Discussion
Although the COVID-19 pandemic subsided after two years of global devastation, sporadic outbreaks continue to recur in different parts of the world, especially during winter seasons.48-50 The mortality rates remain high in hospitalized patients with severe infections. Findings of lung autopsy samples of patients, who died of COVID-19, revealed a wide spectrum of pathologic manifestations including acute DAD, impaired lung repair, and fibrosis. Notably, lung fibrosis was more prominent in the critically ill COVID-19 patients with prolonged hospitalization and extended progression of the disease. The underlying mechanisms of how SARS-CoV-2 infection progresses to fibrotic abnormalities in injured lungs is still largely unknown and has motivated the current study.
Pathophysiological development of pulmonary fibrosis involves a series of events that include massive inflammation, alveolar type II epithelial damage, subsequent surfactant deficiency, and abnormal ECM remodeling, which result in alveolar collapse, collapse induration, and fibrosis.41-52 Alveolar epithelial loss and insufficient epithelial regeneration trigger expression of pro-fibrotic cytokines such as transforming growth factor beta, IL-6, and tumor necrosis factor-alpha, all known to induce fibrotic remodeling.53 Recent observations highlight AT-II epithelial injury, and ER-stress-induced arrest of AT-II cell regeneration as promoters of fibrotic changes in COVID-19 patients.17,54 The present study investigates structural integrity of ECM proteins including elastin and collagen and their correlation with pathologic manifestations of fibrotic abnormalities in the lung autopsy samples of COVID-19 patients.
The most notable result of this study was the overall degradation of elastin in the extracellular matrix of COVID-19 lungs. This degradation included the cleavage and disintegration of the elastin fibers of the alveolar wall, as well as within the critical vascular network in the lung parenchyma. Damaged lungs with pathologic manifestations of fibrosing organizing pneumonia displayed complete degradation or absence of the elastin network. Elastin is a polymer of tropoelastin units that scaffold onto fibrillin-1 along the walls of the alveoli.23 Structurally, elastin provides elasticity and tensile strength essential for normal stretching and contraction of alveoli during respiration. Thus, a loss of elastin fibers potentially destabilizes alveoli promoting their collapse. Elastolysis has been associated with several chronic and acute clinical conditions.34 The dysregulated neutrophil activation and overwhelming NE activity contribute to elastin degradation and disruption of alveolar basement causing emphysema in COPD patients. NE-mediated release of elastin degradative products such as desmosine and isodesmosine are potent inducers of fibrosis because they enhance myofibroblast differentiation, proliferation and collagen expression.28,32,55 In the present study, analysis of lung autopsies displayed a dense neutrophil congregates within the areas of DAD with overwhelming expression of NE and MPO in the airways and alveolar airspaces. Further, an increase in PAD4 expression, indicates active NETosis in the infected lung microenvironment of COVID-19 patients. Consistent with NETosis, high levels of total citrullinated proteins were observed in lung autopsy samples of COVID-19 patients compared to non-COVID-19 patients. In fact numerous clinical reports also found increased neutrophilia, high neutrophil-lymphocyte ratios,56-58 and NETosis with progressive lung damage.59 These results indicate an extensive elastin degradation in a short period of time attests to the shocking aggressiveness of invading neutrophils in the lungs and their attack on the extracellular matrix in the lung parenchyma.
Interestingly, morphometric analysis of alveolar interstitium showing elastin degradation found replaced with excessive collagen deposition thus forming interstitial fibrotic changes. In support of this, collagen:elastin ratios showed diffuse collagen deposition in the lung parenchyma in advanced fibrotic regions, which displayed an absence of elastin fibers and widespread alveolar epithelial disintegration in COVID-19 patients. Thus, a combination of elastolysis with epithelial and endothelial injury disrupts the alveolar-capillary barrier; increased alveolar rigidity may cause remodeling of the alveolar architecture, eventually inducing collagen expression, and, ultimately, engulfing fibrosis. These results support the view that elastolysis combined with epithelial injury precede collagen deposition and likely progress to interstitial fibrosis. The disintegration of alveolar architecture compromises gas exchange at the thin epithelial-capillary network in the alveoli. Thus, the results of this study indicate that elastolysis and alveolitis could contribute to collagenous fibrotic abnormalities, ultimately leading to respiratory dysfunction in severely ill COVID-19 patients.
These findings also draw a direct connection between pulmonary sequestration and degranulation of neutrophils, with the destruction of alveolar architecture. Many views of COVID-19 affected lungs show granulocytes and identify colocalized granule enzymes.18-20 Among these, NE is the obvious candidate for the massive degradation of elastin fibers that precedes fibrotic abnormalities. Clearly, NE can be placed at the scene of matrix dissolution by immuno-fluorescence. However, it is difficult to measure changes in NE more distantly, such as in plasma from affected individuals. This is because NE that is locally released is rapidly inhibited by A1AT through the formation of NE-A1AT complexes. Interestingly, analysis of plasma samples of COVID-19 patients showed high levels of NE-A1AT complexes indicating elevated extracellular release of NE. The formation of NE-A1AT complexes thus may be used for diagnosis of neutrophil activation, NET release, and indicate tissue damage in COVID-19 patients. These observations need further investigation to validate and correlate their association with COVID-19.
In summary, the present study provides insights into the pathologic manifestations of COVID-19 and highlights that elastolytic activity plays a key role in exacerbating pulmonary pathogenesis in COVID-19 patients, especially in the pathologic development of pulmonary fibrosis. Dysregulated neutrophil activity and NETosis contribute to elastin degradation, ECM remodeling, and fibrotic changes in COVID-19. A better understanding of the mechanism of elastin degradation and its significance for the pathology of fibrosis will likely help to identify novel therapeutic targets and prevent pulmonary fibrosis in COVID-19 patients.