RESUMO
Recalcitrant staphylococcal osteomyelitis may be due, in part, to the ability of Staphylococcus aureus to invade bone cells. However, osteoclasts and osteoblasts are now recognized to shape host responses to bacterial infection and we have recently described their ability to produce IFN-ß following S. aureus infection and limit intracellular bacterial survival/propagation. Here, we have investigated the ability of novel, rationally designed, nucleic acid nanoparticles (NANPs) to induce the production of immune mediators, including IFN-ß, following introduction into bone cells. We demonstrate the successful delivery of representative NANPs into osteoblasts and osteoclasts via endosomal trafficking when complexed with lipid-based carriers. Their delivery was found to differentially induce immune responses according to their composition and architecture via discrete cytosolic pattern recognition receptors. Finally, the utility of this nanoparticle technology was supported by the demonstration that immunostimulatory NANPs augment IFN-ß production by S. aureus infected bone cells and reduce intracellular bacterial burden.
Assuntos
Nanopartículas , Ácidos Nucleicos , Osteoblastos , Osteoclastos , Infecções Estafilocócicas , Staphylococcus aureus , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/imunologia , Nanopartículas/química , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Animais , Ácidos Nucleicos/química , Ácidos Nucleicos/farmacologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/tratamento farmacológico , Camundongos , Interferon Tipo I/metabolismo , Humanos , Interferon beta/metabolismo , Adjuvantes Imunológicos/farmacologiaRESUMO
Staphylococcus aureus is the principal causative agent of osteomyelitis, a serious bacterial infection of bone that is associated with progressive inflammatory damage. Bone-forming osteoblasts have increasingly been recognized to play an important role in the initiation and progression of detrimental inflammation at sites of infection and have been demonstrated to release an array of inflammatory mediators and factors that promote osteoclastogenesis and leukocyte recruitment following bacterial challenge. In the present study, we describe elevated bone tissue levels of the potent neutrophil-attracting chemokines CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 in a murine model of posttraumatic staphylococcal osteomyelitis. RNA sequencing (RNA-Seq) gene ontology analysis of isolated primary murine osteoblasts showed enrichment in differentially expressed genes involved in cell migration and chemokine receptor binding and chemokine activity following S. aureus infection, and a rapid increase in the expression of mRNA encoding CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7, in these cells. Importantly, we have confirmed that such upregulated gene expression results in protein production with the demonstration that S. aureus challenge elicits the rapid and robust release of these chemokines by osteoblasts and does so in a bacterial dose-dependent manner. Furthermore, we have confirmed the ability of soluble osteoblast-derived chemokines to elicit the migration of a neutrophil-like cell line. As such, these studies demonstrate the robust production of CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by osteoblasts in response to S. aureus infection, and the release of such neutrophil-attracting chemokines provides an additional mechanism by which osteoblasts could drive the inflammatory bone loss associated with staphylococcal osteomyelitis.
Assuntos
Osteomielite , Infecções Estafilocócicas , Animais , Camundongos , Staphylococcus aureus/metabolismo , Neutrófilos/metabolismo , Quimiocinas/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Osteoblastos , Interleucina-8/metabolismo , Infecções Estafilocócicas/microbiologia , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Quimiocina CCL7/metabolismo , Quimiocina CCL3/metabolismoRESUMO
BACKGROUND: The mechanisms by which glia respond to viral central nervous system (CNS) pathogens are now becoming apparent with the demonstration that microglia and astrocytes express an array of pattern recognition receptors that include intracellular RNA and DNA sensors. We have previously demonstrated that glia express Z-DNA binding protein 1 (ZBP1) and showed that this cytosolic nucleic acid sensor contributes to the inflammatory/neurotoxic responses of these cells to herpes simplex virus-1 (HSV-1). However, the relative contribution made by ZBP1- to HSV-1-mediated cell death in glia has not been determined. METHODS: We have investigated the relative contribution made by ZBP1- to HSV-1-mediated cell death in primary astrocytes derived from mice genetically deficient in this sensor. We have used capture ELISAs and immunoblot analysis to assess inflammatory cytokine production and ZBP1 and phosphorylated mixed lineage kinase domain-like protein (MLKL) expression levels, respectively, following HSV-1 challenge. Furthermore, we have used a commercially available cell viability assay to determine the proportion and rate of cell death in cells following infection with laboratory and neuroinvasive clinical strains of HSV-1, and pharmacological inhibitors of necroptotic and apoptotic pathway components to assess the relative role of each. RESULTS: We show that the loss of ZBP1 in astrocytes results in an increase in the number of viral particles released following HSV-1 infection. Importantly, we have confirmed that HSV-1 induces necroptosis in astrocytes and have established the ability of ZBP1 to mediate this cell death pathway. Interestingly, while ZBP1 is best known for its role in necroptotic signaling, our findings indicate that this sensor can also contribute to virally induced apoptosis in these glia. CONCLUSIONS: Our findings indicate that ZBP1 serves as a restriction factor for HSV-1 infection and is associated with the induction of both necroptotic and apoptotic cell death pathways in primary murine astrocytes. While it remains to be seen whether ZBP1-mediated activation of cell death in astrocytes contributes significantly to host protection or, rather, exacerbates HSV-1 encephalitis pathology, the identification of such a role in resident CNS cells may represent a novel target for therapeutic intervention to reduce HSV encephalitis-associated morbidity and mortality.
Assuntos
Encefalite por Herpes Simples , Herpes Simples , Infecções por Herpesviridae , Herpesvirus Humano 1 , Animais , Apoptose , Astrócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNARESUMO
Nucleic acid nanoparticles (NANPs) have become powerful new platforms as therapeutic and diagnostic tools due to the innate biological ability of nucleic acids to identify target molecules or silence genes involved in disease pathways. However, the clinical application of NANPs has been limited by factors such as chemical instability, inefficient intracellular delivery, and the triggering of detrimental inflammatory responses following innate immune recognition of nucleic acids. Here, we have studied the effects of altering the chemical composition of a circumscribed panel of NANPs that share the same connectivity, shape, size, charge and sequences. We show that replacing RNA strands with either DNA or chemical analogs increases the enzymatic and thermodynamic stability of NANPs. Furthermore, we have found that such composition changes affect delivery efficiency and determine subcellular localization, effects that could permit the targeted delivery of NANP-based therapeutics and diagnostics. Importantly, we have determined that altering NANP composition can dictate the degree and mechanisms by which cell immune responses are initiated. While RNA NANPs trigger both TLR7 and RIG-I mediated cytokine and interferon production, DNA NANPs stimulate minimal immune activation. Importantly, incorporation of 2'F modifications abrogates RNA NANP activation of TLR7 but permits RIG-I dependent immune responses. Furthermore, 2'F modifications of DNA NANPs significantly enhances RIG-I mediated production of both proinflammatory cytokines and interferons. Collectively this indicates that off-target effects may be reduced and/or desirable immune responses evoked based upon NANPs modifications. Together, our studies show that NANP composition provides a simple way of controlling the immunostimulatory potential, and physicochemical and delivery characteristics, of such platforms.
Assuntos
DNA/química , Nanopartículas/química , RNA/química , Transporte Biológico , Linhagem Celular , Citocinas/biossíntese , DNA/metabolismo , Humanos , Fatores Reguladores de Interferon/metabolismo , NF-kappa B/metabolismo , Nanopartículas/metabolismo , Oligonucleotídeos/química , RNA/metabolismo , TermodinâmicaRESUMO
BACKGROUND: Bacterial meningitis and meningoencephalitis are associated with devastating neuroinflammation. We and others have demonstrated the importance of glial cells in the initiation of immune responses to pathogens invading the central nervous system (CNS). These cells use a variety of pattern recognition receptors (PRRs) to identify common pathogen motifs and the cytosolic sensor retinoic acid inducible gene-1 (RIG-I) is known to serve as a viral PRR and initiator of interferon (IFN) responses. Intriguingly, recent evidence indicates that RIG-I also has an important role in the detection of bacterial nucleic acids, but such a role has not been investigated in glia. METHODS: In this study, we have assessed whether primary or immortalized human and murine glia express RIG-I either constitutively or following stimulation with bacteria or their products by immunoblot analysis. We have used capture ELISAs and immunoblot analysis to assess human microglial interferon regulatory factor 3 (IRF3) activation and IFN production elicited by bacterial nucleic acids and novel engineered nucleic acid nanoparticles. Furthermore, we have utilized a pharmacological inhibitor of RIG-I signaling and siRNA-mediated knockdown approaches to assess the relative importance of RIG-I in such responses. RESULTS: We demonstrate that RIG-I is constitutively expressed by human and murine microglia and astrocytes, and is elevated following bacterial infection in a pathogen and cell type-specific manner. Additionally, surface and cytosolic PRR ligands are also sufficient to enhance RIG-I expression. Importantly, our data demonstrate that bacterial RNA and DNA both trigger RIG-I-dependent IRF3 phosphorylation and subsequent type I IFN production in human microglia. This ability has been confirmed using our nucleic acid nanoparticles where we demonstrate that both RNA- and DNA-based nanoparticles can stimulate RIG-I-dependent IFN responses in these cells. CONCLUSIONS: The constitutive and bacteria-induced expression of RIG-I by human glia and its ability to mediate IFN responses to bacterial RNA and DNA and nucleic acid nanoparticles raises the intriguing possibility that RIG-I may be a potential target for therapeutic intervention during bacterial infections of the CNS, and that the use of engineered nucleic acid nanoparticles that engage this sensor might be a method to achieve this goal.
Assuntos
DNA Bacteriano/imunologia , Microglia/imunologia , RNA Bacteriano/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Receptores do Ácido Retinoico/imunologia , Animais , Células Cultivadas , Humanos , Fator Regulador 3 de Interferon/biossíntese , Interferons/biossíntese , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Glia play a key role in immunosurveillance within the central nervous system (CNS) and can recognize a wide range of pathogen-associated molecular patterns (PAMPS) via members of multiple pattern recognition receptor (PRR) families. Of these, the expression of cytosolic/nuclear RNA and DNA sensors by glial cells is of particular interest as their ability to interact with intracellular nucleic acids suggests a critical role in the detection of viral pathogens. The recently discovered DNA sensors cyclic GMP-AMP synthase (cGAS) and interferon gamma-inducible protein 16 (IFI16) have been reported to be important for the recognition of DNA pathogens such as herpes simplex virus-1 (HSV-1) in peripheral human cell types, and we have recently demonstrated that human glia express cGAS and its downstream adaptor molecule stimulator of interferon genes (STING). Here, we have demonstrated that human microglial cells functionally express cGAS and exhibit robust constitutive IFI16 expression. While cGAS serves as a significant component in IRF3 activation and IFN-ß production by human microglial cells in response to foreign intracellular DNA, IFI16 is not required for such responses. Surprisingly, neither of these sensors mediate effective antiviral responses to HSV-1 in microglia, and this may be due, at least in part, to viral suppression of cGAS and/or IFI16 expression. As such, this ability may represent an important HSV immune evasion strategy in glial cells, and approaches that mitigate such suppression might represent a novel strategy to limit HSV-1-associated neuropathology.
Assuntos
DNA Viral/genética , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno/genética , Microglia/virologia , Proteínas Nucleares/genética , Nucleotidiltransferases/genética , Fosfoproteínas/genética , Astrócitos/imunologia , Astrócitos/virologia , Linhagem Celular Transformada , DNA Viral/imunologia , Regulação da Expressão Gênica , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Interferon beta/genética , Interferon beta/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Microglia/imunologia , Proteínas Nucleares/imunologia , Nucleotidiltransferases/imunologia , Fosfoproteínas/imunologia , Cultura Primária de Células , Transdução de SinaisRESUMO
Programmable nucleic acid nanoparticles (NANPs) provide controlled coordination of therapeutic nucleic acids (TNAs) and other biological functionalities. Beyond multivalence, recent reports demonstrate that NANP technology can also elicit a specific immune response, adding another layer of customizability to this innovative approach. While the delivery of nucleic acids remains a challenge, new carriers are introduced and tested continuously. Polymeric platforms have proven to be efficient in shielding nucleic acid cargos from nuclease degradation while promoting their delivery and intracellular release. Here, we venture beyond the delivery of conventional TNAs and combine the stable cationic poly-(lactide-co-glycolide)-graft-polyethylenimine with functionalized NANPs. Furthermore, we compare several representative NANPs to assess how their overall structures influence their delivery with the same carrier. An extensive study of various formulations both in vitro and in vivo reveals differences in their immunostimulatory activity, gene silencing efficiency, and biodistribution, with fibrous NANPs advancing for TNA delivery.
Assuntos
Adjuvantes Imunológicos , Inativação Gênica , Nanopartículas/química , Ácidos Nucleicos , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacocinética , Adjuvantes Imunológicos/farmacologia , Linhagem Celular Tumoral , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Humanos , Ácidos Nucleicos/química , Ácidos Nucleicos/farmacocinética , Ácidos Nucleicos/farmacologiaRESUMO
BACKGROUND: Glia are key regulators of inflammatory responses within the central nervous system (CNS) following infection or trauma. We have previously demonstrated the ability of activated glia to rapidly produce pro-inflammatory mediators followed by a transition to an anti-inflammatory cytokine production profile that includes the immunosuppressive cytokine interleukin (IL)-10 and the closely related cytokine IL-19. IL-24, another member of the IL-10 family, has been studied in a number of inflammatory conditions in the periphery and is known to modulate immune cell activity. However, the ability of glia to produce IL-24 remains unclear and the effects of this pleiotropic cytokine on glial immune functions have not been investigated. METHODS: In this study, we have assessed whether primary murine glia produce IL-24 following stimulation and evaluated the effect of this cytokine on the immune responses of such cells. We have utilized RT-PCR and immunoblot analyses to assess the expression of IL-24 and its cognate receptors by astrocytes following challenge with bacteria or their components. Furthermore, we have determined the effect of recombinant IL-24 on astrocyte immune signaling and responses to clinically relevant bacteria using RT-PCR and specific capture ELISAs. RESULTS: We demonstrate that astrocytes express IL-24 mRNA and release detectable amounts of this cytokine protein in a delayed manner following bacterial challenge. In addition, we have determined that glia constitutively express the cognate receptors for IL-24 and show that such expression can be increased in astrocytes following activation. Importantly, our results indicate that IL-24 exerts an immunosuppressive effect on astrocytes by elevating suppressor of cytokine signaling 3 expression and limiting IL-6 production following challenge. Furthermore, we have demonstrated that IL-24 can also augment the release of IL-10 by bacterially challenged astrocytes and can induce the expression of the potentially neuroprotective mediators, glutamate transporter 1, and cyclooxygenase 2. CONCLUSIONS: The expression of IL-24 and its cognate receptors by astrocytes following bacterial challenge, and the ability of this cytokine to limit inflammatory responses while promoting the expression of immunosuppressive and/or neuroprotective mediators, raises the intriguing possibility that IL-24 functions to regulate or resolve CNS inflammation following bacterial infection in order to limit neuronal damage.
Assuntos
Astrócitos/imunologia , Citocinas/imunologia , Regulação da Expressão Gênica/imunologia , Inflamação/imunologia , Animais , Astrócitos/metabolismo , Infecções Bacterianas/imunologia , Citocinas/biossíntese , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Substance P (SP) is produced at high levels in the central nervous system (CNS), and its target receptor, neurokinin 1 receptor (NK-1R), is expressed by glia and leukocytes. This tachykinin functions to exacerbate inflammatory responses at peripheral sites. Moreover, SP/NK-1R interactions have recently been associated with severe neuroinflammation and neuronal damage. We have previously demonstrated that NK-1R antagonists can limit neuroinflammatory damage in a mouse model of bacterial meningitis. Furthermore, we have since shown that these agents can attenuate Borrelia burgdorferi-induced neuronal and glial inflammatory mediator production in non-human primate brain explants and isolated neuronal cells. METHODS: In the present study, we have assessed the role played by endogenous SP/NK-1R interactions in damaging CNS inflammation in an established rhesus macaque model that faithfully reproduces the key clinical features of Lyme neuroborreliosis, using the specific NK-1R antagonist, aprepitant. We have utilized multiplex ELISA to quantify immune mediator levels in cerebrospinal fluid, and RT-PCR and immunoblot analyses to quantify cytokine and NK-1R expression, respectively, in brain cortex, dorsal root ganglia, and spinal cord tissues. In addition, we have assessed astrocyte number/activation status in brain cortical tissue by immunofluorescence staining and confocal microscopy. RESULTS: We demonstrate that aprepitant treatment attenuates B. burgdorferi-induced elevations in CCL2, CXCL13, IL-17A, and IL-6 gene expression in dorsal root ganglia, spinal cord, and/or cerebrospinal fluid of rhesus macaques at 2 to 4 weeks following intrathecal infection. In addition, we demonstrate that this selective NK-1R antagonist also prevents increases in total cortical brain NK-1R expression and decreases in the expression of the astrocyte marker, glial fibrillary acidic protein, associated with B. burgdorferi infection. CONCLUSIONS: The ability of a centrally acting NK-1R inhibitor to attenuate B. burgdorferi-associated neuroinflammatory responses and sequelae raises the intriguing possibility that such FDA-approved agents could be repurposed for use as an adjunctive therapy for the treatment of bacterial CNS infections.
Assuntos
Encefalite/tratamento farmacológico , Encefalite/etiologia , Neuroborreliose de Lyme/complicações , Morfolinas/uso terapêutico , Antagonistas dos Receptores de Neurocinina-1/uso terapêutico , Análise de Variância , Animais , Aprepitanto , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/microbiologia , Astrócitos/patologia , Borrelia burgdorferi/fisiologia , Quimiocina CCL2/líquido cefalorraquidiano , Quimiocina CXCL13/genética , Quimiocina CXCL13/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalite/patologia , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Macaca mulatta , Masculino , RNA Mensageiro/metabolismo , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Fatores de TempoRESUMO
BACKGROUND: The tachykinin substance P (SP) is recognized to exacerbate inflammation at peripheral sites via its target receptor, neurokinin 1 receptor (NK-1R), expressed by leukocytes. More recently, SP/NK-1R interactions have been associated with severe neuroinflammation and neuronal damage. We have previously demonstrated that NK-1R antagonists can limit neuroinflammatory damage in a mouse model of bacterial meningitis. Furthermore, we have since shown that these agents can attenuate bacteria-induced neuronal and glial inflammatory mediator production in nonhuman primate (NHP) brain explants and isolated neuronal cells, and following in vivo infection. METHODS: In the present study, we have assessed the ability of NHP brain explants, primary human microglia and astrocytes, and immortalized human glial cell lines to express NK-1R isoforms. We have utilized RT-PCR, immunoblot analysis, immunofluorescent microscopy, and/or flow cytometric analysis, to quantify NK-1R expression in each, at rest, or following bacterial challenge. Furthermore, we have assessed the ability of human microglia to respond to SP by immunoblot analysis of NF-kB nuclear translocation and determined the ability of this neuropeptide to augment inflammatory cytokine release and neurotoxic mediator production by human astrocytes using an ELISA and a neuronal cell toxicity assay, respectively. RESULTS: We demonstrate that human microglial and astrocytic cells as well as NHP brain tissue constitutively express robust levels of the full-length NK-1R isoform. In addition, we demonstrate that the expression of NK-1R by human astrocytes can be further elevated following exposure to disparate bacterial pathogens or their components. Importantly, we have demonstrated that NK-1R is functional in both human microglia and astrocytes and show that SP can augment the inflammatory and/or neurotoxic immune responses of glial cells to disparate and clinically relevant bacterial pathogens. CONCLUSIONS: The robust constitutive and functional expression of the full-length NK-1R isoform by human microglia and astrocytes, and the ability of SP to augment inflammatory signaling pathways and mediator production by these cells, support the contention that SP/NK-1R interactions play a significant role in the damaging neuroinflammation associated with conditions such as bacterial meningitis.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Microglia/metabolismo , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Animais , Astrócitos/imunologia , Encéfalo/imunologia , Linhagem Celular , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Macaca mulatta , Meningites Bacterianas/imunologia , Meningites Bacterianas/metabolismo , Microglia/imunologia , Técnicas de Cultura de Órgãos , Receptores da Neurocinina-1/imunologia , Substância P/imunologiaRESUMO
In the past few years, the study of therapeutic RNA nanotechnology has expanded tremendously to encompass a large group of interdisciplinary sciences. It is now evident that rationally designed programmable RNA nanostructures offer unique advantages in addressing contemporary therapeutic challenges such as distinguishing target cell types and ameliorating disease. However, to maximize the therapeutic benefit of these nanostructures, it is essential to understand the immunostimulatory aptitude of such tools and identify potential complications. This paper presents a set of 16 nanoparticle platforms that are highly configurable. These novel nucleic acid based polygonal platforms are programmed for controllable self-assembly from RNA and/or DNA strands via canonical Watson-Crick interactions. It is demonstrated that the immunostimulatory properties of these particular designs can be tuned to elicit the desired immune response or lack thereof. To advance the current understanding of the nanoparticle properties that contribute to the observed immunomodulatory activity and establish corresponding designing principles, quantitative structure-activity relationship modeling is conducted. The results demonstrate that molecular weight, together with melting temperature and half-life, strongly predicts the observed immunomodulatory activity. This framework provides the fundamental guidelines necessary for the development of a new library of nanoparticles with predictable immunomodulatory activity.
Assuntos
Imunomodulação , Microglia/citologia , Ácidos Nucleicos/química , Relação Quantitativa Estrutura-Atividade , Linhagem Celular Tumoral , DNA/química , Humanos , RNA/química , Reprodutibilidade dos TestesRESUMO
RNA nanotechnology employs synthetically modified ribonucleic acid (RNA) to engineer highly stable nanostructures in one, two, and three dimensions for medical applications. Despite the tremendous advantages in RNA nanotechnology, unmodified RNA itself is fragile and prone to enzymatic degradation. In contrast to use traditionally modified RNA strands e.g. 2'-fluorine, 2'-amine, 2'-methyl, we studied the effect of RNA/DNA hybrid approach utilizing a computer-assisted RNA tetra-uracil (tetra-U) motif as a toolkit to address questions related to assembly efficiency, versatility, stability, and the production costs of hybrid RNA/DNA nanoparticles. The tetra-U RNA motif was implemented to construct four functional triangles using RNA, DNA and RNA/DNA mixtures, resulting in fine-tunable enzymatic and thermodynamic stabilities, immunostimulatory activity and RNAi capability. Moreover, the tetra-U toolkit has great potential in the fabrication of rectangular, pentagonal, and hexagonal NPs, representing the power of simplicity of RNA/DNA approach for RNA nanotechnology and nanomedicine community.
Assuntos
DNA/química , Nanopartículas/química , Nanotecnologia/métodos , RNA/química , Uracila/química , Sequência de Bases , Linhagem Celular , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Interferência de RNA , RNA Interferente Pequeno/administração & dosagemRESUMO
A defining feature of viral central nervous system (CNS) infection is the rapid onset of severe neuroinflammation. However, the mechanisms underlying glial responses to replicative neurotropic viruses are only now becoming apparent with the discovery of a number of cytosolic sensors for viral nucleic acids. We have described the expression by murine and human glial cells of two disparate pattern recognition receptors, retinoic acid inducible gene-I (RIG-I) and DNA-dependent activator of interferon regulatory factors (DAI), receptors for viral RNA and DNA moieties, respectively. In the present study, we demonstrate the functional significance of RIG-I expression in primary murine microglia and astrocytes. Our data indicate that murine glial immune responses to a model neurotropic RNA virus, vesicular stomatitis virus, are RIG-I dependent and independent of levels of DAI expression or RNA polymerase III activity. In contrast, maximal glial inflammatory and antiviral responses to the DNA virus herpes simplex virus-1 (HSV-1) are dependent on the expression of both RIG-I and DAI, and require RNA polymerase III activity. These findings indicate that the RNA sensor, RIG-I, acts in parallel with DAI in an RNA polymerase III-dependent manner to initiate glial responses to HSV-1. We therefore suggest that RIG-I plays a significant role in the detection of both RNA and DNA pathogens by microglia and astrocytes.
Assuntos
Astrócitos/imunologia , Citocinas/metabolismo , RNA Helicases DEAD-box/metabolismo , Herpesvirus Humano 1/fisiologia , Microglia/imunologia , Vesiculovirus , Animais , Astrócitos/virologia , Encéfalo/imunologia , Encéfalo/virologia , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Proteína DEAD-box 58 , Modelos Animais de Doenças , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Humanos , Fatores Reguladores de Interferon/metabolismo , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Microglia/virologia , RNA Polimerase III/metabolismo , Proteínas Virais/metabolismoRESUMO
There is growing appreciation that resident glial cells can initiate and/or regulate inflammation following trauma or infection in the central nervous system (CNS). We have previously demonstrated the ability of microglia and astrocytes to respond to bacterial pathogens or their products by rapid production of inflammatory mediators, followed by the production of the immunosuppressive cytokine interleukin (IL)−10. IL-19, another member of the IL-10 family of cytokines, has been studied in the context of a number of inflammatory conditions in the periphery and is known to modulate immune cell activity. In the present study, we demonstrate the constitutive and/or inducible expression of IL-19 and its cognate receptor subunits, IL-19Rα and IL-19Rß (also known as IL-20R1 and IL-20R2, and IL-20RA and IL-20RB), in mouse brain tissue, and by primary murine and human astrocytes. We also provide evidence for the presence of a novel truncated IL-19Rα transcript variant in mouse brain tissue, but not glial cells, that shows reduced expression following bacterial infection. Importantly, IL-19R functionality in glia is indicated by the ability of IL-19 to regulate signaling component expression in these cells. Furthermore, while IL-19 itself had no effect on glial cytokine production, IL-19 treatment of bacterially infected or Toll-like receptor ligand stimulated astrocytes significantly attenuated pro-inflammatory cytokine production. The bacterially induced production of IL-19 by these resident CNS cells, the constitutive expression of its cognate receptor subunits, and the immunomodulatory effects of this cytokine, suggest a novel mechanism by which astrocytes can regulate CNS inflammation.
Assuntos
Astrócitos/metabolismo , Astrócitos/microbiologia , Tolerância Imunológica/fisiologia , Interleucina-10/biossíntese , Streptococcus pneumoniae , Animais , Animais Recém-Nascidos , Astrócitos/imunologia , Células Cultivadas , Feminino , Humanos , Interleucina-10/imunologia , Interleucinas , Camundongos , Camundongos Endogâmicos C57BL , Streptococcus pneumoniae/imunologiaRESUMO
Genomic instability is a key driving force for the development and progression of many age-related neurodegenerative diseases and central nervous system (CNS) cancers. Recently, the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), has been shown to detect and respond to self-DNA accumulation resulting from DNA damaging insults in peripheral cell types. cGAS has been shown to be important in the responses of microglia to DNA viruses and amyloid beta, and we have reported that it underlies the responses of human microglia to exogenous DNA. However, the role of this cytosolic sensor in the detection of self-DNA by glia is poorly understood and its ability to mediate the cellular responses of human microglia to genotoxic DNA damage has not been established. Here, we describe the ability of ionizing radiation and oxidative stress to elicit genomic DNA damage in human microglial cells and to stimulate the production of key inflammatory mediators by these cells in an NF-kB dependent manner. Importantly, we have utilized CRISPR/Cas9 and siRNA-mediated knockdown approaches and a pharmacological inhibitor of the cGAS adaptor protein stimulator of interferon genes (STING) to demonstrate that the cGAS-STING pathway plays a critical role in the generation of these microglial immune responses to such genotoxic insults. Together, these studies support the notion that cGAS mediates the detection of cytosolic self-DNA by microglia, providing a potential mechanism linking genomic instability to the development of CNS cancers and neurodegenerative disorders.
Assuntos
Dano ao DNA , Microglia , Nucleotidiltransferases , Humanos , Dano ao DNA/genética , Inflamação/metabolismo , Proteínas de Membrana/metabolismo , Microglia/metabolismo , NF-kappa B/metabolismo , Nucleotidiltransferases/metabolismo , Estresse OxidativoRESUMO
There is increasing evidence that the tachykinin substance P (SP) can augment inflammatory immune responses within the CNS. We have recently demonstrated that resident CNS cells express high-affinity receptors for this neuropeptide (neurokinin-1 receptors [NK-1R]), and we have shown that SP can significantly augment glial inflammatory responses to clinically relevant Gram-negative bacteria. Furthermore, we provided evidence that endogenous SP/NK-1R interactions are an essential component in the initiation and/or progression of CNS inflammation following in vivo exposure to these pathogens. In this study, we demonstrate that SP similarly enhances inflammatory glial responses to the major Gram-positive causative agent of bacterial meningitis, Streptococcus pneumoniae, and show that endogenous SP/NK-1R interactions play a critical role in the development of CNS inflammation in an in vivo model of pneumococcal meningitis. Importantly, we provide the first demonstration, to our knowledge, that pharmacological targeting of the NK-1R not only prevents the development of damaging inflammation when administered prophylactically, but can also limit or reverse neuroinflammation associated with an established streptococcal CNS infection when delivered therapeutically. We show that an NK-1R antagonist attenuates increases in CNS inflammatory cytokine levels and decreases in immunosuppressive cytokine production associated with an ongoing S. pneumoniae infection. Furthermore, we demonstrate that such a therapeutic intervention reverses infection-associated gliosis and demyelination in the absence of changes in CNS bacterial burden. Together, these results suggest that targeting SP/NK-1R interactions is a strategy worthy of further study for the treatment of microbially induced neuroinflammation.
Assuntos
Inflamação/tratamento farmacológico , Meningite Pneumocócica/tratamento farmacológico , Antagonistas dos Receptores de Neurocinina-1 , Animais , Sistema Nervoso Central/patologia , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Doenças Desmielinizantes , Bactérias Gram-Negativas/efeitos dos fármacos , Inflamação/microbiologia , CamundongosRESUMO
Genomic instability is a key driving force for the development and progression of many neurodegenerative diseases and central nervous system (CNS) cancers. The initiation of DNA damage responses is a critical step in maintaining genomic integrity and preventing such diseases. However, the absence of these responses or their inability to repair genomic or mitochondrial DNA damage resulting from insults, including ionizing radiation or oxidative stress, can lead to an accumulation of self-DNA in the cytoplasm. Resident CNS cells, such as astrocytes and microglia, are known to produce critical immune mediators following CNS infection due to the recognition of pathogen and damage-associated molecular patterns by specialized pattern recognition receptors (PRRs). Recently, multiple intracellular PRRs, including cyclic GMP-AMP synthase, interferon gamma-inducible 16, absent in melanoma 2, and Z-DNA binding protein, have been identified as cytosolic DNA sensors and to play critical roles in glial immune responses to infectious agents. Intriguingly, these nucleic acid sensors have recently been shown to recognize endogenous DNA and trigger immune responses in peripheral cell types. In the present review, we discuss the available evidence that cytosolic DNA sensors are expressed by resident CNS cells and can mediate their responses to the presence of self-DNA. Furthermore, we discuss the potential for glial DNA sensor-mediated responses to provide protection against tumorigenesis versus the initiation of potentially detrimental neuroinflammation that could initiate or foster the development of neurodegenerative disorders. Determining the mechanisms that underlie the detection of cytosolic DNA by glia and the relative role of each pathway in the context of specific CNS disorders and their stages may prove pivotal in our understanding of the pathogenesis of such conditions and might be leveraged to develop new treatment modalities.
Assuntos
Neuroglia , Ácidos Nucleicos , Neuroglia/metabolismo , DNA/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Microglia/metabolismoRESUMO
Staphylococcus aureus infections of bone tissue are associated with inflammatory bone loss. Resident bone cells, including osteoblasts and osteoclasts, can perceive S. aureus and produce an array of inflammatory and pro-osteoclastogenic mediators, thereby contributing to such damage. The neuropeptide substance P (SP) has been shown to exacerbate microbially induced inflammation at sites such as the gut and the brain and has previously been shown to affect bone cell differentiation and activity. Here we demonstrate that the interaction of SP with its high affinity receptor, neurokinin-1 receptor (NK-1R), expressed on murine osteoblasts and osteoclasts, augments the inflammatory responses of these cells to S. aureus challenge. Additionally, SP alters the production of pro- and anti-osteoclastogenic factors by bacterially challenged bone cells and their proteolytic functions in a manner that would be anticipated to exacerbate inflammatory bone loss at sites of infection. Furthermore, we have demonstrated that the clinically approved NK-1R antagonist, aprepitant, attenuates local inflammatory and pro-osteoclastogenic mediator expression in an in vivo mouse model of post-traumatic staphylococcal osteomyelitis. Taken together, these results indicate that SP/NK-1R interactions could play a significant role in the initiation and/or progression of damaging inflammation in S. aureus bone infections and suggest that the repurposing of currently approved NK-1R antagonists might represent a promising new adjunct therapy for such conditions.
Assuntos
Osteomielite , Infecções Estafilocócicas , Animais , Camundongos , Staphylococcus aureus , Substância P/farmacologia , Substância P/metabolismo , Osteoclastos/metabolismo , Osteoblastos/metabolismo , Inflamação/metabolismo , Osteomielite/metabolismo , Antagonistas dos Receptores de Neurocinina-1 , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/metabolismoRESUMO
The immune system has evolved to defend organisms against exogenous threats such as viruses, bacteria, fungi, and parasites by distinguishing between "self" and "non-self". In addition, it guards us against other diseases, such as cancer, by detecting and responding to transformed and senescent cells. However, for survival and propagation, the altered cells and invading pathogens often employ a wide range of mechanisms to avoid, inhibit, or manipulate the immunorecognition. As such, the development of new modes of therapeutic intervention to augment protective and prevent harmful immune responses is desirable. Nucleic acids are biopolymers essential for all forms of life and, therefore, delineating the complex defensive mechanisms developed against non-self nucleic acids can offer an exciting avenue for future biomedicine. Nucleic acid technologies have already established numerous approaches in therapy and biotechnology; recently, rationally designed nucleic acids nanoparticles (NANPs) with regulated physiochemical properties and biological activities has expanded our repertoire of therapeutic options. When compared to conventional therapeutic nucleic acids (TNAs), NANP technologies can be rendered more beneficial for synchronized delivery of multiple TNAs with defined stabilities, immunological profiles, and therapeutic functions. This review highlights several recent advances and possible future directions of TNA and NANP technologies that are under development for controlled immunomodulation.
Assuntos
Nanopartículas , Neoplasias , Ácidos Nucleicos , Humanos , Imunomodulação , Neoplasias/tratamento farmacológico , Imunidade , Nanopartículas/uso terapêutico , Nanopartículas/químicaRESUMO
Introduction: The refractory and recurrent nature of chronic staphylococcal osteomyelitis may be due, at least in part, to the ability of Staphylococcus aureus to invade and persist within bone-forming osteoblasts. However, osteoblasts are now recognized to respond to S. aureus infection and produce numerous immune mediators and bone regulatory factors that can shape the host response. Type I interferons (IFNs) are best known for their antiviral effects, but it is becoming apparent that they impact host susceptibility to a wide range of pathogens including S. aureus. Methods: Here, we have assessed the local expression of IFN-ß by specific capture ELISA in an established in vivo mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis, specific capture ELISAs, and/or immunoblot analyses, were then used to assess the expression of type I IFNs and select IFN stimulated genes (ISGs) in S. aureus infected primary murine osteoblasts. The effect of IFN-ß on intracellular S. aureus burden was assessed in vitro following recombinant cytokine treatment by serial colony counts of liberated bacteria. Results: We report the presence of markedly elevated IFN-ß levels in infected bone tissue in a mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis of S. aureus infected osteoblasts showed enrichment of genes associated with type I IFN signaling and ISGs, and elevated expression of mRNA encoding IFN-ß and ISG products. IFN-ß production was confirmed with the demonstration that S. aureus induces its rapid and robust release by osteoblasts in a dose-dependent manner. Furthermore, we showed increased protein expression of the ISG products IFIT1 and IFIT3 by infected osteoblasts and demonstrate that this occurs secondary to the release of IFN-ß by these cells. Finally, we have determined that exposure of S. aureus-infected osteoblasts to IFN-ß markedly reduces the number of viable bacteria harbored by these cells. Discussion: Together, these findings indicate an ability of osteoblasts to respond to bacteria by producing IFN-ß that can act in an autocrine and/or paracrine manner to elicit ISG expression and mitigate S. aureus infection.