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)

Current existing medicines  Bronchodilators  Combination of Bronchodilators and cortisteroids  Vaccines  Pulmonary Rehabilitation  Oxygen Therapy  Change in Lifestyle

Inflammatory and immune cells involved in chronic obstructive pulmonary disease (COPD) Macrophages and epithelial cells: -chemotactic factors -attract inflammatory cells

T H 17 cells and airway inflammation

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 30 min smoke- free interval The CS exposure animals were divided into two subgroups: Sub-acute exposure: Initiated CS at weeks old Chronic exposure: initiated CS at 6-8 weeks old Control group: exposed to air Mice of each group weeks old when the CS & Air exposure ended

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 T- subsets 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, IL- 23 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

-Increased protein expression of ROR gamma t in COPD patients -Viceversa, decreased protein expression of Foxp3 compared with smokers and nonsmokers Figure 3. Assessing expression of Foxp3 and ROR gamma t protein levels

Figure 4. Immunohistochemistry staining of different proteins All p-values were less than 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

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