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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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ASM is co-transported with CD150 from lysosomal compartments upon DC-SIGN ligation.A. The HA-tag was co-stained in CD150-HA transfected DCs with the cis-Golgi marker GM130 (upper panels), oligomerized MHC II class (FN1, second row), Lamp-1 (third row), and ASM (bottom row) and analyzed by laser confocal microscopy. Intensity profiles and colocalization coefficients are shown for the examples selected from a total of 30 cells analyzed in the right panels. B. CD150-HA expressing DCs were left untreated (upper panels 1–3) or stimulated with mannan for 10 mins (bottom panels) and analyssed for HA-tag and ASM expression by confocal microscopy. The bottom right panel shows 3D reconstruction of 20 z-stacks of mannan stimulated cell in the left panels, the upper right panel shows a representative profile example of CD150-HA/ASM colocalization with the coefficient indicated. A unstimulated cell has been chosen, each 30 unstimulated and mannan exposed CD150-DCs were recruited into the analysis.
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ppat-1001290-g008: ASM is co-transported with CD150 from lysosomal compartments upon DC-SIGN ligation.A. The HA-tag was co-stained in CD150-HA transfected DCs with the cis-Golgi marker GM130 (upper panels), oligomerized MHC II class (FN1, second row), Lamp-1 (third row), and ASM (bottom row) and analyzed by laser confocal microscopy. Intensity profiles and colocalization coefficients are shown for the examples selected from a total of 30 cells analyzed in the right panels. B. CD150-HA expressing DCs were left untreated (upper panels 1–3) or stimulated with mannan for 10 mins (bottom panels) and analyssed for HA-tag and ASM expression by confocal microscopy. The bottom right panel shows 3D reconstruction of 20 z-stacks of mannan stimulated cell in the left panels, the upper right panel shows a representative profile example of CD150-HA/ASM colocalization with the coefficient indicated. A unstimulated cell has been chosen, each 30 unstimulated and mannan exposed CD150-DCs were recruited into the analysis.

Mentions: To gain insight into the nature of the translocating CD150 compartment, we performed marker analyses in DCs transfected to overexpress CD150-HA. Expectedly, CD150-HA was co-detected with the trans-Golgi marker GM130 (Fig. 8A, upper row). CD150-HA does not accumulate in the MIIC loading compartment, as there is little co-segregation with oligomerized MHCII (detected by the FN1 antibody) (Fig. 8A, second row), yet rather in a Lamp-1 positive compartment that also contained ASM (Fig. 8A, third row). CD150 substantially colocalized with ASM in intracellular compartments in unstimulated DCs (Fig. 8B, upper row, first three panels), and both were redistributed to the cell surface on DC-SIGN ligation (Fig. 8B bottom row). Confirming co-segration of both molecules, the degree of colocalization remained identical prior to and after surface recruitment (an example for unstimulated DCs is shown in the pseudo-coloured scatter plot in Fig. 8B, upper row, right panel). These data indicate that CD150 shares an intracytoplasmic lysosomal compartment with ASM from which it is recruited to and displayed at the cell surface on DC-SIGN-mediated ASM activation to enhance MV entry into 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)

ASM is co-transported with CD150 from lysosomal compartments upon DC-SIGN ligation.A. The HA-tag was co-stained in CD150-HA transfected DCs with the cis-Golgi marker GM130 (upper panels), oligomerized MHC II class (FN1, second row), Lamp-1 (third row), and ASM (bottom row) and analyzed by laser confocal microscopy. Intensity profiles and colocalization coefficients are shown for the examples selected from a total of 30 cells analyzed in the right panels. B. CD150-HA expressing DCs were left untreated (upper panels 1–3) or stimulated with mannan for 10 mins (bottom panels) and analyssed for HA-tag and ASM expression by confocal microscopy. The bottom right panel shows 3D reconstruction of 20 z-stacks of mannan stimulated cell in the left panels, the upper right panel shows a representative profile example of CD150-HA/ASM colocalization with the coefficient indicated. A unstimulated cell has been chosen, each 30 unstimulated and mannan exposed CD150-DCs were recruited into the analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040670&req=5

ppat-1001290-g008: ASM is co-transported with CD150 from lysosomal compartments upon DC-SIGN ligation.A. The HA-tag was co-stained in CD150-HA transfected DCs with the cis-Golgi marker GM130 (upper panels), oligomerized MHC II class (FN1, second row), Lamp-1 (third row), and ASM (bottom row) and analyzed by laser confocal microscopy. Intensity profiles and colocalization coefficients are shown for the examples selected from a total of 30 cells analyzed in the right panels. B. CD150-HA expressing DCs were left untreated (upper panels 1–3) or stimulated with mannan for 10 mins (bottom panels) and analyssed for HA-tag and ASM expression by confocal microscopy. The bottom right panel shows 3D reconstruction of 20 z-stacks of mannan stimulated cell in the left panels, the upper right panel shows a representative profile example of CD150-HA/ASM colocalization with the coefficient indicated. A unstimulated cell has been chosen, each 30 unstimulated and mannan exposed CD150-DCs were recruited into the analysis.
Mentions: To gain insight into the nature of the translocating CD150 compartment, we performed marker analyses in DCs transfected to overexpress CD150-HA. Expectedly, CD150-HA was co-detected with the trans-Golgi marker GM130 (Fig. 8A, upper row). CD150-HA does not accumulate in the MIIC loading compartment, as there is little co-segregation with oligomerized MHCII (detected by the FN1 antibody) (Fig. 8A, second row), yet rather in a Lamp-1 positive compartment that also contained ASM (Fig. 8A, third row). CD150 substantially colocalized with ASM in intracellular compartments in unstimulated DCs (Fig. 8B, upper row, first three panels), and both were redistributed to the cell surface on DC-SIGN ligation (Fig. 8B bottom row). Confirming co-segration of both molecules, the degree of colocalization remained identical prior to and after surface recruitment (an example for unstimulated DCs is shown in the pseudo-coloured scatter plot in Fig. 8B, upper row, right panel). These data indicate that CD150 shares an intracytoplasmic lysosomal compartment with ASM from which it is recruited to and displayed at the cell surface on DC-SIGN-mediated ASM activation to enhance MV entry into 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