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)

http://www.nature.com/nri/journal/v8/n3/fig_tab/nri2254_F2.html

Macrophages and epithelial cells:-chemotactic factors-attract inflammatory cells

 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

20 mice exposed to CS of 5 cigarettes

20 mice exposed to CS of 5 cigarettes

20 mice exposed to CS of 5 cigarettes

30 minsmoke free interval

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 26-28

weeks old

Chronic exposure: initiated CS at 6-8

weeks old

Control group: exposed to air

Mice of each group 30-32 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 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 correlatedB: Ratio of Foxp3/ROR gamma t mRNA expression negatively correlated with mean alveoli areaC: Positive correlation with the ratio of Foxp3/ROR gamma t and FEV1%pred

D: ROR gamma t & Foxp3 protein: negatively correlatedE: 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 areaI: 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?