WO2008001379A2

WO2008001379A2 - Activated myeloid cells for promoting tissue repair and detecting damaged tissue

Info

Publication number: WO2008001379A2
Application number: PCT/IL2007/000797
Authority: WO
Inventors: Michal Eisenbach-Schwartz; Oleg Butovsky; Gilad Kunis; Shay Bukshpan
Original assignee: Yeda Research And Development Co. Ltd
Priority date: 2006-06-28
Filing date: 2007-06-28
Publication date: 2008-01-03
Also published as: EP2043666A2; WO2008001379A3

Abstract

A cellular preparation comprising CD11c+ bone marrow-derived myeloid cells is provided for promoting repair of damaged body tissue. When labeled with a suitable imaging agent, the cells can be used to detect and localize a damaged body tissue. The cells are obtained by activation of bone marrow-derived myeloid cells with a cytokine, preferably IL-4.

Description

ACTIVATED MYELOID CELLS FOR PROMOTING TISSUE REPAIR AND DETECTING DAMAGED TISSUE

FIELD OF THE INVENTION

The present invention relates to methods and compositions for promoting tissue repair, for detection of damaged tissues and for delivery of drugs or detectable substances to damaged tissues and, particularly, to bone marrow-derived myeloid cells activated by some cytokines.

Abbreviations used: Aβ, β-amyloid; AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; BDNF, brain-derived neurotrophic factor; BM, bone marrow; BrdU, 5-bromo-2'-deoxyuridine; CFA, complete Freund's adjuvant; Cop 1, Copolymer 1, glatiramer acetate; CSF, cerebrospinal fluid; DCs, dendritic cells; DCX, doublecortin; DT, diphtheria toxin; EAE, experimental autoimmune encephalomyelitis; EGCG, (-)-epigallocatechin-3-gallate (green tea); FCS, fetal calf serum; GA, glatiramer acetate, Cop 1 ; GABA, γ-aminobutyric acid; GAD-67, glutamic acid decarboxylase 67; GABA, γ-aminobutyric acid; GFP, green fluorescent protein; IB4, isolectin B4; ICAM-I, intracellular cell adhesion molecule-; IFA, incomplete Freund's adjuvant; IFN, interferon; IGF-I, insulin-like growth factor 1 ; IL, interleukin; LPS, lipopolysaccharide; MG, microglia; MHC-II, class II major histocompatibility proteins; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; MWM, Morris water maze; NeuN, neuronal nuclear antigen; NPC, neural stem/progenitor cell; PBMC, peripheral bloof mononuclear cells; PBS, phosphate-buffered saline; PDL, poly-D-lysine; Tg, transgenic; Th cell, T-helper cell; STAT-I , signal transducers and activators of transcription 1 ; TNF, tumor necrosis factor. BACKGROUND OF THE INVENTION

Neuroprotection by T cells

Our first observation that systemic immune cells (in the form of T cells directed to certain self-antigens) can protect injured neurons from death came from studies in rodents showing that passive transfer of T cells specific to myelin basic protein reduces the loss of RGCs after a traumatic optic nerve injury (Moalem et al., 1999). We found that these T cells are also effective when directed to either cryptic or pathogenic epitopes of myelin basic protein, as well as to other myelin antigens or their epitopes (Mizrahi et al., 2002). These findings raised a number of critical questions. For example, are myelin antigens capable of protecting the nervous system from any type of acute or chronic insult? Is the observed neuroprotective activity of immune cells merely an anecdotal finding reflecting our experimental conditions, or does it point to the critical participation of the immune system in fighting off injurious conditions in the visual system and in the CNS in general? If the latter, does it mean that neurodegenerative diseases are systemic diseases? If so, can this finding be translated into a systemic therapy that would protect the brain, the eye, and the spinal cord?

In a series of experiments carried out over the last few years we have learned, firstly, that protective T cell response is a physiologically evoked response that might not be sufficient in severe insults or might not always be properly controlled. Moreover, we discovered that the specificity of such protective T cells depends on the site of the insult. Thus, for example, the protective effect of vaccination with myelin-associated antigens is restricted to injuries of the white matter, i.e., to myelinated axons (Mizrahi et al., 2002; Avidan et al., 2004; Schori et al., 2001). If the insult is to the retina, which contains no myelin, myelin antigens have no effect. Secondly, we observed that the injury-induced response of T cells reactive to specific self-antigens residing in the site of stress (eye or brain) is a spontaneous physiological response (Yoles et al., 2001). We then sought to identify the phenotype of the beneficial autoimmune T cells and to understand what determines the balance between a beneficial (neuroprotective) outcome of the T cell-mediated response to a CNS injury and a destructive effect causing autoimmune disease. We also examined ways of translating the beneficial response into a therapy for glaucoma. We found that in immune deficient animals the number of surviving RGCs following an insult in the eye, the spinal cord or the brain is significantly lower than in matched controls with an intact immune system, suggesting that the ability to withstand insult to the CNS depends on the integrity of the immune system and specifically on specific population within the immune system; those that recognize the site-specific self-antigens. Interestingly, the use of steroids caused significant loss of RGCs (Bakalash et al., 2003).

Protective autoimmunity

Some years ago our group formulated the concept of 'protective autoimmunity' (Moalem et al., 1999). Both pro-inflammatory and anti- inflammatory cytokines were found to be critical components of a T cell-mediated beneficial autoimmune response, provided that the timing and the intensity of the T- cell activity was suitably controlled (Butovsky et al., 2005; Shaked et al., 2004), and depending on the nature of the disease (Schwartz et al., 2006). According to our concept, an uncontrolled autoimmunity leads to the commonly known condition of autoimmune diseases associated with overwhelmed activation of microglia (Butovsky et al., 2006a), as will be discussed below. The beneficial effect of the autoreactive T cells was found to be exerted via their ability to induce the CNS- resident microglia to adopt a phenotype capable of presenting antigens (Butovsky et al., 2006a, 2005, 2001 ; Schwartz et al., 2006; Shaked et al., 2004), expressing growth factors (Butovsky et al., 2006a, 2006b, 2005), and buffering glutamate (Shaked et al., 2005).

In attempting to boost the efficacy of the protective autoreactive T cells, we tested many compounds in the search for a safe and suitable antigen for neuroprotection. We then suggested to use glatiramer acetate, also known as Copolymer 1 or Cop- 1 (Kipnis et al., 2000; Avidan et al., 2004; Angelov et al., 2003), a synthetic 4-amino-acid copolymer known to be safe and currently used as a treatment for multiple sclerosis by a daily administration regimen (Copaxone®, Teva Pharmaceutical Industries Ltd., Petach Tikva, Israel). Our studies have demonstrated its low-affinity cross-reaction with a wide range of CNS autoantigens. Because the affinity of cross-reaction is low, the Cop- 1 -activated T cells, after infiltrating the CNS, have the potential to become locally activated with little or no attendant risk of autoimmune disease (Kipnis et al., 2000).

A single injection of Cop- 1 is protective in acute models of CNS insults (Kipnis et al., 2000; Avidan et al., 2004; Kipnis. & Schwartz, 2002), while in chronic models occasional boosting is required for a long-lasting protective effect (Angelov et al., 2003). In the rat model of chronically high intraocular pressure, vaccination with Cop-1 significantly reduces RGC loss even if the pressure remains high. It should be noted that the vaccination does not prevent disease onset, but can slow down its progression by controlling the local extracellular environment of the nerve and retina, making it less hostile to neuronal survival and allowing the RGCs to be better able to withstand the stress (Schori et al., 2001 ; Benner et al., 2004; Kipnis & Schwartz, 2002; Kipnis et al., 2000).

For chronic conditions occasional boosting is needed. For example, in a model of chronically elevated intraocular pressure, weekly administration of adjuvant- free Cop-1 was found to result in neuroprotection (Bakalash et al., 2005). The neuroprotective effect of Cop- 1 has been attributed in part to production of brain-derived neurotrophic factor (BDNF) (Ziemssen et al., 2002).

Microglia and neurodegeneration Microglia are bone marrow-derived glial cells. In addition to astrocytes and oligodendrocytes, microglia represent the third major population of glial cells within the central nervous system (CNS). Microglia are distributed ubiquitously throughout the brain and spinal cord, and one of their main functions is to monitor and sustain neuronal health; they are the immune cells of the CNS, protecting against invading microorganisms, clearing unwanted debris, producing cytokines and cross-talking with the adaptive immune system (Aloisi, 2001, Kreutzberg, 1996). Through their immunoregulatory properties the activated microglia are involved in acute CNS injury, stroke as well as inflammatory and neurodegenerative disease (Streit, 2004; 2005). Microglia are mostly known for their bad reputation in neurodegenerative conditions (Kerschensteiner et al., 1999). Yet, recent studies have pointed out that microglia display a key role not only under pathological conditions and not only destructive effects; microglia are needed for supporting neuronal survival (Butovsky.et al.2006b, 2005; Shaked et al., 2005) and neural cell renewal (Butovsky et al.2006a, 2001 ; Ziv et al., 2006a), and to fighting off neurodegenerative conditions (Butovsky et al., 2006a; Simard et al., 2006).

Several types of microglia are present which may be associated with neurons or with blood vessels, and some of these are antigen-presenting cells (APCs). The nature of microglial activation, either beneficial or harmful, in damaged neural tissue depends on how microglia interpret the threat (Butovsky et al., 2005). Although the presence of microglial cells in normal undamaged neural tissue has been debated for years, it is now an accepted fact (Nimmerjahn et al., 2005), including their presence in the eye. The role of microglia in inflammatory processes is controversial. On the one hand, participation of microglia in inflammatory process of the eye can stimulate mature retinal ganglion cells (RGCs) to regenerate their axons (Yin et al., 2003). On the other hand, the role of microglia in neurodegenerative processes may be detrimental to the neuronal tissue. Roque et al (1999) showed that microglial cells release soluble product(s) that induce degeneration of cultured photoreceptor cells. This controversy may be explained by the contradicting reports regarding the presence of APCs, which are crucial factors of an antigen-specific cell-mediated immune response. Immunological responses in neural retinal microglia are related to early pathogenic changes in retinal pigment epithelium pigmentation and drusen formation. Activated microglia may also be involved in rod cell death in age-related macular degeneration (AMD) and late- onset retinal degeneration. A recent study has proposed that microglia, activated by primary rod cell death, migrate to the outer nuclear layer, remove rod cell debris and may kill adjacent cone photoreceptors (Gupta et al., 2003).

Like blood-derived macrophages, microglia exhibit scavenging of extracellular deposits, and phagocytosis of abnormal amyloid deposits in Alzheimer's disease (AD). Such microglia, while efficiently acting as phagocytic cells, cause neuronal death by the secretion of mediators like tumor necrosis factor alpha (TNF-α) (Butovsky et al., 2005), and thus, while acting as phagocytic cells (Frenkel et al., 2005). they are apparently not efficient enough to fight off the Alzheimer's disease symptoms. In contrast to these resident microglia, microglia derived from the bone marrow of matched wild-type mice can effectively remove plaques (Simard et al., 2006). Moreover, an absence of normally-functioning macrophages lead to the development of clinical AMD. Thus, AMD, like Alzheimer's disease, illustrates a disease in which scavenging of abnormal deposits inevitably induces self-perpetuation of disease progression mediated by the phagocytic cell themselves (Gupta et al., 2003).

Aggregated Aβ induces toxicity on resident microglia and impairs cell renewal

Recent studies performed in our laboratory suggested that microglia exposed to aggregated Aβ_; although effective in removing plaques, are toxic to neurons and impair neural cell renewal (Butovsky et al., 2006a); these effects are reminiscent of the response of microglia to invading microorganisms (as exemplified by their response to LPS) (Butovsky et al., 2005; Schwartz et al., 2006). Such activities are manifested by increased production of TNF-α, down-regulation of insulin-like growth factor (IGF-I), inhibition of the ability to express class II major histocompatibility complex (MHC-II) proteins and CDl Ic (a marker of dendritic cells) and thus to act as antigen-presenting cells (APCs), and failure to support neural tissue survival and renewal ((Butovsky et al., 2006a, 2006b). Further, we found that when microglia encounter aggregated β-amyloid, their ability to remove these aggregates without exerting toxic effects on neighboring neurons or impairing neurogenesis depends upon their undergoing a phenotype switch. A switch in microglial phenotype might take place via a local dialog between microglia and T- cells, which is mediated by T cell-derived cytokines such as IL-4. Addition of IL-4, a cytokine derived from T-helper (Th)-2 cells, to microglia activated by aggregated Aβ can reverse the down-regulation of IGF-I expression, the up-regulation of TNF- α expression, and the failure to act as APCs (Butovsky et al., 2005). The significance of microglia for in-vivo neural cell renewal was demonstrated by enhanced neurogenesis in the rat dentate gyrus after injection of IL-4-activated microglia intracerebroventricularly and by the presence of IGF-I-expressing microglia in the dentate gyrus of rats kept in an enriched environment (Ziv et al., 2006a). In rodents with acute or chronic EAE, injection of IL-4-activated microglia into the cerebrospinal fluid resulted in increased oligodendrogenesis in the spinal cord and improved clinical symptoms. The newly formed oligodendrocytes were spatially associated with microglia expressing MHC-II and IGF-I (Butovsky et al., 2006c). Reference is made to copending International Patent Application No.

PCT/IL2007/ entitled "Method of treatment of age-related macular degeneration" filed by applicant at the Israel PCT Receiving Office (RO/IL) on the same date, the contents thereof being explicitly excluded from the scope of the present invention. Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

The present invention relates, in one aspect, to a method for promoting tissue repair in a patient, said method comprising administering to the patient a therapeutically effective amount of CDl Ic⁺ bone marrow-derived myeloid cells.

In one preferred embodiment the cells express IGF-I and/or BDNF. The CDl I c⁺ bone marrow-derived myeloid cells may be obtained by activation with a cytokine selected from IL-4, 11-13 or a narrow concentration range of IFN-γ of up to 20 ng/ml.

In another aspect, the present invention provides a method for detecting/localizing a damaged tissue, said method comprising administering to an individual in need bone marrow-derived myeloid cells that have been activated with IL-4, IL- 13 or up to 20 ng/ml IFN-γ and labeled with an imaging agent, whereby the labeled cells traffic to the damaged tissue, and imaging the suspected area in the patient, thereby localizing the damaged tissue. The invention further relates to a method for delivering a therapeutic or detectable substance to a damaged tissue or a tumor, said method comprising administering to an individual in need bone marrow-derived myeloid cells that have been activated with IL-4, IL- 13 or up to 20 ng/ml IFN-γ, wherein said cells are cells that have been genetically engineered to express said therapeutic or detectable substance.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A-1F shows that in mice with chronic EAE, GA-immunization induces oligodendrogenesis. Chronic EAE was induced in C57BL/6J mice. GA was administered once (s.c. in IFA) at the day of MOG-immunization (day 0; MOG in CFA). Control mice induced with EAE received a single injection of PBS (s.c. in IFA). From day 19, BrdU was injected for 2.5 days (twice per day) in both MOG- vaccinated group received PBS (n = 5) or co-injected subcutaneously with GA (n = 5). Naϊve mice received the same course of BrdU injection (n = 4). Spinal cords were excised 7 days after the last BrdU injection. (IA) Quantitative analysis of proliferating cells (BrdU⁺) and proliferating microglia (BrdU+/ microglial marker IB4⁺) in both gray matter (GM) and white matter (WM) of the spinal cord analyzed at 300-μm intervals along longitudinal 30-μm sagittal sections (T8-T9). (IB) NG2⁺ (proteoglycan oligodendrocyte marker) or RIP⁺ (mature oligodendrocyte marker) cells co-labeled with BrdU⁺ cells. Data are expressed as means ± SEM per mm³. Asterisks above bars express differences relative to naive mice ( P < 0.05; P < 0.01 ; ^***P < 0.001 ; ANOVA). The P values indicated in the figure represent a comparison of the groups as analyzed by ANOVA. (1C) Representative confocal images of longitudinal sections of spinal cords stained with BrdU and co-stained for IB4, NG2 and RIP. (ID) Newly formed oligodendrocytes are identifiable by co- localization of BrdU and RIP in the white matter of MOG+GA-vaccinated mice. Note that the images are from areas that include both gray matter and white matter; a dashed line shows the border between the two areas. (IE) Proliferating microglia (BrdU⁺/IB4⁺) co-expressing NG2 (arrows). (IF) Appearance of newly formed oligodendrocytes (BrdU⁺/RIP⁺) in close proximity to the central canal (CC) of MOG+GA-vaccinated mice.

Figs. 2A-2B show that GA- vaccination increases the number of CDl Ic⁺ microglia in the white matter of spinal cord of chronic EAE-mice. The spinal cords analyzed in Fig. 1 were also examined for microgliogenesis. (IA) Quantitative analysis of IB4⁺ cell co-labeled with MHC-II or CDl Ic (means ± SEM) from the white matter (WM) per mm^J of PBS- and G A- vaccinated mice (two-tailed Student's Mest; n = 5 per group). Asterisks above bars express the significance of differences relative to PBS-injected mice (^***P < 0.001; two-tailed Student's Mest). (IB) Representative confocal microscopy of longitudinal sagittal sections of spinal cords (T8-T9), stained with IB4 and co-stained with MHC-II and CDl Ic. Significantly more MHC-II⁺ cells are seen, especially in the gray matter, in slices from myelin MOG+GA-vaccinated mice than from PBS-injected mice.

Fig. 3 shows that bone marrow-derived CD l Ic⁺ cells are crucial for EAE development, but their depletion during the onset of the disease exacerbate the disease progression. Lethally irradiated C57BL/J6 mice were reconstituted with syngeneic bone marrow cells of CX₃CRlZ⁰¹⁷^⁺ZCD] Jc^DTR -transgenic mice. After 6 weeks of the transplantation, the mice were vaccinated with MOG. A single s.c. injection of 250 μg GA in IFA alone attenuated MOG-induced EAE in C57BL/6J mice (MOG+GA). Injection of diphtheria toxin (DT) ip at 5 days after MOG- vaccination and given with intervals of 3 and 4 days thereafter (MOG+GA /DT - before onset of EAE) dramatically attenuated EAE pathogenesis. Injection of DT in MOG and MOG+GA group starting on day 12 and given with intervals of 3 and 4 days thereafter (MOG- and MOG+GA /DT - after onset of EAE, respectively) significantly exacerbate clinical score as compared to MOG- and MOG+GA- vaccinated group without DT-treatment.

Figs. 4A-4D show that IL-4, unlike IL-IO, induces MHC-II and CDl Ic in both microglia and bone marrow-derived myeloid cells. Primary culture of mouse microglia and bone marrow-derived myeloid cells were activated with IL-4 (10 ng/ml), IFN-γ (10 ng/ml), IL-IO (10 ng/ml) or in combination of interferon (IFN)- γ+IL-4 or IL-10+IL-4 for 5 days. (4A) Untreated microglia (Control) express hardly any CDl Ic or MHC-II. IL-4 induces microglia to express CDl Ic and MHC-II, whereas IFN-γ induces only MHC-II. A combination of IFN-γ and IL-4 affects both CDl Ic and MHC-II. IL-IO has no effect on the expression, whereas IL-IO suppressed IL-4-induced MHC-II, but not CDl Ic expression. (4B) Similar pattern of activation was observed in mouse bone marrow-derived myeloid cells. Quantitative analysis of expression of CDl Ic and MHC-II (expressed as a percentage of IB-4-labeled microglia) in microglia (4C) and bone marrow-derived myeloid cells (BM) (4D). Results are of three independent experiments in replicate cultures; bars represent means ± SEM. Asterisks above bars denote the significance of differences relative to untreated cells (P < 0.05; ^**P < 0.01; ^***P < 0.001; two- tailed Student's t-test). The P values indicated in the figure represent a comparison of the groups as analyzed by ANOVA.

Fig. 5 shows that IL-4 induces expression of IGF-I and BDNF in bone marrow-derived myeloid cells. Bone marrow-derived myeloid cells were activated as described in Fig. 4. Confocal images represent immunocytochemistry for microglial marker IB4, IGF-I and BDNF. Untreated cells hardly express BDNF and IGF-I. No effect was found after IFN-γ activation, whereas IL-10 significantly increases BDNF but not IGF-I. IL-4 alone or in a combination with IFN-γ induces both IGF-I and BDNF. Addition of IL-IO together with IL-4 has a superior effect on induction of BDNF. Separate confocal channel is shown in right panel. Figs. 6A-6B show that IFN-γ, unlike IL-IO, induces microglial expression of ICAM-I . Microglial culture described in Fig. 4 was analyzed for ICAM-I expression. (6A) After 5 days of culturing, a few cells of untreated microglia (Control) express ICAM- 1. IFN-γ significantly induces microglia to express ICAM- 1, whereas either IL-4 or IL- IO has no effect. IL-4 does not affect IFN-γ-induced ICAM-I, whereas IL-IO significantly inhibited IFN-γ-induced ICAM-I expression. (6B) Quantitative analysis of expression of ICAM- 1 [expressed as intensity per cell in arbitrary units (AU)]. Results are of three independent experiments in replicate cultures; bars represent means ± SEM. Asterisks above bars denote the significance of differences relative to untreated cells (P < 0.05; ^**P < 0.01 ; ^***P < 0.001 ; two- tailed Student's t-test). The P values indicated in the figure represent a comparison of the groups as analyzed by ANOVA

Fig. 7 shows that in mice with chronic EAE, intraventricular Iy injected BM_(IL-4) significantly improves clinical features and induces oligodendrogenesis. Chronic EAE was induced in C57BL/6J mice. EAE scores in mice injected with either non-activated (BM_(-)) or IL-4-activated (BM_(IL-4)) syngeneic bone marrow- derived myeloid cells of CX₁CRl P^Fm /CDl ic^D™-transgenic mice {n = 7 in each group) at 9 days after MOG-vaccination. Additional group of BM_(IL-4)-treated mice received DT (BM_(1L.₄₎ /DT) intraperitoneal Iy (ip) starting on day 9 after MOG- vaccination and thereafter with intervals of 3 days. Control mice induced with EAE received the same regimen of DT (MOG /DT) and used as a control. Injection of

DT, starting from day 15 (third injection) after MOG-vaccination, gradually reduces the beneficial effect of BM₍i_L-4) and exacerbate the clinical manifestation thereafter.

Figs. 8A-8B show that GA increases CDl lc⁺/Lin^' cells in the blood of patients with multiple sclerosis (MS). FACS analysis for cell surface expression of CDl Ic in myeloid cells (Lin^") from human peripheral blood of healthy {n = 10) and untreated- (n = 11) or GA-treated MS patients (n = 12). (8A) Representative FACS analysis of healthy control and both GA-treated and untreated MS patients in remission phase. (8B) Statistical analysis of the examined groups. The P values indicated in the figure represent a comparison of the groups as analyzed by ANOVA.

Figs. 9A-9D demonstrate that IL-4 can counteract the adverse effect of aggregated Aβ on microglial toxicity and promotion of neurogenesis in adult mouse neural progenitor cells (NPC). (Fig. 9A) In-vitro treatment paradigm. (Fig. 9B) Representative confocal microscopic images of NPCs expressing GFP and βlll-T (neuronal marker), co-cultured for 10 days without microglia (MG, control), or with untreated microglia, or with microglia that were pre-activated by aggregated Aβ₍i_₄o₎ (5 μM) (MG(_Aβi-₄₀)) for 48 h and subsequently activated with IFN-γ (10 ng/ml) (MG_(Aβi-4o / iFNγ)) ^{or wim} IL-4 (10 ng/ml) (MG_(Aβi-₄o / iL-4)) or with both IFN- γ (10 ng/ml) and IL-4 (10 ng/ml) (MG_(Aβi-4o / _IFN_Y+_IL-₄))- Note, aggregated Aβ induced microglia adopt an amoeboid morphology, but after IL-4 was added they exhibited a ramified structure. (Fig. 9C) Separate confocal images of NPCs co- expressing GFP and βlll-T adjacent to CDl Ib⁺ microglia. (Fig. 9D) Quantification of cells double-labeled with GFP and βlll-T (expressed as a percentage of GFP⁺ cells) obtained from confocal images. Results are of three independent experiments in replicate cultures; bars represent means ± SEM. Asterisks above bars denote the significance of differences relative to untreated (control) NPCs (P < 0.05; ^***P < 0.001 ; two-tailed Student's t-test). Horizontal lines with P values above them show differences between the indicated groups (ANOVA).

Figs. 10A-10L show that Cop-1 vaccination leads to reduction in β-amyloid and counteracts loss of hippocampal neurons in the brains of transgenic Alzheimer's disease mice: key role of microglia. (Fig. 10A) Representative confocal microscopic images of brain hippocampal slices from non-transgenic (non-Tg), untreated-Tg- Alzheimer' s disease (AD), and Cop-1 -vaccinated Tg-AD mice stained for NeuN (mature neurons) and human Aβ. The non-Tg mouse shows no staining for human Aβ. The untreated-Tg- AD mouse shows an abundance of extracellular Aβ plaques, whereas in the Cop-1 -treated Tg-AD mouse Aβ-immunoreactivity is low. Weak NeuN⁺ staining is seen in the hippocampal CAl and dentate gyrus regions of the untreated-Tg-AD mouse relative to its non-Tg littermate, whereas NeuN⁺ staining in the Cop- 1 -vaccinated Tg-AD mouse is almost normal. (Fig. 10B) Staining for activated microglia using anti-CD l ib antibodies. Images at low and high magnification show a high incidence of microglia double-stained for Aβ and CDl Ib in the CAl and dentate gyrus regions of the hippocampus of an untreated- Tg-AD mouse, but only a minor presence of CDl Ib⁺ microglia in the Cop-1- vaccinated Tg-AD mouse. Arrows indicate areas of high magnification, shown below. (Fig. 10C) CDl Ib⁺ microglia, associated with an Aβ plaque, strongly expressing TNF-α in an untreated-Tg-AD mouse. (Fig. 10D) Staining for MHC-II (a marker of antigen presentation) in a cryosection taken from a Cop- 1 -vaccinated Tg-AD mouse in an area that stained positively for Aβ shows a high incidence of MHC-II⁺ microglia and almost no TNF-Ct⁺ microglia. (Fig. 10E) AU MHC-II⁺ microglia in a brain area that stained positively for Aβ (arrowheads) in a Cop-1- vaccinated Tg-AD mouse co-express CD 1 1 c (a marker of dendritic cells), but only a few CDl lc⁺/MHC-II⁺ microglia are seen in a corresponding area in the brain of an untreated-Tg-AD mouse. (Fig. 10F) MHC-II⁺ microglia in a Cop- 1 -vaccinated Tg- AD mouse co-expresses IGF-I. (Fig. 10G) CD3⁺ T cells are seen in close proximity to an Aβ-plaque and (Fig. 10H) are associated with MHC-II⁺ microglia. Boxed area shows high magnification of an immunological synapse between a T cell (CD3⁺) and a microglial cell expressing MHC-II. (Fig. 101) Histogram showing the total number of Aβ-plaques (in a 30-μm hippocampal slice). (Fig. 10J) Histogram showing staining for Aβ-immunoreactivity. Note the significant differences between Cop- 1 -vaccinated Tg-AD and untreated-Tg-AD mice, verifying the decreased presence of Aβ-plaques in the vaccinated mice. (Fig. 10K) Histogram showing a marked reduction in cells stained for CDl Ib, indicative of activated microglia and inflammation, in the Cop- 1 -vaccinated Tg-AD mice relative to untreated-Tg-AD mice. Note the increase in CDl Ib⁺ microglia with age in the non- Tg littermates. (Fig. 10L) Histogram showing significantly more CD3⁺ cells associated with an Aβ-plaque in Cop- 1 -vaccinated Tg-AD mice than in untreated- Tg-AD mice. Quantification of CD3⁺ cells was analyzed from 30-50 plaques of each mouse tested in this study. Error bars indicate means ± SEM. ^*P < 0.05, ^***P < 0.001 versus non-Tg littermates (Student's /-test). The P values indicated in the figure represent a comparison of the groups as analyzed by ANOVA. All of the mice in this study were included in the analysis (6-8 sections per mouse).

Figs. 1 IA-11C show that Cop-1 vaccination induces microglia to express CDl Ic. (Fig. HA) CDl Ib⁺ microglia co-expressing CDl Ic surround an Aβ-plaque in Cop-1 -vaccinated transgenic Alzheimer (Tg-AD) mice. All of the CDl Ic- expressing microglia are co-labeled for CDl Ib. Separate confocal channel is shown in right panel. (Fig. HB) Histograms showing the number of CDl Ib⁺ cells associated with Aβ-plaque. (Fig. HC) Histograms showing quantification of CDl Ic⁺ cells as a percentage of the total number of CDl Ib⁺ and CDl Ic⁺ cells associated with an Aβ-plaque. For this analysis, cells were counted surrounding 30-50 plaques in each mouse tested. Error bars represent means ± SEM. Asterisks above bars denote the significance of differences between the groups ( P < 0.01; ^***P < 0.001 ; two-tailed Student's Mest).

Figs. 12A-12D show that Cop-1 vaccination induces microglia (MG) to express CD l Ic: role of IL-4. (Fig. 12A) IL-4-activated microglia (MG_(IL-4)) induce CD l Ic expression in a primary culture of mouse microglia 5 days after activation. Untreated microglia (MG_H) express hardly any CDl Ic. (Fig. 12B) Effect of IL-4 (in terms of morphology and CDl Ic expression) on microglia pretreated for 3 days with aggregated Aβ₍i-₄₀₎ (MG_(Aβ)) and assessed 10 days later compared to IL-4 treatment for 10 days without pre-exposure to Aβ. Note that dendritic-like morphology was adopted upon addition of IL-4 to the Aβ-pretreated microglia only, whereas CDl Ic expression was induced by IL-4 both with and without Aβ pretreatment. (Fig. 12C) Quantitative analysis of microglial expression of CDl Ic⁺ microglia (expressed as a percentage of IB-4-labeled microglia) and of CDl Ic intensity per cell, both expressed as a function of time in culture with or without IL- 4. (Fig. 4D) Quantitative analysis of CDl Ic expression (calculated as a percentage of IB-4-labeled microglia) by the cultures shown in (Fig. 12B). Results are of three independent experiments in replicate cultures; bars represent means ± SEM. Asterisks above bars denote the significance of differences relative to untreated microglia at each time point (^***P < 0.001 ; two-tailed Student's Mest).

Figs. 13A-13B show engulfment of aggregated Aβ by activated microglia. Microglia were treated with IL-4 (10 ng/ml) 24 h after seeding (MG_(1L-4)) or were left untreated for 48 h (MG_(-)). The media were then replaced by a labeling medium (DMEM containing 10 mg/ml bovine serum albumin), and aggregated Aβ₍i-₄₀₎ was added (5 μg/ml) for 1 h. Following incubation the cultures were fixed and immunostained with antibodies directed to human Aβ and co-stained for microglia (IB-4). (Fig. 13A) Confocal photomicrographs. (Fig. 13B) Quantitative analysis expressed as intensity per cell. Results of one of two experiments, each containing eight replicates (20-30 cells per replicate) per group, are presented (means ± SD).

Figs. 14A-14E depict enhanced neurogenesis induced by Cop-1 vaccination in the hippocampal dentate gyri of adult transgenic AD mice (Tg). Three weeks after the first Cop-1 vaccination, mice in each experimental group were injected i.p. with BrdU twice daily for 2.5 days. Three weeks after the last injection, their brains were excised and the hippocampi analyzed for BrdU, DCX (a marker of early differentiation of the neuronal lineage), and NeuN (a marker of mature neurons). (Figs. 14A-14C) Histograms showing quantification of the proliferating cells (BrdU⁺). (Fig. 14A) Newly formed mature neurons (BrdU⁺/NeuN⁺) (Fig. 14B), and all pre-mature (DCX⁺-stained) neurons (Fig. 14C). Numbers of BrdU⁺, BrdU⁺/NeuN⁺ and DCX⁺ cells per dentate gyrus (DG), calculated from six equally spaced coronal sections (30 μm) from both sides of the brains of all the mice tested in this study. Error bars represent means ± SEM. Asterisks above bars denote the significance of differences relative to non-Tg littermates (^**P < 0.01; ^***P < 0.001; two-tailed Student's Mest). Horizontal lines with P values above them show differences between the indicated groups (ANOVA). (Fig. 14D) Representative confocal microscopic images of the dentate gyrus showing immunostaining for BrdU/DCX/NeuN in a Cop-1 -vaccinated Tg-AD mouse and in a non-Tg littermate relative to that in an untreated-Tg-AD mouse. (Fig. 14E) Branched DCX⁺ cells are found near MHC-II⁺ microglia located in the subgranular zone (SGZ) of the hippocampal dentate gyrus of a Cop- 1 -vaccinated Tg-AD mouse.

Figs. 15A-15B show that Cop-1 vaccination counteracts cognitive decline in Tg-AD mice. Hippocampus-dependent cognitive activity was tested in the Morris water maze (MWM). (Figs. 15A-15B) Cop- 1 -vaccinated Tg-AD mice {diamond; n = 6) showed significantly better learning/memory ability than untreated-Tg-AD mice {square; n = T) during the acquisition and reversal. Untreated-Tg-AD mice showed consistent and long-lasting impairments in spatial memory tasks. In contrast, performance of the MWM test by the Cop- 1 -vaccinated Tg-AD mice was rather similar, on average, to that of their age-matched naϊve non-Tg littermates {triangle; n = 6) (3-way ANOVA, repeated measures: groups, df (2,16), F = 22.3, P < 0.0002; trials, df (3,48), F = 67.9, P < 0.0001 ; days, df (3,48), F = 3.1, P < 0.035, for the acquisition phase; and groups, df (2, 16), F = 14.9, P < 0.0003; trials, df (3,48), F = 21.7, P < 0.0001; days, df (1, 16), F = 16.9, P < 0.0008, for the reversal phase).

Fig. 16 is a photo of a new apparatus for pre-clinical research. On a Plexiglas plate two thick lead disks are held apart by short lead columns. Attached concentrically and level to the lower disk is a thin Plexiglas ring. Mice are placed on this surface with their heads between the disks. Two removable handles allow positioning the apparatus.

Figs. 17A-17C show that CX3CR1-GFP⁺ bone marrow- derived microglia migrated into the brain after total body γ-irradiation (Fig. 17A). High magnification of the cells are represented in Fig. 17B. (Fig. 17C) CX3CR1-GFP⁺ microglia co- expressing MHC-II and IGF-I (separate channels of confocal image).

Fig. 18 shows CDl Ib⁺ microglia co-expressing ICAM-I associated with Aβ- plaques in the hippocampus of Tg-AD mice at 12 months of age. Top pannels represent separate channels of immunohistochemistry for human Aβ (green), CDl Ib (blue) and ICAM-I (red). Fig. 19 shows that CX3CR1-GFP⁺ bone marrow-derived microglia migrated into diseased spinal cord of SODl -transgenic mice. Confocal microscopy immunohistochemistry for activated microglia labeled with CDl Ib (red) and CDl Ic (blue). Note, all CX3CR1-GFP⁺ cells co-express CDl Ib. Fig. 20 shows that IL-4-activated CX3CR1-GFP⁺ bone marrow-derived myeloid cells injected systemically (iv) migrated into diseased spinal cord of SODl -transgenic mice at the stage of devastation (14-150 days).

Figs. 21A-21H show that microglia treated with IFN-γ differentiated into neuronal-like cells. (21A) ln-vitro treatment paradigm. (21B) Representative confocal microscopic images of microglia stained for CDl Ib and βlll-tubulin (βlll- T; neuronal marker), co-cultured for 10 days in microglial medium in the presence of IFN-γ (lOng/ml) (MG_0FN-γ)) or IL-4 (lOng/ml) (MG₍i_L.₄₎). Note, IFN-γ induced microglia to adopt a morphology of elongated cells co-expressing CDl lb/βlll-T, whereas IL-4-treated microglia exhibited a round-shape morphology as compared to untreated microglia (MG_(-)). (21C, 21D) Separate confocal images of βIII-T⁺ MG₍i_FN-_γ) co-expressing MHC-II (21c) and microglial marker isolectin B4 (IB4) (21D). (21E) Quantification of βIII-T⁺ cells co-labeled for IB4 (expressed as a percentage of IB4⁺ cells) obtained from confocal images of MG_(-), MG_(IFN-γ), MG_(IL. ₄₎ θr microglia treated with both IFN-γ (10ng/ml) or IL-4 (10ng/ml) (MG_(IFN-_Y+IL4)) ^at different time points as indicated. (21F) Purified microglia were stained for IB4⁺. All cells were CDl Ib⁺. The numbers of IB4⁺ microglia (expressed as a percentage of DAPI⁺ cells; DAPI stains DNA in live and dead cells) were verified in all treatments at all time points. Results are of four independent experiments with duplicate or triplicate wells; bars represent means ± SEM. Asterisks above bars denote the significance of differences relative to MG_(-) ( P < 0.05; P < 0.001; two-tailed Student's Mest). Horizontal lines with P values above them show differences between the indicated groups (ANOVA). (21G) At 18 days, MG_(IFN-γ) cultured for additional 10 days in neural medium positively stained for GABA (CDl lb/GABA) and (21H) for GAD67 (CDl lb/βIII-T/GAD) Note, confocal channels are presented separately.

Figs. 22A-22C show time course of CDl Ic expression in microglia activated by IFN-γ and IL-4. Microglia were treated with IFN-γ (10 ng/ml; MGp._τ)) or IL-4 (10 ng/ml; MG_(IL-4)) for 1, 3, 5, 10 and 18 days as described in Fig. 21. MG₍.₎ Were used as controls. (22A) Confocal images of microglia, identified by staining for IB4, immunolabeled for βlll-T (neuronal marker), and CDl Ic after 5 days of treatment. MG_(-) did not express CDl Ic. After exposure to IFN-γ or IL-4 the microglia expressed CDl Ic and exhibited their characteristic morphology. (22B) Co-expression of βlll-T and CDl Ic in microglia activated with IFN-γ (10 ng/ml) for 5 days (IB4/βIII-T/CDl lc). Note confocal channels are presented separately. (22C) Quantitative analysis of the numbers of CDl Ic⁺ microglia (expressed as a percentage of IB4⁺ (microglia marker) cells) were examined in all treatments at all time points. Results are of four independent experiments with duplicate or triplicate wells; bars represent means ± SEM. Asterisks above bars denote the significance of differences relative to MG_(-) (^*P < 0.05; ^**P < 0.01 ; ^***P < 0.001 ; two-tailed Student's f-test). Horizontal lines with P values above them show differences between the indicated groups (ANOVA).

Figs. 23A-23D show that βIII-tubulin⁺ elongated cells are derived from microglia. (23A) Confocal images represent primary culture of microglia from transgenic mice labeled for GFP under the promoter of the chemokine fractalkine receptor CX₃CRl (CX₃CRl ^GFP/+) and co-expressing doublecortin (DCX, a marker of early differentiation of the neuronal lineage) and βlll-T (neuronal marker) after 5 days of treatment with IFN-γ (10ng/ml). Arrows represent the co-expression and elongated morphology of the cells in separate confocal images. (23B) In-vitro treatment paradigm. Primary culture of mixed glial cells and microglia from double- transgenic mice (CX₃CRl ^Gf7V+/CDl lc^D77?) were treated for 2 days with IFN-γ and consequently treated with diphtheria toxin (DTx) for additional 72 h or 6 days, respectively. (23C) Right panel represents confocal images of microglial culture and appearance of elongated GFP^T microglia co-expressing BIII-T; on the right, addition of DTx completely ablates βIII-T⁺ microglia. (23D) Right panel represents confocal images of mixed glial culture and the presence of GFP⁺ microglia and βlll- T⁺ survived neurons; on the left, additional of DTx completely ablate GFP⁺ microglia, but not βIII-T⁺ neurons.

Figs. 24A-24F show that microglia exhibit stem-like features. (24A) Naive GFP⁺-microglia (MG₍.₎) from primary culture of CX₃CRI ''^-transgenic mice generates spheres and co-express CD34, a marker of hematopoietic stem cells and Nestin, a neural stem cells marker. (24B) GFP-expressing neural progenitor cells (NPCs) and GFP⁺ microglial cells dissociated from spheres stained for Nestin and CD34, 24 hours after seeding in medium of differentiation. (24C) At 5 days, Nestin expressed by all microglia irrespective of the treatment. IFN-γ treatment induced elongation of GFP⁺ microglia co-expressed Nestin and DCX (a marker of early differentiation of the neuronal lineage) or (24D) Nestin and βlll-T. Note, confocal channels are presented separately. (24E, 24F) Quantitative analysis of immunoreactivity of Nestin revealed decreased levels of Nestin in MG₍.₎ or MG₍[_FN- _γ) at day 10, whereas MG₍i_L-4) showed significantly increased expression of Nestin. IB-4 is a microglial marker. Results are of four independent experiments with duplicate or triplicate wells; bars represent means ± SEM. Asterisks above bars denote the significance of differences relative to MG_(-) (P < 0.05; ^**P < 0.01 ; ^***P < 0.001 ; two-tailed Student's /-test). AU - arbitrary units.

Figs. 25A-25B show that IL-4, unlike IFN-γ, increased expression of glial markers without inducing morphological features. (25A) Representative confocal images Of GFP⁺ microglia from CX₃CRl^CF/y+-transgenic mice treated with IL-4 (10 ng/ml) and IFN-γ (10 ng/ml) for 10 days and stained for glial fibrillary acidic protein (GFAP, a marker for glial cells). Note, IL-4, unlike IFN-γ triggered expression of GFAP without inducing morphological features of astrocytes. (25B) IL-4 increased the expression of proteoglycan oligodendrocytes marker NG2. The presented data are similar to those of three independent experiments. Fig. 26 shows microglia after long time exposusre to high levels (100ng/ml) of IFN-γ (MG₍i_FNγ-ioo_ng))₅ low levels of IFN-γ (MG_(1FNγ)), IL-4 (MG_(IL-4)), and/or LPS MG(LP₅).

Figs. 27A-27B show that expression of danger signals in SOD1^G93A mice is delayed relative to disease (ALS) progression. SOD 1^G93Λ and control mice were killed at different stages of clinical manifestation of ALS: pre-disease onset (60 d); disease onset (90 d); and disease progression (120 d). At each time point ICAM-I, CDl Ib, and CDl Ic expression in lumbar spinal cord regions were analyzed by immunohistochemistry. Representative confocal images of lumbar spinal cord areas are presented. (27A) ICAM-I and activated microglia (CDl Ib); (27B) expression of CDl Ic. The relevant high-power micrographs are shown below the panels indicating boxed areas.

Figs. 28A-H show that dendritic-like BM-derived myeloid cells expressing IGF-I home to spinal cords in ALS mice. Immunohistochemical analysis of the microglial population in 140-day-old CX₃CRl^GFP-wt SODl chimeric mice in which the SOD bone-marrow cells had been replaced on day 70 by BM cells derived from transgenic mice expressing GFP under the fractalkine receptor-1 CX3CR1. Spinal cords of chimeric- and untreated- S ODl were analyzed for GFP, CDl Ib, and CDl Ic (28A, 28B and 28E, 28F, respectively), and for IGF-I (28C, 28D and 28G, 28H, respectively). (28A, 28C): Representative confocal images of cross-sections of the chimeric-SOD; mice (n=5) (28E, 28G) and untreated- S ODl mice (n=5) (28B, 28D). Boxes show areas of the gray matter (GM) indicated in 28A and 28C, respectively. (28F, 28H) Boxes show areas of the gray matter indicated in 28E and 28G, respectively. Note, corresponding separate confocal channels are shown in the right panels of 28B, 28D, 28F and 28H.

Figs. 29 A-B show that BM_(IL-4) cells from wild-type mice injected systemically into SOD1^G93A diseased mice are recruited exclusively into the ventral horn of the spinal cord. IL-4-activated BM-derived myeloid cells from CX3CR1^GFP- transgenic mice were injected i.v. into SOD1^G93A mice at the stage of progressive disease (at ages 125 days, 130 days, and 136 days). At end stage (approximately 140 days) the mice were killed, and their spinal cords were analyzed for GFP⁺ cells and co-stained for CDl Ib, CDl Ic (29A), and IGF-I (29B).

Figs. 30A-B show that IL-4 induces CDl Ic and IGF-I expression in BM- derived myeloid cells of both SOD and wild-type (WT) mice. Myeloid cells were isolated from BM and treated with IL-4 ( 10 ng/ml) for 72 h. Untreated cells were used as controls. Cells were analyzed by immunocytochemistry for expression of CDl Ic (30A) and IGF-I (30B). No differences were observed in the BM cells derived from SOD and WT mice (three independent experiments, each carried out in triplicate). Q-PCR of BM-derived myeloid cells as described in 3OA, 48 hours after treatment. A significant increase in IGF-I was seen in BM-derived myeloid cells of both SOD and wild-type mice. Data are from 2 independent experiments in replicate cultures; bars represent mean ± SD. **/^J> < 0.01 versus control (two-tailed Student's t test).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to a method for promoting body tissue repair in an individual, said method comprising administering to the individual in need a therapeutically effective amount of CDl Ic⁺ bone marrow- derived myeloid cells. In another aspect, the present invention relates to a method for promoting tissue repair in an individual in need, which comprises mobilizing CDl Ic⁺ bone marrow-derived myeloid cells to the damaged tissue of said individual.

The CDl Ic⁺ bone marrow-derived myeloid cells for use in the methods of the invention may be obtained by activation with at least one cytokine selected from IL-4, IL- 13 or a narrow concentration range of IFN-γ of up to 20 ng/ml.

In one embodiment, the CDl I c⁺ bone marrow-derived myeloid cells, particularly those activated by IL-4, also express IGF-I, BDNF or both IGF-I and BDNF.

The present invention also relates to a method for promoting tissue repair in a patient, said method comprising administering to the patient a therapeutically effective amount of bone marrow-derived myeloid cells that have been activated with a cytokine selected from IL-4, IL- 13 or a narrow concentration range of IFN-γ of up to 20 ng/ml.

In one preferred embodiment, the method of the invention comprises administering to an individual in need IL-4 activated bone marrow-derived myeloid cells. In accordance with this embodiment, the CDl Ic⁺ cells may be

CDl lb⁺/CDl lc⁺ cells, particularly, CDl lb⁺/CDl lc⁺/MHC-lf microglia, a phenotype induced by IL-4.

In this context, it is interesting to note that IL-4 has often been described as an anti-inflammatory cytokine (Chao et al., 1993). However, our results herein strongly argue against this perception and show instead that IL-4 activates microglia to adopt a phenotype that seems to acquire a different morphology and a different activity from those of the innately activated microglia or of the activated microglia commonly seen in neurodegenerative diseases such as Alzheimer's disease (AD) or multiple sclerosis (MS). In MS, for example, unlike in AD, the microglia appear to be overwhelmed by an onslaught of adaptive immunity (Butovsky et al., 2006a). Interestingly, it seems that IL-4 is capable of restoring a favorable activated phenotype even after the microglia have already exhibited phenotypic characteristics of aggregated Aβ ((Butovsky et al, 2005 and shown herein) or been overwhelmed by IFN-γ (Butovsky et al., 2006a). In this regard, IL- 13 has the same effect as IL-4, because it is well established in the field of cytokines that IL-4 and IL- 13 can utilize a common receptor and share many actions such as B-cell activation and suppression of Th-I cells.

Cells in solid tissue experiencing distress due to an insult are capable of communicating with cells of the immune system to direct a beneficial inflammatory response to the site of the insult. One of the actors playing a central role in the molecular mechanism by which this is achieved is the intracellular adhesion molecule- 1 (ICAM-I). ICAM-I is expressed in both hematopoietic and non- hematopoietic cells and mediates adhesive interactions by binding to integrins belonging to the β2 subfamily i.e., CDl la/CD18 (LFA-I), CDl lb/CD18 (Mac-1), and CDl lc/CDlδ. ICAM-I adhesive interactions are critical for the transendothelial migration of leukocytes and the activation of T cells where ICAM- 1 binding functions as a co-activation signal. ICAM-I is present constitutively on the cell surface of a wide variety of cell types including fibroblasts, leukocytes, keratinocytes, endothelial cells, and epithelial cells, and is upregulated in response to a number of inflammatory mediators, including retinoic acid, virus infection, oxidant stresses such as H₂O₂, and the proinflammatory cytokines, IL- lβ, TNF-α, and IFN-γ. Thus, activated blood derived myeloid cells expressing ICAM-I receptors home to insulted tissue in which stressed cells have created an immunological niche manifested by ICAM- I expression.

It is thus clear to a person skilled in the art of immunology that the beneficial effect of IL-4 activated bone marrow-derived myeloid cells observed in neurodegenerative diseases according to the inventions applies also to any tissue of the body that expresses high levels of ICAM-I to signal its distress to the circulating activated cells.

The CDI l⁺ bone marrow-derived myeloid cells subject of the instant invention home to the damaged tissue due to the interaction between the CDl Ic expressed on their surface and ICAM- 1 expressed in cells in distressed tissue. Since ICAM-I is upregulated in most cell types in response to extracellular stress, any damaged body tissue can be repaired by the CDl I⁺ bone marrow-derived myeloid cells of the invention. Examples of these body tissues include, but are not limited to, neural tissue, cardiac tissue, liver tissue, renal tissue, bladder tissue, muscle tissue, intestinal tissue, or visual system tissue.

Injury to the CNS triggers the immediate death of injured neurons, and this is inevitably followed by a series of destructive processes, collectively termed secondary degeneration, which result in the gradually spreading degeneration and death of initially undamaged adjacent neural cells. The processes of secondary degeneration are mediated mainly by destructive self-compounds that emanate from the directly damaged neurons and render the extracellular environment hostile to recovery. CNS disorders and diseases are caused by damage to the CNS, which is exacerbated by secondary degeneration. Thus, CNS disorders and diseases are manifestations of damage inflicted on the CNS tissue, no matter what was the primary cause of the damage. In accordance with the present invention, the CDI l⁺ bone marrow- derived myeloid cells, for example, obtained by activation with a cytokine selected from IL- 4, IL- 13 or up to 20 ng/ml IFN-γ, infiltrate damaged brain tissue and thus can be used to repair brain tissue damage associated with a range of CNS diseases or disorders. Thus, in one preferred embodiment, the CDI l⁺ bone marrow-derived myeloid cells of the invention are used for treatment of neurodegenerative diseases or disorders including, but not limited to, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis (ALS), mental disorders, neuropathies, cognitive dysfunction, dementia, the aging process and senescence. In another preferred embodiment, the cells are used for treatment of injuries in the brain or in the spinal cord.

The methods of the invention are also useful for treatment of ischemia, particularly in tissues especially sensitive to inadequate blood supply such as the heart, the kidneys and the brain. Ischemia in brain tissue, for example due to a cerebrovascular accident such as stroke or head injury, may ultimately kill brain tissue leading to permanent neurologic damage or even death. Ischemia in the heart leads to myocardial infarction or heart attack.

The mental disorders that can be treated by the methods of the invention include psychiatric disorders selected from: (i) anxiety disorders, that include phobic disorders, obsessive-compulsive disorder, post-traumatic stress disorder (PTSD), acute stress disorder and generalized anxiety disorder; (ii) mood disorders, that include depression, dysthymic disorder, bipolar disorders and cyclothymic disorder; (iii) schizophrenia and related disorders such as brief psychotic disorder, schizophreniform disorder, schizoaffective disorder and delusional disorder; (iv) drug use and dependence such as alcoholism, opiate dependence, cocaine dependence, amphetamine dependence, hallucinogen dependence, and phencyclidine use; and (v) memory loss disorders such as amnesia or memory loss associated with Alzheimer's type dementia or with non- Alzheimer's type dementia, e.g. multi-infarct dementia or memory loss associated with Parkinson's disease, Huntington's disease, Creutzfeld- Jakob disease, head trauma, HIV infection, hypothyroidism and vitamin B 12 deficiency.

In another embodiment, the activated bone marrow-derived myeloid cells of the invention are used for treatment of cardiovascular diseases, particularly heart diseases such as myocardial infarction, ischemic heart disease and congestive heart failure (CHF), as known in cellular therapy for such diseases. Despite many recent advances in medical therapy and interventional techniques, these heart diseases remain the major causes of morbidity and mortality in the westen countries. Cellular therapy for treating these and other heart conditions is a growing field of clinical research. In another embodiment, the activated bone marrow-derived myeloid cells of the invention can be used for promoting tissue repair in a patient suffering from an autoimmune disease.

The cells for use in the present invention are preferably autologous, namely, they are obtained from peripheral blood or bone marrow of the individual to be treated.. When cells cannot be obtained from the individual, allogeneic cells from an HLA-matched donor can be used.

The cells are obtained from peripheral blood or bone marrow of the individual or donor and processed by techniques well known in the art. .

Once obtained, the myeloid cells may be cultured until they multiply to the level needed for transplantation into the patient and are then activated with at least one cytokine selected from IL-4, IL- 13 and up to 20 ng/ml IFN-γ for the time necessary to upregulate CDl Ic expression. For example, activation with up to 20 ng/ml IFN-γ may take 2-3 days until the peak of CDl Ic expression is reached.

Thus, in another embodiment, the invention relates to a process for the preparation of a cellular preparation comprising CDl Ic⁺ bone marrow-derived myeloid cells which comprises obtaining myeloid cells from the peripheral blood or from the bone marrow of an individual, and culturing the cells with at least one cytokine selected from IL-4, IL- 13 and up to 20 ng/ml IFN-γ for the time necessary to upregulate CD 1 1 c expression. Prior or after the culture with the cytokine, the cells can be purified by known techniques, for example using a magnetic bead system (e.g., from Miltenyi Biotec, Auburn, CA) and determining the purity. The purity of cell cultures is monitored by flow cytometry using monoclonal antibodies (mAbs) directed to human CDl Ic. The cells are labeled by challenging with a commercially available fluorochrome-conjugated mAb, and then washed with PBS. The fraction of cells positive for CDl Ic is regarded as a measure of culture purity. The parameter is assayed both before and after the incubation/activation stage. The purity of the cell culture should be >80%, preferably 90% , 97% or more CDl Ic⁺ cells.

In the examples, CDl Ic⁺ microglia cells are described. Microglia are immune cells of the CNS that are derived from myeloid progenitor cells, which come from the bone marrow. Thus, microglia are the resident CNS cells whereas the bone marrow-derived myeloid cells are the infiltrating cells. The resident microglia express ICAM-I during distress, and the bone marrow-derived infiltrating myeloid cellssexpress CDl Ic, which enables them to home to the immunological niche defined by the ICAM-I expressing cells.

In another aspect, the invention relates to a method for detecting and localizing a damaged tissue comprising administering bone marrow- derived myeloid cells that have been activated with at least one cytokine selected from IL-4, IL- 13 or up to 20 ng/ml IFN-γ and are labeled with an imaging agent to an individual having or suspected of having a damaged tissue, whereby the labeled cells traffic to the damaged tissue, and imaging the suspected tissue area in the individual, thereby detecting and localizing the damaged tissue.

The cells can be labeled with any agent that allows imaging by any of the imaging techniques. The cells can be detectably labeled with a contrast agent including, without limitation, metals such as gold particles, gadolinium complexes, etc. Alternatively, the cells can be labeled detectably with a radioisotope, including but not limited to ¹²⁵Iodine, ^IjlIodine, 99m-Technecium. The cells can also be detectably labeled using a fluorescence emitting metal such as ¹⁵²Eu, or others of the lanthanide series. One example of imaging agents are contrast agents suitable for magnetic resonance imaging (MRI) such as, but not limited to, diamagnetic agents useful in gastrointestinal imaging, paramagnetic agents such as ions of the metals Gd (preferably gadolinium chelates such as Gd-DTPA), Fe, Mg and Dy, or superparamagnetic and ferromagnetic agents. Other labeling agents are contrast agents for positron emission tomography (PET) or for functional MRI (fMRI).. Labeling of the cells with metal particles may be achieved by incubating cells in a suspension comprising the metal particles wherein the cells spontaneously internalize such particles into the cell's cytosol. Such substances may also be introduced into the cells by a variety of electroporetic techniques (Current Protocols in Immunology, 1997, Eds. Coligan et al., John Wiley & Sons, Inc., NIH). Fluorescence emitting metals or radioactive metals can be attached to the cells using such metal chelating agents as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Labeling of the cells with a radioisotope can be achieved by incubating cells with a radioactive metabolic precursor.

The presence of labeled, activated cells can be detected in the patient using methods known in the art for in-vivo scanning. These methods depend upon the type of label used and the skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include but are not limited to: computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), sonography, radiation responsive surgical instrument (Thurston et al., U.S. Patent 5,441,050), and fluorescence responsive scanning instrument.

In a further aspect, the present invention relates to a method for delivering a therapeutic or detectable substance to a damaged tissue or a tumor, said method comprising administering to a patient in need bone marrow-derived myeloid cells that have been activated with at least one cytokine selected from IL-4, IL- 13 or up to 20 ng/ml IFN-γ, wherein said cells are cells that have been genetically engineered to express said therapeutic or detectable substance.

The present invention is thus directed to methods for the treatment or diagnosis of damaged tissues or tumors by delivering a therapeutic or detectable substance to a damaged site or to the tumor, comprising administering an effective amount of bone marrow-derived myeloid cells that have been activated with at least one cytokine selected from IL-4, IL- 13 or up to 20 ng/ml IFN-γ, said cells expressing a therapeutic or detectable substance, to an individual in need wherein the amount is effective to detect, diagnose, or monitor a site of injury or disease or a tumor in the body or is effective to ameliorate the effects of an injury or disease or to treat the tumor.

In this aspect of the invention, compositions comprising the activated cells are used for delivery of (a) a diagnostic substance or (b) a therapeutic substance to a site of injury or disease of the body or to a tumor.

The cells may be genetically engineered in vitro to insert therein a nucleotide sequence encoding a polypeptide that can be used for terapy or diagnosis, by methods well known in the art. The nucleotide sequence is under the control of necessary elements for transcription and translation such that a biologically active protein encoded by the nucleotide sequence can be either expressed continuously or induced to expression as a result of exposure of the cells to a microenvironment of a kind present at the damaged site. Due to the inherent degeneracy of the genetic code, other nucleotide sequences that encode substantially the same or a functionally equivalent amino acid sequence of a protein, are within the scope of the invention. Preferably, the expression product of said nucleotide sequence is a secretory protein. The recombinant cells which contain a coding sequence and which express a biologically active gene product may be identified by at least four general approaches: (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expression of mRNA transcripts in the cell; and (d) detection of the product encoded by the nucleotide sequence as measured by immunoassay or by its biological activity.

In the first approach, the presence of the coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the coding sequence or portions or derivatives thereof. In the second approach, the recombinant expression system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.). For example, if the coding sequence is inserted within a marker gene sequence of a vector, recombinant cells containing the coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence under the control of the same or different promoter used to control the expression of the coding sequence. Expression of the marker in response to induction or selection indicates expression of the coding sequence. In the third approach, transcriptional activity of a nucleotide sequence can be assessed by hybridization assays. For example, RNA can be isolated and analyzed by Northern blot using a probe having sequence homology to a coding sequence or transcribed noncoding sequence or particular portions thereof. Alternatively, total nucleic acid of the host cell may be extracted and quantitatively assayed for hybridization to such probes. In the fourth approach, the levels of a protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like. The cells may be stably transfected with said nucleotide sequences or may be transiently transfected. Transient transfection may be applicable for acute one-dose therapeutic regimens.

The nucleotide sequences inserted into the cells may encode various substances including, without limitation, therapeutic substances; enzymes which catalyze a therapeutic substance; a regulatory product which stimulates expression of a therapeutic substance in the cells, etc. When used for delivering a therapeutic substance to a neural tissue, the nucleotides may be, for example: nucleotide sequences encoding neurotrophic factors such as NGF; nucleotide sequences encoding enzymes which play a role in CNS nerve regeneration such as the enzyme transglutaminase; nucleotide sequences encoding enzymes which catalyze the production of a neurotransmitter, e.g. enzymes involved in the catalysis of acetylcholine or dopamine, etc. As a result, the cells which localize at the site of CNS injury or disease produce and secrete the needed substances at the site. When used for treatment or diagnosis of a tumor, the nucleotide sequence will encode a polypeptide that can treat or detect/localize the tumor, respectively.

In another aspect, the present invention relates to a method for monitoring the response of a patient being treated for a neurodegenerative or autoimmune disease or disorder to a therapeutic drug for said disease or disorder, said method comprising: (a) determining the level of CDl Ic⁺ myeloid cells in a first sample of peripheral blood taken from the patient prior to treatment with the therapeutic drug; (b) determining the level of the CDl Ic⁺ myeloid cells in at least one blood sample taken from the patient subsequent to the initial treatment with the therapeutic drug; and c) comparing the level of the CDl Ic⁺ myeloid cells in the at least one blood sample of (b) with the level of the CDl Ic⁺ myeloid cells in the first blood sample of (a); wherein an increase in the level of the CDl Ic⁺ myeloid cells in the at least one blood sample of (b) compared to the level of the CDl Ic⁺ myeloid cells in the first blood sample of (a), indicates that the therapeutic drug is effective in treating said neurodegenerative or autoimmune disease or disorder in said patient.

In one embodiment, the method monitors a patient being treated for a neurodegenerative disease or disorder such as, but not limited to, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, age-related macular degeneration, neuropathies, mental disorders, cognitive dysfunction, dementia, and prion diseases. In one embodiment, the neurodegenerative disease is multiple sclerosis.

In another embodiment, the method monitors a patient being treated for an autoimmune disease or disorder such as, but not limited to, Eaton-Lambert syndrome, Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome, autoimmune hemolytic anemia (AIHA), hepatitis, insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), myasthenia gravis, plexus disorders e.g. acute brachial neuritis, polyglandular deficiency syndrome, primary biliary cirrhosis, rheumatoid arthritis, scleroderma, thrombocytopenia, thyroiditis e.g. Hashimoto's disease, Sjogren's syndrome, allergic purpura, psoriasis, mixed connective tissue disease, polymyositis, dermatomyositis, vasculitis, polyarteritis nodosa, polymyalgia rheumatica, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, pemphigus, bullous pemphigoid, dermatitis herpetiformis, inflammatory bowel disease (IBD; e.g. Crohn's disease) and uveitis.

According to this embodiment, the level of CDl Ic⁺ myeloid cells in samples of peripheral blood of individuals with symptoms consistent with neurodegenerative or autoimmune diseases and suspected of having a neurodegenerative or autoimmune disease, is measured and compared with the "normal" level of these cells as established in sample(s) from one or more individuals not having neurodegenerative or autoimmune diseases. Low levels of the CDl Ic⁺ myeloid cells in a sample from an individual with suspected neurodegenerative or autoimmune disease relative to the "normal" level is indicative of said individual having a neurodegenerative or autoimmune disease, while similar levels relative to the "normal" level indicate an absence of such a disease.

The invention further relates to a method for monitoring transition between periods of remission and relapse in relapsing/remitting multiple sclerosis in a patient, comprising:

(a) determining the level of CD 1 1 C⁺ myeloid cells in a first sample of peripheral blood taken from the patient in a period of remission; (b) determining the level of the CDl Ic⁺ myeloid cells in a plurality of blood samples taken from the patient periodically ; and

(c) comparing the level of the CDl Ic⁺ myeloid cells in the blood sample of (b) taken most recently with the level of the CDl Ic⁺ myeloid cells in a previous blood sample of (a) or (b); wherein a decrease in the level of the CDl Ic⁺ myeloid cells in the most recent blood sample compared to the level of the CDl lc+ myeloid cells in the previous blood sample, indicates a transition from a period of remission to a period of relapse.

The method above will permit treatment of the patient close to the period of relapse in an attempt to revert the situation and bring the patient again to the remission period.

The invention further relates to a method for the diagnosis and follow up of a neurodegenerative or autoimmune disease or disorder in an individual, comprising:

(a) determining the level Of CDl Ic⁺ myeloid cells in a sample of peripheral blood taken from an individual suspected of having a neurodegenerative or autoimmune disease;

(b) determining the level Of CDl Ic⁺ myeloid cells in a sample of peripheral blood taken from at least one individual not suffering from a neurodegenerative or autoimmune disease or disorder and; (c) comparing the level of the CDl Ic⁺ myeloid cells in the blood sample taken from said individual in (a) with the level of CDl Ic+ myeloid cells in the blood sample taken from said at least one individual of (b); wherein a lower level of CDl Ic+ myeloid cells in the blood sample of the individual of (a) relative to the level Of CDl Ic⁺ myeloid cells in the blood sample of said at least one individual of (b), is indicative of a neurodegenerative or autoimmune disease or disorder in the individual of (a).

In a further aspect, the present invention provides a cellular preparation comprising CDl Ic⁺ bone marrow-derived myeloid cells and a physiologically acceptable carrier, for promoting repair of damaged body tissue. The cellular preparation comprises CDl Ic⁺ bone marrow-derived myeloid cells that express IGF-I, BDNF or both. In one more preferred embodiment, the CDl Ic⁺ bone marrow- derived myeloid cells express IGF-I.

The CDl Ic⁺ bone marrow-derived myeloid cells are obtained by activation of bone marrow-derived myeloid cells with at least one cytokine selected from the froup consisting of IL-4, IL- 13 and up to 20 ng/ml IFN-γ, more preferably IL-4 or a a mixture of IL-4 and up to 20 ng/ml IFN-γ.

The cellular therapy composition comprises the cells suspended in a physiologically/pharmaceutically acceptable carrier such as PBS or, preferably, in a culture medium such as IMDM or any other suitable cell culture medium such as

RPMI 1640, but other suitable pharmaceutically acceptable carriers will readily be apparent to those skilled in the art.

The activated cells of the invention can be administered by any suitable route used in cellular therapy, for example, systemic infusion, local arterial infusion, venous infusion or they can be administered in situ by direct injection into the damaged tissue, e.g. at the infarct site or at or near a site of injury of the CNS. In one preferred embodiment, the cellular preparation is administered intravenously.

In another embodiment, the invention provides a cellular preparation for detection and localization of damaged body tissue, comprising bone marrow- derived myeloid cells that have been activated with at least one cytokine selected from the group consisting of IL-4, IL- 13 and up to 20 ng/ml IFN-γ and labeled with an imaging agent.

Reference is made to the following patents and patent applications of the applicant, the contents of all these patents and patent applications being hereby incorporated by reference as if fully disclosed herein: WO 99/34827, WO 99/60021,

US 6,844,314, US Patent Application No. 10/810,653, WO 2005/046719, WO

2005/055920, WO 2006/056998.

The invention will now be illustrated by the following non-limiting Examples presented in Sections I to V, each section including a short introduction, materials and methods, examples with reference to the figures and, when suitable, a short discussion.

In Sections I and II, Copolymer 1, which commercial form is also known as glatiramer acetate, is used in the examples. The terms "Cop 1", "Copolymer 1", "glatiramer acetate" and "GA" are used interchangeably in the examples.

SECTION I

Dendritic-like bone marrow-derived myeloid cells activated by IL-4 inhibit clinical manifestation in an animal model of chronic multiple sclerosis and induce oligodendrogenesis

Introduction

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease associated with demyelination and neuronal loss. Studies have shown that inflammation within the CNS blocks neurogenesis (Monje et al., 2003; Ekdahl et al., 2003) and causes structural damage to myelin (Hartung et al., 1992; Olsson, 1995). Moreover, in vitro conditions revealed that inflammation-associated microglia impeded both neurogenesis and oligodendrogenesis from adult stem cells (Butovsky et al., 2006a, 2006b, 2006c). More recent studies have shown, however, that although an uncontrolled local immune response indeed impairs survival of neurons (Chao et al., 1992) and oligodendrocytes (Merrill et al., 1993) and interferes with repair processes (Stirling et al., 2004), a local immune response that is properly controlled can support survival and promote remyelination (Bieber et al., 2003) and recovery (Schwartz et al., 2003; Hauben & Schwartz, 2003). These results are in fact an extension of our pioneering work demonstrating that a local immune response that is well controlled in time, space, and intensity by peripheral adaptive immune processes (mediated by CD4⁺ T-helper cells directed against autoantigens residing at the site of the lesion) is a critical requirement for posttraumatic neuronal survival and repair (Butovsky et al., 2006a, 2005, 2001; Schwartz et al., 2003; Moalem et al., 1999; Shaked et al., 2004). These and other results created the basis for the concept of 'protective autoimmunity¹ (Moalem et al., 1999), formulated by our group. According to this concept both ThI and Th2 cells recognizing CNS antigens are needed for CNS maintenance and repair. The need for a specific subtype, its intensity and duration are determined by the type of the damaged conditions (acute or chronic), and the amount of time transpired following the insult in the case of acute conditions. Moreover, whether or not the T cells are beneficial is determined by their dose. One of the main targets of the T-cell effect was found to be the microglia (Butovsky et al., 2006a, 2006b, 2006c, 2005; Shaked et al., 2005). Thus for example, microglia exposed to low concentrations of IFN-γ exhibit an immune-mediated healing response, microglia exposed to high IFN-γ concentrations are associated with an immune-mediated demyelinating disease (Hartung et al., 1992; Olsson, 1995; Butovsky et al., 2006a), and microglia exposed to IL-4, over a wide range of concentrations, can support neuronal survival (Butovsky et al, 2005). Moreover, IL- 4, via modulation of microglia both in vitro and in vivo, can overcome the destructive effects of high-dose IFN-γ. In vitro, a high dose of IFN-γ, but not a low dose, impairs the ability of microglia to support oligodendrogenesis from adult neural stem cells/progenitor cells (NPCs), and IL-4 counteracts the interference with oligodendrogenesis. When IL-4-activated microglia were stereotaxically injected through the cerebral ventricles into the cerebrospinal fluid (CSF) of rats with acute experimental autoimmune encephalomyelitis (EAE) or of mice with a remitting-relapsing autoimmune disease, the animals demonstrated significantly more oligodendrogenesis and significantly less neurological deficit than did their vehicle-injected diseased controls (Butovsky et al., 2006a). Here we examined whether therapeutic immunomodulation with glatiramer acetate (GA), known to be beneficial in EAE and MS, induces oligodendrogenesis in a mouse model of EAE. If so, whether this effect involves locally activated microglia with activity reminiscence of IL-4-activated microglia. Activated microglia under pathological conditions can be either resident microglia, or bone marrow-derived cells that are attracted to the CNS (Bechmann et al., 2005, Simard et al., 2006). We therefore examined whether systemic injection of IL-4-activated bone marrow-derived myeloid cells would be as effective as immunomodulation therapy in EAE. The correlation between disease conditions and blood levels of IL- 4-activated bone marrow cells in the mouse model encouraged us to investigate whether such a correlation exists in MS patients.

We have recently demonstrated that IL-4 can reverse the destructive effect of overwhelmed activated microglia (MG), known to be associated with MS. Here we show that therapeutic immunomodulation with GA, known to be beneficial in MS, induces oligodendrogenesis in EAE mice. We further demonstrate that this effect involves dendritic-like MG (CDl Ic reminiscence of IL-4-activated MG). Knowing that activated MG under pathological conditions can be resident MG or bone marrow- derived cells we further showed that systemic injection of IL-4 activated bone marrow-derived myeloid cells expressing IGF-I and BDNF diminished the disease development in the mice. Using chimeric mice, in which the bone marrow were replaced with bone marrow derived from transgenic mice expressing diphtheria toxin receptor under CDl Ic promoter, showed that depletion of CDl lc+ of the donor origin by the toxin led to either disease inhibition or was lethal, if given before onset or after onset, respectively. The correlation between disease conditions and blood levels of dendritic-like bone-marrow cells was also found in MS patients.

EXAMPLES Materials and Methods

(i) Animals. Neonatal (PO-Pl) C57B1/6J mice, inbred adult male C57BL/6J mice (8-10 weeks) and heterozygous mutant mouse strain in which the CX₃CRI chemokine receptor gene is replaced with a green fluorescent protein gene (GFP) C57Bl/6-CX₃CRl-GFP (CX₃CRlP^⁺) knock-in mice (Jung et al., 2000) and Diphtheria toxin receptor (DTR) under CDl Ic promoter (CX₃CRl P^FP/Λ /CDIl c^DTR). Mice were supplied by the Animal Breeding Center of The Weizmann Institute of Science (Rehovot, Israel) and were handled according to the regulations formulated by the Weizmann Institute's Animal Care and Use Committee.

(U) Induction of chronic EAE. Induction of chronic EAE in mice was performed, as previously described (Butovsky et al., 2006a). (Ui) Administration of 5-bromo-2'-deoxyuridine and tissue preparation.

The cell-proliferation marker BrdU was dissolved by sonication in PBS and injected i.p. (50 mg/kg body weight) every 12 hours for 2.5 days, starting on day 19 after MOG vaccination in adult male C57BL/6J mice. One week after the last BrdU injection, the animals were deeply anesthetized and perfused transcardially, first with PBS and then with 4% paraformaldehyde. Their spinal cords were removed, postfixed overnight, and then equilibrated in phosphate-buffered 30% sucrose. Free- floating 30-μm longitudinal sections were collected on a freezing microtome (Leica SM2000R) and stored at 4°C prior to immunohistochemistry.

(iv) Immunohistochemistry. For immunohistochemistry, longitudinal sections of the spinal cord or coronal sections of the brain (30-μm) were treated with a permeabilization/blocking solution containing 10% FCS, 2% bovine serum albumin, 1% glycine, and 0.05% Triton X- 100 (Sigma- Aldrich). Primary antibodies were applied for 1 hour in a humidified chamber at room temperature. For BrdU staining, sections were washed with PBS and incubated in 2N HCl at 37⁰C for 30 minutes. Sections were blocked for 1 hour with blocking solution. The tissue was then stained with rat anti-BrdU (1 :200; Oxford Biotechnology) in combination with rabbit anti-NG2 (1 :300) and mouse anti-RIP (1 : 1000) antibodies diluted in PBS containing 0.05% Triton XlOO, 0.1% Tween 20, and 2% horse serum. For labeling of microglia we used IB4 (1 :50). To detect expression of cell-surface MHC-II proteins we used mouse anti MHC-II Abs (1 :50; IQ Products). Expression of IGF-I was detected by goat anti IGF-I Abs (1 : 10-1 : 100; R&D Systems). Sections were incubated with the primary antibody for 24 hours at 4°C, washed with PBS, and incubated with the secondary antibodies in PBS for 1 hour at room temperature while protected from light. Secondary antibodies used for both immunocytochemistry and immunohistochemistry were Cy-3 -conjugated donkey anti-mouse, Cy-3 -conjugated goat anti-rabbit, Cy-5-conjugated goat anti-rat, Cy-2- conjugated goat anti-rat, and Cy-5-conjugated donkey anti-goat. All antibodies were purchased from Jackson ImmunoResearch Laboratories and used at a dilution of 1 :250-500. Control sections (not treated with primary antibody) were used to distinguish specific staining from staining of nonspecific antibodies or autofluorescent components. Sections were then washed with PBS and coverslipped in polyvinyl alcohol with diazabicylo-octane as anti-fading agent.

(v) Quantification and stereological counting procedure. Oligo- dendrogenesis and proliferation of microglia in the spinal cord were evaluated by counting of cells that were double- or triple-labeled with BrdU, and markers of premature oligodendrocytes (NG2), microglia (IB4), antigen-presenting cells (MHC-II), dendritic marker (CDl Ic) or a pre-ensheathing marker of oligodendrocytes (RIP), from sagittal longitudinal sections at segment T8-T9 of the spinal cord. The numbers of cells per mm^J were counted at 300-μm intervals in gray and white matter in each mice (n = 4-7 per group). Specificity of BrdU⁺/NG2⁺ or BrdU⁺/RIP⁺ or BrdU⁺/IB4⁺ or IB4⁺/MHC-II⁺/CDl lc⁺ co-expression was assayed using the confocal microscope (LSM 510) in optical sections at 1-μm intervals. Counting was evaluated automatically using Image-Pro Plus 4.5 software (Media Cybernetics). (vi) Statistical analysis. The in-vitro results were analyzed by the

Tukey-Kramer multiple comparisons test (ANOVA) or Student's r-test and are expressed as means ± SD. In-vivo results were analyzed by Student's Mest or oneway ANOVA and are expressed as means ± SEM. Significance of the EAE score was analyzed by Mann- Whitney test, two-factor repeated measures (ANOVA). (vi)

Example 1. GA induces oligodendrogenesis from endogenous neural stem/progenitor cells in a chronic EAE model

Overwhelming ThI cell-mediated autoimmunity leads to chronic conditions of demyelization, known as autoimmune encephalomyelitis (Hartung et al., 1992; Olsson, 1992). We recently demonstrated that IL-4-activated MG, injected into the CSF of animals with chronic EAE, promoted oligodendrogenesis from endogenous adult NPCs (Butovsky et al., 2006a). GA is an immunomodulator FDA-approved for treatment MS. We therefore examined whether GA treatment of animals, in which EAE was induced, would promote oligodendrogenesis as well. EAE was induced in C57BL/6J mice by immunization with the encephalitogenic MOG peptide (35-55) emulsified in IFA containing Mycobacterium tuberculosis and pertussis toxin (Butovsky et al., 2006a). The animals were separated into two groups, one group received GA in addition to MOG (n = 5) and another group of MOG- vaccinated mice was treated with PBS (n = 5). Additional untreated (naϊve) group was used as a control (n = 4). Eighteen days after MOG-vaccination, all mice were injected intraperitoneally (ip) with BrdU every 12 hours for 2.5 days to identify proliferating cells, and 7 days after the last BrdU injection their spinal cords were examined for the appearance of newly formed oligodendrocytes (Fig. 1). The two groups of MOG- vaccinated mice differed significantly in the numbers of newly formed microglia and oligodendrocytes as compared to naϊve mice. In both grey and white matter EAE was associated with increased BrdU⁺ and BrdlT7lB4⁺ (proliferating microglia) cells which was further increased by the treatment with GA (Fig. IA). In the white matter (but not in the gray matter) of mice with EAE, significantly more newly formed oligodendrocytes (BrdU⁺/NG2⁺/RIP⁺) were observed in the group treated with GA than in the non-treated group (Fig. IB). The results revealed that in non-treated animals in which EAE was induced, similarly to naϊve animals, hardly any oligodendrogenesis could be seen (Fig. 1C). With the aid of scanning confocal microscopy, co-expression of RIP by the newly formed oligodendrocytes was confirmed in the white matter of the GA-treated mice with chronic EAE (Fig. ID). It is noteworthy that some of the newly formed microglia expressed NG2 (Fig. IE). Furthermore, some of the newly oligodendrocytes were found at the margins of the central canal of the GA-treated mice with chronic EAE (Fig. IF). Example 2. Oligodendrogenesis spatially associated with GA-induced CDlIc⁺ microglia

We have recently demonstrated that IL-4- activated microglia express a dendritic-like phenotype characterized by CDl Ic expression (Butovsky et al, 2006c). We therefore examined if CDl Ic⁺ microglia are found in the chronic model of EAE, and if not, whether GA would cause a phenotype change. We analyzed sections from the same spinal cords represented in Fig. 1 for the appearance of microglia expressing MHC-II and CDl Ic. We found abundant microglia expressing both MHC-II and CDl Ic in the GA-treated EAE mice, whereas EAE mice that were not immunized with GA hardly express and CDl Ic (Figs. 2 A, 2B). In naϊve mice no MHC-II or CDl Ic expression on parenchymal MG was detected (Fig. 2B)

Example 3. Bone marrow-derived CDlIc⁺ cells are crucial for EAE development, but their depletion after the onset of the disease has detrimental effect

The CDl Ic found in the spinal cord of GA-immunized animals could be either resident MG that were activated locally by T cell-derived cytokines or recruited bone marrow-derived myeloid cells that were activated peripherally by T cell-derived cytokines. It has been suggested that MG are replenished partly by division of resident cells and partly by immigration of circulating monocytes (Lawson et al., 1992). Moreover, in lethally irradiated mice transplanted with BM cells expressing GFP, the cells immigrated into the brain parenchyma of many regions of the CNS. Nearly all of the infiltrating cells had a highly ramified morphology and colocalized with the MG marker co-expressing CDl Ic (Simard & Rivest, 2004). In addition, in an animal model of Alzheimer's disease, bone marrow-derived microglia are very efficient in restricting amyloid deposits (Simard et al., 2006). On contrary, CDl Ic⁺ perivascular dendritic cells and not parenchymal MG bearing MHC-II are sufficient to present antigen in vivo to primed myelin- reactive T cells in order to induce EAE (Greter et al., 2005). Based on our previous results that IL-4 activated MG expressing CDl Ic injected into the CSF attenuated EAE, we anticipated that in EAE CDl Ic⁺ cells derived from BM recruited to the site of damage needed to fight off the adverse conditions. To detect whether CDl Ic⁺ cells are parenchymal MG or BM-derived cells and contribute to the disease progression, we created chimera mice with BM cells derived from double transgenic mouse model CX₃CR 1^GFP /CD Uc^DTR that express GFP under the promoter of the chemokine fractalkine receptor CX₃CRl (Jung et al., 2000) and DTR (diphtheria toxin receptor) under CDl Ic promoter (Jung et al., 2002). Consequently, heterozygous mice (CX₃CR1^GFP+) express both the DTR and GFP on peripheral monocytes and on a subset of mononuclear phagocytes that include macrophages and dendritic cells (Davalos et al;, 2005, Geissmann et al., 2003). We therefore examined whether ablation of CD l lc-expressing cells derived from bone marrow both before and during the onset of the disease has an impact on clinical disease development. EAE was induced in the chimera mice by immunization with MOG, as described above. The animals were separated into six groups: 1) immunized with MOG (MOG; n = 6), 2) immunized with MOG and injected ip with diphtheria toxin (DT; 250 ng/mice) starting on day 12 after MOG- vaccination. In addition, DT was given with intervals of 3 and 4 days thereafter (MOG/DT; n = 5); 3) immunized with MOG and GA (MOG+GA; n = 4); 4) immunized with MOG+GA and injected ip with DT starting on day 5 after MOG- vaccination and given with intervals of 3 and 4 days thereafter (MOG+GA /DT - before onset of EAE; n = 5); 5) vaccinated with MOG+GA and injected ip with DT starting on day 12 after MOG-vaccination and given with intervals of 3 and 4 days thereafter (MOG+GA /DT - after onset of EAE; n = 5); 6) additional non-vaccinated group injected with DT in parallel to MOG-vaccinated mice was used as a control (n = 6). DT injected 5 days in MOG-vaccinated mice before the onset of the disease and thereafter almost completely prevents the disease manifestation. However, late injection of DT, starting on day 12 and thereafter, in both MOG- and MOG+GA- vaccinated group, significantly exacerbate clinical score as compared to MOG- and MOG+GA-vaccinated group without DT-treatment (Fig. 3). Example 4. IL-4 renders both microglia and bone marrow-derived myeloid cells a dendritic-like phenotype which produces IGF-I and BDNF

To determine the role of cytokines associated with induction of CDl Ic, we analyzed the correlation between bone marrow-derived myeloid cells and brain derived microglia with three key cytokines: IFN-γ, ThI -derived; IL-4, Th2 derived, and IL-IO, known to be associated with regulatory T cells. Both microglia and bone marrow-derived myeloid cells were treated for 5 days with IL-4 (10 ng/ml), IFN-γ (100 ng/ml) or IL-IO (10 ng/ml), or combinations of IL-4 with IFN-γ or IL-10. Low dose of IFN-γ (10ng/ml) induced CDl Ic with a peak intensity at 2-3 days (data not shown). While overwhelming levels of IFN-γ are associated with MS, IL-4 and IL- 10 are associated with the GA treatment (Aharoni et al., 1997; Miller et al., 1998; Neuhaus et al., 2001 ; Jung et al., 2004). We found that high dose of IFN-γ as well as IL- 10 impaired microglial CDl Ic expression whereas IL-4 was able to induce CDl Ic and to overcome the blocking effect of both IFN-γ and IL-10 (Fig. 4A) The same phenotype, which is induced by IL-4 activation, could counteract the disease progression and induce oligodendrogenesis from endogenous NPC cells (Butovsky et al., 2006a). Repetition of the experiments replacing the brain derived microglia with bone marrow-derived myeloid cells revealed similar results (Figs. 4C-D). Quantitative analaysis is shown in Figs. 3d and 3e, respectively. Importantly, the inhibitory effect of IL-10 on CDl Ic expression was found to be dose dependent; IL-4 could barely overcome the inhibitory effect of 100 ng of IL-10 (data not shown).

The above results encouraged us to consider the use of IL-4-activated bone marrow-derived myeloid cells (BM_(1L.₄₎) for the treatment of EAE rather than vaccination with GA. Therefore we further analyzed the bone marrow-derived cells with respect to their ability to produce growth factors. The same bone marrow derived cells described above were analyzed by immunocytochemistry for their expression of IGF-I and BDNF. Immunocytochemical analysis revealed that IL-4 induced expression of both IGF-I and BDNF. IFN-γ at high dose (100 ng/ml) did not induce expression of these two growth factors; however the addition of IL-4 restored the profile of growth factors induced by IL-4 alone. IL-IO at low dose did not induce IGF-I and induced only a low level of BDNF, yet the combination of both IL-4 and of IL- 10 at a low level resulted in a synergistic increase with respect of BDNF expression (Fig. 5).

Example 5. ICAM-I expression

Reports indicate that CDl Ic may play a role as an adhesion molecule which binds to receptors on stimulated epithelium. It associates with CDl 8 to form CDl lc/CD18 complex and binds to ICAM- I (Stacker & Springer, 1991; Frick et al., 2005). Increased expression of ICAM-I and CDl Ib correlated with the disease progression in an animal model of ALS (Alexianu et al., 2001) and AD (Apelt et al., 2002). ICAM-I upregulation in the spinal cords of mice with EAE is dependent upon TNF-α production (Scott et al., 2004). Microglia activated with IFN-γ produce TNF-α in dose dependent manner and have a detrimental effect on neural cell survival (Butovsky et al, 2005) and renewal (Butovsky et al., 2006a). We hypothesized that the immunization-induced increase of microglia co-expressing CDl Ib and ICAM-I in the MOG-vaccinated mice might be attributable to an effect of IFN-γ. Staining of 5-day microglial culture with the ICAM-I marker showed that ICAM-I was abundantly expressed by microglia activated by IFN-γ. Both IL-4 and IL-10, the cytokine of Th2 cells induced by GA-vaccination (Duda et al., 2000, Vieira et al., 2003), were unable to induce ICAM-I . Moreover, IL-10 (10 ng/ml), unlike IL-4, significantly reduced IFN-γ-induced ICAM-I expression (Figs. 6A, 6B). Importantly, IL- 10 at the concentration of 100 ng/ml completely blocked ICAM-I when added together with IFN-γ (10 ng/ml; data not shown). We also examined the possibility that GA directly induced either microglia or bone marrow- derived cells to express ICAM- 1. No effect was found with GA at concentrations of 10 ng/ml, 100 ng/ml, or 1 μg/ml after 3 or 5 days in culture (data not shown). Example 6. IL-4-activated bone marrow-derived myeloid cells prevent EAE induction and induce oligodendrogenesis

The features of BM_(IL-4) described above encouraged us to test their efficacy in vivo using a model of chronic EAE. We addressed the following issues: (i) Can BM_(JL-4) injected systemically bebefit EAE? (ii) If so, do they migrate to the CNS? And (III) Is the benefit due to their migration to the CNS? To achieve these goals, we used bone marrow-derived myeloid cells from C57BL/6J double transgenic mouse model CX₃CR l^/CDl lc⁰™ EAE was induced by MOG in all the experimental animals. One goups was left untreated (MOG; n = 6), two groups received IL-4-activated BM (BM₍[_L-4); n = 7), one group received inactivated BM (BM₍.₎; n = 7), and one of the BM₍i_L-4)-treated grups as well as a non-treated group (n=7) received diphtheria toxin (DT). Bone marrow cells (IxIO⁶) were injected each 6^th day, starting on day nine, a day before the disease onset. BM_(IL-4) almost completely blocked the disease manifestation, whereas BM₍.₎ exacerbated the severity of the disease relative to EAE mice without treatment. Since IL-4 induces CDl Ic expression on BM-derived myeloid cells (Fig. 4B), addition of DT (5 ng/ml) resulted in a complete ablation of the cell derived from the bone marrow of transgenic mice in vitro (data not shown). To verify the beneficial effect of BM_(IL-4), we injected DT (250 ng/mice) into the EAE mice which were treated with BM₍^_-4). DT was injected intraperitoneal^ concurrently with the BM, and thereafter injections were given at intervals of three days. As a control for the effect of the toxin alone, we injected DT in EAE mice without any treatment. The beneficial effect Of BM_(IL-4) was completely wiped out by the DT (Fig. 7).

The beneficial effect of increasing blood levels of IL-4-activated myeloid cells prompted us to continue in two directions, the first to find the site of action and secondly, to assess whether a similar situation exists in MS patients. Example 7. A correlation between blood levels of CDlIc and MS manifestation in patients

The animal studies described above encouraged us to analyze blood levels of myeloid cells (Lin^*) expressing CDl Ic in patients suffering from MS, during remission and relapse, with and without GA treatment. We observed that the levels of myeloid cells expressing CDl Ic (Lin7CDl lc⁺) in MS patients analyzed during remission were significantly lower than in normal healthy individuals and further decreased by 45% during relapse. Treatment with GA caused a significant elevation of CDl Ic when compared to untreated patients in remission and relapse level in GA patients was lower than their remission levels by 30% (Figs. 8A, 8B).

Discussion

The results above lead us to attribute a novel role to IL-4-activated bone marrow-derived myeloid cells in counteracting inflammation-associated neurodegenerative disease. Specifically, we showed that injection of GA, known for its ability to wipe out clinical symptoms of chronic EAE, caused an increase in the numbers of newly formed oligodendrocytes and increased expression of dendritic- like microglia. Moreover, we showed that systemic injection of IL-4-activated bone marrow-derived myeloid cells can replace the vaccination. MS is an inflammatory disease initially viewed as demyelinating disease and lately also associated with a loss of neurons. The disease has been associated with overwhelming ThI cells directed to brain-myelin antigens. Diminution of the disease symptoms has been attributed to several immune-related factors including immunomodulation by GA, IL-4 treatment, T-cell phenotype switch or treatments that block T-cell infiltration.

We have recently demonstrated that IL-4-activated microglia promote oligodendrogenesis in close association with MHC-II expressing microglia. IGF-I that is produced by IL-4-activated microglia is responsible, at least in part, for the increased oligodendrogenesis (Butovsky et al., 2006a). Previous studies of rats and mice with EAE showed that the inflammatory response not only induces proliferation and mobilization of endogenous progenitors (Picard-Riera et al., 2002) but also attracts exogenously delivered adult NPCs (Pluchino et al., 2003), implying that autoimmune brain inflammation, even in the presence of a large proportion of ThI cells and hence of abundant IFN-γ, leads to conditions that attract stem cells. GA is a week agonist of a wide range of CNS antigens among which are myelin proteins. The beneficial effect of GA in EAE or MS has been attributed to several mechanisms none of which however have been connected to the local microglia. Here we show that microglia, as a result of the GA treatment, express a phenotype that is characteristic of dendritic cells. Recent studies have shown that under damage CNS parenchyma can be populated by bone marrow cells. An independent study showed that GA can regulate DC cells. Taken together, it is possible that one way EAE or MS patients benefit from GA is by changing BM- derived myeloid cells which in turn reach the brain parenchyma. It was shown that GA reduces lymphocytes proliferation in MS patients by modulating monocyte- derived dendritic cells (Sanna et al., 2006).

It is important to note that microglia are originated from the bone marrow (Simard & Rivest, 2004). Under pathological conditions bone marrow-derived microglia can benefit the diseased brain in the case of AD (Simard et al., 2006). Therefore the GA-induced dendritic-like microglia found in the brain in the present work could be a result of a local effect of GA-activated T cells that homed to the EAE brain and via their secreted cytokine activated microglia to become dendritic- like cells. Alternatively, the GA treatment could cause enhanced recruitment of bone marrow-derived microglia expressing dendritic-like phenotype. The systemic rise in the DC-activated bone marrow-derived cells could be a result of cytokine or direct GA activation (Sanna et al., 2006; Weber et al., 2004; Kim et al., 2004). Which ever is the mechanism, the present results suggest that the diminished plaques seen in MS patients treated with GA could be an outcome, at least in part of oligodendrocyte renewal.

The treatment with systemically injected IL-4-activated BM indicates that these cells either peripherally or locally participate in disease arresting. The results seen in patients are in apparent correlation. We found low levels of CDl lcTLin^" cells in MS patients and an increase in GA treated patients. Interestingly, in all patients treated or untreated a general trend of lower levels of CDl Ic in relapsing than remission was found. This tendency, taken with the effect of GA on patients and the systemic effect of IL-4-activated BM can be interpreted to propose that IL- 4-activated BM could serve as an immunomodulation therapy for MS and perhaps other neurodegenerative diseases. BM activated by other cytokines seemed to have a different effect. Thus while IFN-γ at low levels could transiently induce CDl Ic (Butovsky et al, 2006c), at high levels it impeded CDl Ic expression. IL-IO commonly viewed as an anti-inflammatory cytokine completely shut off MHC-II and CDl Ic expression. IL-4-activated BM can deliver both BDNF and IGF-I. IL- 10-activated BM can deliver BDNF but not IGF-I. BM activated by the two cytokines can deliver both growth factors. It is thus suggestive that vaccination therapy with GA or any other antigen should be carefully designed when the choice of the regimen is made. Different regimen may differently affect the cytokines and the resulted systemic DC and the resident microglia.

When untreated BM derived cells were injected to the mice that had been immunized with the encephalitogenic MOG peptide not only that no beneficial effect was seen, but even a worsening effect on disease manifestation was observed, further supporting the notion that the phenotype critically affects the outcome. Moreover, it suggests that in the absence of intervention, due to the nature of the induced disease, the myeloid cells by default acquire a negative phenotype. Interestingly, elimination of DT-sensitive myeloid cells in the chimeric mice in which all bone marrow-derived dendritic cells are expressing DTR, revealed that the timing of their elimination relative to disease onset critically determine the outcome. Thus, while early elimination before disease onset prevented the disease manifestation, late elimination exacerbated disease conditions. These results seemed to convey two messages: the dual action of dendritic like cells, and that two populations of dendritic-like cells are involved in the disease development and arrest, both of which are bone marrow-derived. Interestingly, in both induction and arrest of the disease the involved cells are not the resident parenchymal cells but the perivascular, as was previously proposed. It is very likely that the population involved in disease induction is ThI -activated myeloid cells. The ones involved in the disease resolution are the IL-4-activated myeloid cells as our data revealed in both chimeric mice and the ones treated with IL-4 activated bone marrow-derived myeloid cells.

Another important issue which emerged from our studies is that the bone marrow-derived myeloid cells replenish the CNS only under pathological but hardly under physiological conditions. When we created chimeric mice following a whole- body irradiation and then reconstituted the bone marrow with a normal population of bone marrow cells expressing GFP the brain parenchyma was populated with GFP+ cells. However, when the chimeric mice were created by irradiating the body with a brain shield, and thus avoiding any irradiation-induced damage in the brain, no GFP+ cells were seen in the brain. It thus appeared that homing of blood-derived myeloid cells is mainly a result of distress signals creating immunological niche, manifested by ICAM- 1 expression.

Taken together, the present study proposes that active vaccination by weak agonist of CNS antigen via bone marrow-derived myeloid cells, or by direct injection of IL-4-activated myeloid cells, it is possible to arrest uncontrolled inflammatory conditions in the CNS. Also emerging from the present study is that the phenotype of the myeloid cells homing to the brain critically determine disease resolution. This may be applicable to other CNS disease conditions, even noninflammatory; when distress signals are expressed and a local immune niche is created, systemic supply of IL-4-activated BM cells may be a way to arrest disease conditions. SECTION II

Switching of microglia to dendritic-like expressing IGF-I helps the brain to resist Alzheomer's disease

Alzheimer's disease (AD) is an age-related progressive neurodegenerative disorder characterized by memory loss and severe cognitive decline (Hardy & Selkoe, 2002). The clinical features are manifested morphologically by excessive accumulation of extracellular aggregations of amyloid β-peptide (Aβ) in the form of amyloid plaques in the brain parenchyma, particularly in the hippocampus and cerebral cortex, leading to neuronal loss (Selkoe, 1991). In addition, in most mouse models of AD the neurogenesis that normally occurs throughout life in the hippocampus of the adult brain (Eriksson et al., 1998) is disrupted (Haughey et al., 2002). In Alzheimer patients, like in transgenic (PDGF-APPSw, Ind) mice, some increase in neurogenesis takes place but is apparently not sufficient to overcome the disease (Jin et al., 2004a, Jin et al., 2004b). The primary cause of AD remains unknown (Akiyama et al., 2000).

However, a key role in its progression has been attributed to inflammatory processes (Akiyama et al., 2000), particularly those carried out by activated microglia (CDl Ib⁺), representing the innate arm of the immune system in the central nervous system (CNS) and known to be associated with brain senescence (Streit, 2004). Studies from our laboratory over the last few years have shown that recovery from CNS injury is critically dependent on the well-controlled activity of T cells directed to specific CNS autoantigens (Moalem et al., 1999, Kipnis et al., 2002, 55). After homing to the site of damage, these autoreactive T cells evidently regulate microglia in a way that renders them supportive of neuronal survival and neural tissue repair (Butovsky et al., 2001 , 2005). Recent research has established that the microglia do not constitute a single or uniform cell population, but rather a family of cells with diverse phenotypes, some of which have beneficial effects and others that the CNS can barely tolerate and are therefore destructive (Shaked et al., 2004; Butovsky et al, 2005; Schwartz et al., 2006). The phagocytic activity of microglia activated by aggregated Aβ is similar to that activated by lipopolysaccharide (LPS); microglia activated by LPS can act as phagocytes in removal of Aβ-plaques (DiCarlo et al., 2001). A recent study by our group suggested that microglia exposed to aggregated Ap_(I-^_0), although effective in removing plaques, are toxic to neurons and impair neural cell renewal; these effects are reminiscent of the response of microglia to invading microorganisms (as exemplified by their response to LPS) (Butovsky et al, 2005, Schwartz et al., 2006). Such activities are manifested by increased production of TNF-α, down-regulation of IGF-I, inhibition of the ability to express MHC-II proteins and thus to act as APCs, and failure to support neural tissue survival and renewal (Butovsky et al., 2006a, 2006b, 2005). Addition of IL-4, a cytokine derived from Th-2 cells, to microglia activated by aggregated Aβ can reverse the down-regulation of IGF-I expression, the up-regulation of TNF-α expression, and the failure to act as APCs (Butovsky et al, 2005). These and other results prompted us to suspect that when microglia encounter aggregated β-amyloid, their ability to remove these aggregates without exerting toxic effects on neighboring neurons or impairing neurogenesis depends upon their undergoing a phenotype switch.

Our earlier observations in vitro led us to suggest that a switch in microglial phenotype might take place via a local dialog between microglia and T cell-derived cytokines such as IL-4. Here we tested this hypothesis by vaccinating transgenic AD (Tg-AD) mice with copolymer- 1 (Teitelbaum et al., 1996). Cop-1 can weakly cross-react with CNS-resident autoantigens (Kipnis et al., 2000) and can safely simulate the protective and reparative effects of autoreactive T cells (Kipnis et al., 2000; 59; Avidan et al., 2004; Benner et al., 2004). We found that vaccination of double-transgenic AD mice with a high-molecular- weight form of Cop-1 resulted in a switch in microglial phenotype from CDl lb⁺/CDl lc^" to CDl lb⁺/CDl lc⁺ cells, accompanied by reduced plaque formation and increased cell renewal. The vaccinated mice also demonstrated significantly better learning/memory ability than untreated matched controls. EXAMPLES

Materials and Methods

(i) Neural progenitor cell culture. Coronal sections (2 mm thick) of tissue containing the subventricular zone of the lateral ventricle were obtained from the brains of adult C57B1/6J mice. The tissue was minced and then incubated for digestion at 37⁰C, 5% CO₂ for 45 min in Earle's balanced salt solution containing 0.94 mg/ml papain (Worthington, Lakewood, NJ) and 0.18 mg/ml of L-cysteine and EDTA. After centrifugation at 1 10 x g for 15 min at room temperature, the tissue was mechanically dissociated by pipette trituration. Cells obtained from single-cell suspensions were plated (3500 cells/cm²) in 75-cm² Falcon tissue-culture flasks (BD Biosciences, San Diego, CA), in neural stem/progenitor cell (NPC)-culturing medium [Dulbecco's modified Eagles's medium (DMEM)/F12 medium (Gibco/Invitrogen, Carlsbad, CA) containing 2 mM L-glutamine, 0.6% glucose, Merrill et al., 1993.6 μg/ml putrescine, 6.3 ng/ml progesterone, 5.2 ng/ml sodium selenite, 0.02 mg/ml insulin, 0.1 mg/ml transferrin, 2 μg/ml heparin (all from Sigma-Aldrich, Rehovot, Israel), fibroblast growth factor-2 (human recombinant, 20 ng/ml), and epidermal growth factor (human recombinant, 20 ng/ml; both from Peprotech, Rocky Hill, NJ)]. Spheres were passaged every 4-6 days and replated as single cells. Green fluorescent protein (GFP)-expressing NPCs were obtained as previously described (Pluchino et al., 2003).

(H) Primary microglial culture. Brains from neonatal (PO-Pl) C57B1/6J mice were stripped of their meninges and minced with scissors under a dissecting microscope (Zeiss, Stemi DV4, Germany) in Leibovitz-15 medium (Biological Industries, Kibbutz Beit Ha-Emek, Israel). After trypsinization (0.5% trypsin, 10 min, 37°C/5% CO₂), the tissue was triturated. The cell suspension was washed in culture medium for glial cells [DMEM supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, Rehovot), L-glutamine (1 mM), sodium pyruvate (1 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml)] and cultured at 37°C/5% CO₂ in 75-cm² Falcon tissue-culture flasks (BD Biosciences) coated with poly-D-lysine (PDL) (10 mg/ml; Sigma-Aldrich, Rehovot) in borate buffer (2.37 g borax and 1.55 g boric acid dissolved in 500 ml sterile water, pH 8.4) for 1 h, then rinsed thoroughly with sterile, glass-distilled water. Half of the medium was changed after 6 h in culture and every 2^nd day thereafter, starting on day 2, for a total culture time of 10-14 days. Microglia were shaken off the primary mixed brain glial cell cultures (150 rpm, 37°C, 6 h) with maximum yields between days 10 and 14, seeded (10⁵ cells/ml) onto PDL-pretreated 24- well plates (1 ml/well; Corning, New York, NY), and grown in culture medium for microglia [RPMI- 1640 medium (Sigma-Aldrich, Rehovot) supplemented with 10% FCS, L-glutamine (1 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml)]. The cells were allowed to adhere to the surface of a PDL-coated culture flask (30 min, 37°C/5% CO₂), and non-adherent cells were rinsed off.

(Hi) Immunocytochemistry and Immu no histochemistry. Primary antibodies: Bandeiraea simplicifolia isolectin B4 (IB-4; 1 :50; Sigma-Aldrich, Rehovot); mouse anti-β -tubulin (anti-βlll-T) isoform C-terminus antibodies (1 :500; Chemicon, Temecula, CA), rat anti-CD l ib (MACl ; 1 :50; BD-Pharmingen, Franklin Lakes, NJ), hamster anti-CDl lc (1 : 100; eBioscience, San Diego, CA), rat anti-MHC-II Abs (clone IBL-5/22; 1 :50), mouse anti-Aβ (human amino-acid residues 1-17; clone 6E10; Chemicon), rat anti-BrdU (1 :200; Oxford Biotechnology, Kidlington, Oxfordshire, UK), goat anti-doublecortin (anti-DCX) (1 :400; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-neuronal nuclear protein (NeuN; 1 :200; Chemicon), goat anti-IGF-I Abs (1 :20; R&D Systems), goat anti-TNF-α Abs (1 : 100; R&D Systems), rabbit anti-CD3 polyclonal Abs (1 : 100; DakoCytomation, CA). Secondary antibodies: FITC-conjugated donkey anti-goat, Cy- 3 -conjugated donkey anti-mouse, and Cy-3- or Cy-5-conjugated donkey anti-rat, biotin- conjugated anti-hamster antibody and Cy-3- or Cy-5-conjugated streptavidin antibody (all from Jackson ImmunoResearch).

Cover slips from co-cultures of NPCs and mouse microglia were washed with PBS, fixed as described above, treated with a permeabilization/blocking solution containing 10% FCS, 2% bovine serum albumin, 1% glycine, and 0.1%

Triton X-100 (Sigma- Aldrich, Rehovot), and stained with a combination of mouse anti-β-tubulin (anti-βlll-T) isoform C-terminus antibodies (1 :500; Chemicon,

Temecula, CA), rat anti-CD l ib (MAC l ; 1 :50; BD-Pharmingen, Franklin Lakes, NJ) and hamster anti-CDl lc (1 : 100; eBioscience, San Diego, CA). To capture the microglia FITC- or Cy3-conjugated Bandeiraea simplicifolia isolectin B4 (IB-4;

1 :50; Sigma-Aldrich, Rehovot) was used. To detect expression of human Aβ anti-

Aβ (human amino-acid residues 1-17) (mouse, clone 6E10; Chemicon) was used.

For BrdU staining, sections were washed with PBS and incubated in 2N HCl at 37⁰C for 30 min. Sections were blocked for 1 h with blocking solution [PBS containing 20% normal horse serum and 0.1% Triton X-100, or PBS containing mouse immunoglobulin blocking reagent obtained from Vector Laboratories (Burlingame, CA)].

For immunohistochemistry, tissue sections were treated with a permeabilization/blocking solution containing 10% FCS, 2% bovine serum albumin, 1% glycine, and 0.05% Triton X-100 (Sigma-Aldrich, St. Louis). Tissue sections were stained overnight at 4°C with specified combinations of the following primary antibodies: rat anti-BrdU (1 :200; Oxford Biotechnology, Kidlington, Oxfordshire, UK), goat anti-doublecortin (anti-DCX) (1 :400; Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-neuronal nuclear protein (anti- NeuN) (1 :200; Chemicon). Secondary antibodies were FITC-conjugated donkey anti-goat, Cy-3 -conjugated donkey anti-mouse, and Cy-3- or Cy-5-conjugated donkey anti-rat (1 :200; Jackson ImmunoResearch, West Grove, PA). CDl Ib (MAC l ; 1 :50; BD-Pharmingen) or FITC-conjugated IB-4 was used for labeling of microglia. Anti-MHC-II Abs (rat, clone IBL-5/22; 1 :50) was used to detect expression of cell-surface MHC-II proteins. To detect expression of CDl Ic hamster anti-CDl lc (1 : 100; eBioscience, San Diego, CA) was used. Anti-Aβ (human amino-acid residues 1-17) (mouse, clone 6E10; Chemicon) was used to detect expression of human Aβ. Expression of IGF-I was detected by goat anti-IGF-I Abs (1 :20; R&D Systems). Expression of TNF-α was detected by goat anti-TNF-α Abs (1 : 100; R&D Systems). T cells were detected with anti-CD3 polyclonal Abs (rabbit, 1 : 100; DakoCytomation, CA). Propidium iodide (1 μg/ml; Molecular Probes, Invitrogen, Carlsbad, CA), was used for nuclear staining. Control sections (not treated with primary antibody) were used to distinguish specific staining from staining of nonspecific antibodies or autofluorescent components. Sections were then washed with PBS and cover-slipped in polyvinyl alcohol with diazabicyclo-octane as anti-fading agent.

(iv) Transgenic mice. Nineteen adult double-transgenic APP_{K59SN, M596L} ⁺ PS1_ΔE9 mice of the B6C3-Tg (APPswe, PSENldE9) 85Dbo/J strain (Borchelt et al., 1997) were purchased from The Jackson Laboratory (Bar Harbor, ME) and were bred and maintained in the Animal Breeding Center of The Weizmann Institute of Science. All animals were handled according to the regulations formulated by the Weizmann Institute's Animal Care and Use Committee, and all experiments and procedures were approved by the Weizmann Institute's Animal Care and Use Committee.

(v) Genotyping. All mice used in this experiment were genotyped for the presence of the transgenes by PCR as described (Jankowsky et al., 2004).

(vi) Reagents. Recombinant mouse IFN-γ and IL-4 were obtained from R&D Systems (Minneapolis, MN). β-amyloid peptide [fragment 1-40 (Aβi_₄₀)] was purchased from Sigma-Aldrich, St. Louis, MO. The Aβ peptide was dissolved in endotoxin-free water, and Aβ aggregates were formed by incubation of Aβ, as described (Butovsky et al, 2005).

(vii) Copolymer- 1 vaccination. Each mouse was subcutaneously injected five times with a total of 100 μg of high-molecular-weight Cop-1(TV-5010 DS, from batch no. 486220205; Teva Pharmaceutical Industries, Petah Tiqva, Israel) emulsified in 200 μl PBS, from experimental day 0 until day 24, twice during the first week and once a week thereafter. (viii) Behavioral testing. Spatial learning/memory was assessed by performance on a hippocampus-dependent visuo-spatial learning task in the Morris water maze (MWM) and carried out as described in Lichtenwalner et al., 2001.

(ix) Administration of 5-bromo-2'-deoxy uridine and tissue preparation. The cell-proliferation marker 5-bromo-2'-deoxyuridine (BrdU) was dissolved by sonication in phosphate-buffered saline (PBS) and injected intraperitoneally (i.p.) into each mouse (50 mg/kg body weight; 1.25 mg BrdU in 200 μl PBS). Starting from experimental day 22 after the first Cop-1 vaccination, BrdU was injected i.p. twice daily, every 12 h for 2.5 days, to label proliferating cells. Three weeks after the first BrdU injection the mice were deeply anesthetized and perfused transcardially, first with PBS and then with 4% paraformaldehyde. The whole brain was removed, postfixed overnight, and then equilibrated in phosphate-buffered 30% sucrose. Free-floating 30-μm sections were collected on a freezing microtome (Leica SM2000R) and stored at 4°C prior to immunohistochemistry. (x) Co-culturing of neural progenitor cells and microglia. Cultures of treated or untreated microglia were washed twice with fresh NPC-differentiation medium (same as the culture medium for NPCs but without growth factors except for 0.02 mg/ml insulin and with 2.5% FCS) to remove all traces of the tested reagents, then incubated on ice for 15 min and shaken at 350 rpm for 20 min at room temperature. Microglia were removed from the flasks and immediately co- cultured (5 x 10⁴ cells/well) with NPCs (5 x 10⁴ cells/well) for 10 days on cover slips coated with Matrigel™ (BD Biosciences) in 24-well plates, in the presence of NPC-differentiation medium. The cultures were then fixed with 2.5% paraformaldehyde in PBS for 30 min at room temperature and stained for neuronal and glial markers.

(xi) Quantification and stereological counting procedure. A Zeiss LSM 510 confocal laser scanning microscope (χ40 magnification) was used for microscopic analysis. For experiments in vitro fields of 0.053 mm² (n = 8-16 from at least two different cover slips) were scanned for each experimental group. For each marker, 500-1000 cells were sampled. Cells co-expressing GFP and βlll-T were counted. For in-vivo experiments, the numbers of Aβ plaques and CDl Ib⁺ microglia in the hippocampus were counted at 300-μm intervals in 6-8 coronal sections (30 μm) from each mouse. Neurogenesis in the dentate gyrus was evaluated by counting of pre-mature neurons (DCX⁺), proliferating cells (BrdU⁺), and newly formed mature neurons (BrdU⁺/NeuN⁺) in six coronal sections (370 μm apart) per mouse brain. To obtain an estimate of the total number of labeled cells per dentate gyrus, the total number of cells counted in the selected coronal sections from each brain was multiplied by the volume index (the ratio between the volume of the dentate gyrus and the total combined volume of the selected sections). Specificity of BrdU+/NeuN+ co-expression was assayed using the confocal microscope (LSM 510) in optical sections at 1-μm intervals. Quantification of CD3⁺, CDl Ib⁺ and CDl Ic⁺ cells were analyzed from 30-50 Aβ-plaques of each mouse tested in this study. Cell counts, numbers of Aβ plaques, plaque areas, and intensity of NeuN staining per unit area in the dentate gyrus were evaluated automatically using Image-Pro Plus 4.5 software (Media Cybernetics, Carlsbad, CA).

(xii) Statistical analysis . MWM behavior scores were analyzed using 3-way ANOVA. Treatment group and trial block were used as sources of variation to evaluate the significance of differences between mean scores during acquisition trial blocks in the MWM. When the P-value obtained was significant, a pairwise Fisher's least-significant-difference multiple comparison test was run to determine which groups were significantly different.

The in-vitro results were analyzed by two-tailed unpaired Student's f-test and by the Tukey-Kramer multiple comparisons test (ANOVA) and are expressed as means ± SEM. Results in vivo were analyzed by two-tailed unpaired Student's f-test or 1-way ANOVA and are expressed as means ± SEM.

Example 1. Aggregated β-amyloid induces microglia to express a phenotype that blocks neurogenesis, and the blocking is counteracted by IL-4.

Previous in vitro findings from our laboratory have suggested that the microglia found in association with inflammatory and neurodegenerative diseases (e.g. microglia activated by LPS or by aggregated Aβ₍i-₄₀₎) have an impaired ability to present antigen, whereas IL-4-activated microglia, shown to be associated with neural tissue survival, express MHC-II, produce IGF-I, and decrease TNF-α expression (Butovsky et al, 2005). Here we first examined whether Aβ-activated microglia block neurogenesis, and if so, whether T cell-derived cytokines can counteract the inhibitory effect. To this end we co-cultured GFP-expressing neural stem/progenitor cells (NPCs) with microglia that had been pre-incubated for 48 h in their optimal growth medium (Butovsky et al, 2005) in the presence or absence of the aggregated Aβ peptide 1-40 (Aβ₍i_₄₀₎; 5 μM) and subsequently treated for an additional 48 h with IFN-γ (10 ng/ml) or IL-4 (10 ng/ml) or IL-4 together with IFN- γ (10 ng/ml). The choice of Aβ₍i_-4o₎ rather than Aβ_()-42) and it's concentration was based on their previous demonstration that this compound induces cytotoxic activity in microglia (Butovsky et al, 2005). Growth media and cytokine residues were then washed off the co-cultured microglia, and each of the treated microglial preparations was freshly co-cultured with dissociated adult subventricular zone- derived NPC spheres (Butovsky et al., 2006b) on coverslips coated with Matrigel™ in the presence of differentiation medium (Butovsky et al., 2006b) (Fig. 9A). Expression of GFP by NPCs confirmed that any differentiating neurons seen in the cultures were derived from the NPCs rather than from contamination of the primary microglial culture. After 10 days, a few GFP-positive NPCs expressing the neuronal marker βlll-T could be discerned in microglia-free cultures (control). In co-cultures of NPCs with microglia previously activated by incubation with IFN-γ (10 ng/ml; MG(i_FN-γ)) a dramatic increase in numbers of GFP⁺/βIII-T⁺ cells was seen. On the contrary, microglia activated by aggregated Aβ₍₁_₄o) (MG(_Ap i-₄₀>) blocked neurogenesis and decreased the number of NPCs. The addition of IFN-γ to Aβ- activated microglia (MG(_Aβi_-4o/i_FN-γ)X failed to reverse their negative effect on neurogenesis. In contrast, the addition of IL-4 (10 ng/ml) to microglia pretreated with aggregated Aβ₍i_₄₀) (MG(_AP1-40/IL-₄)) partially counteracted the adverse effect of the aggregated Aβ on NPCs survival and differentiation, with the result that these microglia were able to induce NPCs to differentiate into neurons. However, when IFN-γ was added in combination with IL-4 (MG_(Aβi-40/iFN-γ+iL-4))_> their effect in counteracting the negative activity of the Aβ-activated microglia on NPC survival and differentiation was stronger than the effect of IL-4 alone (Fig. 9B). We verified that in all cases the βIII-T⁺ cells also expressed GFP (Fig. 9C). This finding is particularly interesting in view of our earlier demonstration that the order in which threatening stimuli are presented to the microglia critically affects the ability of these cells to withstand them (Butovsky et al, 2005). The quantitative analysis presented in Fig. 9D summarizes the data shown in Fig. 9B, and in addition shows that differentiation in the presence of untreated microglia occurred only to a small extent. Notably, no βIII-T⁺ cells were seen in microglia cultured without NPCs (Butovsky et al., 2006b).

Example 2. T-cell-based vaccination with copolymer-1 modulates immune activity of microglia, eliminates β-amyloid plaque formation, and induces neurogenesis.

The above findings prompted us to examine whether a T cell-based vaccination would alter the default microglial phenotype in AD and hence lead to plaque removal and neurogenesis. The antigen chosen for the vaccination was Cop- 1 and we examined its effect in Tg-AD mice suffering from learning/memory impairment and an accumulation of aggregated Aβ plaques deposited mainly in the cortex and the hippocampus, both characteristic features of early-onset familial AD (Borchelt et al., 1997). The regimen for Cop-1 administration was similar to that used to evoke neuroprotection in a model of chronic elevation of intraocular pressure (Bakalash et al., 2005).

We verified the presence of both transgenes in each mouse by PCR amplification of genomic DNA. Tg-AD mice aged approximately 8 months were then vaccinated subcutaneously with Cop- 1 (n = 6) twice during the first week and once a week thereafter. Age-matched untreated Tg-AD mice (n = 7) and non-Tg littermates (n — 6) served, respectively, as untreated-Tg and wild-type controls. Seven weeks after the first Cop-1 injection all the mice were euthanized and analyzed. Staining of brain cryosections from Tg-AD mice with antibodies specific to human Aβ disclosed numerous plaques in the untreated-Tg-AD mice but very few in the Tg-AD mice vaccinated with Cop-1 (Fig. 10A). No plaques were seen in their respective non-Tg littermates (Fig. 10A).

The above results, coupled with the in-vitro findings, prompted us to look for changes in microglial features in the vaccinated Tg-AD mice. Plaques in the untreated-Tg-AD mice were found to be associated with the abundant appearance of CDl Ib⁺ microglia (Figs. 1OA, 10B) expressing TNF-α (Fig. 10C). Fewer CDl Ib⁺ microglia were detectable in the Cop-1 -vaccinated Tg-AD mice (Fig. 10A). It is important to note that the CDl Ib⁺ microglia in the untreated-Tg-AD mice showed relatively few ramified processes Fig. 10C). Staining with anti-MHC-II antibodies disclosed that in the Cop-1 -vaccinated Tg-AD mice most of the microglia adjacent to residual Aβ plaques expressed MHC-II, and hardly any of them expressed TNF- α (Fig. 10D), whereas in the untreated-Tg-AD mice hardly any microglia expressed MHC-II (Fig. 10E), suggesting that their ability to function as APCs is limited. All of the MHC-II⁺ cells were co-labeled with IB-4 (data not shown), verifying their identification as microglia. The dendritic-like morphology (Fig. 10D) of the MHC- H⁺ microglia seen in the Cop-1 -vaccinated Tg-AD mice encouraged us to examine whether they express the characteristic marker of dendritic cells, namely CDl Ic. CDl Ic⁺ microglia in untreated-Tg-AD mice were only rarely found in association with Aβ⁺ plaques, whereas any residual Aβ-stained plaques seen in the Cop-1- vaccinated mice were surrounded by MHC-II⁺/CDl lc⁺ microglia (Fig. 10E). Notably, these CDl Ic⁺ microglia were also positively stained for CDl Ib (Fig. 11 A); in addition, they were loaded with Aβ, indicative of their engulfment of this peptide (Fig. 10E). Quantitative analysis revealed that the number Of CDl Ib⁺ cells associated with Aβ-plaque significantly decreased in the Cop-1 -vaccinated Tg-AD mice (Fig. 10B), and that as a result of the vaccination 87 % of the CDl Ib⁺ became CDl lb⁺/CDl Ic⁺ cells relative to 25 % in the untreated Tg-AD mice (Fig. 10C). In view of our recent finding that MHC-II⁺ microglia (which are activated by IL-4) abundantly express IGF-I (Butovsky et al, 2005, 2006a, 2006b), we examined IGF-I expression in the vaccinated Tg-AD mice. MHC-II⁺ microglia in these mice were indeed found to express IGF-I (Fig. 10F). Staining for the presence of T cells, identified by anti-CD3 antibodies, revealed that unlike in the untreated-Tg-AD mice, in the Cop- 1 -vaccinated Tg-AD mice there were numerous T cells associated with Aβ-plaques (Fig. 10G). Moreover, most of the T cells in the Cop-1 vaccinated Tg-AD mice were found to be located close to MHC-II⁺ microglia. Any Aβ- immunoreactivity detected in those mice appeared to be associated with the MHC- H⁺ microglia, suggesting the occurrence of an immune synapse between these microglia and CD3⁺ T cells (Fig. 10H). Quantitative analysis confirmed the presence of significantly fewer plaques in the Cop- 1 -vaccinated Tg-AD mice than in the untreated-Tg-AD mice (Fig. 101), and showed that the area occupied by the plaques was significantly smaller in the vaccinated Tg-AD mice than in their age- matched untreated counterparts (Fig. 10J). In addition, significantly fewer CDl Ib⁺ microglia (Fig. 10K) and significantly more T cells associated with Aβ-plaque were observed in the Cop-1 -vaccinated Tg-AD mice than in the corresponding groups of untreated-Tg-AD mice (Fig. 10L).

On the basis of our previous findings, we suspected that the switch from a CDl lb⁺/CDl IcVIGF- 1^" to a CDl lb⁺/CDl lc⁺/IGF-I⁺ microglial phenotype in the Cop-1 vaccinated Tg-AD mice might be attributable to IL-4. This possibility was examined in vitro. Staining of 5-day microglia cultures with the CDl Ic marker showed that CDl Ic was hardly expressed at all by untreated, but was abundantly expressed by microglia activated by IL-4 (Fig. 12A). Moreover, IL-4, even if only added 3 days after the microglia were exposed to Aβ, was able to induce them to express CDl Ic (Fig. 12B). Differential activation of the microglia was also reflected in morphological differences: microglia activated by Aβ exhibited amoeboid morphology, whereas the rounded shape of the CDl Ic⁺ microglia was reminiscent of dendritic cells (Fig. 12B). Most importantly, the amoeboid morphology of the Aβ-stained microglia was reversible on addition of IL-4, when they again took on the morphological appearance of dendritic-like cells (Fig. 12B). The various treatments applied to the microglia did not affect their expression of CDl Ib, suggesting that they did not lose their CDl Ib characteristics when they took on the expression of CDl Ic (Fig. 12B). Quantitative analysis of CDl Ic expression, assessed by the number of CDl Ic⁺ cells and the intensity of their staining as a function of time in culture, revealed that soon after seeding (day 0) untreated microglia expressed low levels of CDl Ic, which gradually disappeared (Fig. 12C). In contrast, the expression of CDl Ic induced by IL-4 was not transient. Quantification of the ability of IL-4 to induce CDl Ic expression even after the microglia were pretreated with Aβ is shown in Fig. 12D.

The correlation between the phenotype that was found to be induced by the Cop-1 vaccination and the IL-4 effect on microglia in vitro prompted us to examine the ability of IL-4-activated microglia to phagocytize aggregated Aβ(j-₄o). Quantitative comparison (by intracellular staining) of immunoreactive Aβ engulfed by IL-4-treated and untreated microglia indicated that IL-4 did not interfere with the ability of microglia to engulf Aβ (Fig. 13).

The observed effects of IL-4 on the expression of CDl Ic, MHC-II, and TNF-α prompted us to examine whether the Cop-1 -vaccinated Tg-AD mice would show increased neurogenesis in vivo. Three weeks before tissue excision all mice had been injected with the proliferating-cell marker BrdU, making it possible to detect new neurons. Quantitative analysis of additional sections from the same areas of the hippocampal dentate gyrus disclosed significantly more BrdLT^1" cells in the Cop-1 -vaccinated Tg-AD mice (Fig. 14A) than in their untreated-Tg counterparts. In addition, compared to the numbers of newly formed mature neurons (BrdU⁺/NeuN⁺) in their respective non-Tg littermates the numbers were significantly lower in the untreated-Tg- AD group, but were similar in the Cop-1- vaccinated Tg-AD group, indicating that the neurogenesis capacity had been at least partially restored by the Cop-1 vaccination (Fig. 14B). Analysis of corresponding sections for DCX, a useful marker for analyzing the absolute number of newly generated pre-mature neurons in the adult dentate gyrus (Rao et al., 2004), disclosed that relative to the non-Tg littermates there were significantly fewer DCX⁺ cells in the dentate gyri of untreated-Tg-AD mice, and slightly but significantly more in the dentate gyri of Tg-AD mice vaccinated with Cop-1 (Fig. 14C). Confocal micrographs illustrate the differences in the numbers of BrdU⁺/NeuN⁺ cells, and in the numbers of DCX⁺ cells and their dendritic processes, between non-Tg littermates, untreated-Tg-AD mice, and Cop-1 -vaccinated Tg-AD mice (Fig. 14D). The results showed that neurogenesis was indeed significantly more abundant in the Cop-1 -treated Tg-AD mice than in the untreated-Tg-AD mice. Interestingly, however, in both untreated and Cop-1 -vaccinated Tg-AD mice the processes of the DCX-stained neurons in the subgranular zone of the dentate gyrus were short, except in those Cop- 1 -vaccinated Tg-AD mice in which the DCX⁺ cells were located adjacent to MHC-II⁺ microglia (Fig. 14E).

Example 3. Copolymer-1 vaccination counteracts cognitive decline in AD. Two weeks before the end of the experiment, all mice were tested in a Morris water maze (MWM) for cognitive activity, as reflected by their performance of a hippocampus-dependent spatial learning/memory task. The MWM performance of the untreated-Tg-AD mice was significantly worse, on average, than that of their age-matched non-Tg littermates (Figs. 15A-15B). However, the performance of Cop-1 -vaccinated Tg-AD mice was superior to that of the untreated-Tg-AD mice and did not differ significantly from that of the non-Tg-AD mice, suggesting that the Cop-1 vaccination had prevented further cognitive loss. Differences in cognitive performance were manifested in both the acquisition (Fig. 15A) and the reversal tasks (Fig. 15B).

Discussion

Alzheimer's disease (AD), in addition to its well-known symptoms of plaque formation, neuronal loss, and cognitive decline, is characterized by a destructive inflammatory response. Using AD double-transgenic mice expressing mutant human genes encoding presenilin 1 and chimeric mouse/human amyloid precursor protein, we showed that switching of the microglia to cells that phenotypically resemble dendritic- like (CDl Ic) cells producing IGF-I, achieved here by a T cell- based vaccination with Cop-1 given according to a specific regimen, resulted in reduction of plaque formation and induction of neurogenesis. The vaccination also led to the attenuation of cognitive decline, assessed by performance in a MWM. In- vitro findings showed that microglia activated by aggregated β -amyloid, and characterized as CDl lb⁺/CDl lc7MHC-II7TNFα⁺ cells, impeded neurogenesis from adult NPCs, whereas CDl lb⁺/CDl 1 c7MHC-II7TNFcf microglia, a phenotype induced by IL-4, counteracted the adverse β-amyloid-induced effect. These results introduce dendritic-like microglia as necessary players in the brain's resistance to Alzheimer's disease.

The results of this study showed that vaccination of Tg-AD mice with GA according to a regimen previously found to lead to neuroprotection resulted in a change in the microglial phenotype from CD l Ib⁺ to CDl lb⁺/CDl Ic⁺, and that this was correlated with plaque removal, neurogenesis, and attenuated cognitive loss.

The vaccinated mice in this study demonstrated attenuated cognitive loss (tested in MWM) and increased neurogenesis. These two aspects of hippocampal plasticity are apparently related to the presence of IGF-I and cognitive activity (Rivera et al., 2005) and cell renewal (Butovsky et al., 2006a, 2006b; Aberg et al., 2000; Lichtenwalner et al., 2001). Reported observations in Tg-AD mice housed in an enriched environment also support a link between mechanisms associated with neurogenesis (Ziv et al., 2006a) and with plaque reduction (Lazarov et al., 2005).

Because aggregated Aβ evidently interferes with the ability of microglia to engage in dialog with T cells, its presence in the brain can be expected to cause loss of cognitive ability and impairment of neurogenesis. Homing of CNS-autoreactive T cells to the site of disease or damage in such cases is critical, but will be effective only if those T cells can counterbalance the destructive activity of the aggregated Aβ. As shown here, IFN-γ by itself is impotent against the activity of microglia that are already committed to an aggregated Aβ phenotype, but is effective when added together with IL-4. Thus, the results of this study strongly suggest that the occurrence of neurogenesis in the adult hippocampus depends on well controlled local immune activity associated with microglial production of growth factors such as IGF-I and BDNF (Ziv et al., 2006a). In line with this notion is the reported finding that neurogenesis is impaired in animals treated with LPS (Monje et al., 2003; Ekdahl et al., 2003), shown to impair microglial production of IGF-I and induce microglial secretion of TNF-α (Butovsky et al, 2005).

SECTION III

Therapeutic and diagnostic potential of bone marrow-derived myeloid cells activated by IL-4 in neurodegenerative and tissue-repair diseases

In view of the findings of the of the previous examples, the next question was whether IL-4-activated myeloid cells derived from the patient's own peripheral blood or from HLA-matched donor may be used for diagnostics and therapy of acute and chronic neurodegenerative diseases. Such cells are homing only to sites that express danger signals in the form of local expression of ICAM- 1 on distressed brain parenchyma. If the tissue is intact or does not provide distressed signals no trafficking of BM cells will take place

BM stem cells give rise to a variety of hematopoietic lineages and repopulate the blood throughout adult life (Fuchs. & Segre, 2000; Weissman, 2000a,b). In the CNS, however, neural stem cells have the ability to give rise to astrocytes and oligodendrocytes but not microglia (Fricker et al., 1999; Gage, 2000). It has been suggested that microglia are replenished partly by division of resident cells and partly by immigration of circulating monocytes (Lawson et al., 1992). However, it was also reported that BM-derived cells rarely crossed the blood-brain barrier (BBB) and transdifferentiated into parenchymal microglia (Hickey et al., 1992). Nevertheless, recent work by Simard and colleagues demonstrated that in lethally irradiated mice transplanted with bone marrow cells expressing GFP, the cells immigrated into the brain parenchyma of many regions of the CNS. Nearly all of the infiltrating cells had a highly ramified morphology and colocalized with the microglial marker (Simard & Rivest, 2004).

BM transplantation to animals is a common procedure in immunology and stem-cell research. The transplantation requires whole body lethal γ-irradiation but does not neccessitate brain irradiation. In the case of the brain it is essential to avoid irradiating the brain which impairs brain neurogenesis and cognitive activity (Monje et al., 2002). Moreover, recent findings demonstrate that sublethal irradiation induces microglial expression of ICAM- I (CD54) (Nordal & Wong, 2004), which has an important role in development and promotion of adhesion. ICAM-I reacts with CDl I/CD 18, CD l ib/CD 18 or CDl lc/CD18 (integrin receptor expressed on monocytes, macrophages and NK cells, moderate on granulocytes, and least on subsets of T and B cells) resulting in immune reaction and/or inflammation (Frick et al., 2005). Endothelial ICAM-I contributes to the extravasations of leukocytes from blood vessels, particularly in areas of inflammation. ICAM-I on APCs contributes to antigen-specific T cell activation, presumably by enhancing interactions between T cells and APCs. ICAM-I does not show a static level of expression, but is upregulated or downregulated depending on conditions in the microenvironment (van de Stolpe & van der Saag, 1996). Endothelial ICAM-I expression increases in response to a variety of different stimuli (Kilgore et al., 1995; Lum & Roebuck., 2001; Roebuck & Finnegan, 1999). Pro-inflammatory T cell cytokines, such as TNF-α and IFN-γ, increase the expression of cellular adhesion molecules on the endothelial cells, particularly ICAM-I, permitting a tight binding of the T cells to the endothelial cells via their ligands on the surface of T cells (Campbell et al., 1998). Based upon the apparent contribution of inflammatory processes to neurodegenerative disease or any pathological processes, including the recruitment of monocytic cells to the Aβ-plaques in an animal model of Alzheimer's disease (AD) (Simard et al., 2006), or to choroid during the progression of age-related macular degeneration (AMD) (Mullins et al., 2006), or in EAE (Scott et al., 2004, Kerschensteiner et al., 1999) it is feasible that molecules such as ICAM-I have a dual role in neurodegeneration and tissue-repair processes. We hypothesized that the BM-derived CDl Ic⁺ microglia described by Simard et al. (Simard & Rivest, 2004) migrated to the CNS upon lethal irradiation which creates danger signals and activates resident microglia and not a physiological function of an organism to replenish existance resident microglia in the CNS as was interpreted by the authors. To this end, we designed an apparatus for shielding mice brains when exposing to whole-body γ-irradiation (Fig. 16) and created chimera mice with BM cells derived from double transgenic mouse model CX₃CR l^GFP/CDl lc^DTR that express GFP under the promoter of the chemokine fractalkine receptor CX₃CRl (Jung et al., 2000) and DTR (diphtheria toxin receptor) under CDl Ic promoter (Jung et al., 2002). Consequently, heterozygous mice (CX₃CRlZ⁰¹⁷^⁺) express both the DTR and GFP on peripheral monocytes, and on a subset of mononuclear phagocytes that include macrophages and dendritic cells (Morris, 1981, van Praag et al., 2000) and by microglia in the CNS (Shaked et al., 2004). In additon, we transplanted BM cells derived from double transgenic mouse model CX₃CRl ^GFP/CDl lc^DTR described above, to the ALS SODl transgenic mice or injected IL-4-activated BM-derived myeloid cells systemically (iv) without γ- irradiation.

EXAMPLES Example 1. BM-derived myeloid cells do not migrate into the intact CNS Under normal laboratory conditions, in chimera mice transplanted with BM from double transgenic mise described above, no GFP⁺ cells were found in the CNS (data not shown). However, chimera mice created accordingly to a classical protocol (whole body lethal γ-irradiation) exhibit the presence of GFP⁺ microglia (Figs. 17A, B) coexpressing MHC-II and IGF-I (Fig. 17C).

Example 2. Migration of bone marrow-derived cells correlates with increased expression of microglial CDllb⁺/ICAM-l⁺ under neurodegenerative conditions

Pathological expression of cell adhesion molecules and functional impairments in related signal transduction mechanisms have been suggested to exhibit a key factor for the development of several neurodegenerative disorders, such as AD and ALS (McGeer et al., 1991). Particularly in Alzheimer's disease, the expression of ICAM- I in Aβ-containing brain tissue has been hypothesized to contribute to plaque formation, tissue remodeling and neurodegeneration (Verbeek et al., 1994). Recent report revealed increased ICAM-I expression in the corona around fibrillary Aβ-plaques and an upregulation of ICAM-I in activated microglial cells located in close proximity to the plaques (Apelt et al., 2002). Moreover, blood- derived microglia massively migrate to the plaques and eliminate amyloid deposits (Simard et al., 2006). In addition, Aβ-peptides injected directly into the hippocampus of wild-type mice recruit and activate blood-derived microglial cells (Simard et al., 2006). In line with our experiment, AD-transgenic mice at 12^th month of age exhibit CDl Ib⁺ microglia associated with Aβ-plaque co-expressing ICAM-I (Fig. 18). It seems feasible that Aβ-microglia interaction creates an immunological niche and attracts BM-derived cells to resist disease progression. Recent report revealed that increased expression of ICAM-I and CDl Ib correlated with the disease progression in an animal model of ALS (Alexianu et al., 2001). To identify the origin of CDl Ib⁺ cells, we created chimera mice, as described above. In these mice, at the stage of devastation (140-150 days of age), bone marrow-derived GFP⁺ myeloid cells co-expressing CDl Ib massively migrated to the site of neuronal damage (particularly populating gray matter, the place of neuronal damage) (Fig. 19). Importantly, by creating this chimeric model, we identified resident activated microglia expressing CDl Ib. Immunohistochemical analysis of the animals at different stages of the disease revealed that the activation of resident microglia (CDl Ib) co-expressing ICAM-I appeared only at 90 days, then the symptoms of the disease are manifested.

Example 3. IL-4 activated bone marrow-derived myeloid cells target degenerative CNS: Implication for MS, AD and ALS

Our recent findings demonstrate that IL-4 renders microglia to a dendritic- like phenotype (CDl Ic) (Butovsky et al, 2006c) producing IGF-I and BDNF (Fig. 18). In-vitro, microglia activated by aggregated β-amyloid (Aβ), and characterized as CDl lb7CDl lc7MHC-ir/TNFα⁺ cells, impeded neurogenesis from adult neural stem cells, whereas CDl lb⁺/CDl lc⁺/MHC-H⁺/TNFα^" microglia, a phenotype induced by IL-4, counteracted the adverse Aβ-induced effect. These results introduce dendritic-like microglia as necessary players in the brain's resistance to AD (Butovsky et al, 2006c).

Therapeutic and diagnostic potential of bone marrow-derived myeloid cells activated with IL-4 is demonstrated in Fig. 20; the cells (GFP⁺) injected systemically (iv), migrated into the spinal cord of ALS-diseased mice. It can thus be summarized from the the results in this section:

1. Bone marrow-derived myeloid cells do not migrate into the intact CNS;

2. Migration of bone marrow-derived cells correlates with increased expression of microglial CDl lb+/IC AM-I + under neurodegenerative conditions;

3. Microglia activated by aggregated β-amyloid (Aβ), and characterized as CDl lb⁺/CDl lc7MHC-H7TNFα⁺ cells, impeded neurogenesis from adult neural stem cells, whereas CDl lb⁺/CD l lc⁺/MHC-Il7TNFof microglia, a phenotype induced by IL-4, counteracted the adverse Aβ-induced effect; and

4. Bone marrow-derived myeloid cells activated with IL-4 migrate into the spinal cord of ALS-diseased mice.

SECTION IV

Microglia activated by IFN-γ or IL-4 differentiated into neuronal-Iike or dendritic-like cells

Studies from our laboratory over the last few years have shown that controlled activity of Th cells directed to autoantigens (autoimmune Th cells) in the CNS is needed for CNS maintenance and repair, a phenomenon designated 'protective autoimmunity' (Moalem et al., 1999). It was further demonstrated that cytokines derived from such T cells are active players in the dialogue between T cells and microglia needed for the protective autoimmunity to be manifested (Butovsky et al., 2006b, 2005, 2001 ; Shaked et al., 2005) and maintenance of cell renewal under physiological conditions (Miller et al., 1998). These data led us to hypothesize that Th-derived cytokine such as IFN-γ or IL-4 may have a role in directing microglia plasticity beyond their innate immune activity.

EXAMPLES Materials and Methods

(i) Animals. Neonatal (PO-Pl) C57B1/6J mice, heterozygous mutant mouse strain in which the CX₃CRl chemokine receptor gene is replaced with a green fluorescent protein gene (GFP) C57BI/6-CX₃CRI-GFP [CX₃CRlZ³^⁺) knock-in mice (Jung et al., 2000), and double transgenic mice, expressing GFP under the CX₃CRI promoter and Diphtheria toxin (DTx) receptor under CDl Ic promoter C57BL/6-CD1 IC-DTR-CX₃CRI-GFP {CD11C^DTR/CX₃CR1J^GFP/+). Mice were supplied by the Animal Breeding Center of The Weizmann Institute of Science and were handled according to the regulations formulated by the Weizmann Institute's Animal Care and Use Committee.

(U) Reagents, β-amyloid fragment (1-40), Lipopolysaccharide (LPS) (containing <1% contaminating proteins; obtained from Escherichia coli 0127:B8) and Diphtheria toxin (DTx; from Corynebacterium diphtheriae) were obtained from Sigma-Aldrich. Recombinant mouse IFN-γ and IL-4 (both containing endotoxin at a concentration below 0.1 ng per μg of cytokine), were obtained from R&D Systems (Minneapolis, MN).

(Ui) Neural progenitor cell culture. Coronal sections (2 mm thick) of tissue containing the subventricular zone of the lateral ventricle were obtained from the brains of adult C57B16/J mice. The tissue was minced and then incubated for digestion at 37⁰C, 5% CO₂ for 45 min in Earle's balanced salt solution containing 0.94 mg/ml papain (Worthington, Lakewood, NJ) and 0.18 mg/ml of L-cysteine and EDTA. After centrifugation at 1 10 x g for 15 min at room temperature, the tissue was mechanically dissociated by pipette trituration. Cells obtained from single-cell suspensions were plated (3500 cells/cm²) in 75-cm² Falcon tissue-culture flasks (BD Biosciences, Franklin Lakes, NJ), in NPC-culturing medium [Dulbecco's modified Eagles's medium (DMEM)/F 12 medium (Gibco/Invitrogen, Carlsbad, CA) containing 2 raM L-glutamine, 0.6% glucose, Merrill et al., 1993.6 μg/ml putrescine, 6.3 ng/ml progesterone, 5.2 ng/ml sodium selenite, 0.02 mg/ml insulin, 0.1 mg/ml transferrin, 2 μg/ml heparin (all from Sigma-Aldrich, Rehovot, Israel), fibroblast growth factor-2 (human recombinant, 20 ng/ml), and epidermal growth factor (human recombinant, 20 ng/ml; both from Peprotech, Rocky Hill, NJ)]. Spheres were passaged every 4-6 days and seeded as single cells. Green fluorescent protein (GFP)-expressing neural progenitor cells (NPCs) were obtained as previously described (Vieira et al., 2003). NPC were collected, centrifugated, 110 x g for 10 min at room temperature and then seeded as single cells (5 ^χ 10⁴ cells/well) on cover slips coated with Poly-L-lysine hydrobromide (PLL; Sigma- Aldrich; 0.125 mg/ml) for 1 h, then rinsed thoroughly with sterile, glass-distilled water and coated with Matrigel (BD Biosciences; 1 : 100 in DMEM) for 1 h. cells were grown in differentiation medium (same as the culture medium for NPCs but without growth factors and with 2.5% FCS).

(iv) Primary microglial culture. Brains from neonatal (PO-Pl) C57B1/6J mice were stripped of their meninges and minced with scissors under a dissecting microscope (Zeiss, Stemi DV4, Germany) in Leibovitz-15 medium (Biological Industries, Beit Ha-Emek, Israel). After trypsinization (0.5% trypsin, 10 min, 37°C/5% CO₂), the tissue was triturated. The cell suspension was washed in culture medium for glial cells [DMEM supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, Rehovot), L-glutamine (1 mM), sodium pyruvate (1 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml)] and cultured at 37°C/5% CO₂ in 75-cm² Falcon tissue-culture flasks (BD Biosciences) coated with poly-D-lysine (PDL) (10 mg/ml; Sigma-Aldrich, Rehovot) in borate buffer (2.37 g borax and 1.55 g boric acid dissolved in 500 ml sterile water, pH 8.4) for 1 h, then rinsed thoroughly with sterile, glass-distilled water. Half of the medium was changed after 6 h in culture and every 2ⁿ day thereafter, starting on day 2, for a total culture time of 10-14 days. Microglia were shaken off the primary mixed brain glial cell cultures (150 rpm, 37°C, 6 h) with maximum yields between days 10 and 14, and seeded (10⁵ cells/ml) onto -pretreated 24-well plates (1 ml/well; Corning, Corning, NY) with Poly-L-lysine hydrobromide (PLL; Sigma- Aldrich; 0.125 mg/ml) and Matrigel (BD Biosciences). The cells were allowed to adhere to the surface (25 min, 37°C/5% CO₂), and non-adherent cells were rinsed off. Cells were grown in culture medium for microglia [RPMI- 1640 medium (Sigma- Aldrich, Rehovot) supplemented with 10% FCS, L-glutamine (1 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml)]. One day after seeding, cells were treated with cytokines (IFN-γ, 10 ng/ml; IFN-γ, 100 ng/ml; IL-4, 10 ng/ml) or LPS (100 ng/ml). After 10 days of treatment, the medium was changed to neuronal medium [Neurobasal medium (Rhenium, Israel) supplemented with L- glutamine (1 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), hepes (1 mM) and B-27 (Rhenium, Israel; 1 :50)]. Cultures were fixed at the different time points with 2.5% paraformaldehyde (PFA) in PBS for 30 min at room temperature and stained for neuronal and glial markers. Cell proliferation rates in vitro were determined by staining with BrdU, 2.5 μM (Sigma-Aldrich, St. Louis).

(v) Diphtheria toxin treatment on primary microglia and brain mixed glial cell cultures. Primary microglia and mixed brain glial cell cultures were isolated from neonatal (PO-P l) C57B1/6J-CD / lc^DTR/CX₃CRl /^⁷⁺ brains as described above. Immediately upon isolation, cells were seeded (10⁵ cells/ml) onto pretreated 24-well plates with PLL and Matrigel in culture medium for glial cells, respectively. Two days after seeding, cell cultures were treated with IFN-γ (10 ng/ml) for 2 days and then, DTx (ng/ml) was added, and the brain mixed glial culture was left for additional 72 hours and the microglial culture was left for additional 6 days, then fixated with 2.5% PFA for 30 min.

(vi) Immunocytochemistry. Cover slips from co-cultures of NPCs and mouse microglia were washed with PBS, fixed as described above, treated with a permeabilization/blocking solution containing 10% FCS, 2% bovine serum albumin, 1% glycine, and 0.1% Triton X- 100 (Sigma-Aldrich, Rehovot), and stained with a the rabbit anti-tubulin β-III-isoform C-terminus antibodies (β-III- tubulin; 1 :2000), rabbit anti-NG2 chondroitin sulfate proteoglycan (NG2; 1 :500), mouse anti-RIP (RIP; 1 : 10000), mouse anti-glutamic acid decarboxylase 67 (GAD; 1 : 1000), mouse anti-nestin (Nestin; 1 : 1000), rat anti-myelin basic protein (MBP; 1 :300), rat anti-MHC class II (MHC-II; 1 :50), (all from Chemicon, Temecula, CA), goat anti-double cortin (DCX; 1 :400; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-glial fibrillary acidic protein (GFAP; 1 : 100; Sigma-Aldrich, St. Louis), rabbit anti γ-Aminobutyric acid (GABA; 1 : 1000; Sigma-Aldrich, St. Louis) and rat anti-CD34 (1 :200; Cedarlane). For labeling of microglia we used either rat anti- CDl Ib (MACl ; 1 :50; BD-Pharmingen, NJ) or FITC-conjugated Bandeiraea simplicifolia isolectin B4 (IB4; 1 :50; Sigma-Aldrich, Rehovot). For total cell quantification fixed cultures were stained with Hoechst 33342 (Sigma-Aldrich, St. Louis; 1:2000).

(vii) Quantification. For microscopic analysis we used a Zeiss LSM 510 confocal laser scanning microscope or a Nikon E800 (all with 4Ox magnification). In the confocal laser scanning microscope we scanned fields of 0.053 mm² (n = 8- 16 from at least two different coverslips) for each experimental group from at least three independent experiments. In the NikonE800 microscope, we scanned fields (n = 5-Merrill et al., 1993 from at least two different coverslips) for each experimental group. Cells were counted using Image-Pro (Media Cybernetics, Silver Spring, MD).

(viii) Statistical analysis. The results were analyzed by two-tailed Student's Mest or by the Tukey-Kramer multiple comparisons test (ANOVA) and are expressed as means ± SD (unless differently indicated).

Example 1. Microglia treated with IFN-γ express markers of early differentiated neurons

Previous studies showed that transient exposure of microglia to Th-derived cytokines conferred them ability to support cell survival (Butovsky et al, 2005, Shaked et al., 2005) and renewal (Butovsky et al., 2006a, 2001). Our hypothesis was that if indeed microglia are capable of differentiation into cells other than activated microglia, then this differentiation might be controlled by long exposure to the same cytokines provided by an adaptive immunity. To test this hypothesis we prepared primary microglia cultures from neonatal C57B1/6J mice (Butovsky et al, 2005). After isolation of the microglia from the mixed glia culture we let them adhere for 15 minutes and then washed away all non-adherent cells (Yokoyama et al., 2004) yielding at least 95% microglia purity quantified by labeling for IB-4 and DAPI (Fig. 21F). Two days after microglial isolation and seeding, the cultures were treated for 10 days with 10 ng/ml IFN-γ (MG,,^), or 10 ng/ml IL-4 (MG_αL-4)), or both MG₍i_FN-_γ+i_L-₄)- After 10 days the medium was changed and microglia were cultured in neuronal medium containing supplement supportive of neuronal survival without the presence of the indicated cytokines (Fig. 21A). The choice of the dosing of the cytokines was based on our previous studies indicating the IFN-γ at dosages above 20 ng/ml imposed on microglia a phenotype associated with a high level of TNF-α, which blocks ability of microglia to express growth factors (Butovsky et al., 2006a, 2001) or to act as antigen presenting cells (Butovsky et al, 2005) or to support cell renewal (Butovsky et al., 2006a, 2001). We analyzed the cells by immunocytochemistry analysis using antibodies directed to activated microglia marker (CD l Ib) and to the neuronal lineage marker (βlll-Tubulin). We found that with time in culture, treatment with 10 ng/ml IFN-γ (Th-I specific cytokine) resulted in the appearance of elongated cells expressing CDl lb⁺/βIII-Tubulin⁺. These cells, however, did not appear in untreated microglial cultures or following treatment with IL-4 (Th-2 specific cytokine) (Fig. 21B, 21E). When the microglia were exposed concomitantly to both IFN-γ and IL-4 they acquired the IFN-γ- induced phenotype and slightly but significantly augmented (Fig. 21E). Moreover, 5 days upon IFN-γ treatement, microglia co-expressed both βlll-Tubulin , MHC-II (Fig. 21C) and IB-4 (Fig. 21D). MHC-II expression disappeared at 10 days of treatment. At any of the tested time points the percentage of IB-4⁺ cells in the culture was not affected by the treatment (Fig. 21F). At 18 days IFN-γ-activated microglia maintained the expression of activated microglial marker CDl Ib, the neural cell marker βIII-T⁺ and in addition expressed γ-aminobutyric acid (GABA) (Fig. 21G), the major inhibitory transmitter in higher brain regions (Soghomonian & Martin, 1998). Because GABA immunoreactivity could potentially result from uptake rather than production of GABA, the cells were also stained for the GABA synthesizing enzyme glutamic acid decarboxylase 67 (GAD-67). Thus, the IFN-γ treated microglia, after 18 days in culture were CDl lb⁺/βIII-T⁺/G AD⁺) (Fig. 21H). Notably, CDl Ib immunoreactivity on βIII-Tubulin⁺ elongated cells was decreased at this stage.

In order to verify that these elongated neuronal-like βIII-Tubulin⁺ cells are not coming from proliferating non-microglial contaminating cells in the primary microglial cultures, we exposed the cultured microglia at different time points to BrdU to assess cell proliferation (Butovsky et al., 2001); we did not find any significant proliferating cells throughout all the phases of the experiment from day 0 of the seeding to day 18 (data not shown).

Example 2. The microglial origin of the elongated βIII-Tubulin⁺ cells

To further establish the microglial origin of the βlll-Tubulin expressing cells we isolated microglia from double transgenic mouse model CX₃CR l^GFP/CDl lc^DTR that express GFP under the promoter of the chemokine fractalkine receptor CX₃CRl (Jung et al., 2000) expressed in microglia (Butovsky et al., 2001, Davalos et al., 2005, Nimmerjahn et al., 2005), and DTR (diphtheria toxin receptor) under CDl Ic promoter (Jung et al., 2002). Expression of GFP in these mice is under the control of the CX₃CRl promoter; consequently, heterozygous mice (CX₃CR 1/⁰^⁺) express both the DT-receptor and GFP on microglial cells in the CNS, on peripheral monocytes, and on a subset of mononuclear phagocytes that include macrophages and dendritic cells (Davalos et al., 2005, Geissmann et al., 2003). Five days following treatment with IFN-γ, GFP⁺ microglia obtained from the double transgenic mice were co-labeled with β-III-Tubulin and doublecortin (DCX), a marker of early differentiated neurons (Kempermann et al, 2004). These cells had morphology of elongated cells (Fig. 23A) indicating that indeed these β-III-Tubulin positive cells are from microglial origin. Additional support in the microglial origin of the βlll-Tubulin expressing cells came from an independent experiment relaying on the induced CDl Ic expression in microglia following cytokine treatment. IFN-γ, at the dosage that we used induces MHC-II expression by microglia (Butovsky et al, 2005) and (Fig. 21C); therefore, we envisioned that such MHC-II-expressing microglia might express, even if only transiently CDl Ic, a marker of dendritic cells (DCs), and if so, by adding the diphtheria toxin (DTx) it would be possible to selectively ablate IFN- γ-responding microglia and thereby the microglia-derived neurons (Gropp et al, 2005). We therefore tested whether at early time points in culture the cells express CDl Ic. In fact, both cytokines induced CDl Ic expression (Fig. 22A). At 5 days in culture all IB4⁺ microglia co-expressed βlll-Tubulin and CdI Ic (Fig. 22B). Importantly, while CD l Ic expression by IFN-γ-activated cells decayed with time in culture, IL-4-activated cells maintained CD l Ic expression throughout the entire time in culture (Fig. 22C,D). Notably, microglia exposed to LPS (100ng/ml) failed to express CDl Ic at all time tested (data not shown). These results prompted us to use DTx in order to test whether the CDl Ic⁺ microglia were indeed the source of the neuronal like cells. In this experiment we prepared primary microglia cultures in medium for microglia (Butovsky et al, 2005) and treated them with IFN-γ (10ng/ml) two days after microglial isolation and seeding. Two days after the addition of the cytokines treatment, DTx was added to the cultures and the cultures were left for additional 6 days before analyzing (Fig. 23B). The DTx treatment resulted in a complete ablation of the β-III-Tubulin expressing cells (Fig. 23C). In order to verify that the β-III-Tubulin expressing cells ablation is not a result of DTx toxic effect on neuronal cells, we treated mixed glia cultures (containing microglia, astroglia, oligodendrocytes and survived neurons) with INF-γ, 2 days later we added the DTx and the culture was left for additional 72 hours before analyzed (Fig. 23D). As can be seen IFN-γ-treated microglia were sensitive to Dtx. whereas βIII-Tubulin⁺ neurons were not. The survived neurons expressed microtubule associated protein MAP2, a marker of mature neuronal cells. In contrast, the CDl lb+/βIII-Tubulin⁺ cells did not express MAP2 at any tested conditions (data not shown). This data supported our hypothesis that indeed the β- III-Tubulin expressing cells were from microglial origin.

Example 3. Stem cell-like nature of microglia Having shown that microglia can give rise to neuronal-like or dendritic-like cells prompted us to examine whether these cells can acquire stem-cell behavior. Interestingly, when GFP⁺ microglia were kept untreated, we found them generating floating spheres expressing Nestin, a neural stem cell marker, and CD34, a marker for hematopoietic stem cells (Krause et al, 1994, Morel et al, 1996) (Fig. 24A). We compared the microglia-derived spheres expressing GFP to adult subventricular zone (SVZ)-derived neural stem cells expressing GFP⁺ (Pluchino et al., 2003) Twenty four hours after dissociation of the spheres, both cultures expressed Nestin, however, only the GFP⁺ microglia expressed CD34 (Fig. 24B). Nestin was expressed by all microglia irrespectively of the treatment up to 5 days in culture (Fig. 24C). Interestingly at day 5 in culture, GFP⁺ microglia treated with IFN-γ (10ng/ml) co-expressed both doublecortin (DCX, a marker of early differentiation of the neuronal lineage) and Nestin (Fig. 24C) and β-III-Tubulin and Nestin (Fig. 24D). From day 10 onward, untreated microglia or microglia treated with IFN-γ, showed decreased levels of Nestin, relative to microglia treated with IL-4 (Fig. 24E,F).

The same GFP⁺ microglia were used to follow the effect of the cytokines on astrocyte and oligodendrocytes lineage. At 10 days, IL-4 but not IFN-γ triggered expression of GFAP without inducing morphological features of astrocytes (Fig. 25A) and increased expression of proteoglycan oligodendrocyte marker NG2 (CDl lb⁺/NG2⁺) (Fig. 25B). No markers for mature oligodendrocytes such as RIP or MBP were identified in IL-4-treated microglia at any tested conditions.

Example 4. Microglia differentiation is impaired when exposed to pathological- associated agents and restored following co-treatment with IL-4.

As microglia function as the immune cells of the CNS it is meaningful to study their functions under pathological conditions manifested by high levels of IFN-γ, LPS or aggregated Aβ peptide. Our previous studies showed that a short exposure of microglia to low levels of IFN-γ confers them a phenotype supportive to neural tissue. In contrast, exposure to high levels of IFN-γ confer them a cytotoxic phenotype, which can be reversed by the addition of IL-4. We examined therefore what would be the fate of microglia, in terms of their differentiation into neuronal-like cells following a long-term exposure to high levels of IFN-γ (100 ng/ml). No differentiation into β-III-Tubulin expressing cells took place, and instead, the microglia become amoeboid resembling microglia treated with 100ng/ml LPS or with aggregated Aβ peptide 1-40 (Aβ₍₁_₄₀₎; 5 μM) which is known to be associated with inflammation (Butovsky et al, 2005). Interestingly, the IFN-γ ability to support differentiation into neuronal-like cells could be restored upon addition of IL-4 (10 ng/ml) (Fig. 26).

Discussion

Microglia play a central role in CNS function in health and disease. In the healthy brain they have been viewed as cells that exist in the resting quiescent state, on alert to act when needed. However, recent study indicated that in healthy CNS microglia are busy and vigilant housekeepers (Nimmerjahn et al., 2005) and their immune activity regulated by autoimmune T cells is correlated with induced neurogenesis (Gage, 2000). There are differing opinions as to how microglia function when called to action in situations of neurodegenerative conditions. They have in general received a bad reputation in this paradoxical situation whereby their activity can cause further damage when tissue protection is needed (reviewed in Svhwartz et al., 2006). Here we show that the scope of microglial pluropotency goes beyond immune or neural activities, the cells can act as a source of stem cells when encountering T-cell-derived cytokines. It is well established that the microglia resides in the brain parenchyma

(Bechmann et al., 2005; Simard & Rivest, 2004; Asheuer et al., 2004) whereas the professional APCs are the dendritic cells residing in the perivascular space (Greter et al., 2005). Views differ as to the role of the dendritic cells in the healthy brain and in the brain parenchyma. In the examples herein we show that resting microglia express low levels of CDl Ic, a marker of dendritic cells. Furthermore, following exposure to LPS, expression of CD l I c is completely impaired, whereas exposure to low dosages of IFN-γ or IL-4 induces expression of CDl Ic. In contrast, prolonged exposure to IFN-γ, results with time in the loss of CDl Ic and expression of features associated with neuronal cells. Microglia encountering IL-4, even for prolonged periods of time, did not express these neuronal features; instead these microglia maintained the dendritic-like cells phenotype that they acquired.

The fact that microglial cells acquire features of neuronal-like cells and concomitantly lose CDl Ic and typical microglial markers such as CX₃CRl, CDl Ib and MHC-II suggests that microglial pluripotency includes ability to act as stem- like cells which give rise to neuronal-like cells. Although we don't know whether the emerging neuronal-like cells function as neurons it is suggestive that they might serve some role in the scaffold needed for the tissue maintenance and renewal. It also emerged from this work that MHC-II found in sites of degenerative conditions are by no means markers of destructive microglia or markers of degenerative conditions. On the contrary, neither absence of IFN-γ or high dose IFN-γ induces MHC-II expression. It thus appeared that CD 1 1 c within the brain might be viewed as a marker of either progenitor neuronal-like cells emerging from microglia, or as a marker of IL-4-activated microglia that supports neural tissue survival and renewal. In fact, using in vitro conditions in which CDl Ic expression is linked to DTR expression revealed that CDl lc-expressing cells are the origin of neuronal cells or the cells that support rather than destroy neuronal cells.

Microglia origin is the hematopoietic cells, and indeed the stem cells that were formed in the microglial culture expressed CD34, a marker of hematopoietic origin, unlike that of adult neural stem cells isolated from SVZ that do not express CD34. It is possible that the adult stem cells found in the healthy brain in neurogenic niches serve as a reservoir of cell renewal under non-pathological conditions. Under pathological conditions, it is very likely that bone marrow- derived local microglia cells, assisted by the T cells, create the "repairing" neurogenic niches. If this is the case, the microglia perform the dual action of forming the niche for the cell renewal and serving as the origin for the renewing cells. Such niches are replenished by bone marrow-derived cells (Brazelton et al., 2000, Mezey et al., 2000, Cogle et al., 2004).

It is still a matter of debate whether hematopoietic stem cells can locally differentiate into neuronal cells (Wagers et al., 2002). It is possible that such trans- differentiation, although not taking place under normal conditions, does take place under pathological conditions assisted by the local immune response. Alternatively, the neuronal-like cells endorse the diseased sites with neuronal-like features needed for the attraction of the resident neural stem cells and for the molecular clues needed for their differentiation into new neurons. Other studies have reported that microglia can become stem cells in in vitro conditions but this has been shown only with a high percentage of serum (Yokoyama et al., 2004).

The microglial-derived neuronal-like cells found in the present study expressed GABA, and GABAergic excitation with GABA_A-R agonists has been found to induce neuronal differentiation of adult progenitor cells (Tozuka et al, 2005). It is therefore possible that the IFN-activated microglia can support neurogenesis from endogenous stem-cell pools by their microglial nature as our previous studies demonstrated, or via their neuronal-like GABAergic nature, or both. Chronic pathological conditions are manifested by high levels of IFN-γ (autoimmune diseases such as multiple sclerosis), or aggregated β-amyloid microglia (e.g. Alzheimer's disease). Our in vitro and in vivo results have suggested that microglia associated with such conditions are cytotoxic (Butovsky et al, 2005) and block cell renewal (Butovsky et al., 2006a, 2001). Compounds associated with neurodegeneration aggregated Aβ^o₎ or high levels of IFN-γ up-regulate the expression of TNF-α by microglia (Butovsky et al., 2006a, 2005). The phenotype of the microglia was correlated with a signal transduction pathway that down-regulates expression of MHC-II through the MHC-II-transactivator which involves STAT-I activation. Moreover, a recent study showed that the LPS receptor (CD 14), a key receptor for innate immunity, interacts with fibrils of Alzheimer's amyloid peptide (Fassbender et al, 2004) and amyloid beta peptide (1-40) (Aβ₍i_-40)) enhances the action of Toll-like receptor (TLR)-2 and -4 agonists (Lotz et al, 2005). STAT-I tyrosine phosphorylation following TLR triggering was shown to be severely impaired by SOCS-I overexpression (Baetz et al, 2004). Our recent study suggested that the behavior of microglia exposed to aggregated Aβ^o₎ is similar to their response to invading microorganisms (as exemplified by their response to LPS) (Butovsky et al, 2005; Schwartz et al., 2006). At the level of signaling pathways, extremely low concentrations of TNF-α ( 1 ng/ml or less) inhibits the IGF-I-induced effect on the proliferation and survival of hematopoietic progenitor cells (Shen et al, 2004) and addition of anti-TNF-α neutralizing antibodies boosted the IFN-γ- induced increase in microglial expression of MHC-II (Butovsky et al, 2005). Protection by IL-4 was attributed to down-regulation of TNF-α and up-regulation of IGF-I (Butovsky et al., 2006a). Analysis of the signal transduction activated by IFN-γ and by IL-4 disclosed that both cytokines had activated genes associated with microglial expression of MHC-II (Butovsky et al, 2005). In the present study we showed that the differentiation of microglia to neuronal-like or to dendritic-like cells by IFN-γ or IL-4, respectively, is via distinctive pathways. IFN-γ, unlike 11-4 activation, could be blocked by STAT-I inhibitors and by IL-10. IFN-γ overwhelmed microglia were shown to be cytotoxic to neural tissue and to impair neurogenesis; both could be reversed by IL-4. Here we are showing also that the ability of microglia to become neural-like cells is also impaired by high levels of IFN-γ and could be reversed by IL-4. These results thus emphasize that both pro-inflammatory and anti-inflammatory cytokines are needed for executing a broad spectrum of microglial activities, yet the balanced levels of these cytokines would critically determine this plasticity.

SECTION V

Lack of early distress signals and deficiency of bone marrow-derived dendritic- like cells in ALS: a possible biological marker

Amyotrophic lateral sclerosis (ALS) is a terminal disease characterized by loss of motor neurons associated with microglial activity. We postulated that the local immune response plays a dual role, by demarcating endangered sites and — if appropriately controlled — by defensive action. Using transgenic ALS mouse model, we show that distress signals appear relatively late and that the late phase of disease progression associated with diminished microglial activity. In chimeric ALS mice whose bone marrow (BM)-derived myeloid cells express GFP, the diseased spinal cord was populated by dendritic-like BM-derived microglia expressing IGF- I. Moreover, IL-4-activated BM-derived myeloid cells expressing both CDl Ic and IGF-I, injected peripherally, homed to spinal cord motor neurons, and maintained their phenotype. Blood from ALS patients (n = 26) contained approximately half as many dendritic-like cells (CDl lc^dm7Lin^~ cells; P < 0.001) as the blood of healthy individuals (n = 13), suggesting the possibility of using these cells as a biological marker. Introduction

Inflammation was recently implicated as a critical mechanism responsible for the progressive nature of neurodegenerative diseases (Minghetti et al, 2005, Wyss- Coray & Mucke, 2002), including Alzheimer's, Parkinson's, and Huntington's diseases, ALS, multiple sclerosis, prion diseases, and many other less common syndromes. The role of inflammation is known to differ in different diseases. All of the abovementioned neurodegenerative diseases typically involve deposits of inclusion bodies that contain abnormal protein folding that is associated with neuronal toxicity (Ross & Poirier, 2004; Taylor et al., 2002). We suggest that the nature and timing of the inflammatory response are determined by the site of protein deposition. Thus, for example, in ALS (Atkin et al., 2006; Stathopulos et al., 2003) or Parkinson's disease (Valente et al., 2004) the misfolded proteins activate a programmed cell-death pathway within the neurons, whereas in Alzheimer's disease the misfolded proteins accumulate externally and are sensed by microglia (Butovsky et al, 2006c; Akiyama & McGeer, 1990).

We suggest that the primary role of the distressed microglia is to convey a 'danger signal' that leads to demarcation of sites of neuronal loss, where immune cells or neural stem cells or both (Butovsky et al., 2006a; Simard et al., 2006; Pluchino et al., 2005) are recruited for repair. In our view, an early immune distress signal is essential for the tissue's effective recruitment of the repair machinery. This is because even in instances where the immune response is too weak and ineffectual or too strong and destructive, if evoked early enough it is at least amenable to modulation. In the present study, using a mouse model of ALS (SODlG93A-transgenic mice), we addressed four key questions. Precisely at what stage does the immunological distress signal appear in this disease? Are the recruited cells resident microglia or cells derived from the bone marrow? If the latter, can they be therapeutically manipulated to deliver the goods? And finally, can the potentially beneficial peripheral cells be used clinically as a biological marker? EXAMPLES Materials and Methods

(i) Animals. We used neonatal (PO-Pl) C57BL/6J mice; inbred adult male C57BL/6J mice (8-10 weeks old); heterozygous mutant C57BL/6J-CX₃CR1/^GFP/+ knock-in mice, in which the CX₃CRl chemokine receptor gene is replaced by a GFP gene (Jung et al., 2000); ALS mice: transgenic mice overexpressing the defective human mutant SODl allele containing the Gly93->Ala (G93A) gene (BoSJL-Tg(SOD 1-G93 A) lGur/J, supplied by The Jackson Laboratories, Bar Harbor, ME, USA); and ALS F2 offspring (F2-SOD1), obtained by two generational backcrosses of heterozygous ALS mice with C57BL/6J mice. Mice were supplied by the Animal Breeding Center of The Weizmann Institute of Science and handled according to the regulations formulated by the Weizmann Institute's Animal Care and Use Committee. (H) Reagents. Recombinant mouse IL-4 (containing endotoxin at a concentration below 0.1 ng per μg cytokine) from R&D Systems.

(Hi) Primary microglial culture was prepared as previously described (Butovsky et al., 2006a).

(iv) Preparation of bone marrow-derived myeloid cells. We harvested bone marrow cells from both the hindlimbs (tibia and femur) and the forelimbs (humerus) of wild-type C57BL/6J mice (8-10 weeks old) or non-mSODl-CX₃CRl/^GFP/+mice (8-10 weeks old) by flushing the bones with Dulbecco's PBS (Sigma- Aldrich) under aseptic conditions. Cells were collected and centrifuged (10 min, 1000 rpm, 4⁰C), resuspended, and then seeded (7x lO⁶ cells) in 10 ml of microglial medium [RPMI- 1640 medium supplemented with 10% FCS, L-glutamine (1 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 μM), penicillin (100 U/ml) and streptomycin (100 U/ml)], and cultured at 37°C/5% CO₂ in 75-cm² tissue-culture flasks coated with poly-D-lysine in borate buffer (pH 8.4) (Butovsky et al., 2006b). Culture medium and floating cells were discarded every 4^th day and fresh microglial medium was added. After 10-14 days of incubation, nonadherent cells were washed out and the flasks with remaining adherent cells were refilled with fresh microglial medium. The cells were then left untreated or were treated for 3 days with recombinant mouse IL-4 (10 ng/ml). Flasks were placed on ice for 20 min and the cells were then harvested with a cell scraper, washed thoroughly with PBS, resuspended (2 x 10⁶/ml in PBS), and injected intravenously (i.v.). Recruitment of IL-4-activated wild-type BM cells was evaluated by transplantation of IL-4- activated BM donor cells from non-mSOD l-CX₃CRl/^GFP/+ mice (2-5 x 10⁵ cells in a volume of 0.1 ml) into the tail vein of non-irradiated diseased F2-SOD1 mice every 5 days, starting from day 125. Purity of the cells was assessed by FACS analysis or by immunocytochemistry after staining with antibodies (Abs) against Mac-1 (CDl Ib) (Pharmingen), and was found to be >95%.

(v) Preparation of human PBMCs. PBMCs were purified by gradient centrifugation. For this purpose, peripheral blood was diluted (1 : 1 ratio) with PBS and stratified on 4 ml Ficoll-Paque (Amersham) in 15-ml conical tubes, which were then centrifuged at 400^χg for 30 min at 20°C. Recovered PBMCs were washed twice with PBS by centrifugation at 100^χg for 10 min at 4⁰C. Viability of the pelletted PBMCs was determined by staining with trypan blue (Sigma).

(vi) FACS analysis of BM-derived myeloid cells. We performed flow cytometric analysis of BM-derived myeloid cells using combinations of the following mAbs: 100 μl anti-CD l lb-PerCP Ab (1 : 100; eBioscence) and co-stained with CDl Ic-APC (1 : 100) and I-A/I-E-PE (MHC-II, clone M5/1 14.15.2; 1 :300). Cells were analyzed by FACScan (Becton-Dickinson); 2 x 10⁴ cells were analyzed for forward scatter (FSC), integrated side scatter (SSC), and phycoerythin (PE), or Cy-chrome-emission. The data were analyzed using Cell Quest software (Becton- Dickinson).

(vii) FACS analysis of human PBMCs. PBMCs were isolated from human blood and double stained with the cell-surface markers mouse anti-human CDl Ic mAb (IgG, PE-labeled; BD Biosciences-Pharmingen) and Lineage Cocktail 1 (Hn 1 ; BD Biosciences-Pharmingen). The latter includes antibody clones against CD3, CD14, CD16, CD19, CD20, and CD56, which, in combination, stain lymphocytes, monocytes, eosinophils, and neutrophils (IgG, FITC-labeled; BD Biosciences-Pharmingen). The percentages of CDl lc^dιm/Lin^~ were determined by flow cytometric analysis using a FACScan (Becton Dickinson) and CELLQUEST software.

(viii) Bone marrow transplantation. To produce CX₃CRl^GFP-wt — »SOD1 chimeric mice, B6SJL-G93 A-SODl IGur mice (70 days old, n = 5) were lethally irradiated with 950 rad from a cobalt source. On the same day, 2.5 * 10⁶ BM donor cells from non-mSOD l-CX₃CRl/^GFP/+ mice were suspended in PBS (total volume 0.1 ml) and transplanted systemically by injection into the tail vein.

(ix) Tissue preparation. We anesthetized mice and perfused them transcardially, first with PBS and then with cold 2.5% paraformaldehyde. Their spinal cords were removed, postfixed overnight, and equilibrated in 1.25% paraformaldehyde, 30% sucrose. Free-floating 30-μm cross-sections were collected on a freezing microtome (SM2000R; Leica) and stored at 4°C prior to immunohistochemistry.

(x) Immunocytochemistry and immunohistochemistry. Cover slips from BM or microglial cultures were washed with PBS, fixed as described above, and stained with the microglial marker FITC-conjugated Bandeiraea simplicifolia IB4 (1 :50 dilution; Sigma-Aldrich), goat anti-IGF-I Ab (1 :50 dilution; R&D Systems), and the dendritic cell marker hamster anti-CDl lc (1 :50; eBioscience). Primary antibodies were applied in PBS with 10% permeabilization/blocking solution [5% FCS, 2% bovine serum albumin, 1% glycine, and 0.1% Triton X-100 (Sigma- Aldrich) in PBS] in a humidified chamber at room temperature for 1 h, washed 3 times with 0.05% Tween (Sigma-Aldrich) in PBS, and incubated with the secondary antibodies in PBS for 1 h in a humidified chamber at room temperature. For immunohistochemistry, cryocrosssections of the lumbar spinal cord (30 μm) were treated with a permeabilization/blocking solution containing 20% horse serum and 0.1% Triton X- 100 (Sigma-Aldrich) in PBS for 1 h at room temperature. Sections were stained with the activated microglia marker rat anti-CDl lb, (MACl; 1 :50 dilution; BD-Pharmingen), hamster anti-CDl lc (1 :50; eBioscience, San Diego), goat anti-IGF-I (1 :50 dilution; R&D Systems), and hamster anti-ICAM- 1 (1 :50 dilution; Chemicon) in PBS with 10% of the permeabilization/blocking solution. Sections were incubated with the primary antibody for 24 h at 4°C, washed with PBS, and incubated with the secondary antibodies in PBS for 1 h at room temperature while being protected from light. Secondary antibodies used for both immunocytochemistry and immunohistochemistry were Cy-3 -conjugated donkey anti-goat and Cy-5-conjugated donkey anti-rat, both used at a dilution of 1 :300. Goat-biotin-conjugated anti-hamster Abs at a dilution of 1 : 100 and Cy-3- or Cy-5-conjugated streptavidin antibody at a dilution of 1 :200 were applied over samples for 15 min at room temperature while being protected from light. All antibodies were purchased from Jackson ImmunoResearch Laboratories. Control sections (no.t treated with primary antibody) were used to distinguish specific staining from staining of nonspecific antibodies or autofluorescent components. Sections were then washed with PBS and cover-slipped in polyvinyl alcohol with diazabicylo-octane as anti-fading agent.

(xi) Real-time quantitative PCR. Total cellular RNA purification from cultured BM cells and cDNA synthesis were performed as described previously (Butovsky et al., 2006a). We assayed the expression of specific mRNAs by fluorescent-based real-time PCR, using selected gene-specific primer pairs. Genes were successfully calibrated to the Q-PCR analyses to obtain optimal efficiencies of PCR reaction kinetics, i.e., extremely close to 1.00 (±0.02). Q-PCR reactions were performed according to the manufacturer's instructions, using Absolute QPCR SYBR^® Green ROX mix (ABgene) containing Thermo-start^® DNA polymerase, dNTPs, MgCl₂, and SYBR Green I dye and ROX reference dye. Q-PCR products were detected by the SYBR Green I dye detector absorbed at 519 nm, obtained in triplicate for each of the cDNA samples using the Rotor-Gene 6 instrument (Corbett Research), and analyzed using Rotor-Gene 6000 software (version 1.7, Corbett). Relative mRNA amounts were evaluated by the relative standard curve method (Livak et al., 2001), assuming similar PCR efficiencies of the gene of interest relative to an endogenous reference gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cytoplasmic β-actin (ACTB) were chosen as reference genes. The amplification cycle was 95⁰C for 5 s, 60⁰C for 20 s, and 72°C for 15 s. The following primers were used: IGF-I: sense, S'-TTCAGTTCGTGTGTGGACCGAG-S'; (SEQ ID NO: 1) antisense, 5'-TCCACAATGCCTGTCTGAGGTG-3^r; (SEQ ID NO:2) for the reference gene GAPDH: sense, 5'-AATGTGTCCGTCGTGGATCTGA-S'; (SEQ ID NO:3) antisense, S'-GATGCCTGCTTCACCACCTTCT-S'; (SEQ ID NO:4) and for the reference gene ACTB: sense 5'-GACGGCCAGGTCATCACTAT-S'; (SEQ ID NO:5) antisense, S'-AAGGAAGGCTGGAAAAGAGC-S'. (SEQ ID NO:6)

At the end of the assay we constructed a melting curve to evaluate the specificity of the reaction.

(xii) Genotyping. DNA from the tails of BoSJL-Tg(SOD 1-G93 A) lGur/J mice was genotyped using the genomic DNA purification kit (Qiagen). Primers (SODl and Tg-SODl) were used as internal standards according to the genotyping protocol of the Jackson Laboratory. PCR was performed with 1 μl of DNA, 0.5 μl of each primer, and 15 μl of ReadyMix PCR Master Mix (ABgene) in 30-μl reactions. The amplification cycle was 95⁰C for 3 min, 36 cycles of 95⁰C for 30 s, 6O⁰C for 30 s, and 72⁰C for 45 s, followed by 72°C for 2 min. Primers for the internal standard SODl were applied at the end of the 7^th cycle. PCR products were subjected to 1.8% agarose gel analysis and were visualized by ethidium bromide staining.

(xiii) Patients and healthy subjects. Blood samples were obtained from 26 patients with confirmed ALS and a control group comprising 13 age- and gender- matched apparently healthy volunteers. Example 1. ICAM-I expression in spinal cord of SODl^G93Λ-transgenic mice.

Studies have shown that expression of ICAM-I on activated microglia can be used as a marker of distress signals (Nordal & Wong, 2004). To determine whether and when distress signals are evoked in SODl^G9jA-transgenic mice, we dissected out the spinal cords of these mice at various stages of the disease. Testing for ICAM-I expression revealed that an immunological niche in the CNS is not formed before onset of clinical symptoms (Fig. 27A), suggesting that the death of motor neurons in ALS mice begins and continues to occur long before any signal for help issuing from the CNS can be detected. Interestingly, activated microglia stained for CDl Ib and co-expressing CDl Ic transiently correlated with increased expression of ICAM-I in 120-day-old ALS mice (Fig. 27B), but disappeared at the end stage (approximately 140 days, as discussed below). Wild-type mice tested under the same conditions failed to show any ICAM-I or CDl lb/CDl lc expression (Fig. 27A, 27B).

Example 2. CDllb⁺/CDllc⁺ bone marrow-derived myeloid cells home to spinal cord of SODl^G93Λ-transgenic mice

In attempting to characterize the nature of the immunological niche created in the CNS under neurodegenerative conditions, we demonstrated hereinbefore that this niche recruits microglia and bone marrow-derived myeloid cells, as well as neural stem cells. Each of these cell types, separately or in combination, can contribute to disease progression or attenuation (Butovsky et al., 2006a, 2006c; Ziv et al., 2006a, 2006b). If the recruited cells are not properly activated they can do more harm than good (Butovsky et al., 2006a, 2006c, 2005). It was therefore important to determine, firstly, whether the cells recruited to this immunological niche within the CNS of SOD mice are CNS-resident microglia or BM-derived cells or both, and then to characterize their phenotype(s). To this end we created chimeric SOD mice by replacing the bone marrow of 70-day-old SOD mice with wild-type BM cells from transgenic mice expressing GFP under the fractalkine receptor CX3CR1 (Jung et al., 2000). GFP-expressing cells in these chimeric mice include all monocytes, dendritic cells and microglia (Cardona et al., 2006). Two months after the chimeric SOD mice were produced, analysis of their spinal cords revealed the presence of numerous GFP-expressing BM-derived myeloid cells co-expressing CDl Ib and CDl I c (Figs. 28A, 28B). In addition, these dendritic-like cells showed abundant co-expression of IGF-I, shown to play a key role in cell survival and renewal in the CNS (Butovsky et al., 2006b, 2005; Aberg et al., 2000; Dudek et al., 1997) (Figs. 28C, 28D). In contrast, analysis of the spinal cords of non-chimeric SOD control mice at this terminal stage of the disease disclosed only small numbers of CDl lb⁺/CDl lc⁺ cells (Figs. 28E, 28F), and the levels of IGF-I expressed by these cells were extremely low (Figs. 28G, 28H). Interestingly, both resident and recruited microglia in the chimeric SOD mice also exhibited the morphology of activated ramified microglia (Figs. 28B, 28D), whereas most of the remaining CDl Ib⁺ cells in the controls had lost their normal morphology (Figs. 28F, 28H). Notably, most of the recruited GFP⁺ cells in the spinal cord were located in the ventral horn, possibly indicating their association with dying neurons (Figs. 28A, 28D).

Example 3. Injected bone marrow-derived myeloid cells home to the spinal cords of SOD mice and maintained their ability to express CDlIc. CDl lc⁺/IGF-I⁺ cells are functionally reminiscent of microglia activated by the cytokine IL-4 (Butovsky et al., 2006a, 2006c). As shown in the former sections herein, IL-4-activated microglia express a dendritic-like phenotype, which is characterized by CDl Ic and IGF-I expression, and can induce both neuroprotection and neural cell renewal (Butovsky et al., 2006a, 2006b, 2006c, 2005). Those results, taken together with the present findings, prompted us to determine whether the incidence of CD 1 1 C⁴VIGF-I⁺ cells in the CNS of diseased SOD mice can be increased by systemic injection of BM-derived myeloid cells pre-activated with IL- 4, and if so, whether they can maintain their phenotype there. CX3CR1 expressing BM-derived myeloid cells were isolated from wild-type mice, activated ex vivo by treatment with IL-4 to express IGF-I, and injected systemically into SODl^G9jA-transgenic mice at the time that their immunological niche was created. We found that the injected cells homed to the spinal cords of the SOD mice and maintained their ability to express CDl Ic (Fig. 29A) and IGF-I (Fig. 29B) there. As in the case of the recruited GFP⁺ cells in SOD chimeric mice, the BM-derived recruited cells expressing IGF-I in these mice homed primarily to the grey matter of the ventral horn, or in other words, to the sites of dying motor neurons (Fig. 29B, inset).

Example 4. BM-derived cells isolated and cultured from end-stage SOD mice and from wild-type mice express similar levels of CDlIc and IGF-I.

These findings prompted us to compare in vitro the CDl Ic expression, as well as the IGF-I expression, in BM-derived cells that were isolated and cultured from end-stage SOD mice and from wild-type mice and then exposed to IL-4. No differences in expression were detectable at either the protein level of CDl Ic (Fig. 30A) or IGF-I (Fig. 30B) or in the mRNA expression of IGF-I (Fig. 30C). A similar picture was obtained when we repeated the in-vitro experiment using primary cultured microglia isolated from newborn wild-type and newborn SOD mice (data not shown).

Discussion

The results of this study of a mouse model of ALS showed that distress signals are expressed only at a relatively late stage of disease progression, and that the expressed signals fail to recruit potentially beneficial microglia. However, injected BM-derived myeloid cells expressing IGF-I can migrate to the CNS and deliver IGF-I to sites of damage there. The relevance of these findings to the human disease was demonstrated by the finding that relative to healthy individuals the blood of patients with ALS contained only half the number of CDl lc-dendritic like cells.

Neurons in ALS begin to die long before their loss is detectable in their immediate neighborhood. By the time clinical signs appear the functional threshold has already been reached, and thereafter the disease progression is rapid. Inflammation has been implicated as a component of the disease progression (Alexianu et al., 2001). Nonspecific anti-inflammatory steroidal treatment has failed, however, to show any benefit (Werdelin et al., 1990). We show hwewin that IGF-I, a key therapeutic factor in ALS, can be expressed by suitably activated microglia, and that such expression is inducible by IL-4 (see also Butovsky et al., 2006a, 2006b, 2006c, 2005). IGF-I has been shown to play a key role in cell survival and renewal in the CNS (Shaked et al., 2004; Butovsky et al, 2005; Aberg et al., 2000; Dudek et al., 1997; Kaspar et al., 2003). That finding, together with the recent demonstration that attenuation of SODl expression by microglia in 90-day- old ALS mice significantly extends their life expectancy (Boillee et al., 2006), can be taken to argue that the microglia in ALS mice make a late negative contribution to disease progression but do not affect disease onset. Our present results are in line with this notion, and support the contention that the local immune cells recruited in ALS have a late and negative effect.

The finding that late manipulation of microglia was effective in slowing down progression of the disease is encouraging in light of our current demonstration that the recruited cells include myeloid cells derived from BM. This implies that relevant peripheral cells, if properly activated in the periphery, might be sufficient to ensure that cells homing to the site of disease progression will not be those having a destructive phenotype, and hence that the homing cells will not only protect the suffering neurons from the primary cause of the disease but will also help to counterbalance the effect of resident destructive cells. Since the recruited BM-derived cells, if suitably activated, not only are not toxic but also express IGF- I, they can be viewed as agents of safe self-delivery of IGF-I to sites of motor neuron loss, thus replacing the need for gene therapy as a means of delivery of this growth factor (Kaspar et al., 2003). Moreover, replacement of the bone marrow of SOD mice with wild-type bone marrow can extend the life expectancy of SOD mice, an effect that might be attributable largely to neurogenesis (Corti et al., 2004). Our present demonstration that injection of IL-4-activated myeloid cells derived from the bone marrow of wild-type mice boosts spontaneously recruited cells that express a beneficial phenotype further supports the delivery of IGF-I to suffering SOD mice as a promising therapeutic measure.

Motor neurons in ALS die by apoptosis (Rabizadeh et al., 1995). Apoptosis is viewed as silent death in that it is not accompanied by signals that activate an immune cascade. The apoptotic death in ALS has been likened to the process that occurs during development, primarily in the nervous system, where growth-factor deprivation acts as the death-inducing signal and eliminates superfluous neurons without intentionally sending a signal for help. The apoptotic death that occurs during development is purposefully programmed, and as there is no need for repair or restoration, the lack of a danger signal can be assumed to be intentional. We argue that in adulthood, however, such internally triggered apoptotic death is potentially lethal, as the tissue might continue to lose neurons without alerting the body for help. In contrast, in the case of apoptotic death induced by an external signal, although the death of neurons might occur via an identical mechanism, it is nevertheless associated with a distress signal that activates the innate immune system. We further suggested that this early signal for help might be sufficient or insufficient; if poorly controlled, however, it is likely to evoke a destructive response. In contrast, in the case of silent death the call for help is significantly delayed, so that by the time it occurs, the local immune response is too late to be of help and is likely to cause further harm.

In the present study we found that when a stress signal in the ALS mice was eventually expressed, cells recruited from the bone marrow or from the CNS itself did not acquire the IGF-expressing, dendritic-like phenotype (Butovsky et al., 2006a, 2006c). The expressed IGF-I indicates that the phenotype is beneficial, but is by no means the only benefit delivered by these cells. In the case of ALS, the findings of this study and of Boillee and colleagues suggest that disease progression is accompanied also by death of the activated microglia that express the danger signal, because the latter microglia express the same mechanism as the one that kills neurons (Boillee et al., 2006). The negative impact of the death of these microglia might thus be threefold: loss of danger signals that call for help, leakage of threatening compounds, and loss of cells which, upon immunomodulation, could potentially deliver the goods.

The origin of the activated microglia seen in the adult CNS has long been a matter of debate. Some authors have suggested that the CNS is populated entirely by microglia derived from bone marrow (Simard & Rivest, 2004). Our accumulated data in several animal models suggest that BM-derived myeloid cells find their way to the adult CNS only in the case of damage or disease, but not to the healthy CNS, and that cells from the periphery home and accumulate only on receiving a danger signal (Butovsky et al., unpublished data). It seems that in the case of ALS, the BM- derived myeloid cells, if recruited, either express the beneficial phenotype only transiently or lack the ability to manifest it at all. This might explain why the loss of neurons was accelerated in this mouse model (Weydt et al., 2004), and why the disease scenario was significantly affected only when SODl expression was attenuated at a relatively late stage (Boillee et al., 2006). It should be emphasized that IL-4-activated, IGF-I-expressing BM-derived myeloid cells that were obtained from wild-type mice and injected systemically into ALS mice did home to sites of CNS damage, where they continued to express IGF-I. That finding suggested that CDI IcVlGF-I⁺ cells are limited in mice with ALS. However, if BM cells that are already endowed with an IGF-producing dendritic-like phenotype are injected into the diseased mice, the acquired phenotype is retained in the diseased environment. Because IGF-I is a key therapeutic factor in ALS (Kaspar et al., 2003), and since it can be supplied by cells that serve to repopulate CNS parenchyma, such cells would appear to be the safest agents for self-delivery of the potential therapy. It is also important to note that the cells home to the motor neuron vicinity and not to the white matter, suggesting self-navigation to an immunological niche that is specifically formed in sites of need.

The significantly reduced incidence of dendritic-like cells expressing CDl Ic in the blood of patients with ALS suggests that a deficiency of these cells might be an important factor contributing to progression of the disease. It is not clear, however, whether BM-derived myeloid cells from ALS patients can be activated ex vivo by IL-4 and used as an autologous cell therapy. If their own cells cannot be suitably activated, it might be necessary to acquire the required ex-v/vo-activated myeloid cells from matched donors. In either case, it might be worth focusing on deficiency of dendritic-like myeloid cells as a possible biological marker, and on IL-4-activated dendritic-like cells as a potential therapy.

REFERENCES

Aberg, M. A., Aberg, N. D., Hedbacker, H., Oscarsson, J. & Eriksson, P. S. Peripheral infusion of IGF-I selectively induces neurogenesis in the adult rat hippocampus. J Neurosci 20, 2896-903 (2000). Aharoni, R., Teitelbaum, D., SeIa, M. & Arnon, R. Copolymer 1 induces T cells of the T helper type 2 that crossreact with myelin basic protein and suppress experimental autoimmune encephalomyelitis. Proc Natl Acad Sci U S A 94, 10821- 6 (1997).

Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G. M., Cooper, N. R., Eikelenboom, P., Emmerling, M., Fiebich, B. L., Finch, C. E., Frautschy, S., Griffin, W. S., Hampel, H., Hull, M. et al. Neurobiol Aging 21, 383- 421 (2000).

Akiyama, H. & McGeer, P. L. J Neuroimmunol 30, 81-93 (1990).

Aktas, O. et al. Green tea epigallocatechin-3-gallate mediates T cellular NF- kappa B inhibition and exerts neuroprotection in autoimmune encephalomyelitis. J Immunol 173, 5794-800 (2004).

Alexianu, M.E., Kozovska, M. & Appel, S.H. Immune reactivity in a mouse model of familial ALS correlates with disease progression. Neurology 57, 1282-9 (2001). Aloisi, F. Immune function of microglia. GHa 36, 165-79 (2001).

Angele, M. K., Ayala, A., Cioffi, W. G., Bland, K. I. & Chaudry, I. H. (1998) Am. J. Physiol. 274, C1530-6.

Angelov, D. N., Waibel, S., Guntinas-Lichius, O., Lenzen, M., Neiss, W. F., Tomov, T. L., Yoles, E., Kipnis, J., Schori, H., Reuter, A. et al. (2003) Proc Natl Acad Sci U S A 100, 4790-5.

Apelt, J., Lessig, J. & Schliebs, R. Beta-amyloid-associated expression of intercellular adhesion molecule- 1 in brain cortical tissue of transgenic Tg2576 mice. Neurosci Lett 329, 1 1 1-5 (2002). Asheuer, M. et al. Human CD34+ cells differentiate into microglia and express recombinant therapeutic protein. Proc Natl Acad Sci U S A 101, 3557-62 (2004).

Atkin, J. D. et al. Induction of the unfolded protein response in familial amyotrophic lateral sclerosis and association of protein disulfide isomerase with superoxide dismutase 1. J Biol Chem (2006).

Avidan, H., Kipnis, J., Butovsky, O., Caspi, R. R. & Schwartz, M. Vaccination with autoantigen protects against aggregated beta-amyloid and glutamate toxicity by controlling microglia: effect of CD4+CD25+ T cells (2004) Eur J Immunol 34, 3434-45.

Baetz, A., Frey, M., Heeg, K. & Dalpke, A. H. Suppressor of cytokine signaling (SOCS) proteins indirectly regulate toll-like receptor signaling in innate immune cells. J Biol Chem 279, 54708-15 (2004).

Bakalash, S., Shlomo, G. B., Aloni, E., Shaked, L, Wheeler, L., Ofri, R. & Schwartz, M. T-cell-based vaccination for morphological and functional neuroprotection in a rat model of chronically elevated intraocular pressure. (2005) J MoI Med 83, 904- 16.

Bard, F., Cannon, C, Barbour, R., Burke, R. L., Games, D., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson- Wood, K. et al. (2000) Nat Med 6, 916-9. Bechmann, I. et al. Circulating monocytic cells infiltrate layers of anterograde axonal degeneration where they transform into microglia. Faseb J 19, 647-9 (2005).

Benner, E. J., Mosley, R. L., Destache, C. J., Lewis, T. B., Jackson-Lewis, V., Gorantla, S., Nemachek, C, Green, S. R., Przedborski, S. & Gendelman, H. E. (2004) Proc Natl Acad Sci U S A 101, 9435-40.

Bieber, A. J., Kerr, S. & Rodriguez, M. Efficient central nervous system remyelination requires T cells. Ann Neurol 53, 680-4 (2003).

Boillee, S. et al. Onset and progression in inherited ALS determined by motor neurons and microglia. Science 312, 1389-92 (2006). Borchelt, D. R., Ratovitski, T., van Lare, J., Lee, M. K., Gonzales, V., Jenkins, N. A., Copeland, N. G., Price, D. L. & Sisodia, S. S. (1997) Neuron 19, 939-45.

Brazelton, T.R., Rossi, F.M., Keshet, G.I. & Blau, H.M. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 290, 1775-9 (2000).

Butovsky, O. et al. Induction and blockage of oligodendrogenesis by differently activated microglia in an animal model of multiple sclerosis. J Clin Invest 116, 905-15 (2006a). Butovsky, O. et al. Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells. MoI Cell Neurosci 31, 149-60 (2006b).

Butovsky, O. et al. Glatiramer acetate fights against Alzheimer's disease by inducing dendritic-like microglia expressing insulin-like growth factor 1. Proc Natl Acad Sci U S A 103, 11784-9 (2006c)

Butovsky, O., Hauben, E. & Schwartz, M. Morphological aspects of spinal cord autoimmune neuroprotection: colocalization of T cells with B7-2 (CD86) and prevention of cyst formation. Faseb J. 15, 1065-7 (2001).

Butovsky, O., Talpalar, A. E., Ben-Yaakov, K. & Schwartz, M. Activation of microglia by aggregated beta-amyloid or lipopolysaccharide impairs MHC-II expression and renders them cytotoxic whereas IFN-gamma and IL-4 render them protective. MoI Cell Neurosci 29, 381-93 (2005).

Campbell, JJ. et al. Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 279, 381-4 (1998). Cardona, A.E. et al. Control of microglial neurotoxicity by the fractalkine receptor. Nat Neurosci (2006).

Carson, M.J., Reilly, C.R., Sutcliffe, J. G. & Lo, D. Mature microglia resemble immature antigen-presenting cells. Glia 22, 72-85 (1998).

Chao, C. C, Molitor, T. W. & Hu, S. (1993) J Immunol 151, 1473-81. Chao, CC, Hu, S., Molitor, T. W., Shaskan, E.G. & Peterson, P.K. Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. J. Immunol. 149, 2736-41 (1992).

Cogle, CR. et al. Bone marrow transdifferentiation in brain after transplantation: a retrospective study. Lancet 363, 1432-7 (2004).

Corti, S. et al. Wild-type bone marrow cells ameliorate the phenotype of SOD1-G93A ALS mice and contribute to CNS, heart and skeletal muscle tissues. Brain 127, 2518-32 (2004).

Davalos, D. et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 8, 752-8 (2005).

DiCarlo, G., Wilcock, D., Henderson, D., Gordon, M. & Morgan, D. (2001) Neurobiol. Aging 22, 1007-12.

Duda, P. W., Schmied, M.C., Cook, S. L., Krieger, J.I. & Hafler, D.A. Glatiramer acetate (Copaxone) induces degenerate, Th2-polarized immune responses in patients with multiple sclerosis. J Clin Invest 105, 967-76 (2000).

Dudek, H. et al. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275, 661-5 (1997).

Ekdahl, C.T., Claasen, J.H., Bonde, S., Kokaia, Z. & Lindvall, O. Inflammation is detrimental for neurogenesis in adult brain. Proc Natl Acad Sci U S A 100, 13632-7 (2003).

Eriksson, P. S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A. M., Nordborg, C, Peterson, D. A. & Gage, F. H. (1998) Nat Med 4, 1313-7.

Fassbender, K. et al. The LPS receptor (CD 14) links innate immunity with Alzheimer's disease. Faseb J 18, 203-5 (2004). Frenkel, D., Maron, R., Burt, D. S. & Weiner, H. L. (2005) J Clin Invest 115,

2423-2433.

Frick, C. et al. Interaction of ICAM-I with beta2-integrin CDl lc/CD18: Characterization of a peptide ligand that mimics a putative binding site on domain D4 of ICAM-I . Eur J Immunol 35, 3610-21 (2005). Fricker, R.A. et al. Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain. J Neurosci 19, 5990-6005 (1999).

Fuchs, E. & Segre, J.A. Stem cells: a new lease on life. Cell 100, 143-55 (2000).

Gage, F.H. Mammalian neural stem cells. Science 287, 1433-8 (2000).

Geissmann, F., Jung, S. & Littman, D.R. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19, 71-82 (2003).

Goolsby, J. et al. Hematopoietic progenitors express neural genes. Proc Natl Acad Sci U S A 100, 14926-31 (2003).

Greter, M. et al. Dendritic cells permit immune invasion of the CNS in an animal model of multiple sclerosis. Nat Med 1 1, 328-34 (2005).

Gropp, E. et al. Agouti-related peptide-expressing neurons are mandatory for feeding. Nat Neurosci 8, 1289-91 (2005). Gupta, N, Brown, KE & Milam, AH. Activated microglia in human retinitis pigmentosa, late-onset retinal degeneration, and age-related macular degeneration. Exp Eye Res 76, 463-71 (2003).

Hailer, N. P., Grampp, A. & Nitsch, R. Proliferation of microglia and astrocytes in the dentate gyrus following entorhinal cortex lesion: a quantitative bromodeoxyuridine-labelling study. Eur J Neurosci 1 1, 3359-64 (1999).

Hardy, J. & Selkoe, D. J. (2002) Science 297, 353-6.

Hartung, H.P. et al. Inflammatory mediators in demyelinating disorders of the CNS and PNS. J Neuroimmunol 40, 197-210 (1992).

Hauben, E. & Schwartz, M. Therapeutic vaccination for spinal cord injury: helping the body to cure itself. Trends Pharmacol. Sci. 24, 7-12 (2003).

Haughey, N. J., Nath, A., Chan, S. L., Borchard, A. C, Rao, M. S. & Mattson, M. P. (2002) J Neurochem 83, 1509-24.

Heppner, F. L., Greter, M., Marino, D., Falsig, J., Raivich, G., Hovelmeyer, N., Waisman, A., Rulicke, T., Prinz, M., Priller, J. et al. Experimental autoimmune encephalomyelitis repressed by microglial paralysis. Nat Med 11, 146-52 (2005) Hickey, W. F., Vass, K. & Lassmann, H. Bone marrow- derived elements in the central nervous system: an immunohistochemical and ultrastructural survey of rat chimeras. J Neuropathol Exp Neurol 51, 246-56 (1992).

Iribarren, P., Cui, Y. H., Le, Y., Ying, G., Zhang, X., Gong, W. & Wang, J. M. (2003) J Immunol 171, 5482-8.

Jankowsky, J. L., Fadale, D. J., Anderson, J., Xu, G. M., Gonzales, V., Jenkins, N. A., Copeland, N. G., Lee, M. K., Younkin, L. H., Wagner, S. L. et al. (2004) Hum MoI Genet 13, 159-70.

Jin, K., Galvan, V., Xie, L., Mao, X. O., Gorostiza, O. F., Bredesen, D. E. & Greenberg, D. A. (2004a) Proc Natl Acad Sci U S A 101, 13363-7.

Jin, K., Peel, A. L., Mao, X. O., Xie, L., Cottrell, B. A., Henshall, D. C. & Greenberg, D. A. (2004b) Proc Natl Acad Sci U S A 101, 343-7.

Jung. S. et al. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. MoI Cell Biol 20, 4106-14 (2000).

Jung, S. et al. In vivo depletion of CDl lc(+) dendritic cells abrogates priming of CD8(+) T cells by exogenous cell-associated antigens. Immunity 17, 21 1-20 (2002).

Jung, S. et al. Induction of IL-IO in rat peritoneal macrophages and dendritic cells by glatiramer acetate. J Neuroimmunol 148, 63-73 (2004).

Kaspar, B.K., Llado, J., Sherkat, N., Rothstein, J.D. & Gage, F.H. Retrograde viral delivery of IGF-I prolongs survival in a mouse ALS model. Science 301, 839- 42 (2003).

Kempermann, G., Jessberger, S., Steiner, B. & Kronenberg, G. Milestones of neuronal development in the adult hippocampus. Trends Neurosci 27, 447-52 (2004).

Kerschensteiner, M., Gallmeier, E., Behrens, L., Leal, V. V., Misgeld, T., Klinkert, W. E., Kolbeck, R., Hoppe, E., Oropeza-Wekerle, R. L., Bartke, I. et al. (1999) J. Exp. Med. 189, 865-70. Kilgore, K.S., Shen, J.P., Miller, B.F., Ward, P.A. & Warren, J.S. Enhancement by the complement membrane attack complex of tumor necrosis factor-alpha-induced endothelial cell expression of E-selectin and ICAM-I . J Immunol 155, 1434-41 (1995). Kim, HJ. et al. Type 2 monocyte and microglia differentiation mediated by glatiramer acetate therapy in patients with multiple sclerosis. J Immunol 172, 7144- 53 (2004).

Kipnis, J., Cohen, H., Cardon, M., Ziv, Y. & Schwartz, M. T cell deficiency leads to cognitive dysfunction: implications for therapeutic vaccination for schizophrenia and other psychiatric conditions (2004) Proc Natl Acad Sci U S A 101, 8180-5.

Kipnis, J., Mizrahi, T., Hauben, E., Shaked, L, Shevach, E. & Schwartz, M. Neuroprotective autoimmunity: naturally occurring CD4+CD25+ regulatory T cells suppress the ability to withstand injury to the central nervous system. (2002) Proc Natl Acad Sci U S A 99, 15620-5.

Kipnis, J. & Schwartz, M. Dual action of glatiramer acetate (Cop-1) in the treatment of CNS autoimmune and neurodegenerative disorders (2002) Trends MoI Med 8, 319-23.

Kipnis, J., Yoles, E., Porat, Z., Cohen, A., Mor, F., SeIa, M., Cohen, I. R. & Schwartz, M. T cell immunity to copolymer 1 confers neuroprotection on the damaged optic nerve: possible therapy for optic neuropathies. (2000) Proc. Natl. Acad. Sci. U S A 97, 7446-51.

Krause, D. S. et al. Characterization of murine CD34, a marker for hematopoietic progenitor and stem cells. Blood 84, 691-701 (1994). Kreutzberg, G. W. Microglia: a sensor for pathological events in the CNS.

Trends Neurosci 19, 312-8 (1996).

Ladeby, R. et al. Proliferating resident microglia express the stem cell antigen CD34 in response to acute neural injury. Glia 50, 121-31 (2005).

Lawson, L.J., Perry, V.H. & Gordon, S. Turnover of resident microglia in the normal adult mouse brain. Neuroscience 48, 405-15 (1992). Lazarov, O., Robinson, J., Tang, Y. P., Hairston, I. S., Korade-Mirnics, Z., Lee, V. M., Hersh, L. B., Sapolsky, R. M., Mimics, K. & Sisodia, S. S. (2005) Cell 120, 701-13.

Lichtenwalner, R. J., Forbes, M. E., Bennett, S. A., Lynch, C. D., Sonntag, W. E. & Riddle, D. R. (2001) Neuroscience 107, 603-13.

Livak, KJ. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-8 (2001).

Lotz, M. et al. Amyloid beta peptide 1-40 enhances the action of Toll-like receptor-2 and -4 agonists but antagonizes Toll-like receptor-9-induced inflammation in primary mouse microglial cell cultures. J Neurochem 94, 289-98 (2005).

Lum, H. & Roebuck, K.A. Oxidant stress and endothelial cell dysfunction. Am J Physiol Cell Physiol 280, C719-41 (2001). McGeer, P.L., McGeer, E.G., Kawamata, T., Yamada, T. & Akiyama, H.

Reactions of the immune system in chronic degenerative neurological diseases. Can J Neurol Sci 18, 376-9 (1991).

Menegazzi, M. et al. Anti-interferon gamma action of epigallocatechin-3- gallate mediated by specific inhibition of STATl activation. Faseb J 15, 1309-11 (2001). [not in original manuscript text]

Merrill, J.E., Ignarro, L.J., Sherman, M.P., Melinek, J. & Lane, T.E. Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide. J Immunol 151, 2132-41 (1993).

Mezey, E., Chandross, KJ. , Harta, G., Maki, R.A. & McKercher, S.R. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 290, 1779-82 (2000).

Miller, A. et al. Treatment of multiple sclerosis with copolymer- 1 (Copaxone): implicating mechanisms of ThI to Th2/Th3 immune-deviation. J Neuroimmunol 92, 1 13-21 (1998). Minghetti, L. Role of inflammation in neurodegenerative diseases. Curr Opin Neurol 18, 315-21 (2005).

Moalem, G. et al. Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat. Med. 5, 49-55 (1999). Monje, M.L., Mizumatsu, S., Fike, J.R. & Palmer, T.D. Irradiation induces neural precursor-cell dysfunction. Nat Med 8, 955-62 (2002).

Monje, M.L., Toda, H. & Palmer, T.D. Inflammatory blockade restores adult hippocampal neurogenesis. Science 302, 1760-5 (2003).

Monsonego, A., Imitola, J., Petrovic, S., Zota, V., Nemirovsky, A., Baron, R., Fisher, Y., Owens, T. & Weiner, H. L. (2006) Proc Natl Acad Sci U S A.

Morel, F., Szilvassy, SJ., Travis, M., Chen, B. & GaIy, A. Primitive hematopoietic cells in murine bone marrow express the CD34 antigen. Blood 88, 3774-84 (1996).

Morris, R. (1984) J Neurosci Methods 1 1, 47-60. Mullins, R.F., Skeie, J.M., Malone, E.A. & Kuehn, M.H. Macular and peripheral distribution of ICAM- 1 in the human choriocapillaris and retina. MoI Vis 12, 224-35 (2006).

Neuhaus, O., Farina, C, Wekerle, H. & Hohlfeld, R. Mechanisms of action of glatiramer acetate in multiple sclerosis. Neurology 56, 702-8 (2001). Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314-8 (2005).

Nordal, R.A. & Wong, CS. Intercellular adhesion molecule-1 and blood- spinal cord barrier disruption in central nervous system radiation injury. J Neuropathol Exp Neurol 63, 474-83 (2004).

Olsson, T. Critical influences of the cytokine orchestration on the outcome of myelin antigen-specific T-cell autoimmunity in experimental autoimmune encephalomyelitis and multiple sclerosis. Immunol Rev 144, 245-68 (1995). Olsson, T. Cytokines in neuroinflammatory disease: role of myelin autoreactive T cell production of interferon-gamma. J Neuroimmunol 40, 211-8 (1992).

Picard-Riera, N. et al. Experimental autoimmune encephalomyelitis mobilizes neural progenitors from the subventricular zone to undergo oligodendrogenesis in adult mice. Proc Natl Acad Sci U S A 99, 13211-6 (2002).

Pluchino, S. et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 422, 688-94 (2003).

Pluchino, S. et al. Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism. Nature 436, 266-71 (2005).

Rabizadeh, S. et al. Mutations associated with amyotrophic lateral sclerosis convert superoxide dismutase from an antiapoptotic gene to a proapoptotic gene: studies in yeast and neural cells. Proc Natl Acad Sci U S A 92, 3024-8 (1995).

Rao, M. S. & Shetty, A. K. (2004) Eur J Neurosci 19, 234-46. Rivera, E. J., Goldin, A., Fulmer, N., Tavares, R., Wands, J. R. & de Ia

Monte, S. M. (2005) J Alzheimers Dis 8, 247-68.

Roebuck, K.A. & Finnegan, A. Regulation of intercellular adhesion molecule- 1 (CD54) gene expression. J Leukoc Biol 66, 876-88 (1999).

Roque, RS, Rosales, AA, Jingjing, L, Agarwal, N & Al-Ubaidi, MR. Retina- derived microglial cells induce photoreceptor cell death in vitro. Brain Res 836, 110-9 (1999).

Ross, CA. & Poirier, M.A. Protein aggregation and neurodegenerative disease. Nat Med 10 Suppl, S 10-7 (2004).

Roybon, L. et al. Failure of transdifferentiation of adult hematopoietic stem cells into neurons. Stem Cells (2006).

Sanna, A. et al. Glatiramer acetate reduces lymphocyte proliferation and enhances IL-5 and IL- 13 production through modulation of monocyte-derived dendritic cells in multiple sclerosis. Clin Exp Immunol 143, 357-62 (2006).

Santambrogio, L. et al. Developmental plasticity of CNS microglia. Proc Natl Acad Sci U S A 98, 6295-300 (2001 ). Schori, H., Kipnis, J., Yoles, E., WoldeMussie, E., Ruiz, G., Wheeler, L. A. & Schwartz, M. Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: implications for glaucoma (2001) Proc. Natl. Acad. Sci. U S A 98, 3398-403. Schwartz, M., Butovsky, O., Bruck, W. & Hanisch, U. K. Microglial phenotype: is the commitment reversible? (2006) Trends Neurosci 29, 68-74.

Schwartz, M., Shaked, L, Fisher, J., Mizrahi, T. & Schori, H. Protective autoimmunity against the enemy within: fighting glutamate toxicity. Trends Neurosci. 26, 297-302 (2003). Scott, G. S. et al. ICAM-I upregulation in the spinal cords of PLSJL mice with experimental allergic encephalomyelitis is dependent upon TNF-alpha production triggered by the loss of blood-brain barrier integrity. J Neuroimmunol 155, 32-42 (2004).

Selkoe, D. J. (1991) Neuron 6, 487-98. Shaked, I. et al. Protective autoimmunity: interferon-gamma enables microglia to remove glutamate without evoking inflammatory mediators. J Neurochem 92, 997- 1009 (2005).

Shaked, I., Porat, Z., Gersner, R., Kipnis, J. & Schwartz, M. Early activation of microglia as antigen-presenting cells correlates with T cell-mediated protection and repair of the injured central nervous system. J Neuroimmunol 146, 84-93 (2004).

Shen, W.H. et al. Tumor necrosis factor alpha inhibits insulin-like growth factor I-induced hematopoietic cell survival and proliferation. Endocrinology 145, 3101-5 (2004). Sheng, J. G., Mrak, R. E. & Griffin, W. S. (1998) Acta Neuropathol (Berl)

95, 229-34.

Simard, A.R. & Rivest, S. Bone marrow stem cells have the ability to populate the entire central nervous system into fully differentiated parenchymal microglia. Faseb J 18, 998-1000 (2004). Simard, A.R., Soulet, D., Gowing, G., Mien, J.P. & Rivest, S. Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron 49, 489-502 (2006).

Soghomonian, JJ. & Martin, D. L. Two isoforms of glutamate decarboxylase: why? Trends Pharmacol Sci 19, 500-5 (1998).

Stacker, S.A. & Springer, T.A. Leukocyte integrin P150,95 (CDl lc/CDlβ) functions as an adhesion molecule binding to a counter-receptor on stimulated endothelium. J Immunol 146, 648-55 (1991 ).

Stathopulos, P. B. et al. Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro. Proc Natl Acad Sci U S A 100, 7021-6 (2003).

Stirling, D. P. et al. Minocycline treatment reduces delayed oligodendrocyte death, attenuates axonal dieback, and improves functional outcome after spinal cord injury. J Neurosci 24, 2182-90 (2004). Streit, W. J. Microglia and Alzheimer's disease pathogenesis. J Neurosci Res

77, 1-8 (2004).

Streit, WJ. Microglia and neuroprotection: implications for Alzheimer's disease. Brain Res Brain Res Rev 48, 234-9 (2005).

Streit, WJ., Walter, S.A. & Pennell, N.A. Reactive microgliosis. Prog Neurobiol 57, 563-81 (1999).

Taylor, J.P., Hardy, J. & Fischbeck, K.H. Toxic proteins in neurodegenerative disease. Science 296, 1991-5 (2002).

Teitelbaum, D., Fridkis-Hareli, M., Arnon, R. & SeIa, M. (1996) J Neuroimmunol 64, 209-17. Townsend, P. A. et al. Epigallocatechin-3-gallate inhibits STAT-I activation and protects cardiac myocytes from ischemia/reperfusion-induced apoptosis. Faseb J 18, 1621-3 (2004). [not in original manuscript text]

Tozuka, Y., Fukuda, S., Namba, T., Seki, T. & Hisatsune, T. GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells. Neuron 47, 803-15 (2005). Valente, E. M. et al. Hereditary early-onset Parkinson's disease caused by mutations in PINKl . Science 304, 1 158-60 (2004). van de Stolpe, A. & van der Saag, P.T. Intercellular adhesion molecule- 1. J MoI Med 74, 13-33 (1996). van Praag, H., Kempermann, G. & Gage, F. H. (2000) Nat Rev Neurosci 1,

191-8.

Verbeek, M.M. et al. Accumulation of intercellular adhesion molecule- 1 in senile plaques in brain tissue of patients with Alzheimer's disease. Am J Pathol 144, 104-16 (1994). Vieira, P. L., Heystek, H. C, Wormmeester, J., Wierenga, E.A. &

Kapsenberg, M.L. Glatiramer acetate (copolymer- 1, Copaxone) promotes Th2 cell development and increased IL-IO production through modulation of dendritic cells. J Immunol 170, 4483-8 (2003).

Wagers, A. J., Sherwood, R.I., Christensen, J.L. & Weissman, LL. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 297, 2256-9 (2002).

Watson, J.L. et al. Green tea polyphenol (-)-epigallocatechin gallate blocks epithelial barrier dysfunction provoked by IFN-gamma but not by IL-4. Am J Physiol Gastrointest Liver Physiol 287, G954-61 (2004). [not in original manuscript text]

Weber, M.S. et al. Multiple sclerosis: glatiramer acetate inhibits monocyte reactivity in vitro and in vivo. Brain 127, 1370-8 (2004).

Weissman, LL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 100, 157-68 (2000a). Weissman, LL. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science 287, 1442-6 (2000b).

Werdelin, L., Boysen, G., Jensen, T.S. & Mogensen, P. Immunosuppressive treatment of patients with amyotrophic lateral sclerosis. Acta Neurol Scand 82, 132- 4 (1990). Weydt, P., Yuen, E. C, Ransom, B.R. & Moller, T. Increased cytotoxic potential of microglia from ALS-transgenic mice. GHa 48, 179-82 (2004).

Wyss-Coray, T. & Mucke, L. Inflammation in neurodegenerative disease— a double-edged sword. Neuron 35, 419-32 (2002). Yin, Y. et al. Macrophage-derived factors stimulate optic nerve regeneration.

J. Neurosci. 23, 2284-93 (2003).

Yokoyama, A., Yang, L., Itoh, S., Mori, K. & Tanaka, J. Microglia, a potential source of neurons, astrocytes, and oligodendrocytes. GHa 45, 96-104 (2004). Yoles, E., Hauben, E., Palgi, O., Agranov, E., Gothilf, A., Cohen, A.,

Kuchroo, V., Cohen, I. R., Weiner, H. & Schwartz, M. Protective autoimmunity is a physiological response to CNS trauma (2001) J Neurosci 21, 3740-8.

Zhang, S. C. & Fedoroff, S. Expression of stem cell factor and c-kit receptor in neural cells after brain injury. Acta Neuropathol (Bed) 97, 393-8 (1999). Ziemssen, T., Kumpfel, T., Klinkert, W. E., Neuhaus, O. & Hohlfeld, R.

(2002) Brain 125, 2381-91.

Ziv, Y., Ron, N., Butovsky, O., Landa, G., Sudai, E., Greenberg, N., Cohen, H., Kipnis, J. & Schwartz, M. Immune cells contribute to the maintenance of neurogenesis and spatial learning abilities in adulthood (2006a) Nat Neurosci 9, 268-75.

Ziv, Y., Avidan, H., Pluchino, S., Martino, G. & Schwartz, M. Synergy between immune cells and adult neural stem/progenitor cells promotes functional recovery from spinal cord injury. Proc Natl Acad Sci U S A 103, 13174-9 (2006b).

Claims

CLAIMS:

1. A method for promoting tissue repair comprising administering to an individual in need a therapeutically effective amount of CDl Ic⁺ bone marrow- derived myeloid cells.

2. The method according to claim 1 , wherein said CDl Ic⁺ bone marrow- derived myeloid cells express IGF-I, BDNF or both.

3. The method according to claim 2, wherein said cells express IGF-I

4. The method according to any of claims 1 to 3 wherein said cells are obtained by activation of bone marrow-derived myeloid cells with at least one cytokine selected from the froup consisting of IL-4, IL- 13 and up to 20 ng/ml IFN-γ.

5. The method according to claim 4, wherein said cytokine is IL-4.

6. The method according to claim 4, wherein said at least one cytokine is a mixture of IL-4 and up to 20 ng/ml IFN-γ.

7. The method according to claim 1, wherein said tissue is a damaged body tissue selected from neural, cardiac, liver, renal, bladder, muscle, intestinal, or visual system tissue.

8. The method according to claim 1, for promoting neural tissue repair in a patient suffering from a neurological disease.

9. The method according to claim 8, wherein sai'd neurological disease is an injury in the brain or in the spinal cord or a neurodegenerative disease or disorder selected from Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, a neuropathy, a mental disorder, a cognitive dysfunction, dementia, or the aging process and senescence.

10. The method according to claim 1, for promoting tissue repair in a patient after an ischemia event in the heart, brain or kidney.

11. The method according to claim 1 , for promoting tissue repair in a patient suffering from a cardiovascular disease.

12. The method according to claim 1 1, wherein the cardiovascular disease is a myocardial infarction, an ischemic heart disease or congestive heart failure.

13. The method according to claim 1, for promoting tissue repair in a patient suffering from an autoimmune disease.

14. The method according to claim 1. wherein the cells are autologous cells obtained from the individual in need.

15. The method according to claim 14, wherein the autologous cells are isolated from the individual's peripheral blood or bone marrow.

16. The method according to claim 1, wherein the cells are allogeneic cells obtained from an HLA-matched donor.

17. A method for detecting and localizing a damaged tissue comprising administering bone marrow-derived myeloid cells that have been activated with at least one cytokine selected from IL-4, IL- 13 or up to 20 ng/ml IFN-γ and are labeled with an imaging agent to an individual having or suspected of having a damaged tissue, whereby the labeled cells traffic to the damaged tissue, and imaging the suspected tissue area in the individual, thereby localizing the damaged tissue.

18. The method according to claim 17, wherein the cells are loaded with a paramagnetic or superparamagnetic contrast agent and the patient is subjected to magnetic resonance imaging (MRI).

19. A method for delivering a therapeutic or detectable substance to a damaged tissue or a tumor, said method comprising administering to a patient in need bone marrow-derived myeloid cells that have been activated with at least one cytokine selected from IL-4, IL- 13 or up to 20 ng/ml IFN-γ, wherein said cells are cells that have been genetically engineered to express said therapeutic or detectable substance.

20. A method for monitoring the response of a patient being treated for a neurodegenerative or autoimmune disease or disorder to a therapeutic drug for said disease or disorder, comprising:

(a) determining the level of CDl Ic⁺ myeloid cells in a first sample of peripheral blood taken from the patient prior to treatment with the therapeutic drug; (b) determining the level of the CDl Ic⁺ myeloid cells in at least one blood sample taken from the patient subsequent to the initial treatment with the therapeutic drug; and c) comparing the level of the CDl Ic⁺ myeloid cells in the at least one blood sample of (b) with the level of the CDl Ic⁺ myeloid cells in the first blood sample of (a); wherein an increase in the level of the CDl Ic⁺ myeloid cells in the at least one blood sample of (b) compared to the level of the CDl Ic⁺ myeloid cells in the first blood sample of (a), indicates that the therapeutic drug is effective in treating said neurodegenerative or autoimmune disease or disorder in said patient.

21. The method according to claim 20, wherein the patient is being treated for a neurodegenerative disease or disorder.

22. The method according to claim 21, wherein the neurodegenerative disease or disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, age- related macular degeneration, a neuropathy, a mental disorder, a cognitive dysfunction, dementia, and a prion disease.

23. The method according to claim 22, wherein the neurodegenerative disease is multiple sclerosis.

I l l

24. The method according to claim 23 for monitoring transition between periods of remission and relapse in a relapsing/remitting multiple sclerosis patient, comprising:

(a) determining the level of CDl Ic⁺ myeloid cells in a first sample of peripheral blood taken from the multiple sclerosis patient in a period of remission;

(b) determining the level of the CD 1 1 C⁺ myeloid cells in a plurality of blood samples taken from the multiple sclerosis patient periodically ; and

(c) comparing the level of the CDl Ic⁺ myeloid cells in the blood sample of (b) taken most recently with the level of the CDl I c⁺ myeloid cells in a previous blood sample of (a) or (b); wherein a decrease in the level of the CDl Ic⁺ myeloid cells in the most recent blood sample compared to the level of the CDl Ic⁺ myeloid cells in the previous blood sample, indicates a transition from a period of remission to a period of relapse.

25. The method according to claim 22, wherein the neurodegenerative disease is amyotrophic lateral sclerosis.

26. The method according to claim 20, wherein the patient is being treated for an autoimmune disease or disorder.

27. The method according to claim 26, wherein the autoimmune disease or disorder is selected from the group consisting of Eaton-Lambert syndrome, Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome, autoimmune hemolytic anemia (AIHA), hepatitis, insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), myasthenia gravis, plexus disorders, polyglandular deficiency syndrome, primary biliary cirrhosis, rheumatoid arthritis, scleroderma, thrombocytopenia, thyroiditis, Sjogren's syndrome, allergic purpura, psoriasis, mixed connective tissue disease, polymyositis, dermatomyositis, vasculitis, polyarteritis nodosa, polymyalgia rheumatica, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, pemphigus, bullous pemphigoid, dermatitis herpetiformis, inflammatory bowel disease and uveitis.

28. A method for the diagnosis of a neurodegenerative or autoimmune disease or disorder in an individual, comprising: (a) determining the level Of CDl Ic⁺ myeloid cells in a sample of peripheral blood taken from an individual suspected of having a neurodegenerative or autoimmune disease;

(b) determining the level Of CDl Ic⁺ myeloid cells in a sample of peripheral blood taken from at least one individual not suffering from a neurodegenerative or autoimmune disease or disorder and;

(c) comparing the level of the CDl Ic⁺ myeloid cells in the blood sample taken from said individual in (a) with the level of CDl Ic+ myeloid cells in the blood sample taken from said at least one individual of (b); wherein a lower level of CDl Ic+ myeloid cells in the blood sample of the individual of (a) relative to the level of CDl Ic⁺ myeloid cells in the blood sample of said at least one individual of (b), is indicative of a neurodegenerative or autoimmune disease or disorder in the individual of (a).

29. A cellular preparation comprising CDl Ic⁺ bone marrow-derived myeloid cells and a physiologically acceptable carrier, for promoting repair of damaged body tissue.

30. The cellular preparation according to claim 29, wherein said CDl Ic⁺ bone marrow-derived myeloid cells express IGF-I, BDNF or both.

31. The cellular preparation according to claim 30, wherein said CDl Ic⁺ bone marrow-derived myeloid cells express IGF-I.

32. The cellular preparation according to claim 30, wherein said CDl Ic⁺ bone marrow-derived myeloid cells are obtained by activation of bone marrow- derived myeloid cells with at least one cytokine selected from the froup consisting of IL-4, IL- 13 and up to 20 ng/ml IFN-γ.

33. The cellular preparation according to claim 32, wherein the at least one cytokine is IL-4.

34. The cellular preparation according to claim 32, wherein the at least one cytokine is a mixture of IL-4 and up to 20 ng/ml IFN-γ.

35. The cellular preparation according to any of claims 29 to 34, for administration by sytemic infusion, local arterial infusion, venous infusion, or direct injection into the tissue.

36. A cellular preparation for detection and localization of damaged body tissue, comprising bone marrow-derived myeloid cells that have been activated with at least one cytokine selected from the group consisting of IL-4, IL- 13 and up to 20 ng/ml IFN-γ and labeled with an imaging agent.