Sphingosine-1-phosphate receptor antagonism enhances proliferation and migration of engrafted neural progenitor cells in a model of viral-induced demyelination.

The oral drug FTY720 affects sphingosine-1-phosphate (S1P) signaling on targeted cells that bear the S1P receptors S1P1, S1P3, S1P4, and S1P5. We examined the effect of FTY720 treatment on the biology of mouse neural progenitor cells (NPCs) after transplantation in a viral model of demyelination. Intracerebral infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in an acute encephalomyelitis, followed by demyelination similar in pathology to the human demyelinating disease, multiple sclerosis. We have previously reported that intraspinal transplantation of mouse NPCs into JHMV-infected animals resulted in selective colonization of demyelinated lesions, preferential differentiation into oligodendroglia accompanied by axonal preservation, and increased remyelination. Cultured NPCs expressed transcripts for S1P receptors S1P1, S1P2, S1P3, S1P4, and S1P5. FTY720 treatment of cultured NPCs resulted in increased mitogen-activated protein kinase phosphorylation and migration after exposure to the chemokine CXCL12. Administration of FTY720 to JHMV-infected mice resulted in enhanced migration and increased proliferation of transplanted NPCs after spinal cord engraftment. FTY720 treatment did not improve clinical disease, diminish neuroinflammation or the severity of demyelination, nor increase remyelination. These findings argue that FTY720 treatment selectively increases NPC proliferation and migration but does not either improve clinical outcome or enhance remyelination after transplantation into animals in which immune-mediated demyelination is initiated by the viral infection of the central nervous system.

Promoting remyelination: utilizing a viral model of demyelination to assess cell-based therapies.

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS. While a broad range of therapeutics effectively reduce the incidence of focal white matter inflammation and plaque formation for patients with relapse-remitting forms of MS, a challenge within the field is to develop therapies that allow for axonal protection and remyelination. In the last decade, growing interest has focused on utilizing neural precursor cells (NPCs) to promote remyelination. To understand how NPCs function in chronic demyelinating environments, several excellent pre-clinical mouse models have been developed. One well accepted model is infection of susceptible mice with neurotropic variants of mouse hepatitis virus (MHV) that undergo chronic demyelination exhibiting clinical and histopathologic similarities to MS patients. Combined with the possibility that an environmental agent such as a virus could trigger MS, the MHV model of demyelination presents a relevant mouse model to assess the therapeutic potential of NPCs transplanted into an environment in which inflammatory-mediated demyelination is established.

FTY720 (fingolimod) modulates the severity of viral-induced encephalomyelitis and demyelination.

BACKGROUND: FTY720 (fingolimod) is the first oral drug approved by the Food and Drug Administration for treatment of patients with the relapsing-remitting form of the human demyelinating disease multiple sclerosis. Evidence suggests that the therapeutic benefit of FTY720 occurs by preventing the egress of lymphocytes from lymph nodes thereby inhibiting the infiltration of disease-causing lymphocytes into the central nervous system (CNS). We hypothesized that FTY720 treatment would affect lymphocyte migration to the CNS and influence disease severity in a mouse model of viral-induced neurologic disease. METHODS: Mice were infected intracranially with the neurotropic JHM strain of mouse hepatitis virus. Infected animals were treated with increasing doses (1, 3 and 10 mg/kg) of FTY720 and morbidity and mortality recorded. Infiltration of inflammatory virus-specific T cells (tetramer staining) into the CNS of FTY720-treated mice was determined using flow cytometry. The effects of FTY720 treatment on virus-specific T cell proliferation, cytokine production and cytolytic activity were also determined. The severity of neuroinflammation and demyelination in FTY720-treated mice was examined by flow cytometry and histopathologically, respectively, in the spinal cords of the mice. RESULTS: Administration of FTY720 to JHMV-infected mice resulted in increased clinical disease severity and mortality. These results correlated with impaired ability to control viral replication (P < 0.05) within the CNS at days 7 and 14 post-infection, which was associated with diminished accumulation of virus-specific CD4+ and CD8+ T cells (P < 0.05) into the CNS. Reduced neuroinflammation in FTY720-treated mice correlated with increased retention of T lymphocytes within draining cervical lymph nodes (P < 0.05). Treatment with FTY720 did not affect virus-specific T cell proliferation, expression of IFN-γ, TNF-α or cytolytic activity. FTY720-treated mice exhibited a reduction in the severity of demyelination associated with dampened neuroinflammation. CONCLUSION: These findings indicate that FTY720 mutes effective anti-viral immune responses through impacting migration and accumulation of virus-specific T cells within the CNS during acute viral-induced encephalomyelitis. FTY720 treatment reduces the severity of neuroinflammatory-mediated demyelination by restricting the access of disease-causing lymphocytes into the CNS but is not associated with viral recrudescence in this model.

Toll-like receptor 4-activated B cells out-compete Toll-like receptor 9-activated B cells to establish peripheral immunological tolerance.

B-cell-induced peripheral T-cell tolerance is characterized by suppression of T-cell proliferation and T-cell-dependent antibody production. However, the cellular interactions that underlie tolerance induction have not been identified. Using two-photon microscopy of lymph nodes we show that tolerogenic LPS-activated membrane-bound ovalbumin (mOVA) B cells (LPS B cells) establish long-lived, highly motile conjugate pairs with responding antigen-specific OTII T cells but not with antigen-irrelevant T cells. Treatment with anti-CTLA-4 disrupts persistent B-cell-T-cell (B-T) contacts and suppresses antigen-specific tolerance. Nontolerogenic CpG-activated mOVA B cells (CpG B cells) also form prolonged, motile conjugates with responding OTII T cells when transferred separately. However, when both tolerogenic and nontolerogenic B-cell populations are present, LPS B cells suppress long-lived CpG B-OTII T-cell interactions and exhibit tolerogenic dominance. Contact of LPS B cells with previously established B-T pairs resulted in partner-swapping events in which LPS B cells preferentially migrate toward and disrupt nontolerogenic CpG mOVA B-cell-OTII T-cell pairs. Our results demonstrate that establishment of peripheral T-cell tolerance involves physical engagement of B cells with the responding T-cell population, acting in a directed and competitive manner to alter the functional outcome of B-T interactions.

Olig1 function is required for remyelination potential of transplanted neural progenitor cells in a model of viral-induced demyelination.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) resulting in cumulative neurologic deficits associated with progressive myelin loss. We have previously shown that transplantation of neural progenitor cells (NPCs) into mice persistently infected with the JHM strain of mouse hepatitis virus (JHMV) results in enhanced differentiation into oligodendrocyte progenitor cells (OPCs) that is associated with remyelination and axonal sparing. The current study examines the contributions of the transcription factor Olig1 on NPC differentiation and remyelination. Under defined conditions, NPCs preferentially differentiate into oligodendroglia whereas NPCs isolated from Olig1-deficient (Olig1-/-) mice exhibit enhanced differentiation into astrocytes. Transplantation of Olig1-/- and Olig1+/+ NPCs into JHMV-infected mice resulted in similar cell survival, proliferation, and selective migration to areas of demyelination. However, only recipients of wild type NPCs exhibited extensive remyelination compared to mice receiving Olig1-/- NPCs. In vivo characterization of NPCs revealed that Olig1+/+ NPCs preferentially differentiated into NG2-positive OPCs and formed processes expressing myelin basic protein that encircled axons. In contrast, the majority of transplanted Olig1-/- NPCs differentiated into GFAP-positive cells consistent with the astrocyte lineage. These results indicate that exogenous NPCs contribute to improved clinical and histological outcome and this is associated with remyelination by this donor population. Further, these findings reveal that Olig1function is required for the remyelination potential of NPCs after transplant, through specification and/or maintenance of oligodendroglial identity.