Vivaria housing conditions expose sex differences in brain oxidation, microglial activation, and immune system states in aged hAPOE4 mice

Abstract

Apolipoprotein E ε4 allele (APOE4) is the predominant genetic risk factor for late-onset Alzheimer's disease (AD). APOE4 mouse models have provided advances in the understanding of disease pathogenesis, but unaccounted variables like rodent housing status may hinder translational outcomes. Non-sterile aspects like food and bedding can be major sources of changes in rodent microflora. Alterations in intestinal microbial ecology can cause mucosal barrier impairment and increase pro-inflammatory signals. The present study examined the role of sterile and non-sterile food and housing on redox indicators and the immune status of humanized-APOE4 knock-in mice (hAPOe4). hAPOE4 mice were housed under sterile conditions until 22 months of age, followed by the transfer of a cohort of mice to non-sterile housing for 2 months. At 24 months of age, the redox/immunologic status was evaluated by flow cytometry/ELISA. hAPOE4 females housed under non-sterile conditions exhibited: (1) higher neuronal and microglial oxygen radical production and (2) lower CD68⁺ microglia (brain) and CD8⁺ T cells (periphery) compared to sterile-housed mice. In contrast, hAPOE4 males in non-sterile housing exhibited: (1) higher MHCII⁺ microglia and CD11b⁺CD4⁺ T cells (brain) and (2) higher CD11b⁺CD4⁺ T cells and levels of lipopolysaccharide-binding protein and inflammatory cytokines in the periphery relative to sterile-housed mice. This study demonstrated that sterile vs. non-sterile housing conditions are associated with the activation of redox and immune responses in the brain and periphery in a sex-dependent manner. Therefore, housing status may contribute to variable outcomes in both the brain and periphery.

Keywords: Alzheimer’s disease; Apolipoprotein; Housing and food sterility; Immune status; Sex differences.

Fig. 1

Oxidant production is increased in non-sterile conditions in hAPOE4 female, but not male, brain cells. Brain cells were dissociated from whole brain samples, stained for type-specific markers, and analyzed by flow cytometry. Panels (A–H) are representative graphs illustrating the brain cell gating strategy. A Brain cells were selected by their SSC and FSC characteristics. B Dead brain cells and C cell doublets were discriminated by their DAPI staining and FSC-A and FSC-H characteristics. Single brain cells were identified by gating expression of specific cell type markers: D Microglia expressing CD11b high and CD45 intermediate, E Astrocytes expressing ACSA-2⁺/CD45⁻/CD11b⁻, neurons expressing CD31⁻/O4⁻/ACSA-2⁻/CD45⁻/CD11b⁻, F oligodendrocyte expressing O4⁺/CD31⁻/ACSA-2⁻/CD45⁻/CD11b⁻ and endothelial cells by expression of CD31⁺/O4⁻/ACSA-2⁻/CD45⁻/CD11b⁻. G Each brain cell type was used for gating mitochondrial oxidative stress (MitoSox⁺). H Microglial cells were gated for expression of MHC-II and CD68. Quantification of mitochondrial oxidative stress in microglia (I) and neurons (J). Data are represented as mean ± SD. p values were calculated using two-way ANOVA (Bonferroni adjusted p-values are shown in Supplementary Table 2; *p ≤ 0.05, **p ≤ 0.01)

Fig. 2

Microglia activation is affected by housing and food status. Quantification of expression of microglial activation markers A MHCII⁺, B CD68⁺, and C MHCII⁺ CD68⁺. Data are represented as mean ± SD. p values calculated using two-way ANOVA (Bonferroni adjusted p-values are shown in Supplementary Table 2; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001)

Fig. 3

Non-sterile environments increase the infiltration of T cells into the male, but not female brain. Male and female APOE4 mouse brains were dissociated and assessed using a single-cell suspension. Brain cells were stained for immune cell markers and analyzed by flow cytometry. A After discriminating dead cells and doublets, immune cells were gated by expressing CD45⁺. Immune cells were classified as B T cells (CD45⁺ CD3⁺), C B cells (CD19⁺/CD45⁺/CD3⁻) and D helper T cells (CD4⁺/CD3⁺/CD45⁺) and cytotoxic T cells (CD8⁺/CD3⁺/CD45⁺). E Helper T cells were gated for expression of CD11b. Quantification of F total T cells, G helper T cells and H CD11b⁺ helper T cells in the brain. Data are represented as mean ± SD. p values were calculated using two-way ANOVA (Bonferroni adjusted p-values are shown in Supplementary Table 2; *p ≤ 0.05, and **p ≤ 0.01)

Fig. 4

Peripheral adaptive immune cells are affected by sterile and non-sterile food and housing conditions. White blood cells (WBC) and splenic cells were isolated and stained immune cell markers and analyzed by flow cytometry. A Quantification of % B cells (CD19⁺/CD45⁺/CD3⁻), B T cell (CD3⁺/CD45⁺), C helper T cell (CD4⁺/CD3⁺/CD45⁺) and D cytotoxic T cells (CD8⁺/CD3⁺/CD45⁺) in spleen and WBC. Data are represented as mean ± SD. p values were calculated using two-way ANOVA (Bonferroni adjusted p-values are shown in Supplementary Table 2; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

Fig. 5

Sterile and non-sterile food and housing effects on the expression of T cell activation and inflammatory markers. Splenic cells and WBCs were stained for B cell and T cell markers (CD3⁺, CD4⁺, CD8⁺), the T cell activation marker CD69⁺, and inflammation marker CD11b⁺ and analyzed by flow cytometry. CD69⁺ was measured in both A CD4⁺ and B CD8⁺ T cells in the blood and in the spleen (C, D). CD11b⁺ was measured in C CD4⁺ and D CD8⁺ T cells and E B cells in the blood and in the spleen. Data are represented as mean ± SD. p values were calculated using two-way ANOVA (Bonferroni adjusted p-values are shown in Supplementary Table 2; *p ≤ 0.05 and **p ≤ 0.01)

Fig. 6

Non-sterile food and housing increase the levels of LPS-binding protein and inflammatory cytokines in the plasma of APOE4 males. The concentration of circulating A LPS-binding protein (LBP), B TNF-α, C IL-1β, D KC/GRO, E IL-6, and F INF-γ- were measured from plasma collected at necropsy. Data are represented as mean ± SD. p values were calculated using two-way ANOVA (Bonferroni adjusted p-values are shown in Supplementary Table 2; *p ≤ 0.05 and **p ≤ 0.01)

Fig. 7

Aged hAPOE4 males are more susceptible to housing and food status than aged hAPOE4 females. hAPOE4 females and males responded differently to sterile and non-sterile housing and food status. Female hAPOE4 mice moved to non-sterile conditions increase in MitoSox + microglia and neurons and did not exhibit an inflammatory response to non-sterile food and housing conditions. However, male hAPOE4 mice exposed to non-sterile food and housing exhibited an increase in the levels of LPS and inflammatory cytokines in the circulatory system, activated microglia, infiltration of T cells into the brain compared to those in sterile food and housing conditions