Regulation of experimental autoimmune encephalomyelitis by TPL-2 kinase.

Tumor progression locus 2 (TPL-2) expression is required for efficient polarization of naive T cells to Th1 effector cells in vitro, as well as for Th1-mediated immune responses. In the present study, we investigated the potential role of TPL-2 in Th17 cells. TPL-2 was found to be dispensable for Th17 cell differentiation in vitro, and for the initial priming of Th17 cells in experimental autoimmune encephalomyelitis (EAE), a Th17 cell-mediated disease model for multiple sclerosis. Nevertheless, TPL-2-deficient mice were protected from EAE, which correlated with reduced immune cell infiltration, demyelination, and axonal damage in the CNS. Adoptive transfer experiments demonstrated that there was no T cell-intrinsic function for TPL-2 in EAE, and that TPL-2 signaling was not required in radiation-sensitive hematopoietic cells. Rather, TPL-2 signaling in radiation-resistant stromal cells promoted the effector phase of the disease. Importantly, using a newly generated mouse strain expressing a kinase-inactive form of TPL-2, we demonstrated that stimulation of EAE was dependent on the catalytic activity of TPL-2 and not its adaptor function to stabilize the associated ubiquitin-binding protein ABIN-2. Our data therefore raise the possibility that small molecule inhibitors of TPL-2 may be beneficial in multiple sclerosis therapy.

TNFR2 on non-haematopoietic cells is required for Foxp3+ Treg-cell function and disease suppression in EAE.

The TNF/TNFR system exerts multiple proinflammatory and immunosuppressive functions in the pathogenesis of chronic inflammation and autoimmunity. In EAE, the experimental model of Multiple Sclerosis (MS), genetic ablation of TNFR2, results in exacerbated immune reactivity and chronic disease course. The underlying mechanism driving this immunosuppressive function of TNFR2 remains unclear. We show here that chronic exacerbated EAE in TNFR2 KO mice is associated with increased Th17-cell responses and reduced numbers of Foxp3(+) Treg cells both in the spinal cord and peripheral lymphoid organs. Treg cells from TNFR2-deficient animals developing EAE show decreased proliferative and suppressive functions, both ex vivo and in vivo, and appear responsible for the exacerbated non-remitting disease, as evidenced by phenotypic rescue following adoptive transfer of Treg cells from WT but not TNFR2(-/-) donors. Reciprocal BM transplantation experiments between WT and TNFR2-deficient mice demonstrated that the capacity of TNFR2 to support Treg-cell expansion and function during EAE is non-intrinsic to Treg or other haematopoietic cells but requires expression of TNFR2 in radiation-resistant cells of the host. These results reveal a previously unsuspected role for non-haematopoietic TNFR2 in modulating Treg-cell expansion and immune suppression during development of autoimmunity and suggest that a similar mechanism may affect chronicity and relapses characterizing human autoimmune disease, including MS.

Differential effects of interleukin-1 alpha and beta on interleukin-6 and chemokine synthesis in neurones.

Interleukin (IL)-1 is a key mediator of neuroinflammation via actions of two agonists IL-1alpha and beta that bind to the IL-1 type I receptor (IL-1RI), and are thought to trigger identical responses. However, evidence suggests that IL-1alpha and beta may have differential actions in the central nervous system (CNS). The objective of this study was to test the hypothesis that IL-1alpha and beta differentially regulate the expression of IL-6 and chemokines KC, IP-10 and MCP-1 in primary neurones. Here we demonstrate that, whilst IL-1beta induced significant synthesis of IL-6 in neurones, IL-1alpha had no effect. In contrast, IL-1alpha and beta induced strong synthesis and constitutive release of chemokines KC, IP-10 and MCP-1 from neurones, and these responses were IL-1RI-dependent. Whilst IL-1beta-induced IL-6 synthesis was dependent on the nSMase/Src kinase signalling cascade, specific inhibitors of nSMase (3-OMS) and Src kinase (PP2) failed to inhibit IL-1alpha- and IL-1beta-induced chemokines synthesis, suggesting the existence of alternative signalling pathway(s) in neurones.

Mechanisms of interleukin-6 synthesis and release induced by interleukin-1 and cell depolarisation in neurones.

Cytokines are important mediators of the immune response to infection and injury and are produced mainly by lymphocytes or monocytes. Many aspects of the acute phase response are mediated by the actions of cytokines such as interleukin (IL)-1 and IL-6 within the brain. IL-1-induced IL-6 expression in neuronal cells has been described previously, but the mechanisms of IL-6 transport and release remain unknown. We show here that IL-1 induces IL-6 gene and protein expression in mouse primary cortical neurones, but that the IL-6 protein is stored intracellularly in the perinuclear area. Depolarisation of IL-1-treated neurones caused the axonal transport and release of IL-6 into the extracellular compartment. The transport and release occurs via an active mechanism (blocked by colchicine) through the Golgi apparatus, but not secretogranin-II vesicles. These results reveal a neuronal-specific mechanism of IL-6 synthesis, transport and release in response to IL-1 and cell depolarisation.

Multiphoton imaging of chick retinal development in relation to gap junctional communication.

Neural progenitor cells in the developing retina extend processes that stretch from the basal vitread surface to the apical ventricular surface. During the cell cycle, the nucleus undergoes interkinetic nuclear migration (INM), moving in a vitread direction during G1, passing through S-phase at its peak and then, on entering G2, returning towards the ventricular surface where it enters M-phase and divides. We have previously shown that individual saltatory movements of the nucleus correlate with transient changes in cytosolic calcium concentration within these progenitor cells and that these events spread to neighbouring progenitors through connexin43 (Cx43) gap junction channels, thereby coordinating the migration of coupled clusters of cells. Disrupting coupling with pharmacological agents, Cx43-specific antisense oligodeoxynucleotides (asODNs) or dominant negative Cx43 (dnCx43) inhibits the sharing of calcium events, reducing the number that each cell experiences and significantly slowing INM. We have developed protocols for imaging migrating progenitor cells by confocal microscopy over relatively short periods, and by multiphoton microscopy over more extended periods that include complete cell cycles. We find that perturbing gap junctional communication not only slows the INM of progenitor cells but also apparently prevents them from changing direction at critical phases of the cell cycle. It also disrupts the migration of young neurons to their appropriate layers after terminal division and leads to their ectopic differentiation. The ability to perform extended time-lapse imaging over 3D volumes in living retina using multiphoton microscopy should now allow fundamental mechanisms governing development of the retinal neuroepithelium to be probed in detail.