In vitro modulation of the LPS-induced

proinflammatory profile of hepatocytes and

macrophages- approaches for intervention in obesity?
Ramiar K. Kheder1, 3, James Hobkirk2, Cordula M. Stover1*
1

Infection, Immunity, Inflammation, University of Leicester, United Kingdom,

Department of Sport, Health and Exercise Science, University of Hull, United Kingdom,

College of Nursing, University of Raparin, Iraq

Submitted to Journal:
Frontiers in Cell and Developmental Biology
Specialty Section:
Lipidology

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ISSN:
2296-634X
Article type:
Original Research Article

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Received on:
11 Mar 2016
Accepted on:
07 Jun 2016

Provisional PDF published on:

07 Jun 2016
Frontiers website link:
www.frontiersin.org

Citation:
Kheder RK, Hobkirk J and Stover CM(2016) In vitro modulation of the LPS-induced proinflammatory
profile of hepatocytes and macrophages- approaches for intervention in obesity?. Front. Cell Dev.
Biol. 4:61. doi:10.3389/fcell.2016.00061
Copyright statement:
2016 Kheder, Hobkirk and Stover. This is an open-access article distributed under the terms of the
Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other
forums is permitted, provided the original author(s) or licensor are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use,
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This Provisional PDF corresponds to the article as it appeared upon acceptance, after peer-review. Fully formatted PDF
and full text (HTML) versions will be made available soon.

Frontiers in Cell and Developmental Biology | www.frontiersin.org

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In vitro modulation of the LPS-induced proinflammatory profile of hepatocytes and

macrophages- approaches for intervention in obesity?
Ramiar Kheder1,2, James Hobkirk3, Cordula Stover1*
1

Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK;

College of Nursing, University of Raparin, Kurdistan Region, Iraq; 3Department of Sport,

Health and Exercise Science, University of Hull, Hull, UK

*corresponding author: Dr. med. Cordula M. Stover, Department of Infection, Immunity and
Inflammation, College of Biological Sciences, Medicine and Psychology, University of
Leicester, University Road, Leicester LE1 9HN, UK; email cms13@le.ac.uk

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Abstract

Low grade endotoxemia is a feature of obesity which is linked to development of steatohepatitis

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in non-alcoholic fatty liver disease. In this study, macrophages (J774) and hepatocytes (HepG2)
were stimulated with lipopolysaccharide (LPS) from E.coli 0111: B4 and analysed for

modulation of this response when preconditioned or stimulated subsequent to LPS, with different
doses of Vitamin D3 or docosahexaenoic acid (DHA) over a time period of one and five days.
Pro-inflammatory TNF and pro-fibrotic TGF released into the supernatants were measured by
ELISA; qPCR was performed for Srebp-1c and PPAR mRNA (genes for products involved in
fatty acid synthesis and catabolism, respectively). Vitamin D3 and DHA exerted a consistent,
dose dependent anti-inflammatory effect and increased PPAR relative to Srebp-1c in both cell
types. By contrast, addition of free fatty acids (FFA, oleic acid/palmitic acid 2:1) caused
aggravation of LPS-induced inflammatory reaction and an increase of Srebp-1c relative to
PPAR. Our results argue in favour of dietary supplementation of Vitamin D3 or DHA (and
avoidance of monounsaturated/saturated fatty acids) to alleviate development of fatty liver
disease.
Key words
Vitamin D3, docosahexaenoic acid, HepG2, J774, lipopolysaccharide

Introduction
Low grade endotoxemia has been detected in circumstances as diverse as a bolus of high fat diet
(1), hemodialysis (2) and surgery (3). Drained via the portal vein, abdominally derived
endotoxin, a pathogen associated molecular pattern, PAMP, directly stimulates the
reticuloendothelial system of the liver but also hepatocytes. Obesity, from which insulin
resistance develops, is linked to increased translocation of endotoxins from the gut (4). Steatosis
is the accumulation of fat in hepatocytes which can lead to an overall increase in liver size, socalled hepatomegaly. Accumulation of lipids may lead to inflammation; this is called nonalcoholic steatohepatitis, to differentiate the disease from alcohol-induced liver injury, either of
which can progress to cirrhosis. It is estimated that there is evidence of non-alcoholic
steatohepatitis in up to a third of populations in the developed world (5). Because of increasing
prevalence of obesity in these populations, fatty liver disease has recently become a paediatric
diagnosis (6). Bacterial overgrowth, increased gut permeability and intestinal dysmotility are

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characteristics of patients with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic
steatohepatitis (NASH). Endotoxin levels were elevated in patients diagnosed with NAFLD
compared to control subjects (7). One approach of modulating hepatic disease activity is to target

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the microbiome (8), but another might be to modulate the effects of bacterial lipopolysaccharide
(LPS) on hepatic cells, which express its receptor, TLR4 (9). Of note, circulating free fatty acids
may also function as endogenous ligands for TLR binding as so-called danger associated
molecular patterns, DAMPs.

Vitamin D3 and docosahexaenoic acid (an omega 3 fatty acid) offer promise for the adjuvant
treatment of NAFLD (10, 11). In fact, Vitamin D3 deficiency has been found in patients with
NAFLD, and omega 3 fatty acid deficiency is associated with the development of insulin
resistance, fatty liver disease, and dyslipidemia (12).
Therefore, the aim of this study was to expose macrophages and hepatocytes to LPS and to
gauge preventative or curative effects of Vitamin D3 and docosahexaenoic acid (DHA) in vitro
while quantifying a detrimental effect of FFA, a mixture of oleic acid and palmitic acid.
Reactions were assessed by measuring TNF and TGFin supernatants of cells for which
mRNA expressions for Srebp-1c and PPAR were determined in parallel.

Material and Methods

Materials
LPS E. coli 0111:B4 was from Invivogen (Toulouse, France), docosahexaenoic acid, oleic acid
and palmitic acid were purchased from Sigma-Aldrich Company Ltd. (Dorset, UK), and Vitamin
D3 was from DSM Nutritional products (Basel, Switzerland).
Cell culture
J774, a mouse macrophage cell line, and HepG2, a human hepatocellular carcinoma cell line,
were grown to semi confluence at 37oC in a humidified atmosphere (5% CO2) in RPMI 1640
medium or DMEM, respectively, supplemented with 10% (v/v) fetal calf serum, 100 g/ml
streptomycin, 100 IU/ml penicillin. They were passaged using trypsin EDTA. Cells were
counted and adjusted to 60 000/ml for J774, 40 000/ml for HepG2 in 25cm2 flasks (5ml) and
treated when 70% confluent.

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Experimental design

The doses of Vitamin D3 (0.4, 2, 4 g/ml) were chosen based on a pilot experiment that

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investigated induction of insulin receptor mRNA in target cells, a described, beneficial effect of
Vitamin D3 aimed at increasing insulin sensitivity (13) (suppl. Fig 1). DHA was efficient at
50M (16 g/ml) to dampen the proinflammatory response of J774 induced by LPS (100ng/ml)
after five days of stimulation (14). Doses of 8, 16, 32 g/ml DHA were chosen for this work.
The composition of FFA was oleic acid/palmitic acid (2:1) and was added at 15 and 30mM (15).
Two basic stimulation models were performed on HepG2 and J774, namely, independently,
preconditioning and subsequent stimulation with LPS as well as initial stimulation with LPS and
posthoc stimulation: In the preconditioning model, J774 and HepG2 were exposed to different
doses of Vitamin D3 (0.4, 2, 4 g/ml), docosahexaenoic acid, DHA (8, 16, 32 g/ml) or FFA,
free fatty acids (oleic acid/palmitic acid 2:1, 15 and 30mM) prior to stimulation with LPS E.coli
0111:B4 (100ng/ml) for 24 hours. In the posthoc stimulation model, J774 and HepG2 were
exposed to different doses of Vitamin D3 (0.4, 2, 4 g/ml), docosahexaenoic acid, DHA (8, 16,
32 g/ml) or FFA, free fatty acids (oleic acid/palmitic acid 2:1, 15 and 30mM) after stimulation
with LPS E.coli 0111:B4 (100ng/ml) for four hours. While Vitamin D3 and DHA are protective
agents, FFA simulates an obesity relevant aggravating agent. Unstimulated controls were run in
parallel.
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cDNA synthesis and qPCR

RNA was prepared using RNeasy Mini Kit (Qiagen, Manchester UK), genomic DNA was
digested and cDNA was synthesised using RevertAid H Minus First Strand cDNA Synthesis Kit
(Thermo Fisher Scientific, Loughborough, UK). Primer sequences are available in Supplemental
Table 1. Their efficiency was tested and sensitivity of amplification verified by linearity of
product yield vs dilutions of cDNA. Real time PCR was used to quantify the relative changes in
gene expression by 2^-Ct method using SYBR Green formulation and Corbett, Rotor gene 6000
real time rotary analyser. GAPDH mRNA expression was found to be stable. Any variation was
corrected by relating expression of gene of interest to GAPDH mRNA expression of the
respective controls as part of the 2^-Ct method (16).
ELISA
ELISAs for mouse and human Transforming growth factor , TGF, were from R&D Systems
(Abingdon, UK), ELISAs for mouse and human Tumour necrosis factor , TNF, were from

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PeproTech EC Ltd (London, UK). Supernatants were used neat and assayed in triplicate.
3,3,5,5-Tetramethylbenzidine (TMB) Liquid Substrate System was used.

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Statistics

For ELISA measurements, standard curves were constructed according to the manufacturers
instructions and levels in supernatants extrapolated using Graph Prism Pad. For qPCR analyses,
the data were presented as the fold change in gene expression normalised to GAPDH and relative
to unstimulated control. Data from triplicate determinations were expressed as means from
averages +/- SD. Unpaired one-way ANOVA with Tukeys multiple comparisons testing was
performed and adjusted p-values expressed.

Results
J774 and HepG2 were used as model cells to investigate in parallel the modulation of their LPS
induced response by the presence of Vitamin D3 and DHA using markers of relevance in the
inflammatory, profibrotic and lipogenic response. Free Fatty Acids, FFA (oleic acid/palmitic
acid 2:1), were used as a stimulus to aggravate LPS-induced reactivity. A special focus was to
capture adaptation of the cells to long term corrective exposure with Vitamin D3 or DHA before
and after LPS stimulation with a low endotoxin dose or the detrimental accumulating effect of
FFA when cells were exposed before or after the LPS stimulus.
The dose of LPS E. coli 0111:B4 to model responses towards low grade endotoxemia was
compared in a pilot experiment to a tenfold higher dose. The dose of 1000ng/ml was found to
negatively impact TLR and VDR mRNA expression in J774, which behaved as acute sensors of
the presence of this PAMP in comparison with HepG2 (Suppl. fig 2). The dose of 100ng/ml LPS
has been used by others to study the anti-inflammatory effect of DHA on macrophages (17). To

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determine that the dose of FFA was not toxic, a pilot experiment investigated the effect of FFA
on a morphological feature of HepG2 cells, namely lipid accumulation. FFA was found to
enhance Oil Red O positive cell inclusions, which could be reduced in the presence of Vitamin

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D3, whilst retaining cell intactness (Suppl. fig 3). DHA (50M) was shown to have a similar
effect on palmitate-induced lipids in HepG2 (18).

Benefical effect of DHA and Vitamin D3 on LPS-induced TNF and TGF production by
macrophages and hepatocytes

A robust induction of TNF production by 24 h stimulation with LPS was observed in J774 and
HepG2 (Fig 1A-D). There was a significant and dose dependent reduction when parallel cultures
of J774 or HepG2 were preconditioned with varying concentrations of Vitamin D3 (0.4, 2, 4
g/ml) or DHA (8, 16, 32 g/ml) for one or five days. Five days preconditioning with Vitamin
D3 (0.4, 2, 4 g/ml) or DHA (8, 16, 32 g/ml) before stimulation with LPS was not more
effective in reducing the inflammatory gene response than preconditioning for one day only. The
TNFresponse in J774 and HepG2 elicited by an acute 4 hour-stimulation with LPS was
significantly reduced at one and five days of subsequent incubation with Vitamin D3 or DHA at
three different doses each (Fig 1E-H). Macrophages at five days stimulation with Vitamin D3 or
DHA seemed particularly depressed in their TNFproduction (Fig 1E, G).

Analysis of the same supernatants for TGFshowed that Vitamin D3 and DHA significantly
reduced TGF production of J774 and HepG2 when preconditioned with these modulators prior
to LPS stimulation (Fig 2A-D). LPS led to a robust production of TGFin macrophages and
hepatocytes. It is known that LPS can trigger TGF production in monocytes (19). Macrophages
at five days stimulation with Vitamin D3 or DHA seemed particularly depressed in their TGF
production (Fig 2A, C). Addition for one day and five days of higher doses of Vitamin D3 (2, 4
g/ml) or DHA (16, 32g/ml) prior to the LPS stimulation reduced the production of TGFby
HepG2 (Fig 2B, D). LPS-induced TGF production by J774 and HepG2 cells was reduced in a
dose dependent manner by the subsequent addition of Vitamin D3 or DHA for one and five days
(Fig 2E-H). HepG2 seemed more sensitive to the action of Vitamin D3 or DHA compared to
J774.
Incubation with FFA elicited a dose dependent TNFrelease from J774 and HepG2 compared to
basal levels (Fig 3A-D). When preconditioned with FFA, macrophages showed a TNF response

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to subsequent LPS stimulation which was of the same magnitude as LPS alone (Fig 3A).
Stimulation with FFA subsequent to LPS, however, led to an impaired TNFresponse (Fig 3C).
In HepG2, stimulation with 30mM FFA for five days prior to LPS exposure, produced TNF

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levels which were reduced compared to those generated by LPS alone (Fig 3B), while

subsequent exposure with FFA after LPS stimulation further increased TNFsecreted by HepG2
compared to LPS stimulated cells (Fig 3D). Regarding TGF, the levels produced by J774 were
highest after 5 days of either preconditioning of cells with 30mM FFA before LPS stimulation,
or posthoc treatment of cells with 30mM FFA after LPS stimulation (Fig 3 E, G). The same
observation was made for HepG2 cells: stimulation with 30mM FFA for five days prior to LPS
exposure, produced TGFlevels which exceeded those generated by LPS alone (Fig 3 F), as did
posthoc exposure with 30mM FFA after LPS (Fig 3H).
Effect of DHA and Vitamin D3 on LPS-induced mRNA expression of Srepb-1c and PPARa by
macrophages and hepatocytes
LPS exposure of J774 and HepG2 stimulated Srebp-1c mRNA expression (Fig 4) as expected
(20). Preconditioning of J774 for one day with Vitamin D3 led to a dose dependent reduction of
Srepb-1c mRNA, which was further reduced after five days preconditioning. This means that
five days of incubation with a minimum dose of Vitamin D3 (0.4g/ml) prevented the induction
of Srepb-1c observed after addition of 100ng/ml LPS for 24 hours (Fig 4A). The same pattern
6

was observed for HepG2 cells, although higher doses of Vitamin D3 (2, 4 g/ml) were efficient
after just one day of preconditioning (Fig 4B). Preconditioning of J774 and HepG2 with DHA
for five days efficiently downregulated Srepb-1c mRNA with a minimum dose studied of 8g/ml
(Fig 4 C, D). One day prestimulation with 8g/ml DHA significantly reduced Srepb-1c mRNA
in HepG2, while higher doses were necessary to achieve this effect in J774 (Fig 4C, D).
Exposure of LPS stimulated J774 with Vitamin D3 or DHA yielded a dose dependent reduction
in Srepb-1c mRNA for both durations of incubation, one and five days (Fig 4 E, G). Higher
doses of Vitamin D3 (2, 4 g/ml) were needed to maintain the reduction in Srepb-1c mRNA
observed at one day posthoc stimulation compared to five days (Fig 4E). DHA, by contrast,
significantly downregulated Srepb-1c mRNA at five days incubation after LPS stimulation
compared to one day incubation (Fig 4G). In the presence of Vitamin D3, LPS-induced Srepb-1c
mRNA expression was effectively downregulated at one and five days stimulation in HepG2
(Fig 4 F), whereas higher doses of DHA had this effect at five days (Fig 4 H).

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An opposing pattern of stimulation is obtained when studying PPARmRNA expression (Fig 5

A-H), whose translation product is involved in the breakdown of fatty acids. LPS-induced
stimulation of PPAR mRNA was very low, contrasting with the robust increase of Srepb-1c

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mRNA. High PPAR mRNA was achieved in conditions of preconditioning with Vitamin D3 for
one day prior to LPS stimulation of J774, whereas this required five days preincubation for
HepG2 (Fig 5 A,B).There was a notable delay in PPAR mRNA response in HepG2 cells
preconditioned with Vitamin D3 (Fig 5B). A dose dependent response in PPARmRNA
expression is recorded when stimulating J774 or HepG2 with LPS and subsequently with
Vitamin D3 or DHA. The highest levels of PPAR mRNA were achieved at five days
preincubation of J774 or HepG2 with 32 g/ml DHA (Fig 5 C,D). After LPS stimulation,
Vitamin D3 (4 g/ml) was able to increase PPAR mRNA expression significantly after one
days treatment in J774 and HepG2 cells (Fig 5E, F). One days posthoc stimulation with DHA
(after LPS stimulation) at doses of 16 and 32g/ml was more efficient at increasing PPAR
mRNA in J774 than in HepG2 (Fig 5G, H).
The sole addition of FFA without LPS led to significantly increased levels of Srebp-1c mRNA
expression at five days stimulation compared to one day stimulation of J774 cells. FFA at
30mM increased Srepb-1c mRNA compared to LPS-induced expression significantly when
added for five days to J774 culture prior to LPS stimulation (Fig 6A). While FFA at 30mM
significantly increased Srebp-1c mRNA at five dayss stimulation compared to the shorter
7

incubation period of one day, additional stimulation with LPS did not further increase Srebp-1c
mRNA in HepG2 cells (Fig 6B). Prior stimulation with LPS of J774 and addition of FFA for one
and five days significantly increased Srebp-1c mRNA (Fig 6 C). HepG2 cells showed an
inconsistent pattern of changes in Srebp-1c mRNA in response to LPS and subsequent
stimulation with FFA at 15 and 30mM (Fig 6 D). FFA given for one and five days had a
significant role in increasing PPARmRNA levels. Subsequent LPS stimulation decreased this
effect (Fig 6E). HepG2 cells showed an inconsistent pattern of changes in PPAR mRNA in
response to FFA and subsequent stimulation with LPS (Fig 6F). Subsequent stimulation of J774
with FFA after four hours LPS led to modulation of PPARmRNA (Fig 6G); a clear reduction
in PPAR mRNA was observed in HepG2 cells exposed to 15mM FFA after LPS stimulation
(Fig 6 H).

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Discussion
Endotoxemia is an important factor in the progression from uncomplicated NAFLD to NASH
(21), which carries the risk of developing cirrhosis. This study analyses in parallel two TLR4
positive cell types of relevance to the liver, the macrophage and hepatocyte, for their regulation
of inflammatory, fibrotic, and steatotic reactants when stimulated with Vitamin D3 or DHA.
These are dietary immune modulators (22, 23) whose adjuvant clinical utility is beginning to be
widely studied in preclinical and clinical studies. The interest of this study lay in investigating a
qualitative difference in ameliorating the effect of endotoxin if Vitamin D3 or DHA were present
prior to this stimulus or available shortly after. This might give an indication whether constant
dietary admixture is recommendable or whether doses of treatment after LPS stimulus were
effective. TNF is a key factor in the development of NAFLD and NASH in both humans and
animals (24). Hasegawa et al. showed that TGF1 levels were increased in patients with NASH
as compared to patients with hepatic steatosis (25). Macrophages determine progression of

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inflammation. This is because activation of macrophages in liver leads to production of

inflammatory mediators such as TNF, Interleukin-1and reactive oxygen species in NASH. These
inflammatory mediators further stimulate hepatocytes and hepatic satellite cells to induce

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hepatocyte steatosis and fibrosis. Macrophages encourage development of steatohepatitis

because of the interaction of chemokine-chemokine receptors on liver cells and Kupffer cells
(26). Steatosis, liver injury and proinflammatory monocyte infiltration was reduced in vivo as a
result of depletion of Kupffer cells (27).

This study finds that preconditioning with Vitamin D3 or DHA prior to LPS stimulation or
incubation following LPS stimulation (posthoc) decreased TNF and TGFproduction of
J774 and of HepG2 significantly. Vitamin D3 and DHA signal via distinct receptors (VDR and
GPR120) but affect TLR4 mediated signalling effects in a similar manner, whether allowing
cells to adapt to the presence of Vitamin D3 or DHA prior to LPS stimulation or treating the
inflammatory state of HepG2 or J774 induced by LPS with Vitamin D3 or DHA subsequently.
Stimulation with obesity relevant FFA elicits elevated levels of inflammatory cytokines in
HepG2 and J774. The use of FFA allowed consideration of development of tolerance in our
system; because FFA bind to TLR4 as endogenous DAMP, it is possible that there is dampening
of LPS mediated effects on subsequent challenge or, vice versa, dampening of FFA effect
because of prior TLR4 engagement with LPS. In our study, there might be evidence of tolerance
development when stimulating J774 with LPS, then subsequently with FFA for one day, because

the elicited TNF response compared to that of FFA stimulation alone and was half that
achieved by LPS stimulation alone (Fig 3C). This was not observed for HepG2.
Vitamin D3 and DHA efficiently downregulated LPS-induced Srebp-1c mRNA expression in
J774 and HepG2 cells regardless of the timing of their addition, whether Vitamin D3 and DHA
were present before (preconditioning) or after (posthoc) LPS stimulation. The translation
product of Srebp-1c mRNA is involved in increasing cholesterol biosynthesis as part of an acute
phase reaction (28). PPAR mRNA expression showed an inverse pattern to that observed for
Srebp-1c mRNA, with regard to LPS-induced regulation and modulation by Vitamin D3 and
DHA. Our data suggest that qPCR can detect a high ratio of Srebp-1c / PPAR in LPSstimulated HepG2 and J774 which is viewed as unfavourable in the obese (29) and is provoked
by the addition of FFA in our study. Hepatic Srebp-1c mRNA levels were significantly elevated,
while hepatic PPARmRNA levels were significantly lower in obese patients undergoing
bariatric surgery compared to non-obese patients undergoing cholecystectomy (29). Relevant to
our study, these obese patients with NAFLD had significantly lower DHA in liver total lipids

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compared to their study controls (29).

Taken together, Vitamin D3 and DHA exerted beneficial effects in the context of endotoxin

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stimulation by reducing TNFand TGFproteins and Srebp-1c mRNA, whilst increasing

PPARmRNAThe translation products of Srebp-1c and PPARmRNA (which were not
measured) are involved in cholesterol biosynthesis and triglyceride turnover, respectively.
Macrophage responses are skewed towards a phenotype that produces less proinflammatory
TNF and profibrotic TGFthereby is less able to support NAFLD development and
progression to NASH (30). Both, lipid metabolism and inflammation are relevant in the
development of insulin resistance (31). Our data lend support to both, curative and preventative
dietary regimes involving Vitamin D3 or DHA to quench the detrimental effects of low grade
endotoxemia. In vivo studies are needed next to address the efficacy of dietary supplementation
with an immune modulator in enhancing the outcome from NAFLD. One recent clinical trial of
supplementation with Vitamin D3 showed significant improvement in some serum markers of
inflammation, but a greater observation time (> 4 months) was likely to be necessary to observe
amelioration in transaminase levels and ultrasonographic grades of NAFLD (32). A metaanalysis
of treatments of patients with NAFLD (for a median duration of six months) with supplements of
omega 3 fatty acids showed significant ultrasonographic improvement of steatosis, likely
involving a reversal of the Srebp-1c / PPAR ratio (33).

10

Conflict of interest statement

There is no conflict of interest to declare.
Authors and Contributors
RK, Substantial contributions to the acquisition, analysis, and interpretation of data for the work,
drafting the work, final approval of the version to be published, agreement to be accountable for
all aspects of the work in ensuring that questions related to the accuracy or integrity of any part
of the work are appropriately investigated and resolved
JH, Substantial contributions to the conception or design of the work, final approval of the
version to be published, agreement to be accountable for all aspects of the work in ensuring that
questions related to the accuracy or integrity of any part of the work are appropriately
investigated and resolved
CS, Substantial contributions to the interpretation of data for the work, drafting the work and
revising it critically for important intellectual content, final approval of the version to be
published, agreement to be accountable for all aspects of the work in ensuring that questions
related to the accuracy or integrity of any part of the work are appropriately investigated and
resolved

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Funding

This study was funded by HCDP (Human Capacity Development Program) Kurdistan region

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government (award to RK).

Acknowledgments

The study contains part of R. Kheders PhD thesis (University of Leicester). Dr S. Byrne is
acknowledged for technical support, and Dr Z. Zwaini is acknowledged for critical reading of the
manuscript.

11

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15

Supplementary material
Supplementary figure 1: Cells (A, J774; B, HepG2) were preconditioned with Vitamin D for one
day, then stimulated with LPS for 24 hrs, RNA was prepared using TriReagent and qPCR for
insulin receptor gene performed on cDNA transcribed from 3g RNA each.
Supplementary figure 2: Cells (J774, A, B; HepG2, C, D) were stimulated for 24 hrs, RNA was
prepared using TriReagent and qPCR for TLR4 (A, C) and VDR (B, D) mRNA expression
performed on cDNA transcribed from 3 g RNA each.
Supplementary figure 3: Micrographs (x40 oil) of Oil Red O stained HepG2 cells grown on
coverslips and exposed to FFA or FFA with Vitamin D3 for 48 hours (A-C). Spectrophotometric
analysis of DMSO solubilised cells after washing; mean SD of 2 independent experiments is
presented (unpaired t-test, p<0.05).

Supplementary table 1. Sequences of primer pairs (with their annealing temperatures) used in
this study

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16

Figure Legends
Fig 1 A-H: TNF response of J774 (A, C, E, G) and HepG2 (B, D, F, H) towards LPS and its
modulation by preconditioning with Vitamin D3 (A, B) or DHA (C, D) or posthoc (subsequent to
LPS) stimulation with Vitamin D3 (E, F) or DHA (G, H). Asterisks indicate significant differences
to the LPS induced response. Results are presented as averages +/- SD from triplicate
determinations. ** p<0.01, **** p< 0.0001 (adjusted p-values)
Fig 2A-H: TGF response of J774 (A, C, E, G) and HepG2 (B, D, F, H) towards LPS and its
modulation by preconditioning with Vitamin D3 (A, B) or DHA (C, D) or posthoc (subsequent to
LPS) stimulation with Vitamin D3 (E, F) or DHA (G, H). Asterisks indicate significant differences
to the LPS induced response. Results are presented as averages +/- SD from triplicate
determinations. * p<0.05 ** p<0.01 , *** p< 0.005, **** p< 0.0001 (adjusted p-values)
Fig 3 A-H: TNF and TGF response of J774 (A, C, E, G) and HepG2 (B, D, F, H) towards LPS
and its modulation by preconditioning with FFA (A, B, E, F) or posthoc (subsequent to LPS)
stimulation with FFA (C, D, G, H). Asterisks indicate significant differences to the LPS induced
response. Results are presented as averages +/- SD from triplicate determinations. * p<0.05, *** p<
0.005, **** p< 0.0001 (adjusted p-values)

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Fig 4A-H: qPCR analysis of J774 (A, C, E, G) and HepG2 (B, D, F, H) for Srebp-1c mRNA
expression induced by LPS and modulated by preconditioning with Vitamin D3 (A, B) or DHA (C,
D) or posthoc (subsequent to LPS) stimulation with Vitamin D3 (E, F) or DHA (G, H). Triplicate
data were averaged, normalised to GAPDH mRNA abundance and expressed in relation to baseline
Srebp-1c mRNA expression. a p<0.0001 compared to LPS, b p<0.0001 compared to one day
precond., c p< 0.002 compared to one day posthoc stimulation (adjusted p-values)

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Fig 5A-H: qPCR analysis of J774 (A, C, E, G) and HepG2 (B, D, F, H) for PPAR mRNA
expression induced by LPS and modulated by preconditioning with Vitamin D (A, B) or DHA (C,
D) or posthoc (subsequent to LPS) stimulation with Vitamin D3 (E, F) or DHA (G, H). Triplicate
data were averaged, normalised to GAPDH mRNA abundance and expressed in relation to baseline
PPAR mRNA expression. a p<0.0001 compared to LPS, b p<0.0001 compared to day 1, cp<0.05
compared to day 1, dp <0.05 compared to LPS, ep <0.005 compared to LPS, fp<0.001 compared to
LPS, gp<0.0005 compared to LPS (adjusted p-values)
Fig 6 A-H: qPCR analysis of J774 (A, C, E, G) and HepG2 (B, D, F, H) for Srebp-1c (A-D) and
PPAR (E-H) mRNA expression induced by LPS and modulated by preconditioning with FFA (AD) or posthoc (subsequent to LPS) stimulation with FFA (E-H). Triplicate data were averaged,
normalised to GAPDH mRNA abundance and expressed in relation to baseline Srebp-1c or PPAR
mRNA expression. ap<0.0001 compared to LPS, bp<0.0001 compared to day 1, cp<0.001
compared to LPS, dp<0.005 compared to LPS, ep<0.005 compared to day 1 (adjusted p-values)

17

Figures

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LPS) stimulation with Vitamin D3 (E, F) or DHA (G, H). Asterisks indicate significant differences
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determinations. ** p<0.01, **** p< 0.0001 (adjusted p-values)
18

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to the LPS induced response. Results are presented as averages +/- SD from triplicate
determinations. * p<0.05 ** p<0.01 , *** p< 0.005, **** p< 0.0001 (adjusted p-values)

19

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stimulation with FFA (C, D, G, H). Asterisks indicate significant differences to the LPS induced
response. Results are presented as averages +/- SD from triplicate determinations. * p<0.05, *** p<
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20

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/m

8
A
H

D
+

+
L

S
P
L

/m

S
P

/m
g

2
3

H
+

l
g

P
L

D
+
L

P
L

/m

A
H

H
D
+

D
+
P
L

/m

2
A

3
A
H

A
H
D
+

S
P
L

6
1

8
S

/m

/m

l
/m

l
/m

S
P
A
H
D
P

p o s th o c

10

fiv e d a y s

p o s th o c

a, b

a, b

S r e b p -1 c fo ld c h a n g e H e p G 2

a, b

8
A
H
D

0.

a
4

/m

/m

p o s th o c

one day

p o s th o c

2
3
A
H

.4

/m

/m

/m

fiv e d a y s

L
+
l
/m

/m

g
D

S r e b p -1 c fo ld c h a n g e H e p G 2

/m

/m

/m

S
P
L

.4

D
V
+

S
P
L

L
+
l

+
l
/m
g

6
D

fiv e d a y s

l
a
n
o
si

10

i
v
o
r
P
a

S
P

S
P

A
H

A
H

a, c

10

+
g

2
3

8
A
H

A
H

/m

l
g

/m

/m

/m
g

2
3

6
1
A
H

l
/m

l
/m

8
A
H
D

S r e b p -1 c fo ld c h a n g e J 7 7 4

a, b

p o s th o c

p o s th o c

.4
0

D
V

D
V

a
a

10

P
L
+

S
P
L

15

one day
p o s th o c

10

p re c o n d .

20

f iv e d a y s

one day

+
l

f iv e d a y s

p re c o n d .

25

p o s th o c

S r e b p - 1 c fo ld c h a n g e J 7 7 4

/m

/m
g

.4
0
D
V

one day
S r e b p - 1 c f o ld c h a n g e H e p G 2

p re c o n d .

one day

P
l
/m

+
/m

/m
4
V

f iv e d a y s

p re c o n d .

.4

4
D
V

l
/m

l
/m

/m

l
/m

2
D
V

one day
4

S re b p -1 c fo ld c h a n g e J 7 7 4

P
L
+

P
L

L
+

S
P

P
L

L
+

L
+

P
L

P
L
g

.4
0
D
V

a ,b

a ,b

a ,b
a

a ,b

10

15

20

p re c o n d .

25

S r e b p - 1 c fo ld c h a n g e J 7 7 4

p re c o n d .

p re c o n d .

S r e b p - 1 c f o ld c h a n g e H e p G 2

p re c o n d .

f iv e d a y s

one day

Fig 4A-H: qPCR analysis of J774 (A, C, E, G) and HepG2 (B, D, F, H) for Srebp-1c mRNA
expression induced by LPS and modulated by preconditioning with Vitamin D3 (A, B) or DHA (C,
D) or posthoc (subsequent to LPS) stimulation with Vitamin D3 (E, F) or DHA (G, H). Triplicate
data were averaged, normalised to GAPDH mRNA abundance and expressed in relation to baseline
Srebp-1c mRNA expression. a p<0.0001 compared to LPS, b p<0.0001 compared to one day
precond., c p< 0.002 compared to one day posthoc stimulation (adjusted p-values)
21

p re c o n d .
a, b

4
a, b
a, b
a
2

P
L

S
P

L
l
/m
g

2
D

/m

/m

L
/m
V

P
L

l
/m
g

/m

/m

S
P

S
L
+

2
3

/m

.4

l
g

2
3
A
H

D
P
L

S
P
L

D
+
S
P

/m

/m

6
1
A

A
H

A
H
D

/m

l
/m

2
3

6
1
A
H
D

/m

8
A
H
D
+
L

S
P
L

/m

/m

/m

l
/m

P
L

/m
g
2
3

A
H
D
+

P
L

6
A
H

D
+

+
S
L

.4

l
/m
g

/m
g

8
A
H
D

H
D
+
L

S
P

l
2

6
A

1
H
D
+

S
L

8
A

A
H
L

/m

l
/m

l
/m

S
P
L
D
+
S
P

/m

/m

a, b
a, b

p o s th o c

f iv e d a y s

p o s th o c

b, f

one day
8

a, b

0 .0

0 .5

15

1 .0

F o ld c h a n g e P P A R H e p G 2

a, b

/m
g

c, g

1 .5

p o s th o c

p o s th o c

p o s th o c

2 .0

F o ld c h a n g e P P A R J 7 7 4

f iv e d a y s

10

/m

l
/m
g

/m

/m

.4

/m

0 .0

F o ld c h a n g e P P A R H e p G 2

p o s th o c

P
L

P
L

i
v
o
r
P
0 .5

one day

g
V

L
+
l
/m

/m
g
2

e
1 .0

20

f iv e d a y s

one day

p o s th o c

F o ld c h a n g e P P A R J 7 7 4

L
+
l
/m
g

4
D

2
V

3
A
D

one day
1 .5

a
a, b

l
a
n
o
si

A
H

H
D

a
10

6
1

8
A

P
L
+
l

+
l
/m
g

6
1
A
H

L
+

L
/m

L
+
l

/m
g

/m
g
H
D

S
P

S
P

S
P
L

8
A
H
D

a, b

p re c o n d .

15

p re c o n d .

fiv e d a y s

p re c o n d .

F o ld c h a n g e P P A R J 7 7 4

fiv e d a y s

F o ld c h a n g e P P A R H e p G 2

one day
5

S
P
/m
g

.4

l
/m

l
/m
g

+
l

S
L

P
+

L
+

+
l
D
V

one day

.4

/m

/m

l
/m
g

2
D
V

S
P

P
L
+

S
P
L
l
/m
g

.4
0
D
V

0 .0

0 .5

a, b

a, b

a
a

1 .0

a, b
8

1 .5

p re c o n d .

.4

a, c

fiv e d a y s

p re c o n d .

10

F o ld c h a n g e P P A R - H e p G 2

one day

p re c o n d .

2 .0

F o ld c h a n g e P P A R J 7 7 4

f iv e d a y s

p re c o n d .

one day

Fig 5A-H: qPCR analysis of J774 (A, C, E, G) and HepG2 (B, D, F, H) for PPAR mRNA
expression induced by LPS and modulated by preconditioning with Vitamin D (A, B) or DHA (C,
D) or posthoc (subsequent to LPS) stimulation with Vitamin D3 (E, F) or DHA (G, H). Triplicate
data were averaged, normalised to GAPDH mRNA abundance and expressed in relation to baseline
PPAR mRNA expression. a p<0.0001 compared to LPS, b p<0.0001 compared to day 1, cp<0.05
compared to day 1, dp <0.05 compared to LPS, ep <0.005 compared to LPS, fp<0.001 compared to
LPS, gp<0.0005 compared to LPS (adjusted p-values)
22

f iv e d a y s

p re c o n d .

p re c o n d .

4
b

M
m
0

M
m

L
+
M
A

F
F

0
3
A

M
3

m
5
A

3
A

A
S
P

F
F

M
m
0

5
1
A

3
A

F
F

F
F

m
0

m
5
1
S

fiv e d a y s
p o s th o c

0 .8

0 .6

0 .4

0 .2

M
m
0
A
F

F
S

P
L

S
P

5
1
A
F

3
A

F
+

M
m

M
m
0

m
5
1

F
F
+

S
P
L

M
m

1
F

0 .0

+
S

one day
p o s th o c

P
L

0
M

A
5
1
A

F
F
+
S

P
L
m

0 .0

M
3
A

A
F
F
+
S
P

p re c o n d .

0 .5

5
1

3
A
F
F
L

fiv e d a y s

p re c o n d .

m
0

5
1
A
F
F

M
m

S
P

F
F

A
A

F
P
L

0 .0

1 .0

one day

1 .0

0 .2

M
m
0
3

1
A
F
F

0 .4

F o ld c h a n g e P P A R H e p G 2

0 .6

p o s th o c

1 .5

F o ld c h a n g e P P A R H e p G 2

0 .8

F
F

m
5

L
m

i
v
o
r
P
one day

1 .0

5
1
A
F

S
P

5
M

A
F
+
A
F
F

0 .0

S
P

L
F

S tre b p -1 c fo ld c h a n g e H e p G 2

m
5

S
P
L

1 .0

0 .5

10

+
a

p o s th o c

S
P

p re c o n d .
a

F o ld c h a n g e P P A R J 7 7 4

f iv e d a y s

p re c o n d .

1 .5

f iv e d a y s

l
a
n
o
si

one day

F o ld c h a n g e P P A R J 7 7 4

one day
p o s th o c

15

M
m

M
5
1
A
F

A
F

A
F
F

+
S
L

M
1
F

+
M

0
3

10

m
5
1

p o s th o c

10

f iv e d a y s

p o s th o c
a

b
a

15

F
F

one day

15

p re c o n d .

M
+

0
3

5
1
A

F
F

S
m

S
M

20

S re b p -1 c fo ld c h a n g e J 7 7 4

+
M
0
3
A

F
F

F
F

P
L

m
0
m
5
1

f iv e d a y s

p re c o n d .

20

one day
25

S re b p -1 c fo ld c h a n g e J 7 7 4

one day

S re b p -1 c fo ld c h a n g e H e p G 2

Fig 6 A-H: qPCR analysis of J774 (A, C, E, G) and HepG2 (B, D, F, H) for Srebp-1c (A-D) and
PPAR (E-H) mRNA expression induced by LPS and modulated by preconditioning with FFA (AD) or posthoc (subsequent to LPS) stimulation with FFA (E-H). Triplicate data were averaged,
normalised to GAPDH mRNA abundance and expressed in relation to baseline Srebp-1c or PPAR
mRNA expression. ap<0.0001 compared to LPS, bp<0.0001 compared to day 1, cp<0.001
compared to LPS, dp<0.005 compared to LPS, ep<0.005 compared to day 1 (adjusted p-values)

23

Supplement

In s u lin r e c e p to r fo ld c h a n g e H e p G 2

y
/m

in

in

it

m
a
it
V

a
d
1
S
P
L
+

L
+
/m
g

.4
0
D
in
m
a
it

a
d
1
S
P

P
L
+
/m

P
L
+
2
G
p
e
H

y
a
d
S

n
0
0
1
S

/m
g

4
D
in
m
a
it

y
a
d
1
P
L
+

L
+
/m
g

2
D
in

m
a
it
V

1
S
P

P
L
+
/m
g

.4
0

D
in
m
a
it

y
d

d
1
S

n
0
0
1
S
P
L
+
4
7
7
J
V

/m

0 .0

0 .5

/m

1 .0

1 .5

B
2 .0

In s u lin r e c e p to r f o ld c h a n g e J 7 7 4

Supplementary figure 1: Cells (A, J774; B, HepG2) were preconditioned with Vitamin D for one
day, then stimulated with LPS for 24 hrs, RNA was prepared using TriReagent and qPCR for
insulin receptor gene performed on cDNA transcribed from 3g RNA each.

l
a
n
o
si

i
v
o
r
P

24

l
a
n
o
si

i
v
o
r
P

Supplementary figure 2
Cells (J774, A, B; HepG2, C, D) were stimulated for 24 hrs, RNA was prepared using TriReagent
and qPCR for TLR4 (A, C) and VDR (B, D) mRNA expression performed on cDNA transcribed
from 3 g RNA each.

25

**

0.05

Absorbance at 510nm

**

0.04
0.03
0.02
0.01

g
/m
l
0.
4

2
g/
m
l

+V
D

VD
+

VD
+

FF
A

30
m
M
FF
A

30
m
M
FF
A

30
m
M

30
m
M

FF
A

FFA 30mM + Vitamin D3 4g/ml

C
on
tr
ol

FFA 30mM

4
g/
m
l

0.00

Control

Supplementary figure 3: Micrographs (x40 oil) of Oil Red O stained HepG2 cells grown on
coverslips and exposed to FFA or FFA with Vitamin D3 for 48 hours (A-C). Spectrophotometric
analysis of DMSO solubilised cells after washing; mean SD of 2 independent experiments is
presented (unpaired t-test, p<0.05).

l
a
n
o
si

i
v
o
r
P

26

Supplemantary table 1. Sequences of primer pairs (with their annealing temperatures) used in this
study
Target

Forward sequences 5'3'

Reverse sequences 5'3'

CCTGGAGAAACCT
GCCAAGTATG
TNF-
GGCAGGTCTACTT
TGGAGTCATTCC
TLR4
CGCTTTCACCTCT
GCCTTCACTACAG
SREBPTCTGCCTTGATGA
1C
AGTGTGG
Vitamin D TACATCCGCTGCC
R
GCCACCCGC
PPAR-
TCGAGGAAGGCAC
TACACC
Insulin R TTTGTCATGGATG
GAGGCTA
GGACCTCTCTCTA
TNF-
ATCAGCCCTC
PPAR-
GCAGAAACCCAG
AACTCAGC
SREBPGGATTGCACTTTC
1C
GAAGACATG
TLR4
AGGATGATGCCAG
GATGATGTC
GAPDH
TCCCTGAGCTGAA
CGGGAAG
Vitamin D CTCATCTGTCAGA
R
ATGAACTCCTTCA
Insulin R AACCAGAGTGAGT
ATGAGGAT

AGAGTGGGAGTTG
CTGTTGAAGTC
ACATTCGAGGCTC
CAGTGAATTCGG
ACACTACCACAAT
AACCTTCCGGCTC
AGCAGCCCCTAGA
ACAAACA
TCAGGAGTCTCAT
TGCC
TCTTCCCAAAGCT
CCTTCAA
CCTCATCTTGGGG
TTGAACT
TCGAGAAGATGAT
CTGACTGCC
ATGGCCCAGTGTA
AGAAACG
ACTCTGGACCTGG
GTGTGCAAG
TCAGGTCCAGGTT
CTTGGTTGAG
GGAGGAGTGGGT
GTCGCTGT
TCACCAAGGACAA
CCGACG
CCGTTCCAGAGCG
AAGTGCTT

GAPDH

Annealing
Temp.
0
C
55

Species

60

Mouse

55

Mouse

55

Mouse

55

Mouse

55

Mouse

55

Mouse

55

Human

55

Human

55

Human

55

Human

55

Human

55

Human

60

Human

Mouse

i
v
o
r
P

l
a
n
o
si

27