Human macrophages are innate immune cells with diverse, functionally distinct phenotypes, namely, pro-inflammatory M1 and anti-inflammatory M2 macrophages. Both are involved in multiple... Show moreHuman macrophages are innate immune cells with diverse, functionally distinct phenotypes, namely, pro-inflammatory M1 and anti-inflammatory M2 macrophages. Both are involved in multiple physiological and pathological processes, including would healing, infection, and cancer. However, the metabolic differences between these phenotypes are largely unexplored at single-cell resolution. To address this knowledge gap, an untargeted live single-cell mass spectrometry-based metabolomic profiling coupled with a machine-learning data analysis approach was developed to investigate the metabolic profile of each phenotype at the single-cell level. Results show that M1 and M2 macrophages have distinct metabolic profiles, with differential levels of fatty acyls, glycerophospholipids, and sterol lipids, which are important components of plasma membrane and involved in multiple biological processes. Furthermore, we could discern several putatively annotated molecules that contribute to inflammatory response of macrophages. The combination of random forest and live single-cell metabolomics provided an in-depth profile of the metabolome of primary human M1 and M2 macrophages at the single-cell level for the first time, which will pave the way for future studies targeting the differentiation of other immune cells. Show less
Ebola virus can trigger a release of pro-inflammatory cytokines with subsequent vascular leakage and impairment of clotting finally leading to multiorgan failure and shock after entering and... Show moreEbola virus can trigger a release of pro-inflammatory cytokines with subsequent vascular leakage and impairment of clotting finally leading to multiorgan failure and shock after entering and infecting patients. Ebola virus is known to directly target endothelial cells and macrophages, even without infecting them, through direct interactions with viral proteins. These interactions affect cellular mechanics and immune processes, which are tightly linked to other key cellular functions such as metabolism. However, research regarding metabolic activity of these cells upon viral exposure remains limited, hampering our understanding of its pathophysiology and progression. Therefore, in the present study, an untargeted cellular metabolomic approach was performed to investigate the metabolic alterations of primary human endothelial cells and M1 and M2 macrophages upon exposure to Ebola virus-like particles (VLP). The results show that Ebola VLP led to metabolic changes among endothelial, M1, and M2 cells. Differential metabolite abundance and perturbed signaling pathway analysis further identified specific metabolic features, mainly in fatty acid-, steroid-, and amino acid-related metabolism pathways for all the three cell types, in a host cell specific manner. Taken together, this work characterized for the first time the metabolic alternations of endothelial cells and two primary human macrophage subtypes after Ebola VLP exposure, and identified the potential metabolites and pathways differentially affected, highlighting the important role of those host cells in disease development and progression. Show less
Macrophages remodel their mechanics during differentiation toward different subtypes and drastically adapt their shapes during phagocytosis or entry to inflamed tissues. Although these functions... Show moreMacrophages remodel their mechanics during differentiation toward different subtypes and drastically adapt their shapes during phagocytosis or entry to inflamed tissues. Although these functions depend on cell mechanical properties, the mechanical behavior of macrophages is still poorly understood and accurate physiologically relevant data on basic mechanical properties of different macrophage subtypes are lacking almost entirely. By combining several complementary single-cell force spectroscopy techniques, whole cell mechanics of M1 (differentiated by granulocyte macrophage colony-stimulating factor [GM-CSF]) and M2 (differentiated by macrophage colony-stimulating factor [M-CSF]) macrophages is systematically analyzed, and it is revealed that M2 macrophages exhibit solid-like behavior, whereas M1 macrophages behave more fluid-like. In addition, the findings indicate that M2 macrophages exhibit increased dynamic motility as compared to M1 macrophages, consistent with their mechanical phenotypes. The technology presented herein can be used to distinguish macrophage subtypes based on their mechanical phenotype, and suggests that mechanical properties of macrophages are linked to their immune function. Show less
While conventional in vitro culture systems and animal models have been used to study the pathogenesis of viral infections and to facilitate development of vaccines and therapeutics for viral... Show moreWhile conventional in vitro culture systems and animal models have been used to study the pathogenesis of viral infections and to facilitate development of vaccines and therapeutics for viral diseases, models that can accurately recapitulate human responses to infection are still lacking. Human organ-on-a-chip (Organ Chip) microfluidic culture devices that recapitulate tissue-tissue interfaces, fluid flows, mechanical cues, and organ-level physiology have been developed to narrow the gap between in vitro experimental models and human pathophysiology. Here, we describe how recent developments in Organ Chips have enabled re-creation of complex pathophysiological features of human viral infections in vitro. Show less
Junaid, A.; Tang, H.Q.; Reeuwijk, A. van; Abouleila, Y.; Wuelfroth, P.; Duinen, V. van; ... ; Mashaghi, A. 2020
Ebola virus, for which we lack effective countermeasures, causes hemorrhagic fever in humans, with significant case fatality rates. Lack of experimental human models for Ebola hemorrhagic fever is... Show moreEbola virus, for which we lack effective countermeasures, causes hemorrhagic fever in humans, with significant case fatality rates. Lack of experimental human models for Ebola hemorrhagic fever is a major obstacle that hinders the development of treatment strategies. Here, we model the Ebola hemorrhagic syndrome in a microvessel-on-a-chip system and demonstrate its applicability to drug studies. Luminal infusion of Ebola virus-like particles leads to albumin leakage from the engineered vessels. The process is mediated by the Rho/ROCK pathway and is associated with cytoskeleton remodeling. Infusion of Ebola glycoprotein (GP(1,2)) generates a similar phenotype, indicating the key role of GP(1,2) in this process. Finally, we measured the potency of a recently developed experimental drug FX06 and a novel drug candidate, melatonin, in phenotypic rescue. Our study confirms the effects of FX06 and identifies melatonin as an effective, safe, inexpensive therapeutic option that is worth investigating in animal models and human trials. Show less
