A Refined Approach to Target the Molecular and Cellular Mechanisms in Pulmonary Fibrosis

A Refined Approach to Target the Molecular and Cellular Mechanisms in Pulmonary Fibrosis 13 Sabita Singh, Joytri Dutta, Archita Ray, Ashish Jaiswal, and Ulaganathan Mabalirajan Abstract The COVID-19 scenario has heated up the entire scientific community including those dealing with pulmonary fibrosis. The fibrotic sequelae of SARS-CoV2 have given value to the anti-fibrotic therapies that are being evaluated to prevent the severity of the pandemic. However, our understanding of the precise mechanism that drives fibrosis and knowledge about effective management of pulmonary fibrosis are still in the state of darkness. A landscape of pulmonary fibrosis (PF) and dismal prognosis continues to mar the progression of society over the past decades. It was in 2014 that two “umbrella” therapies were approved by the FDA for IPF management, nintedanib and pirfenidone, post which there are no significant additions in this field. An interplay between genetic and environmental factors leads to cause microinjuries to the alveolar epithelium. The maladaptive repair process over time contributes to the fibroblast proliferation and epitheliummesenchymal crosstalk leading to the pathogenesis of PF. Although there are several hurdles to combat this deadly disease, our next step is to develop efficacious treatment regimens that can ameliorate survivability and functional quality of life. This chapter presents recent updates in PF pathogenesis and possible novel therapeutic strategies. The authors declare no competing financial interests. Sabita Singh, Joytri Dutta and Archita Ray contributed equally with all other contributors. S. Singh · J. Dutta · A. Ray · A. Jaiswal · U. Mabalirajan (*) Molecular Pathobiology of Respiratory Diseases, Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 K. Dua et al. (eds.), Targeting Cellular Signalling Pathways in Lung Diseases, https://doi.org/10.1007/978-981-33-6827-9_13 311 312 S. Singh et al. Keywords Pulmonary fibrosis · Alveolar epithelial injury · Fibroblast · Epitheliummesenchymal crosstalk 13.1 Introduction 13.1.1 Pulmonary Fibrosis: A Global Challenge Pulmonary fibrosis (PF) is a chronic restrictive lung disease that is highly heterogeneous [1, 2] and refractory to any sort of treatment. The literal meaning of pulmonary fibrosis is lung scarring [2]. It is pathologically characterized by the progressive and irreversible deposition of excessive extracellular matrix (ECM) proteins such as elastin, fibronectin, hyaluronan, and collagen in lung interstitial regions [1] and remodelling of the lung architecture leading to thickening of the alveolar and peribronchial walls [3] and additionally characterized by noticeable clinical, physiological, and radiographic findings. Lung fibrosis develops due to various factors like exposure to various noxious chemicals, smoke, and also certain viral infections [1, 2]. Cancer patients who are undergoing radiotherapy and taking chemotherapy can also develop lung fibrosis. In addition, various toxins present in the air could cause microinjuries in gradual manner to the alveolar epithelium to initiate fibrogenesis in lungs [1]. September is the Global Pulmonary Fibrosis Awareness Month, and the awareness to combat year-on-year increase in mortality rate due to pulmonary fibrosis is the need of the hour. A much-needed awareness among patients with pulmonary fibrosis can avert its progression. However, part of the trouble comes while raising awareness of pulmonary fibrosis because of its complexity of the disease. Indeed, the term pulmonary fibrosis itself is an umbrella term as it comprises numerous similar lung diseases, and these diseases are together also called as interstitial lung diseases [4]. Due to the similarity of various diseases, it is very difficult to calculate the incidence of lung fibrosis. 13.1.2 Historical Perspective of Pulmonary Fibrosis It is well known that dust-rich occupation is strongly related to incidence of numerous lung diseases. Even Hippocrates observed the relation between respiratory problems and metal mining in his times [5, 6]. Later in the 1500s, the relation between the exposure to the dusty environment and the development of severe lung diseases has been reported [5]. The current illustration of pulmonary fibrosis crossed the mind of Hamman and Rich in the early twentieth century [4, 6] in which they found interstitial fibrosis in the lungs of few patients. In contrast to their observations 13 A Refined Approach to Target the Molecular and Cellular Mechanisms in. . . 313 of fast progression in the development of lung fibrosis, many patients have gradual progression. Due to the complexity and multifactorial nature of lung fibrosis, various clinicians and scientists have coined different terminology for the lung fibrosis [5]. 13.1.3 Types of Pulmonary Fibrosis Based on the Known Cause When a clinician sees a patient with the features of lung fibrosis, he cannot immediately identify the cause of lung fibrosis unless if there is any history of exposure to fibrogenic agents. If he can rule out all possible known reasons of lung fibrosis, it would be termed as “idiopathic” [7]. These are the five main known etiologies of pulmonary fibrosis: (a) Iatrogenic: Various medications are implicated in causing PF. Drugs like chemotherapeutics [1], antiarrhythmic (amiodarone), and certain antiinflammatory drugs (methotrexate) cause inflammation, injury, and scarring in the lungs. (b) Radiation-induced: Exposure to radiations can cause lung fibrosis [8]. (c) Environmental: Pulmonary fibrosis is also caused due to environmental cues like exposures to mold spores, agricultural/farming, bacteria, animal droppings (especially from caged/indoor animals), or other known triggers [1, 8]. (d) Autoimmune: With an autoimmune disease, a person’s own immune system attacks the lungs, inflicting inflammation and scarring that can impair lung function and breathing. Lung fibrosis is one of the pathological features of few systemic autoimmune diseases like Sjogren’s syndrome [1, 9]. (e) Occupational: Various work environments which are related to exposure to fibrogenic materials devastate the lung tissue, like silica, beryllium, etc. 13.1.4 Idiopathic Pulmonary Fibrosis (IPF): The Expert’s Conundrum A historical change has been swayed in the definition of idiopathic pulmonary fibrosis (IPF) since Hamman and Rich recounted the pathogenesis of pulmonary fibrosis. IPF, which is a devastating and a progressive form of interstitial lung disease with no known etiology, has now been taken up seriously by the researchers. Earlier it was believed that inflammation caused by exogenous irritants could drive the progression of lung fibrosis. However, this concept has changed as many started believing that the entire pathophysiology roots from alveolar epithelial dysfunction after lung epithelia are subjected to repetitive microinjuries followed by anomalous crosstalk between epithelial and mesenchymal cells, creating a disturbance in the balance of pro-fibrotic and anti-fibrotic mediators [10, 11]. Phenotypically, IPF is characterized as a chronic condition wherein the lung tissue, primarily the interstitium, and the space or tissue around the alveoli of the lungs become increasingly scarred and thick, leading to chronic cough, breathlessness, and, ultimately, respiratory failure and death [10, 12]. Reports suggest that the 314 S. Singh et al. death in IPF patients is solely due to the disease, followed by rapid deterioration of lung health and manifestation of lung cancer and cardiac disorders. Based on intense research using multiple strategies and models, relatively successful drugs like pirfenidone and nintedanib were discovered to reduce the development of lung fibrosis [1]. However, these drugs could not reverse the already established fibrogenesis, and as a result, these drugs cannot restore the lung function completely. Thus, intrinsic insight into the roles of the various mediators contributing in this condition is obligatory for developing considerable number of therapeutic interventions. 13.1.5 The Nexus Between Pulmonary Fibrosis and COVID-19 In December 2019, news and reports surfaced out from Wuhan, China, of the outbreak of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [13]. As of September 1, 2020, nearly 26.6 million people tested positive, and around 875, 000 people have died from coronavirus disease 2019 (COVID-19) worldwide. Patients with confirmed SARS-CoV-2 complain of diverse range of symptoms, from mere sneezing to severe respiratory distress [14]. Reports insinuate that all COVID-19-related serious consequences accentuate pneumonia. Interestingly, both COVID-19 and lung fibrosis are having common risk factors like old age, male gender, and disease comorbid conditions like diabetes and hypertension [13, 14]. Pulmonary fibrosis and COVID-19 have a stark resemblance, and at first sight, it won’t seem evident [15]. It solely depends upon us, what learning we can take from these two dreadful conditions in order to benefit patients who are having permanent scars in their lungs. COVID-19 and pulmonary fibrosis are grievous diseases, and both embark with a lung injury [15]. It is proposed that pulmonary fibrosis is an consequence of acute lung injury caused by viruses. The mechanisms through which SARS-CoV-2 hurls damage to the lung are only partially known [16], but probable contributors include a “cytokine storm” triggered by the viral antigen upon entry into the system resulting in a hyperactive inflammatory reaction, drug-induced pulmonary toxicity, and intubation or invasive ventilation upon hypoxemia, and high airway pressure induces acute lung injury [16]. 13.2 Genetic Factors Involved in the Development of Pulmonary Fibrosis The heritable form of pulmonary fibrosis is called familial pulmonary fibrosis (FPF). It is characterized by a history of pulmonary fibrosis in two or more members of a family. It is an autosomal dominant disease with low penetrance. Around 10% of the IPF cases are FPF [17]. 13 A Refined Approach to Target the Molecular and Cellular Mechanisms in. . . 315 13.2.1 MUC5B Gene Polymorphism A gene polymorphism for MUC5B, a gene encoding a mucin glycoprotein, has been extensively linked to both FPF and sporadic IPF. Homozygosity for the mutant allele (T) in single-nucleotide polymorphism of the MUC5B gene on chromosome 11p15.5 was found to increase the risk of having sporadic or familial PF by 20-fold [18]. This association has also been found in non-idiopathic fibrotic lung conditions like rheumatoid arthritis-associated interstitial lung disease (RA-ILD) and chronic hypersensitivity pneumonitis but not in some other forms of lung fibrosis like scleroderma-associated interstitial lung disease, sarcoidosis, and asbestosis [19]. This gain-of-function variant is associated with a heightened expression of the gene in the bronchoalveolar region. But, how the MUC5B variant increases susceptibility to PF is poorly understood. Overexpression of this gene impairs mucociliary clearance and enhances retention of inhaled particles and lung injury which might culminate into fibrosis in the long run [20]. The overexpression was found to ameliorate lung fibrosis but did not induce spontaneous fibrosis [19]. Quite contrary to expectations, only a small fraction of carriers of this risk allele was actually found to develop the disease indicating that genetic susceptibility alone is not enough to trigger the pathogenesis of PF [18]. 13.2.2 Surfactant Protein Mutations Surfactant protein C (SP-C), a constituent of surfactant, is solely synthesized by the alveolar type 2 epithelial cells (AEC2). More than 60 mutations of the surfactant protein C gene (SPTPC) have been linked to the pathogenesis of PF till date which amply highlights the importance of studying its mechanistic involvement in the disease [21]. The mutant protein has been found to accumulate in the endoplasmic reticulum (ER) causing ER stress and activating unfolded protein response (UPR). Prolonged or severe UPR activation may cause AEC2 cell apoptosis [18]. Recently, Nureki et al. demonstrated in a knock-in murine model of SPTPC mutation that this mutation indeed causes spontaneous PF in vivo. This shows that ER stress induced by the mutant SPTPC in AEC2 cells is involved in development of fibrosis in the lungs [22]. Surfactant protein A (SP-A), a surfactant protein secreted by the airway cells, is formed by the SPA1 and SPA2 proteins encoded by the genes SFTPA1 and SFTPA2, respectively [21]. Mutations in both SFTPA1 and SFTPA2 have been reported to have association with the pathogenesis of PF [21, 23]. SFTPA2 mutant protein was found to be retained in the ER and hence not secreted, subsequently activates UPR [21]. Takezaki et al. have recently shown that a homozygous SFTPA1 mutation induces AEC2 cell necroptosis and initiates spontaneous fibrosis in a knock-in murine model of the mutation [23]. 316 S. Singh et al. 13.2.3 Mutations in Other Genes Rare mutations in telomere-related genes like telomerase reverse transcriptase (TERT), dyskerin pseudouridine synthase 1 (DKC1), and regulator of telomere elongation helicase 1 (RTEL1) have been linked to FPF pathogenesis [17]. Short telomeres have been detected in PF patients in comparison to age-matched controls, irrespective of whether they carry telomerase gene loss-of-function mutations [18]. Gable et al. showed that zinc finger CCHC-type domain containing 8 protein (ZCCHC8) is necessary for telomerase RNA component (TERC) maturation, and its heterozygous loss-of-function mutation leads to short telomere length and FPF [24]. Polymorphism in and around Toll-interacting protein (TOLLIP) gene, a regulator of Toll-like receptors, has been implicated in IPF pathogenesis [25]. The protective role of TOLLIP in PF is further supported by another study that showed TOLLIP to have an inhibitory effect in the pro-fibrotic transforming growth factor (TGFβ) signalling in lung epithelial cells [26]. Genetic variations in desmoplakin (DSP), involved in cell adhesion, and Akinase-anchoring protein (AKAP13), predispose an individual to develop IPF. The expression of these proteins has been found to be elevated in IPF lungs [25]. In recent times, AKAP13 was shown to activate TGFβ (a key mediator of PF) in lung epithelial cells via Rho-αvβ6 pathway, thus providing a functional basis to its link with IPF pathogenesis [27]. 13.3 Various Inducers for the Development of Pulmonary Fibrosis PF can occur in response to a persisting lung condition like chronic inflammation or due to an unknown etiology, known as IPF. It can also occur due to certain fibrogenic agents like ambient particulate matter and soluble chemicals or secondary to some existing conditions like rheumatoid arthritis, sarcoidosis, etc. There is a controversy regarding wrongful labelling some patients as having IPF that might have occurred because of environmental or occupational exposure. Although this can be due to improper diagnosis, it can be also attributed to insufficient association between the intensity of exposure of the etiological factor and severity of the disease. Therefore, occupational or environmental exposures may partially prompt the development of IPF. Some of these factors can be exemplified as organic dust from activities like agriculture, livestock, and farming or exposure to wood dust, mineral dust, asbestos, metal, and ambient particulate matter [28]. 13.3.1 Silica Silica or silicon dioxide (SiO2) exists in a number of crystalline forms or polymorphs. Inhalation of fine crystalline silica of respirable conformation (<10 μm size) predisposes to the risk of developing a lung condition called silicosis. 13 A Refined Approach to Target the Molecular and Cellular Mechanisms in. . . 317 In silicosis, the fine silica particles are deposited in the macrophages. Thus, an inflammatory response ensues that incites the fibroblasts to multiply and produce collagen. The silica is enwrapped by collagen that leads to fibrosis and formation of nodular lesions in the lungs, which are typical of the disease [29]. 13.3.2 Asbestos Asbestos are crystalline mineral fibers that find commercial use due to their durable and heat-resistant nature. There are quite a few types, the chrysotile or “curly” fibers and the straight rod-like crocidolite fibers, which are the most common. Prolonged heavy exposure to asbestos gives rise to a diffuse PF called asbestosis. After inhalation, asbestos is taken up by the alveolar epithelial cells (AECs) and alveolar macrophages that lead to oxidative stress in the lungs. Both reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated on the surface of the fibers through cell-free mechanism. These oxidants damage the cellular macromolecules, especially DNA, triggering the apoptosis of AECs. Epithelial injury is known to provoke repair responses by releasing pro-fibrotic cytokines, leading to fibrosis [30]. Furthermore, macrophages on phagocytosis release pro-fibrotic mediators that trigger deposition of connective tissue by fibroblasts. Since asbestos fibers are naturally resistant to digestion, the macrophages perish, releasing more such cytokines and enticing more macrophages and fibroblasts to generate fibrous tissue [31]. 13.3.3 Iatrogenic Bleomycin-induced pulmonary fibrosis mice model is a well-known animal model for studying PF. Bleomycin is a glycopeptide antibiotic and anticancer drug that has been first isolated in 1966 from Streptomyces verticillus. For almost 50 years, bleomycin has been used in parallel to chemotherapy in squamous cell carcinomas, germ cell cancers, and malignant lymphomas which has proved to be highly effective due to its low immunosuppression and myelosuppression. It also showed potent effects against other types of cancers like cervical cancer, ovarian cancer, melanoma, and sarcoma. However, its high therapeutic efficiency is severely restricted by its potential to cause PF [32]. 13.3.4 Paraquat Paraquat (PQ) is the most common herbicide used worldwide. It accumulates in the lung epithelial cells after exposure and causes significant damage. As a result of this, repair responses are triggered, leading to the development of PF [33]. In one study, collagen deposition was found to occur after 2 h of PQ administration. A separate study has shown epithelial to mesenchymal transition (EMT) to be a central event in 318 S. Singh et al. the development and progression of PF. PQ being extremely toxic to human beings has a high mortality rate of 60–87.5%, PF being detected in most of the survivors [34]. 13.3.5 Polyhexamethylene Guanidine (PHMG) The PHMG is a biocide and disinfectant which was initially thought to be safe and nontoxic. Later, when it was used as a humidifier disinfectant, it was shown to be associated with PF [35]. A study by Kim et al. showed that PHMG can trigger ROS generation, causing airway barrier injury, followed by fibrotic repair response with increased synthesis of collagen and fibronectin and increased ECM deposition [36]. 13.3.6 Carbon Nanotubes (CNTs) The CNTs are 1-atom-wide graphene sheets with tube-like structures. They are extensively used for commercial and industrial purposes owing to some of their useful properties like remarkable strength and high thermal conductivity. Individuals involved in the manufacture of CNT-containing products and the consumers of such items are extensively exposed to this material. CNTs act like airborne fibers due to their nanoscale size and shape that is a like a fiber, thus increasing the risk of being inhaled. The inhaled fibers can accumulate in the airways and the alveoli. They are capable of inducing oxidative stress and pass through the plasma membrane, leading to epithelial cell lesions. Disruption of the epithelial barrier in the airways and type 2 alveolar epithelial cells leads to the secretion of alarmins that elicit type 1 or type 2 immune responses. Activated T helper 2 (Th2) cells and M2 macrophage induce the release of pro-fibrotic factors to promote tissue repair and lung fibrosis in type 2 immune response [37]. 13.3.7 Lung Fibrosis as a Part of Other Diseases Sarcoidosis is an inflammatory disease induced by a yet to be identified antigen, characterized by the formation of granuloma, most frequently in the lungs. Due to its unidentified etiology, specific treatment is still unavailable. About 20% of the patients having sarcoidosis are found to develop PF, which transforms this benign disease into a fatal one. In such cases, fibrosis starts at the fringes of the sarcoid granulomas. Progressive collagen deposition over time gives rise to mature fibrosis that damages the lung parenchyma, ushering in the “end-stage” sarcoidosis [38]. Rheumatoid arthritis (RA) is an inflammatory condition of autoimmune nature, affecting the joints. RA patients have sometimes been found to have interstitial lung disease, named rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Clinically evident RA-ILD has been found in almost 10% of the RA patients [39]. Although the exact mechanism is not yet clear, two theories have been put 13 A Refined Approach to Target the Molecular and Cellular Mechanisms in. . . 319 forward based on available evidence regarding the pathology of this disease. First, external insults like cigarette smoke cause citrullination of proteins in the lungs, which stimulates the synthesis of anti-citrullinated protein antibodies (ACPA). This triggers breakdown of self-tolerance in RA. The second theory propounds that in local or systemic inflammation of chronic nature (as in RA), circulating inflammatory cytokines [interferon γ (IFNγ), tumor necrosis factor α (TNFα)] act on the lungs inducing an increase in expression of adhesion molecules on the pulmonary vascular endothelial cells. These signals attract the circulating monocytes/macrophages to the lungs and also activate lung-resident macrophages and other immune cells. On exposure to external factors like cigarette smoke, antigen presentation (of citrullinated proteins) occurs, triggering the activation of secondary or adaptive immune response and the resultant production of ACPA. The second bout of inflammation also stimulates the release of pro-fibrotic TGFβ and subsequent fibroblast activation and collagen deposition, leading to lung fibrosis [40]. 13.4 Lung Injury: A Predominant Inducer for Pulmonary Fibrosis Injury to the lung tissue can occur as a result of various insults, such as mechanical stretch, exposure to high doses of radiation, microbial infection, or gastroesophageal reflux. 13.4.1 Mechanical Stretch Mechanical ventilation (MV) is an essential supportive therapy for patients having acute respiratory distress syndrome (ARDS), but it is capable of causing lung injury, named as ventilator-induced lung injury (VILI). VILI is caused either by recurrent opening or closure of the alveoli, i.e., the lungs can become derecruited with each expiration and recruited once again in the subsequent inspiration (atelectrauma), MV-associated inflammation (biotrauma), or overdistention of the lungs (volutrauma) [41]. Mechanical stretches to the alveolar epithelial cells lead to disruption of the tight junction and cellular attachments. Biotrauma heightens the production of ECM, triggering the repair response. The loss of alveolar epithelial integrity and overexuberant repair response induces EMT. There is also an increase in circulating fibrocytes and fibroblast proliferation. These events collectively contribute to the development of PF [42]. 13.4.2 Exposure to High Doses of Radiation Treatment by radiation of malignancies present in the lung, breast, and esophagus increases the vulnerability of patients to radiation-induced lung injury (RILI). The sensitivity of the normal lung parenchyma is the deciding factor for the dose of 320 S. Singh et al. radiation in the malignancies of the chest. When the radiation penetrates the soft tissue, the localized aggregation of energy packets induces the breakdown of the strong chemical bonds of mainly water molecules, generating free radicals. These free radicals damage the cellular macromolecules, primarily DNA, and also lipids and proteins. This DNA damage sets in motion the downstream processes, which culminate into cell death in both normal and cancer cells. To increase the therapeutic efficacy and reduce toxicity of radiation, the total dose of radiation has to be broken down into small daily doses. This ensures enough time for recovery of the normal epithelial cells in comparison to malignant cells, owing to their differential DNA repair capacity and radiation sensitivity. Apart from the cytotoxic effects, strong inflammatory responses are also incited which eventually culminates into PF, the final phase of RILI. In this phase, pro-fibrotic mediators are released, and these lead to deposition of collagen by fibroblasts in the alveolar spaces and resultant reduction in lung volume [43]. 13.4.3 Lung Fibrosis as a Post-SARS Sequel Evident from the clinical studies and radiography, many persons who were infected by severe acute respiratory syndrome (SARS) in 2003 epidemic were found to have PF, as fibrosis of varying degrees was noticed on autopsy of the deceased patients. Although such fibrotic changes have also been observed in other respiratory viral infections, it was found to be more frequent in post-SARS coronavirus (SARS-CoV) infection. Increased levels of TGFβ, a pro-fibrotic mediator, have been found in the mouse models of SARS-CoV infection [44]. The SARS-CoV strain has genetic similarity with SARS-CoV-2, the cause of the 2020 pandemic that is wreaking havoc worldwide. Preliminary analysis of the COVID-19 patients on hospital discharge indicates that greater than one-third of these recovered individuals have fibrotic abnormalities [14]. 13.4.4 Link with Gastroesophageal Reflux Gastroesophageal reflux is the motion of gastric fluid in a retrograde direction toward the esophagus [45]. In states of waned consciousness as when a patient is under the influence of general anesthesia or conditions like cerebral vascular ischemia, trauma, and metabolic encephalopathies, the gastric refluxate may aspirate into the lower airways. The aspirate material can be composed of acid, bile, food particulates, pepsin, and microbes [46]. In order to reach the airways, the aspirate has to evade a lot of barriers such as esophageal peristalsis, cough reflex, swallow reflex, and mucociliary barrier [45]. The gastric contents are capable of inflicting injury to the lung epithelium and consequently trigger sterile inflammation [46]. Chronic microaspiration causes recurrent injury which eventually over a period of time results in lung fibrosis [45]. 13 A Refined Approach to Target the Molecular and Cellular Mechanisms in. . . 13.5 321 Alveolar Epithelial Dysfunction as a Converging Point for the Development of Pulmonary Fibrosis The distal lung is formed of two alveolar epithelial cell types, namely, type 1 and type 2. Type 1 alveolar epithelial cells (AEC1) are situated near the endothelial cells and form a surface for gaseous exchange. AEC2 function as stem cells by differentiating into AEC1 cells for repair and renewal of the alveolar epithelium. Besides, they are also responsible for surfactant production [47]. Haschek and Witschi proposed a theory, in the 1990s, challenging the pre-existing notion that lung fibrosis is an inflammatory disease. After that, there was a gradual shift in the concept when it was realized that although inflammation contributes to the disease pathogenesis, it is not the central event that drives PF. AEC2 dysfunction was eventually recognized to have a key role in development of PF [21]. Multiple factors like genetic predisposition, ROS, hypoxia, and infection trigger ER stress in the AECs. There is a buildup of unfolded proteins inside the ER which activates the unfolded protein response. A downstream process ensues with the increase in chaperone proteins in order to aid protein folding. The AEC2 cells in the fibrotic lungs are unable to deal with ER stress owing to defective autophagy, which normally helps to remove the expanded ER [47]. There is also downregulation of lipid synthesis enzymes like stearoyl-CoA desaturase (SCD1), which is needed for reducing ER stress in the AECs [48]. Persistent ER stress leads to the activation of the ER stress-associated transcription factor, C/EBP homologous protein (CHOP), triggering apoptosis of the AEC2 cells [47]. Hypoxic microenvironment in the alveoli has been shown to stabilize hypoxia-inducible factor 1α (HIF1α) that triggers ER stress and CHOP-mediated transcription of apoptotic genes like B cell lymphoma 2 (Bcl2), Bcl2-like protein 11 (Bim), and cation transport regulator-like protein 1 (Chac1) [49]. Kamp et al. showed that oxidative stress induced by asbestos fibers induces ER stress followed by mitochondria-mediated apoptosis via ER Ca2+ release [50]. AEC2 cells, being metabolically active, possess a large number of mitochondria [51]. Mitochondrial fusion proteins mitofusin 1 and mitofusin 2 were shown to be involved in the production of surfactant lipids in AEC2 cells, and their absence triggers spontaneous lung fibrosis. This shows that damaged mitochondria affect the AEC2 surfactant production, thus compromising the integrity of epithelial barrier eventually leading to fibrosis [52]. Instead of apoptosis, AECs can also undergo premature senescence, a permanent form of cell cycle arrest, in response to injury [47]. Senescence in AECs is regulated by the activation of PTEN/NF-kB pathway that drives collagen deposition by fibroblasts and, consequently, fibrosis [53]. Moreover, plasminogen activator inhibitor-1 (PAI-1) has also been shown to induce AEC2 senescence by activating the cell cycle repressor p53-p21-Rb pathway in fibrotic lungs [54]. AEC2 senescence in lung fibrosis has been also attributed to dysfunctional telomere [51]. Senescence limits the capacity of transdifferentiation of AEC2 cells into AEC1 cells as observed in PF. Activation of developmental pathways like Notch and Hedgehog significantly affects AEC2 differentiation in fibrotic lungs [51]. Homeobox only protein X (HOPX), a protein involved in the development of distal lung, was found 322 S. Singh et al. to promote AEC2 to AEC1 differentiation in early stages of alveolar injury but decreases with progression of IPF, leading to the loss of reparative capacity of AEC2 cells [55]. Recently, Wu et al. showed that failure to differentiate into AEC1 generates mechanical tension in AEC2 cells which activates the pro-fibrotic TGFβ pathway [56]. In previous studies, it has been shown that mechanical stress in AECs induces epithelial cell-restricted integrin αvβ6 to bring about conformational changes in the inactive precursor latent complex of TGFβ in the ECM, revealing the active TGFβ that can bind to its receptor on neighboring cells and exert its fibrotic effects [57]. The impact of this mechanical tension progress from the periphery to the center which provides a possible explanation for the periphery to center progression observed in IPF [56]. AEC2 cells have also been observed to go through EMT and drive fibrosis through epithelial-fibroblast crosstalk [58]. Thus, external insults and genetic factors set in motion a series of events in the AECs such as ER stress, impaired autophagy, intrinsic apoptosis, and cellular senescence, triggering an aberrant repair response in the lungs which culminates into fibrosis. 13.6 Role of Inflammatory Cells and Mediators in Pulmonary Fibrosis In general, the role of innate immune cells like macrophage and neutrophil in the development of lung fibrosis is well known. However, the role of inflammation or immunological involvement in IPF is controversial as a number of studies have ruled out the involvement of immunological mechanisms in the IPF development [59]. Based on the earlier observation like the existence of more neutrophils in lung interstitium of IPF patients, it was hypothesized that IPF could be a chronic inflammatory disease. Later, a number of studies could not verify the involvement of inflammation. Initially, it was believed that the infiltration of inflammatory cells is the primary reason for the development of fibrosis. Currently, this concept has been modified in such a way that inflammation may have a role only after the establishment of lung fibrosis [59]. The immunopathogenesis could involve both innate and adaptive immune cells. The neutrophils, macrophages, fibrocytes, monocytes, dendritic cell, mast cells, and type 2 innate lymphoid cells are the innate immune cells, whereas almost all kinds of helper T cells (Th1, Th2, Th17), Treg, and B cells are adaptive immune cells [59]. Macrophages can be of two types: (a) M1 if the macrophages are induced by TNFα or IFNγ and (b) M2 if the macrophages are induced by cytokines like IL-4, IL-13, IL-10, or TGFβ1. While M1 macrophages are formed in stages of acute inflammation, M2 macrophages are formed when the inflammation is getting resolved. During the resolution of inflammation, fibrosis occurs. In IPF, the M2 macrophages are activated, and these macrophages secrete TGFβ1 [59, 60]. Though neutrophils are recruited at the peak stage of inflammation, its role in IPF is beyond the inflammation as it helps in tissue remodelling through their enzymes like 13 A Refined Approach to Target the Molecular and Cellular Mechanisms in. . . 323 neutrophil elastase [59, 60]. Though the role of extracellular neutrophil traps has been shown to be present in a number of fibrotic conditions, its involvement in IPF is not clear yet. Fibrocytes, derived from blood monocytes, are present in the circulation. But when there is an injury, these cells are recruited to the site of injury. When they get activated, these cells further activate the fibroblast to induce the deposition of various ECM proteins. While Th2 cytokines like IL-4 and IL-13 can participate in the development of lung fibrosis by activating M2 macrophages, type 2 ILCs (innate lymphoid cells) also secrete most of these Th2 cytokines along with amphiregulin. IL-13 secreted by type 2 ILCs participate in lung interstitium remodelling along with the deposition of ECM proteins [59]. Similarly, Th2 cells of adaptive immune response also secrete cytokines like IL-4 and IL-13. Both of these cytokines are capable of activating myofibroblasts so that they secrete more ECM protein to cause lung fibrosis. On the other hand, IFNγ secreted by the Th1 cells are known to attenuate the features of lung fibrosis [59]. While B cells can produce autoantibodies against epithelial antigens to initiate an autoimmune response, regulatory T cells have been shown to reduce autoimmune response along with its anti-fibrotic role in lung fibrosis. 13.7 Role of Oxidative Stress in Pulmonary Fibrosis Though the involvement of immunopathogenesis or inflammation in IPF is controversial, the role of oxidative stress in lung fibrosis especially in IPF is well demonstrated [61, 62]. In any event, its causative role in IPF is not proven yet. As IPF affects mostly elder people, it is also important to consider whether aging acts as a factor to induce oxidative stress, because dysfunction of mitochondria, reduction in proteostasis, and other oxidative stress-related mechanisms of aging could be involved in the development of IPF [62]. Oxidative stress participates in a number of ways in the pathogenesis of IPF, starting from the predisposition stage to the progression stage. Both structural cells like alveolar epithelial cells, fibroblasts, vascular endothelial cells, and immune cells like macrophages and neutrophils can generate ROS and RNS. On the other hand, most of these cells also generate various antioxidant enzymes. However, the antioxidant mechanisms are reduced with aging. Each and every abovementioned cell has a unique way of participation in promoting lung fibrosis. For example, oxidative stress helps in the polarization of macrophage to promote lung fibrosis. While ROS can be pro-inflammatory and thus polarize macrophages toward M1, ROS released by dysfunctional mitochondria and ER stress could polarize the macrophage into pro-fibrotic M2 macrophage through TGFβ, platelet-derived growth factor (PDGF), and tissue inhibitor of metalloproteinase [62]. Oxidative stress-induced cellular senescence observed in structural cells is linked to the development of lung fibrosis. Whether the cellular senescence is involved with pro-fibrosis or anti-fibrosis depends on the organ and type of senescent cells. In case of lung fibrosis, senescence of alveolar epithelial cells and myofibroblasts has been well demonstrated. The senescent lung epithelia may 324 S. Singh et al. reduce the rate of re-epithelialization to produce pro-fibrotic cytokines. Senolytics, a new variant of drugs that selectively targets the senescent cells, have been shown to reduce the deposition of ECM proteins [62]. Lung epithelial cell apoptosis can happen due to mitochondrial dysfunction or ER stress due to oxidative stress. The alveolar epithelial apoptosis leads to aberrant activation of fibroblasts and consequently ushering in lung fibrosis [61, 62]. In contrast, the apoptosis of myofibroblasts is reduced in lung fibrotic conditions. This could lead to the accumulation of more number of myofibroblasts in the lung interstitium, and these activated myofibroblasts secrete a variety of ECM proteins to cause dense lung fibrosis. Not only activated myofibroblasts participate in the deposition of ECM proteins, but also senescent fibroblasts can also promote lung fibrosis through attaining senescence-associated secretory phenotype that leads to express a number of pro-fibrotic genes including collagen, fibronectin, etc. [62]. While ROS released by various cells lead to fibrosis through various mechanisms, oxidation of even ECM proteins could lead to worsening of lung fibrosis by hardening of ECM along with resistance to proteolysis [62]. Thus, oxidative stress serves as a pro-fibrotic mechanism in a peculiar and a context-dependent manner. The role of antioxidants in reducing lung fibrosis seems to be controversial due to the additional anti-fibrotic role of ROS. However, finding effective and site-targeted antioxidants may be beneficial in reducing lung fibrosis. 13.8 Role of MMPs in the Development of Pulmonary Fibrosis As mentioned above, alveolar epithelial cells are injured to initiate the development of pulmonary fibrosis. These alveolar epithelial cells secrete multiple mediators that promote fibroblast generation and myofibroblast accumulation which in turn alters the normal lung architecture. Zinc-dependent matrix metalloproteases (MMPs) belong to a larger family known as M10A metallopeptidase family. They degrade the ECM and can also cause casting away of cell membrane proteins. MMPs lead to activation of other mediators like cytokines, growth factors, chemokines, etc. In IPF, imperfect regulation of MMPs has a crucial role. MMP family consists of many genes. On one hand, some MMPs have a protective role against fibrosis. On the other hand, MMPs can promote and also contribute to disease progression [63]. MMPs cause parenchymal remodelling by accumulation of ECM constituents in lung interstitium which further aggravates the progression of the disease. Anti-protease treatment is thus thought to be beneficial to stabilize the balance between excessive synthesis and degradation of ECM [64]. Bleomycin-induced fibrosis rat model had shown initial elevation of MMP-9 level followed by reduction. Similarly, MMP-2 level increases and decreases more rapidly. In BAL (bronchoalveolar lavage) fluid, MMP-2 and MMP-9 activity is seen to be heightened in the early phase of pulmonary fibrosis; however, the level then gradually decreases. The level of change in MMP-9 was more prominent when compared to MMP-2. This may be due to the reason that MMP-9 is secreted by neutrophils. Alveolar macrophages and neutrophils release these MMPs which 13 A Refined Approach to Target the Molecular and Cellular Mechanisms in. . . 325 degrades the basement membrane. The breached basement membrane then allows the infiltration of inflammatory cell, and here the role of MMP-9 seems to be more. MMP-2 is hypothesized to act more in the later fibrosis phase and has a role in the generation of the fibrotic foci [65]. Tissue inhibitor of metalloprotease (TIMP) family proteins blocks the action of MMPs. Myofibroblast residing in the intraalveolar fibrosis showed predominant reaction toward TIMP-2. Thus, TIMP-2 might have a role in irreversible lung structure remodelling observed in IPF. Matrix metalloprotease-1 (MMP-1) level is also found to be overexpressed in IPF. There is a slight paradox regarding MMP-1. In lung obtained from IPF, MMP-1 level is found to be localized in the AEC and not in fibroblasts [66]. Along with MMP-1, the level of MMP-7 (matrilysin) is seen to be increased in pulmonary fibrosis. The level of MMP-7 gets increased in lung and BAL fluid in comparison to control subjects. The surge in MMP-7 and MMP-1 level is correlated with the increase in the severity of disease. These can be the rationale for taking MMP-7 as a biomarker in asymptomatic patients suffering from early interstitial lung disease. Enhanced level of MMP-7, found in peripheral blood in IPF, shows correlation with pulmonary function test. Thus it can be a potential biomarker in IPF pathobiology [67]. MMP-3 has a unique role in promoting IPF. There is heightened expression of MMP-3 mRNA in the IPF lung. Experiments carried out in vitro on epithelial cells have shown that MMP-3 mediates EMT. MMP-3 works via inducing the ß-catenin/ WNT pathway and causes the cleavage of epithelial cell marker E-cadherin. Upon MMP-3 treatment in lung epithelial cells, there is an increase in the expression level of mesenchymal marker, vimentin [68]. Another novel biomarker in fibrotic lung disease is MMP-10. In comparison with control and COPD patients, MMP-10 level is higher in sera of IPF patients. Immunohistochemistry studies have shown that apart from alveolar epithelial cells, macrophages and peribronchial epithelial cells also express MMP-10 [69]. MMP-8 (collagenase-2) is another pro-fibrotic MMP that promotes PF. In IPF, the MMP-8 level gets increased in BAL fluid. Studies revealed that MMP-8 decreases the expression of macrophage inflammatory protein1 α (MIP-1α) and IFNγ-inducible protein 10 (IP-10) in the lung. Fibrocytes have been shown to express MMP-8. In PF, migration of fibrocytes gets increased. Fibrocytes incubated with MMP-8 inhibitor showed a decrease in their migratory activity. This accentuates the fact that MMP-8 heightens PF by regulating the migratory capacity of fibrocytes [70]. Few MMPs exert anti-fibrotic effects. MMP-13 (collagenase-3) is a primary enzyme that causes degradation of cartilages [70]. Compared to donor lungs, an increase in the levels of MMP-13 is observed in lungs of IPF patients. In bleomycinexposed MMP-13 knockout mice, a severe increase in symptoms of fibrosis along with early onset of inflammation is observed [71]. Another anti-fibrotic regulator in IPF is MMP-19. When MMP-19 knockdown mice were exposed to bleomycin, heightened PF characteristics were observed. Another study showed that fibroblasts obtained from MMP-19-deficient mice showed an increase in fibrillar collagen proteins, collagens present in the basement membrane along with an elevation in collagen production. The growth pace of the fibroblast was also found to be increased. Along with these, production of myosin genes gets increased, and an 326 S. Singh et al. increase in myofibroblast is also observed. MMP-19 thus has a controlling effect on IPF by regulating fibroblast growth and its differentiation into myofibroblast [72]. MMP has a regulatory effect on fibrosis. With MMP expression, various pathways also get affected. Thus, MMP can be a potent therapeutic target in PF. But we need to be cautious for the following reasons: (a) dynamicity of MMP in different stages of PF and (b) MMP inhibition that could cause havoc in lung homeostasis as a number of MMPs are required to fight against various infections by recruiting various immune cells and to activate key cytokines and chemokines that are involved in immune cell recruitment. 13.9 Disturbance in Balance of Pro-fibrotic and Anti-fibrotic Molecules Fibrosis is an irreversible degenerative biological phenomenon which encompasses numerous other smaller events. Upon sensing the tissue degradation, scar formation takes place. This happens through excessive production of the ECM proteins and deposition of connective tissue like collagen. The cellular environment also undergoes certain alterations to decide whether the fibrotic pathway will be activated or not. There are certain pro-fibrotic agents that promote fibrosis. They are type 2 CD4 T lymphocyte cells and CD40 ligand-receptor interaction. There are also other mediators like cytokines and growth factors that accelerate the fibrotic process. Anti-fibrotic agent like interferon γ also exists [73]. CD40 receptors are expressed on the surface of fibroblasts. Immune cells like mast cells and T cells express CD40 ligand. In PF, CD40 ligand-receptor interaction stimulates the production of pro-inflammatory mediators and cell adhesion molecules like cyclooxygenase 2 (COX2) which then increases the production of ECM proteins. Prostaglandin 2 (PGE2) synthesis happens in the human fibroblasts which then disturbs the type 1/type 2 cytokine balance. Type 2 cytokine productions are also increased. On the other hand, generation of IL-12 and IFNγ which belong to type 1 cytokine family are inhibited [74]. T lymphocytes are primary inflammatory cells in fibrosis. Compared to wild type, bleomycin-administered T cell-deficient athymic mice showed less inflammation and fibroblast deposition on ECM. Immunosuppressive T regulatory (Treg) cells exert pro-fibrotic effect via pro-fibrotic cytokines like TGFβ and PDGF [6]. There is a balance transpiring between pro-fibrotic and anti-fibrotic cytokine mediators. They are like the two sides of a seesaw. The pro-fibrotic cytokines are TGFβ, TNFα, vasoconstrictor molecule endothelin-1 (ET-1), and interleukins. TGFβ family assists in the production of fibroblast collagen gene. TNFα which gets upregulated in PF promotes the replication of fibroblasts and escalates the synthesis of collagen in the lung. ET-1 found in the lungs of fibrotic patients escalates proliferation of fibroblasts, promotes chemotaxis, and increases pro-collagen synthesis [75]. Interleukin-9 (IL-9) is another anti-inflammatory cytokine that has a role in attenuating lung fibrosis. B lymphocytes have a role in regulating the protective role of IL-9 [6]. Coagulation-regulating proteinases like thrombin and factor VIIa employ pro-fibrotic effect in the remodelled lung. These act by regulating the 13 A Refined Approach to Target the Molecular and Cellular Mechanisms in. . . 327 proteinase-activated receptors 1–4 (PAR-1–PAR-4). PAR-1 which imparts its effect by factor Xa and thrombin is found to have a prominent role in IPF. Experimentally it is seen that inhibiting the pro-inflammatory mediator thrombin prevents fibrosis in the IPF setting. PAR-1-deficient mice are also seen to be insulated from bleomycinmediated injury [76]. In normal lung, there is a balance between pro-fibrotic collagen-synthesizing cytokines and anti-fibrotic collagen-inhibiting cytokines. Such balance existing between the positive and negative collagen-producing cytokines is found to be hampered in fibrotic lung. The synthesis of collagenpositive cytokines is higher in the fibrotic lungs. Pro-fibrotic cytokines work in a redundant manner. These cytokines act on diverse cell phenotypes and affect various mediator responses. Apart from affecting collagen synthesis, cytokines regulate recruitment of adhesion molecules, impact leukocytes, and also affect the inflammatory pattern in PF. These pro-fibrotic cytokines do have a role in accelerating the growth of fibroblast cells at injury sites. Hence, this cytokine balance is very crucial in the pathology of PF. The cytokine-dependent therapeutic strategy is another approach targeting PF. Further studies should aim at finding more anti-fibrotic agents to maintain the positive-negative collagen balance in PF [75]. 13.10 Therapeutic Targets in Pulmonary Fibrosis While numerous clinical trials aimed at developing therapies for pulmonary fibrosis are in the pipeline, pirfenidone and nintedanib have been shown to be relatively successful [77] and are available in the market. Our understanding of the complex pathobiology of pulmonary fibrosis has refined gradually over time, and this has modified the perspective to treatment. Nevertheless, most of these trials conducted have been gloomy, probably due to the milieu of mediators, complexity of the disease, and signalling pathways involved in the fibrotic process of IPF. Thus, a deeper investigation of the cellular and molecular mechanisms involved in the pathobiology is necessary in order to harness the knowledge and utilize them in developing new therapeutic targets and drugs. Below we discuss the evolving therapeutic targets which hold a promising future in combating the development and progression of pulmonary fibrosis. 13.10.1 Leukotriene Receptor Antagonists Leukotrienes, derived from the oxidative metabolism of arachidonic acid, are inflammatory mediators and pro-fibrotic metabolites. In 1996, it was first demonstrated that leukotrienes were found to be elevated in the lungs of IPF patients. Thus, leukotrienes may be targeted for developing therapies for PF. Tipelukast (MN-001), an antagonist of the leukotriene receptor, is currently being explored [78], and it is undergoing a phase 2 trial. Initial data obtained from this study suggests that it is generally safe and well tolerated [78, 79]. 328 S. Singh et al. 13.10.2 Targeting B Lymphocyte Various abnormalities in B cell functions including the presence of increased autoantibodies have been demonstrated in IPF patients indicating the importance of B lymphocyte in pathogenesis of IPF [80]. And, now that there is evidence showing autoimmunity is involved in the genesis of fibrotic condition, drugs targeting autoimmune processes will be an effective therapy. Hence, the CD20 surface molecule of B lymphocytes is being targeted to rescue the lung damage inflicted by the autoantibodies generated during IPF. Rituximab, an antibody targeting the CD20 surface molecule of B lymphocytes, is currently being assessed in IPF patients [78, 79, 81]. Results of these trials are still pending; however, positive results of these investigations will substantiate the efficacy and pharmacokinetics of rituximab treatment in IPF patients. 13.10.3 Protein Kinase Inhibitors The pro-fibrogenic role of several tyrosine kinases including PDGF, vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) has been implicated in lung fibrosis [82]. So, more exploration has been done to identify novel drugs using various relevant kinases as targets. Two such kinases were Rho-associated coiled-coil-containing protein kinase (ROCK2) and c-Jun N-terminal kinases (JNK). So KD025 and CC-90001 were discovered as inhibitors of ROCK2 and JNK, respectively. Both of these inhibitors have reached phase 2 trial [79, 83]. 13.10.4 Phosphoinositide 3-Kinase/Protein Kinase B/Mammalian Target of Rapamycin (PI3K/Akt/mTOR) Pathway Inhibitors A variety of vital cellular functions starting from cell differentiation to proliferation are tightly regulated by this PI3K/Akt/mTOR pathway [84]. The conversion of fibroblasts to myofibroblasts is associated with the increase in the expression of various isoforms of PI3K. Thus, the inhibition of PI3K might halt the fibrosing processes. This hypothesis was proven by the demonstration of the effects of omipalisib, which inhibit PI3K as observed in the phase 1 study [78, 83] Similarly, sirolimus, a mTOR inhibitor, had reached the phase 2 trial with anti-fibrotic properties. On the one hand, PI3K/AKT is the upstream regulator of ER stress, and on the other hand, ER stress-associated proteins were found to be increased in pulmonary fibrosis [84]. These evidences indicate that either ER stress inhibitors or a PI3K inhibitor could halt lung fibrosis [84]. 13 A Refined Approach to Target the Molecular and Cellular Mechanisms in. . . 329 13.10.5 Anti-Integrin Antibodies Integrins are cell adhesion molecules that facilitate the adhesion and movement of cells on ECM. Beyond cell adhesion and migration, integrins were also shown to activate crucial kinases like epidermal growth factor receptor (EGFR) [78]. Reports have demonstrated that integrin family members are the key mediators of tissue fibrosis. The activation of TGFβ needs the help of αvβ6 integrin that is present exclusively in the lung epithelium. Thus, one can expect the inhibition of TGFβ activation along with a reduction in the features of lung fibrosis by inhibiting αvβ6 integrin [78, 79]. In a mice model of lung fibrosis, αvβ6 integrin antibody-mediated partial neutralization has been shown to reduce the features of lung fibrosis. In this context, the humanized monoclonal antibody against the αvβ6 integrin (BG00011) trial (phase 2) is ongoing after a pilot study that was performed to determine the efficacy of the antibody on TGFβ signalling inhibition. The outcomes of the study are still awaited [79]. 13.10.6 Anti-Connective Tissue Growth Factor Connective tissue growth factor (CTGF), a key glycoprotein, is involved in the process of fibrotic conditions along with TGFβ [78, 79]. Reports have suggested that CTGF due to its capability of producing collagen and fibronectin deposition in wound healing is also involved in the production of the extracellular matrix. Excess expression of CTGF upregulates several growth factors such as TGFβ [79]. It activates myofibroblasts which are responsible for fibrosis and tissue remodelling, consequently leading to various pathological conditions. Researchers have recently taken upon targeting CTGF which seems to be a promising therapeutic approach to combat the development of fibrosis. Pamrevlumab is a human monoclonal antibody against CTGF [85]. Preclinical studies and open-label phase 2 trials indicate the effectiveness of pamrevlumab. A randomized phase 3 clinical trial (NCT03955146) is set to start for pamrevlumab. More recently, PBI-4050 was developed and has demonstrated anti-fibrotic activities by reducing levels of CTGF [78, 86]. It has undergone a phase 2 evaluation, open-label, and it seems it does not have any safety issues. 13.10.7 Inhibitors of Autotaxin-Lysophosphatidic Acid Autotaxin (ATX) is an extracellular enzyme that produces lysophosphatidic acid (LPA), a lipid molecule responsible for releasing pro-inflammatory mediators and recruitment of fibroblast and epithelial apoptosis [87]. The autotaxinlysophosphatidic acid (ATX-LPA) pathway has been demonstrated to be involved in various pathological and fibroproliferative disorders, including pulmonary fibrosis [88]. The increased levels of autotaxins have been found in IPF patients indicating that the ATX-LPA pathway could yield successful anti-fibrotic inhibitors 330 S. Singh et al. [83]. Cuozzo and his group have screened a number of inhibitors of autotaxin and obtained a potent inhibitor, X-165 [89]. The FDA has cleared the way for X-165 and has moved into the first clinical test as a therapy candidate. 13.10.8 Pentraxin-2 (PTX-2) Analogues The PTX-2 is a naturally occurring plasma protein that controls various facets of the innate immune system as it has the ability to regulate the conversion of blood monocytes into pro-fibrotic macrophages and is also involved in the process of wound healing [78]. Studies have shown reduced levels of PTX-2 in IPF conditions correlating with the severity of the disease. Administration of PTX-2 blocks the bleomycin-induced fibrotic damage in mice [78, 90]. Preclinical data showed a reduction in fibrotic conditions upon using PRM-151. Based on the phase 1 trial, PRM-151 was seen to significantly increase the blood levels of PTX-2 [78, 79, 90]. A very recent double-blind, phase 2 study (NCT02550873) of PRM-151 has demonstrated a significantly better exercise capacity compared to the placebo as well as a slower decline in lung function. Further studies are needed to evaluate its safety and efficacy. 13.10.9 Targeting the Respiratory Microbiota The doctrine of “lung sterility” which persisted in the medical literature for decades has long been debunked. Researches and evidence now indicate how the lung and microbiota interact and exist. Since the Nobel Prize discovery by Marshall et al. for identifying the bacterium H. pylori and its causative role in gastric inflammatory disease [91], the perspective toward the involvement of microbes in chronic conditions has changed. The exploitation of the flora has now become a pragmatic therapeutic and prophylactic approach for many infectious and inflammatory diseases [91, 92]. Various reports have demonstrated that the severity of IPF is strongly correlated with the presence of certain microbiome in the lungs [92]. 13.11 Pirfenidone and Nintedanib: Success in Pulmonary Fibrosis Though the immunological role in the development of IPF is controversial, it was believed that immunosuppressive therapy was the primary option for IPF therapy earlier [93]. Indeed, corticosteroids and azathioprine were used for the treatment of IPF. Later N-acetyl cysteine was also added in this combination [94]. Until the 2012 PANTHER study, this triple therapy was the preferable mode of therapy in IPF. However, the PANTHER study indicated the higher mortality due to this triple therapy [93]. Then disease-modifying drugs including pirfenidone and nintedanib were used after a number of clinical trials. Though the exact mechanism of 13 A Refined Approach to Target the Molecular and Cellular Mechanisms in. . . 331 pirfenidone is not known, reduction in the proliferation of fibroblasts along with the reduction in the secretion of pro-fibrotic growth factors like TGFβ and PDGF has been demonstrated [93]. Initially, it was approved in Japan followed by many other countries. The anti-fibrotic effects of nintedanib were evident after the 3rd phase of INPULSIS 1 and 2 trials. It is a tyrosine kinase inhibitor as it inhibits tyrosine kinases like PDGF, fibroblast growth factor (FGF), and VEGF [93]. Thus, the possible relative success story behind these two drugs might be attributable to their multiple targets and actions. As one of the long-term complications of COVID-19 is lung fibrosis, many believe that these two drugs could stop the fibrotic complications post-COVID-19 [95]. 13.12 Conclusion The pathogenesis of pulmonary fibrosis seems to be a sort of a labyrinth and is multifactorial. The development of lung fibrosis is similar to wound repair or healing. In spite of so many pathways involved, researchers have succeeded in developing only two drugs, pirfenidone and nintedanib, which are currently approved by the FDA and available in the market to avert the progression of the disease. Immunosuppressants and glucocorticoid medications are given to the IPF patients that have poor efficiency and various toxic effects. In this review, we have highlighted some of the regulatory mechanisms that are linked to the development of lung fibrosis. With this in mind, an intricate understanding of these pathways and the identification of various biomarkers are needed for designing more number of efficacious therapies for this fatal lung disease. 13.13 Future Perspectives In contemplation of the experience from the positive and negative outcomes of the studies, inhibition of the single pathway of IPF might only have a modest effect on the fibrotic condition. The accomplishment of the drugs such as pirfenidone and nintedanib that inhibit multiple pathways of IPF has motivated the researchers and clinicians to design and assess the anti-multifactorial pathogenesis drugs with novel molecular scaffolds to attenuate the progression of IPF. Clinical investigations with anti-fibrotic therapeutics targeting IL-13 and TGFβ, chemokine receptor antagonists, and inhibitors of angiogenesis are required. An integrative effort by researchers and clinicians across various disciplines could make this a climbable mountain (Fig. 13.1). Acknowledgments This work was supported by the project MLP137 (MISSION LUNG) at CSIR-Indian Institute Chemical Biology, Council of Scientific and Industrial Research, Govt. of India. SS, JD, AR, and AJ acknowledge the Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India, for their Ph.D. registrations. 332 S. Singh et al. Fig. 13.1 Schematic diagram to show the pathogenesis of pulmonary fibrosis. The development of IPF has three major stages: (a) predisposition, (b) initiation, and (c) progression. A single or milieu of factors like exogenous environmental cues, drugs, certain viruses, and genetic susceptibility predisposes the individuals to cause alveolar epithelial injury with or without inflammation. The alveolar epithelial injury initiates a multitude of pathways that leads to loss of epithelial cells, conversion of fibroblast to myofibroblasts, and EMT. 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