Title Page
Contents
국문초록 12
ABSTRACT 14
Preface 16
Chapter 1. Role of peroxisome proliferator-activated receptor gamma on function of alternatively activated macrophages 19
I. Introduction 20
II. Objectives 23
III. Materials and Methods 24
3.1. Macrophage polarization and treatment 24
3.2. Quantitative real-time polymerase chain reaction 25
3.3. Flow cytometry 26
3.4. Small interfering RNA transfection 26
3.5. Chromatin immunoprecipitation assay 27
3.6. Western blot analysis 27
3.7. Measurement of intracellular accumulation of Hoechst 33342 28
3.8. Pyrazinamide accumulation assay 28
3.9. Statistical analysis 29
IV. Results 30
4.1. M2 macrophages express high levels of ABCG2 30
4.2. PPARγ is critical for ABCG2 expression in M2 THP-1 cells 37
4.3. STAT6 is required for PPARG expression in M2 THP-1 cells 45
4.4. Pharmacological inhibition of PPARγ can decrease ABCG2-mediated the efflux activity of M2 macrophages 49
V. Discussion 53
Chapter 2. Role of aryl hydrocarbon receptor on function of lung epithelial cells 58
I. Introduction 59
II. Objectives 62
III. Materials and Methods 63
3.1. Mice 63
3.2. Hematopoietic stem cell transplantation 63
3.3. In vitro treatment 64
3.4. Detection of AHR expression in lung tissue 64
3.5. Isolation of lung epithelial cells and in vitro stimulation 65
3.6. Quantitative real-time polymerase chain reaction 66
3.7. Western blot analysis 68
3.8. Chromatin immunoprecipitation assay 68
3.9. Statistical analysis 69
IV. Results 71
4.1. Expression of AHR is independent of the IFN-γ-IDO1 pathway in the lung after HSCT 71
4.2. Inhibition of host AHR activation results in exacerbation of IPS 77
4.3. AHR regulates gene expression of AP-1 family in lung epithelial cells 80
4.4. AHR suppresses inflammatory gene in lung epithelial cells through transcriptional repression of Jund 83
V. Discussion 87
VI. Conclusions 90
References 91
Chapter 1. 8
Table 1. Sequence of primers 25
Chapter 2. 8
Table 2. Sequence of primers 67
Chapter 1. 9
Figure 1. mRNA levels of polarization markers in macrophage subsets. 32
Figure 2. The mRNA expression of ABCB1 and ABCG2 is highly expressed in M2 macrophages. 33
Figure 3. M2 macrophages express high levels of ABCG2 in THP-1 cells. 34
Figure 4. M2 macrophages express high levels of ABCG2 in human macrophages. 35
Figure 5. High efflux activity in M2 macrophages is dependent on ABCG2. 36
Figure 6. Kinetics of ABCG2 expression in THP-1 during M2 polarization. 39
Figure 7. IL-4/IL-13-induced ABCG2 expression requires pre-sensitization of PMA. 40
Figure 8. PPARγ is critical for ABCG2 expression in M2 THP-1 cells. 41
Figure 9. T0070907 suppresses ABCG2 expression in both a ligand-dependent and -independent manner. 42
Figure 10. Inhibition of PGC-1α has no effect on ABCG2 expression. 43
Figure 11. PPARG knockdown in M2 THP-1 cells reduces ABCG2 levels and efflux activity. 44
Figure 12. PPARγ levels in M2 THP-1 cells. 46
Figure 13. PPARG expression in M2 THP-1 cells requires STAT6 signaling. 47
Figure 14. PPARγ induces ABCG2 transcription by binding to ABCG2 promoter. 48
Figure 15. Pharmacological inhibition of PPARγ can decrease ABCG2-mediated the efflux activity of M2 THP-1 cells. 50
Figure 16. Pharmacological inhibition of PPARγ can decrease ABCG2-mediated the efflux activity of human M2 macrophages. 51
Figure 17. T0070907 reduces clinically used anti-tuberculous drug efflux activity of M2 THP-1 cells. 52
Chapter 2. 10
Figure 1. Expression and activation of Ahr is induced in the lung after HSCT. 73
Figure 2. Expression of AHR is induced in the lung epithelial cells and donor T cells after HSCT. 74
Figure 3. The IFN-γ-IDO pathway does not induce AHR expression, but is required for AHR activation. 75
Figure 4. Ahr expression is upregulated by NF-κB activation in lung epithelial cells. 76
Figure 5. Inhibition of AHR decreases expression of Cyp1b1 in CD4+ T cells and lung epithelial cells.[이미지참조] 78
Figure 6. Deficiency of host AHR exacerbates IPS. 79
Figure 7. Overrepresentation of the AP-1 family genes in lungs of Ahr-/- after HSCT.[이미지참조] 81
Figure 8. AHR regulates Jund expression in lung epithelial cells. 82
Figure 9. A549 lung epithelial cells are responsive to IFN-γ for activation of the IDO1-Kyn-AHR pathway. 84
Figure 10. IL-1β-induced JUND expression requires STAT1 and AP-1 signaling. 85
Figure 11. AHR represses inflammatory genes by blocking transcriptional activity of STAT1 and JunD. 86