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DC-SIGN mediated sphingomyelinase-activation and ceramide generation is essential for enhancement of viral uptake in dendritic cells.

Avota E, Gulbins E, Schneider-Schaulies S - PLoS Pathog. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Virology and Immunobiology, University of Würzburg, Wuerzburg, Germany.

ABSTRACT
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.

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DC-SIGN ligation activates SMases and causes membrane ceramide accumulation on DCs.A. to C. Surface ceramide levels on DCs were determined by spot assays (shown in the insets) or flow cytometry (A, B). A. DCs were exposed to a MOCK preparation (open circles left panel), MV alone (black circles, left and middle panels) or after pretreatment with a blocking DC-SIGN antibody (AZ-D1) (black triangles, left panel) or in the presence of EGTA (open circles, middle panel). Right panel: DCs were left untreated (black circles) or exposed to an αCD150-antibody (5C6) prior to MV or MOCK treatment (white circles and black triangles, respectively). B. DCs were exposed to AZ-D1 alone (open circles, left panel) or after addition of a crosslinking antibody (filled circles, both panels). DCs were stimulated with isotype antibody as a negative control (black triangles, left panel) or DC-SIGN was ligated after 2 hrs pretreatment with amitriptyline (open circles, right panel). C. DCs were exposed to mannan for the time intervals indicated and surface levels of ASM (black triangles) and ceramides in the absence (black circles) or presence of EGTA (open circles) were analysed by spot assays (left panel). D. Whole cell lysates (for ASM, left panel) or membrane fractions (for NSM, right panel) of DCs exposed to mannan for the time intervals indicated were used to determine enzyme activities using a commercial kit. DCs were transfected with NSM siRNA (open circles) or not (black circles) (right panel, with effciency of NSM ablation shown by RT-PCR in the inset). E. ASM activity was determined 10 mins following exposure of DCs to MV or a recombinant MV expressing VSV G protein instead of the MV glycoproteins (MGV) at the multiplicities of infection indicated or a mock preparation applied at a concentration corresponding to m.o.i 2) as in D. Experiments shown are representatives of each three independent experiments involving different donors.
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ppat-1001290-g001: DC-SIGN ligation activates SMases and causes membrane ceramide accumulation on DCs.A. to C. Surface ceramide levels on DCs were determined by spot assays (shown in the insets) or flow cytometry (A, B). A. DCs were exposed to a MOCK preparation (open circles left panel), MV alone (black circles, left and middle panels) or after pretreatment with a blocking DC-SIGN antibody (AZ-D1) (black triangles, left panel) or in the presence of EGTA (open circles, middle panel). Right panel: DCs were left untreated (black circles) or exposed to an αCD150-antibody (5C6) prior to MV or MOCK treatment (white circles and black triangles, respectively). B. DCs were exposed to AZ-D1 alone (open circles, left panel) or after addition of a crosslinking antibody (filled circles, both panels). DCs were stimulated with isotype antibody as a negative control (black triangles, left panel) or DC-SIGN was ligated after 2 hrs pretreatment with amitriptyline (open circles, right panel). C. DCs were exposed to mannan for the time intervals indicated and surface levels of ASM (black triangles) and ceramides in the absence (black circles) or presence of EGTA (open circles) were analysed by spot assays (left panel). D. Whole cell lysates (for ASM, left panel) or membrane fractions (for NSM, right panel) of DCs exposed to mannan for the time intervals indicated were used to determine enzyme activities using a commercial kit. DCs were transfected with NSM siRNA (open circles) or not (black circles) (right panel, with effciency of NSM ablation shown by RT-PCR in the inset). E. ASM activity was determined 10 mins following exposure of DCs to MV or a recombinant MV expressing VSV G protein instead of the MV glycoproteins (MGV) at the multiplicities of infection indicated or a mock preparation applied at a concentration corresponding to m.o.i 2) as in D. Experiments shown are representatives of each three independent experiments involving different donors.

Mentions: Membrane ceramide platforms segregate receptors and signalosomes both of which can affect viral entry. DC-SIGN may act to trap or concentrate virions (also including MV) for receptor interaction, and we thus analysed whether MV interaction with this molecule promoted membrane ceramide accumulation on DCs by employing an assay based on immunodetection of an a-ceramide antibody bound to intact cells (spot assay). On MV exposure, DCs responded by an about twofold increase in extrafacial ceramides which peaked at 15 mins and subsequently returned to baseline levels (Fig. 1A, left panel). Ceramide accumulation occurred DC-SIGN dependently, since it was efficiently abrogated upon pre-exposure of DCs with a DC-SIGN-specific antibody (AZ-D1) or EGTA, which prevents Ca2+-dependent DC-SIGN ligand binding (Fig. 1A, left panel and middle panel). In contrast, antibodies blocking MV interaction with its entry receptor, CD150, did not prevent, yet even slightly enhanced MV ceramide induction (Fig. 1A, right panel). Similar to MV, the DC-SIGN binding antibody AZ-D1 increased membrane ceramide display when crosslinked, while this was not observed with the antibody alone nor an isotype control antibody alone (Fig. 1B, left panel; for further experiments, DC-SIGN-specific antibodies were thus used crosslinked). Ceramide production in response to DC-SIGN ligation was sensitive to the ASM inhibitor amitriptyline indicating that activation of this enzyme was involved (Fig. 1B, right panel). To assess activation of SMases directly, we determined their activity after exposure of DCs to mannan, a well-defined DC-SIGN agonist. In line with amitriptyline sensitivity of ceramide generation, ASM surface display raised about 1.8 fold almost immediately following mannan addition, and this was paralleled by a rise in extrafacial ceramides both of which were EGTA sensitive (Fig. 1C, and not shown). Mannan-dependent activation of ASM was further confirmed using a commercial detection assay (Fig. 1D, left panel) which essentially mirrored kinetics and magnitude of the response determined by spot assays. Using the same experimental approach, a rapid, very efficient activation of NSM was also measured (about 5-fold) which peaked after 3 mins and then vanished, and this was entirely prevented upon RNAi mediated silencing of NSM expression (Fig. 1D, right panel). Importantly, ASM activation also occurred on MV exposure of DCs in a dose dependent manner, and this relied on the presence of the MV glycoproteins because it was not observed when a recombinant MV expressing the VSV G protein instead was used (Fig. 1E). Altogether these findings indicate that ligation of DC-SIGN by antibodies, mannan or MV promotes rapid activation of SMases, and ASM-dependent ceramide accumulation in the outer membrane leaflet.


DC-SIGN mediated sphingomyelinase-activation and ceramide generation is essential for enhancement of viral uptake in dendritic cells.

Avota E, Gulbins E, Schneider-Schaulies S - PLoS Pathog. (2011)

DC-SIGN ligation activates SMases and causes membrane ceramide accumulation on DCs.A. to C. Surface ceramide levels on DCs were determined by spot assays (shown in the insets) or flow cytometry (A, B). A. DCs were exposed to a MOCK preparation (open circles left panel), MV alone (black circles, left and middle panels) or after pretreatment with a blocking DC-SIGN antibody (AZ-D1) (black triangles, left panel) or in the presence of EGTA (open circles, middle panel). Right panel: DCs were left untreated (black circles) or exposed to an αCD150-antibody (5C6) prior to MV or MOCK treatment (white circles and black triangles, respectively). B. DCs were exposed to AZ-D1 alone (open circles, left panel) or after addition of a crosslinking antibody (filled circles, both panels). DCs were stimulated with isotype antibody as a negative control (black triangles, left panel) or DC-SIGN was ligated after 2 hrs pretreatment with amitriptyline (open circles, right panel). C. DCs were exposed to mannan for the time intervals indicated and surface levels of ASM (black triangles) and ceramides in the absence (black circles) or presence of EGTA (open circles) were analysed by spot assays (left panel). D. Whole cell lysates (for ASM, left panel) or membrane fractions (for NSM, right panel) of DCs exposed to mannan for the time intervals indicated were used to determine enzyme activities using a commercial kit. DCs were transfected with NSM siRNA (open circles) or not (black circles) (right panel, with effciency of NSM ablation shown by RT-PCR in the inset). E. ASM activity was determined 10 mins following exposure of DCs to MV or a recombinant MV expressing VSV G protein instead of the MV glycoproteins (MGV) at the multiplicities of infection indicated or a mock preparation applied at a concentration corresponding to m.o.i 2) as in D. Experiments shown are representatives of each three independent experiments involving different donors.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040670&req=5

ppat-1001290-g001: DC-SIGN ligation activates SMases and causes membrane ceramide accumulation on DCs.A. to C. Surface ceramide levels on DCs were determined by spot assays (shown in the insets) or flow cytometry (A, B). A. DCs were exposed to a MOCK preparation (open circles left panel), MV alone (black circles, left and middle panels) or after pretreatment with a blocking DC-SIGN antibody (AZ-D1) (black triangles, left panel) or in the presence of EGTA (open circles, middle panel). Right panel: DCs were left untreated (black circles) or exposed to an αCD150-antibody (5C6) prior to MV or MOCK treatment (white circles and black triangles, respectively). B. DCs were exposed to AZ-D1 alone (open circles, left panel) or after addition of a crosslinking antibody (filled circles, both panels). DCs were stimulated with isotype antibody as a negative control (black triangles, left panel) or DC-SIGN was ligated after 2 hrs pretreatment with amitriptyline (open circles, right panel). C. DCs were exposed to mannan for the time intervals indicated and surface levels of ASM (black triangles) and ceramides in the absence (black circles) or presence of EGTA (open circles) were analysed by spot assays (left panel). D. Whole cell lysates (for ASM, left panel) or membrane fractions (for NSM, right panel) of DCs exposed to mannan for the time intervals indicated were used to determine enzyme activities using a commercial kit. DCs were transfected with NSM siRNA (open circles) or not (black circles) (right panel, with effciency of NSM ablation shown by RT-PCR in the inset). E. ASM activity was determined 10 mins following exposure of DCs to MV or a recombinant MV expressing VSV G protein instead of the MV glycoproteins (MGV) at the multiplicities of infection indicated or a mock preparation applied at a concentration corresponding to m.o.i 2) as in D. Experiments shown are representatives of each three independent experiments involving different donors.
Mentions: Membrane ceramide platforms segregate receptors and signalosomes both of which can affect viral entry. DC-SIGN may act to trap or concentrate virions (also including MV) for receptor interaction, and we thus analysed whether MV interaction with this molecule promoted membrane ceramide accumulation on DCs by employing an assay based on immunodetection of an a-ceramide antibody bound to intact cells (spot assay). On MV exposure, DCs responded by an about twofold increase in extrafacial ceramides which peaked at 15 mins and subsequently returned to baseline levels (Fig. 1A, left panel). Ceramide accumulation occurred DC-SIGN dependently, since it was efficiently abrogated upon pre-exposure of DCs with a DC-SIGN-specific antibody (AZ-D1) or EGTA, which prevents Ca2+-dependent DC-SIGN ligand binding (Fig. 1A, left panel and middle panel). In contrast, antibodies blocking MV interaction with its entry receptor, CD150, did not prevent, yet even slightly enhanced MV ceramide induction (Fig. 1A, right panel). Similar to MV, the DC-SIGN binding antibody AZ-D1 increased membrane ceramide display when crosslinked, while this was not observed with the antibody alone nor an isotype control antibody alone (Fig. 1B, left panel; for further experiments, DC-SIGN-specific antibodies were thus used crosslinked). Ceramide production in response to DC-SIGN ligation was sensitive to the ASM inhibitor amitriptyline indicating that activation of this enzyme was involved (Fig. 1B, right panel). To assess activation of SMases directly, we determined their activity after exposure of DCs to mannan, a well-defined DC-SIGN agonist. In line with amitriptyline sensitivity of ceramide generation, ASM surface display raised about 1.8 fold almost immediately following mannan addition, and this was paralleled by a rise in extrafacial ceramides both of which were EGTA sensitive (Fig. 1C, and not shown). Mannan-dependent activation of ASM was further confirmed using a commercial detection assay (Fig. 1D, left panel) which essentially mirrored kinetics and magnitude of the response determined by spot assays. Using the same experimental approach, a rapid, very efficient activation of NSM was also measured (about 5-fold) which peaked after 3 mins and then vanished, and this was entirely prevented upon RNAi mediated silencing of NSM expression (Fig. 1D, right panel). Importantly, ASM activation also occurred on MV exposure of DCs in a dose dependent manner, and this relied on the presence of the MV glycoproteins because it was not observed when a recombinant MV expressing the VSV G protein instead was used (Fig. 1E). Altogether these findings indicate that ligation of DC-SIGN by antibodies, mannan or MV promotes rapid activation of SMases, and ASM-dependent ceramide accumulation in the outer membrane leaflet.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Virology and Immunobiology, University of Würzburg, Wuerzburg, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus