Macro- and micro-scale culture environment differentially regulate the effects of crowding on macrophage function.

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.

Harnessing macrophage plasticity for tissue regeneration.

Macrophages are versatile and plastic effector cells of the immune system, and contribute to diverse immune functions including pathogen or apoptotic cell removal, inflammatory activation and resolution, and tissue healing. Macrophages function as signaling regulators and amplifiers, and influencing their activity is a powerful approach for controlling inflammation or inducing a wound-healing response in regenerative medicine. This review discusses biomaterials-based approaches for altering macrophage activity, approaches for targeting drugs to macrophages, and approaches for delivering macrophages themselves as a therapeutic intervention.

Differential regulation of macrophage inflammatory activation by fibrin and fibrinogen.

Fibrin is a major component of the provisional extracellular matrix formed during tissue repair following injury, and enables cell infiltration and anchoring at the wound site. Macrophages are dynamic regulators of this process, advancing and resolving inflammation in response to cues in their microenvironment. Although much is known about how soluble factors such as cytokines and chemokines regulate macrophage polarization, less is understood about how insoluble and adhesive cues, specifically the blood coagulation matrix fibrin, influence macrophage behavior. In this study, we observed that fibrin and its precursor fibrinogen elicit distinct macrophage functions. Culturing macrophages on fibrin gels fabricated by combining fibrinogen with thrombin stimulated secretion of the anti-inflammatory cytokine, interleukin-10 (IL-10). In contrast, exposure of macrophages to soluble fibrinogen stimulated high levels of inflammatory cytokine tumor necrosis factor alpha (TNF-α). Macrophages maintained their anti-inflammatory behavior when cultured on fibrin gels in the presence of soluble fibrinogen. In addition, adhesion to fibrin matrices inhibited TNF-α production in response to stimulation with LPS and IFN-γ, cytokines known to promote inflammatory macrophage polarization. Our data demonstrate that fibrin exerts a protective effect on macrophages, preventing inflammatory activation by stimuli including fibrinogen, LPS, and IFN-γ. Together, our study suggests that the presentation of fibrin(ogen) may be a key switch in regulating macrophage phenotype behavior, and this feature may provide a valuable immunomodulatory strategy for tissue healing and regeneration. STATEMENT OF SIGNIFICANCE: Fibrin is a fibrous protein resulting from blood clotting and provides a provisional matrix into which cells migrate and to which they adhere during wound healing. Macrophages play an important role in this process, and are needed for both advancing and resolving inflammation. We demonstrate that culture of macrophages on fibrin matrices exerts an anti-inflammatory effect, whereas the soluble precursor fibrinogen stimulates inflammatory activation. Moreover, culture on fibrin completely abrogates inflammatory signaling caused by fibrinogen or known inflammatory stimuli including LPS and IFN-γ. Together, these studies show that the presentation of fibrin(ogen) is important for regulating a switch between macrophage pro- and anti-inflammatory behavior.

Regulation of macrophage polarization and plasticity by complex activation signals.

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.

Macrophage secretion heterogeneity in engineered microenvironments revealed using a microwell platform.

Secreted proteins play a major role in orchestrating the response of cell populations. However, a quantitative understanding of the dynamic changes in protein secretion in response to microenvironmental cues at the single cell level remains elusive. Measurements taken using traditional molecular techniques typically require bulk cultures, and therefore cannot capture the diversity within cell populations. Recent advances in chip-based technologies have shown that single cell measurements can provide important insights into the temporal dynamics of cellular activation and function, but these tools have had limited control of the adhesive cellular microenvironment. Here, we created a single cell cytokine detection platform that allows for controlled physical and adhesive microenvironment. We validated the platform by examining cytokine secretion of macrophages exposed to varying dosages of soluble stimulation and on different adhesive substrates. We also used the platform to demonstrate that cell shape affects single macrophage cytokine secretion. Together, these results show the ability of the microwell system to detect secreted cytokines from individual macrophages in controlled adhesive environments. This technique may be broadly applied to detect secreted products from any adherent cell type.

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy.

Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

Registered report: Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replicating selected results from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012 were selected on the basis of citations and Altimetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from 'Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis' by Goetz and colleagues, published in Cell in 2011 (Goetz et al., 2011). The key experiments being replicated are those reported in Figures 7C (a-d), Supplemental Figure S2A, and Supplemental Figure S7C (a-c) (Goetz et al., 2011). In these experiments, which are a subset of all the experiments reported in the original publication, Goetz and colleagues show in a subcutaneous xenograft model that stromal caveolin-1 remodels the intratumoral microenvironment, which is correlated with increased metastasis formation. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.