RESUMO
Human circulating monocytes are established targets for Zika virus (ZIKV) infection. Because of their important migratory properties toward any tissues, including the central nervous system (CNS), a better understanding of the mechanisms underlying monocyte transmigration upon ZIKV infection is required. Here, we monitored adhesion, migration and transmigration properties of monocytes exposed to ZIKV. We found that ZIKV enhanced monocyte adhesion on collagen compared to mock-exposed samples, and that pharmacological inhibition of mDia and Cdc42 function induced a significant decrease of adhesion in both mock- and ZIKV-exposed monocytes. In contrast, monocyte migration through collagen was inhibited by most of the tested small molecules targeting regulators of actin polymerization, including Rac1, ROCK, Cdc42, mDia, Arp2/3, Myosin-II and LFA-1. ZIKV-exposed monocyte migration showed a very similar profile to that of their mock-exposed counterparts. Finally, assessment of monocyte transmigration through human cerebral microvascular endothelial cells (hCMEC/D3) showed dependency on Rac1, ROCK, and Cdc42, independently of their infection status. In contrast, we identified that BIRT377, an antagonist of LFA-1, significantly inhibited transmigration of ZIKV-exposed but not mock-exposed monocytes. As BIRT377 increased adhesion of ZIKV-exposed monocytes, we propose that LFA-1 might be involved in a post-adhesion step to enhance viro-induced transmigration. These data suggest that ZIKV exposure triggers specific migratory properties of monocytes that are not exploited under physiological conditions. This work provides further insights on virus-host interactions important for viral neuroinvasion and offers novel targets to specifically inhibit the infiltration of infected cells to the CNS. SUMMARY SENTENCE: Monocyte transmigration involves massive actin cytoskeleton reorganization regulated by small Rho GTPases and integrins, which can be subverted by viruses.
RESUMO
Transmigration of circulating monocytes from the bloodstream to tissues represents an early hallmark of inflammation. This process plays a pivotal role during viral neuroinvasion, encephalitis, and HIV-associated neurocognitive disorders. How monocytes locally unzip endothelial tight junction-associated proteins (TJAPs), without perturbing impermeability, to reach the central nervous system remains poorly understood. Here, we show that human circulating monocytes express the TJAP Occludin (OCLN) to promote transmigration through endothelial cells. We found that human monocytic OCLN (hmOCLN) clusters at monocyte-endothelium interface, while modulation of hmOCLN expression significantly impacts monocyte transmigration. Furthermore, we designed OCLN-derived peptides targeting its extracellular loops (EL) and show that transmigration of treated monocytes is inhibited in vitro and in zebrafish embryos, while preserving vascular integrity. Monocyte transmigration toward the brain is an important process for HIV neuroinvasion and we found that the OCLN-derived peptides significantly inhibit HIV dissemination to cerebral organoids. In conclusion, our study identifies an important role for monocytic OCLN during transmigration and provides a proof-of-concept for the development of mitigation strategies to prevent monocyte infiltration and viral neuroinvasion.
Assuntos
Células Endoteliais , Monócitos , Ocludina , Migração Transendotelial e Transepitelial , Peixe-Zebra , Ocludina/metabolismo , Ocludina/genética , Humanos , Monócitos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/virologia , Animais , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Células Endoteliais/efeitos dos fármacos , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , HIV-1/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/metabolismo , Encéfalo/metabolismo , Encéfalo/virologiaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with short- and long-term neurological complications. The variety of symptoms makes it difficult to unravel molecular mechanisms underlying neurological sequalae after coronavirus disease 2019 (COVID-19). Here we show that SARS-CoV-2 triggers the up-regulation of synaptic components and perturbs local electrical field potential. Using cerebral organoids, organotypic culture of human brain explants from individuals without COVID-19 and post-mortem brain samples from individuals with COVID-19, we find that neural cells are permissive to SARS-CoV-2 to a low extent. SARS-CoV-2 induces aberrant presynaptic morphology and increases expression of the synaptic components Bassoon, latrophilin-3 (LPHN3) and fibronectin leucine-rich transmembrane protein-3 (FLRT3). Furthermore, we find that LPHN3-agonist treatment with Stachel partially restored organoid electrical activity and reverted SARS-CoV-2-induced aberrant presynaptic morphology. Finally, we observe accumulation of relatively static virions at LPHN3-FLRT3 synapses, suggesting that local hindrance can contribute to synaptic perturbations. Together, our study provides molecular insights into SARS-CoV-2-brain interactions, which may contribute to COVID-19-related neurological disorders.
Assuntos
Encéfalo , COVID-19 , Homeostase , Organoides , SARS-CoV-2 , Sinapses , Humanos , SARS-CoV-2/fisiologia , COVID-19/virologia , COVID-19/metabolismo , COVID-19/patologia , Encéfalo/virologia , Sinapses/virologia , Sinapses/metabolismo , Organoides/virologia , Vírion/metabolismo , Neurônios/virologia , Neurônios/metabolismo , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genéticaRESUMO
Viral neuroinfections represent a major health burden for which the development of antivirals is needed. Antiviral compounds that target the consequences of a brain infection (symptomatic treatment) rather than the cause (direct-acting antivirals) constitute a promising mitigation strategy that requires to be investigated in relevant models. However, physiological surrogates mimicking an adult human cortex are lacking, limiting our understanding of the mechanisms associated with viro-induced neurological disorders. Here, we optimized the Organotypic culture of Post-mortem Adult human cortical Brain explants (OPAB) as a preclinical platform for Artificial Intelligence (AI)-driven antiviral studies. OPAB shows robust viability over weeks, well-preserved 3D cytoarchitecture, viral permissiveness, and spontaneous local field potential (LFP). Using LFP as a surrogate for neurohealth, we developed a machine learning framework to predict with high confidence the infection status of OPAB. As a proof-of-concept, we showed that antiviral-treated OPAB could partially restore LFP-based electrical activity of infected OPAB in a donor-dependent manner. Together, we propose OPAB as a physiologically relevant and versatile model to study neuroinfections and beyond, providing a platform for preclinical drug discovery.
Assuntos
Antivirais , Hepatite C Crônica , Humanos , Antivirais/farmacologia , Inteligência Artificial , Sistemas Microfisiológicos , EncéfaloRESUMO
Stem cell-derived cerebral organoids are artificially grown miniature organ-like structures mimicking embryonic brain architecture. They are composed of multiple neural cell types with 3D cell layer organization exhibiting local field potential. Measuring the extracellular electrical activity by means of conventional planar microelectrode arrays is particularly challenging due to the 3D architecture of organoids. In order to monitor the intra-organoid electrical activity of thick spheroid-shaped samples, we developed long protruding microelectrode arrays able to penetrate the inner regions of cerebral organoids to measure the local potential of neurons within the organoids. A new microfabrication process has been developed which, thanks to the relaxation of internal stresses of a stack of materials deposited over a sacrificial layer, allows one to build a protruding cantilever microelectrode array placed at the apex of beams which rise vertically, over two hundred microns. These slender beams inserted deeply into the organoids give access to the recording of local field potential from neurons buried inside the organoid. This novel device shall provide valuable tools to study neural functions in greater detail.
Assuntos
Organoides , Células-Tronco , Microeletrodos , Organoides/metabolismo , Neurônios/metabolismoRESUMO
Pseudomonas aeruginosa is a Gram-negative bacterium causing morbidity and mortality in immuno-compromised humans. It produces a lectin, LecB, that is considered a major virulence factor, however, its impact on the immune system remains incompletely understood. Here we show that LecB binds to endothelial cells in human skin and mice and disrupts the transendothelial passage of leukocytes in vitro. It impairs the migration of dendritic cells into the paracortex of lymph nodes leading to a reduced antigen-specific T cell response. Under the effect of the lectin, endothelial cells undergo profound cellular changes resulting in endocytosis and degradation of the junctional protein VE-cadherin, formation of an actin rim, and arrested cell motility. This likely negatively impacts the capacity of endothelial cells to respond to extracellular stimuli and to generate the intercellular gaps for allowing leukocyte diapedesis. A LecB inhibitor can restore dendritic cell migration and T cell activation, underlining the importance of LecB antagonism to reactivate the immune response against P. aeruginosa infection.
Assuntos
Pseudomonas aeruginosa , Migração Transendotelial e Transepitelial , Humanos , Animais , Camundongos , Células Endoteliais/metabolismo , Lectinas/metabolismo , Lectinas/farmacologia , ImunidadeRESUMO
BACKGROUND AND AIMS: Numerous HCV entry factors have been identified, and yet information regarding their spatiotemporal dynamics is still limited. Specifically, one of the main entry factors of HCV is occludin (OCLN), a protein clustered at tight junctions (TJs), away from the HCV landing site. Thus, whether HCV particles slide toward TJs or, conversely, OCLN is recruited away from TJs remain debated. APPROACH AND RESULTS: Here, we generated CRISPR/CRISPR-associated protein 9 edited Huh7.5.1 cells expressing endogenous levels of enhanced green fluorescent protein/OCLN and showed that incoming HCV particles recruit OCLN outside TJs, independently of claudin 1 (CLDN1) expression, another important HCV entry factor located at TJs. Using ex vivo organotypic culture of hepatic slices obtained from human liver explants, a physiologically relevant model that preserves the overall tissue architecture, we confirmed that HCV associates with OCLN away from TJs. Furthermore, we showed, by live cell imaging, that increased OCLN recruitment beneath HCV particles correlated with lower HCV motility. To decipher the mechanism underlying virus slow-down upon OCLN recruitment, we performed CRISPR knockout (KO) of CLDN1, an HCV entry factor proposed to act upstream of OCLN. Although CLDN1 KO potently inhibits HCV infection, OCLN kept accumulating underneath the particle, indicating that OCLN recruitment is CLDN1 independent. Moreover, inhibition of the phosphorylation of Ezrin, a protein involved in HCV entry that links receptors to the actin cytoskeleton, increased OCLN accumulation and correlated with more efficient HCV internalization. CONCLUSIONS: Together, our data provide robust evidence that HCV particles interact with OCLN away from TJs and shed mechanistic insights regarding the manipulation of transmembrane receptor localization by extracellular virus particles.
Assuntos
Hepatite C , Junções Íntimas , Proteína 9 Associada à CRISPR/metabolismo , Claudina-1/genética , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatócitos/metabolismo , Humanos , Ocludina , Vírion , Internalização do VírusRESUMO
The evolution of the COVID-19 pandemic can be monitored through the detection of SARS-CoV-2 RNA in sewage. Here, we measured the amount of SARS-CoV-2 RNA at the inflow point of the main waste water treatment plant (WWTP) of Montpellier, France. We collected samples 4 days before the end of lockdown and up to 70 days post-lockdown. We detected increased amounts of SARS-CoV-2 RNA at the WWTP from mid-June on, whereas the number of new COVID-19 cases in the area started increasing a couple of weeks later. Future epidemiologic investigations shall explain such asynchronous finding.
Assuntos
Encéfalo/virologia , Monócitos/fisiologia , Zika virus/patogenicidade , Adulto , Animais , Encéfalo/patologia , Quimiotaxia de Leucócito/fisiologia , Feminino , Humanos , Evasão da Resposta Imune/fisiologia , Monócitos/virologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/patologia , Complicações Infecciosas na Gravidez/virologia , Carga Viral/fisiologia , Zika virus/fisiologia , Infecção por Zika virus/epidemiologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/patologiaRESUMO
Zika virus (ZIKV) invades and persists in the central nervous system (CNS), causing severe neurological diseases. However the virus journey, from the bloodstream to tissues through a mature endothelium, remains unclear. Here, we show that ZIKV-infected monocytes represent suitable carriers for viral dissemination to the CNS using human primary monocytes, cerebral organoids derived from embryonic stem cells, organotypic mouse cerebellar slices, a xenotypic human-zebrafish model, and human fetus brain samples. We find that ZIKV-exposed monocytes exhibit higher expression of adhesion molecules, and higher abilities to attach onto the vessel wall and transmigrate across endothelia. This phenotype is associated to enhanced monocyte-mediated ZIKV dissemination to neural cells. Together, our data show that ZIKV manipulates the monocyte adhesive properties and enhances monocyte transmigration and viral dissemination to neural cells. Monocyte transmigration may represent an important mechanism required for viral tissue invasion and persistence that could be specifically targeted for therapeutic intervention.