Plasminogen Activator Inhibitor-1 Promotes the Recruitment and Polarization of Macrophages in Cancer.
ABSTRACT: Plasminogen activator inhibitor-1 (PAI-1) has a pro-tumorigenic function via its pro-angiogenic and anti-apoptotic activities. Here, we demonstrate that PAI-1 promotes the recruitment and M2 polarization of monocytes/macrophages through different structural domains. Its LRP1 interacting domain regulated macrophage migration, while its C-terminal uPA interacting domain promoted M2 macrophage polarization through activation of p38MAPK and nuclear factor ?B (NF-?B) and induction of an autocrine interleukin (IL)-6/STAT3 activation pathway. We then show in several experiments in mice that expression of PAI-1 is associated with increased tumorigenicity, increased presence of M2 macrophages, higher levels of IL-6, and increased STAT3 phosphorylation in macrophages. Strong positive correlations between PAI-1, IL-6, and CD163 (M2 marker) expression were also found by meta-analysis of transcriptome data in many human cancers. Altogether, these data provide evidence for a mechanism explaining the paradoxical pro-tumorigenic function of PAI-1 in cancer.
Project description:Resident macrophages in the tumor microenvironment exert a dual role in tumor progression. So far, the mechanism of intratumoral macrophage generation is still largely unknown. In the present study, the importance of macrophages in the pro-tumor role of gastric cancer-derived mesenchymal stromal cells (GC-MSCs) was observed in a mouse xenograft model with macrophage depletion. In gastric cancer tissues, high expression levels of Ym-1, Fizz-1, arginase-1, and CCR-2, as well as a low expression level of iNOS, were verified, and co-localization of GC-MSCs and tumor-associated macrophages (TAMs) was observed by dual immunofluorescence histochemistry. TAMs isolated from gastric cancer tissues predominantly displayed an M2 phenotype. In a co-culture system, the contribution of GC-MSCs to M2 polarization of macrophages was confirmed by the M2-related protein expression, M2-like immunophenotype and cytokine profile of GC-MSC-primed macrophages in vitro. Blockade of IL-6/IL-8 by neutralizing antibodies significantly attenuated the promoting effect of GC-MSCs on M2-like macrophage polarization via the JAK2/STAT3 signaling pathway. In addition, GC-MSC-primed macrophages promoted the migration and invasion of gastric cancer cells, and the process of EMT in gastric cancer cells was significantly enhanced by GC-MSC-primed macrophage treatment. Our study showed that tumor-promoting GC-MSCs contribute to M2 macrophage polarization within the gastric cancer niche through considerable secretion of IL-6 and IL-8. These GC-MSC-primed macrophages can subsequently prompt gastric cancer metastasis via EMT promotion in gastric cancer cells.
Project description:Macrophages contribute to the pathogenesis of rheumatoid arthritis (RA). They can display different states of activation or "polarization," notably the so-called inflammatory "M1" and the various alternative "M2" polarizations, characterized by distinct functions. Data regarding the effects of RA anti-cytokine biological disease-modifying anti-rheumatic drugs (bDMARDs) on macrophage polarization are scarce. We aimed to assess in vitro modulation of macrophage polarization by bDMARDs targeting pro-inflammatory cytokines in RA. We generated monocyte derived macrophages using blood samples from 20 RA patients with active RA and 30 healthy controls. We evaluated in vitro the impact on M1 inflammatory macrophages of: etanercept (ETA), adalimumab (ADA), certolizumab (CZP), tocilizumab (TCZ), and rituximab (RTX). We assessed the impact on macrophage polarization using flow cytometry and RTqPCR to study the expression of surface markers and perform functional studies of cytokine production, phagocytosis, and negative feedback control of inflammation. Among evaluated bDMARDs, anti-TNF agents modulated the polarization of inflammatory macrophages by decreasing inflammatory surface markers (CD40, CD80) and favoring alternative markers (CD16, CD163, MerTK). Anti-TNF agents also induced alternative functions in macrophages activated in inflammatory condition with (i) the inhibition of inflammatory cytokines (TNF, IL-6, IL-12), (ii) an increase in phagocytosis. These findings were mechanistically related to an increase in early IL-10 production, responsible for higher negative feedback control of inflammation involving SOCS3 and Gas6. This IL-10 effect was STAT3-dependent. Anti-TNF agents not only inhibit in vitro inflammatory functions of macrophages, but also favor resolution of inflammation through polarization toward alternative features specifically involving the IL-10/STAT3 axis.
Project description:The Wnt/?-catenin pathway is crucial in normal development and throughout life, but aberrant activation of this pathway has been linked to kidney fibrosis, although the mechanisms involved remain incompletely determined. Here, we investigated the role of Wnt/?-catenin in regulating macrophage activation and the contribution thereof to kidney fibrosis. Treatment of macrophages with Wnt3a exacerbated IL-4- or TGF?1-induced macrophage alternative (M2) polarization and the phosphorylation and nuclear translocation of STAT3 in vitro Conversely, inhibition of Wnt/?-catenin signaling prevented these IL-4- or TGF?1-induced processes. In a mouse model, induced deletion of ?-catenin in macrophages attenuated the fibrosis, macrophage accumulation, and M2 polarization observed in the kidneys of wild-type littermates after unilateral ureter obstruction. This study shows that activation of Wnt/?-catenin signaling promotes kidney fibrosis by stimulating macrophage M2 polarization.
Project description:Macrophages are a major component of inflammatory and tumor microenvironment. We previously reported that embelin suppresses colitis-associated tumorigenesis. Here, the role of macrophage targeting in the anti-inflammatory and anti-tumor properties of embelin was investigated. By using colitis-associated cancer (CAC) model, we demonstrated that embelin significantly depleted colon macrophages by blocking their recruitment. Moreover, embelin attenuated M2-like polarization of macrophages within the tumor microenvironment and eliminated their tumor-promoting functions during the development of CAC. Embelin potently inhibited NF-?B signaling in macrophages and decreased the production of key pro-inflammatory cytokines and tumorigenic factors involved in CAC, such as TNF?, IL-6 and COX-2. In addition, embelin directly reduced the polarization of M2 macrophages in vitro even in the presence of Th2 cytokines. These results suggested that targeting macrophages is, at least in part, responsible for the anti-tumor activity of embelin in CAC. Our observations strengthen the rationale for future validation of embelin in the prevention and treatment of CAC.
Project description:Macrophages are key participants in melanoma growth and survival. In general, macrophages can be classified as M1 or M2 activation phenotypes. Increasing evidence demonstrates that melanoma exosomes also facilitate tumor survival and metastasis. However, the role of melanoma exosomes in directly influencing macrophage function is poorly understood. Herein, we investigated the hypothesis that natural melanoma exosomes might directly influence macrophage polarization. To explore this hypothesis, ELISA, RT-qPCR, and macrophage functional studies were performed in vitro using an established source of melanoma exosomes (B16-F10). ELISA results for melanoma exosome induction of common M1 and M2 cytokines in RAW 264.7 macrophages, revealed that melanoma exosomes do not polarize macrophages exclusively in the M1 or M2 direction. Melanoma exosomes induced the M1 and M2 representative cytokines TNF-? and IL-10 respectively. Further assessment, using an RT-qPCR array with RAW 264.7 and primary macrophages, confirmed and extended the ELISA findings. Upregulation of markers common to both M1 and M2 polarization phenotypes included CCL22, IL-12B, IL-1?, IL-6, i-NOS, and TNF-?. The M2 cytokine TGF-? was upregulated in primary but not RAW 264.7 macrophages. Pro-tumor functions have been attributed to each of these markers. Macrophage functional assays demonstrated a trend toward increased i-NOS (M1) to arginase (M2) activity. Collectively, the results provide the first evidence that melanoma exosomes can induce a mixed M1 and M2 pro-tumor macrophage activation phenotype.
Project description:Macrophages acquire distinct phenotypes during tissue stress and inflammatory responses. Classically, macrophages are categorized into two different subsets named inflammatory M1 and anti-inflammatory M2 macrophages. We herein identified a unique pathogenic macrophage subpopulation, named M17, driven by IL-23 with a distinct gene expression profile including cytokines and membrane molecules. In contrast to M1 and M2-polarized macrophages, M17 macrophages express high levels of CXCR5 on the cell surface and produce large amounts of IL-17A, IL-22 and IFN-γ. IL-23 induces IL-17A expression in macrophages through the signal transducer and activator of transcription 3 (STAT3)-retinoid related orphan receptor-γ T (RORγT) pathway, while induces IFN-γ through T-bet. Importantly, IL-23-induced M17 macrophage polarization promotes the pathogenesis of psoriasis in vivo. The characteristics and significances of M17 macrophage subpopulation in the physiological status and pathogenesis caused by infections, tumors and graft rejection needs to be explored. Overall design: To further demonstrate whether M17 macrophage polarization represents a distinct polarization of macrophages, we detected gene expression profiles of M1, M2 and M17 polarized macrophages by microarray methods.
Project description:Gene delivery from biomaterials can create an environment that promotes and guides tissue formation. However, the immune response induced upon biomaterial implantation can be detrimental to tissue regeneration. Macrophages play a central role in mediating early phases of this response, and functional "polarization" of macrophages towards M1 (inflammatory) or M2 (anti-inflammatory) phenotypes may bias the local immune state at the implant site. Since gene delivery from biomaterial scaffolds can confer transgene expression in macrophages in vivo, we investigated whether transduction of macrophages with an IL-10 encoding lentivirus can (1) induce macrophage polarization toward an M2 phenotype even in an pro-inflammatory environment, and (2) prevent a shift in polarization from M2 to M1 following exposure to pro-inflammatory stimuli. IL-10 lentivirus delivery to pre-polarized M1 macrophages reduced TNF-? production 1.5-fold when compared to cells treated with either a control virus or a bolus delivery of recombinant IL-10 protein. IL-10 lentivirus delivery to naïve macrophages reduced the amount of TNF-? produced following an inflammatory challenge by 2.5-fold compared to cells treated with both the control virus and recombinant IL-10. At a mechanistic level, IL-10 lentivirus delivery mediated sustained reduction in NF-?B activation and, accordingly, reduced transcription of TNF-?. In sum, lentiviral delivery of IL-10 to macrophages represents a promising strategy for directing and sustaining macrophage polarization towards an M2 phenotype in order to promote local immune responses that facilitate tissue engineering.
Project description:BACKGROUND:Tumor-associated macrophages (TAMs) in the tumor microenvironment influence tumor initiation, invasion and metastasis. Several studies have shown that Wnt5a is mainly expressed in the tumor stroma, especially in TAMs. However, whether Wnt5a regulates the polarization and biological function of TAMs in colorectal cancer (CRC) is incompletely understood. METHODS:Immunofluorescence staining was performed to detect CD68 and Wnt5a expression in colorectal tissues from patients (63 CRC specimens VS 20 normal tissues). RT-qPCR, flow cytometry, ELISA and inhibitors were carried out to explore the role of Wnt5a in the polarization of TAMs. Clone formation and transwell assays were performed to determine the effects of Wnt5a-treated macrophages on tumor proliferation, migration and invasion in vitro. Finally, a xenograft model was applied to confirm the effects of Wnt5a+ TAMs on CRC tumorigenesis. RESULTS:We found that high Wnt5a+CD68+/CD68+ TAMs ratio was significantly associated with poor prognosis in CRC patients and Wnt5a+ TAM was an M2-like TAM subtype. Subsequently, we found that Wnt5a induced macrophages to secrete IL-10, which then acted as an autocrine cytokine to induce M2 polarization of these macrophages. IL-10 neutralizing antibody completely reversed the pro-M2 effect of Wnt5a. Mechanistically, the CaKMII-ERK1/2-STAT3 pathway was required for Wnt5a-mediated IL-10 expression in macrophages. Furthermore, Wnt5a-induced M2 macrophages promoted CRC cells proliferation, migration and invasion; knockdown of Wnt5a in TAMs significantly impaired the pro-tumor functions of TAMs. CONCLUSIONS:Our data indicate that Wnt5a could induce M2 polarization of TAMs by regulating CaKMII-ERK1/2-STAT3 pathway-mediated IL-10 secretion, ultimately promoting tumor growth and metastasis of CRC.
Project description:Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern, whose effect on macrophages is not entirely elucidated. Here we identified that eCIRP promotes macrophage endotoxin tolerance. Septic mice had higher serum levels of eCIRP; this was associated with a reduced ex vivo immune response of their splenocytes to LPS. Pretreatment of macrophages with recombinant murine CIRP (rmCIRP) resulted in a tolerance to LPS stimulation as demonstrated by a reduction of TNF-? production. We found that eCIRP increased phosphorylated STAT3 (p-STAT3) in macrophages. A STAT3 inhibitor, Stattic, rescued macrophages from rmCIRP-induced tolerance by restoring the release of TNF-? in response to LPS stimulation. We discovered strong binding affinity between eCIRP and IL-6 receptor (IL-6R) as revealed by Biacore, fluorescence resonance energy transfer (FRET), and their colocalization in macrophages by immunostaining assays. Blockade of IL-6R with its neutralizing Ab inhibited eCIRP-induced p-STAT3 and restored LPS-stimulated TNF-? release in macrophages. Incubation of macrophages with rmCIRP skewed them toward an M2 phenotype, while treatment with anti-IL-6R Ab prevented rmCIRP-induced M2 polarization. Thus, we have demonstrated that eCIRP activates p-STAT3 via a novel receptor, IL-6R, to promote macrophage endotoxin tolerance. Targeting eCIRP appears to be a new therapeutic option to correct immune tolerance in sepsis.
Project description:Tumor-associated macrophages (TAMs) are a major component of leukocytic infiltrate in tumors, which facilitates tumor progression and promotes inflammation. TGF-? promotes the differentiation of non-activated macrophages into a TAM-like (M2-like) phenotype; however, the underlying mechanisms are not clear. In this study, we found that TGF-? induces a M2-like phenotype characterized by up-regulation of the anti-inflammatory cytokine IL-10, and down-regulation of the pro-inflammatory cytokines TNF-? and IL-12. In human THP-1 macrophages, overexpression of SNAIL caused M2-like differentiation by inhibiting pro-inflammatory cytokine release and promoting the expression of M2-specific markers. By contrast, SNAIL knockdown promoted M1 polarization through up-regulation of pro-inflammatory cytokines and abolished TGF-?-mediated M2-polarization of THP-1 macrophages. The SMAD2/3 and PI3K/AKT signaling pathways were crucial for TGF-?-induced SNAIL overexpression in THP-1 cells. These findings suggest that TGF-? skews macrophage polarization towards a M2-like phenotype via SNAIL up-regulation, and blockade of TGF-?/SNAIL signaling restores the production of pro-inflammatory cytokines. This study provides new understanding of the role of SNAIL in M2 polarization of macrophages, and suggests a potential therapeutic target for antitumor immunity.