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Greater impact of dietary fat manipulation than apolipoprotein E genotype on ex vivo cytokine production – Insights from the SATgenε study☆

Highlights • The effect of diet and genotype on inflammatory response was explored in humans.• Cytokine production was not affected by apolipoprotein E (APOE) genotype.• TNF-α concentration significantly increased after a high saturated fat diet.• IL-10 concentration was significantly higher after a low fat diet.• The amount and type of dietary fat modulated cytokine production.


Introduction
The aetiology and progression of cardiovascular disease (CVD) is affected by both environmental and genetic factors [1]. The most widely researched common gene variants, with respect to CVD risk are the apolipoprotein E (APOE) single nucleotide polymorphisms (SNP) [2]. Although the literature is not fully consistent [3,4] APOE4 carriers (approximately 25% of the Caucasian population), have been reported to have a higher risk of CVD. This was originally attributed to elevated blood lipid levels in this subgroup [4]. However, the mechanisms that relate APOE4 to increased CVD risk may be more complex than solely a lipid effect [1,2] with studies largely conducted in transgenic animals or cell lines indicating that the APOE4 allele is related to a more pro-oxidative and pro-inflammatory state compared with the APOE3 allele [1,2].
Dietary fat manipulation may play an important role in the inflammatory response [5,6]; several studies indicated increasing levels of pro-inflammatory cytokines after a high fat meal or chronic consumption of a high fat diet [5,7,8]. However, few studies have focused on the impact of dietary fat composition on these markers in response to iso-energetic diets. Recently, we have reported that APOE genotype influences the C-reactive protein (CRP) response to dietary fat intake, with higher concentrations after diets rich in saturated fat (HSF) and HSF with 3.45 g/d of docosahexaenoic acid (DHA) in APOE4 carriers, relative to a low fat (LF) diet [9]. Yet, there are no studies investigating the combined effect of APOE genotype and fat manipulation on cytokine production in normolipidaemic subjects.
The aim of the present study was to investigate the effect of these three iso-energetic diets differing in fat quantity and quality on ex vivo whole blood culture (WBC) cytokine production according to APOE genotype. The WBC technique measures cytokine production following a pro-inflammatory stimulant and is considered a more physiologically meaningful and informative measure of inflammatory status in humans relative to the more traditional assessment of plasma cytokines in fasting blood.

Subjects and study design
A subset of the normolipidaemic participants from the SATgene study (n = 52/88), who were prospectively recruited according to APOE genotype (n = 26 E3/E3 and n = 26 E3/E4) provided blood samples at the beginning of the study (baseline) and eight weeks after the low fat (LF), high saturated fat (HSF) and HSF with 3.45 g/day DHA (HSF-DHA) diet for the determination of ex vivo cytokine production using whole blood culture. The target macronutrient composition of the diets are shown in Table 1 and a detailed description of study design and dietary manipulation are presented in Carvalho-Wells et al. [9].

Stimulation of whole blood cultures
Blood samples collected in EDTA tubes were diluted 1:9 with RPMI 1640 medium (Sigma, UK) supplemented with 1% antibiotics, 1% L-glutamine and 1% non-essential amino acids (BioScience, UK). Subsequently, the diluted blood sample was cultured in 12-well plates (Greiner bio-one, UK), with 10 or 0.5 lg/ml of bacterial lipopolysaccharide (LPS) (E. coli 026:B6, Sigma, UK), leading to a final concentration of 1 or 0.05 lg/ml, respectively. Cultures were incubated at 37°C for 24 h before centrifugation at 700Âg to isolate the supernatant, which was stored at À20°C until analysis. The monocyte count of each sample was measured by the Pathology Department at the Royal Berkshire Hospital in Reading.

Measurement of the cytokine concentration using the Luminex method
A Human Cytokine 10-Plex Panel (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFN-c, TNF-a, and GM-CSF; Invitrogen, Life Technologies) was used to measure the concentration of cytokines in the whole blood culture supernatant using the Luminex 200. Only IL-1b, IL-6, IL-8, IL-10 and TNF-a were detectable in the whole blood culture samples. Cytokine production was expressed as lg/10 3 monocytes as previously reported by Nagata et al. [10] and Rohleder et al. [11].

Statistical analysis
A one-within, one-between repeated measures ANOVA was used to analyse the effects of the different diets on the whole blood culture cytokine concentrations in the two genotype groups. Logarithmic or square-root transformation was applied to the variables that were not normally distributed. When statistical differences were found, data were further tested by the least significant difference (LSD) post hoc test. The statistical analysis was performed using SPSS version 17.0 (Statistical Package for Social Sciences, SPSS Inc., Chicago, Illinois, USA). P 6 0.05 was considered statistically significant.

Results
Two subjects (APOE4 carriers) were excluded because of missing monocyte population data. Cytokine production by either 0.05 lg/ml or 1 lg/ml LPS-stimulated WBC was not significantly affected by genotype ( Table 2, Fig. 1). In the data for the two genotype groups combined, dietary fat manipulation significantly affected TNF-a and IL-10 production, for both 0.05 lg/ml (P = 0.012 and P = 0.036, respectively, repeated measures ANOVA) and 1 lg/ml of LPS (P = 0.006 and P = 0.049, respectively, repeated measures ANOVA) (Fig. 1). Post hoc analysis revealed that TNF-a production was significantly higher in the WBC supernatant after the subjects consumed the HSF diet compared with baseline (P = 0.004 and P = 0.002, for 0.05 and 1 lg/ml LPS, respectively) and the LF diet (P = 0.020, for 1 lg/ml LPS only) (Fig. 1). The HSF-DHA diet resulted in higher TNF-a concentration in comparison to baseline (P = 0.012 and P = 0.021, for 0.05 and 1 lg/ml LPS, respectively) (Fig. 1). For both LPS concentrations, the consumption of the LF diet resulted in significantly higher concentrations of IL-10, compared with baseline (P = 0.013 and P = 0.015, for 0.05 and 1 lg/ml LPS, respectively) and the HSF-DHA diet (P = 0.026 and P = 0.050, for 0.05 and 1 lg/ml LPS, respectively) (Fig. 1). The levels of IL-1b, IL-6 and IL-8 were not significantly modulated by dietary fat composition (Table 2).

Discussion
The present study investigated the impact of APOE genotype on the ex vivo cytokine response of normolipidaemic subjects to chronic dietary fat manipulation. It has been indicated that APOE has immuno-modulatory properties that could affect the risk of CVD [2]. APOE genotype has been reported to affect macrophage cytokine secretion [1,2] and, thus, may be predicted to influence inflammatory responses. As we previously reviewed [2] animal and cell culture models and limited human study evidence showed that pro-inflammatory cytokine levels, such as TNF-a and IL-6, were higher whereas the anti-inflammatory cytokine IL-10 concentrations were lower in APOE4-expressing cells or genotype groups compared to APOE3 homozygotes. In contrast, in the current study, cytokine production was not significantly affected by APOE genotype suggesting that in normolipidaemic adults APOE genotype may not be a significant determinant of the circulating pro-inflammatory cytokine status [2].  Values represent mean SEM for the data presented according to genotype (E3/E3, n = 26 and E3/E4, n = 24) and the genotype groups combined (All, n = 50). Abbreviations: LPS, lipopolysaccharide; LF, low fat diet; HSF, high saturated fat diet; HSF-DHA, high saturated fat diet with 3.45 g/d docosahexaenoic acid (DHA). Several observational and intervention studies have explored the impact of a high fat diet, as well as high SFA diet, on pro-inflammatory markers [5][6][7][8]. Nappo et al. [7] have reported that a high fat meal (59% fat) increased the postprandial levels of TNF-a, IL-6, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in normal subjects, whereas an isoenergetic high carbohydrate meal had no effect. Longer-term dietary studies have also reported a significant effect of dietary fat manipulation on the inflammatory response. Baer et al. [8] have shown that the consumption of specific saturated fatty acids including lauric, myristic, palmitic and stearic acid for a five-week period was associated with higher levels of CRP, fibrinogen and IL-6. These results are in agreement with our data, where TNF-a, an important pro-inflammatory cytokine involved in the progression of atherosclerosis, significantly increased after consumption of a diet high in SFA (HSF). Previous analysis of our data has also shown that the HSF diet resulted in a significant increase in fasted serum CRP, compared with the LF diet [9]. These data suggests that the deleterious impact of habitual high fat and high saturated fat intake may in part be mediated via an impact on TNF-a production.
Consumption of the LF diet was associated with a significant increase in IL-10 concentrations compared with the baseline. IL-10 is an anti-inflammatory cytokine, which down-regulates many inflammatory pathways that are associated with atherosclerosis [12]. In particular, it inhibits the production of NF-jB and thus, suppresses cytokine production [12]. Moreover, it is associated with beneficial effects in patients with acute coronary disease [13]. To the best of our knowledge, this study is among the first to evaluate the effect of different iso-energetic fat diets on the ex vivo levels of an anti-inflammatory cytokine, such as IL-10 in WBC samples obtained from human subjects. With reference to the supplementation of the HSF diet with long chain n-3 fatty acids (3.45 g/d of DHA) we did not observe any significant changes in cytokine levels between APOE3/E3 and APOE4 carriers nor in our study group as a whole. Although long-chain n-3 fatty acids are purported to be anti-inflammatory, with for example demonstrated benefits on plaque inflammation [14] and the need for use of non-steroidal anti-inflammatory drugs in rheumatoid arthritis patients [15], our results are in general agreement with the majority of human intervention studies, including our main SATgene study, which have not shown a clear effect on circulating inflammatory status [16].
In this study two concentrations of LPS were used for stimulation of WBC (1 lg/ml and 0.05 lg/ml) with both giving comparable results. It is therefore suggested that in future studies it would be advisable to use the more physiological concentration of 0.05 lg/ ml. Moreover, since monocytes are the main site of cytokine production after LPS stimulation in whole blood, a correction with monocytes is considered by many to be the most appropriate method to use [10,11].
In conclusion, dietary fat composition but not APOE genotype significantly influenced ex vivo pro-and anti-inflammatory cytokine production after LPS stimulation of WBC samples obtained from normolipidaemic adults.