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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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SMase activation enhances MV infection of DCs.A. Left panel: DCs were left untreated (black bar) or exposed to amitriptyline (white bar) prior to MV infection. Right panel: An GFP-tagged MV was used for infection of DC cultures where expression of ASM had been siRNA silenced (white bar) or not (black bar) (inset shows efficiency of silencing by RT-PCR). The frequency of cells expressing the MV N protein (left panel) or GFP (right panel) was determined after 12 hrs by flow cytometry. Values shown were generated in each three independent experiments involving different donors, p-values are indicated. B. DCs left untreated (each black bars and black lines) or pre-exposed to amitriptyline (each white bars and grey lines) were exposed to MV and virus binding (after 2 hrs at 4°C, left panel and graph) or uptake (after 16 hrs at 37°C with FIP added following infection) were determined by surface staining for F protein (to detect surface bound virus; left panels) or GFP detection (which is only expressed on MV replication; right panels) by flow cytometry. The arrow marks the histogram for amitriptilyne treated cells. F protein expression or GFP fluorescence in uninfected cells are shown as filled histogram. C. Molt-4 or A549 cells were left untreated (black bars) or pre-exposed to amitriptyline prior to infection with Ed-eGFP (m.o.i. 2,5)(white bars). The percentage of GFP-expressing cells (left panel) and their mean fluorescent intensities (right panel) are indicated. One out of three representative experiments is shown.
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ppat-1001290-g004: SMase activation enhances MV infection of DCs.A. Left panel: DCs were left untreated (black bar) or exposed to amitriptyline (white bar) prior to MV infection. Right panel: An GFP-tagged MV was used for infection of DC cultures where expression of ASM had been siRNA silenced (white bar) or not (black bar) (inset shows efficiency of silencing by RT-PCR). The frequency of cells expressing the MV N protein (left panel) or GFP (right panel) was determined after 12 hrs by flow cytometry. Values shown were generated in each three independent experiments involving different donors, p-values are indicated. B. DCs left untreated (each black bars and black lines) or pre-exposed to amitriptyline (each white bars and grey lines) were exposed to MV and virus binding (after 2 hrs at 4°C, left panel and graph) or uptake (after 16 hrs at 37°C with FIP added following infection) were determined by surface staining for F protein (to detect surface bound virus; left panels) or GFP detection (which is only expressed on MV replication; right panels) by flow cytometry. The arrow marks the histogram for amitriptilyne treated cells. F protein expression or GFP fluorescence in uninfected cells are shown as filled histogram. C. Molt-4 or A549 cells were left untreated (black bars) or pre-exposed to amitriptyline prior to infection with Ed-eGFP (m.o.i. 2,5)(white bars). The percentage of GFP-expressing cells (left panel) and their mean fluorescent intensities (right panel) are indicated. One out of three representative experiments is shown.

Mentions: To assess the overall impact of SMase activation on MV uptake into DCs, these were exposed to amitriptyline prior to infection. Thereby, intracellular accumulation levels of MV N protein 12 hrs following infection were reduced by about 50% (Fig. 4A, left panel) indicating that SMase activation is beneficial for viral DC infection. Consistent with this hypothesis, GFP levels produced from a tagged MV wild-type recombinant virus only on replication (IC323-eGFP) were reduced by about 50% upon siRNA mediated ablation of ASM expression (Fig. 4A, right panel). Pre-exposure to amitriptyline did not affect MV binding to DCs as determined by detection of MV F protein positive cells after 2 hrs at 4°C (Fig. 4B, left panel and graph), yet efficiently reduced intracellular GFP-accumulation after 24 hrs (with FIP added following infection to prevent MV spread) (Fig. 4B, right panel and graph). Amitriptyline did, however, not affect uptake or replication of a recombinant attenuated MV strain into T or epithelial cells (Fig. 4C) (which cannot be infected with IC323-eGFP due to the absence of CD150) indicating that SMase activation or ceramide elevation alone do not necessarily enhance MV infection as occurring in DCs.


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)

SMase activation enhances MV infection of DCs.A. Left panel: DCs were left untreated (black bar) or exposed to amitriptyline (white bar) prior to MV infection. Right panel: An GFP-tagged MV was used for infection of DC cultures where expression of ASM had been siRNA silenced (white bar) or not (black bar) (inset shows efficiency of silencing by RT-PCR). The frequency of cells expressing the MV N protein (left panel) or GFP (right panel) was determined after 12 hrs by flow cytometry. Values shown were generated in each three independent experiments involving different donors, p-values are indicated. B. DCs left untreated (each black bars and black lines) or pre-exposed to amitriptyline (each white bars and grey lines) were exposed to MV and virus binding (after 2 hrs at 4°C, left panel and graph) or uptake (after 16 hrs at 37°C with FIP added following infection) were determined by surface staining for F protein (to detect surface bound virus; left panels) or GFP detection (which is only expressed on MV replication; right panels) by flow cytometry. The arrow marks the histogram for amitriptilyne treated cells. F protein expression or GFP fluorescence in uninfected cells are shown as filled histogram. C. Molt-4 or A549 cells were left untreated (black bars) or pre-exposed to amitriptyline prior to infection with Ed-eGFP (m.o.i. 2,5)(white bars). The percentage of GFP-expressing cells (left panel) and their mean fluorescent intensities (right panel) are indicated. One out of three representative experiments is shown.
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Related In: Results  -  Collection

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ppat-1001290-g004: SMase activation enhances MV infection of DCs.A. Left panel: DCs were left untreated (black bar) or exposed to amitriptyline (white bar) prior to MV infection. Right panel: An GFP-tagged MV was used for infection of DC cultures where expression of ASM had been siRNA silenced (white bar) or not (black bar) (inset shows efficiency of silencing by RT-PCR). The frequency of cells expressing the MV N protein (left panel) or GFP (right panel) was determined after 12 hrs by flow cytometry. Values shown were generated in each three independent experiments involving different donors, p-values are indicated. B. DCs left untreated (each black bars and black lines) or pre-exposed to amitriptyline (each white bars and grey lines) were exposed to MV and virus binding (after 2 hrs at 4°C, left panel and graph) or uptake (after 16 hrs at 37°C with FIP added following infection) were determined by surface staining for F protein (to detect surface bound virus; left panels) or GFP detection (which is only expressed on MV replication; right panels) by flow cytometry. The arrow marks the histogram for amitriptilyne treated cells. F protein expression or GFP fluorescence in uninfected cells are shown as filled histogram. C. Molt-4 or A549 cells were left untreated (black bars) or pre-exposed to amitriptyline prior to infection with Ed-eGFP (m.o.i. 2,5)(white bars). The percentage of GFP-expressing cells (left panel) and their mean fluorescent intensities (right panel) are indicated. One out of three representative experiments is shown.
Mentions: To assess the overall impact of SMase activation on MV uptake into DCs, these were exposed to amitriptyline prior to infection. Thereby, intracellular accumulation levels of MV N protein 12 hrs following infection were reduced by about 50% (Fig. 4A, left panel) indicating that SMase activation is beneficial for viral DC infection. Consistent with this hypothesis, GFP levels produced from a tagged MV wild-type recombinant virus only on replication (IC323-eGFP) were reduced by about 50% upon siRNA mediated ablation of ASM expression (Fig. 4A, right panel). Pre-exposure to amitriptyline did not affect MV binding to DCs as determined by detection of MV F protein positive cells after 2 hrs at 4°C (Fig. 4B, left panel and graph), yet efficiently reduced intracellular GFP-accumulation after 24 hrs (with FIP added following infection to prevent MV spread) (Fig. 4B, right panel and graph). Amitriptyline did, however, not affect uptake or replication of a recombinant attenuated MV strain into T or epithelial cells (Fig. 4C) (which cannot be infected with IC323-eGFP due to the absence of CD150) indicating that SMase activation or ceramide elevation alone do not necessarily enhance MV infection as occurring in DCs.

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