No abstract

Background: Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. Methods: We generated myeloid-specific Stat4ΔLysMLdlr-/-, neutrophil-specific Stat4ΔS100A8 Ldlr-/-, and control Stat4fl/flLdlr-/- mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat Pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. In vivo homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled Stat4ΔLysMLdlr-/- and Stat4fl/flLdlr-/- bone marrow cells into aged atherosclerotic Apoe-/- mice and detected by flow cytometry. Results: STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in Stat4ΔLysMLdlr-/- mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired in vivo neutrophil trafficking to atherosclerotic aorta. Conclusions: Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.

Atherosclerosis is an inflammatory disease of the large and medium size arteries characterized by deposition of oxidized lipids within the vessel. While specific self-antigens are not well characterized, data show that the immune response is involved in atherogenesis. The role of B cells in atherosclerosis is subset specific. FO B cells are proatherogenic, while B1 and MZ B cells serve as protective. B cell receptor signaling is important in B cell activation, differentiation, and functions, while important costimulatory molecules, like CD45, assist in initiating signal propagation. To date, it is unknown whether B cell subsets respond differently to BCR signaling or if CD45-dependent regulation of BCR signaling is subset specific in homeostatic or atherosclerotic conditions. Taking advantage of transgenic mice that express low levels of CD45(CD45L/L), we examined activation of CD45L/L B cells through Ca2+ flux. We show that BCR-induced B cell activation differs between subsets and CD45-dependent modulation of BCR signaling is subset specific. MZ and B1 have high levels of Ca2+ flux, with MZ B cell activation the most affected by CD45 expression. To test the effects of CD45L/L B in atherosclerosis, we performed adoptive transfer of CD45L/L or WT B cells into B cell-deficient atherosclerotic prone mice. After 23–38 wks of western diet feeding, CD45L/L B cell recipients had increased lesion formation despite an increase of protective MZ B cells. This increase in atherogenesis could be due to MZ CD45L/L B cells having reduced activation, disrupting their protective ability. This suggests that the subset-specific modulation of BCR signaling through CD45 plays a role in the regulation of B cell functions in atherosclerosis. This work was supported by American Heart Association AIREA grant 8AIREA33960546.

Atherosclerosis is a chronic inflammatory disease of large and medium size vessels that progresses across a lifespan and has been extensively linked to insufficient sleep. Poor fragmented sleep quality is strongly associated with all-cause mortality. Accumulating evidence demonstrates the independent effects of atherosclerosis and fragmented sleep on chronic inflammation within multiple tissues. However, there is limited data on the combined effects of sleep fragmentation (SF) and atherosclerosis on the inflammatory environments of multiples tissue. To address this question, we used flow cytometry and qPCR to investigate immune alterations within the peripheral blood, bone marrow, spleen, aorta, small intestine, and brains of female Apoe −/− mice following 12 weeks of high fat diet (HFD) feeding and SF. SF was induced by waking up mice at the day time using a moving bar. Importantly, we used an activity control (AC) group of mice that had a moving bar during nighttime. We found that chronic SF accelerated atherogenesis and induced increased myelopoiesis. Particularly, we detected increases in pro-inflammatory monocytes and neutrophils in the bone marrow, blood, spleen, aortas and small intestines of SF vs AC groups of HFD-fed female Apoe−/− mice. Additionally, we detected increase inflammatory markers in the brains of SF vs the control AC mice. Our data suggests that SF accelerates atherosclerosis and alters the immune composition of multiple tissues including the blood, bone marrow, spleen, small intestine, and aorta of atherosclerosis-prone mice. Furthermore, the increased peripheral monocytosis and neutrophilia may serve as a link to the elevated inflammatory markers in the brains of HFD-fed, female SF Apoe−/− mice. Supported by the American Heart Association Pre-Doctoral Fellowship (20PRE35180156)

There are roughly 70 million Americans who report insufficient sleep due to work responsibilities, sleep apnea, or lifestyle choices. Altered duration and poor sleep quality are associated with an increase in brain inflammation, high rates of cardiovascular disease and other inflammatory diseases in humans. It is known that psychological stress is associated with increased atherogenesis, it is also known that sleep fragmentation (SF) can exacerbate and be induced by SF. However, the exact relationship between SF, neuroinflammation, and atherogenesis is not well known. We found that chronic SF led to increased plaque formation in the aortas HFD fed Apoe−/− mice. SF also resulted in alterations in the immune composition of the bone marrow (BM), blood, aortas, and lymph nodes in the HFD fed Apoe−/− mice. Importantly we detected an increased content of myeloid cells in the blood of SF and HFD fed female Apoe−/− mice. There was an increased percentage of CD68+ macrophages in SF Apoe−/− aortas. Additionally, we detected an increase in CD11b expression and inflammatory markers in the brains of mice that were sleep fragmented when compared to control mice. Our data suggest that SF induces elevated myelopoeisis resulting in monocytosis, neutrophilia, and the altered immune composition of the aortas of atherosclerosis-prone mice, resulting in accelerated atherosclerotic plaque burden.