CHRONIC OBSTRUCTIVE PULMONARY DISEASE ZULEYHA OZEN OVERVIEW ● Introduction ● Information about Chronic Obstructive Pulmonary Disease (COPD) ● Inflammatory Responses ● Paper 1 ● Paper 2 ● What is still unknown? ● Future Studies/Specific Aim Disease Overview Pathophysiology of COPD (Chronic Bronchitis and Emphysema) -Chronic Bronchitis: Loss of muco-ciliary clearance Loss of cilia function -Emphysema: Destruction of elastin fibers Proteases, matrix metalloproteinase 9 (MMP9) cause elastin degradation Shortness of Breath Cough/Sputum-(Progressive dyspnea) http://www.wgabinecie.pl/artykul/65-pochp-przewlekla-obturacyjna-choroba-pluc/ Current existing medicines Bronchodilators Combination of Bronchodilators and cortisteroids Vaccines Pulmonary Rehabilitation Oxygen Therapy Change in Lifestyle http://www.ncbi.nlm.nih.gov/books/NBK26827/figure/A4531/?report=objectonly https://www.caymanchem.com/app/template/Article.vm/article/2177 Inflammatory and immune cells involved in chronic obstructive pulmonary disease (COPD) Macrophages and epithelial cells: -chemotactic factors -attract inflammatory cells http://www.nature.com/nri/journal/v8/n3/fig_tab/nri2254_F2.html TH17 cells and airway inflammation http://www.nature.com/nri/journal/v8/n3/fig_tab/nri2254_F5.html Smoke Exposure (n=20/group) Whole body exposed to cigarette smoke (CS) 20 mice exposed to CS of 5 cigarettes 30 min smoke free interval 20 mice exposed to CS of 5 cigarettes 30 min smokefree interval 20 mice exposed to CS of 5 cigarettes 30 min smokefree interval 20 mice exposed to CS of 5 cigarettes The CS exposure animals were divided into two subgroups: Control group: exposed to air Sub-acute exposure: Initiated CS at 26-28 weeks old Mice of each group 30-32 weeks old when the CS & Air exposure ended Chronic exposure: initiated CS at 6-8 weeks old Figure 1. Assessing effect of CS exposure on lung inflammation Total number of alveolar inflammatory cells increased in chronic CS exposure • Sub-acute exposure significantly lower than chronic CS Figure 2A. Prevalence of Th17 cells in lung tissue Prevalence of Th17 cells: -ratio of CD4+ IL-17A+ cells to the total amount of CD4+ T lymphocytes -Th17 prevalence markedly higher in mice with chronic CS and Sub-acute CS Figure 2B. Peripheral blood mononuclear cells -similarly the prevalence of Th17 cells increased in CS exposure Figure 2C. Collective analysis of flow cytometry Figure 3A. Prevalence of T regulatory cells (ratio of CD4+CD25+Foxp3+ cells to the total amount of CD4+ T lymphocytes) in lung tissue Prevalence of T regulatory cells: -markedly higher in sub-acute exposure -drops in chronic CS exposure Figure 3B. Peripheral blood mononuclear cells -similarly the prevalence of T regulatory cells decreased in chronic CS exposure Figure 3C. Collective analysis of flow cytometry Figure 4. Assessing the ratio of Th17 and Treg in lung tissue and peripheral blood -In lung tissues, the ratio of Th/Treg is decreased with sub-acute CS exposure -Increases in chronic CS exposure Ratio of Th17/Treg in peripheral blood: -significantly increased in chronic CS exposure Figure 5. The expression of Foxp3 and ROR gamma t mRNA -Looking at specific transcription factors of both Tsubsets by real time-PCR -Th17 specific transcription factor ROR gamma t mRNA expression: significantly increased in CS exposed -T regulatory specific transcription factor Foxp3 mRNA expression: significantly decreased in CS exposed Table 1: levels of IL-17A, IL-6, IL23 and TGF-beta in serum significantly higher in chronic CS exposure -IL-10 sig. lower in chronic CS Conclusions from paper 1 There is an obvious imbalance between Th/Treg cells in CS exposed mice Prevalence of Th17 and Th17 specific transcription factor, ROR gamma t mRNA: increased Treg cell prevalence and Treg specific transcription factor, Foxp3 mRNA: decreased Thus, leading to an imbalance in the ratio of Th/Treg cell profiles The existing cytokine profile can be further evaluated for specific therapeutic approach Figure 1. Assessing histology of lung tissues Fig.1 Histology of Lung Tissues ● Mean alveoli were expanded and broken ● COPD lung- more inflammatory cells Figure 2. Expression of transcription factors ROR gamma t and Foxp3 • • • Fig.2a. Foxp3 relative mRNA expression level significantly lower in COPD patients Fig.2b. RORyt relative mRNA expression level significantly higher in COPD patients Fig.2c. Ratio of Treg/Thelper cells in the level of mRNA lower in COPD patients Figure 3. Assessing expression of Foxp3 and ROR gamma t protein levels -Increased protein expression of ROR gamma t in COPD patients -Viceversa, decreased protein expression of Foxp3 compared with smokers and nonsmokers Figure 4. Immunohistochemistry staining of different proteins All p-values were less than 0.001 -IL-17+, CCR6+ and IL-23R cells increased -Foxp3 cells in alveolar walls decreased Figure 5. The number of Foxp3+, IL-17+, CCR6+ and IL-23R+ cells in alveolar walls -Foxp3+ and Foxp3+ / IL-17+ cells decreased in cell number in chronic CS exposure -IL-17+, CCR6+ and IL-23R+ cells increased in cell number in chronic CS exposure -Foxp3+ & Foxp3+/IL-17+ cells decreased in alveolar walls of COPD A: ROR gamma t & Foxp3 mRNA expression: negatively correlated B: Ratio of Foxp3/ROR gamma t mRNA expression negatively correlated with mean alveoli area C: Positive correlation with the ratio of Foxp3/ROR gamma t and FEV1%pred D: ROR gamma t & Foxp3 protein: negatively correlated E: Ratio of Foxp3/ROR gamma t in level of protein and mean alveoli area F: Positive correlation with the ratio of Foxp3/ROR gamma t in level of protein and FEV1%pred G: Numbers of Foxp3+/IL-17+ cells: negatively correlated H: Ratio of Foxp3/IL-17+ cells: negatively correlated to the mean alveoli area I: Positive correlation with the ratio of Foxp3+/IL-17+ cells and FEV1%pred Conclusions from paper 2 Decreased ratio of Foxp3/ROR gamma t in patients with COPD and normal smokers persistent with the aggravation of the disease Decreased ratio of Foxp3/ROR gamma t important in pathogenesis of COPD Immune dysregulation, and participation in lung inflammation: leading to destruction in the lung Pro-inflammatory cytokines and chemokine receptors are also evident in development of the disease Their association with the transcription factors ROR gamma t and Foxp3 can be further researched for potential therapeutics What is still unknown ? The regulatory cytokine involvement: TGF-beta levels in the progression of the disease is still unknown Study by Zhou et al. TGF-beta induced Foxp3 leads to inhibition of Th17 cell differentiation In a dose dependent matter, TGF-beta might be a factor seen in the imbalance between Th/T regulatory cells Specific Aim In the research proposal, my area of focus will be to further analyze the negatively correlated relationship between the T-regulatory and T helper cells with regards to changes in the expression of the specific transcription factors and cytokine TGF-beta Implement TGF-beta induced Foxp3 and use of cytokine IL-6 antagonist for therapeutic approach THANK YOU! QUESTIONS?