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1.
Cell Physiol Biochem ; 53(5): 774-793, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31647207

RESUMO

BACKGROUND/AIMS: Deregulation of the complex interaction among host genetics, gut microbiota and environmental factors on one hand and aberrant immune responses on the other hand, are known to be associated with the development of inflammatory bowel disease. Recent studies provided strong evidence that autophagy plays a key role in the etiology of Crohn's disease (CD). Probiotics may exhibit many therapeutic properties, including anti-inflammatory abilities. While successful results have been obtained in ulcerative colitis patients, probiotics remain inefficient in CD for unknown reason. It remains therefore important to better understand their molecular mechanisms of action. METHODS: The activation of autophagy was examined by stimulating bone marrow-derived dendritic cells by the bacteria, followed by confocal microscopy and western blot analysis. The impact of blocking in vitro autophagy was performed in peripheral blood mononuclear cells using 3-methyl adenine or bafilomycin followed by cytokine secretion measurement by ELISA. The role of autophagy in the anti-inflammatory capacities of the bacterial strains was evaluated in vivo using an acute trinitrobenzene sulfonic acid-induced murine model of colitis. The impact of BMDC was evaluated by adoptive transfer, notably using bone marrow cells derived from autophagy-related 16-like 1-deficient mice. RESULTS: We showed that selected lactobacilli and bifidobacteria are able to induce autophagy activation in BMDCs. Blocking in vitro autophagy abolished the capacity of the strains to induce the release of the anti-inflammatory cytokine interleukin-10, while it exacerbated the secretion of the pro-inflammatory cytokine interleukin-1ß. We confirmed in the TNBS-induced mouse model of colitis that autophagy is involved in the protective capacity of these selected strains, and showed that dendritic cells are involved in this process. CONCLUSION: We propose autophagy as a novel mechanism involved in the regulatory capacities of probiotics.


Assuntos
Autofagia , Bifidobacterium/fisiologia , Lactobacillus/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Células da Medula Óssea/citologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Colite/induzido quimicamente , Colite/microbiologia , Colite/patologia , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Feminino , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Macrolídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout
2.
Immunogenetics ; 71(8-9): 519-530, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31520135

RESUMO

Human CD4+ T lymphocytes play an important role in inducing potent immune responses. T cells are activated and stimulated by peptides presented in human leucocyte antigen (HLA)-class II molecules. These HLA-class II molecules typically present peptides of between 12 and 20 amino acids in length. The region that interacts with the HLA molecule, designated as the peptide-binding core, is highly conserved in the residues which anchor the peptide to the molecule. In addition, as these peptides are the product of proteolytic cleavages, certain conserved residues may be expected at the N- and C-termini outside the binding core. To study whether similar conserved residues are present in different cell types, potentially harbouring different proteolytic enzymes, the ligandomes of HLA-DRB1*03:01/HLA-DRB > 1 derived from two different cell types (dendritic cells and EBV-transformed B cells) were identified with mass spectrometry and the binding core and N- and C-terminal residues of a total of 16,568 peptides were analysed using the frequencies of the amino acids in the human proteome. Similar binding motifs were found as well as comparable conservations in the N- and C-terminal residues. Furthermore, the terminal conservations of these ligandomes were compared to the N- and C-terminal conservations of the ligandome acquired from dendritic cells homozygous for HLA-DRB1*04:01. Again, comparable conservations were evident with only minor differences. Taken together, these data show that there are conservations in the terminal residues of peptides, presumably the result of the activity of proteases involved in antigen processing.


Assuntos
Linfócitos B/metabolismo , Células Dendríticas/metabolismo , Antígenos HLA-DR/classificação , Antígenos HLA-DR/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteoma/metabolismo , Motivos de Aminoácidos , Linfócitos B/citologia , Células Cultivadas , Células Dendríticas/citologia , Humanos , Ligantes , Ligação Proteica
3.
Anticancer Res ; 39(9): 4643-4652, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519562

RESUMO

BACKGROUND/AIM: Adenoviral-mediated expression of CD40 ligand (CD40L) on dendritic cells (DCs) activates immune check point CD40/CD40L, enhancing the immunostimulation of DCs and effector cells against human renal carcinoma cells (RCC) and inducing tumor cell apoptosis in vitro. MATERIALS AND METHODS: DCs, isolated from buffy coats from healthy donors, were transduced with adenoviruses carrying human CD40L (Ad-hCD40L). Subsequently maturation marker and cytokine expression were analyzed by fluorescence-activated cell sorting and enzyme-linked immunosorbent assay. RESULTS: Adenoviral transduction induced high expression of soluble CD40L and membrane-bound CD40L, leading to a strong CD40-CD40L interaction in DCs. Interestingly, a T-helper cell type 1 shift of expressed cytokines/chemokines was observed due to the expression of membrane-bound CD40L rather than due to soIuble CD40L alone, which significantly reduced immunoactivation of DCs. However, supernatants of Ad-hCD40L-transduced DCs induced apoptosis of RCC cells. Co-culture of Ad-hCD40L DCs with cytokine-induced killer cells led to a significant stimulation of tumor-specific cytokine-induced killer cells, with increased proliferation and cytotoxicity. CONCLUSION: Use of Ad-hCD40L-transduced DCs is a promising approach to treating RCC.


Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Carcinoma de Células Renais/metabolismo , Células Dendríticas/metabolismo , Imunomodulação , Neoplasias Renais/metabolismo , Adenoviridae/genética , Biomarcadores , Ligante de CD40/genética , Carcinoma de Células Renais/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Neoplasias Renais/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transdução Genética
4.
J Dermatol ; 46(9): 808-811, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31290564

RESUMO

Disseminated granuloma annulare (GA) is a rare granulomatous dermatitis of unknown etiology. Treatment is often challenging and lack of a uniformly effective treatment, adds to the disease morbidity. Tumor necrosis factor (TNF)-α is an important cytokine in granuloma formation and previous reports have shown improvement of disseminated GA with anti-TNF-α therapy. Nevertheless, the underlying mechanism of actions of TNF-α inhibitors in GA remains unclear. Our aim was to evaluate alterations in the inflammatory infiltrate in a patient who experienced complete clearance of GA after treatment with infliximab. A skin biopsy was obtained before and 24 weeks after treatment with infliximab 5 mg/kg at weeks 0, 2, 6, 14 and 24. Immunohistochemical stains were performed in pre- and post-treatment biopsy specimens using CD1a, CD4, CD8, CD11c, CD32, CD68, CD69, CD163, CD183 and human leukocyte antigen (HLA)-DR to characterize alterations of the infiltrates. Parallel with clinical improvement, we observed a marked decrease in myeloid (CD11c) dendritic cells, different macrophage subsets (CD68, CD32, CD163) and T cells. In addition, a marked reduction of activation markers (HLA-DR, CD69) and CD183+ (CXCR3) cells was observed in post-treatment biopsy specimens. In conclusion, the clinical improvement of disseminated GA by infliximab is paralleled by inhibition of activated myeloid dendritic cells, different macrophage subsets and type 1 T cells.


Assuntos
Células Dendríticas/efeitos dos fármacos , Fármacos Dermatológicos/farmacologia , Granuloma Anular/tratamento farmacológico , Infliximab/farmacologia , Macrófagos/efeitos dos fármacos , Idoso , Biópsia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fármacos Dermatológicos/uso terapêutico , Granuloma Anular/imunologia , Granuloma Anular/patologia , Humanos , Infliximab/uso terapêutico , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Receptores CXCR3/imunologia , Receptores CXCR3/metabolismo , Pele/citologia , Pele/efeitos dos fármacos , Pele/patologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia
5.
Nat Commun ; 10(1): 2498, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175312

RESUMO

Allogeneic mesenchymal stem cells (MSCs) exhibit immunoregulatory function in human autoimmune diseases such as systemic lupus erythematosus (SLE), but the underlying mechanisms remain incompletely understood. Here we show that the number of peripheral tolerogenic CD1c+ dendritic cells (DCs) and the levels of serum FLT3L are significantly decreased in SLE patients especially with lupus nephritis, compared to healthy controls. Transplantation of allogeneic umbilical cord-derived MSCs (UC-MSCs) significantly up-regulates peripheral blood CD1c+DCs and serum FLT3L. Mechanistically, UC-MSCs express FLT3L that binds to FLT3 on CD1c+DCs to promote the proliferation and inhibit the apoptosis of tolerogenic CD1c+DCs. Conversely, reduction of FLT3L with small interfering RNA in MSCs abolishes the up-regulation of tolerogenic CD1c+DCs in lupus patients treated with MSCs. Interferon-γ induces FLT3L expression in UC-MSCs through JAK/STAT signaling pathway. Thus, allogeneic MSCs might suppress inflammation in lupus through up-regulating tolerogenic DCs.


Assuntos
Antígenos CD1/imunologia , Células Dendríticas/imunologia , Glicoproteínas/imunologia , Tolerância Imunológica/imunologia , Lúpus Eritematoso Sistêmico/terapia , Proteínas de Membrana/imunologia , Transplante de Células-Tronco Mesenquimais , Adulto , Antígenos CD1/metabolismo , Estudos de Casos e Controles , Células Dendríticas/metabolismo , Feminino , Glicoproteínas/metabolismo , Humanos , Interferon gama/farmacologia , Janus Quinases/efeitos dos fármacos , Janus Quinases/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/terapia , Masculino , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Fatores de Transcrição STAT/efeitos dos fármacos , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transplante Homólogo , Adulto Jovem
6.
Nat Commun ; 10(1): 2759, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227717

RESUMO

Langerhans cells (LC) are thought to be the only mononuclear phagocyte population in the epidermis where they detect pathogens. Here, we show that CD11c+ dendritic cells (DCs) are also present. These cells are transcriptionally similar to dermal cDC2 but are more efficient antigen-presenting cells. Compared to LCs, epidermal CD11c+ DCs are enriched in anogenital tissues where they preferentially interact with HIV, express the higher levels of HIV entry receptor CCR5, support the higher levels of HIV uptake and replication and are more efficient at transmitting the virus to CD4 T cells. Importantly, these findings are observed using both a lab-adapted and transmitted/founder strain of HIV. We also describe a CD33low cell population, which is transcriptionally similar to LCs but does not appear to function as antigen-presenting cells or acts as HIV target cells. Our findings reveal that epidermal DCs in anogenital tissues potentially play a key role in sexual transmission of HIV.


Assuntos
Células Dendríticas/virologia , Células Epidérmicas/virologia , Infecções por HIV/transmissão , HIV-1/imunologia , Apresentação do Antígeno/imunologia , Antígeno CD11c/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Epidérmicas/imunologia , Células Epidérmicas/metabolismo , Epiderme/imunologia , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/patogenicidade , Voluntários Saudáveis , Humanos , Masculino , Cultura Primária de Células , Receptores CCR5/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Linfócitos T/imunologia , Internalização do Vírus
7.
Cancer Sci ; 110(8): 2357-2367, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31169331

RESUMO

Dendritic cells (DCs) are potent and specialized antigen presenting cells, which play a crucial role in initiating and amplifying both the innate and adaptive immune responses against cancer. Tumor cells can escape from immune attack by secreting suppressive cytokines that solely or cooperatively impair the immune function of DCs. However, the underlying mechanisms are not fully defined. Vascular endothelial growth factor (VEGF) has been identified as a major cytokine in the tumor microenvironment. To elucidate the effects of VEGF on the motility and immune function of mature DCs (mDCs), the cells were treated with 50 ng/mL VEGF and investigated by proteomics and molecular biological technologies. The results showed that VEGF can impair the migration capacity and immune function of mDCs through the RhoA-cofilin1 pathway mediated by the VEGF receptor 2, suggesting impaired motility of mDCs by VEGF is one of the aspects of immune escape mechanisms of tumors. It is clinically important to understand the biological behavior of DCs and the immune escape mechanisms of tumor as well as how to improve the efficiency of antitumor therapy based on DCs.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Movimento Celular/imunologia , Células Dendríticas/metabolismo , Transdução de Sinais/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Fatores de Despolimerização de Actina/imunologia , Células Cultivadas , Citocinas/imunologia , Células Dendríticas/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Proteína rhoA de Ligação ao GTP/imunologia
8.
PLoS Genet ; 15(6): e1008228, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31220078

RESUMO

Dendrite growth is constrained by a self-avoidance response that induces retraction but the downstream pathways that balance these opposing mechanisms are unknown. We have proposed that the diffusible cue UNC-6(Netrin) is captured by UNC-40(DCC) for a short-range interaction with UNC-5 to trigger self-avoidance in the C. elegans PVD neuron. Here we report that the actin-polymerizing proteins UNC-34(Ena/VASP), WSP-1(WASP), UNC-73(Trio), MIG-10(Lamellipodin) and the Arp2/3 complex effect dendrite retraction in the self-avoidance response mediated by UNC-6(Netrin). The paradoxical idea that actin polymerization results in shorter rather than longer dendrites is explained by our finding that NMY-1 (non-muscle myosin II) is necessary for retraction and could therefore mediate this effect in a contractile mechanism. Our results also show that dendrite length is determined by the antagonistic effects on the actin cytoskeleton of separate sets of effectors for retraction mediated by UNC-6(Netrin) versus outgrowth promoted by the DMA-1 receptor. Thus, our findings suggest that the dendrite length depends on an intrinsic mechanism that balances distinct modes of actin assembly for growth versus retraction.


Assuntos
Actinas/genética , Proteínas de Caenorhabditis elegans/genética , Células Dendríticas/metabolismo , Netrinas/genética , Neurônios/metabolismo , Citoesqueleto de Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Membrana/genética , Cadeias Pesadas de Miosina/genética , Proteínas do Tecido Nervoso/genética , Miosina não Muscular Tipo IIB/genética
9.
Oncology ; 97(3): 135-148, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31216557

RESUMO

BACKGROUND: We have developed a Wilms' tumor 1 (WT1)-targeting dendritic cell (DC)-based cancer vaccine combined with standard chemotherapy for patients with advanced pancreatic ductal adenocarcinoma (PDA). METHODS: We evaluated predictive markers of overall survival (OS) in PDA patients treated with multiple major histocompatibility complex class I/II-restricted, WT1 peptide-pulsed DC vaccinations (DC/WT1-I/II) in combination with chemotherapy. Throughout the entire period of immunochemotherapy, the plasma levels of soluble factors derived from granulocytes of 7 eligible PDA patients were examined. Moreover, systemic inflammatory response markers (neutrophil-to-lymphocyte ratio [NLR], monocyte-to-lymphocyte ratio [MLR], and granulocyte-to-lymphocyte ratio [GLR]) were assessed. In addition, cytoplasmic WT1 expression in PDA cells was examined. RESULTS: Compared to the 4 non-super-responders (OS <1 year), the remaining 3 super-responders (OS ≥1 year) showed significantly decreased low plasma matrix metalloproteinase-9 levels throughout long-term therapy. The NLR, MLR, and GLR after 5 DC/WT1-I/II vaccinations and 3 cycles of gemcitabine were significantly lower in the super-responders than in the non-super-responders. Furthermore, the cytoplasmic WT1 expression in the PDA cells of super-responders was relatively weak compared to that in the PDA cells of non-super-responders. CONCLUSIONS: Prolonged low levels of a granulocyte-related systemic inflammatory response after the early period of therapy and low cytoplasmic WT1 expression in PDA cells may be markers predictive of OS in PDA patients receiving WT1-targeting immunochemotherapy.


Assuntos
Biomarcadores Tumorais , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Imunoterapia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/mortalidade , Proteínas WT1/imunologia , Biomarcadores , Vacinas Anticâncer/administração & dosagem , Terapia Combinada , Células Dendríticas/metabolismo , Epitopos/imunologia , Feminino , Humanos , Imunofenotipagem , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Peptídeos/imunologia , Peroxidase/metabolismo , Prognóstico , Fator de Crescimento Transformador beta1/metabolismo , Resultado do Tratamento , Vacinação , Proteínas WT1/genética
10.
Nat Immunol ; 20(7): 852-864, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31213723

RESUMO

Dendritic cells (DC) are currently classified as conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Through a combination of single-cell transcriptomic analysis, mass cytometry, in vivo fate mapping and in vitro clonal assays, here we show that, at the single-cell level, the priming of mouse hematopoietic progenitor cells toward the pDC lineage occurs at the common lymphoid progenitor stage, indicative of early divergence of the pDC and cDC lineages. We found the transcriptional signature of a pDC precursor stage, defined here, in the IL-7Rα+ common lymphoid progenitor population and identified Ly6D, IL-7Rα, CD81 and CD2 as key markers of pDC differentiation, which distinguish pDC precursors from cDC precursors. In conclusion, pDCs developed in the bone marrow from a Ly6DhiCD2hi lymphoid progenitor cell and differentiated independently of the myeloid cDC lineage.


Assuntos
Antígenos Ly/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Citometria de Fluxo , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Camundongos , Transcriptoma
11.
Molecules ; 24(9)2019 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-31083610

RESUMO

Optimal targeting of nanoparticles (NP) to dendritic cells (DCs) receptors to deliver cancer-specific antigens is key to the efficient induction of anti-tumour immune responses. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing tètanus toxoid and gp100 melanoma-associated antigen, toll-like receptor adjuvants were targeted to the DC-SIGN receptor in DCs by specific humanized antibodies or by ICAM3-Fc fusion proteins, which acts as the natural ligand. Despite higher binding and uptake efficacy of anti-DC-SIGN antibody-targeted NP vaccines than ICAM3-Fc ligand, no difference were observed in DC activation markers CD80, CD83, CD86 and CCR7 induced. DCs loaded with NP coated with ICAM3-Fc appeared more potent in activating T cells via cross-presentation than antibody-coated NP vaccines. This fact could be very crucial in the design of new cancer vaccines.


Assuntos
Vacinas Anticâncer/metabolismo , Células Dendríticas/metabolismo , Molécula 3 de Adesão Intercelular/metabolismo , Nanopartículas/química , Vacinas Anticâncer/química , Células Cultivadas , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Leucócitos/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Receptores de IgG/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
12.
Vet Immunol Immunopathol ; 211: 19-24, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31084889

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is one of the most common diseases in the global swine industry. PRRSV infection is highly restricted to cells of the monocyte-macrophage lineage. However, the lack of antibodies to swine monocyte-macrophage lineage markers significantly hampers PRRSV research. In this study, we have developed a monoclonal antibody against the swine leukocyte antigen (SLA)-DRα chain and confirmed its reactivity with endogenous expressed SLA-DR in a variety of cell lines and primary swine antigen-presenting cells (PAMs, PBMC and BM-DCs). Moreover, the level of SLA-DR expression after PRRSV infection were evaluated by our homemade Mab and a commercial anti-SLA-DR antibody. Based on our result, the protein level of SLA-DRα expression is increased after PRRSV infection in DC, while the mRNA of both SLA-DRα and SLA-DRß were significantly inhibited by PRRSV replication. In conclusion, we successfully developed a MAb reactive with endogenous SLA-DR in western blotting, and this MAb could be a useful tool for further research and analysis. Moreover, the inconsistency of SLA-DR expression between protein and mRNA levels may suggest a novel role of DC played during the immune response after PRRSV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Células Dendríticas/metabolismo , Cadeias alfa de HLA-DR/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Animais , Western Blotting , Medula Óssea/imunologia , Medula Óssea/metabolismo , Linhagem Celular , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Células HEK293 , Cadeias alfa de HLA-DR/metabolismo , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Proteínas Recombinantes , Suínos/imunologia
13.
Nat Commun ; 10(1): 1731, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043596

RESUMO

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis and vasculopathy. CXCL4 represents an early serum biomarker of severe SSc and likely contributes to inflammation via chemokine signaling pathways, but the exact role of CXCL4 in SSc pathogenesis is unclear. Here, we elucidate an unanticipated mechanism for CXCL4-mediated immune amplification in SSc, in which CXCL4 organizes "self" and microbial DNA into liquid crystalline immune complexes that amplify TLR9-mediated plasmacytoid dendritic cell (pDC)-hyperactivation and interferon-α production. Surprisingly, this activity does not require CXCR3, the CXCL4 receptor. Importantly, we find that CXCL4-DNA complexes are present in vivo and correlate with type I interferon (IFN-I) in SSc blood, and that CXCL4-positive skin pDCs coexpress IFN-I-related genes. Thus, we establish a direct link between CXCL4 overexpression and the IFN-I-gene signature in SSc and outline a paradigm in which chemokines can drastically modulate innate immune receptors without being direct agonists.


Assuntos
DNA Bacteriano/metabolismo , Interferon-alfa/metabolismo , Fator Plaquetário 4/metabolismo , Escleroderma Sistêmico/imunologia , Receptor Toll-Like 9/metabolismo , Adulto , Idoso , Biópsia , Estudos de Casos e Controles , DNA Bacteriano/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Interferon-alfa/imunologia , Cristais Líquidos , Masculino , Pessoa de Meia-Idade , Fator Plaquetário 4/imunologia , Receptores CXCR3/imunologia , Receptores CXCR3/metabolismo , Escleroderma Sistêmico/microbiologia , Escleroderma Sistêmico/patologia , Pele/citologia , Pele/imunologia , Pele/microbiologia , Pele/patologia , Receptor Toll-Like 9/imunologia
14.
Nat Commun ; 10(1): 2220, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101805

RESUMO

Both medullary thymic epithelial cells (mTEC) and dendritic cells (DC) present tissue-restricted antigens (TRA) to thymocytes to induce central tolerance, but the relative contributions of these antigen-presenting cell (APC) subsets remain unresolved. Here we developed a two-photon microscopy approach to observe thymocytes interacting with intact APCs presenting TRAs. We find that mTECs and DCs cooperate extensively to induce tolerance, with their relative contributions regulated by the cellular form of the TRA and the class of major histocompatibility complex (MHC) on which antigen is presented. Even when TRA expression is restricted to mTECs, DCs still present self-antigens at least as frequently as mTECs. Notably, the DC subset cDC2 efficiently acquires secreted mTEC-derived TRAs for cross-presentation on MHC-I. By directly imaging interactions between thymocytes and APCs, while monitoring intracellular signaling, this study reveals that distinct DC subsets and AIRE+ mTECs contribute substantially to presentation of diverse self-antigens for establishing central tolerance.


Assuntos
Tolerância Central/imunologia , Células Dendríticas/imunologia , Timócitos/imunologia , Timo/imunologia , Animais , Apresentação do Antígeno/imunologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Transplante de Medula Óssea , Separação Celular/métodos , Células Dendríticas/metabolismo , Células Epiteliais/imunologia , Feminino , Citometria de Fluxo/métodos , Microscopia Intravital/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Linfócitos T Reguladores/imunologia , Timócitos/metabolismo , Timo/citologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Quimeras de Transplante/imunologia
15.
Mol Med Rep ; 19(6): 5377-5385, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059096

RESUMO

Hyperglycemia promotes the growth and reproduction of bacteria, thereby increasing the probability of infection, which also causes rebound hyperglycemia. Therefore, the interactions of infection and hyperglycemia lead to the progression and deterioration of these diseases. Type 1 diabetes mellitus (T1DM) is an autoimmune disease. Studies have shown that regulatory T cells (Tregs) play a key role in maintaining islet­specific tolerance. Treg deficiency may lead to the development of early pancreatitis and T1DM, and sufficient amounts of Tregs can restore this tolerance, thereby inhibiting the occurrence of T1DM. Moreover, different subpopulations of dendritic cells (DCs) play an important role in activating autoreactive T cells and inducing autoimmune tolerance to autoantigens, which are closely related to the functional diversity caused by different phenotypes, maturation status, and the immune microenvironment of DC subpopulations. In the present study, we used streptozotocin­induced hyperglycemic mice to model T1DM and induced a Salmonella infection in the mouse model, leading to aggravated inflammation, which resulted in an elevated proportion of CD103+CD11b+ DCs and a significantly elevated proportion of CD4+FoxP3+ Tregs in the intestinal lamina propria. After co­culturing CD4+ T cells and DCs, we found that CD103+CD11b+ DCs could significantly promote the proliferation of CD4+ T cells. The elevated proportions of CD4+FoxP3+ Tregs were considered to be correlated with the increased number of CD103+CD11b+ DCs.


Assuntos
Infecções por Salmonella/patologia , Linfócitos T Reguladores/metabolismo , Animais , Antígenos CD/metabolismo , Antígeno CD11b/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Técnicas de Cocultura , Citocinas/sangue , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Inflamação , Cadeias alfa de Integrinas/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/patologia , Salmonella/patogenicidade , Infecções por Salmonella/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia
16.
BMC Cancer ; 19(1): 439, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088527

RESUMO

BACKGROUND: Dendritic cells (DCs) alter their role from being immunostimulatory to immunosuppressive at advanced stages of tumor progression, but the influence of cancer stem cells (CSCs) and their secreted factors on generation and phenotypic change of DCs is unknown. Retinoic acid-inducible gene I (RIG-I) plays a role in regulation of other cellular processes including leukemic stemness besides its antiviral function. METHODS: Short hairpin RNA-mediated gene silencing was employed to generate stable RIG-I-knocked-down human hepatocellular carcinoma (HCC) cell lines. Expression levels of genes and proteins in spheres of those HCC cells were determined by quantitative real-time PCR and Western bot, respectively. Levels of secreted cytokines were measured by ELISA. The surface molecule expression levels of DCs were analyzed using flow cytometry. The ability of DCs to induce proliferation of T cells was assessed by a mixed lymphocyte reaction (MLR) assay. RESULTS: RIG-I-knocked-down HCC cells showed upregulated expression of stem cell marker genes, enhanced secretion of factors suppressing in vitro generation of DCs into the conditioned medium (CM), and induction of a phenotype of tumor-infiltrating DCs (TIDCs) with low levels of DC markers in their tumors in nude mice. Those DCs and TIDCs showed reduced MLR, indicating RIG-I deficiency-induced immunotolerance. The RIG-I-deficient HCC cells secreted more TGF-ß1 than did reference cells. The tumors formed after injection of RIG-I-deficient HCC cells had higher TGF-ß1 contents than did tumors derived from control cells. DC generation and MLR suppressed by the CM of RIG-I-deficient HCC cells were restored by an anti-TGF-ß1 antibody. TGF-ß1-induced phosphorylation of Smad2 and Akt was enhanced in RIG-I-deficient HCC spheres, knockdown of AKT gene expression abolishing the augmentation of TGF-ß1-induced Smad2 phosphorylation. Akt and p-Akt were co-immunoprecipitated with Smad2 in cytoplasmic proteins of RIG-I-deficient spheres but not in those of control spheres, the amounts of co-immunoprecipitated Akt and p-Akt being increased by TGF-ß stimulation. CONCLUSIONS: Our results demonstrate that RIG-I deficiency in HCC cells induced their stemness, enhanced secretion and signaling of TGF-ß1, tolerogenic TIDCs and less generation of DCs, and the results suggest involvement of TGF-ß1 in those RIG-I deficiency-induced tolerogenic changes and involvement of CSCs in DC-mediated immunotolerance.


Assuntos
Carcinoma Hepatocelular/patologia , Proteína DEAD-box 58/deficiência , Células Dendríticas/citologia , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Microambiente Tumoral , Regulação para Cima
17.
Nat Protoc ; 14(6): 1926-1943, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31101906

RESUMO

The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8+ T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2-4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Epitopos/imunologia , Neoplasias/imunologia , Células Cultivadas , Células Dendríticas/metabolismo , Eletroporação/métodos , Epitopos/genética , Humanos , Imunoterapia/métodos , Neoplasias/terapia , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/análise , Receptores de Antígenos de Linfócitos T/imunologia , Transfecção/métodos
18.
Nutrients ; 11(4)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987344

RESUMO

Intestinal bifidobacteria benefit human health by promoting and modulating the gut flora, and boosting therapeutic efficiency for chronic metabolic diseases and cancer. Recently, Bifidobacterium adolescentis strains with high adhesion to intestinal epithelial cells were associated with induction of T-helper 17 (Th17) cells in humans and rodents. Here, two B. adolescentis strains with similar adhesive ability but different aggregation properties were investigated for specific immunoregulatory effects, including the underlying cellular pathway, on macrophage and T-regulatory (Treg)/Th17 axis activation in vitro and in the colon of dextran sodium sulfate (DSS)-colitis mice in vivo. In-vitro, the auto-aggregative B. adolescentis strain IF1-11 induced significantly higher IL-6 and lower IL-10 secretion from immune cells, and it induced abundant Th17 cells. The non-aggregating strain IF1-03 induced significantly higher IL-10, less IL-6 and a high proportion of Treg/Th17 cells compared to total T cells. In vivo, orally administered IF1-03 protected DSS-colitis mice via activation of dendritic cells or macrophages and skewing of Treg/Th17 cells, consistent with Treg cell induction in vitro. IF1-03 exopolysaccharides showed a functional recognition pattern similar to IF1-03 for IL-10 cytokine secretion and Treg cell-differentiation induction, both dependent on the toll-like receptor 2-ERK/p38 MAPK-signaling cascade for macrophage activation. We suggest that B. adolescentis exopolysaccharide-associated enterocyte adhesion/aggregation phenotypes determine strain-specific adaptive immune responses in the gut via the macrophage-regulated Treg/Th17 axis.


Assuntos
Aderência Bacteriana , Bifidobacterium adolescentis/metabolismo , Colite/prevenção & controle , Colo/microbiologia , Microbioma Gastrointestinal , Polissacarídeos Bacterianos/metabolismo , Probióticos/administração & dosagem , Linfócitos T Reguladores/microbiologia , Células Th17/microbiologia , Animais , Bifidobacterium adolescentis/imunologia , Colite/imunologia , Colite/metabolismo , Colite/microbiologia , Colo/imunologia , Colo/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Sulfato de Dextrana , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interações Hospedeiro-Patógeno , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polissacarídeos Bacterianos/imunologia , Células RAW 264.7 , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Receptor 2 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
Artif Cells Nanomed Biotechnol ; 47(1): 1543-1558, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31007088

RESUMO

Mannosylation of nanovaccine is an appropriate strategy for targeting the mannose receptors on DCs. Here, HBsAg and mannose loaded on the surface of iron oxide nanoparticles to increases HBsAg vaccine potency. Nanoparticles are made by co-precipitation method and bonded to the HBsAg and mannose by chemical bonding. The physicochemical properties of nano-vaccines, their toxicity and antigenicity were determined. The synthesized nano-vaccine showed spherical shape with a mean particle size of 60 nm, a zeta potential of -44 mV, an antigen-binding efficiency of around 100% and for mannose 78%. In vitro release of nanoparticles exhibited about 30% at the first day and about 60% until the third day. SDSPAGE analysis confirmed structural integrity of HBsAg loaded on nanoparticles. The HBsAg-loaded LCMNP and MLCMNP nanoparticles had no toxic effects on HEK293 cell line. The quantification of the intracellular Fe by ICP-OES as a criterion of nano-vaccine uptake revealed mannose intensify uptake of MLCMNP. In addition, mannose in the structure of MLCMNP improved IL-6, TNF-α and IFN-γ (>16 fold) cytokines genes expression by macrophage/dendritic cells after exposure in 12 h. Immunization of experimental mice (subcutaneously, two times with 2-week intervals) with 5 µg of HBsAg loaded on MLCMNP nanoparticles increased specific total IgG and IgG2a/IgG1 ratio. In addition, TNF-α, IL-12, IL-2 and IL-4 cytokines in mannosylated nano-vaccine increased versus nano-vaccine group while lymphocyte proliferation and IFN-γ responses in the targeted nano-vaccine group show a tiny increase versus the nano-vaccine group. The results show that mannosylated nano-vaccine promotes higher level of cellular and humoural immune responses against HBsAg nano-vaccine.


Assuntos
Portadores de Fármacos/química , Compostos Férricos/química , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/farmacologia , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Manose/química , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Portadores de Fármacos/toxicidade , Feminino , Células HEK293 , Humanos , Imunoglobulina G/metabolismo , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Vacinas/química , Vacinas/farmacologia
20.
Int J Mol Sci ; 20(8)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31013796

RESUMO

Telomerase, an enzyme responsible for the synthesis of telomeres, is activated in many cancer cells and is involved in the maintenance of telomeres. The activity of telomerase allows cancer cells to replicate and proliferate in an uncontrolled manner, to infiltrate tissue, and to metastasize to distant organs. Studies to date have examined the mechanisms involved in the survival of cancer cells as targets for cancer therapeutics. These efforts led to the development of telomerase inhibitors as anticancer drugs, drugs targeting telomere DNA, viral vectors carrying a promoter for human telomerase reverse transcriptase (hTERT) genome, and immunotherapy targeting hTERT. Among these novel therapeutics, this review focuses on immunotherapy targeting hTERT and discusses the current evidence and future perspectives.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Telomerase/antagonistas & inibidores , Animais , Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Vacinas Anticâncer/imunologia , Terapia Baseada em Transplante de Células e Tecidos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Terapia Genética , Humanos , Imunoterapia , Terapia de Alvo Molecular , Neoplasias/metabolismo , Telomerase/genética , Telomerase/metabolismo , Vacinas de DNA/imunologia
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