LXR Activation Induces a Proinflammatory Trained Innate Immunity-Phenotype in Human Monocytes

Abstract

Objectives: The concept of trained innate immunity describes a long-term proinflammatory memory in innate immune cells. Trained innate immunity is regulated through reprogramming of cellular metabolic pathways including cholesterol and fatty acid synthesis. Here, we have analyzed the role of Liver X Receptor (LXR), a key regulator of cholesterol and fatty acid homeostasis, in trained innate immunity.

Methods and results: Human monocytes were isolated and incubated with different stimuli for 24 h, including LXR agonists, antagonists and Bacillus Calmette-Guerin (BCG) vaccine. After 5 days resting time, cells were restimulated with the TLR2-agonist Pam3cys. LXR activation did not only increase BCG trained immunity, but also induced a long-term inflammatory activation by itself. This inflammatory activation by LXR agonists was accompanied by characteristic features of trained innate immunity, such as activating histone marks on inflammatory gene promoters and metabolic reprogramming with increased lactate production and decreased oxygen consumption rate. Mechanistically, LXR priming increased cellular acetyl-CoA levels and was dependent on the activation of the mevalonate pathway and IL-1β signaling. In contrast to mevalonate pathway inhibition, blocking fatty acid synthesis further increased proinflammatory priming by LXR.

Conclusion: We demonstrate that LXR activation induces a proinflammatory trained immunity phenotype in human monocytes through epigenetic and metabolic reprogramming. Our data reveal important novel aspects of LXR signaling in innate immunity.

Keywords: LXR; acetyl-CoA; inflammation; monocytes; trained innate immunity.

FIGURE 1

LXR agonists induce a proinflammatory phenotype in human monocytes. Monocytes were treated as indicated with BCG, 2 μM T1317 (LXR agonist), 10 μM GW3965 (LXR agonist), 5 μM GSK2033 (LXR antagonist) or vehicle for 24 h, kept for 5 days in complete medium and restimulated with 5 μg/ml Pam3cys or 10 ng/ml LPS for 24 h. IL-6 (A,D,E), TNFα (B,F) and IL-8 (C) were measured in the supernatant. Graphs represent mean values ± SD of six individuals in three different experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 2

LXR priming induces epigenetic reprogramming. (A–D) Monocytes were treated as indicated with 2 μM T1317 (LXR agonist), 10 μM GW3965 (LXR agonist) or vehicle for 24 h and kept for 5 days in complete medium. Chromatin was collected and ChIP assay was performed using an antibody against histone H3 acetylated at lysine 27 (H3K27ac), histone H3 trimethylated at lysine 3 (H3K4me3) or control IgG. Then quantitative real-time PCR was performed with primers for IL-6 (A,C) and TNFα (B,D). (E,F) Monocytes were treated as indicated with 2 μM T1317 (LXR agonist), 20 μM MTA (histone methyltransferase inhibitor) or vehicle for 24 h, kept for 5 days in complete medium and restimulated with 5 μg/ml Pam3cys for 24 h. IL-6 (E) and TNFα (F) were measured in the supernatant. Graphs represent mean values ± SD of six individuals in three different experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 3

LXR priming induces metabolic reprogramming in trained human monocytes. Monocytes were treated as indicated with 2 μM T1317 (LXR agonist), 10 μM GW3965 (LXR agonist), 100 nM Torin1 (mTOR inhibitor), 10 μM LW6 (HIF1α-Inhibitor), 10 μM KC7F2 (HIF1α-Inhibitor) or vehicle for 24 h and kept for 5 days in complete medium. (A,B) Cells were restimulated with 5 μg/ml Pam3cys for 24 h and IL-6 was measured in the supernatant. (C) Oxygen consumption rate (OCR) of trained cells was determined on day 6. (D,E) Graphs depict mean basal (D) and maximal respiration (E). (F) On day 6 cells were lysed and lactate concentration was measured. Graphs represent mean values ± SD of six individuals in three different experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 4

LXR priming depends on the mevalonate pathway. Monocytes were treated as indicated with 2 μM T1317 (LXR agonist), 10 μM GW3965 (LXR agonist), 20 μM Fluvastatin (HMG-CoA reductase inhibitor) or vehicle for 24 h and kept for 5 days in complete medium. (A,B) Cells were restimulated with 5 μg/ml Pam3cys for 24 h and IL-6 (A) and TNFα (B) were measured in the supernatant. (C–F) mRNA levels were analyzed by real-time qPCR 24 h after priming. Graphs represent mean values ± SD of six individuals in three different experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 5

LXR priming increases Acetyl-CoA levels, a novel mediator of inflammatory priming. (A–C) Monocytes were treated as indicated with 2 μM T1317 (LXR agonist), 5 μM GSK2033 (LXR antagonist), 500 μM Acetyl-CoA, 25 μM C75 (FASN-inhibitor) or vehicle for 24 h. (A) cells were lysed and acetyl-CoA concentration measured on day 3. (B,C) cells were kept for 5 days in complete medium and restimulated with 5 μg/ml Pam3cys for 24 h. IL-6 was measured in the supernatant. (D) monocytes were transfected with siRNA against SREBP1 or scrambled siRNA, treated with 2 μM T1317 or vehicle for 24 h and kept for 5 days in complete medium. Then cells were restimulated with 5 μg/ml Pam3cys for 24 h and IL-6 was measured in the supernatant. Graphs represent mean values ± SD of six individuals in three different experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 6

LXR priming is dependent on IL-1β. Monocytes were treated as indicated with 2 μM T1317 (LXR agonist), 10 μM GW3965 (LXR agonist), 5 μM GSK2033 (LXR antagonist), 1 μM Diacerein, 10 ng/ml IL-1β, 200 ng/ml IL1RA (IL-1 receptor antagonist) or vehicle for 24 h. (A) mRNA levels of IL-1β, were analyzed by real-time qPCR. (B) 24 h after priming medium was changed and after 24 h resting time supernatant was collected and the level of IL-1β was measured using ELISA. (C–F) cells were kept for 5 days in complete medium and restimulated with 5 μg/ml Pam3cys for 24 h. IL-6 (C–E) and TNFα (F) were measured in the supernatant. Graphs represent mean values ± SD of six individuals in three different experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.