.0 months (95% CI 1.8-6.3) and 10.8 months (8.1-16.7) from first administration, respectively. Two- and 3-year survival rates were 26.7% and 20.7%, respectively. CV9201 was well-tolerated and immune responses could be detected after treatment supporting further clinical investigation.


Assuntos
Linfócitos B/imunologia , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Imunoterapia/métodos , Neoplasias Pulmonares/terapia , RNA Mensageiro/uso terapêutico , Linfócitos T/imunologia , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/genética , Vacinas Anticâncer/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Células Cultivadas , Feminino , Humanos , Imunoterapia/efeitos adversos , Reação no Local da Injeção/etiologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Análise de Sobrevida
16.
Int J Cancer ; 145(4): 1111-1124, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-30719701

RESUMO

No curative treatment options are available for advanced hepatocellular carcinoma (HCC). Anti-PD1 antibody therapy can induce tumor regression in 20% of advanced HCC patients, demonstrating that co-inhibitory immune checkpoint blockade has therapeutic potential for this type of cancer. However, whether agonistic targeting of co-stimulatory receptors might be able to stimulate anti-tumor immunity in HCC is as yet unknown. We investigated whether agonistic targeting of the co-stimulatory receptor GITR could reinvigorate ex vivo functional responses of tumor-infiltrating lymphocytes (TIL) freshly isolated from resected tumors of HCC patients. In addition, we compared GITR expression between TIL and paired samples of leukocytes isolated from blood and tumor-free liver tissues, and studied the effects of combined GITR and PD1 targeting on ex vivo TIL responses. In all three tissue compartments, CD4+ FoxP3+ regulatory T cells (Treg) showed higher GITR- expression than effector T-cell subsets. The highest expression of GITR was found on CD4+ FoxP3hi CD45RA- activated Treg in tumors. Recombinant GITR-ligand as well as a humanized agonistic anti-GITR antibody enhanced ex vivo proliferative responses of CD4+ and CD8+ TIL to tumor antigens presented by mRNA-transfected autologous B-cell blasts, and also reinforced proliferation, IFN-γ secretion and granzyme B production in stimulations of TIL with CD3/CD28 antibodies. Combining GITR ligation with anti-PD1 antibody nivolumab further enhanced tumor antigen-specific responses of TIL in some, but not all, HCC patients, compared to either single treatment. In conclusion, agonistic targeting of GITR can enhance functionality of HCC TIL, and may therefore be a promising strategy for single or combinatorial immunotherapy in HCC.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma Hepatocelular/imunologia , Proteína Relacionada a TNFR Induzida por Glucocorticoide/imunologia , Neoplasias Hepáticas/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T Reguladores/imunologia , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoterapia/métodos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/imunologia
17.
Immunity ; 50(3): 738-750.e7, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30770248

RESUMO

Systemic immunosuppression greatly affects the chemotherapeutic antitumor effect. Here, we showed that CD19+ extracellular vesicles (EVs) from B cells through CD39 and CD73 vesicle-incorporated proteins hydrolyzed ATP from chemotherapy-treated tumor cells into adenosine, thus impairing CD8+ T cell responses. Serum CD19+ EVs were increased in tumor-bearing mice and patients. Patients with fewer serum CD19+ EVs had a better prognosis after chemotherapy. Upregulated hypoxia-inducible factor-1α (HIF-1α) promoted B cells to release CD19+ EVs by inducing Rab27a mRNA transcription. Rab27a or HIF-1α deficiency in B cells inhibited CD19+ EV production and improved the chemotherapeutic antitumor effect. Silencing of Rab27a in B cells by inactivated Epstein-Barr viruses carrying Rab27a siRNA greatly improved chemotherapeutic efficacy in humanized immunocompromised NOD PrkdcscidIl2rg-/- mice. Thus, decreasing CD19+ EVs holds high potential to improve the chemotherapeutic antitumor effect.


Assuntos
Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Vesículas Extracelulares/imunologia , Animais , Antígenos CD19/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Células HEK293 , Herpesvirus Humano 4/imunologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Células NIH 3T3 , RNA Mensageiro/imunologia , Transcrição Genética/imunologia , Proteínas rab27 de Ligação ao GTP/imunologia
18.
Nucleic Acids Res ; 47(6): 3086-3100, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30624716

RESUMO

Peptides presented on major histocompatibility (MHC) class I molecules form an essential part of the immune system's capacity to detect virus-infected or transformed cells. Earlier works have shown that pioneer translation peptides (PTPs) for the MHC class I pathway are as efficiently produced from introns as from exons, or from mRNAs targeted for the nonsense-mediated decay pathway. The production of PTPs is a target for viral immune evasion but the underlying molecular mechanisms that govern this non-canonical translation are unknown. Here, we have used different approaches to show how events taking place on the nascent transcript control the synthesis of PTPs and full-length proteins. By controlling the subcellular interaction between the G-quadruplex structure (G4) of a gly-ala encoding mRNA and nucleolin (NCL) and by interfering with mRNA maturation using multiple approaches, we demonstrate that antigenic peptides derive from a nuclear non-canonical translation event that is independently regulated from the synthesis of full-length proteins. Moreover, we show that G4 are exploited to control mRNA localization and translation by distinguishable mechanisms that are targets for viral immune evasion.


Assuntos
Antígenos/genética , Antígenos de Histocompatibilidade Classe I/genética , Peptídeos/genética , Biossíntese de Proteínas/genética , Antígenos/imunologia , Núcleo Celular/genética , Núcleo Celular/imunologia , Quadruplex G , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Degradação do RNAm Mediada por Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , Peptídeos/imunologia , Biossíntese de Proteínas/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia
19.
Clin Exp Immunol ; 196(2): 276-286, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30636174

RESUMO

Dysfunction of the immune regulatory system plays an important role in the pathogenesis of rheumatoid arthritis (RA). Vasoactive intestinal peptide (VIP) has multiple bioactivities. This study aims to investigate the role of VIP in the maintenance of the immune regulatory capacity of monocytes (Mos). Human peripheral blood samples were collected from RA patients and healthy control (HC) subjects. Mos and CD14+ CD71- CD73+ CD25+ regulatory Mos (RegMos) were isolated from the blood samples and characterized by flow cytometry. A rat RA model was developed to test the role of VIP in the maintenance of the immune regulatory function of Mos. The results showed that RegMos of HC subjects had immune suppressive functions. RegMos of RA patients expressed less interleukin (IL)-10 and showed an incompetent immune regulatory capacity. Serum levels of VIP were lower in RA patients, which were positively correlated with the expression of IL-10 in RegMos. In-vitro experiments showed that the IL-10 mRNA decayed spontaneously in RegMos, which could be prevented by the presence of VIP in the culture. VIP suppressed the effects of tristetraprolin (TTP) on inducing IL-10 mRNA decay in RegMos. Administration of VIP inhibited experimental RA in rats through restoring the IL-10 expression in RegMos. RegMos have immune suppressive functions. VIP is required in maintaining IL-10 expression in RegMos. The data suggest that VIP has translational potential in the treatment of immune disorders such as RA.


Assuntos
Monócitos/imunologia , Peptídeo Intestinal Vasoativo/imunologia , Adulto , Animais , Artrite Reumatoide/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Fatores Imunológicos/imunologia , Interleucina-10/imunologia , Masculino , RNA Mensageiro/imunologia , Ratos , Ratos Sprague-Dawley
20.
J Immunol ; 202(5): 1635-1643, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30683706

RESUMO

CD163 facilitates regulation and resolution of inflammation and removal of free hemoglobin and is highly expressed in myeloid cells from patients with inflammatory disorders, such as systemic juvenile idiopathic arthritis (SJIA) and macrophage activation syndrome (MAS). Our recent studies indicate that regulation of CD163 mRNA expression is a key functional property of polarized monocytes and macrophages and is mediated at the transcriptional and posttranscriptional level, including via microRNAs. The goal of the current study is to develop a multiparameter flow cytometry panel incorporating detection of CD163 mRNA for polarized monocyte and macrophage populations in disorders such as SJIA and MAS. THP-1 cells and CD14+ human monocytes were stained using fluorochrome-conjugated Abs to myeloid surface markers, along with CD163 mRNA. Staining for mRNA could reliably detect CD163 expression while simultaneously detecting different macrophage populations using Abs targeting CD14, CD64, CD80, CD163, and CD209. This approach was found to be highly sensitive for increased mRNA expression when macrophages were polarized with IL-10 [M(IL-10)], with a strong signal over a broad range of IL-10 concentrations, and showed distinct kinetics of CD163 mRNA and protein induction upon IL-10 stimulation. Finally, this panel demonstrated clear changes in polarization markers in unstimulated monocytes from patients with SJIA and MAS, including upregulated CD163 mRNA and increased CD64 expression. This approach represents a robust and sensitive system for RNA flow cytometry, useful for studying CD163 expression as part of a multimarker panel for human monocytes and macrophages, with broad applicability to the pathogenesis of hyperinflammatory diseases.


Assuntos
Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Citometria de Fluxo , Inflamação/imunologia , Macrófagos/imunologia , Monócitos/imunologia , RNA Mensageiro/análise , Receptores de Superfície Celular/análise , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/imunologia , Células Cultivadas , Humanos , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia
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