In the present study, we investigated a novel immunotherapeutic approach for the reduction of Aβ in a mouse model of AD. We found that induction of EAE in APP-Tg mice reduces amyloid load in the brain in an antibody-independent fashion. This effect could be reproduced without induction of EAE using GA given parenterally or nasally with an adjuvant. GA is an FDA-approved drug used for the treatment of relapsing-remitting MS. The mechanism of action of GA in MS appears to be related to its induction of T cells that secrete antiinflammatory cytokines that mediate bystander suppression in the CNS (26
). Furthermore, GA has been shown to be of benefit in experimental models of CNS trauma (37
), optic nerve injury (38
), amyotrophic lateral sclerosis (39
), and Parkinson disease (40
). The mechanism of action in these models is not known, although it may relate to the induction of T cells that release growth factors and/or IFN-γ (38
In previous studies involving mouse models of AD in which animals were immunized with Aβ in CFA, anti-amyloid antibodies were shown to have a key role both in vitro (11
) and in vivo (4
) in reducing amyloid load. The presence of anti-Aβ antibodies and microglia-like cells that were immunopositive for amyloid has led to the speculation that clearance of Aβ is the result of FcR-mediated phagocytosis by microglia of anti-Aβ and Aβ immune complexes. Since microglia are phagocytic cells, it has been suggested that microglia may function as plaque-attacking scavenger cells (41
We believe that activation of microglia is the final common pathway for Aβ clearance in our immunization experiments. It has been reported that increased expression of M-CSFR in mouse and human microglia accelerates phagocytosis of aggregated Aβ, through increasing both expression of macrophage scavenger receptors and/or microglial expression of FcRγ (42
). As we observed robust clearance of Aβ in B cell–deficient mice that also had increased staining for M-CSFR, the effects we observed are occurring via a non–Fc-mediated mechanism (43
). Microglial activation by subcutaneous or nasal vaccination was correlated with increased numbers of T cells, which may play a role in promoting microglial activation, as there was a correlation between numbers of T cells and numbers of IFN-γ–secreting cells. Previous studies have reported that TGF-β may either increase or reduce Aβ fibril formation in the APP-Tg mouse (45
). We observed a reduction in TGF-β expression in MOG- and GA+IVX-908–treated mice compared with controls. Furthermore, there was a strong correlation between a reduction in TGF-β expression and the percentage of Aβ fibril in the hippocampus region.
In the case of EAE, we postulate that Th1-type myelin-reactive T cells are activated in the periphery by immunization with MOG or PLP plus CFA, and these T cells migrate to the brain, where they release IFN-γ and activate microglia. Encephalomyelitis and paralysis of animals results from damage to myelin and underlying axons. Immunization with BSA/CFA in the periphery did not lead to Aβ clearance because BSA-specific Th1-type cells do not accumulate in the brain. Peripheral immunization with GA in CFA induces GA-specific T cells that have been shown in the EAE model to accumulate in the brain due to cross-reactivity with MBP (26
) and that, we hypothesize, accumulate in the brains of immunized APP-Tg animals. These T cells are then able to secrete IFN-γ and activate microglia but are unable to cause EAE because of altered affinity for MBP and the concomitant secretion of antiinflammatory cytokines (26
). Nasal administration of IVX-908 alone leads to some clearance of Aβ, as it is able to activate microglia through its LPS effects. It was reported that injection of LPS into the hippocampus can cause clearance of Aβ (47
). Nevertheless, IVX-908 alone did not clear Aβ as efficiently as the combination of IVX-908 plus GA, which may be a stronger activator of T cells than LPS. Importantly, in non-Tg mice, no microglia activation was observed when GA was given parenterally with CFA or intranasally with IVX-908. Aβ deposition leads to partial activation of microglia surrounding the plaque, and it appears that this activation is a prerequisite for microglia to be further activated by GA+IVX-908. It is possible that partial activation of microglia is associated with the expression of IFN-γ or Toll-like receptors that prime microglia for further activation by IVX-908 (35
). It was reported (48
) that Protollin could be stimulating the microglial cells both through LPS via TLR-4 and through Por B, which makes up 70% of the proteosome protein and is known to activate APCs via TLR-2.
In previous studies (5
) in which we nasally administrated Aβ peptide without an adjuvant, the mechanism of Aβ clearance was postulated to be the induction of anti-Aβ antibodies, as there was a correlation between antibody levels and Aβ clearance. We also found a small number of cells that secreted IL-10 and TGF-β in the brain. These types of cells are preferably induced following mucosal administration of antigen. In the present work, we immunized nasally using GA or parenterally using myelin antigens or GA in Th1-type adjuvants. The reduction in Aβ levels in the present study was related to cellular mechanisms associated with IFN-γ in the brain and a reduction in the expression of the antiinflammatory cytokine TGF-β (no changes were observed for IL-10). Thus, the mechanism of action leading to Aβ clearance and the antigens used for nasal or parenteral immunization in the present study are quite distinct from those in our prior work.
Moreover, even though there was clearance of Aβ both in EAE and with GA+IVX-908 treatment, GA+IVX-908 treatment was not associated with the neuronal toxicity that was observed in EAE. Furthermore, there was an actual reduction in astrocytosis in the GA+IVX–908 treated animals compared with untreated control animals. In our studies, we did not measure cognitive function, though others have shown improvement in cognitive tasks in animals that have had a lowering of Aβ levels (6
When considering immune-based therapy for the treatment of AD, one must carefully consider the pros and cons of any such therapy, given the untoward side effects that occurred following immunization with Aβ, a therapy designed to induce anti-Aβ antibodies. A major question that arises regarding the antibody-independent strategy we have described is whether the induction of an inflammatory response in the brains of humans that includes a Th1 component will have untoward side effects (50
). It is postulated that the meningoencephalitis observed following Aβ immunization was related to the induction of Aβ-reactive T cells, and we have shown that there is increased T cell reactivity to Aβ in elderly individuals (53
). In our animal studies, in which we induced EAE or nasally administered GA+IVX-908, we did not observe induction of anti-Aβ antibodies or priming of anti-Aβ T cells, though the latter remains a possibility.
Inflammation, in itself, is not a specific hallmark of disease, and as a host defense mechanism, it may have beneficial effects, as we have demonstrated in our studies. Furthermore, inflammatory processes may differ. We observed a clear difference in the type of inflammation in EAE animals and those treated nasally with GA+IVX-908, even though both led to clearance of Aβ. In EAE, there was breakdown of the BBB, no reduction in astrocytosis, evidence of neuronal death, and nitric oxide oxidative stress. None of these effects of inflammation was observed with nasal GA+IVX-908 treatment.
We have demonstrated clearance of Aβ in association with microglia activation. It should be pointed out that microglial activation can have both positive and negative effects (54
). Microglia represent a natural mechanism by which protein aggregates and debris can be removed from the brain, and there are reports that microglial activation following Aβ immunization or stroke may lead to Aβ clearance (20
). In animal studies, Wyss-Coray and colleagues (32
) demonstrated that there is prominent neurodegeneration and increased plaque formation in the complement-inhibited AD mouse model, in which microglia were significantly less activated than in wild-type AD mice at the same age. This supports the concept that activated microglia have a beneficial role in decreasing amyloid load without causing major neurotoxicity in the AD mouse model.
In an AD patient with stroke, Akiyama and McGeer (55
) reported a local reduction in senile plaques in a neocortical region affected by incomplete ischemia and suggested that their findings and those reported by Nicoll et al. (20
) could be related to phagocytosis of amyloid by highly reactive microglia. We have reported elevation of reactive microglia (CD11b+
cells) in ischemic area following stroke model in mouse (29
). Nonetheless, there could be dysfunction of microglia in AD (56
), and activation of microglia could lead to further damage.____
___Although we observed clearance of amyloid fibrils in association with microglial activation, it should be kept in mind that there are differences in the biochemical and solubility characteristics between amyloid deposits in mice and humans (57
). Due to post-translational processes, amyloid peptides in humans are considerably more derivative and thus more insoluble. Despite the stroke example cited above, in humans there are instances of microglial reaction to amyloid, yet no apparent clearance as has been observed with AD patients that have had multiple bouts of inflammatory disease before they die. Thus, the controlled inflammatory stimuli we have produced in our mouse studies may not lead to significant clearance of amyloid in humans.
Despite the fact that we did not observe toxicity in animals given GA+IVX-908, it must be pointed out that AD is a chronic disease, and chronic administration of GA+IVX-908 might cause side effects that we did not observe in our animal studies. Nonetheless, the fact that we observed a decrease in Aβ load even after treatment for 6 weeks raises the possibility that clinical effects may be observed after a short period of therapy in humans. Furthermore, since we also found reduction of Aβ without behavior changes in animals given GA+IVX-908 for 8 months, it is possible that chronic human therapy may be well tolerated.
In summary, our results define a novel immune therapeutic approach for the treatment of AD that is antibody independent and is mediated by activated microglia. By combining an approved immunomodulatory drug used to treat MS with a nasal adjuvant, we were able to activate microglia and clear Aβ with 2 compounds that have been used in humans with no toxicity; however, it should also be pointed out that although IVX-908 and GA have been given to humans without toxicity, they have not been tested given together. Given the potential side effects described above, they should be cautiously applied in human studies. Nonetheless, in view of studies showing that immune-mediated reduction of cerebral Aβ leads to cognitive improvement in APP-Tg mice, our finding that a novel vaccination approach clears Aβ in older Tg mice with no apparent toxicity in animals, provides a new avenue for immune therapy that might prove efficacious in the treatment of AD.