In post-industrial settings, APOE4 is associated with increased cardiovascular and neurological disease risk. However, the majority of human evolutionary history occurred in environments with higher pathogenic diversity and low cardiovascular risk. We hypothesize that in high-pathogen and energy-limited contexts, the APOE4 allele confers benefits by reducing innate inflammation when uninfected, while maintaining higher lipid levels that buffer costs of immune activation during infection. Among Tsimane forager-farmers of Bolivia (N=1266), APOE4 is associated with 30% lower C-reactive protein, and higher total cholesterol and oxidized-LDL. Blood lipids were either not associated, or negatively associated with inflammatory biomarkers, except for associations of oxidized-LDL and inflammation which were limited to high BMI adults. Further, APOE4 carriers maintain higher levels of total and LDL cholesterol at low BMIs. These results suggest the relationship between APOE4 and lipids may be beneficial for pathogen-driven immune responses, and unlikely to increase cardiovascular risk in an active subsistence population.

Biomarker data used in this paper were collected by the THLHP between 2004 - 2015 (see Gurven et al., 2017; Kraft et al., 2020 for details). A Bolivian and Tsimane mobile medical team travel annually or biannually among study communities conducting clinical health assessments and collecting biochemical and anthropometric information from community members that want to participate. This sample includes all data from individuals for whom we have APOE genotyping and measurements of age, sex, and BMI - which is the base criteria for this study. Sample size varies by biomarker and over time for several reasons: sampling strategy varies by data type, absent or sick team personnel needed to collect data, the number of study villages and thus enrolled participants has increased over time, and the data types collected have changed over time (see Kraft et al., 2020). There are also fewer repeat measurements for a subset of biomarkers (i.e. C-reactive protein and oxidized LDL) that were assayed in the U.S., due to them being analyzed as part of a prior project. Specific sample sizes are reported in Table 1, and full tables report sample size for each model.

Ethics

This research has been approved by institutional review boards at the University of New Mexico (#07-157) and University of California Santa Barbara (#3-20-0740), as well as the Tsimane government (Tsimane Gran Consejo) and village leaders. Study participants give consent for each part of the research and data collection prior to participating, during every visit by the THLHP.

APOE genotyping

Whole blood samples were stored in cryovials (Nalgene, USA) and frozen in liquid nitrogen before transfer on dry ice to the University of California-Santa Barbara, where they were stored at -80°C until genotyping. Single nucleotide polymorphism (SNP) genotyping was used to identify APOE allelic variants in blood samples. Samples were shipped on dry ice to University of Southern California (2010 and 2013) and University of Texas-Houston (2016), where DNA was extracted, quantified, and haplotype coded for APO- E2, E3, and E4 alleles using the TaqMan Allelic Discrimination system (Thermo-Fisher Scientific, Carlsbad, CA, USA). Determination of the APOE2/E3/E4 alleles in the Tsimane derived from 2 SNPs of 20-30bp oligonucleotides surrounding the polymorphic site (Cys112Arg/rs429358 and Cys158Arg/rs7412) (Trumble et al., 2017; Vasunilashorn et al., 2011).

Measurement of blood lipids and immune function

Biomarkers were either assayed in the field at the time of collection, or in the Human Biodemography laboratory at UC Santa Barbara in 2016.

Blood was collected by venipuncture in a heparin-coated vacutainer. Immediately following the blood draw, total leukocyte counts and hemoglobin were determined with a QBC Autoread Plus dry hematology system (QBC Diagnostics), with a QBC calibration check performed daily to verify QBC performance. Relative fractions of neutrophils, eosinophils, and lymphocytes were determined manually by microscopy with a hemocytometer by a certified Bolivian biochemist. ESR was calculated following the Westergren method (Westergren, 1957).

Serum was separated and frozen in liquid nitrogen before transfer to the University of California-Santa Barbara where a commercial immunoassay was used to measure oxidized LDL (Mercodia, Winston Salem, NC). Serum high sensitivity C-Reactive Protein (hs-CRP) was assessed via immunoassay (Brindle et al., 2010), and was cross-validated by the University of Washington laboratory, using the protocols utilized for the National Health and Nutrition Evaluation Survey (NHANES). Oxidized LDL and hs-CRP assays use materials from the same lot across all measures. Total and LDL cholesterol levels from serum samples were measured (Stat Fax 1908, Awareness Technology, Palm City, FL) in the THLHP laboratory in San Borja, Beni, Bolivia.

Data is in long-format, as there are numerous repeated measurements per individual (pid). Given that there the number of observations vary by biomarker and individual, there are also many NAs in the data. Mixed effects linear regressions with restricted maximum likelihood estimation should used for analyses, with random intercept effects for individual ID, i.e. 'pid' (to deal with repeat observations). Because Tsimane villages vary in sanitation infrastructure, including access to soap and other hygienic products, and potentially prevalence by pathogen type (e.g. some living very close to the river versus farther out in the forest), individuals may also be clustered by community (i.e. community_id') to account for variation in such community-level factors. Immune and lipid measures are transformed to normalize their skewed distributions. Variables were transformed as follows: CRP, BMI, and triglycerides were natural log-transformed; total leukocytes and subsets (lymphocytes neutrophils, eosinophils), ESR, and remaining cholesterols (total cholesterol, LDL, HDL, ox-LDL) were square-root transformed.