Alessandra Bergami

Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis

Widespread demyelination and axonal loss are the pathological hallmarks of multiple sclerosis. The multifocal nature of this chronic inflammatory disease of the central nervous system complicates cellular therapy and puts emphasis on both the donor cell origin and the route of cell transplantation. We established syngenic adult neural stem cell cultures and injected them into an animal model of multiple sclerosis—experimental autoimmune encephalomyelitis (EAE) in the mouse—either intravenously or intracerebroventricularly. In both cases, significant numbers of donor cells entered into demyelinating areas of the central nervous system and differentiated into mature brain cells. Within these areas, oligodendrocyte progenitors markedly increased, with many of them being of donor origin and actively remyelinating axons. Furthermore, a significant reduction of astrogliosis and a marked decrease in the extent of demyelination and axonal loss were observed in transplanted animals. The functional impairment caused by EAE was almost abolished in transplanted mice, both clinically and neurophysiologically. Thus, adult neural precursor cells promote multifocal remyelination and functional recovery after intravenous or intrathecal injection in a chronic model of multiple sclerosis.

Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism

In degenerative disorders of the central nervous system (CNS), transplantation of neural multipotent (stem) precursor cells (NPCs) is aimed at replacing damaged neural cells. Here we show that in CNS inflammation, NPCs are able to promote neuroprotection by maintaining undifferentiated features and exerting unexpected immune-like functions. In a mouse model of chronic CNS inflammation, systemically injected adult syngeneic NPCs use constitutively activated integrins and functional chemokine receptors to selectively enter the inflamed CNS. These undifferentiated cells survive repeated episodes of CNS inflammation by accumulating within perivascular areas where reactive astrocytes, inflamed endothelial cells and encephalitogenic T cells produce neurogenic and gliogenic regulators. In perivascular CNS areas, surviving adult NPCs induce apoptosis of blood-borne CNS-infiltrating encephalitogenic T cells, thus protecting against chronic neural tissue loss as well as disease-related disability. These results indicate that undifferentiated adult NPCs have relevant therapeutic potential in chronic inflammatory CNS disorders because they display immune-like functions that promote long-lasting neuroprotection.

Inflammation Triggers Synaptic Alteration and Degeneration in Experimental Autoimmune Encephalomyelitis

Neurodegeneration is the irremediable pathological event occurring during chronic inflammatory diseases of the CNS. Here we show that, in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, inflammation is capable in enhancing glutamate transmission in the striatum and in promoting synaptic degeneration and dendritic spine loss. These alterations occur early in the disease course, are independent of demyelination, and are strongly associated with massive release of tumor necrosis factor-α from activated microglia. CNS invasion by myelin-specific blood-borne immune cells is the triggering event, and the downregulation of the early gene Arc/Arg3.1, leading to the abnormal expression and phosphorylation of AMPA receptors, represents a culminating step in this cascade of neurodegenerative events. Accordingly, EAE-induced synaptopathy subsided during pharmacological blockade of AMPA receptors. Our data establish a link between neuroinflammation and synaptic degeneration and calls for early neuroprotective therapies in chronic inflammatory diseases of the CNS.

Intrathecal Delivery of IFN-γ Protects C57BL/6 Mice from Chronic-Progressive Experimental Autoimmune Encephalomyelitis by Increasing Apoptosis of Central Nervous System-Infiltrating Lymphocytes

The exclusive detrimental role of proinflammatory cytokines in demyelinating diseases of the CNS, such as multiple sclerosis, is controversial. Here we show that the intrathecal delivery of an HSV-1-derived vector engineered with the mouse IFN-γ gene leads to persistent (up to 4 wk) CNS production of IFN-γ and inhibits the course of a chronic-progressive form of experimental autoimmune encephalomyelitis (EAE) induced in C57BL/6 mice by myelin oligodendrocyte glycoprotein (MOG)35–55. Mice treated with the IFN-γ-containing vector before EAE onset showed an earlier onset but a milder course of the disease compared with control mice treated with the empty vector. In addition, 83% of IFN-γ-treated mice completely recovered within 25 days post immunization, whereas control mice did not recover up to 60 days post immunization. Mice treated with the IFN-γ-containing vector within 1 wk after EAE onset partially recovered from the disease within 25 days after vector injection, whereas control mice worsened. Recovery from EAE in mice treated with IFN-γ was associated with a significant increase of CNS-infiltrating lymphocytes undergoing apoptosis. During the recovery phase, the mRNA level of TNFR1 was also significantly increased in CNS-infiltrating cells from IFN-γ-treated mice compared with controls. Our results further challenge the exclusive detrimental role of IFN-γ in the CNS during EAE/multiple sclerosis, and indicate that CNS-confined inflammation may induce protective immunological countermechanisms leading to a faster clearance of encephalitogenic T cells by apoptosis, thus restoring the immune privilege of the CNS.

Myeloid microvesicles are a marker and therapeutic target for neuroinflammation.

Microvesicles (MVs) have been indicated as important mediators of intercellular communication and are emerging as new biomarkers of tissue damage. Our previous data indicate that reactive microglia/macrophages release MVs in vitro. The aim of the study was to evaluate whether MVs are released by microglia/macrophages in vivo and whether their number varies in brain inflammatory conditions, such as multiple sclerosis (MS).

Activation of invariant NKT cells by αGalCer administration protects mice from MOG35-55-induced EAE: critical roles for administration route and IFN-γ

Invariant NKT (inv. NKT) cells co‐express an invariant α β T cell receptor and the NK receptor NK1.1 and, upon CD1d‐restricted recognition of the glycosphingolipid antigen α‐galactosyl ceramide (αGalCer), secrete large amounts of regulatory cytokines. We investigated whether αGalCer‐dependent activation of inv. NKT cells protects from experimental autoimmune encephalomyelitis (EAE), an immune‐mediated disease of the central nervous system mimicking multiple sclerosis, induced in C57BL/6 mice by the myelin oligodendrocyte glycoprotein (MOG) encephalitogenic peptide aa 35–55. αGalCer was administered at the time of immunization s.c., mixed with complete Freunds adjuvant and MOG35‐55 peptide, or administered i.p., diluted in PBS. EAE onset was delayed and disease severity was decreased only when αGalCer was s.c. administered. The protective effect of s.c. administration of αGalCer was associated with a markedly enhanced IFN‐γ production by liver‐confined inv. NKT cells which, in turn, suppressed Th1‐cytokine production and fostered secretion of IL‐10 from MOG35–55‐specific T cells. In vivo neutralization of IFN‐γ, but notIL‐4, reversed the protective effect induced by s.c. administration of αGalCer, further confirming the critical regulatory role exerted by IFN‐γ‐producing inv. NKT cells. Our results indicate that αGalCer, properly administered, may elicit an inv. NKT‐cell‐mediated suppressive effect on the effector function of encephalitogenic T cells; this effect is able to ameliorate autoimmunedemyelination.

Interleukin-1β causes synaptic hyperexcitability in multiple sclerosis.

The frequency of inflammatory episodes in the early stages of multiple sclerosis (MS) has been correlated with late neurodegeneration, but the mechanism by which inflammation gives rise to delayed neuronal damage is unknown. Increased activity of the neurotransmitter glutamate is thought to play a role in the inflammation‐driven neurodegenerative process of MS, and therefore we tested whether inflammatory cytokines released during acute MS attacks have the property of enhancing glutamate‐mediated transmission and excitotoxicity in central neurons.

Fibroblast growth factor-II gene therapy reverts the clinical course and the pathological signs of chronic experimental autoimmune encephalomyelitis in C57BL/6 mice.

The development of therapies aimed to promote remyelination is a major issue in chronic inflammatory demyelinating disorders of the central nervous system (CNS) such as multiple sclerosis (MS), where the permanent neurological impairment is due to the axonal loss resulting from recurrent episodes of immune-mediated demyelination. Here, we show that the intrathecal injection of a herpes simplex virus (HSV) type-1 replication-defective multigene vector, engineered with the human fibroblast growth factor (FGF)-II gene (TH:bFGF vector), was able to significantly revert in C57BL/6 mice the clinicopathological signs of chronic experimental autoimmune encephalomyelitis (EAE), the animal model of MS. The treatment with the TH:bFGF vector was initiated within 1 week after the clinical onset of EAE and was effective throughout the whole follow-up period (ie 60 days). The disease-ameliorating effect in FGF-II-treated mice was associated with: (1) CNS production of FGF-II from vector-infected cells which were exclusively located around the CSF space (ependymal, choroidal and leptomeningeal cells); (2) significant decrease (P < 0.01) of the number of myelinotoxic cells (T cells and macrophages) both in the CNS parenchyma and in the leptomeningeal space; and (3) significant increase (P < 0.01) of the number of oligodendrocyte precursors and of myelin-forming oligodendrocytes in areas of demyelination and axonal loss. Our results indicate that CNS gene therapy using HSV-1-derived vector coding for neurotrophic factors (ie FGF-II) is a safe and non-toxic approach that might represent a potential useful ‘alternative’ tool for the future treatment of immune-mediated demyelinating diseases.

Central Nervous System Delivery of Interleukin 4 by a Nonreplicative Herpes Simplex Type 1 Viral Vector Ameliorates Autoimmune Demyelination

Multiple sclerosis (MS) is a T cell-mediated organ-specific inflammatory disease leading to central nervous system (CNS) demyelination. On the basis of results obtained in experimental autoimmune encephalomyelitis (EAE) models, MS treatment by administration of antiinflammatory cytokines such as interleukin 4 (IL-4) is promising but is hampered by the limited access of the cytokines to the CNS and by the pleiotropic effects of systemically administered cytokines. We established a cytokine delivery system within the CNS using non-replicative herpes simplex type 1 (HSV-1) viral vectors engineered with cytokine genes. These vectors injected into the cisterna magna (i.c.) of mice diffuse in all ventricular and subarachnoid spaces and infect with high efficiency the ependymal and leptomeningeal cell layers surrounding these areas, without obvious toxic effects. Heterologous genes contained in the vectors are efficiently transcribed in infected ependymal cells, leading to the production of high amounts of the coded proteins. For example, 4.5 ng of interferon gamma (IFN-gamma) per milliliter is secreted into the cerebrospinal fluid (CSF) up to day 28 postinjection (p.i.) and reaches the CNS parenchyma in bioactive form, as demonstrated by upregulation of MHC class I expression on CNS-resident cells. We then exploited the therapeutic potential of the vectors in EAE mice. An HSV-1-derived vector containing the IL-4 gene was injected i.c. in Biozzi AB/H mice at the time of EAE induction. We found the following in treated mice: (1) delayed EAE onset, (2) a significant decrease in clinical score, (3) a significant decrease in perivascular inflammatory infiltrates and in the number of macrophages infiltrating the CNS parenchyma and the submeningeal spaces, and (4) a reduction in demyelinated areas and axonal loss. Peripheral T cells from IL-4-treated mice were not affected either in their antigen-specific proliferative response or in cytokine secretion pattern. Our results indicate that CNS cytokine delivery with HSV-1 vectors is feasible and might represent an approach for the treatment of demyelinating diseases. Advantages of this approach over systemic cytokine administration are the high cytokine level reached in the CNS, the absence of effects on the peripheral immune system, and the long-lasting cytokine production in the CNS after a single vector administration.

Central nervous system gene therapy with interleukin-4 inhibits progression of ongoing relapsing-remitting autoimmune encephalomyelitis in Biozzi AB/H mice.

Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system (CNS) that might benefit from anti-inflammatory therapies. However, systemic delivery of anti-inflammatory drugs in MS patients has so far been disappointing, mostly due to the limited capacity of these molecules to enter the CNS. We injected into the cisterna magna (i.c.) of Biozzi AB/H mice affected by a relapsing–remitting form of experimental autoimmune encephalomyelitis (EAE), the animal model of MS, a non-replicative herpes simplex virus (HSV) type-1-derived vector containing the interleukin (IL)-4 gene (d120:LacZ:IL-4). CNS delivery of the d120:LacZ:IL-4 vector, after EAE onset, induced the in situ production of IL-4 by CNS-resident cells facing the cerebrospinal fluid (CSF) spaces and reduced by 47% (P < 0.02) the disease-related deaths. compared with mice treated with the control d120:lacz vector, il-4-treated mice also showed a shorter duration of the first eae attack, a longer inter-relapse period, and a reduction in the severity and duration of the first relapse. protection from eae progression in il-4-treated mice was associated with activation of microglia in spinal cord areas where mrna content of the pro-inflammatory chemokines, macrophage chemoattractant protein-1 (mcp-1) and rantes, was reduced and that of the anti-inflammatory cytokine il-4 was increased. finally, cns-infiltrating mononuclear cells from il-4-treated mice produced lower levels of mcp-1 mrna compared with control mice. our results, showing that il-4 gene delivery using hsv-1 vectors induces protection from eae by in situ modulating the cytokine/chemokine-mediated circuits sustaining effector cell functions, indicate that the intrathecal ‘therapeutic’ use of nonreplicative hsv-1-derived vectors containing anti-inflammatory molecules might represent an alternative strategy in inflammatory diseases of the cns.