\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[greek,ngerman,polish,english]{babel}



\usepackage{float}








\begin{document}

\title{Neutrophil Aggregates and Elastin Degradation Compromise Lung
Architecture in Fatal COVID-19}



\author[1]{Teluguakula Narasaraju}%
\author[1]{Indira Neeli}%
\author[2]{Sheila Criswell L}%
\author[3]{Amita Krishnappa}%
\author[4]{Wenzhao Meng}%
\author[1]{Vasuki Silva}%
\author[5]{Galyna Bila}%
\author[5]{Volodymyr Vovk}%
\author[6]{Zolotukhin Serhiy}%
\author[7]{Gary Bowlin L}%
\author[4]{Nuala Meyer}%
\author[4]{Eline T. Luning Prak}%
\author[1]{Marko Radic}%
\author[5]{Rostyslav Bilyy}%
\affil[1]{The University of Tennessee Health Science Center Department of Microbiology Immunology and Biochemistry}%
\affil[2]{The University of Tennessee Health Science Center College of Medicine}%
\affil[3]{Adichunchanagiri Institute of Medical Sciences}%
\affil[4]{University of Pennsylvania Perelman School of Medicine}%
\affil[5]{Lvivs'kyj nacional'nyj medychnyj universytet imeni Danyla Halyc'koho}%
\affil[6]{Lvivs'ka oblasna naukova medichna biblioteka}%
\affil[7]{The University of Memphis Department of Biological Scienes}%


\vspace{-1em}



  
  \date{June 09, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Pulmonary fibrosis, profound alveolitis, and the failure to restore
alveolar epithelial architecture are major causes of respiratory failure
in fatal COVID-19. However, contributing factors to abnormal fibrosis in
critically ill COVID-19 patients are yet to be understood. This study
analyzed the histopathology of lung autopsy samples from eight COVID-19
and six non-COVID-19 post-mortems. The distribution and changes in
extracellular matrix (ECM) proteins, including elastin and collagen in
lung alveoli, were quantitatively assessed through morphometric
analyses. These studies reveal massive degradation of elastin fibers
along the thin-alveolar walls of the lung parenchyma that supersedes
interstitial collagenous fibrosis and intra-alveolar fibrotic
abnormalities. Injured lungs with collapsed alveoli and organized
fibrotic regions exhibited widespread elastolysis. Further,
immunoblotting of lung autopsy extracts validated extensive elastin
degradation. Importantly, loss of elastin was correlated with induction
of neutrophil elastase (NE), a potent protease that degrades ECM, and
increased staining of peptidylarginine deiminase, a marker for
neutrophil extracellular traps release, and extensive epithelial
necrosis. Further, elevated plasma levels of NE-alpha1-antitrypsin
complex in hospitalized COVID-19 patients indicate dysregulated
neutrophil activity. These findings place elastin degradation at the
center of alveolar structural disintegration and argue that elastolysis
and alveolitis lead to abnormal ECM repair and fibrosis in fatal
COVID-19.%
\end{abstract}%



\sloppy


\textbf{Introduction.}

The COVID-19 pandemic demonstrated the potentially devastating outcomes
of respiratory viral infections. Although most infected individuals
recover with relatively mild symptoms, many experience impaired lung
function, require hospitalization, and as many as 2\% of the infected
patients succumb to the disease caused by the coronavirus CoV-2. To gain
greater insight into the course of fatal COVID-19, lung autopsies from
COVID-19 patients have been examined by histopathology. The disease
reveals a complexity of pathologic features, including acute diffuse
alveolar damage (DAD) and organized pneumonia and
fibrosis.\textsuperscript{1-6} Growing evidence indicates that during
disease progression, fibrotic abnormalities become more predominant,
especially in fatal COVID-19.\textsuperscript{7-10} Similarly,
post-acute phase sequelae of COVID-19 chest computed tomography (CT)
scans find a variable degree of fibrotic abnormalities in a large number
of recovered patients.\textsuperscript{11} In fact, pathologic lesions
caused by fibrosis were also noted earlier in fatal avian influenza,
2003-SARS-COV, and Middle East respiratory syndrome coronavirus
infections.\textsuperscript{12-15} However, etiologic factors that drive
virus-inflicted pulmonary fibrosis are yet largely unexplored.

Pulmonary fibrosis develops in a complex interplay between cell death,
inflammation, and abnormal ECM remodeling that lead to alveolar
architectural disorganization, irreversible lung dysfunction, and
death.\textsuperscript{16} Recent studies associate alveolar type II
pneumocyte (AT-II) epithelial injury and endoplasmic reticulum
(ER)-stress-induced impairment of AT-II cell regeneration with
SARS-CoV-2-inflicted fibrotic abnormalities and reveal common features
with idiopathic pulmonary fibrosis (IPF).\textsuperscript{17} Persistent
neutrophil accumulation and release of neutrophil extracellular traps
(NETs) in the lower respiratory tracts and plasma samples of severely
ill COVID-19 patients and associated with acute alveolar
injury.\textsuperscript{18-21} Therefore, factors that drive the
disorganization of ECM proteins and abnormal ECM remodeling toward
fibrotic changes in acute SARS-COV-2 infection deserve greater
attention.

Elastin and collagen are major structural ECM proteins in the
alveoli.\textsuperscript{22} Elastin fibers consist of tropoelastin
polymers, which are cross-linked by lysyl oxidase-mediated conversion of
4 lysine side chains into desmosine.\textsuperscript{23-24} Elastin
fibers align along the walls of the alveoli by association with
fibrillin-1 and facilitate the expansion and recoiling of alveoli during
normal breathing.\textsuperscript{25} Unlike other ECM proteins, elastin
has a half-life of 70-80 years, as synthesis of elastin fibers occurs
during the postnatal lung alveolarization and continues during the first
10 years of life in humans.\textsuperscript{26} The
regeneration/replacement of damaged elastin is inefficient, thus damaged
elastin is often replaced by collagen.\textsuperscript{27-28} Elastin is
a potent substrate for neutrophil elastase (NE), a serine proteinase
produced by activated neutrophils.\textsuperscript{29} NE-mediated
elastolysis releases elastin degradative products (EDP), potent
mediators of inflammation and interstitial
fibrosis.\textsuperscript{30}Cleavage of elastin is linked to the
pathophysiology in bleomycin-induced fibrosis,\textsuperscript{31}
IPF,\textsuperscript{32}and chronic obstructive pulmonary disease
(COPD).\textsuperscript{33}The activity of NE is regulated by an
inhibitor called alpha-1 anti-antitrypsin (A1AT), which complexes with
NE and blocks its activity.\textsuperscript{34-35}

In this study, eight lung autopsy samples from COVID-19 patients and six
non-COVID-19 lungs were compared microscopically. Micro-architecture
with progressive elastin cleavage and collagen deposition in injured
lungs correlated with lung pathophysiology, inflammation, and fibrosis.
These observations reveal extensive degradation of elastin fibers in the
alveolar interstitium that shows severe epithelial damage and massive
neutrophil-specific inflammation. Further, loss of elastin and increased
collagen deposits marked lesions with interstitial collagenous fibrosis
and organized parenchymal fibrosis. Elastin degradation was also
observed in areas of alveolar collapse indicating that elastolysis and
alveolitis contribute to abnormal ECM repair and fibrosis in fatal
COVID-19 patients. As proxy for elastase, NE-A1AT complexes in plasma of
a second group of hospitalized COVID-19 patients were quantitated.
Plasma samples from hospitalized patients had approximately 30-fold
higher NE-A1AT complexes than plasmas from healthy donors, suggesting
that such complexes may provide an early warning of host tissue damage
due to inflammation in respiratory lung infections.

\textbf{Materials and Methods}

Materials.

Citrulline (C7629), 2,3-Butanedione monoxime (B0753), Antipyrine
(A5882), Iron (lll) Chloride (157740), A1AT, Bovine serum albumin
(A9418), and PNPP (S0942) were purchased from Sigma-Aldrich, MO.
Antibodies including anti-NE (MAB9167, Novus Biologicals, CO), anti-A1AT
(Thermo Fisher Scientific, MA), anti- peptidylarginine deiminase 4
(PAD4, ab128086, Abcam, MA), anti-MPO (14569P, Cell-signaling MA),
anti-thyroid transcription factor-1 (TTF-1, SPT24, Biocare~Medical, CA),
anti-elastin (MA5-41583, Thermo Fisher Scientific, MA) and anti-histone
H3 pan antibodies (07-690, Upstate, NY) were used in this study. Soluble
elastin (Elastin Products Company, MO). Secondary antibodies for Western
blot were purchased from Li-Cor Biosciences, NE, and anti-Rabbit IgG-AP
(Southern Biotech, AL). Reagents including EnVision~FLEX Target
Retrieval Solution High pH (Dako/Agilent, CA) and low pH IHC Antigen
Retrieval Solution (Invitrogen, MA), Biocare~Decloaking Chamber
(Biocare~Medical, CA) and Tris-Buffered Saline (TBS) (Dako/Agilent, CA)
were used in immunohistochemistry.

Collection of the lung autopsy samples, plasma, and ethical committee
approvals:

Lung autopsy samples from eight patients, who died of COVID-19, and six
patients, who died of non-COVID-19-related deaths were examined. This
study was conducted in compliance with approval by the ethical committee
of Danylo Halytsky Lviv National Medical University, Ukraine (Protocol
Numbers: 20180226/2; 20211122/9). Patients, who were hospitalized with
positive COVID-19 test, with pneumonia, and with at least one
complicating factor such as diabetes mellitus or hypertension and who
died in the hospital after therapy including oxygen support, constituted
the COVID-19 group. Non-COVID-19 samples were from hospitalized
patients, who had no positive COVID-19 tests at the time of death and
were characterized by oxygen saturation being \textgreater{}95\%.

Plasma samples from hospitalized COVID-19 patients (\emph{n}  = 62) were
obtained following a protocol approved by the institutional review board
(IRB\# 808542), University of Pennsylvania, between April and June
2020\textsuperscript{36}; anonymous discarded plasma samples (n=15; gift
of Donald Siegel, University of Pennsylvania)\textsuperscript{37} and
additional samples (n=11) were obtained following an IRB-approved
protocol from the University of Tennessee Health Science Center.

Histopathology.

Lung tissues were fixed in neutral-buffered formalin and embedded in
paraffin. Lung sections were cut at 4 \selectlanguage{greek}μ\selectlanguage{english}m, dewaxed, rehydrated, and
stained with hematoxylin and eosin. Various lung pathologic features
including acute or organizing diffuse alveolar damage (DAD), alveolar
hemorrhage, thrombosis, and fibrotic remodeling described earlier in
COVID-19 patients\textsuperscript{38-40} were evaluated by an
experienced pathologist (AK) in a blinded manner. Pathological lesions
of acute DAD were characterized by disrupted alveolar epithelium,
epithelial sloughing, formation of hyaline membranes, necrotizing
bronchiolitis, interstitial inflammation, oedema, fibrin deposition,
vasculitis, pulmonary hemorrhage, and microvascular thrombosis. The
organizing form of DAD was defined as loosely organized fibrosis,
interstitial fibrotic changes, and abnormal remodeling of epithelium
with hyperplasic type II pneumocytes and proliferative airway
epithelium. Fibrotic changes were defined as extensive and dense
interstitial fibrosis replacing lung parenchyma and disintegrating
alveolar architecture. The histopathological grading was 0-absent,
1-mild, 2-moderate, and 3-severe. Lung sections were stained with
Verhoff van Gieson (VVG) and orcein for detecting elastin fibers and
Masson's trichrome for identification of collagen depositions.

Immunohistochemistry.

Lung tissue sections were subjected to immunohistochemistry analysis
using Bond\textsuperscript{TM} Polymer Refine Red Detection Kit (Leica
Biosystems, IL) with primary antibodies for the detection of neutrophil
granule proteins including NE (1:400 dilution), myeloperoxidase (MPO,
1:10000 dilution); alveolar type II epithelium marker, TTF-1, (1:1
dilution); and peptidylarginine deiminase 4 (PAD4, 1:300 dilution), a
marker for neutrophil extracellular traps (NETs) induction. Antigen
epitope retrieval was conducted with~EnVision~FLEX Target Retrieval
Solution High pH (K800421-2, Dako/Agilent, USA) and low pH IHC Antigen
Retrieval Solution (00-4955-58, Invitrogen, USA) using
a~Biocare~Decloaking Chamber (Biocare~Medical, USA) for 5 minutes at 120
\selectlanguage{ngerman}°C. Following antigen retrieval, tissue sections were incubated with
primary antibodies as described above, followed by Horse radish
peroxidase-conjugated secondary antibodies. Color was developed using
universal diaminobenzidine chromogen substrate. Whole slide images were
captured using CaseViewer 3DHITECH Ltd. software.

Elastin fiber morphometry

Elastin fibers in the alveolar walls were detected by VVG and orcein
staining and images were captured as described above. Lengths of the
elastin fibers were measured using ImageJ software analysis (National
Institute of Health, April, 2019; ImageJ with 64-bit Java 1.8.0\_172).
Images from 10 randomly selected fields at 400x were captured. The
images were first normalized for scale, the actual lengths of the
elastin fibers were measured in micrometers (\selectlanguage{greek}μ\selectlanguage{english}m) in a blinded manner,
and the curvatures of the elastin fibers along the walls of the alveoli
were evaluated using freehand length measurement.\textsuperscript{41}
The bundle of elastin fibers was measured as a single fiber unless the
fibers were distinctly separated from each other. Elastin fibers often
appear as single detached, fragmented or degraded fibers in COVID-19
patients. These fibers were counted separately to measure their lengths.
All data were exported into an Excel file and the average fiber length
in \selectlanguage{greek}μ\selectlanguage{english}m was determined. Each fiber was counted only once and no extra
weighting was used for larger/wider fibers. Elastin fibers staining in
the airways or surrounding bronchioles or pulmonary blood vessels were
excluded.

Quantification of collagen and measurement of collagen:elastin ratio in
the lungs of COVID-19 patients.

Masson's Trichrome stained lung sections were imaged as described above,
and images were analyzed by ImageJ software. For the measurement of
collagen density, 10 random fields were selected from each lung section.
The collagen staining was processed by selecting Masson's Trichrome
specific staining using color deconvolution plugin software in ImageJ
that separates red, blue, and green components from the image.
Thresholds for collagen staining were established for each slide by
enhancing the contrast to a point at which the collagen staining was
easily identified by blue staining and the density of the collagen was
measured over total lung area from the image.\textsuperscript{42} The
collagen was measured as percent collagen density in total lung area of
the image. No weighting was performed for the intensity of blue
staining. Further, adjacent serial lung sections were stained with
Masson's Trichrome and orcein for quantitative measurement of
collagen:elastin ratio as a fold change after both stains were
normalized to the total lung area in the image captured in the same
areas of the sections.

Protein extraction from formalin-fixed paraffin-embedded (FFPE) lung
tissues and western blot analysis:

FFPE lung tissues (10 \selectlanguage{greek}μ\selectlanguage{english}m sections) from non-COVID-19 and COVID-19
patients were transferred into 1.5 ml centrifuge tubes, dewaxed in
xylene for 10 min and centrifuged. T issue pellets were washed 3x with
xylene and rehydrated with graded concentrations of ethanol (100 \%, 85
\% and 75 \% for 2 min in each). After the ethanol wash, proteins in the
tissue pellets were solubilized in Tris-SDS buffer, pH 8.0, by
homogenization and sonication. Tissue homogenates were centrifuged at
10,000 g for 20 min and the supernatants were measured for total protein
concentrations. Equal amounts of proteins were resolved by 12\% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer
onto nitrocellulose membrane. Membranes were then blocked with 5\%
bovine serum albumin and incubated with anti-elastin (1:1000 dilution)
and anti-histone H3 Pan antibodies (loading control, 1:4000 dilution)
overnight at 4 \textsuperscript{0}C. Next day, membranes were washed in
TBST and incubated with IRDye 800CW~and IRDye 600CW conjugated secondary
antibodies. The membranes were washed 3 times and signal was measured
using the Odyssey Infrared Imaging System (LI-COR Biosciences).

Colorimetric determination of citrulline residues in proteins:

To measure the total citrullinated proteins in the lung homogenates, an
equal amount of proteins from non-COVID-19 and COVID-19 patients were
added to the chromogenic reagent (Diacetyl-monoxime Solution +
Antipyrine Solution + Acid-Ferric Solution, at 1:2:3), mixed well, and
the tubes covered with aluminum foil. For standard, 0.5 to 10 nmoles of
citrulline were prepared as described above. The samples and standards
were heated for 25 min in a boiling water bath and cooled to RT on ice.
The color developed was read at 464
nm.\textsuperscript{43}Quantification of the citrullinated proteins in
the lung homogenates was determined using the citrulline-protein
standard curve.

ELISA for evaluation of the NE-A1AT complexes in the plasma samples:

Standard NE-A1AT complexes were prepared by combining 2.5 \selectlanguage{greek}μ\selectlanguage{english}M NE with 10
\selectlanguage{greek}μ\selectlanguage{english}M A1AT in 100ul of PBS, and incubated at 37\textsuperscript{0}C for 30
and 60 min. After the incubation, aliquots were snap frozen and kept at
-80\textsuperscript{0}C until further use. For ELISA, 96-well plates
were coated with the anti-NE antibody at 1 \selectlanguage{greek}μ\selectlanguage{english}g/ml and incubated overnight
at 4\textsuperscript{0}C. On the following day, the plates were washed
once with PBS Tween-20, and blocked with 1 \% BSA in PBS for 1 hour at
RT. The plates were then incubated with plasma samples collected from
non-COVID-19 or COVID-19 patients for 2 hours at RT, followed by 3
washes, and further incubation with rabbit polyclonal anti-A1AT antibody
at 1:10000 dilutions for 1 hour. Next, the plates were washed 3 times,
and incubated with anti-rabbit-IgG-AP for 1 hour at 1:2000, followed by
3 washes, and further incubation with para-Nitrophenylphosphate
substrate for 30min at RT in the dark. The color developed was read at
405 nm.\textsuperscript{44,45} The NE-A1AT known standard complexes were
used to quantitatecomplexes present in the COVID-19 and non-COVID-19
plasma samples.

Statistical analyses:

Data are expressed as the mean group value \selectlanguage{ngerman}± standard error mean (SEM).
Analysis of the lung autopsies from COVID-19 positive and COVID-19
negative patients for measuring elastin fibers and collagen were
performed using Image J software analysis ((National Institute of
Health, April, 2019; ImageJ with 64-bit Java 1.8.0\_172) Website:
https://imagej.nih.gov/ij/ for performing densitometry measurements.
NIH). Statistical differences between the two groups were determined by
Student's \emph{t} -test with a two-tailed comparison. A p-value of
\emph{\textless{}0.05} was considered statistically significant.

\textbf{Results:}

\textbf{Characteristics of Tissue Donors}

The characteristics of the subjects with fatal COVID-19 are described in
Table 1. Postmortem lung autopsy samples were collected at Lviv Regional
Pathological Anatomy Office, CU ENT «Pulmonology Lviv Regional
Diagnostic Center» from patients who were COVID-19 positive by RT-PCR
and presented with bilateral pneumonia during admission into the
hospital \textbf{(Table 1)} . The mean age of the subjects was 60.4
years with a range of 30 to 80 years (4 female and 4 male patients).
Patients were admitted into the hospital on average 7.1 days from the
onset of symptoms and 7 of 8 subjects were in the hospital for more than
10 days (range 10 to 21 days). One patient died on 5th day after
hospitalization. The biochemical analyses revealed a high percentage of
blood neutrophils (non-COVID-19 patients 57.5 \% vs COVID-19 patients
79.8 \%; \emph{P} value \textless{}0.03) and decreased lymphocytes
(non-COVID-19 patients 36 \% vs COVID-19 patients 14.9 \%, \emph{P}value
\textless{}0.09). Erythrocyte sedimentation rate (ESR), a plasma marker
for inflammatory response, was elevated in COVID-19 patients compared to
non-COVID-19 patients (51.7±10.7 vs 17±9.8; \emph{P} value
\textless{}0.0004). Six lung autopsy samples collected from patients who
died of non-COVID-19 related diseases were also analyzed.

\textbf{Histopathological manifestations of acute DAD and variable
fibrotic abnormalities in COVID-19.}

In comparison of lung autopsy samples of 8 COVID-19 patients and 6
non-COVID-19 patients, progressive pathologic changes, including diffuse
alveolar damage (DAD), fibrosing organizing pneumonia, were
evaluated\textbf{(Figure 1A, B and Table 2)} . DAD was found in all 8/8
COVID-19 patients with major characteristic features of widespread
alveolitis (\textbf{Figure S1A),} sloughing of the bronchiolar
epithelium\textbf{(Figure S1B),} formation of a hyaline membrane, (4/8)
of cases\textbf{(Figure S1C),} and Intra-alveolar edema (5/8)
cases\textbf{(Figure S1D)} . Interestingly, lung fibrosis exhibited a
variable degree of abnormal features in all COVID-19 patients. The onset
of fibrotic changes appeared focally in the alveolar interstitium with
increased deposition of collagenous fibers \textbf{(Figure 1C)} . Mild
to moderate interstitial fibrosis was present in 3/8 cases, while
intra-alveolar fibrosis was noted in all except 2 cases \textbf{(Figure
1D)} . In 5/8 cases, organizing fibrosis was evident, with fibrosis
extending into the surrounding parenchyma, thus causing disorganization
of alveolar architecture and loss of air spaces \textbf{(Figure 1E)} .
The loose myxoid fibroblastic proliferation extended between the alveoli
in an intercommunicating manner, at places forming Masson bodies in
alveolar and bronchiolar regions \textbf{(Figure 1F)} . Formation of
bronchiolitis obliterans organizing pneumonia (BOOP) was noted in one
case \textbf{(Figure 1G).} The squamous cells formed distinct nodules
resembling squamous morules \textbf{(Figure 1H)} . Fibrotic changes also
appear in the perivascular regions with pulmonary blood vessels
surrounded by loosely organized proliferating fibroblasts\textbf{(Figure
1I)} . One specimen showed evidence of high inflammation in the pleura
\textbf{(Figure S1E)} .In two cases, a superimposed bacterial infection
was present, which was characterized by the presence of bacterial
colonies admixed with neutrophils within the alveoli\textbf{(Figure
S1F)} . Vascular thrombi in the small vessels were observed in 6/8 cases
\textbf{(Figure S1G)} .

\textbf{Evidence for elastolytic activity in COVID-19 patients.}

Recent studies linked alveolar epithelial senescence and impaired
regeneration as inducing factors of fibrosis in
COVID-19.\textsuperscript{17} However, the changes in the ECM that
trigges in fibrosis in unknown. These studies reveal widespread
alveolar-elastolysis in COVID-1 patients. Morphometrically, the elastin
fibers in the normal alveoli appear as slender fibers aligned with the
alveolar walls. The fibers often appear as bundles of similar length and
branch at intersections between alveoli in non-COVID-19
lungs\textbf{(Figure 2A)} . Abundant elastin fibers also were observed
at the tips of the alveolar septa. The elastin fibers that align with
alveolar walls are essential in providing mechanical support and elastic
recoil to the air spaces during respiration. The elastin fibers in
COVID-19 lungs exhibited extensive disintegration \textbf{(Figure 2B)} .
The degradation of elastin was most notable along the walls of alveoli
that displayed extensive necrosis of alveolar epithelium. Further,
fragmented elastin formed shorter, and single fibers that separated from
the elastin bundles in the alveolar interstitial lining. The individual
elastin fibers often lost their alignment within the alveolar walls.
Quantitative measurement of the lengths of the elastin fibers displayed
significant reduction in the lengths in the COVID-19
patients\textbf{(Figure 2C)} . In parallel, elastin fibers in the
perivascular regions also displayed extensive cleavage in COVID-19
patients\textbf{(Figure 2D).} The loss of elastin along the blood
vessels is associated with severe endothelial necrosis and distortion of
the blood vessel structures. Thus, a loss of breathing capacity was
compounded by the lack of elasticity in blood vessels carrying blood
needed for gas exchange. In support of these findings, Western blot
analysis of elastin from protein extracted from FFPE lung tissues
displayed degradation of tropoelastin band at 70 kDa. Lung extracts from
COVID-19 negative patients showed a prominent tropoelastin
\textbf{(Figure 2E)} . Densitometry analysis of Western blots using
ImageJ software\textbf{(Figure 2F)} showed a significant reduction in
tropoelastin reactivity in COVID-19 lungs, in agreement with the
morphometric analysis of elastin degradation in the COVID-19 lungs.

\textbf{Collagen} \textbf{accumulates and replaces} \textbf{elastin in
the lung parenchyma of COVID-19 patients.}

Unlike the collagen staining that appeared mainly in the alveolar walls
of non-COVID-19 patients, dense accumulations of collagen were observed
in the alveolar interstitium in COVID-19 patients, accompanied by
thickening of the alveolar septa \textbf{(Figure 3A, B)} . A
quantitative assessment of collagen was determined using ImageJ analysis
by selecting Trichrome-specific signals that show significant increase
in collagen accumulation within the alveolar regions in the COVID-19
patient\textbf{(Figure 3C)} . In normal ECM of alveolar intersitium,
elastin fibers are generally interspersed with collagen fibers that
provides elasticity and mechanical strength to the alveoli. To evaluate
the relation between collagen and elastin in COVID-19 patients,
especially during elastolysis, this study examined expression of
collagen and elastin fibers in the thin-alveolar walls of COVID-19
positive and COVID-19 negative patients. As shown in \textbf{Figure 3
D-E} , epithelial injury and elastin degradation in the alveolar walls
were found replaced with abundant collagen deposition. The thickened
walls of the alveoli show dense interstitial collagenous fibrosis
associated with degradation or complete loss of elastin fibers
\textbf{(Figure 3 F-H)} . Interestingly, the interstitial regions
showing degraded elastin also display epithelial necrosis, indicating
that both elastolysis and epithelial injury are preceding events in
pathologic development of interstitial fibrosis (\textbf{Figure 3 D-E)}
. A recent study identified collapse induration associated with alveolar
epithelial necrosis and denudation of basal lamina in a COVID-19
patient.\textsuperscript{46}Accordingly, this study found widespread
alveolar collapse with hallmarks of necrotic epithelium and narrowing of
the alveolar lumen. Interestingly, the collapsed alveoli also displayed
an extensive degradation of elastin fibers and, conversely, dense
collagen deposition in the collapsed alveolar regions \textbf{(Figure
3I-J)} suggesting that epithelial damage and elastolysis may precede the
fibrotic changes in the injured alveoli. Similarly, perivascular regions
showing loosely proliferating fibroblasts exhibit thick collagen
deposition and degradation of elastin fibers \textbf{(Figure S1H-J)} .
In support of these observations, the collagen:elastin ratio was
increased by four-fold in the lung sections of COVID-19 patients
compared to COVID-19 negative patients \textbf{(Figure 4 A-E)} . The
lung parenchymal regions with advanced fibrosis displayed dense collagen
diffusion with complete absence of elastin fibers, thus indicating an
extensive elastolysis occurring during progression of the fibrosis in
COVID-19 patients. Areas of fibrosing organizing pneumonia displayed
extensive loss of alveolar epithelium that correlates with the loss of
elastin fibers in the lung parenchyma \textbf{(Figure 4 F-H)} .

\textbf{Neutrophil aggregates and active NETosis were prominent features
in the lungs of COVID-19 patients.}

Although neutrophil influx and NET release were reported in COVID-19
patients, their pathogenic role is yet to be understood. Massive
neutrophil aggregates were found within the airways and alveolar spaces
that displayed widespread alveolar architectural
disintegration\textbf{(Figure 5A-D)} , The lung neutrophils displayed
strong immunostaining for granule proteins including NE, MPO, and PAD4
co-localized within the regions of neutrophil aggregates in the
alveoli\textbf{(Figure 5E)} . Accordingly, we measured an increase in
total citrullinated proteins in COVID-19 lungs compared to non-COVID-19
lungs\textbf{(Figure 5F)} . Strong NE staining in the extracellular
space within the disintegrated alveoli is consistent with the
degranulation of neutrophils in the inflammatory microenvironment.

Increased NE-A1AT complexes were found in the plasma samples of COVID-19
patients:

Given the accumulation of neutrophils in COVID-19 patient lungs and the
abundant evidence of NE expression and activity in the tissues, it was
of interest to determine whether plasma from COVID-19 patients showed
indications of increased NE. Because NE activity quickly dissipates, due
to the high levels of the A1AT protease inhibitor that binds to NE and
blocks its activity,\textsuperscript{47} an appealing alternative is to
measure concentrations of NE-A1AT complexes that have an extended
half-life. A sandwich ELISA was developed to determine levels of NE-A1AT
complexes in the plasma samples of COVID-19 patients as well as healthy
individuals. As shown in \textbf{Figure 6,} a significant increase in
NE-A1AT complexes was observed in COVID-19 patients.

\textbf{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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,\textsuperscript{56-58} and NETosis with progressive lung
damage.\textsuperscript{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.\textsuperscript{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.

\textbf{Acknowledgments}

The authors are thankful to the members of ``The PREDICT-19
Consortium'', Australia and the members of the ``NETwork to target
neutrophils in COVID-19 collaborative working group'', USA for their
helpful advice and encouragement. We thank Anna Vaseruk for her help in
collecting clinical data. The authors acknowledge the study participants
and their surrogates and the clinical and research staff who made this
study possible.

\textbf{Funding:}

This work was supported by the ``UTHSC/UofM SARS-CoV-2/COVID-19 Research
CORNET~Award'' to MR and GLB; and the National Research Foundation of
Ukraine grant 2020.02.0131 and the European Union's Horizon 2020
(NeutroCure Project 861878) to RB.

\textbf{Authors Contributions}

\emph{Conceptualization and design of the of the study} : Marko Radic,
Rostyslav Bilyy, Teluguakula Narasaraju. \emph{Conducted the experiment
and analyzed the data} : Rostyslav Bilyy, Teluguakula Narasaraju, Indira
Neeli, Sheila L Criswell, Amita Krishnappa, Wenzhao Meng, Silva, Vasuki,
Galyna Bila, Volodymyr Vovk, Zolotukhin Serhiy. \emph{Wrote the
manuscript, review and editing} : Teluguakula Narasaraju, Marko Radic,
Indira Neeli, Sheila L Criswell, Gary L Bowlin, Nuala Meyer, Eline T.
Luning Prak, Rostyslav Bilyy.

\textbf{Table Legends}

\textbf{Table 1.} \textbf{Summary of the autopsy cases.}

Clinical and biochemical signatures in COVID-19 positive and COVID-19
negative hospitalized patients during admission and progression of the
COVID-19 disease in intensive care units.

\textbf{Table 2} . \textbf{Histopathologic signatures of the lung
autopsies of COVID-19 patients.} Semiquantitative histopathology scores
of the lung autopsy samples from COVID-19 positive and COVID-19 negative
patients.

\textbf{Figure Legends}

\textbf{Figure 1. Lung histopathology analyses of COVID-19 patients.}

Autopsy lung samples collected from COVID-19 patients show major
patterns of (A) widespread diffuse alveolar damage, and (B) fibrosing
organizing pneumonia. Fibrotic changes in COVID-19 patients show
variable patterns including (C) interstitial fibrotic development (black
arrows), (D) intra-alveolar fibrin deposition and organizing fibrosis
showing spreading of fibrosis in alveoli (white arrows) and (E) diffuse
alveolar fibrosis within small airways (arrowhead). (F) The loose myxoid
fibroblastic proliferation extended between the alveoli forming Mason
bodies in alveolar and bronchiolar regions (yellow frame). (G)
Bronchiolitis obliterans organizing pneumonia (BOOP)-like areas (red
frame). (H) The squamous cells formed distinct nodules resembling
squamous morules were noted in COVID-19 patients (blue circles). (I)
Disrupted small blood vessels were also displayed perivascular
fibroblast proliferations (Hash). Scale Bars = 100 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 2. Elastin degradation in the lungs of the COVID-19
patients.}

The quantitative measurement of elastin fibers length in \selectlanguage{greek}μ\selectlanguage{english}m. The elastin
fibers present in the alveolar walls were measured using ImageJ
analysis. (A) Among COVID-19 negative patients. Elastin fibers appear as
bundles in the alveolar walls (red arrows). (B) In COVID-19 patients,
fragmentation of elastin fibers was observed (open red arrow). (C)
Lengths of elastin fibers in COVID-10 positive and COVID-19 negative
patients. (D) Elastolysis (red arrowhead) in the perivascular regions of
the COVID-19 patients compared to COVID-19 negative patients. To
validate elastolysis observed in morphometric analyses, lung tissue
lysates prepared from COVID-19 positive and COVID-19 negative patients
were evaluated by western blot analysis. (E) The lung tissue lysates
from COVID-19 negative (2-5 lanes) and COVID-positive (6-11 lanes), and
soluble elastin (EL) (12) were probed with anti-elastin antibodies and
the immunoblot shows a strong band at 70 kDa representing tropoelastin.
Lung tissue lysates of COVID-19 patients show degradation of
tropoelastin. (F) The densities of tropoelastin were normalized to the
histone protein levels in the lung homogenates. The values of elastin
fibers lengths were represented as means \selectlanguage{ngerman}± SD in lung sections from 8
COVID-19 patients and 6 non-COVID-19 patients (2C). Data was represented
as mean ± SD from lung sections from 6 COVID-19 patients and 4
non-COVID-19 patients (2F). Scale bar=100 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 3. Loss of elastin found replaced with collagenous
fibrosis in alveolar interstitium and perivascular regions.}

The density of collagen distribution in the lung parenchyma was measured
in (A) COVID-19 negative and (B) COVID-19 positive patients using
Masson's trichrome collagen specific staining. (C) The density of
collagen in the lung was quantified using ImageJ analysis as described
in the methods. The values of collagen density were represented as means
\selectlanguage{ngerman}± SD in lung sections from 8 COVID-19 patients and 6 non-COVID-19
patients. (D) Necrotic and denuded alveolar epithelial lining displayed
elastolysis and (E) dense collagen deposition (red arrows - loss of
elastin in the alveolar walls; red asterisk - epithelial denudation;
black arrows - collagen accumulation). (F, G, H) Collagen replacing
elastin fibers in formation of interstitial fibrotic changes in COVID-19
patients evident by H\&E, collagen deposition, and elastin degradation
respectively (red frame). (I, J) Lung parenchyma shows alveolar collapse
(red asterisks) and fibrotic development and reduction of elastin
fibers. Scale bar=100 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 4. Measurement of collagen:elastin ratio.}

The density of collagen and elastin fibers distribution in the lung
parenchyma was measured in COVID-19 positive and COVID-19 negative
patients. Adjacent lung sections stained with Masson's trichrome and
orcein from (A, B) non-COVID-19 patient (A, collagen - black arrow) and
(B, elastin -- black arrowhead). COVID-19 patient lungs displayed
widespread (C) collagen deposition, (D) complete loss of elastin. (E)
The collagen:elastin ratio was quantified using ImageJ analysis as
described in the methods. (F-H) Areas of fibrosing organizing pneumonia
also displayed (G) extensive loss of alveolar epithelium stained with
TTF-1 (yellow line), while undamaged areas show positive staining for
TTF-1 and elastin (red arrow). Scale bar=100 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 5. Neutrophil influx, and NETosis in the lung parenchyma
of COVID-19 lung autopsy cases.}

(A) H\&E staining of lung tissues with massive neutrophil influx. (B)
Neutrophils show high expression of neutrophil elastase. (C) Insert from
panel B depicts extracellular release of NE (white asterisk) in the
alveoli. (D) Lung sections with immunohistochemistry for MPO also
indicate neutrophil activation. (E) PAD4 staining. (F) Increased levels
of total citrullinated proteins in the lungs of COVID-19 patients
compared to COVID-19-negative patients. Scale Bar=100 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 6. Evaluation of NE-A1AT complex in the plasma samples by
ELISA.}

The levels of NE-A1AT complexes were found significantly higher in
COVID-19 patients compared to healthy individuals.

\textbf{Figure S1 Pathological lesions of alveolitis, bronchiolitis,
inflammation thrombosis and pleuritic in COVID-19.} (A) Alveolar
epithelial denudation is evident by H\&E staining (open arrow). (B)
Extensive bronchiolitis with sloughing of bronchiolar epithelium was
noted in all cases (black arrow). (C) Denudation of epithelial lining
and formation of hyaline membranes in COVID-19 patients (black
arrowhead), (D) edema with filling of alveolar air spaces with protein
exudates (asterisks), (E) One specimen showed evidence of pleuritic
(yellow frame). (F) In two cases, bacterial superinfections were noted
with presence of bacterial colonies admixed with neutrophils within the
alveoli (hash). (G) Blood vessels also show prominent fibrin thrombus.
(H- I) Perivascular regions showing loosely proliferating fibroblasts,
accumulation of collagen, and (H) degradation of elastin fibers (red
arrows). Scale bar=100 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{References}

\begin{enumerate}
\tightlist
\item
  Ravaglia C, Doglioni C, Chilosi M, Piciucchi S, Dubini A, Rossi G,
  Pedica F, Puglisi S, Donati L, Tomassetti S, Poletti V. Clinical,
  radiological and pathological findings in patients with persistent
  lung disease following SARS-CoV-2 infection. Eur Respir J.
  2022;60(4):2102411.
\item
  Mauad T, Duarte-Neto AN, da Silva LFF, de Oliveira EP, de Brito JM, do
  Nascimento ECT, de Almeida Monteiro RA, Ferreira JC, de Carvalho CRR,
  do Nascimento Saldiva PH, Dolhnikoff M. Tracking the time course of
  pathological patterns of lung injury in severe COVID-19. Respir Res.
  2021;22(1):32.
\item
  Merdji H, Mayeur S, Schenck M, Oulehri W, Clere-Jehl R, Cunat S,
  Herbrecht JE, Janssen-Langenstein R, Nicolae A, Helms J, Meziani F,
  Chenard MP; CRICS TRIGGERSEP Group (Clinical Research in Intensive
  Care, Sepsis Trial Group for Global Evaluation, Research in Sepsis).
  Histopathological features in fatal COVID-19 acute respiratory
  distress syndrome. Medicina Intensiva. 2021;45(5):261-270.
\item
  Batah SS, Fabro AT. Pulmonary pathology of ARDS in COVID-19: A
  pathological review for clinicians. Respir Med. 2021;176:106239.
\item
  Valdebenito S, Bessis S, Annane D, Lorin de la Grandmaison G,
  Cramer-Bord\selectlanguage{ngerman}é E, Prideaux B, Eugenin EA, Bomsel M. COVID-19 Lung
  Pathogenesis in SARS-CoV-2 Autopsy Cases. Front Immunol.
  2021;12:735922.
\item
  Yao XH, Luo T, Shi Y, He ZC, Tang R, Zhang PP, Cai J, Zhou XD, Jiang
  DP, Fei XC, Huang XQ, Zhao L, Zhang H, Wu HB, Ren Y, Liu ZH, Zhang HR,
  Chen C, Fu WJ, Li H, Xia XY, Chen R, Wang Y, Liu XD, Yin CL, Yan ZX,
  Wang J, Jing R, Li TS, Li WQ, Wang CF, Ding YQ, Mao Q, Zhang DY, Zhang
  SY, Ping YF, Bian XW. A cohort autopsy study defines COVID-19 systemic
  pathogenesis. Cell Res. 2021;31(8):836-846.
\item
  Li Y, Wu J, Wang S, Li X, Zhou J, Huang B, Luo D, Cao Q, Chen Y, Chen
  S, Ma L, Peng L, Pan H, Travis WD, Nie X. Progression to fibrosing
  diffuse alveolar damage in a series of 30 minimally invasive autopsies
  with COVID-19 pneumonia in Wuhan, China. Histopathology.
  2021;78(4):542-555.
\item
  Wells AU, Devaraj A, Desai SR. Interstitial lung disease after
  COVID-19 infection: a catalog of uncertainties.~\emph{Radiology.~}
  2021;299(1):E216--e8.
\item
  Berezowska S, Schmid A, Losmanová T, Trippel M, Blank A, Banz Y, Jakob
  SM, Langer R. Frequency and Significance of Pathologic Pulmonary
  Findings in Postmortem Examinations-A Single Center Experience before
  COVID-19. Diagnostics (Basel). 2021;11(5):894.
\item
  Spagnolo P, Balestro E, Aliberti S, et al. Pulmonary fibrosis
  secondary to COVID-19: a call to arms?~\emph{Lancet Respir Med.~}
  2020;8(8):750--752.~{[}PMC free article{]}~{[}PubMed{]}~{[}Google
  Scholar{]}
\item
  Caruso D, Guido G, Zerunian M, Polidori T, Lucertini E, Pucciarelli F,
  Polici M, Rucci C, Bracci B, Nicolai M, Cremona A, De Dominicis C,
  Laghi A. Post-Acute Sequelae of COVID-19 Pneumonia: Six-month Chest CT
  Follow-up. Radiology. 2021;301:E396-405.
\item
  Huang, W.J., Tang, X.X. Virus infection induced pulmonary
  fibrosis.~\emph{J Transl Med} ~19, 496 (2021).
\item
  Qiao J, Zhang M, Bi J, Wang X, Deng G, He G, Luan Z, Lv N, Xu T, Zhao
  L. Pulmonary fibrosis induced by H5N1 viral infection in mice. Respir
  Res. 2009;10(1):107.
\item
  Wu X, Dong D, Ma D.~Thin-Section Computed Tomography Manifestations
  During Convalescence and Long-Term Follow-Up of Patients with Severe
  Acute Respiratory Syndrome (SARS).~\emph{Med Sci Monit}
  ~2016;22:2793--2799.
\item
  Arabi YM, Balkhy HH, Hayden FG, Bouchama A, Luke T, Baillie JK,
  Al-Omari A, Hajeer AH, Senga M, Denison MR, Nguyen-Van-Tam JS, Shindo
  N, Bermingham A, Chappell JD, Van Kerkhove MD, Fowler RA. Middle East
  Respiratory Syndrome. N Engl J Med. 2017;376(6):584-594.
\item
  Chanda D, Otoupalova E, Smith SR, Volckaert T, De Langhe SP,
  Thannickal VJ. Developmental pathways in the pathogenesis of lung
  fibrosis. Mol Aspects Med. 2019;65:56-69.
\item
  Sinha S, Castillo V, Espinoza CR, Tindle C, Fonseca AG, Dan JM, Katkar
  GD, Das S, Sahoo D, Ghosh P. COVID-19 lung disease shares driver AT2
  cytopathic features with Idiopathic pulmonary fibrosis. EBioMedicine.
  2022 Aug;82:104185.
\item
  Ackermann M, Anders HJ, Bilyy R, Bowlin GL, Daniel C, De Lorenzo R,
  Egeblad M, Henneck T, Hidalgo A, Hoffmann M, Hohberger B, Kanthi Y,
  Kaplan MJ, Knight JS, Knopf J, Kolaczkowska E, Kubes P, Leppkes M,
  Mahajan A, Manfredi AA, Maueröder C, Maugeri N, Mitroulis I, Muñoz LE,
  Narasaraju T, Naschberger E, Neeli I, Ng LG, Radic MZ, Ritis K,
  Rovere-Querini P, Schapher M, Schauer C, Simon HU, Singh J, Skendros
  P, Stark K, Stürzl M, van der Vlag J, Vandenabeele P, Vitkov L, von
  Köckritz-Blickwede M, Yanginlar C, Yousefi S, Zarbock A, Schett G,
  Herrmann M. Patients with COVID-19: in the dark-NETs of neutrophils.
  Cell Death Differ. 2021;28(11):3125-3139.
\item
  Werner J D Ouwendijk, Matthijs P Raadsen, Jeroen J A van Kampen,
  Robert M Verdijk, Jan H von der Thusen, Lihui Guo, Rogier A S Hoek,
  Johannes P C van den Akker, Henrik Endeman, Thomas Langerak, Richard
  Molenkamp, Diederik Gommers, Marion P G Koopmans, Eric C M van Gorp,
  Georges M G M Verjans, Bart L Haagmans, High Levels of Neutrophil
  Extracellular Traps Persist in the Lower Respiratory Tract of
  Critically Ill Patients With Coronavirus Disease 2019,~\emph{The
  Journal of Infectious Diseases} . 2021;223: 1512--1521.
\item
  Seren S, Derian L, Kele\selectlanguage{polish}ş \selectlanguage{english}I, Guillon A, Lesner A, Gonzalez L, Baranek
  T, Si-Tahar M, Marchand-Adam S, Jenne DE, Paget C, Jouan Y, Korkmaz B.
  Proteinase release from activated neutrophils in mechanically
  ventilated patients with non-COVID-19 and COVID-19 pneumonia. Eur
  Respir J. 2021;57(4):2003755.
\item
  Wang Y, Schughart K, Pelaia TM, Chew T, Kim K, Karvunidis T,
  Knippenberg B, Teoh S, Phu AL, Short KR, Iredell J, Thevarajan I,
  Audsley J, Macdonald S, Burcham J, McLean A; PREDICT-19 consortium;
  Tang B, Shojaei M. Blood transcriptome responses in patients correlate
  with severity of COVID-19 disease. Front Immunol. 2023;13:1043219.
\item
  Gagiannis D, Steinestel J, Hackenbroch C, et al. Clinical,
  serological, and histopathological similarities between severe
  COVID-19 and acute exacerbation of connective tissue
  disease-associated interstitial lung disease (CTD-ILD)~\emph{Front
  Immunol.~} 2020;11
\item
  Kozel BA, mecham RP, Rosenbloom J. Elastin. In: Mecham RP,
  editorBiology of Extracellular Matrix. Springer-Verlag;
  Berlin-Heidelberg: 2011. 267--301.
\item
  Davidson JM. Biochemistry and turnover of lung interstitium. Eur
  Respir J. 1990;3: 1048--68.
\item
  Mecham RP. Elastin in lung development and disease pathogenesis.
  Matrix Biol. 2018;73:6-20.
\item
  Deslee G, Woods JC, Moore CM, Liu L, Conradi SH, Milne M, Gierada DS,
  Pierce J, Patterson A, Lewit RA, Battaile JT, Holtzman MJ, Hogge JC,
  Pierce RA. Elastin expression in very severe human COPD. Eur Respir J.
  2009;34:324--31.
\item
  mShapiro SD, Endicott SK, Province MA, Pierce JA, Campbell EJ. Marked
  longevity of human lung parenchymal elastic fibers deduced from
  prevalence of D-aspartate and nuclear weapons-related radiocarbon. J
  Clin Invest. 1991;87(5):1828-34.
\item
  Liu X, Ma S, Turino G, Cantor J. The pattern of elastic Fiber
  breakdown in Bleomycin-induced pulmonary fibrosis may reflect
  microarchitectural changes. Lung. 2017;195:93--9.
\item
  Benjamin JT, Plosa EJ, Sucre JM, van der Meer R, Dave S, Gutor S,
  Nichols DS, Gulleman PM, Jetter CS, Han W, Xin M, Dinella PC,
  Catanzarite A, Kook S, Dolma K, Lal CV, Gaggar A, Blalock JE, Newcomb
  DC, Richmond BW, Kropski JA, Young LR, Guttentag SH, Blackwell TS.
  Neutrophilic inflammation during lung development disrupts elastin
  assembly and predisposes adult mice to COPD. J Clin Invest. 2021;131:
  e139481.
\item
  Starcher B, Peterson B. The kinetics of elastolysis: elastin
  catabolism during experimentally induced fibrosis. Exp Lung Res.
  1999;25(5):407-24.
\item
  de Brouwer B, Drent M, van den Ouweland JMW, Wijnen PA, van Moorsel
  CHM, Bekers O, Grutters JC, White ES, Janssen R. Increased circulating
  desmosine and age-dependent elastinolysis in idiopathic pulmonary
  fibrosis. Respir Res. 2018;19:45.
\item
  Araos J, Ayala P, Meneses M, Contreras R, Cuti\selectlanguage{ngerman}ño A, Montalva R,
  Tazelaar H, Borzone G. Resolution of lung injury after a single event
  of aspiration: a model of bilateral instillation of whole gastric
  fluid. Am J Pathol. 2015;185: 2698--708.
\item
  He J, Turino GM, Lin YY. Characterization of peptide fragments from
  lung elastin degradation in chronic obstructive pulmonary disease. Exp
  Lung Res. 2010;36:548--57.
\item
  Wewers MD, Crystal RG. Alpha-1 antitrypsin augmentation therapy. COPD.
  2013;10 Suppl 1:64-7.
\item
  D'Souza RF, Masson SWC, Woodhead JST, James SL, MacRae C, Hedges CP,
  Merry TL. \selectlanguage{greek}α\selectlanguage{english}1-Antitrypsin A treatment attenuates neutrophil elastase
  accumulation and enhances insulin sensitivity in adipose tissue of
  mice fed a high-fat diet. Am J Physiol Endocrinol Metab.
  2021;321:E560-E570.
\item
  Chang SE, Feng A, Meng W, Apostolidis SA, Mack E, Artandi M, Barman L,
  Bennett K, Chakraborty S, Chang I, Cheung P, Chinthrajah S, Dhingra S,
  Do E, Finck A, Gaano A, Ge\selectlanguage{ngerman}ßner R, Giannini HM, Gonzalez J, Greib S,
  Gündisch M, Hsu AR, Kuo A, Manohar M, Mao R, Neeli I, Neubauer A,
  Oniyide O, Powell AE, Puri R, Renz H, Schapiro J, Weidenbacher PA,
  Wittman R, Ahuja N, Chung HR, Jagannathan P, James JA, Kim PS, Meyer
  NJ, Nadeau KC, Radic M, Robinson WH, Singh U, Wang TT, Wherry EJ,
  Skevaki C, Luning Prak ET, Utz PJ. New-onset IgG autoantibodies in
  hospitalized patients with COVID-19. Nat Commun. 2021;12:5417.
\item
  Bar KJ, Shaw PA, Choi GH, Aqui N, Fesnak A, Yang JB, Soto-Calderon H,
  Grajales L, Starr J, Andronov M, Mastellone M, Amonu C, Feret G,
  DeMarshall M, Buchanan M, Caturla M, Gordon J, Wanicur A, Monroy MA,
  Mampe F, Lindemuth E, Gouma S, Mullin AM, Barilla H, Pronina A, Irwin
  L, Thomas R, Eichinger RA, Demuth F, Luning Prak ET, Pascual JL, Short
  WR, Elovitz MA, Baron J, Meyer NJ, Degnan KO, Frank I, Hensley SE,
  Siegel DL, Tebas P. A randomized controlled study of convalescent
  plasma for individuals hospitalized with COVID-19 pneumonia. J Clin
  Invest. 2021;131:e155114.
\item
  Mauad T, Duarte-Neto AN, da Silva LFF, de Oliveira EP, de Brito JM, do
  Nascimento ECT, de Almeida Monteiro RA, Ferreira JC, de Carvalho CRR,
  do Nascimento Saldiva PH, Dolhnikoff M. Tracking the time course of
  pathological patterns of lung injury in severe COVID-19. Respir Res.
  2021;22:32.
\item
  Merdji H, Mayeur S, Schenck M, Oulehri W, Clere-Jehl R, Cunat S,
  Herbrecht JE, Janssen-Langenstein R, Nicolae A, Helms J, Meziani F,
  Chenard MP; CRICS TRIGGERSEP Group (Clinical Research in Intensive
  Care, Sepsis Trial Group for Global Evaluation, Research in Sepsis).
  Histopathological features in fatal COVID-19 acute respiratory
  distress syndrome. Med Intensiva (Engl Ed). 2021;45:261-270.
\item
  Valdebenito S, Bessis S, Annane D, Lorin de la Grandmaison G,
  Cramer-Bordé E, Prideaux B, Eugenin EA, Bomsel M. COVID-19 Lung
  Pathogenesis in SARS-CoV-2 Autopsy Cases. Front Immunol.
  2021;12:735922.
\item
  Zweers MC, van Vlijmen-Willems IM, van Kuppevelt TH, Mecham RP,
  Steijlen PM, Bristow J, Schalkwijk J. Deficiency of tenascin-X causes
  abnormalities in dermal elastic fiber morphology. J Invest Dermatol.
  2004;122:885-91.
\item
  Kottmann RM, Sharp J, Owens K, Salzman P, Xiao GQ, Phipps RP, Sime PJ,
  Brown EB, Perry SW. Second harmonic generation microscopy reveals
  altered collagen microstructure in usual interstitial pneumonia versus
  healthy lung. Respir Res. 2015;16:61.
\item
  Dwivedi N, Hedberg A, Zheng YY, Neeli I, Satoh M, Morel L, Rekvig OP,
  Radic M. B Cell Tolerance to Deiminated Histones in BALB/c, C57BL/6,
  and Autoimmune-Prone Mouse Strains. Front Immunol. 2017;8:362.
\item
  Neumann S, Hennrich N, Gunzer G, Lang H. Enzyme-linked immunoassay for
  human granulocyte elastase in complex with alpha 1-proteinase
  inhibitor. Adv Exp Med Biol. 1984;167:379-90
\item
  Engelmaier A, Weber A. Sensitive and specific measurement of
  alpha\textsubscript{1}-antitrypsin activity with an elastase complex
  formation immunosorbent assay (ECFISA). J Pharm Biomed Anal.
  2022;209:114476
\item
  Ochs M, Timm S, Elezkurtaj S, Horst D, Meinhardt J, Heppner FL,
  Weber-Carstens S, Hocke AC, Witzenrath M. Collapse induration of
  alveoli is an ultrastructural finding in a COVID-19 patient. Eur
  Respir J. 2021;57(5):2004165
\item
  McElvaney OJ, McEvoy NL, Boland F, McElvaney OF, Hogan G, Donnelly K,
  Friel O, Browne E, Fraughen DD, Murphy MP, Clarke J, Choileáin ON,
  O'Connor E, McGuinness R, Boylan M, Kelly A, Hayden JC, Collins AM,
  Cullen A, Hyland D, Carroll TP, Geoghegan P, Laffey JG, Hennessy M,
  Martin-Loeches I, McElvaney NG, Curley GF. A randomized, double-blind,
  placebo-controlled trial of intravenous alpha-1 antitrypsin for ARDS
  secondary to COVID-19. Med. 2022;3(4):233-248.e6.
\item
  Mahroum N, Seida I, Esirgün SN, Bragazzi NL. The COVID-19 pandemic -
  How many times were we warned before? Eur J Intern Med. 2022;105:8-14.
\item
  Voskarides K. SARS-CoV-2: tracing the origin, tracking the evolution.
  BMC Med Genomics. 2022;15:62.
\item
  Nugent MA. The Future of the COVID-19 Pandemic: How Good (or Bad) Can
  the SARS-CoV2 Spike Protein Get? Cells. 2022;11:855.
\item
  Burkhardt A. Pathogenesis of pulmonary fibrosis. Hum Pathol 1986;17:
  971--973.
\item
  Spencer H. Pathogenesis of interstitial fibrosis of the lung. Progr
  Respir Res. 1975;8: 34-44.
\item
  Froese AR, Shimbori C, Bellaye PS, Inman M, Obex S, Fatima S, Jenkins
  G, Gauldie J, Ask K, Kolb M. Stretch-induced Activation of
  Transforming Growth Factor-\selectlanguage{greek}β\selectlanguage{english}1 in Pulmonary Fibrosis. Am J Respir Crit
  Care Med. 2016;194(1):84-96.
\item
  D'Agnillo F, Walters KA, Xiao Y, Sheng ZM, Scherler K, Park J, Gygli
  S, Rosas LA, Sadtler K, Kalish H, Blatti CA 3rd, Zhu R, Gatzke L,
  Bushell C, Memoli MJ, O'Day SJ, Fischer TD, Hammond TC, Lee RC, Cash
  JC, Powers ME, O'Keefe GE, Butnor KJ, Rapkiewicz AV, Travis WD, Layne
  SP, Kash JC, Taubenberger JK. Lung epithelial and endothelial damage,
  loss of tissue repair, inhibition of fibrinolysis, and cellular
  senescence in fatal COVID-19. Sci Transl Med. 2021;13:eabj7790.
\item
  Nagel D, Clough R, Sime PJ , Kottamann RM. Elastin degradative
  products induce myofibroblast differentiation Am J Resp Crit Care Med
  2019, 199:A5333.
\item
  Gong J, Dong H, Xia QS, Huang ZY, Wang DK, Zhao Y, Liu WH, Tu SH,
  Zhang MM, Wang Q, Lu FE. Correlation analysis between disease severity
  and inflammation-related parameters in patients with COVID-19: a
  retrospective study. BMC Infect Dis. 2020;20(1):963.
\item
  Wang X, Li X, Shang Y, Wang J, Zhang X, Su D, Zhao S, Wang Q, Liu L,
  Li Y, Chen H. Ratios of neutrophil-to-lymphocyte and
  platelet-to-lymphocyte predict all-cause mortality in inpatients with
  coronavirus disease 2019 (COVID-19): a retrospective cohort study in a
  single medical centre. Epidemiol Infect. 2020;148:e211.
\item
  Wang D, Hu B, Hu C, Zhu F, Liu X, Zhang J, Wang B, Xiang H, Cheng Z,
  Xiong Y, Zhao Y, Li Y, Wang X, Peng Z. Clinical Characteristics of 138
  Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia
  in Wuhan, China. JAMA. 2020 Mar 17;323(11):1061-1069. doi:
  10.1001/jama.2020.1585. Erratum in: JAMA. 2021;325:1061--9.
\item
  Barnes BJ, Adrover JM, Baxter-Stoltzfus A, Borczuk A, Cools-Lartigue
  J, Crawford JM, Da\selectlanguage{ngerman}ßler-Plenker J, Guerci P, Huynh C, Knight JS, Loda
  M, Looney MR, McAllister F, Rayes R, Renaud S, Rousseau S, Salvatore
  S, Schwartz RE, Spicer JD, Yost CC, Weber A, Zuo Y, Egeblad M.
  Targeting potential drivers of COVID-19: Neutrophil extracellular
  traps. J Exp Med. 2020 ;217(6):e20200652.
\end{enumerate}

\textbf{Hosted file}

\verb`FIgures Main.pptx`  available at \url{https://authorea.com/users/630776/articles/650421-neutrophil-aggregates-and-elastin-degradation-compromise-lung-architecture-in-fatal-covid-19}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Table-1/Table-1}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

