Immune quorum sensing dictates IFN-I response dynamics in human plasmacytoid dendritic cells.

Type I interferons (IFN-Is) are key in fighting viral infections, but also serve major roles beyond antiviral immunity. Crucial is the tight regulation of IFN-I responses, while excessive levels are harmful to the cells. In essence, immune responses are generated by single cells making their own decisions, which are based on the signals they perceive. Additionally, immune cells must anticipate the future state of their environment, thereby weighing the costs and benefits of each possible outcome, in the presence of other potentially competitive decision makers (i.e., IFN-I producing cells). A rather new cellular communication mechanism called quorum sensing describes the effect of cell density on cellular secretory behaviors, which fits well with matching the right amount of IFN-Is produced to fight an infection. More competitive decision makers must contribute relatively less and vice versa. Intrigued by this concept, we assessed the effects of immune quorum sensing in pDCs, specialized immune cells known for their ability to mass produce IFN-Is. Using conventional microwell assays and droplet-based microfluidics assays, we were able the characterize the effect of quorum sensing in human primary immune cells in vitro. These insights open new avenues to manipulate IFN-I response dynamics in pathological conditions affected by aberrant IFN-I signaling.

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.

Cartilage-derived cells display heterogeneous pericellular matrix synthesis in agarose microgels.

The pericellular matrix (PCM) surrounding chondrocytes is essential for articular cartilage tissue engineering. As the current isolation methods to obtain chondrocytes with their PCM (chondrons) result in a heterogeneous mixture of chondrocytes and chondrons, regenerating the PCM using a tissue engineering approach could prove beneficial. In this study, we aimed to discern the behavior of articular chondrocytes (ACs) in regenerating the PCM in such an approach and whether this would also be true for articular cartilage-derived progenitor cells (ACPCs), as an alternative cell source. Bovine ACs and ACPCs were encapsulated in agarose microgels using droplet-based microfluidics. ACs were stimulated with TGF-ß1 and dexamethasone and ACPCs were sequentially stimulated with BMP-9 followed by TGF-ß1 and dexamethasone. After 0, 3, 5, and 10 days of culture, PCM components, type-VI collagen and perlecan, and ECM component, type-II collagen, were assessed using flow cytometry and fluorescence microscopy. Both ACs and ACPCs synthesized the PCM before the ECM. It was seen for the first time that synthesis of type-VI collagen always preceded perlecan. While the PCM synthesized by ACs resembled native chondrons after only 5 days of culture, ACPCs often made less well-structured PCMs. Both cell types showed variations between individual cells and donors. On one hand, this was more prominent in ACPCs, but also a subset of ACPCs showed superior PCM and ECM regeneration, suggesting that isolating these cells may potentially improve cartilage repair strategies.

Tuning Structural Organization via Molecular Design and Hierarchical Assembly to Develop Supramolecular Thermoresponsive Hydrogels.

The cellular microenvironment is composed of a dynamic hierarchical fibrillar architecture providing a variety of physical and bioactive signals to the surrounding cells. This dynamicity, although common in biology, is a challenge to control in synthetic matrices. Here, responsive synthetic supramolecular monomers were designed that are able to assemble into hierarchical fibrous structures, combining supramolecular fiber formation via hydrogen bonding interactions, with a temperature-responsive hydrophobic collapse, resulting in cross-linking and hydrogel formation. Therefore, amphiphilic molecules were synthesized, composed of a hydrogen bonding ureido-pyrimidinone (UPy) unit, a hydrophobic alkyl spacer, and a hydrophilic oligo(ethylene glycol) tail. The temperature responsive behavior was introduced by functionalizing these supramolecular amphiphiles with a relatively short poly(N-isopropylacrylamide) (PNIPAM) chain (M n ∼ 2.5 or 5.5 kg/mol). To precisely control the assembly of these monomers, the length of the alkyl spacer between the UPy moiety and PNIPAM was varied in length. A robust sol-gel transition, with the dodecyl UPy-PNIPAM molecule, was obtained, with a network elasticity enhancing over 2000 times upon heating above room temperature. The UPy-PNIPAM compounds with shorter alkyl spacers were already hydrogels at room temperature. The sol-gel transition of the dodecyl UPy-PNIPAM hydrogelator could be tuned by the incorporation of different UPy-functionalized monomers. Furthermore, we demonstrated the suitability of this system for microfluidic cell encapsulation through a convenient temperature sol-gel transition. Our results indicate that this novel thermoresponsive supramolecular system offers a modular platform to study and guide single-cell behavior.

Single-cell analysis reveals TLR-induced macrophage heterogeneity and quorum sensing dictate population wide anti-inflammatory feedback in response to LPS.

The role of macrophages in controlling tissue inflammation is indispensable to ensure a context-appropriate response to pathogens whilst preventing excessive tissue damage. Their initial response is largely characterized by high production of tumor necrosis factor alpha (TNFα) which primes and attracts other immune cells, thereafter, followed by production of interleukin 10 (IL-10) which inhibits cell activation and steers towards resolving of inflammation. This delicate balance is understood at a population level but how it is initiated at a single-cell level remains elusive. Here, we utilize our previously developed droplet approach to probe single-cell macrophage activation in response to toll-like receptor 4 (TLR4) stimulation, and how single-cell heterogeneity and cellular communication affect macrophage-mediated inflammatory homeostasis. We show that only a fraction of macrophages can produce IL-10 in addition to TNFα upon LPS-induced activation, and that these cells are not phenotypically different from IL-10 non-producers nor exhibit a distinct transcriptional pathway. Finally, we demonstrate that the dynamics of TNFα and IL-10 are heavily controlled by macrophage density as evidenced by 3D hydrogel cultures suggesting a potential role for quorum sensing. These exploratory results emphasize the relevance of understanding the complex communication between macrophages and other immune cells and how these amount to population-wide responses.

A Microfluidic Approach for Probing Heterogeneity in Cytotoxic T-Cells by Cell Pairing in Hydrogel Droplets.

Cytotoxic T-cells (CTLs) exhibit strong effector functions to leverage antigen-specific anti-tumoral and anti-viral immunity. When naïve CTLs are activated by antigen-presenting cells (APCs) they display various levels of functional heterogeneity. To investigate this, we developed a single-cell droplet microfluidics platform that allows for deciphering single CTL activation profiles by multi-parameter analysis. We identified and correlated functional heterogeneity based on secretion profiles of IFNγ, TNFα, IL-2, and CD69 and CD25 surface marker expression levels. Furthermore, we strengthened our approach by incorporating low-melting agarose to encapsulate pairs of single CTLs and artificial APCs in hydrogel droplets, thereby preserving spatial information over cell pairs. This approach provides a robust tool for high-throughput and single-cell analysis of CTLs compatible with flow cytometry for subsequent analysis and sorting. The ability to score CTL quality, combined with various potential downstream analyses, could pave the way for the selection of potent CTLs for cell-based therapeutic strategies.