Studies on microbial adhesion factors in smoking and chronic obstructive pulmonary disease (COPD)
Creators: name: Shukla, SD
Publisher: name: University Of Tasmania
Year: 2023

description: Background: Chronic obstructive pulmonary disease (COPD) is emerging as the third largest cause of human mortality worldwide, killing over more than 3 million people annually. It is a defined as a chronic inflammatory lung disease characterized by airflow limitation that is not fully reversible. Tobacco smoking is the single most important causative factor for the development of COPD, especially in developed countries. Colonization/infection of the lower respiratory tract with microorganisms in COPD is likely to be one of the important factors driving inflammatory processes in the airways, possibly leading to long-term progression and exacerbations of the disease. Nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumonia (S. pneumoniae) are the most important pulmonary bacteria during both stable phase of disease and exacerbations of COPD, but the underlying mechanisms for their prominence in these conditions remain elusive. Exposure to cigarette smoke has been shown to significantly increase pulmonary tissue bacterial infections (with both NTHi and S. pneumoniae) in cell culture and animal models, as well as human lung samples. Bacterial adherence to lung epithelium and subsequent tissue invasion is known to be a crucial step in the pathogenesis of respiratory infections. Notably, platelet activating factor receptor (PAFr) has been established as the primary attachment molecule for both NTHi and S. pneumoniae. Moreover, intercellular adhesion molecule-1 (ICAM-1) serves as an entry receptor for not only the majority (~60%) of human rhinoviruses (HRVs), but also for NTHi. Although the expression of these microbial receptors have been investigated in smokers, the expression pattern of these adhesion molecules in COPD has not been reported till date. Aims: I have investigated the expression pattern of airway epithelial PAFr and ICAM-1 in both the large and small airways, as well as lung parenchyma in tissues from: smoker with normal spirometry, COPD subjects (smokers and ex-smokers), and a group with small dysfunction only. I have also investigated whether blocking PAFr reduces the bacterial adherence to bronchial cells pretreated with cigarette smoke. Methodology: I evaluated the expression of PAFr and ICAM-1 crosssectionally in both large (bronchial biopsies, EBB and resected lung tissue, RT) and small airways (RT) from: 16 EBB/8 RT smokers with normal lung function (NLFS); 10 with smoking-related small airway narrowing only; 15 EBB/8 RT COPD smokers; 12 EBB/10 RT COPD ex-smokers, and compared these with 15 EBB/9 RT control tissues (NC). Anti-PAFr/ICAM-1 immunostaining on paraffin-embedded tissue was quantified using computeraided image analysis. Moreover, human cultures bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE). PAFr and ICAM- 1 expression levels were determined using immunocytochemistry and qPCR. The epithelial cells were challenged with either NTHi or S. pneumoniae labeled with FITC, and bacterial adhesion was measured using immunofluorescence. The effect of a well-evaluated antagonist of PAFr, WEB- 2086, on binding of the bacterial pathogens to BEAS-2B cells was then assessed. With collaborators, I also carried out in-silico studies of the tertiary structure of PAFr and the binding pocket for PAF and its antagonist WEB- 2086. Results: Large airway epithelium showed markedly enhanced expression of PAFr in COPD-smokers especially (p&lt;0.005) and also COPD-ex-smokers (p&lt;0.002) compared to smokers with normal lung function. Moreover, significantly increased PAFr expression in small airway epithelium of all clinical groups was found compared with controls (p&lt;0.01), but with no difference between smokers with or without COPD. Smoking history (packyear) correlated significantly with PAFr expression in the currently smoking individuals, especially in NLFS (r=0.9; p&lt;0.002). An increase above normal in PAFr-expressing cells in the airway sub-epithelial Rbm was only significant in COPD smokers (p&lt;0.007). A lesser increase in PAFr-expressing cell in alveolar epithelium (compared to airway epithelium) was uniformly found in all clinical groups compared with normal controls (p&lt;0.01). In vitro analyses showed that PAFr expression by bronchial epithelial cells was upregulated by CSE, and significantly associated with increased bacterial (both NTHi and S. pneumoniae) adhesion. WEB-2086, a known PAFr-antagonist, reduced the epithelial adhesion by both NTHi and S pneumoniae to levels observed for non-CSE exposed control cells. Furthermore, WEB-2086 was non-toxic towards the bronchial epithelial cells. Moreover, in silico analyses allowed identification of the binding pocket for PAF/WEB-2086 in the predicted PAFr tertiary structure. Significantly increased ICAM-1-positive cells were found in the epithelium of the both large (p&lt;0.006) and small airways (p&lt;0.004) of all clinical groups, compared to control tissue. Moreover, epithelial ICAM-1 expression was especially upregulated in the airway obstruction (CAL) group compared to smokers with normal lung function (p&lt;0.007, for large airway expression; p&lt;0.02, for small airway expression). Smoking history (pack-year) correlated significantly with ICAM-1 expression in both the large and small airway epithelium in the currently smoking individuals (r=0.49; p&lt;0.03). In the subepithelial Rbm, increase in ICAM-1 expressing cells was only significant in small airways in the CAL group (p&lt;0.02). The staining patterns were quite intriguing: in the CAL group especially, both basal and luminal areas of epithelial cells stained heavily for ICAM-1, but so did goblet cells and submucosal glands. These features were little evident in either normal controls or NLFS subjects. Conclusion: The expression of epithelial PAFr and ICAM-1 is upregulated in both the large and small airways in smokers, but markedly so in COPD. Increased expression of these microbial receptors in the respiratory tract could be crucial in viral and bacterial infections known to be important duringthe course of the disease.