The Journal of Nutrition
A Conjugated Linoleic Acid-Enriched Beef
Diet Attenuates Lipopolysaccharide-Induced
Inflammation in Mice in Part through
PPARg-Mediated Suppression of
Toll-Like Receptor 41–3
Clare M. Reynolds,4Eve Draper,5Brian Keogh,6Arman Rahman,5Aidan P. Moloney,7
Kingston H. G. Mills,8Christine E. Loscher,5and Helen M. Roche4*
4Nutrigenomics Research Group, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield,
Dublin 4, Ireland;5Immunomodulation Research Group, School of Biotechnology, Dublin City University, Dublin 9, Ireland;
6Trinity Centre for Health Sciences, St. James Hospital, Dublin 7, Ireland;7Teagasc, Grange Beef Research Centre, Dunsany,
County Meath, Ireland; and8Immune Regulation Research Group, Trinity College Dublin, Dublin 2, Ireland
Conjugated linoleic acid (CLA) is a PUFA found in beef and dairy products that has immunoregulatory properties. The level
of CLA in beef can be enhancedby feeding cattle fresh grass rather than concentrates. This study determined the effectof
feeding a high-CLA beef diet on inflammation in an in vivo model of septic shock. Mice were fed a high-CLA beef (4.3%
total fatty acid composition) or low-CLA beef diet (0.84% total fatty acid composition) for 6 wk. Lipopolysaccharide (LPS; 3
mg) or sterile PBS was injected i.v. and serum was harvested 6 h after injection. Serum interleukin (IL)-1b, IL-12p70, IL-
12p40, and interferon-g concentrations were significantly reduced in response to the LPS challenge in the high-CLA beef
diet group. Bone marrow-derived dendritic cells (BMDC) from the high-CLA beef diet group had significantly less IL-12 and
more IL-10 in response to ex vivo LPS stimulation. Furthermore, toll-like receptor 4 (TLR4) and CD14 protein and mRNA
expression on BMDC was significantly attenuated in the high-CLA compared with the low-CLA beef diet group.
Complimentary in vitro experiments to determine the specificity of the effect showed that synthetic cis9, trans11-CLA
suppressed surface expression of CD14 and TLR4 on BMDC. Treatment with the PPARg inhibitor GW9662 partially
reversed TLR4 expression in immature BMDC. The results of this study demonstrate that feeding a diet enriched in high-
beef CLA exerts profound antiinflammatory effects in vivo within the context of LPS-induced sepsis. In addition,
downregulation of BMDC TLR4 is mediated through induction of PPARg. J. Nutr. doi: 10.3945/jn.109.113035.
Sepsis is an inflammatory condition that is caused by an over-
whelming dysregulation of the immune system. Endotoxin or
lipopolysaccharide (LPS)9is an essential component of Gram-
Once released into the circulation LPS has potent effects that can
necrosis factor-a (TNFa), interleukin (IL)-1b, IL-6, interferon-g
(IFNg), and IL-12, which play a major role in the pathogenesis of
sepsis. Indeed, circulating levels of these cytokines can be used as
Cells of the innate immune system recognize the LPS
component of Gram negative bacteria through the cell surface
receptor toll-like receptor 4 (TLR4). Once released into the
bloodstream, LPS binds to LPS binding protein and mediates its
effects by signaling through TLR4 and its coreceptor, CD14 (4).
The TLR4 CD14 signaling complex plays a major role in the
pathogenesis of sepsis. In addition, TLR4-deficient mice are
hyporesponsive to LPS and resistant to endotoxin-induced septic
shock (5). In humans, TLR4 polymorphisms have been associ-
ated with increased risk of Gram negative infection and sepsis
(Asp299Gly; 896G) (6).
1Supported by the Food Institutional Research Measure, Department of
Agriculture and Food, Ireland (project no. 5254).
2Author disclosures: K. H. G. Mills, funded by Science Foundation Ireland and is
a cofounder and shareholder in Opsona Therapeutics Ltd, a start-up company
involved in the development of antiinflammatory therapeutics. C. Reynolds, E.
Draper, B. Keogh, A. Rahman, A. P. Moloney, C. E. Loscher, and H. M. Roche, no
conflicts of interest.
3Supplemental Figure 1 and Tables 1 and 2 are available with the online posting
of this paper at jn.nutrition.org.
9Abbreviations used: BMDC, bone marrow-derived dendritic cell; CLA, conjugated
linoleic acid; c9, t11-CLA, cis9, trans11-CLA; DC, dendritic cell; DMSO, dimethyl-
sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulo-
cyte macrophage colony stimulating factor; IFNg, interferon-g; IKKa, I kB kinase;IL,
interleukin; LPS, lipopolysaccharide; NF-kB, nuclear factor-kB; TLR4, Toll-like
receptor 4; TVA, trans-vaccenic acid; TNFa, tumor necrosis factor-a.
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
0022-3166/08 $8.00 ã 2009 American Society for Nutrition.
Manuscript received July 15, 2009. Initial review completed August 9, 2009. Revision accepted October 8, 2009.
Copyright (C) 2009 by the American Society for Nutrition
1 of 7
The Journal of Nutrition. First published ahead of print October 21, 2009 as doi: 10.3945/jn.109.113035.
Dendritic cells (DC) provide the first line of defense against
pathogens and influence the nature of the subsequent adaptive
immune response (7). Their production of IL-12 promotes
differentiation of naı ¨ve Th cells to Th1 cells, whereas IL-10
production promotes regulatory T cell differentiation (8). DC
are critical for initiating the overwhelming cytokine response
during the early phase of sepsis (9). There is evidence that the
number of DC become depleted during sepsis (10,11), the extent
of DC loss correlates with mortality (12), as DC dysfunction and
reduced IL-12 impede clearance of infection (13).
Dietary lipids can modulate the inflammatory response; SFA
are associated with proinflammatory disease states. SFA activate
TLR4, thereby exacerbating inflammatory effects in many
inflammatory disorders (14). In contrast, monounsaturated
fatty acids and PUFA tend to be antiinflammatory (15–17).
Indeed, nutritional intervention with both monounsaturated
fatty acids and PUFA increases survival in animal models and
human cases of septic shock (18,19). Conjugated linoleic acid
(CLA) refers to the positional and geometric isomers of linoleic
acid (20). It is found in beef and dairy products and may have
positive health effects in animal models of inflammation (21,22).
cis9, trans11-CLA (c9, t11-CLA) is the predominant natural
isomer accounting for up to 90% of total dietary CLA (22),
which has been shown to have antiinflammatory properties (23–
25). Feeding cattle on fresh pasture can enhance the level of CLA
and its precursor trans-vaccenic acid (TVA) in beef (26,27).
Therefore, natural beef-derived CLA may be more potent than
c9, t11-CLA is known to inhibit DC maturation and have
antiinflammatory effects on DC following activation with LPS in
vitro (23,28). CLA is known to be a PPARg ligand and it is
possible that its antiinflammatory effects can be attributed to
this nuclear hormone receptor (29,30). Recent evidence has
shown that the PPARg agonist pioglitazone inhibits TLR4
expression in human monocytes (31).
In this study, we examined the antiinflammatory effects of a
high-CLA beef diet in a murine model of septic shock.
Materials and Methods
Materials. LPS from Escherichia coli (serotype 127:B8) and synthetic
c9, t11-CLAwere obtained from Alexis Chemicals. The PPARg inhibitor
GW9662 was purchased from Sigma. c9, t11-CLA and GW9662 were
dissolved in sterile dimethylsulfoxide (DMSO) and stored at 2208C
away from light sources.
Mouse experiments and nutritional intervention. All experiments
were performed in accordance with current EU and Irish Department of
Health guidelines on the use of experimental animals. Female BALB/c
mice were purchased from Harlan, UK. Mice were fed an irradiated diet
from 6 wk of age. The mice were randomly assigned to either a low-CLA
beef diet group or a high-CLA beef diet group for a 6-wk period. Mice
consumed food and water ad libitum. Food consumption and weight
were monitored every second day. Weight and food consumption did not
differ between the groups.All diets were storedat 48C andwere provided
fresh to the mice every second day.
Generation of beef-derived feeds. Charolais-cross steers were fed a
high-concentrate/straw ration to generate the low-CLA beef diet. For the
high-CLA beef diet, Charolais-cross heifers were offered grazed peren-
nial ryegrass supplemented with sunflower oil and fish oil. After 150 d,
psoas muscle, longissimus muscle, and subcutaneous adipose tissue were
collected and pooled within ration type to yield a 35% fat beef product.
This was then freeze-dried and incorporated into the mouse feed.
Fatty acid composition of dietary feeds and adipose tissue. The
composition of the diets, in particular the fatty acid composition of the
high- and low-CLA beef-enriched diets, is presented in Supplemental
Table 1. In addition,the adipose tissuefatty acid composition of mice fed
the high- or low-CLA beef diets was determined as a biomarker of
dietary fat modification. Briefly, lipid was extracted according to Folch
et al. (32), dried lipid samples were methylated, and acidic trans-
esterification was carried out as outlined by Kramer and Zhou (33).
LPS shock model. After 6wk ofconsumingeither ahigh-CLAora low-
CLA beef diet, LPS shock was induced by injecting mice (n = 5 per
dietary group) i.v. with 3 mg LPS or sterile PBS (n = 5 per dietary group)
(Alexis). After 6 h mice, were killed by cervical dislocation. Blood and
adipose tissue were collected. The blood was allowed to clot overnight at
48C and serum removed after centrifugation at 12000 3 g for 5 min.
Serum samples were stored at 2208C and analyzed for the presence of
cytokines using specific immunoassays.
Isolation and culture of bone marrow-derived DC. Bone marrow-
derived DC (BMDC) were prepared by culturing bone marrow cells
obtained from the femurs and tibia of mice in RPMI 1640 medium in the
presence of granulocyte macrophage colony stimulating factor (GM-
CSF) (50 nmol/L; Sigma-Aldrich). Cells from low-CLA and high-CLA
beef diet groups were cultured in the presence of GM-CSF (50 nmol/L;
Sigma-Aldrich). Alternatively, DMSO (vehicle control) or c9, t11-CLA
(50 mmol) was added to the cells on d 1 of culture. The cells were
cultured at 378C in 5% CO2for 3 d, when the supernatant was carefully
removed without disturbing the cell monolayer and replaced with fresh
medium containing GM-CSF. On d 7 of culture, cells were collected,
counted, and used. In other experiments 48 h prior to cell collection, the
PPARg inhibitor GW9662 (10 nmol/L) was added.
Cytokine analysis. Supernatants from the DC activation experiments
were analyzed for IL-12p70 and IL-10 concentrations using commercial
DuoSet ELISA kits (R&D Systems) according to the manufacturer’s
instructions.Serumfrom the LPSshock model experimentswas analyzed
for IL-12p70, IL-12p40, IL-1b, and IFNg using commercial DuoSet
ELISA kits (R&D Systems) according to the manufacturer’s instructions.
mRNA analysis of BMDC. BMDC were isolated from mice fed either a
high-CLA or low-CLA beef diet and cultured in the presence of GM-CSF
(50 nmol/L; Sigma-Aldrich). On d 7 of culture, DC (2 3 106cells) were
cultured in 6-well plates with LPS (100 nmol/L) or medium alone for 0–
12 h. After removal of the supernatants, the cells were harvested and TRI
Reagent (Molecular Research Centre) was added. Total RNA was
extracted from the cells according to the manufacturer’s instructions.
RNA (2 mg) was treated with RNase-free DNase to remove contami-
nating genomic DNA (DNase I Amplification Grade; Invitrogen). Single-
stranded cDNAwas prepared using the High Capacity cDNA archive kit
(Applied Biosystems). mRNA expression was quantified by real-time
PCR on an ABI 7700 Sequence Detection system (Perkin-Elmer Applied
Biosystems). TaqMan real-time PCR was performed for TLR4, IL-10,
and IL-12p35 using Pre-Developed Assay Reagent kits. For each sample,
results were normalized by dividing the amount of target gene by the
amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
expressed relative to the low-CLA beef control treatment for the non-
Analysis of protein expression in BMDC by Western blotting. BMDC
were plated at 2 3 106cells/well in 6-well tissue culture plates. These cells
were then stimulated for 0, 2, or 5 h with 100 nmol/L LPS. Cytosolic and
nuclear extracts were prepared using Nuclear extract kit (Active Motif).
The concentration of protein in the nuclear and cytoplasmic samples was
quantified by Bradford assay (Bio-Rad Laboratories). A total of 15 mg of
protein was separated by SDS-PAGE for Western immunoblotting.
Antibodies were used to detect nuclear factor-kappa B (NF-kB) p65, I kB
and TLR4 (Abcam; 1/1000 dilution) in nuclear and/or cytosolic extracts.
Membranes were washed with PBS-T and incubated for 1 h at room
temperature with anti-rabbit IgG (Santa Cruz Biotechnology; 1:1000–
2 of 7 Reynolds et al.
2000). After further washing, protein complexes were visualized with
Supersignal (Pierce). Membranes were exposed to film for 1–10 min and
processed using an Agfa X-ray processor. Protein bands were quantified
using the GeneSnap acquisition and GeneTools analysis software
(GeneGenius Gel Documentation and Analysis System). Where required,
membranes were stripped by incubating them in stripping buffer (Tris, pH
6.8, b-mercaptoethanol, SDS) for 30 min at 508C before probing with a
Transcription factor assay. NF-kB activity was determined using a
TransAM NF-kB assay (Active Motif), which measured NF-kBp65:DNA
binding. The assay was used in accordance with the manufacturer’s
instructions. For each kit, 2 mg of nuclear protein was incubated in a 96-
well platecontaining the consensus
GGGACTTTCC-39) for 1 h. After washing, a primary antibody that
recognizes an epitope on NF-kBp65 upon DNA binding was applied for
1 h. After subsequent washing, a secondary antibody conjugated to HRP
was added for 1 h, followed by the addition of a chemiluminescent
substrate, the color product of which was quantified by luminescence.
sequence for NF-kB (59-
Effect of c9, t11-CLA on surface marker expression by flow
cytometry. The expression of CD14 and TLR4 on DC was assessed
using an anti-mouse CD14 (anti-mouse, eBioscience), TLR4-MD2 (anti-
mouse, eBioscience), and appropriately labeled isotype-matched anti-
bodies, which acted as controls. After incubation for 30 min at 48C, cells
were washed and immunofluorescence analysis was performed on a
FACsCalibur (BD Biosciences) using Cell Quest software.
Statistics. Data are presented as means 6 SEM. Data that were not
normally distributed were log-transformed before analysis. ANOVAwas
used to determine significant differences between conditions. Two-way
ANOVA was used to determine significant differences between dietary
conditions and LPS-dependant effects and their interaction. When this
indicated significance (P , 0.05), we used post hoc Bonferroni test
analysis to determine which conditions were significantly different from
each other. Cells alone and DMSO (vehicle control) treated cells did not
differ; therefore, DMSO was used as the reference treatment.
Adipose tissue c9, t11-CLA. Enrichment of diet for 6 wk with
CLA lead to a 4.1-fold increase in adipose tissue c9, t11-CLA
and TVA concentrations (P , 0.01). This increase was at the
expense of oleic acid 18:1 (n-9) and palmitic acid (16:0)
(Supplemental Table 2).
LPS-induced serum and BMDC cytokine responses. We
examined the effect of high-CLA and low-CLA beef diets in a
murine model of septic shock. An i.v. injection of LPS signifi-
cantly increased circulating IL-12p70, IL-12p40, IL-1b, and
IFNg concentrations in both the low-CLA beef diet group (P ,
0.01) and the high-CLA beef diet group (P , 0.05) (Table 1).
Following LPS injection, circulating concentrations of IL-12p70
(P , 0.05), IL-12p40 (P , 0.001), IL-1b (P , 0.001), and IFNg
(P , 0.01) were lower in the high-CLA compared with the low-
CLA beef diet group (Table 1).
This study focused on how the high-CLA diet modulated the
inflammatory response of DC. In unstimulated DC, IL-10 and
IL-12p70 mRNA concentrations did not differ between the low-
and high-CLA beef diet-fed mice. Cytokine mRNA expression
increased significantly 6 and 12 h post-LPS stimulation in both
groups. DC from the high-CLA beef diet group had greater IL-
10 mRNA expression 6 h post-LPS (P , 0.05), which normal-
ized to equivalent levels as the low-CLA diet 12 h post-LPS
stimulation (Fig. 1A). Conversely, IL-12p70 mRNA expression
decreased 12 h following LPS stimulation in DC from the high-
CLA beef diet group (P , 0.05) (Fig. 1B).
TLR4 expression in BMDC. TLR4 and CD14 are involved in
the recognition of LPS (34), therefore their expression was
determined on DC from the high- and low-CLA beef diets
following stimulation with LPS for 0–12 h. LPS-stimulated DC
from the high-CLA beef diet group had significantly lower TLR4
protein (Fig. 2A; P , 0.05) and mRNA (Fig. 2C; P , 0.05)
expression compared with the low-CLA beef diet group. In
addition, TLR4 protein concentrations were also decreased in
unstimulated DC (Fig. 2A; P , 0.01).
Prior to LPS stimulation, CD14 protein expression was less
on DC from the high-CLA compared with the low-CLA beef diet
group (Fig. 2B; P , 0.05). Furthermore, when examined 5 h
after stimulation with LPS, there was a significant reduction in
A high-CLA beef diet attenuates LPS-induced septic
shock in BALB/c mice1
diet + LPS
diet + LPS
5.5 6 2
29.1 6 6
19.4 6 0.04
1.22 6 2
25.4 6 8
19.1 6 0.7
33.6 6 14b
1201.3 6 170c
38.4 6 12c
114.8 6 74c
21.2 6 7*
183 6 49**a
12.3 6 10**c
22.4 6 2**a
1Values are means 6 SEM, n = 4–5. Asterisks indicate different from the
corresponding low-CLA: *P , 0.05, **P , 0.01, ***P , 0.002. Letters indicate
different from corresponding control (no LPS):aP , 0.05,bP , 0.01,cP , 0.001. ND,
(B) expression in BMDC from BALB/c mice. Expression of IL-10 (A)
and IL-12p35 (B) were measured using Taqman RT-PCR. mRNA levels
were normalized to GAPDH and the results expressed as fold
induction relevant to unstimulated DC from the low-CLA beef diet
group. The results represent the mean 6 SEM, n = 3. *Different from
low CLA, P , 0.05.
A high-CLA beef diet modulates IL-10 (A) and IL-12p35
CLA suppresses LPS responses via PPARg and TLR4 3 of 7
CD14 protein expression in DC from the high- compared with
the low-CLA beef diet group (Fig. 2C; P , 0.05).
NF-kB activation in BMDC. We examined expression of
nuclear and cytosolic NF-kB and cytosolic IKKa. Cytosolic
NF-kBp65 expression did not differ between the low- and high-
CLA beef diet groups in either unstimulated DC or in DC
stimulated for 2 h with LPS (Fig. 3A). However, following 5 h
LPSactivation, there was greater cytosolic NF-kBp65 expression
in DC from mice in the high-CLA beef diet group (Fig. 3A; P ,
0.05). Nuclear NF-kBp65 was not detected in resting DC.
Stimulation with LPS induced nuclear NF-kBp65 expression in
DC from the mice in the high- and low-CLA beef diet groups;
however, the groups did not differ after LPS stimulation (Fig.
3B). ATransAM assay determined that NF-kBp65 DNA binding
did not differ between diet groups in unstimulated DC.
Following stimulation with LPS for 5 h, there was less NF-
kBp65:DNA binding activity in the high-CLA beef diet mice
(P , 0.05; Fig. 3C). This reduction in NF-kBp65:DNA binding
activity was accompanied by a significant reduction in IKKa
protein levels in both resting and LPS stimulated DC from mice
fed the high-CLA beef diet (Fig. 3D; P , 0.05).
PPARg expression in BMDC. PPARg is a nuclear hormone
receptor that is known to have antiinflammatory effects and has
been shown to reduce NF-kB activation in DC (35). In the
cytosolic fraction, DC from the 2 diet groups did not differ prior
to LPS stimulation; however, PPARg expression was greater 2
and 5 h after LPS stimulation in DC from the mice fed the high-
CLA beef diet (Fig. 4B; P , 0.05). Nuclear PPARg expression in
unstimulated DC was greater in the high-CLA beef diet mice
(Fig. 4A; P , 0.05). Furthermore, the nuclear PPARg protein
level was greater in the LPS-activated DC from the high-
compared with the low-CLA beef diet mice (Fig. 4B; P , 0.05).
Suppression of TLR4 but not CD14 by c9, t11-CLA is
PPARg dependant. It has been demonstrated that activation of
PPARg with the specific agonist pioglitazone can suppress
expression of TLR4 in macrophages from db/db mice and in
peripheral blood monocytes (PBMC) from healthy volunteers
(31). Therefore, we determined whether PPARg activation
played a role in the suppression of TLR4 expression in DC
treated with c9, t11-CLA, which suppressed TLR4 (MFI 61 vs.
MFI 42) in unstimulated DC; this was reversed by the PPARg
inhibitor,GW9662 (MFI 42vs. MFI 72). CLA also decreased the
expression of CD14 in resting DC (MFI 125 vs. MFI 64);
however, this effect was not reversed in the presence of the
PPARg inhibitor, GW9662 (Supplemental Fig. 1).
Sepsis is a biological syndrome that can occur following
infection or injury and is characterized by an initial surge of
proinflammatory cytokines, including TNFa, IL-1b, IL-12, and
IFNg, in the bloodstream (36). This leads to vasodilation,
vascular and endothelial permeability, and hypotension, which
can result in multiple organ failure and death (37). A range of
pharmaceutical treatments, including recombinant human acti-
vated protein C (38), and anti-TNFa monoclonal antibodies
(39) dampen the inflammatory responses and improve survival.
Nevertheless, patient mortality is still very high.
Several studies have demonstrated better clinical outcomes
and reduced mortality following addition of the dietary fatty
acids eicosapentanoic acid and g-linolenic acid to the feeding
regimen of sepsis patients (19,40). However, little is known in
relation to the potential role of another fatty acid, CLA, which is
naturally enriched in beef from animals fed on pasture rather
than concentrates or straw. In addition, there is also significant
enrichment with TVA, which can be endogenously converted to
the c9, t11-CLA isomer in mammals (41). The potential
antiinflammatory benefits of feeding synthetic CLA have been
CD14 (B) expression in BMDC from BALB/c mice. Expression of TLR4
(A) and CD14 (B) protein was measured 0–5 h post-LPS stimulation by
Western blot. Densitometric analysis was conducted on immunoblots.
Results are expressed 6 SEM, n = 3, relative to unstimulated DC from
the low-CLA beef diet group. A representative immunoblot is shown.
(C) TLR4 mRNA expression was measured using Taqman PCR. mRNA
levels were normalized to GAPDH and the results expressed relative
to unstimulated DC from mice fed the low-CLA beef diet group. The
results represent the mean 6 SEM, n = 3. *Different from low CLA,
P , 0.05.
Feeding a high-CLA beef diet suppresses TLR4 (A,C) and
4 of 7Reynolds et al.
demonstrated in vitro and in vivo and within the context of
intestinal inflammation (25,42).The present study demonstrated
that feeding mice with a natural source of CLA derived from
beef could modulate LPS-induced immune responses and
suggested that food products with a high CLA content can act
as a viable, functional nutrient for the prevention and treatment
of septic shock syndromes.
IFNg, IL-12, and IL-1b are elevated in the serum during
septic syndromes and playa key role inthe pathogenesis of sepsis
(43). This study showed that prefeeding mice with a high-CLA
beef diet reduced these proinflammatory cytokines in response
to LPS-induced septic shock. This concurs with the effects of
dietary supplementation with synthetic c9, t11-CLA, which
reduced serum IL-1b in pigs (44) and other cytokines in a range
of cell types, including macrophages (45), epithelial cells (46),
and smooth muscle cells (47).
DC play a critical role in inducing and directing the adaptive
immune response. Furthermore, regulatory cytokines produced
by DC and other cells of the innate immune system in response
to pathogens or their products influence T helper cell responses
(43). Given the key role of the DC in innate and adaptive
immune response, we examined whether a low-CLA and high-
CLA beef diet affected the response of DC to LPS, in particular
its ability to produce IL-10 and IL-12. The active form of IL-12,
IL-12p70, is produced by DC following stimulation with TLR
agonists and promotes naı ¨ve T cells to differentiate into Th1
cells (44). We found that IL-12p35 mRNA (the functional
subunit of IL-12p70) was significantly reduced in BMDC
isolated from mice fed the high-CLA diet, which concurs with
our previous in vitro studies in BMDC (14). The antiinflamma-
tory cytokine IL-10 regulates IL-12 production (45) and has an
important role in controlling septic shock. The balance between
these cytokines has a major influence on the nature of the
immune response mounted following infection. A polymor-
phism in the promoter region of IL-10 (-1082A) reduces IL-10
production and increases susceptibility to sepsis in humans (46).
Furthermore, overexpression of IL-10 improves survival in a
mouse model of sepsis (47). The present study showed that IL-10
mRNA expression was higher in LPS-stimulated DC from mice
fed the high-CLA beef diet 6 h post-LPS stimulation, indicating
early induction of the antiinflammatory effect. This concurs with
evidence that a synthetic 50:50 mixture of c9, t11-CLA and t10,
c12-CLA increased IL-10 levels in the serum, spleen, and thymus
of pigs challenged with LPS (48) and our previous in vitro
finding that synthetic c9, t11-CLA enhanced secretion of IL-10
from DC (14).
TLR4 is a pattern recognition receptor that, along with the
coactivator CD14, is responsible for detecting LPS, a component
of Gram negative bacteria (4). The present study demonstrated
that CLA downregulates TLR4 mRNA and protein expression
in BMDC of mice fed a high-CLA beef diet. Furthermore, TLR4
expression was reduced in immature DC, demonstrating that
CLA may act by dampening cellular responsiveness to LPS.
It has been proposed that PPARg activation may mediate the
antiinflammatory effects of CLA (21,24,30). PPARg ligands,
J2, can attenuate LPS-induced TLR4 expression in murine
macrophages, human PBMC, and the colonic cell line HT-29
(31,49). PPARg ligands also have positive effects in the treat-
ment of sepsis (50,51). In the present study, PPARg expression
on DC was significantly higher in mice fed the high-CLA beef
diet. Furthermore, c9, t11-CLA significantly reduced CD14 and
TLR4 surface expression on DC in vitro. The addition of the
specific PPARg inhibitor,GW9662, reversed the effect ofc9, t11-
CLA in lowering TLR4 but not CD14 expression in immature
DC. This suggests that the antiinflammatory effects of CLA are
partly mediated by PPARg-induced TLR4 downregulation.
NF-kB regulates the inflammatory cytokine response follow-
ing LPS-induced TLR4 activation (52). Previous studies have
shown that CLA mediates its antiinflammatory effects through
creases NF-kB activation (A,B,D) and
IKKa (C) expression in BMDC from
BALB/c mice. NF-kBp65 in the cytosolic
(A) and nuclear (B) fractions and IKKa
cytosolic protein (D) were measured by
Western blotting. Densitometric analysis
was conducted on immunoblots. Results
are expressed 6 SEM, n = 3. A repre-
sentative immunoblot is shown. (C) NF-
kB activation was measured in nuclear
fractions by an NF-kB binding assay.
Values were expressed relative to the
mean luminescence value for unstimu-
lated DC from the low-CLA beef diet
group. *Different from low CLA, P ,
A high-CLA beef diet de-
CLA suppresses LPS responses via PPARg and TLR45 of 7
the NF-kB pathway (53–55). The present study suggests that
small but significant increase in IKKa in DC following stimula-
tion with LPS for 2 h, with significantly lower expression in DC
TransAM assay demonstrated that whereas there was no reduc-
tion in nuclear NF-kBp65, there was a significant reduction in
NF-kBp65 DNA binding. This concurs with a previous in vitro
analysis of BMDC treated with synthetic c9, t11-CLA (14).
The results of this study demonstrate that naturally derived
beef CLA has potent antiinflammatory effects at both a systemic
and cellular level in a mouse model of sepsis. We demonstrated
that feeding mice a high-CLA beef diet dramatically reduced
LPS-induced proinflammatory cytokine concentrations, which
are important in the pathogenesis of septic shock syndromes. DC
are the primary antigen presenting cells in the initial immune
response to infection. Our results suggest that reduced ex vivo
expression of TLR4 on DC after the high-beef CLA diet limits
their ability to sense LPS, which in turn attenuates the NF-kB
pathway. It is also clear that PPARg activation plays a role by
reducing TLR4 expression. Overall, these findings provide clear
evidence that naturally derived CLA has a powerful antiinflam-
matory effect. Further studies should help to underscore the
potential importance of CLA as a viable, functional food in the
treatment and prevention of sepsis.
We thank Aiveen Marron and Peter Dunne for excellent
technical assistance. H.M.R. and C.E.L. designed research.
C.M.R., E.D., B.K., and A.R. conducted research. K.H.G.M.
and A.P.M. provided essential materials. C.M.R. analyzed data.
C.M.R. wrote the paper. H.M.R. had primary responsibility for
final content. All authors read and approved the final manu-
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