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1.
Int J Nanomedicine ; 14: 5229-5242, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371958

RESUMO

Purpose: Dexamethasone (Dex) has long been used as a potent immunosuppressive agent in the treatment of inflammatory and autoimmune diseases, despite serious side effects. In the present study, Dex and model antigen ovalbumin (OVA) were encapsulated with poly(lactic-co-glycolic acid) to deliver Dex and OVA preferentially to phagocytic cells, reducing systemic side effects of Dex. The OVA-specific immune tolerance-inducing activity of the nanoparticles (NPs) was examined. Methods: Polymeric NPs containing OVA and Dex (NP[OVA+Dex]) were prepared by the water-in-oil-in-water double emulsion solvent evaporation method. The effects of NP[OVA+Dex] on the maturation and function of immature dendritic cells (DCs) were examined in vitro. Furthermore, the OVA-specific immune tolerizing effects of NP[OVA+Dex] were confirmed in mice that were intravenously injected or orally fed with the NPs. Results: Immature DCs treated in vitro with NP[OVA+Dex] did not mature into immunogenic DCs but instead were converted into tolerogenic DCs. Furthermore, profoundly suppressed generation of OVA-specific cytotoxic T cells and production of OVA-specific IgG were observed in mice injected with NP[OVA+Dex], whereas regulatory T cells were concomitantly increased. Feeding of mice with NP[OVA+Dex] also induced OVA-specific immune tolerance. Conclusion: The present study demonstrates that oral feeding as well as intravenous injection of poly(lactic-co-glycolic acid) NPs encapsulating both antigen and Dex is a useful means of inducing antigen-specific immune tolerance, which is crucial for the treatment of autoimmune diseases.


Assuntos
Antígenos/imunologia , Materiais Biocompatíveis/química , Dexametasona/farmacologia , Epitopos/imunologia , Tolerância Imunológica , Nanopartículas/química , Administração Oral , Animais , Apresentação do Antígeno/efeitos dos fármacos , Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Imunidade/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Ovalbumina/imunologia , Fagocitose/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo
2.
Nat Immunol ; 20(9): 1110-1128, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406380

RESUMO

In recent years, a population of unconventional T cells called 'mucosal-associated invariant T cells' (MAIT cells) has captured the attention of immunologists and clinicians due to their abundance in humans, their involvement in a broad range of infectious and non-infectious diseases and their unusual specificity for microbial riboflavin-derivative antigens presented by the major histocompatibility complex (MHC) class I-like protein MR1. MAIT cells use a limited T cell antigen receptor (TCR) repertoire with public antigen specificities that are conserved across species. They can be activated by TCR-dependent and TCR-independent mechanisms and exhibit rapid, innate-like effector responses. Here we review evidence showing that MAIT cells are a key component of the immune system and discuss their basic biology, development, role in disease and immunotherapeutic potential.


Assuntos
Apresentação do Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Células T Invariáveis Associadas à Mucosa/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Antígenos/imunologia , Suscetibilidade a Doenças/imunologia , Humanos , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/imunologia
3.
Int J Nanomedicine ; 14: 4867-4880, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308663

RESUMO

Background: The demand for an effective vaccine delivery system that drives a suitable immune response is increasing. The oxidized carbon nanosphere (OCN), a negatively charged carbon nanoparticle, has the potential to fulfill this requirement because it can efficiently deliver macromolecules into cells and allows endosomal leakage. However, fundamental insights into how OCNs are taken up by antigen-presenting cells, and the intracellular behavior of delivered molecules is lacking. Furthermore, how immune responses are stimulated by OCN-mediated delivery has not been investigated. Purpose: In this study, the model protein antigen ovalbumin (OVA) was used to investigate the uptake mechanism and intracellular fate of OCN-mediated delivery of protein in macrophages. Moreover, the immune response triggered by OVA delivered by OCNs was characterized. Methods: Bone-marrow-derived macrophages (BMDMs) from mice were used to study antigen uptake and intracellular trafficking. Mice were immunized using OCN-OVA combined with known adjuvants, and the specific immune response was measured. Results: OCNs showed no cytotoxicity against BMDMs. OCN-mediated delivery of OVA into BMDMs was partially temperature independent process. Using specific inhibitors, it was revealed that intracellular delivery of OCN-OVA does not rely on phagocytosis or the clathrin- and lipid raft/caveolae-mediated pathways. Delivered OVA was found to colocalize with compartments containing MHC class I, but not with early endosomes, lysosomes, and autophagosomes. Immunization of OVA using OCNs in combination with the known adjuvant monophosphoryl lipid A specifically enhanced interferon gamma (IFNγ)- and granzyme B-producing cytotoxic T cells (CTLs). Conclusion: OCNs effectively delivered protein antigens into macrophages that localized with compartments containing MHC class I partially by the temperature independent, but not clathrin- and lipid raft/caveolae-mediated pathways. Increased CD8+ T-cell activity was induced by OCN-delivered antigens, suggesting antigen processing toward antigen presentation for CTLs. Taken together, OCNs are a potential protein antigen delivery system that stimulates the cell-mediated immune response.


Assuntos
Antígenos/administração & dosagem , Carbono/química , Sistemas de Liberação de Medicamentos , Imunidade Celular , Nanopartículas/química , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos/imunologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Linhagem Celular , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Feminino , Imunidade Celular/efeitos dos fármacos , Cinética , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Nanopartículas/toxicidade , Oxirredução , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
4.
Nat Immunol ; 20(8): 963-969, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285625

RESUMO

Over the past several decades, B cell antigen receptor (BCR)-induced signaling pathways have been described in extraordinary molecular detail, mainly from studies of B cell responses to antigens in vitro. BCR signaling has been shown to govern the initiation of transcriptional programs associated with B cell activation and fate decisions, as well as the BCR-dependent processing of antigen and presentation of antigen to T cells. However, although the potential of the BCR to orchestrate B cell behavior was known, there was no clear appreciation of the context in which B cells signal in secondary lymphoid organs in vivo or how that context influences signaling. In this Review, we describe the current view of the cellular consequences of BCR signaling and advances in the understanding of B cell signaling in context in vivo.


Assuntos
Apresentação do Antígeno/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Humanos , Ativação Linfocitária/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia
5.
Cancer Immunol Immunother ; 68(11): 1855-1863, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31309255

RESUMO

Monitoring T cells is of major importance for the development of immunotherapies. Recent sophisticated assays can address particular aspects of the anti-tumor T-cell repertoire or support very large-scale immune screening for biomarker discovery. Robust methods for the routine assessment of the quantity and quality of antigen-specific T cells remain, however, essential. This review discusses selected methods that are commonly used for T-cell monitoring and summarizes the advantages and limitations of these assays. We also present a new functional assay, which specifically detects activated ß2 integrins within a very short time following CD8+ T-cell stimulation. Because of its unique and favorable characteristics, this assay could be useful for implementation into our T-cell monitoring toolbox.


Assuntos
Adesão Celular/imunologia , Integrina alfa2/imunologia , Linfócitos T/imunologia , Antígenos/imunologia , Biomarcadores/metabolismo , Humanos , Imunoterapia/métodos
7.
Scand J Immunol ; 90(3): e12795, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31148206

RESUMO

Antigen-specific molecules of the immune system, namely antibodies, the membrane immunoglobulins (mIgs) of B cells and T cell receptors (TcRs), can all signal their interaction with antigen. There are different mechanisms by which this signalling could occur. These mechanisms can be divided into two general categories: allosteric and non-allosteric. In allosteric mechanisms, the monovalent binding of the antigen to the receptor triggers a conformational change at the binding site that is propagated to an invariant part of the receptor, a change recognized by a sensing unit. We argue allosteric mechanisms are implausible. Non-allosteric mechanisms depend on steric effects due to the antigen's size and/or multivalency. We consider two non-allosteric mechanisms by which the mIg of B cells has been envisaged to signal its interaction with antigen: the popular cross-linking model and the dissociation activation model. We argue, on the basis of both experimental observations and physiological considerations, that the dissociation activation model, developed by Reth and his colleagues, is uniquely plausible.


Assuntos
Anticorpos/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Sistema Imunitário/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Humanos , Receptores de Antígenos de Linfócitos T/imunologia
8.
Anal Bioanal Chem ; 411(17): 3881-3890, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31152222

RESUMO

Aflatoxin B1 (AFB1) is one of the major mycotoxins, which naturally occurs in food and agricultural products. In this study, a cyclic peptide (CVPSKPGLC) mimicking AFB1 was used to develop a biotinylated peptide-based immunoassay (bp-ELISA) for AFB1 determination. This cyclic peptide was isolated from a commercially available phage-displayed random 7-mer cyclic peptide library, and then synthesized chemically. Instead of phage particles, the peptide was biotinylated and used to detect AFB1 by bp-ELISA, with an IC50 of 0.92 ng/mL, which was approximately 60-fold better than that of phage ELISA. Good recoveries (83-102%) were obtained in spiked rice and corn samples, which were further validated by high-performance liquid chromatography-fluorescence detector. As better sensitivities (0.92-1.21 ng/mL) were obtained by bp-ELISA even using selected anti-AFB1 antibodies prepared previously in laboratory, this cyclic peptide is suitable as a substitute for synthetic competitive AFB1 antigens in food contamination monitoring. Graphical abstract.


Assuntos
Aflatoxina B1/imunologia , Antígenos/química , Antígenos/imunologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Biotina/química , Técnicas de Visualização da Superfície Celular , Cromatografia Líquida de Alta Pressão/métodos , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Contaminação de Alimentos/análise , Concentração Inibidora 50 , Limite de Detecção , Espectrometria de Fluorescência/métodos , Estreptavidina/química
9.
Nat Commun ; 10(1): 2423, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160559

RESUMO

The germinal center (GC) reaction in Peyer's patches (PP) requires continuous access to antigens, but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7- B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells, we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs, 40% of antigen-specific SED B cells bind antigen within 2 h, whereas unspecifc cells do not, indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice, but is unperturbed in mice depleted of classical dendritic cells (DC). Thus, we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses, and should be taken into account when developing mucosal vaccines.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Células Epiteliais/imunologia , Nódulos Linfáticos Agregados/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Células Dendríticas/imunologia , Centro Germinativo/imunologia , Imunoglobulina A/imunologia , Ativação Linfocitária , Camundongos
10.
Mol Immunol ; 111: 128-135, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31054406

RESUMO

The main challenge in the development of antibody is to select the appropriate antigen particularly when a truncated protein is used for immunization or as vaccine antigen. In previous studies, fragment selection was mainly based on epitopes and less often on the structure. Fewer studies have paid attention to the prediction of the truncated protein 3D structure and retained its similarity in the native and truncated proteins. Here we used in silico analysis to select two fragments of Pyruvate Kinase M2 (PKM2), as a tumor marker. One fragment, M-tPKM2, had a shorter sequence with one epitope although the predicted 3D structure was similar to the native PKM2. The other fragment, R-tPKM2, had a longer sequence and thus more epitopes, but had a different structure from the native PKM2. Recombinant truncated proteins were expressed in E. coli and purified via affinity chromatography. Secondary structure elements in purified proteins were determined by Circular Dichroism, then they were utilized to develop antibodies in mice. Both antigens could elicit high immune response against themselves (OD450 = 3.326 ± 0.562 for M-tPKM2; OD450 = 3.562 ± 0.110 for R-tPKM2). However, significantly higher response against PKM2 was observed among the mice immunized with M-tPKM2 (p < 0.0001 by One way ANOVA followed by Tukey's post hoc comparison). Also, the monoclonal antibody produced against the M-tPKM2 could recognize the native PKM2 in the MCF7 cells. Our finding suggested that for the purpose of designing an antigen with the ability to produce a potent antibody against the target protein, it is better to select sequences which have a similar structure in truncated and native proteins, even at the cost of having shorter sequences and fewer epitopes.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Epitopos/imunologia , Animais , Linhagem Celular Tumoral , Mapeamento de Epitopos/métodos , Escherichia coli/imunologia , Feminino , Humanos , Imunização/métodos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Piruvato Quinase/imunologia , Proteínas Recombinantes/imunologia
11.
Immunity ; 50(5): 1188-1201.e6, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31053504

RESUMO

Lymph nodes (LNs) play critical roles in adaptive immunity by concentrating in one location the antigens, antigen-presenting cells, and antigen-responsive lymphocytes involved in such responses. Recent studies have revealed nonrandom localization of innate and adaptive immune cells within these organs, suggesting that microanatomical positioning optimizes responses involving sparse cooperating cells. Here, we report that the peripheral localization of LN cDC2 dendritic cells specialized for MHC-II antigen presentation is matched by a similarly biased paracortical distribution of CD4+ T cells directed by the chemoattractant receptor Ebi2. In the absence of Ebi2, CD4+ T cells lose their location bias and are delayed in antigen recognition, proliferative expansion, differentiation, direct effector activity, and provision of help for CD8+ T cell-mediated memory responses, limiting host defense and vaccine responses. These findings demonstrate evolutionary selection for distinct niches within the LN that promote cellular responses, emphasizing the critical link between fine-grained tissue organization and host defense.


Assuntos
Imunidade Adaptativa/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Apresentação do Antígeno/imunologia , Antígenos/imunologia , Diferenciação Celular/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas-G/genética
12.
Parasitol Res ; 118(6): 1701-1710, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31065831

RESUMO

Coccidiosis is a major poultry disease which compromises animal welfare and costs the global chicken industry a huge economic loss. As a result, research entailing coccidial control measures is crucial. Coccidiosis is caused by Eimeria parasites that are highly immunogenic. Consequently, a low dosage of the Eimeria parasite supplied by a vaccine will enable the host organism to develop an innate immune response towards the pathogen. The production of traditional live anticoccidial vaccines is limited by their low reproductive index and high production costs, among other factors. Recombinant vaccines overcome these limitations by eliciting undesired contaminants and prevent the reversal of toxoids back to their original toxigenic form. Recombinant vaccines are produced using defined Eimeria antigens and harmless adjuvants. Thus, studies regarding the identification of potent novel Eimeria antigens which stimulate both cell-mediated and humoral immune responses in chickens are essential. Although the prevalence and risk posed by Eimeria have been well established, there is a dearth of information on genetic and antigenic diversity within the field. Therefore, this paper discusses the potential and efficiency of recombinant vaccines as an anticoccidial control measure. Novel protective Eimeria antigens and their antigenic diversity for the production of cheap, easily accessible recombinant vaccines are also reviewed.


Assuntos
Coccidiose/veterinária , Eimeria/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas Protozoárias/imunologia , Adjuvantes Imunológicos , Animais , Antígenos/administração & dosagem , Antígenos/genética , Antígenos/imunologia , Galinhas/parasitologia , Coccidiose/imunologia , Coccidiose/parasitologia , Coccidiose/prevenção & controle , Eimeria/genética , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/parasitologia , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/genética
13.
Eur J Pharm Biopharm ; 140: 29-39, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31055066

RESUMO

Using subunit vaccines, e.g., based on peptide or protein antigens, to teach the immune system to kill abnormal host cells via induction of cytotoxic T lymphocytes (CTL) is a promising strategy against intracellular infections and cancer. However, customized adjuvants are required to potentiate antigen-specific cellular immunity. One strong CTL-inducing adjuvant is the liposomal cationic adjuvant formulation (CAF)09, which is composed of dimethyldioctadecylammonium (DDA) bromide, monomycoloyl glycerol (MMG) analogue 1 and polyinosinic:polycytidylic acid [poly(I:C)]. However, this strong CTL induction requires intraperitoneal administration because the vaccine forms a depot at the site of injection (SOI) after subcutaneous (s.c.) or intramuscular (i.m.) injection, and depot formation impedes the crucial vaccine targeting to the cross-presenting dendritic cells (DCs) residing in the lymph nodes (LNs). The purpose of the present study was to investigate the effect of polyethylene glycol (PEG) grafting of CAF09 on the ability of the vaccine to induce antigen-specific CTL responses after s.c. administration. We hypothesized that steric stabilization and charge shielding of CAF09 by PEGylation may reduce depot formation at the SOI and enhance passive drainage to the LNs, eventually improving CTL induction. Hence, the vaccine (antigen/CAF09) was post-grafted with a novel type of anionic PEGylated peptides based on GDGDY repeats, which were end-conjugated with one or two PEG1000 moieties, resulting in mono- and bis-PEG-peptides of different lengths (10, 15 and 20 amino acid residues). For comparison, CAF09 was also grafted by inclusion of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(PEG)-2000 (DSPE-PEG2000) in the bilayer structure during preparation. Grafting of CAF09 with either type of PEG resulted in charge shielding, evident from a reduced surface charge. Upon s.c. immunization of mice with the model antigen ovalbumin (OVA) adjuvanted with PEGylated CAF09, stronger CTL responses were induced as compared to immunization of mice with unadjuvanted OVA. Biodistribution studies confirmed that grafting of CAF09 with DSPE-PEG2000 improved the passive drainage of the vaccine to LNs, because a higher dose fraction was recovered in DCs present in the draining LNs, as compared to the dose fraction detected for non-PEGylated CAF09. In conclusion, PEGylation of CAF09 may be a useful strategy for the design of an adjuvant, which induces CTL responses after s.c. and i.m. administration. In the present studies, CAF09 grafted with 10 mol% DSPE-PEG2000 is the most promising of the tested adjuvants, but additional studies are required to further elucidate the potential of the strategy.


Assuntos
Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Lipossomos/química , Polietilenoglicóis/química , Linfócitos T Citotóxicos/imunologia , Vacinas de Subunidades/imunologia , Animais , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Feminino , Imunidade Celular/imunologia , Imunização/métodos , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Fosfatidiletanolaminas/química , Compostos de Amônio Quaternário/química , Distribuição Tecidual
14.
Nat Immunol ; 20(6): 756-764, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110315

RESUMO

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Memória Imunológica , Pele/imunologia , Animais , Antígenos/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Epiderme/imunologia , Epiderme/metabolismo , Imunofluorescência , Humanos , Camundongos , Especificidade de Órgãos/imunologia , Anticorpos de Domínio Único/imunologia , Pele/metabolismo , Vacinas de DNA/genética , Vacinas de DNA/imunologia
15.
Nature ; 568(7752): 405-409, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30944470

RESUMO

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract1-4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (Treg) cells4-8, and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain Treg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce Treg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1ß. Macrophages in the small intestine produce IL-1ß, and activation of this pathway involves MYD88- and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining Treg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn's disease, and this correlated with lower frequencies of Treg cells. Our results reveal a previously unappreciated pathway in which a microbiota- and IL-1ß-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine.


Assuntos
Imunidade Inata/imunologia , Interleucina-2/imunologia , Intestinos/citologia , Intestinos/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos/administração & dosagem , Antígenos/imunologia , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Feminino , Microbioma Gastrointestinal/imunologia , Homeostase/imunologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Interleucina-2/deficiência , Interleucina-2/metabolismo , Intestino Delgado/citologia , Intestino Delgado/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Linfócitos T Reguladores/classificação , Linfócitos T Reguladores/metabolismo
16.
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
17.
Nutrients ; 11(4)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018604

RESUMO

In patients with non-IgE-mediated milk allergy, a cellular mechanism of delayed-type hypersensitivity (DTH) is considered. Recent findings prove that cell-mediated reactions can be antigen-specifically inhibited by extracellular vesicles (EVs) carrying miRNA-150. We sought to establish a new mouse model of DTH to casein and test the possibility of antigen-specific suppression of the inflammatory reaction. To produce soluble antigenic peptides, casein was subjected to alkaline hydrolysis. DTH reaction to casein was induced in CBA, C57BL/6, and BALB/c mice by intradermal (id) injection of the antigen. Cells collected from spleens and lymph nodes were positively or negatively selected and transferred to naive recipients intravenously (iv). CBA mice were tolerized by iv injection of mouse erythrocytes conjugated with casein antigen and following id immunization with the same antigen. Suppressive EVs were harvested from cell cultures and serum of tolerized donors by means of ultrafiltration and ultracentrifugation for further therapeutic utilization. The newly established mouse model of DTH to casein was mediated by CD4+ Th1 cells and macrophages, while EVs produced by casein-tolerized animals effectively suppressed effector cell response, in an miRNA-150-dependent manner. Altogether, our observations contribute to the current understanding of non-IgE-mediated allergy to casein and of the possibilities to downregulate this reaction.


Assuntos
Caseínas/imunologia , Vesículas Extracelulares/química , Hipersensibilidade Tardia , MicroRNAs/metabolismo , Transferência Adotiva , Animais , Antígenos/imunologia , Subpopulações de Linfócitos B/fisiologia , Linfócitos T CD4-Positivos , Regulação da Expressão Gênica/imunologia , Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA
18.
Mater Sci Eng C Mater Biol Appl ; 100: 209-214, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948054

RESUMO

The development of antibody immobilization techniques is essential for creating antibody-based biomaterials. Although numerous methods for antibody immobilization have been demonstrated, low stability and disordered orientation of the immobilized antibody remain an important problem. In this work, an original antibody immobilization technique using a protein film, which achieved a high stability and orientation control of the immobilized antibody, has been described. In this method, an antibody-immobilized albumin film was prepared by adding the cross-linked albumin solution to the substrate, where antibodies were attached in uniform orientation, followed by subsequent drying, and detaching the formed film from the substrate by heating at 120 °C in a dry state. Antibodies in the film showed high antigen-binding capacity, at a level comparable to the oriented immobilized antibody using protein G. The stability of antibodies in the film was found to be significantly high; their antigen-binding capacity was completely retained even after storage at 40 °C in a dry state for one month. Thus, this approach provides useful information to immobilize the antibody on solid surfaces while controlling its orientation and increasing its stability.


Assuntos
Anticorpos Imobilizados/química , Soroalbumina Bovina/química , Animais , Anticorpos Imobilizados/imunologia , Antígenos/imunologia , Proteínas de Bactérias/química , Bovinos , Estabilidade Proteica , Compostos de Sulfidrila/química , Propriedades de Superfície , Temperatura Ambiente
19.
Biomed Res Int ; 2019: 6481654, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30931329

RESUMO

Screening of fetomaternal hemorrhage (FMH) is essential in management of fetomaternal antigen incompatibilities of blood. The objective in this study was to evaluate the ability of automatic blood analyzer (ABA) to screen FMH, also comparing this method with flow cytometry (FCM). The contents of fetal red blood cells and fetal hemoglobin were evaluated by FCM and ABA, respectively, using both blood samples of male adults laced with umbilical cord blood diluted at 1/10, 1/100, 1/1,000, and 1/10,000, or blood from puerperal women collected within 48 hours following delivery. FCM had better performance (area under curve, AUC = 0.8723) than ABA (AUC = 0.6569) in detecting fetal blood laced with blood from male adults. At a critical level of 0.5%, ABA indicated that 27.5% of puerperal women would have FMH while FCM did not detect FMH. Our results showed that ABA overestimates FMH and disagrees with FCM on indicating puerperal women with FMH. ABA is inadequate for being used to screen for or to measure FMH.


Assuntos
Antígenos/sangue , Incompatibilidade de Grupos Sanguíneos/sangue , Transfusão Feto-Materna/sangue , Testes Hematológicos/métodos , Adolescente , Adulto , Antígenos/imunologia , Incompatibilidade de Grupos Sanguíneos/patologia , Feminino , Sangue Fetal/imunologia , Hemoglobina Fetal/imunologia , Transfusão Feto-Materna/imunologia , Transfusão Feto-Materna/patologia , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Período Pós-Parto , Gravidez , Sistema do Grupo Sanguíneo Rh-Hr , Adulto Jovem
20.
Nat Commun ; 10(1): 1198, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867416

RESUMO

Microbe-host interactions are generally homeostatic, but when dysfunctional, they can incite food sensitivities and chronic diseases. Celiac disease (CeD) is a food sensitivity characterized by a breakdown of oral tolerance to gluten proteins in genetically predisposed individuals, although the underlying mechanisms are incompletely understood. Here we show that duodenal biopsies from patients with active CeD have increased proteolytic activity against gluten substrates that correlates with increased Proteobacteria abundance, including Pseudomonas. Using Pseudomonas aeruginosa producing elastase as a model, we show gluten-independent, PAR-2 mediated upregulation of inflammatory pathways in C57BL/6 mice without villus blunting. In mice expressing CeD risk genes, P. aeruginosa elastase synergizes with gluten to induce more severe inflammation that is associated with moderate villus blunting. These results demonstrate that proteases expressed by opportunistic pathogens impact host immune responses that are relevant to the development of food sensitivities, independently of the trigger antigen.


Assuntos
Proteínas de Bactérias/metabolismo , Doença Celíaca/imunologia , Proteínas na Dieta/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Metaloendopeptidases/metabolismo , Receptor PAR-2/imunologia , Adulto , Idoso , Animais , Antígenos/imunologia , Antígenos/metabolismo , Proteínas de Bactérias/genética , Biópsia , Estudos de Casos e Controles , Doença Celíaca/diagnóstico por imagem , Doença Celíaca/microbiologia , Doença Celíaca/patologia , Estudos de Coortes , Colonoscopia , Proteínas na Dieta/metabolismo , Modelos Animais de Doenças , Duodeno/imunologia , Duodeno/metabolismo , Duodeno/microbiologia , Duodeno/patologia , Feminino , Microbioma Gastrointestinal/imunologia , Vida Livre de Germes , Glutens/imunologia , Glutens/metabolismo , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Antígenos HLA-DQ/metabolismo , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteólise , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismo , Receptor PAR-2/metabolismo , Regulação para Cima , Adulto Jovem
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