Modulation of Stiffness-Dependent Macrophage Inflammatory Responses by Collagen Deposition.

Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such interactions can lead to the modulation of cellular activity. Collagen is often used in the culture of immune cells, but the effects of substrate functionalization conditions are not typically considered. Here, we show that the solvent system used to attach collagen onto a hydrogel surface affects its surface distribution and organization, and this can modulate the responses of macrophages subsequently cultured on these surfaces in terms of their inflammatory activation and expression of adhesion and mechanosensitive molecules. Collagen was solubilized in either acetic acid (Col-AA) or N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) (Col-HEP) solutions and conjugated onto soft and stiff polyacrylamide (PA) hydrogel surfaces. Bone marrow-derived macrophages cultured under standard conditions (pH 7.4) on the Col-HEP-derived surfaces exhibited stiffness-dependent inflammatory activation; in contrast, the macrophages cultured on Col-AA-derived surfaces expressed high levels of inflammatory cytokines and genes, irrespective of the hydrogel stiffness. Among the collagen receptors that were examined, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) was the most highly expressed, and knockdown of the Lair-1 gene enhanced the secretion of inflammatory cytokines. We found that the collagen distribution was more homogeneous on Col-AA surfaces but formed aggregates on Col-HEP surfaces. The macrophages cultured on Col-AA PA hydrogels were more evenly spread, expressed higher levels of vinculin, and exerted higher traction forces compared to those of cells on Col-HEP. These macrophages on Col-AA also had higher nuclear-to-cytoplasmic ratios of yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), key molecules that control inflammation and sense substrate stiffness. Our results highlight that seemingly slight variations in substrate deposition for immunobiology studies can alter critical immune responses, and this is important to elucidate in the broader context of immunomodulatory biomaterial design.

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.

Magnetic resonance imaging of tumor-associated-macrophages (TAMs) with a nanoparticle contrast agent.

In the tumor micro-environment, tumor associated macrophages (TAMs) represent a predominant component of the total tumor mass, and TAMs play a complex and diverse role in cancer pathogenesis with potential for either tumor suppressive, or tumor promoting biology. Thus, understanding macrophage localization and function are essential for cancer diagnosis and treatment. Typically, tissue biopsy is used to evaluate the density and polarization of TAMs, but provides a limited "snapshot" in time of a dynamic and potentially heterogeneous tumor immune microenvironment. Imaging has the potential for three-dimensional mapping; however, there is a paucity of macrophage-targeted contrast agents to specifically detect TAM subtypes. We have previously found that sulfated-dextran coated iron oxide nanoparticles (SDIO) can target macrophage scavenger receptor A (SR-A, also known as CD204). Since CD204 (SR-A) is considered a biomarker for the M2 macrophage polarization, these SDIO might provide M2-specific imaging probes for MRI. In this work, we investigate whether SDIO can label M2-polarized cells in vitro. We evaluate the effect of degree of sulfation on uptake by primary cultured bone marrow derived macrophages (BMDM) and found that a higher degree of sulfation led to higher uptake, but there were no differences across the subtypes. Further analysis of the BMDM showed similar SR-A expression across stimulation conditions, suggesting that this classic model for macrophage subtypes may not be ideal for definitive M2 subtype marker expression, especially SR-A. We further examine the localization of SDIO in TAMs in vivo, in the mammary fat pad mouse model of breast cancer. We demonstrate that uptake by TAMs expressing SR-A scales with degree of sulfation, consistent with the in vitro studies. The TAMs demonstrate M2-like function and secrete Arg-1 but not iNOS. Uptake by these M2-like TAMs is validated by immunohistochemistry. SDIO show promise as a valuable addition to the toolkit of imaging probes targeted to different biomarkers for TAMs.

Stiffness- and Bioactive Factor-Mediated Protection of Self-Assembled Cartilage against Macrophage Challenge in a Novel Co-Culture System.

OBJECTIVE: Tissue-engineered cartilage implants must withstand the potential inflammatory and joint loading environment for successful long-term repair of defects. The work's objectives were to develop a novel, direct cartilage-macrophage co-culture system and to characterize interactions between self-assembled neocartilage and differentially stimulated macrophages. DESIGN: In study 1, it was hypothesized that the proinflammatory response of macrophages would intensify with increasing construct stiffness; it was expected that the neocartilage would display a decrease in mechanical properties after co-culture. In study 2, it was hypothesized that bioactive factors would protect neocartilage properties during macrophage co-culture. Also, it was hypothesized that interleukin 10 (IL-10)-stimulated macrophages would improve neocartilage mechanical properties compared to lipopolysaccharide (LPS)-stimulated macrophages. RESULTS: As hypothesized, stiffer neocartilage elicited a heightened proinflammatory macrophage response, increasing tumor necrosis factor alpha (TNF-α) secretion by 5.47 times when LPS-stimulated compared to construct-only controls. Interestingly, this response did not adversely affect construct properties for the stiffest neocartilage but did correspond to a significant decrease in aggregate modulus for soft and medium stiffness constructs. In addition, bioactive factor-treated constructs were protected from macrophage challenge compared to chondrogenic medium-treated constructs, but IL-10 did not improve neocartilage properties, although stiff constructs appeared to bolster the anti-inflammatory nature of IL-10-stimulated macrophages. However, co-culture of bioactive factor-treated constructs with LPS-treated macrophages reduced TNF-α secretion by over 4 times compared to macrophage-only controls. CONCLUSIONS: In conclusion, neocartilage stiffness can mediate macrophage behavior, but stiffness and bioactive factors prevent macrophage-induced degradation. Ultimately, this co-culture system could be utilized for additional studies to develop the burgeoning field of cartilage mechano-immunology.

Multiplexed Detection of Secreted Cytokines at near-Molecular Resolution Elucidates Macrophage Polarization Heterogeneity.

Monitoring the secretion of proteins from single cells can provide important insights into how cells respond to their microenvironment. This is particularly true for immune cells, which can exhibit a large degree of response heterogeneity. Microfabricated well arrays provide a powerful and versatile method to assess the secretion of cytokines, chemokines, and growth factors from single cells, but detection sensitivity has been limited to high levels on the order of 10,000 per cell. Recently, we reported a quantum dot-based immunoassay that lowered the detection limit for the cytokine TNF-α to concentrations to nearly the single-cell level. Here, we adapted this detection method to three additional targets while maintaining high detection sensitivity. Specifically, we detected MCP-1, TGF-ß, IL-10, and TNF-α using quantum dots with different emission spectra, each of which displayed a detection threshold in the range of 1-10 fM or ∼1-2 molecules per well. We then quantified secretion of all four proteins from single macrophage cells that were stimulated toward a pro-inflammatory state with lipopolysaccharide (LPS) or toward a pro-healing state with both LPS and interleukin 4 (IL-4). We found that MCP-1 and TGF-ß were predominantly secreted at high levels only (>10,000 molecules/cell), while a substantial number of cells secreted IL-10 and TNF-α at lower levels that could only be detected using our method. Subsequent principal component and cluster analysis revealed that secretion profiles could be classified as either exclusively pro-inflammatory, including MCP-1 and/or TNF-α, or more subtle responses displaying both pro-healing and pro-inflammatory characters. Our results highlight the heterogeneous and nondiscrete nature of macrophage phenotypes following in vitro stimulation of a cell line. Future work will focus on expanding the multiplexing capacity by extending emission spectra bandwidth and/or spatially barcoding capture antibodies, as well as evaluating the enhanced detection sensitivity capabilities with normal and diseased immune cell populations in vitro and in vivo.

A Src-H3 acetylation signaling axis integrates macrophage mechanosensation with inflammatory response.

Macrophages are mechanosensitive cells that can exquisitely fine-tune their function in response to their microenvironment. While macrophage polarization results in concomitant changes in cell morphology and epigenetic reprogramming, how biophysically-induced signaling cascades contribute to gene regulatory programs that drive polarization remains unknown. We reveal a cytoskeleton-dependent Src-H3 acetylation (H3Ac) axis responsible for inflammation-associated histone hyperacetylation. Inflammatory stimuli caused increases in traction forces, Src activity and H3Ac marks in macrophages, accompanied by reduced cell elongation and motility. These effects were curtailed following disruption of H3Ac-signaling through either micropattern-induced cell elongation or inhibition of H3Ac readers (BRD proteins) directly. Src activation relieves the suppression of p300 histone acetyltransferase (HAT) activity by PKCδ. Furthermore, while inhibition of Src reduced p300 HAT activity and H3Ac marks globally, local H3Ac levels within the Src promoter were increased, suggesting H3Ac regulates Src levels through feedback. Together, our study reveals an adhesome-to-epigenome regulatory nexus underlying macrophage mechanosensation, where Src modulates H3Ac-associated epigenetic signaling as a means of tuning inflammatory gene activity and macrophage fate decisions in response to microenvironmental cues.

Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues.

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 (αM 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.

Isolation and characterization of porcine macrophages and their inflammatory and fusion responses in different stiffness environments.

Evaluating the host immune response to biomaterials is an essential step in the development of medical devices and tissue engineering strategies. To aid in this process, in vitro studies, whereby immune cells such as macrophages are cultured on biomaterials, can often expedite high throughput testing of many materials prior to implantation. While most studies to date utilize murine or human cells, the use of porcine macrophages has been less well described, despite the prevalent use of porcine models in medical device and tissue engineering development. In this study, we describe the isolation and characterization of porcine bone marrow- and peripheral blood-derived macrophages, and their interactions with biomaterials. We confirmed the expression of the macrophage surface markers CD68 and F4/80 and characterized the porcine macrophage response to the inflammatory stimulus, bacterial lipopolysaccharide. Finally, we investigated the inflammatory and fusion response of porcine macrophages cultured on different stiffness hydrogels, and we found that stiffer hydrogels enhanced inflammatory activation by more than two-fold and promoted fusion to form foreign body giant cells. Together, this study establishes the use of porcine macrophages in biomaterial testing and reveals a stiffness-dependent effect on biomaterial-induced giant cell formation.

Mechanically activated ion channel Piezo1 modulates macrophage polarization and stiffness sensing.

Macrophages perform diverse functions within tissues during immune responses to pathogens and injury, but molecular mechanisms by which physical properties of the tissue regulate macrophage behavior are less well understood. Here, we examine the role of the mechanically activated cation channel Piezo1 in macrophage polarization and sensing of microenvironmental stiffness. We show that macrophages lacking Piezo1 exhibit reduced inflammation and enhanced wound healing responses. Additionally, macrophages expressing the transgenic Ca2+ reporter, Salsa6f, reveal that Ca2+ influx is dependent on Piezo1, modulated by soluble signals, and enhanced on stiff substrates. Furthermore, stiffness-dependent changes in macrophage function, both in vitro and in response to subcutaneous implantation of biomaterials in vivo, require Piezo1. Finally, we show that positive feedback between Piezo1 and actin drives macrophage activation. Together, our studies reveal that Piezo1 is a mechanosensor of stiffness in macrophages, and that its activity modulates polarization responses.

YAP-mediated mechanotransduction tunes the macrophage inflammatory response.

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.

Biophysical and epigenetic regulation of cancer stemness, invasiveness and immune action.

PURPOSE OF REVIEW: The tumor microenvironment (TME) is an amalgam of multiple dysregulated biophysical cues that can alter cellular behavior through mechanotransductive signaling and epigenetic modifications. Through this review, we seek to characterize the extent of biophysical and epigenetic regulation of cancer stemness and tumor-associated immune cells in order to identify ideal targets for cancer therapy. RECENT FINDINGS: Recent studies have identified cancer stemness and immune action as significant contributors to neoplastic disease, due to their susceptibility to microenvironmental influences. Matrix stiffening, altered vasculature, and resultant hypoxia within the TME can influence cancer stem cell (CSC) and immune cell behavior, as well as alter the epigenetic landscapes involved in cancer development. SUMMARY: This review highlights the importance of aberrant biophysical cues in driving cancer progression through altered behavior of CSCs and immune cells, which in turn sustains further biophysical dysregulation. We examine current and potential therapeutic approaches that break this self-sustaining cycle of disease progression by targeting the presented biophysical and epigenetic signatures of cancer. We also summarize strategies including the normalization of the TME, targeted drug delivery, and inhibition of cancer-enabling epigenetic players.

Zwitterionically modified alginates mitigate cellular overgrowth for cell encapsulation.

Foreign body reaction (FBR) to implanted biomaterials and medical devices is common and can compromise the function of implants or cause complications. For example, in cell encapsulation, cellular overgrowth (CO) and fibrosis around the cellular constructs can reduce the mass transfer of oxygen, nutrients and metabolic wastes, undermining cell function and leading to transplant failure. Therefore, materials that mitigate FBR or CO will have broad applications in biomedicine. Here we report a group of zwitterionic, sulfobetaine (SB) and carboxybetaine (CB) modifications of alginates that reproducibly mitigate the CO of implanted alginate microcapsules in mice, dogs and pigs. Using the modified alginates (SB-alginates), we also demonstrate improved outcome of islet encapsulation in a chemically-induced diabetic mouse model. These zwitterion-modified alginates may contribute to the development of cell encapsulation therapies for type 1 diabetes and other hormone-deficient diseases.

Biophysical regulation of macrophages in health and disease.

Macrophages perform critical functions for homeostasis and immune defense in tissues throughout the body. These innate immune cells are capable of recognizing and clearing dead cells and pathogens, and orchestrating inflammatory and healing processes that occur in response to injury. In addition, macrophages are involved in the progression of many inflammatory diseases including cardiovascular disease, fibrosis, and cancer. Although it has long been known that macrophages respond dynamically to biochemical signals in their microenvironment, the role of biophysical cues has only recently emerged. Furthermore, many diseases that involve macrophages are also characterized by changes to the tissue biophysical environment. This review will discuss current knowledge about the effects of biophysical cues including matrix stiffness, material topography, and applied mechanical forces, on macrophage behavior. We will also describe the role of molecules that are known to be important for mechanotransduction, including adhesion molecules, ion channels, as well as nuclear mediators such as transcription factors, scaffolding proteins, and epigenetic regulators. Together, this review will illustrate a developing role of biophysical cues in macrophage biology, and also speculate upon molecular targets that may potentially be exploited therapeutically to treat disease.

Pushing the limits of detection for proteins secreted from single cells using quantum dots.

Single cell analysis methods are increasingly being utilized to investigate how individual cells process information and respond to diverse stimuli. Soluble proteins play a critical role in controlling cell populations and tissues, but directly monitoring secretion is technically challenging. Microfabricated well arrays have been developed to assess secretion at the single cell level, but these systems are limited by low detection sensitivity. Semiconductor quantum dots (QD) exhibit remarkably bright and photostable luminescence signal, but to date they have not been evaluated in single cell secretion studies using microfabricated well arrays. Here, we used QDs in a sandwich immunoassay to detect secretion of the soluble cytokine tumor necrosis factor-α (TNF-α) from single cells. To enhance detection sensitivity, we employed two different strategies. First, we used a unique single QD imaging approach, which provided a detection threshold (180 attomolar) that was >100-fold lower than previously reported results using QDs. We also amplified QD binding to each captured TNF-α molecule using the bioorthogonal cycloaddition reaction between trans-cyclooctene and tetrazine, which further lowered detection threshold to 60 attomolar. This is 6 orders of magnitude more sensitive than organic fluorophores that have been used for single cell secretion studies, and far surpasses single molecule resolution within sub-picoliter microwells that are used to assess single cell secretion. Finally, single cell secretion studies were performed using phorbol 12-myristate 13-acetate (PMA) differentiated and lipopolysaccharide (LPS) activated U-937 cells. TNF-α secretion was detected from 3-fold more single cells using the QD-based method in comparison to rhodamine, which was accomplished by extending sensitivity into the range of ∼2 to 10 000 molecules captured per microwell. In future work, we will apply this technique to assess immune cell secretion dynamics under diverse stimuli and disease settings. We will also incorporate multiplexing capabilities to evaluate the secretome at the resolution of single molecules.

Zwitterionic PEG-PC Hydrogels Modulate the Foreign Body Response in a Modulus-Dependent Manner.

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.

Incorporation of a Ligand Peptide for Immune Inhibitory Receptor LAIR-1 on Biomaterial Surfaces Inhibits Macrophage Inflammatory Responses.

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is an inhibitory receptor broadly expressed on immune cells, with its ligands residing within the extracellular matrix protein collagen. In this study, surfaces are modified with a LAIR-1 ligand peptide (LP), and it is observed that macrophages cultured on LAIR-1 LP-conjugated surfaces exhibit significantly reduced secretion of inflammatory cytokines in response to proinflammatory stimuli that reflect an injured environment. These downregulated mediators include TNF-α, MIP-1α, MIP-1ß, MIP-2, RANTES, and MIG. Knockdown of LAIR-1 using siRNA abrogates this inhibition of cytokine secretion, supporting the specificity of the inhibitory effect to this receptor. These results are the first to demonstrate that integration of LAIR-1 ligands with biomaterials could suppress inflammatory responses.

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.