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 CholesterolCirculating Cholesterol(bound by plasma lipoproteins)(bound by plasma lipoproteins)

27-Hydroxycholesterol27-Hydroxycholesterol

cholesterolcholesterol27-Hydroxycholesterol27-Hydroxycholesterol

Excreted as Bile AcidsExcreted as Bile Acidsfrom the Liverfrom the Liver

CholesterolCholesterol

27-Hydroxylase27-Hydroxylase

27-Hydroxycholesterol27-Hydroxycholesterol

MacrophageMacrophage

LXRLXR

ABCA1ABCA1 ABCA1ABCA1

CholesterolCholesterol(bound by plasma (bound by plasma

lipoproteins)lipoproteins)

CholesterolCholesterol

Reverse Cholesterol Transport

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 pro-atherogenic. 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 anti-atherogenic effects respectively via genes involved in RCT.

MethodsCell 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 (5g/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-3-phosphate 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 reduced ABCA1 gene expression to 0.632 ±0.052 vs. 1.473±0.181 of control, p=0.01

TNF- treatment significantly reduced ABCA1 gene expression to 0.785±0.159 vs. control, p=0.04

Infliximab treatment nullified the TNF effect by increasing ABCA1 expression to 1.847±0.167, p=0.01

ABCA1

0.0

0.5

1.0

1.5

2.0

2.5

p=0.01*

p=0.04*

p=0.01*

AB

CA

1/G

APD

H E

xpre

ssio

n

Results LXR gene expression

remained unchanged upon interferon γ treatment 1.390±0.112 vs. 1.117±0.174 to that of the control

TNF- treatment

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

LXR

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

p=0.01*

p=0.01*

LXR

/GA

PDH

Exp

ress

ion

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. ReissK. AnwarS. Carsons E. BelilosWinthrop University Hospital, New York