Study About The Effects Of Human TIM-3 On NF-κB And Inflammation-related Cytokines Expression In Human Bronchial Epithelial Cell Line 16HBE

Allergic asthma is a common complex respiratory tract inflammatory disease related with immunity, and its characteristics are mainly airway hyperreaction and specific IgE or all IgE upregulation to environmental allergen, which is caused by multiple factors such as genetic, environmental and individual immune state. It was reported that chromosome 5q31-33 region was strongly linked to asthma. Human Tim-3 is one member of Tims gene family, which is located in the chromosome 5q31-33 region strongly linked to asthma. At present some researchers think Tim-3 as a good candidate susceptibility gene for asthma, and perform the association study about Tim-3 polymorphism with asthma. The results of study conducted in several populations showed that Tim-3 polymorphism was linked to asthma.Recent study has showed that the two subtype TH1 cells and TH2 cells are regulated by different transcription factors, secrete different cytokines to coordinate and maintain internal environment homeostasis. The imbalance of TH1/TH2 cells and their secretary cytokines is the important factor for the development of asthma. When asthma developes, TH2 cells appears polarization, TH2 cells and their secretary cytokines increase correspondingly. Tim-3 was first identified to be a transmembrane protein expressed on terminally differentiated TH1 cells when they were stimulated by antigen. If Tim-3 binds to the Tim-3 ligand, galectin-9, it play the role to terminate TH1-mediated immunity. Interaction of Tim-3 and its ligand galectin-9 triggers an inhibitory signal to lead to apoptosis of TH1 cells, and thus negatively regulating TH1-mediated immunity. However, expression of Tim-3 is not restricted to T cells. Previous study demonstrated that Tim-3 was expressed in a wide range of human normal and malignant epithelial tissues by RT-PCR, on microglia, monocytes, DCs, M2 macrophages, mast cells, cytotoxic CD8+ T cells, activated CD4+ T cells, Th17 and Treg, which suggests Tim-3 may be associated with several tissue and immune functions. Therefore, it is to be further investigated that whether Tim-3 is expressed in respiratory system and whether Tim-3 is linked with asthma by respiratory system and immune system. In this study, we detect Tim-3 expression in 16HBE cell, GLC-82 cell and A549 cell by using PCR. We have cloned human full-length Tim-3 from peripheral blood monocytes. And we transfect the eukaryotic expression plasmids which respectively expresses human pEGFP-C2-flTim-3 and pEGFP-C2 into human bronchial epithelial cells and screen positive clones by G418. Then we stimulate the transfected 16HBE cells with house dust mite or/and Dexamethasone at some concentration, and detect NF-κB and inflammation-related genes expression level in treated groups to assess the effects of hTim-3 on NF-κB and inflammation-related genes expression level.OBJECTIVE:To Screen the respiratory tract related cell lines for Tim-3 positive expression and make foundation for elucidating that Tim-3 may be associated with respiratory tract related disease. To construct an eukaryotic expression vector pEGFP-C2-hTim-3 for further study of the function of hTim-3. To transfect 16HBE cells by human pEGFP-C2-hTim-3 and pEGFP-C2 and screen the positive clones for further study.METHOD:The human bronchial epithelial cell lines 16HBE, A549 and GLC-82 were cultured in DMEM with high glucose medium supplemented with 10% fetal bovine serum, and were maintained at 37℃and 5% CO2 in saturated humidity air. Monocytes were separated from peripheral blood anticoagulated by EDTA-K2 of healthy adults with Ficoll lymphocyte separation medium. The RNA of the four above cells was extracted by TRIzol and reverse transcripted into cDNA. PCR for detecting mRNA of Tim-3 in respiratory tract related cell lines was performed. The full-length hTim-3 gene was amplified with monocytes cDNA from peripheral blood of healthy adults. The full-length hTim-3 gene cDNA was cloned into pEGFP-C2 plasmid. And the extracted plasmid was verified by PCR, restrictedly enzyme analysis and DNA sequencing. The right plasmid was construction of an eukaryotic expression vector pEGFP-C2-hTim-3. The recombination plasmid pEGFP-C2-hTim-3 and the plasmid pEGFP-C2 were transfected into 16HBE cells by Lipofectamine 2000, respectively. The transient transfection results was observed by the fluorescence microscopy. The transfected 16HBE cells were screened by 0.6 mg/mL G418 and were maintained by 0.4 mg/mL G418 after two weeks. The fluorescence protein expression in 16HBE transfected pEGFP-C2-hTim-3 and pEGFP-C2 was observed by the fluorescence microscopy. The protein expression of Tim-3 in 16HBE transfected pEGFP-C2 and pEGFP-C2-hTim-3 was detected by Western Blot.RESULT:1. The PCR results showed that hTim-3 was expressed in monocytes from human peripheral blood,16HBE, GLC-82 and A549 cells.2. After DNA sequencing, the sequence of the constructed pEGFP-C2-hTim-3 plasmid was in accordance with GeneBank accession nos. BC063431 and AF450242.3. After transient transfection, transfected pEGFP-C2-hTim3 plasmid 16HBE with the green fluorescence protein expression occupied less than 8%; transfected pEGFP-C2 plasmid 16HBE with the green fluorescence protein expression occupied less than 15%. After screening, The fluorescence microscopy observation results presented that the green fluorescence of 16HBE transfected pEGFP-C2-hTim3 plasmid was located on the membrane, suggesting that hTim3 was expressed on the membrane; the green fluorescence of 16HBE transfected pEGFP-C2 plasmid was mainly located on the cytoplasma. The expression of Tim-3 in 16HBE transfected pEGFP-C2-hTim3 plasmid was higher than that in 16HBE transfected pEGFP-C2 plasmid by Real-time PCR and Western Blot analysis (p< 0.01).CONCLUSION:HTim-3 was expressed in monocytes from human peripheral blood, 16HBE, GLC-82 and A549 cells, suggesting that Tim-3 may be associated with respiratory tract-related disease. An eukaryotic expression vector pEGFP-C2-hTim-3 was constructed successfully. The 16HBE cells stably transfected by human pEGFP-C2-hTim-3 were established successfully.OBJECTIVE:To investigate the effect of Tim-3 pathway on NF-κB expression in 16HBE cell treated with house dust mite or/and Dexamethasone, and further elucidate the mechanism of regulation.METHOD:16HBE transfected pEGFP-C2-hTim-3 and 16HBE transfected pEGFP-C2 were treated with house dust mite or/and Dexamethasone at some concentration, respectively. The mRNA of cells were extracted by TRIzol and reverse transcripted to cDNA. The mRNA expression of Tim-3, NF-κB, IFN-γ, galectin-9, T-bet, IL-4 and GATA-3 in different treated cells after transfection were detected by Real-time quantative PCR. The protein of cells were extracted for detecting NF-κB protein expression in different treated cells after transfection by Western Blot analysis. Then we investigated the effect of hTim-3 on NF-κB and some cytokines expression in 16HBE cells treated with house dust mite or/and Dexamethasone and discussed corresponding mechanism.RESULT:1. When 16HBE cells were treated with house dust mite or/and Dexamethasone, expression of Tim-3(p<0.01), NF-κB(p<0.01), IFN-γ(p<0.01), T-bet(p<0.01), galectin-9(p<0.01), IL-4(p<0.01) in 16HBE cells transfected pEGFP-C2-hTim3 plasmid were different obviously from that of 16HBE cell transfected pEGFP-C2 in corresponding groups, and GATA-3 did not change obviously(p>0.05). Furthermore, expression of Tim-3 was positive correlation with expression of IFN-γ, T-bet and galectin-9, correlated negatively with expression of NF-κB and IL-4 in 16HBE cells transfected pEGFP-C2-hTim3 plasmid; however, there were not the strong correlations in 16HBE cell transfected pEGFP-C2. It suggested that effect of Tim-3 in 16HBE cells transfected pEGFP-C2-hTim3 plasmid when treated with house dust mite or/and Dexamethasone on NF-κB probably was influenced by upregulating of IFN-γ, T-bet, galectin-9 expression, and downregulating IL-4.2. When 16HBE cells were treated with house dust mite or/and Dexamethasone, Tim-3 expression in 16HBE cells transfected pEGFP-C2-hTim3 plasmid was negative correlation with NF-κB protein expression, however, there was not the tendency in 16HBE cells transfected pEGFP-C2. It showed that Tim-3 in 16HBE cells transfected pEGFP-C2-hTim3 suppressed protein expression of NF-κB.CONCLUSION:Upregulation of Tim-3 pathway may attenuate NF-κB expression by upregulation expression of IFN-γ, T-bet, galectin-9, and downregulation level of IL-4 in 16HBE cells, thus inhibiting inflammation.OBJECTIVE:To investigate the effect of Tim-3 pathway on inflammation-related factors expression in 16HBE cell treated with house dust mite or/and Dexamethasone, and further elucidate the mechanism of regulation.METHOD:16HBE transfected pEGFP-C2-hTim-3 and 16HBE transfected pEGFP-C2 were treated with house dust mite or/and Dexamethasone at some concentration, respectively. The mRNA of cells were extracted by TRIzol and reverse transcripted to cDNA. The mRNA expression of Tim-3, NF-κB, TNF-α, IL-8, RANTES, ICAM-1 in different treated cells after transfection were detected by Real-time quantative PCR. The supernants of cells were separated for detecting NF-κB, TNF-α, IL-8 protein expression in different treated cells after transfection by ELISA. Then we investigated the effect of Tim-3 on TNF-α, NF-κB and some inflammation-related factors expression in 16HBE cells treated with house dust mite or/and Dexamethasone and discussed corresponding mechanism.RESULT:1. When 16HBE cells were treated with house dust mite or/and Dexamethasone, expression of Tim-3(p<0.01), NF-κB(p<0.01), TNF-α(p<0.01), IL-8(p<0.01), RANTES(p<0.01), ICAM-1 (p<0.01) in 16HBE cells transfected pEGFP-C2-hTim3 plasmid were different obviously from that of 16HBE cell transfected pEGFP-C2 in corresponding groups. Furthermore, expression of Tim-3 was negative correlation with expression of NF-κB, TNF-α, IL-8, RANTES and ICAM-1 in 16HBE cells transfected pEGFP-C2-hTim3 plasmid; however, there were not the strong correlations in 16HBE cell transfected pEGFP-C2. Further, the effects of Tim-3 on IL-8, RANTES and ICAM-1 expression may be achieved in downregulating NF-κB-dependent manner, TNF-a may downregulate NF-κB expression.2. When 16HBE cells were treated with house dust mite or/and Dexamethasone, expression of Tim-3 in 16HBE cells transfected pEGFP-C2-hTim3 plasmid was negative correlation with NF-κB, TNF-αand IL-8 protein expression, however, there was not the tendency in 16HBE cells transfected pEGFP-C2. It showed that upregulation of Tim-3 in 16HBE cells transfected pEGFP-C2-hTim3 suppressed protein expression of NF-κB, TNF-αand IL-8.CONCLUSION:Upregulation of Tim-3 pathway may attenuate the expression of IL-8, RANTES and ICAM-1 in NF-κB-dependent manner, TNF-a may downregulate NF-κB expression, thus inhibiting inflammation development.