Macrophage Polarization Markers Research Articles

Promotion of mandibular distraction osteogenesis by parathyroid hormone via macrophage polarization induced through iNOS downregulation

ObjectiveTo investigate whether Parathyroid hormone (PTH) can promote mandibular distraction osteogenesis by regulating macrophage polarization and the underlying mechanisms of this phenomenon. MethodsForty-eight Rabbits were used to establish the mandibular distraction osteogenesis experimental model, randomly divided into 2 groups. Intermittent post-operative injections of 20 μg/kg PTH and normal saline were administered to the experimental and control groups, respectively. Regenerated new bone was examined using HE staining, osteoclast numbers were determined through tartrate-resistant acid phosphatase (TRAP) staining, and macrophage polarization markers arginase 1 (Arg1) and inducible nitric oxide synthase (iNOS) expressions were elucidated using immunohistochemistry (IHC), the mRNA expression of CD206, CD11C, Arg1 and iNOS were detected using qPCR. ResultsThe bone trabeculae in the experimental group were thicker, with a more homogeneous structure and more new osteoid than in the control group. In the area of distraction osteogenesis, the osteoclast count in the experimental group was higher than in the control group (P < 0.05). IHC results indicated differential expressions of Arg1 and iNOS in the experimental group compared to the control group (P < 0.05). Relative mRNA expressions of CD11c and iNOS were lower in the experimental group than in the control group (P < 0.05), whereas the expressions of CD206 and Arg1 mRNA were higher in the experimental group compared to the control group (P < 0.05). ConclusionIntermittent PTH injections increased macrophage quantity in the mandible generated by distraction osteogenesis, downregulated iNOS, upregulated Arg1, and promoted macrophage polarization from M1 to M2 phenotype, thereby promoting mandibular distraction osteogenesis.

S100A9 promotes renal calcium oxalate stone formation via activating the TLR4-p38/MAPK-LCN2 signaling pathway

ObjectivesTo explore the role of S100A9 protein in renal calcium oxalate (CaOx) stone formation. MethodsCaOx nephrocalcinosis mice were established via intraperitoneal injection of glyoxylate. They were treated with S100A9 deficiency, Paquinimod, or p38 MAPK-IN-1. Vonkossa staining was conducted to observe the deposition of CaOx crystals. Renal expression of inflammation, macrophage polarization, and injury markers was detected using immunohistochemistry and qPCR. Effects of S100A9 on renal tubular epithelial cells (HK−2) were explored by transcriptome sequencing. The mechanism of how S100A9 regulated lipocalin 2 (LCN2) was studied through Western Blot. Flow cytometry was performed to detect the influence of LCN2 on macrophages polarization. ResultsS100A9 deficiency inhibited the renal deposition of CaOx crystals in nephrocalcinosis mice. S100A9 upregulated the expression of LCN2 in HK-2 cells via activating the TLR4-p38/MAPK pathway. LCN2 promoted the migration and M1 polarization of macrophages. S100A9 deficiency downregulated the renal expression of LCN2, IL1-β, Kim-1, F4/80, and CD80 in nephrocalcinosis mice. Paquinimod and p38 MAPK-IN-1 both inhibited the renal deposition of CaOx crystals and downregulated the expression of LCN2, IL1-β, Kim-1, F4/80, iNOS, and CD68 in nephrocalcinosis mice. ConclusionsS100A9 promotes renal inflammatory injury by activating the TLR4-p38/MAPK-LCN2 pathway and then contributes to CaOx stone formation.

Sea Buckthorn Oil Promotes the PI3K-Akt-ERK Signaling Pathway and Macrophage M2 Polarization to Reduce Radiation-induced Skin Injury.

In this work, we explored the role and mechanism of sea buckthorn oil in reducing radiation-induced skin damage. The radiation-induced rat skin injury model was established using strontium-90. Rats were treated with sea buckthorn oil twice a day postirradiation, and skin damage was observed at different times and evaluated using an injury score. Skin pathological changes were observed using hematoxylin and eosin (H&E) staining. Western blotting and immunohistochemistry were used to detect the expression of vascular growth and pathway proteins. ELISA was used to detect the secretion level of inflammatory factors. Immunohistochemistry was used to detect macrophage polarization marker proteins. We found that sea buckthorn oil can alleviate radiation-induced skin damage, accelerate skin vascular regeneration, and promote the up-regulation of vascular endothelial growth factor (VEGF) and its receptor (VEGFR). These results demonstrate the beneficial effects of sea buckthorn oil on radiation-induced skin damage. Furthermore, the levels of IL-1β and TNF-α in the sea buckthorn oil treatment group were significantly lower than those in the control group, while the levels of IL-4 and IL10 were significantly higher (P < 0.05). CD206 expression also increased in the sea buckthorn oil treatment group, while CD16 expression decreased compared to the control group (P < 0.05). Western blotting showed that PI3K, Akt and ERK expression increased in the sea buckthorn oil treatment group (P < 0.05). The beneficial effect of sea buckthorn oil in reducing the inflammatory response in irradiated rats was diminished when they were treated with PI3K inhibitor. We conclude that sea buckthorn oil may regulate macrophage M2 polarization by increasing the PI3K-Akt-ERK signaling pathway, thereby inhibiting the inflammatory response and promoting skin vascular regeneration to prevent and treat radiation-induced skin damage.

PTGR1-mediated immune evasion mechanisms in late-stage triple-negative breast cancer: mechanisms of M2 macrophage infiltration and CD8+ T cell suppression.

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by metabolic dysregulation. Tumor cell immune escape plays an indispensable role in the development of TNBC tumors. Furthermore, in the abstract, we explicitly mention the techniques used and enhance the clarity and impact of our findings. "Based on bioinformatics analysis results, we utilized CRISPR/Cas9 technology to knockout the target gene and established a mouse model of breast cancer. Through experiments such as CCK8, scratch assay, and Transwell assay, we further investigated the impact of target gene knockout on the malignant behavior of tumor cells. Subsequently, we conducted immunohistochemistry and Western Blot experiments to study the expression of macrophage polarization and infiltration-related markers and evaluate the effect of the target gene on macrophage polarization. Next, through co-culture experiments, we simulated the tumor microenvironment and used immunohistochemistry staining to observe and analyze the distribution and activation status of M2 macrophages and CD8+ T cells in the co-culture system. We validated in vivo experiments the molecular mechanism by which the target gene regulates immune cell impact on TNBC progression.

MiR-455/GREM1 axis promotes colorectal cancer progression and liver metastasis by affecting PI3K/AKT pathway and inducing M2 macrophage polarization

BackgroundColorectal cancer is among the most common malignant tumors affecting the gastrointestinal tract. Liver metastases, a complication present in approximately 50% of colorectal cancer patients, are a considerable concern. Recently, studies have revealed the crucial role of miR-455 in tumor pathogenesis. However, the effect of miR-455 on the progression of liver metastases in colorectal cancer remains controversial. As an antagonist of bone morphogenetic protein(BMP), Gremlin 1 (GREM1) may impact organogenesis, body patterning, and tissue differentiation. Nevertheless, the role of miR-455 in regulating GREM1 in colorectal cancer liver metastases and how miR-455/GREM1 axis influences tumour immune microenvironment is unclear.MethodsBioinformatics analysis shows that miR-455/GREM1 axis plays crucial role in liver metastasis of intestinal cancer and predicts its possible mechanism. To investigate the impact of miR-455/GREM1 axis on the proliferation, invasion, and migration of colorectal cancer cells, colony formation assay, wound healing and transwell assay were examined in vitro. The Dual-Luciferase reporter gene assay and RNA pull-down assay confirmed a possible regulatory effect between miR-455 and GREM1. In vivo, colorectal cancer liver metastasis(CRLM) model mice was established to inquiry the effect of miR-455/GREM1 axis on tumor growth and macrophage polarization. The marker of macrophage polarization was tested using immunofluorescence(IF) and quantitative real-time polymerase chain reaction(qRT-PCR). By enzyme-linked immunosorbent assay (ELISA), cytokines were detected in culture medium supernatants.ResultsWe found that miR-455 and BMP6 expression was increased and GREM1 expression was decreased in liver metastase compared with primary tumor. miR-455/GREM1 axis promotes colorectal cancer cells proliferation, migration, invasion via affected PI3K/AKT pathway. Moreover, downregulating GREM1 augmented BMP6 expression in MC38 cell lines, inducing M2 polarization of macrophages, and promoting liver metastasis growth in CRLM model mice.ConclusionThese data suggest that miR-455/GREM1 axis promotes colorectal cancer progression and liver metastasis by affecting PI3K/AKT pathway and inducing M2 macrophage polarization. These results offer valuable insights and direction for future research and treatment of CRLM.

Ghrelin Expression in Atherosclerotic Plaques and Perivascular Adipose Tissue: Implications for Vascular Inflammation in Peripheral Artery Disease.

Atherosclerosis, a leading cause of peripheral artery disease (PAD), is driven by lipid accumulation and chronic inflammation within arterial walls. Objectives: This study investigates the expression of ghrelin, an anti-inflammatory peptide hormone, in plaque morphology and inflammation in patients with PAD, highlighting its potential role in age-related vascular diseases and metabolic syndrome. Methods: The analysis specifically focused on the immunohistochemical expression of ghrelin in atherosclerotic plaques and perivascular adipose tissue (PVAT) from 28 PAD patients. Detailed immunohistochemical staining was performed to identify ghrelin within these tissues, comparing its presence in various plaque types and assessing its association with markers of inflammation and macrophage polarization. Results: Significant results showed a higher prevalence of calcification in fibro-lipid plaques (63.1%) compared to fibrous plaques, with a notable difference in inflammatory infiltration between the two plaque types (p = 0.027). Complicated plaques exhibited increased ghrelin expression, suggesting a modulatory effect on inflammatory processes, although this did not reach statistical significance. The correlation between ghrelin levels and macrophage presence, especially the pro-inflammatory M1 phenotype, indicates ghrelin's involvement in the inflammatory dynamics of atherosclerosis. Conclusions: The findings propose that ghrelin may influence plaque stability and vascular inflammation, pointing to its therapeutic potential in managing atherosclerosis. The study underlines the necessity for further research to clarify ghrelin's impact on vascular health, particularly in the context of metabolic syndrome and age-related vascular alterations.

THP-1 Monocytic Cells Are Polarized to More Antitumorigenic Macrophages by Serial Treatment with Phorbol-12-Myristate-13-Acetate and PD98059.

Background and Objectives: As modulators of the tumor microenvironment, macrophages have been extensively studied for their potential in developing anticancer strategies, particularly in regulating macrophage polarization towards an antitumorigenic (M1) phenotype rather than a protumorigenic (M2) one in various experimental models. Here, we evaluated the effect of PD98059, a mitogen-activated protein kinase kinase MAPKK MEK1-linked pathway inhibitor, on the differentiation and polarization of THP-1 monocytes in response to phorbol-12-myristate-13-acetate (PMA) under various culture conditions for tumor microenvironmental application. Materials and Methods: Differentiation and polarization of THP-1 were analyzed by flow cytometry and RT-PCR. Polarized THP-1 subsets with different treatment were compared by motility, phagocytosis, and so on. Results: Clearly, PMA induced THP-1 differentiation occurs in adherent culture conditions more than nonadherent culture conditions by increasing CD11b expression up to 90%, which was not affected by PD98059 when cells were exposed to PMA first (post-PD) but inhibited when PD98059 was treated prior to PMA treatment (pre-PD). CD11bhigh THP-1 cells treated with PMA and PMA-post-PD were categorized into M0 (HLA-DRlow and CD206low), M1 (HLA-DRhigh and CD206low), and M2 (HLA-DRlow and CD206high), resulting in an increased population of M1 macrophages. The transcription levels of markers of macrophage differentiation and polarization confirmed the increased M1 polarization of THP-1 cells with post-PD treatment rather than with PMA-only treatment. The motility and cytotoxicity of THP-1 cells with post-PD treatment were higher than THP-1 cells with PMA, suggesting that post-PD treatment enhanced the anti-tumorigenicity of THP-1 cells. Confocal microscopy and flow cytometry showed the effect of post-PD treatment on phagocytosis by THP-1 cells. Conclusions: We have developed an experimental model of macrophage polarization with THP-1 cells which will be useful for further studies related to the tumor microenvironment.

Glycerol monolaurate regulates apoptosis and inflammation by suppressing lipopolysaccharide-induced ROS production and NF-κB activation in avian macrophages

Macrophages play a crucial role in both innate and adaptive immunity. However, their abnormal activation can lead to undesirable inflammatory reactions. This study aimed to investigate the effects of glycerol monolaurate (GML), a natural monoester known for its anti-inflammatory and immunoregulatory properties, on avian macrophages using the HD11 cell line. The results indicated that a concentration of 10 μg/mL of GML enhanced the phagocytic activity of HD11 cells (P < 0.05) without affecting cell viability (P > 0.05). GML decreased the expression of M1 macrophage polarization markers, such as CD86 and TNF-α genes (P < 0.05), while increasing the expression of M2 macrophage polarization markers, such as TGF-β1 and IL-10 genes (P < 0.05). GML suppressed ROS production, apoptosis, and the expression of proinflammatory genes (IL-1β and IL-6) induced by LPS (P < 0.05). GML also promoted the expression of TGF-β1 and IL-10 (P < 0.05), both in the presence and absence of LPS exposure. Moreover, GML suppressed the gene expression of TLR4 and NF-κB p65 induced by LPS (P < 0.05), as well as the phosphorylation of NF-κB p65 (P < 0.05). In conclusion, GML exhibited regulatory effects on the polarized state of avian macrophages and demonstrated significant anti-apoptotic and anti-inflammatory properties by suppressing intracellular ROS and the NF-κB signaling pathway.

Construction of a Prognostic Model for Hepatocellular Carcinoma Based on Macrophage Polarization-Related Genes.

The progression of hepatocellular carcinoma (HCC) is related to macrophage polarization (MP). Our aim was to identify genes associated with MP in HCC patients and develop a prognostic model based on these genes. We successfully developed a prognostic model consisting of six MP-related genes (SCN4A, EBF3, ADGRB2, HOXD9, CLEC1B, and MSC) to calculate the risk score for each patient. Patients were then classified into high- and low-risk groups based on their median risk score. The performance of the MP-related prognostic model was evaluated using Kaplan-Meier and ROC curves, which yielded favorable results. Additionally, the nomogram demonstrated good clinical effectiveness and displayed consistent survival predictions with actual observations. Gene Set Enrichment Analysis (GSEA) revealed enrichment of pathways related to KRAS signaling downregulation, the G2M checkpoint, and E2F targets in the high-risk group. Conversely, pathways associated with fatty acid metabolism, xenobiotic metabolism, bile acid metabolism, and adipogenesis were enriched in the low-risk group. The risk score positively correlated with the number of invasion-related genes. Immune checkpoint expression differed significantly between the two groups. Patients in the high-risk group exhibited increased sensitivity to mitomycin C, cisplatin, gemcitabine, rapamycin, and paclitaxel, while those in the low-risk group showed heightened sensitivity to doxorubicin. These findings suggest that the high-risk group may have more invasive HCC with greater susceptibility to specific drugs. IHC staining revealed higher expression levels of SCN4A in HCC tissues. Furthermore, experiments conducted on HepG2 cells demonstrated that supernatants from cells with reduced SCN4A expression promoted M2 macrophage polarization marker, CD163 in THP-1 cells. Reduced SCN4A expression induced HCC-related genes, while increased SCN4A expression reduced their expression in HepG2 cells. The MP-related prognostic model comprising six MPRGs can effectively predict HCC prognosis, infer invasiveness, and guide drug therapy. SCN4A is identified as a suppressor gene in HCC.

Gadolinium retention effect on macrophages - a potential cause of MRI contrast agent Dotarem toxicity.

Gadolinium is a component of the MRI contrast agent Dotarem. Although Dotarem is the least toxic among MRI contrasts used, gadolinium present in Dotarem accumulates for many years in various organs and tissues exerting toxic effects. We showed previously that gadolinium remains in macrophages for at least 7days after exposure to Dotarem. However, very little is known about the effect of gadolinium retention on the immune cells such as macrophages. We studied the effect of 1-day and 7-day retention of gadolinium on various functions and molecular pathways of macrophages. Gadolinium retention for 7days decreased macrophage adhesion and motility and dysregulated the expression of adhesion and fibrotic pathway-related proteins such as Notch1 and its ligand Jagged1, adhesion/migration-related proteins PAK1 and Shp1, immune response-related transcription factors Smad3 and TCF19, and chemokines CXCL10 and CXCL13, and dysregulated the mRNA expression of fibrosis-related genes involved in extracellular matrix (ECM) synthesis, such as Col6a1, Fibronectin, MMP9, and MMP12. It also completely (below a level of detection) shut down the transcription of anti-inflammatory M2 macrophage polarization marker theArg-1.Such changes, if they occur in MRI patients, can be potentially detrimental to the patient's immune system and immune response-related processes.

Macrophage-enriched novel functional long noncoding RNAs LRRC75A-AS1 and GAPLINC regulate polarization and innate immune responses.

Macrophages (Mφs) are functionally dynamic immune cells that bridge innate and adaptive immune responses; however, the underlying epigenetic mechanisms that control Mφ plasticity and innate immune functions are not well elucidated. To identify novel functions of macrophage-enriched lncRNAs in regulating polarization and innate immune responses. Total RNA isolated from differentiating monocyte-derived M1 and M2 Mφs was profiled for lncRNAs expression using RNAseq. Impact of LRRC75A-AS1, GAPLINC and AL139099.5 knockdown was examined on macrophage differentiation,polarization markers, phagocytosis, and antigen processing by flow cytometry and florescence microscopy. Cytokine profiles were examined by multiplex bead array and cytoskeletal signaling pathway genes were quantified by PCR-based array. Gingival biopsies were collected from periodontally healthy and diseased subjects to examine lncRNAs,M1/M2marker expression. Transcriptome profiling of M1 and M2 Mφs identified thousands of differentially expressed known and novel lncRNAs. We characterized three Mφ-enriched lncRNAs LRRC75A-AS1, GAPLINC and AL139099.5 in polarization and innate immunity. Knockdown of LRRC75A-AS1 and GAPLINC downregulated the Mφ differentiation markers and skewed Mφ polarization by decreasing M1 markers without a significant impact on M2 markers. LRRC75A-AS1 and GAPLINC knockdown also attenuated bacterial phagocytosis, antigen processing and inflammatory cytokine secretion in Mφs, supporting their functional role in potentiating innate immune functions. Mechanistically, LRRC75A-AS1 and GAPLINC knockdown impaired Mφ migration by downregulating the expression of multiple cytoskeletal signaling pathways suggesting their critical role in regulating Mφ migration. Finally, we showed that LRRC75A-AS1 and GAPLINC were upregulated in periodontitis and their expression correlates with higher M1 markers suggesting their role in macrophage polarizationin vivo. Our results show that polarized Mφs acquire a unique lncRNA repertoire and identified many previously unknown lncRNA sequences. LRRC75A-AS1 and GAPLINC, which are induced in periodontitis, regulate Mφ polarization and innate immune functions supporting their critical role in inflammation.

Macrophage polarization in lymph node granulomas from cattle and pigs naturally infected with Mycobacterium tuberculosis complex.

Tuberculosis in animals is caused by members of the Mycobacterium tuberculosis complex (MTC), with the tuberculous granuloma being the main characteristic lesion. The macrophage is the main cell type involved in the development of the granuloma and presents a wide plasticity ranging from polarization to classically activated or pro-inflammatory macrophages (M1) or to alternatively activated or anti-inflammatory macrophages (M2). Thus, this study aimed to analyze macrophage polarization in granulomas from cattle and pig lymph nodes naturally infected with MTC. Tuberculous granulomas were microscopically categorized into four stages and a panel of myeloid cells (CD172a/calprotectin), M1 macrophage polarization (iNOS/CD68/CD107a), and M2 macrophage polarization (Arg1/CD163) markers were analyzed by immunohistochemistry. CD172a and calprotectin followed the same kinetics, having greater expression in late-stage granulomas in pigs. iNOS and CD68 had higher expression in cattle compared with pigs, and the expression was higher in early-stage granulomas. CD107a immunolabeling was only observed in porcine granulomas, with a higher expression in stage I granulomas. Arg1+ cells were significantly higher in pigs than in cattle, particularly in late-stage granulomas. Quantitative analysis of CD163+ cells showed similar kinetics in both species with a consistent frequency of immunolabeled cells throughout the different stages of the granuloma. Our results indicate that M1 macrophage polarization prevails in cattle during early-stage granulomas (stages I and II), whereas M2 phenotype is observed in later stages. Contrary, and mainly due to the expression of Arg1, M2 macrophage polarization is predominant in pigs in all granuloma stages.

The Differential Effect of a Shortage of Thyroid Hormone Compared with Knockout of Thyroid Hormone Transporters Mct8 and Mct10 on Murine Macrophage Polarization.

Innate immune cells, including macrophages, are functionally affected by thyroid hormone (TH). Macrophages can undergo phenotypical alterations, shifting between proinflammatory (M1) and immunomodulatory (M2) profiles. Cellular TH concentrations are, among others, determined by TH transporters. To study the effect of TH and TH transporters on macrophage polarization, specific proinflammatory and immunomodulatory markers were analyzed in bone marrow-derived macrophages (BMDMs) depleted of triiodothyronine (T3) and BMDMs with a knockout (KO) of Mct8 and Mct10 and a double KO (dKO) of Mct10/Mct8. Our findings show that T3 is important for M1 polarization, while a lack of T3 stimulates M2 polarization. Mct8 KO BMDMs are unaffected in their T3 responsiveness, but exhibit slight alterations in M2 polarization, while Mct10 KO BMDMs show reduced T3 responsiveness, but unaltered polarization markers. KO of both the Mct8 and Mct10 transporters decreased T3 availability and, contrary to the T3-depleted BMDMs, showed partially increased M1 markers and unaltered M2 markers. These data suggest a role for TH transporters besides transport of TH in BMDMs. This study highlights the complex role of TH transporters in macrophages and provides a new angle on the interaction between the endocrine and immune systems.

PPM1G regulates hepatic ischemia/reperfusion injury through STING-mediated inflammatory pathways in macrophages.

Ischemia/reperfusion injury (IRI) is generally unavoidable following liver transplantation. Here, we investigated the role of protein phosphatase, Mg2+ /Mn2+ dependent 1G (PPM1G) in hepatic IRI. Hepatic IRI was mimicked by employing a hypoxia/reperfusion (H/R) model in RAW 264.7 cells and a 70% warm ischemia model in C57BL/6 mice, respectively. In vitro, expression changes of tumor necrosis factor-α and interleukin were detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and enzyme-linked immunosorbent assay. The protein expressions of PPM1G and the stimulator of interferon genes (STING) pathway components were analyzed by western blot. Interaction between PPM1G and STING was verified by coimmunoprecipitation (CO-IP). Immunofluorescence was applied for detection of p-IRF3. Flow cytometry, qRT-PCR and western blot were utilized to analyze markers of macrophage polarization. In vivo, histological analyses of mice liver were carried out by TUNEL and H&E staining. Changes in serum aminotransferases were also detected. Following H/R intervention, a steady decline in PPM1G along with an increase in inflammatory cytokines in vitro was observed. Addition of plasmid with PPM1G sequence limited the release of inflammatory cytokines and downregulated phosphorylation of STING. CO-IP validated the interaction between PPM1G and STING. Furthermore, inhibition of PPM1G with lentivirus enhanced phosphorylation of STING and its downstream components; meanwhile, p65, p38, and Jnk were also surged to phosphorylation. Expression of INOS and CD86 was surged, while CD206, Arg-1, and IL-10 were inhibited. In vivo, PPM1G inhibition further promoted liver damage, hepatocyte apoptosis, and transaminases release. Selective inhibition of STING with C-176 partially reversed the activation of STING pathway and inflammatory cytokines in vitro. M1 markers were also suppressed by C-176. In vivo, C-176 rescued liver damage and transaminase release caused by PPM1G inhibition. PPM1G suppresses hepatic IRI and macrophage M1 phenotype by repressing STING-mediated inflammatory pathways.

The mechanism by which cannabidiol (CBD) suppresses TNF-α secretion involves inappropriate localization of TNF-α converting enzyme (TACE)

Cannabidiol (CBD) is a phytocannabinoid derived from Cannabis sativa that exerts anti-inflammatory mechanisms. CBD is being examined for its putative effects on the neuroinflammatory disease, multiple sclerosis (MS). One of the major immune mediators that propagates MS and its mouse model experimental autoimmune encephalomyelitis (EAE) are macrophages. Macrophages can polarize into an inflammatory phenotype (M1) or an anti-inflammatory phenotype (M2a). Therefore, elucidating the impact on macrophage polarization with CBD pre-treatment is necessary to understand its anti-inflammatory mechanisms. To study this effect, murine macrophages (RAW 264.7) were pre-treated with CBD (10 µM) or vehicle (ethanol 0.1 %) and were either left untreated (naive; cell media only), or stimulated under M1 (IFN-γ + lipopolysaccharide, LPS) or M2a (IL-4) conditions for 24 hr. Cells were analyzed for macrophage polarization markers, and supernatants were analyzed for cytokines and chemokines. Immunofluorescence staining was performed on M1-polarized cells for the metalloprotease, tumor necrosis factor-α-converting enzyme (TACE), as this enzyme is responsible for the secretion of TNF-α. Overall results showed that CBD decreased several markers associated with the M1 phenotype while exhibiting less effects on the M2a phenotype. Significantly, under M1 conditions, CBD increased the percentage of intracellular and surface TNF-α but decreased secreted TNF-α. This phenomenon might be mediated by TACE as staining showed that CBD sequestered TACE intracellularly. CBD also prevented RelA nuclear translocation. These results suggest that CBD may exert its anti-inflammatory effects by reducing M1 polarization and decreasing TNF-α secretion via inappropriate localization of TACE and RelA.

Effects and mechanisms of Zhizi Chuanxiong herb pair against atherosclerosis: an integration of network pharmacology, molecular docking, and experimental validation

BackgroundThe Zhizi Chuanxiong herb pair (ZCHP) can delay pathological progression of atherosclerosis (AS); however, its pharmacological mechanism remains unclear because of its complex components. The purpose of current study is to systematically investigate the anti-AS mechanism of ZCHP.MethodsThe databases of TCMSP, STITCH, SwissTargetPrediction, BATMAN-TCM, and ETCM were searched to predict the potential targets of ZCHP components. Disease targets associated with AS was retrieved from the GEO database. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analyses were executed using DAVID 6.8. Molecular docking method was employed to evaluate the core target binding to blood components, and animal experiments were performed to test action mechanism.ResultsA ZCHP-components-targets-AS network was constructed by using Cytoscape, included 11 main components and 52 candidate targets. Crucial genes were shown in the protein–protein interaction network, including TNF, IL-1β, IGF1, MMP9, COL1A1, CCR5, HMOX1, PTGS1, SELE, and SYK. KEGG enrichment illustrated that the NF-κB, Fc epsilon RI, and TNF signaling pathways were important for AS treatment. These results were validated by molecular docking. In ApoE−/− mice, ZCHP significantly reduced intima-media thickness, pulse wave velocity, plaque area, and serum lipid levels while increasing the difference between the end-diastolic and end-systolic diameters. Furthermore, ZCHP significantly decreased the mRNA and protein levels of TNF-α and IL-1β, suppressed NF-κB activation, and inhibited the M1 macrophage polarization marker CD86 in ApoE−/− mice.ConclusionThis study combining network pharmacology, molecular biology, and animal experiments showed that ZCHP can alleviate AS by suppressing the TNF/NF-κB axis and M1 macrophage polarization.Graphical

Study on the Role and Mechanism of SLC3A2 in Tumor-Associated Macrophage Polarization and Bladder Cancer Cells Growth.

Background: Solute carrier family 3 member 2 (SLC3A2) is highly expressed in various types of cancers, including bladder cancer (BLCA). However, the role and mechanism of SLC3A2 in the onset and progression of BLCA are still unclear. Methods: The interfering plasmid for SLC3A2 was constructed and transfected into BLCA cells. Cell proliferation, invasion, and migration abilities were assessed to evaluate the impact of SLC3A2 silencing on BLCA cell growth. M1 and M2 macrophage polarization markers were detected to evaluate macrophage polarization. The levels of reactive oxygen species (ROS), lipid peroxidation, and Fe2+, as well as the expression of ferroptosis-related proteins, were measured to assess the occurrence of ferroptosis. Ferroptosis inhibitors were used to verify the mechanism. Results: The experimental results showed that SLC3A2 was highly expressed in BLCA cell lines. The proliferation, invasion, and migration of BLCA cells were reduced after interfering with SLC3A2. Interference with SLC3A2 led to increase the expression of M1 macrophage markers and decreased the expression of M2 macrophage markers in M0 macrophages co-cultured with tumor cells. Additionally, interference with SLC3A2 led to increased levels of ROS, lipid peroxidation, and Fe2+, downregulated the expression of solute carrier family 7 member11 (SLC7A11) and glutathione peroxidase 4 (GPX4), while upregulated the expression of acyl-coA synthetase long chain family member 4 (ACSL4) and transferrin receptor 1 (TFR1) in BLCA cells. However, the impact of SLC3A2 interference on cell proliferation and macrophage polarization was impeded by ferroptosis inhibitors. Conclusion: Interference with SLC3A2 inhibited the growth of BLCA cells and the polarization of tumor-associated macrophages by promoting ferroptosis in BLCA cells.

NLRX1 Prevents M2 Macrophage Polarization and Excessive Renal Fibrosis in Chronic Obstructive Nephropathy.

Chronic kidney disease often leads to kidney dysfunction due to renal fibrosis, regardless of the initial cause of kidney damage. Macrophages are crucial players in the progression of renal fibrosis as they stimulate inflammation, activate fibroblasts, and contribute to extracellular matrix deposition, influenced by their metabolic state. Nucleotide-binding domain and LRR-containing protein X (NLRX1) is an innate immune receptor independent of inflammasomes and is found in mitochondria, and it plays a role in immune responses and cell metabolism. The specific impact of NLRX1 on macrophages and its involvement in renal fibrosis is not fully understood. To explore the specific role of NLRX1 in macrophages, bone-marrow-derived macrophages (BMDMs) extracted from wild-type (WT) and NLRX1 knockout (KO) mice were stimulated with pro-inflammatory and pro-fibrotic factors to induce M1 and M2 polarization in vitro. The expression levels of macrophage polarization markers (Nos2, Mgl1, Arg1, and Mrc1), as well as the secretion of transforming growth factor β (TGFβ), were measured using RT-PCR and ELISA. Seahorse-based bioenergetics analysis was used to assess mitochondrial respiration in naïve and polarized BMDMs obtained from WT and NLRX1 KO mice. In vivo, WT and NLRX1 KO mice were subjected to unilateral ureter obstruction (UUO) surgery to induce renal fibrosis. Kidney injury, macrophage phenotypic profile, and fibrosis markers were assessed using RT-PCR. Histological staining (PASD and Sirius red) was used to quantify kidney injury and fibrosis. Compared to the WT group, an increased gene expression of M2 markers-including Mgl1 and Mrc1-and enhanced TGFβ secretion were found in naïve BMDMs extracted from NLRX1 KO mice, indicating functional polarization towards the pro-fibrotic M2 subtype. NLRX1 KO naïve macrophages also showed a significantly enhanced oxygen consumption rate compared to WT cells and increased basal respiration and maximal respiration capacities that equal the level of M2-polarized macrophages. In vivo, we found that NLRX1 KO mice presented enhanced M2 polarization markers together with enhanced tubular injury and fibrosis demonstrated by augmented TGFβ levels, fibronectin, and collagen accumulation. Our findings highlight the unique role of NLRX1 in regulating the metabolism and function of macrophages, ultimately protecting against excessive renal injury and fibrosis in UUO.

The Nutritional Intervention of Ingredients from Food Medicine Homology Regulating Macrophage Polarization on Atherosclerosis.

The polarization of macrophages plays a crucial regulatory role in a range of physiological and pathological processes involving macrophages. There are numerous concerns with macrophage polarization in atherosclerosis; however, most focus on modulating macrophage polarization to improve the microenvironment, and the mechanism of action remains unknown. In recent years, the advantages of natural and low-toxicity side effects of food medicine homology-derived substances have been widely explored. Few reports have started from ingredients from food medicine homology to regulate the polarization of macrophages so that early intervention can reduce or delay the process of atherosclerosis. This review summarizes the classification of macrophage polarization and related markers in the process of atherosclerosis. It summarizes the regulatory role of ingredients from food medicine homology in macrophage polarization and their possible mechanisms to provide ideas and inspiration for the nutritional intervention in vascular health.

E3 Ubiquitin Ligase TRIM21 Enhances Macrophage-Mediated Bortezomib Resistance By Inducing M2 Polarization in Multiple Myeloma

Background: Multiple myeloma (MM) is a malignant plasma cell hematologic tumor with an increasing incidence and has become the second most common hematologic malignancy after non-Hodgkin lymphoma. Macrophage is an abundant component of the MM bone marrow microenvironment and contributes to the enhancement of MM drug resistance. Our previous study found that the expression of the E3 ubiquitin ligase TRIM21 was reduced in MM cells, and MM cells with low expression of TRIM21 showed reduced sensitivity to Bortezomib (Bort). This study aims to investigate the role and mechanism of TRIM21 in macrophage-mediated MM Bort resistance. Objective ：Explore the effect and mechanism of TRIM21 in mediating multiple myeloma Bortezomib resistance. Methods ：1. Immunofluorescence to analyze the expression of TRIM21 on macrophages in bone marrow biopsy specimens from patients with MM; 2. QPCR, western blot (WB) and flow cytometry (FCM) to detect the level of macrophage polarization markers after constructing overexpression or knockdown of TRIM21; 3. WB and FCM to detect the sensitivity of MM cells to Bort after co-culture of MM cells and macrophage; 4. Transcriptome sequencing to identify TRIM21-mediated pathway changes in macrophage; 5. QPCR and WB to verify the effect of TRIM21 on macrophage polarization after treatment with pathway inhibitors. Results:1. Immunofluorescence analysis showed increased expression of TRIM21 in macrophage from relapsed and refractory MM (RRMM) patients compared to newly diagnosed multiple myeloma (NDMM) patients;2. QPCR, WB and FCM results showed that overexpression of TRIM21 in macrophage led to decreased polarization markers of M1 phenotype and increased the polarization markers of M2 phenotype. In contrast, knockdown of TRIM21 increased M1 polarization and decreased M2 polarization; 3. WB and FCM results demonstrated that co-culture of MM cells with TRIM21-overexpressing macrophages significantly enhanced MM cell tolerance to Bortezomib and decreased MM cell apoptosis; 4. Transcriptome sequencing revealed activation of the JAK-STAT3 pathway in macrophages after TRIM21 overexpression; 5. QPCR and WB results showed that the macrophage polarization effect induced by TRIM21 overexpression was reversed after treatment with the STAT3 inhibitor C188-9. Conclusions: Our study shows that TRIM21 is overexpressed in macrophages in patients with relapsed and refractory MM. In addition, TRIM21 induces M2 polarization by activating the JAK-STAT3 pathway, thereby enhancing the protective effect of macrophage on MM cells. These findings provide a new insight into the role of TRIM21 in MM drug resistance and lay an important theoretical foundation for targeting macrophages in MM. Key words: TRIM21, Macrophage, Bortezomib, JAK-STAT3