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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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MV causes CD150 surface display in an amitriptyline sensitive manner.WGA and CD150 were codetected on CD150-DCs left untreated (upper row) or exposed to MV with (bottom row) or without a 2 hrs amitriptyline pre-treatment (middle row). Colocalization coefficients were determined (right intensity profiles) and are indicated for the examples shown within the panels, and for each 30 cells analyzed in the right graph where they were scored into no (white bars), intermediate (grey bars) or high (black bars) degree of WGA/CD150 co-segregation.
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ppat-1001290-g007: MV causes CD150 surface display in an amitriptyline sensitive manner.WGA and CD150 were codetected on CD150-DCs left untreated (upper row) or exposed to MV with (bottom row) or without a 2 hrs amitriptyline pre-treatment (middle row). Colocalization coefficients were determined (right intensity profiles) and are indicated for the examples shown within the panels, and for each 30 cells analyzed in the right graph where they were scored into no (white bars), intermediate (grey bars) or high (black bars) degree of WGA/CD150 co-segregation.

Mentions: MV binding to DC-SIGN causes SMase activation on DCs which, in turn, promotes MV infection. We thus analysed whether this might relate to DC-SIGN-dependent alterations of membrane distribution of CD150. Because expression levels of this protein are low on DCs ([9], [10] and see below), we initially analysed the impact of DC-SIGN ligation on CD150 expression in Raji cells expressing high levels of endogenous CD150, and on stable transfection, DC-SIGN (Raji-DC-SIGN) (Fig. 5A, upper and second row). In untreated Raji-DC-SIGN cells, both molecules revealed a punctate expression pattern overall covering the cell surface (Fig. 5). DC-SIGN-ligation caused enhanced co-clustering of DC-SIGN and CD150 in large platforms (after 5 mins, Fig. 5A, third row), which subsequently protruded from the cell surface (after 10 mins, Fig. 5A, fourth row), revealing that DC-SIGN signaling indeed promotes redistribution of CD150. Suggesting a role of SMase activation in this process, DC-SIGN enriched protrusions emanating from the cell surface (prominent formation of which may relate to very low phospholipid scramblase levels of Raji cells [42]) were locally also enriched for ceramides (Fig. 5A, bottom row). As reported erlier, efficient MV uptake into DCs relies on both DC-SIGN (for trapping) and CD150 (particularly for fusion) [9], [10]. In line with earlier findings, interference of DC-SIGN interaction by mannan, an antibody or EGTA reduced MV binding to DCs by approximately 50% ([9] and Fig. S1), and blocking of either DC-SIGN and CD150 alone or in combination strongly interferes with MV uptake and replication (Fig. 5B). On immature DCs, expression of endogenous CD150 was generally low as reported [9], [10], yet increased surface display ligation within 15 mins after DC-SIGN was detectable by flow cytometry (Fig. 5C). To follow CD150 redistribution in response to DC-SIGN ligation in DCs in detail, we generated C-terminally HA-tagged CD150 which, when overexpressed in HeLa cells, did not differ with regard to subcellular distribution, surface expression level, glycosylation and DRM association from the unmodified protein (not shown). When transfected into DCs (CD150-HA-DCs), transgenic CD150, similar as the endogenous in DCs, mainly localized to intracellular compartments, while DC-SIGN, expectedly appeared in clusters at the cell surface [17], [19], [43] with little detectable overlap of both molecules (Fig. 6A, upper panels and right graph). Mirroring our findings in Raji-DC-SIGN cells, DC-SIGN ligation by mannan promoted both surface translocation, clustering of CD150-HA and co-clustering with DC-SIGN in DCs peaking between 10 and 15 mins after exposure (Fig. 6A, middle and bottom panels, and right graph) indicating that DC-SIGN-signaling indeed causes clustering and surface recruitment of this molecule. The latter was further confirmed by using a surface biotinylation/streptavidin precipitation approach with CD150-HA-DCs where exposure to mannan substantially increased the amounts of CD150 pulled down by streptavidin-beads (Fig. 6B, right lanes). The amounts of cytosolic CD150 were also slightly elevated on mannan exposure indicating that the CD150 storage compartment might reveal differential sensitivity to detergent lysis on DC-SIGN signaling (Fig. 6B, left lanes). DC-SIGN-dependent CD150-HA surface recruitment involved transport and membrane fusion of exocytic vesicles in a SNARE-dependent manner as indicated by its sensitivity to N-ethylmaleimide (Fig. 6C), and, also ASM activation as it was essentially abolished on pre-exposure to amitriptyline (Fig. 6D). Importantly, ASM inhibition also interfered with MV-induced CD150 surface recruitment as determined by WGA/CD150 co-segregation levels (Fig. 7).


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)

MV causes CD150 surface display in an amitriptyline sensitive manner.WGA and CD150 were codetected on CD150-DCs left untreated (upper row) or exposed to MV with (bottom row) or without a 2 hrs amitriptyline pre-treatment (middle row). Colocalization coefficients were determined (right intensity profiles) and are indicated for the examples shown within the panels, and for each 30 cells analyzed in the right graph where they were scored into no (white bars), intermediate (grey bars) or high (black bars) degree of WGA/CD150 co-segregation.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001290-g007: MV causes CD150 surface display in an amitriptyline sensitive manner.WGA and CD150 were codetected on CD150-DCs left untreated (upper row) or exposed to MV with (bottom row) or without a 2 hrs amitriptyline pre-treatment (middle row). Colocalization coefficients were determined (right intensity profiles) and are indicated for the examples shown within the panels, and for each 30 cells analyzed in the right graph where they were scored into no (white bars), intermediate (grey bars) or high (black bars) degree of WGA/CD150 co-segregation.
Mentions: MV binding to DC-SIGN causes SMase activation on DCs which, in turn, promotes MV infection. We thus analysed whether this might relate to DC-SIGN-dependent alterations of membrane distribution of CD150. Because expression levels of this protein are low on DCs ([9], [10] and see below), we initially analysed the impact of DC-SIGN ligation on CD150 expression in Raji cells expressing high levels of endogenous CD150, and on stable transfection, DC-SIGN (Raji-DC-SIGN) (Fig. 5A, upper and second row). In untreated Raji-DC-SIGN cells, both molecules revealed a punctate expression pattern overall covering the cell surface (Fig. 5). DC-SIGN-ligation caused enhanced co-clustering of DC-SIGN and CD150 in large platforms (after 5 mins, Fig. 5A, third row), which subsequently protruded from the cell surface (after 10 mins, Fig. 5A, fourth row), revealing that DC-SIGN signaling indeed promotes redistribution of CD150. Suggesting a role of SMase activation in this process, DC-SIGN enriched protrusions emanating from the cell surface (prominent formation of which may relate to very low phospholipid scramblase levels of Raji cells [42]) were locally also enriched for ceramides (Fig. 5A, bottom row). As reported erlier, efficient MV uptake into DCs relies on both DC-SIGN (for trapping) and CD150 (particularly for fusion) [9], [10]. In line with earlier findings, interference of DC-SIGN interaction by mannan, an antibody or EGTA reduced MV binding to DCs by approximately 50% ([9] and Fig. S1), and blocking of either DC-SIGN and CD150 alone or in combination strongly interferes with MV uptake and replication (Fig. 5B). On immature DCs, expression of endogenous CD150 was generally low as reported [9], [10], yet increased surface display ligation within 15 mins after DC-SIGN was detectable by flow cytometry (Fig. 5C). To follow CD150 redistribution in response to DC-SIGN ligation in DCs in detail, we generated C-terminally HA-tagged CD150 which, when overexpressed in HeLa cells, did not differ with regard to subcellular distribution, surface expression level, glycosylation and DRM association from the unmodified protein (not shown). When transfected into DCs (CD150-HA-DCs), transgenic CD150, similar as the endogenous in DCs, mainly localized to intracellular compartments, while DC-SIGN, expectedly appeared in clusters at the cell surface [17], [19], [43] with little detectable overlap of both molecules (Fig. 6A, upper panels and right graph). Mirroring our findings in Raji-DC-SIGN cells, DC-SIGN ligation by mannan promoted both surface translocation, clustering of CD150-HA and co-clustering with DC-SIGN in DCs peaking between 10 and 15 mins after exposure (Fig. 6A, middle and bottom panels, and right graph) indicating that DC-SIGN-signaling indeed causes clustering and surface recruitment of this molecule. The latter was further confirmed by using a surface biotinylation/streptavidin precipitation approach with CD150-HA-DCs where exposure to mannan substantially increased the amounts of CD150 pulled down by streptavidin-beads (Fig. 6B, right lanes). The amounts of cytosolic CD150 were also slightly elevated on mannan exposure indicating that the CD150 storage compartment might reveal differential sensitivity to detergent lysis on DC-SIGN signaling (Fig. 6B, left lanes). DC-SIGN-dependent CD150-HA surface recruitment involved transport and membrane fusion of exocytic vesicles in a SNARE-dependent manner as indicated by its sensitivity to N-ethylmaleimide (Fig. 6C), and, also ASM activation as it was essentially abolished on pre-exposure to amitriptyline (Fig. 6D). Importantly, ASM inhibition also interfered with MV-induced CD150 surface recruitment as determined by WGA/CD150 co-segregation levels (Fig. 7).

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