Extracellular Vesicles Obtained From Lung Adenocarcinoma Cells Cultured Under Intermittent Hypoxia Induce M2 Macrophage Polarization via miR-20a-5p Delivery

The aim of this paper was to investigate the mechanism of saffron extract (SE) in promoting the polarization of macrophages towards M2 and thereby promoting burn wound healing by modulating the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/NOD-like receptor family pyrin domain-containing 3 (NLRP3) cascade. A burn model was induced in mice, which were treated with SE to burn wounds. Wound characteristics were observed, and wound healing rate and wound contraction rate were measured. Wounds were examined pathologically using hematoxylin-eosin staining, macrophage surface markers were identified by immunofluorescence staining, and cytokine levels were measured with enzyme-linked immunosorbent assay. In vitro experiments were conducted to investigate the effects of SE on lipopolysaccharide-induced polarization of mouse macrophages (RAW 264.7) and on proliferation and migration of mouse embryonic fibroblasts (NIH3T3). Proteins related to the Nrf2/HO-1/NLRP3 cascade were measured by Western blot. SE effectively promoted burn wound healing, inhibited inflammatory response during wound healing and promoted macrophage M2 polarization in burn model mice. SE induced macrophage M2 polarization in vitro and promoted NIH3T3 cell proliferation and migration. Regulation of the Nrf2/HO-1/NLRP3 cascade by SE was observed, and Nrf2 knockdown negated the SE-driven M2 polarization of macrophages and the increased proliferation and migration of NIH3T3 cells in a co-culture environment. SE promotes burn wound healing by modulating the Nrf2/HO-1/NLRP3 cascade to promote macrophage M2 polarization.

Macrophages can exhibit a spectrum of activation states ranging from a pro-inflammatory and antitumorigenic M1 (CD80+; IL-12high; iNOShigh) to a pro-tumorigenic M2 [CD163+; IL-10high; CD206+; Arg+ (mouse)] phenotype. Plasminogen activator inhibitor-1 (PAI-1) is overexpressed in many cancers and although it acts as a protease inhibitor it is paradoxically correlated with poor outcome. PAI-1 exerts its pro-tumorigenic role via pro-angiogenic and anti-apoptotic functions. Increasing evidence points towards the importance of PAI-1 in inflammation and tumorigenesis, but its influence on the immune component of the tumor microenvironment has not yet been investigated. Here we report a novel observation that PAI-1 is a regulator of macrophage polarization. We observe that in PAI-1 KO mice xenotransplanted with 3 human cancer cell lines (HT1080, A549, HCT116) in which PAI-1 was knocked down (KD), tumors were less infiltrated with macrophages with lower Arg expression compared to WT mice implanted with PAI-1 pos. tumors. We demonstrate that recombinant PAI-1 through its uPA interactive domain increased the expression of M2 polarization markers (CD163, IL-10) and decreased or did not affect the expression of M1 polarization markers (iNOS, CD80) in human peripheral blood monocytes. Further investigation demonstrated that treatment of monocytes with PAI-1 induced a rapid (2 h.) increase in IL-6 mRNA and secretion (4 h.) of the protein that was followed by phosphorylation (8-24 h.) of signal transduction and activation of transcription 3 (STAT3) in monocytes. Further linking STAT3 activation to PAI-1, we observed a decreased pSTAT3 in monocytes co-cultured with HT1080 cells upon PAI-1 downregulation. We found that STAT3 activation was downstream of IL-6 and responsible for M2 polarization, as the blockage of the IL-6 receptor with a function blocking antibody (tocilizumab) or inhibition of STAT3 activation by a JAK2/STAT3 inhibitor (ruxolitinib) prevented M2 polarization of monocytes by PAI-1. The contribution of PAI-1 to M2 polarization of macrophages was tested in PAI-1 KO mice implanted with human tumors in which PAI-1 expression was controlled by doxycycline. These experiments demonstrated that induction of PAI-1 in established tumors increased the presence of tumor associated macrophages (TAM) (F4/80+) and decreased the presence of M1 (iNOS+) TAM. Further supporting the presence of an autocrine PAI-1/IL-6 pathway in macrophage polarization in human cancers, a meta-analysis of gene expression array data indicated strong correlations between PAI-1 and IL-6 expression in breast (P=<0.05) and colon (P<0.01) cancers and between PAI-1 and CD163 in colon cancer (P<0.0001). The data thus identify a new pro-tumorigenic function for PAI-1 in the communication between tumor cells and macrophages where tumor-derived PAI-1 activates an autocrine IL-6/STAT3 pathway that promotes M2 polarization. Citation Format: Marta H. Kubala, Veronica R. Placencio, Yves A. DeClerck. Tumor-derived PAI-1 promotes macrophage M2 polarization by stimulating an autocrine IL-6 /STAT3 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3061. doi:10.1158/1538-7445.AM2017-3061

Exosomes, the cargo of circRNA, are crucial in cancer cell-tumor microenvironment communication. circSMARCC1 exerts a pro-tumor effect. However, this role has not been previously reported in gastric cancer (STAD). This study explores the role of STAD cell-derived exosomal circSMARCC1 in promoting macrophage M2 polarization. In this study, exosomes from the peripheral blood of STAD patients and AGS cells were extracted and identified. THP-1 cells were differentiated into macrophages with phorbol 12-myristate 13-acetate (PMA) and polarized to the M2 phenotype by IL-4 and IL-13. circSMARCC1 expression was analyzed in STAD tissues, cell lines, and patient-derived exosomes. The biological function of circSMARCC1 in STAD cells was evaluated by CCK-8, EdU, and Transwell assays. The role of circSMARCC1 and U2 small nuclear RNA auxiliary factor 2 (U2AF2) in regulating macrophage M2 polarization was verified by bioinformatics methods, qRT-PCR, Western blot, ELISA, and a nude mouse tumor-bearing model. Our findings demonstrated that circSMARCC1 was upregulated in STAD and associated with macrophage immune infiltration. circSMARCC1 knockdown suppressed the malignant phenotypes of STAD cells and M2 macrophage polarization, whereas its overexpression led to the contrary result. Animal experiments further confirmed circSMARCC1 regulated tumor growth and macrophage M2 polarization. Importantly, exosomal circSMARCC1 in STAD patients was increased. Knockdown of circSMARCC1 in AGS cells reduced its level in secreted exosomes and inhibited M2 polarization, whereas overexpression produced the opposite effect. Mechanistically, circSMARCC1 upregulated U2AF2 expression through exosomes to promote macrophage M2 polarization. Collectively, STAD cell-derived exosomes induced macrophage M2 polarization to promote STAD progression through circSMARCC1.

To explore the oncogenic mechanism of FOXM1 in the tumor microenvironment (TME) regarding triple-negative breast cancer (TNBC) promotion, the mRNA and protein levels of target genes in TNBC cells and their exosomes were detected by RT-qPCR and western blot. A co-culture model of TNBC cells and THP-1/M0 macrophages was established to detect the impact of co-culture on FOXM1 expression and the direction of macrophage polarization. A bioinformatics website was used to predict FOXM1 binding sites in the IDO1 promoter, which were further validated using dual-luciferase reporter and chromatin immunoprecipitation assays. Next, after erastin-induced ferroptosis, we conducted cell viability assays, apoptosis assays and other experiments to investigate whether the FOXM1/IDO1 axis regulates M2 macrophage polarization through ferroptosis. We found that FOXM1 was abundant in exosomes derived from TNBC cells, and that TNBC cells upregulated FOXM1 expression in THP-1 cells through exosomes to promote M2 macrophage polarization. Furthermore, FOXM1 upregulated IDO1 in M2-type tumor-associated macrophages (TAMs) by stimulating its transcription. Finally, FOXM1/IDO1 inhibited ferroptosis, promoting M2 macrophage polarization, thereby advancing TNBC progression. In conclusion, FOXM1 carried by TNBC cell-derived exosomes activated IDO1 transcription in TAMs to inhibit ferroptosis, promoting M2 polarization of TAMs and exerting carcinogenic effects.

<b>Objective:</b> Intermittent hypoxia (IH)&nbsp;is a hallmark of obstructive sleep apnea. Herein, we investigated the effects of extracellular vesicles derived from intermittent hypoxic lung cancer cells on macrophage polarization. <b>Methods:</b> A549 cells were exposed to normoxia or IH (48 cycles, 5 min of 1% O₂ hypoxia, followed by 5 min of normoxia). Cancer cell–derived EVs were isolated and cocultured with macrophages differentiated from THP-1. <b>Results:</b> EVs derived from IH group highly expressed miR-20a-5p. Macrophages treated with EVs from IH group or miR-20a-5p-rich EVs showed M2 phenotype. Luciferase reporter analysis confirmed that PTEN was a target gene of miR-20a-5p. Transfection of miR-20a-5p mimics down-regulated PTEN expression, upregulated M2 polarization markers expressions and promoted Akt phosphorylation in macrophages, whereas the miR-20a-5p inhibitor had the opposite influence. <b>Conclusions:</b> EVs derived from intermittent hypoxic lung cancer cells promote M2 macrophage polarization through miR-20a-5p/PTEN/Akt pathway.

Glioblastoma (GBM) cell-derived extracellular vesicles (EVs) have been demonstrated to modulate tumor microenvironment. In the present study, we attempted to discuss the role of hsa-microRNA-27a-3p (miR-27a-3p) delivered by GBM-EVs in M2 macrophage polarization. The isolated GBM-EVs were co-cultured with macrophages. After co-culture under normoxia/hypoxia, the effect of EV-derived hsa-miR-27a-3p on GBM cell biological processes was analyzed. Additionally, the target genes of hsa-miR-27a-3p were predicted. Moreover, the binding of enhancer of zeste homologue 1 (EZH1) to lysine-specific demethylase 3A (KDM3A) promoter region and the interaction between KDM3A and connective tissue growth factor (CTGF) were analyzed. GBM mouse models were established to verify the functions of EV-derived hsa-miR-27a-3p in vivo. We found increased hsa-miR-27a-3p in GBM tissues as well as GBM-EVs, which induced M2 polarization, thus promoting proliferative, migrative and invasive potentials of GBM cells. hsa-miR-27a-3p targeted EZH1 and promoted KDM3A expression to elevate the CTGF expression. GBM-EV-delivered hsa-miR-27a-3p promoted the KDM3A-upregulated CTGF by downregulating EZH1, thereby promoting M2 macrophage polarization and development of GBM in vivo. We demonstrated that EV-derived hsa-miR-27a-3p may promote M2 macrophage polarization to induce GBM.

BackgroundExpression of Oct4 maintains cancer stem cell (CSC)-like properties in lung cancer cells and is correlated with poor prognosis of lung adenocarcinoma. M2-type tumor-associated macrophages (TAMs) promote cancer cell migration and metastasis. Tumor microenvironments promote monocyte differentiation into M2 TAMs via a complex cytokine-based connection. We explored the role of Oct4 in cytokine secretion in lung cancer and its impact on M2 TAM polarization.MethodsMonocytes co-cultured with the conditioned medium from Oct4-overexpressing lung cancer cells were used to investigate M2 TAM differentiation. The inflammatory factors in the conditioned medium of Oct4-overexpressing A549 cells were examined using human inflammation antibody arrays. The correlations of Oct4, macrophage colony-stimulating factor (M-CSF), and M2 TAMs were validated in lung cancer cells, syngeneic mouse lung tumor models, and clinical samples of non-small cell lung cancer (NSCLC).ResultsOct4-overexpressing A549 cells expressed elevated levels of M-CSF, which contributed to increased M2 macrophages and enhanced tumor migration. Overexpression of Oct4 enhanced tumor growth and reduced the survival of lung tumor-bearing mice, which was correlated with increased number of M2 macrophages in lung cancer. Notably, NSCLC patients with high expression levels of Oct4, M-CSF, and M2 TAMs had the poorest recurrence-free survival. A positive correlation between Oct4, M-CSF, and M2 TAMs was observed in the tumor tissue of NSCLC patient. Treatment with all-trans retinoic acid exerted anti-tumor effects and reduced M2 TAMs in tumor-bearing mice.ConclusionsOur results indicate that Oct4 expressed by lung cancer cells promotes M2 macrophage polarization through upregulation of M-CSF secretion, leading to cancer growth and metastasis. Our findings also implicate that the Oct4/M-CSF axis in M2 macrophage polarization may be potential therapeutic targets for lung cancer.

Exosomes from ectopic endometrial stromal cells promote M2 macrophage polarization by delivering miR-146a-5p

BackgroundHepatocellular carcinoma (HCC) cells-secreted exosomes (exo) could stimulate M2 macrophage polarization and promote HCC progression, but the related mechanism of long non-coding RNA distal-less homeobox 6 antisense 1 (DLX6-AS1) with HCC-exo-mediated M2 macrophage polarization is largely ambiguous. Thereafter, this research was started to unearth the role of DLX6-AS1 in HCC-exo in HCC through M2 macrophage polarization and microRNA (miR)-15a-5p/C-X-C motif chemokine ligand 17 (CXCL17) axis.MethodsDLX6-AS1, miR-15a-5p and CXCL17 expression in HCC tissues and cells were tested. Exosomes were isolated from HCC cells with overexpressed DLX6-AS1 and co-cultured with M2 macrophages. MiR-15a-5p/CXCL17 down-regulation assays were performed in macrophages. The treated M2 macrophages were co-cultured with HCC cells, after which cell migration, invasion and epithelial mesenchymal transition were examined. The targeting relationships between DLX6-AS1 and miR-15a-5p, and between miR-15a-5p and CXCL17 were explored. In vivo experiment was conducted to detect the effect of exosomal DLX6-AS1-induced M2 macrophage polarization on HCC metastasis.ResultsPromoted DLX6-AS1 and CXCL17 and reduced miR-15a-5p exhibited in HCC. HCC-exo induced M2 macrophage polarization to accelerate migration, invasion and epithelial mesenchymal transition in HCC, which was further enhanced by up-regulated DLX6-AS1 but impaired by silenced DLX6-AS1. Inhibition of miR-15a-5p promoted M2 macrophage polarization to stimulate the invasion and metastasis of HCC while that of CXCL17 had the opposite effects. DLX6-AS1 mediated miR-15a-5p to target CXCL17. DLX6-AS1 from HCC-exo promoted metastasis in the lung by inducing M2 macrophage polarization in vivo.ConclusionDLX6-AS1 from HCC-exo regulates CXCL17 by competitively binding to miR-15a-5p to induce M2 macrophage polarization, thus promoting HCC migration, invasion and EMT.

ObjectiveTo investigate the lung cancer-promoting mechanism of mesenchymal stem cell-secreted extracellular vesicles (MSC-EV).MethodsEV were isolated from culture media of human bone marrow-derived MSCs that were pre-challenged with or without hypoxia (referred to as H-EV and N-EV, respectively). After treatment with N-EV or H-EV, A549 and H23 cell proliferation, apoptosis, trans-well invasion and epithelial-to-mesenchymal transition (EMT) were examined. Polarization of human primary monocytes-derived macrophages with or without N-EV or H-EV induction were analyzed by flow cytometry and ELISA. PTEN, PDCD4 or RECK gene was overexpressed in A549 cells, while miR-21-5p was knocked down in MSCs, A549 or H23 lung cancer cells or primary monocytes by miR-21-5p inhibitor transfection. Protein level of PTEN, PDCD4, RECK, AKT or STAT3 as well as phosphorylation level of AKT or STAT3 protein were assayed by western blot. Tumorigenicity of A549 and H23 cells with or without MSC-EV co-injection was assayed on immunocompromised mice. The xenograft tumor were examined for cell proliferation, angiogenesis, apoptosis and intra-tumoral M1/M2 macrophage polarization.ResultsComparing to N-EV, H-EV treatment significantly increased A549 and H23 cell proliferation, survival, invasiveness and EMT as well as macrophage M2 polarization. MiR-21-5p knocked down significantly abrogated the cancer-promoting and macrophage M2 polarizing effects of H-EV treatment. H-EV treatment downregulated PTEN, PDCD4 and RECK gene expression largely through miR-21-5p. Overexpressing PTEN, PDCD4 and RECK in A549 cells significantly reduced the miR-21-5p-mediated anti-apoptotic and pro-metastatic effect of H-EV, while overexpressing PTEN in monocytes significantly reduced macrophage M2 polarization after induction with the presence of H-EV. H-EV co-injection significantly increased tumor growth, cancer cell proliferation, intra-tumoral angiogenesis and M2 polarization of macrophages in vivo partially through miR-21-5p.ConclusionsIncreased miR-21-5p delivery by MSC-EV after hypoxia pre-challenge can promote lung cancer development by reducing apoptosis and promoting macrophage M2 polarization.

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.

It has been documented that M2 macrophage polarization plays a suppressive role in atherosclerosis in diabetes mellitus (DM). In addition, prostaglandin E2 (PGE2) is implicated in the development of M2 macrophage polarization. Therefore, the study aimed to investigate the specific mechanism of PGE2 in M2 macrophage polarization in diabetic coronary atherosclerosis (DMAS). Initially, clinical samples were obtained and DMAS mouse model was established. The expression of BDNF was determined, and M1 and M2 macrophage polarizations were evaluated. Then, the levels of BDNF and PGE2 were modified in DMAS mice and the serum indicator, atherosclerotic plaque, lipid uptake by PBMCs, as well as M1 and M2 macrophage polarization were determined. Macrophages were isolated and the effects of PGE2 and the CREB/BDNF/TrkB signaling pathway on M2 macrophage polarization were explored. BDNF was downregulated and macrophages were differentiated into M1 in DMAS patients and mice. BDNF and PGE2 were observed to promote M2 macrophage polarization, where atherosclerotic plaque and lipid uptake by PBMCs were reduced, and DMAS was alleviated in mice. Overexpression of BDNF activated the CREB/BDNF/TrkB signaling pathway and stimulated M2 macrophage polarization in macrophages. PGE2 stimulated M2 macrophage polarization by inducing KLF4 via the activation of the CREB/BDNF/TrkB signaling pathway. This study demonstrates that PGE2 promotes M2 macrophage polarization by activating the CREB/BDNF/TrkB signaling pathway, thus alleviating DMAS.

Cancer-derived small extracellular vesicles (sEVs) are emerging as crucial mediators of intercellular communication between cancer cells and M2-tumor-associated macrophages (M2-TAMs) via transferring lncRNAs. We previously reported that miR-134 blocks the expression of its targeting protein LAMC2 via the PI3K/AKT pathway and inhibits cancer stem cell (CSC) migration and invasion in oral squamous cell carcinoma (OSCC). This study hypothesize that OSCC-CSC-derived small extracellular vesicles (OSCC-CSC-sEVs) transfer a ceRNA of miR-134 and consequently promote M2 macrophage polarization by targeting LAMC2 via the PI3K/AKT pathway through in vitro and in vivo experiment methods. The results showed that sEVs derived from CD133+CD44+ OSCC cells promoted M2 polarization of macrophages by detecting several M2 macrophage markers (CD163, IL-10, Arg-1, and CD206+CD11b+). Mechanistically, we revealed that the lncRNA UCA1, by binding to miR-134, modulated the PI3K/AKT pathway in macrophages via targeting LAMC2. Importantly, OSCC-CSC-sEV transfer of UCA1, by targeting LAMC2, promoted M2 macrophage polarization and inhibited CD4+ T-cell proliferation and IFN-γ production in vitro and in vivo. Functionally, we demonstrated that M2-TAMs, by transferring exosomal UCA and consequently targeting LAMC2, enhanced cell migration and invasion of OSCC in vitro and the tumorigenicity of OSCC xenograft in nude mice. In conclusion, our results indicated that OSCC-CSC-sEV transfer of UCA1 promotes M2 macrophage polarization via a LAMC2-mediated PI3K/AKT axis, thus facilitating tumor progression and immunosuppression. Our findings provide a new understanding of OSCC-CSC molecular mechanisms and suggest a potential therapeutic strategy for OSCC through targeting CSC-sEVs and M2-TAMs.

Histone lactylation regulates PRKN-Mediated mitophagy to promote M2 Macrophage polarization in bladder cancer.

The polarization of macrophages plays a critical role in the immune response to infectious diseases, with M2 polarization shown to be particularly important in various pathological processes. However, the specific mechanisms of M2 macrophage polarization in Mycobacterium tuberculosis (Mtb) infection remain unclear. In particular, the roles of Granulin (GRN) and tumor necrosis factor receptor 2 (TNFR2) in the M2 polarization process have not been thoroughly studied. To investigate the effect of macrophage M2 polarization on Mtb infection and the mechanism of GRN and TNFR2 in M2 polarization. Forty patients with pulmonary tuberculosis (PTB) and 40 healthy volunteers were enrolled in this study, and peripheral blood samples were taken to detect the levels of TNFR2 and GRN mRNA by Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR); monocytes were isolated and then assessed by Flow Cytometry (FC) for M1 and M2 macrophage levels. To further validate the function of TNFR2 in macrophage polarization, we used interleukin 4 (IL-4) to induce mouse monocyte macrophages RAW264.7 to M2 polarized state. The expression of TNFR2 was detected by Western Blot and RT-qPCR. Next, we constructed a GRN knockdown plasmid and transfected it into IL-4-induced mouse monocyte macrophage RAW264.7, and detected the expression of TNFR2, M1 macrophage-associated factors tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and interleukin 6 (IL-6), and the M2 macrophage-associated factors CD206, IL-10, and Arginase 1 (Arg1); Immunofluorescence staining was used to monitor the expression of CD86+ and CD206+, and FC was used to analyze the macrophage phenotype. Subsequently, immunoprecipitation was used to detect the binding role of GRN and TNFR2. Finally, the effects of GRN and TNFR2 in macrophage polarization were further explored by knocking down GRN and simultaneously overexpressing TNFR2 and observing the macrophage polarization status. The results of the study showed elevated expression of TNFR2 and GRN and predominance of M2 type in macrophages in PTB patients compared to healthy volunteers (p < 0.05). Moreover, TNFR2 was highly expressed in M2 macrophages (p < 0.05). Additionally, GRN knockdown was followed by elevated expression of M1 polarization markers TNF-α, iNOS and IL-6 (p < 0.05), decreased levels of M2 polarization-associated factors CD206, IL-10 and Arg1 (p < 0.05), and macrophage polarization towards M1. Subsequently, we found that GRN binds to TNFR2 and that GRN upregulates TNFR2 expression (p < 0.05). In addition, knockdown of GRN elevated M1 polarization marker expression, decreased M2 polarization marker expression, and increased M1 macrophages and decreased M2 macrophages, whereas concurrent overexpression of TNFR2 decreased M1 polarization marker expression, elevated M2 polarization marker expression, and decreased M1 macrophages and increased M2 macrophages. TNFR2 and GRN are highly expressed in PTB patients and GRN promotes macrophage M2 polarization by upregulating TNFR2 expression.