Cellular Heterogeneity of Activated Primary Human Macrophages and Associated Drug-Gene Networks: From Biology to Precision Therapeutics.

BACKGROUND: Interferon-γ (IFNγ) signaling plays a complex role in atherogenesis. IFNγ stimulation of macrophages permits in vitro exploration of proinflammatory mechanisms and the development of novel immune therapies. We hypothesized that the study of macrophage subpopulations could lead to anti-inflammatory interventions. METHODS: Primary human macrophages activated by IFNγ (M(IFNγ)) underwent analyses by single-cell RNA sequencing, time-course cell-cluster proteomics, metabolite consumption, immunoassays, and functional tests (phagocytic, efferocytotic, and chemotactic). RNA-sequencing data were analyzed in LINCS (Library of Integrated Network-Based Cellular Signatures) to identify compounds targeting M(IFNγ) subpopulations. The effect of compound BI-2536 was tested in human macrophages in vitro and in a murine model of atherosclerosis. RESULTS: Single-cell RNA sequencing identified 2 major clusters in M(IFNγ): inflammatory (M(IFNγ)i) and phagocytic (M(IFNγ)p). M(IFNγ)i had elevated expression of inflammatory chemokines and higher amino acid consumption compared with M(IFNγ)p. M(IFNγ)p were more phagocytotic and chemotactic with higher Krebs cycle activity and less glycolysis than M(IFNγ)i. Human carotid atherosclerotic plaques contained 2 such macrophage clusters. Bioinformatic LINCS analysis using our RNA-sequencing data identified BI-2536 as a potential compound to decrease the M(IFNγ)i subpopulation. BI-2536 in vitro decreased inflammatory chemokine expression and secretion in M(IFNγ) by shrinking the M(IFNγ)i subpopulation while expanding the M(IFNγ)p subpopulation. BI-2536 in vivo shifted the phenotype of macrophages, modulated inflammation, and decreased atherosclerosis and calcification. CONCLUSIONS: We characterized 2 clusters of macrophages in atherosclerosis and combined our cellular data with a cell-signature drug library to identify a novel compound that targets a subset of macrophages in atherosclerosis. Our approach is a precision medicine strategy to identify new drugs that target atherosclerosis and other inflammatory diseases.

Systems Approach to Discovery of Therapeutic Targets for Vein Graft Disease: PPARα Pivotally Regulates Metabolism, Activation, and Heterogeneity of Macrophages and Lesion Development.

BACKGROUND: Vein graft failure remains a common clinical challenge. We applied a systems approach in mouse experiments to discover therapeutic targets for vein graft failure. METHODS: Global proteomics and high-dimensional clustering on multiple vein graft tissues were used to identify potential pathogenic mechanisms. The PPARs (peroxisome proliferator-activated receptors) pathway served as an example to substantiate our discovery platform. In vivo mouse experiments with macrophage-targeted PPARα small interfering RNA, or the novel, selective activator pemafibrate demonstrate the role of PPARα in the development and inflammation of vein graft lesions. In vitro experiments further included metabolomic profiling, quantitative polymerase chain reaction, flow cytometry, metabolic assays, and single-cell RNA sequencing on primary human and mouse macrophages. RESULTS: We identified changes in the vein graft proteome associated with immune responses, lipid metabolism regulated by the PPARs, fatty acid metabolism, matrix remodeling, and hematopoietic cell mobilization. PPARα agonism by pemafibrate retarded the development and inflammation of vein graft lesions in mice, whereas gene silencing worsened plaque formation. Pemafibrate also suppressed arteriovenous fistula lesion development. Metabolomics/lipidomics, functional metabolic assays, and single-cell analysis of cultured human macrophages revealed that PPARα modulates macrophage glycolysis, citrate metabolism, mitochondrial membrane sphingolipid metabolism, and heterogeneity. CONCLUSIONS: This study explored potential drivers of vein graft inflammation and identified PPARα as a novel potential pharmacological treatment for this unmet medical need.

Evolution of Cooperation in the Presence of Higher-Order Interactions: From Networks to Hypergraphs.

Many real systems are strongly characterized by collective cooperative phenomena whose existence and properties still need a satisfactory explanation. Coherently with their collective nature, they call for new and more accurate descriptions going beyond pairwise models, such as graphs, in which all the interactions are considered as involving only two individuals at a time. Hypergraphs respond to this need, providing a mathematical representation of a system allowing from pairs to larger groups. In this work, through the use of different hypergraphs, we study how group interactions influence the evolution of cooperation in a structured population, by analyzing the evolutionary dynamics of the public goods game. Here we show that, likewise to network reciprocity, group interactions also promote cooperation. More importantly, by means of an invasion analysis in which the conditions for a strategy to survive are studied, we show how, in heterogeneously-structured populations, reciprocity among players is expected to grow with the increasing of the order of the interactions. This is due to the heterogeneity of connections and, particularly, to the presence of individuals standing out as hubs in the population. Our analysis represents a first step towards the study of evolutionary dynamics through higher-order interactions, and gives insights into why cooperation in heterogeneous higher-order structures is enhanced. Lastly, it also gives clues about the co-existence of cooperative and non-cooperative behaviors related to the structural properties of the interaction patterns.

A disease-driver population within interstitial cells of human calcific aortic valves identified via single-cell and proteomic profiling.

Cellular heterogeneity of aortic valves complicates the mechanistic evaluation of the calcification processes in calcific aortic valve disease (CAVD), and animal disease models are lacking. In this study, we identify a disease-driver population (DDP) within valvular interstitial cells (VICs). Through stepwise single-cell analysis, phenotype-guided omic profiling, and network-based analysis, we characterize the DDP fingerprint as CD44highCD29+CD59+CD73+CD45low and discover potential key regulators of human CAVD. These DDP-VICs demonstrate multi-lineage differentiation and osteogenic properties. Temporal proteomic profiling of DDP-VICs identifies potential targets for therapy, including MAOA and CTHRC1. In vitro loss-of-function experiments confirm our targets. Such a stepwise strategy may be advantageous for therapeutic target discovery in other disease contexts.

Effective approach to epidemic containment using link equations in complex networks.

Epidemic containment is a major concern when confronting large-scale infections in complex networks. Many studies have been devoted to analytically understand how to restructure the network to minimize the impact of major outbreaks of infections at large scale. In many cases, the strategies are based on isolating certain nodes, while less attention has been paid to interventions on the links. In epidemic spreading, links inform about the probability of carrying the contagion of the disease from infected to susceptible individuals. Note that these states depend on the full structure of the network, and its determination is not straightforward from the knowledge of nodes' states. Here, we confront this challenge and propose a set of discrete-time governing equations that can be closed and analyzed, assessing the contribution of links to spreading processes in complex networks. Our approach allows a scheme for the containment of epidemics based on deactivating the most important links in transmitting the disease. The model is validated in synthetic and real networks, yielding an accurate determination of epidemic incidence and critical thresholds. Epidemic containment based on link deactivation promises to be an effective tool to maintain functionality of networks while controlling the spread of diseases, such as disease spread through air transportation networks.

Assessing reliable human mobility patterns from higher order memory in mobile communications.

Understanding how people move within a geographical area, e.g. a city, a country or the whole world, is fundamental in several applications, from predicting the spatio-temporal evolution of an epidemic to inferring migration patterns. Mobile phone records provide an excellent proxy of human mobility, showing that movements exhibit a high level of memory. However, the precise role of memory in widely adopted proxies of mobility, as mobile phone records, is unknown. Here we use 560 million call detail records from Senegal to show that standard Markovian approaches, including higher order ones, fail in capturing real mobility patterns and introduce spurious movements never observed in reality. We introduce an adaptive memory-driven approach to overcome such issues. At variance with Markovian models, it is able to realistically model conditional waiting times, i.e. the probability to stay in a specific area depending on individuals' historical movements. Our results demonstrate that in standard mobility models the individuals tend to diffuse faster than observed in reality, whereas the predictions of the adaptive memory approach significantly agree with observations. We show that, as a consequence, the incidence and the geographical spread of a disease could be inadequately estimated when standard approaches are used, with crucial implications on resources deployment and policy-making during an epidemic outbreak.

Strategical incoherence regulates cooperation in social dilemmas on multiplex networks.

Cooperation is a very common, yet not fully-understood phenomenon in natural and human systems. The introduction of a network within the population is known to affect the outcome of cooperative dynamics, allowing for the survival of cooperation in adverse scenarios. Recently, the introduction of multiplex networks has yet again modified the expectations for the outcome of the Prisoner's Dilemma game, compared to the monoplex case. However, much remains unstudied regarding other social dilemmas on multiplex, as well as the unexplored microscopic underpinnings of it. In this paper, we systematically study the evolution of cooperation in all four games in the T-S plane on multiplex. More importantly, we find some remarkable and previously unknown features in the microscopic organization of the strategies, that are responsible for the important differences between cooperative dynamics in monoplex and multiplex. Specifically, we find that in the stationary state, there are individuals that play the same strategy in all layers (coherent), and others that don't (incoherent). This second group of players is responsible for the surprising fact of a non full-cooperation in the Harmony Game on multiplex, never observed before, as well as a higher-than-expected cooperation rates in some regions of the other three social dilemmas.