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Sabina Halappanavar 1*, Monita Sharma2, Hakan Wallin3, Ulla Vogel3, Kristie Sullivan4, Amy J. Clippinger2
1Environmental Health Science and Research Bureau, Health Canada, Ottawa.
2PETA International Science Consortium Ltd., London, United Kingdom.
3Physicians Committee for Responsible Medicine, Washington, DC.
4National Research Centre for the Working Environment, Copenhagen, Denmark.
*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
Sabina Halappanavar (email point of contact)
- Monita Sharma
- Sabina Halappanavar
|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 Do not cite||EAGMST Under Review||1.32||Included in OECD Work Plan|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 January 28, 2019 11:15
|Increased, interaction with the resident cell membrane components||January 15, 2018 17:02|
|Increased, secretion of proinflammatory and profibrotic mediators||January 15, 2018 16:27|
|Increased, recruitment of inflammatory cells||January 05, 2018 12:52|
|Increased, loss of alveolar capillary membrane integrity||January 05, 2018 12:59|
|Increased, activation of T (T) helper (h) type 2 cells||January 05, 2018 13:03|
|Increased, fibroblast proliferation and myofibroblast differentiation||January 05, 2018 13:08|
|Increased, extracellular matrix deposition||January 05, 2018 13:14|
|Pulmonary fibrosis||January 16, 2018 09:23|
|Interaction with the cell membrane leads to Increased proinflammatory mediators||January 05, 2018 13:18|
|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||January 01, 2018 16:49|
|Carbon nanotubes, Multi-walled carbon nanotubes, single-walled carbon nanotubes, carbon nanofibres||January 01, 2018 17:52|
This adverse outcome pathway (AOP) describes the linkages between the interaction of substances with the cellular membrane components and the lung fibrosis. Lung fibrosis is a dysregulated or an exaggerated tissue repair process. It denotes the presence of scar tissue in the alveolar capillary region of the lung where gas exchange occurs; it can be localised or more diffuse involving bronchi and pleura. The process involves intricate dynamics between several inflammatory and immune response cells, and the microenvironment of the alveolar-capillary membrane consisting of both immune and non-immune cells, and the lung interstitium, in the presence of sustained or repeated toxicant stimuli. Regardless of the type of stimulus, 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 and pro-fibrotic mediators are secreted (Key Event (KE) 1) 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, the purpose of which is to clear the invading pathogen or toxic substance. In the presence of continuous stimulus or persistent toxic substances, tissue injury ensues leading to the alveolar capillary membrane integrity loss (KE3) and activation of adaptive immune response. The purpose of the adaptive immune response is to resolve the inflammation and initiate healing process, involving activation of the T Helper type 2 cell signalling (KE4), during which anti-inflammatory and pro-repair/fibrotic molecules continue to be secreted. Once the healing process is initiated, fibroblast proliferation and myofibroblast differentiation is induced (KE5) leading to synthesis and deposition of extracellular matrix or collagen (KE6). Exaggerated collagen deposition leads to alveolar septa thickening, decrease in total lung volume, and lung fibrosis (Adverse Outcome). It is important to note that many of the individual KEs occur in parallel, early after exposure to fibrogenic stimuli and thus, it is difficult to establish key event relationships (KERs). The eventual clinical manifestation of the disease is influenced by the physical-chemical properties of the substance and duration of exposure.
Lung fibrosis can be induced by many substances, microorganisms or by over expression of specific inflammatory mediators such as cytokines and chemokines. This AOP is also applicable to materials such as nanomaterials that induce an inflammatory response as well as possess unique properties that allow for significant chronicity of the response, which takes place deep within the lung, beyond the airways and within the alveoli. Lung fibrosis occurs in humans and the key biological events involved are similar as the ones observed in animals. Thus, this AOP provides a detailed mechanistic account of the process of lung fibrosis across species.
Summary of the AOP
Events: Molecular Initiating Events (MIE)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||1495||Increased, interaction with the resident cell membrane components||Interaction with the 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||Increased, 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)
|Carbon nanotubes, Multi-walled carbon nanotubes, single-walled carbon nanotubes, carbon nanofibres|
Life Stage Applicability
Overall Assessment of the AOP
Overall assessment of AOP
Assessment of the Weight-of-Evidence supporting the AOP
Concordance of dose-response relationships
The pathway presented in this AOP is qualitative. There is some evidence on dose-response relationships; however, dose-response relationships for each individual KE are not available. Although there is empirical evidence to support the occurrence of each individual KE in the pathway of lung fibrosis, their essentiality to the final AO is not always experimentally investigated in the same studies or separately. For example, Gilhodes J-C (2017) showed that single intratracheal instillation of 0.25, 0.5, 0.75, and 1 mg/kg of bleomycin in mice induced alveolar thickening at 14 days post-exposure in a dose-dependent manner. However, this study did not investigate the occurrence of other KEs. In vivo exposure to different types of CNTs has been shown to induce all individual KEs involving inflammation and alveolar thickening in both mice and rats (Aiso et al., 2010; Dong et al., 2014; Lam et al., 2004; Mangum et al., 2006; Muller et al., 2005; Park et al., 2011; Porter et al., 2010, 2013; Shvedova et al., 2005). The inflammatory response following treatment with CNTs is typically characterised by the recruitment of inflammatory cells, and secretion of pro-inflammatory mediators; the lung fibrosis is characterised by the histopathological analysis for collagen deposition, fibrous lesions, and proliferation of fibroblasts. However, these studies did not evaluate the KERs.
The dose and the time-response of CNT-induced lung fibrosis is investigated in many studies. For example, pharyngeal aspiration of 10, 20, 40, or 80 µg/mouse MWCNT induced lung fibrosis in a dose-dependent manner which was apparent as early as 7 days post-exposure at 40 µg/mouse dose and persisted up to 56 days post-exposure (porter DW, 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 RR, 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 DW, 2013). Lung inflammation and fibrosis was observed in mice intratracheally instilled with 162 µg/mouse MWCNTs at 28 days post-exposure Fibrotic changes in lungs persisted up to 1 year post-exposure to 40 or 80 µg/mouse MWCNTs. All of the studies involving CNTs showed elevated levels of pro-inflammatory mediators, pro-inflammatory cells and cytotoxicity in BALF.
Barbarin et al (2005) showed that silica particles induce early inflammatory KEs in both rats and mice; however, the magnitude of the inflammatory response was much severe in rats compared to mice. Regardless, the extent of fibrosis as measured by collagen deposition and alveolar thickening was the same between the two species. Again, a clear dose-response relationship between KEs and the end AOP was not established. Silica-induced lung injury involves all the KEs described in this AOP including the associative events of chronic inflammation and ROS generation (Hubbard AK, 2005). Although the fibrotic pathology induced by silica involves acute inflammatory phase and Th2 response, the role of these KEs in progression of the disease itself is not conclusive (Barbarin C, 2005; Brombacher MP, 2007).
Studies have shown that inhibition of inflammatory events early after exposure to bleomycin (Gasse P, 2007) or CNTs (Nikota J, 2017) attenuate fibrotic response in mice. These studies provide the necessary evidence supporting the essentiality of inflammatory KEs to the end AO.
There is enough empirical evidence to show the temporal associations between the individual KEs leading to the AO.
Strength, consistency, and specificity of association of adverse outcome and initiating event
Depending on the type of substance and its physical-chemical property, the type of interactions with resident cells differs. Asbestos fibres are shown to bind directly to cellular macromolecules including proteins and membrane lipids, which is influenced by their surface properties such as surface charge (reviewed in Hanley GD, 1995). The alarmin HMGB1 is released from damaged or nectrotic cells in cell culture models and in animals following exposure to asbestos and is suggested to be involved in the inflammatory events elicited by asbestos (Yang H, 2010). Interaction of CNTs with HMGB1-RAGE is implicated in pro-inflammatory and genotoxic effects of CNTs (Hiraku Y, 2016). Mechanical stress and membrane damage following cellular uptake of long and stiff CNTs by lysosomes is involved in cell injury and consequent adverse effects (Zhu W, 2016). CNT-induced inflammatory response in vitro is mediated by IL-1, absence of which negatively impacts gap junctional intercellular communication (Arnoldussen YJ, 2016). The levels of IL-1a are increased in BALF of mice immediately after exposure to MWCNTs (Nikota J, 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 final AO. The inconsistency could be due to the fact that early defence mechanisms involving DAMPs is fundamental for survival, which may necessitate redundancy in signalling pathways involved. 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 J, 2017) and thus, IL-1a mediated signalling is suggested to be involved in MWCNT-induced lung inflammation and fibrosis (Rydman EM, 2015). However, inhibition of IL-1a signalling alone did not alter the MWCNT-induced fibrotic response in mice (Nikota J, 2017). This study further showed that simultaneous inhibition of both acute inflammatory events and Th2 –mediated signalling may be required to suppress lung fibrosis induced by MWCNTs (Nikota J, 2017). Disengagement between innate immune responses including MIE, KE1 and KE2, and ultimate lung fibrosis has been shown in a mouse model following exposure to silica (Re SL, 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 suggested to be involved in bleomycin-induced lung inflammation and fibrosis; inhibition of IL1-R1 signalling attenuates the bleomycin pathology (Gasse P, 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 MIE and individual KEs, and thus evidence supporting the AOP is strong. Although, there is inconsistency in empirical evidence supporting the KERs, the AOP is established with the existing biological evidence. Thus, the 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 for any known substance. However, many may argue that innate immune response involving acute inflammation and associated KEs may not be necessary for lung fibrosis. This raises the question if the MIE should be a KE that involves lung injury or activation of cells that produce collagen.
Uncertainties, inconsistencies and data gap
As mentioned earlier, the AOP is based on the existing knowledge and describes the sequence of events that are shown to occur following exposure to many fibrogenic substances. However, it is mostly qualitative and additional studies are needed to support the essentiality of the KEs.
As described above, 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. Regardless of the type of interactions or the substance characteristics, the end outcome of lung fibrosis follows the same sequence of events.
Essentiality of the Key Events (key event relationships)
Weight of evidence summary
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: Increased, substance interaction with the resident cell membrane component leads
to increased pro-inflammatory mediators
KE1- KE2: Increased, pro-inflammatory mediators lead to increased recruitment of pro-inflammatory cells
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 C, 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, IL-1 is a pleotropic cytokine and impacts nearly every cell in the body. Members of the IL-1 signalling pathway are evolutionarily conserved across many species including mammals, insects, plants, and yeast. Certain members of the IL-1 signalling pathway seem to have been present in the unicellular ancestral organisms of both plants and animals, suggesting that IL-1 represents an ancient defence system that provided protection against pathogen invasion. In higher vertebrates, IL-1 signalling plays a vital role in defence against infection and injury (O’Neill LAJ and Greene C, 1998). IL-1a is an alarmin. When released from the damaged cells, IL-1a stimulates host-derived alarmin/IL-1a synthesis, thus perpetuating and sustaining the response. The stressor can also trigger the IL-1R1 signalling without the initial cell death. In such cases, the external stressors trigger translocation of intracellular pro-IL-1a onto the plasma membrane, where IL-1a appears as membrane bound molecule ready to bind IL-1R1 on the neighbouring nonhematopoietic cells or resident macrophages, activating the IL-1R1 cascade. Binding of IL-1a to IL-1R1 can release NF-κb resulting in its translocation to nucleus and transactivation of pro-inflammatory genes including cytokines, growth factors and acute phase genes, and increased 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 CJ, 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. Thus secreted mediators signal the recruitment of neutrophils, which are the first cell types to be recruited in acute inflammatory conditions. Neutrophil influx in sterile inflammation is driven mainly by IL-1a (Rider P, 2011). IL-1 mediated signalling regulates neutrophil influx in silica-induced acute lung inflammation (Horning V, 2008). IL1 signalling also mediates neutrophil influx in other tissues and organs including liver, peritoneum. Other types of cells including macrophages, eosinophils, lymphocytes are also recruited in a signal-specific manner. Recruitment of leukocytes (neutrophils mainly) 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 V, 2008; Girtsman TA, 2014; Gasse P, 2007; Nikota J, 2017; Suwara MI, 2013; Rabolli V, 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 J, 2017), cigarette smoke (Halappanavar S, 2013), silica crystals (Rabolli V, 2014; Re SL, 2014) and bleomycin (Gasse P, 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 J, 2017). The inconsistency could be due to the fact that early defence mechanisms involving DAMPs is fundamental for survival, which may necessitate redundancy in signalling pathways involved. 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.
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 that greater concentrations or doses induce higher release of IL-1a and concomitant IL-1R1 signalling, resulting in a higher neutrophil influx in lungs. However, these studies demonstrate strong temporal relationships between the individual KEs.
KE2 – KE3
Increased, recruitment of proinflammatory cells leads to loss of alveolar capillary membrane integrity
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 C, 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 (Robert M, 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 (Robert M, 2009). In rodents treated with bleomycin, the damaged ACM resembles that seen in the fibrotic human lung (Grandel NR, 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 O, 2017), which leads to ACM integrity loss. Neutrophils are the dominant cell population during acute inflammation and communicate with other immune cell types such as platelets, monocytes, dendritic cells, and different types of lymphocytes via the mediators they synthesise and secrete (Nathan C, 2002). However, the exact mechanisms by which acute neutrophilic inflammation leads to chronic inflammation vary and depend on the type of tissue injury or properties of the toxic substance. Some of the common links between neutrophils and chronic inflammation include: 1) neutrophils deposit a variety of granule proteins on the endothelium, some of which act as chemotactic for monocytes, 2) it is shown that neutrophils also secrete alarmins such as S1008, S1009, and high-mobility group box 1, which drive myeloid cell recruitment and induce recruitment of pro-inflammatory macrophages, 3) prolonged life span of neutrophils - normally, death of neutrophils marks the beginning of resolution phase of the acute inflammation, during which dead neutrophils are cleared by macrophages and normal homeostasis is established, and 4) neutrophils committed to apoptosis also activate signals that degrade pro-inflammatory mediators, and thus deplete and dampen the inflammatory stimulus. Clearance of neutrophils from the inflammatory site triggers resolution of inflammation and consequently, the tissue repair process (Nathan C, 2002).
In the presence of persisting or repeated tissue injury, these 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. It has been shown that in 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 can lead to inflammation, pulmonary injury and subsequently, fibrosis in experimental bleomycin models (Chaudhary NI, 2006). ROS released by neutrophils via the multicomponent enzyme nicotinamide adenine dinucleotide phosphate oxidase 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 S 2012; Koli K, 2008).
Exposure to high doses of insoluble materials such as ENMs 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 2004), which in turn, is dependent on activation of NF-κB and ROS synthesis (Shi et al 1998; Cassel et al 2008; Kawasaki 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 W, 2001). Several studies have reported that anti-oxidant treatment attenuates the bleomycin-induced oxidative burden and subsequent pulmonary fibrosis (Wang, HD, 2002; Serrano-Mollar A, 2003; Punithavathi, D, 2000).
Uncertainties or inconsistencies
Although there is enough evidence to suggest a role for persistent inflammation 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, the 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 SN, 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 (Bin Ma, 2016).
Loss of alveolar capillary membrane integrity leads to activation of Th2 type cell signalling
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 and pro-inflammatory and fibrotic mediators including GATA-3, IL-13 and Arg-1 are increased in lung-irradiation induced fibrosis (Wynn TA, 2004; Brush J, 2007; Han G, 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, shows that Th2 response associated genes are upregulated in fibrotic lungs (Nikota J, 2016). Exposure of mice lacking STAT6 transcription factor to fibrosis causing doses of MWCNTs resulted in abrogated expression of Th2 genes and reduced lung fibrosis (Nikota J, 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 MS and Wynn TA, 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 E, 2005). Chemokines associated with the Th2 response in airway epithelial cells include CCL1, CCL17, CCL20, and CCL22 (Lekkerkerker N, 2012).
Weight of evidence studies establishing the KER are very scarce and data is not available to establish the quantitative dose- or time- response relationships.
Activation of Th2 type cell signalling leads to fibroblast proliferation and myofibroblast differentiation
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 TGF-β, 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 TGFb1 in macrophages and its absence results in reduced TGFb1 expression and decrease in collagen deposition (Fichtner-Feigl S, 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 L and Wynn TA, 2011).
Weight of evidence
C57BL/6 mice exposed to MWCNTs for seven days showed altered expression of a wide variety of Th2 cytokines including IL-4 and IL-13 (Dong J and Ma Q, 2016). The study implicated IL-4 and IL-13 signalling as well as activation of transcription factors STAT6 and GATA3 are necessary for MWCNT-induced lung fibrosis in rodents. In agreement with this study, in a meta-analysis, comparison of gene expression profiles of lungs of mice exposed to different types of nanomaterials to the lung transcriptomics data from mouse models of lung inflammation and lung fibrosis showed significant similarities between Th2-cytokine model and MWCNT-induced lung fibrosis model (Nikota J, 2016). Moreover, exposure of STAT6 transgenic mice to MWCNTs suppressed the expression of Th2-mediated gene expression and the overall fibrotic response to MWCNTs (Nikota J, 2017).
Uncertainties or inconsistencies
Since a vast number of pro-inflammatory mediators with redundant functions are involved in these processes, targeting a single molecule or a pathway is not sufficient enough to impact the KE or the eventual adverse outcome.
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
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-1b and TGF-β, 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 IA, 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 TGF-β, 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 U, 2012; Wallace WA, 2007).
Weight of evidence, Uncertainties or inconsistencies, Quantitative understanding of the linkage
There is some evidence to show that inhibition of fibroblast proliferation and differentiation by counteracting the activity of TGF-β attenuates bleomycin-induced lung fibrosis (Chen Y-L, 2013; Guan R, 2016). Several studies have shown that inhibition of TGF-β involved both 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.
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 redundancy in the signalling pathways involved. Therefore, the results of the essentiality experiments may show incongruence based on the individual protein, gene or a pathway selected for inhibition.
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 lacking.
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 of other organs. Moreover, 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 this some of the KEs in this AOP can be extended to any study investigating inflammation mediated adverse outcomes.
Are the initiating and key evets 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.
Domain of Applicability
Essentiality of the Key Events
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)
Considerations for potential applications of the AOP
Efforts are ongoing to develop a predictive in vitro model to assess the fibrogenic potential of substances. Additional work is needed to optimise these cell systems to mimic the in vivo conditions. 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, design and development of relevant in vitro models for screening, prioritising, and assessing the chemicals’ potential to induce fibrosis. This in turn will facilitate uptake of data derived from alternative toxicity testing methods for the purposes of regulatory decision making. The assessment of the extent of perturbation of the MIE, KE1, and KE2 can help rank the fibrogenic capacity of a substance, all of which are influenced by the physical-chemical properties of a substance.
We thank Dr. Stephen Edwards and Dr. Dan Villeneuve for their prompt and able technical assistance in smooth uploading of this AOP. Many thanks to Dr. Brigitte Landesmann for the critical review of the AOP and for valuable input. Thanks to Dr. Carole Yauk for her support and critical comments on the AOP. Funding for this project was provided by Health Canada’s Genomics Research and Development Initiative. Dr. Ula Vogel received support from SmartNanoTox, European Union’s Horizon 2020 research and innovation programme under grant agreement No. 686098. We thank the members of the SmartNanoTox consortium members for their constructive comments on the AOP.
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