Inflammatory

1. International Immunopharmacology 96 (2021) 107608
Available online 12 April 2021
1567-5769/© 2021 Elsevier B.V. All rights reserved.
Review
Inflammatory mediators in various molecular pathways involved in the
development of pulmonary fibrosis
M. Fathimath Muneesa, Sadiya B. Shaikh, T.M. Jeena, Yashodhar P. Bhandary *
Yenepoya Research Centre, Yenepoya University, Deralakatte, Mangalore 575018, Karnataka, India
A R T I C L E I N F O
Keywords:
Inflammation
Idiopathic pulmonary fibrosis
Cytokines
Epithelial-mesenchymal transition
Fibrinolytic system
Apoptosis
A B S T R A C T
Idiopathic pulmonary fibrosis (IPF) is a type of interstitial lung disease (ILD) that is marked by scarring of lung
tissue, ultimately leading to respiratory failure. The survival rate of IPF is disappointing and to date demonstrates
a clinical quandary. The exact etiology of the disease remains under discussion. According to the recent hy­
pothesis, inflammatory mediators cause severe damage to the alveolar epithelium leading to the impairment of
the alveolar structure. The role of inflammation in the development of the IPF has been controversial for years.
There are two schools of thought regarding the role of inflammation. One group of researchers claims that cell
death and fibroblast dysfunction are the primary causes and inflammation is just a secondary cause of IPF. The
other group claims inflammation to be the primary cause. Studies using human subjects have also reported
inflammation as a critical element in IPF. Inflammatory cytokines serve a major role in commencing the in­
flammatory response in the lungs. Several cytokines are reported to be involved in different molecular mecha­
nisms underlying IPF, some of which also contribute additionally by acting as growth factors. The present review
addressed to explore the contribution of various inflammatory cytokines, growth factors, and various other in­
flammatory molecules activating the major molecular pathways involved during the development of IPF.
1. Introduction
Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung
disease (ILD) that involves the irreversible scarring of the tissue because
of inflammation, the impaired wound healing process, and ultimately
fibrosis. Fibrosis is due to the excessive deposition of extracellular ma­
trix (ECM) proteins like fibronectin and collagens in the interstitial space
and alveolar septa [1]. This results in the alveolar structural damage
reducing the number of functional alveoli and thereby resulting in
impaired gas exchange. Thus the disease is associated with reduced lung
function and gradual respiratory failure.
The first step of diagnosis of IPF involves ruling out the known causes
of ILDs. The next step is to check for the usual interstitial pneumonia
(UIP) pattern on High-resolution computer tomography (HRCT) and
Abbreviations: AIM2, Absent In Melanoma; AKT, Also known as Protein Kinase B (PKB); ARDS, Acute Respiratory Distress Syndrome; ASC, Apoptosis-Associated
Speck-Like Protein Containing A Caspase-Recruitment Domain; ATII, Alveolar Type II; BAL, Bronchoalveolar Lavage Fluid; bFGF, Basic Fibroblast Growth Factor;
CCL, CC Chemokine Ligand; CCN3, Cellular Communication Network Factor 3; CCR, CC Chemokine Receptor; CD28, Cluster Of Differentiation 28; CXCL, Chemokine
(C-X-C Motif) Ligand; CXCR, Chemokine (C-X-C Motif) Receptor; dNLR, Derived Neutrophil To Lymphocyte Ratio; ECM, Extracellular Matrix; EMT, Epithelial-
Mesenchymal Transition; ERK, Extracellular Signal-Regulated Kinases; FVC, Forced Vital Capacity; GDF, Growth/Differentiation Factor; HRCT, High-Resolution
Computed Tomography; ICAM-1, Intercellular Adhesion Molecule 1; IFN-γ, Interferon Gamma; IGF, Insulin-Like Growth Factor; IKK, Inhibitor Of NF-Κb Kinase; IL,
Interleukin; ILD, Interstitial Lung Disease; IPF, Idiopathic Pulmonary Fibrosis; JAK, Janus Kinase; LPS, Lipopolysaccharide; MAPK, Mitogen-Activated Protein Kinase;
MCP, Monocyte Chemoattractant Protein; M-CSF, Macrophage Colony-Stimulating Factor; MMP, Matrix Metalloproteinases; NF-KB, Nuclear Factor Kappa B (NF-Κb);
NLRP3, NLR Family Pyrin Domain Containing 3; NOD, Nucleotide-Binding And Oligomerization Domain; NUR77, Also Known As Nerve Growth Factor IB (NGFIB);
PAI-1, Plasminogen Activator Inhibitor; PBMCs, Peripheral Blood Mononuclear Cells; PDGF, Platelet-Derived Growth Factor; PGE2, Prostaglandin E2; PI3K, Phos­
phoinositide 3-Kinase; PMN, Polymorphonuclear Leukocytes; SIRI, Systemic Inflammation Response Index; SMA, Smooth Muscle Actin; SMAD, Mothers Against
Decapentaplegic Homolog; SNAIL-1, Zinc finger protein SNAI1; STAT, Signal Transducer And Activator Of Transcription; TGF-β, Transforming Growth Factor-Beta;
TIMP, Tissue Inhibitors Of Matrix Metalloproteinases; TLR, Toll-Like Receptor; TNF-α, Tumor Necrosis Factor; TRAF, Tumor Necrosis Factor Receptor–Associated
Factor; UIP, Usual Interstitial Pneumonia; uPA, Urokinase Plasminogen Activator; uPAR, Urokinase Plasminogen Activator Receptor; Wnt, Wingless-Related Inte­
gration Site.
* Corresponding author.at: Yenepoya Research Centre, Yenepoya University, Mangalore 575 018, Karnataka, India.
E-mail address: yash28bhandary@gmail.com (Y.P. Bhandary).
Contents lists available at ScienceDirect
International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp
https://doi.org/10.1016/j.intimp.2021.107608
Received 10 December 2020; Received in revised form 23 February 2021; Accepted 21 March 2021

2. International Immunopharmacology 96 (2021) 107608
2
surgical lung biopsy. The characteristic histopathological feature in
surgical lung biopsies of IPF patients is the prominent fibrotic foci whose
appearance is due to the accumulated fibroblasts and myofibroblasts.
The median survival rate of an IPF patient after diagnosis is 3–5 years
[2]. Currently, there are no clinically effective treatment options against
IPF except lung transplantation [3]. The molecular mechanisms under­
lying the disease are also not clearly understood. But in the last few
years, researchers across the world have reached significant landmarks
associated with the disease. Initially, the inflammation process triggered
by an injury was considered to be the primary cause for the initiation
and progression of fibrosis. When there is an injury to the alveoli, the
resident macrophages release cytokines to attract fibroblasts, epithelial
and endothelial cells for promoting wound healing. But if there is
persistent injury, the neutrophils and the monocytes are recruited to the
sites. These inflammatory cells generate reactive oxygen species which
leads to an imbalance in oxidants and anti-oxidants. Eventually, the
inflammatory cells gathered secrete growth factors and other mediators
contributing to fibrosis [4,5]. But currently, the role of inflammation is a
controversial topic among researchers as many studies showed results
contradicting the inflammation theory. Various cellular processes play a
prime role in the pathogenesis of lung fibrosis (Fig. 1). The present re­
view discusses the role of inflammatory molecules in the core molecular
pathways involved during the development of lung fibrosis. Here, we
also highlight the controversial role of inflammation in IPF.
2. Inflammatory mediators in epithelial-mesenchymal
transition
The fibrotic foci in the parenchyma of a fibrotic lung are accumu­
lated with mesenchymal cells. These mesenchymal cells include fibro­
blasts and myofibroblasts, which are highly proliferative and produce an
excessive amount of ECM. The origin of the mesenchymal cells may be
the circulating fibrocytes or alveolar epithelial cells (AEC) that under­
went epithelial-mesenchymal transition (EMT) [6]. EMT is a process in
which the epithelial cells undergo morphological change and attain
motility due to the change in cytoskeletal arrangements and dissociation
of membrane-associated adherens junctions and desmosomes.
Myofibroblasts (or activated fibroblasts) and macrophages secrete
chemokines and growth factors that activate the ERK-MAPK (extrac­
ellularsignal-regulated kinase-mitogen activated protein kinase),
Smads, and the PI3K-AKT (Phosphatidylinositol-3-kinase-Protein kinase
B) signaling pathways to induce EMT [7]. Cytokines like transforming
growth factor-β (TGF-β), tumor necrosis factor (TNF-α), and interleukin-
1β (IL-1β) are reported to induce EMT in the mesenchymal (myofibro­
blast-like) phenotype in the fibrotic foci [8]. TGF-β is the most studied
and most crucial cytokine playing role in this process. High expression of
TGF-β in bronchoalveolar lavage (BAL) fluid and at the regions of
epithelial cells lining the fibrotic foci have been reported [9]. TGF-β
mediated EMT is through Mothers against decapentaplegic homolog 2
(SMAD-2) pathway but not MAPK pathways. TGF-β induces elevated
expression of matrix metalloproteinase 2 (MMP-2). This could disinte­
grate the basement membrane and in turn, induce motility of the cells
[10]. Shaikh et al. 2020., reported the potential contribution of TGF-β1
promoting p53-fibrinolytic system during alveolar EMT in A549 cells
[11].There are notable similarities between lung fibrosis and lung can­
cer concerning genetic and epigenetic alterations, EMT, fibroblasts in­
vasion, and some signal transduction pathways [12,13].
Elevated levels of IL-17A are reported in IPF and bleomycin-induced
pulmonary fibrosis (BLM-PF) models [14,15]. IL-17A mediates EMT
through TGF-β mediated ERK1/2 and Smad2/3 activation [16]. Studies
also reported the participation of IL-17A in activation of TGF-β via smad
dependent and independent signaling in alveolar epithelial cells during
ALI in vivo [17]. In a recent study, it was reported that IL-17A and
growth/differentiation factor 15 (GDF15) induced EMT of lung epithe­
lial cells in response to cigarette smoke [18]. IL-6 promotes growth and
EMT in CD133 + A549 cells by targeting Hhg (Hedgehog protein), Bcl-2
(B cell lymphoma 2), and Erk/MEK [19,20]. Zhang et al.,demonstrated
that inhibition of IL-18 in BLM-PF led to an increase in epithelial marker,
E-cadherin. The study also concluded that IL-18 regulated EMT by up-
regulating Snail-1 [21].
NOD-like receptor protein 3 (NLRP3) inflammasome is a multi­
protein complex that activates caspase-1 which in turn leads to the
secretion of pro-inflammatory cytokines, IL-1β and IL-18, and their
maturation. The components of this protein complex are a sensor
molecule, NLRP3, an adaptor molecule apoptosis-associated speck-like
protein containing a caspase recruitment domain (ASC), and pro-cas­
pase-1[22,23]. It is found to be involved in acute respiratory distress
syndrome (ARDS), chronic obstructive pulmonary disease (COPD), IPF,
asthma, and silicosis [24]. NLRP3 inflammasome mediates EMT through
TAK1-MAPK-Snail/NF-κB pathway [25]. IL-1β can activate EMT related
pathways like Wnt/GSK3β (Wingless-Related Integration Site/glycogen
synthase kinase 3β) and TGF-/Smad signaling pathways [26,27]. The
expression and activation of another inflammasome named absent in
melanoma 2 (AIM2) are enhanced in IPF peripheral blood mononuclear
cells (PBMCs) as well as in a lung fibrosis exacerbation model [28,29].
AIM2, upon activation in PBMCs, causes the release of pro-fibrotic cy­
tokines IL-1α, IL-18, and TGF-β in a TLR4-/caspase-1-/caspase-8-/cal­
pain-independent fashion and also releases caspase-4 [29]. Recent
studies reports that AIM2 inflammasome-dependent inflammation plays
apivotal role during progression of pulmonary fibrosis [30].
Fig 1. Cellular factors responsible for lung fibrosis: Various cellular processes like apoptosis of alveolar epithelial cells, EMT, inflammatory cells infiltration,
myofibroblast invasion or differentiation, and DNA damage play a major role in the pathogenesis of lung fibrosis.
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3. International Immunopharmacology 96 (2021) 107608
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CXCR3 is the receptor for chemokine ligand CXCL9. Beirne et al.,
demonstrated the elevated expression of CXCR3 in the proximity of cells
that have undergone EMT in IPF surgical lung biopsies. Their study
concluded the antifibrotic activity of CXCL9 via abrogation of the TGFβ
signaling pathway by the reduction of phosphorylation of Smad2 and
Smad3 in AECs [31]. The expression of IL-22, belonging to the IL-10
family, is shown to be reduced in BLM-PF mice models. IL-22 inhibits
EMT of AECs [32].
In a study conducted on HCl-induced epithelial remodeling, it was
found that monocytes interact with epithelial cells through ICAM-1
(Intracellular adhesion molecule-1) and enhance EMT and induce the
release of IL-8 and PDGF [33]. IL-8 interacts with CXCR1/CXCR2 to
activate the MAPK pathway and is shown to favor fibrosis [34,35].
IL-37 is an antifibrotic interleukin [36–38]. IL-37 and CCL22 co-
localizes and inhibits proliferation and EMT by abrogating IL-6/Signal
Transducer and Activator of Transcription-3 (STAT3) signaling
pathway in A549 cells [37,39]. IL-37 also binds to Smad3 and down­
regulates different signaling pathways, including focal adhesion kinase
(FAK), STAT, p53, MAP kinase p38α, and mTOR (mammalian target of
rapamycin)[40]. IL-27 is also a negative regulator of EMT in AEC
through the JAK/STAT and TGF-β/Smad signalling pathways [41].
3. Inflammatory mediators in apoptosis of epithelial cells
Apoptosis plays a significant role in the pathogenesis of PF. Epithelial
cells undergo apoptosis in patients with IPF and also in PF animal
models. Intratracheal administration of BLM-induced elevates expres­
sion of fas on alveolar epithelium [42]. The abnormal activation of AEC
is sufficient to initiate fibrotic activities [43]. Alveolar type II (AT-II)
epithelial cells are the ones that undergo apoptosis. AT-II cells have the
capability of being transformed into type 1 cells [44]. Consistent with
the theory, it was proved that collagen deposition could be reduced by
the installation of apoptotic inhibitors in animal models [45]. The
presence of epithelial cell apoptosis in normal lungs suggests that
apoptosis can probably be an initiating event in fibrogenesis [46].
Epithelial cells are important because they produce mediators that are
antifibrotic. For instance, prostaglandin E2 (PGE2) produced by
epithelial cells inhibit fibroblast proliferation. Plasminogen activators
and metalloproteinases expressed by them aid in fibrin and ECM
degradation [47]. Epithelial cells probably play as a barrier to cytokines
secreted by activated macrophages and thereby protecting the under­
lying tissue. Thus apoptosis of epithelial cells could play a crucial role in
the onset of fibrosis [48].
TNF-α induces AEC apoptosis dependent on angiotensin II in the
BLM-PF model [49,50]. It activates the IKK ( IκB kinase ) and NF-KB
(nuclear factor kappa light chain enhancer of activated B cells) com­
plex. CD28 (Cluster of differentiation-23) is a costimulatory protein for
the activation of T-cells expressed on antigen-presenting cells. CD28
deficiency abrogates blast exposure-induced lung tissue apoptosis [49].
In multiple myeloma, the CD28 pathway is associated with the phos­
phorylation of PI3K/Akt and inactivation of the FoxO1 [51]. CD28 in­
duces the expression of IL-17A and other cytokines, IL-6 and IL-8, in
multiple sclerosis T lymphocytes [52]. It activates RelA/NF-κB which in
turn induces IL-6 expression that leads to the activation and nuclear
translocation of tyrosine-phosphorylated STAT3 (pSTAT3). pSTAT3 as­
sociates with RelA/NF-κB by binding to the specific sequences in the
proximal promoter of the human IL-17A gene, thereby inducing its
expression. Class 1A PI3K regulates CD28-mediated RelA/NF-κB and
STAT3 recruitments and trans-activation of IL-17A promoter [53]. IL-
17A induces the expression of p53 augmenting apoptosis of AEC
[54,55].
Elevated levels of nephroblastoma overexpressed protein (NOV; also
known as CCN3), an inflammatory mediator, was observed in the
plasma of patients with severe pneumonia-induced ARDS when
compared to the healthy controls [56]. CCN3 brings about pro-apoptotic
effects on AECs by the activation of the TGF-β signaling pathway, NF-κB
signaling pathway, and Bcl-2/caspase-3 pathway [57]. CCN3 could play
a pro-fibrotic role by inducing apoptosis of AECs and the destruction of
epithelial integrity, ultimately leading to IPF. The inflammatory cyto­
kines such as IL-17A, IL-6, TNF-α, IL-33, and IL-37 act via various
signaling pathways through cell surface receptors playing a pivotal role
in regulating alveolar apoptosis and EMT causing alveolar damage and
ultimately leading to the development of lung fibrosis (Fig. 2).
4. Inflammatory mediators in the impaired fibrinolytic system
Extravascular accumulation of fibrin is implicated in the progression
of PF [58]. When an injury occurs in the lung tissue, the vascular
permeability increases; due to which the coagulation and fibrinolytic
factors leak out to the impaired tissue [59]. Thrombosis and fibrin
deposition in the alveolar space are common features in a fibrotic lung
[60].
The balance between fibrinolysis and coagulation plays a critical role
in tissue homeostasis. The main components of the lung fibrinolytic
system are urokinase plasminogen activators (uPA), urokinase plas­
minogen activator receptor (uPAR), and plasminogen activator inhibitor
Type-1 (PAI-1). uPA and uPAR promote fibrinolysis whereas PAI-1 in­
hibits fibrinolysis. Increased level of PAI-1 due to inflammatory pro­
cesses is observed in pleural effusion [61]. Park et al., observed
increased levels of PAI-1 in the plasma and the injured lungs of patients
with PF. The study also demonstrated PAI-1 as a potential “don’t eat me
signals” for viable and apoptotic neutrophils [62]. The persistence of
apoptotic neutrophils in the fibrotic lung may worsen the condition by
releasing intracellular toxic substances to the surroundings through
necrosis or autolysis of the apoptotic bodies.
Regulation of coagulation and fibrinolytic system by inflammatory
mediators is studied extensively in certain pathological conditions like
sepsis. Stimulation of human adipocytes with IL-β or TNF-α or TGF-β
elevates the expression of PAI-1 [63]. Chiu et al., suggested that the
increased expression of pro-inflammatory cytokines may lead to fibri­
nolytic imbalance and result in fibrin deposition in the pleura of the
pediatric para-pneumonic lung [64]. Akpan et al., noted that interferon-
γ (IFN-γ) and IL-10 increased fibrinolysis inactive tuberculosis which
involves chronic inflammation [65]. A recent study proved that IL-17A
enhanced the expression of PAI-1 and p53 [55] and downregulates the
expression of uPA and uPAR in acute lung injury (ALI) in vivo [54]. p53
is reported to destabilize the mRNAs of uPA and uPAR and stabilization
of mRNA of PAI-1[66,67].Tiwari et al., stated that PAI-1 attracts poly­
morphonuclear (PMN) cells and other inflammatory cells to alveolar
space through induction of expression of chemokines by AECs [68]. IL-
1β and IL-12 have also been suggested to play important roles in the
fibrinolytic system [69]. IGF-II (Insulin-like growth factor-II) impairs
MMP and tissue inhibitor of metalloproteinases (TIMP) balance [70]. IL-
5 stimulates eosinophils to adhere to fibrinogen and results in degran­
ulation which ultimately leads to the secretion of IL-13, IL1a, IL-4, TNF,
and CCL24 [71]. Soluble fibrinogen interacts with CD11b on neutrophils
and activates focal adhesion kinase and ERK1/2 to regulate degranula­
tion, phagocytosis, and apoptosis [72]. TGF-β is a strong inducer of PAI-
1. Downstream molecules of the TGF-β signaling pathway, SMAD 3 and
4 proteins, bind to consensus sites in the TGF beta-inducible elements in
the promoter of PAI-1 promoter [73]. Numerous inflammatory mole­
cules induce PAI-1 expression further giving rise to various inflamma­
tory responses such as fibrin deposition, activation of macrophages,
ECM deposition, increasing fibronectin and collagen levels during lung
fibrosis (Fig. 3).
Lipopolysaccharides (LPS) mediated expression of PAI-1 is
augmented by TNF-α.Nuclear receptor-77 (Nur77) is a transcriptional
factor generated in response to TNF-α. Nur77 induces transcription
of PAI-1 by binding to NGFI-B responsive element (NBRE) in the PAI-1
gene in a ligand-independent mechanism [73]. IL-33 participates in
the production of profibrotic cytokines and modulates the expression of
collagen IV, MMP-9, TIMP-1, tumor necrosis factor receptor (TNFR)-
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4. International Immunopharmacology 96 (2021) 107608
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associated factor 6 (TRAF-6), and NF-κB. It creates an imbalance be­
tween MMP-9 and TIMP-1 and thereby modulating the deposition of
MMP [74].
PAI-1 also influences inflammatory responses. It can increase the
infiltration of neutrophils, macrophages, and myofibroblasts; thereby
contributing to the progression of fibrosis (Fig. 4) [75,76]. In short,
fibrinolytic system is capable of regulating inflammatory responses and
vice-versa.
5. Inflammatory mediators in accumulation of myofibroblasts
Accumulation of fibroblasts is one of the significant observations in
PF. The fibroblasts in fibrotic lung express α-smooth muscle actin
(α-SMA) and ECM, which leads to damage to the alveolar architecture
[77]. They are key sources of collagen and profibrotic cytokines like
TGF-β. The role of inflammatory cytokines in the proliferation and
accumulation of fibroblasts has been implicated in several studies. Han
et al. noted that basic fibroblast growth factor (bFGF) induced activation
of ERK5 by enhancing its phosphorylation. ERK5 is an important protein
for cell proliferation, differentiation, and tissue repair. bFGF-induces
PAI-1 and cell proliferation in lung fibroblasts [78]. TGF-β is the most
reported growth factor cytokine which exhibits the property of induc­
tion of differentiation into myofibroblasts. TGF-β is elevated in human
subjects with IPF as well as in animal models of PF. It promotes the
activation and differentiation of normal lung fibroblasts to fibrotic lung
fibroblasts [79,80]. Fibrotic lung fibroblasts express α-SMA and ECM in
excess which in turn leads to changes in the alveolar architecture.
There is increased expression of CCR7 by fibroblasts, mononuclear,
and epithelial cells in IPF lung [81]. The ligand of CCR7, CCL21, en­
hances the proliferation and migration of fibroblasts. Immuno-
neutralization of either of the proteins inhibits migration of fibro­
blasts. This indicates that the migration of fibroblasts is dependent on
both, the ligand and the receptor [82]. CXCL16/CXCR6 accelerates
fibrosis by augmenting proliferation, migration, and collagen synthesis
of human pulmonary fibroblasts through the activation of the PI3K/
AKT/FOXO3a signaling pathway [83]. IL-11, a cytokine from the IL-6
Fig 2. Inflammatory cytokines mediated pathways activating alveolar apoptosis and EMT leading to the pathogenesis of pulmonary fibrosis: Pro-Inflammatory
cytokines are responsible for activation of alveolar inflammatory pathways. IL-17AR operates IL-17A activating the prime modulator of EMT TGF-β1 activating
smad2/3 complex leading to phosphorylation of smad 2/3 in the nucleus. IL-17A also induces EGFR stimulating MAPK/ERK 1/2 elevating the expression of p53 and
favors its translocation in the nucleus inducing phosphorylation of p53 causing alveolar injury. IL-6R facilitates the binding of P-JAK stimulating P-STAT1 and P-
STAT3 further translocating in the nucleus promoting JAK-STAT pathway activation. The receptor of TNF-α binds to TRAF2 elevating IKK expressions forming the
IKK-NFKB complex inducing phosphorylation of NFKB in the nucleus. TRAF6 binds to the receptor of IL-33 activating MAPK and NFKB further expressing NFKB
phosphorylation in the nucleus, however, this mechanism is found to be blocked by the anti-inflammatory cytokine IL-37. All the above cytokines are responsible to
induce alveolar epithelial injury and activate the biological process such as apoptosis and EMT leading to the pathogenesis of pulmonary fibrosis. On the other hand,
BLM injury stimulates NLRP3 inflammasome activating pro-caspase 3 and active caspase 3 promoting alveolar apoptosis causing lung epithelial injury ultimately
leading to pulmonary fibrosis.
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5. International Immunopharmacology 96 (2021) 107608
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family, has mitogenic property and inhibits fas-induced apoptosis of
lung fibroblasts [84,85]. Oncostatin M, another cytokine of the IL-6
family, also has mitogenic property and induces collagen expression in
lung fibroblasts. Oncostatin also inhibits the expression of α-SMA
induced by the TGFβ signaling pathway [86,87]. IGF-II induces trans­
differentiation of normal lung fibroblasts into myofibroblasts by acti­
vating the TGFβ receptor [70]. As reported by Tager et al., CXCL10
inhibits the accumulation of fibroblasts and thereby plays a protective
role in PF. It indicates that overexpression of CXCL10 could resolve PF
[88].
6. Inflammatory mediators in immune cell infiltration
Elevated levels of cytokines that play a role in chemotaxis and acti­
vation of neutrophils, lymphocytes, and monocytes are observed in tis­
sue and fluid from the lungs of IPF patients. IL-8, monocyte
chemoattractant protein (MCP-1/CCL2), and macrophage inflammatory
protein-1α (MIP-1α/CCL3) are examples of these cytokines. Baran et al.,
(2007), reported elevated levels of macrophage colony-stimulating
factor (M− CSF) in the fluid from the lungs of IPF patients. The study
also demonstrated M− CSF mediated recruitment of macrophages in the
BLM-PF model [89].Cytokine-like-factor-1(CLF-1) augments the infil­
tration of CD4 + T cells in BLM-induced fibrotic lung [90]. Recent study
demonstrated the potentiality of the pro-inflammatory cytokine IL-17A
in elevation of AMPKα/COX-2 expressions via enhancing inflammatory
molecules like NF-κB-p65, NF-κB-p105, CXCL1 and IL-1β etc [91]. IL-6,
an activator of JAK-STAT pathway, promotes migration and infiltration
by inducing the expression of MMP1 and MMP-9 through PGE-2 and
COX2 signaling [92]. IL-1β upregulates CXCL5 and causes infiltration of
neutrophils and macrophages. CXCL5 is, in turn, a substrate for MMPs 2
and 9 which activate CXCL5 by cleaving it [93]. Anti TNF-α adminis­
tration in nitrogen mustard induced pulmonary injury model, reduced
the numbers of M1 macrophages Ym1+
M2 macrophages in the lung
[94]. In conclusion, inflammatory mediators like CXCL5, IL-1β and TNF-
α play potential role in infiltration of immune cells which in turn leads to
enhanced inflammation and gradually contributes to fibrosis.
7. Therapeutics targeting inflammation
Inflammation can worsen the condition in PF and thereby leading to
early mortality. The therapy against ILD includes anti-inflammatory and
immunosuppressive agents, with anti-fibrotic agents introduced lately
for the treatment of IPF. Treatment with anti-inflammatory drugs even
before the process of irreversible parenchymal fibrosis could probably
ease IPF. Pirfenidone used widely against IPF is an immunosuppressant
with anti-inflammatory as well as anti-fibrotic features [95]. It can also
modulate cross-talk between DCs and T-cells to a reduce the production
of pro-inflammatory cytokines, decrease Th-cell proliferation and im­
pairs Th2-cell polarization [96–98]. Nintedanib, another widely used
drug against IPF, reduces BAL lymphocytes and neutrophils but not
macrophages. It also lowers the levels of interleukin-1b, keratinocyte
chemoattractant, TIMP-1, and lung collagen [99]. Thalidomide, pos­
sessing immunomodulatory potentials, could improve cough and res­
piratory quality in IPF patients [100]. Anti-inflammatory and anti-
fibrotic properties of dabigatranetexilate resolve pulmonary fibrosis in
experimental models. It reduces the infiltration of inflammatory cells
and pro-fibrotic protein expression [101]. Statin therapy, which is
mainly used to manage cardiovascular disorders, reduces inflammation
and release of pro-inflammatory cytokines in lungs. It is also potent in
inhibiting fibroblast activity and hinders the advancement of the disease
to fibrotic stage; thereby reducing mortality. There are also studies
showing adverse effect of statins against ILDs [102–106]. A high dose of
a non-steroidal anti-inflammatory drug, ibuprofen is reported to slow
down the progression of lung disease in cystic fibrosis [107]. Cortico­
steroids when used at the active stage of inflammation could bring about
a significant response in delaying the progress of the disease [108].
Fig 3. Inflammatory mediators in the fibrinolytic system leading to pulmonary
fibrosis: Inflammatory irritants induce reactive oxygen species (ROS) activating
the inflammatory cytokines IL-6, IL-17A, and TNF-α increasing the expression
levels of p53 further recruiting the fibrinolytic molecules. ie, decrease expres­
sions of uPA and uPAR and increase levels of PAI-1 promoting fibrin deposition
causing fibroblast accumulation eventually causing pulmonary fibrosis.
Fig 4. PAI-1 influences various inflammatory responses: Various inflammatory
cytokines like IL-6, IL-1β, TGF-β1, IL-17A, and TNF-α elevates expression levels
of PAI-1 stimulating several inflammatory responses like fibrin deposition, in­
duction of macrophages and PMN, activation of myofibroblast, ECM deposition,
and increase in fibronectin and collagen levels favors in the development of
pulmonary fibrosis.
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6. International Immunopharmacology 96 (2021) 107608
6
These medications are efficient against inflammation, but have potential
side effects and some of their benefits are unproven. Generous resolution
of inflammation in lungs is extremely important, as compromised res­
olution could lead to chronic inflammatory stage and ultimately fibrosis.
8. The controversy of the role of inflammation in IPF
The role of inflammation in PF has been a topic of discussion for
years and is still under controversy. Since there is no effective response
to the immunosuppressive medications in PF patients, some experts
claim that inflammation doesn’t play a role in the pathogenesis of the
disease. But several studies involving human subjects as well as animal
models delineate the role of inflammation in the fibrotic process. Some
researchers state that inflammation is just a secondary cause for the
disease. Ga67
uptake is used as a measure of inflammation at a specific
site. Mura et al., observed that although there is a prominent decline in
lung function parameters during the progression of PF, there was no
significant correlation between Ga67
uptake and the disease progression.
They concluded that inflammation is prominent in advanced stages of
fibrosis but does not play a pivotal role in the progression of the disease
[109]. A similar study conducted by Grijm et al., also indicates that
effective reduction in inflammation (as assessed using Ga67
scintig­
raphy) did not improve the clinical outcome of the disease [110]. Pa­
tients suffering from IPF have periods of an acute decline in lung
function parameters known as acute exacerbations. Increased levels of
neutrophils, pro-inflammatory cytokines, and M2 cytokines (an indica­
tion of macrophage activation) were noted during acute exacerbations
in BALF of IPF patients. The study concluded that acute exacerbation is
not a subsidiary event but is mediated by M2 macrophage activation
[111]. In a very recent study, it was reported that blood inflammation
indexes like neutrophil to lymphocyte ratio (NLR), derived neutrophil to
lymphocyte ratio (dNLR), monocyte to lymphocyte ratio (MLR), sys­
temic inflammation response index (SIRI), and aggregate index of sys­
temic inflammation (AISI) independently associated with the existence
of IPF after adjusting for age, gender, body mass index and smoking
status [113].
In a study by Balestro et al., pulmonary fibrosis patients were clas­
sified into slow or rapid progressors of PF based on the FVC fall per year.
The morphometric analysis of lung explants of slow progressors with
acute exacerbation and rapid progressors with or without acute exac­
erbations in PF showed significant inflammatory cell infiltrates. They
also observed that the number of innate immune cells (neutrophils and
macrophages) and that of adaptive immune cells (CD4+, CD8+, and B
cells) were high in rapid progressors than in slow progressors, pointing
at the influencing role of inflammation in the progression of PF [112].
In this review, we advocate that inflammation has a decisive role in
IPF. Delineating the mechanisms of the disease by using high throughput
technologies, the understanding of the disease pathogenesis will be
concrete and wider and also open the way for novel therapeutics and
diagnostics.
9. Conclusion
Inflammation does play role in the development of the disease. This
is demonstrated in different animal models as well as in studies in
human IPF patients. As the models used and modes of induction of the
disease in these models differ from study to study, there is non-
uniformity in the investigation and results obtained. Numerous cyto­
kines are shown to be involved in the disease. But their mechanistic role
in the disease is not studied extensively. With the usage of sophisticated
technologies in biochemistry and molecular biology, we expect delin­
eation of the mechanisms underlying the disease and thereby, probably,
the discovery of novel diagnostic and therapeutic approaches for pa­
tients suffering from IPF.
Authors contributions
The manuscript has been prepared and reviewed by Ms. Fathimath
Muneesa M in correspondence with Dr. Yashodhar Prabhakar Bhandary.
Sadiya B Shaikh added some more points and prepared the figures.
Jeena TM added some information and reviewed the entire manuscript.
Acknowledgments
Authors would like to acknowledge Yenepoya Research Centre,
Yenepoya University, Department of Biotechnology Grant (BT/
PR25198/MED/30/1896/2017), and Indian Council of Medical
Research (No.59/12/2015/Online/BMS/TRM) for supporting our
research.
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