Transcutaneous beta-amyloid immunization reduces cerebral beta-amyloid deposits without T cell infiltration and microhemorrhage

Abstract

Alzheimer's disease (AD) immunotherapy accomplished by vaccination with beta-amyloid (Abeta) peptide has proved efficacious in AD mouse models. However, "active" Abeta vaccination strategies for the treatment of cerebral amyloidosis without concurrent induction of detrimental side effects are lacking. We have developed a transcutaneous (t.c.) Abeta vaccination approach and evaluated efficacy and monitored for deleterious side effects, including meningoencephalitis and microhemorrhage, in WT mice and a transgenic mouse model of AD. We demonstrate that t.c. immunization of WT mice with aggregated Abeta(1-42) plus the adjuvant cholera toxin (CT) results in high-titer Abeta antibodies (mainly of the Ig G1 class) and Abeta(1-42)-specific splenocyte immune responses. Confocal microscopy of the t.c. immunization site revealed Langerhans cells in areas of the skin containing the Abeta(1-42) immunogen, suggesting that these unique innate immune cells participate in Abeta(1-42) antigen processing. To evaluate the efficacy of t.c. immunization in reducing cerebral amyloidosis, transgenic PSAPP (APPsw, PSEN1dE9) mice were immunized with aggregated Abeta(1-42) peptide plus CT. Similar to WT mice, PSAPP mice showed high Abeta antibody titers. Most importantly, t.c. immunization with Abeta(1-42) plus CT resulted in significant decreases in cerebral Abeta(1-40,42) levels coincident with increased circulating levels of Abeta(1-40,42), suggesting brain-to-blood efflux of Abeta. Reduction in cerebral amyloidosis was not associated with deleterious side effects, including brain T cell infiltration or cerebral microhemorrhage. Together, these data suggest that t.c. immunization constitutes an effective and potentially safe treatment strategy for AD.

Fig. 1.

Generation of immune responses in WT mice t.c.-immunized with aggregated Aβ_1–42 peptide plus CT. (A) Aβ antibody titers were measured by ELISA. Data are presented as mean ± SD (n = 10) of Aβ antibodies (ng/ml plasma). One-way ANOVA followed by post hoc comparison revealed significant differences in anti-Aβ titers when comparing week 4 to weeks 8, 12, or 16 (∗∗, P < 0.001). (B and C) IgG isotypes were determined by an Ig isotyping assay and are represented as ratios (mean ± SD; n = 10) of IgG1 to IgG2a (B) or IgG1 to IgG2b (C). One-way ANOVA followed by post hoc comparison revealed significant differences between the ratio of IgG1 and IgG2a versus IgG1 and IgG2b at each week shown (∗∗, P < 0.001). (D) Splenocytes were individually isolated and cultured from WT mice t.c.-immunized with Aβ_1–42/CT, CT alone, or PBS (control). These cells were stimulated with Con A (5 μg/ml) or Aβ_1–42 (20 μg/ml) for 48 h. Cultured supernatants were collected from these cells for IFN-γ, IL-2, and IL-4 cytokine analyses by ELISA. Data are presented as relative fold mean ± SD (n = 10) of each cytokine over PBS control. One-way ANOVA followed by post hoc comparison revealed significant differences between groups for levels of each of three cytokines [IFN-γ, IL-2, and IL-4 (∗∗, P < 0.001)] after in vitro Aβ_1–42 challenge. As noted, there was also a significant difference in cytokine levels between IL-4 and either IFN-γ or IL-2 after Aβ_1–42 challenge (##, P < 0.001). (E) To characterize dermal immune responses to Aβ/CT t.c. immunization, skin tissues were prepared from nontransgenic C57BL/6 mice t.c.-immunized for 18 h with PBS (control, Top), CT alone (Middle), or Aβ/CT (Bottom) as indicated and then analyzed by laser scanning confocal microscopy with the indicated antibodies (antibody 4G8 was used to reveal Aβ). Note the presence of CD207⁺CD11c⁺ LCs in Aβ-positive regions in the Aβ/CT t.c.-immunized group. DAPI (blue signal) was used as a nuclear counterstain in merged images shown to the right. (Scale bar: 50 μm.)

Fig. 2.

Increased systemic Aβ after Aβ_1–42/CT t.c. immunization of PSAPP mice. For Aβ analysis, blood samples were individually collected from Aβ/CT or CT alone t.c.-immunized PSAPP mice at the time points indicated. (A) Plasma Aβ antibody titers were measured by ELISA. Data are presented as mean ± SD (n = 9) of Aβ antibodies (pg/ml plasma) (∗∗, P < 0.001) and between time points within the Aβ/CT t.c.-immunized group as indicated (##, P < 0.001). (B and C) Plasma Aβ_1–40,42 peptides were measured separately by Aβ ELISA. Data are presented as mean ± SD (n = 9) of Aβ_1–40 or Aβ_1–42 (pg/ml plasma). ∗, P < 0.05; ∗∗, P < 0.001. Arrows indicate each t.c. immunization with respect to the time of blood sample collection.

Fig. 3.

Reduction of cerebral Aβ/β-amyloid pathology in PSAPP mice t.c.-immunized with Aβ_1–42/CT. (A and B) Detergent-soluble Aβ_1–40,42 peptides (A) and insoluble Aβ_1–40,42 prepared from 5 M guanidine extraction (B) were separately measured in brain homogenates by ELISA. Data are presented as mean ± SD (n = 9) of Aβ_1–40 or Aβ_1–42 (pg/mg protein), and reductions for each group are indicated. (C) A significant inverse correlation (P < 0.001) between plasma and brain-soluble Aβ levels was revealed. Plasma/brain Aβ levels are presented as percentage mean ± SD (n = 9) of soluble circulating/brain Aβ at 16 weeks after t.c. immunization of PSAPP mice with Aβ/CT over CT control mice. (D) Mouse brain coronal paraffin sections were stained with monoclonal anti-human Aβ antibody 4G8. (Left) Aβ_1–42/CT t.c. immunized PSAPP mice. (Right) CT t.c.-immunized PSAPP mice. (Top) The cingulate cortex (CC). (Middle) The hippocampus (H). (Bottom) The entorhinal cortex (EC). (E) Percentages (plaque burden, area plaque/total area) of Aβ antibody-immunoreactive Aβ plaques (mean ± SD; n = 9) were calculated by quantitative image analysis, and reductions for each mouse brain area analyzed are indicated. (F) Mouse brain sections from the indicated regions were stained with Congo red. (Left) Aβ_1–42/CT t.c.-immunized PSAPP mice. (Right) CT t.c.-immunized PSAPP mice. (G) Percentages of Congo red-stained plaques (mean ± SD; n = 9) were quantified by image analysis, and reductions for each brain region are indicated.

Fig. 4.

Absence of T cell infiltration or brain microhemorrhage in Aβ/CT t.c.-immunized mice. (A–C) Brain sections were stained for CD3 as an indicator of T cell infiltration. (D–F) Staining for hemosiderin was also performed to identify microhemorrhage in mice immunized with Aβ/CT (B and E) or CT alone (C and F). (A) For CD3 staining, the positive control consisted of CD3-positive brain sections from experimental autoimmune encephalomyelitis mice. (D) For microhemorrhage, experimental sections were compared with sections from mouse brains suffering microhemorrhage. Each panel is representative of staining repeated in triplicate for each brain section for either CD3 or hemosiderin. The brain region shown for each panel is the neocortex. (Magnification: CD3 staining, ×10; microhemorrhage, ×20.)