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Intern
    Graduiertenkolleg 2157

    Membrane and protein microdomains governing measles virus transmission at entry and exit interfaces


    State of the art

    Infections with measles virus (MV) continue to be of high medical relevance. They are still associated with high morbidity/mortality rates due to complications mainly affecting the respiratory tract and the CNS. The complications arise predominantly, because of the secondary infections, which establish due to the profound immunosuppressive potential of the virus. Measles pathogenesis substantially segregates with usage of specific receptors mediating trapping on and/or entry into its primary host cells as a prerequisite for trafficking to target tissues but also for exiting the host at late stages of the infection. Immune cells, especially dendritic cells (DCs) are widely considered of key importance in early (to immmune and endothelial cells), and late viral transmission to epithelial cells. The relevance of the respective receptors for transmission (DC-SIGN, CD150 on immune cells, nectin-4 on epithelial cells) has mainly been documented in 2D models (often with transformed target cells) and by in situ analyses on paraffin embedded fixed material of infected animals. An experimental system revealing transmission between immune cells and from immune cells to epi/endothelial cells directly in a more complex environment, which would allow for detailed analysis of compartimentalization of essential membrane components and cytoskelatal dynamics in this process is still missing.

    Previous Work

    DCs generated from monocyte precursors trap MV via DC-SIGN and transiently upregulate CD150 to enable viral entry by promoting activation of acid sphingomyelinase (ASM) [4]. Biological consequences of DC-SIGN-induced activation of the neutral sphingomyelinase (NSM) have so far not been addressed. We have also shown that MV-infection of DCs interferes with DC functional maturation [1, 2], but also promotes early production of soluble semaphorin 3A which has been implicated in repulsion and loss of actin dynamics in target cells [3, 4, 5, 7]. These particular regulations may be highly important both in DC migration in tissues and adhesion to and interaction with target cells for viral transmission also including endothelial, epithelial and T cells.

    In 2D cultures, MV transmission to T cells can occur independently of DC infection (DC-SIGN-trapped virus, referred to as trans-infection) or by virus produced from infected DCs (cis-infection). This relies on the formation of stable synaptic DC/T cell interfaces (virological synapses, VS) which organize entry receptor(s) and other surface molecules (believed to stabilize the VS) as well as cytoskeletal activity [6]. In 2D interaction systems between autologous MV infected DCs and pre-activated primary T cells we observed the formation of VS concentrating CD150, LFA-1, DC-SIGN, CD81, pERM, moesin and substance P receptor most likely serving as tranmissison structures. Long filopodial actin based connecting structures also containing MV proteins were seen as well [6].

    Figure: Transmission of MV from infected DCs (A, red, B, green) to T cells oddurs via filopodial bridges (A) and synapse-like structures (A and B, often involving polyconjugates) in 2D cultures.
    C. Ceramide is co-detected with DC-SIGN in MV-exposed DCs (A and B: from [6, 8]).


    Work Plan

    Understanding of molecular mechanisms, structures and subcellular components essential for MV transmission from immune to target cells requires 3D systems to study the dynamics of cell interactions, conjugate formation and mode and efficiency of viral transfer involving genetically manipulated donor and target cells and tagged, genetically engineered viruses. For DC/T cell transmission as relevant in host entry this will involve 3D collagen matrix systems (already established in the group) where questions such as motility of MV-infected DCs and their ability to recruit naïve/activated T cells into conjugates can be studied.

    Questions to be addressed in 2D and transferred to 3D systems will include the role of 1) individual VS concentrated receptors in transmission by RNAi ablation strategies, 2) actin dynamics especially with regard to modulations by sphingomyelinase activation/suppression, and 3) of semaphorins on transmission. This will reveal alterations of DC mobility and ability to conjugate T cells in response to MV trapping/infection (which defines frequency, type and kinetics of VS formation and the role of VS components, actin dynamics and repulsive proteins in the stabilization/transmission process).

    The impact of MV acquisition on T cell integrity and motility and the role of lipid raft integrity and Ca2+ mobilization (as important for transmission/fusion for certain viruses in vitro) will be studied. The ability of T cells to acquire a front/rear polarization and to segregate functionally important receptors can be detected after immigration, recovery and subsequent fixation both in and separated from conjugates. In host exit, MV appears to infect respiratory epithelial cells from the basolateral side using nectin-4 as an entry receptor. Experimentally not yet proven, infected immune cells (eventually DCs or T cells) transmit virus to nectin-4+ cells in the respiratory epithelium. Initially, 2D systems consisting of co-cultures of DCs or T cells (where MV infection may well modulate adhesion or formation of actin-based protrusions) with Calu-3 or primary bronchus epithelial cells would be used to study MV transmission with regard to conjugate formation, receptor sorting, actin dynamics and sensitivity to sphingomyelinase modulation as for the host entry system detailed above.

    A protocol to reconstitute DCs of various maturation stages into the 3D airway mucosa model generated within the Steinke/Gross project is currently being established and will be extended by reconstitution by other immune cell types (T cells or macrophages). Because endothelial cells are also targets of MV infection in vivo, and their infection can be mediated by infected lymphocytes in 2D systems, the 3D model of the blood-cerebrospinal fluid (B-CSF) barrier to be developed in the Schubert-Unkmeir/Walles collaborative project can be explored using various MV-infected donor cell population.


    References

    1. Shishkova et al. (2007) Immune synapses formed with MV infected DCs are unstable and fail to sustain T cell activation. Cell Microbiol. 9, 1974-86. PubMed
    2. Abt et al. (2009). MV modulates chemokine release and chemotactic responses of DCs. J Gen Virol 90, 909-14. PubMed
    3. Gassert et al. (2009) Induction of Membrane Ceramides: A Novel Strategy to Interfere with T Lymphocyte Cytoskeletal Reorganisation in  Viral Immunosuppression. PloS Pathogens, 5, e1000623. PubMed
    4. Avota et al. (2011) DC-SIGN mediated sphingomyelinase-activation and ceramide generation is essential for enhancement of viral uptake in DCs. Plos Pathogens 10.1371/journal.ppat.1001290. PubMed
    5. Tran-Van et al. (2011) MV modulates DC/T cell communication at the level of plexinA1/neuropilin-1 recruitment and activity. Eur J Immunol 41, 151-163. PubMed
    6. Koethe et al. (2012) MV transmission from DCs to T cells: formation of synapse like interfaces concentrating viral and cellular components. J Virol 86, 9773-9781. PubMed
    7. Mueller et al. (2014). The role of the neutral sphingomyelinase in physiological and MV mediated T cell suppression. PLoS Pathog. 10(12):e1004574. PubMed
    8. Avota et al. (2013). Membrane dynamics and interactions in measles virus dendritic cell infections. Cell. Microbiol. 15(2):161-9. PubMed