Lnc-CHRM4-2:1 Inhibits M2 Polarization and Efferocytosis of Macrophages by Downregulating MerTK and SLC2A1 in Rheumatoid Arthritis.

Our current study is conducted with intention to explore the regulatory mechanism of mesenchymal stem cell (MSC)-derived extracellular vesicle (EV)-miR-744-5p in glioma. Expression patterns of TGFB1, TGFBR1, and miR-744-5p were determined. EVs were isolated from human MSCs, which were characterized. Then, macrophages were co-cultured with MSCs with ectopic miR-744-5p expression to explore its role in cell proliferation, invasion, and migration capabilities. A nude mouse model of glioma xenograft was developed to observe the tumorigenesis and metastasis ability of glioma in vivo. TGFB1 and TGFBR1 were upregulated in glioma. TGFB1 promoted M2 polarization of macrophages through theMAPK signaling, thereby promoting the progression of glioma. MSC-EVs suppressed TGFB1 expression in macrophages and inhibited M2 polarization of macrophages. MSC-EVs-miR-744-5p/TGFB1/MAPK axis inhibited M2 polarization of macrophages and reduced the malignant phenotypes of glioma cells. In vivo experiments verified that MSC-EVs-miR-744-5p inhibited the polarization of macrophage M2 and prevented glioma progression. Taken together, MSC-EVs-miR-744-5p may suppress the MAPK signaling activity by downregulating TGFB1, and then inhibit polarization of macrophages M2, thereby preventing the progression of glioma. Graphical Headlights 1. TGFB1 promotes the M2 polarization of macrophages via the MAPK signaling. 2. miR-744-5p carried by MSC-EVs targets and inhibits TGFB1. 3. MSC-EV-miR-744-5p inhibits M2 polarization of macrophages to prevent glioma progression. 4. miR-744-5p loaded by MSC-EVs may be a preventive strategy against glioma.

Acute lung injury (ALI), recognized as a prevalent and severe respiratory disorder, represents a critical medical condition. During ALI, Vascular Endothelial Growth Factor A (VEGFA)-mediated M1/M2 macrophage polarization is crucial, yet its specific regulatory mechanisms remain unclear. THP-1 cells were treated with lipopolysaccharide (LPS)/interferon-γ (IFN-γ). VEGFA expression in the serum of ALI patients was identified using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Flow cytometry was employed to identify the expression of M1 and M2 polarization markers. Inflammatory cytokines were detected by ELISA. Bioinformatics was employed to predict the m6A sites on VEGFA mRNA, and Western blot was conducted to examine the protein levels. Methylated RNA immunoprecipitation (MeRIP), and RIP assays were used to verify the modification and binding between RBM15 and VEGFA. Actinomycin D assay was performed to evaluate the mRNA stability. An ALI mouse model was established to assess the in vivo role of the RBM15/VEGFA axis. HE staining was used to assess the lung tissue injury of mice. Meanwhile, myeloperoxidase (MPO) activity was measured using colorimetry. VEGFA exhibited elevated expression in the serum of ALI patients and LPS/IFN-γ-induced THP-1/M0 cells. Additionally, VEGFA inhibitor and silencing VEGFA restrained M1 polarization and contributed to M2 polarization of LPS/IFN-γ-induced THP-1/M0 cells accompanied by the reduction in the levels of pro-inflammatory cytokines (TNF-α, IL-12, IL-6, and IL-1β) and the relative increase in the anti-inflammatory cytokine (IL-10). Moreover, VEGFA expression was promoted by RBM15 through m6A modification. The RBM15/VEGFA axis promoted the M1 polarization while inhibiting the M2 polarization of LPS/IFN-γ-induced THP-1/M0 cells. ALI was alleviated by inhibiting the RBM15/VEGFA axis in vivo. RBM15 enhanced VEGFA expression through m6A modification, thereby promoting M1 polarization, inhibiting M2 polarization of macrophages, and facilitating the progression of ALI.

Objective To explore the mechanism of ubiquitin specific peptidase 21 (USP21) increasing the stability of forkhead box protein M1 (FOXM1) and promoting M2 polarization of macrophages in endometriosis (EM). Methods Eutopic endometrial stromal cells (EESC) collected from patients and normal endometrial stromal cells (NESC) from routine health examiners were cultured in vitro, and the expression levels of USP21 and FOXM1 were detected using RT-qPCR and Western blot. EESCs were co-cultured with macrophages. M1 polarization markers of interleukin 6 (IL-6) and CXC chemokine ligand 10 (CXCL10) and M2 polarization markers of CD206 and fibronectin 1 (FN1) were tested using RT-qPCR. M2 marker CD206 was further detected by flow cytometry. IL-6, tumor necrosis factor-alpha (TNF-α), IL-10, and transforming growth factor-beta (TGF-β) levels in cell supernatant were detected by ELISA. Co-immunoprecipitation was used to assess the interaction between USP21 and FOXM1, and the ubiquitination level of FOXM1. FOXM1 protein stability was detected through cycloheximide (CHX) assay. Results USP21 and FOXM1 expression levels in the EESC group were significantly increased compared with those in the NESC group; compared with the NESC + M0 group, the EESC + M0 group showed no significant difference in the expression of M1 polarization markers (IL-6 and CXCL10), but increased expression of M2 polarization markers (CD206 and FN1), along with notably increased number of M2 macrophages; there was no significant difference in IL-6 and TNF-α levels, but increased levels of IL-10 and TGF-β in the cell supernatant. The above findings indicated that the deubiquitinase USP21 was highly expressed in EM, promoting M2 polarization of macrophages. Knocking down USP21 or FOXM1 can inhibit M2 polarization of EM macrophages. USP21 interacted with FOXM1 in EESC, leading to a decrease in FOXM1 ubiquitination level and an increase in FOXM1 protein stability. Overexpression of FOXM1 reversed the inhibitory effect of knocking down USP21 on M2 polarization of EM macrophages. Conclusion The deubiquitinase USP21 interacts with FOXM1 to increase the stability of FOXM1 and promote M2 polarization of EM macrophages.

BackgroundSphingosine kinase 1 (SphK1) has distinct roles in the activation of Kupffer cells (KCs) and hepatic stellate cells (HSCs) in liver fibrosis. This study aims to examine the role of...

BACKGROUNDM1 polarization of macrophages is an important pathological process in myocardial ischemia reperfusion injury, which is the major obstacle for the treatment of acute myocardial infarction. Currently, the strategies and mechanisms of inhibiting M1 polarization are poorly explored. This study aims to investigate the role of soluble death receptor 5-Fc (sDR5-Fc) in regulating M1 polarization of macrophages under extreme conditions and explore the mechanisms from the aspect of glycolysis.METHODSExtreme conditions were induced in RAW264.7 cells. Real-time quantitative polymerase chain reaction and western blot were used to detect the expression of mRNA and proteins, respectively. Cell counting kit-8 was used to investigate the proliferation activity of cells. Expression levels of inflammatory cytokines were determined by enzyme-linked immunosorbent assay.RESULTSWe found that sDR5-Fc rescues the proliferation of macrophages under extreme conditions, including nutrition deficiency, excessive peroxide, and ultraviolet irradiation. In addition, administration of sDR5-Fc inhibits the M1 polarization of macrophages induced by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ), as the expression of M1 polarization markers CD86, CXC motif chemokine ligand 10, matrix metalloproteinase 9, and tumor necrosis factor-α, as well as the secretion of inflammatory factors interleukin (IL)-1β and IL-6, were significantly decreased. By further investigation of the mechanisms, the results showed that sDR5-Fc can recover the LPS and IFN-γ induced pH reduction, lactic acid elevation, and increased expression of hexokinase 2 and glucose transporter 1, which were markers of glycolysis in macrophages.CONCLUSIONSsDR5-Fc inhibits the M1 polarization of macrophages by blocking the glycolysis, which provides a new direction for the development of strategies in the treatment of myocardial ischemia reperfusion injury.

Decidual macrophages (dMϕs) play critical roles in the establishment of microhomeostasis at the maternal-fetal interface during pregnancy. Impaired macrophage polarization during early pregnancy is associated with recurrent spontaneous abortion (RSA). In the present study, the SEC5 expression level was found to be significantly decreased in primary dMϕs of patients with RSA, and downregulation of SEC5 expression inhibited M2 polarization and STAT6 phosphorylation, whereas SEC5 overexpression in the Mϕs promoted M2 polarization and STAT6 phosphorylation in vitro. We subsequently found that SEC5 interacted with STAT6 in THP-1-derived Mϕs. The abundance of phosphorylated STAT6 (pSTAT6) protein was obviously increased, with a predominant distribution in the nucleus, after M2 polarization of Mϕs, and SEC5 protein was colocalized with pSTAT6. Moreover, a significantly reduced pSTAT6 expression level was observed in the dMϕs of patients with RSA. M2 polarization of Mϕs showed a stimulatory effect on the proliferation and invasion of human extravillous trophoblasts (EVTs) in vitro, and downregulation of SEC5 expression in Mϕs effectively reversed this effect. In a mouse model of LPS-induced early pregnancy loss, the uterine SEC5 expression level and the number of M2-Mϕs at the maternal-fetal interface were significantly reduced. More interestingly, heterozygous SEC5-deficient (SEC5−/+) pregnant mice were more sensitive to LPS-induced pregnancy loss. Taken together, these data indicate that SEC5 participates in the regulation of M2 polarization of Mϕs by interacting with STAT6 and that decreased SEC5 expression inhibits the M2 polarization of dMϕs and results in early pregnancy loss by interfering with the physical activities of EVTs and immunotolerance at the maternal-fetal interface.

The effects of mesenchymal stem cells (MSCs) on different types of diseases are controversial, and the inner mechanisms remain unknown, which retards the utilization of MSCs in disease therapy. In this study, we aimed to elucidate the mechanisms of MSCs‐extracellular vesicles (EVs) carrying transforming growth factor‐beta 1 (TGF‐β1) in M2 polarization in mouse macrophages via the microRNA‐132 (miR‐132)/E3 ubiquitin ligase myc binding protein 2 (Mycbp2)/tuberous sclerosis complex 2 (TSC2) axis. Mouse MSCs were isolated for adipogenic and osteogenic induction, followed by co‐culture with mouse macrophages RAW264.7. Besides, mouse macrophages RAW264.7 were co‐cultured with MSCs‐EVs in vitro, where the proportion of macrophages and inflammation were detected by flow cytometry and ELISA. The experimental data revealed that MSCs‐EVs promoted M2 polarization of macrophages, and elevated interleukin (IL)‐10 expression and inhibited levels of IL‐1β, tumour necrosis factor (TNF)‐α and IL‐6. MSC‐EV‐treated macrophages RAW264.7 increased TGF‐β1 expression, thus elevating miR‐132 expression. MiR‐132 directly bound to Mycbp2, as confirmed by luciferase activity assay. Meanwhile, E3 ubiquitin ligase Mycbp2 could ubiquitinate TSC2 protein. Furthermore, silencing TGF‐β1 inhibited M2 polarization of MSC‐EV‐treated macrophages. Taken conjointly, this study provides evidence reporting that MSC‐secreted EVs carry TGF‐β1 to promote M2 polarization of macrophages via modulation of the miR‐132/Mycbp2/TSC2 axis.

Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by high morbidity and mortality rates. Sepsis-induced ARDS involves excessive inflammatory responses, which are modulated by macrophages. This study aimed to elucidate the effect of Recombinant Mouse IL-27 Protein on macrophage ferroptosis and polarization, as well as its impact on sepsis-induced ARDS. A cecal ligation and puncture (CLP)-induced sepsis model was established using wild-type (WT) or IL27R-/- mice. Then, the mice were randomly divided into 4 groups: a control group, a CLP group, an IL-27 + CLP combination group, and an IL-27, CLP, and Oltipraz combination group. RAW 264.7 cells and BMDMs were used to further determine the role and mechanism of IL-27 in vitro. In vitro, IL-27 alone did not alter the expression of proteins linked to the ferroptosis pathway or macrophage polarization. Contrastingly, the combination of IL-27 with LPS further amplified LPS-induced alterations in the ferroptosis pathway, thereby promoting macrophage M1 polarization and inhibiting M2 polarization. Additionally, IL-27 + LPS increased ROS levels in macrophages. A sepsis-induced ARDS mouse model was then established via CLP. In vivo, IL-27 exacerbated CLP-induced lung injury in WT mice. Additionally, it decreased the expression levels of ferroptosis-related proteins (Nrf2, HO-1, GPX4) and increased those of Ptgs2 in the lung tissue of septic mice. Besides, GSH and SOD levels in lung tissue were also reduced. Moreover, IL-27 also promoted M1 polarization and inhibited M2 polarization in macrophages. In IL27R-/- mice, the effects of IL-27 were abrogated. Oltipraz inhibited IL-27-induced changes by up-regulating Nrf2 expression. Overall, this present study demonstrated that the combination of IL-27 and LPS-induced macrophage ferroptosis, promoted macrophage M1 polarization, and inhibited M2 polarization by inhibiting the Nrf2/HO-1 pathway. Oltipraz may alleviate ARDS-related lung injury by up-regulating Nrf2 expression and concurrently inhibiting macrophage ferroptosis.

Rheumatoid arthritis (RA) is characterized by the massive infiltration of various chronic inflammatory cells in synovia. In synovial fluid of patients with RA, M1 macrophages are dominant among all subtypes of macrophages, the mechanisms of macrophages polarization imbalance in RA has not been fully illuminated. The prostaglandin E2 (PGE2) augments M2 polarization in part via the cyclic adenosine monophosphate (cAMP)-cyclic AMP responsive element binding (CREB) signaling. However, previous study found constant stimulus of PGE2 on fibroblast-like synovial cells of adjuvant arthritis rats induced the decrease of cAMP, which is primarily caused by G protein-coupled receptor kinase 2 (GRK2)-induced EP4 over- desensitization. Whether GRK2 mediated-EP4 over-desensitization reduces the level of cAMP and inhibits M2 polarization in RA is unclear. Here we observed M1 macrophages were dominant in peritoneal macrophages (PMs), bone-marrow-derived macrophages (BMMs) and synovial macrophages of collagen-induced arthritis (CIA) mice. PGE2 stimulated M2 polarization via the EP4-cAMP-CREB in normal mice, while failed to promote M2 polarization in the PMs of CIA mice. Further, we found the EP4 over-desensitization stimulated by PGE2 induced abnormal PGE2-cAMP-CREB signaling as well as the imbalance of macrophage polarization. Targeted disruption of GRK2 in Raw264.7 (RAW) through GRK2 siRNA or CRISPR/Cas9 downregulated the M1 macrophage markers, upregulated the M2 macrophage markers and the EP4 membrane localization. The reduced M1/M2 ratio and increased p-CREB expression were observed in BMMs and PMs of GRK2+/− mice. This study highlighted a novel role of GRK2 in regulating macrophages function in RA and provided new idea for precision treatment of RA.

Osteosarcoma (OS) is a malignant tumor whose chemoresistance severely compromises therapeutic efficacy. This study aims to investigate the molecular mechanism by which TIMM23 mediates M2 polarization of macrophages through mitophagy and regulates TIMM23-PARGP1 fusion gene expression and chemoresistance in OS. Single-cell transcriptomic analysis revealed a strong interaction between macrophages and tumor cells. By integrating bulk RNA sequencing data and weighted gene co-expression network analysis (WGCNA) co-expression network analysis, we identified TIMM23 as a key gene associated with macrophage polarization. Using STAR-Fusion, we further detected the TIMM23-PARGP1 fusion gene, which was validated via fluorescence in situ hybridization (FISH). CRISPR/Cas9 was employed to generate TIMM23-knockout macrophages, and a lentiviral system was used to create TIMM23-overexpressing macrophages. Flow cytometry demonstrated that TIMM23 promotes M2 polarization, while confocal and transmission electron microscopy confirmed its role in regulating mitophagy in M2 macrophages. These findings indicate that TIMM23 promotes M2 polarization through mitophagy modulation. Next, a co-culture model of macrophages and OS cells was established. MTT, Cell Counting Kit-8 (CCK-8), EdU, Transwell, and TUNEL assays were performed to evaluate tumor cell behavior and chemosensitivity. The results showed that TIMM23-induced M2 polarization upregulated TIMM23-PARGP1 expression in OS cells, thereby enhancing their proliferation, migration, and invasion while inhibiting apoptosis and reducing the effectiveness of chemotherapeutic agents. In vivo experiments further confirmed the role of TIMM23 in promoting M2 polarization and fusion gene expression, leading to increased chemoresistance and tumor growth. In conclusion, TIMM23 enhances OS chemoresistance and tumor progression by promoting M2 macrophage polarization via mitophagy and upregulating TIMM23-PARGP1 fusion gene expression.Graphical . Molecular mechanism diagram illustrating the involvement of TIMM23 in mitophagy-mediated M2 polarization of macrophages and the impact of TIMM23-PARGP1 fusion gene on chemoresistance in OS (Created with BioRender.com).

miR-146a-5p enhances embryo survival in unexplained recurrent spontaneous abortion by promoting M2 polarization of decidual macrophages

Dickkopf-1 (DKK1) is a secretory antagonist that can bind with the Wnt coreceptor to desensitize cells to canonical Wnt ligands. DKN-01 is a specific antibody targeting secreted DKK1, which has been investigated as a monotherapy or combination therapy for various malignant tumors, including gastric cancer (GC). Tumor-associated macrophages (TAMs) with high plasticity usually present M2 phenotype, which can promote tumor progression. The aim of this study was to investigate the effect of DKN-01 on macrophage polarization in GC and the underlying molecular mechanism. To ascertain the effect of DKN-01 on GC tumor growth, we established a tumor-bearing mouse model and found that DKN-01 treatment suppressed tumor growth efficiently. Through RNA-seq and pathway enrichment analysis, we identified that the differentially expressed genes after DKN-01 treatment are associated with tumor immune-related pathways. Macrophage polarization was assessed using immunohistochemistry and quantitative real-time polymerase chain reaction. DKN-01 and knockdown of DKK1 promoted M1 polarization and inhibited M2 polarization of macrophages, while DKK1 overexpression got the opposite results. Moreover, DKN-01 activated the cGAS/STING pathway, while the inactivation of cGAS-STING pathway using RU.521 reversed the inhibition of tumor growth in vivo and macrophage M2 polarization caused by DKN-01. This study reveals that DKN-01 suppresses GC tumor growth through activating cGAS-STING pathway to block macrophage M2 polarization.

Discovery of new macrophage M2 polarization modulators as multiple sclerosis treatment agents that enable the inflammation microenvironment remodeling

Pro-proliferative, M2-like polarization of macrophages is a critical step in the development of fibrosis and remodeling in chronic lung diseases such as pulmonary fibrosis and pulmonary hypertension. Macrophages in healthy and diseased lungs express gremlin 1 (Grem1), a secreted glycoprotein that acts in both paracrine and autocrine manners to modulate cellular function. Increased Grem1 expression plays a central role in pulmonary fibrosis and remodeling, however, the role of Grem1 in M2-like polarization of macrophages has not previously been explored. The results reported here show that recombinant Grem1 potentiated M2-like polarization of mouse macrophages and bone marrow-derived macrophages (BMDMs) in response to the Th2 cytokines IL4 and IL13. Genetic depletion of Grem1 in BMDMs inhibited M2 polarization while exogenous gremlin 1 could partially rescue this effect. Taken together, these findings reveal that gremlin 1 is required for M2-like polarization of macrophages.NEW & NOTEWORTHY We show here that gremlin 1 potentiated M2 polarization of mouse bone marrow-derived macrophages (BMDMs) in response to the Th2 cytokines IL4 and IL13. Genetic depletion of Grem1 in BMDMs inhibited M2 polarization while exogenous gremlin 1 partially rescued this effect. Taken together, these findings reveal a previously unknown requirement for gremlin 1 in M2 polarization of macrophages and suggest a novel cellular mechanism promoting fibrosis and remodeling in lung diseases.

Lung cancer, a prevalent and aggressive disease, is characterized by recurrence and drug resistance. It is essential to comprehend the fundamental processes and discover novel therapeutic objectives for augmenting treatment results. Based on our research findings, we have identified a correlation between methylation of cg09897064 and decreased expression of ZBP1, indicating a link to unfavorable prognosis in patients with lung cancer. Furthermore, these factors play a role in macrophage polarization, with ZBP1 upregulated in M1 macrophages compared to both M0 and M2 polarized macrophages. We observed cg09897064 methylation in M2 polarization, but not in M0 and M1 polarized macrophages. ATACseq analysis revealed closed chromatin accessibility of ZBP1 in M0 polarized macrophages, while open accessibility was observed in both M1 and M2 polarized macrophages. Our findings suggest that ZBP1 is downregulated in M0 polarized macrophages due to closed chromatin accessibility and downregulated in M2 polarized macrophages due to cg09897064 methylation. Further investigations manipulating cg09897064 methylation and ZBP1 expression through overexpression plasmids and shRNAs provided evidence for their role in modulating macrophage polarization and tumor growth. ZBP1 inhibits M2 polarization and suppresses tumor growth, while cg09897064 methylation promotes M2 polarization and macrophage-induced tumor growth. In mechanism investigations, we found that cg09897064 methylation impairs CEBPA binding to the ZBP1 promoter, leading to decreased ZBP1 expression. Clinical experiments were conducted to validate the correlation between methylation at cg09897064, ZBP1 expression, and macrophage M2 polarization. Targeting these factors may hold promise as a strategy for developing innovative checkpoint inhibitors in lung cancer treatment.