Mechanisms of Trained Innate Immunity in oxLDL Primed Human Coronary Smooth Muscle Cells

Abstract

Objective: Damage and pathogen associated molecular patterns such as oxidized low-density lipoprotein (oxLDL) or bacillus Calmette-Guerin (BCG) vaccine can induce long term pro-inflammatory priming in monocytes and macrophages due to metabolic and epigenetic reprogramming-an emerging new concept called trained innate immunity. Vascular smooth muscle cells express pattern recognition receptors involved in trained innate immunity in monocytes. Here we investigated whether the mechanisms of trained innate immunity also control a proinflammatory phenotype in human coronary smooth muscle cells. Methods: Human coronary smooth muscle cells were primed with oxLDL or BCG for 24 h. After a resting time of 4 to 7 days, the cells were restimulated with either PAM3cys4, LPS or TNFα and cytokine production or mRNA expression were measured. Then, mechanisms of monocyte trained innate immunity were analyzed in smooth muscle cells, including receptors, intracellular pathways as well as metabolic and epigenetic reprogramming. Results: Priming with oxLDL or BCG lead to a significantly increased production of IL6, IL8 and MCP-1 following restimulation. OxLDL priming had little effect on the expression of macrophage or SMC marker genes. Proinflammatory priming of smooth muscle cells induced mTOR-HIF1α-signaling and could be blocked by mTOR-, TLR2-, and TLR4-inhibition. Finally, metabolic and epigenetic mechanisms of trained innate immunity in monocytes could be replicated in smooth muscle cells, including increased glucose consumption, lactate production, responsiveness to 6-fluoromevalonate and mevalonate treatment and inhibition of priming by the histone methyltransferase inhibitor methylthioadenosine (MTA). Conclusion: We demonstrate for the first time that mechanisms of the so called trained innate immunity control a proinflammatory phenotype in non-immune cells of the vascular wall. Our findings warrant further research into the specificity of trained innate immunity as an immune cell response as well as the mechanisms of vascular smooth muscle cells inflammation.

Keywords: inflammation; mTOR; oxLDL; smooth muscle cells; trained innate immunity.

Figure 1

OxLDL induces a dose-dependent proinflammatory priming effect in SMCs. (A) SMCs were treated with different doses of oxLDL for 24 h, on day 5 cells were restimulated with 5 μg/ml PAM3cys4 for 24 h and IL6 levels were analyzed in the supernatant. (B) SMCs were treated with 10 μg/ml oxLDL for 3 to 24 h and restimulated with 5 μg/ml PAM3cys4 on day 5. IL6 levels were analyzed in the supernatant. (C,D) SMCs were treated with 10 μg/ml oxLDL for 24 h. On day 5 cells were restimulated with 5 μg/ml PAM3cys4 for 24 h and IL8 levels (C) and MCP-1 levels (D) were analyzed in the supernatant. (E) SMCs were treated with 10 μg/ml oxLDL for 24 h and restimulated with 5 μg/ml PAM3cys4 on day 8 (^*p < 0.05, SEM, all experiments were repeated at least 3 times).

Figure 2

OxLDL priming induces mRNA levels of inflammatory cytokines. (A–C) SMCs were treated with 10 μg/ml oxLDL for 24 h, and restimulated with 5 μg/ml PAM3cys4 for 6 h on day 5. mRNA levels were analyzed by real-time qPCR. (D,E) SMCs were treated with the indicated doses of oxLDL for 24 h and restimulated with 10 ng/ml TNFα (D) or 50 ng/ml LPS for 24 h. IL6 levels were analyzed in the supernatant (^*p < 0.05, SEM, all experiments were repeated at least 3 times).

Figure 3

BCG induces a dose-dependent proinflammatory priming effect in SMCs. (A,C,E) SMCs were treated with the indicated doses of BCG for 24 h and restimulated with 5 μg/ml PAM3cys4 for 6 h on day 5. mRNA levels of Il6, IL8, and MCP1 were analyzed by real-time qPCR. (B,D,F) SMCs were treated with the indicated doses doses of BCG for 24 h, on day 5 cells were restimulated with 5 μg/ml PAM3cys4 for 24 h and IL6 levels (B), IL8 levels (D), and MCP-1 levels (F) were analyzed in the supernatant (^*p < 0.05, SEM, all experiments were repeated at least 3 times).

Figure 4

OxLDL priming has little effect on the expression of macrophage and SMC marker genes. (A–D) SMCs were treated with 10 μg/ml oxLDL for 24 h. mRNA levels were analyzed by real-time qPCR (^*p < 0.05, SEM, all experiments were repeated at least 3 times).

Figure 5

Inhibition of TLR2-, TLR4-, and mTOR-signaling blocks oxLDL priming. (A) SMCs were pretreated with 5 μM of the TLR2-inhibitor CU-CPT22 or 1 μM of the TLR4-inhibitor TAK242 or both for 30 min, followed by 10 μg/ml oxLDL for 24 h. Cells were restimulated on day 5 with 5 μg/ml PAM3 for 24 h and IL6 levels were analyzed in the supernatant. (B,C) SMCs were treated with 10 μg/ml oxLDL for 4 h. Cell lysates were analyzed by western blot. (D) SMCs were pretreated with 10 nM of the mTOR-inhibitor Torin1 or 1 μM of the mTOR-inhibitor OSI27 for 30 min, followed by 10 μg/ml oxLDL for 24 h. Cells were restimulated on day 5 with 5 μg/ml PAM3cys4 for 24 h and IL6 levels were analyzed in the supernatant (^*p < 0.05, SEM, all experiments were repeated at least 3 times).

Figure 6

OxLDL priming involves metabolic and epigenetic mechanisms. (A,B) SMCs were treated with 10 μg/ml oxLDL for 24 h. After 3 days resting time cells were harvested and HIF1α protein levels were analyzed by western blot. (C) SMCs were treated with 10 μg/ml oxLDL for 24 h. After 3 days resting time cells were lysed and lactate concentration measured. (D) SMCs were treated with 10 μg/ml oxLDL for 24 h. After 3 days resting time fresh medium was applied and after another 24 h glucose concentration was measured in the supernatant. (E–G): SMCs were pretreated with 3 mM mevalonate (E) or 1 mM 6-fluoromevalonate (F) or 10 μM of the histone methyltransferase inhibitor MTA (G) for 30 min, followed by 10 μg/ml oxLDL for 24 h. Cells were restimulated on day 5 with 5 μg/ml PAM3cys4 for 24 h and IL6 levels were analyzed in the supernatant (^*p < 0.05, SEM, all experiments were repeated at least 3 times).