Tie2 Signaling Cooperates with TNF to Promote the Pro-Inflammatory Activation of Human Macrophages Independently of Macrophage Functional Phenotype

Angiopoietin (Ang) -1 and -2 and their receptor Tie2 play critical roles in regulating angiogenic processes during development, homeostasis, tumorigenesis, inflammation and tissue repair. Tie2 signaling is best characterized in endothelial cells, but a subset of human and murine circulating monocytes/macrophages essential to solid tumor formation express Tie2 and display immunosuppressive properties consistent with M2 macrophage polarization. However, we have recently shown that Tie2 is strongly activated in pro-inflammatory macrophages present in rheumatoid arthritis patient synovial tissue. Here we examined the relationship between Tie2 expression and function during human macrophage polarization. Tie2 expression was observed under all polarization conditions, but was highest in IFN-γ and IL-10 –differentiated macrophages. While TNF enhanced expression of a common restricted set of genes involved in angiogenesis and inflammation in GM-CSF, IFN-γ and IL-10 –differentiated macrophages, expression of multiple chemokines and cytokines, including CXCL3, CXCL5, CXCL8, IL6, and IL12B was further augmented in the presence of Ang-1 and Ang-2, via Tie2 activation of JAK/STAT signaling. Conditioned medium from macrophages stimulated with Ang-1 or Ang-2 in combination with TNF, sustained monocyte recruitment. Our findings suggest a general role for Tie2 in cooperatively promoting the inflammatory activation of macrophages, independently of polarization conditions.     

Prostaglandin E3 attenuates macrophage-associated inflammation and prostate tumour growth by modulating polarization

Alternative polarization of macrophages regulates multiple biological processes. While M1-polarized macrophages generally mediate rapid immune responses, M2-polarized macrophages induce chronic and mild immune responses. In either case, polyunsaturated fatty acid (PUFA)-derived lipid mediators act as both products and regulators of macrophages. Prostaglandin E₃ (PGE₃) is an eicosanoid derived from eicosapentaenoic acid, which is converted by cyclooxygenase, followed by prostaglandin E synthase successively. We found that PGE₃ played an anti-inflammatory role by inhibiting LPS and interferon-γ-induced M1 polarization and promoting interleukin-4-mediated M2 polarization (M2a). Further, we found that although PGE₃ had no direct effect on the growth of prostate cancer cells in vitro, PGE₃ could inhibit prostate cancer in vivo in a nude mouse model of neoplasia. Notably, we found that PGE₃ significantly inhibited prostate cancer cell growth in a cancer cell-macrophage co-culture system. Experimental results showed that PGE₃ inhibited the polarization of tumour-associated M2 macrophages (TAM), consequently producing indirect anti-tumour activity. Mechanistically, we identified that PGE₃ regulated the expression and activation of protein kinase A, which is critical for macrophage polarization. In summary, this study indicates that PGE₃ can selectively promote M2a polarization, while inhibiting M1 and TAM polarization, thus exerting an anti-inflammatory effect and anti-tumour effect in prostate cancer.     

Lipotoxic hepatocytes promote nonalcoholic fatty liver disease progression by delivering microRNA-9-5p and activating macrophages

M1-polarized macrophages are involved in chronic inflammatory diseases, including nonalcoholic fatty liver disease (NAFLD). However, the mechanisms responsible for the activation of macrophages in NAFLD have not been fully elucidated. This study aimed at investigating the physiological mechanisms by which extracellular vesicles (EVs)-encapsulated microRNA-9-5p (miR-9-5p) derived from lipotoxic hepatocytes might activate macrophages in NALFD. After blood sample and cell collection, EVs were isolated and identified followed by co-culture with macrophages. Next, the palmitic acid-induced cell and high fat diet-induced mouse NALFD models were established to explore the in vitro and in vivo effects of EVs-loaded miR-9-5p on NAFLD as evidenced by inflammatory cell infiltration and inflammatory reactions in macrophages. Additionally, the targeting relationship between miR-9-5p and transglutaminase 2 (TGM2) was identified using dual-luciferase reporter gene assay. miR-9-5p was upregulated in the NAFLD-EVs, which promoted M1 polarization of THP-1 macrophages. Furthermore, miR-9-5p could target TGM2 to inhibit its expression. Downregulated miR-9-5p in NAFLD-EVs alleviated macrophage inflammation and M1 polarization as evidenced by reduced levels of macrophage inflammatory factors, positive rates of CD86⁺ CD11b⁺, and levels of macrophage surface markers in vitro. Moreover, the effect of silencing of miR-9-5p was replicated in vivo, supported by reductions in TG, TC, AST and ALT levels and attenuated pathological changes. Collectively, lipotoxic hepatocytes-derived EVs-loaded miR-9-5p downregulated the expression of TGM2 and facilitated M1 polarization of macrophages, thereby promoting the progression of NAFLD. This highlights a potential therapeutic target for treating NAFLD.     

N6-methyladenosine demethylase FTO promotes M1 and M2 macrophage activation

Macrophage polarization is the driving force of various inflammatory diseases, especially those involved in M1/M2 imbalance. N6-methyladenosine (m⁶A) is the most prevalent internal mRNA modification in eukaryotes that affects multiple biological processes, including those involved developmental arrest and immune response. However, the role of m⁶A in macrophage polarization remains unclear. This study found that FTO silencing significantly suppressed both M1 and M2 polarization. FTO depletion decreased the phosphorylation levels of IKKα/β, IκBα and p65 in the NF-κB signaling pathway. The expression of STAT1 was downregulated in M1-polarized macrophages while the expression of STAT6 and PPAR-γ decreased in M2 polarization after FTO knockdown. The actinomycin D experiments showed that FTO knockdown accelerated mRNA decay of STAT1 and PPAR-γ. Furthermore, the stability and expression of STAT1 and PPAR-γ mRNAs increased when the m⁶A reader YTHDF2 was silenced. In conclusion, our results suggest that FTO knockdown inhibits the NF-κB signaling pathway and reduces the mRNA stability of STAT1 and PPAR-γ via YTHDF2 involvement, thereby impeding macrophage activation. These findings indicated a previously unrecognized link between FTO and macrophage polarization and might open new avenues for research into the molecular mechanisms of macrophage polarization-related diseases.     

Ginsenoside Rb1 enhances atherosclerotic plaque stability by skewing macrophages to the M2 phenotype

Atherosclerosis (AS) is characterized as progressive arterial plaque, which is easy to rupture under low stability. Macrophage polarization and inflammation response plays an important role in regulating plaque stability. Ginsenoside Rb1 (Rb1), one of the main active principles of Panax Ginseng, has been found powerful potential in alleviating inflammatory response. However, whether Rb1 could exert protective effects on AS plaque stability remains unclear. This study investigated the role of Rb1 on macrophage polarization and atherosclerotic plaque stability using primary peritoneal macrophages isolated from C57BL/6 mice and AS model in ApoE^?/? mice. In vitro, Rb1 treatment promoted the expression of arginase‐I (Arg‐I) and macrophage mannose receptor (CD206), two classic M2 macrophages markers, while the expression of iNOS (M1 macrophages) was decreased. Rb1 increased interleukin‐4 (IL‐4) and interleukin‐13 (IL‐13) secretion in supernatant and promoted STAT6 phosphorylation. IL‐4 and/or IL‐13 neutralizing antibodies and leflunomide, a STAT6 inhibitor attenuated the up‐regulation of M2 markers induced by Rb1. In vivo, the administration of Rb1 promoted atherosclerotic lesion stability, accompanied by increased M2 macrophage phenotype and reduced MMP‐9 staining. These data suggested that Rb1 enhanced atherosclerotic plaque stability through promoting anti‐inflammatory M2 macrophage polarization, which is achieved partly by increasing the production of IL‐4 and/or IL‐13 and STAT6 phosphorylation. Our study provides new evidence for possibility of Rb1 in prevention and treatment of atherosclerosis.     

Renal cell carcinoma-derived exosomes deliver lncARSR to induce macrophage polarization and promote tumor progression via STAT3 pathway

Tumor-derived exosomes play a pivotal role in regulating tumor progression by mediating crosstalk between tumor cells and immune cells such as macrophages within the tumor microenvironment. Macrophages can adopt two distinct polarization statuses and switch between M1 or M2 activation phenotypes in response to the different external stimuli. However, the role of tumor derived exosomes in the macrophage phenotypic switch and tumor development have not been elucidated in renal cell carcinoma (RCC). Here we found that high macrophage infiltration was associated with worse prognosis in RCC patients, therefore we propose our hypothesis that RCC derived exosomes might directly influence macrophage polarization and thus promote tumor progression. Both cell-based in vitro models and orthotopic transplantation in vivo tumor models were constructed and ELISA, flow cytometry, and macrophage functional studies were performed to investigate whether and how RCC-derived exosomes regulate macrophage polarization and tumor growth. The results found that these exosomes promote macrophage polarization, cytokine release, phagocytosis, angiogenesis, and tumor development. Further study revealed high amount of a recently discovered lncRNA called lncARSR in RCC-derived exosomes. Overexpression of lncARSR induced phenotypic and functional changes of macrophages in vitro and promoted tumor growth in vivo, while knockdown of lncARSR by siRNA disrupted the exosomes-mediated macrophage polarization. LncARSR interacts directly with miR-34/miR- 449 to increase STAT3 expression and mediate macrophage polarization in RCC cells. Together, RCC-derived exosomes facilitate the development of tumor through inducing macrophage polarization via transferring lncARSR, suggesting that RCC-derived exosomes, lncARSR and STAT3 are the potential therapeutic targets for treatment of RCC.     

MCTR1 enhances the resolution of lipopolysaccharide‐induced lung injury through STAT6‐mediated resident M2 alveolar macrophage polarization in mice

Acute respiratory distress syndrome (ARDS) is a fatal disease characterized by excessive infiltration of inflammatory cells. MCTR1 is an endogenously pro‐resolution lipid mediator. We tested the hypothesis that MCTR1 accelerates inflammation resolution through resident M2 alveolar macrophage polarization. The mice received MCTR1 via intraperitoneal administration 3 days after LPS stimulation, and then, the bronchoalveolar lavage (BAL) fluid was collected 24 hours later to measure the neutrophil numbers. Flow cytometry was used to sort the resident and recruited macrophages. Post‐treatment with MCTR1 offered dramatic benefits in the resolution phase of LPS‐induced lung injury, including decreased neutrophil numbers, reduced BAL fluid protein and albumin concentrations and reduced histological injury. In addition, the expression of the M2 markers Arg1, FIZZ1, Remlα, CD206 and Dectin‐1 was increased on resident macrophages in the LPS + MCTR1 group. Resident macrophage depletion abrogated the therapeutic effects of MCTR1, and reinjection of the sorted resident macrophages into the lung decreased neutrophil numbers. Finally, treatment with MCTR1 increased STAT6 phosphorylation. The STAT6 inhibitor AS1517499 abolished the beneficial effects of MCTR1. In conclusion, MCTR1 promotes resident M2 alveolar macrophage polarization via the STAT6 pathway to accelerate resolution of LPS‐induced lung injury.     

Major role of adipocyte prostaglandin E2 in lipolysis-induced macrophage recruitment

Obesity induces accumulation of adipose tissue macrophages (ATMs), which contribute to both local and systemic inflammation and modulate insulin sensitivity. Adipocyte lipolysis during fasting and weight loss also leads to ATM accumulation, but without proinflammatory activation suggesting distinct mechanisms of ATM recruitment. We examined the possibility that specific lipid mediators with anti-inflammatory properties are released from adipocytes undergoing lipolysis to induce macrophage migration. In the present study, we showed that conditioned medium (CM) from adipocytes treated with forskolin to stimulate lipolysis can induce migration of RAW 264.7 macrophages. In addition to FFAs, lipolytic stimulation increased release of prostaglandin E₂ (PGE₂) and prostaglandin D₂ (PGD₂), reflecting cytosolic phospholipase A₂ α activation and enhanced cyclooxygenase (COX) 2 expression. Reconstituted medium with the anti-inflammatory PGE₂ potently induced macrophage migration while different FFAs and PGD₂ had modest effects. The ability of CM to induce macrophage migration was abolished by treating adipocytes with the COX2 inhibitor sc236 or by treating macrophages with the prostaglandin E receptor 4 antagonist AH23848. In fasted mice, macrophage accumulation in adipose tissue coincided with increases of PGE₂ levels and COX1 expression. Collectively, our data show that adipocyte-originated PGE₂ with inflammation suppressive properties plays a significant role in mediating ATM accumulation during lipolysis.     

Molecular mechanism for adiponectin-dependent M2 macrophage polarization: link between the metabolic and innate immune activity of full-length adiponectin

The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.     

IL-27 affects helper T cell responses via regulation of PGE2 production by macrophages

IL-27 is a heterodimeric cytokine that regulates both innate and adaptive immunity. The immunosuppressive effect of IL-27 largely depends on induction of IL-10-producing Tr1 cells. To date, however, effects of IL-27 on regulation of immune responses via mediators other than cytokines remain poorly understood. To address this issue, we examined immunoregulatory effects of conditional medium of bone marrow-derived macrophages (BMDMs) from WSX-1 (IL-27Rα)-deficient mice and found enhanced IFN-γ and IL-17A secretion by CD4⁺ T cells as compared with that of control BMDMs. We then found that PGE₂ production and COX-2 expression by BMDMs from WSX-1-deficient mice was increased compared to control macrophages in response to LPS. The enhanced production of IFN-γ and IL-17A was abolished by EP2 and EP4 antagonists, demonstrating PGE₂ was responsible for enhanced cytokine production. Murine WSX-1-expressing Raw264.7 cells (mWSX-1-Raw264.7) showed phosphorylation of both STAT1 and STAT3 in response to IL-27 and produced less amounts of PGE₂ and COX-2 compared to parental RAW264.7 cells. STAT1 knockdown in parental RAW264.7 cells and STAT1-deficiency in BMDMs showed higher COX-2 expression than their respective control cells. Collectively, our result indicated that IL-27/WSX-1 regulated PGE₂ secretion via STAT1–COX-2 pathway in macrophages and affected helper T cell response in a PGE₂-mediated fashion.     

Hypoxic glioma-derived exosomes promote M2-like macrophage polarization by enhancing autophagy induction

Exosomes participate in intercellular communication and glioma microenvironment modulation, but the exact mechanisms by which glioma-derived exosomes (GDEs) promote the generation of the immunosuppressive microenvironment are still unclear. Here, we investigated the effects of GDEs on autophagy, the polarization of tumor-associated macrophages (TAMs), and glioma progression. Compared with normoxic glioma-derived exosomes (N-GDEs), hypoxic glioma-derived exosomes (H-GDEs) markedly facilitated autophagy and M2-like macrophage polarization, which subsequently promoted glioma proliferation and migration in vitro and in vivo. Western blot and qRT-PCR analyses indicated that interleukin 6 (IL-6) and miR-155-3p were highly expressed in H-GDEs. Further experiments showed that IL-6 and miR-155-3p induced M2-like macrophage polarization via the IL-6-pSTAT3-miR-155-3p-autophagy-pSTAT3 positive feedback loop, which promotes glioma progression. Our study clarifies a mechanism by which hypoxia and glioma influence autophagy and M2-like macrophage polarization via exosomes, which could advance the formation of the immunosuppressive microenvironment. Our findings suggest that IL-6 and miR-155-3p may be novel biomarkers for diagnosing glioma and that treatments targeting autophagy and the STAT3 pathway may contribute to antitumor immunotherapy.Subject terms: Cancer microenvironment, Autophagy     

Schistosomal-derived lysophosphatidylcholine triggers M2 polarization of macrophages through PPARγ dependent mechanisms

Mansonic schistosomiasis is a disease caused by the trematode Schistosoma mansoni, endemic to tropical countries. S. mansoni infection induces the formation of granulomas and potent polarization of Th2-type immune response. There is great interest in understanding the mechanisms used by this parasite that causes a modulation of the immune system. Recent studies from our group demonstrated that lipids of S. mansoni, including lysophosphatidylcholine (LPC) have immunomodulatory activity. In the present study, our aim was to investigate the role of lipids derived from S. mansoni in the activation and polarization of macrophages and to characterize the mechanisms involved in this process. Peritoneal macrophages obtained from wild type C57BL/6mice or bone marrow derived macrophages were stimulated in vitro with lipids extracted from adult worms of S. mansoni. We demonstrated that total schistosomal-derived lipids as well as purified LPC induced alternatively activated macrophages/M2 profile observed by increased expression of arginase-1, mannose receptor, Chi3l3, TGFβ and production of IL-10 and PGE₂ 24 h after stimulation. The involvement of the nuclear receptor PPARγ in macrophage response against LPC was investigated. Through Western blot and immunofluorescence confocal microscopy we demonstrated that schistosomal-derived LPC induces increased expression of PPARγ in macrophages. The LPC-induced increased expression of arginase-1 were significantly inhibited by the PPAR-γ antagonist GW9662. Together, these results demonstrate an immunomodulatory role of schistosomal-derived LPC in activating macrophages to a profile of the type M2 through PPARγ-dependent mechanisms, indicating a novel pathway for macrophage polarization triggered by parasite-derived LPC with potential implications to disease pathogenesis.