Cytoskeletal Dynamics: Concepts in Measles Virus Replication and Immunomodulation

In common with most viruses, measles virus (MV) relies on the integrity of the cytoskeleton of its host cells both with regard to efficient replication in these cells, but also retention of their motility which favors viral dissemination. It is, however, the surface interaction of the viral glycoprotein (gp) complex with receptors present on lymphocytes and dendritic cells (DCs), that signals effective initiation of host cell cytoskeletal dynamics. For DCs, these may act to regulate processes as diverse as viral uptake and sorting, but also the ability of these cells to successfully establish and maintain functional immune synapses (IS) with T cells. In T cells, MV signaling causes actin cytoskeletal paralysis associated with a loss of polarization, adhesion and motility, which has been linked to activation of sphingomyelinases and subsequent accumulation of membrane ceramides. MV modulation of both DC and T cell cytoskeletal dynamics may be important for the understanding of MV immunosuppression at the cellular level

Induction of Membrane Ceramides: A Novel Strategy to Interfere with T Lymphocyte Cytoskeletal Reorganisation in Viral Immunosuppression

Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase–dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to β1 integrin ligation or αCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics

Impact of ceramide accumulation on T lymphocyte cytoskeletal reorganisation,immune synapse formation and activation

Ceramide sind biologisch aktive Sphingolipide, die verschiedene zelluläre Signalwege regulieren, meist im Zusammenhang mit der Induktion von Apoptose oder der Regulation des Zellzyklus. Darüber hinaus wurde in der Literatur beschrieben, dass Ceramide die Zytoskelettdynamik unterschiedlicher Zelltypen beeinflussen, die Bedeutung von Ceramiden für die Funktion von T-Zellen wurde allerdings bisher wenig untersucht. In der vorliegenden Arbeit konnte gezeigt werden, dass die exogene Akkumulation von Ceramiden ebenso wie die Generierung von Ceramiden durch bSMase die Adhärenz von T-Zellen an FN bzw. ICAM-1 beeinträchtigt. Des Weiteren konnte eine verminderte T-Zell-Polarisierung auf FN sowie eine reduzierte Chemotaxis und Motilität ceramidmodifizierter T-Zellen in Antwort auf SDF-1 nachgewiesen werden. In Übereinstimmung mit der Unfähigkeit ceramidmodifizierter Zellen morphologisch zu polarisieren wird ferner die Relokalisation von Oberflächenmolekülen und intrazellulärer Proteine durch die Akkumulation von Ceramiden gestört. Überdies konnte in dieser Arbeit gezeigt werden, dass Ceramide mit dem Aktivierungsstatus von Akt und ERM-Proteinen interferieren, da eine verminderte stimulationsabhängige Phosphorylierung von Akt und ERM-Proteinen in ceramidmodifizierten Zellen nachgewiesen wurde. Ein wesentlicher Schritt im Verlauf der T-Zell-Aktivierung ist die Ausbildung einer immunologischen Synapse mit dendritischen Zellen. In dieser Arbeit konnte gezeigt werden, dass, obwohl ceramidreiche Membrandomänen von der Kontaktstelle ausgeschlossen werden, Konjugatfrequenz und Architektur der IS durch die Induktion von Ceramiden nicht beeinflusst werden, da eine normale Verteilung von CD3 und des MTOC beobachtet wurde. Allerdings wird die Funktionalität der Konjugate durch die Induktion von Ceramiden beeinträchtigt. Ceramidmodifizierte Zellen waren nur eingeschränkt in der Lage Orai1 und Stim1 zur Kontaktfläche mit DCs zu translozieren. In Übereinstimmung mit diesen Befunden wurde auch ein verminderter Calcium-Einstrom sowie eine verminderte Proliferation infolge der Akkumulation von Ceramiden detektiert. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass Ceramide wesentliche Prozesse im Verlauf der T-Zell-Aktivierung beeinflussen, so dass die pathogeninduzierte Generierung von Ceramiden einen möglichen Mechanismus darstellt, die Funktion von T-Zellen zu beeinträchtigen.Ceramides are sphingolipids regulating various signalling pathways mainly associated with the induction of apoptosis and cell cycle arrest. Besides their established role in these processes several lines of evidence suggest that ceramides also regulate cytoskeletal dynamics in non-hematopoietic cells, but their role in T lymphocyte function has not yet been addressed. In this study we show that accumulation of membrane ceramides affects several processes required for accurate T cell function. Treatment of T cells with bSMase or exogenously added ceramides interfered with T cell adhesion to FN and ICAM-1 as well as T cell polarisation, chemotaxis and motility in response to SDF-1. In line with the impairment to morphologically polarise, relocation of surface receptors as well as intracellular signalling molecules was also impaired upon ceramide treatment of T cells. Moreover, increase in cellular ceramide levels interfered with cellular signalling pathways as revealed by reduced phosphorylation levels of Akt and ERM proteins. T cell activation requires the formation of an IS with DCs. In this study we could show, that ceramides, although excluded from the DC/T cell interface, do not interfere with conjugate frequency or synapse organisation, since a normal distribution of CD3 and the MTOC was observed. Nevertheless, ceramide generation interfered with the translocation of Orai1 and Stim1 to the interface and in line with this observation a reduced calcium-influx in T cells was detected upon bSMase exposure. In addition to the decrease in cytosolic calcium levels ceramides also reduced the ability of T cells to proliferate in response to CD3/CD28 stimulation. Therefore the induction of ceramides by certain pathogens, including MV, might be a possible mechanism to interfere with essential processes required for T cell activation

Ceramide accumulation abolishes redistribution of CXCR4 and affects T cell chemotactic motility.

<p>(A) T cells left untreated (first column) or exposed to MV (second column) alone or after a 2 h pretreatment with amitriptilyne (third column) or to bSMase (fourth column) were seeded onto FN and stained for f-actin and CXCR4. Size bars represent 5 µm. (B and C) T cells were left untreated (each: left panel) or pretreated with amitriptyline ((B), right panel) or not before exposure to MV for 2 h at 4°C ((B), middle panel) or treated with bSMase ((C), middle and right panels) for 2 hrs at 37°C and seeded onto FN with addition of SDF-1 (1,5 µg/ml). Migration of cells on FN in response to SDF-1 was recorded over a time period of 10 min and the individual velocities of cells and their trajectories were calculated using ImageJ software. Tracks of cells faster than an arbitrary velocity threshold of 7 µm/min in B, or 9 µm/min in C, are indicated in black, those slower in red in each panel. Examples shown are representative of three individual experiments including different donors with a total of at least 120 cells recorded for each experimental setup.</p

SMase activation accounts for MV-induced prevention of T cell polarisation and loss of membrane protrusions.

<p>(A) Human T cells were transfected with a NSM2-specific or a scrambled siRNA for 96 hrs or exposed to amitriptyline (ami) or GW4869 2 hrs prior to MOCK (right panel, white bars) or MV (right panel, black bars) exposure for 2 hrs at 4°C and subsequently seeded onto FN-coated slides. Cells were fixed after 30 min and stained for f-actin and CD43 (exemplified in the left panel, with the scale bar representing 5 µm) and the percentage of cells revealing a front-rear polarisation with CD43 enriched in the uropod was determined. At least 100 cells per culture were recruited into the analysis. (B) Human T cell cultures exposed to MOCK or MV (pre-exposed to amitriptyline for 24 hrs or not) were seeded onto FN coated slides and analysed by SEM (left panel; bars represent 5 µm). Right panel: Cells in each culture were scored into morphological categories (non-, partially or fully polarized; see insets for examples; 100 cells per culture were counted) and the respective percentage of cells exposed to MV (black bars) or MOCK (white bars) was determined. (C) Left panel: Spleen cells from wild-type (upper and second row) or AsmKO mice (middle to bottom row) were exposed to MOCK (upper and third row), MV (second and fourth row) or C₁₆ ceramide (bottom row) prior to seeding onto FN and subsequent SEM analysis. Right, upper panel: Wild-type or AsmKO spleen cells exposed to MV (black bars) or MOCK (white bars) were seeded onto FN and the percentages of cells forming membrane protrusions in each culture were determined by SEM. Right, bottom panel: AsmKO cells exposed to MOCK (white bars) or C₁₆ ceramide (black bars) were seeded onto FN and percentages of cells revealing membrane protrusions or with smoot appearance were determined. Both right panels: At least 100 cells were recruited into the analysis per culture. Levels of significance were determined using a t-test (p = 0.01).</p