The therapeutic effect of mesenchymal stem cell transplantation in experimental autoimmune encephalomyelitis is mediated by peripheral and central mechanisms

Stem cells are currently seen as a treatment for tissue regeneration in neurological diseases such as multiple sclerosis, anticipating that they integrate and differentiate into neural cells. Mesenchymal stem cells (MSCs), a subset of adult progenitor cells, differentiate into cells of the mesodermal lineage but also, under certain experimental circumstances, into cells of the neuronal and glial lineage. Their clinical development, however, has been significantly boosted by the demonstration that MSCs display significant therapeutic plasticity mainly occurring through bystander mechanisms. These features have been exploited in the effective treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis where the inhibition of the autoimmune response resulted in a significant amelioration of disease and decrease of demyelination, immune infiltrates and axonal loss. Surprisingly, these effects do not require MSCs to engraft in the central nervous system but depend on the cells' ability to inhibit pathogenic immune responses both in the periphery and inside the central nervous system and to release neuroprotective and pro-oligodendrogenic molecules favoring tissue repair. These results paved the road for the utilization of MSCs for the treatment of multiple sclerosis.

Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that can be isolated from many adult connective tissues. Th e cells grow as plasticadherent fi broblast-like cells that proliferate in vitro, maintaining pluripotency after prolonged culture. Under appropriate stimulus, MSCs can diff erentiate in vitro and in vivo into cells of the mesodermal lineage, such as bone, fat and cartilage cells.
MSCs have mainly been characterized after isolation from the bone marrow, where they are likely to represent the precursor cells for stromal tissue in close physical association with hematopoietic stem cells involved in hematopoiesis and maintenance of the homeostasis of the hematopoietic stem cell niche [1]. In the bone marrow the existence of a population of neural-crestderived stem cells was also shown, thus providing an expla nation for the reported ability of bone-marrowderived stem cells to also generate, to some extent, neural cells [2].
Despite evidence showing that MSCs can trans diff erentiate into multiple cell types in vitro and in vivo, the real contribution of MSCs to tissue repair -through signi fi cant engraftment and diff erentiation into biologically and functionally relevant tissue-specifi c cell typesis still elusive [3]. In the bone marrow, MSCs provide a sheltering microenvironment contributing to the preserva tion of hematopoietic stem cells by shielding them from diff erentiation and apoptotic stimuli and regulating their quiescence, proliferation and diff erentiation. Owing to their ability to support hematopoiesis, MSCs were fi rst utilized to enhance immune reconstitution when transplanted together with hematopoietic stem cells. Th e translation of the capacity of MSCs to diff erentiate into other tissues was fi rst exploited for reparative purposes, for example, in bone and heart diseases. Th e observation

Abstract
Stem cells are currently seen as a treatment for tissue regeneration in neurological diseases such as multiple sclerosis, anticipating that they integrate and diff erentiate into neural cells. Mesenchymal stem cells (MSCs), a subset of adult progenitor cells, diff erentiate into cells of the mesodermal lineage but also, under certain experimental circumstances, into cells of the neuronal and glial lineage. Their clinical development, however, has been signifi cantly boosted by the demonstration that MSCs display signifi cant therapeutic plasticity mainly occurring through bystander mechanisms. These features have been exploited in the eff ective treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis where the inhibition of the autoimmune response resulted in a signifi cant amelioration of disease and decrease of demyelination, immune infi ltrates and axonal loss. Surprisingly, these eff ects do not require MSCs to engraft in the central nervous system but depend on the cells' ability to inhibit pathogenic immune responses both in the periphery and inside the central nervous system and to release neuroprotective and pro-oligodendrogenic molecules favoring tissue repair. These results paved the road for the utilization of MSCs for the treatment of multiple sclerosis.

© 2010 BioMed Central Ltd
The therapeutic eff ect of mesenchymal stem cell transplantation in experimental autoimmune encephalomyelitis is mediated by peripheral and central mechanisms that bone-marrow-derived MSCs suppressed T-cell proliferation in vitro [4] and in vivo [5], however, unexpectedly drove attention to their exploitation for the treatment of immune-mediated diseases; for example, in those diseases where their ability of modulating the immune response could combine with the ability to integrate into damaged tissues and foster repair. Experimental autoimmune encephalo myelitis (EAE), a model for multiple sclerosis, has been the fi rst experimental autoimmune disease success fully treated with MSCs [6].

Experimental autoimmune encephalomyelitis is an example of immune-mediated disease
EAE can be actively induced in susceptible inbred rodents by immunization with diff erent neural antigens mainly derived from myelin, including myelin basic protein, proteolipid protein (PLP) and myelin oligodendro cyte protein (MOG) in complete Freud's adjuvant. Disease induction with PLP in SJL mice, and likewise MOG in C57BL/6 mice, requires the use of pertussis toxin that facilitates immune cell entry into the central nervous system (CNS) and contributes to T-cell tolerance breaking. EAE can be also induced in naïve mice by the intravenous passive transfer of encephalitogenic myelinspecifi c T cells. In fact, EAE is considered a prototypical MHC class-II-restricted CD4 + T-cell-mediated disease. During the induction phase, myelin-reactive CD4 + T cells are primed and expanded in the peripheral lymphoid organs. Th e eff ector phase involves migration of activated myelin-specifi c T cells to the CNS, where they cross the blood-brain barrier and require myelin peptides presented by local antigen-presenting cells and dendritic cells for full reactivation [7].
Several lines of evidence indicate that many subsets of T cells play diff erent roles in the onset, maintenance and recovery of EAE, T-helper-type 17 cells and regulatory T cells being among the main contributors to the fi nal outcome [8]. Not only T cells but also B cells producing demyelinating antibodies and macrophages are key eff ector cells in EAE pathogenesis. Typical EAE lesions resemble patterns of demyelination, infl ammatory cell perivascular infi ltrates, reactive microgliosis and astrocytosis, observed in multiple sclerosis lesions [9].

Systemic eff ect of the intravenous delivery of mesenchymal stem cells in experimental autoimmune encephalomyelitis
In the study by Zappia and colleagues we demonstrated that intravenous injection of syngeneic MSCs into C57BL/6 mice immunized with peptide 35 to 55 of MOG signifi cantly improved the clinical severity of EAE, in parallel decreasing CNS infl ammation and demyelination [6]. More impor tantly, we demonstrated that one injection of MSCs at disease onset or at the peak of disease suffi ces to induce peripheral tolerance, as demonstrated by the inability of T cells isolated from lymph nodes of MSC-treated mice, but not from control animals, to proliferate when stimulated with the immunizing antigen MOG. We also observed a dosedependent eff ect that reached maximum effi cacy and negligible mortality at the dose of 1 × 10 6 MSCs. No clinical eff ect was observed when MSCs were infused during the chronic phase of EAE, suggesting that multiple injections may not provide further advantages if permanent tissue damage has occurred [6]. In another study, Zhang and colleagues demonstrated that intravenous administration of human MSCs could improve the clinical course of PLP-induced EAE in SJL mice through some level of engraftment in the CNS and subsequent release of neurotrophic factors promoting oligodendrogenesis [10]. Th ese results highlighted that MSCs can cross MHC boundaries and exert their therapeutic eff ect also in the CNS, regardless of a very limited engraft ment. Following these pioneer works, in the last years several studies have focused on the mechanisms underlying the therapeutic eff ect of MSC transplantation on EAE.
Th e concept that MSCs ameliorate EAE through the induction of peripheral immune tolerance was further nourished by the demonstration that intravenous adminis tration of allogeneic MSCs in PLP-immunized mice inhibits the production of myelin-specifi c antibodies compared with controls [11]. In addition, the exposition of encephalitogenic T cells to MSCs in vitro signifi cantly decreases their ability to passively transfer EAE to healthy syngeneic mice [11]. Many other studies have confi rmed that MSCs can modulate the peripheral immune response to myelin antigens [12][13][14][15][16][17][18][19]. Th ese in vivo results have been corroborated by detailed in vitro studies dissecting the mechanisms of action of MSCs on T lymphocytes, B lymphocytes, dendritic cells, natural killer cells and other immune cells [20].

Mesenchymal stem cells are neuroprotective
It is important to underline that eff ects of MSCs on EAE are not exclusively due to their immunomodulatory activity, as many groups have shown that MSCs can also protect neurons and spare axons with no or very limited evidence of engraftment and/or transdiff erentiation into neural cells [11][12][13]15,16,21]. Th ese fi ndings posed the question of whether the observed neuroprotection in EAE is due to the peripheral eff ects suppressing the immune response that damages myelin or to a direct protective and reparative activity that follows their engraftment in the CNS.
Several lines of evidence suggest that, somehow, MSCs have a direct eff ect on neural cells. Th ey have been shown to enhance remyelination in vivo [15,16], provide in vitro soluble cues that infl uence fate determination of neural cells [16,22], display a potent antioxidant eff ect in vivo [23,24] and display a neuroprotective eff ect [25] mediated by the release of antiapoptotic molecules in vitro [26] and in vivo [27]. Th ese neuroprotective eff ects may well explain the remarkable eff ect obtained with the administration of MSCs in experimental models of stroke [28] and spinal cord injury [29]. Th ere is uncertainty, however, regarding the ability of MSCs to colonize the CNS after peripheral delivery due to their scarce ability to pass the lung fi lter following intravenous administration [30] and due to the lack of reliable labels or defi nitive markers for MSCs [31].
Irrespective of these aspects, the current view suggests that MSCs may exert their neuroprotective eff ect at distance through the release of trophic molecules, possibly aff ecting microglia activation [27] and inducing local neurogenesis [15,16,32].

Does local administration provide signifi cant advantage compared with systemic infusion?
To enhance the possibility for MSCs to engraft in the CNS and provide optimal therapeutic eff ects locally, Kassis and colleagues demonstrated, following intraventricular injection of MSCs, the expression of neural markers by a few transplanted labeled cells mainly in the proximity of infl ammatory lesions -suggesting that some level of transdiff erentiation was achieved [12]. Similarly, Barhum and colleagues showed that intraventricular adminis tration in vitro of MSCs modifi ed to produce neuro trophins successfully attenuated EAE [19].
We therefore evaluated whether local injection of a high number of MSCs may provide some advantage over intravenous systemic administration by comparing two diff erent routes of cell delivery in C57Bl/6 mice following immunization with the myelin antigen, peptide 35 to 55 of MOG. Th e intratechal delivery of 1 × 10 6 MSCs at the onset of the fi rst clinical symptoms (around day 10) resulted in a signifi cant amelioration of EAE compared with intra techally PBS-injected animals. No signifi cant diff erence was observed, however, when we compared the clinical course of mice intravenously injected with those treated intratechally ( Figure 1 and Table 1). No signifi cant diff er ence was also observed when the extent of spinal cord demyelinating lesions was compared ( Figure 1). As expected, the number of Luciferasetransfected MSCs, detected after 24 hours in the CNS of intratechally injected mice, was higher than in those where MSCs were delivered intravenously. After 40 days, however, the number of Luciferase-positive cells was clearly diminished with no statistical diff erence between the two groups ( Figure 1). Th ese results favor the current hypo thesis that MSCs act by diff erent mechanisms, mainly paracrinally on cells both at a distance and at the site of tissue damage, without the requirement of longterm engraftment [33].

Intravenous injection of mesenchymal stem cells also modulates the immune response in the CNS
A major issue still unsolved by the above-described studies was whether intravenously injected MSCs could also impact the immune response inside the CNS. It is well known that, following intravenous administration, MSCs inhibit infi ltration of T cells and macrophages in mice with EAE [6]. Th ese results, however, are likely to be an eff ect of the cells' tolerogenic ability exerted in the periphery on encephalitogenic T cells, as demonstrated by the inhibition of EAE following passive transfer of myelin-specifi c T cells [11].
To address this question we isolated T cells infi ltrating the brain of EAE-aff ected mice treated either intravenously or intratechally with MSCs and we measured by intracellular fl ow cytometry and real-time PCR the expression of the transcription factor FOXP3, a specifi c marker of regulatory T cells previously demonstrated to be expanded in the lymphoid organs of mice with collagen-induced arthritis treated with MSCs [34]. We observed not only that the intratechal delivery of MSCs induced an expansion of FoxP3 + T cells in the brain of EAE-aff ected mice compared with controls, but also that a similar result was observed in intravenously injected mice (Figure 2). Such a result probably depends on increased recruitment of this subset from the peripheral blood. To our surprise we observed, in the T cells isolated from the brain of both groups of MSC-treated mice compared with controls, an increase in the expression of  IL-17, a cytokine that plays an important role in the pathogenesis of autoimmune diseases ( Figure 2). Th ese results may be explained by the recent demonstration that MSCs can induce T-helper-type 17 cells to acquire a regulatory phenotype [35], and may also clarify the observation that human MSCs were shown to increase Thelper-type 17 responses in vitro [36].

Conclusions
Overall, many studies have confi rmed that MSCs, either from syngenic or xenogeneic sources, are eff ective in the treatment of EAE and dissected their mechanisms of action, probably in a much deeper fashion than in any other experimental disease. Th e results discussed in the present article demonstrate that MSCs can repair neural tissues as they display a broad therapeutic activity that acts both on immune and neural cells but feebly involves their transdiff erentiation. Interestingly, despite a limited ability to engraft in the nervous system, MSCs can clearly modulate the immune response not only in the peripheral lymphoid organs [6] but also within the CNS. Based on these studies and the available clinical experience obtained in several human conditions, MSCs can be considered an appealing therapeutic option for multiple sclerosis individuals with ongoing infl ammatory disease refractory to conventional therapies [37,38].

Competing interests
The authors declare that they have no competing interests.