International Journal of

Molecular Sciences
Article

Expression and Sequence Variants of Inflammatory

Genes; Effects on Plasma Inflammation Biomarkers
Following a 6-Week Supplementation with Fish Oil
Hubert Cormier 1,2 , Iwona Rudkowska 3,4 , Simone Lemieux 1,2 , Patrick Couture 1,4 and
Marie-Claude Vohl 1,2, *
1

2
3
4

Institute of Nutrition and Functional Foods (INAF), Laval University, Quebec City, QC G1V 0A6, Canada;
[email protected] (H.C.); [email protected] (S.L.);
[email protected] (P.C.)
School of Nutrition, Laval University, Quebec City, QC G1V 0A6, Canada
Endocrinology and Nephrology, Department of Kinesiology, CHU de Qubec Research Center,
Laval University, Quebec City, QC G1V 0A6, Canada; [email protected]
Centre Hospitalier Universitaire (CHU) de Qubec Research Center, Laval University, Quebec City,
QC G1V 4G2, Canada
Correspondence: [email protected]; Tel.: +1-418-656-2131

Academic Editor: Marcello Iriti

Received: 3 February 2016; Accepted: 7 March 2016; Published: 15 March 2016

Abstract: (1) Background: A growing body of literature suggest that polymorphisms (SNPs) from
inflammation-related genes could possibly play a role in cytokine production and then interact
with dietary n-3 fatty acids (FAs) to modulate inflammation. The aim of the present study was to
test whether gene expression of selected inflammatory genes was altered following an n-3 PUFA
supplementation and to test for genediet interactions modulating plasma inflammatory biomarker
levels. (2) Methods: 191 subjects completed a 6-week n-3 FA supplementation with 5 g/day of fish
oil. Gene expression of TNF- and IL6 was assessed in peripheral blood mononuclear cells (PBMCs)
using the TaqMan technology. Genotyping of 20 SNPs from the TNF-LTA gene cluster, IL1, IL6 and
CRP genes was performed. (3) Results: There was no significant reduction of plasma IL-6, TNF- and
C-reactive protein (CRP) levels after the 6-week fish oil supplementation. TNF- and IL6 were slightly
overexpressed in PBMCs after the supplementation (fold changes of 1.05 0.38 and 1.18 0.49,
respectively (n = 191)), but relative quantification (RQ) within the 0.5 to 2.0 fold are considered
as nonbiologically significant. In a MIXED model for repeated measures adjusted for the effects of
age, sex and BMI, gene by supplementation interaction effects were observed for rs1143627, rs16944,
rs1800797, and rs2069840 on IL6 levels, for rs2229094 on TNF- levels and for rs1800629 on CRP
levels (p < 0.05 for all). (4) Conclusions: This study shows that a 6-week n-3 FA supplementation
with 5 g/day of fish oil did not alter gene expression levels of TNF- and IL6 in PBMCs and did not
have an impact on inflammatory biomarker levels. However, genediet interactions were observed
between SNPs within inflammation-related genes modulating plasma inflammatory biomarker levels.
Keywords: omega-3 polyunsaturated fatty acids; polymorphism; inflammation; nutrigenetics

1. Introduction
Evidence suggests that fatty acids (FAs) can modulate adipokine production, thus accelerating and
influencing an individuals inflammatory response [1]. Omega-3 (n-3) FAs have potent anti-inflammatory
effects. n-3 FAs may affect inflammatory processes through modulation of eicosanoid metabolism and
by regulating transcription factors genes involved in inflammation [2]. The resolution of inflammation
involves active biochemical compounds, termed resolvins and protectins, which enable inflamed
Int. J. Mol. Sci. 2016, 17, 375; doi:10.3390/ijms17030375

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tissues to restore homeostasis [3]. However, these effects have been demonstrated in in vitro studies [4,5].
The extent of these effects in vivo is not clearly established and remains unclear. Dietary FAs, in
particular saturated FAs in addition to n-3 and omega-6 FAs could potentially modulate the expression
of genes encoding cytokines, possibly altering plasma cytokine levels as well [6].
A review by Joffe et al. suggested that polymorphisms (SNPs) within inflammation-related
genes may interact with environmental factors, such as dietary intakes, to modulate an individuals
susceptibility to develop obesity and its comorbidities [6]. Interaction effects between dietary FAs
and variations in inflammation-related genes such as tumor necrosis factor (TNF-) and interleukin 6
(IL6) may influence obesity phenotypes [6]. A growing body of literature suggest that SNPs from
inflammation-related genes could possibly play a role in cytokine production and then interact with
dietary n-3 FAs to modulate inflammation [7].
n-3 FAs are beneficial for inflammation-related diseases such as rheumatoid arthritis, inflammatory
bowel diseases and asthma [8]. Accordingly, marine n-3 FAs have been shown to decrease expression
levels of inflammation-related genes as well as plasma concentrations of cytokine and C-reactive
protein (CRP) [6]. However, there are no extensive studies showing a strong relationship between
expression of inflammation-related genes and n-3 FA supplementation. Previous research from
our laboratory has demonstrated changes in inflammatory pathways in human peripheral blood
mononuclear cells (PBMCs) after an n-3 FA supplementation via a transcriptomic approach [9].
Similarly, Bouwens et al. (2009), have examined the effects of high doses of n-3 FA supplementation
on whole-genome gene expression profiles in PBMCs, and reported a decrease in expression of genes
involved in inflammatory- and atherogenic-related pathways. Although, according to their results,
TNF- and IL6 gene expression levels were not underexpressed after the supplementation [10].
The aim of the present study was to test whether gene expression of inflammation-related genes
is altered following an n-3 FA supplementation and to test for possible genediet interactions with
SNPs within these genes modulating plasma inflammatory biomarker levels. We hypothesized that
a 6-week marine n-3 FA supplementation decreases gene expression of selected inflammation-related
genes and also decreases plasma levels of inflammatory biomarkers such as CRP, TNF- and IL-6
under the influence of SNPs within inflammation-related genes.
2. Results
Population characteristics have been previously described here [11] for the total cohort and
here [12] for this specific population included in this reanalysis. Briefly, participants have a higher
BMI following the 6-week n-3 FA supplementation (p = 0.006), but the absolute difference remains
extremely low at 0.1 kg/m2 , with no effects on waist circumference. After the supplementation, TG
levels decreased, as expected (p < 0.0001). Table 1 reports the descriptive characteristics of study
participants at baseline.
Table 1. Characteristics of the study participants at baseline (n = 191).
Characteristics

All (n = 191)

Men (n = 95, 49.7%)

Women (n = 96, 50.3%)

Age
Waist circumference, cm
BMI, kg/m2

30.8 8.7
93.2 10.6
27.2 3.6

30.9 8.1
94.8 10.9
27.5 3.6

30.8 9.3
91.6 10.1
27.9 3.5

Values are means SD.

Pre- and post-supplementation inflammatory marker levels are presented in Table 2. There was
no significant difference observed in inflammatory marker levels. However, a large inter-individual
variability was observed in the inflammatory response to a fish oil supplementation. For instance,
45.0%, 47.6% and 48.2% of study participants increased their plasma levels of CRP, TNF- and IL-6
respectively after the 6-week supplementation.

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Table 2. Pre- and post-supplementation inflammatory marker levels (n = 191).

Biomarkers

Pre n-3 FA

Post n-3 FA

p-Value 1

CRP levels (mg/L) 2

TNF- levels (g/mL) 3
IL-6 levels, (g/mL) 2

1.78 2.09
1.70 1.48
1.34 1.13

1.81 2.07
1.68 1.30
1.28 0.98

0.95
0.69
0.54

Values are means SD. * p < 0.05; 1 p-value are derived from a MIXED procedure for repeated measures
adjusted for age, sex and BMI (except for the BMI and the waist circumference that were adjusted only for age
and sex); 2 values were log(10) transformed; 3 values were normalised using the Box-Cox transformation.

Energy intakes were lower after the n-3 FA supplementation (p = 0.003) as shown in Table 3.
When looking at macronutrient distribution, there was a shift towards an increase in total fat together
with a decrease in carbohydrates (absolute difference of 24.8 g/day) and proteins (absolute difference
of 5.4 g/day). In fat intake, PUFA intakes were higher (p = 0.001) as a result of taking 5 g/day of fish
oil supplements while saturated FA intakes were lower with an absolute difference of 3.7 g/day.
Table 3. Dietary intakes pre- and post-supplementation with n-3 PUFAs (n = 191).
Dietary Intakes

Pre-Suppl.

Post-Suppl.

p-Value 1

(Including n-3 FA Supplements)

Energy, (kcal)
Carbohydrate, (% of TEI)
Carbohydrate, (g/day)
Protein, (% of TEI)
Protein, (g/day)
Total fat, (% of TEI)
Total fat, (g/day)
SFA, (% of TEI)
SFA, (g/day)
MUFA, (% of TEI)
MUFA, (g/day)
PUFA, (% of TEI)
PUFA, (g/day)
1

2290 599
50.5 7.2
288.9 79.8
17.4 3.3
98.6 30.6
32.5 6.0
85.0 29.7
11.1 3.1
29.1 12.1
11.8 2.8
30.8 11.8
5.9 2.1
15.3 6.7

2196 570
48.5 7.8
264.1 78.1
16.9 3.1
93.2 30.1
35.2 6.3
86.8 29.9
10.3 3.0
25.4 10.5
12.0 3.3
29.7 12.5
6.9 2.1
16.9 6.7

0.003
0.001
<0.0001
0.15
0.002
<0.0001
0.56
0.001
<0.0001
0.41
0.17
<0.0001
0.001

p-value provided by a paired t-test. TEI stands for Total energy intakes; SFA stands for Saturated fat.

Partial Spearman correlations between EPA, DHA or total n-3 FA levels (in % of total FA) from
plasma phospholipids and inflammatory marker levels adjusted for baseline data (both FAs and
inflammatory marker levels), age, sex and BMI are presented in Figure 1. Briefly, total plasma n-3 FA
levels negatively correlates with CRP (r = 0.15, p = 0.04), TNF- (r = 0.17, p = 0.02), and IL-6 levels
(r = 0.15, p = 0.04). Looking only at plasma EPA levels, a negative correlation was found with TNF-
levels (r = 0.18, p = 0.01) while trends were observed with CRP and IL-6 levels. Plasma DHA levels
tended to be negatively correlated with CRP and IL-6 levels (p < 0.10, for all).
Figure 2 shows a change in the expression of inflammation-related genes. Indeed, using the
2CT calculation method, TNF- and IL6 were slightly overexpressed in PBMCs after the 6-week
n-3 FA supplementation (fold changes of 1.05 0.38 and 1.18 0.49, respectively), but relative
quantification (RQ) within the 0.5 to 2.0 fold are considered as non-significant.

Int.
Int. J.J.Mol.
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375
Int. J. Mol. Sci. 2016, 17, 375

8
8

10
10

Spearman:
rSpearman:
= -0.18
-0.18
pr == 0.01
p = 0.01

10
10
5
5
0
00
0

2
2

EPA
(t1FA]
)
[% of
total
[% of total FA]

4
6
4
DHA
(t )6
1

8
8

TNF-
levels
[g/ml]
TNF-
levels
[g/ml]

15
15

Spearman:
rSpearman:
= -0.13
-0.13
pr == 0.07
p = 0.07

2
2

Spearman:
rSpearman:
= -0.11
-0.11
pr == 0.13
p = 0.13

5
5
0
00
0

15
15

15
15

20
20

TNF-
levels
[g/ml]
TNF-
levels
[g/ml]

CRP
levels
[mg/L]
CRP
levels
[mg/L]

[% of
totaln-3
FA]
Total
[% of total FA]

4
4
2
2
0
00
0

10
10

Spearman:
rSpearman:
= -0.14
-0.14
pr == 0.05
p = 0.05

6
6

2
2

2
2

4
6
4
DHA
(t )6

8
8

10
10

10
10
8
8
6
6
4
4
2
2
0
00
0

Spearman:
rSpearman:
= -0.17
-0.17
pr == 0.02
p = 0.02

5
5
0
00
0

5
5

10

8
8

10
10

F) DHA and IL-6 levels

F) DHA and IL-6 levels

Spearman:
rSpearman:
= -0.13
-0.13
pr == 0.08
p = 0.08

2
2

4
6
4
DHA
(t )6

8
8

10
10

DHA
)
[% of
total(t1FA]
[% of total FA]

H) Total n-3 and TNF- levels

H) Total n-3 and TNF- levels

10
10

4
EPA
(t1) 6
EPA
(t1FA]
)
[% of
total
[% of total FA]

DHA
)
[% of
total(t1FA]
[% of total FA]

Spearman:
rSpearman:
= -0.15
-0.15
pr == 0.04
p = 0.04

10
Total10n-3

10
10

10
10

G) Total n-3 and CRP levels

G) Total n-3 and CRP levels

5
5

8
8

E) DHA and TNF- levels

E) DHA and TNF- levels

DHA
)
[% of
total(t1FA]
[% of total FA]

10
10
8
8
6
6
4
4
2
2
0
00
0

4
6
4
EPA
(t ) 6

8
8

EPA
(t1FA]
)
[% of
total
[% of total FA]

D) DHA and CRP levels

D) DHA and CRP levels

CRP
levels
[mg/L]
CRP
levels
[mg/L]

10
10
8
8
6
6
4
4
2
2
0
00
0

C) EPA and IL-6 levels

C) EPA and IL-6 levels

levels
[g/ml]
IL-6IL-6
levels
[g/ml]

4
6
4
EPA
(t ) 6

B) EPA and TNF- levels

B) EPA and TNF- levels

15

15
Total10n-3
[% of
totaln-3
FA]
Total
[% of total FA]

20
20

10
10
8
8
6
6
4
4
2
2
0
00
0

I) Total n-3 and IL-6 levels

I) Total n-3 and IL-6 levels

levels
[g/ml]
IL-6IL-6
levels
[g/ml]

2
2

15
15

levels
[g/ml]
IL-6IL-6
levels
[g/ml]

Spearman:
rSpearman:
= -0.12
-0.12
pr == 0.09
p = 0.09

TNF-
levels
[g/ml]
TNF-
levels
[g/ml]

A) EPA and CRP levels

A) EPA and CRP levels

CRP
levels
[mg/L]
CRP
levels
[mg/L]

10
10
8
8
6
6
4
4
2
2
0
00
0

of 15
15
44 of
4 of 15

Spearman:
rSpearman:
= -0.15
-0.15
pr == 0.04
p = 0.04

5
5

10

15

15
Total10n-3
[% of
totaln-3
FA]
Total
[% of total FA]

20
20

Figure 1. Partial Spearman correlations between EPA, DHA or total n-3 PUFA (in % of total FA) from
Figure
1. Partial
PartialSpearman
Spearmancorrelations
correlations
between
EPA,
DHA
or total
n-3 PUFA
of FA)
totalfrom
FA)
Figure 1.
between
EPA,
DHA
or total
n-3 PUFA
(in %(in
of %
total
phospholipids and inflammatory marker levels controlled for baseline data (both FAs and
from
phospholipids
inflammatory
marker
levels
controlled
baselinedata
data(both
(both FAs
FAs and
phospholipids
and and
inflammatory
marker
levels
controlled
forfor
baseline
and
inflammatory marker levels), age, sex and BMI. Panels (AC) show correlations between EPA (in %
inflammatory
marker levels),
levels), age,
age, sex
sex and
and BMI.
BMI. Panels
Panels (AC)
(AC) show
show correlations
correlations between
between EPA
EPA (in
(in %
%
inflammatory marker
of total FA) and inflammatory markers; Panels (DF) show correlations between DHA (in % of total
of
total
FA)
and
inflammatory
markers;
Panels
(DF)
show
correlations
between
DHA
(in
%
of
total
of total FA) and inflammatory markers; Panels (DF) show correlations between DHA (in % of total
FA)
and inflammatory
markers; and
panels (GI)
show correlations
between total
% of
total
FA)
total n-3
n-3 (in
(in
FA) and
and inflammatory
inflammatory markers;
markers; and
and panels
panels (GI)
(GI) show
show correlations
correlations between
between total
n-3
(in %
% of
of total
total
FA)
and
inflammatory
markers;
straight
lines:
unadjusted
regression
slope,
and
dotted
lines:
FA)
markers;
straight
lines:lines:
unadjusted
regression
slope, and
dotted
adjusted
FA) and
andinflammatory
inflammatory
markers;
straight
unadjusted
regression
slope,
andlines:
dotted
lines:
adjusted
regression
slope
withinflammatory
baseline inflammatory
marker
levels,n-3
baseline
n-3 from phospholipids
regression
slope with
baseline
marker levels,
baseline
from phospholipids
levels, age,
adjusted regression
slope
with baseline inflammatory
marker
levels, baseline
n-3 from phospholipids
levels,
age,
sex
and
BMI
(n
=
191).
sex
and
BMI
(n
=
191).
levels, age, sex and BMI (n = 191).

Figure 2.
2. IL6
IL6and
andTNF-
TNF-gene
geneexpressions
expressions after
after aa 6-week
6-week n-3
n-3 FA
FA supplementation
supplementation (n
(n == 191).
191).
Figure
Figure 2. IL6 and TNF- gene expressions after a 6-week n-3 FA supplementation (n = 191).

All
All selected
selected SNPs
SNPs were
were in
in HWE
HWE and
and LD
LD plots
plots from
from Haploview
Haploview v4.2
v4.2 for
for each
each gene
gene are
are presented
presented
All selected SNPs were in HWE and LD plots from Haploview v4.2 for each gene are presented
in
in Figure
Figure 3.
3. For
For SNPs
SNPswithin
withinthe
theTNF-LTA
TNF-LTA gene
gene cluster,
cluster, 66 SNPS
SNPS covered
covered 93%
93% of
of the
the known
known genetic
genetic
in Figure 3. For SNPs within the TNF-LTA gene cluster, 6 SNPS covered 93% of the known genetic
variability,
forIL6,
IL6,5 5SNPS
SNPS
covered
100%,
for IL-1,
5 SNPs
covered
for4 CRP,
SNPs
variability, for
covered
100%,
for IL-1,
5 SNPs
covered
100%,100%,
and forand
CRP,
SNPs 4covered
variability, for IL6, 5 SNPS covered 100%, for IL-1, 5 SNPs covered 100%, and for CRP, 4 SNPs
covered
100%.
Table all
4 reports
all the
selected
within
the five inflammation-related
100%. Table
4 reports
the selected
SNPs
withinSNPs
the five
inflammation-related
genes studied.genes
covered 100%. Table 4 reports all the selected SNPs within the five inflammation-related genes
studied.
studied.

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Figure 3.
3. LD
lighter
the
shade
of of
grey,
the
lesser
is
Figure
LD plots
plotsof
ofselected
selectedinflammation-related
inflammation-relatedgenes.
genes.The
The
lighter
the
shade
grey,
the
lesser
thethe
correlation
between
two
SNPs.
(A)(A)
CRP
gene;
(B) (B)
TNF-
gene;
(C) (C)
LTALTA
gene;
(D) (D)
IL6 IL6
gene;
(E)
is
correlation
between
two
SNPs.
CRP
gene;
TNF-
gene;
gene;
gene;
IL-1
gene.
(E) IL-1 gene.

In a repeated MIXED model adjusted for the effects of age, sex, and BMI, several genediet
In a repeated MIXED model adjusted for the effects of age, sex, and BMI, several
interactions impacting inflammatory marker levels were observed following the n-3 FA
genediet interactions impacting inflammatory marker levels were observed following the n-3 FA
supplementation, as shown in Table 5. Figure 4 shows the genediet interaction on plasma TNF-
supplementation, as shown in Table 5. Figure 4 shows the genediet interaction on plasma TNF- levels
levels according to rs2229094 where carriers of the mutated allele increased their plasma TNF-
according to rs2229094 where carriers of the mutated allele increased their plasma TNF- levels after
levels after the 6-week n-3 FA supplementation while wild type homozygotes decreased theirs. Also,
the 6-week n-3 FA supplementation while wild type homozygotes decreased theirs. Also, significant
significant differences were observed in the genotype distribution of rs2229094 between positive and
differences were observed in the genotype distribution of rs2229094 between positive and negative
negative responders according to delta TNF- levels where positive responders decreased their
responders according to delta TNF- levels where positive responders decreased their plasma TNF-
plasma TNF- levels after the supplementation were an increase was observed in negative
levels after the supplementation were an increase was observed in negative responders. There was
responders. There was a higher proportion of C/C HMZ that were negative responders (6.8%) vs.
a higher proportion of C/C HMZ that were negative responders (6.8%) vs. positive responders (2.1%)
positive responders (2.1%) (p = 0.0003). An interaction between rs2229094 (TNF-LTA) and the 6-week
(p = 0.0003). An interaction between rs2229094 (TNF-LTA) and the 6-week n-3 FA supplementation
n-3 FA supplementation is of particular interest because that SNP is located in an exon and is
is of particular interest because that SNP is located in an exon and is responsible for an amino acid
responsible for an amino acid change (Cys13Arg). Moreover, analyses with SIFT argue for potential
change (Cys13Arg). Moreover, analyses with SIFT argue for potential functional effect of this SNP as
functional effect of this SNP as the amino acid change was considered damaged using homologues
the amino acid change was considered damaged using homologues in protein alignment (score = 0.04),
in protein alignment (score = 0.04), but tolerated using orthologues in protein alignment.
but tolerated using orthologues in protein alignment.

Int. J. Mol. Sci. 2016, 17, 375

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Table 4. Selected SNPs within TNF-LTA, IL-6, IL-1, and CRP genes.
Genes

dbSNP No.

Sequence b

Position

AA

CC

CA

CG

CT

GA

GG

45 (21.4)

163 (77.6)

29 (13.8)

3 (1.4)

GT

AT

TT

n (%)
TNF-LTA
TNF-LTA
TNF-LTA
TNF-LTA
TNF-LTA
TNF-LTA
IL6
IL6
IL6
IL6
IL6
IL1B
IL1B
IL1B
IL1B
IL1B
CRP
CRP
CRP
CRP
a

rs1041981
rs2857706
rs1800629
rs2239704
rs3093662
rs2229094
rs2069861
rs2069840
rs2069837
rs2069827
rs1800797
rs1143634
rs1143633
rs16944
rs3136558
rs1143627
rs1800947
rs3093059
rs1130864
rs1205

CA[A/C]CC
TA[A/G]GT
TG[A/G]GG
GC[G/T]GG
AC[A/G]GA
TG[C/T]GT
AA[C/T]AA
AA[C/G]TT
TA[A/G]AT
TC[G/T]AT
GG[A/G]TG
TT[C/T]GA
CC[A/G]CC
TC[A/G]GG
GA[C/T]CT
GC[C/T]AT
CT[C/G]TC
AT[C/T]GG
AA[C/T]GG
CA[C/T]AG

Missense [Thr][Asn]
Intron
nearGene-5
UTR-5
Intron
Missense [Cys][Arg]
nearGene-3
Intron
Intron
nearGene-5
nearGene-5
Cds-synon [Phe][Phe]
Intron
nearGene-5
Intron
nearGene-5
Cds-synon [Leu][Leu]
nearGene-5
UTR-3
UTR-3

22 (10.5)
2 (1.0)
49 (23.3)
178 (84.8)

97 (46.2)
151 (7.2)
72 (34.3)
19 (9.1)
177 (84.3)
95 (45.2)

91 (43.3)
52 (24.8)

7 (3.3)

89 (42.4)
74 (35.2)
33 (15.7)

117 (55.7)
0 (0.0)

181 (86.2)

92 (43.8)
28 (13.3)

73 (34.8)

111 (52.9)

23 (11.0)
1 (0.5)
172 (81.9)
26 (12.4)

94 (44.8)
82 (39.1)

84 (40.0)
100 (47.6)

129 (61.4)

70 (33.3)

32 (15.2)
28 (13.3)
8 (3.8)
29 (13.8)
190 (90.5)
2 (1.0)
102 (48.6)
99 (47.1)

77 (36.7)
81 (38.6)
20 (9.5)
32 (15.2)
84 (40.0)
89 (42.4)

38 (18.1)

0 (0.0)
11 (5.2)

125 (59.5)
100 (47.6)
0 (0.0)
176 (83.6)
24 (11.4)
22 (10.5)

dbSNP No. from HapMap Data Rel 28 Phase II + III, 10 August on NCBI b36 Assembly dbSNP b126 database; b Genes sequences from dbSNP short genetics variations NCBI
reference assembly.

Int. J. Mol. Sci. 2016, 17, 375

7 of 15

Table 5. Genediet interaction on inflammatory markers levels.

Gene

Biomarker

SNPs

Genotype by Suppl.

SE

p-Value for
the Genotype

p-Value for
the n-3 Suppl.

p-Value for the

Interaction

IL-6 levels (g/mL)

rs1143627

CC n-3 suppl.
CT n-3 suppl.
TT n-3 suppl.

0.014 0.025
0.037 0.014
0.014 0.013

0.91

0.78

0.02

IL-6 levels (g/mL)

rs16944

AA n-3 suppl.
AG n-3 suppl.
GG n-3 suppl.

0.018 0.025
0.038 0.014
0.014 0.013

0.87

0.87

0.02

IL-6 levels (g/mL)

rs1800797

AA n-3 suppl.
AG n-3 suppl.
GG n-3 suppl.

0.025 0.015
0.023 0.013
0.016 0.025

0.30

0.64

0.05

IL-6 levels (g/mL)

rs2069840

CC n-3 suppl.
CG n-3 suppl.
GG n-3 suppl.

0.010 0.013
0.016 0.014
0.063 0.029

0.11

0.28

0.04

TNF- levels, (g/mL)

rs2229094

CC n-3 suppl.
CT n-3 suppl.
TT n-3 suppl.

0.022 0.012
0.005 0.006
0.009 0.005

0.85

0.20

0.03

CRP levels (mg/L)

rs1800629

GG n-3 suppl.
[AA+GA] n-3 suppl.

0.012 0.016
0.058 0.030

0.37

0.17

0.04

IL-1

IL6

TNF-LTA
gene family

: regression coefficient. The MIXED model for repeated measures included the SNP, the time and the interaction term with adjustments for the effects of age, sex and BMI.

Int. J. Mol. Sci. 2016, 17, 375

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Int. J. Mol. Sci. 2016, 17, 375

7 of 15

TNF- levels [g/ml]

T/ T (n=108)
C/ T (n=66)
C/ C (n=17)

Before

After

Figure
4. Genediet
onplasma
plasma
TNF-
levels
according
to rs2229094
Figure
4. Genedietinteraction
interaction on
TNF-
levels
according
to rs2229094
(TNF-) (TNF-)
(n = 191). (n = 191).
3. Discussion
3. Discussion
We observed in the present study that n-3 FAs may interact with SNPs from

We observed in the present study that n-3 FAs may interact with SNPs from inflammation-related
inflammation-related genes to modulate plasma cytokine levels. There was no reduction of plasma
genesIL-6
to modulate
plasma
cytokine
was no
reduction
of plasmawith
IL-6SNPs
and TNF-
and TNF-
as well
as CRPlevels.
levels, There
but several
genediet
interactions
within as well as
CRP inflammation-related
levels, but several genediet
interactions
inflammation-related
genes and n-3
genes and n-3
FAs have with
been SNPs
found within
potentially
modulating inflammatory
marker
levels.
These
findings are
consistent with
the well-known
anti-inflammatory
propertiesare
of consistent
FAs have
been
found
potentially
modulating
inflammatory
marker
levels. These findings
but the amplitude
of the results
may differofaccording
an the
individuals
genotype.
with n-3
the FAs,
well-known
anti-inflammatory
properties
n-3 FAs,tobut
amplitude
of the In
results may
addition
to
nutrigenetic
effects,
baseline
plasma
EPA,
DHA
or
total
n-3
FA
levels
are
negatively
differ according to an individuals genotype. In addition to nutrigenetic effects, baseline plasma EPA,
associated with plasma cytokine and CRP levels (Figure 1).
DHA or total n-3 FA levels are negatively associated with plasma cytokine and CRP levels (Figure 1).
Ferrucci et al. have reported that plasma levels of n-3 FAs were independently associated with
Ferrucci
et al.ofhave
reported thatmarkers
plasmaand
levels
of n-3
FAs of
were
independentlymarkers
associated with
lower levels
pro-inflammatory
higher
levels
anti-inflammatory
lowerindependent
levels of pro-inflammatory
markers
higher
levels status,
of anti-inflammatory
independent
of confounders such
as age, and
sex, BMI,
smoking
education, energymarkers
intake, and
potentially confounding
drugsex,
treatment
others
[13]. They
observed that
lowerintake,
EPA and
totalpotentially
of confounders
such as age,
BMI,among
smoking
status,
education,
energy
and
n-3 FAs were
associated
with
higherothers
IL-6 and
TNF-
levels
(for totalthat
n-3 lower
FAs only)
[13].
Our
results
confounding
drug
treatment
among
[13].
They
observed
EPA
and
total
n-3 FAs were
are moving in the same direction as shown by the correlations between baseline FA levels with the
associated with higher IL-6 and TNF- levels (for total n-3 FAs only) [13]. Our results are moving
principal inflammatory biomarkers (Figure 1).
in the same
direction as shown by the correlations between baseline FA levels with the principal
Trebble et al. have shown in 16 healthy subjects, that the dose-response relationships between
inflammatory
biomarkers
(Figure 1).
n-3 FA, phospholipid
composition
and cytokine production by PBMCs is U-shaped meaning that an
Trebble
et al.levels
have
in 16
healthy
that phospholipids,
the dose-response
relationships
intermediate
of shown
EPA within
plasma
and subjects,
cell membrane
resulting
from an n-3 between
FA phospholipid
supplementationcomposition
of <2.0 g/day, and
may cytokine
be associated
with a greater
inhibitory
effect on TNF-
n-3 FA,
production
by PBMCs
is U-shaped
meaning that
release
than
higher
EPA
concentrations
resulting
from
n-3
FA
supplementary
intakes
of
>2.0
g/day
an intermediate levels of EPA within plasma and cell membrane phospholipids, resulting from an n-3
[14]. Rees et al. have suggested that there is a threshold for an anti-inflammatory effect of EPA
FA supplementation of <2.0 g/day, may be associated with a greater inhibitory effect on TNF- release
somewhere between 1.35 and 2.7 g/day [15]. However, in the present study, there was no significant
than reduction
higher EPA
concentrations
from
n-3 FA
supplementary
intakes
of >2.0the
g/day [14].
in plasma
CRP, IL-6 orresulting
TNF- levels
despite
the high
doses of n-3 FAs
used during
Rees protocol
et al. have
suggested
that there
is a threshold
for an levels
anti-inflammatory
of EPA
somewhere
(Table
4). This could
be explained
by low baseline
of inflammatoryeffect
markers,
by the
exclusion
individuals
with
plasma
CRP levels
>10.0
mg/L, or
by thethere
intermediate
of EPA reduction
between
1.35 of
and
2.7 g/day
[15].
However,
in the
present
study,
was nodoses
significant
given
to
study
participants.
Moreover,
there
could
be
a
differential
effect
of
each
of
the
n-3
in plasma CRP, IL-6 or TNF- levels despite the high doses of n-3 FAs used duringFAs
the protocol
impacting inflammatory marker levels.
(Table
4). This could be explained by low baseline levels of inflammatory markers, by the exclusion
Moreover, besides the independent influence of dietary FAs on cytokine levels, some SNPs in
of individuals
with plasma CRP levels >10.0 mg/L, or by the intermediate doses of EPA given to
the TNF-LTA gene family may be related to the inter-individual variability observed in plasma
studyTNF-
participants.
could
differential
effect of
each isofassociated
the n-3 FAs
levels and Moreover,
TNF- gene there
expression
[6].beInathis
study, rs2229094
(TNF-)
withimpacting
inflammatory
marker
levels.
plasma TNF-
levels.
This SNP is of particular interest due to its exonic location and the presence of
a missense mutation
that is located
at a of
conserved
studies
havesome
shown
Moreover,
besides (Cys13Arg)
the independent
influence
dietaryresidue.
FAs onRecent
cytokine
levels,
SNPs in the
that
rs2229094
was
associated
with
type
2
diabetes
[16],
CRP
levels
[16],
sepsis
[17],
Crohns
disease
TNF-LTA gene family may be related to the inter-individual variability observed in plasma TNF-
[18], and cancer risk [19]. According to Huang et al., four functional SNPs of the LTA gene, including
levels and TNF- gene expression [6]. In this study, rs2229094 (TNF-) is associated with plasma
rs2229094, may exert possible regulatory effects on gene expression and cytokine production [19].
TNF- levels. This SNP is of particular interest due to its exonic location and the presence of a missense
mutation (Cys13Arg) that is located at a conserved residue. Recent studies have shown that rs2229094
was associated with type 2 diabetes [16], CRP levels [16], sepsis [17], Crohns disease [18], and cancer
risk [19]. According to Huang et al., four functional SNPs of the LTA gene, including rs2229094, may
exert possible regulatory effects on gene expression and cytokine production [19].
Another SNPs in the TNF-LTA gene family was associated with inflammatory responses. Indeed,
we observed an interaction between rs1800629 and n-3 FAs modulating CRP levels where carriers

Int. J. Mol. Sci. 2016, 17, 375

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of the mutated allele had significantly higher CRP levels than the wild-type genotype after fish oil
supplementation (Table 5). Song et al. have found that the mutated allele of rs1800629 was associated
with increased TNF- production in PBMCs from healthy subjects after stimulation with LPS [20].
Studies have reported that dietary fat intake could alter the relationship between TNF 308G>A (also
referred to as rs1800629) with adiposity and serum lipid concentrations. The main results of these
studies are that TNF 308G>A was associated with an increased risk of obesity and dyslipidaemia, and
carriers of the mutated allele appeared to be more responsive to dietary fat intake [21,22]. Meydani et al.
reported that the production of the cytokines IL-1, TNF-, and IL-6 by mononuclear cells was reduced
after the consumption of the low-fat, high-fish diet [23].
Evidence have shown that carriers of the mutated allele had a 2-fold increase in the TNF-
transcriptional activity, thus playing a role in the altered TNF- gene expression possibly leading to
an increase in cytokine production [24]. Moreover, Wilson et al. have found that rs1800629 may exert
direct effects on TNF- gene regulation, potentially leading to high TNF- phenotype (expression
levels, transcriptional activity, inflammatory marker levels) and more severe infection diseases in
TNF2 homozygotes [25]. For example, Antonicelli et al. reported that carriers of TNF- gene 308G>A
were more likely to be affected by severe ischemic damage in a case-control study including elderly
Italian individuals with and without coronary heart disease [26]. However, in this study, there were no
significant differences observed in TNF- gene expression nor in plasma TNF- levels according to the
rs1800629 genotype both in dominant and additive models. Although the relative change in TNF-
levels after the supplementation was not statistically significant between genotype groups owning to
the small sample size, the difference was clinically relevant and seems to be in agreement with the
actual literature (delta TNF-G/G: 28.0% vs. A/G: 3.3% vs. A/A 1.3%). This heterogeneity observed
in the response to fish oil could be partly explained by the replacement of arachidonic acids by n-3 FAs
into cell membranes leading to the production of diverse eicosanoids. Accordingly, Grimble et al. have
suggested that the overall effect on TNF- production (inhibition or stimulation) probably depends on
the balance among the different stimulatory and inhibitory eicosanoids produced from arachidonic
acid and EPA [7].
Although gene expression of inflammation-related genes is often decreased following an increase
of the n-3 FAs in the diet [2729], several studies reported no significant decrease in plasmatic levels of
inflammatory biomarkers, such as TNF-, IL-6 or CRP [13,30]. In the present study, TNF- and IL6
genes were slightly overexpressed in PBMCs after the 6-week n-3 FA supplementation, but relative
quantification (RQ) within the 0.5 to 2.0 fold are considered not significant. There was no clear effect
of the 6-week n-3 FA supplementation on the expression of the two selected inflammation-related
genes (IL6 and TNF-) on a metabolically healthy, but slightly overweight population, even with
the use of triplicates to ensure a better reduction in biological variance. These results failed to
demonstrate changes in expression levels of TNF- and IL6 when looking at these two genes specifically
using a real-time PCR approach. However, Rudkowska et al. and Bouwens et al. have shown
using transcriptomic approaches that inflammation-related pathways in PBMCs were changed to the
anti-inflammatory direction after an n-3 FA supplementation [9,10].
Strengths and Limitations
Several limitations of the present study need to be addressed. Participants were relatively young
(mean age of 30.8 8.7 years) and they had low inflammatory biomarker levels at baseline. The patients
were healthy and we excluded participants having plasma CRP levels > 10 mg/L. We did not observe
difference in gene expression of inflammation-related genes after the supplementation. This could be
attributable to the use of the 2CT calculation method assuming that the endogenous control gene
and target gene have both similar efficiencies. Also, this study did not allow to isolate the effect of
a single FA and its potential genediet interactions on inflammatory markers due to the composition
of the n-3 FA fish oil capsules given to participants that contained EPA and DHA.

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4. Experimental Section
4.1. Subjects
A total of 254 unrelated subjects from the greater Quebec City metropolitan area were recruited
through emails sent to University students and employees via advertisements in local newspapers.
Inclusion criteria were as follow: (1) to be aged between 18 and 50 years; (2) being non-smoker;
(3) having a body mass index (BMI) between 25 and 40 kg/m2 ; and (4) being free of pharmacologic
lipid lowering treatment and/or metabolic disorders. Subjects who had taken n-3 FA supplements
six months prior to the beginning of the study were excluded. A total of 210 subjects completed the
supplementation protocol. Individuals with CRP levels >10 mg/L were excluded, bringing the total down
to 191 eligible individuals. This experimental protocol #C09-05-030 was approved by the CHU de Quebec
ethics committee on 16 September 2009. This clinical trial was registered at clinicaltrials.gov (NCT01343342).
4.2. Study Design and Diets
In order to minimize the intra- and inter-variability in dietary intakes, a 2-week run-in period
preceded the supplementation. During this run-in period, a registered dietitian gave individual dietary
instructions in order to ensure that participants were in a stable condition before the beginning of
the study. Participants received recommendations by a registered dietitian in order to follow the
recommendations of the Eating Well with Canadas Food Guide [31]. Participants were also asked to
maintain their body weight stable throughout the whole research protocol. After the run-in period,
each participant received n-3 FA capsules in sufficient quantity for the next six weeks. They were
instructed to take five capsules/d of fish oil (total of 5 g/day of fish oil), providing a total of 3.03.3 g
of n-3 FAs including 1.92.2 g of eicosapentaenoic acid (EPA) and 1.1 g of docosahexaenoic acid
(DHA). Before the run-in period, each participant completed a validated food-frequency questionnaire
(FFQ) [32] supervised by a registered dietitian. This 91-item FFQ is based on typical food items found
in North America. Moreover, they were asked to complete two 3-day food recordsprior to and
after the n-3 FA supplementation period. Dietary intakes data were analyzed using Nutrition Data
system for Research software v.2011 (Nutrition Coordinating Center (NCC), University of Minnesota,
Minneapolis, MN, USA).
4.3. Anthropometric Measurements
Body weight, height and waist circumference were measured at every visit in accordance with the
Airlie Conference on the Standardization of anthropometric measurements [33]. BMI was calculated as
weight per meter squared (kg/m2 ).
4.4. Biochemical Parameters
Because the study design was not intended to look specifically at inflammation, only a small
subset of inflammatory biomarkers were available for this reanalysis. Blood samples were collected
after a 12 h overnight fast and 48h alcohol abstinence, from an antecubital vein into vacutainer
tubes containing EDTA. Plasma CRP was measured by nephelometry (Dade Behring, Deerfield, IL,
USA) using a sensitive assay, as described previously [34]. Plasma concentrations of IL6 and TNF-
were measured with high-sensitivity ELISA kits including: Human IL6 Quantikine HS ELISA Kit
Minneapolis, MN, USA (R & D Systems, Minneapolis, MN, USA (HS600B)) and Human TNF-
Quantikine HS ELISA Kit (R & D Systems (HSTA00D)) [34].
4.5. Measurement of FA Composition in Plasma Phospholipids
FA composition of plasma phospholipids was determined by gas chromatography. Venous blood
was drawn into EDTA tubes, then separated by centrifugation at 500 g for 6 min and stored at 80 C
for subsequent analyses. Plasma lipids were extracted with chloroform:methanol (2:1, by volume)

Int. J. Mol. Sci. 2016, 17, 375

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according to a modified Folch method [35]. Total phospholipids were then isolated with isopropyl
ether:acetic acid (96:4) by thin layer chromatography [36]. Isolated plasma phospholipids were then
methylated [37]. FA profiles were obtained after methylation in methanol/benzene 4:1 (v/v) [37]
and capillary gas chromatography using a temperature gradient on a HP5890 gas chromatograph
(Hewlett Packard, Toronto, ON, Canada) equipped with a HP-88 capillary column (100 m 0.25 mm
i.d. 0.20 m film thickness; Agilent Technologies, Palo Atto, CA, USA) coupled with a flame
ionization detector. Helium was used as carrier gas using a split ratio of 1:80). FA were identified
according to their retention time as well as the following methylated FAs C22:5n-6 (Larodan AB,
Malm, Sweden) and C22:5n-3 (Supelco Inc., Bellefonte, PA, USA). Finally, phospholipids FA profiles
were determined using the relative percentage areas of total FAs.
4.6. SNP Selection and Genotyping
SNPs in the TNF-LTA gene cluster (6 SNPs), IL6 (5 SNPs) and CRP (4 SNPs) were identified using
the International HapMap Project SNP database, based on the National Center for Biotechnology
Information (NCBI) B36 assembly Data Rel 28. phase II + III, build 126. The TNF-LTA gene cluster is
made of two genes that are located very close to each other on chromosome 6 (LTA location: Chr6p21.3,
31,572,099 . . . 31,574,324; TNF- location: Chr6p21.3, 31,575,567 . . . 31,578,336). LTA is also referred to
as member 1 of the TNF-superfamily. Five hundred kilo-base pairs (kbp) downstream of each gene
and 2500 kbp upstream of each gene were added to cover the 51 UTR and 31 UTR regions. Gene Tagger
procedure in Haploview v4.2 was used to determine SNPs using a minor allele frequency (MAF) 5%
and pairwise tagging (r2 0.8). Subsequently, the linkage disequilibrium (LD) was examined for
all SNPs using the LD Plot procedure in Haploview v4.2. The SIGMA GenElute Gel Extraction Kit
(Sigma-Aldrich Co., St. Louis, MO, USA) has been used to extract genomic DNA. Selected SNPs of
the TNF-LTA gene family (rs1041981, rs2857706, rs1800629, rs2239704, rs3093662, and rs2229094),
IL6 (rs2069861, rs2069840, rs2069837, rs2069827, and rs1800797), IL1 (rs1143633, rs1143634, rs16944,
rs3136558, rs1143627) and CRP (rs1800947, rs3093059, rs1130864, and rs1205) have been genotyped
using validated primers and TaqMan probes (Thermo Fisher Scientific, Waltham, MA, USA) [38]. DNA was
mixed with TaqMan Universal PCR Master Mix (Thermo Fisher Scientific), with a gene-specific primer
and with probe mixture (pre-developed TaqMan SNP Genotyping Assays; Thermo Fisher Scientific) in
a final volume of 10 L. Genotypes were assessed using a 7500 RT-PCR System and the ABI Prism
Sequence Detection System v2.0.5 was used to analyse the data (Thermo Fisher Scientific).
4.7. Gene Expression
Gene expression of IL6 and TNF- genes was measured following the 6-week n-3 FA supplementation.
Complementary DNA (cDNA) was mixed with TaqMan Universal PCR Master Mix (Thermo Fisher
Scientific) and a gene-specific primer and probe mixture in a final volume of 20 L. All samples were
run on a 7500 Fast Real Time PCR System (Thermo Fisher Scientific) using the following thermal
cycling profile: 50 C (2 min), 95 C (10 min), followed by 40 steps of 95 C for 15 s and 60 C for
60 s. The real-time PCR data were imported into Expression Suite Software v1.0. These samples were
analysed in triplicate and calibrated to the GAPDH gene (endogenous control; GAPDH: Hs99999905_ml).
Relative quantification estimations were achieved using the 2CT calculation method [39].
4.8. Statistical Analyses
This study is a reanalysis of the Fatty Acid Sensor (FAS) Study that primarily wanted to understand
how genes and environment act together on the cardiometabolic risk profile. Data were analyzed with
SAS Genetics v9.3. Values that were not normally distributed were log(10) -transformed, negative
reciprocal or normalised by a Box-Cox transformation (TNF-) before analysis. Subjects were
categorised as positive ( < 0%) or negative ( 0%) responders based on their relative change
of plasma TNF-, CRP or IL-6 levels after the supplementation. The MIXED procedure for repeated
measures was used to test for significant differences in metabolic characteristics between men and

Int. J. Mol. Sci. 2016, 17, 375

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women at baseline and for differences between various nutrient intakes prior to and after n-3 FA
supplementation and to test for the effects of the genotype, the supplementation and the genotype
by supplementation (time) interaction on inflammatory marker levels when age, sex and BMI were
included in the model. Partial Spearman correlations were calculated between EPA, DHA or total n-3
of plasma phospholipids (in % of total FAs) and inflammatory markers. The ALLELE procedure was
used to verify the departure from Hardy-Weinberg equilibrium (HWE) and to calculate the minor allele
frequency. Individuals with CRP levels > 10 mg/L were excluded bringing the total to 191 eligible
individuals. SIFT web-based software was used to predict the effect of amino acid substitution and
all tests were run under default threshold values. Finally, the FREQ procedure was used to verify
differences in genotype frequency distribution between positive and negative responders to the n-3 FA
supplementation. Statistical significance was defined as p 0.05.
5. Conclusions
Overall, this study shows that a 6-week n-3 FA supplementation with 5 g of fish oil daily
did not alter gene expression levels of TNF- and IL6 in PBMCs and did not have an impact
on inflammatory biomarker levels. However, significant genediet interactions were observed
between SNPs within inflammation-related genes modulating plasma inflammatory biomarker levels.
These genediet interactions may potentially explain the large inter-individual variability observed in
plasma inflammatory response following an n-3 FA supplementation.
Acknowledgments: The authors would like to thank Catherine Raymond, Ann-Marie Paradis, lisabeth Thifault,
Catherine Ouellette, Vronique Garneau, Frdric Gunard and Annie Bouchard-Mercier who contributed to
the success of this study. This work was supported by an operating grant from Canadian Institutes of Health
Research (CIHR) (MOP-110975). Hubert Cormier received a doctoral research award from the Canadian Institutes
of Health Research (CIHR). Iwona Rudkowska is recipient of a Junior 1 scholarship from the Fonds de recherche
du Qubec-Sant. Marie-Claude Vohl holds a Tier 1 Canada Research Chair in Genomics Applied to Nutrition
and Health.
Author Contributions: Hubert Cormier performed statistical analysis, interpreted data and wrote the paper;
Simone Lemieux and Marie-Claude Vohl designed research; Patrick Couture was responsible for the medical
follow-up; Iwona Rudkowska participated to the selection of genes; Hubert Cormier undertook laboratory work;
Hubert Cormier and Marie-Claude Vohl have primary responsibility for the final content. All authors read and
approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript:
n-3
FAs
SNPs
TNF-
IL6
CRP
PBMCs
BMI
EPA
DHA
FFQ
TG
MAF
HWE

omega-3
fatty acids
single nucleotide polymorphisms
tumor necrosis factor alpha
interleukin 6
C-reactive protein
peripheral blood mononuclear cells
body mass index
eicosapentaenoic acid
docosahexaenoic acid
food-frequency questionnaire
triglycerides
minor allele frequency
Hardy-Weinberg equilibrium

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