Unit: Cytokine Signalling - CNRS URA 1961
Director: Pellegrini Sandra
Our projects aim at understanding the complex signaling networks induced by type I interferons (IFN-a/b). What distinguishes these cytokines from other helical cytokines is the existence of a multiplicity of IFN subtypes binding to the same receptor, the extreme pleiotropy and cell specificity of the biological responses. By studying receptor dynamics in response to different IFN subtypes and in distinct cellular backgrounds, we hope to unravel molecular mechanisms and signal integration that underline the pleiotropic responses to the type I IFNs, notably IFN-α and IFN-β. Current focus is also on further characterization of novel Jak-interacting proteins that will broaden our understanding of signaling pathways initiated by cytokines and may also reveal additional functions of Jak enzymes.
Dynamics and traffic of the type I IFN receptor (Z. Marijanovic, J. Ragimbeau)
The four members of the mammalian Janus or Jak family of tyrosine kinases are non-covalently associated with cell surface receptors of helical bundled cytokines. In the heterodimeric type I IFN receptor, Tyk2 associates with the IFNAR1 subunit and positively influences its plasma membrane expression. We are interested in the intracellular fate of the IFN receptor subunits following ligand binding and in the role that Jak proteins play in post-binding events. Binding of IFN-α stimulates internalization of IFNAR1, a process that requires its serine phosphorylation and the recruitment of a ubiquitin E3 ligase. We have studied the dynamics of IFNAR1 upon ligand stimulation, with the aim to determine whether Tyk2 regulates it at any step. Using various approaches, we have shown that IFN-induced internalization of IFNAR1 does not require active Tyk2. Conversely, Tyk2 kinase activity is required to efficiently direct ligand-engaged IFNAR1 towards the lysosomal compartment and we are presently looking into the precise role exerted by Tyk2 in this context. The traffic of the other subunit of the IFN receptor (IFNAR2) is under study with particular attention to its dynamics in different cell types and in response to two IFN subtypes (α2 and β), to highlight potential relationships between regulation of traffic and IFN subtype-specific signaling.
Study of Jamip1 in T lymphocytes (V. Libri, D. Schulte)
We identified Jamip1 (Jak and microtubule interacting protein) for its ability to bind to the FERM homology domain of Tyk2. Jamip1 belongs to a novel family of three genes conserved in vertebrates and is predominantly expressed in neural tissues and lymphoid organs. Jamip proteins lack known domains and are rich in coiled coils. Jamip1 comprises a N-ter region that targets the protein to microtubule polymers and, when overexpressed in fibroblasts, profoundly perturbs the microtubule network. The C-ter domain interacts with Jak kinases and can be target of multiple phosphorylation. The restricted Jamip1expression in lymphocytes and neurons, and its association with the microtubule cytoskeleton suggest a specialized function in dynamic processes such as cell polarization, local segregation of molecules or complexes, and/or in trafficking of secretory organelles/endocytic vesicles along microtubule tracks towards the immune synapse in lymphocytes and from/towards the neuronal synapse. We are interested in elucidating the function of Jamip1 in T lymphocytes. Activation of helper and cytotoxic T lymphocytes by antigen-presenting cells involves polarization of the lymphocyte towards the contact area and the formation of the immunological synapse. Cytoskeletal rearrangements are necessary for the stability of this interaction, for the maturation of the immunological synapse and for sustained T cell signaling. The microtubule organizing center (MTOC) reorients towards the synapse after TCR engagement and this may play a crucial role in the accumulation of TCR at the central synapse, the segregation of signaling proteins and the relocation of the secretory machinery that leads to polarized delivery of lymphokines, cytolytic granules. Using RNA interference and lentiviral transduction, we are exploring the involvement of Jamip1 in the killing activity of cytotoxic T lymphocytes.
Dendritic cells and IFN subtype-specific response (J. Ragimbeau, collaboration with E.M. Coccia, Istituto Superiore di Sanità, Rome, Italy)
Following viral and bacterial infections or stimulation with TLR agonists, type I IFN is rapidly and transiently secreted from monocyte-derived DC and this autocrine-acting IFN can affect the biology of DC. Maturing DC acquire the capacity of migrating from infected tissues to lymph nodes, when their capacity to release IFN is exhausted. In this new location, they may come across a new micro-environment with elevated amounts of IFN-a/b released by plasmacytoid DC. We measured the IFN responsiveness of DC at different stages of maturation and in particular we studied whether LPS-matured DC respond to environmental IFNα and IFN-β, once they reach the lymph node. To mimick migration, extensive washes were perfomed on LPS-matured cells in order to clear the released IFN. We found that such mature DC fully respond to IFN-β but not to IFN-α and that desensitization to IFN-α correlates with low level of the IFNAR1 subunit. This suggests that the level of IFNAR1 may determine subtype-specific responses. This work also suggests that, depending on the initial maturation stimulus, migrating DC will display distinctive abilities to respond to IFN-α and IFN-β encountered in lymphoid organs.
Keywords: signaling, cytokine, interferon-alpha/beta, receptor, tyrosine kinase, trafic, cytoskeleton, lymphocyte, immunology