Peripherally administered human umbilical cord blood cells reduce parenchymal and vascular beta-amyloid deposits in Alzheimer mice

Abstract

Modulation of immune/inflammatory responses by diverse strategies including amyloid-beta (Abeta) immunization, nonsteroidal anti-inflammatory drugs, and manipulation of microglial activation states has been shown to reduce Alzheimer's disease (AD)-like pathology and cognitive deficits in AD transgenic mouse models. Human umbilical cord blood cells (HUCBCs) have unique immunomodulatory potential. We wished to test whether these cells might alter AD-like pathology after infusion into the PSAPP mouse model of AD. Here, we report a marked reduction in Abeta levels/beta-amyloid plaques and associated astrocytosis following multiple low-dose infusions of HUCBCs. HUCBC infusions also reduced cerebral vascular Abeta deposits in the Tg2576 AD mouse model. Interestingly, these effects were associated with suppression of the CD40-CD40L interaction, as evidenced by decreased circulating and brain soluble CD40L (sCD40L), elevated systemic immunoglobulin M (IgM) levels, attenuated CD40L-induced inflammatory responses, and reduced surface expression of CD40 on microglia. Importantly, deficiency in CD40 abolishes the effect of HUCBCs on elevated plasma Abeta levels. Moreover, microglia isolated from HUCBC-infused PSAPP mice demonstrated increased phagocytosis of Abeta. Furthermore, sera from HUCBC-infused PSAPP mice significantly increased microglial phagocytosis of the Abeta1-42 peptide while inhibiting interferon-gammainduced microglial CD40 expression. Increased microglial phagocytic activity in this scenario was inhibited by addition of recombinant CD40L protein. These data suggest that HUCBC infusion mitigates AD-like pathology by disrupting CD40L activity.

FIG. 1.

Cerebral Aβ/β-amyloid pathology is reduced in PSAPP and Tg2576 mice peripherally infused with HUCBCs. Mouse paraffin-embedded coronal brain sections from the cingulate cortex (CC), hippocampus (H), and entorhinal cortex (EC) were stained with monoclonal human Aβ antibody 4G8 (A) or Congo Red (C). Percentages (plaque area/total area) of Aβ antibody-immunoreactive deposits (B) or of Congo Red-stained deposits (D) were calculated by quantitative image analysis (mean ± SD; n = 10, 5 females and 5 males per group). (E) Aβ ELISA analysis was carried out for both levels of detergent-soluble Aβ_1-40,42 (top panel) or 5 M guanidine-extracted Aβ_1-40,42 (bottom panel). Data are represented as mean ± SD of Aβ_1-40,42 (pg/mg protein). Mouse paraffin-embedded coronal brain sections from hippocampal regions of Tg2576 mice were stained with Congo Red (F). Positions of the hippocampal subfields CA1, CA3, and dentate gyrus (DG) are indicated (upper left panel). Arrows indicate Aβ deposit-affected vessels. (G) Percentages (% labeled area) of Congo Red-stained plaques/vessels were quantified by image analysis (mean ± SD; n = 10, 5 females and 5 males), and percentage reduction is indicated.

FIG. 1.

Cerebral Aβ/β-amyloid pathology is reduced in PSAPP and Tg2576 mice peripherally infused with HUCBCs. Mouse paraffin-embedded coronal brain sections from the cingulate cortex (CC), hippocampus (H), and entorhinal cortex (EC) were stained with monoclonal human Aβ antibody 4G8 (A) or Congo Red (C). Percentages (plaque area/total area) of Aβ antibody-immunoreactive deposits (B) or of Congo Red-stained deposits (D) were calculated by quantitative image analysis (mean ± SD; n = 10, 5 females and 5 males per group). (E) Aβ ELISA analysis was carried out for both levels of detergent-soluble Aβ_1-40,42 (top panel) or 5 M guanidine-extracted Aβ_1-40,42 (bottom panel). Data are represented as mean ± SD of Aβ_1-40,42 (pg/mg protein). Mouse paraffin-embedded coronal brain sections from hippocampal regions of Tg2576 mice were stained with Congo Red (F). Positions of the hippocampal subfields CA1, CA3, and dentate gyrus (DG) are indicated (upper left panel). Arrows indicate Aβ deposit-affected vessels. (G) Percentages (% labeled area) of Congo Red-stained plaques/vessels were quantified by image analysis (mean ± SD; n = 10, 5 females and 5 males), and percentage reduction is indicated.

FIG. 2.

β-Amyloid-associated microgliosis and astrocytosis are reduced in HUCBC infused-PSAPP mice. (A) Immunofluorescence was performed on mouse brain coronal paraffin sections prepared from PSAPP mice infused with HUCBC or PBS. The red signal indicates Aβ⁺ (top panels); green indicates CD40⁺ (middle panels), and merged images (bottom panels) reveal co-localization of CD40 and Aβ. DAPI (blue) was used as a nuclear counterstain. (B) Immunofluorescence intensity for Aβ and CD40 was determined. (C) Western blot analysis shows reduced CD40 expression in brain homogenates from PSAPP/HUCBC versus PSAPP/PBS mice as indicated (actin was used as an internal reference control). Densitometry analysis shows the ratio of CD40 to actin as indicated below the figure. (D) Immunohistochemistry analysis shows GFAP staining (top panel), and immunofluorescence (bottom panel) reveals co-localization of GFAP (red signal) and Aβ (green signal). (E) Morphometric analysis results (mean GFAP/β-amyloid double positive plaques per mouse ± SD) are shown for the neocortex and the hippocampus of PSAPP/HUCBC versus PSAPP/PBS mice. Percent reduction of plaques double positive for GFAP and Aβ in PSAPP/HUCBC mice is indicated.

FIG. 3.

HUCBC infusion results in CD40-dependent increased plasma Aβ levels in PSAPP mice. ELISA analysis results are shown from blood (plasma) for Aβ_1-40 (A), Aβ_1-42 (B), sCD40L (C), and IgM/IgG (D). Data are presented as mean ± SD (n = 10) for Aβ_1-40, Aβ_1-42, or sCD40L (pg/ml plasma). Arrows below the panels show the time for each peripheral infusion with HUCBC or PBS. (D) Data are presented as a ratio of IgM to IgG in blood (plasma) from mice at the 6th month following the treatment. Aβ ELISA analysis for Aβ_1-40 (E) and Aβ_1-42 (F) in blood (plasma) derived from PSAPP/CD40^+/+ or PSAPP/CD40^−/− mice at the 2nd month, following the third HUCBC infusion. Data in E and F are presented as mean ± SD (n = 4, 2 males and 2 females) of Aβ_1-40 or Aβ_1-42 (pg/ml plasma).

FIG. 4.

HUCBC infusion promotes anti-inflammatory/Th2 responses and decreases sCD40L in the CNS. ELISA results are shown for plasma-derived (A), splenocyte culture-derived (B), brain tissue-derived cytokines (C), and brain tissue-derived sCD40L (D). Data are presented as mean ± SD (n = 10) values of cytokines (pg/ml plasma or medium) (A and B) or fold increase of cytokines over control (untreated) mice (C and D).

FIG. 5.

HUCBCs modulate microglial CD40 expression and promote Aβ microglial/macrophage phagocytic activity. (A) FACS analysis for CD40 expression in primary wild-type neonatal microglial cells treated with cultured medium from HUCBCs or human adult mononuclear cells (HAMNCs), or serum from individual PSAPP/HUCBC or PSAPP/PBS mice following IFN-γ challenge. Data are presented as percentage of CD40 expressing cells (mean ± SD; n = 5). (B–D) Microglial/macrophage phagocytosis assay results for extracellular and cell-associated FITC-Aβ_1-42, which was detected using a fluorometer. Data are represented as the relative fold of mean ± SD fluorescence over control for each sample (n = 4 for each condition presented). Primary microglial cells from adult PSAPP/HUCBC or PSAPP/PBS mice (B), wild-type neonatal microglia (C), and primary peripheral macrophages (D) are shown.

FIG. 5.

HUCBCs modulate microglial CD40 expression and promote Aβ microglial/macrophage phagocytic activity. (A) FACS analysis for CD40 expression in primary wild-type neonatal microglial cells treated with cultured medium from HUCBCs or human adult mononuclear cells (HAMNCs), or serum from individual PSAPP/HUCBC or PSAPP/PBS mice following IFN-γ challenge. Data are presented as percentage of CD40 expressing cells (mean ± SD; n = 5). (B–D) Microglial/macrophage phagocytosis assay results for extracellular and cell-associated FITC-Aβ_1-42, which was detected using a fluorometer. Data are represented as the relative fold of mean ± SD fluorescence over control for each sample (n = 4 for each condition presented). Primary microglial cells from adult PSAPP/HUCBC or PSAPP/PBS mice (B), wild-type neonatal microglia (C), and primary peripheral macrophages (D) are shown.