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Aop: 173

AOP Title

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

Short name:

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Substance interaction with the lung cell membrane leading to lung fibrosis

Graphical Representation

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Click to download graphical representation template

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Authors

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

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

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 and Nanotoxicology Laboratory

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

Tunney's Pasture Bldg. 8 (P/L 0803A),

50 Colombine Driveway, Ottawa, Ontario, K1A 0K9 Canada.

sabina.halappanavar@canada.ca

Email: sabina.halappanavar@canada.ca

Point of Contact

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Sabina Halappanavar   (email point of contact)

Contributors

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  • Monita Sharma
  • Sabina Halappanavar

Status

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Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite EAGMST Under Review 1.32 Included in OECD Work Plan


This AOP was last modified on November 11, 2019 21:59

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Revision dates for related pages

Page Revision Date/Time
Interaction with the lung resident cell membrane components November 08, 2019 08:32
Increased, secretion of proinflammatory and profibrotic mediators October 30, 2019 11:28
Increased, recruitment of inflammatory cells October 30, 2019 12:23
Loss of alveolar capillary membrane integrity October 31, 2019 12:01
Increased, activation of T (T) helper (h) type 2 cells October 31, 2019 13:09
Increased, fibroblast proliferation and myofibroblast differentiation November 01, 2019 08:57
Increased, extracellular matrix deposition November 01, 2019 11:14
Pulmonary fibrosis November 01, 2019 11:51
Interaction with the lung cell membrane leads to Increased proinflammatory mediators November 01, 2019 13:03
Increased proinflammatory mediators leads to Recruitment of inflammatory cells January 05, 2018 13:18
Recruitment of inflammatory cells leads to Loss of alveolar capillary membrane integrity January 05, 2018 13:19
Loss of alveolar capillary membrane integrity leads to Activation of Th2 cells January 05, 2018 13:19
Activation of Th2 cells leads to Increased cellular proliferation and differentiation January 05, 2018 13:20
Increased cellular proliferation and differentiation leads to Increased extracellular matrix deposition January 05, 2018 13:20
Increased extracellular matrix deposition leads to Pulmonary fibrosis January 16, 2018 09:35
Bleomycin October 29, 2019 13:08
Carbon nanotubes, Multi-walled carbon nanotubes, single-walled carbon nanotubes, carbon nanofibres January 01, 2018 17:52

Abstract

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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 lung cells leading to fibrosis. 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 an exaggerated tissue repair process. It denotes the presence of scar tissue in the localised alveolar capillary region of the lung where gas exchange occurs; it can be localised or more diffuse involving bronchi and pleura. It involves the presence of sustained or repeated exposure to 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) in the process of tissue repair. As a consequence, a myriad of pro-inflammatory mediators are secreted (KE1) that signal the recruitment of pro-inflammatory cells into the lungs (KE2). 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) and activation of adaptive immune response, the T Helper type 2 cell signalling (KE4), during which anti-inflammatory and pro-repair/fibrotic molecules are secreted. The repair and healing process stimulates fibroblast proliferation and myofibroblast differentiation (KE5), leading to synthesis and deposition of extracellular matrix or collagen (KE6). Excessive collagen deposition culminates in alveolar septa thickening, decrease in total lung volume and lung fibrosis (Adverse Outcome).

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 over expression 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 substances of diverse properties and provides a detailed mechanistic account of the process of lung fibrosis across species.


Background (optional)

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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 foundation for the later pathology. 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. Recently, an AOP for stressor-induced pulmonary inflammation resulting in lung emphysema has been initiated and is currently under development. Here, a mechanism underlying stressor-induced lung fibrosis that involves a pro-inflammatory component is described.

Although this AOP is applicable to a broad group of chemicals of diverse properties, the AOP was specifcally assembled keeping in mind, a novel class of engineered materials (nanomaterials) exhibiting sophisticated properties that have been shown to induce lung fibrosis via this mechanism. Thus, it demonstrates the applicability of the AOP framework to nanotoxicology.  

Given the fundametal role of inflammation in organ homeostasis, well characterized AOPs targetting 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.  

 


Summary of the AOP

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Events: Molecular Initiating Events (MIE)

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Key Events (KE)

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Adverse Outcomes (AO)

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Sequence Type Event ID Title Short name
1 MIE 1495 Interaction with the lung resident cell membrane components Interaction with the lung cell membrane
2 KE 1496 Increased, secretion of proinflammatory and profibrotic mediators Increased proinflammatory mediators
3 KE 1497 Increased, recruitment of inflammatory cells Recruitment of inflammatory cells
4 KE 1498 Loss of alveolar capillary membrane integrity Loss of alveolar capillary membrane integrity
5 KE 1499 Increased, activation of T (T) helper (h) type 2 cells Activation of Th2 cells
6 KE 1500 Increased, fibroblast proliferation and myofibroblast differentiation Increased cellular proliferation and differentiation
7 KE 1501 Increased, extracellular matrix deposition Increased extracellular matrix deposition
8 AO 1458 Pulmonary fibrosis Pulmonary fibrosis

Relationships Between Two Key Events
(Including MIEs and AOs)

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Title Adjacency Evidence Quantitative Understanding
Interaction with the lung cell membrane leads to Increased proinflammatory mediators adjacent High Not Specified
Increased proinflammatory mediators leads to Recruitment of inflammatory cells adjacent High High
Recruitment of inflammatory cells leads to Loss of alveolar capillary membrane integrity adjacent High High
Loss of alveolar capillary membrane integrity leads to Activation of Th2 cells adjacent High Moderate
Activation of Th2 cells leads to Increased cellular proliferation and differentiation adjacent High High
Increased cellular proliferation and differentiation leads to Increased extracellular matrix deposition adjacent High High
Increased extracellular matrix deposition leads to Pulmonary fibrosis adjacent High High

Network View

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Stressors

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Life Stage Applicability

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Life stage Evidence
Adult High

Taxonomic Applicability

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Term Scientific Term Evidence Link
human Homo sapiens High NCBI
mouse Mus musculus High NCBI
rat Rattus norvegicus High NCBI

Sex Applicability

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Sex Evidence
Unspecific High

Overall Assessment of the AOP

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Attached file: Table 1   weight of evidence

Overall assessment

Ideopathic pulmonary fibrosis (IPF) is a complex, progressive disease of unknown etiology that is most commonly observed in humans. Lung fibrosis in humans is also observed following exposure to pharmacological agents such as bleomycin, following inhalation of silica, asbestos, cigarette smoke, coal dust and following microbials and allergen exposure. Regardless of the etiology, lung fibrosis in humans is characterised by the presence of inflammatory lesions, excessive extracellular matrix deposition, 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 functions. Some other considerations of relevance to this AOP:

This AOP represents fibrotic mechanism that involves a strong inflammatory component. Exposure to pro-fibrotic stressors such as, bleomycin, silica, asbestos, CNTs, radiation or models of overexpression of cytokines 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, Hebda and Wells, 2012). Asbestosis and silicosis, 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, cats, donkeys, pigs and other animals have been studied to investigate different types of fibrosis. 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.

Assessment of the Weight-of-Evidence supporting the AOP

Concordance of dose and time-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 fibrosis in rodents. 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 (over 2000 microarrays) mouse lungs exposed individually mouse 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 MWCNT 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 (10mg/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 was 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 adverse outcome 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; Dörger et al., 2001; Brown et al., 2007; Kim J-E 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 Hanley, 1995). 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 alarmins from cells.

The alarmin HMGB1 is released from damaged or nectrotic 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-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-1a 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 MIE 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 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-1a secretion in BALF of mice (Nikota et al., 2017) and thus, IL-1a mediated signalling is involved in MWCNT-induced lung inflammation and fibrosis (Rydman et al., 2015). Inhibition of IL-1a 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 and KE2) and Th2 –mediated signalling (KE4) is required to suppress lung fibrosis induced by MWCNTs (Nikota et al., 2017). Disengagement between innate immune responses including MIE, KE1 and KE2, and ultimate lung fibrosis is shown in a 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 mouse models lacking individual members of 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, the alarmin IL-1a and IL-1R1 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). Thus, the results supporting the KERs are not consistent.

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 that may be described

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 PPARg resulting in lung fibrosis was proposed (Jeong et al., 2019). The alternate AOP for fibrosis placed activation of TGFb1 upstream of inflammatory events (KE2, KE3), which is contrary to its perceived role in downstream events leading to fibroblast proliferation and differentiation, and extracellular matrix deposition. The stressors identified in this study were also different, suggesting the PPARg 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 extracellular matrix deposition. For example, overexpression of TGFb1 can promote excessive ECM deposition and fibrosis in rodents independent of inflammation.

Uncertainties, inconsistencies and data gap

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.

Domain of Applicability

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This AOP applies to the following:

Stressors

1. Stressors that persist in the lung environment for a long duration of time causing chronic injury (silica, coal dust). Repeated exposure to stressors such as bleomycin, cigarette smoke, other pharamcological drugs that cause chronic lung injury. 

2. The long and rigid fibres or high aspect ratio fibres (asbestos, CNTs).

3. Stressors that indcue a strong inflammatory component (bleomycin, silica, silica dust, etc).

4. Stressors exhibiting unique physical-chemical properties including shape (fibres, particles), crystal structure (crystalline silica), etc.

Sex/Gender and age

Ideopathic pulmonary fibrosis (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. 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, cats, donkeys, pigs and other animals have been studied to investigate different types of fibrosis. 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.

Other applications

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


Essentiality of the Key Events

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Essentiality of the Key Events (key event relationships)

Please refer to Table-1.

Although the MIE, KE1, and KE2 occur in sequence and are described as separate KEs, the animal or cell culture experiments are generally not designed to measure these events separately. As a result, there is not enough empirical support to build individual KERs. Thus, in the KER description below, the following KERs will be considered together.

MIE – KE1: Substance interaction with the resident lung cell membrane components leads to increased pro-inflammatory mediators

KE1- KE2: Increased pro-inflammatory mediators leads to increased recruitment of pro-inflammatory cells

KER description

Innate immune response is the first line of defence in any organism against invading infectious pathogens and toxic substances. It involves tissue triggered startle response to cellular stress and is described by a complex set of interactions between the toxic stimuli, soluble macromolecules and cells (reviewed in Nathan, 2002). The process culminates in a functional change defined as inflammation, purpose of which is to resolve infection and promote healing. In lungs, the interaction of toxic substances with resident cells results in cellular stress, death or necrosis leading to release of intracellular components such as alarmins (DAMPs, IL-1a, HMGB1). Released alarmins (danger sensors) bind cell surface receptors such as Interleukin 1 Receptor 1 (IL-1R1), Toll Like Receptors (TLRs) or others leading to activation of innate immune response signalling.

For example, binding of IL-1a to IL-1R1 can release Nuclear Factor (NF)-κb resulting in its translocation to nucleus and transactivation of pro-inflammatory genes including cytokines, growth factors and acute phase genes. The signalling also stimulates secretion of a variety of pro-inflammatory mediators. Overexpression of IL-1a in cells induces increased secretion of pro-inflammatory mediators. Products of necrotic cells are shown to stimulate the immune system in an IL-1R1-dependent manner (Chen et al., 2007).

The secreted alarmins activate resident cells pre-stationed in the tissues such as mast cells or macrophages leading to propagation of the already initiated immune response by releasing more eicosanoids, cytokines, chemokines and other pro-inflammatory mediators. The secreted mediators, in turn, signal the recruitment of neutrophils, which are the first cell types to be recruited in acute inflammatory conditions. Other types of cells including macrophages, eosinophils, lymphocytes are  recruited in a signal-specific manner. Neutrophil influx in sterile inflammation is driven mainly by IL-1a (Rider et al., 2011). IL-1 mediated signalling regulates neutrophil influx  (Horning et al., 2008). IL-1 signalling also mediates neutrophil influx in other tissues and organs including liver and peritoneum.  Recruitment of leukocytes induces critical cytokines associated with the Th2 immune response, including TNF-α, IL-1β, and IL-13.

Weight of Evidence

Both empirical evidence and biological plausibility are strong. Increased expression of IL-1a or IL-1b following lung exposure to MWCNTs, bleomycin, micro silica particles, silica crystals, and polyhexamethyleneguanidine phosphate has been shown to be associated with neutrophil influx in rodents (Horning et al., 2008; Girtsman et al., 2014; Gasse et al., 2007; Nikota et al., 2017; Suwara et al., 2013; Rabolli et al., 2014). Inhibition of IL-1 function by knocking out the expression of IL-1R1 using IL-1R1 KO mice or via treatment with IL-1a or IL-1b neutralising antibodies results in complete abrogation of lung neutrophilic influx following exposure to MWCNTs (Nikota et al, 2017), cigarette smoke (Halappanavar et al., 2013), silica crystals (Rabolli et al., 2014; Re et al., 2014) and bleomycin (Gasse et al., 2017). In transgenic mice lacking IL1R1, Myd88 signalling or the IL-33 receptor St2, early inflammatory responses are suppressed following silica or bleomycin treatment (Dong, et al., 2014; Gasse et al., 2017).

Uncertainties or inconsistencies

Attenuation or complete abrogation of KE1 and KE2 following inflammogenic stimuli is observed in rodents lacking functional IL-1R1 or other cell surface receptors that engage innate immune response upon stimulation. However, following exposure to MWCNTs, it has been shown that absence of IL-1R1 signalling is compensated for eventually and neutrophil influx is observed at a later post-exposure time point (Nikota et al., 2017). In another study, acute neutrophilic inflammation induced by MWCNT was suppressed at 24 hr in mice deficient in IL1R1 signalling; however, these mice showed exacerbated neutrophilic influx and fibrotic response at 28 days post-exposure (Girtsman et al., 2014). The early defence mechanisms involving DAMPs is fundamental for survival, which may necessitate activation of compensatory signalling pathways . As a result, inhibition of a single biological pathway mediated by an individual cell surface receptor may not be sufficient to completely abrogate the lung inflammatory response. Forced suppression of pro-inflammatory and immune responses early after exposure to substances that cannot be effectively cleared from lungs, may enhance the injury and initiate other pathways leading to exacerbated response.

Quantitative understanding of the linkage

A majority of the in vivo studies are conducted with only one dose and thus, it is difficult to derive quantitative dose-response relationships based on the existing data. However, it is clear from the studies referenced above that greater concentrations or doses of pro-fibrotic substances results in higher release of alarmins, and consequently, higher pro-inflammatory signalling. The above studies also  demonstrate strong temporal relationships between the individual KEs.

KE2 – KE3

Increased recruitment of pro-inflammatory cells leads to loss of alveolar capillary membrane integrity

KER description

Acute lung injury followed by normal repair of the ACM results in rapid resolution of the tissue injury and restoration of tissue integrity and function. The irreversible loss of alveolar membrane integrity occurs when 1) acute inflammation is not able to get rid of the toxic substance or invading pathogen (this happens following exposure to a toxic substance that is persistent or when the host is repeatedly exposed to the substance over a long period of time), 2) acute inflammation, originally incited to protect the host from external stimuli and to maintain normal homeostasis, by itself damages the host, resulting in tissue injury, and 3) the host fails to initiate a resolution response, which is essential to override the self-perpetuating inflammation response (Nathan, 2002). Loss of type-1 epithelial cells and endothelial cells, the collapse of alveolar structures and fusion of basement membranes, and persistent proliferation of type II alveolar epithelial cells on a damaged ECM, mark this phase (Strieter and Mehrad, 2009). The lung tissues from patients diagnosed with idiopathic pulmonary fibrosis show ultrastructural damage to the ACM with type-1 pneumocyte and endothelial cell injury (Strieter and Mehrad, 2009).  In rodents treated with bleomycin, the damaged ACM resembles that seen in the fibrotic human lung (Grandel, 1998). 

Role of ROS synthesis and chronic inflammation in the loss of ACM integrity

In general, chronic or persistent inflammation occurs after prolonged acute inflammation (Soehnlein et al., 2017), which leads to ACM integrity loss. Neutrophils are the dominant cell population during acute inflammation. Clearance of neutrophils from the inflammatory site triggers resolution of inflammation and consequently, the tissue repair process (Nathan, 2002). Failure to trigger neutrophil deathand continued secretion of damaging enzymes by the neutrophils contributes to the propagation of inflammatory response and cell injury.

In the presence of persisting or repeated tissue injury, the macrophages induce a large amount of pro-inflammatory cytokines that can prolong the lifespan of neutrophils resulting in prolongation of the acute inflammatory phase. The pro-inflammatory cytokines such as IL-1b, TNF-α, and others secreted by macrophages are shown to delay neutrophil death. The other factor that can delay neutrophil death is ROS, synthesised by neutrophils, which can activate specific membrane receptors that inhibit neutrophil apoptosis. Humans suffering from sepsis exhibiting neutropenia (deficiency of neutrophils) have fewer macrophages in their BALF compared to the healthy human population. Excessive production of ROS leads to inflammation, pulmonary injury and subsequently, fibrosis in experimental bleomycin models (Chaudhary, Schnapp and Park, 2006). ROS released by neutrophils via the multicomponent enzyme nicotinamide adenine dinucleotide phosphate oxidase (NAPDH) is a known contributor to tissue injury and mediator of both lung and liver fibrosis. ROS can activate TGF-β directly or indirectly via proteases, and TGF-β itself further induces ROS production through NADPH oxidase catalytic subunit NOX4 (Caielli, Banchereau and Pascual, 2012; Koli et al., 2008).

Weight of evidence

Exposure to high doses of insoluble nanomaterials can impair the macrophage-mediated clearance process, initiating chronicity of inflammation characterized by cytokine release, ROS synthesis and the tissue damage cascade (Palecanda and Kobzik, 2001) and subsequently leading to tissue injury. For example, exposure to crystalline silica generates oxidative stress, increased release of pro-inflammatory cytokines (e.g. TNF-α, IL-1, IL-6), activation of transcription factors (e.g. NF-κB, AP-1), and other cell signalling pathways including MAP and ERK kinase (Hubbard et al., 2001; Hubbard et al., 2002; Fubini and Hubbard 2003). In silicosis, TNF-α is suggested to play a critical role in the observed pathogenicity (Castranova et al., 2004), which in turn, is dependent on activation of NF-κB and ROS synthesis (Shi et al.,1998; Cassel et al.,2008; Kawasaki et al., 2015). It has been proposed that IPF is a disorder of elevated oxidative stress, with the existence of an oxidant-antioxidant imbalance in distal alveolar air spaces (MacNee, 2001). Several studies have reported that anti-oxidant treatment attenuates the bleomycin-induced oxidative burden and subsequent pulmonary fibrosis (Wang et al., 2002; Serrano-Mollar  et al., 2003; Punithavathi, et al., 2000).

Mice deficient in Nalp3 showed reduced inflammation, lower cytokine production and dampened fibrotic response following exposure to asbestos or silica (Dostert et al., 2008). SWCNT exposure induces alveolar macrophage activation, enhanced oxidative stress, increased and persistent expression of pro-inflammatory mediators associated with chronic inflammation and severe granuloma formation in mice (Chou et al., 2008). Bleomycin treatment induces increased lung weight, epithelial cell death, inflammation, increased hydroxyproline content, collagen accumulation and fibrotic lesions in mice, all of which were elevated in mice deficient in Nrf2 (Cho et al., 2004). MWCNT-induced fibrotic response is the result of interplay between oxidative stress and inflammation, which determines the severity of the fibrotic pathology. Mice lacking Nrf2 (the nuclear factor erythroid 2-related factor 2), that is associated with mounting anti-oxidant defense against oxidative stress, exhibit exuberant fibrotic responses to MWCNT (Dong and Ma, 2016).

Uncertainties or inconsistencies

Although there is enough evidence to suggest a role for persistent inflammation and oxidative stress in ACM integrity loss, a direct relationship is hard to establish as studies involving inhibition of early pro-inflammatory cellular influx alter other immune cell types, thereby altering the end outcome.

Quantitative understanding of the linkage

In the context of lung fibrosis, data supporting quantitative dose-response relationships between the individual KEs is scarce. A majority of the mechanistic studies investigating the role of inflammation in lung fibrosis report acute neutrophilic inflammation and how altering neutrophil influx acutely after exposure to a toxic substance alters the end fibrotic outcome. However, these studies do not characterise the impact on immediate downstream KEs including the loss of ACM integrity or chronic inflammation in the absence of acute neutrophilia. Few studies have shown such concordance. For example, in mice exposed to different doses of bleomycin, total number of cells in BALF increased in a dose-dependent manner with predominant neutrophil phenotype at 7 days post-exposure and macrophage dominance at 24 days post-exposure (Kim et al., 2010). Other studies have shown that upon onset of chronic inflammation, secondary stimuli such as persisting toxic substance can make the injured tissue highly sensitive to acute inflammatory stimuli and may in turn fuel the ongoing chronic inflammation and affect the disease process (Ma et al., 2016).

KE3-KE4

Loss of alveolar capillary membrane integrity leads to activation of Th2 type cell signalling

KER description

During the tissue injury-mediated immune response, naïve CD4+ Th cells differentiate into two major functional subsets: Th1 and Th2 type. Both Th1 and Th2 secrete distinct cytokines that promote proliferation and differentiation of their respective T cell population and inhibit proliferation and differentiation of the opposing subset. Th2 cytokines including pro-inflammatory and fibrotic mediators such as GATA-3, IL-13 and Arg-1 are increased in lung-irradiation induced fibrosis (Wynn, 2004; Brush et al., 2007; Han et al., 2011). Th2 immune response is implicated in allergen-mediated lung fibrosis. Meta-analysis of gene expression data collected from lungs of mice exposed to various fibrogenic substances including MWCNTs, showed that the expression and function of Th2 response associated genes and pathways are altered in fibrotic lungs (Nikota Jet al., 2016). Exposure of mice lacking STAT6 transcription factor to MWCNTs resulted in abrogated expression of Th2 genes and reduced lung fibrosis (Nikota et al., 2017). IL-4, the archetypal Th2 cytokine is a pro-fibrotic cytokine and is elevated in IPF and lung fibrosis. Overexpression of pro-fibrotic Th2 cytokine IL-13 results in subepethelial fibrosis with eosinophilic inflammation (Wilson and Wynn, 2009). In silica-induced pulmonary fibrosis in mice, T regulatory lymphocytes are recruited to the lungs where they increase expression of platelet-derived growth factor (PDGF) and TGF-β (Maggi et al., 2005). Chemokines associated with the Th2 response in airway epithelial cells include CCL1, CCL17, CCL20, and CCL22 (Lekkerkerker et al., 2012).

Weight of evidence

Studies establishing this KER are very scarce and data is not available to establish the quantitative dose- or time- response relationships.

In mice lacking both TNFα receptor 1 (TNF-R1) and receptor 2 (TNF-R2) or in wild type mice treated with anti-TNFα, bleomycin-induced lung fibrosis is attenuated (Ortiz, 1998; Piguet, 1989). Persistent activation of TNF-α and IL1-β results in elevated secretion of pro-inflammatory cytokines that are tissue damaging.  Over expression of IL-1β induces acute lung injury and lung fibrosis in mice (Kold, 2001). TNFα and IL1β are the therapeutic targets in IPF and asbestosis (Zhang et al., 1993). Overexpression of TNFa induces spontaneous fibrosis in mouse lungs (Miyaki et al., 1995). In cases of infestation with parasitic worm helminths, chronic injury activates a large immune response, resulting in secretion of pro-inflammatory mediators that can inflict cell and tissue damage. Effective treatment involves control of immune-response mediated damage (reviewed in Jackson et al., 2009).

Inconsistencies

Exogenous delivery of TNFα to mouse lungs with established fibrosis, reduced the fibrotic burden. Exogenous treatment with TNFα slowed the M2 macrophage polarisation. TNFα deficient mice showed prolonged pro-fibrotic response and M2 polarisation following bleomycin treatment (Redente et al., 2014).  

KE4-KE5

Activation of Th2 type cell signalling leads to fibroblast proliferation and myofibroblast differentiation

KER description

The wound healing process involves an inflammatory phase, during which the damage tissue/wound is provisionally filled with ECM. This phase is characterised by secretion of cytokines/chemokines, growth factors and recruitment of inflammatory cells, fibroblasts and endothelial cells. The activated Th1/Th2 response and increased pool of specific cytokines and growth factors such as IL-1b, IL-6, IL-13, and TGFβ, induce fibroblast proliferation. Th2 cells can directly stimulate fibroblasts to synthesise collagen with IL-1 and IL-13. Th2 cytokines IL-13 and IL-4, known to mediate the fibrosis process induce phenotypic transition of human fibroblasts (Hashimoto S, 2001). IL-13 is shown to inhibit MMP-mediated matrix degradation resulting in excessive collagen deposition by downregulating the synthesis and expression of matrix degrading MMPs. IL-13 is also suggested to induce TGFβ1 in macrophages and its absence results in reduced TGFβ1 expression and decrease in collagen deposition (Fichtner-Feigl et al., 2006). These cytokines are suggested to initiate polarisation of macrophages to M2 phenotype. Th2 cells that synthesise IL-4 and IL-13 induce synthesis of Arg-1 in M2 macrophages. The Arg-1 pathway stimulates synthesis of proline for collagen synthesis required for fibrosis (Barron and Wynn, 2011).

Weight of evidence

A majority of the weight of evidence studies assess collagen synthesis as a proxy to fibroblast proliferation and myofibroblast differentiation. A few studies have shown that Th2 cytokine IL-4 stimulates fibroblast proliferation (Sempowski et al., 1994) and production of ECM components (Postlethwaite et al., 1992). In human studies, the progression of idiopathic pulmonary fibrosis is also associated with a sustained IL-4 production (Wallace and Howie, 1999; Ando et al., 1999). Th2 cytokines induce expression and activity of TGFb1, levels of which are elevated in BALF of patients suffering from lung interstitial diseases, is a potent inducer of myofibroblast differentiation and collagen synthesis (Redington et al., 1997; Kurosaka et al., 1998). Exposure of STAT6 deficient mice to MWCNTs, suppressed acute lung inflammation, expression of Th2-mediated gene expression, reduced vimentin positive cells (marker of fibroblasts), levels of collagen synthesis and reduced the overall fibrotic response to MWCNTs (Nikota et al., 2017). Mice deficient in IL-33r (St2, Th2 response cytokine) or mice treated with anti-IL33 antibody, showed reduced lung inflammation, reduced collagen production and fibrotic pathology induced by bleomycin. IL-33 deficient mice treated with bleomycin showed reduced levels of IL-1 and other pro-inflammatory cytokines. Mice administered exogenously with mature IL-33 enhanced bleomycin-induced lung inflammation, collagen synthesis and fibrotic lesions (Dong et al., 2014).

Uncertainties or inconsistencies

Due to multifarious functions of several cytokines involved in the process of inflammation and repair, the timing of when a pathway is intervened in an experiment is important in the assessment of the KER studies. For example, exposure to pro-fibrotic bleomycin stimulates IL-4 production during the acute inflammatory phase, which is suggested to limit the recruitment of T lymphocytes and production of damaging cytokines such as TNFα, IFNγ, and nitric oxide, playing a tissue protective role. However, production of IL-4 during the chronic phase of tissue repair and healing, favours fibrosis manifestation. Treatment of IL4 -/- mice with low doses of bleomycin induced fewer fibrotic lesions compared to IL-4 +/+ mice. However, treatment of high doses of bleomycin induced more lethality in IL-4 -/- mice compared to the wild type mice (Huaux et al., 2003). Moreover, the KEs represented in the AOP can function in parallel in a positive feedback loop, perpetuating and magnifying the response at each stage. The resulting microenvironment may contain same molecules in different proportions exhibiting different functions. Thus, the complexity of the process and the functional heterogeneity of the molecular players involved, makes it nearly impossible to establish KERs using a targeted deletion of one single gene or a pathway in a study, which is how most of the studies are designed.

Quantitative understanding of the linkage

A majority of the in vivo studies are conducted with only one dose and thus, it is difficult to derive quantitative dose-response relationships based on the existing data.

KE5 – KE6

Fibroblast proliferation and myofibroblast differentiation leads to ECM/collagen deposition

KER description

When activated, fibroblasts migrate to the site of tissue injury and build a provisional ECM, which is then used as a scaffold for tissue regeneration. Activated fibroblasts in turn produce IL-13, IL-6, IL-1β and TGFβ, propagating the response. In the second phase, which is the proliferative phase, angiogenesis is stimulated to provide vascular perfusion to the wound. During this phase more fibroblasts are proliferated and they acquire a-smooth muscle actin expression and become myofibroblasts. Thus, myofibroblasts exhibit features of both fibroblasts and smooth muscle cells. The myofibroblasts synthesise and deposit ECM components that eventually replace the provisional ECM. Because of their contractile properties, they play a major role in contraction and closure of the wound tissue (Darby et al., 2014). Apart from secreting ECM components, myofibroblasts also secrete proteolytic enzymes such as metalloproteinases and their inhibitors tissue inhibitor of metalloproteinases, which play a role in the final phase of the wound healing which is scar formation phase or tissue remodelling.

During this final phase, new synthesis of ECM is suppressed to allow remodelling. The wound is resolved with the secretion of procollagen type 1 and elastin, and infiltrated cells including inflammatory cells, fibroblasts and myofibroblasts are efficiently removed by cellular apoptosis. However, in the presence of continuous stimulus resulting in excessive tissue damage, uncontrolled healing process is initiated involving exaggerated expression of pro-fibrotic cytokines and growth factors such as TGFβ, excessive proliferation of fibroblasts and myofibroblasts, increased synthesis and deposition of ECM components, inhibition of reepithelialisation, all of which lead to replacement of the normal architecture of the alveoli and fibrosis (Satoshi et al., 2012; Wallace et al., 2007).

Weight of evidence, Uncertainties or inconsistencies, Quantitative understanding of the linkage

Mice infused subcutaneously with bleomycin showed pronounced lung fibrosis, characterised by the elevated levels of TGFβ1 and collagen genes (Hoyt et al., 1988). Radiation induced lung fibrosis was shown to precede high levels of TGFβ1 expression (Eunhee et al., 1996). Mice lacking TGFβ-receptor II showed resistance to bleomycin-induced lung fibrosis (Li et al., 2011). Inhibition of fibroblast proliferation and differentiation by counteracting the activity of TGF-β attenuates bleomycin-induced lung fibrosis (Chen et al., 2013; Guan et al., 2016). Adenoviral vector-mediated gene transfer based transient overexpression of TGFb1 in lungs of mice induced progressive lung fibrosis (Bonniaud et al., 2004). Targeted inhibition of Wnt/b-catenin signalling by a small molecule drug inhibited the mesenchymal-myofibroblast transition and repressed matrix gene expression leading to attenuated lung fibrosis (Cao et al., 2018). Several studies have shown that inhibition of TGF-β involved in fibroblast activation and collagen deposition results in attenuated fibrotic response in lungs; however, results are inconsistent.

More studies are required to support the quantitative KER.

KE5 – KE6

Excessive ECM/collagen deposition leads to alveolar septa thickness (fibrosis)

Fibrosis by definition is the end result of a healing process. It involves a series of lung remodelling and reorganisation events leading to permanent alteration in the lung architecture and a fixed scar tissue or fibrotic lesion (Wallace WA, 2007). Excessive deposition of ECM or collagen is the hallmark of this disease and there is ample evidence to support this KER.

Quantitative considerations

Since the adverse outcome of lung fibrosis involves multiple cell types, cell - cell interactions and cell–biomolecule interactions, it is difficult to recapitulate the entire process in one model. Therefore an integrated approach, such as one consisting of cell systems that assess individual KEs and quantitative relationships between the KEs, is needed to predict the AO in humans. 

 


Evidence Assessment

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Quantitative Understanding

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Quantitative considerations

Since the adverse outcome of lung fibrosis involves multiple cell types, cell - cell interactions and cell–biomolecule interactions, it is difficult to recapitulate the entire process in one model. Therefore an integrated approach, such as one consisting of cell systems that assess individual KEs and quantitative relationships between the KEs, is needed to predict the AO in humans.


Considerations for Potential Applications of the AOP (optional)

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Considerations for potential applications of the AOP

Pulmonary fibrosis is a progressive debilitating disease with no cure. A number of environmental and occupational agents, such as cigarette smoke, 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 nanomaterial, 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. Especially, in the field of nanotoxicology, considering the vast number of nanomaterials and their property variants that require testing, the AOP will allow rapid screening and identification of potentially pro-fibrogenic materials. This AOP is 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 nanomaterials to cause inhalation hazard.

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 organisms’ 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 a pathway selected for intervention.

How well characterised is the AOP?

The adverse outcome 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 C, 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 adverse outcome.

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. 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 adverse outcomes.

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

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