Liraglutide prevents lupus-associated diffuse alveolar hemorrhage via inhibiting lymphocyte infiltration 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.

Macrophage phenotypes have been linked to progression and prognosis of cutaneous melanoma. However, the association between Warburg effect in A375 melanoma and macrophages polarization, as well as the underlying mechanisms, remains less well documented. The present study aimed to investigate the effect of lactate derived from A375 melanoma on macrophage polarization, melanoma phenotype responses and the underlying mechanisms. Flow cytometry was performed to evaluate the expression of M1 and M2 markers, cell cycle and apoptosis. Levels of transforming growth factor β (TGF-β) and tumor necrosis factor α (TNF-α) were determined with enzyme-linked immunosorbent assay (ELISA) kit. Proliferation and invasion were assessed by CCK8 and transwell assays, respectively. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were analyzed using an XF96 extracellular flux analyzer. Protein expressions were determined by Western blotting. Our results revealed that melanoma A375 conditioned medium (A375-CM) induced peripheral blood mononuclear cells (PBMCs) to polarize toward anti-inflammatory M2 macrophages. M2 markers CD206 and ARG1 expression increased, as did TGF-β secretion. Conversely, M1 marker CD68 expression decreased. Furthermore, hypoxia promoted macrophage M2 polarization induced by A375-CM. Elevated lactate level in PIG1-conditioned medium (PIG1-CM) induced M2 polarization, whereas the lactate transport inhibitor AZD3965 suppressed this effect in PBMCs cultured with A375-CM. Additionally, lactate derived from melanoma regulated M1/M2 polarization by the tricarboxylic acid (TCA) cycle instead of glycolysis. Significantly, polarized macrophages altered melanoma phenotypes including proliferation, clone formation, cell cycle, apoptosis, migration and invasion via TCA cycle and TGF-β. Our data collectively demonstrate that lactate derived from melanoma facilitates polarization of M2 macrophages, which subsequently leads to modifications in melanoma phenotypes via TCA cycle and TGF-β signaling.

TcpC is a multifunctional virulence factor of Uropathogenic Escherichia coli (UPEC). Macrophages can differentiate into two different subsets M1 and M2 that play distinct roles in anti-infection immunity. Here, we investigate the influence of TcpC on M1/M2 polarization and the potential mechanisms. Our data showed that M1 markers CD86 and iNOS were significantly inhibited, while the M2 markers CD163, CD206 and Arg-1 were enhanced in macrophages in kidneys from the TcpC-secreting wild-type CFT073 (CFT073wt)-infected pyelonephritis mouse model, compared with those in macrophages in kidneys from TcpC knockout CFT073 mutant (CFT073Δtcpc)-infected mice. CFT073wt or recombinant TcpC (rTcpC) treatment inhibits LPS + IFN-γ-induced CD80, CD86, TNF-α and iNOS expression, but promotes IL-4-induced CD163, CD206, Arg-1 and IL-10 expression in both human and mouse macrophage cell lines THP-1 and J774A.1. Moreover, rTcpC significantly attenuated LPS + IFN-γ-induced phosphorylation of p38, ERK, p50 and p65 but enhanced IL-4-induced phosphorylation of Akt and STAT6. These data suggest that TcpC inhibits M1 but promotes M2 macrophage polarization by down-regulation of p38, ERK/NF-κB and up-regulation of the Akt/STAT6 signaling pathway, respectively. Our findings not only illuminate the regulatory effects of TcpC on macrophage M1/M2 polarization and its related signaling pathways, but also provide a novel mechanism underlying TcpC-mediated immune evasion of macrophage-mediated innate immunity.Simple SummaryWe investigate the influence of TcpC, a multifunctional virulence factor of Uropathogenic Escherichia coli (UPEC), on M1/M2 macrophage polarization and the potential mechanisms. TcpC-secreting wild-type CFT073 (CFT073wt) or recombinant TcpC (rTcpC) treatment inhibits LPS + IFN-γ-induced CD80, CD86, TNF-α and iNOS expression, but promotes IL-4-induced CD163, CD206, Arg-1 and IL-10 expression in CFT073wt-infected pyelonephritis model mouse and both human and mouse macrophage cell lines THP-1 and J774A.1, respectively. Moreover, rTcpC significantly attenuated LPS + IFN-γ-induced phosphorylation of p38, ERK, p50 and p65 but en-hanced IL-4-induced phosphorylation of Akt and STAT6. These data suggest that TcpC inhibits M1 but promotes M2 macrophage polarization by down-regulation of p38, ERK/NF-κB and up-regulation of the Akt/STAT6 signaling pathway, respectively. Our findings not only illuminate the regulatory effects of TcpC on macrophage M1/M2 polarization and its related signaling pathways, but also provide a novel mechanism underlying TcpC-mediated immune evasion of macrophage-mediated innate immunity.

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.

Urolithin A, a Gut Metabolite, Induces Metabolic Reprogramming of Adipose Tissue by Promoting M2 Macrophage Polarization and Mitochondrial Function (OR12-02-19)

Myocardial ischemia-reperfusion injury (MIRI) is a common pathological phenomenon during the treatment of acute myocardial infarction. Recent studies suggest that macrophage polarization plays a crucial role in MIRI progression. However, whether quercetin mitigates MIRI by modulating macrophage polarization and the underlying molecular mechanisms remain unclear. The protective effects of quercetin against MIRI were assessed using TTC-Evans blue staining, echocardiography, and myocardial enzyme assays. Histological changes, including myocardial fibrosis, were evaluated via HE and Masson staining. Western blot, qPCR, and immunofluorescence were performed to analyze macrophage M1/M2 polarization. Additionally, co-immunoprecipitation (Co-IP) assays were conducted to determine whether quercetin regulates M2 polarization through autophagy modulation. Quercetin significantly reduced infarct size, improved cardiac function, and alleviated inflammation and myocardial fibrosis in a dose-dependent manner. Western blot and immunofluorescence analyses showed that quercetin downregulated M1 markers while upregulating M2 markers and enhancing IL-10 secretion. In vitro experiments further confirmed that quercetin promoted M2 macrophage polarization under H/R conditions, thereby attenuating cardiomyocyte injury through a paracrine mechanism. Mechanistically, quercetin facilitated autophagic flux by reducing the binding affinity between Bcl-2 and Beclin-1, leading to enhanced M2 macrophage polarization, an effect partially reversed by 3-MA. This study provides the first evidence that quercetin exerts cardioprotective effects in MIRI by promoting M2 macrophage polarization. Furthermore, it elucidates a novel molecular mechanism in which quercetin regulates autophagy to drive M2 polarization, offering experimental support for quercetin as a potential therapeutic strategy for MIRI.

Several components of Cayratia japonica (CJ) such as rutin and quercetin have shown anti-inflammatory effect, yet its function in ulcerative colitis (UC) remains to be clarified. This study focuses on the modulatory effect of CJ on UC as well as molecular mechanism by which CJ regulates macrophage polarization in UC. The targets related to CJ components and UC were, respectively, obtained through in silico analysis, and their intersection targets were selected for pathway enrichment analysis. RAW264.7 cells were stimulated with lipopolysaccharide (LPS) to induce M1 macrophages. Expression of the macrophage polarization M1 marker CD11b and M2 marker CD206 was measured to determine the phenotype of macrophages. The mouse model was treated with dextran sodium sulfate (DSS) to induce UC to observe the effects of CJ on UC in vivo. The in silico analysis suggested the crucial significance of TLR4 and its downstream MAPK/NF-κB pathways in the modulatory effect of CJ on UC. Furthermore, experimental data revealed that CJ could promote M2 macrophage polarization but alleviate immune inflammation and reduce colon damage in DSS-evoked UC model. Additionally, CJ can inhibit the expression of TLR4/MAPK/NF-κB signaling pathway to enhance the M2-like polarization. Hence, CJ may exert anti-inflammatory effects and an inhibitory role in UC by inhibiting the TLR4/MAPK/NF-κB pathway and subsequent M1-like macrophage polarization.

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

lncRNAs are related to the progression of various diseases, including oral squamous cell carcinoma (OSCC), which is a common squamous cell carcinoma of the head and neck. Tumor-associated macrophages and tumor cells are significant components of tumor microenvironment. M2 polarization of tumor-associated macrophages is a crucial actor in tumor malignancy and metastasis. In this study, we studied the molecular mechanism of lncRNA DCST1-AS1 in OSCC. Here, we reported that DCST1-AS1 was significantly increased in OSCC cells. We found that loss of DCST1-AS1 obviously inhibited the proliferation, migration, and invasion of OSCC cells and xenograft tumor growth. Meanwhile, silencing of DCST1-AS1 also repressed the percentage of macrophages expressing M2 markers CD206 and CD11b. DCST1-AS1 shRNA enhanced the percentage of macrophages expressing M1 markers CD80 and CD11c. Then, we observed that loss of DCST1-AS1 suppressed OSCC progression via inactivating NF-κB signaling. As well established, NF-κB signaling exerts critical roles in tumor progression, and our study proved that DCST1-AS1 could regulate NF-κB signaling. We proved that blocking the NF-κB pathway using antagonists greatly downregulated OSCC progression and M2 macrophage polarization induced by the overexpression of DCST1-AS1. To sum up, we reported that DCST1-AS1 plays an important role in modulating OSCC tumorigenicity and M2 macrophage polarization through regulating the NF-κB pathway.

Hepatic ischemia-reperfusion injury (IRI) is an important factor affecting the prognosis of liver transplantation patients. The role of Arrb2 in liver injury is unclear. Our study aimed to determine the role of Arrb2 in hepatic IRI and to identify its underlying mechanisms. An analysis of clinical samples was conducted to assess the association between Arrb2 expression and the prognosis of liver transplantation. A 70% hepatic ischemia/reperfusion model in mice was established to verify the mechanism of Arrb2 in hepatocytes promoting M2 macrophage polarization in attenuating hepatic IRI by regulating 6-ketoLCA. A model of hypoxia/reoxygenation in vitro was established to investigate the molecular mechanism of 6-ketoLCA in promoting M2 macrophage polarization and pharmacological screening. Arrb2 in hepatocytes has been shown to provide significant liver protection against hepatic IRI, primarily through promoting the polarization of liver macrophages to M2. Arrb2 remodels bile acids and upregulates 6-ketoLCA through Cyp7a1 in hepatocytes, promoting M2 polarization of macrophages, thereby alleviating hepatic IRI. Mechanistically, TGR5 plays a crucial role in promoting the induction of M2 polarization in macrophages by 6-ketoLCA. Pharmacological screening indicates that dutasteride enhances the activity of the Arrb2 promoter and upregulates Arrb2 expression in hepatocytes, thereby mitigating hepatic IRI. Arrb2 in hepatocytes attenuates hepatic IRI by promoting macrophages toward the M2 phenotype through bile acid. Moreover, dutasteride, a selective agonist of Arrb2, emerges as a promising targeted therapeutic agent for the clinical management of liver injury.

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.

Electroacupuncture (EA) at the Neiguan acupoint (PC6) has shown significant cardioprotective effects. Sympathetic nerves play an important role in maintaining cardiac function after myocardial infarction (MI). Previous studies have found that EA treatment may improve cardiac function by modulating sympathetic remodeling after MI. However, the mechanism in how EA affects sympathetic remodeling and improves cardiac function remains unclear. The aim of this study is to investigate the cardioprotective mechanism of EA after myocardial ischemic injury by improving sympathetic remodeling and promoting macrophage M2 polarization. We established a mouse model of MI by occluding coronary arteries in male C57/BL6 mice. EA treatment was performed at the PC6 with current intensity (1 mA) and frequency (2/15 Hz). Cardiac function was evaluated using echocardiography. Heart rate variability in mice was assessed via standard electrocardiography. Myocardial fibrosis was evaluated by Sirius red staining. Levels of inflammatory factors were assessed using RT‐qPCR. Sympathetic nerve remodeling was assessed through ELISA, western blotting, immunohistochemistry, and immunofluorescence staining. Macrophage polarization was evaluated using flow cytometry. Our results indicated that cardiac systolic function improved significantly after EA treatment, with an increase in fractional shortening and ejection fraction. Myocardial fibrosis was significantly mitigated in the EA group. The sympathetic nerve marker tyrosine hydroxylase and the nerve sprouting marker growth‐associated Protein 43 were significantly reduced in the EA group, indicating that sympathetic remodeling was significantly reduced. EA treatment also promoted macrophage M2 polarization, reduced levels of inflammatory factors TNF‐α, IL‐1β, and IL‐6, and decreased macrophage‐associated nerve growth factor in myocardial tissue. To sum up, our results suggest that EA at PC6 attenuates sympathetic remodeling after MI to promote macrophage M2 polarization and improve cardiac function.

Systemic lupus erythematosus (SLE)-associated diffuse alveolar hemorrhage (DAH) is a rare but extremely harmful condition. The current study sought to dissect the mechanisms underlying the effects of human umbilical cord mesenchymal stem cell (HUCMSC)-derived exosomes on M2 macrophage polarization in SLE-associated DAH via the microRNA (miR)-146a-5p/NOTCH1 axis. A DAH mouse model was established using pristane. Exosomes were isolated from HUCMSCs transfected or untransfected with the miR-146a-5p antagonist or agonist and their NCs and then injected into DAH mice. Additionally, miR-146a-5p was overexpressed in macrophages. Expression of miR-146a-5p, NOTCH1, M1 macrophage markers, and M2 macrophage markers was measured in mice and macrophages, and inflammatory factor levels were detected. Mouse lung injuries were evaluated, so was the binding of miR-146a-5p to NOTCH1. Rescue experiments were conducted in mice and macrophages using NOTCH1 shRNA and pcDNA3.1-NOTCH1, respectively. NOTCH1 expression was enhanced in DAH mice. HUCMSC-derived exosomes reduced NOTCH1 expression, bleeding, inflammation, and M1 macrophage polarization but elevated M2 macrophage polarization in lung tissues of DAH mice. Mechanistically, NOTCH1 is negatively targeted by miR-146a-5p. miR-146a-5p overexpression diminished M1 marker and inflammatory factor levels but enhanced M2 marker levels in macrophages, which was nullified by NOTCH1 overexpression. HUCMSC-derived exosomes with miR-146a-5p inhibition increased NOTCH1 expression, worsened bleeding and inflammation, and augmented M1 macrophage polarization while decreasing M2 macrophage polarization in lung tissues of DAH mice, which was abrogated by silencing NOTCH1. HUCMSC-derived exosomes diminished NOTCH1 expression to accelerate M2 macrophage polarization via delivery of miR-146a-5p, thus alleviating SLE-associated DAH in mice.

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.

Allergic asthma is an inappropriate Th2 immune response in the lungs to innocuous inhaled antigens. This results in M2 polarization of alveolar macrophages (AM), which number correlates with asthma severity in humans. Sex differences in asthma suggest that sex hormones modify lung inflammation and macrophage polarization. Since estrogen enhances M2 polarization and androgens have been used as experimental asthma therapy, we hypothesized that androgens [testosterone and dihydrotestosterone (DHT)] and androgen receptor (AR) suppress M2 macrophage polarization. Here, we demonstrate that although androgen reconstitution in castrated mice reduced inflammation of the bronchoalveolar space in a mouse model of allergic lung inflammation, it enhanced the production of the eosinophil-recruiter and M2 marker YM1 in AM, indicating a cell-specific role for androgens. DHT also enhanced IL-4-induced M2 macrophage polarization in vitro. Using mice lacking the AR in monocytes/macrophages (ARfloxLysMCre), we found that ARfloxLysMCre males had less eosinophil recruitment and lung inflammation in our model of allergic lung inflammation. No differences were found in females. This decreased inflammation was due to impaired M2 polarization and diminished production of eosinophil-recruiting chemokines by AM rather to a regulatory profile of AM lacking AR. Furthermore, macrophages lacking AR did not show an increase in IL-4-induced M2 polarization in vitro with DHT treatment. These data reveal an unexpected role for androgen/AR in promoting M2 macrophage polarization, and suggest possible mechanisms to counteract diseases that are promoted by M2 macrophages and affect mainly men, such as eosinophilic esophagitis and prostate cancer.