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1.
Nat Cell Biol ; 23(3): 219-231, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33649477

RESUMO

Regulation of haematopoietic stem and progenitor cell (HSPC) fate is crucial during homeostasis and under stress conditions. Here we examine the aetiology of the Flt3 ligand (Flt3L)-mediated increase of type 1 conventional dendritic cells (cDC1s). Using cellular barcoding we demonstrate this occurs through selective clonal expansion of HSPCs that are primed to produce cDC1s and not through activation of cDC1 fate by other HSPCs. In particular, multi/oligo-potent clones selectively amplify their cDC1 output, without compromising the production of other lineages, via a process we term tuning. We then develop Divi-Seq to simultaneously profile the division history, surface phenotype and transcriptome of individual HSPCs. We discover that Flt3L-responsive HSPCs maintain a proliferative 'early progenitor'-like state, leading to the selective expansion of multiple transitional cDC1-primed progenitor stages that are marked by Irf8 expression. These findings define the mechanistic action of Flt3L through clonal tuning, which has important implications for other models of 'emergency' haematopoiesis.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteínas de Membrana/farmacologia , RNA-Seq , Análise de Célula Única , Transcriptoma/efeitos dos fármacos , Animais , Linhagem da Célula , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo
2.
Int J Mol Sci ; 22(4)2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33671896

RESUMO

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). MS and its animal model called experimental autoimmune encephalomyelitis (EAE) immunopathogenesis involve a plethora of immune cells whose activation releases a variety of proinflammatory mediators and free radicals. Vitamin D3 (VitD) is endowed with immunomodulatory and antioxidant properties that we demonstrated to control EAE development. However, this protective effect triggered hypercalcemia. As such, we compared the therapeutic potential of VitD and paricalcitol (Pari), which is a non-hypercalcemic vitamin D analog, to control EAE. From the seventh day on after EAE induction, mice were injected with VitD or Pari every other day. VitD, but not Pari, displayed downmodulatory ability being able to reduce the recruitment of inflammatory cells, the mRNA expression of inflammatory parameters, and demyelination at the CNS. Lower production of proinflammatory cytokines by lymph node-derived cells and IL-17 by gut explants, and reduced intestinal inflammation were detected in the EAE/VitD group compared to the EAE untreated or Pari groups. Dendritic cells (DCs) differentiated in the presence of VitD developed a more tolerogenic phenotype than in the presence of Pari. These findings suggest that VitD, but not Pari, has the potential to be used as a preventive therapy to control MS severity.


Assuntos
Antioxidantes/administração & dosagem , Colecalciferol/administração & dosagem , Encefalomielite Autoimune Experimental/prevenção & controle , Ergocalciferóis/administração & dosagem , Fatores Imunológicos/administração & dosagem , Profilaxia Pós-Exposição/métodos , Animais , Antioxidantes/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Colecalciferol/farmacologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/imunologia , Ergocalciferóis/farmacologia , Feminino , Fatores Imunológicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Esclerose Múltipla/prevenção & controle , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento
3.
Int J Nanomedicine ; 16: 1889-1899, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33707944

RESUMO

Introduction: Sonodynamic therapy (SDT) has good targeting and non-invasive advantages in the treatment of solid cancers, and checkpoint blockade immunotherapy is also a promising treatment to cure cancer. However, their antitumor effects are not sufficient due to some inherent factors. Some studies that combined SDT with immunotherapy or nanoparticles have managed to enhance its efficiency to treat cancers. Methods: In this work, an effective therapeutic strategy that can potentiate the antitumor efficacy of anti-PD-L1 antibody (aPD-L1) is developed by the use of cascade immuno-sonodynamic therapy (immuno-SDT). Titanium dioxide (TiO2), a nanostructured agent for SDT, sonosensitizer Chlorin e6 (Ce6), and immunological adjuvant CpG oligonucleotide (CpG ODN), are used to construct a multifunctional nanosonosensitizer (TiO2-Ce6-CpG). Then, we conducted in vitro and in vivo experiments to explore the antitumor effect of TiO2-Ce6-CpG under ultrasound (US) treatment. Results: The characterization tests showed that the nanosonosensitizers are polycrystalline structure with homogeneous sizes, resulting in a good drug loading efficiency. The innovative nanosonosensitizers (TiO2-Ce6-CpG) can not only effectively inhibit tumor growth but also stimulate the immune system to activate the adaptive immune responses, using the TiO2-Ce6 to augment SDT and the immune adjuvant CpG to enhance the immune response. After combined with the aPD-L1, the synergistic effect could not only efficiently inhibit the primary tumor growth but also lead to an inhibition of the non-irradiated pre-existing distant tumors by inducing a strong tumor-specific immune response. Conclusion: In this study, we present an effective strategy for tumor treatment by combining nanosonosensitizer-augmented SDT and aPD-L1 checkpoint blockade. This work provides a promising strategy and offers a new vision for treating malignant tumors.


Assuntos
/uso terapêutico , Imunoterapia , Nanopartículas/química , Neoplasias/imunologia , Neoplasias/terapia , Terapia por Ultrassom , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Terapia Combinada , Células Dendríticas/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Feminino , Humanos , Imunidade/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Neoplasias/patologia , Oligodesoxirribonucleotídeos/química , Porfirinas/química , Espécies Reativas de Oxigênio/metabolismo , Titânio/química
4.
J Vis Exp ; (168)2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33616089

RESUMO

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation. Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (αDEC-205) for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Antígenos CD/imunologia , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Lectinas Tipo C/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Receptores de Superfície Celular/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos CD/metabolismo , Apresentação Cruzada , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Técnicas In Vitro , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor/metabolismo , Ovalbumina/imunologia , Receptores de Superfície Celular/metabolismo
5.
Yonsei Med J ; 62(2): 137-148, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33527793

RESUMO

PURPOSE: In organ transplantation, the need for immune modulation rather than immune suppression has been emphasized. In this study, we investigated whether combinatorial treatments of with thalidomide (TM) and dexamethasone (DX) might be new approaches to induce systemic immunomodulation on T cells and other immune cells that regulate the expression of co-inhibitory molecules. MATERIALS AND METHODS: Naïve splenic T cells from C57BL/6 mice were sort-purified and cultured in vitro for CD4+ T cell proliferation and regulatory T cell (Treg) conversion in the presence of TM or/and DX. Expression of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1) in proliferated and converted T cells was quantified by flow cytometry. We also quantified in vivo expression of CTLA-4 and PD-1 on splenic CD4+ T cells and other immune cells isolated from TM- or/and DX-treated mice. Mixed lymphocytes reactions (MLR) were performed to evaluate the capacity of immune cells in carrying out immune responses. RESULTS: CTLA-4 expressions in effector T cells in vivo and in Tregs in vivo/vitro significantly increased upon TM/DX combinatorial treatment. Corresponding to increased CTLA-4 expression in T cells, the expression of ligand molecules for CTLA-4 significantly increased in splenic dendritic cells in TM/DX-treated groups. In addition, MLR results demonstrated that splenocytes isolated from TM/DX-treated mice significantly suppressed the proliferation of T cells isolated from other strains. CONCLUSION: Based on these results, we suggest that TM/DX combinatorial treatments might be efficient immunomodulatory methods for regulating T cell immunity.


Assuntos
Dexametasona/farmacologia , Imunomodulação/efeitos dos fármacos , Linfócitos T/imunologia , Talidomida/farmacologia , Animais , Antígeno CTLA-4/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Citometria de Fluxo , Ativação Linfocitária/efeitos dos fármacos , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/metabolismo , Baço/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T Reguladores/imunologia
6.
Int J Biol Macromol ; 171: 112-122, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33418037

RESUMO

The aim of this study was to investigate the primary structure of an acetylated Cyclocarya paliurus polysaccharide (Ac-CPP0.1) and its protective effect on H2O2-treated dendritic cells. The backbone of Ac-CPP0.1 was →3)-ß-D-Galp-(1→, with some branches α-L-Araf-(1→ residues at O-6 and O-5, ß-D-Galp-(1→ and 3,5,6)-ß-D-Galf-(1 residues at O-4 and acetyl groups were substituted at the O-2 and O-6 positions of 3)-ß-D-Galp-(1 residues. The CPP0.1 and Ac-CPP0.1 significantly increased the levels of superoxide dismutase, glutathione peroxidase and catalase on H2O2-treated dendritic cells. Meanwhile, both CPP0.1 and Ac-CPP0.1 up-regulated the expression of Nrf2 (NF-E2-related factor 2) and down-regulated the Keap1 (Kelch-like ECH-associated protein-1), but Ac-CPP0.1 had a better effect on antioxidant capacity. These results indicated that potential application of Ac-CPP0.1 as an antioxidant agent.


Assuntos
Antioxidantes/farmacologia , Células Dendríticas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Juglandaceae/química , Polissacarídeos/farmacologia , Acetilação , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Compostos de Bifenilo/antagonistas & inibidores , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Catalase/genética , Catalase/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Picratos/antagonistas & inibidores , Extratos Vegetais/química , Folhas de Planta/química , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Cultura Primária de Células , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
7.
PLoS Pathog ; 17(1): e1009168, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33444400

RESUMO

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ's effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1ß and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.


Assuntos
Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/fisiologia , Resinas Acrílicas/química , Adjuvantes Imunológicos/farmacologia , Apresentação do Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , NADPH Oxidase 2/fisiologia , Animais , Apresentação do Antígeno/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
8.
PLoS One ; 16(1): e0244439, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33444326

RESUMO

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease representing a serious unmet medical need. The disease is associated with the loss of self-tolerance and exaggerated B cell activation, resulting in autoantibody production and the formation of immune complexes that accumulate in the kidney, causing glomerulonephritis. TLR7, an important mediator of the innate immune response, drives the expression of type-1 interferon (IFN), which leads to expression of type-1 IFN induced genes and aggravates lupus pathology. Because the lysosomal peptide symporter slc15a4 is critically required for type-1 interferon production by pDC, and for certain B cell functions in response to TLR7 and TLR9 signals, we considered it as a potential target for pharmacological intervention in SLE. We deleted the slc15a4 gene in C57BL/6, NZB, and NZW mice and found that pristane-challenged slc15a4-/- mice in the C57BL/6 background and lupus prone slc15a4-/- NZB/W F1 mice were both completely protected from lupus like disease. In the NZB/W F1 model, protection persisted even when disease development was accelerated with an adenovirus encoding IFNα, emphasizing a broad role of slc15a4 in disease initiation. Our results establish a non-redundant function of slc15a4 in regulating both innate and adaptive components of the immune response in SLE pathobiology and suggest that it may be an attractive drug target.


Assuntos
Lúpus Eritematoso Sistêmico/patologia , Proteínas de Membrana Transportadoras/metabolismo , Animais , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Imidazóis/farmacologia , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/mortalidade , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NZB , Camundongos Knockout , Taxa de Sobrevida , Terpenos/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo
9.
Nat Commun ; 12(1): 105, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397973

RESUMO

Environmental triggers have important functions in multiple sclerosis (MS) susceptibility, phenotype, and trajectory. Exposure to early life trauma (ELT) has been associated with higher relapse rates in MS patients; however, the underlying mechanisms are not well-defined. Here we show ELT induces mechanistic and phenotypical alterations during experimental autoimmune encephalitis (EAE). ELT sustains downregulation of immune cell adrenergic receptors, which can be attributed to chronic norepinephrine circulation. ELT-subjected mice exhibit interferon-ß resistance and neurodegeneration driven by lymphotoxin and CXCR2 involvement. These phenotypic changes are observed in control EAE mice treated with ß1 adrenergic receptor antagonist. Conversely, ß1 adrenergic receptor agonist treatment to ELT mice abrogates phenotype changes via restoration of immune cell ß1 adrenergic receptor function. Our results indicate that ELT alters EAE phenotype via downregulation of ß1 adrenergic signaling in immune cells. These results have implications for the effect of environmental factors in provoking disease heterogeneity and might enable prediction of long-term outcomes in MS.


Assuntos
Regulação para Baixo , Interferon beta/metabolismo , Esclerose Múltipla/complicações , Degeneração Neural/complicações , Receptores Adrenérgicos beta 1/metabolismo , Transdução de Sinais , Estresse Psicológico/complicações , Agonistas de Receptores Adrenérgicos beta 1/farmacologia , Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Animais , Biomarcadores/metabolismo , Encéfalo/imunologia , Encéfalo/patologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/patologia , Feminino , Complexo de Golgi/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Esclerose Múltipla/sangue , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Degeneração Neural/sangue , Degeneração Neural/imunologia , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Norepinefrina/sangue , Fenótipo , Índice de Gravidade de Doença , Regulação para Cima/efeitos dos fármacos
10.
Int J Nanomedicine ; 16: 403-420, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33469292

RESUMO

Background: Therapeutic tumor vaccines are one of the most promising strategies and have attracted great attention in cancer treatment. However, most of them have shown unsatisfactory immunogenicity, there are still few available vaccines for clinical use. Therefore, there is an urgent demand to develop novel strategies to improve the immune efficacy of antitumor vaccines. Purpose: This study aimed to develop novel adjuvants and carriers to enhance the immune effect of MUC1 glycopeptide antigen-based antitumor vaccines. Methods: An antitumor vaccine was developed, in which MUC1 glycopeptide was used as tumor-associated antigen, α-GalCer served as an immune adjuvant and AuNPs was a multivalent carrier. Results: Immunological evaluation results indicated that the constructed vaccines enabled a significant antibody response. FACS analysis and immunofluorescence assay showed that the induced antisera exhibited a specific binding with MUC1 positive MCF-7 cells. Moreover, the induced antibody can mediate CDC to kill MCF-7 cells. Besides stimulating B cells to produce MUC1-specific antibodies, the prepared vaccines also induced MUC1-specific CTLs in vitro. Furthermore, the vaccines significantly delayed tumor development in tumor-bearing mice model. Conclusion: These results showed that the construction of vaccines by presenting α-GalCer adjuvant and an antigen on gold nanoparticles offers a potential strategy to improve the antitumor response in cancer immunotherapy.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Galactosilceramidas/farmacologia , Ouro/farmacologia , Nanopartículas Metálicas/química , Mucina-1/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antineoplásicos/imunologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Galactosilceramidas/síntese química , Galactosilceramidas/química , Humanos , Soros Imunes/metabolismo , Melanoma/imunologia , Melanoma/patologia , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Baço/patologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
11.
Methods Mol Biol ; 2248: 155-165, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33185874

RESUMO

Immunomodulation with anti-TNFα biologics is highly effective in the treatment of various immune-mediated inflammatory diseases, even though 2-5% of patients treated can develop paradoxical psoriasiform skin lesions. We recently analyzed three patients affected by severe hidradenite suppurativa (HS), and who developed paradoxical psoriasiform reactions following treatment with the TNF-α blockers. Psoriasiform skin reactions showed immunological and immunohistochemical features common to acute psoriasis, characterized by cellular players of innate immunity, such as plasmacytoid dendritic cells (pDC), neutrophils, mast cells, macrophages, and monocytes. In addition, IFN-ß and IFN-α2a, two type I IFNs typical of early psoriasis, were highly expressed in paradoxical skin reactions. Concomitantly, the lymphotoxin (LT)-α and LT-ß were overproduced. Detection of innate immunity cells was carried out on skin sections from HS patients, by immunohistochemistry (IHC) by using antibodies (Abs) against markers identifying specific leukocyte subpopulations. Anti-BDCA2, anti-CD15, anti-CD117, anti-CD68, anti-CD11c, and anti-CD3 Abs were employed to detect pDC, neutrophils, mast cells, macrophages, monocytes/dendritic cells, and T lymphocytes, respectively. In parallel, skin expression of the innate immunity soluble mediators IL-36γ, IFN-ß, IFN-κ, LT-α and LT-ß was also evaluated by IHC by using specific Abs. In this chapter, we describe the methods and protocols to detect the in situ expression and localization of innate immunity molecules and leukocyte subpopulations in skin lesions where inflammatory and psoriasiform reactions are evoked by anti-TNF- α biological therapy.


Assuntos
Produtos Biológicos/efeitos adversos , Fatores Imunológicos/efeitos adversos , Psoríase/etiologia , Psoríase/metabolismo , Fator de Necrose Tumoral alfa/efeitos adversos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Psoríase/patologia
12.
Carbohydr Polym ; 252: 117132, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33183591

RESUMO

Transdermal immunomodulation is of increasing interest as an efficient drug delivery method. It non-invasively delivers drugs directly to skin-resident immune cells, thereby avoiding the first-pass metabolism. Herein, we prepared ovalbumin-conjugated hyaluronic acid-methacrylate (HAMA-OVA) and schizophyllan-methacrylate (SPGMA) hybrid nanogels and investigated their suitability for topical delivery. The particle size was controlled to between 100 and 300 nm using ultrasonication and filtering processes. The nanogels penetrated the porcine stratum corneum layer and were deposited in the dermis via hybridization with HAMA. In addition, the hybridized SPGMA promoted the internalization of the nanogels into dendritic cells (DCs; JAWSII), which resulted in an improvement in the ovalbumin delivery efficiency. In molecular biological assessments, the hybrid nanogels upregulated the DC activation marker interleukin-6 and induced DC maturation, indicating antigen-presenting behavior. These results suggest that HAMA/SPGMA hybrid nanogels are a promising topical delivery carrier for immunomodulation and vaccinations.


Assuntos
Células Dendríticas/efeitos dos fármacos , Portadores de Fármacos/química , Nanogéis/química , Administração Cutânea , Animais , Linhagem Celular , Ácido Hialurônico/química , Fatores Imunológicos/química , Imunomodulação , Metacrilatos/química , Camundongos , Ovalbumina/química , Estudo de Prova de Conceito , Sizofirano/química , Absorção Cutânea , Suínos , beta-Glucanas/química
13.
Carbohydr Polym ; 251: 117098, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33142636

RESUMO

In this study, we synthesized negatively charged chitosan sulfate and positively charged hydroxypropyltrimethyl ammonium chloride chitosan (HACC), and then prepared chitosan derivatives with positive and negative ions as nanoparticles (NPs) by ovalbumin encapsulation using the polyelectrolyte method. NPs with different substitution sites and molecular weights (MW) were prepared by varying conditions. We then determined the zeta potential average, diameter, encapsulation effect, and their immunostimulatory effects on dendritic cells (DCs). The results showed that chitosan-derivative NPs ranged in size from 153.33 to 320.90 nm; all NPs were positive, with charges ranging from 17.10 to 39.30 mV and the encapsulation rates of 65 %-75 %. Three NPs greatly promoted the expression and secretion of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and interleukin-1ß (IL-1ß) in DC cells: C2,3,6 chitosan sulfate-HACC (C2,3,6-HACC; 200 kDa), C3,6 chitosan sulfate-HACC (C3,6-HACC; 200 kDa) and C6 chitosan sulfate-HACC (C6-HACC; 50 kDa). We also found that 200-kDa C2,3,6-HACC and 50-kDa C6-HACC NPs greatly increased secretion of the major histocompatibility complex-II (MHC-II), CD40, CD80, and CD86, indicating that these NPs promote effective antigen presentation, further increasing immunity effects. Finally, we applied laser confocal photography and determined that NPs entered the cell to promote the regulation of cellular immune activity; this discovery lays a foundation for further research on their mechanism of their action. Therefore, C2,3,6-HACC and C6-HACC NPs have the potential as immunological adjuvants.


Assuntos
Quitosana/análogos & derivados , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Nanopartículas/química , Compostos de Amônio Quaternário/química , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Quitosana/química , Quitosana/farmacologia , Citocinas/imunologia , Humanos , Imunomodulação/efeitos dos fármacos
14.
Methods Mol Biol ; 2223: 281-293, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226601

RESUMO

Allergic disease is on the rise and yet the underlying cause and risk factors are not fully understood. While lifesaving in many circumstances, the use of antibiotics and the subsequent disruption of the microbiome are positively correlated with the development of allergies. Here, we describe the use of the antibiotic vancomycin in combination with the papain-induced mouse model of allergic disease that allows for the assessment of microbiome perturbations and the impact on allergy development.


Assuntos
Antibacterianos/farmacologia , Asma/imunologia , Macrófagos Alveolares/efeitos dos fármacos , Microbiota/efeitos dos fármacos , Coloração e Rotulagem/métodos , Vancomicina/farmacologia , Animais , Animais Recém-Nascidos , Asma/induzido quimicamente , Asma/genética , Asma/microbiologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/patologia , Modelos Animais de Doenças , Amarelo de Eosina-(YS)/química , Feminino , Hematoxilina/química , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Pulmão/patologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Papaína/administração & dosagem , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia
15.
Phytomedicine ; 80: 153392, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33113503

RESUMO

BACKGROUND: Acacetin 7-O-ß-D-glucoside (tilianin) is a major constituent of Agastache rugosa, a traditional medicine that has long been used for the treatment of gastrointestinal disorders. Tilianin has a wide variety of pharmacological properties such as cardioprotective, neuroprotective, and anti-atherogenic activities. We recently discovered that tilianin has the ability to suppress MUC5AC expression in vitro. In addition, we have established an in vivo model of allergic asthma using house dust mite (HDM) that can be applied to tilianin. PURPOSE: We investigated the effects of tilianin on airway inflammation in a HDM-induced asthma mouse model and associated mechanisms. METHODS: Tilianin was treated in splenocytes cultured in Th0 condition and HDM-stimulated bone marrow-derived dendritic cells (BMDCs), and their mRNA expression and cytokines production were determined by quantitative real-time PCR and ELISA. To evaluate the effects of tilianin in an allergic asthma model, mice were sensitized and challenged with HDM. Tilianin was administered prior to challenge by oral gavage and airway hyper-reactivity (AHR) to methacholine, inflammatory cell infiltration, cytokine levels, and airway remodeling were assessed. RESULTS: Tilianin inhibited the production of Th2-related cytokines in splenocytes, which play pivotal roles in allergic airway inflammation. When treated in HDM-stimulated BMDCs, tilianin decreased Th2-skewing cytokine IL-33 and transcription factor IRF4. On the contrary, tilianin increased Th1-skewing regulators, IL-12 and IRF1. In an HDM-induced asthmatic mouse model, tilianin attenuated AHR and airway inflammation. Tilianin suppressed the expression of Th2-related cytokines, IL-13 and IL-33 in lung tissues. As seen in HDM-stimulated BMDCs, tilianin also downregulated the expression of the transcription factor IRF4 but not IRF1. CONCLUSION: Taken together, these results suggest that tilianin attenuates HDM-induced allergic airway inflammation by inhibiting Th2-mediated inflammation through the selective inhibition of the IRF4-IL-33 axis in dendritic cells.


Assuntos
Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Flavonoides/farmacologia , Glicosídeos/farmacologia , Fatores Reguladores de Interferon/metabolismo , Células Th2/efeitos dos fármacos , Remodelação das Vias Aéreas , Animais , Asma/imunologia , Asma/metabolismo , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Feminino , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/etiologia , Fatores Reguladores de Interferon/imunologia , Interleucina-33/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Camundongos Endogâmicos BALB C , Pyroglyphidae/patogenicidade , Células Th2/imunologia , Células Th2/metabolismo
16.
Methods Mol Biol ; 2223: 49-65, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226586

RESUMO

Egg allergy is one of the most common food allergies in children, being the most important allergenic proteins found in the egg white (EW). Allergy to EW shows a complex phenotype that involves a multifaceted reaction that can only be assessed in vivo. Although other routes of sensitization have been described, oral exposure to food antigens is one of the most suitable in humans. In mice, oral administration of allergenic proteins results in the development of tolerance, and the use of adjuvants, such as cholera toxin (CT), is required to promote Th2-biased immune responses over tolerogenic responses. In this regard, among the mouse strains that readily display Th2 responses, Balb/c has been widely used. Here, we describe a frequently used protocol of oral EW sensitization by using CT as an adjuvant and we explain in detail the methods that we have developed to analyze the sensitizing and eliciting capacity of EW proteins including evaluation of signs, measurement of serum levels of specific immunoglobulins, mast cell degranulation, cytokine secretion profile of allergen-reactive T cells, phenotyping of mesenteric lymph node- and spleen-derived dendritic and T cells by flow cytometry, and quantification of intestinal gene expression.


Assuntos
Células Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Hipersensibilidade a Ovo/imunologia , Clara de Ovo/química , Imunofenotipagem/métodos , Células Th2/efeitos dos fármacos , Adjuvantes Imunológicos/administração & dosagem , Administração Oral , Animais , Biomarcadores/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Galinhas , Toxina da Cólera/administração & dosagem , Células Dendríticas/citologia , Células Dendríticas/imunologia , Hipersensibilidade a Ovo/sangue , Hipersensibilidade a Ovo/genética , Hipersensibilidade a Ovo/patologia , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Imunoglobulinas/sangue , Imunoglobulinas/classificação , Imunoglobulinas/imunologia , Interleucinas/genética , Interleucinas/imunologia , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Células Th2/citologia , Células Th2/imunologia
17.
Methods Mol Biol ; 2223: 101-114, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226590

RESUMO

Mouse models of allergic asthma have been utilized to establish the role of T helper type 2 (Th2) cells in driving lung inflammation, airway hyperresponsiveness, and obstruction. Here, we present the allergic asthma models, in which mice are hypersensitized to ovalbumin (OVA) and house dust mite (HDM). These models mimic the major characteristics of human asthma including the eosinophilic inflammation and hyperactivity of the airway, overproduction of Th2 cytokines in the lung, and elevated total and allergen-specific immunoglobulin E (IgE) in serum.


Assuntos
Asma/imunologia , Células Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Ovalbumina/administração & dosagem , Pyroglyphidae/imunologia , Hipersensibilidade Respiratória/imunologia , Células Th2/efeitos dos fármacos , Adjuvantes Imunológicos/administração & dosagem , Alérgenos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Animais , Asma/induzido quimicamente , Asma/genética , Asma/patologia , Biomarcadores/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/patologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Eosinófilos/patologia , Citometria de Fluxo/métodos , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Expressão Gênica , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interleucinas/genética , Interleucinas/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pyroglyphidae/química , Testes de Função Respiratória , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/patologia , Células Th2/imunologia , Células Th2/patologia
18.
Cell Death Dis ; 11(12): 1062, 2020 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-33311488

RESUMO

Reversing the highly immunosuppressive tumor microenvironment (TME) is essential to achieve long-term efficacy with cancer immunotherapy. Despite the impressive clinical response to checkpoint blockade in multiple types of cancer, only a minority of patients benefit from this approach. Here, we report that the oncolytic virus M1 induces immunogenic tumor cell death and subsequently restores the ability of dendritic cells to prime antitumor T cells. Intravenous injection of M1 disrupts immune tolerance in the privileged TME, reprogramming immune-silent (cold) tumors into immune-inflamed (hot) tumors. M1 elicits potent CD8+ T cell-dependent therapeutic effects and establishes long-term antitumor immune memory in poorly immunogenic tumor models. Pretreatment with M1 sensitizes refractory tumors to subsequent checkpoint blockade by boosting T-cell recruitment and upregulating the expression of PD-L1. These findings reveal the antitumor immunological mechanism of the M1 virus and indicated that oncolytic viruses are ideal cotreatments for checkpoint blockade immunotherapy.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Vírus Oncolíticos/metabolismo , Linfócitos T/imunologia , Animais , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Morte Celular Imunogênica/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Inflamação/genética , Injeções Intravenosas , Camundongos Endogâmicos C57BL , Vírus Oncolíticos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
19.
Sci Rep ; 10(1): 21626, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318509

RESUMO

MZB1 is an endoplasmic reticulum (ER)-resident protein that plays an important role in the humoral immune response by enhancing the interaction of the µ immunoglobulin (Ig) heavy chain with the chaperone GRP94 and by augmenting the secretion of IgM. Here, we show that MZB1 is also expressed in plasmacytoid dendritic cells (pDCs). Mzb1-/- pDCs have a defect in the secretion of interferon (IFN) α upon Toll-like receptor (TLR) 9 stimulation and a reduced ability to enhance B cell differentiation towards plasma cells. Mzb1-/- pDCs do not properly expand the ER upon TLR9 stimulation, which may be accounted for by an impaired activation of ATF6, a regulator of the unfolded protein response (UPR). Pharmacological inhibition of ATF6 cleavage in stimulated wild type pDCs mimics the diminished IFNα secretion by Mzb1-/- pDCs. Thus, MZB1 enables pDCs to secrete high amounts of IFNα by mitigating ER stress via the ATF6-mediated UPR.


Assuntos
Células Dendríticas/metabolismo , Interferon-alfa/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Imunidade Humoral , Cadeias mu de Imunoglobulina/imunologia , Cadeias mu de Imunoglobulina/metabolismo , Interferon-alfa/imunologia , Camundongos , Chaperonas Moleculares/imunologia , Cultura Primária de Células , Transdução de Sinais
20.
Proc Natl Acad Sci U S A ; 117(52): 32962-32969, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33318219

RESUMO

Clinical investigations have shown that a nonimmunogenic "cold" tumor is usually accompanied by few immunopositive cells and more immunosuppressive cells in the tumor microenvironment (TME), which is still the bottleneck of immune activation. Here, a fluorine assembly nanocluster was explored to break the shackles of immunosuppression, reawaken the immune system, and turn the cold tumor "hot." Once under laser irradiation, FS@PMPt produces sufficient reactive oxygen species (ROS) to fracture the ROS-sensitive linker, thus releasing the cisplatin conjugated PMPt to penetrate into the tumors and kill the regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Meanwhile, ROS will induce potent immunogenic cell death (ICD) and further promote the accumulation of dendritic cells (DCs) and T cells, therefore not only increasing the infiltration of immunopositive cells from the outside but also reducing the immunosuppressive cells from the inside to break through the bottleneck of immune activation. The FS@PMPt nanocluster regulates the immune process in TME from negative to positive, from shallow to deep, to turn the cold tumor into a hot tumor and provoke a robust antitumor immune response.


Assuntos
Antineoplásicos/síntese química , Flúor/química , Fatores Imunológicos/síntese química , Nanoconjugados/química , Microambiente Tumoral/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Dendrímeros/química , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Fatores Imunológicos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Platina/química , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia
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