On a Western diet, APOE ɛ4 is associated with low innate immune sensing, but not APOE ɛ3
Authors: Janina Dose, Anke Schloesser, Guillermo G. Torres, Geetha Venkatesh, Robert Häsler, Friederike Flachsbart, Wolfgang Lieb, Almut Nebel, Gerald Rimbach, Patricia Huebbe
Journal: Journal of Allergy and Clinical Immunology (2018)

Apolipoprotein E (APOE), a polymorphic constituent of lipoproteins in humans, exerts functions within and beyond lipid metabolism. APOE isoforms, encoded by the alleles ɛ2, ɛ3, and ɛ4, seem to affect immunological processes and host defense differentially. In developed countries, APOE ɛ4 is associated with increased risk of age-related chronic diseases. APOE ɛ4 was earlier characterized as proinflammatory.1Gale S.C. Gao L. Mikacenic C. Coyle S.M. Rafaels N. Murray Dudenkov T. et al.APOɛ4 is associated with enhanced in vivo innate immune responses in human subjects.J Allergy Clin Immunol. 2014; 134: 127-134Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar Gale et al1Gale S.C. Gao L. Mikacenic C. Coyle S.M. Rafaels N. Murray Dudenkov T. et al.APOɛ4 is associated with enhanced in vivo innate immune responses in human subjects.J Allergy Clin Immunol. 2014; 134: 127-134Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar have recently demonstrated an enhanced innate immune response in the presence of APOE ɛ4 after stimulation with Toll-like receptor (TLR) ligands in humans and APOE targeted replacement mice. However, if there is no direct acute inflammatory stimulus, but rather a chronic low-grade induction of the immune response via a Western diet (WD), our data indicate that APOE ɛ4 is associated with a lower expression of markers important in innate immune sensing. We analyzed the expression of all 10 human TLRs (TLR1-TLR10) in relation to the APOE genotype in whole blood (n = 127; see Table E1 in this article's Online Repository at www.jacionline.org). RNAseq-based transcriptome data showed significantly lower expression of TLR1 (P = .002), TLR4 (P = .009), TLR6 (P = .005), TLR8 (P = .009), and TLR10 (P = .065) and a trend for TLR2 (P = .100) in APOE ɛ4/ɛ3 heterozygous versus APOE ɛ3/ɛ3 homozygous individuals (Fig 1). TaqMan gene expression data from a second set of human samples were supportive, showing 5% (TLR6) to 32% (TLR10) lower expression in APOE ɛ4/ɛ3 heterozygous versus APOE ɛ3/ɛ3 homozygous individuals (see Tables E1 and E2 in this article's Online Repository at www.jacionline.org). We are first in examining an association of the APOE genotype with expression levels of all human TLRs. It should be noted that we observed APOE genotype-dependent differences without having been able to correct for dietary fat intake or intake of anti-inflammatory drugs by study participants, recent infections, or levels of cell subpopulations in the samples. More comparable studies in humans are warranted with very high numbers that would allow a comparison between APOE ɛ3/ɛ3 and APOE ɛ4/ɛ4 homozygous individuals; unfortunately, the prevalence of APOE ɛ4/ɛ4 homozygosity is generally very low in Europeans. In mechanistic experiments using APOE targeted replacement mice (n = 4-10/genotype/treatment), we chose a WD (high in fat, sugar, and cholesterol) over direct LPS stimulation, because our interest was not the response to an acute inflammatory stimulus, but the potential consequences of the Western lifestyle. WD-induced low-grade systemic endotoxemia is typical for Western populations. Because dietary restriction (DR) may affect immune function positively, our studies further encompassed a 30% restricted WD (WD-DR) feeding regimen and a healthier control diet (CD; lower in fat, sugar, and cholesterol; higher in starch). On ad libitum WD, APOE ɛ4/ɛ4 mice showed lower liver weights than APOE ɛ3/ɛ3 mice (P = .004; Fig 2, A). Hepatic expression of the LPS sensors lipopolysaccharide binding protein (Lbp), monocyte differentiation antigen Cd14 (Cd14), and Tlr4 was also lower in APOE ɛ4/ɛ4 mice. APOE ɛ3/ɛ3 mice exhibited 1.3-fold higher Lbp (P = .009), 2.7-fold higher Cd14 (P = .002), and 1.5-fold higher Tlr4 (P = .014) mRNA levels than APOE ɛ4/ɛ4 mice (Fig 2, B). Concordantly, we detected a higher transcription of Tnf-α (P = .002), Il6 (P = .048), and Cd36 (P = .088) in APOE ɛ3/ɛ3 mice (Fig 2, B). In plasma, LBP was approximately 16% higher expressed in APOE ɛ3/ɛ3 versus APOE ɛ4/ɛ4 mice (P = .034; Fig 2, C). Strikingly, CD14 levels were more than 2-fold higher in APOE ɛ3/ɛ3 mice (P = .000007; Fig 2, D). APOE ɛ3/ɛ3 mice reacted to changes in diet availability (WD vs WD-DR) or nutrient content (WD vs CD), with an overall stronger reduction in the expression of LPS sensors than APOE ɛ4/ɛ4 mice, which exhibited low expression relatively independent of the dietary regimen (see Table E3 in this article's Online Repository at www.jacionline.org). Part of the effects may be mediated by an APOE genotype-dependent differential nuclear factor kappa B activation (see Fig E1 in this article's Online Repository at www.jacionline.org). A higher activity of the LPS detoxifying enzyme intestinal alkaline phosphatase (IAP) in APOE ɛ4/ɛ4 versus APOE ɛ3/ɛ3 mice may influence the differential immune activation. On ad libitum WD, IAP was 130% more active in feces of APOE ɛ4/ɛ4 versus APOE ɛ3/ɛ3 mice (P = .006; Fig 2, E). On WD-DR, its activity appeared 60% higher (see Fig E2, A in this article's Online Repository at www.jacionline.org). In contrast, hepatic alkaline phosphatase (ALP) activity was approximately 55% higher in APOE ɛ3/ɛ3 versus APOE ɛ4/ɛ4 mice (P = .0001; Fig 2, F). In response to DR or CD feeding, ALP activity decreased approximately 56% and 65%, respectively, in APOE ɛ3/ɛ3 mice (P = .00003 and P = .004 for WD vs WD-DR and WD vs CD, respectively), whereas ALP activity in APOE ɛ4/ɛ4 mice remained unchanged (Fig E2, B). In APOE ɛ3/ɛ3 mice, hepatic ALP activity correlated with body weight, but not so in APOE ɛ4/ɛ4 mice (Fig E2, C). Taken together, our data suggest lower innate immune sensing in the presence of APOE ɛ4 at least in the absence of an acute direct stimulus. APOE ɛ3/ɛ3 mice are more prone to diet-induced obesity.2Huebbe P. Dose J. Schloesser A. Campbell G. Glüer C.C. Gupta Y. et al.Apolipoprotein E (APOE) genotype regulates body weight and fatty acid utilization— studies in gene-targeted replacement mice.Mol Nutr Food Res. 2015; 59: 334-343Crossref PubMed Google Scholar, 3Arbones-Mainar J.M. Johnson L.A. Altenburg M.K. Maeda N. Differential modulation of diet-induced obesity and adipocyte functionality by human apolipoprotein E3 and E4 in mice.Int J Obes. 2008; 32: 1595-1605Crossref PubMed Scopus (74) Google Scholar Therefore, a higher inflammatory load in APOE ɛ3/ɛ3 mice as observed in our experiments is reasonable. Our results appear to contradict the accepted concept of a proinflammatory nature of APOE ɛ4; however, the response of APOE ɛ4 to an inflammatory stimulus may depend on the environment and stimulus type. An acute infection can hardly be compared with WD-induced low-grade chronic endotoxemia. Furthermore, LPS administered to isolated cells or injected systemically in vivo does not encounter IAP; that is, it cannot be detoxified before entry into the circulation. We hypothesize that in APOE ɛ4/ɛ4 mice, gut-derived LPS is largely detoxified in the intestine, leading to less LPS in plasma and consequently to low systemic immune activation, reflected in low expression of LPS sensors and low hepatic ALP activity. In APOE ɛ3/ɛ3 mice, it appears that more LPS passes on to the liver, which requires high hepatic ALP activity. CD14 deficiency has been found to lead to lower body and liver weight in mice,4Cani P.D. Amar J. Iglesias M.A. Poggi M. Knauf C. Bastelica D. et al.Metabolic endotoxemia initiates obesity and insulin resistance.Diabetes. 2007; 56: 1761-1772Crossref PubMed Scopus (4184) Google Scholar and IAP deficiency to enhanced body weight gain in WD-fed mice.5Narisawa S. Huang L. Iwasaki A. Hasegawa H. Alpers D.H. Millán J.L. Accelerated fat absorption in intestinal alkaline phosphatase knockout mice.Mol Cell Biol. 2003; 23: 7525-7530Crossref PubMed Scopus (169) Google Scholar Concordantly, our APOE ɛ4/ɛ4 mice were thinner and displayed lower liver weights compared with APOE ɛ3/ɛ3 mice. Interestingly, IAP is implicated in gut homeostasis.6Lallès J.P. Intestinal alkaline phosphatase: multiple biological roles in maintenance of intestinal homeostasis and modulation by diet.Nutr Rev. 2010; 68: 323-332Crossref PubMed Scopus (253) Google Scholar Therefore, our data provide evidence for a beneficial effect of APOE ɛ4 in intestinal health as has been suggested before.7Azevedo O.G. Bolick D.T. Roche J.K. Pinkerton R.F. Lima A.A. Vitek M.P. et al.Apolipoprotein E plays a key role against cryptosporidial infection in transgenic undernourished mice.PLoS One. 2014; 9: e89562Crossref PubMed Scopus (26) Google Scholar To strengthen our hypothesis, it would be crucial to determine the actual LPS load in plasma in future studies. Furthermore, our results encourage studies focusing on immune-relevant cells such as macrophages and monocytes or natural killer cells, in which potential read-outs could be the TLR4 and/or CD14 expression and the distribution and/or numbers of cells under various dietary regimens in relation to the APOE genotype. Our results are in agreement with previous investigations that showed APOE ɛ4 to be associated with higher levels of the anti-inflammatory and immunomodulatory vitamin D and adiponectin.2Huebbe P. Dose J. Schloesser A. Campbell G. Glüer C.C. Gupta Y. et al.Apolipoprotein E (APOE) genotype regulates body weight and fatty acid utilization— studies in gene-targeted replacement mice.Mol Nutr Food Res. 2015; 59: 334-343Crossref PubMed Google Scholar In addition, levels of C-reactive protein are lower in APOE ɛ4 carriers versus noncarriers,8Hubacek J.A. Peasey A. Pikhart H. Stavek P. Kubinova R. Marmot M. et al.APOE polymorphism and its effect on plasma C-reactive protein levels in a large general population sample.Hum Immunol. 2010; 71: 304-308Crossref PubMed Scopus (50) Google Scholar which may be indicative of an anti-inflammatory milieu. In summary, all these findings suggest a protective role of APOE ɛ4 in metabolic disorders. Indeed, in a recent study, WD feeding led to higher insulin and plasma lipid levels, impaired glucose tolerance, and higher body weights in APOE ɛ3/ɛ3 versus APOE ɛ4/ɛ4 mice,9Slim K.E. Vauzour D. Tejera N. Voshol P.J. Cassidy A. Minihane A.M. The effect of dietary fish oil on weight gain and insulin sensitivity is dependent on APOE genotype in humanized targeted replacement mice.FASEB J. 2017; 31: 989-997Crossref PubMed Scopus (12) Google Scholar although contrasting findings exist.3Arbones-Mainar J.M. Johnson L.A. Altenburg M.K. Maeda N. Differential modulation of diet-induced obesity and adipocyte functionality by human apolipoprotein E3 and E4 in mice.Int J Obes. 2008; 32: 1595-1605Crossref PubMed Scopus (74) Google Scholar Human studies are still rare and inconclusive. We hope that our data stimulate further research into the role of the APOE genotype in metabolic disorders. German study participants were recruited with the help of the biobank PopGen in Kiel. Recruitment of individuals was conducted as described previously.E1Nebel A. Croucher P.J.P. Stiegeler R. Nikolaus S. Krawczak M. Schreiber S. No association between microsomal triglyceride transfer protein (MTP) haplotype and longevity in humans.Proc Natl Acad Sci U S A. 2005; 102: 7906-7909Crossref PubMed Scopus (87) Google Scholar Both human studies were approved by the Ethics Committee of Kiel University, and written informed consent was obtained from all individuals. Study population 1E2Häsler R. Venkatesh G. Tan Q. Flachsbart F. Sinha A. Lieb W. et al.Genetic interplay between human longevity and metabolic pathways – a large-scale eQTL study.Aging Cell. 2017; 16: 716-725Crossref PubMed Scopus (12) Google Scholar consisted of 127 individuals; study population 2 encompassed 142 study participants. APOE genotyping for the SNPs rs429358 and rs7412 was performed using TaqMan SNP Genotyping Assays (Thermo Fisher Scientific Germany BV & Co KG, Braunschweig, Germany). APOE genotype distributions are presented in Table E1. Extraction of DNA and RNA was performed as described in Häsler et al.E2Häsler R. Venkatesh G. Tan Q. Flachsbart F. Sinha A. Lieb W. et al.Genetic interplay between human longevity and metabolic pathways – a large-scale eQTL study.Aging Cell. 2017; 16: 716-725Crossref PubMed Scopus (12) Google Scholar For study population 1, TLR expression (TLR1-TLR10) was analyzed using quantile-normalized whole blood transcriptome data generated as described previously.E2Häsler R. Venkatesh G. Tan Q. Flachsbart F. Sinha A. Lieb W. et al.Genetic interplay between human longevity and metabolic pathways – a large-scale eQTL study.Aging Cell. 2017; 16: 716-725Crossref PubMed Scopus (12) Google Scholar TLR expression levels (TLR1, 4, 5, 8, and 10) of the individuals of study population 2 were analyzed by TaqMan gene expression assays (Thermo Fisher Scientific Germany BV & Co KG). TaqMan gene expression data were normalized by the mean expression of the housekeeping genes actin beta (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (RN18S) and analyzed with the 2−ΔΔCT method.E3Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−(Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123336) Google Scholar Mice studies were conducted according to the German regulations of animal welfare and were approved by the local animal ethical committee. Mice, homozygous for human APOE ɛ3/ɛ3 and APOE ɛ4/ɛ4, respectively, were purchased from Taconic (Ry, Denmark). All mice were 4 to 6 weeks old on delivery. The genetic difference between APOE ɛ3/ɛ3 and APOE ɛ4/ɛ4 targeted replacement (TR) mice is less than 2%, reflecting the typical genomic variance due to targeted gene replacement.E4Huebbe P. Dose J. Schloesser A. Campbell G. Glüer C.C. Gupta Y. et al.Apolipoprotein E (APOE) genotype regulates body weight and fatty acid utilization—studies in gene-targeted replacement mice.Mol Nutr Food Res. 2015; 59: 334-343Crossref PubMed Scopus (40) Google Scholar The mice were housed in macrolon cages under a 12-hour light/dark cycle (22°C, 55%-60% humidity). Purified semisynthetic diets were purchased from Ssniff (Soest, Germany; for composition of the diets see Table E4) and were provided in a pelleted form. Study 1 (WD vs WD-DR): After delivery and 2 weeks of acclimation, 24 male APOE ɛ3/ɛ3 and APOE ɛ4/ɛ4 TR mice (12 of each APOE genotype) were assigned to 2 different dietary regimens. The assignment to the groups was carried out as described previously.E5Schloesser A. Campbell G. Glüer C.C. Rimbach G. Huebbe P. Restriction on an energy-dense diet improves markers of metabolic health and cellular aging in mice through decreasing hepatic mTOR activity.Rejuvenation Res. 2015; 18: 30-39Crossref PubMed Scopus (20) Google Scholar Half of the mice of each APOE genotype were fed an energy-dense high-fat and high-sugar diet (WD) ad libitum to induce mild metabolic endotoxemia. The other half was fed WD-DR on the same WD, which means that mice obtained 30% less food than did their ad libitum–fed counterparts. Implementation of WD-DR was performed as described previously.E5Schloesser A. Campbell G. Glüer C.C. Rimbach G. Huebbe P. Restriction on an energy-dense diet improves markers of metabolic health and cellular aging in mice through decreasing hepatic mTOR activity.Rejuvenation Res. 2015; 18: 30-39Crossref PubMed Scopus (20) Google Scholar Mice were housed individually with free access to tap water. Feces were collected in the final week of the feeding experiment. Mice were sacrificed at an age of 7.5 months. A small proportion of the liver was collected in RNAlater (Qiagen, Hilden, Germany) for RNA analyses and stored at −20°C. The remaining liver tissue was immediately snap-frozen in liquid nitrogen and stored at −80°C. Heparin-blood was collected by heart puncture directly after cervical dislocation. Blood was incubated on ice for 30 minutes and then centrifuged (10 minutes, 2000g, 4°C). Plasma was stored at −80°C until ELISA measurements. Study 2 (WD vs CD): Study 2 was carried out as published previously.E4Huebbe P. Dose J. Schloesser A. Campbell G. Glüer C.C. Gupta Y. et al.Apolipoprotein E (APOE) genotype regulates body weight and fatty acid utilization—studies in gene-targeted replacement mice.Mol Nutr Food Res. 2015; 59: 334-343Crossref PubMed Scopus (40) Google Scholar Isolation of RNA from liver tissue of APOE ɛ3/ɛ3 and APOE ɛ4/ɛ4 TR mice was performed as described previouslyE6Chin D. Hagl S. Hoehn A. Huebbe P. Pallauf K. Grune T. et al.Adenosine triphosphate concentrations are higher in the brain of APOE3- compared to APOE4-targeted replacement mice and can be modulated by curcumin.Genes Nutr. 2014; 9: 397Crossref PubMed Scopus (33) Google Scholar with some modifications. RNA was isolated using the NucleoSpin miRNA kit (Macherey-Nagel GmbH & Co KG, Dueren, Germany) according to the manufacturer's instructions. The concentration and quality of the isolated RNA was measured on a NanoDrop ND-2000 spectrophotometer (VWR International GmbH, Erlangen, Germany). Primers for quantitative RT-PCR were purchased from Eurofins MWG (Ebersberg, Germany). Primer3 Input software version 0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) was used for primer design (for primer sequences, see Table E5). One-step quantitative RT-PCR was carried out on a Rotorgene 6000 cycler (Corbett Life Science, Sydney, Australia) using the SensiFAST SYBR No-ROX One-Step Kit with SYBR green detection (Bioline, Luckenwalde, Germany). Relative mRNA quantities of target genes in the samples were determined from known relative quantities of standard RNA. Gene expression levels of target genes were normalized by the mean expression of housekeeping genes (mean of Rn18s and Eef2 levels for mouse study 1; mean of Actb, Gapdh, Eef2, B2m and Rn18s for mouse study 2, respectively). The values for the WD groups from mouse study 1 and 2 were pooled after normalization. CD14 and LBP plasma levels were determined using commercially available ELISA kits (Mouse CD14 Quantikine ELISA Kit, R&D Systems, Minneapolis, Minn; Mouse LBP ELISA Kit, Uscn Life Science Inc, Hubei, China). For CD14 measurement, plasma samples were diluted by a factor of 40, and for the determination of LBP plasma levels by 100. Hepatic and fecal ALP activities were determined using the ALP activity assay kit (Abcam, Cambridge, UK) according to the manufacturer's instructions with some modifications. Liver tissue and fecal samples were homogenized in assay buffer at a ratio of 1:10 and centrifuged (10 minutes, 4800g, 4°C). Liver samples were used at a dilution of 10 in the assay, and fecal samples at a dilution of 2000. An external standard curve with p-nitrophenylphosphate was used for the calculation of ALP activity in the samples. ALP activity in liver samples was normalized to the protein content. Protein content of liver homogenates was determined with the Thermo Scientific Pierce BCA Protein Assay Kit (Thermo Scientific Inc, Rockford, Ill). For fecal samples, results were calculated on the basis of weighed portion of feces. Cytosolic (phospho-nuclear factor kappa B inhibitor alpha [pIκBα]) and nuclear extracts (nuclear factor kappa B [NF-κB p65]) were prepared from mouse liver tissue as described previously.E7Graeser A.C. Boesch-Saadatmandi C. Lippmann J. Wagner A.E. Huebbe P. Storm N. et al.Nrf2-dependent gene expression is affected by the proatherogenic apoE4 genotype—studies in targeted gene replacement mice.J Mol Med. 2011; 89: 1027-1035Crossref PubMed Scopus (24) Google Scholar Protein concentrations of tissue extracts were determined with the Thermo Scientific Pierce BCA Protein Assay Kit. Western blot analysis was carried out as described previously.E5Schloesser A. Campbell G. Glüer C.C. Rimbach G. Huebbe P. Restriction on an energy-dense diet improves markers of metabolic health and cellular aging in mice through decreasing hepatic mTOR activity.Rejuvenation Res. 2015; 18: 30-39Crossref PubMed Scopus (20) Google Scholar Identification of target proteins was performed using respective primary (New England Biolabs GmbH, Frankfurt am Main, Germany [IκBα, pIκBα]; Santa Cruz Biotechnology Inc, Heidelberg, Germany [NF-κB p65]) and secondary (BioRad, Munich, Germany) antibodies. Protein bands were visualized in a ChemiDoc XRS system (BioRad) using ECL reagents (Fisher Scientific, Schwerte, Germany). Band intensities were calculated with Image Lab 4.1 Software (BioRad). Target protein expression was normalized by total protein load per lane, measured as PVDF membrane fluorescence. Statistical analysis was performed using the statistics software R.E8R Core Team A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna2014Google Scholar Mouse data were tested for normal distribution by Shapiro-Wilk test. Data were analyzed by linear regression considering sex as confounding variable. Outlier correction in mouse data was performed with the R outliers package. An outlier was excluded from the analysis if the following 2 criteria were fulfilled: (1) group size more than 6 and (2) outlier with significant P value (P < .05) after chi-square test for outlier. Human transcriptome and TaqMan gene expression data were analyzed by a linear regression model considering age and sex as confounders. Results obtained from transcriptome analysis were corrected for multiple testing using the Benjamini-Hochberg method.E9Benjamini Y. Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing.J Roy Statist Soc Ser B. 1995; 57: 289-300Google Scholar P values of less than .05 were considered statistically significant. Correlation coefficients with respect to the association between body weight of APOE ɛ3/ɛ3 and APOE ɛ4/ɛ4 TR mice and hepatic ALP activity were calculated on the basis of Pearson.Fig E2Alkaline phosphatase activity in feces and liver of APOE TR mice subjected to different dietary regimens. A, IAP activity in feces. B, Hepatic ALP activity. C, Correlation of hepatic ALP activity with body weight. A-C, The values for the WD groups were pooled from 2 mouse studies. Values are means + SEM (n = 4-10); linear regression, gender-corrected data. P values are given above the respective bars (Fig E2, A and B) and next to the graph (Fig E2, C), respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1APOE genotype distributions in study populationsVariableStudy population 1Study population 2n127142APOE genotype (n) (%) APOE ɛ2/ɛ20 (0.0)1 (0.7) APOE ɛ2/ɛ43 (2.4)7 (4.9) APOE ɛ2/ɛ320 (15.7)25 (17.6) APOE ɛ3/ɛ374 (58.3)79 (55.6) APOE ɛ4/ɛ329 (22.8)28 (19.7) APOE ɛ4/ɛ41 (0.8)2 (1.4) Open table in a new tab Table E2Expression of TLRs 1, 4, 6, 8, and 10 assessed by TaqMan gene expression assays in whole blood of APOE ɛ4/ɛ3 heterozygous vs APOE ɛ3/ɛ3 homozygous individuals in study population 2Gene2ˆCt values∗Values are means ± SEM; values normalized by the mean of the housekeeping genes.P value†Multilinear regression, adjusted for age and gender.Fold change‡APOE ɛ3/ɛ3 set as reference (set to 1).APOE ɛ3/ɛ3APOE ɛ4/ɛ3TLR15.17 × 10−09 ± 5.48 × 10−104.30 × 10−09 ± 5.14 × 10−10.382.85TLR41.15 × 10−09 ± 1.12 × 10−109.37 × 10−10 ± 1.13 × 10−10.321.88TLR69.85 × 10−09 ± 9.47 × 10−108.71 × 10−09 ± 7.52 × 10−10.632.94TLR85.25 × 10−11 ± 5.08 × 10−124.02 × 10−11 ± 5.28 × 10−12.281.86TLR104.35 × 10−10 ± 4.64 × 10−113.65 × 10−10 ± 6.46 × 10−11.630.68∗ Values are means ± SEM; values normalized by the mean of the housekeeping genes.† Multilinear regression, adjusted for age and gender.‡ APOE ɛ3/ɛ3 set as reference (set to 1). Open table in a new tab Table E3APOE genotype-dependent effects of WD-DR or CD feeding on markers of inflammation in liver and plasma of APOE TR miceGene/proteinAPOE ɛ3/ɛ3APOE ɛ4/ɛ4WDWD-DRCDWDWD-DRCDMeanMeanP value∗Compared with the WD-fed group (linear regression, sex considered a confounding variable); n = 4-10. Values for the WD groups pooled from 2 mouse studies. ELISA measurements performed in duplicate for more than half of the samples in each group. IAP and ALP activity levels measured in duplicate. All values are means ± SEM. P values given above the respective bars.MeanP value∗Compared with the WD-fed group (linear regression, sex considered a confounding variable); n = 4-10. Values for the WD groups pooled from 2 mouse studies. ELISA measurements performed in duplicate for more than half of the samples in each group. IAP and ALP activity levels measured in duplicate. All values are means ± SEM. P values given above the respective bars.MeanMeanP value∗Compared with the WD-fed group (linear regression, sex considered a confounding variable); n = 4-10. Values for the WD groups pooled from 2 mouse studies. ELISA measurements performed in duplicate for more than half of the samples in each group. IAP and ALP activity levels measured in duplicate. All values are means ± SEM. P values given above the respective bars.MeanP value∗Compared with the WD-fed group (linear regression, sex considered a confounding variable); n = 4-10. Values for the WD groups pooled from 2 mouse studies. ELISA measurements performed in duplicate for more than half of the samples in each group. IAP and ALP activity levels measured in duplicate. All values are means ± SEM. P values given above the respective bars.Lbp mRNA1.08 ± 0.051.08 ± 0.12.8410.78 ± 0.13.2670.82 ± 0.071.24 ± 0.33.4150.79 ± 0.12.205Cd14 mRNA2.56 ± 0.361.30 ± 0.46.0380.45 ± 0.14.0330.96 ± 0.201.47 ± 0.69.5750.41 ± 0.09.240Tlr4 mRNA1.69 ± 0.161.08 ± 0.20.0490.57 ± 0.10.0131.12 ± 0.121.22 ± 0.38.7250.71 ± 0.10.501Tnf-α mRNA1.47 ± 0.160.77 ± 0.15.0031.05 ± 0.41.8850.81 ± 0.110.83 ± 0.33.5880.61 ± 0.21.761Il6 mRNA1.25 ± 0.230.93 ± 0.12.5402.07 ± 0.60.8450.87 ± 0.091.33 ± 0.33.1752.26 ± 0.75.078Cd36 mRNA1.84 ± 0.100.45 ± 0.104 × 10−070.98 ± 0.21.00021.57 ± 0.220.59 ± 0.34.1591.46 ± 0.29.174CD14 (ng/mL) Plasma122 ± 7.7758.6 ± 6.09.000543.3 ± 5.23.000257.4 ± 4.0752.1 ± 5.66.46646.8 ± 9.72.420LBP (μg/mL) Plasma1.58 ± 0.121.54 ± 0.10.1091.30 ± 0.10.00091.32 ± 0.091.30 ± 0.16.8541.41 ± 0.13.891∗ Compared with the WD-fed group (linear regression, sex considered a confounding variable); n = 4-10. Values for the WD groups pooled from 2 mouse studies. ELISA measurements performed in duplicate for more than half of the samples in each group. IAP and ALP activity levels measured in duplicate. All values are means ± SEM. P values given above the respective bars. Open table in a new tab Table E4Composition of purified semisynthetic experimental dietsVariableWDCDGross energy (MJ/kg)22.118.4Nutrients (%) Crude protein (from casein)17.117.1 Crude fat21.2 (from milk fat)5.1 (from soy oil) Crude fiber5.05.0 Crude ash4.54.2 Nitrogen-free extracts49.364.5 Starch (from corn starch)14.539.0 Sugar (from corn starch)32.823.3 Cholesterol (mg/kg)2.07— Open table in a new tab Table E5Nucleotide sequences of primers used for quantitative real-time PCRGeneGeneIDPrimer, forwardPrimer, reverseAnn.(°C)Cd1412475GAATCTACCGACCATGGAGCCTGGACCAATCTGGCTTCGG59Lbp16803TGGAGCTCCTTGGAACAGTGCATTAGTGACCACGCCAAGCC59Tlr421898GGACTCTGATCATGGCACTGTTAGGAACTACCTCTATGCAGGGAT61Tnf-α21926TCGTAGCAAACCACCAAGTGAGATAGCAAATCGGCTGACG55Cd3612491CAA AAC GAC TGC AGG TCA ACCCA ATG GTC CCA GTC TCA TT55Il616193TAGTCCTTCCTACCCCAATTTCCTTGGTCCTTAGCCACTCCTTC61Actb∗Housekeeping gene.22461GACAGGATGCAGAAGAGATTTGATCCACATCTGCTGGAAGGT55Gapdh∗Housekeeping gene.14433CCGCATCTTCTTGTGCAGTGGCAACAATCTCCACTTTGC57B2m∗Housekeeping gene.12010GTCGCTTCAGTCGTCAGCATGTATGTTCGGCTGCCCATTC57Eef2∗Housekeeping gene.13629GCGTGCCAAGAAAGTAGAGGAAGATGGGGTCCAGGATGAG59Rn18s∗Housekeeping gene.19791GGTAACCCGTTGAACCCCATCAACGCAAGCTTATGACCCG59Actb, Actin, beta; Ann., annealing temperature; B2m, beta-2 microglobulin; Eef2, eukaryotic translation elongation factor 2; Rn18S, 18S ribosomal RNA.∗ Housekeeping gene. Open table in a new tab Actb, Actin, beta; Ann., annealing temperature; B2m, beta-2 microglobulin; Eef2, eukaryotic translation elongation factor 2; Rn18S, 18S ribosomal RNA.