Oxidized LDL Levels Are Increased in HIV Infection and May Drive Monocyte Activation

Abstract

Background: HIV infection is associated with increased cardiovascular risk, and this risk correlates with markers of monocyte activation. We have shown that HIV is associated with a prothrombotic monocyte phenotype, which can be partially mitigated by statin therapy. We therefore explored the relationship between oxidized low-density lipoprotein (oxLDL) particles and monocyte activation.

Methods: We performed phenotypic analysis of monocytes using flow cytometry on fresh whole blood in 54 patients with HIV and 24 controls without HIV. Plasma levels of oxLDL, soluble CD14, IL-6, and soluble CD163 were measured by enzyme-linked immunosorbent assay. In vitro experiments were performed using flow cytometry.

Results: Plasma levels of oxLDL were significantly increased in HIV infection compared with controls (60.1 units vs. 32.1 units, P < 0.001). Monocyte expression of the oxLDL receptors, CD36 and Toll-like receptor 4, was also increased in HIV. OxLDL levels correlated with markers of monocyte activation, including soluble CD14, tissue factor expression on inflammatory monocytes, and CD36. In vitro stimulation with oxLDL, but not to low-density lipoprotein, resulted in expansion of inflammatory monocytes and increased monocyte expression of tissue factor, recapitulating the monocyte profile we find in HIV disease.

Conclusions: OxLDL may contribute to monocyte activation, and further study in the context of HIV disease is warranted.

Figure 1. Plasma levels of oxLDL are increased in HIV-1 infected patients and correlate with levels of sCD14 and tissue factor expression on inflammatory monocytes

Plasma samples from all donors were thawed and levels A) oxidized LDL B) IL-6, C) sCD14, and D) sCD163 were measured by ELISA. There is a direct correlation between plasma levels of oxLDL and E) sCD14 and oxLDL and F) the proportion of inflammatory (CD14+CD16+) monocytes in HIV disease. Monocytes were assessed by flow cytometry.

Figure 2. Expression of TLR4 and CD36 are increased on monocyte subsets from HIV-1 infected patients

Whole blood samples were obtained from 54 HIV-1 infected donors and 24 healthy donors and the relative proportions of monocyte subsets were analyzed by flow cytometry. Three monocyte subsets were identified by size and granularity and by CD14 and CD16 expression. Representative histograms and summary data showing expression of A) Toll-Like Receptor 4 or B) CD36 on monocyte subsets from uninfected donors and HIV-1 infected patients are displayed.

Figure 3. Exposure of whole blood samples to oxLDL, but not LDL, results in proportional increases in inflammatory (CD14+CD16+) monocytes

Whole blood was obtained from HIV-1 uninfected subjects and was exposed to lipopolysaccharide (LPS, 20 ng/mL), LDL (50 μg/mL), or oxLDL (50 μg/mL) for 3 hours. Surface expression of CD14 and CD16 was measured on monocyte subsets by flow cytometry. Exposure to LPS or oxLDL, but not LDL resulted in an increase in the proportional representation of inflammatory monocytes. A) Representative dot plots B) Summary data. (repeated measures ANOVA, bonfierroni post tests; *** p<0.001)

Figure 4. Exposure of whole blood samples to oxLDL, but not LDL, results in increased surface expression of TF on classical and inflammatory monocyte subsets

Whole blood was obtained from HIV-1 uninfected subjects and was exposed to lipopolysaccharide (LPS, 20 ng/mL), LDL (50 μg/mL), or oxLDL (50 μg/mL) for 3 hours. Surface expression tissue factor was measured on monocyte subsets by flow cytometry. OxLDL induces TF expression on classical and inflammatory monocytes. (repeated measures ANOVA, bonfierroni post tests; * p<0.05, *** p<0.001)