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AOP: 173

Title

A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the AO. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE.  More help

Substance interaction with the pulmonary resident cell membrane components leading to pulmonary fibrosis

Short name
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Substance interaction with the pulmonary cell membrane leading to pulmonary fibrosis
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Handbook Version v1.0

Graphical Representation

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Authors

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Sabina Halappanavar 1*, Monita Sharma2, Silvia Solorio-Rodriguez1, Hakan Wallin3, Ulla Vogel3, Kristie Sullivan4, Amy J. Clippinger2

1Environmental Health Science and Research Bureau, Health Canada, Ottawa, Canada.

2PETA International Science Consortium Ltd., London, United Kingdom.

3National Research Centre for the Working Environment, Copenhagen, Denmark.

4Physicians Committee for Responsible Medicine, Washington, DC.

*Point of contact

Sabina Halappanavar, PhD

Research Scientist, Genomics, Nanotoxicology and Alternative Methods Laboratory

Environmental Health Science and Research Bureau, ERHSD, HECSB, Health Canada

Sir Frederick G Banting Research Centre,

251 Sir Frederick Banting Driveway, Building 22

Ottawa, ON, Canada, K1A 0K9

Email: sabina.halappanavar@hc-sc.gc.ca

Point of Contact

The user responsible for managing the AOP entry in the AOP-KB and controlling write access to the page by defining the contributors as described in the next section.   More help
Sabina Halappanavar   (email point of contact)

Contributors

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  • Sabina Halappanavar

Coaches

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  • Cinzia La Rocca

OECD Information Table

Provides users with information concerning how actively the AOP page is being developed and whether it is part of the OECD Workplan and has been reviewed and/or endorsed. OECD Project: Assigned upon acceptance onto OECD workplan. This project ID is managed and updated (if needed) by the OECD. OECD Status: For AOPs included on the OECD workplan, ‘OECD status’ tracks the level of review/endorsement of the AOP . This designation is managed and updated by the OECD. Journal-format Article: The OECD is developing co-operation with Scientific Journals for the review and publication of AOPs, via the signature of a Memorandum of Understanding. When the scientific review of an AOP is conducted by these Journals, the journal review panel will review the content of the Wiki. In addition, the Journal may ask the AOP authors to develop a separate manuscript (i.e. Journal Format Article) using a format determined by the Journal for Journal publication. In that case, the journal review panel will be required to review both the Wiki content and the Journal Format Article. The Journal will publish the AOP reviewed through the Journal Format Article. OECD iLibrary published version: OECD iLibrary is the online library of the OECD. The version of the AOP that is published there has been endorsed by the OECD. The purpose of publication on iLibrary is to provide a stable version over time, i.e. the version which has been reviewed and revised based on the outcome of the review. AOPs are viewed as living documents and may continue to evolve on the AOP-Wiki after their OECD endorsement and publication.   More help
OECD Project # OECD Status Reviewer's Reports Journal-format Article OECD iLibrary Published Version
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This AOP was last modified on November 30, 2023 08:11

Revision dates for related pages

Page Revision Date/Time
Substance interaction with the lung resident cell membrane components May 17, 2023 15:10
Increased, secretion of proinflammatory mediators May 17, 2023 15:18
Increased, recruitment of inflammatory cells May 12, 2023 17:03
Loss of alveolar capillary membrane integrity May 17, 2023 15:35
Increased, activation of T (T) helper (h) type 2 cells May 12, 2023 16:28
Increased, fibroblast proliferation and myofibroblast differentiation May 17, 2023 15:51
Pulmonary fibrosis May 12, 2023 17:09
Accumulation, Collagen May 17, 2023 15:55
Interaction with the lung cell membrane leads to Increased proinflammatory mediators August 29, 2023 09:00
Increased proinflammatory mediators leads to Recruitment of inflammatory cells May 18, 2023 12:46
Recruitment of inflammatory cells leads to Loss of alveolar capillary membrane integrity May 18, 2023 13:35
Loss of alveolar capillary membrane integrity leads to Activation of Th2 cells May 16, 2023 09:19
Activation of Th2 cells leads to Increased cellular proliferation and differentiation May 18, 2023 14:33
Increased cellular proliferation and differentiation leads to Accumulation, Collagen May 18, 2023 15:03
Accumulation, Collagen leads to Pulmonary fibrosis May 18, 2023 14:04
Bleomycin October 29, 2019 13:08
Carbon nanotubes, Multi-walled carbon nanotubes, single-walled carbon nanotubes, carbon nanofibres January 01, 2018 17:52

Abstract

A concise and informative summation of the AOP under development that can stand-alone from the AOP page. The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance. More help

This AOP describes the qualitative linkages between interactions of substances (e.g. physical, chemical or, receptor-mediated) with the membrane components (e.g. receptors, lipids) of pulmonary (lung) cells leading to pulmonary fibrosis. The terms 'lung' and 'pulmonary' mean the same and are used throughout the AOP description in an interchangeable manner. This AOP represents a pro-fibrotic mechanism that involves a strong inflammatory component. It demonstrates the applicability of the AOP framework for nanotoxicology and describes a mechanism that is common to both chemical and nanomaterial-induced lung fibrosis. Lung fibrosis is a dysregulated or exaggerated tissue repair process. It denotes the presence of scar tissue in the localized alveolar capillary region of the lung where gas exchange occurs; it can be localized or more diffuse involving, bronchi and pleura. It involves the presence of sustained or repeated exposure to a stressor and intricate dynamics between several inflammatory and immune response cells, and the microenvironment of the alveolar-capillary region consisting of both immune and non-immune cells, and the lung interstitium. The interaction between the substance and components of the cellular membrane leading to release of danger signals/alarmins marks the first event, which is a molecular initiating event (MIE; Event 1495) in the process of tissue repair. As a consequence, a myriad of pro-inflammatory mediators are secreted (Key Event (KE) 1; Event 1496) that signal the recruitment of pro-inflammatory cells into the lungs (KE2; Event 1497). The MIE, KE1 and KE2 represent the same functional changes that are collectively known as inflammation. In the presence of continuous stimulus or persistent stressor, non-resolving inflammation and ensuing tissue injury, leads to the alveolar capillary membrane integrity loss (KE3; Event 1498) and activation of adaptive immune response, T helper type 2 cell signalling (KE4; Event 1499), during which anti-inflammatory and pro-repair/fibrotic molecules are secreted. The repair and healing process stimulates fibroblast proliferation and myofibroblast differentiation (KE5; Event 1500), leading to synthesis and accumulation of extracellular matrix or collagen (KE6; Event 68). Excessive collagen deposition culminates in alveolar septa thickening, decrease in total lung volume, and pulmonary fibrosis (Adverse Outcome (AO); Event 1458). At the individual level, pulmonary fibrosis will lead to death, which is the ultimate AO (Mortality, increased); however, it is not discussed in the AOP description. Thus, for this AOP, pulmonary fibrosis is the final AO (Event 1458).

Lung fibrosis is frequently observed in miners and welders exposed to metal dusts, making this AOP relevant to occupational exposures. Other stressors include pharmacological products, fibres, chemicals, microorganisms or overexpression of specific inflammatory mediators. Novel technology-enabled stressors, such as nanomaterials possess properties that promote fibrosis via this mechanism. Lung fibrosis occurs in humans and the key biological events involved are the same as the ones observed in experimental animals. Thus, this AOP is applicable to a broad group of stressors of diverse properties and provides a detailed mechanistic account of the process of lung fibrosis across species.

Acknowledgements: The lead author would like to acknowledge the able assistance of Andrey Boyadzhiev of Health Canada, Ottawa, Ontario, Canada, in formatting the response document and preparing some responses to external reviewers' comments and questions, and Professor Carole Yauk for her expert advice and kind support throughout the development and review process of AOP173. Lastly, the author acknowledges the funding received through the Genomics Research and Development Initiative and the Chemicals Management Plan of Health Canada.

AOP Development Strategy

Context

Used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development.The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. More help

There is a high potential for inhalation exposure to toxicants in various occupational settings and polluted environments. Extensive investigation of pulmonary toxicity following inhalation of chemical and particulate stressors have demonstrated that these toxicants mount an exuberant inflammatory response early after exposure that, when unresolved, lays the foundation for later pathologies. Although inflammation is a normal immune reaction of the organism designed to effectively eliminate the invading threat, chronic and unresolved tissue inflammation is detrimental. Unresolved lung inflammation in humans plays a causative role in many debilitating and even lethal adverse health effects, such as decreased lung function, emphysema, fibrosis, and cancer. The various pathways, mechanisms, and biological processes associated with the pulmonary inflammatory process are well characterized in experimental animals and, to a great extent, in humans. Here, a mechanism underlying stressor-induced pulmonary fibrosis that involves a pro-inflammatory component is described.

Pulmonary fibrosis is a chronic lung pathology, which when not treated, results in lethality. It is characterized by the excessive extracellular matrix (ECM) and collagen deposition and restructuring. Numerous respiratory diseases, such as pneumoconiosis, silicosis, asbestosis, bronchiolitis obliterans (BO) (‘popcorn lung’), and chronic beryllium disease have pulmonary fibrosis as a main or secondary symptom. In addition, exposure to pharmaceuticals and environmental contaminants such as bleomycin and arsenic via inhalation, oral or intravenous routes also induces the adverse otucome (AO) of pulmonary fibrosis. Idiopathic pulmonary fibrosis (IPF) is the most common type of pulmonary fibrosis in humans and involves alveolar regions of the lung consisting of type 2 alveolar epithelial cells (AEC2s), type 1 alveolar epithelial cells (AEC1s) and mesenchymal cells. AEC1s are responsible for gas exchange and AEC2s synthesise surfactant. The AEC2s are capable of self-renewal and differentiate to AEC1s regularly during normal tissue maintenance (Barkauskas and Noble, 2014). In pro-fibrotic conditions, AEC2s fail to regenerate AEC1s lost by injury and do not respond normally to epithelial injury, undergoing hyperplasia. As a result, human patients suffering from IPF have dysregulated levels of surfactant proteins normally secreted by AEC2s (Barlo et al.,2009; Phelps et al., 2004). Genetic studies have associated mutations in genes encoding surfactant proteins and the development of a familial type of lung fibrosis. Furthermore, immunohistochemical staining of human IPF lung slices shows AEC death as well as proliferation adjacent to fibrotic foci (Uhal et al., 1998). AEC2s are hyperplastic and are located on top of the fibrotic lesions in the lung in human specimens (Katzenstein and Myers, 1998). In animal models of bleomycin-induced pulmonary fibrosis, abnormal AEC2s are incapable of protecting the basement membrane destroyed by cell death, leading to aberrant repair and deposition of ECM, resulting in fibrosis (Rock et al., 2011). Targeted removal of AEC2s in mouse lungs results in full manifestion of the fibrotic disease (Sisson et al., 2010). In certain infectious conditions, epithelial cell stress and dysfunction leading to inefficient repair capacity or transcriptional reprogramming of epithelial cells to secrete pro-fibrotic and pro-inflammatory factors leads to lung fibrosis (Lawson et al., 2008; Lawson et al., 2011). Mesenchymal cells are the other main type of cell, which contribute to fibrosis development. The dysregulated proliferation of fibroblasts and myofibroblast differentiation leading to excessive ECM deposition in the fibrotic scar is the result of disrupted cross-talk between epithelial and mesenchymal cells (Barkauskas and Noble 2014). Myofibroblasts exhibiting contractile properties of smooth muscle cells and expressing Alpha-smooth muscle actin (α-SMA) and vimentin, are the types of mesenchymal cells that are most commonly associated with excessive collagen secretion in pro-fibrotic phenotypes (Todd et al., 2012). Myofibroblasts can arise mainly from differentiation of tissue resident fibroblasts, translocation of bone marrow (BM) derived fibrocytes into the lung, or from epithelial-to-mesenchymal transformation (EMT; a type of trans-differentiation) (Hung, 2020; Todd et al., 2012). These cells are critical to the normal process of wound healing, and are the main cells contributing to collagen deposition in both normal wear-and-tear repair processes and in disease promoting conditions. Following successful wound healing, myofibroblasts de-differentiate and disappear (Friedman, 2012). Myofibroblasts persistence is suggested to play a key role in progressive pulmonary fibrosis in humans. There is evidence for both EMT derived myofibroblasts and BM derived fibrocytes in human pulmonary fibrotic conditions. Air epithelial biopsies from human patients suffering from BO following lung transplant show significantly increased staining for mesenchymal markers (Vimentin and α-SMA), decreased staining for e-cadherin, and co-localization of epithelial and mesenchymal markers as compared to stable patients (Borthwick et al., 2009). With respect to BM derived fibrocytes, these cells have been proposed as an indicator for poor prognosis in human IPF patients, and research has shown that the amount of fibrocytes in the human IPF lung correlates with the amount of fibroblastic foci (Andersson-Sjöland et al., 2008; Moeller et al., 2009). Additional cell types involved in fibrotic process include endothelial cells and immune cells such as macrophages, neutrophils, and T helper (Th) cells. Endothelial cells contribute to the fibrotic process through EMT, as evidenced in bleomycin model systems in which endothelial cells in fibrotic conditions take on the characteristics of myofibroblasts (Kato et al., 2018). Macrophages present in the alveolar space as well as macrophages recruited to the lung during the fibrotic process also contribute to the inflammatory environment and potentiate the AO of pulmonary fibrosis. Direct interaction of fibrotic stressors, such as multi-walled carbon nanotubes (MWCNTs), silica, and asbestos, with the macrophage cell membrane can occur through scavenger receptors as well as through receptors such as Macrophage receptor with collagenous structure (MARCO) (Li and Cao, 2018; Murphy et al., 2015). This can induce macrophage cell injury through frustrated or incomplete phagocytosis which leads to the production of alarmins such as Interleukin (IL)-1β and reactive oxygen species (ROS), and profibrotic mediators such as Tumour necrosis factor alpha (TNF-α), Transforming growth factor beta (TGF-β), and Platelet derived growth factor (PDGF) (Dong and Ma, 2016; Li and Cao, 2018). The injured resident macrophages contribute to the initial acute phase pro-inflammatory response leading to recruitment of additional immune cells to the lung. Depending on the fibrotic stressor, different populations of immune cells can be initially recruited to the site of action. The recruitment of neutrophils into the lung space potentiates the inflammatory response and tissue damage. Furthermore, in conditions of acute lung injury, which can precede the development of a fibrotic phenotype, neutrophil recruitment to the lung through trans-epithelial migration can induce the formation of lesions in the epithelium and contribute to the loss of alveolar capillary membrane (ACM) integrity (Zemans et al., 2009). Finally, Th cells recruited to the lung potentiate the inflammatory environment, and through the induction of a Th type 2 (Th2) response, stimulate the proliferation of fibroblasts and differentiation of myofibroblasts driving the development of a fibrotic phenotype (Shao et al., 2008; Wynn, 2004).

Although this AOP is applicable to a broad group of stressors, the AOP was specifically assembled keeping in mind, a novel class of engineered nanomaterials (NMs) exhibiting sophisticated properties that have been shown to induce lung fibrosis via this mechanism. Specifically, nanomaterial properties such as aspect ratio, tube/fiber rigidity, crystallinity and persistence are suggested to play a role in the induction of pulmonary fibrosis. Thus, it demonstrates the applicability of the AOP framework to nanotoxicology.

Given the fundamental role of inflammation in organ homeostasis, well characterized AOPs targeting the pathological outcomes of unregulated inflammatory responses are important and will guide the development of appropriate assays to measure the key events that are predictive of inflammation-mediated chronic health impacts, and aid in screening a large array of inhalation toxicants that are inflammogenic, for their potential to induce lung diseases.

Strategy

Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components.  Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). More help

The development of AOP173 was initiated in 2014, mostly focussing on the available literature. Since then, the first representation of the AOP and its components including KEs have undergone several changes, mainly because of the results of the specific KE validation experiments conducted in the primary author's laboratory and the evolving literature. The KE titles were also changed to keep the KE ontology consistent across all AOPs, as well as to allow their use by the other AOP developers.

The AOP173 is written in a narrative review fashion, as the quantitative literature supporting all KERs is limited. Also, more targeted experiments are planned and being conducted currently to validate the AOP.

Summary of the AOP

This section is for information that describes the overall AOP.The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help

Events:

Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help
Key Events (KE)
A measurable event within a specific biological level of organisation. More help
Adverse Outcomes (AO)
An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help
Type Event ID Title Short name
MIE 1495 Substance interaction with the lung resident cell membrane components Interaction with the lung cell membrane
KE 1496 Increased, secretion of proinflammatory mediators Increased proinflammatory mediators
KE 1497 Increased, recruitment of inflammatory cells Recruitment of inflammatory cells
KE 1498 Loss of alveolar capillary membrane integrity Loss of alveolar capillary membrane integrity
KE 1499 Increased, activation of T (T) helper (h) type 2 cells Activation of Th2 cells
KE 1500 Increased, fibroblast proliferation and myofibroblast differentiation Increased cellular proliferation and differentiation
KE 68 Accumulation, Collagen Accumulation, Collagen
AO 1458 Pulmonary fibrosis Pulmonary fibrosis

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarizes all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP.Each table entry acts as a link to the individual KER description page. More help

Network View

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (WoE) summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Life Stage Applicability

The life stage for which the AOP is known to be applicable. More help
Life stage Evidence
Adult High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available. More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI
mouse Mus musculus High NCBI
rat Rattus norvegicus High NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help
Sex Evidence
Unspecific High

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). More help

Pulmonary fibrosis is the thickening and scarring of lung tissue, caused by excessive deposition of collagen/ECM. The most common fibrotic disease of the lung in humans is IPF, a complex, progressive disease of unknown etiology with often poor prognosis. Pulmonary fibrosis in humans is also observed following exposure to pharmacological agents such as bleomycin, following inhalation of silica, asbestos, cigarette smoke (CS), coal dust and following exposure to microbials and allergens. Regardless of the etiology, lung fibrosis in humans is characterised by the presence of inflammatory lesions, excessive ECM/collagen deposition, and reduced lung volume and function. Mechanistically, using animals, it has been shown that key biological events that play a critical role in the onset and progression of the disease are similar in humans and animals. The main differences are limited to anatomical and physiological aspects of lung and its functions.

Some other considerations of relevance to this AOP:

This AOP represents a fibrotic mechanism that involves a strong inflammatory component. Exposure to pro-fibrotic stressors such as, bleomycin, silica, asbestos, carbon nanotubes (CNTs), radiation or models of cytokine overexpression involve a profound inflammatory response. IPF in humans is more commonly observed in male subjects. A study in mice showed that male mice developed lung fibrosis more readily following exposure to bleomycin compared to female mice and that age is a risk factor, with aged male mice showing exuberant fibrosis (Redente et al., 2011). Scar formation is reduced in fetal wounds (Yates et al., 2012). Asbestosis and silicosis, (two types of fibrotic disease) are clinically manifested in aged humans. Thus, the AOP presented here is applicable to lung fibrosis observed in adults predominantly.

Different animal species have been used to study the pathology of fibrotic disease; with mice being the most common and rats the second most used. Australian sheep, horse, dogs, cats, donkeys, pigs and other animals have been studied to investigate different types of fibrosis. There are some limitations, however, in these animal systems with respect to modelling human pulmonary fibrosis. The most commonly used model, the bleomycin mouse model, presents a rapidly developing fibrotic phenotype which undergoes at least partial resolution following 28 days (Tashiro et al., 2017). Higher order organisms, like dogs, cats, and horses offer a chance to examine naturally occurring pulmonary fibrosis, with closer resemblance to human IPF, with a natural cough reflex (Williams and Roman, 2015). However, inherent limitations in these models, such as their outbred nature and lack of systematic characterization (Williams and Roman, 2015) make them poor candidates for routine fibrosis research. Regardless of the species or the type of fibrosis investigated, the key characteristic events that define the disease process are the same with few species-specific anatomical, physiological and histological differences. Thus, cross-species applicability for this AOP is strong.

Domain of Applicability

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. More help

Sex/Gender and Age:

IPF in humans is more commonly observed in male subjects. Male mice develop lung fibrosis more readily following exposure to bleomycin compared to female mice and that age is a risk factor, with aged male mice showing exuberant fibrosis (Redente et al., 2011). Scar formation is reduced in fetal wounds (Yates et al., 2012). Asbestosis and silicosis, forms of fibrotic disease are clinically manifested in aged humans. This may be due to the fact that in humans, the disease progression is rather slow and takes time to manifest. Thus, the AOP presented here is applicable to lung fibrosis observed in adult males predominantly.

Taxonomy:

Different animal species have been used to study the pathology of fibrotic disease; with mice being the most common and rats the second most used. Australian sheep, horse, dogs, cats, donkeys, pigs and other animals have been studied to investigate different types of fibrosis. There are some limitations, however, in these animal systems with respect to modelling human pulmonary fibrosis. The most commonly used model, the bleomycin mouse model, presents a rapidly developing fibrotic phenotype which undergoes at least partial resolution following 28 days post-exposure (Tashiro et al., 2017). Higher order organisms, like dogs, cats, and horses offer a chance to examine naturally occurring pulmonary fibrosis, with closer resemblance to human IPF in animals with a natural cough reflex (Williams and Roman, 2015). However, inherent limitations in these models, such as their outbred nature and lack of systematic characterization (Williams and Roman, 2015) make them poor candidates for routine fibrosis research. Regardless of the species or the type of fibrosis investigated, the key characteristic events that define the disease process are the same with few species-specific anatomical, physiological and histological differences. Thus, cross-species applicability for this AOP is strong.

Types of Stressors:

Persistent and soluble stressors can induce fibrotic pathologies in humans (as well as in model animals) in concordance with the AOP presented. Asbestos exposure in humans has long been known to induce pulmonary fibrosis (asbestosis) due to chronic inflammation induced from persistent fibres deposited within the lung (Kamp and Weitzman 1997). Similarly, human exposure to silica (crystalline silica dust) leads to the development of silicosis in concordance with the AOP presented (Ding et al., 2002). Furthermore, the soluble chemotherapeutic compound bleomycin has long been known to induce pulmonary fibrosis in humans (in line with this AOP) as a side effect of intravenous administration (Froudarakis et al., 2013). In addition to these model stressors, exposure to various metals including uranium, arsenic, cadmium, and soluble copper can lead to fibrotic outcomes in humans (Assad et al., 2019). Occupational exposure to cobalt can induce interstitial lung disease in humans, which can progress to fibrotic outcomes (Adams et al., 2017). In male mice exposed via inhalation to cadmium oxide nanoparticles (NPs), increases in the pro-fibrotic and pro-inflammatory mediators IL-1β, TNF-α , and Interferon gamma (IFN-γ) were noted one day post-exposure, with accompanying pulmonary inflammation (Blum et al., 2014). In another study, intratracheal instillation of cadmium chloride (CdCl2in mice induced peribronchiolar fibrosis through activation of myofibroblasts via the Suppressor of mothers against decapentaplegic (SMAD) signalling (Li et al., 2017). As with the aforementioned cadmium NPs, murine animals exhibit pronounced acute inflammation and immune cell infiltration after pulmonary exposure to copper oxide NPs (Gosens et al., 2016), which can progress to a fibrotic phenotype in some model systems after 28 days with marked increases of TGF-β1 detected in the bronchoalveolar lavage fluid (BALF), activation of myofibroblasts, and pronounced deposition of ECM (Lai et al., 2018). In mice, intratracheal instillation of cobalt NPs results in pronounced infiltration of neutrophils and macrophages into the alveolar and interstitial space, and increased amounts of C-X-C motif chemokine ligand (CXCL)1 in the BALF 1-7 days post-exposure; pronounced pulmonary fibrosis was detected at 4 months post-exposure marked by increased collagen deposition and bronchiolization of the alveolar epithelium (Wan et al., 2017).

Essentiality of the Key Events

The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently, evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence. The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs. More help

The essentiality of the MIE; Event 1495 was rated as moderate. Molecular interaction is an essential step but it is dynamic in nature. The interaction can be specific, non-specific or both depending on the stressor. Also, NMs, one type of stressors may adopt a molecular corona in biological environments, which can mediate cellular interactions. Efforts are currently made to develop each individual interaction described in MIE as group MIEs and the associated KERs. 

The essentiality of KE1; Event 1496 and KE2; Event 1497 was rated as moderate, due to the redundant nature of the inflammatory response and the inherent challenges in abrogating this response without inducing another pathology in the model system.

For KE3; Event 1498, the essentiality was also listed as moderate, due to the fact that attenuation or abrogation of this response isn’t practical, and as such the supporting evidence is indirect.

For KE4; Event 1499 and KE6; Event 68, the essentiality was rated as high due to the plethora of experimental evidence showing that modulation of these responses modifies the AO and downstream KEs. For additional information, please consult the Evidence Assessment Call Table below.

Evidence Assessment

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. More help

Concordance of Dose-Response Relationships:

The AOP presented here is qualitative. There is some evidence on dose-response relationships; however, dose-response relationships for each individual KE are not available. In Labib et al., 2016, Benchmark Dose (BMD) analysis of MWCNT-induced gene expression changes in lungs of mice and canonical pathways associated with each of the KEs identified in this AOP was conducted and the resulting BMD values were correlated with BMD values derived for the apical endpoints that measured histologically manifested fibrotic lesions in rodents (National Institute for Occupational Safety and Health, 2013). The study showed that low doses of MWCNTs induce early KEs of inflammation and immune response at the acute post-exposure timepoints, and histological manifestation of fibrosis required higher MWCNT doses and was only evident at the later timepoints. Similarly, in another study, the meta-analyses of transcriptomics data gathered from mouse lungs (over 2000 microarrays) exposed individually to a variety of pro-fibrotic agents showed that the gene expression profiles from the high dose MWCNT-exposed samples collected at sub-chronic timepoints were strongly associated with the Th2 response signalling observed in mouse fibrotic disease models compared to the low dose early timepoint MWCNT samples (Nikota et al., 2016). These studies showed temporal and dose-response relationships between KEs. 

In another study, pharyngeal aspiration of 10, 20, 40, or 80 µg/mouse MWCNTs induced lung fibrosis in a dose-dependent manner, which became apparent as early as 7 days post-exposure at 40 µg/mouse dose and persisted up to 56 days post-exposure (Porter et al., 2010). Pharyngeal aspiration of 10, 20, 40, or 80 µg/mouse MWCNTs induced significant alveolar septa thickness over time (1, 7, 28, and 56 days post-exposure) in 40 and 80 µg dose groups (Mercer et al., 2011). Similarly, inhalation of MWCNTs (10 mg/m3, 5h/day) for 2, 4, 8, or 12 days showed dose-dependent lung inflammation and lung injury with the development of lung fibrosis in mice (Porter et al., 2013). Lung inflammation and fibrosis were observed in mice intratracheally instilled with 162 µg/mouse MWCNTs at 28 days post-exposure (Nikota et al., 2017). The above studies involving CNTs showed elevated levels of pro-inflammatory mediators, pro-inflammatory cells and cytotoxicity in BALF.

Strength, Consistency, and Specificity of Association of AO and Initiating Event:

This AOP describes a non-specific MIE. Typically, in an experimental setting, the MIE itself is not assessed. Rather, the outcomes of MIE engagement or MIE trigger are assessed. Depending on the type of stressor and its physical-chemical property, the type of interactions between the stressor and the lung resident cells differ. High aspect ratio fibres such as asbestos and CNTs induce frustrated phagocytosis, acute cell injury (Boyles et al., 2015; Brown et al., 2007; Dörger et al., 2001; Kim et al., 2010; Poland et al., 2008), leading to inflammation, immune responses and fibrosis. Asbestos and silica crystals engage scavenger receptors present on the macrophages (Murthy et al., 2015), resulting in acute cell injury and inflammatory cascade, leading eventually to the AO. Bleomycin binds high affinity bleomycin binding sites present on rat alveolar macrophage surfaces, leading to macrophage activation (Denholm and Phan, 1990). Asbestos fibres also bind directly to cellular macromolecules including proteins and membrane lipids, which is influenced by their surface properties such as surface charge (reviewed in Agency for Toxic Substances and Disease Registry 2001). These studies demonstrate the types of interactions between cells and the pro-fibrotic stressors, which are often not measured in animal or cell culture experiments. Instead, the consequences or outcomes of triggering the MIE are measured, which are the release of danger associated molecular patterns (DAMPs) or alarmins from cells.

The alarmin High mobility group box 1 (HMGB1) is released from damaged or necrotic cells in cell culture models and in animals following exposure to asbestos and is involved in the inflammatory events elicited by asbestos (Yang et al., 2010), which plays a critical role in asbestosis. CNTs interact with HMGB1-Receptor for advanced glycation end-products (RAGE), which is implicated in pro-inflammatory and genotoxic effects of CNTs (Hiraku et al., 2016). Mechanical stress and membrane damage following cellular uptake of long and stiff CNTs by lysosomes results in cell injury and consequent adverse effects (Zhu, et al., 2016). CNT-induced inflammatory response in vitro is mediated by IL-1, absence of which negatively impacts gap junctional intercellular communication (Arnoldussen et al., 2016). The levels of IL-1α are increased in BALF of mice immediately after exposure to MWCNT doses that induce fibrosis (Nikota et al., 2017).

Although there is enough empirical evidence to suggest the occurrence of the MIE; Event 1495 following exposure to pro-fibrogenic substances, there is incongruence in supporting its essentiality to the eventual AO. The inconsistency could be due to the fact that early defence mechanisms involving DAMPs is fundamental for the organism’s survival, which may necessitate multifaceted signalling pathways. As a result, inhibition of a single biological pathway of the innate immune response may not be sufficient to completely abrogate the lung fibrotic response. For example, MWCNTs induce IL-1α secretion in BALF of mice (Nikota et al., 2017) and thus, IL-1α mediated signalling is involved in MWCNT-induced lung inflammation and fibrosis (Rydman et al., 2015). Inhibition of IL-1α signalling alone does not alter the MWCNT-induced fibrotic response in mice (Nikota et al., 2017). This study further showed that simultaneous inhibition of both acute inflammatory events (KE1; Event 1496 and KE2; Event 1497) and Th2–mediated signalling (KE4; Event 1499) is required to suppress lung fibrosis induced by MWCNTs (Nikota et al., 2017). Disengagement between innate immune responses (MIE; Event 1495, KE1; Event 1496 and KE2; Event 1497) and lung fibrosis is shown in mice following exposure to silica (Re et al., 2014). In this study, the role of innate immune responses in lung fibrosis were characterised in 11 separate knockout (KO) mouse models lacking individual members of the IL-1 family. The study supported the earlier hypothesis of Nikota et al., 2017 that inhibition of a single pathway may not be sufficient to attenuate the fibrotic response. On the contrary, IL-1α and IL-1 receptor (IL1-R)1 mediated signalling are shown to be involved in bleomycin-induced lung inflammation and fibrosis; inhibition of IL1-R1 signalling attenuated the bleomycin pathology (Gasse et al, 2007).

Biological Plausibility, Coherence, and Consistency of the Experimental Evidence:

As described above, there is significant evidence to support the occurrence of the MIE and individual KEs, and thus, evidence supporting the KEs involved in this AOP is strong. However, there is inconsistency in empirical evidence supporting the KERs. Again, this may be due to the redundancy in pathways involved in the early immune responses to injury and repair. Despite the incongruences, AOP presented is coherent and logical.

Alternative Mechanisms:

The AOP as presented is the most agreed upon sequence of biological events occurring in the process of lung fibrosis that involves robust inflammation following exposure to a variety of stressors of different physical-chemical properties. However, in a recent study, using ToxCast data, a different MIE that involves inhibition of Peroxisome proliferator-activated receptor gamma (PPAR-γ) resulting in lung fibrosis was proposed (Jeong et al., 2019). This alternate AOP for fibrosis placed activation of TGF-β1 upstream of inflammatory events (KE2; Event 1497, KE3; Event 1498), which is contrary to its perceived role in downstream events leading to fibroblast proliferation and differentiation, and ECM deposition. The stressors identified in this study were also different, suggesting the PPAR-γ inhibition may be selective to a group of chemicals. The other alternative mechanisms may involve bypassing of the initial inflammatory KEs that directly trigger activation of fibroblast proliferation and differentiation leading to ECM deposition. For example, overexpression of TGF-β1 can promote excessive ECM deposition and fibrosis in rodents independent of inflammation (Hardie et al., 2004)

Further mechanisms may involve the targeted inhibition of receptor tyrosine kinases by compounds like Gefitinib, Imatinib, and Sorafenib, as well as some monoclonal antibodies which affect receptors for growth factors like Platelet derived growth factor (PDGF), Endothelial growth factor (EGF), and Vascular endothelial growth factor (VEGF). This is thought to directly impair the regenerative capacity of lung epithelial cells (MIE; Event 1495 to KE3; Event 1497), resulting in an aberrant wound healing response (Li et al., 2018). Finally, one more alternative mechanism involves pulmonary fibrosis in the context of BO. In this condition, the fibrotic phenotype is brochiolocentric and not alveolocentric – with the main insult involving the bronchiolar epithelium and an inability of the basal cells to replace lost bronchio epithelial cells.  Stressors, such as soluble diacetyl used in popcorn flavouring and e-cigarette vape liquids, can cause BO in humans. A recent human case study of a Canadian youth admitted to hospital with BO following vaping flavoured liquid containing diacetyl, as well as tetrahydrocannabinol, shows septal thickening, type II pneumocyte hyperplasia, immune cell infiltration and myofibroblast proliferation & incorporation into pulmonary septa (Landmann et al., 2019). Pulmonary exposures in murine model systems indicate that diacetyl induces pronounced damage to the airway epithelium, and that repair processes result in a compositionally different epithelium (Reviewed in Brass and Palmer, 2017). In a study using rat models, inhalation of 200 ppm of diacetyl resulted in bronchiolar fibrosis, with chronic inflammation accompanying the fibrotic outcomes (Morgan et al., 2016).

Evidence Assessment Summary:

The MIE; Event 1495 and KE1; Event 1496 – KE2; Event 1497 occur in sequence, however most in vivo and in vitro experiments are not designed to measure these events separately. This is an area of focus for future pulmonary fibrosis research.

Support for Essentiality of KEs

MIE; Event 1495:  Interaction with the lung resident cell membrane components

Persistent fibres like CNTs and asbestos are known to induce frustrated or incomplete phagocytosis in resident lung cells following respiratory exposure. Particles such as silica, as well as asbestos fibers engage scavenger receptors on the surface of macrophages leading to activation and inflammation. The soluble pro-fibrotic compound bleomycin binds to as-of-yet uncharacterised sites on macrophages, leading to similar activation.

Essentiality: Moderate. While the specific receptors involved vary depending on the stressor, and there is evidence of compensation in the context of KO models, over 20 years of research has shown that interaction between the fibrotic stressor and the resident lung cells is crucial for downstream responses. (Behzadi et al., 2017; Denholm and Phan 1990; Mossman and Churg 1998). 

KE 1; Event 1496:  Increased, secretion of proinflammatory mediators

Injured and activated resident lung cells release pro-inflammatory and fibrotic mediators, such as cytokines, chemokines, growth factors and ROS, into the surrounding environment.

Essentiality: Moderate. It is accepted that one of the main mechanisms underlying pulmonary fibrosis involves a profound inflammatory component. This has been shown in animal models exposed to fibrotic stressors such as bleomycin, MWCNTs, silica, and asbestos. The exact nature of the secreted mediators, and the essentiality of specific mediators requires further research. (Park and Im, 2019; Rabolli et al., 2014; Rahman et al., 2017).

KE 2; Event 1497:  Increased, recruitment of inflammatory cells

Inflammatory cells migrate into the lung according to the pro-inflammatory stimuli released.

Essentiality: High. The migration of inflammatory immune cells relies upon secretion of chemotactic stimuli in response to a stressor. KO models have shown reduced recruitment of immune cells to the lung in response to fibrotic stressors such as bleomycin. However, compensation has been noted due to the redundant nature of these molecules. (Gasse et al., 2007; Girtsman et al., 2014; Rabolli et al., 2014)

KE 3; Event 1498:  Loss of alveolar capillary membrane integrity

Significant alveolar damage from the inflammatory environment (including chronic inflammation and oxidative stress) results in the loss of ACM integrity.

Essentiality: Moderate. While it is generally recognized that damage to the ACM is integral to the development of fibrosis, evidence from KO models is lacking. Indirect evidence using bleomcyin has shown that animals deficient in Nuclear factor erythroid 2-related factor 2 (Nrf2), and therefore presenting a weakened antioxidant response, have higher levels of ACM injury and more pronounced fibrosis as compared to Nrf2 competent mice. This was assessed by proxy, using lactate dehydrogenase release into the BALF and the presence of pulmonary injury markers as a proxy for ACM injury. (Cho et al., 2004; Kikuchi et al., 2010)

KE 4; Event 1499:  Increased, activation of T (T) helper (h) type 2 cells

Th cells present in, and recruited to the lung environment commit to Th2 differentiation, which then release cytokines like IL-4, IL-5, and IL-13 and potentiate a Th2 driven response.

Essentiality: High. Induction of a Th2 response stimulates fibroblast proliferation and pulmonary fibrogenesis. Overexpression of Th2 type cytokine IL-13 stimulates pulmonary fibrosis in the absence of external stressors. IL-13 can directly activate TGF-β1 and initiates fibroblast proliferation and differentiation in pulmonary fibrosis. In mice deficient in Signal transducer and activator of transcription 6 (STAT6) with reduced Th2 response, MWCNT-induced fibrotic response involving fibroblast proliferation, and eventual formation of fibrotic lesions, were reduced. There is some inconsistency, as IL-4 deficient mice had a lower fibrotic response compared to wild-type after bleomycin treatment, however with higher rate of mortality. This highlights that the timing of the Th2 response is important for the manifestation of fibrosis. (Huaux et al., 2003; Lee et al., 2001; Nikota et al., 2017; Sempowski et al., 1994; Zhu et al., 1999)

KE 5; Event 1500:  Increased, fibroblast proliferation and myofibroblast differentiation

Fibroblasts originally present in the lung, and recruited to the lung, or which transdifferentiate from epithelial and endothelial cells proliferate and undergo differentiation into a collagen secreting myofibroblast phenotype which expresses α-SMA. This is the main effector cell responsible for secretion of ECM components in pulmonary fibrosis, and represents a nexus KE.

Essentiality: High. The proliferation of fibroblasts and differentiation into myofibroblasts is integral to the development of pulmonary fibrosis. Inhibition or attenuation of fibroblast proliferation and differentiation using TGF-β antagonism attenuates fibrosis in bleomycin mice models. Targeted inhibition of the Wingless/integrated β (Wnt/β)-catenin pathway inhibited myofibroblasts transition and reduced the overall fibrotic phenotype. (Cao et al., 2018; Chen et al., 2013; Guan et al., 2016; Kuhn and McDonald, 1991)

KE 6; Event 68:  Accumulation, collagen

The balance between ECM synthesis and destruction is disrupted, with a sustained increase in the deposition of ECM bearing compositional differences as compared to the native matrix.

Essentiality: High. A sustained imbalance between ECM synthesis and destruction is a prerequisite for the development of pulmonary fibrosis, and as such this KE is essential to the AO. (Bateman et al., 1981; McKleroy et al., 2013)

AO; Event 1458:  Pulmonary fibrosis

Destruction of lung architecture and alveolar capillaries due to increased and aberrant deposition of ECM in the context of prolonged inflammation results in pulmonary fibrosis.

Essentiality: N/A. This is the AO of this AOP, and therefore, is essential.

Support for Biological Plausibility of KERs

MIE --> KE1; Relationship 1702

Injury and activation resulting from the interaction of pro-fibrotic stressors with the membranes of resident lung cells results in the secretion of pro-inflammatory cytokines, chemokines, growth factors, and ROS from the resident epithelial or immune cell.

Biological plausibility: High. There is a mechanistic relationship between the MIE and KE1 which has been evidenced in a number of both in vitro and in vivo model systems in response to stressors such as asbestos, silica, bleomycin, CNTs, and metal oxide NPs. (Behzadi et al., 2017; Denholm and Phan 1990; Mossman and Churg 1998)

KE1 --> KE2; Relationship 1703

The secreted pro-inflammatory and pro-fibrotic mediators induce chemotactic recruitment of immune cells to the lung, in a signal-specific manner. Increases in the presence of macrophages, neutrophils, and eosinophils within pulmonary air spaces is commonly seen in the process of fibrosis, depending on the fibrotic stressor in question.

Biological plausibility: High. There are very well established functional relationships between the secreted signalling molecules and the chemotactic effects on pro-inflammatory and pro-fibrotic cells. (Harris, 1954; Petri and Sanz, 2018)

KE2 --> KE3; Relationship 1704

Inflammatory cells recruited to the lung potentiate further injury to the ACM through ROS production and direct damage, persistent inflammation, or an insufficient wound healing response. AEC1s are lost, AEC2s exhibit enhanced proliferation, ECM changes are notable and alveoli collapse.

Biological plausibility: High. There is a mechanistic relationship between an increase in pro-inflammatory cells and mediators, and damage to the ACM. (Bhalla et al., 2009; Ward, 2003; Zemans et al., 2009)

KE3 --> KE4; Relationship 1705

Continued loss of ACM integrity, together with oxidative stress and chronic inflammation induce a Th2 response in the lung. Th cells differentiate into Th2 cells in response to stimuli such as IL-6 and IL-4, which increase the secretion of IL-4 and IL-13. Increased Th2 cells in the lung polarize macrophages to the M2 phenotype which further suppresses Th1 cell differentiation.

Biological Plausibility: High. There is a mechanistic relationship between ACM injury (tissue damage), and the induction of a Th2 response (responsible for wound healing). (Gieseck et al., 2018; Wynn, 2004)

KE4 --> KE5; Relationship 1706

The increased population of Th2 cells and M2 polarized macrophages increases secretion of pro-fibrotic mediators, like TGF-β1, IL-4, and IL-13 which activate lung resident fibroblasts, as well as fibroblasts and fibrocytes recruited to the lung, and potentiate endo/epithelial to mesenchymal transition. This induces their proliferation and differentiation into a contractile myofibroblast phenotype capable of ECM synthesis and deposition.

Biological plausibility: High. There is a widely understood functional relationship between Th2 response related mediators, and their ability to induce proliferation and differentiation of fibroblasts. (Dong and Ma, 2018; Shao et al., 2008; Wynn, 2004; Wynn and Ramalingam, 2012)

KE5 --> KE6; Relationship 2625

Differentiated myofibroblasts represent the main effector cell responsible for the deposition of ECM during lung fibrosis. In the context of continuous stimuli and elevated levels of TGF-β, myofibroblasts are persistently activated and deposit excessive amounts of collagen in the lung.

Biological plausibility: High. There is an accepted mechanistic relationship between activated myofibroblasts, and the capacity to secrete collagen. (Hinz, 2016ab; Hu and Phan, 2013)

KE6 --> AO; Relationship 1629

Persistent myofibroblast activation and continued deposition of ECM cause destruction of alveolar structures and normal lung architecture. Reductions in lung function are noted, and pulmonary fibrosis develops.

Biological plausibility: High. By definition, pulmonary fibrosis is characterized by excessive deposition of ECM and destruction of native lung architecture. Thus, the plausibility of this association is undisputed. (Fukuda et al., 1985; Richeldi et al., 2017; Thannickal et al., 2004)

Empirical Support for KERs

MIE --> KE1; Relationship 1702

Direct interaction with the membrane is not a typically assessed endpoint in fibrosis research, except when dealing with fibrous stressors. Specific receptors involved in the initial immune cell activation are not wholly understood, even for model fibrotic stressors such as bleomycin. Limited in vitro studies have shown toll-like receptors are involved in silica and zinc nanoparticle macrophage recognition, which stimulates secretion of inflammatory factors. Similarly, bleomycin has been shown to bind to high affinity sites on the surface of macrophages, which stimulates secretion of growth factors and monocyte chemotactic molecules.

Empirical Support: Moderate. There are limited in vitro studies which show a temporal and dose-dependent relationship between these two events, using the upregulation of specific surface receptors as a proxy for direct membrane interaction. (Chan et al., 2018; Denholm and Phan 1990; Roy et al., 2014)

KE1 --> KE2; Relationship 1703

There are many studies which have empirically shown a relationship between secreted mediators and recruitment of immune cells to the lung. A paper by Chen et al., 2016, showed that increases in the levels of CXCL1, CXCL2, and CXCL5 in the lung preceded neutrophil recruitment following in vivo treatment with carbon NPs. In an in vitro study, Schremmer et al., 2016 exposed rat alveolar macrophages to nano silica and noted increases in C-C motif chemokine ligand (CCL)4, CXCL1, CXCL3, and TNF-α in the supernatant. This supernatant was able to induce chemotaxis in unexposed macrophages.

Empirical Support: Moderate. There are many studies which show temporal and dose-dependent recruitment of immune cells following increases in pro-inflammatory mediators. However, these mediators exhibit pleiotropy, and knockdown or KO of a single pathway or mediator can result in compensation and recruitment of immune cells at a later time, as is seen in Nikota et al., 2017. (Chen et al., 2016; Nikota et al., 2017; Schremmer et al., 2016)

KE2 --> KE3; Relationship 1704

The chronic inflammatory environment and oxidative stress potentiated by an increase of immune cells in the lung is well known to precede significant alveolar damage. However, the variety of infiltrating leukocytes differs depending on the stressor in question. In a study with crystalline silica, Umbright et al., 2017, were able to show that increases in pulmonary leukocytes at 3 weeks, preceded increases in total albumin (loss of ACM integrity) at 6 weeks. In another publication by Zeidler-Erdely et al., 2011, mice exposed to stainless steel welding fumes had an increased amount of alveolar macrophages 1 day post-exposure, while alveolar damage (as measured by total protein) was not evident until 4 days post-exposure.

Empirical Support: Moderate. There is both temporal and dose-response evidence to suggest that an increased amount of pro-inflammatory immune cells potentiates alveolar capillary damage. However, few studies assessing these KEs include multiple concentrations and timepoints, and as such, these KEs are typically reported as occurring together (i.e. damage is detected along with an increase in cell abundance). (Umbright et al., 2017; Zeidler-Erdely et al., 2011)

KE3 --> KE4; Relationship 1705

Few studies have directly assessed the ACM integrity loss on the induction of a Th2 response. In one publication, He et al., 2016 showed that ROS induced by a specific superoxide dismutase induces M2 polarization in asbestosis, and inhibition of signalling by Jumonji domain-containing protein D3 (Jmjd3) reduces ROS, M2 polarization, and fibrosis. In another study using NRF2 KO mice, a significant Th2 bias is observed following bleomycin treatment, with enhanced fibrosis noted. Discrepancies are present, for instance where many groups have found that TNF-α receptor (TNF-R)1 and TNF-R2 are associated with fibrosis, and even though TNF-α is a therapeutic target for IPF and asbestosis in humans, other groups have reported the opposite and that its exogenous delivery can reduce the fibrotic burden.

Empirical Support: Moderate. There is limited in vitro and in vivo evidence to support a direct relationship between these two KEs, with some inconsistencies with respect to the specific mediators in question. (Ortiz et al., 1998; Piguet, 1989; Redente et al., 2014)

KE4 --> KE5; Relationship 1706

Activation of a Th2 response is known to activate lung fibroblasts. Research by Hashimoto  et al. 2001, indicates that the Th2 cytokines IL-4 and IL-13 induce differentiation of human fibroblasts to myofibroblasts. Furthermore, IL-13 has been shown to directly activate TGF-β in vivo, and lead to pulmonary fibrosis.

Empirical Support: High. There is a plethora of dose and time response evidence which shows that Th2 cytokines induce the activation and proliferation of fibroblasts. (Hashimoto et al., 2001; Lee et al., 2001)

KE5 --> KE6; Relationship 2625 

While it is difficult to show the accumulation and incorporation of ECM in vitro, the levels of soluble collagen can be assessed. Many publications have reported secretion of soluble matrix components by activated myofibroblasts. For example, research by Li et al. 2017, has shown that soluble cadmium can induce fibrosis in mice, and that in vitro treatment of fibroblasts with cadmium induces expression of α-SMA (hallmark of myofibroblasts), as well as soluble collagen.

Empirical Support: High. It is generally accepted knowledge that activated myofibroblasts are collagen secreting cells. (Blaauboer et al., 2014; Hinz, 2016a; Li et al., 2017)

KE6 --> AO; Relationship 1629

Pulmonary fibrosis results from excessive accumulation of collagen and ECM in the lungs, in the context of prolonged inflammation, injury, and an aberrant healing response. IPF is the most common form in humans, with a poor prognosis overall.

Empirical Support: High. Excessive ECM deposition is the defining characteristic of pulmonary fibrosis, and the evidence to support this relationship is unequivocal. (Meyer, 2017; Thannickal et al., 2004; Williamson et al., 2015; Zisman et al., 2005)

Known Modulating Factors

Modulating factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help
Modulating Factor (MF) Influence or Outcome KER(s) involved
     

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors.  More help

The presented AOP is mostly qualitative and additional studies are needed to support the essentiality of the KEs and to build KERs. However, it is important to note that it is difficult to experimentally demonstrate the relevance of earlier KEs to the end outcome of fibrosis because of the redundancy in pathways involved. The mode or type of interactions between the resident cell membrane and a substance is dependent on the specific physical-chemical characteristics of the substance (e.g. for NMs, aspect ratio, crystallinity, persistence, surface charge, size, etc.). There has been an attempt to determine quantitatively the dose at which the events in AOP 173 are induced with respect to CNTs (Labib et al., 2016; reproduced below). In this manuscript, researchers applied global transcriptomic analysis and BMD modelling to determine the dose at which the MIE, KE1, KE2, KE4, KE5, and KE6 are induced using samples from three separate studies and compared the results to the apical BMD of the AO of pulmonary fibrosis. From the results shown, it can be seen that the BMD intervals of transcriptional pathway induction for each KE largely overlap but are representative of the BMD of AO induction. These results serve to highlight the parallel nature of the KEs in AOP 173, with many of the events occurring concurrently in addition to occurring sequentially.

Quantitative concordance table for AOP 173 KERs. Data is reproduced from Labib et al., 2016 (Figure 4., Additional file 4: Table S3). CNT: carbon nanotube. N/A: Not assessed

Stressor

Species

Time Point

MIEa

(1495)

KE1a

(1496)

KE2a

(1497)

KE3

(1498)

KE4a

(1499)

KE5a

(1500)

KE6a

(68)

AO

(1458)

Mitsui 7 CNT

Mouse

24 Hr

4 – 9

3 - 7

9 – 13

N/A

5 – 11

10 – 21

9 – 13

N/A

Mitsui 7 CNT

Mouse

3 / 7 day

11 – 22

6 – 22

14 – 24

N/A

9 – 16

15 – 26

17 – 34

N/A

Mitsui 7 CNT

Mouse

28 day

No Effect

14 – 26

36 – 51

N/A

14 – 26

11 – 20

No Effect

N/A

Mitsui 7 CNT

Mouse

56 day

N/A

N/A

N/A

N/A

N/A

N/A

N/A

14 – 27b

NRCWE-026

CNT

Mouse

24 Hr

No effect

8 – 15

20 – 37

N/A

8 – 15

21 – 39

No Effect

N/A

NRCWE-026

CNT

Mouse

3 / 7 day

16 – 28

16 – 27

19 – 33

N/A

15 – 24

16 – 26

19 – 36

N/A

NRCWE-026

CNT

Mouse

28 day

No Effect

No Effect

No Effect

N/A

12 – 20

No Effect

No Effect

N/A

NM-401

CNT

Mouse

24 Hr

No Effect

3 – 20

8 – 22

N/A

8 – 22

13 – 22

18 – 29

N/A

NM-401

CNT

Mouse

3 / 7 day

11 - 17

12 - 19

12 - 20

N/A

7 - 20

14 - 22

13 – 21

N/A

NM-401

CNT

Mouse

28 day

20 - 37

17 - 28

No Effect

N/A

No Effect

13 - 21

18 – 31

N/A

a: BMD (Benchmark dose lower confidence (BMDL)) intervals in µg / lung based on transcriptional pathway induction.

b: BMDL – BMD interval in µg / lung based on alveolar thickness.

The MIE of substance interaction with the lung cell membrane is intentionally kept broad and vague, to reflect the many interactions pro-fibrotic substances can have with the plasma membrane of cells. The presented AOP, while applicable to both soluble and persistent stressors, is specifically applicable to substances which induce fibrosis through immune responses. NMs are a group of such substances, which interact with organisms and cells via a dynamic biomolecular corona that is dependant on the biological microenvironment. While great strides have been made in recent years to characterize and understand this corona and how it impacts cellular recognition, further research is needed in order to accurately describe the specific interactions necessary for the initiation of fibrosis pathogenesis. Indeed, this is also true for model soluble stressors such as bleomycin, for which cellular binding and uptake is incompletely understood.

The specific mediators involved in the first KE (KE1; Event 1496), and the threshold necessary for progression to subsequent KEs is incompletely understood. KO models have shown that ablation of alarmins, such as IL-1, changes the initial trajectory of pulmonary fibrosis, however, compensation from other pathways makes it difficult to determine its essentiality to the end pathogenesis. 

In addition, other factors and events such as chronic lung inflammation, oxidative stress and macrophage polarization are suggested to play an important role in the progression of the disease trajectory. These events are not included as KEs in the main AOP because chronic lung inflammation defines temporality of the inflammation process and at present, sensitive markers that distinguish between acute and chronic inflammation are not available. Similarly, the role of ROS and oxidative stress in potentiating pulmonary fibrosis is also ambiguous. Many pro-fibrotic substances induce the formation of ROS and subsequent oxidative stress, as do many non-fibrotic stressors. While it is hard to deny that ROS and oxidative stress serve an important role in fibrosis by increasing cellular injury, potentiating an environment of chronic inflammation & damage, and activation of pro-fibrotic factors like TGF-β1, a causal relationship between the two has not been established.  Furthermore, antioxidant treatment in IPF patients has been largely unsuccessful, indicating a lack of knowledge of the specific redox mechanisms involved. Recent research has indicated a potential role of specific redox mechanisms, such as mitochondrial ROS and nitrogen oxides derived ROS; however, further research is needed to elucidate their role in potentiating pulmonary fibrosis. The development of newer fibrosis model systems that can better capitulate the human condition will assist in clarifying this aspect. Lastly, many studies show influence of macrophage polarization in lung fibrosis; however, collective evidence supporting its inclusion as a main KE in the AOP is lacking. 

Given their influence on the progression and prognosis of the disease, the three events are described in detail below and depicted in a schematic in relation to this AOP. As the new data becomes available, these events will be used to connect multiple AOPs in a network and develop necessary KEs and KERs, respectively.

Chronic Inflammation: 

In the presence of continuous stimulus (e.g., presence of biopersistent toxic fibres such as asbestos, MWCNTs) or following repeated stimulus (e.g., repeated exposure to silica or coal dust), the ensuing lung cell injury fuels the inflammatory mechanisms leading to accumulation of immune cells, prolonged inflammation and aggravated tissue damage. This sustained and perpetuated immunological response is termed as chronic inflammation. During this phase, active inflammation, tissue injury and destruction, and tissue repair processes proceed in tandem. Thus, the causative substance must contain unique physical-chemical properties that grant the material biopersistance in the pulmonary environment or the pulmonary system has to be repeatedly exposed to the same substance that perpetuates the tissue injury leading to loss of ACM. Although, increases in number of neutrophils are observed during chronic inflammation, mononuclear phagocytes (circulating monocytes, tissue macrophages) and lymphoid cells mark this phase. The macrophages, components of mononuclear phagocyte system, are the predominant cells in chronic inflammation. Activated macrophages release a variety of cytokines, chemokines, growth factors, and ROS that, which when uncontrolled, lead to extensive tissue injury. The other types of inflammatory cells involved in chronic inflammation include eosinophils in allergen-induced lung fibrosis, lymphocytes and epithelial cells. Chronic inflammation exists to potentiate the KEs associated with inflammation and tissue injury, rather than acting as a separate KE itself. 

Knockdown and KO models have shown that attenuation of the inflammatory response also attenuates the downstream fibrotic phenotype. Compensation from other inflammatory pathways makes complete abrogation of this response difficult. Furthermore, the essentiality of chronic inflammation leading to fibrotic phenotypes like IPF is questionable, as treatment with anti-inflammatory agents like corticosteroids does not have substantial benefits for patients. (Strieter and Mehrad, 2009; Ueha et al., 2012; Wilson and Wynn, 2009). 

Oxidative stress (KE1392) 

In KE1392 (AOPwiki), ‘Oxidative stress’ is described as an imbalance in the production of ROS and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell. As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al. 2009) and can be used as indicators of the presence of oxidative stress in the cell. In the context of pulmonary fibrosis, oxidative stress potentiates the inflammatory response (KE1-2) and injury to the respiratory epithelium (KE3), and contribute to the differentiation and activation of myofibroblasts (KE5). The exact species of ROS, the specific cell types, and the perturbed oxidative stress related pathways vary depending on the type of pulmonary fibrosis, and even among different human patients suffering from the same fibrosing disease (ex. IPF). Increased levels of ROS have been shown to activate TGF-β1 and induce apoptosis of AECs. Furthermore, oxidative stress induces secretion of pro-inflammatory mediators (mitochondrial DNA, Nalp3 inflammasome-related molecules) from the injured epithelium as well as from resident immune cells like macrophages. This potentiates additional recruitment of immune cells to the site of injury, further compounding the inflammatory response, and inducing further production of ROS by effector cells like neutrophils. Clinical studies in IPF patients have consistently found higher levels of ROS biomarkers in the BALF, serum, as well as in exhaled condensate. Furthermore, increases in ROS and oxidative stress are associated with BO, a fibrosing disease of the bronchioles instead of the alveolar tissue. While there is strong evidence for the involvement of ROS in the pathogenesis of pulmonary fibrosis, it acts to potentiate multiple KEs rather than acting as a key event itself. Oxidative stress is both causative and the consequence of observed responses in a feedforward type mechanism. 

Multiple studies using knockdown and KO mammalian models have shown that oxidative stress is involved in the development of pulmonary fibrosis. However, its essentiality in its pathogenesis is not conclusive, as antioxidant treatment offers no significant benefit in patients with IPF, the most common type of pulmonary fibrosis in humans. Furthermore, uncertainties remain concerning the exact molecular mechanisms underlying oxidative stress in the context of pulmonary fibrosis. (Checa and Aran, 2020; Cheresh et al., 2013; Dostert et al., 2008; Madill et al., 2009ab; Veith et al., 2019; https://aopwiki.org/aops/411; https://aopwiki.org/aops/424; https://aopwiki.org/aops/425). 

Macrophage polarization 

Depending on the lung microenvironment (damaged cells, microbial products, activated lymphocytes), the precursor monocytes differentiate into distinct types of macrophages. Classically activated (M1) macrophages and alternatively activated (M2) macrophages are the important ones to consider in the context of this AOP. The M1 macrophages produce high levels of pro-inflammatory cytokines, mediate resistance to pathogens, induce generation of high levels of ROS and reactive nitrogen species, and Th type 1 (Th1) responses. M1 macrophages produce IL-1, IL-12, IL-23 and induce Th1 cell infiltration and activation. The M2 macrophages secrete anti-inflammatory mediators, by which they play a role in regulation of inflammation. M2 polarization is mediated by Th2 cytokines such as IL-4 and IL-13, which in turn, promotes M2 activation. M2 macrophages express immunosuppressive molecules such as IL-10, Arginase (Arg)-1 and -2, which suppress the induction of Th1 cells that produce the anti-fibrotic cytokine IFN-γ The activity of M2 is associated with tissue remodelling, immune regulation, tumor promotion, tissue regeneration and effective phagocytic activity. 

Inhibition of M2 polarization through genetic depletion of surface receptors such as MARCO, attenuates the fibrotic phenotype. Depletion of interstitial macrophages bearing the M2 phenotype has been shown to block radiation-induced lung fibrosis. (Meziani et al., 2018; Murthy et al., 2015; Stahl et al., 2013).

Schematic depicting how chronic inflammation, oxidative stress and macrophage polarization connect to the main KEs in the AOP 173 through feedback loop. MIE: Molecular Initiating Event. KEs: Key Events. AO: Adverse Outcome.

 

Considerations for Potential Applications of the AOP (optional)

Addressess potential applications of an AOP to support regulatory decision-making.This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. More help

This AOP is applicable to occupational exposures as lung fibrosis is frequently observed in miners and welders exposed to metal dusts.

Pulmonary fibrosis is a progressive debilitating disease with no cure. A number of environmental and occupational agents, such as CS, agriculture or farming, wood dust, metal dust, stone and sand dust, play a causative role in the development of lung fibrosis. More recently, laboratory experiments in animals have shown that exposure to NMs, novel technology-enabled materials of sophisticated properties induce lung fibrosis. Fibrosis also develops in other organs (skin, liver, kidney, heart and pancreas) and the underlying mechanisms are similar. Thus, this AOP is applicable to screening of a broad group of suspected inhalation toxicants and allows the development of in silico and in vitro testing strategies for chemicals suspected to cause inhalation toxicity. Indeed, recent efforts aimed at collating all AOPs with potential relevance to NM risk assessment has led to the production of an AOP network which identified shared KEs of relevance to multiple AOs (Halappanavar et al., 2020). From this list, KE1 and KE2 from this AOP are among the most commonly shared between the various AOPs in the network. Shared KEs such as these can be prioritized for in vitro bio-assay development and tier-1 testing strategies. In a recent review, AOP 173 was used as a case study to define a testing strategy consisting of a slew of targeted bio-assay alternatives that can be used to screen for the in vivo occurrence of a number of the contained KEs (Halappanavar et al., 2021). These recent efforts serve to highlight the utility of AOP 173 in guiding the development of rapid screening strategies as well as research recommendations spanning across multiple AOPs with shared events.

This AOP is also currently being used by the various European Union nano research consortia to inform the design and development of relevant in vitro and in silico models for screening, prioritising, and assessing the potential of NMs to cause inhalation hazard. Specifically, this AOP has recently informed the development of a Nano Quantitative Structure Activity Relationship (NanoQSAR) model of CNT induced pulmonary inflammation, which found that the transcriptional response is associated with the aspect ratio of the nano fibres (Jagiello et al., 2021). Furthermore, this AOP can also inform the creation of biomarkers for fibrosis, such as the preliminary 17-gene pro-fibrotic biomarker panel, which was produced using global transcriptional datasets from mice exposed to CNTs (Rahman et al., 2020). Although in a preliminary stage, this signature composed of 17 genes can be used to assess the response of the MIE (Event 1495), KE1 (Event 1496), KE2 (Event 1497), KE4 (Event 1499), and KE5 (Event 1500), based on the differential expression of key bioinformatics-informed transcripts.

Given the fact that a number of pharmacological agents and allergens cause fibrosis via a similar mechanism; the mechanistic representation of the lung fibrotic process in an AOP format, clearly identifying the individual KEs potentially involved in the disease process, enables visualisation of the possible avenues for therapeutic interference in humans.

Confidence in the AOP

Mechanistically, there is enough evidence to support the occurrence of each individual KE in the process of lung fibrosis as described. There is also enough evidence to support each KERs. However, as mentioned earlier, the early KEs constitute an organism's defence system and thus exhibit high heterogeneity in the signalling pathways and biological networks involved. Therefore, the results of the essentiality experiments may show incongruence based on the individual protein, gene or pathway selected for intervention.

How well characterised is the AOP?

The AO is established and there is some quantitative data for some stressors.

How well are the initiating and other key events causally linked to the outcome?

The occurrence of each individual KE in the process leading to lung fibrosis is well accepted and established. However, individual studies mainly focus on a single KE and its relationship with the end AO. Quantitative data to support individual KERs is scarce.

What are the limitations in the evidence in support of the AOP?

As described earlier, attempts have been made to establish an in vitro model to predict the occurrence of fibrosis. However, the model has not been validated for screening the potential fibrogenic substances; the model has been used to identify drug targets that can effectively inhibit the progression to fibrosis (Chen et al., 2009). This is mainly due to the inability to accurately capture the responses induced by different cell types involved, and the intricate dynamics between the cell types, biological pathways and the biomolecules involved. Studies conducted to date have mainly focussed on the AO.

Is the AOP specific to certain tissues, life stages/age classes?

Fibrosis is a disease that affects several organ systems in an organism including lung, liver, heart, kidney, skin, and eye. The hallmark events preceding the end AO are similar to the one described here for lung fibrosis and involve similar cell types and biomolecules. Thus, the AOP can be extended to represent fibrosis in other organs. An AOP for liver fibrosis already exists and the KE 68 (collagen, accumulation) is shared by several fibrosis-related AOPs. The AOP is mainly applicable to adults as evidence to support applicability to different life stages is lacking. Lung fibrosis is thought to be a disease of male subjects. The early inflammatory KEs represented in this AOP constitute functional changes that describe inflammation in general. Several diseases are known to be mediated by inflammation and thus, early KEs in this AOP can be extended to any study investigating inflammation mediated AOs.

Are the initiating and key events expected to be conserved across taxa?

The events and pathways captured in this AOP are suggested to be conserved across different species and the process itself is influenced by the physical-chemical properties of the toxic substance.

References

List of the literature that was cited for this AOP. More help
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