M2a Phenotypes Research Articles

TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype and is associated with increased survival in cancer patients with high tumor macrophage content.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can either induce cell death or activate survival pathways after binding to death receptors (DRs) DR4 or DR5. TRAIL is investigated as a therapeutic agent in clinical trials due to its selective toxicity to transformed cells. Macrophages can be polarized into pro-inflammatory/tumor-fighting M1 macrophages or anti-inflammatory/tumor-supportive M2 macrophages and an imbalance between M1 and M2 macrophages can promote diseases. Therefore, identifying modulators that regulate macrophage polarization is important to design effective macrophage-targeted immunotherapies. The impact of TRAIL on macrophage polarization is not known. Primary human monocyte-derived macrophages were pre-treated with either TRAIL or with DR4 or DR5-specific ligands and then polarized into M1, M2a, or M2c phenotypes in vitro. The expression of M1 and M2 markers in macrophage subtypes was analyzed by RNA sequencing, qPCR, ELISA, and flow cytometry. Furthermore, the cytotoxicity of the macrophages against U937 AML tumor targets was assessed by flow cytometry. TCGA datasets were also analyzed to correlate TRAIL with M1/M2 markers, and the overall survival of cancer patients. TRAIL increased the expression of M1 markers at both mRNA and protein levels while decreasing the expression of M2 markers at the mRNA level in human macrophages. TRAIL also shifted M2 macrophages towards an M1 phenotype. Our data showed that both DR4 and DR5 death receptors play a role in macrophage polarization. Furthermore, TRAIL enhanced the cytotoxicity of macrophages against the AML cancer cells in vitro. Finally, TRAIL expression was positively correlated with increased expression of M1 markers in the tumors from ovarian and sarcoma cancer patients and longer overall survival in cases with high, but not low, tumor macrophage content. TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype via both DR4 and DR5. Our study defines TRAIL as a new regulator of macrophage polarization and suggests that targeting DRs can enhance the anti-tumorigenic response of macrophages in the tumor microenvironment by increasing M1 polarization.

Rabies virus uniquely reprograms the transcriptome of human monocyte-derived macrophages.

Macrophages are amongst the first immune cells that encounter rabies virus (RABV) at virus entry sites. Activation of macrophages is essential for the onset of a potent immune response, but insights into the effects of RABV on macrophage activation are scarce. In this study we performed high-throughput sequencing on RNA extracted from macrophages that were exposed to RABV for 48 hours, and compared their transcriptional profiles to that of non-polarized macrophages (M0), and macrophages polarized towards the canonical M1, M2a and M2c phenotypes. Our analysis revealed that RABV-stimulated macrophages show high expression of several M1, M2a and M2c signature genes. Apart from their partial resemblance to these phenotypes, unbiased clustering analysis revealed that RABV induces a unique and distinct polarization program. Closer examination revealed that RABV induced multiple pathways related to the interferon- and antiviral response, which were not induced under other classical polarization strategies. Surprisingly, our data show that RABV induces an activated rather than a fully suppressed macrophage phenotype, triggering virus-induced activation and polarization. This includes multiple genes with known antiviral (e.g. APOBEC3A, IFIT/OAS/TRIM genes), which may play a role in anti-RABV immunity.

Assessing the response of human primary macrophages to defined fibrous architectures fabricated by melt electrowriting.

The dual role of macrophages in the healing process depends on macrophage ability to polarize into phenotypes that can propagate inflammation or exert anti-inflammatory and tissue-remodeling functions. Controlling scaffold geometry has been proposed as a strategy to influence macrophage behavior and favor the positive host response to implants. Here, we fabricated Polycaprolactone (PCL) scaffolds by Melt Electrowriting (MEW) to investigate the ability of scaffold architecture to modulate macrophage polarization.Primary human macrophages unpolarized (M0) or polarized into M1, M2a, and M2c phenotypes were cultured on PCL films and MEW scaffolds with pore geometries (square, triangle, and rhombus grid) characterized by different angles.M0, M2a, and M2c macrophages wrapped along the fibers, while M1 macrophages formed clusters with rounded cells. Cell bridges were formed only for angles up to 90°. No relevant differences were found among PCL films and 3D scaffolds in terms of surface markers. CD206 and CD163 were highly expressed by M2a and M2c macrophages, with M2a macrophages presenting also high levels of CD86. M1 macrophages expressed moderate levels of all markers. The rhombus architecture promoted an increased release by M2a macrophages of IL10, IL13, and sCD163 compared to PCL films. The proangiogenic factor IL18 was also upregulated by the rhombus configuration in M0 and M2a macrophages compared to PCL films.The interesting findings obtained for the rhombus architecture represent a starting point for the design of scaffolds able to modulate macrophage phenotype, prompting investigations addressed to verify their ability to facilitate the healing process in vivo.

Cortical bone stem cells modify cardiac inflammation after myocardial infarction by inducing a novel macrophage phenotype.

Acute damage to the heart, as in the case of myocardial infarction (MI), triggers a robust inflammatory response to the sterile injury that is part of a complex and highly organized wound-healing process. Cortical bone stem cell (CBSC) therapy after MI has been shown to reduce adverse structural and functional remodeling of the heart after MI in both mouse and swine models. The basis for these CBSC treatment effects on wound healing are unknown. The present experiments show that CBSCs secrete paracrine factors known to have immunomodulatory properties, most notably macrophage colony-stimulating factor (M-CSF) and transforming growth factor-β, but not IL-4. CBSC therapy increased the number of galectin-3+ macrophages, CD4+ T cells, and fibroblasts in the heart while decreasing apoptosis in an in vivo swine model of MI. Macrophages treated with CBSC medium in vitro polarized to a proreparative phenotype are characterized by increased CD206 expression, increased efferocytic ability, increased IL-10, TGF-β, and IL-1RA secretion, and increased mitochondrial respiration. Next generation sequencing revealed a transcriptome significantly different from M2a or M2c macrophage phenotypes. Paracrine factors from CBSC-treated macrophages increased proliferation, decreased α-smooth muscle actin expression, and decreased contraction by fibroblasts in vitro. These data support the idea that CBSCs are modulating the immune response to MI to favor cardiac repair through a unique macrophage polarization that ultimately reduces cell death and alters fibroblast populations that may result in smaller scar size and preserved cardiac geometry and function.NEW & NOTEWORTHY Cortical bone stem cell (CBSC) therapy after myocardial infarction alters the inflammatory response to cardiac injury. We found that cortical bone stem cell therapy induces a unique macrophage phenotype in vitro and can modulate macrophage/fibroblast cross talk.

Use of integrated metabolomics, transcriptomics, and signal protein profile to characterize the effector function and associated metabotype of polarized macrophage phenotypes.

MΦs display remarkable plasticity and the ability to activate diverse responses to a host of intracellular and external stimuli. Despite extensive characterization of M1 MΦs and a broad set of M2 MΦs, comprehensive characterization of functional phenotype and associated metabotype driving this diverse MΦ activation remains. Herein, an ex vivo model was utilized to produce 6 MΦ functional phenotypes. Isolated CD14+ PBMCs were differentiated into resting M0 MΦs, and then polarized into M1 (IFN-γ/LPS), M2a (IL-4/IL-13), M2b (IC/LPS), M2c (IL-10), and M2d (IL-6/LIF) MΦs. The MΦs were profiled using a bioanalyte matrix of 4 cell surface markers, ∼50 secreted proteins, ∼800 expressed myeloid genes, and ∼450 identified metabolites relative to M0 MΦs. Signal protein and expressed gene profiles grouped the MΦs into inflammatory (M1 and M2b) and wound resolution (M2a, M2c, and M2d) phenotypes; however, each had a unique metabolic profile. While both M1 and M2b MΦs shared metabotype profiles consistent with an inflammatory signature; key differences were observed in the TCA cycle, FAO, and OXPHOS. Additionally, M2a, M2c, and M2d MΦs all profiled as tissue repair MΦs; however, metabotype differences were observed in multiple pathways including hexosamine, polyamine, and fatty acid metabolism. These metabolic and other key functional distinctions suggest phagocytic and proliferative functions for M2a MΦs, and angiogenesis and ECM assembly capabilities for M2b, M2c, and M2d MΦs. By integrating metabolomics into a systems analysis of MΦ phenotypes, we provide the most comprehensive map of MΦ diversity to date, along with the global metabolic shifts that correlate to MΦ functional plasticity in these phenotypes.

Influence of Secretome of Different Functional Phenotypes of Macrophages on Proliferation, Differentiation, and Collagen-Producing Activity of Dermal Fibroblasts In Vitro.

We studied the effect of conditioned media of GM-CSF-differentiated human macrophages polarized in M1(LPS), M2a(IL-4), M2c(dexamethasone), and M2(low serum) phenotypes on proliferation, differentiation, and collagen-producing activity of dermal fibroblasts. It was found that M1(LPS) and M2a(IL-4) were characterized by moderate influence on functional activity of fibroblasts. At the same time, soluble factors of M2c(dexamethasone) significantly enhanced the proliferative response of fibroblasts, but not their differentiation and type I collagen production. On the contrary, M2(low serum) generated under conditions of growth factors deficiency had a pronounced stimulating effect on the differentiation of fibroblasts and production of type I collagen by these cells, but moderately stimulated the fibroblast proliferation. Thus, the secretory activity of various functional phenotypes of macrophages is an important mechanism of fibrogenesis regulation.

Mesenchymal stem cell-inspired microgel scaffolds to control macrophage polarization.

There is a desire in regenerative medicine to create biofunctional materials that can control and direct cell function in a precise manner. One particular stem cell of interest, human mesenchymal stem cells (hMSCs), can function as regulators of the immunogenic response and aid in tissue regeneration and wound repair. Here, a porous hydrogel scaffold assembled from microgel subunits was used to recapitulate part of this immunomodulatory behavior. The scaffolds were used to culture a macrophage cell line, while cytokines were delivered exogenously to polarize the macrophages to either a pro‐inflammatory (M1) or alternatively activated (M2a) phenotypes. Using a cytokine array, interleukin 10 (IL‐10) was identified as one key anti‐inflammatory factor secreted by hMSCs in pro‐inflammatory conditions; it was elevated (125 ± 25 pg/ml) in pro‐inflammatory conditions compared to standard medium (6 ± 10 pg/ml). The ability of hMSC laden scaffolds to reverse the M1 phenotype was then examined, even in the presence of exogenous pro‐inflammatory cytokines. Co‐culture of M1 and M2 macrophages with hMSCs reduced the secretion of TNFα, a pro‐inflammatory cytokine even in the presence of pro‐inflammatory stimulatory factors. Next, IL‐10 was supplemented in the medium or tethered directly to the microgel subunits; both methods limited the secretion of pro‐inflammatory cytokines of encapsulated macrophages even in pro‐inflammatory conditions. Cumulatively, these results reveal the potential of biofunctional microgel‐based scaffolds as acellular therapies to present anti‐inflammatory cytokines and control the immunogenic cascade.

Substrate stiffness directs the phenotype and polarization state of cord blood derived macrophages

Cord blood (CB) mononuclear cell populations have demonstrated significant promise in biomaterials-based regenerative therapies; however, the contributions of monocyte and macrophage subpopulations towards proper tissue healing and regeneration are not well understood, and the phenotypic responses of macrophage to microenvironmental cues have not been well-studied. In this work, we evaluated the effects of cytokine stimulation and altered substrate stiffness. Macrophage derived from CB CD14+ monocytes adopted distinct inflammatory (M1) and anti-inflammatory (M2a and M2c) phenotypes in response to cytokine stimulation (M1: lipopolysaccharide (LPS) and interferon (IFN-γ); M2a: interleukin (IL)-4 and IL-13; M2c: IL-10) as determined through expression of relevant cell surface markers and growth factors. Cytokine-induced macrophage readily altered their phenotypes upon sequential administration of different cytokine cocktails. The impact of substrate stiffness on macrophage phenotype was evaluated by seeding CB-derived macrophage on 3wt%, 6wt%, and 14wt% poly(ethylene glycol)-based hydrogels, which exhibited swollen shear moduli of 0.1, 3.4, and 10.3 kPa, respectively. Surface marker expression and cytokine production varied depending on modulus, with anti-inflammatory phenotypes increasing with elevated substrate stiffness. Integration of specific hydrogel moduli and cytokine cocktail treatments resulted in the differential regulation of macrophage phenotypic biomarkers. These data suggest that CB-derived macrophages exhibit predictable behaviors that can be directed and finely tuned by combinatorial modulation of substrate physical properties and cytokine profiles.

Docosahexaenoic acid impacts macrophage phenotype subsets and phagolysosomal membrane permeability with particle exposure

ABSTRACT Inhalation of particles results in pulmonary inflammation; however, treatments are currently lacking. Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid shown to exhibit anti-inflammatory capabilities. The impact of DHA on particle-induced inflammation is unclear; therefore, the aim of this study was to examine the hypothesis that DHA downregulates macrophage inflammatory responses by altering phagolysosomal membrane permeability (LMP) and shifting macrophage phenotype. Isolated Balb/c alveolar macrophages (AM) were polarized into M1, M2a, M2b, or M2c phenotypes in vitro, treated with DHA, and exposed to a multi-walled carbon nanotube (MWNCT) or crystalline silica (SiO2). Results showed minimal cytotoxicity, robust effects for silica particle uptake, and LMP differences between phenotypes. Docosahexaenoic acid prevented these effects to the greatest extent in M2c phenotype. To determine if DHA affected inflammation similarly in vivo, Balb/c mice were placed on a control or 1% DHA diet for 3 weeks, instilled with the same particles, and assessed 24 hr following instillation. Data demonstrated that in contrast to in vitro findings, DHA increased pulmonary inflammation and LMP. These results suggest that pulmonary responses in vivo may not necessarily be predicted from single-cell responses in vitro.

A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages

BackgroundEleutherococcus senticosus or Siberian ginseng is a medicinal plant containing adaptogenic substances believed to regulate immune responses. Both, the root and stem bark are commonly used in traditional medicines. PurposeThe purpose of the present study is to chemically characterize E. senticosus root and bark extracts and to compare their effects on functions of human primary macrophages. Study design and methodsHPLC-DAD-MS analysis was used to characterize chemical constituents of alcoholic extracts from E. senticosus root and bark. The data obtained and available databases were combined for network pharmacology analysis. Involvement of predicted pathways was further functionally confirmed by using monocyte-derived human macrophages and endotoxin-free E. senticosus root and bark extracts. ResultsChemical analysis showed that the root extract contained more syringin, caffeic acid, and isofraxidin than the bark extract. At variance, bark extract contained more sesamin and oleanolic acid. Coniferyl aldehyde and afzelin were below the limit of quantification in both extracts. Network pharmacology analysis indicated that constituents of E. senticosus might affect the immune cell phenotype and signaling pathways involved in cell metabolism and cytoskeleton regulation. Indeed, both extracts promoted actin polymerization, migration, and phagocytosis of E. coli by macrophages pointing to macrophage polarization towards the M2 phenotype. In addition, treatment with E. senticosus root and bark extracts decreased phosphorylation of Akt on Ser473 and significantly reduced expression of the hemoglobin scavenger receptor CD163 by macrophages. Neither extract affected expression of CD11b, CD80, or CD64 by macrophages. In addition, macrophages treated with the bark extract, but not with the root extract, exhibited activated p38 MAPK and NF-κB and released increased, but still moderate, amounts of proinflammatory TNF-α and IL-6, anti-inflammatory IL-10, and chemotactic CCL1, which all together point to a M2b-like macrophage polarization. Differently, the root extract increased the IL-4-induced expression of anti-inflammatory CD200R. These changes in monocytes are in agreement with an increased M2a macrophage polarization. ConclusionThe ability of E. senticosus root and bark extracts to promote polarization of human macrophages towards anti-inflammatory M2a and M2b phenotypes, respectively, might underlay the immunoregulatory activities and point to potential wound healing promoting effects of this medicinal plant.

Exosomes Derived From M2b Macrophages Attenuate DSS-Induced Colitis.

Macrophages are commonly classified as M1 macrophages or M2 macrophages. The M2 macrophages are further sub-categorized into M2a, M2b, M2c, and M2d subtypes. The M2a, M2b, and M2c subtypes play roles in anti-inflammatory activity, tissue remodeling, type 2 T helper cell (Th2) activation, and immunoregulation. Previous studies have shown that macrophage exosomes can affect some disease processes. Exosomes are 30–150-nm lipid bilayer membrane vesicles derived from most living cells, with important biological functions. The role of exosomes in preventing the development of autoimmune diseases, including inflammatory bowel disease (IBD), has evoked increasing interest. Here, we analyze the roles of exosomes derived from M2a, M2b, and M2c macrophage phenotypes in dextran sulfate sodium (DSS)-induced colitis. Exosomes were isolated from the supernatant of different types of macrophages and identified via transmission electron microscopy (TEM), western blotting, and NanoSight. The results showed that M2b macrophage exosomes significantly attenuated the severity of DSS-induced colitis in mice. The number of regulatory T (Treg) cells in the spleens of mice with colitis and levels of IL-4 both increased following treatment with M2b macrophage exosomes. In addition, key cytokines associated with colitis (IL-1β, IL-6, and IL-17A) were significantly suppressed, following treatment with M2b macrophage exosomes. The M2b macrophage exosomes exerted protective effects on DSS-induced colitis, mainly mediated by the CC chemokine 1 (CCL1)/CCR8 axis. These findings provide a novel approach for the treatment of IBD.

Gene Regulatory Network Modeling of Macrophage Differentiation Corroborates the Continuum Hypothesis of Polarization States.

Macrophages derived from monocyte precursors undergo specific polarization processes which are influenced by the local tissue environment: classically activated (M1) macrophages, with a pro-inflammatory activity and a role of effector cells in Th1 cellular immune responses, and alternatively activated (M2) macrophages, with anti-inflammatory functions and involved in immunosuppression and tissue repair. At least three different subsets of M2 macrophages, namely, M2a, M2b, and M2c, are characterized in the literature based on their eliciting signals. The activation and polarization of macrophages is achieved through many, often intertwined, signaling pathways. To describe the logical relationships among the genes involved in macrophage polarization, we used a computational modeling methodology, namely, logical (Boolean) modeling of gene regulation. We integrated experimental data and knowledge available in the literature to construct a logical network model for the gene regulation driving macrophage polarization to the M1, M2a, M2b, and M2c phenotypes. Using the software GINsim and BoolNet, we analyzed the network dynamics under different conditions and perturbations to understand how they affect cell polarization. Dynamic simulations of the network model, enacting the most relevant biological conditions, showed coherence with the observed behavior of in vivo macrophages. The model could correctly reproduce the polarization toward the four main phenotypes as well as to several hybrid phenotypes, which are known to be experimentally associated to physiological and pathological conditions. We surmise that shifts among different phenotypes in the model mimic the hypothetical continuum of macrophage polarization, with M1 and M2 being the extremes of an uninterrupted sequence of states. Furthermore, model simulations suggest that anti-inflammatory macrophages are resilient to shift back to the pro-inflammatory phenotype.

Effect of Zr Addition on the Corrosion of Ti in Acidic and Reactive Oxygen Species (ROS)-Containing Environments.

The effect of systematic Zr additions on the corrosion behavior of Ti was studied in both acidic and reactive oxygen species (ROS) containing environments, including macrophage cell culture, simulating inflammation associated with metallic implants. Electrochemical measurements on commercially pure (CP) Ti, Zr, and TiZr alloys showed that increasing Zr additions progressively enhanced Ti passivity in both acidic (HCl) and oxidative (H2O2) environments. However, a Ti50Zr alloy was found with increased pitting susceptibility. Corrosion was also evaluated using mass-spectrometry to determine metal ion release following exposure of the alloys to THP-1 macrophage cell cultures, transformed into either their M1 (inflammatory states) or M2a (tissue repair states) phenotypes. The magnitude of ion release was reduced with increasing Zr contents, consistent with electrochemical observations. Nevertheless, optimized Zr content in Ti should balance both passivity and pitting resistance.

Accumulation and localization of macrophage phenotypes with human intervertebral disc degeneration

Background ContextChronic inflammation is an important component of intervertebral disc (IVD) degeneration, but there is limited knowledge about the identity and source of inflammatory cells involved with the degenerative processes. Macrophages can exhibit multiple phenotypes and are known inflammatory regulators in many tissues, but their phenotypes have not been characterized in IVD degeneration. PurposeWe aimed to characterize accumulation and localization of macrophages in IVD degeneration. Study Design/SettingThis is an exploratory study to characterize macrophage phenotypes in human cadaver IVDs and the effects of injury and degeneration using multiple immunohistochemistry methods. Outcome MeasuresPercent positivity of immunohistochemical markers specific for CCR7, CD163, and CD206, and qualitative assessments of dual immunofluorescence and immunostaining localization were the outcome measures. MethodsMacrophages were identified in human cadaveric IVDs with immunohistochemistry using cell surface markers CCR7, CD163, and CD206, which are associated with proinflammatory M1, remodeling M2c, and anti-inflammatory M2a phenotypes, respectively. Variations in the accumulation and localization of macrophage markers with degenerative grade across subjects and within donors are described. ResultsCells expressing all three macrophage markers were found in all degenerative IVDs, but not in the healthiest IVDs. Cells expressing CCR7 and CD163, but not CD206, significantly increased with degenerative grade. Many cells also co-expressed multiple macrophage markers. Across all degenerative grades, CCR7+ and CD163+ were significantly more present in unhealthy nucleus pulposus (NP), annulus fibrosus (AF), and end plate (EP) regions exhibiting structural irregularities and defects. Positively stained cells in the NP and AF closely resembled resident IVD cells, suggesting that IVD cells can express macrophage cell surface markers. In the EP, there were increasing trends of positively stained cells with atypical morphology and distribution, suggesting a source for exogenous macrophage infiltration into the IVD. ConclusionsChronic inflammatory conditions of IVD degeneration appear to involve macrophages or macrophage-like cells, as expression of multiple macrophage markers increased with degeneration, especially around unhealthy regions with defects and the EP. Knowledge of macrophage phenotypes and their localization better elucidates the complex injury and repair processes in IVDs and may eventually lead to novel treatments.

Cardiac Progenitor Cell Recruitment Drives Fetal Cardiac Regeneration by Enhanced Angiogenesis

In contrast to adults, the fetal response to myocardial infarction (MI) is regenerative, requiring the recruitment of cardiac progenitor cells to replace infarcted myocardium. Macrophage contribution to tissue repair depends on their phenotype: M1 are proinflammatory and initiate angiogenesis; M2a are profibrotic and contribute to blood vessels maturation; and M2c are proremodeling and proangiogenesis. The goal of the present study was to expand on this work by examining cardiac progenitor cells recruitment, and the role of macrophages in promoting angiogenesis and cardiac regeneration in the fetal heart after MI. Fetal and adult sheep underwent MI and were sacrificed 3 or 30 days after MI. Some fetal hearts received stromal cell-derived factor-1α-inhibitor treatment. The microvasculature was evaluated by micro-computed tomography, gene expression was evaluated by real-time polymerase chain reaction, and vessels counts were evaluated by immunohistochemistry. Micro-computed tomography analysis showed restoration of microvasculature in fetal hearts after MI. Vascular endothelial growth factor-α increased, and the expression of tissue markers associated with the M1, M2a, and M2c macrophage phenotypes were elevated at day 3 after MI, but returned to baseline by 30 days after MI. In contrast, adult hearts after MI exhibited low vascular endothelial growth factor-α and persistent upregulation of all macrophage markers, consistent with prolonged inflammation, fibrosis, and remodeling. Inhibition of stromal cell-derived factor-1α in fetal infarcts prevented angiogenesis, decreased vascular endothelial growth factor-α, and was associated with a sustained increase in M1, M2a, and M2c markers afterMI. Changes in angiogenesis and macrophage phenotype-related gene expression after MI are important for the fetal regenerative response to MI and are mediated at least in part by cardiac progenitor cells recruitment.

Transcriptome analysis of IL-10-stimulated (M2c) macrophages by next-generation sequencing

Alternatively activated “M2” macrophages are believed to function during late stages of wound healing, behaving in an anti-inflammatory manner to mediate the resolution of the pro-inflammatory response caused by “M1” macrophages. However, the differences between two main subtypes of M2 macrophages, namely interleukin-4 (IL-4)-stimulated “M2a” macrophages and IL-10-stimulated “M2c” macrophages, are not well understood. M2a macrophages are characterized by their ability to inhibit inflammation and contribute to the stabilization of angiogenesis. However, the role and temporal profile of M2c macrophages in wound healing are not known. Therefore, we performed next generation sequencing (RNA-seq) to identify biological functions and gene expression signatures of macrophages polarized in vitro with IL-10 to the M2c phenotype in comparison to M1 and M2a macrophages and an unactivated control (M0). We then explored the expression of these gene signatures in a publicly available data set of human wound healing. RNA-seq analysis showed that hundreds of genes were upregulated in M2c macrophages compared to the M0 control, with thousands of alternative splicing events. Following validation by Nanostring, 39 genes were found to be upregulated by M2c macrophages compared to the M0 control, and 17 genes were significantly upregulated relative to the M0, M1, and M2a phenotypes (using an adjusted p-value cutoff of 0.05 and fold change cutoff of 1.5). Many of the identified M2c-specific genes are associated with angiogenesis, matrix remodeling, and phagocytosis, including CD163, MMP8, TIMP1, VCAN, SERPINA1, MARCO, PLOD2, PCOCLE2 and F5. Analysis of the macrophage-conditioned media for secretion of matrix-remodeling proteins showed that M2c macrophages secreted higher levels of MMP7, MMP8, and TIMP1 compared to the other phenotypes. Interestingly, temporal gene expression analysis of a publicly available microarray data set of human wound healing showed that M2c-related genes were upregulated at early times after injury, similar to M1-related genes, while M2a-related genes appeared at later stages or were downregulated after injury. While further studies are required to confirm the timing and role of M2c macrophages in vivo, these results suggest that M2c macrophages may function at early stages of wound healing. Identification of markers of the M2c phenotype will allow more detailed investigations into the role of M2c macrophages in vivo.

Guanylate-binding protein 5 is a marker of interferon-γ-induced classically activated macrophages.

Macrophage activation is the main immunological process occurring during the development of several diseases, and the heterogeneity of macrophage activation or differentiation has been suggested to be involved in disease progression. In the present study, we attempted to identify molecules specifically expressed on human classically activated macrophages (M1) to investigate the significance of the M1-like phenotype in human diseases. Human monocyte-derived macrophages were differentiated into M1, M2a, M2b and M2c phenotypes, and also M1(−) (the M1 phenotype differentiated with interferon-γ) to eliminate the strong effects of lipopolysaccharides (LPS) on the gene expression profile. The gene expression profiles of those macrophage phenotypes were analyzed by a cDNA microarray analysis and were used for a bioinformatics examination to identify the markers of the M1 phenotype that are expressed in both M1 and M1(−). The gene expression profiles of murine macrophages were also evaluated. We identified guanylate-binding protein 5 (GBP5), which is associated nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 (NLRP3)-mediated inflammasome assembly in the M1 macrophages of both humans and mice. Notably, the expression of GBP5 protein was detected in cultured M1(−) as well as in M1 macrophages by western blotting, which means that GBP5 is a more generalized marker of the M1 phenotype compared with the M1 markers that can be induced by LPS stimulation. GBP5 is a useful candidate marker of the M1 phenotype.

Down syndrome individuals with Alzheimer's disease have a distinct neuroinflammatory phenotype compared to sporadic Alzheimer's disease

Down syndrome (DS) is the most common genetic cause of intellectual disability and is primarily caused by the triplication of chromosome 21. The overexpression of amyloid precursor protein gene may be sufficient to drive Alzheimer's disease (AD) neuropathology that is observed in virtually all individuals with DS by the age of 40 years. There is relatively little information about inflammation in the DS brain and how the genetics of DS may alter inflammatory responses and modify the course of AD pathogenesis in this disorder. Using the macrophage classification system of M1, M2a, M2b, and M2c inflammatory phenotypes, we have shown that the early stages of AD are associated with a bias toward an M1 or M2a phenotype. In later stages of AD, markers of M1, M2a and M2c are elevated. We now report the inflammatory phenotype in a DS autopsy series to compare this with the progression in sporadic AD. Tissue from young DS cases (under 40 years of age, pre-AD) show a bias toward M1 and M2b states with little M2a or M2c observed. Older DS cases (over 40 with AD pathology) show a distinct bias toward an M2b phenotype. Importantly, this is distinct from sporadic AD where the M2b phenotype has been rarely, if ever observed in postmortem studies. Stimulated by immune complex activation of microglial cells and toll-like receptor activation, the M2b phenotype represents a unique neuroinflammatory state in diseased brain and may have significant implications for therapeutic intervention for persons with DS.

213 Functional Characterisation of Monocyte Derived Macrophage Phenotypes for their Role in Atherosclerosis

IntroductionCardiovascular disease, especially atherosclerosis and related pathologies, is one of the leading causes of death globally. It is well established that macrophages play crucial role in the formation and progression...

Macrophage activation and skeletal muscle healing following traumatic injury

Following injury to different tissues, macrophages can contribute to both regenerative and fibrotic healing. These seemingly contradictory roles of macrophages may be related to the markedly different phenotypes that macrophages can assume upon exposure to different stimuli. We hypothesized that fibrotic healing after traumatic muscle injury would be dominated by a pro-fibrotic M2a macrophage phenotype, with M1 activation limited to the very early stages of repair. We found that macrophages accumulated in lacerated mouse muscle for at least 21 days, accompanied by limited myofibre regeneration and persistent collagen deposition. However, muscle macrophages did not exhibit either of the canonical M1 or M2a phenotypes, but instead up-regulated both M1- and M2a-associated genes early after injury, followed by down-regulation of most markers examined. Particularly, IL-10 mRNA and protein were markedly elevated in macrophages from 3-day injured muscle. Additionally, though flow cytometry identified distinct subpopulations of macrophages based on high or low expression of TNFα, these subpopulations did not clearly correspond to M1 or M2a phenotypes. Importantly, cell therapy with exogenous M1 macrophages but not non-activated macrophages reduced fibrosis and enhanced muscle fibre regeneration in lacerated muscles. These data indicate that manipulation of macrophage function has potential to improve healing following traumatic injury.