RESUMO
In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO4) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO4 illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.
Assuntos
Galactose Oxidase , Vacinas Pneumocócicas , Polissacarídeos Bacterianos , Streptococcus pneumoniae , Animais , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Galactose Oxidase/química , Galactose Oxidase/imunologia , Galactose Oxidase/metabolismo , Glicoconjugados , Camundongos , Vacinas Pneumocócicas/química , Vacinas Pneumocócicas/imunologia , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/imunologia , Sorogrupo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/imunologia , Vacinas ConjugadasRESUMO
Nucleic acid vaccines introduce the genetic materials encoding antigenic proteins into host cells. If these proteins are directed into the secretory pathway with a signal/leader sequence, they will be exposed to the host's glycosylation machinery, and, if their amino acid sequences contain consensus sequons for N-linked glycosylation, they may become glycosylated. The presence of host glycans on the proteins of microbial origin may prevent a strong protective immune response either through hindering access to key epitopes by lymphocytes or through altering immune responses by binding to immunoregulatory glycan-binding receptors on immune cells. Ag85A expressed by Mycobacterium tuberculosis (Mtb) is a bacterial surface protein that is commonly used in nucleic acid vaccines in multiple clinical trials. Here we show that, when Ag85A is expressed in mammalian cells, it is glycosylated, does not induce a strong humoral immune response in mice, and does not activate Ag85A-specific lymphocytes as highly as Ag85A natively expressed by the bacterium. Our study indicates that host glycosylation of the vaccine target can impede its antigenicity and immunogenicity. Glycosylation of the antigenic protein targets therefore must be carefully evaluated in designing nucleic acid vaccines.
Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Imunogenicidade da Vacina , Processamento de Proteína Pós-Traducional , Vacinas contra a Tuberculose/imunologia , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Glicosilação , Células HEK293 , Humanos , Linfócitos/imunologia , CamundongosRESUMO
The pneumococcal serine-rich repeat protein (PsrP) is a high-molecular-weight, glycosylated adhesin that promotes the attachment of Streptococcus pneumoniae to host cells. PsrP, its associated glycosyltransferases (GTs), and dedicated secretion machinery are encoded in a 37-kb genomic island that is present in many invasive clinical isolates of S. pneumoniae PsrP has been implicated in establishment of lung infection in murine models, although specific roles of the PsrP glycans in disease progression or bacterial physiology have not been elucidated. Moreover, enzymatic specificities of associated glycosyltransferases are yet to be fully characterized. We hypothesized that the glycosyltransferases that modify PsrP are critical for the adhesion properties and infectivity of S. pneumoniae Here, we characterize the putative S. pneumoniaepsrP locus glycosyltransferases responsible for PsrP glycosylation. We also begin to elucidate their roles in S. pneumoniae virulence. We show that four glycosyltransferases within the psrP locus are indispensable for S. pneumoniae biofilm formation, lung epithelial cell adherence, and establishment of lung infection in a mouse model of pneumococcal pneumonia.IMPORTANCE PsrP has previously been identified as a necessary virulence factor for many serotypes of S. pneumoniae and studied as a surface glycoprotein. Thus, studying the effects on virulence of each glycosyltransferase (GT) that builds the PsrP glycan is of high importance. Our work elucidates the influence of GTs in vivo We have identified at least four GTs that are required for lung infection, an indication that it is worthwhile to consider glycosylated PsrP as a candidate for serotype-independent pneumococcal vaccine design.
Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/patogenicidade , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Proteínas de Bactérias/genética , Feminino , Glicosiltransferases/genética , Humanos , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , VirulênciaRESUMO
Streptococcus pneumoniae remains a leading cause of bacterial pneumonia despite the widespread use of vaccines. While vaccines are effective at reducing the incidence of most serotypes included in vaccines, a rise in infection due to nonvaccine serotypes and moderate efficacy against some vaccine serotypes have contributed to high disease incidence. Additionally, numerous isolates of S. pneumoniae are antibiotic or multidrug resistant. Several conserved pneumococcal proteins prevalent in the majority of serotypes have been examined for their potential as vaccines in preclinical and clinical trials. An additional, yet-unexplored tool for disease prevention and treatment is the use of human monoclonal antibodies (MAbs) targeting conserved pneumococcal proteins. Here, we isolated the first human MAbs (PhtD3, PhtD6, PhtD7, PhtD8, and PspA16) against the pneumococcal histidine triad protein (PhtD) and the pneumococcal surface protein A (PspA), two conserved and protective antigens. MAbs to PhtD target diverse epitopes on PhtD, and MAb PspA16 targets the N-terminal segment of PspA. The PhtD-specific MAbs bind to multiple serotypes, while MAb PspA16 serotype breadth is limited. MAbs PhtD3 and PhtD8 prolong the survival of mice infected with pneumococcal serotype 3. Furthermore, MAb PhtD3 prolongs the survival of mice in intranasal and intravenous infection models with pneumococcal serotype 4 and in mice infected with pneumococcal serotype 3 when administered 24 h after pneumococcal infection. All PhtD and PspA MAbs demonstrate opsonophagocytic activity, suggesting a potential mechanism of protection. Our results identify new human MAbs for pneumococcal disease prevention and treatment and identify epitopes on PhtD and PspA recognized by human B cells.
Assuntos
Anticorpos Monoclonais/farmacologia , Interações Hospedeiro-Patógeno/imunologia , Hidrolases/antagonistas & inibidores , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Relação Dose-Resposta Imunológica , Epitopos/imunologia , Humanos , Hidrolases/imunologia , Ligação Proteica , SorogrupoRESUMO
Cancer-associated fibroblasts (CAFs) are present in many types of tumors and play a pivotal role in tumor progression and immunosuppression. Fibroblast-activation protein (FAP), which is overexpressed on CAFs, has been indicated as a universal tumor target. However, FAP expression is not restricted to tumors, and systemic treatment against FAP often causes severe side effects. To solve this problem, a photodynamic therapy (PDT) approach was developed based on ZnF16Pc (a photosensitizer)-loaded and FAP-specific single chain variable fragment (scFv)-conjugated apoferritin nanoparticles, or αFAP-Z@FRT. αFAP-Z@FRT PDT efficiently eradicates CAFs in tumors without inducing systemic toxicity. When tested in murine 4T1 models, the PDT treatment elicits anti-cancer immunity, causing suppression of both primary and distant tumors, i.e. abscopal effect. Treatment efficacy is enhanced when αFAP-Z@FRT PDT is used in combination with anti-PD1 antibodies. Interestingly, it is found that the PDT treatment not only elicits a cellular immunity against cancer cells, but also stimulates an anti-CAFs immunity. This is supported by an adoptive cell transfer study, where T cells taken from 4T1-tumor-bearing animals treated with αFAP PDT retard the growth of A549 tumors established on nude mice. Overall, our approach is unique for permitting site-specific eradication of CAFs and inducing a broad spectrum anti-cancer immunity.
RESUMO
PURPOSE: Streptococcus pneumoniae (Spn) serotype 3 (Spn3) is considered one of the most virulent serotypes with resistance to conventional vaccine and treatment regimens. Pn3Pase is a glycoside hydrolase that we have previously shown to be highly effective in degrading the capsular polysaccharide of type 3 Spn, sensitizing it to host immune clearance. To begin assessing the value and safety of this enzyme for future clinical studies, we investigated the effects of high doses of Pn3Pase on host cells and immune system. METHODS: We assessed the enzyme's catalytic activity following administration in mice, and performed septic infection models to determine if prior administration of the enzyme inhibited repeat treatments of Spn3-challenged mice. We assessed immune populations in mouse tissues following administration of the enzyme, and tested Pn3Pase toxicity on other mammalian cell types in vitro. RESULTS: Repeated administration of the enzyme in vivo does not prevent efficacy of the enzyme in promoting bacterial clearance following bacterial challenge, with insignificant antibody response generated against the enzyme. Immune homeostasis is maintained following high-dose treatment with Pn3Pase, and no cytotoxic effects were observed against mammalian cells. CONCLUSIONS: These data indicate that Pn3Pase has potential as a therapy against Spn3. Further development as a drug product could overcome a great hurdle of pneumococcal infections.
Assuntos
Proteínas de Bactérias/farmacologia , Glicosídeo Hidrolases/farmacologia , Paenibacillus/enzimologia , Infecções Pneumocócicas/tratamento farmacológico , Streptococcus pneumoniae/efeitos dos fármacos , Animais , Cápsulas Bacterianas/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/uso terapêutico , Modelos Animais de Doenças , Feminino , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/uso terapêutico , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Infecções Pneumocócicas/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
Most pathogenic bacteria express surface carbohydrates called capsular polysaccharides (CPSs). CPSs are important vaccine targets because they are easily accessible and recognizable by the immune system. However, CPS-specific adaptive humoral immune responses can only be achieved by the covalent conjugation of CPSs with carrier proteins to produce glycoconjugate vaccines. We previously described a mechanism by which a model glycoconjugate vaccine can activate the adaptive immune system and demonstrated that the mammalian CD4+ T cell repertoire contains a population of carbohydrate-specific T cells. In this study, we use glycoconjugates of type 3 Streptococcus pneumoniae CPS (Pn3P) to assess whether the carbohydrate-specific adaptive immune response exemplified in our previous study can be applied to the conjugates of this lethal pathogen. In this article, we provide evidence for the functional roles of Pn3P-specific CD4+ T cells utilizing mouse immunization schemes that induce Pn3P-specific IgG responses in a carbohydrate-specific T cell-dependent manner.
Assuntos
Cápsulas Bacterianas/imunologia , Linfócitos T CD4-Positivos/imunologia , Glicoconjugados/imunologia , Imunidade Humoral , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Imunidade Adaptativa , Animais , Cápsulas Bacterianas/química , Linfócitos T CD4-Positivos/metabolismo , Carboidratos/imunologia , Feminino , Glicoconjugados/administração & dosagem , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Streptococcus pneumoniae/patogenicidade , Vacinação , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologiaRESUMO
Despite a century of investigation, Streptococcus pneumoniae remains a major human pathogen, causing a number of diseases, such as pneumonia, meningitis, and otitis media. Like many encapsulated pathogens, the capsular polysaccharide (CPS) of S. pneumoniae is a critical component for colonization and virulence in mammalian hosts. This study aimed to evaluate the protective role of a glycoside hydrolase, Pn3Pase, targeting the CPS of type 3 S. pneumoniae, which is one of the most virulent serotypes. We have assessed the ability of Pn3Pase to degrade the capsule on a live type 3 strain. Through in vitro assays, we observed that Pn3Pase treatment increases the bacterium's susceptibility to phagocytosis by macrophages and complement-mediated killing by neutrophils. We have demonstrated that in vivo Pn3Pase treatment reduces nasopharyngeal colonization and protects mice from sepsis caused by type 3 S. pneumoniae Due to the increasing shifts in serotype distribution, the rise in drug-resistant strains, and poor immune responses to vaccine-included serotypes, it is necessary to investigate approaches to combat pneumococcal infections. This study evaluates the interaction of pneumococcal CPS with the host at molecular, cellular, and systemic levels and offers an alternative therapeutic approach for diseases caused by S. pneumoniae through enzymatic hydrolysis of the CPS.
Assuntos
Cápsulas Bacterianas/metabolismo , Glicosídeo Hidrolases/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Fagocitose/fisiologia , Infecções Pneumocócicas/fisiopatologia , Polissacarídeos Bacterianos/metabolismo , Streptococcus pneumoniae/metabolismo , Animais , Humanos , Hidrólise , CamundongosRESUMO
During hematopoiesis, hematopoietic stem cells constantly differentiate into granulocytes and macrophages via a distinct differentiation program that is tightly controlled by myeloid lineage-specific transcription factors. Mice with a null mutation of IFN regulatory factor 8 (IRF8) accumulate CD11b(+)Gr1(+) myeloid cells that phenotypically and functionally resemble tumor-induced myeloid-derived suppressor cells (MDSCs), indicating an essential role of IRF8 in myeloid cell lineage differentiation. However, IRF8 is expressed in various types of immune cells, and whether IRF8 functions intrinsically or extrinsically in regulation of myeloid cell lineage differentiation is not fully understood. In this study, we report an intriguing finding that, although IRF8-deficient mice exhibit deregulated myeloid cell differentiation and resultant accumulation of CD11b(+)Gr1(+) MDSCs, surprisingly, mice with IRF8 deficiency only in myeloid cells exhibit no abnormal myeloid cell lineage differentiation. Instead, mice with IRF8 deficiency only in T cells exhibited deregulated myeloid cell differentiation and MDSC accumulation. We further demonstrated that IRF8-deficient T cells exhibit elevated GM-CSF expression and secretion. Treatment of mice with GM-CSF increased MDSC accumulation, and adoptive transfer of IRF8-deficient T cells, but not GM-CSF-deficient T cells, increased MDSC accumulation in the recipient chimeric mice. Moreover, overexpression of IRF8 decreased GM-CSF expression in T cells. Our data determine that, in addition to its intrinsic function as an apoptosis regulator in myeloid cells, IRF8 also acts extrinsically to repress GM-CSF expression in T cells to control myeloid cell lineage differentiation, revealing a novel mechanism that the adaptive immune component of the immune system regulates the innate immune cell myelopoiesis in vivo.
Assuntos
Linhagem da Célula/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fatores Reguladores de Interferon/imunologia , Células Mieloides/imunologia , Mielopoese/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Proliferação de Células , Quimera , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Camundongos , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Mielopoese/efeitos dos fármacos , Mielopoese/genética , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/transplanteRESUMO
The Fas-FasL effector mechanism plays a key role in cancer immune surveillance by host T cells, but metastatic human colon carcinoma often uses silencing Fas expression as a mechanism of immune evasion. The molecular mechanism under FAS transcriptional silencing in human colon carcinoma is unknown. We performed genome-wide chromatin immunoprecipitation sequencing analysis and identified that the FAS promoter is enriched with H3K9me3 in metastatic human colon carcinoma cells. The H3K9me3 level in the FAS promoter region is significantly higher in metastatic than in primary cancer cells, and it is inversely correlated with Fas expression level. We discovered that verticillin A is a selective inhibitor of histone methyltransferases SUV39H1, SUV39H2, and G9a/GLP that exhibit redundant functions in H3K9 trimethylation and FAS transcriptional silencing. Genome-wide gene expression analysis identified FAS as one of the verticillin A target genes. Verticillin A treatment decreased H3K9me3 levels in the FAS promoter and restored Fas expression. Furthermore, verticillin A exhibited greater efficacy than decitabine and vorinostat in overcoming colon carcinoma resistance to FasL-induced apoptosis. Verticillin A also increased DR5 expression and overcame colon carcinoma resistance to DR5 agonist drozitumab-induced apoptosis. Interestingly, verticillin A overcame metastatic colon carcinoma resistance to 5-fluorouracil in vitro and in vivo. Using an orthotopic colon cancer mouse model, we demonstrated that tumor-infiltrating cytotoxic T lymphocytes are FasL(+) and that FasL-mediated cancer immune surveillance is essential for colon carcinoma growth control in vivo. Our findings determine that H3K9me3 of the FAS promoter is a dominant mechanism underlying FAS silencing and resultant colon carcinoma immune evasion and progression.
Assuntos
Neoplasias do Colo/etiologia , Resistencia a Medicamentos Antineoplásicos/genética , Inativação Gênica , Histonas/metabolismo , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Receptor fas/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Metilação de DNA , Modelos Animais de Doenças , Proteína Ligante Fas/metabolismo , Fluoruracila/farmacologia , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Antígenos de Histocompatibilidade/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Indóis/farmacologia , Concentração Inibidora 50 , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metilação/efeitos dos fármacos , Metiltransferases/metabolismo , Camundongos , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Inducible nitric oxide synthase (iNOS) metabolizes L-arginine to produce nitric oxide (NO) which was originally identified in myeloid cells as a host defense mechanism against pathogens. Recent studies, however, have revealed that iNOS is often induced in tumor cells and myeloid cells in the tumor microenvironment. Compelling experimental data have shown that iNOS promotes tumor development in certain cellular context and suppresses tumor development in other cellular conditions. The molecular mechanisms underlying these contrasting functions of iNOS is unknown. Because iNOS is often induced by inflammatory signals, it is therefore likely that these contrasting functions of iNOS could be controlled by the inflammatory signaling pathways, which remains to be determined. METHODS: iNOS is expressed in colon carcinoma and myeloid cells in the tumor microenvironment. Colon carcinoma and myeloid cell lines were used to elucidate the molecular mechanisms underlying iNOS expression. Chromatin immunoprecipitation and electrophoretic mobility shift assay were used to determine the IFNγ-activated pSTAT1 and NF-κB association with the chromatin DNA of the nos2 promoter. RESULTS: We show here that iNOS is dramatically up-regulated in inflammed human colon tissues and in human colon carcinoma as compared to normal colon tissue. iNOS is expressed in either the colon carcinoma cells or immune cells within the tumor microenvironment. On the molecular level, the proinflammatory IFNγ and NF-κB signals induce iNOS expression in human colon cancer cells. We further demonstrate that NF-κB directly binds to the NOS2 promoter to regulate iNOS expression. Although neither the IFNγ signaling pathway nor the NF-κB signaling pathway alone is sufficient to induce iNOS expression in myeloid cells, IFNγ and NF-κB synergistically induce iNOS expression in myeloid cells. Furthermore, we determine that IFNγ up-regulates IRF8 expression to augment NF-κB induction of iNOS expression. More interestingly, we observed that the p65/p65 and p50/p50 homodimers, not the canonical p65/p50 heterodimer, directly binds to the nos2 promoter to regulate iNOS expression in myeloid cells. CONCLUSIONS: IFNγ-induced IRF8 acts in concert with NF-κB to regulate iNOS expression in both colon carcinoma and myeloid cells. In myeloid cells, the NF-κB complexes that bind to the nos2 promoter are p65/p65 and p50/p50 homodimers.
Assuntos
Neoplasias do Colo/metabolismo , Fatores Reguladores de Interferon/fisiologia , Subunidade p50 de NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Transcrição RelA/fisiologia , Animais , Linhagem Celular Tumoral/metabolismo , Colo/metabolismo , Humanos , Fatores Reguladores de Interferon/metabolismo , Interferon gama/fisiologia , Camundongos , Células Mieloides/metabolismo , Regiões Promotoras Genéticas , Microambiente Tumoral/fisiologiaRESUMO
Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that accumulate in response to tumor progression. Compelling data from mouse models and human cancer patients showed that tumor-induced inflammatory mediators induce MDSC differentiation. However, the mechanisms underlying MDSC persistence is largely unknown. Here, we demonstrated that tumor-induced MDSCs exhibit significantly decreased spontaneous apoptosis as compared with myeloid cells with the same phenotypes from tumor-free mice. Consistent with the decreased apoptosis, cell surface Fas receptor decreased significantly in tumor-induced MDSCs. Screening for changes of key apoptosis mediators downstream the Fas receptor revealed that expression levels of IRF8 and Bax are diminished, whereas expression of Bcl-xL is increased in tumor-induced MDSCs. We further determined that IRF8 binds directly to Bax and Bcl-x promoter in primary myeloid cells in vivo, and IRF8-deficient MDSC-like cells also exhibit increased Bcl-xL and decreased Bax expression. Analysis of CD69 and CD25 levels revealed that cytotoxic T lymphocytes (CTLs) are partially activated in tumor-bearing hosts. Strikingly, FasL but not perforin and granzymes were selectively activated in CTLs in the tumor-bearing host. ABT-737 significantly increased the sensitivity of MDSCs to Fas-mediated apoptosis in vitro. More importantly, ABT-737 therapy increased MDSC spontaneous apoptosis and decreased MDSC accumulation in tumor-bearing mice. Our data thus determined that MDSCs use down-regulation of IRF8 to alter Bax and Bcl-xL expression to deregulate the Fas-mediated apoptosis pathway to evade elimination by host CTLs. Therefore, targeting Bcl-xL is potentially effective in suppression of MDSC persistence in cancer therapy.
Assuntos
Apoptose , Farmacorresistência Bacteriana/genética , Regulação Neoplásica da Expressão Gênica , Células Mieloides/citologia , Neoplasias/metabolismo , Proteína bcl-X/metabolismo , Animais , Linfócitos T CD8-Positivos/citologia , Caspase 8/metabolismo , Humanos , Fatores Reguladores de Interferon/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteína X Associada a bcl-2/metabolismo , Receptor fas/metabolismoRESUMO
BACKGROUND: Ceramide is a bioeffector that mediates various cellular processes, including apoptosis. However, the mechanism underlying ceramide function in apoptosis is apparently cell type-dependent and is not well-understood. We aimed at identifying molecular targets of ceramide in metastatic human colon and breast cancer cells, and determining the efficacy of ceramide analog in suppression of colon and breast cancer metastasis. METHODS: The activity of and mechanism underlying ceramide as a cytotoxic agent, and as a sensitizer for Fas-mediated apoptosis was analyzed in human cell lines established from primary or metastatic colon and breast cancers. The efficacy of ceramide analog LCL85 in suppression of metastasis was examined in preclinical mouse tumor models. RESULTS: Exposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to Fas-mediated apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to Fas-mediated apoptosis. LCL85 resembles Smac mimetic BV6 in sensitization of colon carcinoma cells to Fas-mediated apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. LCL85 also decreased xIAP1 and cIAP1 protein levels and sensitized metastatic human breast cancer cells to Fas-mediated apoptosis. Silencing xIAP and cIAP1 with specific siRNAs significantly increased the metastatic human colon carcinoma cell sensitivity to Fas-mediated apoptosis, suggesting that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells and ceramide induces IAP protein degradation to sensitize the tumor cells to apoptosis induction. Consistent with its apoptosis sensitization activity, subtoxic doses of LCL85 suppressed colon carcinoma cell metastatic potential in an experimental lung metastasis mouse model, as well as breast cancer growth and spontaneous lung metastasis in an orthotopic breast cancer mouse model. CONCLUSION: We have identified xIAP and cIAP1 as molecular targets of ceramide and determined that ceramide analog LCL85 is an effective sensitizer in overcoming resistance of human cell lines established from metastatic colon and breast cancers to apoptosis induction to suppress metastasis in vivo.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Ceramidas/farmacologia , Neoplasias do Colo/tratamento farmacológico , Proteínas Inibidoras de Apoptose/metabolismo , Propanolaminas/farmacologia , Compostos de Piridínio/farmacologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/secundário , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/secundário , Progressão da Doença , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Proteínas Inibidoras de Apoptose/genética , Camundongos , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transfecção , Ubiquitina-Proteína Ligases , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor fas/metabolismoRESUMO
The COVID-19 pandemic dramatically demonstrated the need for improved vaccination strategies and therapeutic responses to combat infectious diseases. However, the efficacy of vaccines has not yet been demonstrated in combination with commonly used immunosuppressive drug regimens. We sought to determine how common pharmaceutical drugs used in autoimmune disorders can alter immune responses to the SARS-CoV-2 spike protein vaccination. We treated mice with five immunosuppressant drugs (cyclophosphamide, leflunomide, methotrexate, methylprednisolone, and mycophenolate mofetil), each with various mechanisms of action prior to and following immunization with SARS-CoV-2 spike protein. We assessed the functionality of antibody responses to spike protein and compared immune cell populations in mice that received no treatment with those that received continuous or temporarily suspended immune suppressive therapy. All tested immunosuppressants significantly reduced the antibody titers in serum and functional antibody response against SARS-CoV-2 spike protein in immunized mice. Temporarily halting selected immunosuppressants (methylprednisolone and methotrexate, but not cyclophosphamide) improved antibody responses significantly. Through proof-of-principle experiments utilizing a mouse model, we demonstrated that immune suppression in autoimmune disorders through pharmaceutical treatments may impair vaccine response to SARS-CoV-2, and temporary suspension of immunosuppressant treatment may be necessary to mount an effective antibody vaccine response. This work provides feasibility for future clinical assessment of the impact of immunosuppressants on vaccine efficacy in humans.
Assuntos
Tratamento Farmacológico da COVID-19 , Preparações Farmacêuticas , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , Humanos , Imunossupressores , Camundongos , Pandemias , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vacinação , Eficácia de VacinasRESUMO
Capsular polysaccharides (CPSs), with which most pathogenic bacterial surfaces are decorated, have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide. Pneumococcal conjugate vaccines (PCVs) are administered globally to prevent invasive pneumococcal disease (IPD). While PCVs have played important roles in controlling IPD in all age groups, their empirical, and labor-intensive chemical conjugation yield poorly characterized, heterogeneous, and variably immunogenic vaccines, with poor immune responses in high-risk populations such as the elderly and patients with weak immune systems. We previously developed a method that bypasses the dependency of chemical conjugation and instead exploits prokaryotic glycosylation systems to produce pneumococcal conjugate vaccines. The bioconjugation platform relies on a conjugating enzyme to transfer a bacterial polysaccharide to an engineered carrier protein all within the lab safe bacterium E. coli. In these studies, we demonstrate that a serotype 8 pneumococcal bioconjugate vaccine is highly immunogenic and elicits functionally protective anti-serotype 8 antibody responses. Specifically, using multiple models we show that mice immunized with multiple doses of a serotype 8 bioconjugate vaccine elicit antibody responses that mediate opsonophagocytic killing, protect mice from systemic infection, and decrease the ability of serotype 8 pneumococci to colonize the nasopharynx and disseminate. Collectively, these studies demonstrate the utility of bioconjugation to produce efficacious pneumococcal conjugate vaccines.
Assuntos
Infecções Pneumocócicas , Vacinas Pneumocócicas , Animais , Anticorpos Antibacterianos , Proteínas de Transporte , Escherichia coli , Camundongos , Polissacarídeos Bacterianos , Vacinas ConjugadasRESUMO
Capsular polysaccharides (CPSs) are major virulence factors that decorate the surfaces of many human bacterial pathogens. In their pure form or as glycoconjugate vaccines, CPSs are extensively used in vaccines deployed in clinical practice worldwide. However, our understanding of the structural requirements for interactions between CPSs and antibodies is limited. A longstanding model based on comprehensive observations of antibody repertoires binding to CPSs is that antibodies expressing heavy chain variable gene family 3 (VH3) predominate in these binding interactions in humans and VH3 homologs in mice. Toward understanding this highly conserved interaction, we generated a panel of mouse monoclonal antibodies (MAb) against Streptococcus pneumoniae serotype 3 CPS, determined an X-ray crystal structure of a protective MAb in complex with a hexasaccharide derived from enzymatic hydrolysis of the polysaccharide, and elucidated the structural requirements for this binding interaction. The crystal structure revealed a binding pocket containing aromatic side chains, suggesting the importance of hydrophobicity in the interaction. Through mutational analysis, we determined the amino acids that are critical in carbohydrate binding. Through elucidating the structural and functional properties of a panel of murine MAbs, we offer an explanation for the predominant use of the human VH3 gene family in antibodies against CPSs with implications in knowledge-based vaccine design. IMPORTANCE Infectious diseases caused by pathogenic bacteria are a major threat to human health. Capsular polysaccharides (CPSs) of many pathogenic bacteria have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide, with various degrees of success. Immunization with a glycoconjugate vaccine elicits T cell help for B cells that produce IgG antibodies to the CPS. Thus, it is important to develop an in-depth understanding of the interactions of carbohydrate epitopes with the antibodies. Structural characterization of the ligand binding of polysaccharide-specific antibodies laid out in this study may have fundamental biological implications for our comprehension of how the humoral immune system recognizes polysaccharide antigens, and in future knowledge-based vaccine design.
Assuntos
Anticorpos Antibacterianos/imunologia , Cápsulas Bacterianas/química , Polissacarídeos Bacterianos/imunologia , Polissacarídeos Bacterianos/metabolismo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Monoclonais , Cápsulas Bacterianas/classificação , Cápsulas Bacterianas/imunologia , Cristalização , Feminino , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Modelos Estruturais , Polissacarídeos Bacterianos/química , Sorogrupo , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/patogenicidade , VacinaçãoRESUMO
Group B Streptococcus (GBS) is a leading cause of neonatal infections and invasive diseases in nonpregnant adults worldwide. Developing a protective conjugate vaccine targeting the capsule of GBS has been pursued for more than 30 years; however, it has yet to yield a licensed product. In this study, we present a novel bioconjugation platform for producing a prototype multivalent GBS conjugate vaccine and its subsequent analytical and immunological characterizations. Using a glycoengineering strategy, we generated strains of Escherichia coli that recombinantly express the type Ia, type Ib, and type III GBS capsular polysaccharides. We then combined the type Ia-, Ib-, and III-capsule-expressing E. coli strains with an engineered Pseudomonas aeruginosa exotoxin A (EPA) carrier protein and the PglS oligosaccharyltransferase. Coexpression of a GBS capsule, the engineered EPA protein, and PglS enabled the covalent attachment of the target GBS capsule to an engineered serine residue on EPA, all within the periplasm of E. coli. GBS bioconjugates were purified, analytically characterized, and evaluated for immunogenicity and functional antibody responses. This proof-of-concept study signifies the first step in the development of a next-generation multivalent GBS bioconjugate vaccine, which was validated by the production of conjugates that are able to elicit functional antibodies directed against the GBS capsule.
Assuntos
Escherichia coli , Infecções Estreptocócicas , Adulto , Anticorpos Antibacterianos , Escherichia coli/genética , Humanos , Recém-Nascido , Infecções Estreptocócicas/prevenção & controle , Streptococcus agalactiae/genética , Vacinas CombinadasRESUMO
The inherent molecular complexity of human pathogens requires that mammals evolved an adaptive immune system equipped to handle presentation of non-conventional MHC ligands derived from disease-causing agents, such as HIV-1 envelope (Env) glycoprotein. Here, we report that a CD4+ T cell repertoire recognizes a glycopeptide epitope on gp120 presented by MHCII pathway. This glycopeptide is strongly immunogenic in eliciting glycan-dependent cellular and humoral immune responses. The glycopeptide specific CD4+ T cells display a prominent feature of Th2 and Th17 differentiation and exert high efficacy and potency to help Env trimer humoral immune responses. Glycopeptide-induced CD4+ T cell response prior to Env trimer immunization elicits neutralizing antibody development and production of antibodies facilitating uptake of immunogens by antigen-presenting cells. Our identification of gp120 glycopeptide-induced, T cell-specific immune responses offers a foundation for developing future knowledge-based vaccines that elicit strong and long-lasting protective immune responses against HIV-1 infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Imunidade Humoral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Citocinas/metabolismo , Epitopos de Linfócito T/química , Glicopeptídeos/química , Glicopeptídeos/imunologia , Anticorpos Anti-HIV/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Celular , Imunização , Camundongos , Polissacarídeos/imunologia , Células Th17/imunologia , Células Th2/imunologiaRESUMO
Although accumulation of myeloid-derived suppressor cells (MDSC) is a hallmark of cancer, the underlying mechanism of this accumulation within the tumor microenvironment remains incompletely understood. We report here that TNFα-RIP1-mediated necroptosis regulates accumulation of MDSCs. In tumor-bearing mice, pharmacologic inhibition of DNMT with the DNA methyltransferease inhibitor decitabine (DAC) decreased MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes. DAC-induced decreases in MDSC accumulation correlated with increased expression of the myeloid cell lineage-specific transcription factor IRF8 in MDSCs. However, DAC also suppressed MDSC-like cell accumulation in IRF8-deficient mice, indicating that DNA methylation may regulate MDSC survival through an IRF8-independent mechanism. Instead, DAC decreased MDSC accumulation by increasing cell death via disrupting DNA methylation of RIP1-dependent targets of necroptosis. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter was hypermethylated in tumor-induced MDSCs in vivo. DAC treatment dramatically increased TNFα levels in MDSC in vitro, and neutralizing TNFα significantly increased MDSC accumulation and tumor growth in tumor-bearing mice in vivo. Recombinant TNFα induced MDSC cell death in a dose- and RIP1-dependent manner. IL6 was abundantly expressed in MDSCs in tumor-bearing mice and patients with human colorectal cancer. In vitro, IL6 treatment of MDSC-like cells activated STAT3, increased expression of DNMT1 and DNMT3b, and enhanced survival. Overall, our findings reveal that MDSCs establish a STAT3-DNMT epigenetic axis, regulated by autocrine IL6, to silence TNFα expression. This results in decreased TNFα-induced and RIP1-dependent necroptosis to sustain survival and accumulation. SIGNIFICANCE: These findings demonstrate that targeting IL6 expression or function represent potentially effective approaches to suppress MDSC survival and accumulation in the tumor microenvironment.
Assuntos
Comunicação Autócrina/efeitos dos fármacos , Interleucina-6/farmacologia , Células Supressoras Mieloides/efeitos dos fármacos , Necroptose/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Supressoras Mieloides/fisiologia , Necroptose/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , DNA Metiltransferase 3BRESUMO
A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.