Circadian Control of Pulmonary Endothelial Signaling occurs via the NADPH oxidase 2-NLRP3 pathway

Abstract

Circadian rhythms refer to cell autonomous oscillations that occur with a 24-hr periodicity. These rhythms are ubiquitous and thus, vascular endothelial cells that line the vascular bed are also subjected to circadian regulation. While the circadian control of vascular function has been demonstrated in the context of various pathologies, the relevance and functional implication of clock control over pulmonary vasculature has never been investigated. As the pulmonary endothelium is a crucial site for the inflammatory response to a lung specific pathogen, we investigated the role of the circadian clock in mediation the response of the pulmonary endothelium to inflammation. We hypothesized that the pulmonary endothelium is under circadian control and that this rhythm is disrupted by inflammatory stimuli via redox mediated signaling processes involving the NLRP3 inflammasome. Methods: Circadian rhythms were monitored in pulmonary artery segments and endothelial cells isolated from mPer2luciferase transgenic mice in the presence of an inflammatory stimuli (LPS). Reactive oxygen species (ROS) production in LPS treated cells was measured by fluorescence microscopy using the cell permeant dye CellROX Green. NLRP3 inflammasome was monitored post-mortem (0-72 h post LPS instillation) by measuring the expression of the NLRP3 subunit in wild type and Bmal1-/- and Cry1/2-/- mice. Inflammation was quantified in these mice by measuring PMN adherence and intercellular adhesion molecule (ICAM-1). Results: We observed that the circadian rhythm of the pulmonary vasculature was altered LPS. LPS also led to ROS production in these cells; ROS increased 3 h post LPS treatment, peaked by 36 h and returned to baseline values by 72 h. ROS were inhibited by pretreating the cells with the NADPH oxidase 2 (NOX2) inhibitor dipheneylene iodonium (DPI). Addition of DPI, prior to LPS pretreatment also restored the circadian rhythmicity of the pulmonary endothelium. The increase in NLRP3 along the vessel wall (post LPS treatment) was resolved by 72 h in lungs of wild type mice but not in Bmal1-/- and Cry1-/-Cry2-/- lungs. Inflammation (ICAM-1 and PMN) was also resolved in wild type but not in mice wherein the circadian clock had been disrupted genetically. Conclusion: Our data indicate that pro-inflammatory stimuli reprogram circadian rhythms in the pulmonary endothelium via ROS via the NOX2-NLRP3 pathway. Disruption of the clock mediates a sustained increase in ROS via this Nox2-NLRP3 pathway in endothelial cells, thus offering a novel mechanism for mitigating the effects of clock disruption.

NOX2 inhibition enables retention of the circadian clock in BV2 microglia and primary macrophages

“…NOX2 is associated with the progression of various neurodegenerative diseases ( 14 , 17 , 32 , 54 , 79 – 84 ). Notably, the inhibition of NOX2 in vascular endothelial cells using diphenyleneiodonium chloride (DPI) restored PER2 rhythmicity under LPS stimulation ( 85 ). While these findings linked the circadian clock and oxidative stress, we have shown herein that gene expression of p47 phox and gp91 phox are timed by the clock, indicating that NOX2 is directly timed by the microglial circadian clock.…”

NOX2 Inhibition Enables Retention of the Circadian Clock in BV2 Microglia and Primary Macrophages

“…NOX2 is associated with the progression of various neurodegenerative diseases (13,16,30,46,70–75). Notably, the inhibition of NOX2 in vascular endothelial cells using diphenyleneiodonium chloride (DPI) restored PER2 rhythmicity under LPS stimulation (76). While these findings linked the circadian clock and oxidative stress, we have shown herein that gene expression of p47 phox and gp91 phox are timed by the clock, indicating that NOX2 is directly timed by the microglial circadian clock.…”