Aop: 173


Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [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 lung resident cell membrane components leading to lung fibrosis

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Substance interaction with the lung cell membrane leading to lung fibrosis

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help


List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

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.


Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Sabina Halappanavar   (email point of contact)


List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Monita Sharma
  • Sabina Halappanavar


The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
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
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

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


In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). 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 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)

This optional subsection should be 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. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. 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 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

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


Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
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)

This table summarises 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.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help


The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help

Life Stage Applicability

Identify the life stage for which the KE 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 in relation to this KE. 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 authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Unspecific High

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help
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

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

This AOP applies to the following:


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.


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

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. 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.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

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).


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).


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).  


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

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

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)

At their discretion, the developer may include in this section discussion of the 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. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

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.


List the bibliographic references to original papers, books or other documents used to support the AOP. More help
  1. Aiso, S., Yamazaki, k., Umeda, Y., Asakura, M., Kasai, T., Takaya, M., Toya, T., Koda, S., Nagano, K., Arito, H. and Fukushima, S. (2010). Pulmonary Toxicity of Intratracheally Instilled Multiwall Carbon Nanotubes in Male Fischer 344 Rats. Industrial Health, 48(6), pp.783-795.
  2. Aiyappa Palecanda and Lester Kobzik (2001). Receptors for Unopsonized Particles: The Role of Alveolar Macrophage Scavenger Receptors. Current Molecular Medicine, 1(5), pp.589-595.
  3. Ando, M., Miyazaki, E., Fukami, T., Kumamoto, T. and Tsuda, T. (1999). Interleukin-4-producing cells in idiopathic pulmonary fibrosis: An immunohistochemical study. Respirology, 4(4), pp.383-391.
  4. Arnoldussen, Y., Anmarkrud, K., Skaug, V., Apte, R., Haugen, A. and Zienolddiny, S. (2016). Effects of carbon nanotubes on intercellular communication and involvement of IL-1 genes. Journal of Cell Communication and Signaling, 10(2), pp.153-162.
  5. Ashcroft, G., Yang, X., Glick, A., Weinstein, M., Letterio, J., Mizel, D., Anzano, M., Greenwell-Wild, T., Wahl, S., Deng, C. and Roberts, A. (1999). Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nature Cell Biology, 1(5), pp.260-266.
  6. Barbarin, V., Nihoul, A., Misson, P., Arras, M., Delos, M., Leclercq, I., Lison, D. and Huaux, F. (2005). The role of pro- and anti-inflammatory responses in silica-induced lung fibrosis. Respiratory Research, 6(1).
  7. Barron, L. and Wynn, T. (2011). Fibrosis is regulated by Th2 and Th17 responses and by dynamic interactions between fibroblasts and macrophages. American Journal of Physiology-Gastrointestinal and Liver Physiology, 300(5), pp.G723-G728.
  8. Bonniaud, P., Kolb, M., Galt, T., Robertson, J., Robbins, C., Stampfli, M., Lavery, C., Margetts, P., Roberts, A. and Gauldie, J. (2004). Smad3 Null Mice Develop Airspace Enlargement and Are Resistant to TGF-β-Mediated Pulmonary Fibrosis. The Journal of Immunology, 173(3), pp.2099-2108.
  9. Brush, J., Lipnick, S., Phillips, T., Sitko, J., McDonald, J. and McBride, W. (2007). Molecular Mechanisms of Late Normal Tissue Injury. Seminars in Radiation Oncology, 17(2), pp.121-130.
  10. Caielli, S., Banchereau, J. and Pascual, V. (2012). Neutrophils come of age in chronic inflammation. Current Opinion in Immunology, 24(6), pp.671-677.
  11. Cao, H., Wang, C., Chen, X., Hou, J., Xiang, Z., Shen, Y. and Han, X. (2018). Inhibition of Wnt/β-catenin signaling suppresses myofibroblast differentiation of lung resident mesenchymal stem cells and pulmonary fibrosis. Scientific Reports, 8(1).
  12. Chen, C., Kono, H., Golenbock, D., Reed, G., Akira, S. and Rock, K. (2007). Identification of a key pathway required for the sterile inflammatory response triggered by dying cells. Nature Medicine, 13(7), pp.851-856.
  13. Chen, C., Peng, Y., Wang, Z., Fish, P., Kaar, J., Koepsel, R., Russell, A., Lareu, R. and Raghunath, M. (2009). The Scar-in-a-Jar: studying potential antifibrotic compounds from the epigenetic to extracellular level in a single well. British Journal of Pharmacology, 158(5), pp.1196-1209.
  14. Chen, Y., Zhang, X., Bai, J., Gai, L., Ye, X., Zhang, L., Xu, Q., Zhang, Y., Xu, L., Li, H. and Ding, X. (2013). Sorafenib ameliorates bleomycin-induced pulmonary fibrosis: potential roles in the inhibition of epithelial–mesenchymal transition and fibroblast activation. Cell Death & Disease, 4(6), pp.e665-e665.
  15. Chaudhary, N., Schnapp, A. and Park, J. (2006). Pharmacologic Differentiation of Inflammation and Fibrosis in the Rat Bleomycin Model. American Journal of Respiratory and Critical Care Medicine, 173(7), pp.769-776.
  16. Cho, H., Reddy, S., Yamamoto, M. and Kleeberger, S. (2004). The transcription factor NRF2 protects against pulmonary fibrosis. The FASEB Journal, 18(11), pp.1258-1260.
  17. Chou, C., Hsiao, H., Hong, Q., Chen, C., Peng, Y., Chen, H. and Yang, P. (2008). Single-Walled Carbon Nanotubes Can Induce Pulmonary Injury in Mouse Model. Nano Letters, 8(2), pp.437-445.
  18. Cassel, S. and Sutterwala, F. (2010). Sterile inflammatory responses mediated by the NLRP3 inflammasome. European Journal of Immunology, 40(3), pp.607-611.
  19. Castranova, V. (2004). Signaling Pathways Controlling The Production Of Inflammatory Mediators in Response To Crystalline Silica Exposure: Role Of Reactive Oxygen/Nitrogen Species. Free Radical Biology and Medicine, 37(7), pp.916-925.
  20. Desmouliere, A., Darby, I., Laverdet, B. and Bonté, F. (2014). Fibroblasts and myofibroblasts in wound healing. Clinical, Cosmetic and Investigational Dermatology, p.301.
  21. Dong, J. and Ma, Q. (2015). Suppression of basal and carbon nanotube-induced oxidative stress, inflammation and fibrosis in mouse lungs by Nrf2. Nanotoxicology, 10(6), pp.699-709.
  22. Dong, J. and Ma, Q. (2016). In vivo activation of a T helper 2-driven innate immune response in lung fibrosis induced by multi-walled carbon nanotubes. Archives of Toxicology, 90(9), pp.2231-2248.
  23. Dong, J., Porter, D., Batteli, L., Wolfarth, M., Richardson, D. and Ma, Q. (2014). Pathologic and molecular profiling of rapid-onset fibrosis and inflammation induced by multi-walled carbon nanotubes. Archives of Toxicology, 89(4), pp.621-633.
  24. Dostert, C., Petrilli, V., Van Bruggen, R., Steele, C., Mossman, B. and Tschopp, J. (2008). Innate Immune Activation Through Nalp3 Inflammasome Sensing of Asbestos and Silica. Science, 320(5876), pp.674-677.
  25. Fichtner-Feigl, S., Strober, W., Kawakami, K., Puri, R. and Kitani, A. (2005). IL-13 signaling through the IL-13α2 receptor is involved in induction of TGF-β1 production and fibrosis. Nature Medicine, 12(1), pp.99-106.
  26. Fubini, B. and Hubbard, A. (2003). Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radical Biology and Medicine, 34(12), pp.1507-1516.
  27. Gasse, P., Mary, C., Guenon, I., Noulin, N., Charron, S., Schnyder-Candrian, S., Schnyder, B., Akira, S., Quesniaux, V., Lagente, V., Ryffel, B. and Couillin, I. (2007). IL-1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice. Journal of Clinical Investigation.
  28. Gharib, S., Johnston, L., Huizar, I., Birkland, T., Hanson, J., Wang, Y., Parks, W. and Manicone, A. (2013). MMP28 promotes macrophage polarization toward M2 cells and augments pulmonary fibrosis. Journal of Leukocyte Biology, 95(1), pp.9-18.
  29. Girtsman, T., Beamer, C., Wu, N., Buford, M. and Holian, A. (2012). IL-1R signalling is critical for regulation of multi-walled carbon nanotubes-induced acute lung inflammation in C57Bl/6 mice. Nanotoxicology, 8(1), pp.17-27.
  30. Gilhodes, J., Julé, Y., Kreuz, S., Stierstorfer, B., Stiller, D. and Wollin, L. (2017). Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis. PLOS ONE, 12(1), p.e0170561.
  31. Guan, R., Wang, X., Zhao, X., Song, N., Zhu, J., Wang, J., Wang, J., Xia, C., Chen, Y., Zhu, D. and Shen, L. (2016). Emodin ameliorates bleomycin-induced pulmonary fibrosis in rats by suppressing epithelial-mesenchymal transition and fibroblast activation. Scientific Reports, 6(1).
  32. Halappanavar, S., Nikota, J., Wu, D., Williams, A., Yauk, C. and Stampfli, M. (2013). IL-1 Receptor Regulates microRNA-135b Expression in a Negative Feedback Mechanism during Cigarette Smoke–Induced Inflammation. The Journal of Immunology, 190(7), pp.3679-3686.
  33. Hashimoto, S., Gon, Y., Takeshita, I., Maruoka, S. and Horie, T. (2001). IL-4 and IL-13 induce myofibroblastic phenotype of human lung fibroblasts through c-Jun NH2-terminal kinase–dependent pathway. Journal of Allergy and Clinical Immunology, 107(6), pp.1001-1008.
  34. Hiraku, Y., Guo, F., Ma, N., Yamada, T., Wang, S., Kawanishi, S. and Murata, M. (2015). Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation. Particle and Fibre Toxicology, 13(1).
  35. Hornung, V., Bauernfeind, F., Halle, A., Samstad, E., Kono, H., Rock, K., Fitzgerald, K. and Latz, E. (2008). Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nature Immunology, 9(8), pp.847-856.
  36. Huaux, F., Liu, T., McGarry, B., Ullenbruch, M. and Phan, S. (2003). Dual Roles of IL-4 in Lung Injury and Fibrosis. The Journal of Immunology, 170(4), pp.2083-2092.
  37. Hubbard, A., Timblin, C., Rincon, M. and Mossman, B. (2001). Use of Transgenic Luciferase Reporter Mice To Determine Activation of Transcription Factors and Gene Expression by Fibrogenic Particles. Chest, 120(1), pp.S24-S25.
  38. Hubbard, A., Timblin, C., Shukla, A., Rincón, M. and Mossman, B. (2002). Activation of NF-κB-dependent gene expression by  silica in lungs of luciferase reporter mice. American Journal of Physiology-Lung Cellular and Molecular Physiology, 282(5), pp.L968-L975.
  39. Jackson, J., Friberg, I., Little, S. and Bradley, J. (2009). Review series on helminths, immune modulation and the hygiene hypothesis: Immunity against helminths and immunological phenomena in modern human populations: coevolutionary legacies?. Immunology, 126(1), pp.18-27.
  40. Kolb, M., Margetts, P., Anthony, D., Pitossi, F. and Gauldie, J. (2001). Transient expression of IL-1β induces acute lung injury and chronic repair leading to pulmonary fibrosis. Journal of Clinical Investigation, 107(12), pp.1529-1536.
  41. Karmouty-Quintana, H., Philip, K., Acero, L., Chen, N., Weng, T., Molina, J., Luo, F., Davies, J., Le, N., Bunge, I., Volcik, K., Le, T., Johnston, R., Xia, Y., Eltzschig, H. and Blackburn, M. (2015). Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension. The FASEB Journal, 29(1), pp.50-60.
  42. Kawasaki, H. (2015). A mechanistic review of silica-induced inhalation toxicity. Inhalation Toxicology, 27(8), pp.363-377.
  43. Kim, S., Lee, J., Yang, H., Cho, J., Kwon, S., Kim, Y., Her, J., Cho, K., Song, C. and Lee, K. (2010). Dose-response Effects of Bleomycin on Inflammation and Pulmonary Fibrosis in Mice. Toxicological Research, 26(3), pp.217-222.
  44. Koli, K., Myllärniemi, M., Keski-Oja, J. and Kinnula, V. (2008). Transforming Growth Factor-β Activation in the Lung: Focus on Fibrosis and Reactive Oxygen Species. Antioxidants & Redox Signaling, 10(2), pp.333-342.
  45. Lam, C. (2003). Pulmonary Toxicity of Single-Wall Carbon Nanotubes in Mice 7 and 90 Days After Intratracheal Instillation. Toxicological Sciences, 77(1), pp.126-134.
  46. Li, D., Guabiraba, R., Besnard, A., Komai-Koma, M., Jabir, M., Zhang, L., Graham, G., Kurowska-Stolarska, M., Liew, F., McSharry, C. and Xu, D. (2014). IL-33 promotes ST2-dependent lung fibrosis by the induction of alternatively activated macrophages and innate lymphoid cells in mice. Journal of Allergy and Clinical Immunology, 134(6), pp.1422-1432.e11.
  47. Li, M., Krishnaveni, M., Li, C., Zhou, B., Xing, Y., Banfalvi, A., Li, A., Lombardi, V., Akbari, O., Borok, Z. and Minoo, P. (2011). Epithelium-specific deletion of TGF-β receptor type II protects mice from bleomycin-induced pulmonary fibrosis. Journal of Clinical Investigation, 121(1), pp.277-287.
  48. Lijnen, P., Petrov, V., Rumilla, K. and Fagard, R. (2003). Transforming growth factor-beta1 promotes contraction of collagen gel by cardiac fibroblasts through their differentiation into myofibroblasts. Methods and Findings in Experimental and Clinical Pharmacology, 25(2), p.79.
  49. Lo Re, S., Yakoub, Y., Devosse, R., Uwambayinema, F., Couillin, I., Ryffel, B., Marbaix, E., Lison, D. and Huaux, F. (2014). Uncoupling between Inflammatory and Fibrotic Responses to Silica: Evidence from MyD88 Knockout Mice. PLoS ONE, 9(7), p.e99383.
  50. Ma, B., Whiteford, J., Nourshargh, S. and Woodfin, A. (2016). Underlying chronic inflammation alters the profile and mechanisms of acute neutrophil recruitment. The Journal of Pathology, 240(3), pp.291-303.
  51. MacNee, W. (2001). Oxidative stress and lung inflammation in airways disease. European Journal of Pharmacology, 429(1-3), pp.195-207.
  52. Maggi, E., Cosmi, L., Liotta, F., Romagnani, P., Romagnani, S. and Annunziato, F. (2005). Thymic regulatory T cells. Autoimmunity Reviews, 4(8), pp.579-586.
  53. Mercer, R., Hubbs, A., Scabilloni, J., Wang, L., Battelli, L., Friend, S., Castranova, V. and Porter, D. (2011). Pulmonary fibrotic response to aspiration of multi-walled carbon nanotubes. Particle and Fibre Toxicology, 8(1), p.21.
  54. Misson, P., Brombacher, F., Delos, M., Lison, D. and Huaux, F. (2007). Type 2 immune response associated with silicosis is not instrumental in the development of the disease. American Journal of Physiology-Lung Cellular and Molecular Physiology, 292(1), pp.L107-L113.
  55. Miyazaki, Y., Araki, K., Vesin, C., Garcia, I., Kapanci, Y., Whitsett, J., Piguet, P. and Vassalli, P. (1995). Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis. A mouse model of progressive pulmonary fibrosis. Journal of Clinical Investigation, 96(1), pp.250-259.
  56. Muller, J., Huaux, F., Moreau, N., Misson, P., Heilier, J., Delos, M., Arras, M., Fonseca, A., Nagy, J. and Lison, D. (2005). Respiratory toxicity of multi-wall carbon nanotubes. Toxicology and Applied Pharmacology, 207(3), pp.221-231.
  57. Nathan, C. (2002). Points of control in inflammation. Nature, 420(6917), pp.846-852.
  58. Nikota, J., Banville, A., Goodwin, L., Wu, D., Williams, A., Yauk, C., Wallin, H., Vogel, U. and Halappanavar, S. (2017). Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: insights from an adverse outcome pathway framework. Particle and Fibre Toxicology, 14(1).
  59. N. Lekkerkerker, A., Aarbiou, J., van Es, T. and A.J. Janssen, R. (2012). Cellular Players in Lung Fibrosis. Current Pharmaceutical Design, 18(27), pp.4093-4102.
  60. O'Neill, L. and Greene, C. (1998). Signal transduction pathways activated by the IL-1 receptor family: ancient signaling machinery in mammals, insects, and plants. Journal of Leukocyte Biology, 63(6), pp.650-657.
  61. Ortiz, L., Lasky, J., Hamilton, R., Holian, A., Hoyle, G., Banks, W., Peschon, J., Brody, A., Lungarella, G. and Friedman, M. (1998). Expression of TNF and the Necessity of TNF Receptors in Bleomycin-Induced Lung Injury in Mice. Experimental Lung Research, 24(6), pp.721-743.
  62. Park, E., Roh, J., Kim, S., Kang, M., Han, Y., Kim, Y., Hong, J. and Choi, K. (2011). A single intratracheal instillation of single-walled carbon nanotubes induced early lung fibrosis and subchronic tissue damage in mice. Archives of Toxicology, 85(9), pp.1121-1131.
  63. Piguet, P. (1989). Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis. Journal of Experimental Medicine, 170(3), pp.655-663.
  64. Porter, D., Hubbs, A., Chen, B., McKinney, W., Mercer, R., Wolfarth, M., Battelli, L., Wu, N., Sriram, K., Leonard, S., Andrew, M., Willard, P., Tsuruoka, S., Endo, M., Tsukada, T., Munekane, F., Frazer, D. and Castranova, V. (2012). Acute pulmonary dose–responses to inhaled multi-walled carbon nanotubes. Nanotoxicology, 7(7), pp.1179-1194.
  65. Postlethwaite, A., Holness, M., Katai, H. and Raghow, R. (1992). Human fibroblasts synthesize elevated levels of extracellular matrix proteins in response to interleukin 4. Journal of Clinical Investigation, 90(4), pp.1479-1485.
  66. Porter, D., Hubbs, A., Mercer, R., Wu, N., Wolfarth, M., Sriram, K., Leonard, S., Battelli, L., Schwegler-Berry, D. and Friend, S. (2010). Mouse pulmonary dose- and time course-responses induced by exposure to multi-walled carbon nanotubes. Toxicology, 269(2-3), pp.136-147.
  67. Punithavathi, D., Venkatesan, N. and Babu, M. (2000). Curcumin inhibition of bleomycin-induced pulmonary fibrosis in rats. British Journal of Pharmacology, 131(2), pp.169-172.
  68. Rabolli, V., Badissi, A., Devosse, R., Uwambayinema, F., Yakoub, Y., Palmai-Pallag, M., Lebrun, A., De Gussem, V., Couillin, I., Ryffel, B., Marbaix, E., Lison, D. and Huaux, F. (2014). The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles. Particle and Fibre Toxicology, 11(1).
  69. Redente, E., Jacobsen, K., Solomon, J., Lara, A., Faubel, S., Keith, R., Henson, P., Downey, G. and Riches, D. (2011). Age and sex dimorphisms contribute to the severity of bleomycin-induced lung injury and fibrosis. American Journal of Physiology-Lung Cellular and Molecular Physiology, 301(4), pp.L510-L518.
  70. Redente, E., Keith, R., Janssen, W., Henson, P., Ortiz, L., Downey, G., Bratton, D. and Riches, D. (2014). Tumor Necrosis Factor-α Accelerates the Resolution of Established Pulmonary Fibrosis in Mice by Targeting Profibrotic Lung Macrophages. American Journal of Respiratory Cell and Molecular Biology, 50(4), pp.825-837.
  71. Redington, A., Madden, J., Frew, A., Djukanovic, R., Roche, W., Holgate, S. and Howarth, P. (1997). Transforming Growth Factor- β 1 in Asthma. American Journal of Respiratory and Critical Care Medicine, 156(2), pp.642-647.
  72. Rider, P., Carmi, Y., Guttman, O., Braiman, A., Cohen, I., Voronov, E., White, M., Dinarello, C. and Apte, R. (2011). IL-1α and IL-1β Recruit Different Myeloid Cells and Promote Different Stages of Sterile Inflammation. The Journal of Immunology, 187(9), pp.4835-4843.
  73. Rydman, E., Ilves, M., Vanhala, E., Vippola, M., Lehto, M., Kinaret, P., Pylkkänen, L., Happo, M., Hirvonen, M., Greco, D., Savolainen, K., Wolff, H. and Alenius, H. (2015). A Single Aspiration of Rod-like Carbon Nanotubes Induces Asbestos-like Pulmonary Inflammation Mediated in Part by the IL-1 Receptor. Toxicological Sciences, 147(1), pp.140-155.
  74. Serrano-Mollar, A., Closa, D., Prats, N., Blesa, S., Martinez-Losa, M., Cortijo, J., Estrela, J., Morcillo, E. and Bulbena, O. (2003). In vivoantioxidant treatment protects against bleomycin-induced lung damage in rats. British Journal of Pharmacology, 138(6), pp.1037-1048.
  75. Shi, X., Castranova, V., Halliwell, B. and Vallyathan, V. (1998). Reactive oxygen species and silica‐induced carcinogenesis. Journal of Toxicology and Environmental Health, Part B, 1(3), pp.181-197.
  76. Shvedova, A., Kisin, E., Mercer, R., Murray, A., Johnson, V., Potapovich, A., Tyurina, Y., Gorelik, O., Arepalli, S., Schwegler-Berry, D., Hubbs, A., Antonini, J., Evans, D., Ku, B., Ramsey, D., Maynard, A., Kagan, V., Castranova, V. and Baron, P. (2005). Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. American Journal of Physiology-Lung Cellular and Molecular Physiology, 289(5), pp.L698-L708.
  77. Soehnlein, O., Steffens, S., Hidalgo, A. and Weber, C. (2017). Neutrophils as protagonists and targets in chronic inflammation. Nature Reviews Immunology, 17(4), pp.248-261.
  78. Strieter, R. and Mehrad, B. (2009). New Mechanisms of Pulmonary Fibrosis. Chest, 136(5), pp.1364-1370.
  79. Suwara, M., Green, N., Borthwick, L., Mann, J., Mayer-Barber, K., Barron, L., Corris, P., Farrow, S., Wynn, T., Fisher, A. and Mann, D. (2013). IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts. Mucosal Immunology, 7(3), pp.684-693.
  80. Ueha, S., Shand, F. and Matsushima, K. (2012). Cellular and Molecular Mechanisms of Chronic Inflammation-Associated Organ Fibrosis. Frontiers in Immunology, 3.
  81. Wallace, W. and Howie, S. (1999). Immunoreactive interleukin 4 and interferon-? expression by type II alveolar epithelial cells in interstitial lung disease. The Journal of Pathology, 187(4), pp.475-480.
  82. Wallace, W., Fitch, P., Simpson, A. and Howie, S. (2006). Inflammation-associated remodelling and fibrosis in the lung - a process and an end point. International Journal of Experimental Pathology, 88(2), pp.103-110.
  83. Wang, H., Yamaya, M., Okinaga, S., Jia, Y., Kamanaka, M., Takahashi, H., Guo, L., Ohrui, T. and Sasaki, H. (2002). Bilirubin Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats. American Journal of Respiratory and Critical Care Medicine, 165(3), pp.406-411.
  84. Wilson, M. and Wynn, T. (2009). Pulmonary fibrosis: pathogenesis, etiology and regulation. Mucosal Immunology, 2(2), pp.103-121.
  85. Wynn, T. (2004). Fibrotic disease and the TH1/TH2 paradigm. Nature Reviews Immunology, 4(8), pp.583-594.
  86. Xie, C. (2011). Th2-like immune response in radiation-induced lung fibrosis. Oncology Reports.
  87. Yang, H., Rivera, Z., Jube, S., Nasu, M., Bertino, P., Goparaju, C., Franzoso, G., Lotze, M., Krausz, T., Pass, H., Bianchi, M. and Carbone, M. (2010). Programmed necrosis induced by asbestos in human mesothelial cells causes high-mobility group box 1 protein release and resultant inflammation. Proceedings of the National Academy of Sciences, 107(28), pp.12611-12616.
  88. Yates, C., Hebda, P. and Wells, A. (2012). Skin Wound Healing and Scarring: Fetal Wounds and Regenerative Restitution. Birth Defects Research Part C: Embryo Today: Reviews, 96(4), pp.325-333.
  89. Yi, E., Bedoya, A., Lee, H., Chin, E., Saunders, W., Kim, S., Danielpour, D., Remick, D., Yin, S. and Ulich, T. (1996). Radiation-induced lung injury in vivo: Expression of transforming growth factor?Beta precedes fibrosis. Inflammation, 20(4), pp.339-352.
  90. Zhu, W., von dem Bussche, A., Yi, X., Qiu, Y., Wang, Z., Weston, P., Hurt, R., Kane, A. and Gao, H. (2016). Nanomechanical mechanism for lipid bilayer damage induced by carbon nanotubes confined in intracellular vesicles. Proceedings of the National Academy of Sciences, 113(44), pp.12374-12379.