Objective:
This study tested the hypothesis that mitochondrial DNA damage could trigger NLRP3 inflammasome activation during inflammation, and LOX-1 may play a critical role in this process.
Methods and Results:
We performed studies in cultured human THP1 macrophages exposed to ox-LDL or LPS,which are often used as inflammation stimuli
in vitro
. We examined and confirmed the increase in LOX-1 expression when cells were treated with ox-LDL or LPS. Parallel groups of cells were treated with LOX-1 Ab to bind LOX-1. In accordance with our previous studies in endothelial cells and smooth muscle cells, LOX-1 Ab markedly reduced ox-LDL- as well as LPS-stimulated LOX-1 expression. To assess mitochondrial ROS generation, MitoSOX™ Red mitochondrial superoxide indicator was used. Both fluorescence staining and flow cytometry analysis showed that LPS induced (more than ox-LDL) mitochondrial ROS generation. Pretreatment with LOX-1 Ab significantly attenuated mitochondrial ROS generation in response to ox-LDL or LPS. Then we observed mtDNA damage in THP1 cells exposed to ox-LDL or LPS. Importantly, pretreatment with LOX-1 Ab protected mtDNA from damage in response to both stimuli. This was also confirmed by q-PCR (mtDNA/nDNA ratio) analysis. Further, ox-LDL or LPS induced the expression of phos-NF-kB p65, caspase-1 p10 and p20, and cleaved proteins IL-1β and IL-18. Of note, NLRP3 inflammasome was activated in response to ox-LDL or LPS in a similar manner. Pretreatment of cells with LOX-1 Ab treatment blocked or significantly attenuated these inflammatory responses.
Conclusions:
These observations based on
in vitro
observations indicate that LOX-1 via ROS generation plays a key role in mtDNA damage which then leads to NLRP3 inflammasome activation during inflammation.
Mitochondrial DNA damage is associated with damage accrual and disease duration in patients with Systemic Lupus Erythematosus
