RESUMO
Sialic acids are negatively charged carbohydrates that cap the glycans of glycoproteins and glycolipids. Sialic acids are involved in various biological processes including cell-cell adhesion and immune recognition. In dendritic cells (DCs), the major antigen-presenting cells of the immune system, sialic acids emerge as important regulators of maturation and interaction with other lymphocytes including T cells. Many aspects of how sialic acids regulate DC functions are not well understood and tools and model systems to address these are limited. Here, we have established cultures of murine bone marrow-derived DCs (BMDCs) that lack sialic acid expression using a sialic acid-blocking mimetic Ac53FaxNeu5Ac. Ac53FaxNeu5Ac treatment potentiated BMDC activation via toll-like receptor (TLR) stimulation without affecting differentiation and viability. Sialic acid blockade further increased the capacity of BMDCs to induce antigen-specific CD8+ T cell proliferation. Transcriptome-wide gene expression analysis revealed that sialic acid mimetic treatment of BMDCs induces differential expression of genes involved in T cell activation, cell-adhesion, and cell-cell interactions. Subsequent cell clustering assays and single cell avidity measurements demonstrated that BMDCs with reduced sialylation form higher avidity interactions with CD8+ T cells. This increased avidity was detectable in the absence of antigens, but was especially pronounced in antigen-dependent interactions. Together, our data show that sialic acid blockade in BMDCs ameliorates maturation and enhances both cognate T cell receptor-MHC-dependent and independent T cell interactions that allow for more robust CD8+ T cell responses.
Assuntos
Células da Medula Óssea/imunologia , Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/imunologia , Células Dendríticas/imunologia , Ácido N-Acetilneuramínico/imunologia , Animais , Células da Medula Óssea/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Adesão Celular/genética , Adesão Celular/imunologia , Comunicação Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Células Dendríticas/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Ácido N-Acetilneuramínico/antagonistas & inibidores , Ácido N-Acetilneuramínico/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismoRESUMO
Eradicating influenza A virus (IAV) is difficult, due to its genetic drift and reassortment ability. As the infectious cycle is initiated by the influenza glycoprotein, hemagglutinin (HA), which mediates the binding of virions to terminal sialic acids moieties, HA is a tempting target of anti-influenza inhibitors. However, the complexity of the HA structure has prevented delineation of the structural characterization of the HA protein-ligand complex. Our computational strategy efficiently analyzed > 200,000 records of compounds held in the United States National Cancer Institute (NCI) database and identified potential HA inhibitors, by modeling the sialic acid (SA) receptor binding site (RBS) for the HA structure. Our modeling revealed that compound NSC85561 showed significant antiviral activity against the IAV H1N1 strain with EC50 values ranging from 2.31 to 2.53 µM and negligible cytotoxicity (CC50 > 700 µM). Using the NSC85561 compound as the template to generate 12 derivatives, robust bioassay results revealed the strongest antiviral efficacies with NSC47715 and NSC7223. Virtual screening clearly identified three SA receptor binding site inhibitors that were successfully validated in experimental data. Thus, our computational strategy has identified SA receptor binding site inhibitors against HA that show IAV-associated antiviral activity.
Assuntos
Antivirais/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/metabolismo , Ácido N-Acetilneuramínico/antagonistas & inibidores , Animais , Sítios de Ligação/fisiologia , Cães , Células Madin Darby de Rim Canino , Ligação Proteica/fisiologiaRESUMO
Sialic acid sugars on the surface of cancer cells have emerged as potent immune modulators that contribute to the immunosuppressive microenvironment and tumor immune evasion. However, the mechanisms by which these sugars modulate antitumor immunity as well as therapeutic strategies directed against them are limited. Here we report that intratumoral injections with a sialic acid mimetic Ac53FaxNeu5Ac block tumor sialic acid expression in vivo and suppress tumor growth in multiple tumor models. Sialic acid blockade had a major impact on the immune cell composition of the tumor, enhancing tumor-infiltrating natural killer cell and CD8+ T-cell numbers while reducing regulatory T-cell and myeloid regulatory cell numbers. Sialic acid blockade enhanced cytotoxic CD8+ T-cell-mediated killing of tumor cells in part by facilitating antigen-specific T-cell-tumor cell clustering. Sialic acid blockade also synergized with adoptive transfer of tumor-specific CD8+ T cells in vivo and enhanced CpG immune adjuvant therapy by increasing dendritic cell activation and subsequent CD8+ T-cell responses. Collectively, these data emphasize the crucial role of sialic acids in tumor immune evasion and provide proof of concept that sialic acid blockade creates an immune-permissive tumor microenvironment for CD8+ T-cell-mediated tumor immunity, either as single treatment or in combination with other immune-based intervention strategies.Significance: Sialic acid sugars function as important modulators of the immunosuppressive tumor microenvironment that limit potent antitumor immunity.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3574/F1.large.jpg Cancer Res; 78(13); 3574-88. ©2018 AACR.
Assuntos
Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Melanoma Experimental/terapia , Ácido N-Acetilneuramínico/antagonistas & inibidores , Evasão Tumoral/efeitos dos fármacos , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral/transplante , Feminino , Glicosilação/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Imunoterapia Adotiva/métodos , Injeções Intralesionais , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ácido N-Acetilneuramínico/análise , Ácido N-Acetilneuramínico/imunologia , Ácido N-Acetilneuramínico/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Oligodesoxirribonucleotídeos/uso terapêutico , Evasão Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 are human pathogens responsible for bloody diarrhea and renal failures. EHEC employ a type 3 secretion system to attach directly to the human colonic epithelium. This structure is encoded by the locus of enterocyte effacement (LEE) whose expression is regulated in response to specific nutrients. In this study, we show that the mucin-derived sugars N-acetylglucosamine (NAG) and N-acetylneuraminic acid (NANA) inhibit EHEC adhesion to epithelial cells through down-regulation of LEE expression. The effect of NAG and NANA is dependent on NagC, a transcriptional repressor of the NAG catabolism in E. coli. We show that NagC is an activator of the LEE1 operon and a critical regulator for the colonization of mice intestine by EHEC. Finally, we demonstrate that NAG and NANA as well as the metabolic activity of Bacteroides thetaiotaomicron affect the in vivo fitness of EHEC in a NagC-dependent manner. This study highlights the role of NagC in coordinating metabolism and LEE expression in EHEC and in promoting EHEC colonization in vivo.
Assuntos
Acetilglucosamina/antagonistas & inibidores , Aderência Bacteriana/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Fosfoproteínas/genética , Proteínas Repressoras/genética , Animais , Bacteroides thetaiotaomicron/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/metabolismo , Escherichia coli Êntero-Hemorrágica/patogenicidade , Células Epiteliais/microbiologia , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Células HCT116 , Células HeLa , Humanos , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Ácido N-Acetilneuramínico/antagonistas & inibidores , Óperon , Fosfoproteínas/metabolismo , Proteínas Repressoras/fisiologiaRESUMO
Sialic acid sugars cover the surface of dendritic cells (DCs) and have been suggested to impact several aspects of DC biology. Research into the role of sialic acids in DCs, however, is complicated by the limited number of tools available to modulate sialic acid expression. Here we report on a synthetic, fluorinated sialic acid mimetic, Ac53FaxNeu5Ac, which potently blocks sialic acid expression in human monocyte-derived DCs (moDCs). Sialic acid blockade enhanced the responsiveness of moDCs to Toll-like receptor (TLR) stimulation as measured by increased maturation marker expression and cytokine production. Consequently, the T-cell activation capacity of Ac53FaxNeu5Ac-treated moDCs was strongly increased. In addition to sialic acids, moDCs also expressed the sialic acid-binding immunoglobulin-like lectins (Siglecs) -3, -5, -7, -9 and -10, immune inhibitory receptors recognizing these sialic acids. Treatment with Ac53FaxNeu5Ac abrogated putative cis and trans interactions between sialic acids and Siglec-7/-9. Together, these data indicate that sialic acids limit the activation of moDCs via the TLR pathway, potentially by interacting with Siglec-7 or Siglec-9. Metabolic sialic acid blockade with Ac53FaxNeu5Ac could therefore potentially be used to generate more potent DC-based vaccines for induction of robust anti-viral or anti-tumor immune responses.
Assuntos
Células Dendríticas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ácido N-Acetilneuramínico/farmacologia , Ácidos Siálicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomimética , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Humanos , Lectinas/metabolismo , Lipopolissacarídeos/imunologia , Teste de Cultura Mista de Linfócitos , Monócitos/imunologia , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/antagonistas & inibidores , Poli I-C/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Receptores Toll-Like/metabolismoRESUMO
Sialic acid sugars are overexpressed by cancer cells and contribute to the metastatic cascade at multiple levels. Therapeutic interference of sialic acids, however, has been difficult to pursue because of the absence of dedicated tools. Here we show that a rationally designed sialic acid-blocking glycomimetic (P-3F(ax)-Neu5Ac) successfully prevents cancer metastasis. Formulation of P-3F(ax)--Neu5Ac into poly(lactic-co-glycolic acid nanoparticles coated with antityrosinase-related protein-1 antibodies allowed targeted delivery of P-3F(ax)--Neu5Ac into melanoma cells, slow release, and long-term sialic acid blockade. Most importantly, intravenous injections of melanoma-targeting P-3F(ax)--Neu5Ac nanoparticles prevented metastasis formation in a murine lung metastasis model. These findings stress the importance of sialoglycans in cancer metastasis and advocate that sialic acid blockade using rationally designed glycomimetics targeted to cancer cells can effectively prevent cancer metastases. This targeting strategy to interfere with sialic acid-dependent processes is broadly applicable not only for different types of cancer but also in infection and inflammation.
Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Portadores de Fármacos/química , Melanoma Experimental/patologia , Ácido N-Acetilneuramínico/antagonistas & inibidores , Ácidos Siálicos/química , Ácidos Siálicos/farmacologia , Animais , Anticorpos/química , Anticorpos/imunologia , Feminino , Ácido Láctico/química , Neoplasias Pulmonares/secundário , Glicoproteínas de Membrana/imunologia , Camundongos , Nanopartículas/química , Metástase Neoplásica , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoAssuntos
Adenoviridae/efeitos dos fármacos , Antivirais/química , Antivirais/farmacologia , Córnea/virologia , Ciclobutanos/química , Ciclobutanos/farmacologia , Ácido N-Acetilneuramínico/antagonistas & inibidores , Ácidos Siálicos/química , Ácidos Siálicos/farmacologia , Antivirais/síntese química , Linhagem Celular , Cristalografia por Raios X , Ciclobutanos/síntese química , Humanos , Conformação Proteica , Ácidos Siálicos/síntese química , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Epilepsy can be described as a group of neurological disorders, characterized by recurrent episodes of convulsive seizures, loss of consciousness, sensory disturbances, abnormal behavior, or all of these. Altered glutathione metabolism in association with increased oxidative stress has been implicated in the pathogenesis of many diseases such as seizures. It is therefore reasonable to propose that sialic acid levels can be affected by this pathological state or, alternately, by seizures. The present study showed that the sialic acid levels were significantly different between the experimental groups as well as in the subgroup analysis. The results suggest that glutathione may have a neuroprotective effect by decreasing sialic acid levels in mice brain.
Assuntos
Encéfalo/metabolismo , Epilepsia/metabolismo , Glutationa/farmacologia , Ácido N-Acetilneuramínico/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Animais , Convulsivantes/administração & dosagem , Relação Dose-Resposta a Droga , Epilepsia/induzido quimicamente , Feminino , Masculino , Camundongos , Ácido N-Acetilneuramínico/metabolismo , Pentilenotetrazol/administração & dosagem , Distribuição TecidualRESUMO
Exposure for 24 h of mucus-secreting HT-29 cells to the sugar analogue GalNAc-alpha-O-benzyl results in inhibition of Galbeta1-3GalNAc:alpha2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their secretion (Huet, G., I. Kim, C. de Bolos, J.M. Loguidice, O. Moreau, B. Hémon, C. Richet, P. Delannoy, F.X. Real., and P. Degand. 1995. J. Cell Sci. 108:1275-1285). To determine the effects of prolonged inhibition of sialylation, differentiated HT-29 populations were grown under permanent exposure to GalNAc-alpha-O-benzyl. This results in not only inhibition of mucus secretion, but also in a dramatic swelling of the cells and the accumulation in intracytoplasmic vesicles of brush border-associated glycoproteins like dipeptidylpeptidase-IV, the mucin-like glycoprotein MUC1, and carcinoembryonic antigen which are no longer expressed at the apical membrane. The block occurs beyond the cis-Golgi as substantiated by endoglycosidase treatment and biosynthesis analysis. In contrast, the polarized expression of the basolateral glycoprotein GP 120 is not modified. Underlying these effects we found that (a) like in mucins, NeuAcalpha2-3Gal-R is expressed in the terminal position of the oligosaccharide species associated with the apical, but not the basolateral glycoproteins of the cells, and (b) treatment with GalNAc-alpha-O-benzyl results in an impairment of their sialylation. These effects are reversible upon removal of the drug. It is suggested that alpha2-3 sialylation is involved in apical targeting of brush border membrane glycoproteins and mucus secretion in HT-29 cells.