Inflammation impairs reverse cholesterol transport in vivo

Abstract

Background: Inflammation is proposed to impair reverse cholesterol transport (RCT), a major atheroprotective function of high-density lipoprotein (HDL). The present study presents the first integrated functional evidence that inflammation retards numerous components of RCT.

Methods and results: We used subacute endotoxemia in the rodent macrophage-to-feces RCT model to assess the effects of inflammation on RCT in vivo and performed proof of concept experimental endotoxemia studies in humans. Endotoxemia (3 mg/kg SC) reduced (3)H-cholesterol movement from macrophage to plasma and (3)H-cholesterol associated with HDL fractions. At 48 hours, bile and fecal counts were markedly reduced consistent with downregulation of hepatic expression of ABCG5, ABCG8, and ABCB11 biliary transporters. Low-dose lipopolysaccharide (0.3 mg/kg SC) also reduced bile and fecal counts, as well as expression of biliary transporters, but in the absence of effects on plasma or liver counts. In vitro, lipopolysaccharide impaired (3)H-cholesterol efflux from human macrophages to apolipoprotein A-I and serum coincident with reduced expression of the cholesterol transporter ABCA1. During human (3 ng/kg; n=20) and murine endotoxemia (3 mg/kg SC), ex vivo macrophage cholesterol efflux to acute phase HDL was attenuated.

Conclusions: We provide the first in vivo evidence that inflammation impairs RCT at multiple steps in the RCT pathway, particularly cholesterol flux through liver to bile and feces. Attenuation of RCT and HDL efflux function, independent of HDL cholesterol levels, may contribute to atherosclerosis in chronic inflammatory states including obesity, metabolic syndrome, and type 2 diabetes.

Conflict of interest statement

Disclosures

The other authors report no potential conflicts of interest.

Figures

Figure 1. LPS (3mg/kg, SQ) impairs macrophage 3H-cholesterol efflux into plasma in vivo

(A) Plasma counts, measured over time across multiple studies, were normalized within each individual study and percentage reduction at indicated time-points post-LPS presented (n=54). A representative FPLC profile (one of three) is presented and revealed (B) no change in cholesterol mass at 4h and (C) increased LDL and no change in HDL-cholesterol mass at 48h after endotoxin. LPS reduced 3H-cholesterol associated with HDL fractions at (D) 4h and (E) 48h. Immunoblot analysis of FPLC fractions revealed a modest reduction in apoA-I and increase in apoB and apoE levels at (F) 4h and (G) 48h following LPS (3mg/kg) (*p<0.05, **p<0.01, ***p<0.001).

Figure 2. HDL remodeling during endotoxemia impairs HDL acceptor function

(A) Endotoxemia (3mg/kg) induced hepatic serum amyloid A (SAA) mRNA. (B) Plasma SAA protein levels markedly increased following LPS (n=6). FPLC profiling demonstrated the majority of SAA was associated with HDL fractions at (C) 4h and (D) 48h; further SAA was detectable in the apoE rich “shoulder” region at 48h (Fractions 22–28). (E) Ex vivo cholesterol efflux from 3H-cholesterol-labeled, acLDL-loaded J774 macrophages to HDL isolated from LPS treated mice (n=5) was reduced at all time-points post-LPS (*p<0.05, **p<0.01, ***p<0.001).

Figure 3. Endotoxin impairs 3H-cholesterol movement through liver to bile and feces

(A) No significant effect on liver counts was observed at 48h following LPS; (B) Reduced mRNA expression of ABCA1 and SR-BI was observed across multiple studies with no effect on ABCG1 expression. (C) LPS markedly reduced bile counts at 48h. This coincided with (D) reduced mRNA expression of hepatic transporters ABCG5, ABCG8 and ABCB11 and the bile-acid synthesis enzyme CYP7A1, as well as (E) reduced 3H-cholesterol levels in feces (n=54). (F) Immunoblot analysis of liver membrane lysates revealed no change in SR-BI, ABCA1 and ABCG1 protein expression but a marked reduction in ABCG5/8 heterodimer expression at 24h. Densitometry data, normalized to β-actin, is shown for SR-BI and ABCG5/8 heterodimer (*p<0.05, **p<0.01, ***p<0.001).

Figure 4. Low-dose endotoxin selectively impairs cholesterol transport through liver to bile and feces

Low-dose endotoxin (0.3mg/kg) (A) induced no significant increase in plasma TNFα protein levels, (B) did not reduce 3H-cholesterol plasma levels and had no effect on (C) FPLC lipid profiles and (D) HDL-associated counts at 24h. (E) 3H-cholesterol tracer counts in liver were not affected. However, low-dose LPS reduced (F) bile and (G) fecal counts as well as (H) hepatic ABCG5, ABCG8 and ABCB11 mRNA expression (n=6, *p<0.05, **p<0.01, ***p<0.001).

Figure 5. Human acute phase HDL has reduced acceptor capacity for macrophage 3H-cholesterol ex vivo

Human experimental endotoxemia (3ng/kg, IV bolus) had non-significant effects on plasma levels of (A) HDL-cholesterol (F=0.39, p=0.91) and moderately decreased (B) ApoA-I at later time-points. (C) A reduction in plasma phospholipid and (D) a marked induction of plasma SAA levels and hsCRP was observed as early as 6h post LPS (n=20). (E) Ex vivo3H-cholesterol efflux from J774 macrophages to acute phase HDL (2.8% PEG supernatant) was reduced at 24h post-LPS (n=20). (F) 3H-cholesterol efflux from LPS treated (100ng/ml for 4h) human macrophages in vitro was reduced to apoA-I (20μg/ml) and serum (2.5%) but not to HDL3 (50μg/ml). (G) LPS reduced mRNA expression of ABCA1, ABCG1 and SR-BI in human macrophages (cells derived from three people); (H) Immunoblot analysis revealed a marked reduction in ABCA1, a moderate reduction in SR-BI and little change in ABCG1 protein levels (Figure 5H) (*p<0.05, **p<0.01, ***p<0.001).