Sex and APOE Genotype Alter the Basal and Induced Inflammatory State of Primary Microglia from APOE Targeted Replacement Mice

Abstract Background: Sex and APOE4 genotype are significant risk factors for Alzheimer’s disease (AD), with females demonstrating increased risk that is modulated by APOE genotype. However, the mechanism(s) responsible for this interaction are still a matter of debate. Neuroinflammation represents a plausible mechanism for this interaction, but most experimental studies have not incorporated both sex and APOE genotype. Here, we sought to assess the response of sex-specific APOE3 and APOE4 primary microglia (PMG) in comparison to traditional mixed sex cultures. Methods: Mixed sex and sex-specific PMG were generated from neonatal targeted replacement APOE3 and APOE4 pups. Purity over 98% was consistently obtained for cultures and the presence or absence of the Sry (Sex-determining region Y) gene by qPCR was used to determine male-specific microglia. PMG were treated with 10 ng/mL or a combination of lipopolysaccharide (LPS) and interferon-gamma (IFNg). Pro- and anti-inflammatory cytokine profiles were assessed by quantitative RT-PCR and multiplex MSD assays.Results: Mixed sex APOE4 PMG were found to have higher basal mRNA expression of pro-inflammatory cytokines including TNFa, and iNOS compared to APOE3 PMG which was accompanied by increased levels of secreted TNFa, but not nitric oxide. In sex-specific cultures, basal expression of IL1b, TNFa, IL6, and iNOS was 2-3 fold higher in APOE4 female PMG compared to APOE4 males, with both being higher compared to APOE3 cells. Similar results were found for protein levels of these cytokines. Following an inflammatory stimulus, the expression of pro-inflammatory cytokines was upregulated in the order E4 female > E4 male > E3 female > E3 male in sex-specific cultures. Similar results were found with secreted cytokine levels.Conclusions: These data indicate that the APOE4 genotype and female sex, together contribute to a greater inflammatory response in PMG isolated from targeted replacement APOE mice. These data are consistent with clinical data and sex-specific PMG may provide a platform for exploring mechanisms of genotype and sex differences in AD-related to neuroinflammation and neurodegeneration.