GM-CSF-activated STAT5A regulates macrophage functions and inflammation in atherosclerosis.

Introduction: Inhibition of STAT5 was recently reported to reduce murine atherosclerosis. However, the role of STAT5 isoforms, and more in particular STAT5A in macrophages in the context of human atherosclerosis remains unknown. Methods and results: Here, we demonstrate reciprocal expression regulation of STAT5A and STAT5B in human atherosclerotic lesions. The former was highly upregulated in ruptured over stable plaque and correlated with macrophage presence, a finding that was corroborated by the high chromosomal accessibility of STAT5A but not B gene in plaque macrophages. Phosphorylated STAT5 correlated with macrophages confirming its activation status. As macrophage STAT5 is activated by GM-CSF, we studied the effects of its silencing in GM-CSF differentiated human macrophages. STAT5A knockdown blunted the immune response, phagocytosis, cholesterol metabolism, and augmented apoptosis terms on transcriptional levels. These changes could partially be confirmed at functional level, with significant increases in apoptosis and decreases in lipid uptake and IL-6, IL-8, and TNFa cytokine secretion after STAT5A knockdown. Finally, inhibition of general and isoform A specific STAT5 significantly reduced the secretion of TNFa, IL-8 and IL-10 in ex vivo tissue slices of advanced human atherosclerotic plaques. Discussion: In summary, we identify STAT5A as an important determinant of macrophage functions and inflammation in the context of atherosclerosis and show its promise as therapeutic target in human atherosclerotic plaque inflammation.

Culture density influences the functional phenotype of human macrophages.

Macrophages (MΦ) are commonly cultured in vitro as a model of their biology and functions in tissues. Recent evidence suggests MΦ to engage in quorum sensing, adapting their functions in response to cues about the proximity of neighboring cells. However, culture density is frequently overlooked in the standardization of culture protocols as well as the interpretation of results obtained in vitro. In this study, we investigated how the functional phenotype of MΦ was influenced by culture density. We assessed 10 core functions of human MΦ derived from the THP-1 cell line as well as primary monocyte-derived MΦ. THP-1 MΦ showed increasing phagocytic activity and proliferation with increasing density but decreasing lipid uptake, inflammasome activation, mitochondrial stress, and secretion of cytokines IL-10, IL-6, IL-1ß, IL-8, and TNF-α. For THP-1 MΦ, the functional profile displayed a consistent trajectory with increasing density when exceeding a threshold (of 0.2 x 103 cells/mm2), as visualized by principal component analysis. Culture density was also found to affect monocyte-derived MΦ, with functional implications that were distinct from those observed in THP-1 MΦ, suggesting particular relevance of density effects for cell lines. With increasing density, monocyte-derived MΦ exhibited progressively increased phagocytosis, increased inflammasome activation, and decreased mitochondrial stress, whereas lipid uptake was unaffected. These different findings in THP-1 MΦ and monocyte-derived MΦ could be attributed to the colony-forming growth pattern of THP-1 MΦ. At the lowest density, the distance to the closest neighboring cells showed greater influence on THP-1 MΦ than monocyte-derived MΦ. In addition, functional differences between monocyte-derived MΦ from different donors could at least partly be attributed to differences in culture density. Our findings demonstrate the importance of culture density for MΦ function and demand for awareness of culture density when conducting and interpreting in vitro experiments.

Effective Transfection and Gene Silencing of Primary Murine Macrophages with Small Interfering RNA.

Transfection of murine primary macrophages to silence genes can be a challenging procedure because this cell type has developed mechanisms to evade cellular intrusion. The introduction of small interfering RNA (siRNA) encapsulated in liposomes to the cell to decrease gene expression is one of the methods that can be used to achieve gene silencing. There are different commercially available compounds to introduce siRNA into the cell, including Lipofectamine RNAiMAX and HiPerfect. The chapter will describe a method for gene silencing in mouse primary macrophages using liposome-based transfection of siRNA.

Heterogeneity of atherosclerotic plaque macrophage origin, phenotype and functions: Implications for treatment.

Macrophages are key players in atherosclerotic lesions, regulating the local inflammatory milieu and plaque stability by the secretion of many inflammatory molecules, growth factors and cytokines. Monocytes have long been considered to be the main source of plaque macrophages. However, recent findings provide evidence for proliferation of local macrophages or transdifferentiation from other vascular cells as alternative sources. Recent years of research focused on the further identification and characterisation of macrophage phenotypes and functions. In this review we describe the advances in our understanding of monocyte and macrophage heterogeneity and its implications for specific therapeutic interventions, aiming to reduce the ever growing significant risk of cardiovascular events without any detrimental side effects on the patient's immune response.