Immune senescence in aged APP/PS1 mice

Abstract

Objectives: To evaluate the linkage between age and deficits in innate and adaptive immunity which heralds both Alzheimer's disease (AD) onset and progression. The pathobiological events which underlie and tie these outcomes remain not fully understood.

Methods: To investigate age-dependent immunity in AD, we evaluated innate and adaptive immunity in coordinate studies of regulatory T cell (Treg) function, T cell frequencies, and microglial integrity. These were assessed in blood, peripheral lymphoid tissues, and the hippocampus of transgenic (Tg) amyloid precursor protein/presenilin 1 (APP/PS1) against non-Tg mice. Additionally, immune arrays of hippocampal tissue were performed at 4, 6, 12, and 20 months of age.

Results: APP/PS1 mice showed progressive impairment of Treg immunosuppressive function with age. There was partial restoration of Treg function in 20-month-old mice. Ingenuity pathway analyses of hippocampal tissues were enriched in inflammatory, oxidative, and cellular activation pathways that paralleled advancing age and AD-pathobiology. Operative genes in those pathways included, but were not limited to triggering receptor on myeloid cells 1 (TREM1), T helper type 1 (Th1), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways. Interleukin-17 (IL-17), nitric oxide, acute phase, and T cell receptor signaling pathways were also perturbed. Significant inflammation was observed at 6- and 12-months. However, at 20-months, age associated partial restoration of Treg function reduced inflammatory phenotype.

Conclusions: Impaired Treg function, inflammation and oxidative stress were associated with AD pathology. Age associated partial restoration of Treg function in old mice reduced the hippocampal inflammatory phenotype. Restoring Treg suppressive function can be a therapeutic modality for AD.

Keywords: Alzheimer’s disease; Treg; aging; neuroinflammation; oxidative stress.

Figure 1:

Amyloid deposition and microglia activation throughout AD progression. Immunohistochemistry (pan-Aβ) and immunofluorescence (Thioflavin-S) were performed in APP/PS1 mice at 4-, 6-, 12-, and 20-months of age to determine the area occupied by Aβ plaques in cortical and hippocampal brain sections. (A) Representative images showing total amyloid plaques in sections of cortical and hippocampal brain regions (scale bar=100 μm). (B) Percentage of area within cortices and hippocampi occupied by total Aβ plaques. (C) Representative images showing dense amyloid plaques in sections of cortical and hippocampal brain regions (scale bar=100 μm). (D) Percentage of area within cortices and hippocampi occupied by dense Aβ plaques. Numbers of reactive Iba1+ microglia cells in cortical and hippocampal brain regions were quantified by immunohistochemistry. (E) Representative images showing Iba1+-reactive cells in cortical and hippocampal (scale bar=100 μm). (F) Number of reactive microglia as Iba1+ ameboid cells in cortical and hippocampal tissues. Statistical differences between groups were determined using one-way ANOVA followed by Dunnett’s multiple comparisons test for n=4 mice/group whereby ^*p≤0.05, ^**p≤0.001, ^***p≤0.0001, and ^****p≤0.00001.

Figure 2:

Treg frequency and function throughout progressive AD. (A) Flow cytometry gating strategies for T lymphocyte analysis from APP/PS1 and non-Tg controls. Frequencies of CD4+ and CD8+ T cells within total T lymphocyte population in blood (B), spleen (C), and lymph nodes (D) for 4-, 6-, 12-, and 20-month old APP/PS1 and non-Tg mice. (E) Frequencies of CD4+CD25+FOXP3+ Tregs within CD3+CD4+ T cells in blood, spleen, and lymph nodes of non-Tg and Tg mice at different ages. (F) Inhibition of CFSE-stained-Tresp (CD4+CD25-) proliferation by Tregs from APP/PS1 at different ages. Significant differences (table) in slope and intercept were determined by linear regression analysis. (B–F) Data are represented as mean ± SEM for n=3–4 mice per group. Statistical significance between groups was determined using Student’s t-test or one-way ANOVA followed by Tukey’s multiple comparison test. The p value ≤0.05 was considered significant; ^*p≤0.05, ^**p≤0.001, ^***p≤0.0001, and ^****p≤0.00001. M, months; non-Tg, control; Tg, APP/PS1.

Figure 3:

Canonical pathway enrichments of immune-related genes in hippocampi of non-Tg control mice. Hippocampal tissues were obtained from non-Tg control mice at 4-, 6-, 12- and 20-months of age (4M, 6M, 12M, and 20M). Canonical pathway enrichment analysis for hippocampal tissues was performed using IPA (Qiagen) for 6M, 12M, and 20M non-Tg control mice compared to 4M non-Tg controls. (A) Data was represented using −log₁₀ (p value), where p<0.05 was considered significant. (B) Data represented using z-score where z-score ≤−2 or ≥2 were considered significant. Inhibited pathways are represented in green, whereas activated pathways are represented in red.

Figure 4:

Canonical pathway enrichments of immune-related genes in hippocampi of APP/PS1 mice. Hippocampal tissues were obtained from APP/PS1 mice at 4-, 6-, 12- and 20-months of age (4M, 6M, 12M, and 20M). Canonical pathway enrichment analysis was performed using IPA (Qiagen) for 6M, 12M, and 20M APP/PS1 mice and compared to 4M APP/PS1 mice. (A) Data represented as −log₁₀ (p value), whereby p values ≤0.05 was considered significant. (B) Data represented as z-score, whereby z-scores ≤−2 or ≥2 were considered significant. Inhibited pathways are represented in green, whereas activated pathways are represented in red.

Figure 5:

Canonical pathway enrichments of immune-related genes for APP/PS1 mice compared to age-matched non-Tg control mice. Hippocampal tissues were obtained from APPP/PS1 and non-Tg control mice at 4-, 6-, 12- and 20-months of age (4M, 6M, 12M, and 20M). Canonical pathway enrichment analysis was performed using IPA (Qiagen) for APP/PS1 mice of different ages compared to age-matched non-Tg control mice (4M, 6M, 12M, and 20M). (A) Data was represented using −log₁₀ (p value) and p values ≤0.05 were considered significant. (B) Data represented as z-scores whereby z-scores ≤−2 or ≥2 were considered significant. Inhibited pathways are represented in green, whereas activated pathways are represented in red.