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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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Related in: MedlinePlus

ASM activity is essential for DC-SIGN signaling.A. Surface expression levels of CD83 and CD86 were determined on DCs left untreated (iDCs, white bars) or pre-exposed to amitriptyline (black bars) or not (grey bars) prior to LPS stimulation (mDCs) after 24 hrs by flow cytometry. B. Levels of p-c-Raf1 and p-ERK were determined in DCs left untreated (right graphs: each black circles) or exposed to amitriptyline for 2 hrs (right graphs: each open circles) and subsequently activated by PMA/ionomycin (upper panels and right graphs, obtained after densitometric quantification) or AZ-D1 (followed by crosslinking) for the time intervals indicated (bottom panels and right graphs). ERK detection served as loading control.
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ppat-1001290-g002: ASM activity is essential for DC-SIGN signaling.A. Surface expression levels of CD83 and CD86 were determined on DCs left untreated (iDCs, white bars) or pre-exposed to amitriptyline (black bars) or not (grey bars) prior to LPS stimulation (mDCs) after 24 hrs by flow cytometry. B. Levels of p-c-Raf1 and p-ERK were determined in DCs left untreated (right graphs: each black circles) or exposed to amitriptyline for 2 hrs (right graphs: each open circles) and subsequently activated by PMA/ionomycin (upper panels and right graphs, obtained after densitometric quantification) or AZ-D1 (followed by crosslinking) for the time intervals indicated (bottom panels and right graphs). ERK detection served as loading control.

Mentions: DC-SIGN signaling includes activation of c-Raf-1 and ERK [11], [22], [41]. To asses if DC-SIGN signaling involves SMase activation, cells were pre-exposed to amitriptyline which per se did not affect DC viability (not shown) or LPS-induced upregulation of CD86 or CD83 after 24 hrs (Fig. 2A). PMA/ionomycin-dependent activation of c-Raf-1 or ERK as determined by detection of p-c-Raf-1 or pERK within 30 mins was unaffected on pre-exposure of DCs to amitriptyline (Fig. 2B, upper panels). In line with earlier findings obtained upon ManLam-, antibody or HIV exposure [11], [22], [41], DC-SIGN ligation by crosslinked AZ-D1 caused c-Raf-1 and ERK activation (Fig. 2B, bottom panels). In contrast to that induced by PMA/ionomycin, however, α-DC-SIGN induced c-Raf-1 and ERK phosphorylation was sensitive to amitriptyline indicating that DC-SIGN signaling involves ASM activation (Fig. 2B, bottom panels). DC-SIGN signaling does not confer NF-κB activation, yet apparently modulates that induced upon TLR ligation [11]. As revealed both by an ELISA kit based detection system or nuclear translocation of p65, TLR4 ligation by LPS indeed promoted NF-κB activation after 60 mins. (Fig. 3A). Mannan exposure did, however, reduce magnitude of NF-κB activation measured by either method indicating that DC-SIGN ligation interferes with TLR4 signaling (Fig. 3A and B). Interestingly, however, ablation of DC-SIGN signaling by amitriptyline or an NSM inhibitor, GW4896, apparently enhanced LPS-induced NF-κB activation as reflected by efficient nuclear accumulation of p65, and enhanced levels of activation as determined by ELISA (Fig. 3B and C). Overall, these findings support the interpretation that DC-SIGN membrane signaling essentially involves ceramide generation, and may act to dampen rather than to enhance TLR-dependent NF-κB activation and thereby production of pro-inflammatory cytokines.


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)

ASM activity is essential for DC-SIGN signaling.A. Surface expression levels of CD83 and CD86 were determined on DCs left untreated (iDCs, white bars) or pre-exposed to amitriptyline (black bars) or not (grey bars) prior to LPS stimulation (mDCs) after 24 hrs by flow cytometry. B. Levels of p-c-Raf1 and p-ERK were determined in DCs left untreated (right graphs: each black circles) or exposed to amitriptyline for 2 hrs (right graphs: each open circles) and subsequently activated by PMA/ionomycin (upper panels and right graphs, obtained after densitometric quantification) or AZ-D1 (followed by crosslinking) for the time intervals indicated (bottom panels and right graphs). ERK detection served as loading control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040670&req=5

ppat-1001290-g002: ASM activity is essential for DC-SIGN signaling.A. Surface expression levels of CD83 and CD86 were determined on DCs left untreated (iDCs, white bars) or pre-exposed to amitriptyline (black bars) or not (grey bars) prior to LPS stimulation (mDCs) after 24 hrs by flow cytometry. B. Levels of p-c-Raf1 and p-ERK were determined in DCs left untreated (right graphs: each black circles) or exposed to amitriptyline for 2 hrs (right graphs: each open circles) and subsequently activated by PMA/ionomycin (upper panels and right graphs, obtained after densitometric quantification) or AZ-D1 (followed by crosslinking) for the time intervals indicated (bottom panels and right graphs). ERK detection served as loading control.
Mentions: DC-SIGN signaling includes activation of c-Raf-1 and ERK [11], [22], [41]. To asses if DC-SIGN signaling involves SMase activation, cells were pre-exposed to amitriptyline which per se did not affect DC viability (not shown) or LPS-induced upregulation of CD86 or CD83 after 24 hrs (Fig. 2A). PMA/ionomycin-dependent activation of c-Raf-1 or ERK as determined by detection of p-c-Raf-1 or pERK within 30 mins was unaffected on pre-exposure of DCs to amitriptyline (Fig. 2B, upper panels). In line with earlier findings obtained upon ManLam-, antibody or HIV exposure [11], [22], [41], DC-SIGN ligation by crosslinked AZ-D1 caused c-Raf-1 and ERK activation (Fig. 2B, bottom panels). In contrast to that induced by PMA/ionomycin, however, α-DC-SIGN induced c-Raf-1 and ERK phosphorylation was sensitive to amitriptyline indicating that DC-SIGN signaling involves ASM activation (Fig. 2B, bottom panels). DC-SIGN signaling does not confer NF-κB activation, yet apparently modulates that induced upon TLR ligation [11]. As revealed both by an ELISA kit based detection system or nuclear translocation of p65, TLR4 ligation by LPS indeed promoted NF-κB activation after 60 mins. (Fig. 3A). Mannan exposure did, however, reduce magnitude of NF-κB activation measured by either method indicating that DC-SIGN ligation interferes with TLR4 signaling (Fig. 3A and B). Interestingly, however, ablation of DC-SIGN signaling by amitriptyline or an NSM inhibitor, GW4896, apparently enhanced LPS-induced NF-κB activation as reflected by efficient nuclear accumulation of p65, and enhanced levels of activation as determined by ELISA (Fig. 3B and C). Overall, these findings support the interpretation that DC-SIGN membrane signaling essentially involves ceramide generation, and may act to dampen rather than to enhance TLR-dependent NF-κB activation and thereby production of pro-inflammatory cytokines.

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