Objective To evaluate therapeutic efficacy and safety of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) inhalation in patients with recurrent pulmonarv alveolar proteinosis (PAP). Methods Three cases of recurrent PAP were treated by GM-CSF inhalation after whole lung lavage. The clinical data of the pulmonary function and SpO 2, the clinical symptoms and pulmonary lesions were compared before and after treatment. Results The pulmonary function and manifestations were improved obviously after GM-CSF inhalation. Also the ground-glass opacity was improved in high-resolution CT. The pulmonary function and SpO 2 increased obviously after received GM-CSF inhalation. There were no any adverse reactions in 3 cases. Conclusion GM-CSF inhalation therapy is effective and safe in recurrent PAP, but the long-term effect remains to be seen.
Objective To review the osteoimmunomodulatory effects and related mechanisms of inorganic biomaterials in the process of bone repair. Methods A wide range of relevant domestic and foreign literature was reviewed, the characteristics of various inorganic biomaterials in the process of bone repair were summarized, and the osteoimmunomodulatory mechanism in the process of bone repair was discussed. Results Immune cells play a very important role in the dynamic balance of bone tissue. Inorganic biomaterials can directly regulate the immune cells in the body by changing their surface roughness, surface wettability, and other physical and chemical properties, constructing a suitable immune microenvironment, and then realizing dynamic regulation of bone repair. Conclusion Inorganic biomaterials are a class of biomaterials that are widely used in bone repair. Fully understanding the role of inorganic biomaterials in immunomodulation during bone repair will help to design novel bone immunomodulatory scaffolds for bone repair.
Objective To investigate the effect of human tooth bone graft materials on the proliferation, differentiation, and morphology of macrophages, and to understand the biocompatibility and cytotoxicity of human tooth bone graft materials. Methods Fresh human teeth were collected to prepare human tooth bone graft materials, the adhesion of mouse mononuclear macrophages RAW264.7 to human bone graft materials was observed under confocal microscopy. Scanning electron microscopy was used to observe the morphology of human tooth bone graft materials, OSTEONⅡ synthetic highly resorbable bone grafting materials, and untreated tooth powder (dental particles without preparation reagents). Different components of the extract were prepared in 4 groups: group A (DMEM medium containing 10% fetal bovine serum), group B (human tooth bone graft materials), group C (OSTEONⅡ synthetic highly resorbable bone grafting materials), group D (untreated tooth powder without preparation reagents). The 4 groups of extracts were co-cultured with the cells, and the cytotoxicity was qualitatively determined by observing the cell morphological changes by inverted microscope. The cell proliferation and differentiation results and cell relative proliferation rate were determined by MTT method to quantitatively determine cytotoxicity. The cell viability was detected by trypanosoma blue staining, and tumor necrosis factor α (TNF-α ) and interleukin 6 (IL-6) expressions were detected by ELISA. Results Scanning electron microscopy showed that the surface of the human tooth bone graft material and the OSTEONⅡ synthetic highly resorbable bone grafting materials had a uniform pore structure, while the untreated tooth particle collagen fiber structure and the demineralized dentin layer collapsed without specific structure. Confocal microscopy showed that the cells grew well on human tooth bone graft materials. After co-culture with the extract, the morphology and quantity of cells in groups A, B, and C were normal, and the toxic reaction grades were all grade 0, while group D was grade 3 reaction. MTT test showed that the cytotoxicity of groups B and C was grade 0 or 1 at each time point, indicating that the materials were qualified. The cytotoxicity was grade 2 in group D at 1 day after culture, and was grade 4 at 3, 5, and 7 days. Combined with cell morphology analysis, the materials were unqualified. The trypanosoma blue staining showed that the number of cells in groups A, B, and C was significantly higher than that in group D at each time point (P<0.05), but no significant difference was found among groups A, B, and C (P<0.05). ELISA test showed that the levels of TNF-α and IL-6 in groups A, B, and C were significantly lower than those in group D (P<0.05), but no significant difference was found among groups A, B, and C (P<0.05). Conclusion The human tooth bone graft materials is co-cultured with mice mononuclear macrophages without cytotoxicity. The extract has no significant effect on cell proliferation and differentiation, does not increase the expression of inflammatory factors, has good biocompatibility, and is expected to be used for clinical bone defect repair.
In the tumor microenvironment, tumor-associated macrophage, as polarized macrophages M2 phenotype, can promote tumor progression and affect the prognosis of cancer. Significant attention has been drawn towards tumor-associated macrophage in recent years. In this review, we describe the polarization state of macrophages determined by tumor microenvironment and the recruitment of tumor-associated macrophage. We also pay special attention to the interaction between tumor-associated macrophages and tumors, discuss and summarize various targeted therapy strategies for tumor-associated macrophages, aiming to provide a reference for the future development of these novel and effective anti-cancer treatments.
Objective To investigate the role of alveolar macrophages ( AMs ) in airway inflammation of smoke-induced COPD rat model and its possible regulating mechanism. Methods Twelve Wistar rats were randomly divided into a COPD group and a control group. The rat model of COPD was established with smoke exposure and LPS intrathacheal instillation. Bronchoalveolar lavage fluid ( BALF)was collected for measurement of total and differential cell counts. Then AMs were isolated and identified byimmunofluorescence. Western blot was employed to analyze the cytoplasmic and nuclear NF-κB p65 expression of AMs. The concentrations of TNF-α,macrophage inflammatory protein 2 ( MIP-2) and IL-10 in cell culture supernatantwere assayed by ELISA.Results The scores of bronchitis and mean liner intercepts in the COPD group were significantly higher than those in the control group [ 4. 33 ±1. 16 vs. 1. 33 ±0. 58,P =0. 016; ( 168. 77 ±11. 35) μm vs. ( 93. 61 ±4. 16) μm, P = 0. 000) ] . The total cell count in BALF of the COPD group was significantly higher than that in the control group ( P lt; 0. 05) , and the AMs and neutrophils were predominant [ ( 72. 00 ±2. 22) % and ( 18. 29 ±8. 34) % ] . The cytoplasmic NF-κB p65 expression of AMs in the COPD group was significantly lower , while the nuclear NF-κB p65 expression was significantly higher ( P lt; 0. 05) compared with the control group. The ELISA results showed that the concentrations of TNF-αand MIP-2 in culture supernatant of AMs in the COPD group were significantly higher than those in the control group ( P lt;0. 05) , while the concentration of IL-10 was not significantly different between the two groups ( P gt;0. 05) . Conclusions COPD rat model was established successfully with smoke exposure and LPS intratracheal instillation with a profile of macrophage-based chronic inflammation and increased secretion of TNF-αand MIP-2. The mechanismis closely related to activation of NF-κB.
ObjectiveTo investigate the regulatory role of MSC-derived exosomes in obliterative bronchiolitis after lung transplantation. MethodsThe murine lung transplantation model was established with male C57BL/6 mice, and the mice were divided into a sham group (sham, n=6), a surgery group (OB, n=6), and a treatment group (OB+MSC-exo, n=6). The in vitro model was created by stimulating RAW264.7 with lipopolysaccharide+nigericin (LPS+Nigericin), and comprised a PBS group, a LPS+Nigericin group, and a LPS+Nigericin+MSC-exo group. Immunofluorescence and hematoxylin-eosin (HE) staining were used to analyze gasdermin D (GSDMD) expression, as well as lumen stenosis in lung grafts. Bioinformatics methods were employed to predict and screen target gene collagen type V alpha 1 (COL5A1). Q-PCR was used to measure mRNA expression levels of interleukin (IL)-1β, IL-18, IL-6, tumor necrosis factor-α (TNF-α), and COL5A1 in lung grafts and macrophages. Western blot was performed to detect Cleaved-Caspase 1 protein expression in lung grafts and GSDMD protein expression in macrophages. ResultsImmunofluorescence and HE staining revealed that in vivo infusion of MSC-exo reduced GSDMD expression in grafts, ameliorated tracheal epithelial cilia loss and lumen stenosis, and decreased Cleaved-Caspase 1 protein as well as IL-1β and IL-18 mRNA expression. MSC-exo treatment or COL5A1 knockdown reduced IL-1β, IL-18, IL-6, and TNF-α mRNA expression in macrophages, with comparable efficacy. MSC-exo infusion also decreased the number of COL5A1+ cells and mRNA expression levels in lung grafts. ConclusionMSC-derived exosomes alleviate obliterative bronchiolitis after lung transplantation by inhibiting COL5A1.
Objective To investigate the regulatory effects of SHP2 inhibition on the secretion of macrophage-associated inflammatory factors in KRAS-mutant lung cancer cells and to elucidate the underlying mechanisms by which this inhibition remodels the tumor immune microenvironment. Methods Three KRAS-mutant lung cancer cell lines were treated with the SHP2 inhibitor SHP099. The levels of phosphorylated SHP2 and ERK were assessed by Western blot. The expression levels of related inflammatory factors were analyzed using Luminex assay and qRT-PCR assay. Transcriptome sequencing was performed to identify differentially expressed genes and conduct KEGG pathway enrichment analysis. The expression of CXCL8 was validated by flow cytometry and Western blot. Survival analysis and gene set correlation analysis were conducted based on the TCGA database. Results SHP099 significantly inhibited the expression of p-SHP2 and p-ERK proteins, and reduced the secretion of multiple macrophage-related inflammatory factors. qRT-PCR confirmed a decrease in CXCL8 mRNA levels. Transcriptome analysis revealed significant enrichment of the rheumatoid arthritis pathway. Flow cytometry and Western blot validated a significant reduction in CXCL8 protein expression. Survival analysis showed that patients with KRAS-mutant lung adenocarcinoma and high CXCL8 expression had a shorter overall survival, and CXCL8 was positively correlated with M2 macrophage marker genes. Conclusion Targeted inhibition of SHP2 can suppress the expression of some macrophage-related inflammatory factors in KRAS-mutant lung cancer cells, with the most significant inhibition of CXCL8 expression. The mechanism may involve SHP2 regulating the transcription factor AP-1.
ObjectiveTo summarize the research progress of tumor-associated macrophages (TAM) in immunotherapy and drug resistance of gastric cancer, and provide new ideas for the treatment of gastric cancer. MethodThe literatures about tumor-associated macrophages in immunotherapy and drug resistance of gastric cancer at home and abroad in recent years were searched and reviewed. ResultsThe incidence and mortality of gastric cancer in China were significantly higher than those in other countries. Surgical treatment remained the primary approach for gastric cancer, and targeted therapy combined with immunotherapy had become the standard first-line treatment for advanced gastric cancer. TAM were a large population of immune cells present in the tumor immune microenvironment and had emerged as novel therapeutic targets and prognostic indicators in individualized treatment strategies. As the relationship between TAM and malignant tumors was further elucidated, TAM was expected to become a key target for the development of novel cancer therapeutics. However, some patients developed resistance during treatment. Recent preclinical and clinical studies had demonstrated that targeting TAM had yielded promising results in gastric cancer treatment. ConclusionsThe mechanism of TAM and the key factors driving the phenotypic changes of TAM in the microenvironment of gastric cancer remain to be further study. How to inhibit the tumor promoting effect of TAM will provide new clues for the future treatment of gastric cancer.
ObjectiveTo study the effect and mechanism of lipopolysaccharide (LPS) on osteoclasts formation and its bone resorption function.MethodsBone 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.ResultsThe 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).ConclusionLPS at concentration of 100 ng/mL can promote the expression of Connexin43, resulting in increased osteoclastogenesis and enhanced osteoclastic bone resorption.
ObjectiveTo investigate the effect of succinate induced polarization of MH-S murine alveolar macrophage cells on hyperoxia-induced epithelial-mesenchymal transition (EMT) of MLE-12 mouse alveolar epithelial cells. Methods Determine the exposure time: MLE-12 cells was cultured in an incubator with 95%O2 for different time to establish a cell model of acute hyperoxia-induced lung injury. The relative expression of EMT-related proteins (E-cadherin, N-cadherin, vimentin) was determined by Western blotting. Co-culture of MLE-12 and MH-S to explore the influence of MH-S on EMT: MLE-12 was divided into hyperoxia group for 0h, hyperoxia group for 48h and co-cultured with MH-S hyperoxia group for 48h (Co). The relative expression of EMT-related proteins was determined by Western blotting. Determination of succinate concentration and its effect on MH-S polarization and succinate receptor GPR91: MLE-12 was cultured in different concentrations of succinate medium for 24h, and the cell viability was determined by CCK-8. MH-S was divided into control group (C) and succinate group (S). Group C was cultured for 24h, and group S was added with succinate at the above concentration. The relative expression of GPR91 and polarization-related factor mRNA in MH-S was measured by RT-qPCR, and the expression of macrophage polarization-related proteins (CD11b, CD206, CD86) was measured by flow cytometry. Study on the effect of succinate on EMT by cell co-culture: MLE-12 and MH-S were co-cultured in a Transwell chamber and divided into control group (Co), succinate group (SUC) and GPR91 inhibitor group (I). Results Expression of EMT-related proteins in four groups of MLE-12 at different times: Compared with 0h, the expression of vimentin and N-cadherin in 24h and 48h increased, while the expression of E-cadherin in 48 h and 72 h decreased (P<0.05), and there was no significant difference in other groups. The follow-up experiment was conducted under hyperoxia conditions for 48h. Influence of MH-S on EMT: The expression of vimentin and N-cadherin in Co group was higher than that in 48h, and the expression of E-cadherin was lower than that in 48h (P<0.05). After 24 h of intervention with different concentrations of succinate on MLE-12, compared with the 0mmol/L, the cell viability of 2.5mmol/L, 1mmol/L and 500 μmol/L increased (P<0.05), and there was no significant difference in other groups, so the 1mmol/L succinate concentration was selected for subsequent experiment. Compared with group C, the expression of GPR91 mRNA in group S increased, and the expression of iNOS and CD86 mRNA in group S increased (P<0.05), but there was no significant difference in other groups. The analysis of flow cytometry showed that 1mmol/L succinate could increase the number and proportion of CD86+CD206– alveolar macrophages. Compared with Co group, the expression of vimentin and N-cadherin in SUC group increased, while the expression of E-cadherin decreased. Compared with SUC group, the expression of vimentin and N-cadherin in group I decreased, while the expression of E-cadherin increased (P<0.05). Conclusion Succinate can induce mouse alveolar macrophages polarization to M1 through GPR91, enhance EMT of mouse alveolar epithelial cell injury model under hyperoxia, and promote the formation of pulmonary fibrosis.