RESUMEN
Conversion of human pluripotent stem cells from primed to naïve state is accompanied by altered transcriptome and methylome, but glycosphingolipid (GSL) profiles in naïve human embryonic stem cells (hESCs) have not been systematically characterized. Here we showed a switch from globo-(SSEA-3, SSEA-4, and Globo H) and lacto-series (fucosyl-Lc4Cer) to neolacto-series GSLs (SSEA-1 and H type 2 antigen), along with marked down-regulation of ß-1,3-galactosyltransferase (B3GALT5) upon conversion to naïve state. CRISPR/Cas9-generated B3GALT5-knockout (KO) hESCs displayed an altered GSL profile, increased cloning efficiency and intracellular Ca2+, reminiscent of the naïve state, while retaining differentiation ability. The altered GSLs could be rescued through overexpression of B3GALT5. B3GALT5-KO cells cultured with 2iLAF exhibited naïve-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. In addition, B3GALT5-KO rendered hESCs more resistant to calcium chelator in blocking entry into naïve state. Thus, loss of B3GALT5 induces a distinctive state of hESCs displaying unique GSL profiling with expression of neolacto-glycans, increased Ca2+, and conducive for transition to naïve pluripotency.
Asunto(s)
Diferenciación Celular , Galactosiltransferasas/metabolismo , Glicoesfingolípidos/metabolismo , Células Madre Pluripotentes/metabolismo , Antígenos Embrionarios Específico de Estadio/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular , Células Madre Embrionarias , Galactosiltransferasas/genética , Técnicas de Silenciamiento del Gen , HumanosRESUMEN
Natural killer T (NKT) cell is a distinct population of T lymphocytes that can rapidly release massive amount of Th1 and Th2 cytokines upon the engagement of their T cell receptor with glycolipids presented by CD1d. The secreted cytokines can promote cell-mediated immunity to kill tumor cells and intracellular pathogens, or suppress autoreactive immune cells in autoimmune diseases. Thus, NKT cell is an attractive target for developing new therapeutics to manipulate immune system. The best-known glycolipid to activate NKT cells is α-galactosylceramide (α-GalCer), which has been used as a prototype for designing new NKT stimulatory glycolipids. Many analogues have been generated by modification of the galactosyl moiety, the acyl chain or the phytosphingosine chain of α-GalCer. Some of the analogues showed greater abilities than α-GalCer in polarizing immune responses toward Th1 or Th2 dominance. Among them, several analogues containing phenyl groups in the lipid tails were more potent in inducing Th1-skewed cytokines and exhibited greater anticancer efficacy than α-GalCer. Analyses of the correlation between structure and activity of various α-GalCer analogues on the activation of iNKT cell revealed that CD1d-glycolipid complexes interacted with the same population of iNKT cell expressing similar T-cell receptor Vß as α-GalCer. On the other hand, those phenyl glycolipids with propensity for Th1 dominant responses showed greater binding avidity and stability than α-GalCer for iNKT T-cell receptor when complexed with CD1d. Thus, it is the avidity and stability of the ternary complexes of CD1d-glycolipid-iNKT TCR that dictate the polarity and potency of immune responses. These findings provide a key to the rationale design of immune modulating glycolipids with desirable Th1/Th2 polarity for clinical application. In addition, elucidation of α-GalCer-induced anergy, liver damage and accumulation of myeloid derived suppressor cells has offered explanation for its lacklustre anti-cancer activities in clinical trials. On other hand, the lack of such drawbacks in glycolipid analogues containing phenyl groups in the lipid tails of α-GalCer coupled with the greater binding avidity and stability of CD1d-glycolipid complex for iNKT T-cell receptor, account for their superior anti-cancer efficacy in tumor bearing mice. Further clinical development of these phenyl glycolipids is warranted.
Asunto(s)
Galactosilceramidas/inmunología , Inmunidad Celular , Activación de Linfocitos/inmunología , Células T Asesinas Naturales/inmunología , Animales , Humanos , RatonesRESUMEN
Strategies for cancer immunotherapy include activating immune system for therapeutic benefit or blockade of immune checkpoints. To harness innate immunity to fight cancer, α-galactosylceramide (α-GalCer) has been used to activate NKT cells. Unfortunately, administration of α-GalCer causes long-term NKT cell anergy, but the molecular mechanism is unclear. In this study, we showed that α-GalCer-triggered egr2/3, which induced programmed death 1 and cbl-b in NKT cells, leading to NKT cell anergy. We also uncovered the induction of the immunosuppressive myeloid-derived suppressor cells (MDSCs) in the spleen by α-GalCer that might attenuate its antitumor efficacy. The accumulation of MDSC was accompanied by 20-fold rise in their arg-1 mRNAs and enhanced expression of programmed death 1/programmed death ligand 1. Furthermore, α-GalCer-induced egr-2/3 in hepatic NKT cells upregulated their TRAIL in addition to Fas ligand (FasL) and induced alarm signaling molecule IL-33 in Kupffer cells, presumably because of liver damage triggered by TRAIL/FasL. We further demonstrated that IL-33-stimulated macrophages produce G-CSF, which in turn, boosted MDSCs. Thus, α-GalCer-induced FasL/TRAIL and IL-33 provided a novel mechanism underlying α-GalCer-induced hepatotoxicity and MDSC accumulation. In contrast, analogs of α-GalCer containing phenyl group in the lipid tail could neither induce NKT anergy nor enhance MDSCs accumulation. Furthermore, tumor-infiltrating MDSCs in mice injected repeatedly with α-GalCer were 2-fold higher than those treated with phenyl-glycolipids. These results not only revealed the induction of MDSC via IL-33 as a new mechanism for α-GalCer-elicited immunosuppression but also provided one of the mechanisms underlying the superior antitumor potency of phenyl-glycolipids. Our findings have important implications for the development of NKT-stimulatory glycolipids as vaccine adjuvants and anticancer therapeutics.
Asunto(s)
Antígeno B7-H1/metabolismo , Galactosilceramidas/inmunología , Células Mieloides/inmunología , Células T Asesinas Naturales/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Animales , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Arginasa/genética , Antígeno B7-H1/biosíntesis , Línea Celular Tumoral , Anergia Clonal/inmunología , Proteína 2 de la Respuesta de Crecimiento Precoz/biosíntesis , Proteína 3 de la Respuesta de Crecimiento Precoz/biosíntesis , Proteína Ligando Fas/biosíntesis , Femenino , Galactosilceramidas/uso terapéutico , Factor Estimulante de Colonias de Granulocitos/biosíntesis , Factor Estimulante de Colonias de Granulocitos/metabolismo , Terapia de Inmunosupresión , Inmunoterapia , Interleucina-33 , Interleucinas/metabolismo , Macrófagos del Hígado/metabolismo , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Mieloides/citología , Células T Asesinas Naturales/inmunología , Receptor de Muerte Celular Programada 1/biosíntesis , Proteínas Proto-Oncogénicas c-cbl/biosíntesis , ARN Mensajero/biosíntesis , Bazo/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Regulación hacia ArribaRESUMEN
Invariant natural killer T cell (NKT) cells (iNKT cells) produce both T-helper 1 (Th1) and T-helper 2 cytokines in response to α-Galactosylceramide (α-GalCer) stimulation and are thought to be the important effectors in the regulation of both innate and adaptive immunity involved in autoimmune disorders, microbial infections, and cancers. However, the anticancer effects of α-GalCer were limited in early clinical trial. In this study, several analogs of α-GalCer, containing phenyl groups in the lipid tails were found to stimulate murine and human iNKT cells to secrete Th1-skewed cytokines and exhibit greater anticancer efficacy in mice than α-GalCer. We explored the possibility of different Vß usages of murine Vα14 iNKT or human Vα24 iNKT cells, accounting for differential cytokine responses. However, T-cell receptor Vß analysis revealed no significant differences in Vß usages by α-GalCer and these phenyl glycolipid analogs. On the other hand, these phenyl glycolipids showed greater binding avidity and stability for iNKT T-cell receptor when complexed with CD1d. These findings suggest that CD1d-phenyl glycolipid complexes may interact with the same population of iNKT cells but with higher avidity and stability to drive Th1 polarization. Thus, this study provides a key to the rational design of Th1 biased CD1d reactive glycolipids in the future.
Asunto(s)
Antígenos CD1d/metabolismo , Neoplasias Experimentales/terapia , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Células TH1/inmunología , Animales , Antígenos CD1d/química , Línea Celular Tumoral , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Femenino , Galactosilceramidas/química , Galactosilceramidas/inmunología , Glucolípidos/química , Glucolípidos/inmunología , Humanos , Inmunoterapia , Técnicas In Vitro , Ligandos , Activación de Linfocitos , Sustancias Macromoleculares , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células T Asesinas Naturales/inmunología , Neoplasias Experimentales/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/químicaRESUMEN
Synopsis: A sugar-lipid molecule called OAcGD2 is a novel marker for breast cancer stem cells. Treatment with anti-OAcGD2 mAb8B6 may have superior anticancer efficacy by targeting cancer stem cells, thereby reducing metastasis and recurrence of cancer. Background: Cancer stem cells (CSCs) that drive tumor progression and disease recurrence are rare subsets of tumor cells. CSCs are relatively resistant to conventional chemotherapy and radiotherapy. Eradication of CSCs is thus essential to achieve durable responses. GD2 was reported to be a CSC marker in human triple-negative breast cancer, and anti-GD2 immunotherapy showed reduced tumor growth in cell lines. Using a specific anti-OAcGD2 antibody, mAb8D6, we set out to determine whether OAcGD2+ cells exhibit stem cell properties and mAb8D6 can inhibit tumor growth by targeting OAcGD2+CSCs. Method: OAcGD2 expression in patient-derived xenografts (PDXs) of breast cancer was determined by flow cytometric analyses using mAb8D6. The stemness of OAcGD2+ cells isolated by sorting and the effects of mAb8B6 were assessed by CSC growth and mammosphere formation in vitro and tumor growth in vivo using PDX models. Result: We found that the OAcGD2 expression levels in six PDXs of various molecular subtypes of breast cancer highly correlated with their previously defined CSC markers in these PDXs. The sorted OAcGD2+ cells displayed a greater capacity for mammosphere formation in vitro and tumor initiation in vivo than OAcGD2- cells. In addition, the majority of OAcGD2+ cells were aldehyde dehydrogenase (ALDH+) or CD44hiCD24lo, the known CSC markers in breast cancer. Treatment of PDXs-bearing mice with mAb8B6, but not doxorubicin, suppressed the tumor growth, along with reduced CSCs as assessed by CSC markers and in vivo tumorigenicity. In vitro, mAb8B6 suppressed proliferation and mammosphere formation and induced apoptosis of OAcGD2+ breast cancer cells harvested from PDXs, in a dose-dependent manner. Finally, administration of mAb8B6 in vivo dramatically suppressed tumor growth of OAcGD2+ breast CSCs (BCSCs) with complete tumor abrogation in 3/6 mice. Conclusion: OAcGD2 is a novel marker for CSC in various subtypes of breast cancer. Anti-OAcGD2 mAb8B6 directly eradicated OAcGD2+ cells and reduced tumor growth in PDX model. Our data demonstrate the potential of mAb8B6 as a promising immunotherapeutic agent to target BCSCs.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Neoplasias de la Mama/patología , Gangliósidos/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Animales , Apoptosis/efectos de los fármacos , Biomarcadores , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bromelain has been reported to have anti-inflammatory and immunomodulatory effects. However, the anti-inflammatory mechanism of bromelain is unclear. Therefore, we investigated the effect of bromelain on cytokine production from lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) and monocytic leukemia THP-1 cells. The result showed that bromelain (50-100 microg/ml) significantly and reversibly reduced tumor necrosis factor (TNF)-alpha interleukin- (IL)-1beta and IL-6 from LPS-induced PBMC and THP-1 cells. This effect was correlated with reduced LPS-induced TNF-alpha mRNA and NF-kappaB activity in THP-1 cells. In addition, bromelain dose-dependently inhibited LPS-induced prostaglandin E(2), thromboxane B(2) and COX-2 mRNA but not COX-1 mRNA. Importantly, bromelain degraded TNF-alpha and IL-1beta molecules, reduced the expression of surface marker CD14 but not Toll-like receptor 4 from THP-1 cells. Taken together, the results suggest that the suppression of signaling pathways by bromelain's proteolytic activity may contribute to the anti-inflammatory activity of bromelain.
Asunto(s)
Bromelaínas/farmacología , Citocinas/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/inmunología , Línea Celular Tumoral , Ciclooxigenasa 2/inmunología , Ciclooxigenasa 2/metabolismo , Dinoprostona/inmunología , Dinoprostona/metabolismo , Humanos , Interleucina-1/inmunología , Interleucina-1/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Leucocitos Mononucleares/metabolismo , Receptores de Lipopolisacáridos/inmunología , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Bromelain has been reported to have anti-inflammatory and immunomodulatory effects. It has been cross-linked with organic acids and polysaccharides by gamma irradiation. The cross-linked (CL)-bromelain preparation resisted an acidic environment of pH 3 for 2 h and preserved 80% of its enzyme activity. Pretreatment of rats with CL-bromelain intragastrically for 7 days significantly reduced serum cytokine production induced by injected i.p. with 2.5 mg/kg of lipopolysaccharide (LPS). Bromelain significantly reduced serum glutamate-oxalacetate transaminase induced by LPS. The anti-inflammatory effect of CL-bromelain was correlated with reduced LPS-induced NF-kappaB activity and cyclooxygenase 2 (COX-2) mRNA expression in rat livers. In addition, CL-bromelain dose-dependently inhibited LPS-induced COX-2 mRNA and prostaglandin E2 (PGE2) in BV-2 microglial cells. CL-Bromelain also suppressed the LPS-activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). In conclusion, the anti-inflammatory effects of the CL-bromelain preparation in vivo and in vitro suggest its therapeutic potentials.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Bromelaínas/química , Bromelaínas/farmacología , Citocinas/biosíntesis , Lipopolisacáridos/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Reactivos de Enlaces Cruzados , Ciclooxigenasa 2/genética , Citocinas/sangre , Dinoprostona/metabolismo , Activación Enzimática/efectos de los fármacos , Masculino , Microscopía Electrónica de Rastreo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Glycosphingolipids (GSLs) bearing the α-galactosyl headgroup and the acyl chain terminated with a phenyl derivative were found to be more potent than α-galactosyl ceramide (αGalCer) to stimulate both murine and human invariant natural killer T (iNKT) cells and to induce an antibody isotope switch to IgG. In this study, we replaced the galactosyl group with glucose (αGlc) and its fluoro-analogs and found that phenyl GSLs with αGlc (C34-Glc) and its fluoro-analog 6F-C34-Glc were stronger than those with αGal in stimulating human iNKT cells but weaker in mice. Their activities have a strong correlation with the binding avidities of the ternary interaction between the iNKT-cell receptor (iNKTCR) and CD1d-GSL complex. It was the iNKTCR rather than CD1d that dictated the species-specific responses. C34-Glc was further used as an adjuvant for a SSEA4-crm-197 vaccine, and after immunization in mice, the vaccine was highly effective against Lewis lung carcinoma.
Asunto(s)
Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Glucolípidos/química , Glucolípidos/farmacología , Activación de Linfocitos/efectos de los fármacos , Células T Asesinas Naturales/efectos de los fármacos , Animales , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/farmacología , Línea Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Células T Asesinas Naturales/inmunologíaRESUMEN
Reactive oxygen species (ROS) has been proposed to play a pathogenic role in neuronal injury. Sesame antioxidants that inhibit lipid peroxidation and regulate cytokine production may suppress ROS generation. In this study, we focused on the effect of sesamolin on H2O2-induced neurotoxicity and ROS production in the murine microglial cell line BV-2. Results indicate that the H2O2 elicited BV-2 cell death in a concentration- and time-dependent manner. ROS generation in BV-2 cells was time-dependently increased by the H2O2 treatment. Sesamolin reduced ROS generation in BV-2 cells. p38 mitogen-activated protein kinase (MAPK) and caspase-3 were also activated in BV-2 cells under H2O2 stress. Sesamolin was able to inhibit H2O2-induced p38 MAPK and caspase-3 activation and cell death. In addition, sesamolin preserved superoxide dismutase and catalase activities in BV-2 cells under H2O2 stress. In conclusion, sesamolin protects microglia against H2O2-induced cell injury and this protective effect was accompanied by its inhibition of p38 MAPK and caspase-3 activation and ROS production.
Asunto(s)
Dioxoles/farmacología , Peróxido de Hidrógeno/toxicidad , Microglía/efectos de los fármacos , Microglía/enzimología , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Antioxidantes/metabolismo , Caspasa 3 , Caspasas/metabolismo , Línea Celular , Activación Enzimática/efectos de los fármacos , Humanos , Oxidación-Reducción/efectos de los fármacos , Fosforilación , Especies Reactivas de Oxígeno/metabolismoRESUMEN
H5N1 influenza virus is a highly pathogenic virus, posing a pandemic threat. Previously, we showed that phenyl analogs of α-galactosylceramide (α-GalCer) displayed greater NKT stimulation than α-GalCer. Here, we examined the adjuvant effects of one of the most potent analogs, C34, on consensus hemagglutinin based DNA vaccine (pCHA5) for H5N1 virus. Upon intramuscular electroporation of mice with pCHA5 with/without various α-GalCer analogs, C34-adjuvanted group developed the highest titer against consensus H5 and more HA-specific IFN-γ secreting CD8 cells (203±13.5) than pCHA5 alone (152.6±13.7, p<0.05). Upon lethal challenge of NIBRG-14 virus, C34-adjuvanted group (84.6%) displayed higher survival rate than pCHA5 only group (46.1%). In the presence of C34 as adjuvant, the antisera displayed broader and greater neutralizing activities against virions pseudotyped with HA of clade 1, and 2.2 than pCHA5 only group. Moreover, to simulate an emergency response to a sudden H5N1 outbreak, we injected mice intramuscularly with single dose of a new consensus H5 (pCHA5-II) based on 1192 full-length H5 sequences, with C34 as adjuvant. The latter not only enhanced the humoral immune response and protection against virus challenge, but also broadened the spectrum of neutralization against pseudotyped HA viruses. Our vaccine strategy can be easily implemented for any H5N1 virus outbreak by single IM injection of a consensus H5 DNA vaccine based on updated HA sequences using C34 as an adjuvant.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Glucolípidos/farmacología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Células Asesinas Naturales/efectos de los fármacos , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Electroporación , Femenino , Glucolípidos/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Inyecciones Intramusculares , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/prevención & control , Análisis de Supervivencia , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
Alpha-galactosylceramide (alpha-GalCer), a glycolipid that stimulates natural killer T (NKT) cells to produce both T helper (Th)1 and Th2 cytokines, has shown antitumor effects in mice but failed in clinical trials. We evaluated 16 analogs of alpha-GalCer for their CD1-mediated T cell receptor (TCR) activation of naïve human NKT cells and their anticancer efficacy. In vitro, glycolipids containing an aromatic ring in their acyl tail or sphingosine tail were more effective than alpha-GalCer in inducing Th1 cytokines/chemokines, TCR activation, and human NKT cell expansion. None of these glycolipids could directly stimulate human dendritic cell maturation, except for a glycolipid with an aromatic ring on the sphingosine tail. Here, we show that glycolipids activated the TCR of NKT cells with phosphorylation of CD3epsilon, ERK1/2, or CREB, which correlated with their induction of Th1 cytokines. Notably, the extent of NKT cell activation when glycolipid was presented by antigen-presenting cells was greater than when glycolipid was presented by non-antigen-presenting cells. In vivo, in mice bearing breast or lung cancers, the glycolipids that induced more Th1-biased cytokines and CD8/CD4 T cells displayed significantly greater anticancer potency than alpha-GalCer. These findings indicate that alpha-GalCer analogs can be designed to favor Th1-biased immunity, with greater anticancer efficacy and other immune-enhancing activities than alpha-GalCer itself.