Elena Brambilla

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.

Delayed post-ischaemic neuroprotection following systemic neural stem cell transplantation involves multiple mechanisms

Recent evidence suggests that neural stem/precursor cells (NPCs) promote recovery in animal models with delayed neuronal death via a number of indirect bystander effects. A comprehensive knowledge of how transplanted NPCs exert their therapeutic effects is still lacking. Here, we investigated the effects of a delayed transplantation of adult syngenic NPCs--injected intravenously 72 h after transient middle cerebral artery occlusion--on neurological recovery, histopathology and gene expression. NPC-transplanted mice showed a significantly improved recovery from 18 days post-transplantation (dpt) onwards, which persisted throughout the study. A small percentage of injected NPCs accumulated in the brain, integrating mainly in the infarct boundary zone, where most of the NPCs remained undifferentiated up to 30 dpt. Histopathological analysis revealed a hitherto unreported very delayed neuroprotective effect of NPCs, becoming evident at 10 and 30 dpt. Tissue survival was associated with downregulation of markers of inflammation, glial scar formation and neuronal apoptotic death at both mRNA and protein levels. Our data highlight the relevance of very delayed degenerative processes in the stroke brain that are intimately associated with inflammatory and glial responses. These processes may efficaciously be antagonized by (stem) cell-based strategies at time-points far beyond established therapeutic windows for pharmacological neuroprotection.

Persistent inflammation alters the function of the endogenous brain stem cell compartment.

Endogenous neural stem/precursor cells (NPCs) are considered a functional reservoir for promoting tissue homeostasis and repair after injury, therefore regenerative strategies that mobilize these cells have recently been proposed. Despite evidence of increased neurogenesis upon acute inflammatory insults (e.g. ischaemic stroke), the plasticity of the endogenous brain stem cell compartment in chronic CNS inflammatory disorders remains poorly characterized. Here we show that persistent brain inflammation, induced by immune cells targeting myelin, extensively alters the proliferative and migratory properties of subventricular zone (SVZ)-resident NPCs in vivo leading to significant accumulation of non-migratory neuroblasts within the SVZ germinal niche. In parallel, we demonstrate a quantitative reduction of the putative brain stem cells proliferation in the SVZ during persistent brain inflammation, which is completely reversed after in vitro culture of the isolated NPCs. Together, these data indicate that the inflamed brain microenvironment sustains a non cell-autonomous dysfunction of the endogenous CNS stem cell compartment and challenge the potential efficacy of proposed therapies aimed at mobilizing endogenous precursors in chronic inflammatory brain disorders.

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.

Human neural stem cells ameliorate autoimmune encephalomyelitis in non-human primates†

Transplanted neural stem/precursor cells (NPCs) display peculiar therapeutic plasticity in vivo. Although the replacement of cells was first expected as the prime therapeutic mechanism of stem cells in regenerative medicine, it is now clear that transplanted NPCs simultaneously instruct several therapeutic mechanisms, among which replacement of cells might not necessarily prevail. A comprehensive understanding of the mechanism(s) by which NPCs exert their therapeutic plasticity is lacking. This study was designed as a preclinical approach to test the feasibility of human NPC transplantation in an outbreed nonhuman primate experimental autoimmune encephalomyelitis (EAE) model approximating the clinical and complex neuropathological situation of human multiple sclerosis (MS) more closely than EAE in the standard laboratory rodent.

Immune regulatory neural stem/precursor cells protect from central nervous system autoimmunity by restraining dendritic cell function.

Background The systemic injection of neural stem/precursor cells (NPCs) provides remarkable amelioration of the clinico-pathological features of experimental autoimmune encephalomyelitis (EAE). This is dependent on the capacity of transplanted NPCs to engage concurrent mechanisms of action within specific microenvironments in vivo. Among a wide range of therapeutic actions alternative to cell replacement, neuroprotective and immune modulatory capacities of transplanted NPCs have been described. However, lacking is a detailed understanding of the mechanisms by which NPCs exert their therapeutic plasticity. This study was designed to identify the first candidate that exemplifies and sustains the immune modulatory capacity of transplanted NPCs. Methodology/Principal Findings To achieve the exclusive targeting of the peripheral immune system, SJL mice with PLP-induced EAE were injected subcutaneously with NPCs and the treatment commenced prior to disease onset. NPC-injected EAE mice showed significant clinical improvement, as compared to controls. Exogenous NPCs lacking the expression of major neural antigens were reliably (and for long-term) found at the level of draining lymph nodes, while establishing sophisticated anatomical interactions with lymph node cells. Importantly, injected NPCs were never found in organs other than lymph nodes, including the brain and the spinal cord. Draining lymph nodes from transplanted mice showed focal up-regulation of major developmental stem cell regulators, such as BMP-4, Noggin and Sonic hedgehog. In lymph nodes, injected NPCs hampered the activation of myeloid dendritic cells (DCs) and steadily restrained the expansion of antigen-specific encephalitogenic T cells. Both ex vivo and in vitro experiments identified a novel highly NPC-specific–BMP-4-dependent–mechanism hindering the DC maturation. Conclusion/Significance The study described herein, identifies the first member of the TGF β/BMP family of stem cell regulators as a novel tolerogenic factor released by NPCs. Full exploitation of this pathway as an efficient tool for vaccination therapy in autoimmune inflammatory conditions is underway.

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.

Magnetic Resonance‐Based Tracking and Quantification of Intravenously Injected Neural Stem Cell Accumulation in the Brains of Mice with Experimental Multiple Sclerosis

Eliciting the in situ accumulation and persistence patterns of stem cells following transplantation would provide critical insight toward human translation of stem cell‐based therapies. To this end, we have developed a strategy to track neural stem/precursor cells (NPCs) in vivo using magnetic resonance (MR) imaging. Initially, we evaluated three different human‐grade superparamagnetic iron oxide particles for labeling NPCs and found the optimal labeling to be achieved with Resovist. Next, we carried out in vivo experiments to monitor the accumulation of Resovist‐labeled NPCs following i.v. injection in mice with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. With a human MR scanner, we were able to visualize transplanted cells as early as 24 hours post‐transplantation in up to 80% of the brain demyelinating lesions. Interestingly, continued monitoring of transplanted mice indicated that labeled NPCs were still present 20 days postinjection. Neuropathological analysis confirmed the presence of transplanted NPCs exclusively in inflammatory demyelinating lesions and not in normal‐appearing brain areas. Quantification of transplanted cells by means of MR‐based ex vivo relaxometry (R2*) showed significantly higher R2* values in focal inflammatory brain lesions from EAE mice transplanted with labeled NPCs as compared with controls. Indeed, sensitive quantification of low numbers of NPCs accumulating into brain inflammatory lesions (33.3–164.4 cells per lesion; r2 = .998) was also obtained. These studies provide evidence that clinical‐grade human MR can be used for noninvasive monitoring and quantification of NPC accumulation in the central nervous system upon systemic cell injection.

IL-12 is involved in the induction of experimental autoimmune myasthenia gravis, an antibody- mediated disease

IL‐12 has been shown to be involved in the pathogenesis of Th1‐mediated autoimmune diseases, but its role in antibody‐mediated autoimmune pathologies is still unclear. We investigated the effects of exogenous and endogenous IL‐12 in experimental autoimmune myasthenia gravis (EAMG). EAMG is an animal model for myasthenia gravis, a T cell‐dependent, autoantibody‐mediated disorder of neuromuscular transmission caused by antibodies to the muscle nicotinic acetylcholine receptor (AChR). Administration of IL‐12 with Torpedo AChR (ToAChR) to C57BL/6 (B6) mice resulted in increased ToAChR‐specific IFN‐γ production and increased anti‐ToAChR IgG2a serum antibodies compared with B6 mice primed with ToAChR alone. These changes were associated with earlier and greater neurophysiological evidence of EAMG in the IL‐12‐treated mice, and reduced numbers of AChR. By contrast, when IL‐12‐deficient mice were immunized with ToAChR, ToAChR‐specific Th1 cells and anti‐ToAChR IgG2a serum antibodies were reduced compared to ToAChR‐primed normal B6 mice, and the IL‐12‐deficient mice showed almost no neurophysiological evidence of EAMG and less reduction in AChR. These results indicate an important role of IL‐12 in the induction of an antibody‐mediated autoimmune disease, suggest that Th1‐dependent complement‐fixing IgG2a anti‐AChR antibodies are involved in the pathogenesis of EAMG, and help to account for the lack of correlation between anti‐AChR levels and clinical disease seen in many earlier studies.