Evaluation of the immunomodulatory effect of Bougainvillea xbuttiana (Var. Orange) on the cytokine production induced by Botrhops jararaca venom in macrophages / Evaluación del efecto inmunomodulador de Bougainvillea xbuttiana (var. naranja) sobre la producción de citoquinas de macrófagos inducida pelo veneno de Bothrops jararaca

To study the effect of 50% ethanol extract of Bougainvillea xbuttiana on the enzymatic activity, cell via bility and cytokine production provoked by the venom of Bothrops jararaca in macro - phages. Three assays were used to study the effects of B. xbuttiana extract on the damage pro - duced by B. jararaca : Enzymatic activity was detected by measuring the proteoly tic and phos - pholipase A2; macrophages cytotoxicity was determined by the MTT method; levels of cytokine were evaluated using ELISA and a biological assay. After treatment with 300 µg/mL B. xbuttiana extract for 30 min, the proteolytic and phospholipase A2 activities of the venom were reduced to 95 and 61%, respectively. In macrophages cultures treated with B. xbuttiana extract combined with venom, the production of TNF - α, IL - 6 and IFN - γ was reduced, whereas IL - 10 was potenti - ated. Our results support the potential effect of the B. xbuttiana extract as a complementary therapy against the toxicity caused by the venom of B . jararaca snakes

Estudiar el efecto del extracto etanólico al 50% de Bougainvillea xbuttiana sobre la actividad enzimática viabilidad celular y producci ón de citoquinas provocada por el veneno de Bothrops jararaca en macrófagos Se utilizaron tres ensayos para estudiar los efectos del extracto de B. xbuttiana sobre el daño producido por B. jararaca : Se detectó actividad enzimática mediante la medición del proteolítico y fosfolipasa A2; la citotoxicidad de los macrófagos se determinó por el método MTT; Los niveles de citoquinas se evaluaron utilizando ELISA y un ensayo biológico. Después del tratamiento con 300 µg/mL de extracto de B. xbuttiana durante 30 mi n, las actividades proteolíticas y de fosfolipasa A2 del veneno se redujeron a 95 y 61%, respectivamente. En cultivos de macrófagos tratados con extracto de B. xbuttiana combinado con veneno, la producción de TNF - α, IL - 6 e IFN - γ se redujeron, mientras que IL - 10 se potenció. Nuestros resultados apoyan el efecto potencial del extracto de B. xbuttiana como terapia complementaria frente a la toxicidad provocada por el veneno de B. jararaca .

Role and mechanism of macrophage-mediated osteoimmune in osteonecrosis of the femoral head / 中国修复重建外科杂志

OBJECTIVE@#To summarize the research progress on the role of macrophage-mediated osteoimmune in osteonecrosis of the femoral head (ONFH) and its mechanisms.@*METHODS@#Recent studies on the role and mechanism of macrophage-mediated osteoimmune in ONFH at home and abroad were extensively reviewed. The classification and function of macrophages were summarized, the osteoimmune regulation of macrophages on chronic inflammation in ONFH was summarized, and the pathophysiological mechanism of osteonecrosis was expounded from the perspective of osteoimmune, which provided new ideas for the treatment of ONFH.@*RESULTS@#Macrophages are important immune cells involved in inflammatory response, which can differentiate into classically activated type (M1) and alternatively activated type (M2), and play specific functions to participate in and regulate the physiological and pathological processes of the body. Studies have shown that bone immune imbalance mediated by macrophages can cause local chronic inflammation and lead to the occurrence and development of ONFH. Therefore, regulating macrophage polarization is a potential ONFH treatment strategy. In chronic inflammatory microenvironment, inhibiting macrophage polarization to M1 can promote local inflammatory dissipation and effectively delay the progression of ONFH; regulating macrophage polarization to M2 can build a local osteoimmune microenvironment conducive to bone repair, which is helpful to necrotic tissue regeneration and repair to a certain extent.@*CONCLUSION@#At present, it has been confirmed that macrophage-mediated chronic inflammatory immune microenvironment is an important mechanism for the occurrence and development of ONFH. It is necessary to study the subtypes of immune cells in ONFH, the interaction between immune cells and macrophages, and the interaction between various immune cells and macrophages, which is beneficial to the development of potential therapeutic methods for ONFH.

Single-Cell Mapping of Brain Myeloid Cell Subsets Reveals Key Transcriptomic Changes Favoring Neuroplasticity after Ischemic Stroke / 神经科学通报·英文版

Interactions between brain-resident and peripheral infiltrated immune cells are thought to contribute to neuroplasticity after cerebral ischemia. However, conventional bulk sequencing makes it challenging to depict this complex immune network. Using single-cell RNA sequencing, we mapped compositional and transcriptional features of peri-infarct immune cells. Microglia were the predominant cell type in the peri-infarct region, displaying a more diverse activation pattern than the typical pro- and anti-inflammatory state, with axon tract-associated microglia (ATMs) being associated with neuronal regeneration. Trajectory inference suggested that infiltrated monocyte-derived macrophages (MDMs) exhibited a gradual fate trajectory transition to activated MDMs. Inter-cellular crosstalk between MDMs and microglia orchestrated anti-inflammatory and repair-promoting microglia phenotypes and promoted post-stroke neurogenesis, with SOX2 and related Akt/CREB signaling as the underlying mechanisms. This description of the brain's immune landscape and its relationship with neurogenesis provides new insight into promoting neural repair by regulating neuroinflammatory responses.

Lipopolysaccharide stimulates macrophages to secrete exosomes containing miR-155-5p to promote activation and migration of hepatic stellate cells / 南方医科大学学报

OBJECTIVE@#To observe the effect of exosomes secreted by lipopolysaccharides (LPS)-stimulated macrophages on hepatic stellate cell activation and migration and explore the underlying molecular mechanism.@*METHODS@#Human monocyte THP-1 cells were induced to differentiate into macrophages using propylene glycol methyl ether acetic acid (PMA, 100 ng/mL, 24 h) followed by stimulation with LPS, and the culture supernatant of macrophages was collected for extraction of the exosomes by ultracentrifugation. The expression of miR-155-5p in the exosomes was detected using qRT-PCR. A Transwell co-culture system was used to observe the effects of the macrophage-derived exosomes on LX2 cell (a hepatic stellate cell line) proliferation, migration, oxidative stress and the expression of fibrosis biomarkers, which were also observed in LX2 cells transfected with miR-155-5p-mimics or miR-155-5p-inhibitors. Western blotting was used to detect the expressions of SOCS1 and its downstream signal pathway proteins.@*RESULTS@#Treatment with the exosomes from LPS-stimulated macrophages significantly enhanced the proliferation and migration ability of LX2 cells and increased the levels of oxidative stress and expressions of the fibrosis markers such as type Ⅰ collagen (P < 0.05). The expression of miR-155-5p in the exosomes secreted by macrophages was significantly increased after LPS treatment (P < 0.01). LX2 cells overexpressing miR-155-5p also exhibited significantly enhanced proliferation and migration with increased oxidative stress levels and expression of type Ⅰ collagen (P < 0.05), and interference of miR-155-5p expression produced the opposite effects. Western blotting showed that miR-155-5p overexpression obviously inhibited SOCS1 expression and promoted p-Smad2/3, Smad2/3 and RhoA protein expressions in LX2 cells (P < 0.05).@*CONCLUSION@#LPS stimulation of the macrophages increases miR-155-5p expression in the exosomes to promote activation and migration and increase oxidative stress and collagen production in hepatic stellate cells.

Akt2 inhibitor promotes M2 macrophage polarization in rats with periapical inflammation by reducing miR-155-5p expression / 南方医科大学学报

OBJECTIVE@#To investigate the effect of Akt2 inhibitor on macrophage polarization in the periapical tissue in a rat model of periapical inflammation.@*METHODS@#Rat models of periapical inflammation were established in 28 normal SD rats by opening the pulp cavity of the mandibular first molars, followed by injection of normal saline and Akt2 inhibitor into the left and right medullary cavities, respectively. Four rats without any treatment served as the healthy control group. At 7, 14, 21 and 28 days after modeling, 7 rat models and 1 control rat were randomly selected for observation of inflammatory infiltration in the periapical tissues by X-ray and HE staining. Immunohistochemistry was used to detect the expression and localization of Akt2, macrophages and the inflammatory mediators. RT-PCR was performed to detect the mRNA expressions of Akt2, CD86, CD163, inflammatory mediators, miR-155-5p and C/EBPβ to analyze the changes in macrophage polarization.@*RESULTS@#X-ray and HE staining showed that periapical inflammation was the most obvious at 21 days after modeling in the rats. Immunohistochemistry and RT-PCR showed that compared with those in the control rats, the expressions of Akt2, CD86, CD163, miR-155-5p, C/EBPβ, and IL-10 increased significantly in the rat models at 21 days (P < 0.05). Compared with saline treatment, treatment with the Akt2 inhibitor significantly decreased the expression levels of Akt2, CD86, miR-155-5p and IL-6 and the ratio of CD86+M1/CD163+M2 macrophages (P < 0.05) and increased the expression levels of CD163, C/EBPβ and IL-10 in the rat models (P < 0.05).@*CONCLUSION@#Inhibition of Akt2 can delay the progression of periapical inflammation in rats and promote M2 macrophage polarization in the periapical inflammatory microenvironment possibly by reducing miR-155-5p expression and activating the expression of C/EBPβ in the Akt signaling pathway.

Macrophage heterogeneity role in NAFLD and NASH disease progression / 中华肝脏病杂志

Nonalcoholic fatty liver disease (NAFLD) is a type of metabolic stress liver injury that is closely associated with insulin resistance and genetic susceptibility. The continuum of liver injury in NAFLD can range from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) and even lead to cirrhosis and liver cancer. The pathogenesis of NAFLD is complicated. Pro-inflammatory cytokines, lipotoxicity, and gut bacterial metabolites play a key role in activating liver-resident macrophages (Kupffer cells, KCs) and recruiting circulating monocyte-derived macrophages (MoDMacs) to deposit fat in the liver. With the application of single-cell RNA-sequencing, significant heterogeneity in hepatic macrophages has been revealed, suggesting that KCs and MoDMacs located in the liver exert distinct functions in regulating liver inflammation and NASH progression. This study focuses on the role of macrophage heterogeneity in the development and occurrence of NAFLD and NASH, in view of the fact that innate immunity plays a key role in the development of NAFLD.

Regulation and influence of mitochondria on macrophages during hepatitis B virus infection / 中华肝脏病杂志

Hepatitis B virus (HBV) infection is an important public health concern, as approximately 3.5% of the world's population is currently chronically infected. Chronic HBV infection is the primary cause of cirrhosis, hepatocellular carcinoma, and deaths related to liver disease globally. Studies have found that in HBV infection, viruses can directly or indirectly regulate mitochondrial energy metabolism, oxidative stress, respiratory chain metabolites, and autophagy, thereby altering macrophage activation status, differentiation types, and related cytokine secretion type and quantity regulations. Therefore, mitochondria have become an important signal source for macrophages to participate in the body's immune system during HBV infection, providing a basis for mitochondria to be considered as a potential therapeutic target for chronic hepatitis B.

Regulation of colony-stimulating factor 1 receptor inhibitor pexidartinib on the senescence of mouse bone marrow-derived macrophages stimulated by lipopolysaccharide / 中华口腔医学杂志

Objective: To investigate the effects of colony-stimulating factor 1 receptor (CSF-1R) inhibitor pexidartinib (PLX3397) on the senescence of bone marrow-derived macrophages (BMDM) stimulated by lipopolysaccharide (LPS). Methods: BMDM were isolated and cultured from femurs and tibiae of 10 male C57BL/6 mice aged 6-8 weeks (obtained from Laboratory Animal Center of Guizhou Medical University). They were divided into blank control group, LPS group (treated with 1 μg/ml LPS for 24 h) as well as low, medium and high concentration PLX3397 pretreatment groups (treated with 100, 500 and 1 000 nmol/L PLX3397 for 4 h respectively followed by 1 μg/ml LPS for 24 h). The corresponding markers of macrophages were detected by flow cytometry. Cell viability was detected by cell counting kit-8 and cellular senescence was detected by senescence-associated-β-galactosidase (SA-β-gal) staining. Meanwhile, protein expressions of cycle-dependent kinase inhibitor p16, p21 and CSF-1R were detected by Western blotting, and the expressions of p16 and p21 were detected by intracellular immunofluorescence. Real-time fluorescence quantitative PCR (RT-qPCR) was used to investigate the mRNA levels of senescence-associated secretory phenotype (SASP) genes including interleukin (IL), IL-1β, chemokine-1/10 (CXCL-1/10), matrix metalloproteinase-8 (MMP-8), and transforming growth factor-β (TGF-β). Results: The rate of SA-β-gal positive staining in medium and high concentration PLX3397 pretreatment groups [(39.33±4.93)% and (36.33±3.06)% respectively] were significantly downregulated compared with LPS group [(52.00±3.00)%] (P=0.020, P=0.005). The expression of CSF-1R protein in low, medium and high concentration PLX3397 pretreatment groups were (0.74±0.18, 0.61±0.07, 0.54±0.06), all of which were significantly lower than that in LPS group (1.16±0.08) (P=0.013, P=0.002, P<0.001). The expression levels of CSF-1R mRNA in low, medium and high concentration PLX3397 pretreatment groups (1.04±0.06, 0.90±0.05, 1.18±0.08) showed similar trend (2.90±0.25) (P<0.001). The average fluorescence intensity of p16 in all PLX3397 pretreatment groups were 49.76±3.65, 48.21±1.72, 47.99±1.26 respectively, which were significantly lower than that in LPS group (66.88±5.85) (P=0.001, P<0.001, P<0.001). The average fluorescence intensity of p21 in medium and high concentration PLX3397 pretreatment groups were (34.43±3.62, 30.13±0.86), significantly lower than that in LPS group (46.82±5.33) (P=0.043, P=0.007). The expression of p16 protein in low, medium and high concentration PLX3397 pretreatment groups (0.56±0.04, 0.55±0.04, 0.35±0.19) were significantly lower than that in LPS group (0.98±0.10) (P=0.003, P=0.002, P<0.001), as well the expression of p21 protein (0.69±0.20, 0.42±0.08, 0.26±0.14) (P=0.032, P=0.002, P<0.001). According to the results of RT-qPCR, the expressions of IL-6, IL-1β, CXCL-1, CXCL-10 and MMP-8 in PLX3397 pretreatment groups were significantly lower than those in LPS group (P<0.001), while the expression of TGF-β increased (P<0.001). Conclusions: LPS could induce the cell senescence, increase the secretion of SASP and aggravate local inflammation by activating the CSF-1R on the cell surface of bone marrow-derived macrophages. CSF-1R inhibitor PLX3397 might attenuate CSF-1R activation associated with LPS and inhibit the senescence of bone marrow-derived macrophages induced by LPS.

Infiltration and significance of M1 macrophage in breast cancer: an analysis of METABRIC database and clinical validation / 中华肿瘤杂志

Objective: To investigate the differences of immune microenvironment between stage T1N3 and stage T3N0 breast cancer patients and explore the relationship between M1 macrophage infiltration and lymph node metastasis in breast cancer. Methods: Clinical information and RNA-sequencing (RNA-Seq) expression data of stage T1N3 (n=9) and stage T3N0 (n=11) breast cancer patients were extracted from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) databases. Using CIBERSORT, the proportions of 22 types of immune cells were calculated, and then the differences of immune cell infiltration between stage T1N3 and T3N0 patients were compared. From 2011 to 2022, pathologic specimens were collected from breast cancer patients who underwent curative resection at the Cancer Hospital, Chinese Academy of Medical Sciences, including 77 at stage T1N3 and 58 at stage T3N0.The METABRIC database analysis results were verified by examining the density of M1 macrophages in tissues using dual-staining immunohistochemistry. Results: METABRIC data analysis showed M1 macrophage was the highest proportion, 15.85% in stage T1N3 breast cancer; M2 macrophage was the highest proportion, 13.07% in stage T3N0 breast cancer.M1 macrophage proportions were statistically different between patients with stage T1N3 and stage T3N0 (P=0.010). The dual-staining immunohistochemistry analysis of breast cancer tissues showed M1 macrophage density (median) of 62.0 and 38.0 cells/mm(2) for stage T1N3 and T3N0, respectively. The difference was statistically significant (P=0.002). Conclusion: The density of M1 macrophages is notably higher in stage T1N3 patients and is associated with lymph node metastasis.

Bioinformatics analysis and key gene verification of sepsis myocardial macrophage microarray data based on GEO database / 中华心血管病杂志

Objective: Bioinformatics analysis was used to screen differentially expressed genes (DEGs) in macrophages of sepsis myocardial injury and to verify key genes. Methods: Experiment 1 (gene chip and bioinformatics analysis): The gene chip data GSE104342 of cardiac macrophages in septic mice was downloaded from Gene Expression Omnibus database. DEGs were obtained by R language analysis. DAVID online database was used to obtain gene ontology and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of DEGs. STRING online database was used for protein-protein interaction network analysis of DEGs, and then key genes were screened by using Cytoscape software and molecular complex detection (MCODE) plug-ins. Experiment 2 (sepsis model construction and related protein verification): Ten male C57BL/6 mice, aged 8-14 weeks. Five mice were randomly selected as control group, and 5 mice were selected as the sepsis group by building a mice sepsis model in vivo. Echocardiography was used to detect the cardiac function. Hematoxylin-eosin staining was used to assess the cardiac morphology. TUNEL staining was used to evaluate cardiomyocyte apoptosis. Immunofluorescence staining was used to detect the expression of differentiation antigen cluster 206 (CD206),inducible nitric oxide synthases (iNOS),F4/80,suppressor of cytokine signaling 3 (Socs3) ,interleukin 1 receptor antagonist (Il1rn) and chemokine C-C motif ligand 7 (Ccl7) protein. RAW264.7 macrophages were cultured in vitro and divided into 2 groups: LPS groupstimulated by lipopolysaccharide (LPS, 1 mg/L) and blank control group treated with equal-volume phosphate buffer solution. Reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the expression of Socs3, Il1rn and Ccl7 in vitro. Results: Experiment 1: 24 647 genes were screened in GSE104342 dataset and 177 genes (0.72%) were differential expression, including 120 up-regulated genes and 57 down-regulated genes. Gene ontology enrichment analysis showed that DEGs were mainly involved in inflammatory response, immune response, apoptosis regulation and antigen processing and presentation. KEGG signaling pathway analysis showed that DEGs in cardiac macrophages of septic mice were mainly enriched in cytokine-cytokine receptor interaction, tumor necrosis factor signaling pathway, NOD like receptor signaling pathway. Three hub genes were obtained by STRING and Cytoscape analysis, including Socs3, Il1rn and Ccl7. Experiment 2: In vivo, it was found that compared with the control group, the cardiac function of the sepsis mice decreased significantly, the myocardial cells were significantly edema, inflammatory cell infiltration, myocardial fiber rupture, some myocardial nuclei dissolved and disappeared, and the cardiomyocyte apoptosis increased, suggesting that the sepsis myocardial injury model of mice was successfully constructed. Compared with the control group, the expression of CD206 in the myocardium of septic mice was down-regulated, the expression of iNOS, F4/80, Socs3, Il1rn and Ccl7 were up-regulated. In addition, there was co-localization between Socs3, Il1rn, Ccl7 and F4/80 protein. Compared with the blank control group, the expression of Socs3, Il1rn and Ccl7 significantly upregulated after LPS intervention in vitro by RT-PCR. Conclusions: The selected key genes Socs3, Il1rn and Ccl7 were up-regulated in myocardial macrophages of septic mice. Socs3, Il1rn and Ccl7 are expected to become new targets for the diagnosis and treatment of sepsis cardiac injury.

Platelet-rich Plasma Induces M2 Macrophage Polarization via Regulating AMPK Singling Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the role of platelet-rich plasma (PRP) in inducing the M2 macrophage polarization via regulating AMPK singling pathway.@*METHODS@#The expressions of M1 marker CD11c and M2 marker CD206 in macrophages of blank control group, LPS group, LPS+PRP group, and LPS+PRP+Compound C group were detected by flow cytometry. Western blot was used to observe the effects of PRP on the expression of AMPK-mTOR signaling pathway-related proteins at different times (12 h, 18 h and 24 h) after LPS treatment. RNA interference technology was used to silence the expression of AMPK in macrophages, and the expression of TGF-β protein was subsequently examined by Western blot.@*RESULTS@#LPS significantly reduced the expression of CD206 and increased the expression of CD11c (P <0.05). After the addition of PRP, the expression of CD206 was significantly increased (P <0.05), while the expression of CD11c was significantly decreased (P <0.05). Compared with LPS group, PRP treatment significantly increased the expressions of p-AMPK and p-ULK1 proteins at 12 h, 18 h and 24 h, while significantly decreased the expression of p-mTOR protein (P <0.05). After the addition of AMPK inhibitor Compound C, the expression of CD206 was significantly reduced (P <0.05) and the expression of CD11c was significantly increased compared with LPS+PRP group (P <0.05). After silencing the expression of AMPK in macrophages, the promotion effect of PRP on TGF-β was significantly reduced (P <0.05).@*CONCLUSION@#PRP can stimulate the transformation of macrophages to M2 type via AMPK signalling pathway.

Research Progress on the Mechanism of Macrophages Regulating Hematopoiesis in Bone Marrow Microenvironment--Review / 中国实验血液学杂志

Bone marrow macrophage is an important component of bone marrow microenvironment, which is closely related to hematopoietic regulation and hematopoietic stem cell transplantation(HSCT). Recent studies have shown that bone marrow macrophage is an important part of hematopoietic stem cell niche, which can help regulate the mobilization and function of hematopoietic stem/progenitor cells. After HSCT, the microenvironment of bone marrow is damaged and a large number of macrophages infiltrate into the bone marrow. Regulating the macrophage-related signal pathways can promote the recovery of hematopoiesis and the reconstruction of hematopoietic function. Co-culture of macrophages and hematopoietic stem cells (HSC) in vitro significantly increased the number of HSCs and their ability of clone formation, which suggests that macrophages play an important role in the regulation of hematopoiesis in the hematopoietic microenvironment of bone marrow. This paper reviews the recent research progress on the role of macrophages in bone marrow hematopoietic microenvironment.

Hepatocyte steatosis activates macrophage inflammatory response accelerating atherosclerosis development / 浙江大学学报·医学版

OBJECTIVES@#To investigate the mechanism of comorbidity between non-alcoholic fatty liver disease (NAFLD) and atherosclerosis (AS) based on metabolomics and network pharmacology.@*METHODS@#Six ApoE－/－ mice were fed with a high-fat diet for 16 weeks as a comorbid model of NAFLD and AS (model group). Normal diet was given to 6 wildtype C57BL/6J mice (control group). Serum samples were taken from both groups for a non-targeted metabolomics assay to identify differential metabolites. Network pharmacology was applied to explore the possible mechanistic effects of differential metabolites on AS and NAFLD. An in vitro comorbid cell model was constructed using NCTC1469 cells and RAW264.7 macrophage. Cellular lipid accumulation, cell viability, morphology and function of mitochondria were detected with oil red O staining, CCK-8 assay, transmission electron microscopy and JC-1 staining, respectively.@*RESULTS@#A total of 85 differential metabolites associated with comorbidity of NAFLD and AS were identified. The top 20 differential metabolites were subjected to network pharmacology analysis, which showed that the core targets of differential metabolites related to AS and NAFLD were STAT3, EGFR, MAPK14, PPARG, NFKB1, PTGS2, ESR1, PPARA, PTPN1 and SCD. The Kyoto Encyclopedia of Genes and Genomes showed the top 10 signaling pathways were PPAR signaling pathway, AGE-RAGE signaling pathway in diabetic complications, alcoholic liver disease, prolactin signaling pathway, insulin resistance, TNF signaling pathway, hepatitis B, the relax in signaling pathway, IL-17 signaling pathway and NAFLD. Experimental validation showed that lipid metabolism-related genes PPARG, PPARA, PTPN1, and SCD were significantly changed in hepatocyte models, and steatotic hepatocytes affected the expression of macrophage inflammation-related genes STAT3, NFKB1 and PTGS2; steatotic hepatocytes promoted the formation of foam cells and exacerbated the accumulation of lipids in foam cells; the disrupted morphology, impaired function, and increased reactive oxygen species production were observed in steatotic hepatocyte mitochondria, while the formation of foam cells aggravated mitochondrial damage.@*CONCLUSIONS@#Abnormal lipid metabolism and inflammatory response are distinctive features of comorbid AS and NAFLD. Hepatocyte steatosis causes mitochondrial damage, which leads to mitochondrial dysfunction, increased reactive oxygen species and activation of macrophage inflammatory response, resulting in the acceleration of AS development.

Advances in macrophage-targeting nanoparticles for the diagnosis and treatment of inflammatory bowel disease / 浙江大学学报·医学版

The pathogenesis of inflammatory bowel disease (IBD) is not fully elucidated. However, it has been considered that inflammatory macrophages may be involved in the imbalance of the intestinal mucosal immunity to regulate several signaling pathways, leading to IBD progression. The ratio of M1 to M2 subtypes of activated macrophages tends to increase in the inflamed intestinal section. There are challenges in the diagnosis and treatment of IBD, such as unsatisfactory specificity of imaging findings, low drug accumulation in the intestinal lesions, unstable therapeutic efficacy, and drug-related systemic toxicity. Recently developed nanoparticles may provide a new approach for the diagnosis and treatment of IBD. Nanoparticles targeted to macrophages can be used as contrast agents to improve the imaging quality or used as a drug delivery vector to increase the therapeutic efficiency of IBD. This article reviews the research progress on macrophage-targeting nanoparticles for the diagnosis and treatment of IBD to provide a reference for further research and clinical application.

Establishment of a method for separating macrophage migrasomes / 细胞与分子免疫学杂志

Objective To establish an efficient method for isolating migrasomes from RAW264.7 macrophages and identifying these isolated migrasomes. Methods Scanning electron microscopy was used to observe the morphological characteristics of migrasomes produced by RAW264.7 cells. A 0.45 μm filter was employed for reverse filtration and elution to isolate the migrasomes. The morphological characteristics of the migrasomes were then observed using transmission electron microscopy. Western blot analysis was performed to determine the expression of characteristic markers of the migrasomes. The RNA carried by the migrasomes was analysed by using LabChip bioanalyzer. Results Scanning electron microscopy revealed that the migrasomes, with membranous structures, were attached to the tip or bifurcation of the retraction fiber formed in the tail of RAW264.7 cells. Transmission electron microscopy showed that the isolated migrasomes had a typical oval vesicle-like structure with wrinkled membrane surfaces. Western blot analysis confirmed the expression of the characteristic markers phosphatidylinositol glycan anchor biosynthesis class K (PIGK), epidermal growth factor domain-specific O-linked N-acetylglucosamine transferase (EOGT) and tetraspanin 4 (TSPAN4) in the migrasomes, while the EV (extracellular vesicle) markers tumor susceptibility gene 101 (TSG101) and Arabidopsis homolog of apoptosis-linked gene 2-interacting protein X (ALIX) were not detected. Furthermore, the isolated migrasomes were found to be rich in small RNA, which were approximately 25-200 nt in length. Conclusion A method for the extraction of well-structured and high quality migrasomes from macrophages is established.

Research progress on tumor-associated macrophages in colorectal cancer / 细胞与分子免疫学杂志

Colorectal cancer is a common malignant tumor in gastrointestinal tract. Its onset and development are associated with its own characteristics as well as the tumor microenvironment (TME) in which tumor-associated macrophages (TAMs) are the most abundant immune cells. After being recruited to the tumor site and stimulated by different signals in TME, TAMs can grow into two different subtypes, namely M1 and M2. TAMs are mainly manifested as M1 macrophages in the early stage of colorectal cancer, mediating the immune response to inhibit tumor growth. In the late stage, TAMs mainly grow into M2 macrophages, showing the ability to suppress immunity, stimulate the proliferation of tumor cells and tumor angiogenesis, and promote the invasion and metastasis of tumor cells. It has been found that intervention in TAMs polarization can regulate its relationship with the onset and development of colorectal cancer.

Silencing of SMAD family member 3 promotes M2 polarization of macrophages and the expression of SMAD7 in rheumatoid arthritis / 细胞与分子免疫学杂志

Objective To investigate the effect of SMAD family member 3(SMAD3) silenced by small interfering RNA (siRNA) on macrophage polarization and transforming growth factor β1 (TGF-β1)/ SMAD family signaling pathway in rheumatoid arthritis (RA). Methods RA macrophages co-cultured with rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) were used as a cell model. TGF-β1 was used to stimulate macrophages, and SMAD3-specific siRNA (si-SMAD3) and negative control siRNA (si-NC) were transfected into human RA macrophages co-cultured in TranswellTM chamber. The expression of SMAD3 mRNA was detected by real-time fluorescence quantitative PCR, and the expression of TGF-β1, SMAD3 and SMAD7 protein was detected by Western blot analysis. The contents of TGF-β1 and IL-23 in cell culture supernatant were determined by ELISA. Cell proliferation was detected by CCK-8 assay. TranswellTM chamber was used to measure cell migration. Results Compared with the model group and the si-NC group, the expression of TGF-β1, SMAD3 mRNA and protein in RA macrophages decreased significantly after silencing SMAD3. In addition, the secretion of IL-23 decreased significantly, and the cell proliferation activity and cell migration were inhibited, with high expression of SMAD7. Conclusion Knockdown of SMAD3 can promote M2 polarization and SMAD7 expression in RA macrophages.

Effects and mechanism of knocking down lncRNA H19 to inhibit lipid accumulation in human THP-1 cells-derived macrophages / 细胞与分子免疫学杂志

Objective To investigate the effects of long noncoding RNA H19 on lipid accumulation of macrophages under high fat stress and its mechanism. Methods Human THP-1 cells-derived macrophages were incubated with ox-LDL, and the effects of H19 siRNA intervention on lipid accumulation was observed. The THP-1 cells were divided into control group (conventional culture), ox-LDL group, siRNA negative control (NC siRNA) combined with ox-LDL treatment group, and H19 siRNA combined with ox-LDL treatment group. Oil red O staining was used to determine the lipid accumulation in cells, and cholesterol concentration was analyzed by enzymatic method; ATP assay kit for detecting celluar ATP content; colorimetry was used to detect the levels of oxidative stress indicators and ELISA was used to detect the levels of monocyte chemoattractant protein-1 (MCP-1) in the cell supernatant. Western blot analysis was used to detect the protein expression of ATP binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor α (PPARα), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and nuclear factor κB p-p65 (NF-κB p-p65). Results Knockdown H19 significantly inhibited intracellular lipid accumulation, decreased total cholesterol (TC) and cholesterol ester (CE) content, and decreased CE/TC ratio. Knockdown H19 significantly alleviated cell damage including an increase in ATP content, a decrease in oxidative stress levels and a decrease in MCP-1 levels, which caused by high-fat stress. H19 siRNA upregulated expression of ABCA1, PPARα and PGC-1α in THP-1 derived macrophages, downregulated NF-κB signal pathway. Conclusion Knockdown H19 upregulates PGC-1α expression in THP-1 cells and downregulates NF-κB pathway, which promotes cholesterol reverse transport, reduces inflammatory reaction and inhibits lipid accumulation.

Proanthocyanidins alleviate lipopolysaccharide-induced inflammatory response by up-regulating SIRT1 expression and inhibiting NF-κB pathway in mouse RAW264.7 macrophages / 细胞与分子免疫学杂志

Objective To investigate the role of proanthocyanidins (PC) in lipopolysaccharide (LPS)-induced inflammatory response and its possible mechanism in RAW264.7 macrophages. Methods RAW264.7 macrophages were cultured and treated with PBS and different concentrations of PC for 24 hours, followed by 1 μg/mL LPS for 6 hours. Real-time PCR was used to detect the mRNA expression of interleukin1β (IL-1β), IL-6, monocyte chemoattractant protein 1 (MCP-1), tumor necrotic factor α (TNF-α), IL-4 and arginase 1 (Arg1) in RAW264.7 macrophages. Flow cytometry was used to detect the effects of PBS group, LPS group and PC combined with LPS group on M1 and M2 polarization of macrophages. The protein expressions of silenced information regulator 1 (SIRT1), nuclear factor kappa B p65(NF-κB p65) and acetylated NF-κB p65 (Ace-p65) were detected by Western blot analysis after different concentrations of PC treatment. Co-immunoprecipitation assay was used to detect the binding effect of SIRT1 to NF-κB p65 in macrophages treated with PC. Results Compared with PBS group, the mRNA expression of macrophage pro-inflammatory cytokines IL-1β, IL-6, MCP-1 and TNF-α decreased and the mRNA expression of anti-inflammatory factors IL-4 and Arg1 increased in PC group. Compared with LPS group, PC combined with LPS group could significantly inhibit M1 polarization and promote M2 polarization of macrophages. With the increase of PC concentration, the expression of SIRT1 was up-regulated, and NF-κB p65 protein did not change significantly. The expression of Ace-p65 protein decreased significantly when treated with high concentration of PC. Conclusion PC can significantly alleviate the LPS-induced inflammatory response by up-regulating the expression of SIRT1 and inhibiting NF-κB pathway in RAW264.7 macrophages.

Nano-vesicles derived from M1 macrophages affect the development of mouse endometriosis by polarizing M2 macrophages to M1 phenotype / 细胞与分子免疫学杂志

Objective To explore whether nano-vesicles derived from M1 macrophages (M1-NVs) can reprogram M2 macrophages into M1 phenotype and further affect the development of endometriosis (EMS). Methods Extracellular vesicles (EVs) were isolated from macrophage culture supernatant by differential centrifugation. Immunofluorescence cytochemistry was used to detect the expression of vimentin, CD31 and F4/80 to identify endometrial stromal cells (EMS-ESCs), HUVECs and polarized peritoneal macrophages of EMS patients. M1-NVs were prepared by filtering cell suspension through (5, 1, 0.4, 0.22)μm polycarbonate membrane filters after syringe aspiration at 0-4 DegreesCelsius. Flow cytometry was used to analyze the polarization of RAW264.7 mouse peritoneal macrophages in vitro, and reverse transcription PCR (RT-qPCR) was employed to detect mRNA expression of VEGF, CD86, interleukin-6 (IL-6), IL-1β, tumor necrosis factor α (TNF-α), arginase 1 (Arg1), CD163, CD206, and IL-10. PKH67-labeled M1-NVs were co-cultured with EMS-ESCs, HUVECs and macrophages. And tubule formation experiments were conducted to assess the impact of M1-NVs on the tubule formation of HUVECs. TranswellTM invasion and migration assays were employed to evaluate changes in the migration and invasion abilities of EMS-ESCs. Results By monitoring the contents of NVs, it was found that NVs contained much more protein and other bioactive particles than the same amount of EVs; immunofluorescence staining results showed that PKH67 labeled M1-NVs were internalized by EMS-ESCs, HUVECs and macrophages when co-cultured. The results of flow cytometry and RT-qPCR multi-target analysis showed that after treatment with different concentrations of M1-NVs or M0-NVs, 20 μg/mL of M1-NVs could effectively reprogram M2 macrophages into M1 macrophages compared with M0-NVs. TransewellTM results showed that compared with the blank group and M0-NVs group, the number of EMS-ESCs migrating from the upper chamber to the lower chamber after M1-NV treatment was significantly reduced, while the number of EMS-ESCs treated with M2NVs increased significantly. The invasion situation was similar to the migration situation, indicating that M1-NVs directly or indirectly inhibited invasion, migration and tubule formation of EMS-ESCs in vitro. Conclusion M1-NVs reprogrammes M2 macrophages into M1 macrophages by internalization of primary cells and macrophages, thereby inhibiting invasion, migration and angiogenesis of EMS-ESCs, and further hindering the occurrence and development of EMS.