Macrophages are versatile cells of the innate immune system that perform diverse functions by responding to dynamic changes in their microenvironment. While the effects of soluble cues, including ...cytokines and chemokines, have been widely studied, the effects of physical cues, including mechanical stimuli, in regulating macrophage form and function are less well understood. In this study, we examined the effects of static and cyclic uniaxial stretch on macrophage inflammatory and healing activation. We found that cyclic stretch altered macrophage morphology and responses to IFNγ/LPS and IL4/IL13. Interestingly, we found that both static and cyclic stretch suppressed IFNγ/LPS induced inflammation. In contrast, IL4/IL13 mediated healing responses were suppressed with cyclic but enhanced with static stretch conditions. Mechanistically, both static and cyclic stretch increased expression of the integrin CD11b (α
integrin), decreased expression of the mechanosensitive ion channel Piezo1, and knock down of either CD11b or Piezo1 through siRNA abrogated stretch-mediated changes in inflammatory responses. Moreover, we found that knock down of CD11b enhanced the expression of Piezo1, and conversely knock down of Piezo1 enhanced CD11b expression, suggesting the potential for crosstalk between integrins and ion channels. Finally, stretch-mediated differences in macrophage activation were also dependent on actin, since pharmacological inhibition of actin polymerization abrogated the changes in activation with stretch. Together, this study demonstrates that the physical environment synergizes with biochemical cues to regulate macrophage morphology and function, and suggests a role for CD11b and Piezo1 crosstalk in mechanotransduction in macrophages.
Reducing the foreign body response (FBR) to implanted biomaterials will enhance their performance in tissue engineering. Poly(ethylene glycol) (PEG) hydrogels are increasingly popular for this ...application due to their low cost, ease of use, and the ability to tune their compliance via molecular weight and cross-linking densities. PEG hydrogels can elicit chronic inflammation in vivo, but recent evidence has suggested that extremely hydrophilic, zwitterionic materials and particles can evade the immune system. To combine the advantages of PEG-based hydrogels with the hydrophilicity of zwitterions, we synthesized hydrogels with comonomers PEG and the zwitterion phosphorylcholine (PC). Recent evidence suggests that stiff hydrogels elicit increased immune cell adhesion to hydrogels, which we attempted to reduce by increasing hydrogel hydrophilicity. Surprisingly, hydrogels with the highest amount of zwitterionic comonomer elicited the highest FBR. Lowering the hydrogel modulus (165 to 3 kPa), or PC content (20 to 0 wt %), mitigated this effect. A high density of macrophages was found at the surface of implants associated with a high FBR, and mass spectrometry analysis of the proteins adsorbed to these gels implicated extracellular matrix, immune response, and cell adhesion protein categories as drivers of macrophage recruitment. Overall, we show that modulus regulates macrophage adhesion to zwitterionic-PEG hydrogels, and demonstrate that chemical modifications to hydrogels should be studied in parallel with their physical properties to optimize implant design.
Cell polarity is orchestrated by numerous extracellular cues, and guides events such as chemotaxis, mitosis and wound healing. In scrape-wound assays of cell monolayers, wound-edge cells orient their ...centrosomes towards the wound, a process that appears to depend on the formation of new cell-extracellular-matrix adhesions as cells spread into the wound. In direct contrast to scrape-wounded cells, isolated cells without cell-cell contacts failed to polarize, suggesting that asymmetry of cell-cell adhesions resulting from monolayer disruption might contribute to polarization. By using micropatterned substrates to engineer such asymmetries in kidney epithelial cells, we found that cell-cell contact induced displacement of the nucleus towards the contact, and also caused centrosomal reorientation and lamellipodial ruffling to the distal side of the nucleus. Upon release from micropatterned constraints, cells exhibited directed migration away from the cell-cell contact. Disrupting E-cadherin engagement randomized nuclear position and lamellipodial ruffling in patterned cultures, and abrogated scrape-wound-induced cell reorientation, but not migration rate. Polarity that was induced by cell-cell contact required an intact actin cytoskeleton and Cdc42 activity, but not RhoA or Rac signaling. Together, these findings demonstrate a novel role for cell-cell adhesion in polarization, and have implications for wound healing and developmental patterning.
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.
The extracellular matrix (ECM) is a complex and dynamic structural scaffold for cells within tissues and plays an important role in regulating cell function. Recently it has become appreciated that ...the ECM contains bioactive motifs that can directly modulate immune responses. This review describes strategies for engineering immunomodulatory biomaterials that utilize natural ECM‐derived molecules and have the potential to harness the immune system for applications ranging from tissue regeneration to drug delivery. A top‐down approach utilizes full‐length ECM proteins, including collagen, fibrin, or hyaluronic acid‐based materials, as well as matrices derived from decellularized tissue. These materials have the benefit of maintaining natural conformation and structure but are often heterogeneous and encumber precise control. By contrast, a bottom‐up approach leverages immunomodulatory domains, such as Arg–Gly–Asp (RGD), matrix metalloproteinase (MMP)‐sensitive peptides, or leukocyte‐associated immunoglobulin‐like receptor‐1(LAIR‐1) ligands, by incorporating them into synthetic materials. These materials have tunable control over immune cell functions and allow for combinatorial approaches. However, the synthetic approach lacks the full natural context of the original ECM protein. These two approaches provide a broad range of engineering techniques for immunomodulation through material interactions and hold the potential for the development of future therapeutic applications.
This review describes strategies for engineering immunomodulatory biomaterials that utilize natural extracellular matrix (ECM) derived molecules and have the potential to harness the immune system for applications ranging from tissue regeneration to drug delivery. The incorporation of full‐length ECM proteins, ECM‐derived peptides, or a mixture of both, into biomaterials provides a broad range of engineering techniques for immunomodulation through material interactions.
The immune system performs critical functions to defend against invading pathogens and maintain tissue homeostasis. Immune cells reside within or are recruited to a host of mechanically active ...tissues throughout the body and, as a result, are exposed to varying types and degrees of mechanical stimuli. Despite their abundance in such tissues, the role of mechanical stimuli in influencing immune cell function and the molecular mechanisms responsible for mechanics-mediated changes are still poorly understood. The recent emergence of mechanically-gated ion channels, particularly Piezo1, has provided an exciting avenue of research within the fields of mechanobiology and immunology. Numerous studies have identified roles for mechanically-gated ion channels in mechanotransduction within various different cell types, with a few recent studies in immune cells. These initial studies provide strong evidence that mechanically-gated ion channels play pivotal roles in regulating the immune system. In this review, we discuss characteristics of ion channel mediated mechanotransduction, review the current techniques used to quantify and visualize ion channel activity in response to mechanical stimuli, and finally we provide an overview of recent studies examining the role of mechanically-gated ion channels in modulating immune cell function.
Abstract The non-specific host response to implanted biomaterials is often a key challenge of medical device design. To evaluate biocompatibility, measuring the release of reactive oxygen species ...(ROS) produced by inflammatory cells in response to biomaterial surfaces is a well-established method. However, the detection of ROS in response to materials implanted in vivo has not yet been demonstrated. Here, we develop a bioluminescence whole animal imaging approach to observe ROS released in response to subcutaneously-implanted materials in live animals. We compared the real-time generation of ROS in response to two representative materials, polystyrene and alginate, over the course of 28 days. High levels of ROS were observed near polystyrene, but not alginate implants, and persisted throughout the course of 28 days. Histological analysis revealed that high levels of ROS correlated not only with the presence of phagocytic cells at early timepoints, but also fibrosis at later timepoints, suggesting that ROS may be involved in both the acute and chronic phase of the foreign body response. These data are the first in vivo demonstration of ROS generation in response to implanted materials, and describe a novel technique to evaluate the host response.
Macrophages are versatile cells of the immune system that play an important role in both advancing and resolving inflammation. Macrophage activation has been described as a continuum, and different ...stimuli lead to M1, M2, or mixed phenotypes. In addition, macrophages expressing markers associated with both M1 and M2 function are observed in vivo. Using flow cytometry, we examine how macrophage populations respond to combined M1 and M2 activation signals, presented either simultaneously or sequentially. We demonstrate that macrophages exposed to a combination of LPS, IFN-γ, IL-4, and IL-13 acquire a mixed activation state, with individual cells expressing both M1 marker CD86 and M2 marker CD206 instead of polarizing to discrete phenotypes. Over time, co-stimulated macrophages lose expression of CD86 and display increased expression of CD206. In addition, we find that exposure to LPS/IFN-γ potentiates the subsequent response to IL-4/IL-13, whereas pre-polarization with IL-4/IL-13 inhibits the response to LPS/IFN-γ. Mathematical modeling of candidate regulatory networks indicates that a complex inter-dependence of M1- and M2-associated pathways underlies macrophage activation. Specifically, a mutual inhibition motif was not by itself sufficient to reproduce the temporal marker expression data; incoherent feed-forward of M1 activation as well as both inhibition and activation of M2 by M1 were required. Together these results corroborate a continuum model of macrophage activation and demonstrate that phenotypic markers evolve with time and with exposure to complex signals.
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.
Spatial patterns of cellular growth generate mechanical stresses that help to push, fold, expand, and deform tissues into their specific forms. Genetic factors are thought to specify patterns of ...growth and other behaviors to drive morphogenesis. Here, we show that tissue form itself can feed back to regulate patterns of proliferation. Using microfabrication to control the organization of sheets of cells, we demonstrated the emergence of stable patterns of proliferative foci. Regions of concentrated growth corresponded to regions of high tractional stress generated within the sheet, as predicted by a finite-element model of multicellular mechanics and measured directly by using a micromechanical force sensor array. Inhibiting actomyosin-based tension or cadherin-mediated connections between cells disrupted the spatial pattern of proliferation. These findings demonstrate the existence of patterns of mechanical forces that originate from the contraction of cells, emerge from their multicellular organization, and result in patterns of growth. Thus, tissue form is not only a consequence but also an active regulator of tissue growth.
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