The endothelium is the gatekeeper of vessel health, and its dysfunction is pivotal in driving atherogenesis. Here, we present a protocol to replicate endothelial-macrophage crosstalk during atherogenesis, called the "atherogenesis-on-chip" model, based on the Emulate dual-channel perfusion system. We describe a model for studying endothelial-macrophage interactions during atherogenesis in human aortic endothelial cells and human macrophages using qPCR and secretome analysis, fluorescence microscopy, and flow cytometry. This protocol could be adapted toward more complex plaque microenvironment or other disease settings.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Severe forms of COVID-19, the disease caused by SARS-CoV-2, are characterized by acute respiratory distress syndrome, robust lung inflammation and death in some patients. Strong evidence has been accumulating that polymorphonuclear neutrophilic granulocytes (PMN) play an important role in the pathophysiology of severe COVID-19. SARS-CoV-2 directly induces in vitro PMN activation, mainly the release of neutrophil extracellular traps (NETs). However, the viral components inducing this PMN response remain unclear.
In this work human PMN responses were assessed in vitro in response to the spike (S) protein of two different SARS-CoV-2 variants, anti-S IgG1 antibodies or immune complexes formed by them. Production of reactive oxygen species (ROS) was measured by Diogenes-based chemiluminescence. Release of myeloperoxidase (MPO) was assessed by ELISA while secretion of a list of cytokines and growth factors was determined by high-performance multiplex cytokine assay.
We show that the SARS-CoV-2 Omicron variant S protein and anti-spike IgG1, either alone or together, stimulate ROS production in human PMNs. We also observed that the SARS-CoV-2 Wuhan S protein and anti-S IgG1 antibody together trigger MPO release from PMNs. Based on the relevance of SARS-CoV-2 and influenza co-infections, we have also investigated the impact of influenza virus infection on the previous PMN responses to S proteins or anti-S antibodies. We did not detect any significant effect of influenza co-infection on ROS generation in PMNs. Our data also show that PMN stimulation by S proteins induced the release of different chemokines, growth factors, regulatory and proinflammatory cytokines. Overall, our findings show that the SARS-CoV-2 S protein, an anti-spike IgG1 antibody or their immune complex, promote oxidative responses of PMNs in a variant-dependent manner, contributing to a better understanding of the role of PMN responses during SARS-CoV-2 infection.
Copyright © 2023 Almeida, Fantone, Sarr, Ashtiwi, Channell, Grenfell, Martins-Filho and Rada.

DNA methylation is important to establish a cell’s developmental identity. It also modulates cellular responses to endogenous developmental stimuli or environmental changes. We designed an in vitro myeloid differentiation model to analyze the genetic and developmental contribution to methylome dynamics using whole-genome bisulfide sequencing and transcriptome sequencing. Using a recursive partitioning approach, we identified 34,502 differentially methylated regions (DMRs) associated with genetic background and/or developmental stimuli. Specifically, 23,792 DMRs (69%) were significantly associated with inter-individual variations, of which 82% were associated with genetic polymorphisms in cis . Notably, inter-individual variations further modified 57 of 212 (26%) developmental DMRs with transcriptomic responses. Our study presents a novel analytical approach to determine the bona fide genetic contribution embedded in outlier patterns of CpG-SNPs in individual methylomes. This approach can be used to study genetic and epigenetic mechanisms underlying differential responses to developmental stimuli, environmental changes, and inter-individual differences in drug responses.