RESUMEN
Aedes aegypti mosquitoes are responsible for transmitting many medically important viruses such as those that cause Zika and dengue. The inoculation of viruses into mosquito bite sites is an important and common stage of all mosquito-borne virus infections. We show, using Semliki Forest virus and Bunyamwera virus, that these viruses use this inflammatory niche to aid their replication and dissemination in vivo. Mosquito bites were characterized by an edema that retained virus at the inoculation site and an inflammatory influx of neutrophils that coordinated a localized innate immune program that inadvertently facilitated virus infection by encouraging the entry and infection of virus-permissive myeloid cells. Neutrophil depletion and therapeutic blockade of inflammasome activity suppressed inflammation and abrogated the ability of the bite to promote infection. This study identifies facets of mosquito bite inflammation that are important determinants of the subsequent systemic course and clinical outcome of virus infection.
Asunto(s)
Infecciones por Arbovirus/inmunología , Virus Bunyamwera/fisiología , Inflamación/inmunología , Mordeduras y Picaduras de Insectos/inmunología , Neutrófilos/inmunología , Virus de los Bosques Semliki/fisiología , Replicación Viral , Animales , Movimiento Celular , Células Cultivadas , Culicidae/inmunología , Humanos , Inmunidad Innata , Inflamasomas/metabolismo , Inflamación/virología , Mordeduras y Picaduras de Insectos/virología , Ratones , Neutrófilos/virologíaRESUMEN
Viruses transmitted by Aedes mosquitoes are an increasingly important global cause of disease. Defining common determinants of host susceptibility to this large group of heterogenous pathogens is key for informing the rational design of panviral medicines. Infection of the vertebrate host with these viruses is enhanced by mosquito saliva, a complex mixture of salivary-gland-derived factors and microbiota. We show that the enhancement of infection by saliva was dependent on vascular function and was independent of most antisaliva immune responses, including salivary microbiota. Instead, the Aedes gene product sialokinin mediated the enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having a vertebrate-like tachykinin sequence and is absent from Anopheles mosquitoes, which are incompetent for most arthropod-borne viruses, whose saliva was not proviral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have the potential to limit disease severity following infection with Aedes-mosquito-borne viruses.
Asunto(s)
Aedes , Infecciones por Arbovirus , Arbovirus , Saliva , Taquicininas , Virosis , Aedes/genética , Aedes/virología , Animales , Infecciones por Arbovirus/transmisión , Arbovirus/genética , Arbovirus/metabolismo , Saliva/virología , Taquicininas/genética , Taquicininas/metabolismo , Virosis/transmisiónRESUMEN
Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/virología , Rimantadina/farmacología , Proteínas de la Matriz Viral/antagonistas & inhibidores , Zanamivir/farmacología , Antivirales/farmacología , Farmacorresistencia Viral , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Gripe Humana/tratamiento farmacológico , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
OBJECTIVES: Plasmacytoid dendritic cells (pDC) have been implicated in the pathogenesis of autoimmune diseases, such as scleroderma (SSc). However, this has been derived from indirect evidence using ex vivo human samples or mouse pDC in vivo. We have developed human-specific pDC models to directly identify their role in inflammation and fibrosis, as well as attenuation of pDC function with BDCA2-targeting to determine its therapeutic application. METHODS: RNAseq of human pDC with TLR9 agonist ODN2216 and humanised monoclonal BDCA2 antibody, CBS004. Organotypic skin rafts consisting of fibroblasts and keratinocytes were stimulated with supernatant from TLR9-stimulated pDC and with CBS004. Human pDC were xenotransplanted into Nonobese diabetic/severe combined immunodeficiency (NOD SCID) mice treated with Aldara (inflammatory model), or bleomycin (fibrotic model) with CBS004 or human IgG control. Skin punch biopsies were used to assess gene and protein expression. RESULTS: RNAseq shows TLR9-induced activation of human pDC goes beyond type I interferon (IFN) secretion, which is functionally inactivated by BDCA2-targeting. Consistent with these findings, we show that BDCA2-targeting of pDC can completely suppress in vitro skin IFN-induced response. Most importantly, xenotransplantation of human pDC significantly increased in vivo skin IFN-induced response to TLR agonist and strongly enhanced fibrotic and immune response to bleomycin compared with controls. In these contexts, BDCA2-targeting suppressed human pDC-specific pathological responses. CONCLUSIONS: Our data indicate that human pDC play a key role in inflammation and immune-driven skin fibrosis, which can be effectively blocked by BDCA2-targeting, providing direct evidence supporting the development of attenuation of pDC function as a therapeutic application for SSc.
Asunto(s)
Células Dendríticas/inmunología , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Esclerodermia Localizada/inmunología , Esclerodermia Localizada/patología , Animales , Células Dendríticas/patología , Modelos Animales de Enfermedad , Fibrosis , Xenoinjertos , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Esclerodermia Localizada/metabolismo , Piel/inmunología , Piel/metabolismo , Piel/patologíaRESUMEN
Some free fatty acids derived from milk and vegetable oils are known to have potent antiviral and antibacterial properties. However, therapeutic applications of short- to medium-chain fatty acids are limited by physical characteristics such as immiscibility in aqueous solutions. We evaluated a novel proprietary formulation based on an emulsion of short-chain caprylic acid, ViroSAL, for its ability to inhibit a range of viral infections in vitro and in vivo. In vitro, ViroSAL inhibited the enveloped viruses Epstein-Barr, measles, herpes simplex, Zika and orf parapoxvirus, together with Ebola, Lassa, vesicular stomatitis and severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) pseudoviruses, in a concentration- and time-dependent manner. Evaluation of the components of ViroSAL revealed that caprylic acid was the main antiviral component; however, the ViroSAL formulation significantly inhibited viral entry compared with caprylic acid alone. In vivo, ViroSAL significantly inhibited Zika and Semliki Forest virus replication in mice following the inoculation of these viruses into mosquito bite sites. In agreement with studies investigating other free fatty acids, ViroSAL had no effect on norovirus, a non-enveloped virus, indicating that its mechanism of action may be surfactant disruption of the viral envelope. We have identified a novel antiviral formulation that is of great interest for the prevention and/or treatment of a broad range of enveloped viruses, particularly those of the skin and mucosal surfaces.
Asunto(s)
Antivirales , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Virus , Infección por el Virus Zika , Virus Zika , Animales , Antivirales/farmacología , Lípidos , Ratones , Internalización del VirusRESUMEN
Chemokines are the principal regulators of leukocyte migration and are essential for initiation and maintenance of inflammation. Atypical chemokine receptor 2 (ACKR2) binds and scavenges proinflammatory CC-chemokines, regulates cutaneous T-cell positioning, and limits the spread of inflammation in vivo Altered ACKR2 function has been implicated in several inflammatory disorders, including psoriasis, a common and debilitating T-cell-driven disorder characterized by thick erythematous skin plaques. ACKR2 expression is abnormal in psoriatic skin, with decreased expression correlating with recruitment of T-cells into the epidermis and increased inflammation. However, the molecular mechanisms that govern ACKR2 expression are not known. Here, we identified specific psoriasis-associated microRNAs (miRs) that bind ACKR2, inhibit its expression, and are active in primary cultures of human cutaneous cells. Using both in silico and in vitro approaches, we show that miR-146b and miR-10b directly bind the ACKR2 3'-UTR and reduce expression of ACKR2 transcripts and protein in keratinocytes and lymphatic endothelial cells, respectively. Moreover, we demonstrate that ACKR2 expression is further down-regulated upon cell trauma, an important trigger for the development of new plaques in many psoriasis patients (the Koebner phenomenon). We found that tensile cell stress leads to rapid ACKR2 down-regulation and concurrent miR-146b up-regulation. Together, we provide, for the first time, evidence for epigenetic regulation of an atypical chemokine receptor. We propose a mechanism by which cell trauma and miRs coordinately exacerbate inflammation via down-regulation of ACKR2 expression and provide a putative mechanistic explanation for the Koebner phenomenon in psoriasis.
Asunto(s)
Regulación hacia Abajo , Regulación de la Expresión Génica , Queratinocitos/metabolismo , MicroARNs/metabolismo , Interferencia de ARN , Receptores de Quimiocina/antagonistas & inhibidores , Regiones no Traducidas 3' , Células Cultivadas , Biología Computacional , Células Endoteliales/citología , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Epigénesis Genética , Sistemas Especialistas , Genes Reporteros , Células HEK293 , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Queratinocitos/citología , Queratinocitos/inmunología , Queratinocitos/patología , Especificidad de Órganos , Psoriasis/inmunología , Psoriasis/metabolismo , Psoriasis/patología , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Proteínas Recombinantes/metabolismo , Piel/inmunología , Piel/lesiones , Piel/metabolismo , Piel/patología , Resistencia a la TracciónRESUMEN
Cell therapy regimens are frequently compromised by low-efficiency cell homing to therapeutic niches. Improvements in this regard would enhance effectiveness of clinically applicable cell therapy. The major regulators of tissue-specific cellular migration are chemokines, and therefore selection of therapeutic cellular populations for appropriate chemokine receptor expression would enhance tissue-homing competence. A number of practical considerations preclude the use of Abs in this context, and alternative approaches are required. In this study, we demonstrate that appropriately labeled chemokines are at least as effective in detecting their cognate receptors as commercially available Abs. We also demonstrate the utility of biotinylated chemokines as cell-sorting reagents. Specifically, we demonstrate, in the context of CCR7 (essential for lymph node homing of leukocytes), the ability of biotinylated CCL19 with magnetic bead sorting to enrich for CCR7-expressing cells. The sorted cells demonstrate improved CCR7 responsiveness and lymph node-homing capability, and the sorting is effective for both T cells and dendritic cells. Importantly, the ability of chemokines to detect CCR7, and sort for CCR7 positivity, crosses species being effective on murine and human cells. This novel approach to cell sorting is therefore inexpensive, versatile, and applicable to numerous cell therapy contexts. We propose that this represents a significant technological advance with important therapeutic implications.
Asunto(s)
Quimiocina CCL19/química , Citometría de Flujo/métodos , Receptores CCR7/química , Animales , Quimiocina CCL19/inmunología , Femenino , Humanos , Masculino , Ratones , Receptores CCR7/inmunologíaRESUMEN
UNLABELLED: The encephalitic response to viral infection requires local chemokine production and the ensuing recruitment of immune and inflammatory leukocytes. Accordingly, chemokine receptors present themselves as plausible therapeutic targets for drugs aimed at limiting encephalitic responses. However, it remains unclear which chemokines are central to this process and whether leukocyte recruitment is important for limiting viral proliferation and survival in the brain or whether it is predominantly a driver of coincident inflammatory pathogenesis. Here we examine chemokine expression and leukocyte recruitment in the context of avirulent and virulent Semliki Forest virus (SFV) as well as West Nile virus infection and demonstrate rapid and robust expression of a variety of inflammatory CC and CXC chemokines in all models. On this basis, we define a chemokine axis involved in leukocyte recruitment to the encephalitic brain during SFV infection. CXCR3 is the most active; CCR2 is also active but less so, and CCR5 plays only a modest role in leukocyte recruitment. Importantly, inhibition of each of these receptors individually and the resulting suppression of leukocyte recruitment to the infected brain have no effect on viral titer or survival following infection with a virulent SFV strain. In contrast, simultaneous blockade of CXCR3 and CCR2 results in significantly reduced mortality in response to virulent SFV infection. In summary, therefore, our data provide an unprecedented level of insight into chemokine orchestration of leukocyte recruitment in viral encephalitis. Our data also highlight CXCR3 and CCR2 as possible therapeutic targets for limiting inflammatory damage in response to viral infection of the brain. IMPORTANCE: Brain inflammation (encephalitis) in response to viral infection can lead to severe illness and even death. This therefore represents an important clinical problem and one that requires the development of new therapeutic approaches. Central to the pathogenesis of encephalitis is the recruitment of inflammatory leukocytes to the infected brain, a process driven by members of the chemokine family. Here we provide an in-depth analysis of the chemokines involved in leukocyte recruitment to the virally infected brain and demonstrate that simultaneous blockade of two of these receptors, namely, CXCR3 and CCR2, does not alter viral titers within the brain but markedly reduces inflammatory leukocyte recruitment and enhances survival in a murine model of lethal viral encephalitis. Our results therefore highlight chemokine receptors as plausible therapeutic targets in treating viral encephalitis.
Asunto(s)
Infecciones por Alphavirus/inmunología , Quimiocinas/metabolismo , Encefalitis Viral/inmunología , Leucocitos/inmunología , Fiebre del Nilo Occidental/inmunología , Animales , Encéfalo/patología , Encéfalo/virología , Modelos Animales de Enfermedad , Femenino , Ratones Endogámicos C57BL , Receptores de Quimiocina/inmunología , Virus de los Bosques Semliki/inmunología , Análisis de Supervivencia , Carga Viral , Virus del Nilo Occidental/inmunologíaRESUMEN
The mechanisms by which CC chemokine receptor (CCR)7 ligands are selectively presented on lymphatic endothelium in the presence of inflammatory chemokines are poorly understood. The chemokine-scavenging receptor D6 is expressed on lymphatic endothelial cells (LEC) and contributes to selective presentation of CCR7 ligands by suppressing inflammatory chemokine binding to LEC surfaces. As well as preventing inappropriate inflammatory cell attachment to LECs, D6 is specifically involved in regulating the ability of LEC to discriminate between mature and immature dendritic cells (DCs). D6 overexpression reduces immature DC (iDC) adhesion to LECs, whereas D6 knockdown increases adhesion of iDCs that displace mature DCs. LEC D6 expression is regulated by growth factors, cytokines, and tumor microenvironments. In particular, interleukin-6 and interferon-γ are potent inducers, indicating a preferential role for D6 in inflamed contexts. Expression of the viral interleukin-6 homolog from Kaposi sarcoma-associated herpesvirus is also sufficient to induce significant D6 upregulation both in vitro and in vivo, and Kaposi sarcoma and primary effusion lymphoma cells demonstrate high levels of D6 expression. We therefore propose that D6, which is upregulated in both inflammatory and tumor contexts, is an essential regulator of inflammatory leukocyte interactions with LECs and is required for immature/mature DC discrimination by LECs.
Asunto(s)
Células Endoteliales/metabolismo , Receptores CCR10/genética , Receptores CCR10/fisiología , Animales , Células CHO , Comunicación Celular/genética , Comunicación Celular/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Cricetinae , Cricetulus , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/fisiología , Células Endoteliales/inmunología , Células HEK293 , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Receptores CCR10/análisis , Receptores CCR10/metabolismo , Transfección , Receptor de Quimiocina D6RESUMEN
The inflammatory response is normally limited by mechanisms regulating its resolution. In the absence of resolution, inflammatory pathologies can emerge, resulting in substantial morbidity and mortality. We have been studying the D6 chemokine scavenging receptor, which played an indispensable role in the resolution phase of inflammatory responses and does so by facilitating removal of inflammatory CC chemokines. In D6-deficient mice, otherwise innocuous cutaneous inflammatory stimuli induce a grossly exaggerated inflammatory response that bears many similarities to human psoriasis. In the present study, we have used transcriptomic approaches to define the molecular make up of this response. The data presented highlight potential roles for a number of cytokines in initiating and maintaining the psoriasis-like pathology. Most compellingly, we provide data indicating a key role for the type I interferon pathway in the emergence of this pathology. Neutralizing antibodies to type I interferons are able to ameliorate the psoriasis-like pathology, confirming a role in its development. Comparison of transcriptional data generated from this mouse model with equivalent data obtained from human psoriasis further demonstrates the strong similarities between the experimental and clinical systems. As such, the transcriptional data obtained in this preclinical model provide insights into the cytokine network active in exaggerated inflammatory responses and offer an excellent tool to evaluate the efficacy of compounds designed to therapeutically interfere with inflammatory processes.
Asunto(s)
Interferón Tipo I/metabolismo , Psoriasis/inmunología , Receptores CCR10/genética , Animales , Femenino , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interferón Tipo I/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Ésteres del Forbol/toxicidad , Psoriasis/inducido químicamente , Psoriasis/genética , Psoriasis/patología , Transcripción Genética , Receptor de Quimiocina D6RESUMEN
Flaviviruses, including Zika virus (ZIKV), are a significant global health concern, yet no licensed antivirals exist to treat disease. The small Membrane (M) protein plays well-defined roles during viral egress and remains within virion membranes following release and maturation. However, it is unclear whether M plays a functional role in this setting. Here, we show that M forms oligomeric membrane-permeabilising channels in vitro, with increased activity at acidic pH and sensitivity to the prototypic channel-blocker, rimantadine. Accordingly, rimantadine blocked an early stage of ZIKV cell culture infection. Structure-based channel models, comprising hexameric arrangements of two trans-membrane domain protomers were shown to comprise more stable assemblages than other oligomers using molecular dynamics (MD) simulations. Models contained a predicted lumenal rimantadine binding site, as well as a second druggable target region on the membrane-exposed periphery. In silico screening enriched for repurposed drugs/compounds predicted to bind to either one site or the other. Hits displayed superior potency in vitro and in cell culture compared with rimantadine, with efficacy demonstrably linked to virion-resident channels. Finally, rimantadine effectively blocked ZIKV viraemia in preclinical models, supporting that M constitutes a physiologically relevant target. This could be explored by repurposing rimantadine, or development of new M-targeted-therapies.
RESUMEN
BACE1 is well-known for its role in the development of Alzheimer's disease. Recent publications, including our own, have demonstrated a role for this enzyme in other chronic diseases. The aim of this study was to investigate the role of BACE1 in the autoimmune disease systemic sclerosis (SSc). BACE1 protein levels were elevated in the skin of patients with SSc. Inhibition of BACE1 with small-molecule inhibitors or small interfering RNA blocked SSc and fibrotic stimuli-mediated fibroblast activation. Furthermore, we show that BACE1 regulation of dermal fibroblast activation is dependent on ß-catenin and Notch signaling. The neurotropic factor brain-derived neurotrophic factor negatively regulates BACE1 expression and activity in dermal fibroblasts. Finally, sera from patients with SSc show higher ß-amyloid and lower brain-derived neurotrophic factor levels than healthy controls. The ability of BACE1 to regulate SSc fibroblast activation reveals a therapeutic target in SSc. Several BACE1 inhibitors have been shown to be safe in clinical trials for Alzheimer's disease and could be repurposed to ameliorate fibrosis progression.
RESUMEN
Lymphatic endothelial cells are important for efficient flow of antigen-bearing fluid and antigen-presenting cells (APCs) from peripheral sites to lymph nodes (LNs). APC movement to LNs is dependent on the constitutive chemokine receptor CCR7, although how conflicting inflammatory and constitutive chemokine cues are integrated at lymphatic surfaces during this process is not understood. Here we reveal a previously unrecognized aspect of the regulation of this process. The D6 chemokine-scavenging receptor, which is expressed on lymphatic endothelial cells (LECs), maintains lymphatic surfaces free of inflammatory CC-chemokines and minimizes interaction of inflammatory leukocytes with these surfaces. D6 does not alter the level of CCR7 ligands on LECs, thus ensuring selective presentation of homeostatic chemokines for interaction with CCR7(+) APCs. Accordingly, in D6-deficient mice, inflammatory CC-chemokine adherence to LECs results in inappropriate perilymphatic accumulation of inflammatory leukocytes at peripheral inflamed sites and draining LNs. This results in lymphatic congestion and impaired movement of APCs, and fluid, from inflamed sites to LNs. We propose that D6, by suppressing inflammatory chemokine binding to lymphatic surfaces, and thereby preventing inappropriate inflammatory leukocyte adherence, is a key regulator of lymphatic function and a novel, and indispensable, contributor to the integration of innate and adaptive immune responses.
Asunto(s)
Líquidos Corporales/inmunología , Movimiento Celular/inmunología , Células Endoteliales/inmunología , Ganglios Linfáticos/inmunología , Receptores de Quimiocina/inmunología , Inmunidad Adaptativa/inmunología , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Quimiocina CCL2/inmunología , Quimiocina CCL2/metabolismo , Quimiocina CCL5/inmunología , Quimiocina CCL5/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Inmunidad Innata/inmunología , Leucocitos/citología , Leucocitos/inmunología , Linfa/inmunología , Linfa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Receptores CCR2/inmunología , Receptores CCR2/metabolismo , Receptores CCR7/inmunología , Receptores CCR7/metabolismo , Receptores de Quimiocina/genéticaRESUMEN
Toll-like receptors orchestrate rapid local protective innate-immune responses to invading pathogens and optimize leukocyte priming of subsequent adaptive responses. Paradoxically, systemic excess of the TLR2 ligand, bacterial lipoprotein (BLP), suppresses peripheral inflammatory responses. Here, we demonstrate that this phenomenon is regulated via the TLR2-dependent, cell-autonomous down-regulation of inflammatory chemokine receptor expression on a variety of leukocyte subsets. Remarkably, BLP mediated no effect on constitutive chemokine receptor expression. By tracking adoptively transferred wild-type and TLR2(-/-) leukocytes in vivo, we observed that BLP mediated chemokine receptor switching directed leukocytes away from inflamed sites toward secondary lymphoid organs. These data highlight a novel role for TLR ligands, such as BLP, in regulating leukocyte retention and migration away from innate immune lesions via discrete constitutive and inflammatory chemokine receptor regulation.
Asunto(s)
Movimiento Celular/fisiología , Inflamación/prevención & control , Leucocitos/fisiología , Lipoproteínas/administración & dosificación , Receptores de Quimiocina/metabolismo , Piel/inmunología , Receptor Toll-Like 2/fisiología , Animales , Células Cultivadas , Quimiocinas/metabolismo , Femenino , Citometría de Flujo , Inflamación/inducido químicamente , Inflamación/metabolismo , Interferón gamma/farmacología , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Immune responses in the central nervous system (CNS) are carefully regulated. Despite the absence of most immune processes and a substantive blood brain barrier, potent immune responses form during infection and autoimmunity. Astrocytes are innate immune sentinels that ensheath parenchymal blood vessels and sit at the gateway to the CNS parenchyma. Viral and bacterial infections trigger the influx of distinct leukocyte subsets. We show that astrocytes alone are sufficient for distinguishing between these two main types of infection and triggers release of relevant chemokines that relate to the microbe recognised. Bacterial-associated molecules induced the preferential expression of CCL2, CXCL1, CCL20 and CCL3 whilst a virus-associated dsRNA analogue preferentially up-regulated CXCL10 and CCL5. Thus, astrocytes can respond to infection in a distinct and appropriate manner suggesting they have the capacity to attract appropriate sets of leukocytes into the brain parenchyma. Astrocytes themselves are unable to respond to these chemokines since they were devoid of most chemokine receptors but expressed CXCR4, CXCR7 and CXCR6 at rest. Stimulation with TGF-beta specifically up-regulated CXCR6 expression and may explain how TGF-beta/CXCL16-expressing gliomas are so effective at attracting astroglial cells.
Asunto(s)
Astrocitos/inmunología , Astrocitos/microbiología , Quimiocinas/metabolismo , Inmunidad Innata , Redes y Vías Metabólicas/inmunología , Animales , Astrocitos/virología , Células Cultivadas , Ratones , Receptores de Quimiocina/metabolismoRESUMEN
D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.
Asunto(s)
Factor de Transcripción GATA1/inmunología , Regulación de la Expresión Génica/inmunología , Células Madre Hematopoyéticas/inmunología , Leucocitos/inmunología , Modelos Biológicos , Receptores CCR10/inmunología , Animales , Quimiocinas/genética , Quimiocinas/inmunología , Quimiocinas/metabolismo , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Regulación de la Expresión Génica/genética , Células Madre Hematopoyéticas/citología , Humanos , Leucocitos/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Ratones , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Receptores CCR10/biosíntesis , Receptores CCR10/genética , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/farmacología , Receptor de Quimiocina D6RESUMEN
D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.
Asunto(s)
Factor de Transcripción GATA1/fisiología , Regulación de la Expresión Génica , Leucocitos/metabolismo , Receptores CCR10/genética , Animales , Células Cultivadas , Células Dendríticas , Células Endoteliales , Humanos , Inflamación/inmunología , Ratones , Receptor de Quimiocina D6RESUMEN
Progressive multi-focal leukoencephalopathy (PML) is a potentially fatal encephalitis caused by JC polyomavirus (JCV). PML principally affects people with a compromised immune system, such as patients with multiple sclerosis (MS) receiving treatment with natalizumab. However, intrathecal synthesis of lipid-reactive IgM in MS patients is associated with a markedly lower incidence of natalizumab-associated PML compared to those without this antibody repertoire. Here we demonstrate that a subset of lipid-reactive human and murine IgMs induce a functional anti-viral response that inhibits replication of encephalitic Alpha and Orthobunyaviruses in multi-cellular central nervous system cultures. These lipid-specific IgMs trigger microglia to produce IFN-ß in a cGAS-STING-dependent manner, which induces an IFN-α/ß-receptor 1-dependent antiviral response in glia and neurons. These data identify lipid-reactive IgM as a mediator of anti-viral activity in the nervous system and provide a rational explanation why intrathecal synthesis of lipid-reactive IgM correlates with a reduced incidence of iatrogenic PML in MS.
Asunto(s)
Autoanticuerpos/líquido cefalorraquídeo , Inmunoglobulina M/líquido cefalorraquídeo , Leucoencefalopatía Multifocal Progresiva/inmunología , Lípidos/inmunología , Esclerosis Múltiple , Animales , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Humanos , Huésped Inmunocomprometido/inmunología , Inmunoglobulina M/inmunología , Factores Inmunológicos/efectos adversos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Natalizumab/efectos adversos , Ratas , Ratas Sprague-DawleyRESUMEN
Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The abundance of genetically distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made the development of virus-specific treatments challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical TLR7 agonist suppressed both the local and subsequent systemic course of infection with a variety of arboviruses from the Alphavirus, Flavivirus, and Orthobunyavirus genera. Clinical outcome was improved in mice after infection with a model alphavirus. In the absence of treatment, antiviral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, stimulating the more populous skin-resident macrophages with a TLR7 agonist elicited protective responses in key cellular targets of virus that otherwise proficiently replicated virus. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have identified a putative new strategy for limiting disease after infection with a variety of genetically distinct arboviruses.