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INFLIXIMAB REVERSES SUPPRESSION OF
CHOLESTEROL EFFLUX PROTEINS BY TNF-α : A
MECHANISM FOR MODULATION OF
ATHEROGENESIS
S. Seshadri, K. Anwar, E. Belilos, S. Carsons,
A. B. Reiss
Winthrop University Hospital, New York
Disclosures
 None!
Introduction: Characteristics of Premature
Atherosclerosis in Rheumatoid Arthritis (RA)
 Patients with RA have an increased risk for early atherosclerosis
 Atherosclerosis is a major co-morbid condition in RA with an increased
incidence of cardiovascular events, including myocardial infarctions and
cardiac deaths
 This increased risk is mediated by the presence of both traditional risk
factors and factors unique to those with systemic inflammatory disorders
such as RA
 RA is a T-helper cell-driven disease characterized by increased activity of
numerous cytokines such as TNF-α, IL-1, IL-6. TNF-α is a pivotal
proinflammatory cytokine implicated in the pathogenesis of atherosclerosis
in RA
Introduction: Characteristics of Premature
Atherosclerosis in Rheumatoid Arthritis (RA)
 One of the major biological approaches to treatment of RA include agents
that interfere with cytokine function such as Anti-TNF’s ( Infliximab,
Adalimumab, Etanercept)
 Strict control of inflammation with anti-TNF therapy may reduce the risk of
development of cardiovascular disease in RA
 Infliximab is a chimeric (mouse/human), monoclonal anti-TNF antibody
commonly used in RA.
 Majority of clinical studies have looked at the effect of infliximab on lipid
profiles in RA and have shown favorable results but the effects of TNF-α
blockade on lipid patterns are still unclear. The mechanisms of action of
such treatment have not been fully explored.
Introduction: Pathological stages of atherogenesis
 Endothelial injury- initiating event in atherogenesis
 Monocyte recruitment by injured endothelium which then penetrate from
endothelial surface into arterial intima.
 In the arterial intima, monocytes become macrophages and internalize
modified LDL (oxidized LDL), becoming foam cells.
 Fatty streak formation- foam cells cluster under the endothelial lining to
form a fatty streak, the first overt sign of atherosclerotic change.
 Fibrous plaque maturation- plaque consists of a lipid-rich core, bounded on
the luminal surface by a fibrous cap. Integrity of the cap determines the
stability of the plaque.
Introduction: Cholesterol Balance in the Vessel
Wall
 Cholesterol is the major component of
atherosclerotic plaque.
 Cholesterol accumulation within
atherosclerotic plaque occurs when
cholesterol influx into the arterial wall
exceeds efflux.
 Accumulation of excess cholesterol in
macrophages generates lipid laden foam
cells, key participants in atherosclerosis.
 Cholesterol is returned to the liver for
excretion in a process called reverse
cholesterol transport (RCT).
Circulating Cholesterol
(bound by plasma lipoproteins)
Reverse Cholesterol Transport
Cholesterol
ABCA1
Cholesterol
ABCA1
27-Hydroxylase
27-Hydroxycholesterol
LXR
Cholesterol
(bound by plasma
lipoproteins)
Macrophage
cholesterol
27-Hydroxycholesterol
27-Hydroxycholesterol
Excreted as Bile Acids
from the Liver
Reverse Cholesterol Transport
Genes/Proteins involved:
27-Hydroxylase (27-OH):
 Causes solubilization of cholesterol for transport out of the cell
 Induces expression of the cholesterol efflux molecule ABCA1
LXR:
 Sensor of the lipid environment in the cell
 Also induces expression of the cholesterol efflux molecule ABCA1
ABCA1:
 Cholesterol efflux molecule
Purpose of the study
 TNF-α is a pleiotropic and pro-inflammatory cytokine that has well-
established effects on lipid metabolism and has been reported to be proatherogenic. It contributes to atherogenesis through various mechanisms.
 Recent studies have shown that treatment with anti-TNF agents have
modified cardiovascular burden in patients with rheumatoid arthritis. Most
show an increase in HDL and total cholesterol, but the long-term effects of
TNF-α blockade on lipid patterns are still unclear.
 The mechanisms of action of such treatment have not been fully explored.
We investigated the effect of TNF-α and Infliximab on reverse cholesterol
transport (RCT).
Purpose of the study
• RCT is mediated by specific proteins including ATP binding cassette
transporter A1 (ABCA1) and liver X receptor α (LXR). These two proteins
counteract foam cell formation by ridding cells of excess cholesterol in a
process known as cellular efflux.
• We hypothesize that TNF-α and Infliximab exhibit their pro- and antiatherogenic effects respectively via genes involved in RCT.
Methods
Cell Culture
 THP-1 monocytes were cultured at 370C in a 5% CO2 atmosphere to a
density of 106 cells/ml in growth medium consisting of RPMI 1640
supplemented with 10% Fetal Bovine Serum , 50 units/ml penicillin, and
50 units/ml streptomycin.
Experimental Conditions
 When THP-1 cells reached 106 cells/ml, media was aspirated and cells
were rinsed three times with Dulbecco Phosphate Buffered Saline
(DPBS) without calcium and magnesium.
 THP-1 monocytes were then incubated (106/ml, 18hr, 37°C, 5% CO2,
n=3 per condition) under 4 conditions: 1) untreated control, 2)
Interferon  (500U/ml), 3) TNF- (100U/ml), and 4) TNF- +
Infliximab (5g/ml).
 Total RNA was extracted with TRIzol reagent and was reverse
transcribed to cDNA using reverse transcriptase in the presence of
RNase inhibitor and oligo dT primers (conversion of RNA to cDNA).
Methods
ABCA1 and LXR message by RT-PCR
 Equal amounts of cDNA were taken from each RT reaction for real-time
PCR using specific primers for ABCA1, LXR and glyceraldehyde-3phosphate dehydrogenase (GAPDH) . All experimental results were
normalized to the mean density of GAPDH (control).
Statistical Analysis of Experimental Data
 Statistical analysis was performed using Graph pad Prism, version 5.01.
Pair wise multiple comparison was made between control and treatment
conditions using unpaired t tests, two tailed 95% confidence intervals,
significance p<0.05.
Results
 As previously reported, Interferon
ABCA1
p=0.01
*
2.0
 reduced ABCA1 gene expression
to 0.632 ±0.052 vs. 1.473±0.181 of
control, p=0.01
1.5
p=0.01
*
1.0
p=0.04
*
 TNF- treatment significantly
reduced ABCA1 gene expression
to 0.785±0.159 vs. control, p=0.04
0.5
am
ab
F
lix
TN
N
IF
on
t
ro
l
0.0
 Infliximab treatment nullified the
TN
F
+
In
f
C
ABCA1/GAPDH
Expression
2.5
TNF effect by increasing ABCA1
expression to 1.847±0.167, p=0.01
Results
 LXR gene expression
remained unchanged upon
interferon γ treatment
1.390±0.112 vs. 1.117±0.174 to
that of the control
LXR
1.75
LXR/GAPDH
Expression
1.50
1.25
1.00
p=0.01
*
0.75
 TNF- treatment
p=0.01
*
0.50
0.25
0.00
l
ro
t
n
Co
N
IF
F
N
T
F
N
T
ab
+
am
lf ix
In
significantly reduced LXR
gene expression to
0.412±0.025, p=0.01
 Infliximab treatment
nullified the TNF effect by
increasing LXR expression to
0.747±0.076, p=0.01
Results - Summary
 As previously reported, Interferon  reduced ABCA1 gene expression
compared to that of control( p=0.01).
 LXR gene expression remained unchanged upon interferon γ treatment
compared to that of the control.
 TNF- treatment significantly reduced both ABCA1 (p=0.04) and
LXR gene expression (p=0.01) .
 Infliximab treatment nullified the TNF effect by increasing both
ABCA1(p=0.01) and LXR expression (p=0.01)
Conclusions
 TNF-α has been previously shown to be pro-atherogenic but the
mechanisms involved are yet to be elucidated.
 Our data show that TNF treatment of monocytes dramatically and
significantly reduces ABCA1 and LXR gene expression leading to a
pro-atherogenic effect.
 Infliximab, an anti-TNF drug commonly used in rheumatology reversed
the effects of TNF by increasing both ABCA1 and LXR gene
expression, thereby, potentially ameliorating RCT.
 This is the first report indicating the anti-atherogenic effects of
Infliximab on monocyte like cells.
Acknowledgements
 A. B. Reiss
 K. Anwar
 S. Carsons
 E. Belilos
Winthrop University Hospital, New York