The APOE4 genotype alters the response of microglia and macrophages to 17beta-estradiol

Abstract

The apolipoprotein E4 (APOE4) gene is a well-known risk factor for Alzheimer's disease (AD) and other neurological disorders. Post-menopausal women with AD who express at least one APOE4 gene have more severe neuropathology and worsened cognitive scores than their non-expressing counterparts. Since 17beta-estradiol down-regulates inflammation as part of its neuroprotective role, we examined the effect of 17beta-estradiol on the response of microglia to immune activation as a function of APOE genotype. Our data show that the anti-inflammatory activity of 17beta-estradiol is significantly reduced in APOE4 targeted replacement mice compared to APOE3 mice. A significant interaction between APOE genotype and the response to 17beta-estradiol was observed for NO and cytokine production by immune activated microglia. The genotype specific effect was not restricted to brain macrophages since peritoneal macrophages from APOE4 ovariectomized mice also demonstrated a significant difference in 17beta-estradiol responsiveness. ERbeta protein levels in APOE4 microglia were higher than APOE3 microglia, suggesting a difference in post-translational protein regulation in the presence of the APOE4 gene. Overall, our data indicate that the APOE genotype may be a critical component in assessing the effectiveness of 17beta-estradiol's action and may impact the neuroprotective role of 17beta-estradiol and of hormone replacement therapy on brain function when the APOE4 gene is expressed.

Figure 1. APOE genotype influences the effect of 17β-estradiol on immune activated microglia

Cultured microglia from either APOE3 or APOE4 mice were pretreated with varying doses of 17β-estradiol (0.1 to 5 nM) followed by the addition of 100U/ml IFNγ and 100 ng/ml LPS. A–C: Supernatant nitrite and cytokine levels were measured in the same cell supernatants collected from a single representative APOE3 or APOE4 litter group. Significance was determined using 2-way ANOVA with the Bonferroni post-hoc test. (A) Supernatant nitrite levels demonstrated a significant interaction between APOE genotype and 17β-estradiol dose; p = 0.022; ** = p < 0.01 for 1 nM 17β-estradiol compared to LPS+IFNγ alone in APOE 3 microglia; * = p < 0.05 for 1 or 5 nM 17β-estradiol compared to LPS+IFNγ alone for APOE4 microglia. (B) Supernatant TNFα levels demonstrated a significant effect of genotype (p = 0.0001) and of estrogen dose (p = 0.037). (C) Supernatant IL-6 levels demonstrated a significant effect of genotype (p = 0.0001). D–F: Data represent the average relative change (±SEM) from LPS+IFNγ alone (=1.0) for 6–12 different litter groups, 3 wells cultured per litter group. (D) The change in supernatant nitrite levels demonstrated a significant interaction between APOE genotype and estrogen response (p= 0.0134);* = p < 0.05 for APOE3 vs APOE4; *** = p < 0.001 for APOE3 vs APOE4 (E) The change in supernatant TNFα levels demonstrated a significant interaction between genotype and estrogen dose (p < 0.0001); ** = p < 0.01 for APOE3 vs APOE4; *** = p < 0.001 for APOE3 vs APOE4. (F) The change in supernatant IL-6 levels demonstrated a significant interaction between genotype and estrogen dose (p < 0.0005); *** = p < 0.001 for APOE3 vs APOE4.

Figure 2. ICI 182,780 blocks 17β-estradiol in APOE3 microglia

Microglia from APOE3 and APOE4 mice were pretreated with 17β-estradiol in the presence and absence of ICI 182,780 followed by the addition of LPS+ IFNγ. Average (±SEM) supernatant nitrite levels were determined and the effect of 17β-estradiol and ICI 182,780 treatment compared between APOE3 and APOE4 mice. Significance was determined using 2-way ANOVA with the Bonferroni post-hoc test. A significant interaction was observed between genotype and the response to treatment (p = 0.005); *** = p < 0.001 for treatments compared to LPS+ IFNγ alone.

Figure 3. Expression of mRNA and protein for ERα and ERβ in microglia from APOE3 and APOE4 mice

(A and B) Quantitative RT-PCR was used to measure the average fold change (±SEM) in ERα and ERβ mRNA in microglia using untreated APOE3 as the calibrator. (A) Basal (untreated) levels of ERα mRNA were significantly higher in APOE4 microglia. Significance was determined using 2-way ANOVA with the Bonferroni post-hoc test. A significant effect of genotype was observed (p = 0.0025) for n = 7–12 cultures from a minimum of 3 different litter groups per genotype; ** = p < 0.01 for APOE4 compared to APOE3. (B) No significant difference was observed between untreated or treated APOE3 and APOE4 ERβ mRNA levels. A significant effect of LPS+IFNγ treatment compared to untreated was observed for both APOE3 and APOE4 mRNA (*** = p< 0.001). (C) ERα (top panel) and ERβ (middle and bottom panels) protein levels in untreated microglial lysates were determined using Western blot (labeled- Mg) from APOE3 (labeled- 3) and APOE4 (labeled- 4) mice. For ERβ, blots are presented from 3 different untreated microglial litter groups. Permanently transfected neuronal cells derived from the N2a cell line expressing human APOE3 (labeled -3) or human APOE4 (labeled -4) [6] were used as controls to demonstrate ER antibody specificity. N2a cells only express ERα and do not express ERβ. GADPH served as a loading control.

Figure 4. Peritoneal macrophages from ovariectomized APOE4 mice also demonstrate decreased responsiveness to exogenous treatment with 17β-estradiol

Cultured peritoneal macrophages from ovariectomized APOE3 or APOE4 mice were pretreated with 1nM 17β-estradiol followed by the addition of 100 ng/ml LPS plus 100 U/ml IFNγ (4A: LPS + IFNγ) or 50μg/ml PIC + 100 U/ml IFNγ (4B; PIC + IFNγ) in the continuing presence of 1 nM 17β-estradiol. Data are presented as the average supernatant nitrite levels (± SEM) and a 2-way ANOVA with the Bonferroni post-hoc test was used to determine significant differences. A significant interaction was observed between APOE genotype and the 17β-estradiol dose for either LPS + IFNγ or PIC + IFNγ-stimulated conditions (p < 0.0001). *** = p < 0.001 compared to LPS + IFNγ alone; # = p < 0.05 for APOE3 compared to APOE4; ## = p < 0.01 for APOE3 compared to APOE4.

Figure 5. Nitrite production in peritoneal macrophages derived from ovariectomized APOE3 and APOE4 mice after replacement of endogenous estrogen

Peritoneal macrophages from ovariectomized (OVX) mice and OVX mice treated with a slow release estrogen pellet (OVX + Est Pellet) were immune activated with LPS+IFNγ (A) or PIC+IFNγ (B). Average (±SEM) supernatant nitrite concentrations were determined and significant differences were calculated using 2-way ANOVA with the Bonferroni post-hoc test. A significant interaction was observed between APOE genotype and the treatment condition for both types of immune induction (p < 0.0001). # = p < 0.05 for APOE3 compared to APOE4; ## = p < 0.01 for APOE3 compared to APOE4; * = p < 0.05 for OVX + Est pellet compared to OVX alone; *** = p < 0.001 for OVX + Est pellet compared to OVX alone.

Figure 6. TNFα levels in peritoneal macrophages derived from ovariectomized APOE3 and APOE4 mice after replacement of endogenous estrogen

Average supernatant levels of TNFα (±SEM) were measured in PIC+IFNγ or LPS+IFNγ-treated macrophages cultured from OVX or OVX + Est. Pellet mice. For treatment with LPS+IFNγ (A), a significant effect of genotype (p = 0.024) or of treatment was observed (p < 0.0001). No significant interaction was found. For treatment with PIC+IFNγ (B), a significant interaction between genotype and treatment was observed (p < 0.0001). # = p < 0.05 for APOE3 compared to APOE4; ## = p < 0.01 for APOE3 compared to APOE4; *** = p < 0.001 for OVX + pellet compared to OVX alone.