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.