M2c Phenotypes Research Articles

Glycoprotein non-metastatic melanoma protein B expression correlates with the prognosis of acute liver injury/failure.

Background: Glycoprotein non-metastatic melanoma protein B (GPNMB) is expressed in macrophages during recovery from acute liver injury (ALI) in carbon tetrachloride (CCl4)-induced liver injury model mice. In this retrospective study, we assessed whether GPNMB levels in the serum and injured liver correlate with liver injury severity and prognosis in patients with ALI or acute liver failure (ALF). Methods: The study involved 56 patients with ALI or ALF who visited the Kagoshima University Hospital. Serum GPNMB level was measured over time, and the localization, proportion, origin, and phenotype of GPNMB-expressing cells in the injured liver were assessed. Finally, the phenotypes of human monocyte-derived macrophages and peripheral blood mononuclear cells (PBMCs) of patients with ALI and ALF were analyzed. Results: Peak GPNMB levels were significantly higher in patients with ALF and hepatic encephalopathy (HE), as well as in those who underwent liver transplantation or died, than in others. The peak GPNMB level correlated with prothrombin activity, prothrombin time-international normalized ratio, Model for End-stage Liver Disease score, and serum hepatocyte growth factor level. GPNMB was expressed in CD68-positive macrophages, and its level increased with the severity of liver injury. The macrophages showed the same polarization as M2c macrophages induced with interleukin-10 from human monocytes. Moreover, PBMCs from patients with ALF exhibited an immunosuppressive phenotype. Conclusion: We found that GPNMB levels in the serum and injured liver, which increased in patients with ALF, especially in those with HE, correlated with the severity of liver injury and prognosis of ALI and ALF. Moreover, GPNMB-positive macrophages exhibited the M2c phenotype. Our results indicate that persistently high GPNMB levels may be a prognostic marker in patients with ALI and ALF.

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.

Effective-mononuclear cell (E-MNC) therapy alleviates salivary gland damage by suppressing lymphocyte infiltration in Sjögren-like disease.

Introduction: Sjögren syndrome (SS) is an autoimmune disease characterized by salivary gland (SG) destruction leading to loss of secretory function. A hallmark of the disease is the presence of focal lymphocyte infiltration in SGs, which is predominantly composed of T cells. Currently, there are no effective therapies for SS. Recently, we demonstrated that a newly developed therapy using effective-mononuclear cells (E-MNCs) improved the function of radiation-injured SGs due to anti-inflammatory and regenerative effects. In this study, we investigated whether E-MNCs could ameliorate disease development in non-obese diabetic (NOD) mice as a model for primary SS. Methods: E-MNCs were obtained from peripheral blood mononuclear cells (PBMNCs) cultured for 7days in serum-free medium supplemented with five specific recombinant proteins (5G culture). The anti-inflammatory characteristics of E-MNCs were then analyzed using a co-culture system with CD3/CD28-stimulated PBMNCs. To evaluate the therapeutic efficacy of E-MNCs against SS onset, E-MNCs were transplanted into SGs of NOD mice. Subsequently, saliva secretion, histological, and gene expression analyses of harvested SG were performed to investigate if E-MNCs therapy delays disease development. Results: First, we characterized that both human and mouse E-MNCs exhibited induction of CD11b/CD206-positive cells (M2 macrophages) and that human E-MNCs could inhibit inflammatory gene expressions in CD3/CD28- stimulated PBMNCs. Further analyses revealed that Msr1-and galectin3-positive macrophages (immunomodulatory M2c phenotype) were specifically induced in E-MNCs of both NOD and MHC class I-matched mice. Transplanted E-MNCs induced M2 macrophages and reduced the expression of T cell-derived chemokine-related and inflammatory genes in SG tissue of NOD mice at SS-onset. Then, E-MNCs suppressed the infiltration of CD4-positive T cells and facilitated the maintenance of saliva secretion for up to 12weeks after E-MNC administration. Discussion: Thus, the immunomodulatory actions of E-MNCs could be part of a therapeutic strategy targeting the early stage of primary SS.

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.

MSC-derived sEV-loaded hyaluronan hydrogel promotes scarless skin healing by immunomodulation in a large skin wound model

Designing hydrogel-based constructs capable of adjusting immune cell functions holds promise for skin tissue regeneration. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) have attracted increasing attention owing to their anti-inflammatory and proangiogenic effects. Herein, we constructed a biofunctional hydrogel in which MSC-derived sEVs were incorporated into the injectable hyaluronic acid hydrogel, thus endowing the hydrogel with immunomodulatory effects. When implanted onto the wound site in a mouse large skin injury model, this functional hydrogel facilitates wound healing and inhibits scar tissue formation by driving macrophages towards an anti-inflammatory and anti-fibrotic (M2c) phenotype. Further investigation showed that the M2c-like phenotype induced by MSC-derived sEVs markedly inhibited the activation of fibroblasts, which could result in scarless skin wound healing. Taken together, these results suggest that modulation of the immune response is a promising and efficient approach to prevent fibrotic scar formation.

Tilapia Piscidin 4 (TP4) Reprograms M1 Macrophages to M2 Phenotypes in Cell Models of Gardnerella vaginalis-Induced Vaginosis.

Gardnerella vaginalis is associated with bacterial vaginosis (BV). The virulence factors produced by G. vaginalis are known to stimulate vaginal mucosal immune response, which is largely driven by activated macrophages. While Tilapia piscidin 4 (TP4), an antimicrobial peptide isolated from Nile tilapia, is known to display a broad range of antibacterial functions, it is unclear whether TP4 can affect macrophage polarization in the context of BV. In this study, we used the culture supernatants from G. vaginalis to stimulate differentiation of THP-1 and RAW264.7 cells to an M1 phenotype. The treatment activated the NF-κB/STAT1 signaling pathway, induced reactive nitrogen and oxygen species, and upregulated inflammatory mediators. We then treated the induced M1 macrophages directly with a non-toxic dose of TP4 or co-cultured the M1 macrophages with TP4-treated vaginal epithelial VK2 cells. The results showed that TP4 could not only decrease pro-inflammatory mediators in the M1 macrophages, but it also enriched markers of M2 macrophages. Further, we found that direct treatment with TP4 switched M1 macrophages toward a resolving M2c phenotype via the MAPK/ERK pathway and IL-10-STAT3 signaling. Conversely, tissue repair M2a macrophages were induced by TP4-treated VK2 cells; TP4 upregulated TSG-6 in VK2 cells, which subsequently activated STAT6 and M2a-related gene expression in the macrophages. In conclusion, our results imply that TP4 may be able to attenuate the virulence of G. vaginalis by inducing resolving M2c and tissue repair M2a macrophage polarizations, suggesting a novel strategy for BV therapy.

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.

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.

The investigation of immunomodulatory effects of adipose tissue mesenchymal stem cell educated macrophages on the CD4 T cells

Mesenchymal stem cells (MSCs) are strong immunomodulatory cells investigated in numerous clinical studies on fatal pathologies, such as graft versus host disease and autoimmune diseases; e.g., systemic lupus erythematosus, Crohn's disease, and ulcerative colitis. Macrophages are one of the critical cells linking the innate and adaptive immune system, and it has been shown that MSCs can differentiate between pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype of macrophages. However, it has not yet been fully clarified whether these differentiated macrophages are functional. In this study, we compared the immunomodulatory effects on the CD4 T cells of M1, M2a and M2c macrophages with the macrophages that directly and indirectly cultured with MSCs. We analyzed the changes in CD14, CD64, CD80, CD163 and CD200R expression to evaluate macrophage phenotypes, and the changes in CD4, IFN-g, IL-4, IL-17a and FoxP3 expression to evaluate T helper subsets using the FACS method. The changes in IL-1b, IL-4, IL-10, IL-12p70, IL-17a and IFN-g in the media supernatants were analyzed using the Luminex method. We also performed WST-1 and Caspase-3 ELISA analyses to observe the proliferation and apoptosis status of the T cells. MSCs were found to differentiate macrophages into a distinctive phenotype, which was close to the M2c phenotype, but was not considered as an M2c cell due to the low expression of CD163, a characteristic marker for M2c. While MEM-D, MEM-ID and MSCs showed similar inhibitory effects on the Th2 and Th17 cells, the most significant increase in Treg cell frequencies was seen in MEM-D cells. Macrophages can alter their phenotypes and functions according to the stimuli from the environment. The fact that macrophages educated with MSCs suppressed the production of all the cytokines we evaluated even after the removal of MSCs suggests that these cells may be differentiated by MSCs into a suppressive macrophage subgroup. However, the Treg cell activation caused by direct interactions between MSCs and macrophage cells may be the most prominent observation of this study compared to previous work. As a result, according to our data, the interactions between MSCs and macrophages may lead to differentiation of macrophage cells into an immunosuppressive phenotype, and these macrophages may suppress the T lymphocyte subgroups at least as effectively as MSCs. However, our data obtained from in vitro experiments should be supported by future in vivo studies.

Chronic Hepatitis C Virus Infection Impairs M1 Macrophage Differentiation and Contributes to CD8+ T-Cell Dysfunction.

Chronic hepatitis C virus (HCV) infection causes generalized CD8+ T cell impairment, not limited to HCV-specific CD8+ T-cells. Liver-infiltrating monocyte-derived macrophages (MDMs) contribute to the local micro-environment and can interact with and influence cells routinely trafficking through the liver, including CD8+ T-cells. MDMs can be polarized into M1 (classically activated) and M2a, M2b, and M2c (alternatively activated) phenotypes that perform pro- and anti-inflammatory functions, respectively. The impact of chronic HCV infection on MDM subset functions is not known. Our results show that M1 cells generated from chronic HCV patients acquire M2 characteristics, such as increased CD86 expression and IL-10 secretion, compared to uninfected controls. In contrast, M2 subsets from HCV-infected individuals acquired M1-like features by secreting more IL-12 and IFN-γ. The severity of liver disease was also associated with altered macrophage subset differentiation. In co-cultures with autologous CD8+ T-cells from controls, M1 macrophages alone significantly increased CD8+ T cell IFN-γ expression in a cytokine-independent and cell-contact-dependent manner. However, M1 macrophages from HCV-infected individuals significantly decreased IFN-γ expression in CD8+ T-cells. Therefore, altered M1 macrophage differentiation in chronic HCV infection may contribute to observed CD8+ T-cell dysfunction. Understanding the immunological perturbations in chronic HCV infection will lead to the identification of therapeutic targets to restore immune function in HCV+ individuals, and aid in the mitigation of associated negative clinical outcomes.

MSC-derived sEVs enhance patency and inhibit calcification of synthetic vascular grafts by immunomodulation in a rat model of hyperlipidemia

Vascular grafts often exhibit low patency rates in clinical settings due to the pathological environment within the patients requiring the surgery. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) have attracted increasing attention. These sEVs contain many potent signaling molecules that play important roles in tissue regeneration, such as microRNA and cytokines. In this study, a sEVs-functionalized vascular graft was developed, and in vivo performance was systematically evaluated in a rat model of hyperlipidemia. Electrospun poly (ε-caprolactone) (PCL) vascular grafts were first modified with heparin, to enhance the anti-thrombogenicity. MSC-derived sEVs were loaded onto the heparinized PCL grafts to obtain functional vascular grafts. As-prepared vascular grafts were implanted to replace a segment of rat abdominal artery (1 cm) for up to 3 months. Results showed that the incorporation of MSC-derived sEVs effectively inhibited thrombosis and calcification, thus enhancing the patency of vascular grafts. Furthermore, regeneration of the endothelium and vascular smooth muscle was markedly enhanced, as attributed to the bioactive molecules within the sEVs, including vascular endothelial growth factor (VEGF), miRNA126, and miRNA145. More importantly, MSC-derived sEVs demonstrated a robust immunomodulatory effect, that is, they induced the transition of macrophages from a pro-inflammatory and atherogenic (M1) phenotype to an anti-inflammatory and anti-osteogenic (M2c) phenotype. This phenotypic switch was confirmed in both in vitro and in vivo analyses. Taken together, these results suggest that fabrication of vascular grafts with immunomodulatory function can provide an effective approach to improve vascular performance and functionality, with translational implication in cardiovascular regenerative medicine.

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.

M2C Polarization by Baicalin Enhances Efferocytosis via Upregulation of MERTK Receptor.

Baicalin is the main active ingredient primary isolated from the Chinese herb, Scutellaria baicalensis Georgi. Although baicalin can induce M2 macrophage polarization, we still do not know the subtype of macrophages polarized by baicalin. In this study, we characterized that murine bone marrow derived macrophages induced by M-CSF can be further polarized into M2C phenotype by baicalin. The signatures of M2C macrophages for mRNA expression like interferon regulatory factor 4 (IRF4), interleukin-10 (IL-10), MERTK and PTX3 were up-regulated. Moreover, we observed the concomitantly decreasing of tumor necrosis factor alpha (TNF- ), interferon regulatory factor 5 (IRF5), IL-6. In contrast, M2 macrophages polarized by IL-4 increased gene transcript of arginase-1 (Arg-1) and surface marker of CD206 indicates that their identity as M2A rather than M2C subtypes. Interestingly, the phagocytosis as well as efferocytosis activity were significantly enhanced in M2C macrophage polarized by baicalin and these capacities were associated with the expression of MERTK receptor. Finally, we conclude that baicalin induced M2C macrophages polarization with both elevations of efferocytosis and anti-inflammatory activity.

Keratin biomaterials augment anti-inflammatory macrophage phenotype in vitro.

Biomaterials made from human hair keratins have previously been shown to elicit anti-inflammatory responses from naïve macrophages and polarize them toward an M2 phenotype. In this work we show for the first time that primary human cells respond similarly, that it is the M2c phenotype that predominates, that a sub-fraction of hydrolyzed keratin peptides are most likely responsible for the response, and that immobilization of the keratin peptides to a surface is required. Keratin biomaterials have been used to regenerate several tissues such as skin, muscle, bone, nerve, and cornea, in vitro and in animal studies. Our current findings will help guide the development of keratin-based biomaterials that seek to direct responses toward regenerative outcomes by attenuating inflammation.

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.

Punicalagin, a PTP1B inhibitor, induces M2c phenotype polarization via up-regulation of HO-1 in murine macrophages

Current data have shown that punicalagin (PUN), an ellagitannin isolated from pomegranate, possesses anti-inflammatory and anti-oxidant properties; however, its direct targets have not yet been reported. This is the first report that PTP1B serves as a direct target of PUN, with IC50 value of 1.04μM. Results from NPOI further showed that the Kon and Koff of PUN-PTP1B complex were 3.38e2M−1s−1 and 4.13e-3s−1, respectively. The active site Arg24 of PTP1B was identified as a key binding site of PUN by computation simulation and point mutation. Moreover, inhibition of PTP1B by PUN promoted an M2c-like macrophage polarization and enhanced anti-inflammatory cytokines expression, including IL-10 and M-CSF. Based on gene expression profile, we elucidated that PUN treatment significantly up-regulated 275 genes and down-regulated 1059 genes. M1-like macrophage marker genes, such as Tlr4, Irf1/2, Hmgb1, and Stat1 were down-regulated, while M2 marker genes, including Tmem171, Gpr35, Csf1, Il1rn, Cebpb, Fos, Vegfα, Slc11a1, and Bhlhe40 were up-regulated in PUN-treated macrophages. Hmox-1, a gene encoding HO-1 protein, was preferentially expressed with 16-fold change. Inhibition of HO-1 obviously restored PUN-induced M2 polarization and IL-10 secretion. In addition, phosphorylation of both Akt and STAT3 contributed to PUN-induced HO-1 expression. This study provided new insights into the mechanisms of PUN-mediated anti-inflammatory and anti-oxidant activities and provided new therapeutic strategies for inflammatory diseases.

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.