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Abstract Background Elevated plasma cholesterol promotes the formation of atherosclerotic lesions in which monocyte-derived lipid-laden macrophages are frequently found. To analyze, if circulating monocytes already show increased lipid content and differences in lipoprotein metabolism, we compared monocytes from patients with Familial Hypercholesterolemia (FH) with those from healthy individuals. Methods Cholesterol and oxidized cholesterol metabolite serum levels of FH and of healthy, gender/age matched control subjects were measured by combined gas chromatography – mass spectroscopy. Monocytes from patients with FH and from healthy subjects were isolated by antibody-assisted density centrifugation. Gene expression profiles of isolated monocytes were measured using Affymetrix HG-U 133 Plus 2.0 microarrays. We compared monocyte gene expression profiles from FH patients with healthy controls using a Welch T-test with correction for multiple testing (p < 0.05; Benjamini Hochberg correction, False Discovery Rate = 0.05). The differential expression of FH associated genes was validated at the mRNA level by qRT-PCR and/or at the protein level by Western Blot or flow cytometry. Functional validation of monocyte scavenger receptor activities were done by binding assays and dose/time dependent uptake analysis using native and oxidized LDL. Results Using microarray analysis we found in FH patients a significant up-regulation of 1,617 genes and a down-regulation of 701 genes compared to monocytes from healthy individuals. These include genes of proteins that are involved in the uptake, biosynthesis, disposition, and cellular efflux of cholesterol. In addition, plasma from FH patients contains elevated amounts of sterols and oxysterols. An increased uptake of oxidized as well as of native LDL by FH monocytes combined with a down-regulation of NPC1 and ABCA1 explains the lipid accumulation observed in these cells. Conclusion Our data demonstrate that circulating FH monocytes show differences in cell physiology that may contribute to the early onset of atherosclerosis in this disease.

The development of therapeutic substances to treat diseases of the central nervous system is hampered by the tightness and selectivity of the blood-brain barrier. Moreover, testing of potential drugs is time-consuming and cost-intensive. Here, we established a new microfluidically supported, biochip-based model of the brain endothelial barrier in combination with brain cortical spheroids suitable to detect effects of neuroinflammation upon disruption of the endothelial layer in response to inflammatory signals. Unilateral perfusion of the endothelial cell layer with a cytokine mix comprising tumor necrosis factor, IL-1β, IFNγ, and lipopolysaccharide resulted in a loss of endothelial von Willebrand factor and VE-cadherin expression accompanied with an increased leakage of the endothelial layer and diminished endothelial cell viability. In addition, cytokine treatment caused a loss of neocortex differentiation markers Tbr1, Tbr2, and Pax6 in the cortical spheroids concomitant with reduced cell viability and spheroid integrity. From these observations, we conclude that our endothelial barrier/cortex model is suitable to specifically reflect cytokine-induced effects on barrier integrity and to uncover damage and impairment of cortical tissue development and viability. With all its limitations, the model represents a novel tool to study cross-communication between the brain endothelial barrier and underlying cortical tissue that can be utilized for toxicity and drug screening studies focusing on inflammation and neocortex formation. [ABSTRACT FROM AUTHOR]

The study was undertaken to investigate whether the two major monocyte subsets defined by the surface markers CD14+CD16+ and CD14++CD16- show differences in their responses to hypercholesterolemia. Monocytes were rapidly isolated from the blood of hypercholesterolemic, low-density lipoprotein (LDL) receptor-defective familial hypercholesterolemia (FH) patients and from control persons. Using flow cytometry and uptake, adhesion, and phagocytosis assays as well as laser scanning microscopy, we found significant differences between the monocyte subsets. FH-CD14+CD16+ monocytes exhibit an increased uptake of oxidized LDL (oxLDL) via CD36, whereas FH-CD14++CD16- monocytes preferentially take up native LDL (nLDL). FH-CD14+CD16+ monocytes have an increased expression of surface proteins CD68, stabilin-1, and CD11c and a higher adherence to activated endothelial cells in response to oxLDL and nLDL stimulation. In addition, all CD14+CD16+ monocytes have an increased ability for phagocytosis and a higher resistance to phagocytosis impairment by oxLDL compared with CD14++CD16- monocytes. We conclude that FH-CD14+CD16+ monocytes have specialized functions in the uptake of oxLDL at activated endothelial cell surfaces, and we hypothesize that these functions are critical for the clearance of oxLDL deposits and apoptotic cells from the vessel wall under hyperlipidemic conditions. [ABSTRACT FROM AUTHOR]

This corrects the article DOI: 10.1038/srep21868 [ABSTRACT FROM AUTHOR]

Harvesting cultivated macrophages for tissue engineering purposes by enzymatic digestion of cell adhesion molecules can potentially result in unintended activation, altered function, or behavior of these cells. Thermo-responsive polymer is a promising tool that allows for gentle macrophage detachment without artificial activation prior to subculture within engineered tissue constructs. We therefore characterized different species of thermo-responsive polymers for their suitability as cell substrate and to mediate gentle macrophage detachment by temperature shift. Primary human monocyte- and THP-1-derived macrophages were cultured on thermo-responsive polymers and characterized for phagocytosis and cytokine secretion in response to lipopolysaccharide stimulation. We found that both cell types differentially respond in dependence of culture and stimulation on thermo-responsive polymers. In contrast to THP-1 macrophages, primary monocyte–derived macrophages showed no signs of impaired viability, artificial activation, or altered functionality due to culture on thermo-responsive polymers compared to conventional cell culture. Our study demonstrates that along with commercially available UpCell carriers, two other thermo-responsive polymers based on poly(vinyl methyl ether) blends are attractive candidates for differentiation and gentle detachment of primary monocyte–derived macrophages. In summary, we observed similar functionality and viability of primary monocyte–derived macrophages cultured on thermo-responsive polymers compared to standard cell culture surfaces. While this first generation of custom-made thermo-responsive polymers does not yet outperform standard culture approaches, our results are very promising and provide the basis for exploiting the unique advantages offered by custom-made thermo-responsive polymers to further improve macrophage culture and recovery in the future, including the covalent binding of signaling molecules and the reduction of centrifugation and washing steps. Optimizing these and other benefits of thermo-responsive polymers could greatly improve the culture of macrophages for tissue engineering applications. [ABSTRACT FROM AUTHOR]

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.