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【Objective】 To discuss the regulating effect of stored red blood cells (RBCs) transfusion on BMDMs in inflammatory conditions, and the relationship between stored RBCs transfusion and inflammatory response induced by bacterial infection. 【Methods】 Forty C57BL/6 male mice of 6-8 weeks (18-22 g/mouse) were randomly divided into experimental group and control group. Each mouse was infected with 200 µL Pseudomonas aeruginosa injecting into the tail vein, and 400 µL fresh (storage >14 d) and stored RBCs (storage 0.05). F4/80 of experimental group and control group 2, 4 and 8 hours after RBCs infused were 1.83±0.11 vs 0.75±0.06, 0.46±0.06 vs 0.33±0.06 (P0.05), respectively. iNOS, TNF-α, MCP1 of M1 in liver of experimental group and control group 2, 4 and 8 hours after RBCs infused were respectively: iNOS 3.44±0.20 vs 2.46±0.08, 9.25±0.55 vs 2.67±0.12, 2.80±0.08 vs 2.39 ±0.01; TNF-α 1.69±0.22 vs 1.13±0.03, 1.44±0.24 vs 0.96±0.09, 1.31±0.05 vs 0.96±0.06; MCP1 4.96±0.08 vs 4.28±0.27, 4.63±0.04 vs 2.07±0.09, 2.28±0.19 vs 1.33±0.03 (P0.05). 【Conclusion】 Stored RBCs infusion can greatly promote the M1 polarization of BMDMs in liver.
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The bile acid-responsive G-protein-coupled receptor TGR5 is expressed in monocytes and macrophages, and plays a critical role in regulating inflammatory response. Our previous work has shown its role in promoting the progression of non-small cell lung cancer (NSCLC), yet the mechanism remains unclear. Here, using Tgr5-knockout mice, we show that TGR5 is required for M2 polarization of tumor-associated macrophages (TAMs) and suppresses antitumor immunity in NSCLC via involving TAMs-mediated CD8+ T cell suppression. Mechanistically, we demonstrate that TGR5 promotes TAMs into protumorigenic M2-like phenotypes via activating cAMP-STAT3/STAT6 signaling. Induction of cAMP production restores M2-like phenotypes in TGR5-deficient macrophages. In NSCLC tissues from human patients, the expression of TGR5 is associated with the infiltration of TAMs. The co-expression of TGR5 and high TAMs infiltration are associated with the prognosis and overall survival of NSCLC patients. Together, this study provides molecular mechanisms for the protumor function of TGR5 in NSCLC, highlighting its potential as a target for TAMs-centric immunotherapy in NSCLC.
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@#Objective To investigate the role of tumor necrosis factor alpha stimulated gene/inducible protein 6（TSG-6）on free silica（SiO2 ）-induced secretion of pro-inflammatory cytokine interleukin（IL）-1β by bone-marrow-derived macrophages （BMDMs）. Methods i）BMDMs isolated from bone marrow were divided into eight groups：the control group was untreated； lipopolysaccharide （LPS） group was stimulated by LPS with a concentration of 50 µg/L；The TSG-6 control group was stimulated by 100 µg/L of recombinant mouse TSG-6. SiO2 model group was pre-stimulated with LPS for four hours，and then stimulated with SiO2 suspension at a final concentration of 250 mg/L；Different dose of TSG-6 groups were firstly treated with above concentrations of LPS and SiO2 suspension，then 10，20，50 and 100 µg/L of recombinant mouse TSG-6 were added respectively；After 16 hours of culture，the cells were collected and the level of IL-1β in BMDMs supernatant was detected by enzyme-linked immunosorbent assay（ELISA）to screen optimal concentration of TSG-6. ii）The cells of the control group，LPS group，SiO2 model group，TSG-6 optimal concentration group and TSG-6 control group were collected. The expression of IL-1β and components of its related pathways in BMDMs was detected by Western blot，including IL-1β，pro-IL-1β，caspase-1，pro dcaspase-1，asc type amino acid transport and NOD-like receptor protein 3（NLRP3）. Results i）Compared with the control group，the expression of IL-1β in SiO2 model group was increased significantly（P<0.01）. Compared with SiO2 model group，the expression of IL-1β in 20，50，100 µg/L dose of TSG-6 groups were decreased significantly（all P<0.01），and the optimal concentration of TSG-6 was found to be 100 µg/L. ii）Compared with the control group and LPS group，the relative expression levels of IL-1β，caspase-1 and NLRP3 in SiO2 model group were increased significantly （all P<0.05）. Compared with SiO2 model group，the expression levels of IL-1β、caspase-1 and NLRP3 were decreased in 100 µg/L TSG-6 group（all P<0.05）. Conclusion TSG-6 could inhibit BMDMs to secret pro-inflammatory cytokine IL-1β by down-regulating the expression of NLRP3 and caspase-1.
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Objective: To study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function. Methods: Bone marrow-derived macrophages (BMMs) were extracted from the marrow of femur and tibia of 4-week-old male C57BL/6 mice. Flow cytometry was used to detect BMMs. The effect of different concentrations of LPS (0, 100, 200, 500, 1 000, 2 000 ng/mL) on BMMs activity was examined by cell counting kit 8 (CCK-8) activity test. In order to investigate the effect of LPS on osteoclastogenesis, BMMs were divided into macrophage colony-stimulating factor (M-CSF) group, M-CSF+receptor activator of nuclear factor κB ligand (RANKL) group, M-CSF+RANKL+50 ng/mL LPS group, M-CSF+RANKL+100 ng/mL LPS group. After the completion of culture, tartrate resistant acid phosphatase (TRAP) staining was used to observe the formation of osteoclasts. In order to investigate the effect of LPS on the expression of Connexin43, BMMs were divided into the control group (M-CSF+RANKL) and the LPS group (M-CSF+RANKL+100 ng/mL LPS); and the control group (M-CSF+RANKL), 50 ng/mL LPS group (M-CSF+RANKL+50 ng/mL LPS), and 100 ng/mL LPS group (M-CSF+RANKL+100 ng/mL LPS). The expressions of Connexin43 mRNA and protein were detected by Western blot and real-time fluorescent quantitative PCR, respectively. In order to investigate the effect of LPS on osteoclast bone resorption, BMMs were divided into M-CSF group, M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group. Bone absorption test was used to detect the ratio of bone resorption area. Results: The flow cytometry test confirmed that the cultured cells were BMMs, and CCK-8 activity test proved that the 100 ng/mL LPS could promote the proliferation of BMMs, showing significant differences when compared with the 0, 200, 500, 1 000, and 2 000 ng/mL LPS ( P<0.05). TRAP staining showed no osteoclast formation in M-CSF group. Compared with M-CSF+RANKL group, the osteoclasts in M-CSF+RANKL+50 ng/mL LPS group and M-CSF+RANKL+100 ng/mL LPS group were larger with more nuclei, while the osteoclasts in M-CSF+RANKL+100 ng/mL LPS group were more obvious, and the differences in the ratio of osteoclast area between groups were statistically significant ( P<0.05). Western blot result showed that the relative expression of Connexin43 protein in LPS group was significantly higher than that in control group ( P<0.05). Real-time fluorescent quantitative PCR showed that the relative expression of Connexin43 mRNA in control group, 50 ng/mL LPS group, and 100 ng/mL LPS group increased gradually, and the differences between groups were statistically significant ( P<0.05). Bone resorption test showed that osteoclast bone resorption did not form in M-CSF group, but the ratio of bone resorption area increased gradually in M-CSF+RANKL group, M-CSF+RANKL+50 ng/mL LPS group, and M-CSF+RANKL+100 ng/mL LPS group, and the differences between groups were statistically significant ( P<0.05). Conclusion: LPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.
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To study which subgroup of bone marrow derived cells formed myofibroblasts and the mechanism that transforming growth factor β1 (TGFβ1) regulates the formation of bone marrow derived macrophages into myofibroblasts during renal fibrosis.Chimeric mice were generated by lethally irradiation of C57 mice followed by transfusion of green fluorescent protein (GFP) labeled bone marrow cells.Complete marrow reconstitution was developed until 12 weeks after transplantation.The mice were randomly divided into Sham operation group,unilateral ureteral obstruction (UUO) 3 days group,UUO5 days group,UUO7 days group and UUO7 with TGFβ1 treatment group.Each group had four mice.Flow cytometry was used to evaluate cell components.Compared with Sham operation group the proportions of GFP + CD+14 oα-smooth muscle actin(α-SMA) + cells,GFP + CD+44 CD+105 α-SMA+ cells and GFP+ F4/80+ α-SMA+ cells in each UUO group were progressively increased and the transformation rate in UUO7 day group was the highest.The GFP+ F4/80 + α-SMA+ cells accounted for the largest population.TGFβ1 promoted the transformation of bone marrow derived macrophages into myofibroblasts.Compared with Sham operation group or UUO7 day group,the proportion of GFP+ F4/80+ α-SMA+ cells increased in UUO7 day TGFβ1 treatment group.Compared with Sham operation group (or UUO7 days group) the protein expressions of F4/80,oα-SMA,Collagen Ⅰ increased in UUO7 with TGFβ1 group.Bone marrow derived macrophages are considered as the main type of myofibroblast precursors during the development of renal fibrosis.TGFβ1 regulates the transformation of bone marrow-derived macrophages into myofibroblasts.This process contributes to progressive renal fibrosis and deterioration of renal function.
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Objective To investigate the regulatory effects and possible mechanisms of autophagy induction and bactericidal activity in muring bone marrow derived macrophages ( BMDMs) . Methods Murine BMDMs were isolated and cultured in vitro. Then standard strain of E. coli was used to infect BMDMs and cellular autophagy levels and AMPK activation were detected. We next modulated functions of AMPK by pretreating cells with the specific agonist or antagonist of AMPK. Then cellular AMPK activation, autophagy levels and bactericidal activity were observed after E. coli infection. Results In E. coli infected BMDMs,autophagy related molecules like LC3B and Beclin1 were upregu-lated,accompanied with elevated LC3Ⅱ/Ⅰratio. Phosphorylation of AMPK was also upregulated by E. coli treatment. Enhanced AMPK activi-ty by its agonist leads to increased cellular autophagy and bactericidal activity,whereas inhibition of AMPK by its suppressor downregulated autophagy and dampened bactericidal activity. Conclusion AMPK and its related signaling pathway is required for anti-bacterial response in macrophage,which is dependent on its function in upregulating autophagy related molecules and inducing autophagy.
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PURPOSE: This study focused on in vitro cell differentiation and surface characteristics in a magnesium coated titanium surface implanted on using a plasma ion source. MATERIALS AND METHODS: 40 commercially made pure titanium discs were prepared to produce Ti oxide machined surface (M) and Mg-incorporated Ti oxide machined surface (MM). Surface properties were analyzed using a scanning electron microscopy (SEM). On each surface, alkaline phosphatase (ALP) activity, alizarin red S staining for mineralization of MC3T3-E1 cells, and quantitative analysis of osteoblastic gene expression, were evaluated. Actin ring formation assay and gene expression analysis of TRAP and GAPDH performing RT-PCR were performed to characterize osteoclast differentiation on mouse bone marrow-derived macrophages (BMMs). RESULTS: MM showed similar surface morphology and surface roughness with M, but was slightly smoother after ion implantation at the micron scale. M was more hydrophobic than MM. No significant difference between surfaces on ALP activity at 7 and 14 days were observed. Real-time PCR analyses showed similar levels of mRNA expression of the osteoblast phenotype genes; osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen 1 (Col 1) in cell grown on MM at 7, 14 and 21 days. Alizarin red S staining at 21 days showed no significant difference. BMMs differentiation increased in M and MM. Actin ring formation assay and gene expression analysis of TRAP showed osteoclast differentiation to be more active on MM. CONCLUSION: Both M and MM have a good effect on osteoblastic cell differentiation, but MM may speed the bone remodeling process by activating on osteoclast differentiation.
Subject(s)
Animals , Mice , Actins , Alkaline Phosphatase , Bone Remodeling , Cell Differentiation , Collagen , Gene Expression , Integrin-Binding Sialoprotein , Macrophages , Magnesium , Microscopy, Electron, Scanning , Osteoblasts , Osteocalcin , Osteoclasts , Osteopontin , Phenotype , Plasma , Real-Time Polymerase Chain Reaction , RNA, Messenger , Surface Properties , TitaniumABSTRACT
Objective To investigate the differences of glycoprotein non-metastatic melanoma b (Gpnmb) expression between M1 and M2 bone marrow-derived macrophages (BMMφs) in mouse.Meth-ods Primary BMMφs were cultured and then identified by immunofluorescence staining for F 4/80 and flow cytometry testing of CD11b.Interferon-γand lipopolysaccharide were used to induce differentiation of BMMφs towards M1 macrophages and interleukin-4 was adopted to induce differentiation of M 2 macropha-ges.Realtime PCR was performed to analyze mRNA expressions of tumor necrosis factor (TNF-α), induc-ible NO synthase (iNOS), macrophage mannose receptor (MMR), arginase-1 (Arg-1) and Gpnmb.Pro-teins of Gpnmb and MMR were detected by double immunofluorescence staining , Western blot and flow cy-tometry.Results (1) Immunofluorescence staining showed high expression of F 4/80 in BMMφs and flow cytometry results showed that CD11b was expressed in 92.7%±6.1% of BMMφs, suggesting that primary BMMφs were successfully cultured.(2) Compared with M0 BMMφs, mRNAs of TNF-αand iNOS were highly up-regulated in M1 BMMφs (both P<0.01), and mRNAs of MMR and Arg-1 were highly up-regula-ted in M2 BMMφs (both P<0.01), indicating that differentiation of BMMφs towards M1 and M2 BMMφs were successfully induced .(3) Expressions of Gpnmb mRNA and Gpnmb protein were predominantly up-regulated in M2 BMMφs in comparison with those in M0 and M1 BMMφs (both P<0.01).Gpnmb and MMR were co-expressed in M2 BMMφs and 83.2%±9.7% of MMR positive BMMφs expressed Gpnmb. Conclusion Gpnmb expression is significantly increased in M 2 macrophages than that in M 1 macrophages in vitro, indicating that Gpnmb which takes part in the differentiation of macrophages might be used as a marker for identification of M 1 and M2 macrophages .
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Osteoclasts, derived from multipotent myeloid progenitor cells, play homeostatic roles in skeletal modeling and remodeling, but may also destroy bone in pathological conditions such as osteoporosis and rheumatoid arthritis. Osteoclast development depends critically on a differentiation factor, the receptor activator of NF-kappaB ligand (RANKL). In this study, we found that the hexane soluble fraction of the common fig Ficus carica (HF6-FC) is a potent inhibitor of osteoclastogenesis in RANKL-stimulated RAW264.7 cells and in bone marrow-derived macrophages (BMMs). HF6-FC exerts its inhibitory effects by suppression of p38 and NF-kappaB but activation of ERK. In addition, HF6-FC significantly decreased the expression of NFATc1 and c-Fos, the master regulator of osteoclast differentiation. The data indicate that components of HF6-FC may have therapeutic effects on bone-destructive processes such as osteoporosis, rheumatoid arthritis, and periodontal bone resorption.