Understanding cellular changes and mediators of airway remodelling in COPD

\\(Background\\) \\(and\\) \\(Aims:\\) Chronic obstructive pulmonary disease (COPD) is emerging as a substantial global health problem, with an estimated annual mortality of over 3 million people, which is the third largest disease cause worldwide. COPD is an irreversible chronic, slowly progressive airway obstructive respiratory disease. Cigarette smoking mainly causes it, primarily through small airway fibrosis, narrowing and ultimately obliteration, accompanied by more generalized but non-obstructive chronic bronchitis‚ÄövÑvp throughout the airways, and in some individuals complicated by the later development of lung parenchymal destruction (emphysema). Approximately 50% of smokers develop COPD eventually. In COPD research, there has been a long-held belief that airway disease progression is due to inflammation.‚ÄövÑvp Although this may be true in the airway lumen with innate immunity activated by the effect of smoke or secondary to infection, the accurate picture for inflammatory cells in the airway wall, where the pathophysiological COPD remodeling occurs, is uncertain and especially so in mild to moderate COPD patient's, i.e., earlier disease. This study follows up on a previous finding from our group of possibly decreased cellularity in the airway wall of patients with COPD. Thus, in the current studies, along with total airway wall cellularity, I evaluated the contribution of the main immune cells such as neutrophils, macrophages, mast cells, CD4 and CD8 cells in the large and small airway wall of mild-moderate COPD current (CS) and ex-smokers (ES), plus normal lung function smokers (NLFS), and compared them to non-smoker controls (NC). My studies further delved into the macrophage sub-populations in smokers/COPD and ascertained whether abnormal differential switching occurs in the macrophage population in the small airway wall compared to the lumen. I also measured macrophage-related cytokine profiles in airway luminal bronchoalveolar lavage fluid (BALF) and investigated whether the microenvironment dictated these changes. I evaluated cell dysfunction in small airway wall cells by assessing their degranulating potential via expression of a degranulating-marker lysosome-associated membrane protein -1 (LAMP-1). While analyzing the CD68+ population of macrophages, I noticed that many cells staining strongly were not macrophages as expected, but spindle-shaped cells likely to be a population of fibroblasts; indeed the literature strongly suggests that CD68 can also stain fibroblasts, but this has until now been ignored in respiratory research. I further focused on the non-inflammatory cell components in the airway wall, investigated the relationship between alpha-smooth muscle actin+ myofibroblasts with an expression of epithelial-mesenchymal transition markers (a focus of our group for several years), key extracellular matrix proteins (collagen I and fibronectin) and airway wall thickness. I also related abnormalities to lung function disturbance in COPD. In the final phase of my Ph.D. studies, I also managed to complete some preliminary investigations on abnormal lysosomal accumulation and catabolic changes in the airway epithelium of COPD patients and related these to indices of airway remodeling. \\(Methodology:\\) I evaluated both large airway endobronchial biopsies (ebb) and small airway lung resected tissues (RT) from COPD-CS and ES, NLFS and NC. I immune-stained with human anti-CD68+ for macrophages, neutrophil elastase for neutrophils, mast cell tryptase for mast cells, CD4+ and CD8+ for T cell populations. I conducted total cell counts as well as differential counts in the airway wall lamina propria (LP), epithelium and reticular basement membrane. For the large airways, I standardized cell counts in the LP by taking the depth of 150 for LA and up to 100 microns deep SA while excluding smooth muscle layers. Further, subpopulations of macrophages were evaluated in the SA wall (epithelium and LP) and BAL samples from the same cohort of patients and were stained with CD163 and Arginase-1 antibodies for detection of M2 macrophages and dual stained with anti-CD68+ and anti-iNOS antibodies for M1 populations (CD68+ only cells were designated M0). Cells were quantified and normalized as per mm2 of SA wall and as per mm of length for the epithelium, respectively. Bronchoalveolar lavage (BAL) cytospin macrophages were similarly stained and enumerated by randomly selecting 12 fields and quantified as per ml of original BAL fluid extracted. BAL cytokines to profile for both M1/Th1 and M2/Th2 cytokines used FACS multiplexing strategies. The degranulation capacity of mast cells and total degranulating cells in the small airway wall were measured by dual staining mast cells with anti-mast cell tryptase and LAMP-1. Epithelial and sub-epithelial LAMP-1 were assessed per area and represented as percent degranulation. I measured the total airway wall thickness of SA and divided it into the epithelium, reticular basement membrane (Rbm) LP, smooth muscle layer, and sub-mucosal adventitia, which meets the alveolar tissue. Further, I quantitated the ˜í¬±-SMA+ cells present in the SA lamina propria and adventitia, while the ECM markers (collagen-1 and fibronectin) were quantitated as a percentage of area staining in the same areas. \\(Results:\\) I confirmed hypo-cellularity in the airway wall in both large and small airways in smokers and in COPD; with LA wall cellularity least in the COPD-current smoker (CS), while SA cellularity was similar across smoker/COPD groups. LA neutrophils were decreased in COPD-CS, while SA neutrophil counts were unchanged. In contrast, a small but significant increase was observed in SA CD8+ cells in both normal smokers and COPD-CS but not in LA. Ratiometric analysis of CD4+ and CD8+ T cells showed a dominance of the CD8+ phenotype in the LP area of LA, but not SA. Compared to controls, LA macrophage numbers in COPD were significantly lower, with SA macrophage numbers unchanged. Further evaluation of the SA macrophage subpopulations showed a significant increase in pro-inflammatory M1s in the small airway walls of NLFS and COPD compared to controls with a reciprocal decrease in M2 macrophages, which remained unchanged among pathological groups. However, luminal macrophages went the other way, with a dominant M2 phenotype in both NLFS and COPD subjects. BAL cytokine profiles were skewed towards M2 with an increase in CCL22, IL-4, IL-13, and IL-10 in both NLFS and COPD. A decrease in degranulating mast cell and total degranulating cells was observed in the SA wall via monitoring intracellular LAMP-1 expression. Further, and unexpectedly, this lysosomal LAMP-1 expression was also found to be markedly increased in the epithelium of small airways in COPD. This increase in LAMP-1+ lysosomes significantly correlated with a decrease in lung function increased airway obstruction and increased in airway thickness. Among the non-inflammatory cell populations characterized, I found a significant and marked increase in ˜í¬±SMA+ myofibroblast numbers in the small airway tissue in both smokers and COPD compared to normal controls, which directly correlated with decreased airway caliber measures in COPD patients. A significant increase was observed in the ECM proteins collagen-1 and fibronectin in the small airway wall of smoker and COPD groups. The proliferation of myofibroblast directly co-related to this increased collagen and fibronectin deposition and airway wall thickening, which suggested an active role in airway remodeling. The increase in the myofibroblast population also correlated with increases in expression of the EMT markers S100A4 and vimentin and lung function. \\(Conclusions:\\) These current studies confirmed our group`s novel finding of hypocellularity in the airway wall of COPD patients, both in large and small airways. Analysis of the literature suggests that this has hardly been looked previously and probably not as rigorously as this investigation. These changes corresponded to decreases in most airway inflammatory and immune cells. Overall, the contribution of inflammatory/immune cells to total airway wall cells was small, again something else never previously documented, with the majority likely to be stromal cells. Macrophage subpopulation showed abnormal differential switching in both the airway wall and lumen, though in different directions. Lack of degranulation activity in the cells of the airway wall in COPD indicated dysfunctionality, which could be crucial in viral and bacterial infections known to be essential disease exacerbations in particular. A proliferation of myofibroblast was present in small airways and strongly associated with both remodelling, active EMT and loss of airflow in COPD.