Introduction
Idiopathic pulmonary fibrosis (IPF) is a chronic,
fibrosing interstitial pneumonia of unknown etiology (1). A subset of patients with IPF may
experience acute exacerbations (AE), diagnosed based on established
clinical criteria: A previous or concurrent diagnosis of IPF; acute
worsening of dyspnea (typically over <1 month); high-resolution
computed tomography findings of new bilateral ground-glass
opacities and/or consolidation superimposed on a usual interstitial
pneumonia pattern; and exclusion of cardiac failure or fluid
overload (2). These exacerbations
lead to acute and clinically notable respiratory deterioration. The
annual incidence of AE-IPF ranges from 5 to 10% (3,4),
with mortality rates between 56 and 100% (5). Without lung transplantation, the
median survival time after an AE is 3-4 months (6,7).
Current international guidelines provide only a weak recommendation
for glucocorticoid therapy in AE-IPF, and lung transplantation
remains the only effective long-term treatment for these critically
ill patients (1,8-10).
The etiology of AE-IPF is multifactorial, including
bacterial or viral infections, drug toxicity, aspiration, intrinsic
defects and surgical-related injuries (10-12).
Viral infection often serves as an initiating event (13). Research suggests that latent
viruses can modify the secretory phenotype of lung cells, promoting
a pro-fibrotic environment even when the latent viral load is low
or after mRNA COVID-19 vaccination (14-16).
Other studies have shown that the bacterial load in bronchoalveolar
lavage fluid (BALF) from patients with AE-IPF is considerably
higher when compared with that from stable patients (13). Nevertheless, the exact pathogenesis
of AE-IPF remains unclear. Studies indicate that it may involve
increased epithelial cell apoptosis and excessive inflammatory
responses (17-19).
Notably, type 2 alveolar epithelial cells (AEC2s), which function
as alveolar stem cells, exhibit heightened apoptosis and loss of
self-renewal capacity, playing a pivotal role in pulmonary fibrosis
progression (20-22).
To investigate the mechanisms of AE-IPF, several
animal models have been established. These models typically involve
initial treatment with bleomycin (BLM) or other toxic agents to
induce pulmonary fibrosis via alveolar epithelial cell injury
(23,24); alternatively, TGF-β1 transgenic
mice are used to directly establish pulmonary fibrosis (25). Subsequently, mice are challenged
with bacterial, viral or chemical agents to trigger an AE of
pulmonary fibrosis (AE-PF) (18,19,23,26).
This approach leads to notable alveolar damage, including
epithelial cell shedding, widening of the alveolar septa, extensive
inflammatory cell infiltration and increased collagen deposition
(18,19,27).
However, published AE-PF models vary considerably in the choice of
agents and the timing of the AE-inducing challenge (17). For example, some models use more
toxic agents (24,26), and in certain studies, the AE
trigger is administered as early as day 7 after BLM treatment, when
lung inflammation is most pronounced but clear fibrosis has not yet
developed (27,28). In other studies, AE is induced on
day 21, when the fibrotic changes are fully established, a
reasonable approach. Nevertheless, fibrosis in the BLM model begins
to resolve spontaneously around day 28, so observation at day 35
may yield inaccurate results because natural resolution could
confound the assessment of AE effects (29). Moreover, the pathological changes
in lung tissue across these models are inconsistent; some show
marked destruction of alveolar architecture and significant
fibrosis, while others predominantly exhibit inflammation (30,31).
Because the intratracheal BLM mouse model remains
the most widely used model of pulmonary fibrosis according to
international guidelines published in 2017(7), the present study established
pulmonary fibrosis by intratracheal BLM injection on day 0. On day
14 after the initial BLM treatment, when fibrotic changes are more
evident, mice were rechallenged with replication-deficient
adenoviral vectors (ADV), lipopolysaccharide (LPS) or a second dose
of BLM. The present study systematically compared the
pathophysiological features of these three AE-PF models and
evaluated their suitability as experimental platforms for
investigating the mechanisms underlying acute exacerbations in
patients with IPF.
Materials and methods
Animals
Wild-type male C57BL/6 mice (aged 6-8 weeks;
weighing 22.3-25.1 g) were obtained from Jiangsu Gempharmatech Co.,
Ltd. The animals were housed under a 12-h light/dark cycle with
controlled humidity (50±15%) and temperature (22±2˚C), with food
and water provided ad libitum. Animal health and behavior were
assessed twice daily (9:00 AM and 5:00 PM) throughout the study.
Humane endpoints were predefined as: i) Body weight loss >30% of
initial weight, ii) hunched posture, iii) piloerection, iv) labored
breathing or v) inability to reach food or water. Humane endpoints
were predefined; however, no animals met these criteria during the
study and were euthanized accordingly. All surviving animals were
euthanized at the experimental endpoint for tissue collection. All
efforts were made to minimize suffering and distress. No analgesics
or anesthetics were used during the disease course to avoid
interference with inflammatory and fibrotic responses, except for
the intratracheal instillation procedure.
A total of 75 mice (15 per group) were initially
assigned to the five groups: control (Ctrl), BLM, BLM+ADV, BLM+LPS
and BLM+BLM. On day 0, mice in the Ctrl group received an
intratracheal injection of 50 µl PBS, whereas mice in the four
BLM-treated groups received intratracheal BLM (3 mg/kg in 50 µl
PBS; Nippon Kayaku Co., Ltd.). For intratracheal instillations,
mice were briefly anesthetized with isoflurane (3% for induction;
1.5% for maintenance) delivered via a nose cone. During the first
14 days following BLM administration, three mice died in each of
the four BLM-treated groups, leaving 12 mice per group prior to the
second challenge on day 14. On day 14, mice in the Ctrl and BLM
groups received an intratracheal injection of 50 µl PBS. Mice in
the AE groups (BLM+ADV, BLM+LPS and BLM+BLM) received,
respectively: ADV (1x108 PFU in 50 µl PBS; cat. no.
OP0524; OBiO Technology (Shanghai) Corp.), LPS (1 mg/kg in 50 µl
PBS; cat. no. L2880-10 MG; Escherichia coli O55:B5;
MilliporeSigma) or a second dose of BLM (3 mg/kg in 50 µl PBS). No
further mortality was observed in the BLM group after day 14 (12
mice survived to day 21). By contrast, mortality occurred in all
three AE groups, with final survival numbers of 8 (BLM+ADV), 6
(BLM+LPS) and 8 (BLM+BLM) mice, respectively, as shown in Fig. 1C. All mortalities were attributed
to respiratory distress secondary to exacerbated lung injury.
 | Figure 1Mouse models of AE-PF. (A) Flow chart
of AE-PF mouse models. An intratracheal injection of BLM was
performed on day 0, followed by a second intratracheal injection of
ADV, LPS or an additional dose of BLM on day 14. (The mice were
randomly divided into Ctrl group, BLM group and three AE groups. AE
groups include three subgroups: BLM+ADV group, BLM+LPS group and
BLM+BLM group. (B) The weight changes over 21 days. The weights of
mice in three AE groups were significantly reduced in comparison
with the BLM group at Day 21. (C) The survival of mice from day 14
to day 21 in BLM, BLM+ADV, BLM+LPS and BLM+BLM groups were 100%
(12/12), 66.7% (8/12), 50.0% (6/12) and 66.7% (8/12), respectively.
*P<0.05, **P<0.01,
***P<0.001. AE, acute exacerbation; Ctrl, control;
BLM, bleomycin; ADV, replication-defective adenoviral vectors; LPS,
lipopolysaccharide. |
On day 20, mice underwent chest micro-computed
tomography (micro-CT) imaging. On day 21, the animals were
euthanized by intraperitoneal injection of sodium pentobarbital
(150 mg/kg). Mortality was confirmed by cessation of heartbeat and
respiration, as well as lack of response to toe pinch. Samples of
lung tissue, BALF and peripheral blood were then collected
(Fig. 1A). All experimental
protocols were approved by the Ethics Committee of Nanjing Drum
Tower Hospital, Nanjing University Medical School (Nanjing, China;
approval no. 2016-160-01).
Assays of hydroxyproline content of
lung tissue
Lung tissue samples (50 mg) were precisely weighed
and recorded. Hydroxyproline content was measured using alkaline
hydrolysis according to the manufacturer's instructions (cat. no.
A030-2-1; Nanjing Jiancheng Bioengineering Institute). The
absorbance was measured at 550 nm, and the hydroxyproline
concentration in lung tissues was calculated using the formula
provided in the kit instructions.
Histology and immunofluorescence
Paraffin-embedded lung tissue specimens were cut
into 5-µm-thick sections. Sections were stained with H&E for
histopathological evaluation. The severity of lung injury was
assessed using a modified Ashcroft scale (32). All scoring was performed by three
independent certified pathologists blinded to group allocation and
the average score was used for analysis. Lung fibrosis was
evaluated on sections stained with Masson's trichrome. For
apoptosis detection, tissue sections were subjected to TUNEL
staining according to the manufacturer's instructions (cat. no.
KTA2011; Abbkine Scientific Co., Ltd.) and visualized using a
fluorescence microscope (Thunder; Leica Microsystems GmbH). For
immunofluorescence staining of alveolar epithelial cells, sections
were incubated overnight at 4˚C with a pro-surfactant protein C
(SPC) antibody (cat. no. ab211326; Abcam). Antigens were visualized
using FITC-conjugated secondary antibodies (cat. no. 33112ES60;
Shanghai Yeasen Biotechnology Co., Ltd.) incubated for 1 h at room
temperature. Nuclei were counterstained with DAPI (cat. no. KGA215;
Jiangsu KeyGen Biotech Co., Ltd.).
BALF cytospin and semi-quantitative
cell analysis
BALF was centrifuged at 500 x g for 5 min at 4˚C.
The cell pellet was resuspended in 200 µl of PBS. A 50 µl aliquot
of the cell suspension was used for cytospin preparation. The
slides were air-dried and stained with Giemsa stain (cat. no.
C0131; Beyotime Biotechnology) according to the manufacturer's
instructions. For each mouse, five representative microscopic
fields (x400 magnification) were randomly selected and captured. In
each field, the total number of nucleated cells was counted, and
cells were classified into macrophages, lymphocytes and neutrophils
based on standard morphological criteria: Macrophages by their
large size, irregular nuclei and abundant cytoplasm; lymphocytes by
their small size, round nuclei and scant cytoplasm; neutrophils by
their segmented nuclei and pale pink cytoplasm. For each field, the
relative proportion of each cell type was calculated as: Proportion
of cell type=(number of that cell type in the field)/(total cells
in the same field). These proportions are expressed as decimals
(range 0-1). The data from the five fields per mouse were averaged
for subsequent statistical analysis. Total cell count per mouse was
defined as the average total nucleated cells per 400x field. All
counting was performed by three independent investigators blinded
to group allocation and the average values were used for
analysis.
Micro-CT imaging and scoring
Chest radiological assessment was performed using a
micro-CT system (Hiscan Micro CT; Suzhou Hiscan Information
Technology Co., Ltd.). Image acquisition was standardized by
positioning at three anatomical landmarks: The tracheal
bifurcation, the level of the maximum cardiothoracic ratio and 1 mm
above the diaphragmatic apex. Lung abnormalities were assessed
using a modified Müller scoring system (33). At each of the three standardized
levels, ground-glass opacities were scored on a 0-5 scale as
follows: 0, no abnormality; 1, ≤5% affected area; 2, 5-25; 3,
25-50; 4, 50-75; 5, >75%. The scores from the three levels were
averaged to obtain the mean micro-CT score (range 0-5) for each
animal. All scoring was performed by three independent
investigators blinded to group allocation, and the average score
was used for analysis.
ELISA
Serum and BALF supernatants were centrifuged at
1,500 x g for 5 min at 4˚C, and the pellets were discarded. Levels
of IL-1β, IL-6 and TNF-α were determined using ELISA kits according
to the manufacturer's instructions (cat. nos. MM-0040M1; MM-0163M1
and MM-0132M1; Jiangsu Enzyme Immunoassay Co., Ltd.). Absorbance
was measured at 450 nm using a microplate reader and cytokine
concentrations were calculated from standard curves.
Reverse transcription-quantitative PCR
(RT-qPCR)
Total RNA was extracted from lung tissues using
TRIzol® reagent (Invitrogen; Thermo Fisher Scientific,
Inc.) and reverse-transcribed into complementary DNA (cDNA) using
the HiScript II Reverse Transcriptase kit (cat. no. R323-01; Vazyme
Biotech Co., Ltd.). The thermocycling conditions for reverse
transcription were as follows: 37˚C for 30 min and 85˚C for 5 sec.
qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (cat.
no. Q711-02; Vazyme Biotech Co., Ltd.) on a QuantStudio 6 Flex
Real-Time PCR System (Thermo Fisher Scientific, Inc.). The
thermocycling conditions for qPCR were as follows: Initial
denaturation at 95˚C for 20 sec, followed by 40 cycles of 95˚C for
10 sec and 60˚C for 30 sec, with a final melt curve analysis step
to verify amplicon specificity. GAPDH was used as the internal
reference gene, and its expression was confirmed to be stable
across all experimental groups. Target gene mRNA levels were
quantified using the comparative 2-ΔΔCq method (34), with all samples normalized to the
internal control GAPDH and calibrated to the average Ct value of
the control group. Data were analyzed using QuantStudio™
Real-Time PCR Software (version 1.3; Thermo Fisher Scientific,
Inc.). The primer sequences used for amplification are provided in
Table SI.
Western blotting
Proteins were extracted from lung tissues using
lysis buffer (cat. no. KGP2100; Jiangsu KeyGen Biotech Co., Ltd.).
Protein concentration was determined using the bicinchoninic acid
(BCA) method with a BCA protein assay kit (cat. no. P0012; Beyotime
Biotechnology). Equal amounts of protein (10 µg per lane) were
separated by SDS-PAGE using 12% gels, then transferred onto PVDF
membranes. After blocking with Tris-buffered saline containing 0.1%
Tween-20 (TBST) and 5% non-fat milk for 1 h at room temperature,
the membranes were incubated with primary antibodies overnight at
4˚C. Subsequently, the membranes were incubated with HRP-conjugated
secondary antibodies for 1 h at room temperature. The dilutions of
all antibodies are provided in Table
SII. Protein signals were visualized using a Tanon 5200
chemiluminescence imaging system. The antibodies used for western
blotting are listed in Table
SII.
Statistical analysis
All data are presented as mean ± SEM. Normality of
distribution was assessed using the Shapiro-Wilk test (α=0.05) and
visual inspection of Q-Q plots. For datasets meeting normality
assumptions, comparisons between two groups were analyzed using an
unpaired two-tailed Student's t-test, and comparisons among more
than two groups were analyzed using one-way ANOVA followed by
Dunnett's post hoc test for multiple comparisons (comparing each AE
group to the BLM-only control). For data not meeting normality
assumptions, the Kruskal-Wallis test with Dunn's post hoc
correction was used. Outliers were identified using Grubbs' test
(α=0.05) and confirmed by reviewing experimental records. No
outliers were excluded from the final analysis. Post hoc power
analysis was performed to confirm that the sample sizes were
adequate to detect meaningful differences (power >0.99 for the
primary outcome measure). P<0.05 was considered to indicate a
statistically significant difference. Exact P-values are reported
for P≥0.01, while P<0.01 and P<0.001 are indicated as such.
The western blotting images were analyzed by ImageJ software
(National Institutes of Health). Statistical analyses were
performed by GraphPad Prism version 8 (Dotmatics).
Results
Weights and survival of AE-PF
mice
Mice in the BLM group exhibited a gradual decrease
in body weight compared with the control group. Following
intratracheal rechallenge with ADV, LPS or a second dose of BLM on
day 14 after the initial BLM treatment, mice in all three AE groups
showed more pronounced weight loss and lower survival rates when
compared with mice receiving a single dose of BLM (Fig. 1B and C).
On day 21, the mean body weights of the ADV group
(17.41±1.59 g; n=8), LPS group (16.52±0.61 g; n=6) and two-dose BLM
group (16.83±1.30 g; n=8) were significantly reduced compared with
that of the single-dose BLM group (20.96±2.23 g; n=12; all
P<0.01; Fig. 1B).
Following administration of the AE-inducing agents,
the survival rate of the BLM+LPS group (50.0%) was significantly
lower when compared with that of the BLM group (100%; P=0.019).
Although the survival rates of the BLM+ADV group (66.7%) and the
BLM+BLM group (66.7%) were lower when compared with that of the BLM
group, the differences did not reach statistical significance. No
significant differences in survival rates were observed among the
three AE subgroups (Fig. 1C).
Chest Micro-CT and lung histopathology
of AE-PF mouse models
Micro-CT scans were performed on day 20 in
BLM-treated mice. Compared with the BLM group, mice in all three AE
groups exhibited larger areas of ground-glass opacities in the
lungs. According to the modified Müller scoring criteria (33), the mean CT lesion scores in the
BLM+ADV (4.67±0.41), BLM+LPS (4.56±0.19) and BLM+BLM (4.20±0.30)
groups were significantly higher when compared with those in the
BLM group (2.78±0.84; all P<0.01). No significant differences
were observed among the three AE subgroups (Fig. 2A and B).
 | Figure 2The chest imaging and pathology in
AE-PF mouse models by intratracheally instillation with ADV, LPS
and a second dose of BLM. (A) Chest micro-CT scans showed the lung
injury area. (B) Modified Müller scoring system (33) CT lesion area scores (n=6). (C)
Gross lung tissue specimens showed that the severity of lung injury
was more exacerbated in three AE groups compared with BLM group.
(D) H&E and Masson's trichrome staining of lung tissues
indicated that the AE groups exhibited significantly more severe
lung injury and fibrosis compared with the BLM group. (E) Ashcroft
score of lung tissues in HE or Masson staining on day 21 (each
group, n=6). (F) Quantification of blue-stained area percentage of
lung tissues in Masson's trichrome staining (each group, n=6).
*P<0.05, **P<0.01 and
****P<0.0001. Scale bar, 100 µm. AE, acute
exacerbation; CT, computer tomography; Ctrl, control; BLM,
bleomycin; ADV, adenoviral vectors; LPS, lipopolysaccharide. |
Gross lung tissue specimens from the Ctrl group
exhibited normal morphology, soft texture and no congestion or
edema. By contrast, lungs from the BLM group showed focal injury
characterized by congestion, edema and consolidation. Importantly,
lung injury was more severe in all three AE groups, as evidenced by
larger lesional areas and, in some cases, complete lung
consolidation (Fig. 2C).
Histopathological examination of lung tissues with H&E staining
revealed alveolar septal congestion and edema, excessive
inflammatory cell infiltration and alveolar epithelial cell damage
in AE mice. In comparison, the BLM group showed less inflammatory
cell infiltration and a smaller extent of damage (Fig. 2D). The modified Ashcroft scores
(32) in the BLM+ADV (6.87±0.61),
BLM+LPS (6.27±0.28) and BLM+BLM (6.47±0.51) groups were
significantly higher when compared with those in the single-dose
BLM group (3.93±0.60; all P<0.0001). No significant differences
were observed among the three AE subgroups (Fig. 2E).
Masson's trichrome staining showed mild collagen
deposition around the trachea or blood vessels in the control group
and more extensive deposition in the single-dose BLM group.
Compared with both control and single-dose BLM groups, all three AE
groups exhibited markedly increased collagen deposition surrounding
the bronchi, within the alveolar septa and in remodeled lung
regions (Fig. 2D). Quantitative
analysis revealed that the proportion of Masson's staining area was
significantly higher in each AE group when compared with in the
single-dose BLM group (P<0.0001 for ADV and two-dose BLM groups;
P=0.017 for LPS group). Furthermore, among the AE subgroups, both
the BLM+ADV and BLM+BLM groups had significantly higher scores when
compared with the BLM+LPS group (P<0.01 and P=0.011,
respectively; Fig. 2F).
The expressions of fibrotic markers
are significantly upregulated in the lungs of AE-PF mouse
models
The present study observed a significant increase in
total collagen content, as determined by hydroxyproline assay, in
the BLM+ADV group (0.75±0.09 µg/mg), BLM+LPS group (0.59±0.07
µg/mg) and BLM+BLM group (0.77±0.06 µg/mg) compared with the
single-dose BLM group (0.29±0.04 µg/mg; P<0.001; P<0.01 and
P<0.001, respectively). The BLM+ADV and BLM+BLM groups showed
significantly higher hydroxyproline levels when compared with the
BLM+LPS group (P=0.041 and P=0.044, respectively; Fig. 3A).
 | Figure 3The pulmonary fibrosis of three AE
group mice was significantly aggravated compared with the BLM
group. (A) The hydroxyproline contents in both BLM+ADV and BLM+BLM
groups of mice were higher than in the BLM+LPS group animals (each
group, n=6). (B) Western blotting analysis revealed that the
expressions of fibrosis markers Fibronectin, MMP-7, TGF-β1,
Collagen I and α-SMA in the AE groups were significantly
upregulated compared with the BLM group. (C) Semi-quantification of
Fibronectin protein expression relative to GAPDH. (D)
Semi-quantification of MMP-7 protein expression relative to GAPDH.
(E) Semi-quantification of TGF-β1 protein expression relative to
GAPDH. (F) Semi-quantification of Collagen I protein expression
relative to GAPDH. (G) Semi-quantification of α-SMA protein
expression relative to GAPDH (each group, n=6). The relative mRNA
expressions of fibrosis markers (H) Fibronectin, (I) MMP-7, (J)
TGF-β1, (K) Collagen I and (L) α-SMA in the AE groups were
significantly increased compared with the BLM group by qPCR (each
group, n=6). *P<0.05, **P<0.01,
***P<0.001, ****P<0.0001. Scale bar, 50
µm. AE, acute exacerbation; BLM, bleomycin; ADV, adenoviral
vectors; LPS, lipopolysaccharide; α-SMA: α-smooth muscle actin. |
Similarly, western blotting revealed that the
expression levels of the profibrotic markers fibronectin, MMP7,
TGF-β1, collagen I and α-smooth muscle action (α-SMA) were
significantly upregulated in all three AE subgroups compared with
the single-dose BLM group. Collagen I expression in the lungs of
the BLM+ADV group was significantly higher when compared with that
in the BLM+LPS group (P<0.01) and the BLM+BLM group (P=0.026),
respectively. Likewise, α-SMA protein expression in lung tissue was
significantly higher in the BLM+ADV group when compared with that
in the BLM+LPS group (P<0.01). No significant differences in
other indices were observed among the three AE-PF subgroups
(Fig. 3B-G).
RT-qPCR showed that the mRNA expression levels of
fibronectin, MMP7, TGF-β1, collagen I and α-SMA were significantly
increased in the BLM group compared with the control group. All
three AE groups had significantly higher mRNA levels of these
markers when compared with the BLM group. No significant
differences in fibronectin mRNA levels were detected among the
three AE subgroups. The mRNA level of MMP7 was significantly higher
in the BLM+BLM group when compared with that in the BLM+LPS group
(P<0.01). The mRNA levels of TGF-β1 were significantly higher in
the BLM+ADV group (P<0.0001) and the BLM+BLM group (P<0.0001)
compared with the BLM+LPS group. Collagen I mRNA levels in the
BLM+ADV group were significantly higher when compared with those in
the BLM+LPS group (P<0.01) and the BLM+BLM group (P=0.019). The
mRNA level of α-SMA in the BLM+ADV group was significantly higher
when compared with that in both the BLM+BLM and BLM+LPS groups
(both P<0.0001; Fig. 3H-L).
Excessive inflammation is observed in
AE-PF mice models
Analysis of inflammatory cytokine levels in BALF and
serum by ELISA showed that IL-1β, TNF-α and IL-6 were significantly
elevated in the BLM group compared with the control group, but
remained significantly lower when compared with those in the three
AE groups. When comparing cytokine levels among the three AE
subgroups, BALF IL-1β levels were significantly higher in the
BLM+ADV and BLM+BLM groups than in the BLM+LPS group (P<0.01 and
P=0.021, respectively), whereas no significant differences were
observed in serum IL-1β levels (Fig.
4A and D). TNF-α levels in
both BALF and serum were significantly higher in the BLM+LPS and
BLM+BLM groups compared with the BLM+ADV group (all P<0.0001;
Fig. 4B and E). Furthermore, serum TNF-α levels in the
BLM+LPS group were significantly higher when compared with those in
the two-dose BLM group (P<0.001; Fig. 4E). No significant differences in
IL-6 levels were observed among the AE subgroups in either BALF or
serum (Fig. 4C and F).
 | Figure 4The levels of inflammatory cytokines
in BALF and serum were significantly elevated in AE-PF mice by
ELISA. The levels of (A) IL-1β, (B) TNF-α and (C) IL-6 in BALF were
significantly elevated in three AE-PF group mice compared with BLM
group. The findings of the BLM group were significantly increased
when compared with the Ctrl group (each group, n=6). The levels of
(D) IL-1β, (E) TNF-α and (F) IL-6 in serum were significantly
elevated in three AE-PF groups of mice compared with BLM group. The
findings of the BLM group were significantly increased when
compared with the Ctrl group (each group, n=6).
*P<0.05, **P<0.01,
***P<0.001, ****P<0.0001. AE, acute
exacerbation; BALF, bronchoalveolar lavage fluid; Ctrl, control;
BLM, bleomycin; ADV, adenoviral vectors; LPS,
lipopolysaccharide. |
Giemsa staining of BALF cytospin preparations
revealed distinct cellular compositions across the groups (Fig. S1). Compared with the control
group, the total cell count was significantly increased in the BLM
group, and further elevated in all three AE groups. The proportion
of lymphocytes was significantly higher in the BLM+ADV group when
compared with that in the BLM+LPS and BLM+BLM groups. The
percentages of macrophages in the BLM+LPS and BLM+BLM groups were
significantly higher when compared with those in the BLM+ADV group.
The proportion of neutrophils in the BLM+LPS group was
significantly greater when compared with that in the BLM+ADV and
BLM+BLM groups.
Apoptosis is increased in lung tissues
of AE-PF mice
The enhanced apoptosis of alveolar epithelial cells
plays a key role in the pathogenesis of AE-IPF (35). The present study therefore
investigated apoptosis in lung tissues of the mouse models. TUNEL
assays revealed no clear positive signals in the control group. The
percentage of TUNEL-positive cells in the BLM group was
significantly higher when compared with that in the control group
(P=0.049). Conspicuous and aggregated apoptotic signals were
observed in lung tissues of all three AE groups. Notably, mice in
the BLM+ADV, BLM+LPS and BLM+BLM groups exhibited significantly
increased apoptosis compared with the BLM group (P<0.01; P=0.035
and P=0.032, respectively) (Fig.
5A and B). No statistically
significant differences were observed among the three AE subgroups,
although the BLM+ADV subgroup showed a trend toward higher
values.
 | Figure 5Apoptosis was significantly enhanced
in AE-PF mice, particularly in the BLM+ADV group. (A)
Representative TUNEL staining images of lung tissues from each
group. (B) Quantification of TUNEL-positive cells showed that the
percentages in the AE groups were significantly higher than in the
BLM group, particularly in the BLM+ADV group (each group, n=6). (C)
Quantification of the Bax/Bcl2 ratio revealed a significant
increase in the BLM+ADV group compared with both the BLM+LPS and
BLM+BLM groups (each group, n=6). (D) Representative western
blotting bands showing the protein expression of Bcl2 and Bax in
each group. (E) The findings of quantitative-PCR analysis showed
the Bax/Bcl2 ratio was significantly enhanced in three AE-PF group
mice when compared with BLM group (each group, n=6).
*P<0.05, **P<0.01,
***P<0.001, ****P<0.0001. Scale bar: 50
µm. AE, acute exacerbation; BLM, bleomycin; ADV, adenoviral
vectors; LPS, lipopolysaccharide. |
The present study further analyzed the protein and
mRNA expression of apoptosis-related molecules in the AE-PF mouse
models. In the BLM group, the pro-apoptotic molecule Bax was
significantly increased compared with the control group, but its
level was significantly lower when compared with that in the three
AE groups (Fig. 5D). By contrast,
the expression level of the anti-apoptotic factor Bcl2 was
significantly reduced in the BLM group compared with the control
group, and the AE groups showed a further decrease relative to the
BLM group (Fig. 5D). The Bax/Bcl2
ratio, reflecting apoptotic propensity, was highest in the BLM+ADV
group among the three AE subgroups. This group exhibited
significantly higher Bax/Bcl2 ratios when compared with the BLM+LPS
group (P<0.01) and the BLM+BLM group (P=0.022; Fig. 5C). qPCR analysis also demonstrated
that the Bax/Bcl2 ratio was significantly higher in the BLM+ADV
group compared with that in the BLM+LPS group (P=0.020; Fig. 5E).
The expression of AEC2 markers is
significantly downregulated, suggesting that the damage to AEC2 is
aggravated in the lung of AE-PF mice
Western blotting analysis demonstrated a significant
decrease in E-cadherin expression in all three AE groups compared
with the single-dose BLM group (Fig.
6A and B). The present study
also analyzed the expression of the AEC2 marker SPC in lung
tissues. SPC expression was significantly reduced in the BLM group
and further decreased in mice from the AE groups, indicating that
AEC2s were substantially damaged following the induction of acute
exacerbation (Fig. 6C and D).
 | Figure 6Epithelial cell injury was
significantly enhanced in the AE-PF mice. (A) Western blotting
analysis of E-cadherin protein expression. (B) Quantification of
E-cadherin expression relative to GAPDH (each group, n=6). (C)
Representative immunofluorescence images of SPC staining. (D)
Quantification of SPC fluorescence intensity (each group, n=6).
*P<0.05, **P<0.01,
***P<0.001. Scale bar, 50 µm. AE, acute exacerbation;
BLM, bleomycin; ADV, adenoviral vectors; LPS, lipopolysaccharide;
SPC, pro-surfactant protein C. |
Discussion
The pathogenesis of AE-IPF remains unclear, and it
is uncertain whether acute exacerbations represent an inherent
acceleration of the underlying fibrotic disease or a reaction to
external, often unidentified triggers that lead to acute
respiratory distress syndrome (ARDS) (36). Existing studies have identified
viral, bacterial and toxic injuries as contributors to AE-IPF
(10,17,37,38).
The underlying mechanisms may include increased apoptosis of AEC2s,
excessive lung inflammation and extracellular matrix deposition
(17). Pathologically, AE-IPF
often presents as new diffuse alveolar damage, occasionally
accompanied by hyaline membrane formation, superimposed on a
background of usual interstitial pneumonia (2). Multiple animal models of AE-PF
induced by viruses, bacteria, drugs or surgery have been reported
(19,23,26,28,29).
However, each model possesses inherent limitations, including
suboptimal timing for AE induction, imperfect experimental methods,
prolonged experimental design cycles or an oversimplified
assumption that AE is solely caused by external insults, thereby
overlooking potential contributions from the disease process itself
(17). Consequently, there is an
urgent need to systematically compare and analyze these different
animal models to identify a more robust and clinically relevant
platform, thereby laying the groundwork for subsequent preclinical
research.
The conventional animal model for studying pulmonary
fibrosis is established by intratracheal instillation of a single
dose of BLM, which has proven instrumental in elucidating the
mechanisms underlying alveolar epithelial cell injury and
subsequent fibrosis progression (17,39).
Therefore, the present study developed AE-PF mouse models based on
BLM-induced pulmonary fibrosis using three distinct triggers: ADV,
LPS and a second dose of BLM. Analysis demonstrated that all three
triggers effectively induced clinical, imaging and
histopathological exacerbations in the lungs, accompanied by
molecular marker changes in inflammation, apoptosis and fibrosis
that recapitulate features observed in AE-IPF. However, the degrees
of apoptosis, pulmonary fibrosis and inflammatory response in lung
tissue differed among the three AE-PF models. Compared with the
BLM+LPS group, BLM+ADV mice exhibited more pronounced apoptosis and
lung fibrosis but lower TNF-α levels in both BALF and serum.
Compared with mice receiving two doses of BLM, those treated with
ADV in combination with BLM showed significantly higher levels of
apoptosis, collagen I and α-SMA mRNA, while TNF-α levels in both
BALF and serum were significantly lower. Compared with the BLM+LPS
group, the two-dose BLM group exhibited more severe lung fibrosis,
with similar levels of lung apoptosis and BALF TNF-α. The causes
and mechanisms underlying the differences among these three AE-PF
models warrant further investigation.
Adenovirus is highly contagious and often leads to
outbreaks of respiratory infection in closed or crowded
environments (40). Weng et
al (37) analyzed serum
samples from 41 patients with AE-IPF and 108 patients with stable
IPF, reporting that 7.3% of AE-IPF patients tested positive for
adenovirus-specific IgM antibodies, compared with only 0.9% of
stable patients with IPF (37).
Latent adenovirus can persist in lymphoid or other tissues for
extended periods, with the possibility of reactivation in severely
immunosuppressed patients (41).
In light of this, the present study used replication-deficient ADV,
which retains immunogenicity but lacks replicative capacity, to
induce AE-PF in BLM-treated mice. To the best of our knowledge,
this approach has not been reported previously.
The mechanisms by which ADV infection exacerbates
pulmonary fibrosis may involve two interrelated pathways (17,42).
First, ADV itself may directly damage the lung parenchyma,
potentially triggering aberrant wound healing responses that lead
to AE-PF. Second, ADV infection activates the immune system,
recruiting macrophages and Th2 cells to the site of injury. These
cells release large amounts of pro-inflammatory and pro-fibrotic
factors, including TGF-β, TNF-α, IL-1 and IL-6, through signaling
pathways such as the TNF-α/NF-κB pathway, the IL-6/STAT pathway and
the TGF-β/SMAD pathway, ultimately contributing to the pathogenesis
of AE-PF.
LPS is commonly used in various preclinical models
to induce conditions such as ARDS or peritoneal inflammation
(43-46).
Given the pathological features of ARDS observed in lung tissue
from patients with AE-IPF, Kimura et al (28) established a mouse model of AE-PF by
administering BLM followed by LPS. However, they collected
specimens on day 1 after LPS treatment (day 8 after BLM treatment),
a stage when lung inflammation is most pronounced but fibrosis
remains relatively mild (28). Guo
et al (47) induced an
AE-PF model in BLM-treated mice using LPS on day 14 and assessed
the effects on day 15, reporting no significant progression of
pulmonary fibrosis (47). By
contrast, in the present AE-PF model, LPS was administered on day
14 after BLM treatment, during the stage of established lung
fibrosis, and lung samples were collected on day 21. Administering
triggering agents and collecting samples at appropriate time points
in BLM-treated mice will help improve researchers' understanding of
the pathophysiological processes and explore the pathogenesis of
AE-IPF.
A systematic review and meta-analysis revealed that
patients with AE-IPF with elevated neutrophil counts in BALF have a
high risk of mortality (48). A
previous study showed that germ-free BLM-treated mice exhibit a
similar degree of lung injury compared with wild-type BLM-treated
mice; however, germ-free mice are protected from mortality
(38). Another single-center
retrospective study of patients with IPF reported that among seven
patients with bacterial infections, one developed AE-IPF and two
died during a 60-month follow-up period. Among 12 patients with
viral infections, one developed AE-IPF, but no mortalities occurred
(49). These findings may explain
why, among the three AE-PF models, the mortality rate was highest
in the BLM+LPS group. This is likely attributable to the
inflammatory response associated with bacterial infection and LPS,
which can lead to a high mortality rate.
To elucidate the molecular mechanisms underlying
acute exacerbations induced by ‘idiopathic’ AE or non-infectious
factors, previous studies have demonstrated that administering BLM
two or more times can successfully establish mouse models of AE-PF
(29,50,51).
The two-dose BLM model has been established by administration on
days 0 and 21, with outcome assessment on day 35. The multiple-dose
model has been administered once every 2 weeks for a total of six
doses. However, the duration of these studies is excessively long,
ranging from 5 to 12 weeks. The present findings demonstrated that
administering the same dose of BLM on day 14 after the initial dose
yielded model outcomes assessed on day 21 that closely resembled
those of the long-cycle mouse model. These manifestations included
significantly increased lung fibrosis and apoptosis, along with
elevated levels of inflammatory cytokines such as TNF-α, IL-6 and
IL-1β. Accordingly, the present study successfully induced AE-PF in
mice using this two-dose BLM administration regimen. Thus, this
approach offers a stable, reliable, short-duration and reproducible
method for establishing an animal model of non-infectious AE-PF,
thereby providing a valuable tool for future research.
Among the three AE models evaluated in the present
study, the ADV-triggered BLM model most comprehensively
recapitulated the key pathological features of human AE-IPF. First,
the BLM+ADV group exhibited the most severe fibrotic phenotype, as
reflected by the highest levels of collagen I, α-SMA and TGF-β, as
well as the highest fibrosis scores on histological assessment.
Although hydroxyproline content was slightly lower than that in the
BLM+BLM group, this discrepancy may reflect differences in collagen
turnover or maturation, as hydroxyproline measures total collagen
content, whereas collagen I and α-SMA reflect active fibrogenesis.
Collectively, these findings indicate a robust and active fibrotic
response in the BLM+ADV model. Second, this group also exhibited
the most pronounced alveolar epithelial apoptosis, which is
recognized as a key driver of AE-IPF pathogenesis. Alveolar
epithelial injury is considered to trigger aberrant wound healing
and fibroblast activation, contributing to the rapid decline in
lung function. Third, the ADV group displayed a moderate yet
significant increase in cytokines (Table SIII). Previous studies have
reported elevated levels of multiple cytokines in patients with
AE-IPF, including IL-1β, TNF-α and IL-6 (52-55),
findings that are well aligned with the pathological features of
human AE-IPF. Human AE-IPF is characterized by a complex interplay
of fibrosis, epithelial injury and inflammation. Collectively,
these findings suggest that the ADV-triggered BLM model provides a
translational platform that captures the core pathological features
of AE-IPF, including robust fibrogenesis (elevated collagen I,
α-SMA, TGF-β and fibrosis scores), prominent epithelial apoptosis
and a moderate inflammatory response. This model may therefore be
particularly suitable for studying the mechanisms underlying acute
exacerbations and for evaluating targeted therapeutic
interventions.
The present study has some limitations. The
mortality rates observed in the AE models (33.3-50%) are consistent
with the high mortality reported in patients with AE-IPF (56-100%)
(5), supporting the translational
relevance of these models. While this high mortality is clinically
reflective, it should be considered as a practical consideration
for future preclinical studies. Although the bleomycin-induced
fibrosis model recapitulates key histopathological features of
human IPF, it does not fully replicate the chronic, progressive
nature of the disease. Moreover, species differences in immune
response and lung anatomy, as well as the short observation period
(21 days), may limit the translation of the AE phenotype to the
long-term human disease course. In clinical practice, new
ground-glass opacities and/or consolidations superimposed on
pre-existing pulmonary fibrosis are typically observed on chest
imaging in patients with AE-IPF. However, the present study only
compared chest CT changes between AE-PF mice and BLM-treated
animals at a single time point, without longitudinal tracking of
the same animals before and after the second challenge. The present
study acknowledges that incorporating serial chest CT imaging and
longitudinal BALF cytological classification in the same mice would
better facilitate understanding of the progression and evolution of
pulmonary fibrosis. Although the sample sizes (n=6-8 per group)
were sufficient to detect statistically significant differences, as
confirmed by post hoc power analysis, they remain relatively
modest. Future studies with larger cohorts may help further
validate these findings and allow for more detailed subgroup
analyses. The mechanisms underlying acute exacerbation in mouse
models of pulmonary fibrosis require further investigation.
The present study demonstrated that three triggers,
ADV, LPS and a second dose of BLM, effectively induce AE-PF in
BLM-treated mice by promoting enhanced apoptosis, excessive
inflammation and fibrosis progression. Although the severity of
apoptosis, inflammation and lung fibrosis progression differed
among the three AE-PF models, the protocol of rechallenging mice on
day 14 after conventional BLM treatment may serve as a standardized
AE-PF model. In comparison, the ADV-induced BLM mouse model may
represent a superior platform for studying AE-PF.
Supplementary Material
Cellular composition of BALF across
groups. (A) Representative images of Giemsa-stained BALF cells from
each group (x400 magnification). Red arrows indicate lymphocytes,
black arrows indicate macrophages and blue arrows indicate
neutrophils. Scale bar, 50 μm. (B) Total cell count per
mouse in BALF under x400 magnification (each group, n=6). (C-E)
Percentages of (C) lymphocytes, (D) macrophages and (E) neutrophils
in total BALF cells (each group, n=6). **P<0.01,
***P<0.001, ****P<0.0001. BALF,
bronchoalveolar lavage fluid.
Primer sequences for reverse
transcription-quantitative PCR for mice.
Antibodies used in the present
study
Comparison of fibrosis, inflammation
and apoptosis across models.
Acknowledgements
Not applicable.
Funding
Funding: This work was supported by National Natural Science
Foundation of China (grant nos. 82570105, 82070064, 81670059 and
81200049). and Fundings for Clinical Trials from the Affiliated
Drum Tower Hospital, Medical School of Nanjing University (grant
no. 2022-LCYJ-MS-11).
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
XuY and MZ were responsible for the experiments and
drafting the manuscript. ML, HZ and QL analyzed the images of
histopathology and supplemented experiments. XiY contributed to the
experimental design, performed the experiments, acquired and
analyzed the primary data, and prepared the figures. CJ
participated in data analysis and interpretation, and prepared the
figures. XH contributed to the study conception, supervised the
experiments and critically reviewed and revised the draft. MC
designed the study and reviewed and approved the final manuscript.
XuY and MC confirm the authenticity of all the raw data. All
authors have read and approved the final manuscript.
Ethics approval and consent to
participate
The present study was approved by the Ethics
Committee of Nanjing Drum Tower Hospital, Nanjing University
Medical School (Nanjing, China; approval no. 2016-160-01).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
References
|
1
|
Raghu G, Remy-Jardin M, Richeldi L,
Thomson CC, Inoue Y, Johkoh T, Kreuter M, Lynch DA, Maher TM,
Martinez FJ, et al: Idiopathic pulmonary fibrosis (an update) and
progressive pulmonary fibrosis in adults: An official
ATS/ERS/JRS/ALAT clinical practice guideline. Am J Respir Crit Care
Med. 205:e18–e47. 2022.PubMed/NCBI View Article : Google Scholar
|
|
2
|
Raghu G, Remy-Jardin M, Myers JL, Richeldi
L, Ryerson CJ, Lederer DJ, Behr J, Cottin V, Danoff SK, Morell F,
et al: Diagnosis of idiopathic pulmonary fibrosis. An official
ATS/ERS/JRS/ALAT clinical practice guideline. Am J Respir Crit Care
Med. 198:e44–e68. 2018.PubMed/NCBI View Article : Google Scholar
|
|
3
|
Naccache JM, Jouneau S, Didier M, Borie R,
Cachanado M, Bourdin A, Reynaud-Gaubert M, Bonniaud P, Israël-Biet
D, Prévot G, et al: Cyclophosphamide added to glucocorticoids in
acute exacerbation of idiopathic pulmonary fibrosis (EXAFIP): A
randomised, double-blind, placebo-controlled, phase 3 trial. Lancet
Respir Med. 10:26–34. 2022.PubMed/NCBI View Article : Google Scholar
|
|
4
|
Fabbrizzi A, Nannini G, Lavorini F,
Tomassetti S and Amedei A: Microbiota and IPF: Hidden and detected
relationships. Sarcoidosis Vasc Diffuse Lung Dis.
38(e2021028)2021.PubMed/NCBI View Article : Google Scholar
|
|
5
|
Marchioni A, Tonelli R, Ball L, Fantini R,
Castaniere I, Cerri S, Luppi F, Malerba M, Pelosi P and Clini E:
Acute exacerbation of idiopathic pulmonary fibrosis: Lessons
learned from acute respiratory distress syndrome? Crit Care.
22(80)2018.PubMed/NCBI View Article : Google Scholar
|
|
6
|
Juarez MM, Chan AL, Norris AG, Morrissey
BM and Albertson TE: Acute exacerbation of idiopathic pulmonary
fibrosis-a review of current and novel pharmacotherapies. J Thorac
Dis. 7:499–519. 2015.PubMed/NCBI View Article : Google Scholar
|
|
7
|
Jenkins RG, Moore BB, Chambers RC,
Eickelberg O, Königshoff M, Kolb M, Laurent GJ, Nanthakumar CB,
Olman MA, Pardo A, et al: An official american thoracic society
workshop report: Use of animal models for the preclinical
assessment of potential therapies for pulmonary fibrosis. Am J
Respir Cell Mol Biol. 56:667–679. 2017.PubMed/NCBI View Article : Google Scholar
|
|
8
|
Khor YH: Antifibrotic therapy for
idiopathic pulmonary fibrosis: Combining real world and clinical
trials for totality of evidence. Chest. 160:1589–1591.
2021.PubMed/NCBI View Article : Google Scholar
|
|
9
|
Kreuter M and Maher TM: Treatment of acute
exacerbation of idiopathic pulmonary fibrosis. A call to arms. Am J
Respir Crit Care Med. 201:1030–1032. 2020.PubMed/NCBI View Article : Google Scholar
|
|
10
|
Collard HR, Ryerson CJ, Corte TJ, Jenkins
G, Kondoh Y, Lederer DJ, Lee JS, Maher TM, Wells AU, Antoniou KM,
et al: Acute exacerbation of idiopathic pulmonary fibrosis. An
international working group report. Am J Respir Crit Care Med.
194:265–275. 2016.PubMed/NCBI View Article : Google Scholar
|
|
11
|
Kreuter M, Polke M, Walsh SLF, Krisam J,
Collard HR, Chaudhuri N, Avdeev S, Behr J, Calligaro G, Corte T, et
al: Acute exacerbation of idiopathic pulmonary fibrosis:
International survey and call for harmonisation. Eur Respir J.
55(1901760)2020.PubMed/NCBI View Article : Google Scholar
|
|
12
|
McElroy AN, Invernizzi R, Laskowska JW,
O'Neill A, Doroudian M, Moghoofei M, Mostafaei S, Li F, Przybylski
AA, O'Dwyer DN, et al: Candidate role for Toll-like receptor 3
L412F polymorphism and infection in acute exacerbation of
idiopathic pulmonary fibrosis. Am J Respir Crit Care Med.
205:550–562. 2022.PubMed/NCBI View Article : Google Scholar
|
|
13
|
Molyneaux PL, Cox MJ, Wells AU, Kim HC, Ji
W, Cookson WO, Moffatt MF, Kim DS and Maher TM: Changes in the
respiratory microbiome during acute exacerbations of idiopathic
pulmonary fibrosis. Respir Res. 18(29)2017.PubMed/NCBI View Article : Google Scholar
|
|
14
|
Stoolman JS, Vannella KM, Coomes SM, Wilke
CA, Sisson TH, Toews GB and Moore BB: Latent infection by
γherpesvirus stimulates profibrotic mediator release from multiple
cell types. Am J Physiol Lung Cell Mol Physiol. 300:L274–L285.
2011.PubMed/NCBI View Article : Google Scholar
|
|
15
|
Ghincea A, Ryu C and Herzog EL: An acute
exacerbation of idiopathic pulmonary fibrosis after BNT162b2 mRNA
COVID-19 vaccination: A case report. Chest. 161:e71–e73.
2022.PubMed/NCBI View Article : Google Scholar
|
|
16
|
Bando T, Takei R, Mutoh Y, Sasano H,
Yamano Y, Yokoyama T, Matsuda T, Kataoka K, Kimura T and Kondoh Y:
Acute exacerbation of idiopathic pulmonary fibrosis after
SARS-CoV-2 vaccination. Eur Respir J. 59(2102806)2022.PubMed/NCBI View Article : Google Scholar
|
|
17
|
Ye X, Zhang M, Gu H, Liu M, Zhao Y, Shi Y,
Wu S, Jiang C, Ye X, Zhu H, et al: Animal models of acute
exacerbation of pulmonary fibrosis. Respir Res.
24(296)2023.PubMed/NCBI View Article : Google Scholar
|
|
18
|
D'Alessandro-Gabazza CN, Yasuma T,
Kobayashi T, Toda M, Abdel-Hamid AM, Fujimoto H, Hataji O, Nakahara
H, Takeshita A, Nishihama K, et al: Inhibition of lung
microbiota-derived proapoptotic peptides ameliorates acute
exacerbation of pulmonary fibrosis. Nat Commun.
13(1558)2022.PubMed/NCBI View Article : Google Scholar
|
|
19
|
Cho SJ, Moon JS, Nikahira K, Yun HS,
Harris R, Hong KS, Huang H, Choi AMK and Stout-Delgado H:
GLUT1-dependent glycolysis regulates exacerbation of fibrosis via
AIM2 inflammasome activation. Thorax. 75:227–236. 2020.PubMed/NCBI View Article : Google Scholar
|
|
20
|
Liang J, Zhang Y, Xie T, Liu N, Chen H,
Geng Y, Kurkciyan A, Mena JM, Stripp BR, Jiang D and Noble PW:
Hyaluronan and TLR4 promote surfactant-protein-C-positive alveolar
progenitor cell renewal and prevent severe pulmonary fibrosis in
mice. Nat Med. 22:1285–1293. 2016.PubMed/NCBI View Article : Google Scholar
|
|
21
|
Xie T, Liang J, Stripp B and Noble PW:
Cell-cell interactions and communication dynamics in lung fibrosis.
Chin Med J Pulm Crit Care Med. 2:63–71. 2024.PubMed/NCBI View Article : Google Scholar
|
|
22
|
Huang G, Geng Y, Kulur V, Liu N, Liu X,
Taghavifar F, Liang J, Noble PW and Jiang D: Arrestin beta 1
regulates alveolar progenitor renewal and lung fibrosis. J Respir
Biol Transl Med. 1(10006)2024.PubMed/NCBI View Article : Google Scholar
|
|
23
|
Ashley SL, Jegal Y, Moore TA, van Dyk LF,
Laouar Y and Moore BB: γ-Herpes virus-68, but not Pseudomonas
aeruginosa or influenza A (H1N1), exacerbates established murine
lung fibrosis. Am J Physiol Lung Cell Mol Physiol. 307:L219–L230.
2014.PubMed/NCBI View Article : Google Scholar
|
|
24
|
Kim MS, Kim SH, Jeon D, Kim HY, Han JY,
Kim B and Lee K: Low-dose cadmium exposure exacerbates
polyhexamethylene guanidine-induced lung fibrosis in mice. J
Toxicol Environ Health A. 81:384–396. 2018.PubMed/NCBI View Article : Google Scholar
|
|
25
|
D'Alessandro-Gabazza CN, Kobayashi T,
Yasuma T, Toda M, Kim H, Fujimoto H, Hataji O, Takeshita A,
Nishihama K, Okano T, et al: A Staphylococcus pro-apoptotic peptide
induces acute exacerbation of pulmonary fibrosis. Nat Commun.
11(1539)2020.PubMed/NCBI View Article : Google Scholar
|
|
26
|
Yang L, Lin Z, Wang Y, Li C, Xu W, Li Q,
Yao W, Song Z and Liu G: Nickle(II) ions exacerbate
bleomycin-induced pulmonary inflammation and fibrosis by activating
the ROS/Akt signaling pathway. Environ Sci Pollut Res Int.
25:4406–4418. 2018.PubMed/NCBI View Article : Google Scholar
|
|
27
|
Chen S, Zhang X, Yang C, Wang S and Shen
H: Essential role of IL-17 in acute exacerbation of pulmonary
fibrosis induced by non-typeable Haemophilus influenzae.
Theranostics. 12:5125–5137. 2022.PubMed/NCBI View Article : Google Scholar
|
|
28
|
Kimura T, Nojiri T, Hosoda H, Shintani Y,
Inoue M, Miyazato M, Okumura M and Kangawa K: Exacerbation of
bleomycin-induced injury by lipopolysaccharide in mice:
Establishment of a mouse model for acute exacerbation of
interstitial lung diseases. Eur J Cardiothorac Surg. 48:e85–e91.
2015.PubMed/NCBI View Article : Google Scholar
|
|
29
|
Wei YR, Qiu H, Wu Q, Du YK, Yin ZF, Chen
SS, Jin YP, Zhao MM, Wang C, Weng D and Li HP: Establishment of the
mouse model of acute exacerbation of idiopathic pulmonary fibrosis.
Exp Lung Res. 42:75–86. 2016.PubMed/NCBI View Article : Google Scholar
|
|
30
|
Jia K, Wu J, Li Y, Liu J, Liu R, Cai Y,
Zhang Y and Li X: A novel pulmonary fibrosis murine model with
immune-related liver injury. Animal Model Exp Med. 6:274–282.
2023.PubMed/NCBI View Article : Google Scholar
|
|
31
|
McMillan TR, Moore BB, Weinberg JB,
Vannella KM, Fields WB, Christensen PJ, van Dyk LF and Toews GB:
Exacerbation of established pulmonary fibrosis in a murine model by
gammaherpesvirus. Am J Respir Crit Care Med. 177:771–780.
2008.PubMed/NCBI View Article : Google Scholar
|
|
32
|
Hübner RH, Gitter W, El Mokhtari NE,
Mathiak M, Both M, Bolte H, Freitag-Wolf S and Bewig B:
Standardized quantification of pulmonary fibrosis in histological
samples. Biotechniques. 44:507–511, 514-517. 2008.PubMed/NCBI View Article : Google Scholar
|
|
33
|
Müller NL, Mawson JB, Mathieson JR, Abboud
R, Ostrow DN and Champion P: Sarcoidosis: Correlation of extent of
disease at CT with clinical, functional, and radiographic findings.
Radiology. 171:613–618. 1989.PubMed/NCBI View Article : Google Scholar
|
|
34
|
Livak KJ and Schmittgen TD: Analysis of
relative gene expression data using real-time quantitative PCR and
the 2(-Delta Delta C(T)) method. Methods. 25:402–408.
2001.PubMed/NCBI View Article : Google Scholar
|
|
35
|
Konishi K, Gibson KF, Lindell KO, Richards
TJ, Zhang Y, Dhir R, Bisceglia M, Gilbert S, Yousem SA, Song JW, et
al: Gene expression profiles of acute exacerbations of idiopathic
pulmonary fibrosis. Am J Respir Crit Care Med. 180:167–175.
2009.PubMed/NCBI View Article : Google Scholar
|
|
36
|
Biondini D, Balestro E, Sverzellati N,
Cocconcelli E, Bernardinello N, Ryerson CJ and Spagnolo P: Acute
exacerbations of idiopathic pulmonary fibrosis (AE-IPF): An
overview of current and future therapeutic strategies. Expert Rev
Respir Med. 14:405–414. 2020.PubMed/NCBI View Article : Google Scholar
|
|
37
|
Weng D, Chen XQ, Qiu H, Zhang Y, Li QH,
Zhao MM, Wu Q, Chen T, Hu Y, Wang LS, et al: The role of infection
in acute exacerbation of idiopathic pulmonary fibrosis. Mediators
Inflamm. 2019(5160694)2019.PubMed/NCBI View Article : Google Scholar
|
|
38
|
O'Dwyer DN, Ashley SL, Gurczynski SJ, Xia
M, Wilke C, Falkowski NR, Norman KC, Arnold KB, Huffnagle GB,
Salisbury ML, et al: Lung microbiota contribute to pulmonary
inflammation and disease progression in pulmonary fibrosis. Am J
Respir Crit Care Med. 199:1127–1138. 2019.PubMed/NCBI View Article : Google Scholar
|
|
39
|
Redente EF, Black BP, Backos DS, Bahadur
AN, Humphries SM, Lynch DA, Tuder RM, Zemans RL and Riches DWH:
Persistent, progressive pulmonary fibrosis and epithelial
remodeling in mice. Am J Respir Cell Mol Biol. 64:669–676.
2021.PubMed/NCBI View Article : Google Scholar
|
|
40
|
Zhao S, Wu X, Tan Z, Li L, Ou J, Lin Y,
Song H, Feng L, Seto D, Wu J, et al: Generation of human embryonic
stem cell-derived lung organoids for modeling infection and
replication differences between human adenovirus types 3 and 55 and
evaluating potential antiviral drugs. J Virol.
97(e0020923)2023.PubMed/NCBI View Article : Google Scholar
|
|
41
|
Lynch JP and Kajon AE: Adenovirus:
Epidemiology, global spread of novel types, and approach to
treatment. Semin Respir Crit Care Med. 42:800–821. 2021.PubMed/NCBI View Article : Google Scholar
|
|
42
|
Huang WJ and Tang XX: Virus infection
induced pulmonary fibrosis. J Transl Med. 19(496)2021.PubMed/NCBI View Article : Google Scholar
|
|
43
|
Zhou Y, Jin T, Gao M, Luo Z, Mutahir S,
Shi C, Xie T, Lin L, Xu J, Liao Y, et al: Aqueous extract of
Platycodon grandiflorus attenuates lipopolysaccharide-induced
apoptosis and inflammatory cell infiltration in mouse lungs by
inhibiting PI3K/Akt signaling. Chin Med. 18(36)2023.PubMed/NCBI View Article : Google Scholar
|
|
44
|
Li J, Lu K, Sun F, Tan S, Zhang X, Sheng
W, Hao W, Liu M, Lv W and Han W: Panaxydol attenuates ferroptosis
against LPS-induced acute lung injury in mice by Keap1-Nrf2/HO-1
pathway. J Transl Med. 19(96)2021.PubMed/NCBI View Article : Google Scholar
|
|
45
|
D'Alessio FR: Mouse models of acute lung
injury and ARDS. In: Alper S, Janssen W (eds), Lung Innate Immunity
and Inflammation. Methods in Molecular Biology, vol 1809. Humana
Press, New York, NY, pp341-350, 2018.
|
|
46
|
Seemann S, Zohles F and Lupp A:
Comprehensive comparison of three different animal models for
systemic inflammation. J Biomed Sci. 24(60)2017.PubMed/NCBI View Article : Google Scholar
|
|
47
|
Guo B, Liu W, Ji X, Xi B, Meng X, Xie W,
Sun Y, Zhang M, Liu P, Zhang W, et al: CSF3 aggravates acute
exacerbation of pulmonary fibrosis by disrupting alveolar
epithelial barrier integrity. Int Immunopharmacol.
135(112322)2024.PubMed/NCBI View Article : Google Scholar
|
|
48
|
Pitre T, Lupas D, Ebeido I, Colak A, Modi
M, Kachkovski GV, Montesi SB, Khor YH, Kawano-Dourado L, Jenkins G,
et al: Prognostic factors associated with mortality in acute
exacerbations of idiopathic pulmonary fibrosis: A systematic review
and meta-analysis. Respir Med. 222(107515)2024.PubMed/NCBI View Article : Google Scholar
|
|
49
|
Moghoofei M, Mostafaei S, Kondori N,
Armstrong ME and Babaei F: Bacterial and viral coinfection in
idiopathic pulmonary fibrosis patients: The prevalence and possible
role in disease progression. BMC Pulm Med. 22(60)2022.PubMed/NCBI View Article : Google Scholar
|
|
50
|
Yegen CH, Haine L, Da Costa Ferreira K,
Marchant D, Bernaudin JF, Planès C, Voituron N and Boncoeur E: A
new model of acute exacerbation of experimental pulmonary fibrosis
in mice. Cells. 11(3379)2022.PubMed/NCBI View Article : Google Scholar
|
|
51
|
Fikry H, Saleh LA and Gawad SA:
Therapeutic effect of adipose-derived mesenchymal stem cells
(AD-MSCs) compared to pirfenidone on corticosteroid resistance in a
mouse model of acute exacerbation of idiopathic pulmonary fibrosis.
Histol Histopathol. 37:1065–1083. 2022.PubMed/NCBI View Article : Google Scholar
|
|
52
|
Emura I and Usuda H: Acute exacerbation of
IPF has systemic consequences with multiple organ injury, with
SRA+ and TNF-α+ cells in the systemic
circulation playing central roles in multiple organ injury. BMC
Pulm Med. 16(138)2016.PubMed/NCBI View Article : Google Scholar
|
|
53
|
Papiris SA, Tomos IP, Karakatsani A,
Spathis A, Korbila I, Analitis A, Kolilekas L, Kagouridis K,
Loukides S, Karakitsos P and Manali ED: High levels of IL-6 and
IL-8 characterize early-on idiopathic pulmonary fibrosis acute
exacerbations. Cytokine. 102:168–172. 2018.PubMed/NCBI View Article : Google Scholar
|
|
54
|
Li X, Zhou Y, Zou R, Chen H, Liu X, Qiu X,
Xiao Y, Cai H and Dai J: Associations of serological biomarkers of
sICAM-1, IL-1β, MIF, and su-PAR with 3-month mortality in acute
exacerbation of idiopathic pulmonary fibrosis. Mediators Inflamm.
2020(4534272)2020.PubMed/NCBI View Article : Google Scholar
|
|
55
|
Tomos I, Dimakopoulou K, Manali ED,
Papiris SA and Karakatsani A: Long-term personal air pollution
exposure and risk for acute exacerbation of idiopathic pulmonary
fibrosis. Environ Health. 20(99)2021.PubMed/NCBI View Article : Google Scholar
|
