Presentations

Abstract

Introduction: Cardiovascular disease (CVD) is the leading cause of death worldwide. Atherosclerosis is a multifactorial disease of the large and medium sized arteries and is the major etiological process underlying the development of CVD. It is characterized by an accumulation of modified lipids, vascular dysfunction, and a dysregulated immune response. Sleep fragmentation (SF) is common in modern life effecting ~30% of the population. Recent studies have highlighted the importance of proper sleep by showing that decreased sleep duration and poor sleep quality are associated with high rates of cardiovascular disease (CVD), unstable plaque formation, and increased risk of all-cause mortality. Specifically, mechanistic studies have revealed disturbed sleep results in unexpected premature death in some male mice through the accumulation of reactive oxygen species (ROS) in the small intestines and the hyperactivation of myeloid lineage immune cells, particularly neutrophils. Neutrophils are the most abundant immune cell in circulation and are vital for mounting a proper immune response against pathogens, but chronic neutrophil activation is detrimental and a main driver of chronic inflammatory disease progression. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor known for its role in driving Th1 and Th17 differentiation. We have shown STAT4 is expressed in neutrophils and is a critical regulator of neutrophil activation and function in healthy and infectious conditions. Stat4 deficient neutrophils exhibit reduced ROS production, neutrophil extracellular trap (NET) formation, and migratory ability resulting in decreased survival of animals during bacterial challenge. Interestingly, when this model was used to evaluate atherosclerosis, the deletion of Stat4 in myeloid cells played a protective role in detrimental inflammation and reduced atherosclerosis and improved plaque stability in atherosclerotic Ldlr-/- mice. These studies highlighted the context-dependent role of Stat4 in neutrophils in various disease conditions. Studies in our lab have shown that SF accelerates atherosclerosis and destabilizes atherosclerotic plaques in a neutrophil dependent manner. Specifically, Sleep fragmentation induces hyper activation neutrophils reflected by enhanced ROS production, neutrophil extracellular trap (NET) formation and chemotaxis. We have also shown SF o results in an accumulation of bacterial by-products in circulation with associated intestinal ROS accumulation and premature death of animals fed a high fat diet. Therefore, we hypothesized that Stat4 is involved in the neutrophil-dependent destabilization of plaques seen in sleep fragmented, atherosclerotic mice.

Methods: Eight-week-old, female Stat4ΔLysMLdlr-/- and control Stat4fl/fl Ldlr-/- mice were fed a high fat/cholesterol (DDC) diet and either sleep fragmented or allowed sufficient sleep for 16 weeks. Survival was tracked throughout the study via Kaplan-Meier analysis. After 16 weeks, body weight, and aortic atherosclerotic lesion formation were measured. Cholesterol (WAKO), LPS (ELISA), and cytokine (FACS) levels were measured in plasma. Immune populations in the blood, peritoneal cavity and small intestines were determined via vetscan and flow cytometry on Cytek Aurora. ROS production was measured in the small intestines using DHE and fluorescent microscopy.

Results: Survival studies revealed a increased susceptibility to SF-induced Premature death in 40% of atherosclerotic mice which lacked Stat4 in myeloid cells (Stat4ΔLysMLdlr-/-). Interestingly, while some Stat4ΔLysMLdlr-/- mice died, those which survived the study had improved atherosclerotic lesion formation following 16 weeks of DDC feeding and SF. No difference in BW or cholesterol was seen between genotypes or sleep conditions following 16 weeks of DDC-feeding. In line with previous data, female Stat4ΔLysMLdlr-/- mice had fewer circulating neutrophils compared to Stat4fl/flLdlr-/- controls, regardless of sleep condition. Importantly, 16 weeks of DDC-feeding increased ROS production in the small intestines of SF Stat4ΔLysMLdlr-/- mice vs SF Stat4fl/flLdlr-/- mice. There was also increased inflammation in the small intestines and the surrounding peritoneal cavity of SF Stat4ΔLysMLdlr-/- mice vs SF Stat4fl/flLdlr-/- mice

Conclusion: Myeloid specific Stat4 deletion reduced circulating neutrophil counts with improved atherosclerosis when compared to Stat4fl/flLdlr-/- mice. However, it appears that Stat4 may be important for the survival of mice when intestinal inflammation and barrier breakdown is present. These data suggest that Stat4 is involved in SF-induced myeloid activation and deleting Stat4 in myeloid cells reduces atherosclerosis but if makes the animal vulnerable to any additional immune challenges. Current studies are ongoing to reveal the specific mechanisms involved in the Stat4-dependent reduction of atherosclerosis in SF and to unveil the mechanism responsible for premature death observed.