Toll-like receptor 4-dependent upregulation of cytokines in a transgenic mouse model of Alzheimer's disease.

BACKGROUND: Abeta deposits in the brains of patients with Alzheimer's disease (AD) are closely associated with innate immune responses such as activated microglia and increased cytokines. Accumulating evidence supports the hypothesis that innate immune/inflammatory responses play a pivotal role in the pathogenesis of AD: either beneficial or harmful effects on the AD progression. The molecular mechanisms by which the innate immune system modulates the AD progression are not well understood. Toll-like receptors (TLRs) are first-line molecules for initiating the innate immune responses. When activated through TLR signaling, microglia respond to pathogens and damaged host cells by secreting chemokines and cytokines and express the co-stimulatory molecules needed for protective immune responses to pathogens and efficient clearance of damaged tissues. We previously demonstrated that an AD mouse model homozygous for a destructive mutation of TLR4 has increases in diffuse and fibrillar Abeta deposits as well as buffer-soluble and insoluble Abeta in the brain as compared with a TLR4 wild-type AD mouse model. Here, we investigated the roles of TLR4 in Abeta-induced upregulation of cytokines and chemokines, Abeta-induced activation of microglia and astrocytes and Abeta-induced immigration of leukocytes. METHODS: Using the same model, levels of cytokines and chemokines in the brain were determined by multiplex cytokine/chemokine array. Activation of microglia and astrocytes and immigration of leukocytes were determined by immunoblotting and immunohistochemistry followed by densitometry and morphometry, respectively. RESULTS: Levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-10 and IL-17 in the brains of TLR4 wild-type AD mice were significantly higher than those in TLR4 wild-type non-transgenic littermates. Such increases in cytokines were not found in TLR4 mutant AD mice as compared with TLR4 mutant non-transgenic littermates. Although expression levels of CD11b (a microglia marker) and GFAP (a reactive astrocyte marker) in the brains of TLR4 mutant AD mice were higher than those in TLR4 wild type AD mice, no difference was found in levels of CD45 (common leukocyte antigen). CONCLUSION: This is the first demonstration of TLR4-dependent upregulation of cytokines in an AD mouse model. Our results suggest that TLR4 signaling is involved in AD progression and that TLR4 signaling can be a new therapeutic target for AD.

Enhancing Th2 immune responses against amyloid protein by a DNA prime-adenovirus boost regimen for Alzheimer's disease.

Accumulation of aggregated amyloid beta-protein (Abeta) in the brain is thought to be the initiating event leading to neurodegeneration and dementia in Alzheimer's disease (AD). Therefore, therapeutic strategies that clear accumulated Abeta and/or prevent Abeta production and its aggregation are predicted to be effective against AD. Immunization of AD mouse models with synthetic Abeta prevented or reduced Abeta load in the brain and ameliorated their memory and learning deficits. The clinical trials of Abeta immunization elicited immune responses in only 20% of AD patients and caused T-lymphocyte meningoencephalitis in 6% of AD patients. In attempting to develop safer vaccines, we previously demonstrated that an adenovirus vector, AdPEDI-(Abeta1-6)11, which encodes 11 tandem repeats of Abeta1-6 can induce anti-inflammatory Th2 immune responses in mice. Here, we investigated whether a DNA prime-adenovirus boost regimen could elicit a more robust Th2 response using AdPEDI-(Abeta1-6)11 and a DNA plasmid encoding the same antigen. All mice (n=7) subjected to the DNA prime-adenovirus boost regimen were positive for anti-Abeta antibody, while, out of 7 mice immunized with only AdPEDI-(Abeta1-6)11, four mice developed anti-Abeta antibody. Anti-Abeta titers were indiscernible in mice (n=7) vaccinated with only DNA plasmid. The mean anti-Abeta titer induced by the DNA prime-adenovirus boost regimen was approximately 7-fold greater than that by AdPEDI-(Abeta1-6)11 alone. Furthermore, anti-Abeta antibodies induced by the DNA prime-adenovirus boost regimen were predominantly of the IgG1 isotype. These results indicate that the DNA prime-adenovirus boost regimen can enhance Th2-biased responses with AdPEDI-(Abeta1-6)11 in mice and suggest that heterologous prime-boost strategies may make AD immunotherapy more effective in reducing accumulated Abeta.

Nasal inoculation of an adenovirus vector encoding 11 tandem repeats of Abeta1-6 upregulates IL-10 expression and reduces amyloid load in a Mo/Hu APPswe PS1dE9 mouse model of Alzheimer's disease.

BACKGROUND: One of the pathological hallmarks of Alzheimer's disease (AD) is deposits of amyloid beta-peptide (Abeta) in neuritic plaques and cerebral vessels. Immunization of AD mouse models with Abeta reduces Abeta deposits and improves memory and learning deficits. Because recent clinical trials of immunization with Abeta were halted due to brain inflammation that was presumably induced by a T-cell-mediated autoimmune response, vaccination modalities that elicit predominantly humoral immune responses are currently being developed. METHODS: We have nasally immunized a young AD mouse model with an adenovirus vector encoding 11 tandem repeats of Abeta1-6 fused to the receptor-binding domain (Ia) of Pseudomonas exotoxin A (PEDI), AdPEDI-(Abeta1-6)(11), in order to evaluate the efficacy of the vector in preventing Abeta deposits in the brain. We also have investigated immune responses of mice to AdPEDI-(Abeta1-6)(11). RESULTS: Nasal immunization of an AD mouse model with AdPEDI-(Abeta1-6)(11) elicited a predominant IgG1 response and reduced Abeta load in the brain. The plasma IL-10 level in the AD mouse model was upregulated after immunization and, upon the stimulation with PEDI-(Abeta1-6)(11), marked IL-10 responses were found in splenic CD4(+) T cells from C57BL/6 mice that had been immunized with AdPEDI-(Abeta1-6)(11). CONCLUSIONS: These results suggest that the induction of Th2-biased responses with AdPEDI-(Abeta1-6)(11) in mice is mediated in part through the upregulation of IL-10, which inhibits activation of dendritic cells that dictate the induction of Th1 cells.

Role of toll-like receptor signalling in Abeta uptake and clearance.

Deposits of amyloid beta-protein (Abeta) in neuritic plaques and cerebral vessels are a pathological hallmark of Alzheimer's disease. Fibrillar Abeta deposits are closely associated with inflammatory responses such as activated microglia in brain with this disease. Increasing lines of evidence support the hypothesis that activated microglia, innate immune cells in the CNS, play a pivotal role in the progression of the disease: either clearing Abeta deposits by phagocytic activity or releasing cytotoxic substances and pro-inflammatory cytokines. Toll-like receptors (TLRs) are a family of pattern-recognition receptors in the innate immune system. Exogenous and endogenous TLR ligands activate microglia. To investigate the role of TLR4 in the amyloidogenesis in vivo, we determined the amounts of cerebral Abeta in Alzheimer's disease mouse models with different genotypes of TLR4 using three distinct methods. We show that mouse models (Mo/Hu APPswe PS1dE9 mice) homozygous for a destructive mutation of TLR4 (Tlr(Lps-d)/Tlr(Lps-d)) had increases in diffuse and fibrillar Abeta deposits by immunocytochemistry, fibrillar Abeta deposits by thioflavine-S staining and buffer-soluble and insoluble Abeta by ELISA in the cerebrum, as compared with TLR4 wild-type mouse models. Although the differences in these parameters were less significant, mouse models heterozygous for the mutation (Tlr(Lps-d)/) showed co-dominant phenotypes. Consistent with these observations in vivo, cultured microglia derived from Tlr(Lps-d)/Tlr(Lps-d) mice failed to show an increase in Abeta uptake after stimulation with a TLR4 ligand but not with a TLR9 ligand in vitro. Furthermore, activation of microglia (BV-2 cell) with a TLR2, TLR4 or TLR9 ligand, markedly boosted ingestion of Abeta in vitro. These results suggest that TLR signalling pathway(s) may be involved in clearance of Abeta-deposits in the brain and that TLRs can be a therapeutic target for Alzheimer's disease.

Anti-Abeta single-chain antibody delivery via adeno-associated virus for treatment of Alzheimer's disease.

Immunization of mouse models of Alzheimer disease (AD) with amyloid-peptide (Abeta) reduces Abeta deposits and attenuates their memory and learning deficits. Recent clinical trials were halted due to meningoencephalitis, presumably induced by T cell mediated and/or Fc-mediated immune responses. Because injection of anti-Abeta F(ab')(2) antibodies also induces clearance of amyloid plaques in AD mouse models, we have tested a novel gene therapy modality where an adeno-associated virus (AAV) encoding anti-Abeta single-chain antibody (scFv) is injected into the corticohippocampal regions of AD mouse models. One year after injection, expression of scFv was readily detectable in the neurons of the hippocampus without discernible neurotoxicity. AD mouse models subjected to AAV injection had much less amyloid deposits at the injection sites than the mouse models subjected to PBS injection. Because the scFv lacks the Fc portion of the immunoglobulin molecule, this modality may be a feasible solution for AD without eliciting inflammation.

Induction of anti-inflammatory immune response by an adenovirus vector encoding 11 tandem repeats of Abeta1-6: toward safer and effective vaccines against Alzheimer's disease.

Induction of an immune response to amyloid beta-protein (Abeta) is effective in treating animal models of Alzheimer's disease. Human clinical trials of vaccination with synthetic Abeta (AN1792), however, were halted due to brain inflammation, presumably induced by T cell-mediated immune responses. We have developed an adenovirus vector as a "possibly safer" vaccine. Here, we show that an adenovirus vector encoding 11 tandem repeats of Abeta1-6 can induce an immune response against amyloid beta-protein. Much higher titers against amyloid beta-protein were observed when an adenovirus vector encoding GM-CSF was co-administered. Immunoglobulin isotyping revealed a predominant IgG1 response, indicating anti-inflammatory Th2 type. Immunohistochemical analysis revealed no inflammation-related pathology in the brain of mice immunized with the adenovirus vector. Induced antibodies strongly reacted with amyloid plaques in the brain, demonstrating functional activity of the antibodies. Thus, the adenovirus vector encoding 11 tandem repeats of Abeta1-6 may be a safer alterative to peptide-based vaccines.