Abstract
Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease with a
very poor prognosis as it has a 2.5 to 5 years mean survival after
proper diagnosis. Even nintedanib and pirfenidone cannot halt the
progression, though they slow the progression of IPF. Hence, there is a
need to understand the novel pathophysiology. Phospholipase A2 (PLA2)
could be the ideal candidate to study in IPF, as they have a role in
both inflammation and fibrosis. In the present study, we have shown the
expression profile of various secretory Phospholipase A2 (PLA2)
isoforms by analyzing publicly available transcriptome data of single
cells from the lungs of healthy individuals and IPF patients. Among 11
members of sPLA2, PLA2G2A is found to be increased in the fibroblasts
and mesothelial cells while PLA2G5 is found to be increased in the
fibroblasts of IPF patients. We identified a subset of fibroblasts
expressing high PLA2G2A with moderate expression of PLA2G5 and which
are specific to IPF only; we named it as PLA2G2A+ IPF fibroblast.
Pathway analysis revealed that these PLA2G2A+ IPF fibroblast have
upregulation of both inflammatory and fibrosis-related pathways like
the TGF-β signaling pathway, IL-17 signaling, the arachidonic acid
metabolism pathway and ECM-receptor interaction. In addition to this,
we found elevated levels of sPLA2-IIA in plasma samples of IPF patients
in our cohort. PLA2G3, PLA2G10 and PLA2G12B are found in to be
increased in certain epithelial cells of IPF patients. Thus, these
findings indicate that these five isoforms have a disease-dominant role
along with innate immune roles as these isoforms are found
predominantly in structural cells of IPF patients. Further, we have
targeted sPLA2 in mice model of bleomycin-induced lung fibrosis by
pBPB, a known sPLA2 inhibitor. pBPB treatment attenuated lung fibrosis
induced by bleomycin along with a reduction in TGF-β and deposition of
extracellular matrix in lung. Thus, these findings indicate that these
sPLA2 isoforms especially PLA2G2A may serve as a therapeutic target in
lung fibrosis.
Keywords: idiopathic pulmonary fibrosis, parabromophenacyl bromide,
secretory phospholipase A2, bleomycin, Group-II secretory phospholipase
A2
1. Introduction
Idiopathic pulmonary fibrosis (IPF) is a gradually progressive lung
deteriorating disease with a very poor prognosis, with mere 2.5 to 5
years mean survival. IPF mostly affects older individuals; however,
various studies had found that IPF may affect young individuals. IPF
was considered an inflammatory disease principally. In contrast, recent
literature based on clinical and experimental evidences emphasized that
it could be an epithelial-driven disease [[46]1,[47]2,[48]3]. The
development of IPF features could be due to the convergence of multiple
factors like genetics, aging, and environmental factors and this
convergence could activate the epithelium to initiate the disease by
secreting various profibrotic mediators that cause migration and
activations of fibroblasts. This also causes conversion of fibroblasts
into myofibroblasts that secrete abundant extracellular matrix proteins
to cause lung fibrosis. Epithelial apoptosis in response to lung injury
plays a crucial role in the development of lung fibrosis. LPA-LPA1
signaling is one of the pathways that are known to promote epithelial
cell death with a resistance to fibroblast cell death; to promote the
development of pulmonary fibrosis after lung injury [[49]4]. LPA
(lysophosphatidic acid), a pro-fibrotic, inflammatory mediator and also
a downstream metabolite of Phospholipase A2 (PLA2) signaling was found
to be increased in both BAL fluid of IPF patients and bleomycin-treated
mice. LPA-LPA1 signaling has been shown to be crucial for fibroblast
recruitment and vascular leakage in a bleomycin-induced mice model of
pulmonary fibrosis [[50]4,[51]5,[52]6]. This indicates that PLA2 may
play a critical role in lung fibrosis via LPA-LPA1 signaling.
Phospholipase A2 (PLA2), key enzymes involved in breakdown of membrane
phospholipids to release free fatty acids like arachidonic acid, has
been shown to be the cause of lung injury and inflammation [[53]7].
They are present in multiple forms: secretory phospholipase A2 (sPLA2),
cytosolic phospholipase A2 (cPLA2), Ca^2+ independent (iPLA2) and
lipoprotein associated phospholipase A2, based on biochemical
characteristics [[54]7,[55]8]. The cPLA2 null mice had shown
attenuation of pulmonary fibrosis induced by bleomycin with reduction
in thromboxane and leukotrienes [[56]9]. The cPLA2 has been shown to be
not only crucial in lung fibrosis but also in fibrosis of other organs.
For example, high-fat-diet-fed mice are resistant to developing liver
fibrosis in cPLA2 alpha knockout mice [[57]10]. Indeed, cPLA2 KO mice
had not merely reduced the fibrosis, but also inflammation, indicating
that PLA2 is involved in early stages of fibrosis, as fibrosis is the
end product of inflammation. This also indicates that cPLA2 may also be
involved in the generation of pro-inflammatory mediators. In addition
to cPLA2, it has been demonstrated that sPLA2-IIA promotes the
conversion of fibroblast into myofibroblasts via EGFR activation to
cause cardiac fibrosis [[58]11]. Compared to cPLA2, certain sPLA2
isoforms are not only found in blood, but are also responsible for
controlling other cells and foreign elements like bacteria, thanks to
substrate availability in the bacterial membrane
[[59]12,[60]13,[61]14,[62]15,[63]16,[64]17]. Thus, sPLA2 is not only
important in prognosis of the disease, but also could be a therapeutic
target.
In spite of its diversity, each PLA2 is unique, owing to different
cellular distribution and substrate. Studying the status of one PLA2
alone is not sufficient to understand the complete pathophysiology of
IPF. In addition, it had been demonstrated that exogenous sPLA2 had
been shown to activate the expression of cPLA2 in neutrophils through
LTB4 (Leukotriene B4) that leads to cause phosphorylation of cPLA2
[[65]15]. Similarly, sPLA2 had been shown to activate cPLA2 in
astrocytoma cells [[66]16] and keratinocytes [[67]17]. These indicate
that sPLA2 could be an important upstream regulator of cPLA2. So, it
could be important to study the status and cellular distribution of
various forms of sPLA2. In this study, for the first time, we have
elucidated the expression profile of various sPLA2 by analyzing
transcriptome of single cells from control healthy individuals and IPF
patients from three datasets that are publicly available as described
by Habermann et al. [[68]18], Tsukui et al. [[69]19] and Reyfman et al.
[[70]20]. Single-cell transcriptomic data analysis revealed an increase
in expression of PLA2G2A (protein name is sPLA2-IIA) and PLA2G5
(protein name is sPLA2-V) in a subset of fibroblasts of IPF patients as
compared to healthy individuals. These fibroblasts are present in IPF
individuals only and highly expressing PLA2G2A, so we named them as
PLA2G2A+ IPF fibroblast. TGF-β signaling pathway, IL-17 signaling,
arachidonic acid metabolism pathway, ECM-receptor interaction and other
fibrosis-related pathways are upregulated in these PLA2G2A+ IPF
fibroblast.
We have earlier explored the anti-asthma potential of parabromophenacyl
bromide (pBPB), a known sPLA2 inhibitor both in the acute and chronic
model of asthma [[71]21,[72]22]. Therefore, we have also studied the
effect of pBPB on bleomycin-induced lung fibrosis in mice. Inhibition
of sPLA2 by pBPB treatment had shown the attenuation of certain
features of pulmonary fibrosis along with reduction in TGF-β and
deposition of extracellular matrix in lung. These findings might be
important in developing effective therapeutic strategies for IPF.
2. Materials and Methods
2.1. Single-Cell RNA-Sequencing Data Acquisition
We have used three datasets for data mining of publicly available data
present on NCBI gene expression omnibus: (A) [73]GSE135893, (B)
[74]GSE132771 and (C) [75]GSE122960 of single-cell RNA sequencing that
was reported by Habermann et al. [[76]18], Tsukui et al. [[77]19] and
Reyfman et al. [[78]20], respectively. We acquired Single-Cell
transcriptome data of eight, three, and six healthy individuals
(control) and eight, three and four IPF patients (IPF) from datasets
A–C, respectively ([79]Supplementary Tables S1 and S2). As mentioned in
the original publications, all the studies requiring the clinical
sample were approved by Institutional Review Board. For diagnosis of
pulmonary fibrosis, they had used American Thoracic Society/European
Respiratory Society criteria [[80]18,[81]19,[82]20]. The detailed
description on acquiring the lung samples from healthy individuals and
IPF patients were are available in the original publications. The
demographic details of all clinical samples as described by Habermann
et al. [[83]18] and Reyfman et al. [[84]20] can be found on
[85]Supplementary Tables S1 and S2, respectively.
2.2. Single-Cell RNA-Sequencing Data Analysis
All algorithm used for analysis of single cell RNA seq data was based
on Seurat; R toolkit for single cell genomics with custom modification
[[86]23,[87]24,[88]25,[89]26]. Seurat version 4 was used for analysis
and visualization of single-cell RNA-sequencing data on R Studio. The
combined raw data of all the samples obtained from the gene expression
omnibus were read on RStudio and Seurat object for the same has been
created. For dataset A, a subset for the datasets of eight controls and
eight IPF patients with 73,778 cells were created and subject
identifier information has been added to the metadata. However,
individual cells with less than 200 or more than 4500 unique gene count
and more than 25% of reads arising from mitochondrial genes were
filtered out. Normalization and scaling were done using SCTransform
that normalizes the single cell UMI-data by variance-stabilizing
transformation [[90]27]. The principal component analysis (PCA) was
used for dimensional reduction based on the top 3000 most variable
genes. Variable genes were determined using the FindVariableGenes
function of Seurat. The FindClusters function of Seurat was used on PCA
reduced genes (dims = 1:20). Uniform manifold approximation and
projection (UMAP) was used for visualization of clusters on a 2-D map.
Differentially expressed genes were determined by the FindMarkers
function of Seurat, which is based on the non-parametric Wilcoxon Rank
sum test. To further study the status of various sPLA2, a feature
plot/dot plot/violin plot was generated for each subgroup of Seurat
objects. The FindMarkers function of Seurat was used to determine all
the differentially expressed genes on a subcluster of fibroblast
(PLA2G2A IPF fibroblast), highly expressing PLA2G2A.
Two more datasets were analyzed for validation of dataset A. Dataset B
and C were also analyzed using Seurat version 4 on RStudio. For
analysis of dataset B, cells with less than 300 unique gene and more
than 10% mitochondrial gene were excluded. For dataset C analysis,
cells with less than 300 unique gene and more than 35% mitochondrial
gene were excluded. Log-normalization was performed. FindVariableGenes
function of Seurat was used to detect variable genes on dimensionality
reduced data. On PCA-reduced genes, graph-based clustering was
performed using the FindClusters function of Seurat (at resolution =
0.6 and dims = 1:20). UMAP was used for visualization of clusters on a
2-D map. Differentially expressed genes were determined by the
FindMarkers function of Seurat, which is based on a non-parametric
Wilcoxon Rank sum test. Mesenchymal cells were identified by canonical
markers ([91]Supplementary Figure S3A–D and Supplementary Figure S5A–D
for data set B and C respectively) and further analyzed. Based on
expression of markers, cells were annotated ([92]Supplementary Figures
S4 and S3A and Supplementary Figures S6 and S4A for data set B and C
respectively). To further validate the results for dataset A, UMAP,
feature plot and violin plot were created for PLA2G2A and violin plot
for PLA2G5.
2.3. Pathway Enrichment Analysis
FindMarkers function of Seurat was used to determine all the
differentially expressed genes on a subcluster of fibroblast (PLA2G2A
IPF fibroblast), highly expressing PLA2G2A. All the significantly
differentially expressed genes in PLA2G2A+ IPF fibroblast as compared
to all other mesenchymal cells with logFC > 0.35 were used as an input
for KEGG pathway enrichment analysis using the most widely used gene
set enrichment tool WebGestalt 2019 [[93]28]. Statistical significance
of pathways was based on p-value. The normalized enrichment score and
p-value of all the significantly enriched pathways are provided in
[94]Table 1.
Table 1.
The significantly enriched pathways in PLA2G2A+ IPF fibroblasts.
S. No. Pathway Normalized Enrichment Score p-Value
1. TGF-beta signaling
pathway 1.9917 <2.2 ×10^−16
2. Longevity regulating
pathway 1.8124 <2.2 × 10^−16
3. Legionellosis 1.8419 0.0011287
4. AGE-RAGE signaling pathway in diabetic complications 1.7161
0.0063559
5. IL-17 signaling
pathway 1.7311 0.0066445
6. Antigen processing and presentation 1.7329 0.0081112
7. ECM-receptor
interaction 1.7329 0.0086674
8. Amino sugar and nucleotide sugar metabolism 1.6986 0.0093085
9. Arachidonic acid
metabolism 1.7057 0.010127
10. Amoebiasis 1.7010 0.011737
[95]Open in a new tab
2.4. Patient Samples and Animals
The institutional human ethical committee approval was obtained from
Kalinga Institute of Medical Sciences, Bhubaneswar, for collection and
use of sera samples from control individuals and IPF patients. Human
sera samples from IPF patients (n = 10) and healthy individuals (n =
20) were acquired. IPF was diagnosed based on the criteria of American
Thoracic Society/European Respiratory Society. The demographic details
of the patients/healthy individuals are given in [96]Supplementary
Table S3. Sera samples were utilized for estimation of sPLA2-IIA. All
the samples used in this study are from female individuals with no
smoking history. No statistically significant difference was observed
between the age distribution of both the groups. The diagnosis of IPF
based on the accepted guidelines like the exclusion of known causes of
ILD, like environmental exposures both from domestic and occupation,
drugs etc., and also by having HRCT patter of UIP.
All mentioned animal experimental protocols in this study were as per
CPCSEA (Committee for the Purpose of Control and Supervision of
Experiments on animal guidelines), and comply with the ARRIVE
guidelines and approved by Animal Ethics Committee, CSIR-Institute of
Genomics and Integrative Biology, Delhi, India. C57bl/6 female mice 5–6
weeks old, weighing 18–24 g were acquired from CSIR-Central Drug
Research Institute, Lucknow, India and acclimatized for at least 1 week
under standard laboratory conditions (25 ± 2 °C, 55% humidity) before
starting experiments. We used female mice, since they are less
aggressive and do not require care to separate dominant one [[97]29].
2.5. Mice Model of Pulmonary Fibrosis
The bleomycin-induced mice model of pulmonary fibrosis is most widely
accepted model of pulmonary fibrosis, since it mimics many features of
human idiopathic pulmonary fibrosis [[98]30,[99]31]. To determine at
which time point after instilling bleomycin fibrosis starts developing,
so that we can start giving pBPB, we developed time kinetics mice model
of bleomycin-induced pulmonary fibrosis. Wild type female C57bl/6 were
randomly divided into five groups: Group 1: Control/VEH (PBS treated
mice), Group 2: Bleomycin Day11 (Bleomycin-treated mice, sacrificed on
day 11), Group 3: Bleomycin day17 (Bleomycin-treated mice, sacrificed
on day 17), Group 4: Bleomycin day21 (Bleomycin-treated mice,
sacrificed on day 21) and Group 5: Bleomycin day25 (Bleomycin-treated
mice, sacrificed on day 25) (n = 4–6 for each group). Bleomycin
(Sigma-Aldrich, Saint Louis, MO, USA, cat. No. B2434) was reconstituted
in PBS pH 7.4. For the development of lung fibrosis, mice were
instilled with bleomycin (1 unit/kg) through orotracheal route on day
0. Mice were first anesthetized by brief isoflurane. After anesthesia,
mice were placed on a wood support at an angle of 45 degree, and
carefully, the tongue of the mouse was grasped in an upward and
leftward position using blunt-end forceps. Now 1 mg/kg of bleomycin was
instilled using a pipette. Mice were maintained on same position for 15
sec. and then placed near a warm pad for recovery [[100]32]. Mice were
euthanized by xylazine and thiopentone at various time points (Days 11,
17, 21, and 25).
2.6. Treatment with pBPB
To observe the effect of pBPB in bleomycin-induced mice model of
pulmonary fibrosis, based on results from above time kinetics
experiment; we chose most widely used 21 days model of
bleomycin-induced pulmonary fibrosis. In this experiment mice were
divided into three groups: Group 1. Control/VEH (PBS treated mice),
Group 2: Bleomycin/VEH (Bleomycin-treated mice), Group 3:
Bleomycin/pBPB (Bleomycin + pBPB treated mice) (n = 6 for each group).
Mice were anesthetized with isoflurane and bleomycin (1 mg/kg) was
instilled via orotracheal route on day 0. pBPB was given orally, twice
a day from day12 to bleomycin instilled mice as described in our
previous studies. Briefly, pBPB (Sigma) was dissolved in 50% ethanol
and given orally 1 mg/kg twice a day from day 12 up to the completion
of model.
2.7. BAL Fluid Analysis
Mice were anaesthetized and tracheotomized on day 21, 1 mL PBS was
instilled into the trachea and recovered PBS (approximately 600 µL) was
collected, centrifuged at 1000× g to get cell pellets and supernatants;
cell pellets were used to count total leucocyte cells as demonstrated
by our lab earlier [[101]33]. Supernatant was used to estimate total
protein, TGF-β and IL-17 ELISA.
2.8. Lung Histopathology and Morphometry
Lungs were harvested, some portion was fixed with 10% formalin,
embedded in paraffin, sectioned with microtome and stained with various
stains such as hematoxylin and eosin for assessing fibrosis, Masson’s
trichrome (MT) staining for observing collagen deposition in lungs.
Pulmonary fibrosis scoring was done based on modified Ashcroft scoring,
as described by Hubner et al. [[102]34]. Briefly, individual tissue
slides were observed in a blindfolded manner and scores were given
based on the extent of fibrosis. Images of MT stained slides have been
taken and morphometry for quantitation of collagen content was done
using Image J software [[103]33,[104]35]. Lung injury were scored based
on Semi-quantitative Morphological Index (SMI). Three lung sections
from each mouse were observed under light microscope in a blind fold
manner and scores were given as described earlier. Briefly, Normal
lung: 0; minimal areas of inflammation: 1; more frequent inflammatory
areas and fibrosis: 2; all the three sections lung lesions: 3;
extensive lesions on at least 2–3 sections: 4; majority of area covered
by inflammation and fibrosis: 5 [[105]36,[106]37,[107]38].
2.9. Elastin Measurement in Lung Lysate
Total lung lysate was prepared by homogenizing the 50 mg lungs in 500
μL RIPA buffer containing dithiothreitol (Sigma) and protease inhibitor
cocktail (Sigma Aldrich, Darmstadt, Germany) containing various
protease inhibitors like Aprotinin, AEBSF, E-64, Bestatin, Pepstatin A,
and Leupeptin followed by centrifugation. Supernatant was utilized as
total lung lysate for estimation of elastin and TGF-β after protein
estimation. Elastin was measured using a biochemical assay “Fastin
Elastin assay” (Biocolor, Carrickfergus, UK). This assay is based on
Fastin dye reagent (5,10,15,20-tertraphenyl-21,23-porphine
tetra-sulfonate in a citrate buffer), which binds with elastin present
in the sample. Briefly, elastin was first precipitated and dye was
added to form an elastin–dye complex. The complex was dissolved by dye
dissociation reagent and absorbance was taken at 513 nm wavelength in
microplate reader.
2.10. sPLA2 Activity Assay
sPLA2 activity was measured in sera of mice as per the manufacture’s
instruction (Cayman, Ann Arbor, MI, USA, cat. 765001) with custom
modifications. Briefly, the substrate, ethanolic solution of
diheptanoyl thio-phosphatidylcholine, was dried under the stream of
nitrogen and reconstituted in assay buffer (containing 10 Mm CaCl[2],
100 Mm of KCl and 0.3 M Triton X-100). DTNB (5,
5′-dithio-bis-(2-nitrobenzoic acid)) added to samples (mice sera 20
μL/assay buffer) or bee venom PLA2, a positive control and in the
96-well plate followed by addition of reconstituted substrate.
Absorbance was taken every minute at 414 nm after shaking the plate.
Two time points were selected based on the linear portion of the plot
made using absorbance values as a function of time. The following
equation was used to determine the change in absorbance:
[MATH: ΔA414=A414 (Time 2)− A414 (Time 1)Tim
e 2 (
min.)− Time 1 (min.) :MATH]
[MATH:
sPLA2 Activity (µ<
mi>mol/min/mL)=ΔA414/min.10.66 mM−1<
/mn>×0.130<
mrow> mL0.01
mL× Sample dilution :MATH]
where 10.66 mM^−1 is the extinction coefficient of DTNB at 414 nm
(pathlength = 0.784 cm), 0.130 mL for sera is total volume of the
reaction.
2.11. ELISA
TGF-β 1 in total lung lysate/BAL fluid (mice), IL-17 in BAL fluid
(mice) and sPLA2-IIA in plasma samples of IPF patients and controls
were measured using ELISA as per manufacturer’s (ThermoFisher
Scientific, Vienna, Austria /Cayman, An Arbor, MO, USA) instructions.
Briefly, capture antibody against TGF-β1 or IL-17 or sPLA2-IIA (1:250)
was coated in a 96-well plate and kept overnight; it was then washed
with 0.05% tween-20 in PBS and blocked with 1% BSA. Then samples (total
lung protein or plasma) were added and after 2 h, wells were washed and
detection antibody (1:250) was added, followed by a washing step and
HRP was added further. After a final wash, the plate was developed with
TMB and O.D. was acquired at 450 nm.
2.12. Statistics
All the experiments were performed at least three times with six mice
per group. All in-vivo data are shown as mean ± SEM and is
representative of three independent experiments. All the graphs were
generated using GraphPad prism. Two groups and three groups were
compared based on unpaired Students t-test and one way ANOVA
respectively. A p-value of 0.05 was considered as significant. In any
case, the individual p value for each experiment is being mentioned in
the respective legend.
3. Results
3.1. Expression Profiles of Various Secretory Phospholipase A2 Isoforms in
IPF Patients and Controls
To study the expression profiles of various secretory phospholipase A2
forms, single cell RNA seq. raw data were acquired from gene expression
omnibus [[108]GSE135893, [[109]18]]. The 73,778 single cells from eight
control and eight IPF patients were analyzed in the data set A.
Cells were broadly annotated based on the expression of markers and the
Seurat object was then split into four subgroups ([110]Supplementary
Figure S1A–E):
* (a)
EPCAM+ epithelial cells (clusters 1 and 4),
* (b)
PTPRC+ immune cells (clusters 0, 2, 6 and 7),
* (c)
PECAM1+ and EPAS1+ endothelial cells (cluster 3) and
* (d)
EPCAM- PTPRC- and PECAM1- mesenchymal cells (cluster 5).
For each subgroup Seurat object, a similar dimensional reduction,
clustering and UMAP visualization approach were applied to get
individual UMAP for each subgroup. Each cluster was annotated manually
based on the expression of cell type markers. For integrative analysis,
all the four annotated subgroup Seurat objects were merged into one
large Seurat object followed by SCTransform normalization, scaling,
dimensional reduction and UMAP visualization. After merging, using
Seurat package in R, we observed 21 cell type identities (annotated
based on expression of canonical markers) ([111]Supplementary Figures
S2, S7–S9) ([112]Figure 1A), and all these types were coming under 4
major sub-groups like epithelial, immune, endothelial and mesenchymal
cells. We observed a relatively novel cell type (PLA2G2A+ IPF
fibroblast) that is discussed in a later portion. Among all these
compartments, IPF patients have shown a predominant expression of sPLA2
in most of the structural cells like fibroblasts, mesothelial cells,
certain types of epithelial cells (ciliated, KRT5−/KRT17+ cells)
([113]Figure 1C). There are 11 isoforms of sPLA2 (IB, IIA, IIC, IID,
IIE, IIF, III, V, X, XIIA and XIIB). However, we found prominent
expressions of sPLA2-IB (PLA2G1B), sPLA2-IIA (PLA2G2A), sPLA2-III
(PLA2G3), sPLA2-V (PLA2G5) sPLA2-X (PLA2G10) and sPLA2-XIIA (PLA2G12A)
in the cells present in the lung as shown in [114]Figure 1C, though
each type had different cell distribution. In addition to these six
isoforms, we observed PLA2G2C, PLA2G2D and PLA2G12B also to be
expressed in lungs, but expression was much less, so we have not
explored them further. Moreover PLA2G2C is a pseudo gene in humans,
while PLA2G12B is expressed in an inactive form [[115]39]. Mesenchymal
cells, particularly in fibroblasts, have a predominant expression of
PLA2G2A and PLA2G5. While mesothelial cells have a predominant
expression of PLA2G2A, epithelial cells have PLA2G1B, PLA2G3, PLA2G10
and PLA2G12A indicating that each cell type might use unique secretory
PLA2 isoform for its function ([116]Figure 1C). Interestingly, all
these six dominant isoforms were found to be present only in structural
cells like fibroblast and epithelial cells that are key players in lung
fibrosis. Various immune cells including monocytes, dendritic cells,
plasma and B-cells have also shown expressions of PLA2G5 and PLA2G12B,
but the percent expression is low ([117]Figure 1C).
Figure 1.
[118]Figure 1
[119]Open in a new tab
Analysis of single cell RNA seq. data reveals the expression profile of
various sPLA2 in data set A ([120]GSE135893). (A) Uniform Manifold
Approximation and Projection (UMAP) after analyzing and annotating the
clusters; Single cell RNA seq data from 8 control (healthy individuals)
and 8 IPF patients, using Seurat as described by Habermann et al.,
showing various cell types. (B) UMAP plot showing cells, colored by
their origin either from control or IPF patients (C) Dot plot showing
cellular distribution of PLA2G1B, PLA2G2A, PLA2G2C, PLA2G2D PLA2G3,
PLA2G5, PLA2G10, PLA2G12A and PLA2G12B in lung. (Circle size indicates
percentage of cells expressing the given gene; intensity of blue color
indicates extent of expression).
3.2. Single Cell RNA-seq Analysis of Mesecnymal Cells Reveals PLA2G2A High
Fibroblast in IPF Petients
From the above results, it is clear that PLA2G2A is expressed in
mesenchymal cells, particularly in fibroblasts and mesothelial cells
whereas PLA2G5 is expressed in fibroblasts. However, we wanted to study
the expressions of PLA2G2A and PLA2G5 at the single cell level in the
mesenchymal cell subgroup Seurat object of IPF patients compared to
controls. We observed six sub clusters after re-analysis of mesenchymal
cells subgroup Seurat object ([121]Figure 2A). Based on known markers
and expression of signature genes, these clusters are annotated as (a)
DCN (Decorin) high fibroblast [LUM+ (lumican, a proteoglycan) and DCN
high cells), (b) DCN low fibroblast (LUM+ and DCN low cells), (c)
Myofibroblast [LUM and ACTA2+ (smooth muscle α actin) cells], (d)
PLA2G2A IPF fibroblasts (LUM and PLA2G2A high cells), (e) smooth muscle
cells (ACTA2+ cells) and (f) mesothelial cells (UPK3B+ and CALB2+)
([122]Supplementary Figure S2A–E). During annotation, we identified a
sub-cluster of fibroblasts highly expressing PLA2G2A, and this
population is specific to IPF patients only ([123]Figure 2B–D). Since
PLA2G2A was the most upregulated gene in this population, and this
population is specific to IPF patients, we named it as PLA2G2A+ IPF
fibroblasts ([124]Figure 2A–D). To study the expression of both PLA2G2A
([125]Figure 2D) and PLA2G5 ([126]Figure 2E) in mesenchymal cells, a
violin plot was generated. We found high expression of PLA2G2A and
PLA2G5 in PLA2G2A+ IPF fibroblasts. In addition to fibroblasts,
mesothelial cells have also shown a high expression of PLA2G2A in IPF
patients ([127]Figure 2D–E).
Figure 2.
[128]Figure 2
[129]Open in a new tab
Analyzing expression of PLA2G2A and PLA2G5 in mesenchymal cells in data
set A ([130]GSE135893). (A) UMAP visualization of mesenchymal cells,
various clusters displaying different types of mesenchymal cells. (B)
UMAP plot showing cells, colored by their origin either from Control or
IPF patients. (C) Feature plot displaying expression of PLA2G2A. (D)
Violin plot showing expression of PLA2G2A in various subclusters of
mesenchymal cells. (E) Violin plot of expression of PLA2G5 in
mesenchymal cells. (F) Enrichment analysis of pathways differentially
regulated in PLA2G2A+ IPF fibroblasts. (G) DOT plot showing the
upregulation of key ECM genes in mesenchymal cells. (H) DOT plot
showing the upregulation of key inflammatory genes in mesenchymal
cells.
The pathway enrichment analysis revealed a significant upregulation of
TGF-β signaling pathway, IL−17 signaling, arachidonic acid metabolism
pathway, ECM-receptor interaction and other fibrosis-related pathways
indicating the active role of PLA2G2A+ IPF fibroblasts in IPF
([131]Figure 2F and [132]Table 1). In addition to this, analysis of top
100 genes that are differentially regulated in PLA2G2A+ IPF fibroblasts
has shown upregulation of multiple key extra cellular matrix (ECM)
genes such FBLN1, COL1A2, COL1A1, MFAP5, FBN1, COL3A1, COL6A1, COL6A2
and COL14A1 ([133]Figure 2G). Moreover, a number of chemokines and
cytokines involved in inflammation including CCL2, CXCL12, CXCL1 and
CXCL2 are upregulated in PLA2G2A+ IPF fibroblasts ([134]Figure 2H).
This indicates that PLA2G2A+ IPF fibroblasts has both inflammatory and
fibrotic properties.
We wanted to validate the existence of PLA2G2A+ IPF fibroblast in other
datasets that are publicly available as mentioned in Methods. In the
second dataset (dataset B, [135]GSE132771) [[136]19], we studied the
expressions of PLA2G2A and PLA2G5 in the mesenchymal cells of IPF
patients compared to controls. Particularly, mesenchymal cells are
extracted based on the markers of various mesenchymal cells from all
the lung cells ([137]Supplementary Figure S3A–D) Based on known markers
and expression of signature genes, these clusters are annotated as (a)
UPK3B+ and CALB2+ mesothelial cells; (b) ACTA2+ smooth muscle cells (c)
LUM+ and ACTA2+ Myofibroblasts, (d) DCN+ and LUM+ fibroblasts and (e)
LUM+ PLA2G2A high “PLA2G2A IPF fibroblasts” ([138]Supplementary Figure
S4A–E). We observed five sub clusters ([139]Figure 3A). Similar to
first dataset, this dataset had also shown “PLA2G2A IPF fibroblasts”
almost exclusively in IPF patients. Even though mesothelial cells are
very scarce, IPF patients had shown dominant expression of PLA2G2A in
mesothelial cells compared to controls ([140]Figure 3B–D). PLA2G5
([141]Figure 3E) is also found to be expressed in PLA2G2A IPF
fibroblasts in IPF patients.
Figure 3.
[142]Figure 3
[143]Open in a new tab
Analyzing expression of PLA2G2A and PLA2G5 in mesenchymal cells in data
set B ([144]GSE132771). (A) UMAP visualization of mesenchymal cells,
various clusters displaying different types of mesenchymal cells. (B)
UMAP plot showing cells, colored by their origin either from Control or
IPF patients. (C) Feature plot displaying expression of PLA2G2A. (D)
Violin plot showing expression of PLA2G2A in various subclusters of
mesenchymal cells. (E) Violin plot of expression of PLA2G5 in
mesenchymal cells.
In the third dataset (dataset C, [145]GSE122960) [[146]20] also, we
extracted mesenchymal cells ([147]Supplementary Figure S5A–D) and found
five sub-clusters like DCN low fibroblasts, DCN high fibroblasts,
smooth muscle cells, PLA2G2A IPF fibroblasts and mesothelial cells
([148]Figure 4A, and [149]Supplementary Figure S6A–E). Both “PLA2G2A
IPF fibroblasts” and mesothelial cells had shown dominant expression of
PLA2G2A in IPF patients compared to controls ([150]Figure 4B–D),
whereas PLA2G5 ([151]Figure 4E) is relatively scarce compared to
earlier datasets.
Figure 4.
[152]Figure 4
[153]Open in a new tab
Analyzing expression of PLA2G2A and PLA2G5 in mesenchymal cells in data
set C ([154]GSE122960). (A) UMAP visualization of mesenchymal cells,
various clusters displaying different types of mesenchymal cells. (B)
UMAP plot showing cells, colored by their origin either from Control or
IPF patients. (C)Feature plot displaying expression of PLA2G2A. (D)
Violin plot showing expression of PLA2G2A in various subclusters of
mesenchymal cells. (E) Violin plot of expression of PLA2G5 in
mesenchymal cells.
3.3. PLA2G1B, PLA2G3 PLA2G10 and PLA2G12A Isoforms Have Shown Differential
Expression in IPF Patients
We found expression of PLA2G1B, PLA2G3, PLA2G10 and PLA2G12A in
epithelial cells, therefore the differential expression of PLA2G1B,
PLA2G3,PLA2G10 and PLA2G12A were assessed in epithelial cells by dot
plot. Further sub-clustering of epithelial cells results in six
sub-clusters ([155]Figure 5A). Based on known markers and expression of
signature genes, these clusters are annotated as Club cells
[secretoglobin family 1A member 1 (SCGB1A1) + and secretoglobin family
3A member 1 (SCGB3A1)+)], Ciliated cells [Forkhead Box J1 (FOXJ1+)],
AT1 cells [advanced glycosylation end-product specific receptor (AGER)+
alveolar type 1 cells), Basal cells [Keratin 5 (KRT5) + and KRT17+
cells), AT2 cells [surfactant protein-C (SFTPC)+ alveolar type 2 cells]
and KRT5−/KRT17+ (KRT5 negative and KRT17+ cells) ([156]Supplementary
Figure S7A–G). The KRT5−/KRT17+ cells are one of the recently
identified epithelial population having pro-fibrotic roles
[[157]18,[158]40]. PLA2G10 was found to be increased both in ciliated
cells and KRT5−/KRT17+ cells in IPF individuals while AT2 has shown
reduction in expression of PLA2G10 ([159]Figure 5B). We observed
increased expression of PLA2G3 in club cells and KRT5−/KRT17+ cells in
IPF individuals as compared to control ([160]Figure 5C). Expression of
PLA2G1B was found to be decreased in AT cells of IPF patients
([161]Figure 5D). PLA2G12A has shown increased expression in club and
ciliated cells, while it was reduced in AT1 and AT2 cells in IPF
patients ([162]Figure 5E) as compared to healthy individuals.
Figure 5.
[163]Figure 5
[164]Open in a new tab
Analyzing expression of PLA2G10 and PLA2G3 in epithelial cells (A) UMAP
showing the distribution of various clusters of epithelial cells. (B–E)
Dot plot visualization of expression of PLA2G10, PLA2G3, PLA2G1B and
PLA2G12A in epithelial cells from Control and IPF patients.
3.4. Expression of sPLA2-IIA Increased in the Plasma Samples of IPF Patients
We selected sPLA2-IIA for validation of single cell RNA seq data
analysis, as we found predominant and increased expression of PLA2G2A
(sPLA2-IIA) in IPF patients as compared to healthy individuals. There
were 20 healthy controls and 10 IPF patients ([165]Supplementary Table
S3), and we have measured sPLA2-IIA in sera of both controls and
patients by ELISA as mentioned in Methods. We found elevated levels of
sPLA2-IIA in the sera of IPF patients as compared to healthy
individuals ([166]Figure 6A).
Figure 6.
[167]Figure 6
[168]Open in a new tab
sPLA2-IIA levels are found to be increased in human IPF patients. ELISA
that measures sPLA2-IIA in human sera of both IPF patients and healthy
controls. (Values are expressed in means ± SEM, **** p value = 0.0001).
3.5. Development of Bleomycin-Induced Pulmonary Fibrosis with Time Kinetics
We found increased expression of various sPLA2 in IPF patients as
compared to healthy individuals while most of the sPLA2 has a
pro-inflammatory role and the end stage of a number of inflammatory
conditions is fibrosis [[169]41,[170]42,[171]43]. Therefore, we
hypothesized that sPLA2 could be a potential therapeutic target for
pulmonary fibrosis. Earlier, we showed that pBPB attenuates certain
features of both acute as well as chronic asthma in the Ova-induced
mice model. We have shown that pBPB reduced sub-epithelial fibrosis and
reduced levels of TGF-β in mice model of allergic asthma
[[172]21,[173]22]. On the other hand, pBPB is PLA2 inhibitor and PLA2
may activate fibrosis via the arginase/TGF-β pathway [[174]21,[175]22],
therefore we hypothesized that pBPB may attenuate certain features of
pulmonary fibrosis. The bleomycin mice model is a widely accepted model
for lung fibrosis, mimicking many features of idiopathic pulmonary
fibrosis. To find out at which time point fibrosis starts developing so
that we can start giving pBPB, we did a time kinetics study in which we
have instilled bleomycin to mice and sacrificed mice at various time
points as shown in [176]Figure 7A. We found that after day11 of
bleomycin instillation, inflammation started resolving and fibrosis
started developing. To validate whether bleomycin-treated mice have
developed fibrosis, we performed various assays. We performed H and E
staining to observe the morphology of lung section. From day11,
alveolar disruption starts appearing, though we could observe fibrotic
loci after day17 time point ([177]Figure 7B). Collagen deposition is
one of the hallmark features of pulmonary fibrosis. To observe collagen
deposition, we performed Masson’s trichrome staining, in which collagen
is stained by aniline blue stain. In the early time point i.e., from
Day 11 only, we observed collagen deposition in the lungs of
bleomycin-treated mice ([178]Figure 7C). Since elastin fibers are also
accumulated in the lungs of bleomycin-induced fibrosis mice, so to
assess the same, we performed a fastin elastin assay in Day 21 groups
and controls and found that elastin levels are upregulated as compared
to control mice ([179]Figure 7D). We found that at all the time points
starting from day 11, most of the features of pulmonary fibrosis are
upregulated as compared to the control, while day 21 is the most widely
accepted model, so we used the same in this study.
Figure 7.
[180]Figure 7
[181]Open in a new tab
Development of mice model of bleomycin-induced pulmonary fibrosis (A)
Experimental protocol for developing mice model of pulmonary fibrosis.
(B) H & E staining (bars = 100 µm) showing the morphology of lungs from
control and bleomycin-treated mice. (C) MT staining (bars = 100 µm)
depicting collagen deposition in lungs of control and bleomycin-treated
mice. (D) Elastin assay in total lung lysate from control and
bleomycin-treated mice. ** p = 0.003.
3.6. pBPB Attenuates Pulmonary Fibrosis in Bleomycin-Induced Mice
As we found increased expression of PLA2-IIA in the sera of IPF
patients and increased activity of sPLA2 in lungs of bleomycin-treated
mice, we wanted to study the effect of inhibition of sPLA2 by pBPB.
Bleomycin-administered mice were treated with pBPB (orally, 1 mg/kg,
twice a day) from Day 12–Day 21 as shown in [182]Figure 8A. The pBPB
treatment reduced the activity of sPLA2 ([183]Figure 8B). TGF-β plays a
vital role in pathogenesis of pulmonary fibrosis, therefore we assessed
levels of the same. TGF-β was found to be reduced in
Bleomycin/pBPB-treated mice as compared to only Bleomycin/VEH-treated
mice ([184]Figure 8C). Then we performed hematoxylin and eosin staining
in the formalin fixed lung sections. As shown in [185]Figure 8D, pBPB
treatment reduces fibrotic loci as compared to only bleomycin-treated
mice. The semi-quantitative measurement of fibrosis by modified
Ashcroft scoring suggested reduced fibrosis in Bleomycin/pBPB-treated
mice as compared to only Bleomycin/VEH-treated mice. ([186]Figure 8E).
The lung injury score by Semi-quantitative Morphological Index further
supported the above findings ([187]Supplementary Figure S10). We wanted
to determine whether pBPB reduces collagen deposition in the lungs of
bleomycin-treated mice by performing MT staining. In bleomycin-treated
mice, collagen deposition was observed, but in Bleomycin/pBPB treated
mice, collagen deposition was found to be reduced as compared to only
bleomycin-treated mice ([188]Figure 8F,G). Further BAL fluid analysis
has shown reduction in total protein content and infiltration of
inflammatory cells in BAL fluid of Bleomycin/pBPB treated mice as
compared to Bleomycin/VEH treated mice ([189]Supplementary Figure
S11A,B). We found decreased levels of inflammatory cytokine, IL-17 and
pro-fibrotic cytokine; TGF-β in BAL fluid of Bleomycin/pBPB treated
mice as compared to Bleomycin/VEH treated mice ([190]Supplementary
Figure S11C,D).
Figure 8.
[191]Figure 8
[192]Open in a new tab
pBPB ameliorates pulmonary fibrosis in Bleomycin-treated mice (A)
Schematic showing the mice experimental plan, mice were treated with
bleomycin (orotracheal instillation) and from day 12 onwards, pBPB was
given orally; twice a day and after euthanasia, mice were sacrificed on
day 21. There were three groups and n = 6 in each group. (B) sPLA2
activity and (C) TGF-β1 ELISA was performed in mice sera samples and
total lung lysates of bleomycin-treated mice, respectively, **** p =
0.0001 for control versus Bleomycin/VEH group and **** p = 0.0001 for
Bleomycin/VEH versus Bleomycin/pBPB group, and ** p = 0.003 for control
versus Bleomycin/VEH group, * p = 0.014 for Bleomycin/VEH versus
Bleomycin/pBPB group,sPLA2 activity and TGF-β1, respectively. (D) and
(E) H and E staining (bars = 100 µm) revealed attenuation in pulmonary
inflammation in Bleomycin/pBPB treated mice, Ashcroft score. *** p =
0.0004 for control versus Bleomycin/VEH group and ** p = 0.0056 for
Bleomycin/VEH versus Bleomycin/pBPB group (F) and (G) MT staining (bars
= 100 µm) results has shown reduced collagen deposition in lungs of
Bleomycin/pBPB treated mice as compared to Bleomycin/VEH mice,
semi-quantitative morphometry to assess collagen deposition in lungs. n
= 6. **** p = 0.0001 for control versus Bleomycin/VEH group and **** p
= 0.0001 for Bleomycin/VEH versus Bleomycin/pBPB group.
4. Discussion
The entire PLA2 superfamily has more than 30 members; among them, there
are 11 isoforms of sPLA2 (IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA
and XIIB) [[193]7]. However, we found predominant expression of
PLA2G1B, PLA2G2A, PLA2G3, PLA2G5, PLA2G10 and PAL2G12A in the cells
present in the lung as shown in [194]Figure 1C, though each type had
different cellular distribution. This differential cellular
distribution and known differential selectivity of each of these
isoforms indicate that each sPLA2 seems to have distinct
pathophysiological role [[195]39]. The sPLA2 isoforms have been shown
to have an important role in regulation of inflammation, remodeling of
membrane and also digesting exogenous phospholipids present in food and
microbes. Interestingly, all these six dominant isoforms were found to
be present only in structural cells like fibroblasts (PLA2G2A and
PLA2G5), mesothelial cells (PLA2G2A) and epithelial cells (PLA2G1B,
PLA2G3, PLA2G10 and PLA2G12A) that are key players in lung fibrosis.
All these findings indicate the possible innate immune role of sPLA2
secreted by fibroblasts mesothelial cells and epithelial cells.
In this regard, we found relatively unique and specific expression of
PLA2G2A in a subset of fibroblast (PLA2G2A+ IPF fibroblast) that is
very specific to IPF ([196]Figure 2A–D) and also validated the same in
two different single cell-RNA seq data sets ([197]Figure 3A–E and
[198]Figure 4A–E). We do not know the biological significance of this
PLA2G2A high fibroblast in IPF patients. Compared to other sPLA2
isoforms, sPLA2-IIA (PLA2G2A) is only found to be increased in the
circulation [[199]39], and sPLA2-IIA is otherwise called “bactericidal”
or “inflammatory” PLA2 as it is has substrate specificity on
phosphatidylethanolamine that is mostly found in the bacterial
membranes and has a high cationic property to cause hydrolysis of the
bacterial membrane. In addition to acting on foreign microbes,
sPLA2-IIA also acts on membranes of extracellular mitochondria that are
released at the site of inflammation either from leukocytes or
platelets. Upon being targeted by sPLA2-IIA, these mitochondria act on
neutrophils that further adhere to the vessel walls and this step is
crucial in innate immune mechanisms [[200]39,[201]44]. In this context,
it is well demonstrated that neutrophil and its components promote not
only lung inflammation but also initiate fibrogenesis in various
chronic lung diseases including IPF [[202]45].
Further, sPLA2-IIA has been shown to be linked to the degradation of
surfactant proteins that are rich in phosphatidylglycerol, which is one
of the favorable substrate for sPLA2-IIA [[203]7]. This indicates that
sPLA2-IIA can cause lung collapse. Evidently, a number of studies have
demonstrated increased levels of sPLA2-IIA in various secretions like
BAL fluid and the sera of patients with acute respiratory distress
syndrome [[204]7], severe COVID-19 patients [[205]46], and also in
alveolar fluids of patients with IPF and bleomycin lung (lungs of
patients who had been administered bleomycin as a therapy) [[206]47].
The significantly elevated levels of sPLA2-IIA in the plasma of
COVID-19 patients were found to be well correlated with mortality
[[207]46]. Further, they have identified sPLA2-IIA as one of the
critical variables among the 80 indices for predicting COVID-19
mortality. In the present study, we found an elevated level of
sPLA2-IIA in IPF patients compared to healthy individuals. There are
some limitations in our study. All patients are females and some of the
patients are younger. It is known that IPF mostly affects older
individuals. However, multiple studies had found that IPF may affect
young individuals [[208]48,[209]49,[210]50,[211]51,[212]52]. Leuschner
et al. has reviewed the medical records from February 2011 to February
2015, and they found that out of 440 patients with interstitial lung
diseases (ILD), 23% patients were IPF with an age group of ≤50 years
[[213]29]. Nadrous et al. had shown that the younger IPF patients
(range 28–49 years) had the same poor prognosis as the old patients
[[214]31]. Study by Ganesh Raghu et al. had shown incidence and
prevalence of IPF in younger IPF patients, though they have shown
reduced in younger patients [[215]32]. The reason behind IPF in younger
individuals may be familial [[216]52].
sPLA2-IIA has been shown to stimulate cardiac fibroblasts to
transdifferentiate into myofibroblasts through EGFR activation
[[217]11]. However, the pro-fibrotic role of sPLA2-IIA has not been
demonstrated in lung fibrosis. sPLA2-IIA is also secreted by synovial
fibroblasts of rheumatoid arthritis, and can amplify the synovial
inflammation [[218]53]. In addition to this, sPLA2-IIA converts
mononuclear cells present in the atherosclerotic plaques into
aggressive cells with increased adhesion and migration properties along
with the characters of monocyte-derived dendritic cells. These findings
indicate that sPLA2-IIA can act as a link between innate and acquired
immunity to accelerate the atherosclerosis-related complications
[[219]54]. We also found upregulation of both inflammation- and
fibrosis-related pathways in PLA2G2A+ IPF fibroblast in IPF like the
TGF-β signaling pathway, IL-17 signaling, the arachidonic acid
metabolism pathway and the ECM-receptor interaction ([220]Figure 2F and
[221]Table 1). Multiple ECM genes are significantly upregulated in
PLA2G2A+ fibroblasts ([222]Figure 2G); this indicates that the
fibroblast population may be crucial for the excessive deposition of
extracellular matrix in the fibrotic lungs. All these findings indicate
that the PLA2G2A+ fibroblasts population in IPF may have both fibrotic
and inflammatory properties. In general, fibroblasts are not considered
as active immune cells, though it is well known that its activation
during tissue injury is strongly associated with the fibrosis of the
concerned tissue/organ. The possible reason for such ignorance seems to
be non-availability of specific markers for the fibroblasts. However,
single-cell studies indicate that fibroblasts are like a heterogeneous
sub-population of a variety of cells like mesenchymal stromal cells,
pericytes etc. Thus, the activation of these fibroblasts with more
proliferation and the secretion of various pro-inflammatory mediators
including sPLA2-IIA convert these fibroblasts into aggressive
inflammatory fibroblasts that cross-talk with various immune cells to
precipitate the fibrosis. Thus, it has been suggested that
identification of such markers that are behind such transformation
could lead to very focused therapeutic strategies to treat
fibro-proliferative and fibro-contractive diseases. As sPLA2-IIA seems
to have such property of changing the harmless fibroblasts to more
inflammatory fibroblasts, targeting this could be beneficial in lung
fibrosis [[223]55]. Multiple studies have shown that sPLA2-IIA can
interact and activate integrins [[224]56,[225]57,[226]58]. While
integrins are well known for their role in activation of TGF-β and
fibroblast migration in pulmonary fibrosis, this could be the possible
mechanism by which sPLA2-IIA can participate in the pathogenesis of
pulmonary fibrosis [[227]59,[228]60,[229]61,[230]62] ([231]Figure 9).
Figure 9.
[232]Figure 9
[233]Open in a new tab
Schematic diagram showing the cellular distribution and possible role
of sPLA2 in IPF patients and its inhibition in Bleomycin-induced mice.
Single-cell transcriptome analysis revealed increased expression of
PLA2G2A and PLA2G5 in fibroblasts, PLA2G10 in KRT5−/KRT17+ cells and
ciliated cells, PLA2G3 in KRT5−/KRT17+ cells and club cells and
PLA2G12A in club cells and ciliated cells of IPF patients. PLA2G2A and
PLA2G5 can interact and activate integrins, which in turn activate
TGF-β, the master regulator of pulmonary fibrosis. sPLA2 is involved in
arachidonic acid metabolism and resulting metabolites can drive
pulmonary inflammation and fibrosis. pBPB inhibit sPLA2 and attenuate
certain features of fibrosis including collagen deposition and TGF-β in
bleomycin-treated mice.
We found increased expression of PLA2G5 in PLA2G2A+ IPF fibroblast
population ([234]Figure 2E and [235]Figure 3E) and this increased
PLA2G5 can target PC (Phosphatidylcholine) and surfactant proteins
present in plasma membrane. PLA2G5 is less-studied compared to PLA2G2A;
it has an inflammatory role via arachidonic acid mobilization and
eicosanoid generation [[236]7]. It has greater affinity to bind to the
phosphatidyl choline of the plasma membrane and pulmonary surfactant,
thus it can hydrolyze lung surfactant [[237]7], while the surfactant
disruption can cause alveolar injury and thus participate in the
pathogenesis of IPF [[238]63]. Thus, our study further supports the
hypothesis that sPLA2s, like PLA2G2A and PLA2G5, may act as a
prognostic marker in IPF. sPLA2-V is otherwise called Th2-prone sPLA2
due to its nature of potentiating allergic airway inflammation. The
decreased expression of IL-4 and IgE synthesis in PLA2G5 knockout mice
indicates its dominant role in Th2-mediated immunity. In addition to
its expression in Th2 cells, it is also found to be expressed in airway
epithelial cells and thus is involved in the promotion of airway injury
with degradation of surfactants [[239]39]. In general, the Th2 immune
response is known to coordinate the tissue repair and fibrosis through
the involvement of multiple immune and non-immune cells types including
fibroblasts and epithelial cells.
A number of studies have indicated that airway epithelium act as an
innate cell as it has to face the external environment, and the innate
immune role of fibroblasts might be interesting as they are located
just beneath the basement membrane and away from the external
environment compared to epithelial cells. More investigations are
required to find the possible mechanisms for the upregulation of
sPLA2-IIA in IPF patients and to see whether sPLA2-IIA can be taken as
an effective therapeutic target to prevent the lung fibrosis.
KRT5−/KRT17+ cells are recently identified basal-like epithelial cell
phenotype, highly enriched in IPF patients and known for their role in
ECM production [[240]18,[241]40]. Accumulating evidences suggested that
both the club cells and KRT5−/KRT17+ cells are known to play a
pro-fibrotic role [[242]18,[243]64,[244]65], and we also found an
increased expression of PLA2G3 in club cells and KRT5−/KRT17+ cells of
IPF patients as compared to control and healthy individuals, which
further supports the same hypothesis. PLA2G10 is also found to be
increased in ciliated cell and KRT5−/KRT17+ cells in IPF patients as
compared to control healthy individuals, indicating the pro-fibrotic
role of these cells in IPF [[245]18,[246]66]. Expression of PLA2G12A
was found to be increased in club and ciliated cells of IPF patients as
compared to control healthy individuals. However, decreased expression
of PLA2G10, PLA2G3, PLA2G1B and PLA2G12A in AT1 and AT2 cells of IPF
patients compared to controls indicated that different isoforms may
have different disease modulatory roles. In fact, it has been suggested
that each sPLA2 isoform is having tissue specific roles as it acts on a
variety of specific phospholipid substrates (34). Multiple studies have
observed the hydrolysis of surfactant phospholipids by various isoforms
of sPLA2 (like PLA2G1B, PLA2G2A, PLA2G5 and PLA2G10)
[[247]63,[248]67,[249]68]. While surfactant protein-B (SP-B), produced
by AT2 cells, is known to inhibit hydrolysis of surfactant
phospholipids by sPLA2, AT2 hyperplasia is the key pathological feature
of pulmonary fibrosis [[250]69]. Thus, one can expect more synthesis of
SP-B in pulmonary fibrosis. Moreover, increased surfactant may lead to
impaired lung function during pulmonary fibrosis [[251]63]These also
indicate the need for identification of isoform specific inhibitors for
effective therapeutic strategies.
sPLA2-X is also considered as asthmatic sPLA2 (34). It has been
demonstrated that sPLA2-X is secreted by airway epithelium, and
further, this released sPLA2-X acts on the phospholipids present in the
immune cells like eosinophils and releases lysophospholipid, thus
increasing the generation of CysLT from eosinophils. Importantly, these
entire events are inhibited by inhibition of cPLA2 alpha [[252]70].
These indicate that sPLA2 seems to have a dominant role compared to
cPLA2alpha, and on the other hand, it seems that structural cells like
airway epithelia augment airway inflammation through sPLA2
[[253]70,[254]71,[255]72].
The physiological function of PLA2G12A is not clear yet, though it is
highly expressed in various tissues. It is both structurally and
functionally different from other sPLA2 and has shown much less sPLA2
activity [[256]39]. A genome RNA expression profiling study had shown
upregulation of PLA2G12A in addition to PLA2G1B and PLA2G2D in patients
with pulmonary arterial hypertension secondary to IPF [[257]73].
We found increased expression of PLA2G2D in macrophages and ciliated
cells in IPF patients ([258]Figure 1C), indicating its innate immune
role while earlier literature had shown polymorphism in PLA2G2D is
co-related with body weight loss in COPD patients [[259]39]. However
further studies are required to demonstrate the role of PLA2G2D in lung
diseases.
Thus, sPLA2 seems to have a disease-associated role in IPF. Next, we
wanted to determine the possible therapeutic effect of sPLA2 in the
human-relevant mice model of IPF. The American Thoracic Society
recommends the use of male mice to develop Bleomycin-induced lung
fibrosis. However, the NIH recommends the use of both male and female
mice to develop Bleomycin-induced lung fibrosis. In any event, a number
of studies had used female mice for developing Bleomycin-induced lung
fibrosis [[260]74,[261]75,[262]76,[263]77,[264]78]. For example, study
by Ruscitti et al. had shown the assessment of lung fibrosis by
Micro-CT correlates with the histological manifestation of fibrosis in
bleomycin-induced female mice [[265]74]. Gharaee-Kermani et al.
[[266]79] demonstrated that female mice are having exaggerated lung
fibrotic response compared to male animals in bleomycin model. They
further demonstrated the reduced fibrosis in the ovariectomized,
bleomycin-treated rats whereas estradiol replacement restored the lung
fibrotic response that was comparable to intact female mice. In the
present study, we also observed that C57BL/6 female mice develop
multiple features of pulmonary fibrosis ([267]Figure 7).
While there is always a debate regarding the role of animal models in
human translational approaches, it will be ideal to explore the
pathways that are common to both human and animal models. In this
context, a number of commonly expressed genes were found upon
comparison between human lung fibrotic condition and the rodent model
of bleomycin-induced lung fibrosis, indicating that these genes can be
taken as a target in human-translational strategies. One such common
gene is PLA2G2A, which was found to be upregulated significantly in the
lungs of both rodent and human fibrotic conditions [[268]80]. pBPB is a
known sPLA2 inhibitor, and in our previous studies, we have shown the
anti-asthmatic role of pBPB along with reduction in sub-epithelial
fibrosis [[269]21,[270]22]. Though we have selected pBPB based on our
earlier findings in the asthma study [[271]21,[272]22], there are newer
pharmaceutical agents like varespladib ([273]LY315920) and its prodrug
methyl-varespladib ([274]LY333013) that had shown very effective sPLA2
inhibition even at nanomolar concentrations [[275]81,[276]82], which
should be explored for their anti-fibrotic activities in lung fibrosis
in future. It has been demonstrated that pBPB alkylates the histidine
residue in the active site of sPLA2 to inactivate it without affecting
the cPLA2 activation [[277]16,[278]83,[279]84]. However, there is no
report to demonstrate its effect on specific isoforms of sPLA2.
There is a considerable overlap between asthma and lung fibrosis in the
context of sPLA2. Arachidonic acid metabolites such as leukotrienes and
thromboxane are increased in both the diseases asthma and lung fibrosis
and are well known for their pro-inflammatory roles. Our previous study
has shown that pBPB; a known sPLA2 inhibitor attenuated asthma along
with reduction in sub-epithelial fibrosis in a mice model
[[280]21,[281]22]. We have chosen pBPB, a known sPLA2 inhibitor, in
this present study. We have observed that pBPB treatment reduces
pulmonary inflammation, extracellular matrix deposition (collagen) and
TGF-β in bleomycin-treated mice ([282]Figure 8, [283]Supplementary
Figure S10 and S11), which indicates the possible therapeutic role for
pBPB in pulmonary fibrosis. Though we have focused on sPLA2, pBPB is a
nonspecific inhibitor of PLA2 as it inhibits a number of enzymes such
as phospholipase C and GSK-3β [[284]85]. The GSK-3β is also a
serine/threonine kinase, and its signaling is found to be essential in
the conversion of pulmonary fibroblasts into myofibroblasts [[285]85].
The inhibition of GSK-3 also has been shown to reduce the progression
of pulmonary fibrosis [[286]86]. It is difficult to conclude whether
the pBPB mediated effects are only by inhibiting sPLA2. In any case,
there is a need for a very specific inhibitor for sPLA2-IIA. More
investigations are required to find the sPLA2-IIA-specific inhibitor
along with more in-depth characterization of the PLA2G2A
high-fibroblast population in IPF patients.
5. Conclusions
In summary, we have revealed the PLA2G2A high fibroblast population
with both fibrotic and inflammatory properties in the lungs of IPF
patients. Expression of PLA2G2A and PLA2G5 were found to be increased
in fibroblasts of IPF patients whereas PLA2G3, PLA2G10 and PLA2G12A
were increased in certain key epithelial cells like club cells,
KRT5−/KRT17+ cells and ciliated cells ([287]Figure 9). In addition to
this, we found elevated sera levels of sPLA2-IIA in IPF patients.
Further, inhibition of sPLA2 by pBPB ameliorates pulmonary fibrosis in
bleomycin-induced mice ([288]Figure 9).
Acknowledgments