LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis

Abstract

Aims: Lectin-like ox-LDL scavenger receptor-1 (LOX-1) and mitochondrial DNA (mtDNA) damage play a key role in a variety of cardiovascular diseases, including atherosclerosis, hypertension, and inflammation. We posited that damaged mtDNA could trigger autophagy and NLRP3 inflammasome activation, and LOX-1 may play a critical role in this process.

Methods and results: In order to examine this hypothesis, cultured human THP-1 macrophages exposed to lipopolysaccharide (LPS) were applied to study the link between LOX-1, mtDNA damage, autophagy, and NLRP3 inflammasome expression. Our data showed that LPS markedly induced LOX-1 expression, reactive oxygen species (ROS) generation, autophagy, mtDNA damage, and NLRP3 inflammasome. LOX-1 inhibition with a binding antibody or siRNA inhibited ROS generation, autophagy and mtDNA damage, and a decreased expression of NLRP3 inflammasome. To study the LOX-1-NLRP3 inflammasome signalling, we performed studies using ROS inhibitors and an autophagy inducer, and found that both decreased the expression of NLRP3. On the other hand, autophagy inhibitor enhanced the expression of NLRP3 inflammasome. Knockdown of DNase II inhibited autophagy and NLRP3 inflammasome, providing further support for our hypothesis. Finally, we confirmed the relationship between LOX-1, ROS, mtDNA damage, autophagy, and NLRP3 inflammasome activation in primary macrophages.

Conclusions: This study based on THP-1 macrophages and primary macrophages indicates that LOX-1-mediated autophagy and mtDNA damage play an essential role in NLRP3 inflammasome activation in inflammatory disease states.

Keywords: Autophagy; LOX-1; Mitochondrial DNA; NLRP3; ROS.

Figure 1

LPS induces expression of LOX-1 and NLRP3, ROS generation and autophagy, and increases mtDNA damage in THP-1 macrophages. (A) LPS induces expression of LOX-1 and NLRP3. (B) LPS induces cellular ROS (DCFDA staining and flow cytometry). (C) LPS induces conversion of LC3-I to LC3-II (autophagic flux analysis). (D) LPS increases mtDNA damage (qPCR analysis). Cells were treated with 1–100 ng/mL of LPS for 24 h. Note that the effects of LPS are concentration-dependent. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05 vs. Control.

Figure 2

LOX-1 inhibition reduces ROS generation, autophagy, mtDNA damage, and NLRP3 inflammasome expression. (A) LOX-1 inhibition by pre-treatment with a specific antibody or siRNA transfection blocks LOX-1 expression. (B) LOX-1 inhibition reduces cellular ROS generation. (C) LOX-1 inhibition decreases autophagic flux. (D and E) LOX-1 inhibition protects mtDNA from damage and inhibits NLRP3 expression. Cells were pre-treated with 10 μg/mL of LOX-1 antibody (Ab) or transfected with 20 nM of siRNA directed at LOX-1 for 24 h; then the cells were treated with 10 ng/mL of LPS for another 24 h. ssc, scrambled siRNA control. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05.

Figure 3

ROS inhibition decreases expression of LOX-1 and NLRP3 inflammasome and protects mtDNA from damage. (A) ROS inhibitors (apocynin and YCG063) decrease ROS generation. (B) ROS inhibitors reduce mtDNA damage and autophagy (C) as well as expression of LOX-1 and NLRP3 inflammasome (D). Cells were treated with 10 ng/mL of LPS for 24 h in the presence or absence of 1 mM apocynin and 10 µM YCG063. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05.

Figure 4

Autophagy inhibitor enhances the expression of LOX-1 and NLRP3 inflammasome, and aggravates mtDNA damage, while autophagy inducer has an opposite effect. (A) Autophagy inhibitor 3-methyladenine enhances, while autophagy inducer rapamycin inhibits LC3-II expression. (B–D) Pre-treatment with 3-methyladenine enhances, while rapamycin inhibits cellular ROS generation and the expression of NLRP3 inflammasome and mtDNA damage. Cells were treated with 10 ng/mL of LPS for 24 h in the presence or absence of autophagy inhibitor (5 mM 3-methyladenine) and autophagy inducer (10 nM rapamycin). Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05.

Figure 5

DNase II knockdown enhances the expression of LOX-1 and NLRP3 inflammasome, and aggravates mtDNA damage. (A) Expression of DNase II is inhibited by siRNA transfection. (C–E) DNase II knockdown enhances ROS generation, mtDNA damage, autophagy, and expression of NLRP3. Cells were transfected with 20 nM DNase II siRNA for 24 h, then the cells were treated with 10 ng/mL of LPS for another 24 h. ssc, scrambled siRNA control. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05.

Figure 6

LPS and mtROS generation. (A) LPS induces mtROS generation in a dose-dependent manner. Cells were treated with 1–100 ng/mL of LPS for 24 h before measurement of mtROS. (B) LOX-1 knockdown by antibody or siRNA transfection inhibits mtROS generation. Cells were pre-treated with 10 μg/mL of LOX-1 antibody (Ab) or transfected with 20 nM its siRNA for 24 h, then the cells were treated with 10 ng/mL of LPS for another 24 h. (C) ROS inhibitors apocynin and YCG063 inhibit mtROS generation. Cells were treated with 10 ng/mL of LPS for 24 h in the presence or absence of 1 mM apocynin and 10 µM YCG063. (D) Autophagy inducer rapamycin inhibits, while autophagy inhibitor 3-methyladenine enhances mtROS generation. Cells were treated with 10 ng/mL of LPS for 24 h in the presence or absence of 10 nM rapamycin and 5 mM 3-methyladenine. (E) DNase II knockdown by its siRNA transfection increases mtROS generation. Cells were transfected with 20 nM DNase II siRNA for 24 h, then the cells were treated with 10 ng/mL of LPS for another 24 h. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05.

Figure 7

Study in mice primary peritoneal macrophages confirms the results in THP-1 cells. (A and B) LOX-1 knockdown inhibits mtDNA damage and expression LC3-II, P62, and NLRP3 inflammasome expression. Cells were transfected with LOX-1 siRNA for 24 h, then the cells were treated with LPS for another 24 h. (C to E) Both ROS inhibitors apocynin and YCG063 decrease mtROS generation, mtDNA damage, as well as expression of LC3-II, P62, LOX-1, and NLRP3 inflammasome expression. Cells were treated with LPS for 24 h in the presence or absence of 0.01 mM apocynin and 10 µM YCG063. Bar graphs represent data in mean ± SD based on five experiments, *P < 0.05. (F) Proposed signalling pathway linking LOX-1 to mtDNA damage, autophagy, and NLRP3 activation. There appears to be a positive feedback loop between LOX-1 and ROS. Activation of both LOX-1 and ROS induces mtDNA damage. Though most of damaged DNA can be removed by autophagy and DNase II degradation, some damaged mtDNA that persists may result in activation of NLRP3 inflammasome.