Phosphatidylserine enhances anti-inflammatory effects of reconstituted HDL in macrophages via distinct intracellular pathways.

Phosphatidylserine (PS) is a minor phospholipid constituent of high-density lipoprotein (HDL) that exhibits potent anti-inflammatory activity. It remains indeterminate whether PS incorporation can enhance anti-inflammatory effects of reconstituted HDL (rHDL). Human macrophages were treated with rHDL containing phosphatidylcholine alone (PC-rHDL) or PC and PS (PC/PS-rHDL). Interleukin (IL)-6 secretion and expression was more strongly inhibited by PC/PS-rHDL than PC-rHDL in both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated macrophages. siRNA experiments revealed that the enhanced anti-inflammatory effects of PC/PS-rHDL required scavenger receptor class B type I (SR-BI). Furthermore, PC/PS-rHDL induced a greater increase in Akt1/2/3 phosphorylation than PC-rHDL. In addition, PC/PS but not PC-rHDL decreased the abundance of plasma membrane lipid rafts and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Finally, when these rHDL formulations were administered to dyslipidemic low-density lipoprotein (LDL)-receptor knockout mice fed a high-cholesterol diet, circulating IL-6 levels were significantly reduced only in PC/PS-rHDL-treated mice. In parallel, enhanced Akt1/2/3 phosphorylation by PC/PS-rHDL was observed in the mouse aortic tissue using immunohistochemistry. We concluded that the incorporation of PS into rHDLs enhanced their anti-inflammatory activity by modulating Akt1/2/3- and p38 MAPK-mediated signaling through SR-BI in stimulated macrophages. These data identify PS as a potent anti-inflammatory component capable of enhancing therapeutic potential of rHDL-based therapy.

DRAG in situ barcoding reveals an increased number of HSPCs contributing to myelopoiesis with age.

Ageing is associated with changes in the cellular composition of the immune system. During ageing, hematopoietic stem and progenitor cells (HSPCs) that produce immune cells are thought to decline in their regenerative capacity. However, HSPC function has been mostly assessed using transplantation assays, and it remains unclear how HSPCs age in the native bone marrow niche. To address this issue, we present an in situ single cell lineage tracing technology to quantify the clonal composition and cell production of single cells in their native niche. Our results demonstrate that a pool of HSPCs with unequal output maintains myelopoiesis through overlapping waves of cell production throughout adult life. During ageing, the increased frequency of myeloid cells is explained by greater numbers of HSPCs contributing to myelopoiesis rather than the increased myeloid output of individual HSPCs. Strikingly, the myeloid output of HSPCs remains constant over time despite accumulating significant transcriptomic changes throughout adulthood. Together, these results show that, unlike emergency myelopoiesis post-transplantation, aged HSPCs in their native microenvironment do not functionally decline in their regenerative capacity.

Erythropoietin directly remodels the clonal composition of murine hematopoietic multipotent progenitor cells.

The cytokine erythropoietin (EPO) is a potent inducer of erythrocyte development and one of the most prescribed biopharmaceuticals. The action of EPO on erythroid progenitor cells is well established, but its direct action on hematopoietic stem and progenitor cells (HSPCs) is still debated. Here, using cellular barcoding, we traced the differentiation of hundreds of single murine HSPCs, after ex vivo EPO exposure and transplantation, in five different hematopoietic cell lineages, and observed the transient occurrence of high-output myeloid-erythroid-megakaryocyte-biased and myeloid-B-cell-dendritic cell-biased clones. Single-cell RNA sequencing analysis of ex vivo EPO-exposed HSPCs revealed that EPO induced the upregulation of erythroid associated genes in a subset of HSPCs, overlapping with multipotent progenitor (MPP) 1 and MPP2. Transplantation of barcoded EPO-exposed MPP2 confirmed their enrichment in myeloid-erythroid-biased clones. Collectively, our data show that EPO does act directly on MPP independent of the niche and modulates fate by remodeling the clonal composition of the MPP pool.

Phospholipid transfer to high-density lipoprotein (HDL) upon triglyceride lipolysis is directly correlated with HDL-cholesterol levels and is not associated with cardiovascular risk.

BACKGROUND AND AIMS: While low concentrations of high-density lipoprotein-cholesterol (HDL-C) represent a well-established cardiovascular risk factor, extremely high HDL-C is paradoxically associated with elevated cardiovascular risk, resulting in the U-shape relationship with cardiovascular disease. Free cholesterol transfer to HDL upon lipolysis of triglyceride-rich lipoproteins (TGRL) was recently reported to underlie this relationship, linking HDL-C to triglyceride metabolism and atherosclerosis. In addition to free cholesterol, other surface components of TGRL, primarily phospholipids, are transferred to HDL during lipolysis. It remains indeterminate as to whether such transfer is linked to HDL-C and cardiovascular disease. METHODS AND RESULTS: When TGRL was labelled with fluorescent phospholipid 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), time- and dose-dependent transfer of DiI to HDL was observed upon incubations with lipoprotein lipase (LPL). The capacity of HDL to acquire DiI was decreased by -36% (p<0.001) in low HDL-C patients with acute myocardial infarction (n = 22) and by -95% (p<0.001) in low HDL-C subjects with Tangier disease (n = 7), unchanged in low HDL-C patients with Type 2 diabetes (n = 17) and in subjects with high HDL-C (n = 20), and elevated in subjects with extremely high HDL-C (+11%, p<0.05) relative to healthy normolipidemic controls. Across all the populations combined, HDL capacity to acquire DiI was directly correlated with HDL-C (r = 0.58, p<0.001). No relationship of HDL capacity to acquire DiI with both overall and cardiovascular mortality obtained from epidemiological studies for the mean HDL-C levels observed in the studied populations was obtained. CONCLUSIONS: These data indicate that the capacity of HDL to acquire phospholipid from TGRL upon LPL-mediated lipolysis is proportional to HDL-C and does not reflect cardiovascular risk in subjects widely differing in HDL-C levels.

Free cholesterol transfer to high-density lipoprotein (HDL) upon triglyceride lipolysis underlies the U-shape relationship between HDL-cholesterol and cardiovascular disease.

BACKGROUND: Low concentrations of high-density lipoprotein cholesterol (HDL-C) represent a well-established cardiovascular risk factor. Paradoxically, extremely high HDL-C levels are equally associated with elevated cardiovascular risk, resulting in the U-shape relationship of HDL-C with cardiovascular disease. Mechanisms underlying this association are presently unknown. We hypothesised that the capacity of high-density lipoprotein (HDL) to acquire free cholesterol upon triglyceride-rich lipoprotein (TGRL) lipolysis by lipoprotein lipase underlies the non-linear relationship between HDL-C and cardiovascular risk. METHODS: To assess our hypothesis, we developed a novel assay to evaluate the capacity of HDL to acquire free cholesterol (as fluorescent TopFluor® cholesterol) from TGRL upon in vitro lipolysis by lipoprotein lipase. RESULTS: When the assay was applied to several populations markedly differing in plasma HDL-C levels, transfer of free cholesterol was significantly decreased in low HDL-C patients with acute myocardial infarction (-45%) and type 2 diabetes (-25%), and in subjects with extremely high HDL-C of >2.59 mmol/L (>100 mg/dL) (-20%) versus healthy normolipidaemic controls. When these data were combined and plotted against HDL-C concentrations, an inverse U-shape relationship was observed. Consistent with these findings, animal studies revealed that the capacity of HDL to acquire cholesterol upon lipolysis was reduced in low HDL-C apolipoprotein A-I knock-out mice and was negatively correlated with aortic accumulation of [3H]-cholesterol after oral gavage, attesting this functional characteristic as a negative metric of postprandial atherosclerosis. CONCLUSIONS: Free cholesterol transfer to HDL upon TGRL lipolysis may underlie the U-shape relationship between HDL-C and cardiovascular disease, linking HDL-C to triglyceride metabolism and atherosclerosis.

Oxidative stress pathways involved in cytotoxicity and genotoxicity of titanium dioxide (TiO2) nanoparticles on cells constitutive of alveolo-capillary barrier in vitro.

The health risks of nanoparticles remain a serious concern given their prevalence from industrial and domestic use. The primary route of titanium dioxide nanoparticle exposure is inhalation. The extent to which nanoparticles contribute to cellular toxicity is known to associate induction of oxidative stress. To investigate this problem further, the effect of titanium dioxide nanoparticles was examined on cell lines representative of alveolo-capillary barrier. The present study showed that all nanoparticle-exposed cell lines displayed ROS generation. Macrophage-like THP-1 and HPMEC-ST1.6R microvascular cells were sensitive to endogenous redox changes and underwent apoptosis, but not alveolar epithelial A549 cells. Genotoxic potential of titanium dioxide nanoparticles was investigated using the activation of γH2AX, activation of DNA repair proteins and cell cycle arrest. In the sensitive cell lines, DNA damage was persistent and activation of DNA repair pathways was observed. Moreover, western blot analysis showed that specific pathways associated with cellular stress response were activated concomitantly with DNA repair or apoptosis. Nanoparticles-induced oxidative stress is finally signal transducer for further physiological effects including genotoxicity and cytotoxicity. Within activated pathways, HSP27 and SAPK/JNK proteins appeared as potential biomarkers of intracellular stress and of sensitivity to endogenous redox changes, respectively, enabling to predict cell behavior.