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1.
J Immunol ; 210(9): 1209-1221, 2023 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-36961448

RESUMEN

Autosomal recessive PRKCD deficiency has previously been associated with the development of systemic lupus erythematosus in human patients, but the mechanisms underlying autoimmunity remain poorly understood. We introduced the Prkcd G510S mutation that we previously associated to a Mendelian cause of systemic lupus erythematosus in the mouse genome, using CRISPR-Cas9 gene editing. PrkcdG510S/G510S mice recapitulated the human phenotype and had reduced lifespan. We demonstrate that this phenotype is linked to a B cell-autonomous role of Prkcd. A detailed analysis of B cell activation in PrkcdG510S/G510S mice shows an upregulation of the PI3K/mTOR pathway after the engagement of the BCR in these cells, leading to lymphoproliferation. Treatment of mice with rapamycin, an mTORC1 inhibitor, significantly improves autoimmune symptoms, demonstrating in vivo the deleterious effect of mTOR pathway activation in PrkcdG510S/G510S mice. Additional defects in PrkcdG510S/G510S mice include a decrease in peripheral mature NK cells that might contribute to the known susceptibility to viral infections of patients with PRKCD mutations.


Asunto(s)
Autoinmunidad , Lupus Eritematoso Sistémico , Humanos , Animales , Ratones , Serina-Treonina Quinasas TOR/metabolismo , Linfocitos B , Proliferación Celular
2.
PLoS Pathog ; 15(2): e1007589, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30818370

RESUMEN

Human T Lymphotropic virus (HTLV) infection can persist in individuals resulting, at least in part, from viral escape of the innate immunity, including inhibition of type I interferon response in infected T-cells. Plasmacytoid dendritic cells (pDCs) are known to bypass viral escape by their robust type I interferon production. Here, we demonstrated that pDCs produce type I interferons upon physical cell contact with HTLV-infected cells, yet pDC activation inversely correlates with the ability of the HTLV-producing cells to transmit infection. We show that pDCs sense surface associated-HTLV present with glycan-rich structure referred to as biofilm-like structure, which thus represents a newly described viral structure triggering the antiviral response by pDCs. Consistently, heparan sulfate proteoglycans and especially the cell surface pattern of terminal ß-galactoside glycosylation, modulate the transmission of the immunostimulatory RNA to pDCs. Altogether, our results uncover a function of virus-containing cell surface-associated glycosylated structures in the activation of innate immunity.


Asunto(s)
Células Dendríticas/fisiología , Infecciones por HTLV-I/metabolismo , Citocinas , Galactósidos/metabolismo , Glicosilación , Infecciones por HTLV-I/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Virus Linfotrópico T Tipo 2 Humano/inmunología , Virus Linfotrópico T Tipo 2 Humano/patogenicidad , Humanos , Inmunidad Innata/fisiología , Interferón Tipo I/inmunología , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Células Jurkat , Linfocitos T/inmunología , Linfocitos T/fisiología
3.
PLoS Pathog ; 13(9): e1006610, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28957419

RESUMEN

IFITMs are broad antiviral factors that block incoming virions in endosomal vesicles, protecting target cells from infection. In the case of HIV-1, we and others reported the existence of an additional antiviral mechanism through which IFITMs lead to the production of virions of reduced infectivity. However, whether this second mechanism of inhibition is unique to HIV or extends to other viruses is currently unknown. To address this question, we have analyzed the susceptibility of a broad spectrum of viruses to the negative imprinting of the virion particles infectivity by IFITMs. The results we have gathered indicate that this second antiviral property of IFITMs extends well beyond HIV and we were able to identify viruses susceptible to the three IFITMs altogether (HIV-1, SIV, MLV, MPMV, VSV, MeV, EBOV, WNV), as well as viruses that displayed a member-specific susceptibility (EBV, DUGV), or were resistant to all IFITMs (HCV, RVFV, MOPV, AAV). The swapping of genetic elements between resistant and susceptible viruses allowed us to point to specificities in the viral mode of assembly, rather than glycoproteins as dominant factors of susceptibility. However, we also show that, contrarily to X4-, R5-tropic HIV-1 envelopes confer resistance against IFITM3, suggesting that viral receptors add an additional layer of complexity in the IFITMs-HIV interplay. Lastly, we show that the overall antiviral effects ascribed to IFITMs during spreading infections, are the result of a bimodal inhibition in which IFITMs act both by protecting target cells from incoming viruses and in driving the production of virions of reduced infectivity. Overall, our study reports for the first time that the negative imprinting of the virion particles infectivity is a conserved antiviral property of IFITMs and establishes IFITMs as a paradigm of restriction factor capable of interfering with two distinct phases of a virus life cycle.


Asunto(s)
Antígenos de Diferenciación/metabolismo , Virión , Replicación Viral , Línea Celular , VIH-1/fisiología , Interacciones Huésped-Patógeno , Humanos , Internalización del Virus
4.
PLoS Pathog ; 10(10): e1004434, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25340500

RESUMEN

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.


Asunto(s)
Células Dendríticas/virología , Virus del Dengue , Proteínas del Envoltorio Viral/metabolismo , Citoesqueleto de Actina/metabolismo , Evolución Biológica , Línea Celular , Humanos , Inmunidad Innata/inmunología , Fusión de Membrana/fisiología
5.
Methods Mol Biol ; 2618: 289-315, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36905525

RESUMEN

Dendritic cells (DCs) are key regulators of both innate and adaptive immunity via varied functions, including cytokine production and antigen presentation. Plasmacytoid DC (pDC) is a DC subset specialized in the production of type I and III interferons (IFNs). They are thus pivotal players of the host antiviral response during the acute phase of infection by genetically distant viruses. The pDC response is primarily triggered by the endolysosomal sensors Toll-like receptors, which recognize nucleic acids from pathogens. In some pathologic contexts, pDC response can also be triggered by host nucleic acids, hereby contributing to the pathogenesis of autoimmune diseases, such as, e.g., systemic lupus erythematosus. Importantly, recent in vitro studies from our laboratory and others uncovered that pDCs sense viral infections when a physical contact is established with infected cells. This specialized synapse-like feature enables a robust type I and III IFN secretion at the infected site. Therefore, this concentrated and confined response likely limits the correlated deleterious impacts of excessive cytokine production to the host, notably due to tissue damages. Here we provide a pipeline of methods for ex vivo studies of pDC antiviral functions, designed to address how pDC activation is regulated by cell-cell contact with virally infected cells and the current approaches enabling to decipher the underlying molecular events leading to an efficient antiviral response.


Asunto(s)
Interferón Tipo I , Ácidos Nucleicos , Inmunidad Innata , Antivirales , Interferones , Células Dendríticas , Interferón Tipo I/metabolismo
6.
Nat Commun ; 14(1): 694, 2023 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-36755036

RESUMEN

Type I and III interferons (IFN-I/λ) are important antiviral mediators against SARS-CoV-2 infection. Here, we demonstrate that plasmacytoid dendritic cells (pDC) are the predominant IFN-I/λ source following their sensing of SARS-CoV-2-infected cells. Mechanistically, this short-range sensing by pDCs requires sustained integrin-mediated cell adhesion with infected cells. In turn, pDCs restrict viral spread by an IFN-I/λ response directed toward SARS-CoV-2-infected cells. This specialized function enables pDCs to efficiently turn-off viral replication, likely via a local response at the contact site with infected cells. By exploring the pDC response in SARS-CoV-2 patients, we further demonstrate that pDC responsiveness inversely correlates with the severity of the disease. The pDC response is particularly impaired in severe COVID-19 patients. Overall, we propose that pDC activation is essential to control SARS-CoV-2-infection. Failure to develop this response could be important to understand severe cases of COVID-19.


Asunto(s)
COVID-19 , Interferón Tipo I , Humanos , SARS-CoV-2/metabolismo , Antivirales/metabolismo , Células Dendríticas/metabolismo , Interferón lambda
7.
Commun Biol ; 5(1): 1115, 2022 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-36271143

RESUMEN

Zika virus (ZIKV) infection can cause important developmental and neurological defects in Humans. Type I/III interferon responses control ZIKV infection and pathological processes, yet the virus has evolved various mechanisms to defeat these host responses. Here, we established a pipeline to delineate at high-resolution the genetic evolution of ZIKV in a controlled host cell environment. We uncovered that serially passaged ZIKV acquired increased infectivity and simultaneously developed a resistance to TLR3-induced restriction. We built a mathematical model that suggests that the increased infectivity is due to a reduced time-lag between infection and viral replication. We found that this adaptation is cell-type specific, suggesting that different cell environments may drive viral evolution along different routes. Deep-sequencing of ZIKV populations pinpointed mutations whose increased frequencies temporally coincide with the acquisition of the adapted phenotype. We functionally validated S455L, a substitution in ZIKV envelope (E) protein, recapitulating the adapted phenotype. Its positioning on the E structure suggests a putative function in protein refolding/stability. Taken together, our results uncovered ZIKV adaptations to the cellular environment leading to accelerated replication onset coupled with resistance to TLR3-induced antiviral response. Our work provides insights into Zika virus adaptation to host cells and immune escape mechanisms.


Asunto(s)
Infección por el Virus Zika , Virus Zika , Humanos , Virus Zika/genética , Receptor Toll-Like 3 , Interferones , Antivirales
8.
Cell Host Microbe ; 25(5): 730-745.e6, 2019 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-31003939

RESUMEN

Type I interferon (IFN-I) is critical for antiviral defense, and plasmacytoid dendritic cells (pDCs) are a predominant source of IFN-I during virus infection. pDC-mediated antiviral responses are stimulated upon physical contact with infected cells, during which immunostimulatory viral RNA is transferred to pDCs, leading to IFN production via the nucleic acid sensor TLR7. Using dengue, hepatitis C, and Zika viruses, we demonstrate that the contact site of pDCs with infected cells is a specialized platform we term the interferogenic synapse, which enables viral RNA transfer and antiviral responses. This synapse is formed via αLß2 integrin-ICAM-1 adhesion complexes and the recruitment of the actin network and endocytic machinery. TLR7 signaling in pDCs promotes interferogenic synapse establishment and provides feed-forward regulation, sustaining pDC contacts with infected cells. This interferogenic synapse may allow pDCs to scan infected cells and locally secrete IFN-I, thereby confining a potentially deleterious response.


Asunto(s)
Antivirales/metabolismo , Adhesión Celular , Células Dendríticas/inmunología , Inmunidad Innata , Factores Inmunológicos/metabolismo , Interferón Tipo I/metabolismo , Virosis/inmunología , Línea Celular , Técnicas de Cocultivo , Virus del Dengue/inmunología , Hepacivirus/inmunología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Receptor Toll-Like 7/metabolismo , Virus Zika/inmunología
10.
Sci Rep ; 8(1): 10889, 2018 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-30022130

RESUMEN

Plasmacytoid dendritic cells (pDCs) are specialized in the production of interferons (IFNs) in response to viral infections. The Flaviviridae family comprises enveloped RNA viruses such as Hepatitis C virus (HCV) and Dengue virus (DENV). Cell-free flaviviridae virions poorly stimulate pDCs to produce IFN. By contrast, cells infected with HCV and DENV potently stimulate pDCs via short-range delivery of viral RNAs, which are either packaged within immature virions or secreted exosomes. We report that cells infected with Yellow fever virus (YFV), the prototypical flavivirus, stimulated pDCs to produce IFNs in a TLR7- and cell contact- dependent manner. Such stimulation was unaffected by the presence of YFV neutralizing antibodies. As reported for DENV, cells producing immature YFV particles were more potent at stimulating pDCs than cells releasing mature virions. Additionally, cells replicating a release-deficient YFV mutant or a YFV subgenomic RNA lacking structural protein-coding sequences participated in pDC stimulation. Thus, viral RNAs produced by YFV-infected cells reach pDCs via at least two mechanisms: within immature particles and as capsid-free RNAs. Our work highlights the ability of pDCs to respond to a variety of viral RNA-laden carriers generated from infected cells.


Asunto(s)
Cápside , Células Dendríticas/inmunología , Interferones/metabolismo , ARN Viral/metabolismo , Virión/inmunología , Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/inmunología , Adulto , Anciano , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN Viral/genética , Virión/metabolismo , Fiebre Amarilla/metabolismo , Fiebre Amarilla/virología , Adulto Joven
11.
Elife ; 72018 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-29914621

RESUMEN

Type I interferon (IFN-I) responses are critical for the control of RNA virus infections, however, many viruses, including Dengue (DENV) and Chikungunya (CHIKV) virus, do not directly activate plasmacytoid dendritic cells (pDCs), robust IFN-I producing cells. Herein, we demonstrated that DENV and CHIKV infected cells are sensed by pDCs, indirectly, resulting in selective IRF7 activation and IFN-I production, in the absence of other inflammatory cytokine responses. To elucidate pDC immunomodulatory functions, we developed a mouse model in which IRF7 signaling is restricted to pDC. Despite undetectable levels of IFN-I protein, pDC-restricted IRF7 signaling controlled both viruses and was sufficient to protect mice from lethal CHIKV infection. Early pDC IRF7-signaling resulted in amplification of downstream antiviral responses, including an accelerated natural killer (NK) cell-mediated type II IFN response. These studies revealed the dominant, yet indirect role of pDC IRF7-signaling in directing both type I and II IFN responses during arbovirus infections.


Asunto(s)
Fiebre Chikungunya/inmunología , Dengue/inmunología , Interacciones Huésped-Patógeno/inmunología , Factor 3 Regulador del Interferón/inmunología , Factor 7 Regulador del Interferón/inmunología , Interferón Tipo I/inmunología , Animales , Fiebre Chikungunya/genética , Fiebre Chikungunya/mortalidad , Fiebre Chikungunya/patología , Virus Chikungunya/crecimiento & desarrollo , Virus Chikungunya/inmunología , Virus Chikungunya/patogenicidad , Células Dendríticas/inmunología , Células Dendríticas/virología , Dengue/genética , Dengue/mortalidad , Dengue/patología , Virus del Dengue/crecimiento & desarrollo , Virus del Dengue/inmunología , Virus del Dengue/patogenicidad , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Factor 3 Regulador del Interferón/deficiencia , Factor 3 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/deficiencia , Factor 7 Regulador del Interferón/genética , Interferón Tipo I/genética , Interferón gamma/genética , Interferón gamma/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Viral/antagonistas & inhibidores , ARN Viral/genética , ARN Viral/inmunología , Transducción de Señal , Bazo/inmunología , Bazo/virología , Análisis de Supervivencia
12.
Cell Host Microbe ; 12(4): 558-70, 2012 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-23084922

RESUMEN

Viral nucleic acids often trigger an innate immune response in infected cells. Many viruses, including hepatitis C virus (HCV), have evolved mechanisms to evade intracellular recognition. Nevertheless, HCV-permissive cells can trigger a viral RNA-, TLR7-, and cell-contact-dependent compensatory interferon response in nonpermissive plasmacytoid dendritic cells (pDCs). Here we report that these events are mediated by transfer of HCV-RNA-containing exosomes from infected cells to pDCs. The exosomal viral RNA transfer is dependent on the endosomal sorting complex required for transport (ESCRT) machinery and on Annexin A2, an RNA-binding protein involved in membrane vesicle trafficking, and is suppressed by exosome release inhibitors. Further, purified concentrated HCV-RNA-containing exosomes are sufficient to activate pDCs. Thus, vesicular sequestration and exosomal export of viral RNA may serve both as a viral strategy to evade pathogen sensing within infected cells and as a host strategy to induce an unopposed innate response in replication-nonpermissive bystander cells.


Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Exosomas/metabolismo , Hepacivirus/inmunología , Inmunidad Innata , ARN Viral/inmunología , ARN Viral/metabolismo , Células Cultivadas , Células Dendríticas/virología , Exosomas/virología , Hepatocitos/virología , Humanos
13.
PLoS One ; 7(1): e30788, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22303456

RESUMEN

The mitogen-activated protein kinases (MAPK) ERK1 and ERK2 are among the major signal transduction molecules but little is known about their specific functions in vivo. ERK activity is provided by two isoforms, ERK1 and ERK2, which are ubiquitously expressed and share activators and substrates. However, there are not in vivo studies which have reported a role for ERK1 or ERK2 in HSCs and the bone marrow microenvironment. The present study shows that the ERK1-deficient mice present a mild osteopetrosis phenotype. The lodging and the homing abilities of the ERK1(-/-) HSC are impaired, suggesting that the ERK1(-/-)-defective environment may affect the engrafment of HSCs. Serial transplantations demonstrate that ERK1 is involved in the maintenance of an appropriate medullar microenvironment, but that the intrinsic properties of HSCs are not altered by the ERK1(-/-) defective microenvironment. Deletion of ERK1 impaired in vitro and in vivo osteoclastogenesis while osteoblasts were unaffected. As osteoclasts derive from precursors of the monocyte/macrophage lineage, investigation of the monocytic compartment was performed. In vivo analysis of the myeloid lineage progenitors revealed that the frequency of CMPs increased by approximately 1.3-fold, while the frequency of GMPs significantly decreased by almost 2-fold, compared with the respective WT compartments. The overall mononuclear-phagocyte lineage development was compromised in these mice due to a reduced expression of the M-CSF receptor on myeloid progenitors. These results show that the cellular targets of ERK1 are M-CSFR-responsive cells, upstream to osteoclasts. While ERK1 is well known to be activated by M-CSF, the present results are the first to point out an ERK1-dependent M-CSFR regulation on hematopoietic progenitors. This study reinforces the hypothesis of an active cross-talk between HSCs, their progeny and bone cells in the maintenance of the homeostasis of these compartments.


Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nicho de Células Madre , Animales , Densidad Ósea , Médula Ósea/patología , Huesos/enzimología , Huesos/patología , Compartimento Celular , Diferenciación Celular , Linaje de la Célula , Movimiento Celular , Proliferación Celular , Microambiente Celular , Eliminación de Gen , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 3 Activada por Mitógenos/deficiencia , Monocitos , Osteoblastos/enzimología , Osteoblastos/patología , Osteoclastos/enzimología , Osteoclastos/patología , Osteogénesis
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