RESUMO
Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Ceramidas/farmacologia , Ceramidas/metabolismo , Replicação Viral , Antivirais/farmacologiaRESUMO
Bacterial pore-forming toxins compromise plasmalemmal integrity, leading to Ca2+ influx, leakage of the cytoplasm, and cell death. Such lesions can be repaired by microvesicular shedding or by the endocytic uptake of the injured membrane sites. Cells have at their disposal an entire toolbox of repair proteins for the identification and elimination of membrane lesions. Sphingomyelinases catalyze the breakdown of sphingomyelin into ceramide and phosphocholine. Sphingomyelin is predominantly localized in the outer leaflet, where it is hydrolyzed by acid sphingomyelinase (ASM) after lysosomal fusion with the plasma membrane. The magnesium-dependent neutral sphingomyelinase (NSM)-2 is found at the inner leaflet of the plasmalemma. Because either sphingomyelinase has been ascribed a role in the cellular stress response, we investigated their role in plasma membrane repair and cellular survival after treatment with the pore-forming toxins listeriolysin O (LLO) or pneumolysin (PLY). Jurkat T cells, in which ASM or NSM-2 was down-regulated [ASM knockdown (KD) or NSM-2 KD cells], showed inverse reactions to toxin-induced membrane damage: ASM KD cells displayed reduced toxin resistance, decreased viability, and defects in membrane repair. In contrast, the down-regulation of NSM-2 led to an increase in viability and enhanced plasmalemmal repair. Yet, in addition to the increased plasmalemmal repair, the enhanced toxin resistance of NSM-2 KD cells also appeared to be dependent on the activation of p38/MAPK, which was constitutively activated, whereas in ASM KD cells, the p38/MAPK activation was constitutively blunted.-Schoenauer, R., Larpin, Y., Babiychuk, E. B., Drücker, P., Babiychuk, V. S., Avota, E., Schneider-Schaulies, S., Schumacher, F., Kleuser, B., Köffel, R., Draeger, A. Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins.
Assuntos
Toxinas Bacterianas/farmacologia , Membrana Celular/metabolismo , Proteínas de Choque Térmico/farmacologia , Proteínas Hemolisinas/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Estreptolisinas/farmacologia , Proteínas de Bactérias/farmacologia , Transporte Biológico , Sistemas CRISPR-Cas , Cálcio/metabolismo , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Humanos , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
By hydrolyzing its substrate sphingomyelin at the cytosolic leaflet of cellular membranes, the neutral sphingomyelinase 2 (NSM2) generates microdomains which serve as docking sites for signaling proteins and thereby, functions to regulate signal relay. This has been particularly studied in cellular stress responses while the regulatory role of this enzyme in the immune cell compartment has only recently emerged. In T cells, phenotypic polarization by co-ordinated cytoskeletal remodeling is central to motility and interaction with endothelial or antigen-presenting cells during tissue recruitment or immune synapse formation, respectively. This review highlights studies adressing the role of NSM2 in T cell polarity in which the enzyme plays a major role in regulating cytoskeletal dynamics.
Assuntos
Polaridade Celular , Receptores de Antígenos de Linfócitos T/imunologia , Esfingomielina Fosfodiesterase/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Humanos , Transdução de Sinais , Linfócitos T/metabolismoRESUMO
Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.
Assuntos
Azidas/metabolismo , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Esfingolipídeos/metabolismo , Linfócitos T/metabolismo , Azidas/química , Células Cultivadas , Humanos , Ativação Linfocitária , Transporte Proteico , Agregação de Receptores , Transdução de Sinais , Esfingolipídeos/química , Linfócitos T/imunologiaRESUMO
Though mostly defective, human endogenous retroviruses (HERV) can retain open reading frames, which are especially expressed in the placenta. There, the envelope (env) proteins of HERV-W (Syncytin-1), HERV-FRD (Syncytin-2), and HERV-K (HML-2) were implicated in tolerance against the semi-allogenic fetus. Here, we show that the known HERV env-binding receptors ASCT-1 and -2 and MFSD2 are expressed by DCs and T-cells. When used as effectors in coculture systems, CHO cells transfected to express Syncytin-1, -2, or HML-2 did not affect T-cell expansion or overall LPS-driven phenotypic DC maturation, however, promoted release of IL-12 and TNF-α rather than IL-10. In contrast, HERV env expressing choriocarcinoma cell lines suppressed T-cell proliferation and LPS-induced TNF-α and IL-12 release, however, promoted IL-10 accumulation, indicating that these effects might not rely on HERV env interactions. However, DCs conditioned by choriocarcinoma, but also transgenic CHO cells failed to promote allogenic T-cell expansion. This was associated with a loss of DC/T-cell conjugate frequencies, impaired Ca(2+) mobilization, and aberrant patterning of f-actin and tyrosine phosphorylated proteins in T-cells. Altogether, these findings suggest that HERV env proteins target T-cell activation indirectly by modulating the stimulatory activity of DCs.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Retrovirus Endógenos/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Proteínas do Envelope Viral/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Células CHO , Comunicação Celular/imunologia , Diferenciação Celular , Linhagem Celular , Coriocarcinoma/genética , Coriocarcinoma/imunologia , Coriocarcinoma/metabolismo , Cricetulus , Citocinas/biossíntese , Células Dendríticas/citologia , Retrovirus Endógenos/genética , Feminino , Expressão Gênica , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Humanos , Fenótipo , Gravidez , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Linfócitos T/metabolismo , Proteínas do Envelope Viral/genéticaRESUMO
T cell paralysis is a main feature of measles virus (MV) induced immunosuppression. MV contact mediated activation of sphingomyelinases was found to contribute to MV interference with T cell actin reorganization. The role of these enzymes in MV-induced inhibition of T cell activation remained equally undefined as their general role in regulating immune synapse (IS) activity which relies on spatiotemporal membrane patterning. Our study for the first time reveals that transient activation of the neutral sphingomyelinase 2 (NSM2) occurs in physiological co-stimulation of primary T cells where ceramide accumulation is confined to the lamellum (where also NSM2 can be detected) and excluded from IS areas of high actin turnover. Genetic ablation of the enzyme is associated with T cell hyper-responsiveness as revealed by actin dynamics, tyrosine phosphorylation, Ca2+-mobilization and expansion indicating that NSM2 acts to suppress overshooting T cell responses. In line with its suppressive activity, exaggerated, prolonged NSM2 activation as occurring in co-stimulated T cells following MV exposure was associated with aberrant compartmentalization of ceramides, loss of spreading responses, interference with accumulation of tyrosine phosphorylated protein species and expansion. Altogether, this study for the first time reveals a role of NSM2 in physiological T cell stimulation which is dampening and can be abused by a virus, which promotes enhanced and prolonged NSM2 activation to cause pathological T cell suppression.
Assuntos
Vírus do Sarampo/fisiologia , Esfingomielina Fosfodiesterase/fisiologia , Fatores Supressores Imunológicos/fisiologia , Linfócitos T/fisiologia , Linfócitos T/virologia , Actinas/fisiologia , Antígenos CD28/fisiologia , Complexo CD3/fisiologia , Células Cultivadas , Ceramidas/fisiologia , Humanos , Lipídeos de Membrana/fisiologia , Proteínas de Membrana/fisiologiaRESUMO
The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains.
Assuntos
Encéfalo/irrigação sanguínea , Ceramidas/metabolismo , Endotélio Vascular/microbiologia , Interações Hospedeiro-Patógeno , Neisseria meningitidis/patogenicidade , Esfingomielina Fosfodiesterase/metabolismo , Regulação para Cima , Aderência Bacteriana/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/microbiologia , Linhagem Celular Transformada , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/metabolismo , Meningite Meningocócica/enzimologia , Meningite Meningocócica/metabolismo , Meningite Meningocócica/microbiologia , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Microvasos/microbiologia , Mutação , Neisseria meningitidis/fisiologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Propriedades de Superfície/efeitos dos fármacos , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Disseminated gonococcal infection (DGI) is a rare but serious complication caused by the spread of Neisseria gonorrhoeae in the human host. Gonococci associated with DGI mainly express the outer membrane protein PorBIA that binds to the scavenger receptor expressed on endothelial cells (SREC-I) and mediates bacterial uptake. We recently demonstrated that this interaction relies on intact membrane rafts that acquire SREC-I upon attachment of gonococci and initiates the signalling cascade that finally leads to the uptake of gonococci in epithelial cells. In this study, we analysed the role of sphingomyelinases and their breakdown product ceramide. Gonococcal infection induced increased levels of ceramide that was enriched at bacterial attachment sites. Interestingly, neutral but not acid sphingomyelinase was mandatory for PorBIA -mediated invasion into host cells. Neutral sphingomyelinase was required to recruit the PI3 kinase to caveolin and thereby activates the PI3 kinase-dependent downstream signalling leading to bacterial uptake. Thus, this study elucidates the initial signalling processes of bacterial invasion during DGI and demonstrates a novel role for neutral sphingomyelinase in the course of bacterial infections.
Assuntos
Endocitose , Interações Hospedeiro-Patógeno , Neisseria gonorrhoeae/fisiologia , Porinas/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Células Cultivadas , Ceramidas/metabolismo , Humanos , Transdução de SinaisRESUMO
Viruses exploit membranes and their components such as sphingolipids in all steps of their life cycle including attachment and membrane fusion, intracellular transport, replication, protein sorting and budding. Examples for sphingolipid-dependent virus entry are found for: human immunodeficiency virus (HIV), which besides its protein receptors also interacts with glycosphingolipids (GSLs); rhinovirus, which promotes the formation of ceramide-enriched platforms and endocytosis; or measles virus (MV), which induces the surface expression of its own receptor CD150 via activation of sphingomyelinases (SMases). While SMase activation was implicated in Ebola virus (EBOV) attachment, the virus utilizes the cholesterol transporter Niemann-Pick C protein 1 (NPC1) as 'intracellular' entry receptor after uptake into endosomes. Differential activities of SMases also affect the intracellular milieu required for virus replication. Sindbis virus (SINV), for example, replicates better in cells lacking acid SMase (ASMase). Defined lipid compositions of viral assembly and budding sites influence virus release and infectivity, as found for hepatitis C virus (HCV) or HIV. And finally, viruses manipulate cellular signaling and the sphingolipid metabolism to their advantage, as for example influenza A virus (IAV), which activates sphingosine kinase 1 and the transcription factor NF-κB.
Assuntos
Esfingolipídeos/metabolismo , Fenômenos Fisiológicos Virais , Linhagem Celular , Ceramidas/metabolismo , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Internalização do VírusRESUMO
HIV-1 mediates pro-survival signals and prevents apoptosis via the phosphatidylinositol-3-kinase (PI3K) pathway. This pathway, however, also affects phosphorylation of serine-arginine (SR) proteins, a family of splicing regulatory factors balancing splice site selection. We now show that pharmacologic inhibition of PI3K signalling alters the HIV-1 splicing pattern of both minigene- and provirus-derived mRNAs. This indicates that HIV-1 might also promote PI3K signalling to balance processing of its transcripts by regulating phosphorylation of splicing regulatory proteins.
Assuntos
Regulação Viral da Expressão Gênica , HIV-1/genética , Interações Hospedeiro-Patógeno , Fosfatidilinositol 3-Quinases/metabolismo , Precursores de RNA/metabolismo , Splicing de RNARESUMO
Measles virus (MV) efficiently causes generalized immunosuppression which accounts to a major extent for cases of measles-asscociated severe morbidity and mortality. MV infections alter many functions of antigen presenting cells (APC) (dendritic cells (DCs)) and lymphocytes, yet many molecular targets of the virus remain poorly defined. Cellular interactions and effector functions of DCs and lymphocytes are regulated by surface receptors. Associating with other proteins involved in cell signaling, receptors form part of receptosomes that respond to and transmit external signals through dynamic interctions with the cytoskeleton. Alterations in the composition and metabolism of membrane sphingolipids have a substantial impact on both processes. In this review we focus on the regulation of sphingomyelinase activity and ceramide release in cells exposed to MV and discuss the immunosuppressive role of sphingomyelin breakdown induced by MV.
Assuntos
Vírus do Sarampo/patogenicidade , Esfingomielinas/metabolismo , Citoesqueleto de Actina/metabolismo , Ceramidas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Tolerância Imunológica , Vírus do Sarampo/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Viral entry, compartmentalization and transmission depend on the formation of membrane lipid/protein microdomains concentrating receptors and signalosomes. Dendritic cells (DCs) are prime targets for measles virus (MV) infection, and this interaction promotes immune activation and generalized immunosuppression, yet also MV transport to secondary lymphatics where transmission to T cells occurs. In addition to MV trapping, DC-SIGN interaction can enhance MV uptake by activating cellular sphingomyelinases and, thereby, vertical surface transport of its entry receptor CD150. To exploit DCs as Trojan horses for transport, MV promotes DC maturation accompanied by mobilization, and restrictions of viral replication in these cells may support this process. MV-infected DCs are unable to support formation of functional immune synapses with conjugating T cells and signalling via viral glycoproteins or repulsive ligands (such as semaphorins) plays a key role in the induction of T-cell paralysis. In the absence of antigen recognition, MV transmission from infected DCs to T cells most likely involves formation of polyconjugates which concentrate viral structural proteins, viral receptors and with components enhancing either viral uptake or conjugate stability. Because DCs barely support production of infectious MV particles, these organized interfaces are likely to represent virological synapses essential for MV transmission.
Assuntos
Membrana Celular/metabolismo , Células Dendríticas/metabolismo , Vírus do Sarampo/metabolismo , Sarampo/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Membrana Celular/química , Membrana Celular/virologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Sarampo/imunologia , Sarampo/transmissão , Sarampo/virologia , Vírus do Sarampo/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Transdução de Sinais , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Esfingomielina Fosfodiesterase/metabolismo , Linfócitos T/patologia , Linfócitos T/virologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Replicação ViralRESUMO
Ceramides generated by the activity of the neutral sphingomyelinase 2 (nSMase2) play a pivotal role in stress responses in mammalian cells. Dysregulation of sphingolipid metabolism has been implicated in numerous inflammation-related pathologies. However, its influence on inflammatory cytokine-induced signaling is yet incompletely understood. Here, we used proximity labeling to explore the plasma membrane proximal protein network of nSMase2 and TNFα-induced changes thereof. We established Jurkat cells stably expressing nSMase2 C-terminally fused to the engineered ascorbate peroxidase 2 (APEX2). Removal of excess biotin phenol substantially improved streptavidin-based affinity purification of biotinylated proteins. Using our optimized protocol, we determined nSMase2-proximal biotinylated proteins and their changes within the first 5 min of TNFα stimulation by quantitative mass spectrometry. We observed significant dynamic changes in the nSMase2 microenvironment in response to TNFα stimulation consistent with rapid remodeling of protein networks. Our data confirmed known nSMase2 interactors and revealed that the recruitment of most proteins depended on nSMase2 enzymatic activity. We measured significant enrichment of proteins related to vesicle-mediated transport, including proteins of recycling endosomes, trans-Golgi network, and exocytic vesicles in the proximitome of enzymatically active nSMase2 within the first minutes of TNFα stimulation. Hence, the nSMase2 proximal network and its TNFα-induced changes provide a valuable resource for further investigations into the involvement of nSMase2 in the early signaling pathways triggered by TNFα.
Assuntos
Esfingomielina Fosfodiesterase , Fator de Necrose Tumoral alfa , Humanos , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Células Jurkat , Esfingomielina Fosfodiesterase/metabolismo , Transdução de Sinais , Membrana Celular/metabolismoRESUMO
Transmission of measles virus (MV) to T cells by its early CD150(+) target cells is considered to be crucial for viral dissemination within the hematopoietic compartment. Using cocultures involving monocyte-derived dendritic cells (DCs) and T cells, we now show that T cells acquire MV most efficiently from cis-infected DCs rather than DCs having trapped MV (trans-infection). Transmission involves interactions of the viral glycoprotein H with its receptor CD150 and is therefore more efficient to preactivated T cells. In addition to rare association with actin-rich filopodial structures, the formation of contact interfaces consistent with that of virological synapses (VS) was observed where viral proteins accumulated and CD150 was redistributed in an actin-dependent manner. In addition to these molecules, activated LFA-1, DC-SIGN, CD81, and phosphorylated ezrin-radixin-moesin proteins, which also mark the HIV VS, redistributed toward the MV VS. Most interestingly, moesin and substance P receptor, both implicated earlier in assisting MV entry or cell-to-cell transmission, also partitioned to the transmission structure. Altogether, the MV VS shares important similarities to the HIV VS in concentrating cellular components potentially regulating actin dynamics, conjugate stability, and membrane fusion as required for efficient entry of MV into target T cells.
Assuntos
Células Dendríticas/virologia , Sinapses Imunológicas/virologia , Vírus do Sarampo/fisiologia , Vírus do Sarampo/patogenicidade , Linfócitos T/virologia , Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Dendríticas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Técnicas In Vitro , Lectinas Tipo C/metabolismo , Sarampo/imunologia , Sarampo/transmissão , Sarampo/virologia , Vírus do Sarampo/imunologia , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Linfócitos T/imunologia , Proteínas Virais/imunologiaRESUMO
As pattern recognition receptor on dendritic cells (DCs), DC-SIGN binds carbohydrate structures on its pathogen ligands and essentially determines host pathogen interactions because it both skews T cell responses and enhances pathogen uptake for cis infection and/or T cell trans-infection. How these processes are initiated at the plasma membrane level is poorly understood. We now show that DC-SIGN ligation on DCs by antibodies, mannan or measles virus (MV) causes rapid activation of neutral and acid sphingomyelinases followed by accumulation of ceramides in the outer membrane leaflet. SMase activation is important in promoting DC-SIGN signaling, but also for enhancement of MV uptake into DCs. DC-SIGN-dependent SMase activation induces efficient, transient recruitment of CD150, which functions both as MV uptake receptor and microbial sensor, from an intracellular Lamp-1+ storage compartment shared with acid sphingomyelinase (ASM) within a few minutes. Subsequently, CD150 is displayed at the cell surface and co-clusters with DC-SIGN. Thus, DC-SIGN ligation initiates SMase-dependent formation of ceramide-enriched membrane microdomains which promote vertical segregation of CD150 from intracellular storage compartments along with ASM. Given the ability to promote receptor and signalosome co-segration into (or exclusion from) ceramide enriched microdomains which provide a favorable environment for membrane fusion, DC-SIGN-dependent SMase activation may be of general importance for modes and efficiency of pathogen uptake into DCs, and their routing to specific compartments, but also for modulating T cell responses.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Ceramidas/metabolismo , Células Dendríticas/virologia , Lectinas Tipo C/metabolismo , Vírus do Sarampo/crescimento & desenvolvimento , Receptores de Superfície Celular/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Apresentação de Antígeno , Antígenos CD/genética , Western Blotting , Moléculas de Adesão Celular/genética , Células Cultivadas , Células Dendríticas/metabolismo , Citometria de Fluxo , Humanos , Imunoprecipitação , Lectinas Tipo C/genética , Sarampo , Vírus do Sarampo/genética , Vírus do Sarampo/imunologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores Virais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Linfócitos T/virologiaRESUMO
Besides their essential role in the immune system, sphingolipids and their metabolites are potential key regulators in the life cycle of obligatory intracellular pathogens such as viruses. They are involved in lateral and vertical segregation of receptors required for attachment, membrane fusion and endocytosis, as well as in the intracellular replication, assembly and release of viruses. Glycosphingolipids may themselves act as receptors for viruses, such as Galactosylceramide for human immunodeficiency virus (HIV). In addition, sphingolipids and their metabolites are inseparably interwoven in signal transduction processes, dynamic alterations of the cytoskeleton, and the regulation of innate and intrinsic responses of infected target cells. Depending on the nature of the intracellular pathogen, they may support or inhibit infections. Understanding of the underlying mechanisms depending on the specific virus, immune control, and type of disease may open new avenues for therapeutic interventions.
Assuntos
Transdução de Sinais , Esfingolipídeos/metabolismo , Viroses/metabolismo , Vírus/patogenicidade , Animais , Antivirais/farmacologia , Desenho de Fármacos , Interações Hospedeiro-Patógeno , Humanos , Transdução de Sinais/efeitos dos fármacos , Viroses/tratamento farmacológico , Viroses/virologia , Internalização do Vírus , Replicação Viral , Vírus/efeitos dos fármacosRESUMO
Measles virus (MV)-infected DC fail to promote T-cell expansion, and this could explain important aspects of measles immunosuppression. The efficiency of the immune synapse (IS) is determined by the formation of stable, stimulatory conjugates involving a spatially and timely controlled architecture. PlexinA1 (plexA1) and its co-receptor neuropilin (NP-1) have been implicated in IS efficiency, while their repulsive ligand, SEMA3A, likely acts in terminating T-cell activation. Conjugates involving MV-infected DC and T cells are unstable and not stimulatory, and thus we addressed the potential role of plexA1/NP-1 and semaphorins (SEMAs) in this system. MV does not grossly affect expression levels of plexA1/NP-1 on T cells or DC, yet prevents their recruitment towards stimulatory interfaces. Moreover, MV infection promoted early release of SEMA3A from DC, which caused loss of actin based protrusions on T cells as did the plexA4 ligand SEMA6A. SEMA3A/6A differentially modulated chemokinetic migration of T cells and conjugation with allogeneic DC. Thus, MV targets SEMA receptor function both at the level of IS recruitment, and by promoting a timely inappropriate release of their repulsive ligand, SEMA3A. To the best of our knowledge, this is the first example of viral targeting of SEMA receptor function in the IS.
Assuntos
Comunicação Celular/imunologia , Células Dendríticas/imunologia , Vírus do Sarampo/imunologia , Proteínas do Tecido Nervoso/imunologia , Neuropilina-1/imunologia , Receptores de Superfície Celular/imunologia , Semaforinas/imunologia , Movimento Celular/imunologia , Células Dendríticas/virologia , Humanos , Sinapses Imunológicas/virologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/virologiaRESUMO
Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected, we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells in vitro. Infection of human CD34(+) HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs, where infection is established in CD34(+) CD150(-) and CD34(+) CD150(+) (in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection, as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability, expansion, and colony-forming activity of either CD150(+) or CD150(-) HS/PCs in vitro, it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether, these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression.
Assuntos
Antígenos CD34/imunologia , Células-Tronco Hematopoéticas/citologia , Vírus do Sarampo/fisiologia , Animais , Separação Celular , Citometria de Fluxo , Células-Tronco Hematopoéticas/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NODRESUMO
SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2-RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.
Assuntos
Ceramidase Ácida , Tratamento Farmacológico da COVID-19 , Ceramidase Ácida/genética , Ceramidase Ácida/metabolismo , Fluoxetina , Humanos , Pandemias , RNA , SARS-CoV-2RESUMO
Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase-dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to beta1 integrin ligation or alphaCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics.