RESUMEN
Certain types of cationic lipids have shown promise in cancer immunotherapy, but their mechanism of action is poorly understood. In this study, we describe the properties of an immunotherapeutic consisting of the pure cationic lipid enantiomer R-1,2-dioleoyl-3-trimethyl-ammonium-propane (R-DOTAP) formulated with modified viral or self-peptide Ags. R-DOTAP formulations with peptide Ags stimulate strong cross-presentation and potent CD8 T cell responses associated with a high frequency of polyfunctional CD8 T cells. In a human papillomavirus tumor model system, a single s.c. injection of tumor-bearing mice with R-DOTAP plus human papillomavirus Ags induces complete regression of large tumors associated with an influx of Ag-specific CD8 T cells and a reduction of the ratio of regulatory/Ag-specific CD8 T cells. R-DOTAP also synergizes with an anti-PD1 checkpoint inhibitor, resulting in a significant inhibition of B16 melanoma tumor growth. We found that R-DOTAP stimulates type I IFN production by dendritic cells in vivo and in vitro. s.c. injection of R-DOTAP results in an IFN-dependent increase in draining lymph node size and a concomitant increase in CD69 expression. Using knockout mice, we show that type I IFN is required for the induction of CD8 T cell activity following administration of R-DOTAP plus Ag. This response requires Myd88 but not TRIF or STING. We also show that R-DOTAP stimulates both TLR7 and 9. Collectively, these studies reveal that R-DOTAP stimulates endosomal TLRs, resulting in a Myd88-dependent production of type I IFN. When administered with Ag, this results in potent Ag-specific CD8 T cell responses and antitumor activity.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Melanoma/terapia , Nanopartículas/metabolismo , Papillomaviridae/fisiología , Infecciones por Papillomavirus/terapia , Neoplasias Cutáneas/terapia , Linfocitos T Citotóxicos/inmunología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Ácidos Grasos Monoinsaturados/química , Humanos , Interferón Tipo I/metabolismo , Activación de Linfocitos , Melanoma/inmunología , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Nanopartículas/química , Infecciones por Papillomavirus/inmunología , Compuestos de Amonio Cuaternario/química , Neoplasias Cutáneas/inmunología , Linfocitos T Citotóxicos/trasplanteRESUMEN
The integration of inflammatory signals is paramount in controlling the intensity and duration of immune responses. Eicosanoids, particularly PGE2, are critical molecules in the initiation and resolution of inflammation and in the transition from innate to acquired immune responses. Microsomal PGE synthase 1 (mPGES1) is an integral membrane enzyme whose regulated expression controls PGE2 levels and is highly expressed at sites of inflammation. PGE2 is also associated with modulation of autoimmunity through altering the IL-23/IL-17 axis and regulatory T cell (Treg) development. During a type II collagen-CFA immunization response, lack of mPGES1 impaired the numbers of CD4+ regulatory (Treg) and Th17 cells in the draining lymph nodes. Ag-experienced mPGES1-/- CD4+ cells showed impaired IL-17A, IFN-γ, and IL-6 production when rechallenged ex vivo with their cognate Ag compared with their wild-type counterparts. Additionally, production of PGE2 by cocultured APCs synergized with that of Ag-experienced CD4+ T cells, with mPGES1 competence in the APC compartment enhancing CD4+ IL-17A and IFN-γ responses. However, in contrast with CD4+ cells that were Ag primed in vivo, exogenous PGE2 inhibited proliferation and skewed IL-17A to IFN-γ production under Th17 polarization of naive T cells in vitro. We conclude that mPGES1 is necessary in vivo to mount optimal Treg and Th17 responses during an Ag-driven primary immune response. Furthermore, we uncover a coordination of autocrine and paracrine mPGES1-driven PGE2 production that impacts effector T cell IL-17A and IFN-γ responses.
Asunto(s)
Comunicación Autocrina , Dinoprostona/metabolismo , Comunicación Paracrina , Prostaglandina-E Sintasas/genética , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Animales , Epítopos de Linfocito T/inmunología , Regulación de la Expresión Génica , Inmunización , Inmunomodulación , Activación de Linfocitos/inmunología , Ratones , Fenotipo , Prostaglandina-E Sintasas/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP4 de Receptores de Prostaglandina E/genéticaRESUMEN
Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that specifically catalyzes the conversion of PGH2 to PGE2. We showed that mPGES-1 null mice had a significantly reduced incidence and severity of collagen-induced arthritis compared with wild-type (WT) mice associated with a marked reduction in Abs to type II collagen. In this study, we further elucidated the role of mPGES-1 in the humoral immune response. Basal levels of serum IgM and IgG were significantly reduced in mPGES-1 null mice. Compared with WT mice, mPGES-1 null mice exhibited a significant reduction of hapten-specific serum Abs in response to immunization with the T cell-dependent (TD) Ag DNP-keyhole limpet hemocyanin. Immunization with the T cell-independent type 1 Ag trinitrophenyl-LPS or the T cell-independent type 2 Ag DNP-Ficoll revealed minimal differences between strains. Germinal center formation in the spleen of mPGES-1 null and WT mice were similar after immunization with DNP-keyhole limpet hemocyanin. To determine whether the effect of mPGES-1 and PGE2 was localized to hematopoietic or nonhematopoietic cells, we generated bone marrow chimeras. We demonstrated that mPGES-1 deficiency in nonhematopoietic cells was the critical factor for reduced TD Ab production. We conclude that mPGES-1 and PGE2-dependent phenotypic changes of nonhematopoietic/mesenchymal stromal cells play a key role in TD humoral immune responses in vivo. These findings may have relevance to the pathogenesis of rheumatoid arthritis and other autoimmune inflammatory diseases associated with autoantibody formation.
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Artritis Experimental/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Oxidorreductasas Intramoleculares/deficiencia , Linfocitos T/inmunología , Animales , Artritis Experimental/genética , Trasplante de Médula Ósea , Células Cultivadas , Colágeno/inmunología , Femenino , Inmunidad Humoral , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Prostaglandina-E SintasasRESUMEN
Heat shock transcription factor 1 (HSF1) is a major transcriptional regulator of the heat shock response in eukaryotic cells. HSF1 is evoked in response to a variety of cellular stressors, including elevated temperatures, oxidative stress, and other proteotoxic stressors. Previously, we demonstrated that HSF1 is activated in naive T cells at fever range temperatures (39.5°C) and is critical for in vitro T cell proliferation at fever temperatures. In this study, we demonstrated that murine HSF1 became activated to the DNA-binding form and transactivated a large number of genes in lymphoid cells strictly as a consequence of receptor activation in the absence of apparent cellular stress. Microarray analysis comparing HSF1(+/+) and HSF1(-/-) gene expression in T cells activated at 37°C revealed a diverse set of 323 genes significantly regulated by HSF1 in nonstressed T cells. In vivo proliferation studies revealed a significant impairment of HSF1(-/-) T cell expansion under conditions mimicking a robust immune response (staphylococcal enterotoxin B-induced T cell activation). This proliferation defect due to loss of HSF1 is observed even under nonfebrile temperatures. HSF1(-/-) T cells activated at fever temperatures show a dramatic reduction in cyclin E and cyclin A proteins during the cell cycle, although the transcription of these genes was modestly affected. Finally, B cell and hematopoietic stem cell proliferation from HSF1(-/-) mice, but not HSF1(+/+) mice, were also attenuated under stressful conditions, indicating that HSF1 is critical for the cell cycle progression of lymphoid cells activated under stressful conditions.
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Proteínas de Unión al ADN/metabolismo , Activación de Linfocitos , Estrés Fisiológico , Linfocitos T/inmunología , Factores de Transcripción/metabolismo , Animales , Ciclo Celular , División Celular , Proliferación Celular , Células Cultivadas , Ciclina A/biosíntesis , Ciclina E/biosíntesis , Proteínas de Unión al ADN/genética , Enterotoxinas/inmunología , Fiebre/inmunología , Regulación de la Expresión Génica , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/genéticaRESUMEN
Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.
RESUMEN
It is clear that new approaches are needed to promote broadly protective immunity to viral pathogens, particularly those that are prone to mutation and escape from antibody-mediated immunity. Prototypic pathogens of this type are influenza and SARS-CoV-2, where the receptor-binding protein exhibits extremely high variability in its receptor-binding regions. T cells, known to target many viral proteins, and within these, highly conserved peptide epitopes, can contribute greatly to protective immunity through multiple mechanisms but are often poorly recruited by current vaccine strategies. Here, we have studied a promising novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP), which was previously recognized for its ability to generate anti-tumor immunity through the induction of potent cytotoxic CD8 T cells. Using a preclinical mouse model, we have assessed an R-DOTAP nanoparticle adjuvant system for its ability to promote CD4 T cell responses to vaccination with recombinant influenza protein. Our studies revealed that R-DOTAP consistently outperformed a squalene-based adjuvant emulsion, even when it was introduced with a potent TLR agonist CpG, in the ability to elicit peptide epitope-specific CD4 T cells when quantified by IFN-γ and IL-2 ELISpot assays. Clinical testing of R-DOTAP containing vaccines in earlier work by others has demonstrated an acceptable safety profile. Hence, R-DOTAP can offer exciting opportunities as an immune stimulant for next-generation prophylactic recombinant protein-based vaccines.
Asunto(s)
COVID-19 , Vacunas contra la Influenza , Gripe Humana , Nanopartículas , Animales , Ratones , Humanos , Gripe Humana/prevención & control , Hemaglutininas , Escualeno , Linfocitos T CD4-Positivos , SARS-CoV-2 , Adyuvantes Inmunológicos , Vacunas Sintéticas , Vacunación , CationesRESUMEN
Adjuvants are essential components of subunit vaccines added to enhance immune responses to antigens through immunomodulation. Very few adjuvants have been approved for human use by regulatory agencies due to safety concerns. Current subunit vaccine adjuvants approved for human use are very effective in promoting humoral immune responses but are less effective at promoting T-cell immunity. In this study, we evaluated a novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) as an immunomodulator for subunit vaccines capable of inducing both humoral- and cellular-mediated immunity. Using recombinant protein antigens derived from SARS-CoV2 spike or novel computationally optimized broadly reactive influenza antigen (COBRA) proteins, we demonstrated that R-DOTAP nanoparticles promoted strong cellular- and antibody-mediated immune responses in both monovalent and bivalent vaccines. R-DOTAP-based vaccines induced antigen-specific and polyfunctional CD8+ and CD4+ effector T cells and memory T cells, respectively. Antibody responses induced by R-DOTAP showed a balanced Th1/Th2 type immunity, neutralizing activity and protection of mice from challenge with live SARS-CoV2 or influenza viruses. R-DOTAP also facilitated significant dose sparing of the vaccine antigens. These studies demonstrate that R-DOTAP is an excellent immune stimulator for the production of next-generation subunit vaccines containing multiple recombinant proteins.
Asunto(s)
COVID-19 , ARN Viral , Animales , Humanos , Ratones , Adyuvantes Inmunológicos , Cationes , COVID-19/prevención & control , Ácidos Grasos Monoinsaturados , Inmunidad , Lípidos , SARS-CoV-2 , Vacunas Sintéticas/genética , Anticuerpos Antivirales/inmunologíaRESUMEN
Heat shock factor 1 (HSF1) is a stress-induced transcription factor that promotes expression of genes that protect mammalian cells from the lethal effects of severely elevated temperatures (>42°C). However, we recently showed that HSF1 is activated at a lower temperature (39.5°C) in T cells, suggesting that HSF1 may be important for preserving T cell function during pathogen-induced fever responses. To test this, we examined the role of HSF1 in clearance of Listeria monocytogenes, an intracellular bacterial pathogen that elicits a strong CD8(+) T cell response in mice. Using temperature transponder microchips, we showed that the core body temperature increased approximately 2°C in L. monocytogenes-infected mice and that the fever response was maintained for at least 24 h. HSF1-deficient mice cleared a low-dose infection with slightly slower kinetics than did HSF1(+/+) littermate controls but were significantly more susceptible to challenges with higher doses of bacteria. Surprisingly, HSF1-deficient mice did not show a defect in CD8(+) T cell responses following sublethal infection. However, when HSF1-deficient mice were challenged with high doses of L. monocytogenes, increased levels of serum tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) compared to those of littermate control mice were observed, and rapid death of the animals occurred within 48 to 60 h of infection. Neutralization of TNF-α enhanced the survival of HSF1-deficient mice. These results suggest that HSF1 is needed to prevent the overproduction of proinflammatory cytokines and subsequent death due to septic shock that can result following high-dose challenge with bacterial pathogens.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Fiebre/metabolismo , Listeria monocytogenes , Listeriosis/metabolismo , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Linfocitos T CD8-positivos/fisiología , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Genotipo , Factores de Transcripción del Choque Térmico , Interferón gamma/sangre , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The purpose of this study was to design novel nanocapsules (NCs) with surface-chelated nickel (Ni-NCs) as a vaccine delivery system for histidine (His)-tagged protein antigens. Ni-NCs were characterized for binding His-tagged model proteins through high-affinity non-covalent interactions. The mean diameter and zeta potential of the optimized Ni-NCs were 214.9 nm and -14.8 mV, respectively. The optimal binding ratio of His-tagged Green Fluorescent Protein (His-GFP) and His-tagged HIV-1 Gag p41 (His-Gag p41) to the Ni-NCs was 1:221 and 1:480 w/w, respectively. Treatment of DC2.4 cells with Ni-NCs did not result in significant loss in the cell viability up to 24h (<5%). We further evaluated the antibody response of the Ni-NCs using His-Gag p41 as a model antigen. Formulations were administered subcutaneously to BALB/c mice at day 0 (prime) and 14 (boost) followed by serum collection on day 28. Serum His-Gag p41-specific antibody levels were found to be significantly higher at 1 and 0.5 µg doses of Gag p41-His-Ni-NCs (His-Gag p41 equivalent) compared with His-Gag p41 (1 µg) adjuvanted with aluminum hydroxide (AH). The serum IgG2a levels induced by Gag p41-His-Ni-NCs (1 µg) were significantly higher than AH adjuvanted His-Gag p41. The Ni-NCs alone did not result in the elevation of systemic IL-12/p40 and CCL5/RANTES inflammatory cytokine levels upon subcutaneous administration in BALB/c mice. In conclusion, the proposed Ni-NCs can bind His-tagged proteins and have the potential to be used as antigen delivery system capable of generating strong antigen-specific antibodies at doses much lower than with aluminum-based adjuvant and causing no significant elevation of systemic pro-inflammatory IL-12/p40 and CCL5/RANTES cytokines.
Asunto(s)
Antígenos/inmunología , Histidina/química , Nanocápsulas , Vacunas/administración & dosificación , Animales , Antígenos/administración & dosificación , Citocinas/inmunología , Células Dendríticas/inmunología , Femenino , Proteínas Fluorescentes Verdes/administración & dosificación , Proteínas Fluorescentes Verdes/inmunología , Inflamación/inmunología , Inyecciones Subcutáneas , Lípidos/química , Ratones , Ratones Endogámicos BALB C , Níquel/química , Tamaño de la Partícula , Vacunas/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/administración & dosificación , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
The present studies were aimed at investigating the engineering of NPs with protein-conjugated-surfactant at their surface. In order to increase the immunogenicity of a protein antigen, Brij 78 was functionalized by tresyl chloride and then further reacted with the primary amine of the model proteins ovalbumin (OVA) or horseradish peroxide (HRP). The reaction yielded Brij 78-OVA and Brij 78-HRP conjugates which were then used directly to form NP-OVA or NP-HRP using a one-step warm oil-in-water microemulsion precursor method with emulsifying wax as the oil phase, and Brij 78 and the Brij 78-OVA or Brij 78-HRP conjugate as surfactants. Similarly, Brij 700 was conjugated to HIV p24 antigen to yield Brij 700-p24 conjugate. The utility of these NPs for enhancing the immune responses to protein-based vaccines was evaluated in vivo using ovalbumin (OVA) as model protein and p24 as a relevant HIV antigen. In separate in vivo studies, female BALB/c mice were immunized by subcutaneous (s.c.) injection with NP-OVA and NP-p24 formulations along with several control formulations. These results suggested that with multiple antigens, covalent attachment of the antigen to the NP significantly enhanced antigen-specific immune responses. This facile covalent conjugation and incorporation method may be utilized to further incorporate other protein antigens, even multiple antigens, into an enhanced vaccine delivery system.
Asunto(s)
Adyuvantes Inmunológicos , Lípidos/química , Nanopartículas , Polietilenglicoles/química , Proteínas/química , Sulfonas/química , Adyuvantes Inmunológicos/química , Animales , Formación de Anticuerpos , Sistemas de Liberación de Medicamentos , Femenino , Proteína p24 del Núcleo del VIH/química , Proteína p24 del Núcleo del VIH/inmunología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/química , Ovalbúmina/inmunología , Proteínas/inmunología , Vacunas/química , Vacunas/inmunologíaRESUMEN
Lipid-based nanoparticles (NPs) with a small amount of surface-chelated nickel (Ni-NPs) were developed to easily formulate the HIV his-tagged Tat protein, as well as to formulate and co-deliver two HIV antigens (his-p24 and his-Nef) on one particle. Female BALB/c mice were immunized by s.c. injection with his-Tat/Ni-NP formulation (1.5 µg Tat-his/mouse) and control formulations on day 0 and 14. The day 28 anti-Tat specific IgG titer with his-Tat/Ni-NP was significantly greater than that with Alum/his-Tat. Furthermore, splenocytes from his-Tat/Ni-NP immunized mice secreted significantly higher IFN-γ than those from mice immunized with Alum/his-Tat. Although Ni-NPs did not show better adjuvant activity than Tat-coated anionic NPs made with sodium dodecyl sulfate (SDS/NPs), they were less toxic than SDS/NPs. The initial results indicated that co-immunization of mice using his-p24/his-Nef/Ni-NP induced greater antibody response compared to using Alum/his-p24/his-Nef. Co-delivery of two antigens using Ni-NPs also increased the immunogenicity of individual antigens compared to delivery of a single antigen by Ni-NPs. In conclusion, Ni-NPs are an efficient delivery system for HIV vaccines including both single antigen delivery and multiple antigen co-delivery.
RESUMEN
Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase and specifically catalyzes the conversion of PGH(2) to PGE(2). The present study demonstrates the effect of genetic deletion of mPGES-1 on the developing immunologic responses and its impact on the clinical model of bovine collagen-induced arthritis. mPGES-1 null and heterozygous mice exhibited decreased incidence and severity of arthritis compared with wild-type mice in a gene dose-dependent manner. Histopathological examination revealed significant reduction in lining hyperplasia and tissue destruction in mPGES-1 null mice compared with their wild-type littermates. mPGES-1 deficient mice also exhibited attenuation of mechanical nociception in a gene dose-dependent manner. In addition, mPGES-1 null and heterozygous mice showed a marked reduction of serum IgG against type II collagen, including subclasses IgG1, IgG2a, IgG2b, IgG2c, and IgG3, compared with wild-type mice, which correlated with the reduction in observed inflammatory features. These results demonstrate for the first time that deficiency of mPGES-1 inhibits the development of collagen-induced arthritis, at least in part, by blocking the development of a humoral immune response against type II collagen. Pharmacologic inhibition of mPGES-1 may therefore impact both the inflammation and the autoimmunity associated with human diseases such as rheumatoid arthritis.
Asunto(s)
Artritis Experimental/enzimología , Artritis Experimental/terapia , Colágeno Tipo II/inmunología , Ciclooxigenasa 1/deficiencia , Ciclooxigenasa 1/genética , Inmunoglobulina G , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Microsomas/enzimología , Índice de Severidad de la Enfermedad , Animales , Artritis Experimental/genética , Artritis Experimental/inmunología , Bovinos , Colágeno Tipo II/administración & dosificación , Ciclooxigenasa 1/fisiología , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/fisiología , Femenino , Eliminación de Gen , Tamización de Portadores Genéticos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulina G/fisiología , Inmunoglobulina M/biosíntesis , Incidencia , Masculino , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , ARN Mensajero/biosíntesisRESUMEN
Heat shock factor-1 (HSF1) is a transcription factor that serves as the major temperature-inducible sensor for eukaryotic cells. In most cell types, HSF1 becomes activated to the DNA binding form at 42 degrees C and mediates the classical heat shock response, protecting the cells from subsequent lethal temperatures. We have recently demonstrated that HSF1 is activated at a lower temperature in T lymphocytes than in most other cell types (39 degrees C vs 42 degrees C), within the physiological range of fever. In this study, we show that T cell activation at fever temperatures not only activates HSF1 but induces the up-regulation of the HSF1 protein and the HSF1-regulated protein, HSP70i. T cells from HSF1 knockout mice proliferate normally under optimal conditions but are impaired in proliferation at physiological fever temperatures and low CO2 concentrations, conditions that do not impair wild-type T cells. This defect in proliferation appears to be mediated by a block in the G1/S transition of the cell cycle and is independent of HSP70. Elevated temperature and low CO2 concentrations resulted in a dramatic reduction of the intracellular reactive oxygen species (ROS) levels in both normal and knockout T cells. Wild-type T cells were able to restore ROS levels to normal within 5 h, whereas HSF1-/- T cells were not. These results suggest that the proliferation defect seen in T cells from HSF1-/- mice at fever temperatures was because of dysregulated ROS levels and that HSF1 is important in maintaining ROS homeostasis and cell cycle progression under the stressful conditions encountered during fever.
Asunto(s)
Temperatura Corporal/inmunología , Proteínas de Unión al ADN/fisiología , Fiebre/inmunología , Linfocitos T/inmunología , Factores de Transcripción/fisiología , Animales , Proliferación Celular , Proteínas de Unión al ADN/genética , Fase G1/genética , Fase G1/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico , Activación de Linfocitos , Ratones , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismo , Fase S/genética , Fase S/inmunología , Factores de Transcripción/genéticaRESUMEN
PURPOSE: The purpose of these studies was to prepare nanoparticles (NPs) with a small amount of surface-chelated nickel for obtaining enhanced binding of histidine-tagged (his-tag) proteins compared to non-histidine-tagged protein binding to charged nanoparticles. MATERIALS AND METHODS: NPs were prepared from oil-in-water microemulsion precursors using emulsifying wax, 3 mM Brij 78 and 0.1 mM DOGS-NTA-Ni lipid (referred to as Ni-NPs). The amount of lipid entrapped in the NPs was quantitated by atomic emission spectroscopy (AES). The Ni-NPs were investigated for binding to two his-tag proteins, green fluorescent protein (GFP) and his-tag HIV-1 Gag p24. In vivo studies in mice were carried out to evaluate the immune responses obtained to his-tag Gag p24 bound to Ni-NPs. RESULTS: AES studies demonstrated that approximately 5% of the DOGS-NTA-Ni lipid used was entrapped in the NPs. The optimal binding ratio his-tag GFP and his-tag Gag p24 to Ni-NPs was found to be 1:33.7 and 1:35.4 w/w, respectively. This interaction was stable at 37 degrees C in PBS, pH 7.4 over 4 h and the interaction of his-tag GFP with the Ni-NPs was enhanced compared to control NPs prepared with no Ni on the surface (NTA-NPs). The in vivo studies demonstrated enhanced serum IgG and IgG2a responses to his-tag Gag p24 bound to Ni-NPs compared to protein adjuvanted with Alum or adsorbed on the surface of control NTA-NPs. CONCLUSIONS: Ni-NPs can be used to bind strongly to his-tag proteins. This system was demonstrated to have potential applications in vaccine delivery for enhancing immune responses to protein-based vaccines.
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Antígenos/química , Histidina/química , Nanopartículas , Níquel/química , Proteínas/química , Western Blotting , Recuento de Linfocito CD4 , Proliferación Celular/efectos de los fármacos , Química Farmacéutica , Emulsiones , Ensayo de Inmunoadsorción Enzimática , Proteínas Fluorescentes Verdes/química , Proteína p24 del Núcleo del VIH/química , Concentración de Iones de Hidrógeno , Inmunoglobulina G/biosíntesis , Interferón gamma/metabolismo , Polietilenglicoles , Unión Proteica , Espectrofotometría Atómica , Bazo/citología , Replicación Viral/efectos de los fármacos , CerasRESUMEN
HIV-1 Tat has been identified as an attractive target for vaccine development and is currently under investigation in clinical trials as both a therapeutic and preventative vaccine for HIV-1. It is well known that protein based vaccines produce poor immune responses by themselves and therefore require adjuvants to enhance immune responses. We have previously reported on the use of anionic nanoparticles (NPs) for enhancing cellular and humoral immune responses to Tat (1-72). The purpose of this study was to further evaluate the immune response of HIV-1 Tat (1-72) coated on anionic nanoparticles compared to alum using various doses of Tat (1-72). Nanoparticles were effective at generating comparable antibody titers at both 1 and 5 microg doses of Tat (1-72), whereas the antibody titers significantly decreased at the lower dose of Tat (1-72) using alum. Anti-sera from Tat (1-72) immunized mice reacted greatest to the N-terminal and basic regions of Tat, with the NP groups showing stronger reactivity to these regions compared to alum. Moreover, the anti-sera from all Tat (1-72) immunized groups contained Tat-neutralizing antibodies and were able to significantly inhibit Tat-mediated long terminal repeat (LTR) transactivation.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Compuestos de Alumbre/farmacología , Productos del Gen tat/inmunología , Anticuerpos Anti-VIH/sangre , Nanoestructuras , Vacunas contra el SIDA/inmunología , Animales , Relación Dosis-Respuesta Inmunológica , Mapeo Epitopo , Femenino , Duplicado del Terminal Largo de VIH , Inmunización , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Activación TranscripcionalRESUMEN
The induction of heat shock protein gene expression in response to stress is critical for the ability of organisms to cope with and survive exposure to these stresses. However, most studies on HSF1-mediated induction of hsp70 gene expression have utilized immortalized cell lines and temperatures above the physiologically relevant range. For these reasons much less is known about the heat shock response as it occurs in mammalian cells within tissues in the intact organism. To gain insight into this area we determined the temperature thresholds for activation of HSF1 DNA binding in different mouse tissues. We have found that HSF1 DNA binding activity and hsp70 synthesis are induced in spleen cells at significantly lower temperatures relative to cells of other tissues, with a temperature threshold for activation (39 degrees C) that is within the physiological range for fever. Furthermore, we found that the lowered temperature set point for induction of the stress response in spleen is specific to T-lymphocytes residing within this tissue and is not exhibited by B-lymphocytes. This lowered threshold is also observed in T-lymphocytes isolated from lymph nodes, suggesting that it is a general property of T-lymphocytes, and is seen in different mouse strains. Fever is an early event in the immune response to infection, and thus activation of the cellular stress response in T-lymphocytes by fever temperatures could serve as a way to give these cells enough time to express hsps in anticipation of their function in the coming immune response. The induced hsps likely protect these cells from the stressful conditions that can exist during the immune response, for example increasing their protection against stress-induced apoptosis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Linfocitos T/metabolismo , Linfocitos T/patología , Animales , Linfocitos B/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/química , Fiebre , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Estrés Fisiológico , Temperatura , Factores de Tiempo , Factores de TranscripciónRESUMEN
A significant emphasis has been placed on the development of adjuvants and/or delivery systems to improve both antibody production and cell-mediated immune responses. We previously reported on a novel anionic nanoparticle, which led to enhanced humoral and T helper type-1 (Th1) biased immune responses in mice when coated with cationized model antigen. Tat (1-72) is a conserved regulatory HIV-1 protein. It was hypothesized that HIV vaccine strategies employing Tat (1-72) may be a promising approach. Although previous reports have suggested that Tat (1-86) may be immunosuppressive, it was demonstrated in this present study that Tat (1-72) was not immunosuppressive when co-administered to mice with ovalbumin (OVA). Tat (1-72) was coated on novel anionic nanoparticles. BALB/c mice were immunized with Tat (5 microg)-coated nanoparticles (15 microg) by subcutaneous injection on days 0 and 14. Antibody and cytokine release were determined on day 28 and compared to Tat (5 microg) adjuvanted with Alum (15 microg) as a Th2 control, Tat (5 microg) adjuvanted with Lipid A (50 microg) as a Th1 control. Immunization of BALB/c mice with Tat-coated nanoparticles resulted in antibody levels (IgG and IgM) comparable to those elicited from Tat and Alum. However, Tat-coated nanoparticles led to a Th1 biased immune response. The IFN-gamma release from splenocytes with Tat-coated nanoparticles was comparable to that from mice immunized with Tat and Lipid A, and 3.3-fold greater than that from mice immunized with Tat and Alum. These studies warrant further investigation of these nanoparticles to enhance both antibody and cellular-based immune responses.