Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
1.
Proc Natl Acad Sci U S A ; 116(6): 1958-1967, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30670663

RESUMO

Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein-glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.


Assuntos
Proteínas de Transporte/química , Análise em Microsséries/métodos , Polissacarídeos/química , Polissacarídeos/imunologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Células CHO , Cricetulus , Glicômica , Humanos , Sistema Imunitário , Lectinas , Oligossacarídeos , Polissacarídeos/classificação , Ligação Proteica , Proteínas Recombinantes , Especificidade da Espécie
2.
EMBO J ; 34(7): 881-95, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25666591

RESUMO

Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3(-/-) mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD.


Assuntos
Antígenos CD/imunologia , Proteínas de Bactérias/imunologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Lactobacillus acidophilus/imunologia , Lectinas Tipo C/imunologia , Animais , Antígenos CD/genética , Proteínas de Bactérias/genética , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Lactobacillus acidophilus/genética , Lectinas Tipo C/genética , Lipopolissacarídeos/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Ligação Proteica/genética , Ligação Proteica/imunologia , Ácidos Teicoicos/genética , Ácidos Teicoicos/imunologia
3.
Toxicology ; 483: 153374, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36396002

RESUMO

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells forming the hepatic sinusoidal wall. Besides their high endocytic potential, LSECs have been demonstrated to markedly contribute to liver homeostasis and immunity, and may partially explain unexpected hepatotoxicity of drug candidates. However, their use for in vitro investigations is compromised by poor cell yields and a limited proliferation capacity. Here, we report the transient expansion of primary human LSECs from three donors by lentiviral transduction. Transduced ("upcyte®") LSECs were able to undergo at least 25 additional population doublings (PDs) before growth arrest due to senescence. Expanded upcyte® LSECs maintained several characteristics of primary LSECs, including expression of surface markers such as MMR and LYVE-1 as well as rapid uptake of acetylated LDL and ovalbumin. We further investigated the suitability of expanded upcyte® LSECs and proliferating upcyte® hepatocytes for detecting acetaminophen toxicity at millimolar concentrations (0, 0.5, 1, 2, 5, 10 mM) in static 2D cultures and a microphysiological 3D model. upcyte® LSECs exhibited a higher sensitivity to acetaminophen-induced toxicity compared to upcyte® hepatocytes in 2D culture, however, culturing upcyte® LSECs together with upcyte® hepatocytes in a co-culture reduced APAP-induced toxicity compared to 2D monocultures. A perfused Dynamic42 3D model was more sensitive to acetaminophen than the 2D co-culture model. Cytotoxicity in the 3D model was evident by decreased cellular viability, elevated LDH release, reduced nuclei counts and impaired cell morphology. Taken together, our data demonstrate that transient expansion of LSECs represents a suitable method for generation of large quantities of cells while maintaining many characteristics of primary cells and responsiveness to acetaminophen.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Células Endoteliais , Humanos , Células Endoteliais/metabolismo , Acetaminofen/toxicidade , Fígado/metabolismo , Hepatócitos/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo
4.
Cell Rep ; 28(1): 30-38.e5, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269448

RESUMO

Malaria represents a major cause of death from infectious disease. Hemozoin is a Plasmodium-derived product that contributes to progression of cerebral malaria. However, there is a gap of knowledge regarding how hemozoin is recognized by innate immunity. Myeloid C-type lectin receptors (CLRs) encompass a family of carbohydrate-binding receptors that act as pattern recognition receptors in innate immunity. In the present study, we identify the CLR CLEC12A as a receptor for hemozoin. Dendritic cell-T cell co-culture assays indicate that the CLEC12A/hemozoin interaction enhances CD8+ T cell cross-priming. Using the Plasmodium berghei Antwerpen-Kasapa (ANKA) mouse model of experimental cerebral malaria (ECM), we find that CLEC12A deficiency protects mice from ECM, illustrated by reduced ECM incidence and ameliorated clinical symptoms. In conclusion, we identify CLEC12A as an innate sensor of plasmodial hemozoin.


Assuntos
Hemeproteínas/imunologia , Lectinas Tipo C/imunologia , Malária Cerebral/imunologia , Plasmodium berghei/patogenicidade , Receptores Mitogênicos/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada , Células Dendríticas/imunologia , Modelos Animais de Doenças , Granzimas/metabolismo , Imunidade Inata , Lectinas Tipo C/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores Mitogênicos/genética , Linfócitos T
5.
Trends Biotechnol ; 35(4): 334-346, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28277249

RESUMO

Glycosylation is an integral post-translational modification present in more than half of all eukaryotic proteins. It affects key protein functions, including folding, stability, and immunogenicity. Glycoengineering approaches, such as the use of bacterial N-glycosylation systems, or expression systems, including yeasts, insect cells, and mammalian cells, have enabled access to defined and homogenous glycoproteins. Given that glycan structures on proteins can be recognized by host lectin receptors, they may facilitate cell-specific targeting and immune modulation. Myeloid C-type lectin receptors (CLRs) expressed by antigen-presenting cells are attractive targets to shape immune responses. Multivalent glycan display on nanoparticles, liposomes, or dendrimers has successfully enabled CLR targeting. In this review, we discuss novel strategies to access defined glycan structures and highlight CLR targeting approaches for immune modulation.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Glicoproteínas , Lectinas Tipo C , Polissacarídeos , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Glicoproteínas/química , Glicoproteínas/imunologia , Glicoproteínas/fisiologia , Glicosilação , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Camundongos , Polissacarídeos/química , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Engenharia de Proteínas
6.
ACS Chem Biol ; 11(8): 2347-56, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27314276

RESUMO

Targeting antigens to dendritic cell subsets is a promising strategy to enhance the efficacy of vaccines. C-type lectin receptors (CLRs) expressed by dendritic cells are particularly attractive candidates since CLR engagement may promote cell uptake and may further stimulate antigen presentation and subsequent T cell activation. While most previous approaches have involved antibody-mediated CLR-targeting, glycan-based CLR targeting has become more and more attractive in recent years. In the present study, we show that small structural glycan modifications may markedly influence CLR recognition, dendritic cell targeting, and subsequent T cell activation. A biantennary N-glycan (G0) and its analogous O-2 core xylosylated N-glycan (XG0) were synthesized, covalently conjugated to the model antigen ovalbumin, and analyzed for binding to a set of murine CLR-Fc fusion proteins using lectin microarray. To evaluate whether the differential binding of G0 and XG0 to CLRs impacted dendritic cell targeting, uptake studies using murine dendritic cells were performed. Finally, effects of the ovalbumin glycoconjugates on T cell activation were measured in a dendritic cell/T cell cocultivation assay. Our results highlight the utility of glycan-based dendritic cell targeting and demonstrate that small structural differences may have a major impact on dendritic cell targeting efficacy.


Assuntos
Células Dendríticas/imunologia , Glicoproteínas/metabolismo , Lectinas Tipo C/metabolismo , Receptores Mitogênicos/metabolismo , Xilose/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/metabolismo , Ligação Proteica
7.
Methods Mol Biol ; 1331: 173-87, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26169741

RESUMO

Modern vaccines such as recombinant proteins or nucleic acids are usually of pure origin, enhancing their tolerability and overall safety. However, this purity often renders them less immunogenic, creating the need for potent adjuvants. Carbohydrates are promising candidates to fulfill this role as they enable direct targeting of dendritic cells and modulation of adaptive immunity. C-type lectin receptors (CLRs) comprise a major group of carbohydrate binding receptors. As they are predominantly expressed by cells of innate immunity, CLR targeting can enhance or dampen early stages of cytokine secretion and antigen presentation, thus modulate the activation and differentiation of T cells. Here, we provide a protocol for the identification of novel CLR ligands by glycan array using recombinant CLR-Fc chimeras followed by the covalent conjugation of carbohydrate CLR ligands to the model antigen ovalbumin (OVA). The resulting glycoconjugates are subsequently used to evaluate T cell activation in vitro and immunomodulation in vivo.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos/imunologia , Carboidratos/imunologia , Imunidade Adaptativa/imunologia , Animais , Formação de Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Células CHO , Linhagem Celular , Cricetulus , Humanos , Imunidade Inata/imunologia , Imunomodulação/imunologia , Lectinas Tipo C/imunologia , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Polissacarídeos/imunologia , Linfócitos T/imunologia , Vacinas/imunologia
8.
Cell Host Microbe ; 18(4): 471-7, 2015 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-26468750

RESUMO

The antimicrobial peptide LL-37 is generated upon proteolytic cleavage of cathelicidin and limits invading pathogens by directly targeting microbial membranes as well as stimulating innate immune cell function. However, some microbes evade LL-37-mediated defense. Notably, group A Streptococcus (GAS) strains belonging to the hypervirulent M1T1 serogroup are more resistant to human LL-37 than other GAS serogroups. We show that the GAS surface-associated M1 protein sequesters and neutralizes LL-37 antimicrobial activity through its N-terminal domain. M1 protein also binds the cathelicidin precursor hCAP-18, preventing its proteolytic maturation into antimicrobial forms. Exogenous M1 protein rescues M1-deficient GAS from killing by neutrophils and within neutrophil extracellular traps and neutralizes LL-37 chemotactic properties. M1 also binds murine cathelicidin, and its virulence contribution in a murine model of necrotizing skin infection is largely driven by its ability to neutralize this host defense peptide. Thus, cathelicidin resistance is essential for the pathogenesis of hyperinvasive M1T1 GAS.


Assuntos
Antígenos de Bactérias/metabolismo , Peptídeos Catiônicos Antimicrobianos/antagonistas & inibidores , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Evasão da Resposta Imune , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Ligação Proteica , Dermatopatias Bacterianas/microbiologia , Dermatopatias Bacterianas/patologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Catelicidinas
9.
J Control Release ; 175: 36-42, 2014 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-24368301

RESUMO

Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties.


Assuntos
Carboidratos/imunologia , Fatores Imunológicos/imunologia , Lectinas Tipo C/imunologia , Análise em Microsséries/métodos , Animais , Carboidratos/administração & dosagem , Carboidratos/química , Células Cultivadas , Técnicas de Cocultura , Citocinas/imunologia , Células Dendríticas/imunologia , Feminino , Humanos , Imunização , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/química , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/administração & dosagem , Ovalbumina/química , Ovalbumina/imunologia , Ligação Proteica , Proteínas Recombinantes de Fusão/imunologia
10.
PLoS One ; 9(7): e103281, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25068517

RESUMO

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract. Though its exact etiology is still unclear, it is proposed that an imbalance in the intestinal homeostasis leads to a disturbed interaction between commensal microbiota and the mucosal immune system. Previous studies have shown that both innate and adaptive immunity are involved in an overwhelming colon inflammation, and thus contribute to the pathogenesis of IBD. In innate immunity, several pattern recognition receptors such as Toll-like receptors, NOD-like receptors or C-type lectin receptors (CLRs) are involved in IBD pathogenesis. Myeloid CLRs are mainly expressed by antigen-presenting cells and bind to glycan structures present on self or foreign antigens. The Macrophage-restricted C-type lectin (MCL) and the Dendritic cell immunoreceptor (DCIR) are two poorly characterized members of the CLR family. In this study, we investigated the role of MCL and DCIR in the pathogenesis of murine colitis. Both CLRs bound to intestinal microbiota to a different extent. They modulated the production of pro-inflammatory cytokines by antigen-presenting cells upon stimulation with heat-killed microbiota and impacted subsequent T cell responses. To analyze whether MCL and DCIR contribute to the pathogenesis of IBD, the dextran sulfate sodium (DSS) murine colitis model was employed. MCL-/- as well as DCIR-/- mice exhibited only a slightly increased severity of disease compared to wild-type mice indicating a limited role for MCL and DCIR in the regulation of intestinal immunity.


Assuntos
Colite/metabolismo , Lectinas Tipo C/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Colite/induzido quimicamente , Colite/genética , Colite/imunologia , Colite/microbiologia , Colite/patologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Expressão Gênica , Lectinas Tipo C/genética , Ativação Linfocitária , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Microbiota , Ligação Proteica , Linfócitos T/imunologia , Linfócitos T/metabolismo
11.
Front Immunol ; 4: 196, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23882266

RESUMO

Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern-recognition receptors (PRRs) sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs) are PRRs recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR specific intracellular adhesion molecule-3 grabbing non-integrin homolog-related 3 (SIGNR3) in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homolog of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze whether this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium-induced colitis model was employed. SIGNR3(-/-) mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3(-/-) mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA