The present study shows an overall assessment of the cellular responses of bronchial epithelial cells and lung fibroblasts after inflammatory stimuli associated with chronic lung disease. Our results show that 1) IL-1β stimulates different biological pathways involved in chronic lung disease (IL-8 and MCP-1), except apoptosis and specific inflammatory pathways (NF-Κb) biomarkers; 2) The involvement of IL-1ß stimulation in systemic inflammatory and proteinase imbalance biomarkers is higher in lung fibroblasts; 3) Under hypoxia conditions, there is a systemic inflammatory decrease in fibroblasts; 4) simvastatin maintains this downregulation in fibroblasts; 5) as a consequence, the two cell types show a different inflammatory response.
The evaluation of biological pathways that underlie cellular physiology represents a vital step in our understanding of the normal cellular mechanisms, which may be implicated in the initial pathophysiology of chronic lung diseases such as COPD. These results highlight the biological pathways initially stimulated which could potentially trigger the inflammatory response responsible for the eventual onset of chronic lung disease. It is important to bear in mind that, for this study, we used commercial cell lines, which are involved in the remodeling of lung damage, induced by inflammatory stimulus. Therefore, although the pathways explored are the ones, which participate in the pathogenesis of chronic lung disease, we have actually studied them in “normal” cells in culture. Due to changes in these different pathways in patients who develop chronic lung disease 12, the cells in the respiratory system of patients with this disease may respond differently. For this reason, our results should now be replicated with primary cultures obtained from patients with chronic lung disease in order to clarify their involvement in the pathogenesis of the disease.
Fibroblasts, together with protein imbalance, seem to be the main factors involved in the inflammatory response in chronic lung disease. According to our results, both bronchial epithelial cells and lung fibroblasts participate, each in a different way, in the inflammatory process, which begins in the bronchial epithelium with the release of IL-8, MCP-1 in bronchial epithelial cells and lung fibroblasts and of CRP and SAA in bronchial epithelial cells. Subsequently, the activated macrophages release metalloproteinase 9 and 12, which contribute to the degradation of elastin in the lung parenchyma. However, this inflammatory process in lung fibroblasts could be attenuated by simvastatin. Interestingly, that biomarker associated with a disease-specific response (NF-κB) or with more advanced disease was not significantly changed.
The fact that IL-1β stimulation is not always associated with increased protein levels of pro-inflammatory mediators is of particular interest. Epigenetic post-transcriptional regulation of protein synthesis has been well documented in cell physiology and is directly related to chronic lung disease pathogenesis 13–15. Osei et all. found that co-cultures of lung fibroblast with bronchial epithelial cells significantly increased the expression of miR-146-a-5p. They demonstrated that miR-146a-5p expression played an anti-inflammatory role, downstream of the IL-1 pathway, subsequently reducing IL-8 release from lung fibroblast 16. This regulation mechanism may explain our findings of the release of IL-8 in lung fibroblast stimulated with IL-1β.
Systemic inflammation in chronic lung disease, defined as increased levels of inflammatory markers from different biological pathways 17, has become another target for chronic lung disease-related research and its origin is a challenge for researchers. Not only may proteins originating from the lung exert systemic effects, but there is also a lack of correlation between airway cytokine concentrations and those in the circulation 18, and investigators have been unable to find an association between the inflammatory load of induced sputum and plasma 19. This may be due to hepatic hyperstimulation during chronic lung disease, such as COPD, and this lung-liver feedback should be explored further. Serum amyloid A (SAA), an acute phase protein which occurs in high levels in the blood during infection, was considered to be produced by hepatocytes and subsequently secreted into serum 20. However, one published study has demonstrated that SAA mRNA 21 is normally expressed in the epithelial components of a variety of human organs, and tissues could be released locally in certain organ-specific diseases 22. Moreover, SAA can also be produced by macrophages and other extrahepatic cells as well as in the lung 23,24. Our previous findings show that this SAA expression was different between pulmonary cell types 25. Our results show SAA mRNA expression but no protein expression in lung fibroblast. In contrast, we observed a protein expression of SAA, but no SAA mRNA expression in bronchial epithelial cells. This may be due to differences in the posttranscriptional regulation depending on the cell type. The existence of several SAA receptors has been demonstrated, such as toll-like receptor (TLR) 2 and TLR4, although the structure of SAA differs from the ligands classically associated with these receptors. Furthermore, SAA has ability to activate TLR2 and TLR4, which is of particular interest, given that these TLRs play important roles in the inflammatory response, such as in the modulation of airway epithelial cell regeneration 26. On the other hand, SAA preferentially activates the histone H3 demethylase Jmjd3, and as a result, SAA not only triggers transcription factor activation but also influences gene expression through epigenetic regulation 27,28. Our findings could be explained by several mechanisms, such as the modulation of TLR activity or epigenetic mechanisms. Both of these may act differentially between lung fibroblast and bronchial epithelial cells, leading to increased TLR mRNA expression and a higher uptake of SAA protein, or an inhibition of SAA protein synthesis in lung fibroblast cells, respectively. In addition, our results could indicate an overlap in bronchial epithelial cells between SAA kinetics and the kinetic of the C-reactive protein, which would be in line with previous studies 29.
The interactions between epithelial cells, fibroblasts and the extracellular matrix of the airway wall are intimately involved in a number of functions within the lung 30. MMP play a key role in chronic lung disease pathogenesis, as well as degrading matrix proteins, and its presence is required for normal matrix processing and lung repair 31. In addition, MMP-12 plays a pivotal role in the inflammatory process that leads to lung injury 32. MMP-12 also plays a role in inflammatory lung disease and tissue remodeling 33,34. Hence, an increased expression of MMP-9 and MMP-12 may cause an unwanted degradation of lung tissue. According to our results, both cell types, bronchial epithelial and lung fibroblast, participate actively and differentially during the initial injury in chronic lung disease. Given the potential importance of MMP-12 in chronic lung disease, it is of interest to show that, after an inflammatory stimulus, its production and release occur earlier in fibroblasts than in the epithelial cells. Our findings suggest that the fibroblasts tend to act as sentinel cells in the event of injury, orchestrating the early phases by secreting MMP-12. In addition, fibroblasts could modulate the response of epithelial cells, which is in line with previous studies 35,36. These differences between MMPs may be of major importance when considering the role of a given MMP and, particularly, in selecting an MMP to inhibit together with a synthetic inhibitor.
We explored the effect of hypoxia as a stimulus that would be representative of acute pulmonary diseases, in which hypoxemia would occur as a consequence of the severity of the disease. Although current COPD recommendations no longer consider respiratory failure as a component when measuring the severity of the disease 37, patients with COPD and secondary hypoxemia should be classed as particularly serious cases from a clinical perspective, and they have serious prognostic implications 38. Hypoxia and inflammation are intimately linked 39. Some studies have shown “in vitro” evidence that hypoxia may aggravate airway inflammation through an effect on immune cells such as macrophages 40 or monocytes 41, and our study suggests that inflammation before hypoxia does not worsen local inflammation near lung fibroblasts and bronchial epithelial cells. We observed that exposure of normal lung fibroblasts, in culture, to hypoxia resulted in decreased SAA mRNA expression. Nevertheless, patients with high SAA had greater dyspnea and more frequent interstitial lung disease 42. Therefore, our results suggest that lung fibroblasts can be considered a cellular model, used to analyze the underlying mechanisms in the pathogenesis of overlap syndrome, or to improve hypoxia-induced inflammation, which could be facilitated by high altitude acclimatization. This expression of SAA mRNA remains downregulated after treatment with simvastatin. However, the effect of the statins depends on the underlying stimulus, mechanisms, microenvironmental conditions 43 and cell type used in the experiment. The relationship between pulmonary disease and simvastatin is complex 44. A recent systematic review identified articles evaluating the clinical efficacy of statin therapy in chronic lung disease. Although statin treatment was associated with improvements in exercise capacity, lung function and health status, the authors found no associations with inflammatory markers 45. Multiple retrospective studies have shown that statins are beneficial in chronic lung disease because their anti-inflammatory effects include suppression of the upregulation of pro-inflammatory cytokines, chemokines, adhesion molecules and MMPs by inflammatory cells 46,47, and their potential role in respiratory disease has been hypothesized 48. Interestingly, the impact of simvastatin on epithelial cell lines is less obvious, despite a few reports that simvastatin inhibits alveolar destruction 49 and affects alveolar recovery 50.
On the other hand, the role of increased apoptotic alveolar cells in the peripheral lung is relevant in severe stable COPD patients with pulmonary emphysema 51,52. Our results did not show any differences in the rate of apoptosis and necrosis when we compared cells stimulated with and without IL-1β. The role of apoptosis in cell physiology is probably related to particular presentations of the disease, in which there is an increased destruction of lung parenchyma, such as in emphysema. The rate of apoptosis is higher in epithelial cells because, during the pathogenesis of chronic lung disease, there is destruction of the lung tissue and development of pulmonary emphysema, while fibroblasts increase their proliferation and ability to produce fibrotic tissue 53. Therefore, our model is probably not ideal for studying emphysema.
In summary, the present study is an overall assessment of the cellular behavior of lung epithelial cells and lung fibroblasts after inflammatory stimuli associated with chronic lung disease. We have shown that the different biological pathways involved in chronic lung disease are increased after stimulation with IL-1β. These results highlight the initially stimulated biological pathways that could potentially trigger the inflammatory response responsible for the eventual onset of chronic lung disease. Future studies should replicate this approach with primary cell cultures obtained from patients with chronic lung disease in order to clarify their involvement in the pathogenesis of the disease at more advanced stages.