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Cardiac adaptation to hypoxic injury is regulated by dynamic interactions between cardiomyocytes and macrophages, yet the impacts of immune phenotypes on cardiac structure and contractility remain poorly understood. To address this, we developed the immuno-heart on a chip, a novel in vitro platform to investigate cardiomyocyte–macrophage interactions under normoxic and hypoxic conditions. By integrating neonatal rat ventricular myocytes (NRVMs) and bone marrow-derived macrophages—polarized to pro-inflammatory (M1) or pro-healing (M2/M2*) phenotypes—we elucidated the dual protective and detrimental roles macrophages play in modulating cardiomyocyte cytoskeletal architecture and contractility. Pro-inflammatory stimulation reduced cardiomyocyte structural metrics (z-line length, fraction, and integrity) in normoxic co-cultures. Under hypoxia, M1-stimulated NRVM monocultures exhibited declines in cytoskeletal organization—quantified by actin and z-line orientational order parameters. Relative to monocultures, M1-stimulated co-cultures attenuated hypoxia-induced active stress declines but produced weaker normoxic stresses. In contrast, pro-healing stimulation improved normoxic z-line metrics and preserved post-hypoxia cytoskeletal organization but reduced normoxic contractility. Notably, M2-stimulated macrophages restored normoxic contractility and preserved post-hypoxia systolic stress, albeit with increased diastolic stress. RNAseq analysis of M2-stimulated co-cultures identified upregulated structural and immune pathways driving these hypoxia-induced changes. Cytokine profiles revealed stimulation-specific and density-dependent tumor necrosis factor-alpha and interleukin-10 secretion patterns. Together, these findings quantitatively link clinically relevant macrophage phenotypes and cytokines to distinct changes in cardiac structure and contractility, offering mechanistic insights into immune modulation of hypoxia-induced dysfunction. Moreover, the immuno-heart on a chip represents an innovative framework to guide the development of future therapies that integrate immune and cardiac targets to enhance patient outcomes. 

Abstract Macrophages hold vital roles in immune defense, wound healing, and tissue homeostasis, and have the exquisite ability to sense and respond to dynamically changing cues in their microenvironment. Much of our understanding of their behavior has been derived from studies performed using in vitro culture systems, in which the cell environment can be precisely controlled. Recent advances in miniaturized culture platforms also offer the ability to recapitulate some features of the in vivo environment and analyze cellular responses at the single‐cell level. Since macrophages are sensitive to their surrounding environments, the specific conditions in both macro‐ and micro‐scale cultures likely contribute to observed responses. In this study, we investigate how the presence of neighboring cells influence macrophage activation following proinflammatory stimulation in both bulk and micro‐scale culture. We found that in bulk cultures, higher seeding density negatively regulated the average TNF‐α secretion from individual macrophages in response to inflammatory agonists, and this effect was partially caused by the reduced cell‐to‐media volume ratio. In contrast, studies conducted using microwells to isolate single cells and groups of cells revealed that increasing numbers of cells positively influences their inflammatory activation, suggesting that the absolute cell numbers in the system may be important. In addition, a single inflammatory cell enhanced the inflammatory state of a small group of cells. Overall, this work helps to better understand how variations of macroscopic and microscopic culture environments influence studies in macrophage biology and provides insight into how the presence of neighboring cells and the soluble environment influences macrophage activation. 

Abstract Biomaterial wound dressings, such as hydrogels, interact with host cells to regulate tissue repair. This study investigates how crosslinking of gelatin-based hydrogels influences immune and stromal cell behavior and wound healing in female mice. We observe that softer, lightly crosslinked hydrogels promote greater cellular infiltration and result in smaller scars compared to stiffer, heavily crosslinked hydrogels. Using single-cell RNA sequencing, we further show that heavily crosslinked hydrogels increase inflammation and lead to the formation of a distinct macrophage subpopulation exhibiting signs of oxidative activity and cell fusion. Conversely, lightly crosslinked hydrogels are more readily taken up by macrophages and integrated within the tissue. The physical properties differentially affect macrophage and fibroblast interactions, with heavily crosslinked hydrogels promoting pro-fibrotic fibroblast activity that drives macrophage fusion through RANKL signaling. These findings suggest that tuning the physical properties of hydrogels can guide cellular responses and improve healing, offering insights for designing better biomaterials for wound treatment. 

Innate immune cells are responsible for eliminating foreign infectious agents and cellular debris, and their ability to perceive, respond to, and integrate biochemical and mechanical cues from their microenvironment eventually determines their behavior. In response to tissue injury, pathogen invasion, or a biomaterial implant, immune cells activate many pathways to initiate inflammation in the tissue. In addition to common inflammatory pathways, studies have demonstrated the role of the mechanosensitive proteins and transcriptional coactivators YAP and TAZ (YAP/TAZ) in inflammation and immunity. We review our knowledge of YAP/TAZ in controlling inflammation and immunity in innate immune cells. Furthermore, we discuss the roles of YAP/TAZ in inflammatory diseases, wound healing, and tissue regeneration and how they integrate mechanical cues with biochemical signaling during disease progression. Last, we comment on possible approaches that can be exploited to harness the therapeutic potential of YAP/TAZ in inflammatory diseases. 

Macrophages are innate immune cells that help wounds heal. Here, we study the potential immunomodulatory effects of negative-pressure wound therapy (NPWT) materials on the macrophage inflammatory response. We compared the effects of two materials, Granufoam™ (GF) and Veraflo Cleanse™ (VC), on macrophage function in vitro. We find that both materials cause reduced expression of inflammatory genes, such as TNF and IL1B, in human macrophages stimulated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFNγ). Relative to adherent glass control surfaces, VC discourages macrophage adhesion and spreading, and may potentially sequester LPS/IFNγ and cytokines that the cells produce. GF, on the other hand, was less suppressive of inflammation, supported macrophage adhesion and spreading better than VC, and sequestered lesser quantities of LPS/IFNγ in comparison to VC. The control dressing material cotton gauze (CT) was also immunosuppressive, capable of TNF-α retention and LPS/IFNγ sequestration. Our findings suggest that NPWT material interactions with cells, as well as soluble factors including cytokines and LPS, can modulate the immune response, independent of vacuum application. We have also established methodological strategies for studying NPWT materials and reveal the potential utility of cell-based in vitro studies for elucidating biological effects of NPWT materials. 

Macrophages are innate immune cells that adhere to the extracellular matrix within tissues. However, how matrix properties regulate their function remains poorly understood. Here, we report that the adhesive microenvironment tunes the macrophage inflammatory response through the transcriptional coactivator YAP. We find that adhesion to soft hydrogels reduces inflammation when compared to adhesion on stiff materials and is associated with reduced YAP expression and nuclear localization. Substrate stiffness and cytoskeletal polymerization, but not adhesive confinement nor contractility, regulate YAP localization. Furthermore, depletion of YAP inhibits macrophage inflammation, whereas overexpression of active YAP increases inflammation. Last, we show in vivo that soft materials reduce expression of inflammatory markers and YAP in surrounding macrophages when compared to stiff materials. Together, our studies identify YAP as a key molecule for controlling inflammation and sensing stiffness in macrophages and may have broad implications in the regulation of macrophages in health and disease.