RESUMEN
Current influenza virus vaccines are focused on humoral immunity and are limited by the short duration of protection, narrow cross-strain efficacy, and suboptimal immunogenicity. Here, we combined two chemically and biologically distinct adjuvants, an oil-in-water nanoemulsion (NE) and RNA-based agonists of RIG-I, to determine whether the diverse mechanisms of these adjuvants could lead to improved immunogenicity and breadth of protection against the influenza virus. NE activates TLRs, stimulates immunogenic apoptosis, and enhances cellular antigen uptake, leading to a balanced TH1/TH2/TH17 response when administered intranasally. RIG-I agonists included RNAs derived from Sendai and influenza viral defective interfering RNAs (IVT DI, 3php, respectively) and RIG-I/TLR3 agonist, poly(I:C) (pIC), which induce IFN-Is and TH1-polarized responses. NE/RNA combined adjuvants potentially allow for costimulation of multiple innate immune receptor pathways, more closely mimicking patterns of activation occurring during natural viral infection. Mice intranasally immunized with inactivated A/Puerto Rico/8/1934 (H1N1) (PR/8) adjuvanted with NE/IVT DI or NE/3php (but not NE/pIC) showed synergistic enhancement of systemic PR/8-specific IgG with significantly greater avidity and virus neutralization activity than the individual adjuvants. Notably, NE/IVT DI induced protective neutralizing titers after a single immunization. Hemagglutinin stem-specific antibodies were also improved, allowing recognition of heterologous and heterosubtypic hemagglutinins. All NE/RNAs elicited substantial PR/8-specific sIgA. Finally, a unique cellular response with enhanced TH1/TH17 immunity was induced with the NE/RNAs. These results demonstrate that the enhanced immunogenicity of the adjuvant combinations was synergistic and not simply additive, highlighting the potential value of a combined adjuvant approach for improving the efficacy of vaccination against the influenza virus.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Portadores de Fármacos/química , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/prevención & control , ARN Interferente Pequeño/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Administración Intranasal , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Perros , Emulsiones , Femenino , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunidad Mucosa , Inmunogenicidad Vacunal , Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Ratones , Nanopartículas/química , Poli I-C/administración & dosificación , Cultivo Primario de Células , ARN Interferente Pequeño/inmunología , Vacunación/métodosRESUMEN
The co-precipitation of calcium phosphate nanoparticles (CaPs) in the presence of nucleotide chains such as polynucleotides (i.e., plasmid DNA and siRNA) and oligonucleotides has been extensively used for pre-clinical gene or drug delivery and immunotherapy studies. However, the exact role of these molecules in mineralization and tuning the physicochemical characteristics of the synthesized CaPs is still not entirely clear. In this study, we evaluated the effects of three different CpG oligodeoxynucleotides (ODN) and two representative nucleic acids (siRNA and DNA), when used as templates for the formation of CaPs. We examined the influence of CpGs with naturally-occurring phosphodiester or modified phosphorothioate backbones on the homogeneous formation of CaPs from a modified simulated body fluid solution. The hydrodynamic size, size polydispersity, morphology and surface charge of the CaPs were used as the most critical checkpoints to unravel the involved mechanisms. Our results show that the characteristics of CaPs are highly dependent on the composition, backbone, sequence and concentrations of the CpGs. The CpG type and concentration control the size distribution of the mineralized CaPs and their immunostimulation performance as verified by the activation of dendritic cells and secretion of the pro-inflammatory interleukin-6 (IL-6) cytokine, type I interferon-α (IFN-α) and co-stimulatory CD80, CD86 and CD40 markers. This study paves the way for better design of more efficient CaPs loaded with different types of CpGs for immunostimulation applications as vaccine adjuvants.
Asunto(s)
Adyuvantes Inmunológicos/química , Fosfatos de Calcio/química , Nanopartículas/química , Oligodesoxirribonucleótidos/química , Línea Celular , Precipitación Química , ADN/química , ADN/inmunología , Células Dendríticas/inmunología , Humanos , Estructura Molecular , Oligodesoxirribonucleótidos/inmunología , ARN Interferente Pequeño/química , ARN Interferente Pequeño/inmunologíaRESUMEN
As one of the intractable challenges in the clinic, the treatment of acute liver failure (ALF) is limited due to high mortality and resource cost. RNA interference (RNAi) provides a new modality for the anti-inflammatory therapy of ALF, while its therapeutic efficacy is greatly hampered by the lack of effective carriers to cooperatively overcome the various systemic barriers. Herein, we developed macrophage-targeting and reactive oxygen species (ROS)-responsive polyplexes to enable efficient systemic delivery of TNF-α siRNA (siTNF-α) to attenuate hepatic inflammation in mice bearing ALF. Se-PEI, obtained from the cross-linking of 600 Da polyethylenimine (PEI) via the ROS-responsive diselenide bond, was developed to condense siTNF-α, and the obtained polyplexes were further coated with carboxylated mannan (Man-COOH). Man-COOH coating allowed active targeting of polyplexes to macrophages with over-expressed mannose receptors (MRs), and it shielded the surface positive charges to enhance the serum stability of polyplexes. Se-PEI could be degraded by ROS in inflammatory macrophages to promote intracellular siRNA release to potentiate the gene knockdown efficiency, and in the meantime reduce the material cytotoxicity associated with high molecular weight. As such, i.v. injected Man-COOH/Se-PEI/siTNF-α polyplexes afforded notable TNF-α silencing by â¼80% in inflamed liver tissues at 500 µg siRNA per kg, and notably reduced serum TNF-α levels to achieve potent anti-inflammatory performance against ALF.
Asunto(s)
Antiinflamatorios/farmacología , Fallo Hepático Agudo/terapia , Hígado/inmunología , Terapia Molecular Dirigida , Polietileneimina/química , ARN Interferente Pequeño/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Antiinflamatorios/química , Galactosamina/administración & dosificación , Regulación de la Expresión Génica , Humanos , Inyecciones Intravenosas , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Lipopolisacáridos/administración & dosificación , Hígado/metabolismo , Hígado/patología , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/inmunología , Fallo Hepático Agudo/patología , Masculino , Mananos/inmunología , Mananos/metabolismo , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/inmunología , Ratones , Ratones Endogámicos C57BL , Células RAW 264.7 , ARN Interferente Pequeño/inmunología , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Selenio/química , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Macrophage polarization is flexible, and involves in different signaling pathways and various transcription factors. Suppressor of cytokine signaling (SOCS) is an important inhibitor of cytokine signaling pathways and also a key physiological regulator for natural and acquired immunity systems. Following transfection of SOCS1 short hairpin (sh)RNA into mouse macrophage cells, reverse transcriptionquantitative polymerase chain reaction demonstrated that the mRNA levels of Janus kinase (JAK)1 and signal transducer and activator of transcription (STAT)1 increased significantly. In addition, western blotting indicated that JAK1, STAT1 and pSTAT1 expression was significantly enhanced. Fludarabine can inhibit phosphorylation of STAT1 and SOCS1 expression. When fludarabine was added and SOCS1 shRNA was transfected, the inhibition of fludarabine was weakened, and pSTAT1 expression was upregulated. Flow cytometry detection indicated that, following the downregulation of SOCS1 expression, M1type cells significantly increased, but the proportion of M2type cells did not change significantly. Fludarabine can reduce the effect of SOCS1 shRNA on promoting M1type cell polarization, and macrophages can polarize into both M1 and M2 phenotypes. Further ELISA results presented that, when downregulating SOCS1 expression, interleukin (IL)4 and IL10 expression was both downregulated, and tumor necrosis factor (TNF)α and interferon (IFN)γ expression was significantly upregulated. When adding fludarabine or injecting with the traditional Chinese medicine Xuebijing, IL4 and IL10 expression was both significantly upregulated, and TNFα and IFNγ expression was significantly downregulated. When adding fludarabine and downregulating SOCS1, IL4, IL10, TNFα and IFNγ expression presented no significant changes. The above results indicated that, when SOCS1 expression is downregulated, it will activate the JAK1/STAT1 pathway, and thereby promote the polarization of macrophages into M1 type. The findings are of great importance for understanding occurrence, development and treatment of various immunerelated diseases.
Asunto(s)
Janus Quinasa 1/inmunología , Macrófagos Peritoneales/inmunología , Factor de Transcripción STAT1/inmunología , Transducción de Señal/inmunología , Proteína 1 Supresora de la Señalización de Citocinas/inmunología , Animales , Antiinflamatorios/farmacología , Diferenciación Celular , Medicamentos Herbarios Chinos/farmacología , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Janus Quinasa 1/genética , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Fosforilación , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Factor de Transcripción STAT1/agonistas , Factor de Transcripción STAT1/genética , Proteína 1 Supresora de la Señalización de Citocinas/antagonistas & inhibidores , Proteína 1 Supresora de la Señalización de Citocinas/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Vidarabina/análogos & derivados , Vidarabina/antagonistas & inhibidores , Vidarabina/farmacologíaRESUMEN
BACKGROUND: The immunomodulatory and anti-inflammatory functions of mesenchymal stem cells (MSCs) have been demonstrated in several autoimmune/inflammatory diseases, but their contribution to allergic conjunctivitis and underlying antiallergic mechanisms remain elusive. OBJECTIVE: We sought to explore the clinical application of MSCs to experimental allergic conjunctivitis (EAC) and its underlying antiallergic mechanisms. METHODS: Culture medium from TNF-α-stimulated, bone marrow-derived MSCs (MSC-CMT) was administered topically to mice with EAC, and the related allergic symptoms and biological changes were evaluated. Murine spleen-derived B cells, bone marrow-derived mast cells (MCs), and lung vascular endothelial cells were cultured in vitro to investigate the antiallergic MSC-CMT mechanisms. RESULTS: Topical instillation of MSC-CMT significantly attenuated the clinical symptoms of short ragweed pollen-induced EAC, with a significant decrease in inflammatory cell frequency, nuclear factor κB p65 expression, and TNF-α and IL-4 production. In vitro MSC-CMT significantly inhibited the activation of MCs and B-cell IgE release and reduced histamine-induced vascular hyperpermeability. During EAC, MSC-CMT treatment also decreased IgE production, histamine release, enrichment and activation of MCs, and conjunctival vascular hyperpermeability. The MSC-CMT-mediated inhibition of B cells, MCs, and histamine and its antiallergic effects during EAC were abrogated when MSCs were pretreated with COX2 small interfering RNA. CONCLUSIONS: Our findings provide compelling evidence that MSC-CMT inhibits EAC through COX2-dependent multiple antiallergic mechanisms and support the use of MSC-CMT as a novel strategy for treating allergic conjunctivitis.
Asunto(s)
Conjuntiva/efectos de los fármacos , Conjuntivitis Alérgica/tratamiento farmacológico , Medios de Cultivo Condicionados/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/inmunología , Ambrosia/química , Ambrosia/inmunología , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/patología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/inmunología , Diferenciación Celular , Conjuntiva/inmunología , Conjuntiva/patología , Conjuntivitis Alérgica/inducido químicamente , Conjuntivitis Alérgica/genética , Conjuntivitis Alérgica/inmunología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/inmunología , Regulación de la Expresión Génica , Histamina/metabolismo , Inmunoglobulina E/genética , Interleucina-4/genética , Interleucina-4/inmunología , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos BALB C , Polen/inmunología , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Understanding of the roles of noncoding RNAs (ncRNAs) within complex organisms has fundamentally changed. It is increasingly possible to use ncRNAs as diagnostic and therapeutic tools in medicine. Regarding disease pathogenesis, it has become evident that confinement to the analysis of protein-coding regions of the human genome is insufficient because ncRNA variants have been associated with important human diseases. Thus, inclusion of noncoding genomic elements in pathogenetic studies and their consideration as therapeutic targets is warranted. We consider aspects of the evolutionary and discovery history of ncRNAs, as far as they are relevant for the identification and selection of ncRNAs with likely therapeutic potential. Novel therapeutic strategies are based on ncRNAs, and we discuss here RNA interference as a highly versatile tool for gene silencing. RNA interference-mediating RNAs are small, but only parts of a far larger spectrum encompassing ncRNAs up to many kilobasepairs in size. We discuss therapeutic options in cardiovascular medicine offered by ncRNAs and key issues to be solved before clinical translation. Convergence of multiple technical advances is highlighted as a prerequisite for the translational progress achieved in recent years. Regarding safety, we review properties of RNA therapeutics, which may immunologically distinguish them from their endogenous counterparts, all of which underwent sophisticated evolutionary adaptation to specific biological contexts. Although our understanding of the noncoding human genome is only fragmentary to date, it is already feasible to develop RNA interference against a rapidly broadening spectrum of therapeutic targets and to translate this to the clinical setting under certain restrictions.
Asunto(s)
Enfermedades Cardiovasculares/terapia , Terapia Genética/métodos , Terapia Molecular Dirigida/métodos , Interferencia de ARN , ARN no Traducido/uso terapéutico , Animales , Enfermedades Cardiovasculares/genética , Dependovirus/genética , Dependovirus/inmunología , Evaluación Preclínica de Medicamentos , Estabilidad de Medicamentos , Predicción , Terapia Genética/efectos adversos , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Vectores Genéticos/uso terapéutico , Estudio de Asociación del Genoma Completo , Humanos , MicroARNs/efectos adversos , MicroARNs/inmunología , MicroARNs/fisiología , MicroARNs/uso terapéutico , Terapia Molecular Dirigida/efectos adversos , Procesamiento Postranscripcional del ARN , ARN Interferente Pequeño/efectos adversos , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/fisiología , ARN Interferente Pequeño/uso terapéutico , ARN no Traducido/efectos adversos , ARN no Traducido/clasificación , ARN no Traducido/inmunología , ARN no Traducido/farmacología , ARN no Traducido/fisiología , Especificidad por Sustrato , Transcriptoma , Investigación Biomédica TraslacionalRESUMEN
RNA interference (RNAi) has advanced into clinical trials. In spite of the progress made in systemic RNAi delivery to the liver and solid tumors, delivery of RNAi to leukocytes remains challenging and less advanced. Manipulating leukocyte function with RNAi holds great promise for streamlining the drug discovery process by facilitating in vivo drug target validation and for facilitating the development of RNAi-based therapy platforms for leukocyte-implicated diseases, such as blood cancer, inflammation, and leukocyte-tropic viral infections. In this review, progress in delivery strategies of RNAi payloads to leukocytes, which are notoriously difficult cells to transduce with RNAi, is discussed with special emphasis on the challenges and potential opportunities for manipulating leukocyte function with RNAi.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Inmunomodulación , Leucocitos/inmunología , Interferencia de ARN , ARN Interferente Pequeño/inmunología , Animales , Terapia Biológica/tendencias , Ensayos Clínicos como Asunto , Descubrimiento de Drogas , Humanos , ARN Interferente Pequeño/genéticaRESUMEN
Small interfering RNAs (siRNAs) are promising new active compounds in gene medicine but the induction of non-specific immune responses following their delivery continues to be a serious problem. With the purpose of avoiding such effects chemically modified siRNAs are tested in screening assay but often only examining the expression of specific immunologically relevant genes in selected cell populations typically blood cells from treated animals or humans. Assays using a relevant physiological state in biological models as read-out are not common. Here we use a fish model where the innate antiviral effect of siRNAs is functionally monitored as reduced mortality in challenge studies involving an interferon sensitive virus. Modifications with locked nucleic acid (LNA), altritol nucleic acid (ANA) and hexitol nucleic acid (HNA) reduced the antiviral protection in this model indicative of altered immunogenicity. For LNA modified siRNAs, the number and localization of modifications in the single strands was found to be important and a correlation between antiviral protection and the thermal stability of siRNAs was found. The previously published sisiRNA will in some sequences, but not all, increase the antiviral effect of siRNAs. The applied fish model represents a potent tool for conducting fast but statistically and scientifically relevant evaluations of chemically optimized siRNAs with respect to non-specific antiviral effects in vivo.
Asunto(s)
Antivirales/química , ARN Interferente Pequeño/química , Animales , Antivirales/inmunología , Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Humanos , Modelos Animales , Motivos de Nucleótidos , Oligonucleótidos/química , Oncorhynchus mykiss , Interferencia de ARN , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/farmacología , TemperaturaAsunto(s)
Terapia Genética/métodos , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Ensayos Clínicos como Asunto/economía , Evaluación de Medicamentos/economía , Evaluación Preclínica de Medicamentos/economía , Industria Farmacéutica/economía , Industria Farmacéutica/tendencias , Drogas en Investigación/economía , Terapia Genética/economía , Humanos , Degeneración Macular/terapia , Premio Nobel , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/farmacología , Infecciones del Sistema Respiratorio/terapiaRESUMEN
Chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids in the early 1970s pioneered a new age of therapy in treating chronic inflammatory airway diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction, inflammation and hyper-responsiveness. Fast forward 40 years: corticosteroids are still an important therapeutic intervention; however, they exhibit limited use in moderate to severe asthma and COPD. Oligonucleotide therapies are an emerging class which include the antisense, the RNAi (siRNA and miRNA), the immunomodulatory, the aptamer and the decoy approaches. As these approaches are rather recent in the respiratory field, most are still early in development. Nevertheless, with limitations of current small molecule therapies and the hurdles faced with biologics, the use of oligonucleotides is relevant and the door is open to the development of this category of therapeutics. This review focuses on the major classes of oligonucleotides that are currently in late stage preclinical or clinical development for the treatment of asthma and COPD, and discusses the implications for their use as therapies for respiratory diseases.