Oxidized lipids enhance RANKL production by T lymphocytes: implications for lipid-induced bone loss

Abstract

Osteoporosis is a systemic disease that is associated with increased morbidity, mortality and health care costs. Whereas osteoclasts and osteoblasts are the main regulators of bone homeostasis, recent studies underscore a key role for the immune system, particularly via activation-induced T lymphocyte production of receptor activator of NFkappaB ligand (RANKL). Well-documented as a mediator of T lymphocyte/dendritic cell interactions, RANKL also stimulates the maturation and activation of bone-resorbing osteoclasts. Given that lipid oxidation products mediate inflammatory and metabolic disorders such as osteoporosis and atherosclerosis, and since oxidized lipids affect several T lymphocyte functions, we hypothesized that RANKL production might also be subject to modulation by oxidized lipids. Here, we show that short term exposure of both unstimulated and activated human T lymphocytes to minimally oxidized low density lipoprotein (LDL), but not native LDL, significantly enhances RANKL production and promotes expression of the lectin-like oxidized LDL receptor-1 (LOX-1). The effect, which is also observed with 8-iso-Prostaglandin E2, an inflammatory isoprostane produced by lipid peroxidation, is mediated via the NFkappaB pathway, and involves increased RANKL mRNA expression. The link between oxidized lipids and T lymphocytes is further reinforced by analysis of hyperlipidemic mice, in which bone loss is associated with increased RANKL mRNA in T lymphocytes and elevated RANKL serum levels. Our results suggest a novel pathway by which T lymphocytes contribute to bone changes, namely, via oxidized lipid enhancement of RANKL production. These findings may help elucidate clinical associations between cardiovascular disease and decreased bone mass, and may also lead to new immune-based approaches to osteoporosis.

Figure 1. RANKL production by T lymphocytes exposed to oxidized lipids

A. Unstimulated T lymphocytes were plated in culture medium with various concentrations of MM-LDL (0, 1, 10, 25, 50, 100ug/ml), and incubated for 72 hours. Cell viability was determined by counting the number of live cells under light microscope analysis and trypan blue exclusion (n = 8). B. Measurement of RANKL production by ELISA of unstimulated T lymphocytes exposed to various concentrations of MM-LDL (0, 1, 25, 50, 100ug/ml) (n = 4). Error bars indicate SD. C. T lymphocytes were stimulated with CD2/CD3/CD28 antibody-coated beads in the presence of 25 ug/ml of either nLDL or MM-LDL, and after 72 hours, RANKL (pg/ml) in the culture supernatant was evaluated by ELISA. Representative ELISA results from one donor. D. Fold-increase (compared to buffer control) of RANKL production by activated T lymphocytes treated with oxidized lipids (n = 18). Error bars indicate SD. E. Representative ELISA results for RANKL (pg/ml) production by unstimulated T lymphocytes cultured for 72 hours in the presence of 25 ug/ml of either nLDL or MM-LDL. F. Fold-increase (compared to buffer control) in RANKL production by unstimulated T lymphocytes treated with oxidized lipids (n = 15). Error bars indicate SD. G. Relative expression (compared to GAPDH) of RANKL (left) and OPG (right) transcript in unstimulated T lymphocytes treated with 25 ug/ml nLDL or MM-LDL for 24 hours, as detected by QT- PCR (n = 4). Error bars indicate SD. H. Relative expression (compared to GAPDH) of RANKL transcript in T lymphocytes from mice fed either chow (n = 4) or high fat diet (n = 4) for 11 months as detected by QT- PCR. Error bars indicate SD. I. Serum levels of RANKL (pg/ml), detected by ELISA from mice fed either chow (n = 4) or high fat (n = 4) for 11 months. Error bars indicate SD. * – p ≤ 0.05. ** – p ≤ 0.01.

Figure 2. iso-PGE₂ induces RANKL production by unstimulated T lymphocytes

A. T lymphocytes were exposed to various concentrations of iso-PGE₂ (0, 10, 25, 50 uM) and incubated for 72 hours. RANKL production in culture supernatant was measured by ELISA. Graph represents fold increase over DMSO control (n = 3). Error bars indicate SD. B. T lymphocytes were cultured with 25 uM iso-PGE₂ for 72 hours and RANKL measurement in culture supernatant was evaluated by ELISA. Graph represents fold-increase over DMSO control (n = 8). Error bar indicate SD. C. Relative expression (compared to GAPDH) of RANKL (left) and OPG (right) transcript in unstimulated T lymphocytes treated with 25 uM iso-PGE₂ for 24 hours, as detected by QT- PCR (n = 4). Error bar indicate SD. * – p ≤ 0.01.

Figure 3. Mechanism of oxidized lipid-induced RANKL production in T lymphocytes

A. Activated T lymphocytes: T lymphocytes were pre-incubated with NFκB inhibitor 1 for one hour prior to stimulation with CD2/CD3/CD28 antibody-coated beads in the presence of 25 ug/ml nLDL or MM-LDL. Cells were harvested after 72 hours of treatment, and RANKL in culture supernatant was evaluated by ELISA. Graph represents fold-increase over buffer (n = 4). Error bars indicate SD.; p = 0.052 for MM-LDL no inhibitor vs MM-LDL with inhibitor 1. B. Unstimulated T lymphocytes: T lymphocytes were preincubated with either NFκB inhibitor 1 or inhibitor 2 prior to treatment with oxidized lipids. RANKL (pg/ml) in culture supernatant was evaluated by ELISA after 72 hours of treatment. Data is presented as fold-increase over buffer (n = 4). Error bars indicate SD. p ≤ 0.05 for MM-LDL no inhibitor vs MM-LDL with inhibitor 1 and MM-LDL no inhibitor vs MM-LDL with inhibitor 2. C. Nuclear localization of activated NFκB: Unstimulated T lymphocytes were incubated with 25 ug/ml of MM-LDL for 1 or up to 2 hours, and nuclear extracts were tested in wells containing the NFκB consensus binding site. Assay specificity was determined by competition with wild-type NFκB oligonucleotide (WT p65). Data is presented from 4 independent experiments. Error bars indicate SD. p ≤ 0.05 for buffer vs. MM-LDL.

Figure 4. LOX-1 expression on T lymphocytes treated with oxidized lipids

A. T lymphocytes, unstimulated or activated with 6 ug/ml PHA, were exposed to 25 ug/ml nLDL, 25 ug/ml MM-LDL or 25 uM iso-PGE₂ for 72 hours. Representative histogram analysis of LOX-1 expression from one donor. B. Fold increase (compared to buffer control) of LOX-1 expression by T lymphocytes, unstimulated (n = 9) and activated (n = 10), treated with oxidized lipids. Error bars indicate SD. C. Relative expression (compared to GAPDH) of LOX-1 transcripts in unstimulated (n = 5) and activated (n = 4) T lymphocytes treated with 25 ug/ml nLDL, 25 ug/ml MM-LDL or 25 uM iso-PGE₂ for 24 hours as detected by QT- PCR. Error bars indicate SD. * – p ≤ 0.05. ** – p ≤ 0.01.