AOP ID and Title:

Short Title: Substance interaction with the pulmonary cell membrane leading to pulmonary fibrosis

Authors

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

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

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

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

⁴Physicians Committee for Responsible Medicine, Washington, DC.

 

*Point of contact

Sabina Halappanavar, PhD

Research Scientist, Genomics, Nanotoxicology and Alternative Methods Laboratory

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

Sir Frederick G Banting Research Centre,

251 Sir Frederick Banting Driveway, Building 22

Ottawa, ON, Canada, K1A 0K9

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

 

Status

Abstract

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

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

Acknowledgements: The lead author would like to acknowledge the able assistance of Andrey Boyadhziev of Health Canada, Ottawa, Ontario, Canada, in formatting the response document and preparing some responses to external reviewers' comments and questions.

Background

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

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

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

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

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Bleomycin

Bleomycin is a potent anti-tumour drug, routinely used for treating various types of human cancers (Umezawa et al., 1967; Adamson, 1976). Lung injury and lung fibrosis are the major adverse effects of this drug in humans (Hay J et al., 1991). Bleomycin is shown to induce lung fibrosis in experimental animals - in dogs (Fleischman et al., 1971), mice (Adamson IY and Bowden DH, 1974), hamsters (Snider GL et al., 1978) and is widely used as a model chemical to study the mechanisms of fibrosis in humans (reviewed in Moeller et al., 2008; Gilhodes et al., 2017).

1. Adamson, I. (1976). Pulmonary Toxicity of Bleomycin. Environmental Health Perspectives, 16, p.119.
2. Adamson, IYR. and Bowden, DH. (1974). The Pathogenesis of Bleomycin-Induced Pulmonary Fibrosis in Mice. The American Journal of Pathology. 77(2), pp185-198.
3. Fleischman, R., Baker, J., Thompson, G., Schaeppi, U., Illievski, V., Cooney, D. and Davis, R. (1971). Bleomycin-induced interstitial pneumonia in dogs. Thorax, 26(6), pp.675-682.
4. 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.
5. Hay, J., Shahzeidi, S. and Laurent, G. (1991). Mechanisms of bleomycin-induced lung damage. Archives of Toxicology, 65(2), pp.81-94.
6. Moeller, A., Ask, K., Warburton, D., Gauldie, J. and Kolb, M. (2008). The bleomycin animal model: A useful tool to investigate treatment options for idiopathic pulmonary fibrosis?. The International Journal of Biochemistry & Cell Biology, 40(3), pp.362-382.
7. Snider GL., Celli, BR., Goldstein, RH., O'Brien, JJ. and Lucey, EC. (1978). Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Lung volumes, volume-pressure relations, carbon monoxide uptake, and arterial blood gas studied. Am Rev Respir Dis. Feb; 117(2). pp289-97.
8. Umezawa, H., Ishizuka, M., Maeda, K. and Takeuchi, T. (1967). Studies on bleomycin. Cancer, 20(5), pp.891-895.

Carbon nanotubes, Multi-walled carbon nanotubes, single-walled carbon nanotubes, carbon nanofibres

CNTs are high aspect ratio materials and cause lung fibrosis in experimental animals (Muller et al., 2005; Porter DW et al., 2010). In an intelligence bulletin published by NIOSH on ‘Occupational exposure to carbon nanotubes and nanofibers’, NIOSH reviewed 54 individual animal studies investigating the pulmonary toxicity induced by CNTs and reported that half of those studies consistently showed lung fibrosis (NIOSH bulletin, 2013). Multiwalled carbon nanotubes induce lung fibrosis in mice (Nikota et al., 2017; Rahman et al., 2017). However, the evidence is inconsistent and the occurrence of fibrotic pathology is influenced by the specific physical-chemical properties of CNTs (length, rigidity), their dispersion in exposure vehicle, and the mode of exposure.

1. 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.
2. NIOSH (2013). Occupational exposure to carbon nanotubes and nanofibers: current intelligence bulletin 65.
3. 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.
4. 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).
5. Rahman L, Jacobsen NR, Aziz SA, Wu D, Williams A, Yauk CL, White P, Wallin H, Vogel U, Halappanavar S. Multi-walled carbon nanotube-induced genotoxic, inflammatory and pro-fibrotic responses in mice: Investigating the mechanisms of pulmonary carcinogenesis. Mutat Res. 2017 Nov;823:28-44.

Overall Assessment of the AOP

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

Some other considerations of relevance to this AOP:

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

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

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

Sex/Gender and Age:

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

Taxonomy:

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

Types of Stressors:

Persistent and soluble stressors can induce fibrotic pathologies in humans (as well as in model animals) in concordance with the AOP presented. Asbestos exposure in humans has long been known to induce pulmonary fibrosis (asbestosis) due to chronic inflammation induced from persistent fibres deposited within the lung (Kamp and Weitzman 1997). Similarly, human exposure to silica (crystalline silica dust) leads to the development of silicosis in concordance with the AOP presented (Ding et al., 2002). Furthermore, the soluble chemotherapeutic compound bleomycin has long been known to induce pulmonary fibrosis in humans (in line with this AOP) as a side effect of intravenous administration (Froudarakis et al., 2013). In addition to these model stressors, exposure to various metals including uranium, arsenic, cadmium, and soluble copper can lead to fibrotic outcomes in humans (Assad et al., 2019). Occupational exposure to cobalt can induce interstitial lung disease in humans, which can progress to fibrotic outcomes (Adams et al., 2017). In male mice exposed via inhalation to cadmium oxide nanoparticles (NPs), increases in the pro-fibrotic and pro-inflammatory mediators IL-1β, TNF-α , and Interferon gamma (IFN-γ) were noted one day post-exposure, with accompanying pulmonary inflammation (Blum et al., 2014). In another study, intratracheal instillation of cadmium chloride (CdCl₂) in mice induced peribronchiolar fibrosis through activation of myofibroblasts via the Suppressor of mothers against decapentaplegic (SMAD) signalling (Li et al., 2017). As with the aforementioned cadmium NPs, murine animals exhibit pronounced acute inflammation and immune cell infiltration after pulmonary exposure to copper oxide NPs (Gosens et al., 2016), which can progress to a fibrotic phenotype in some model systems after 28 days with marked increases of TGF-β1 detected in the bronchoalveolar lavage fluid (BALF), activation of myofibroblasts, and pronounced deposition of ECM (Lai et al., 2018). In mice, intratracheal instillation of cobalt NPs results in pronounced infiltration of neutrophils and macrophages into the alveolar and interstitial space, and increased amounts of C-X-C motif chemokine ligand (CXCL)1 in the BALF 1-7 days post-exposure; pronounced pulmonary fibrosis was detected at 4 months post-exposure marked by increased collagen deposition and bronchiolization of the alveolar epithelium (Wan et al., 2017).

Essentiality of the Key Events

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

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

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

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

Weight of Evidence Summary

Concordance of Dose-Response Relationships:

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

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

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

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

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

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

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

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

Alternative Mechanisms:

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

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

Evidence Assessment Summary:

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

Quantitative Consideration

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

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

Stressor	Species	Time Point	MIEa (1495)	KE1a (1496)	KE2a (1497)	KE3 (1498)	KE4a (1499)	KE5a (1500)	KE6a (68)	AO (1458)
Mitsui 7 CNT	Mouse	24 Hr	4 – 9	3 - 7	9 – 13	N/A	5 – 11	10 – 21	9 – 13	N/A
Mitsui 7 CNT	Mouse	3 / 7 day	11 – 22	6 – 22	14 – 24	N/A	9 – 16	15 – 26	17 – 34	N/A
Mitsui 7 CNT	Mouse	28 day	No Effect	14 – 26	36 – 51	N/A	14 – 26	11 – 20	No Effect	N/A
Mitsui 7 CNT	Mouse	56 day	N/A	N/A	N/A	N/A	N/A	N/A	N/A	14 – 27b
NRCWE-026 CNT	Mouse	24 Hr	No effect	8 – 15	20 – 37	N/A	8 – 15	21 – 39	No Effect	N/A
NRCWE-026 CNT	Mouse	3 / 7 day	16 – 28	16 – 27	19 – 33	N/A	15 – 24	16 – 26	19 – 36	N/A
NRCWE-026 CNT	Mouse	28 day	No Effect	No Effect	No Effect	N/A	12 – 20	No Effect	No Effect	N/A
NM-401 CNT	Mouse	24 Hr	No Effect	3 – 20	8 – 22	N/A	8 – 22	13 – 22	18 – 29	N/A
NM-401 CNT	Mouse	3 / 7 day	11 - 17	12 - 19	12 - 20	N/A	7 - 20	14 - 22	13 – 21	N/A
NM-401 CNT	Mouse	28 day	20 - 37	17 - 28	No Effect	N/A	No Effect	13 - 21	18 – 31	N/A

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

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

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

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

The role of ROS and oxidative stress in potentiating pulmonary fibrosis is also ambiguous. Many pro-fibrotic substances induce the formation of ROS and subsequent oxidative stress, as do many non-fibrotic stressors. While it is hard to deny that ROS and oxidative stress serve an important role in fibrosis by increasing cellular injury, potentiating an environment of chronic inflammation & damage, and activation of pro-fibrotic factors like TGF-β1, a causal relationship between the two has not been established.  Furthermore, antioxidant treatment in IPF patients has been largely unsuccessful, indicating a lack of knowledge of the specific redox mechanisms involved. Recent research has indicated a potential role of specific redox mechanisms, such as mitochondrial ROS and nitrogen oxides derived ROS, however further research is needed to elucidate their role in potentiating pulmonary fibrosis. The development of newer fibrosis model systems which better capitulate the human condition will assist in clarifying this aspect.

Considerations for Potential Applications of the AOP (optional)

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

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

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

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

Confidence in the AOP

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

How well characterised is the AOP?

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

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

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

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

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

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

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

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

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

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Appendix 1

List of MIEs in this AOP

Event: 1495: Substance interaction with the lung resident cell membrane components

Short Name: Interaction with the lung cell membrane

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

As stated earlier, there are many different ways by which pro-fibrotic stressors can interact with the components of cell membrane and often involve multiple interactions at the same time. Few studies investigate the exact interaction between the stressor and the cellular membrane components. Asbestos and silica crystals engage scavenger receptors present on the macrophages (Murthy et al., 2015). Bleomycin binds high affinity bleomycin binding sites present on rat alveolar macrophage surfaces, leading to macrophage activation (Denholm and Phan, 1990). However, the consequences of such interactions such as, the release of PRR agonists DAMPs (alarmins) from dying or injured cells, increased  gene or protein synthesis downstream of receptor binding or in the case of NMs, their cellular uptake, are measured routinely as indicative of occurrence of such interactions (Nel et al., 2009; Cheng et al., 2013). Because of the phys-chem properties such as surface charge, NMs and asbestos like materials can bind to cellular macromolecules and cell surface/membrane components, which in turn, facilitate their uptake and intracellular sequestration by the cells (NIOSH, 2011a; Pascolo et al., 2013). Several DAMPs that can be effectively measured in biological samples and cultured cells include High Mobility Group Binding 1 (HMGB1) protein, Heat Shock proteins (HSPs), uric acid, annexins, and S100 proteins (Bianchi, 2007). Of all DAMPs, interleukin (IL)-1α is the most commonly measured alarmin. IL-1α is the principal pro-inflammatory moiety and is a designated ‘alarmin’ in the cell that alerts the host to injury or damage (Di Paolo and Shayakhmetov, 2016). It is shown that administration of necrotic cells to mice results in neutrophilic inflammation that was entirely mediated by IL-1α released from the dying or necrosed cells and consequent activation of IL-1 Receptor 1 (IL-1R1) signalling (Suwara et al., 2014). IL-1α is released following exposure to MWCNTs (Nikota et al., 2017) and silica (Rabolli et al., 2014). Although IL1-b is not a designated alarmin, its secretion following exposure to stressors is routinely assessed and is linked to initiation of cell or tissue injury.

Other high aspect ratio fibres such as asbestos and CNTs induce frustrated phagocytosis and acute cell injury (Boyles et al., 2015; Dörger et al., 2001; Brown et al., 2007; Kim et al., 2010; Poland et al., 2008), leading to DAMP release (Nikota et al, 2017), inflammation and immune responses.

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Human, mouse, rat.

Although the expression of DAMPs following exposure to pro-fibrotic substances is not assessed across species, it is known that alarmins are released after trauma or injury, and their release is important for initiating the inflammatory response in all species including humans. The immediate acute inflammatory response involving DAMP signalling is also observed in human idiopathic pulmonary fibrosis (IPF); however, anti-inflammatory drugs have proven ineffective for treating IPF. Danger signalling axis including uric acid, adenosine triphosphate and IL-33/ST2 has been proven to promote lung fibrosis in animals.

Key Event Description

The human lung consists of approximately 40 different resident cell types that play different roles during homeostasis, injury, repair and disease states (Franks et al., 2008; Luettich et al., 2021). Of these, resident airway epithelial cells, alveolar/interstitial macrophages and dendritic cells are well characterised for their ability to sense the danger upon interaction with harmful substances and relay the message to mount the necessary immune/inflammatory response. The resident macrophages are present in all tissues, and in a steady state, macrophages contribute to epithelial integrity, survey the tissue for invading pathogens or chemicals and maintain an immunosuppressive environment. Their main function is to clear the incoming irritants and microbes. They are named differently based on the tissue type and their specific functions (Kierdorf et al., 2015).

Substance interactions:

The chemicals or pathogens interact with cellular membrane to gain access to the organisms’ interior. A predominant interaction mechanism involves the recognition of innate immune response agonists by pattern recognition receptors (PRRs) present on resident cells such as epithelial and alveolar macrophages. PRRs are also present on other immune and parenchymal cells. PRRs can be activated by two classes of ligands. Pathogen associated molecular patterns (PAMPs) are microbial molecules derived from invading pathogens. PAMPs will not be discussed further as pathogens are not the focus for the AOP presented here. The other class of ligands are called danger associated molecular patterns (DAMPs) that include cellular fragments, nucleic acids, small molecules, proteins and even cytokines released from injured or dying cells (Bianchi, 2007). Most fibrogenic stressors discussed in this AOP act via DAMPs-driven PRR activation. High aspect ratio (HAR) materials such as asbestos or carbon nanotubes (CNTs) pierce the cellular membrane of epithelial cells or resident macrophages resulting in cell injury or non-programmed cellular death. Alveolar macrophages trying to engulf HAR fibres that are long and stiff undergo frustrated phagocytosis because of their inability to engulf the piercing fibres and subsequently lead to cell injury (Boyles et al., 2015; Brown et al., 2007; Donaldson K et al., 2010; Dörger et al., 2001; Mossman and Churg, 1998). The cellular debris from injured or dying cell then serves as ligands for PRRs (Nakayama, 2018), leading to cell activation. In case of pro-fibrotic insoluble particles such as silica, coal dust and nanomaterials (NMs), the particle adsorbed opsonins such as immunoglobulins, complement proteins, or serum proteins act as ligands to the receptors on the macrophage cell surface (Behzadi et al., 2017). The tissue response to these materials resembles that observed following foreign body invasion in lungs.

Toll-like receptors (TLRs) are highly conserved PRRs that are associated with fibrogenic stressors (Desai et al., 2018). Inhibition of TLR-4 is protective against bleomycin-induced fibrosis (Li et al., 2015). However, the exact role and mechanisms by which TLRs mediate lung fibrosis are yet to be uncovered and some studies have shown TLRs to be protective against lung fibrosis (Desai et al., 2018). Asbestos and silica crystals are suggested to engage scavenger receptors present on the macrophages. Mice deficient in class A scavenger macrophage receptor with collagenous structure (MARCO) are shown to induce reduced fibrogenic response following chrysotile asbestos exposure; although, the direct binding of MARCO by asbestos is not investigated in the study (Murthy et al., 2015). In case of soluble substances such as bleomycin, paraquat (Dinis-Oliveira et al., 2008) (N,N'-dimethyl-4, 4′-bipyridinium dichloride) and other soluble fibrogenic chemicals, direct damage of lung epithelial cells and resulting cellular debris or secreted cytokines (DAMPs) serve as triggers for downstream cascading pro-inflammatory events, tissue injury and fibrosis. Engagement of PRRs and consequent cell activation is observed in various organisms including flies and mammals (Denholm and Phan, 1990; Matzinger, 2002).

How it is Measured or Detected

Detection of DAMPs or homeostasis-altering molecular processes:

Cellular interaction with substances or particles can be measured by assessing the release of DAMPs from stressed, injured or dying cells - indicative of binding of PRRs on the cell surface. Release of DAMPs is reflective of substance interaction with resident cells and their activation, a key step in the process of inflammation.

The release of DAMPs can be measured by the techniques listed in the published literature (Nikota et al., 2017; Rabolli et al., 2014; Suwara et al., 2014).

Targeted enzyme-linked immunosorbent assays (ELISA) (routinely used and recommended):

ELISA  – permits quantitative measurement of antigens in biological samples. For example, in a cytokine ELISA (sandwich ELISA), an antibody (capture antibody) specific to a cytokine is immobilised on microtitre wells (96-well, 386-well, etc.). Experimental samples or samples containing a known amount of the specific recombinant cytokine are then reacted with the immobilised antibody. Following removal of unbound antibody by thorough washing, plates are reacted with the secondary antibody (detection antibody) that is conjugated to an enzyme such as horseradish peroxidase, which when bound, will form a sandwich with the capture antibody and the cytokine (Amsen and De Visser, 2009). The secondary antibody can be conjugated to biotin, which is then detected by addition of streptavidin linked to horseradish peroxidase. A chromogenic substrate can also be added, which is the most commonly used method. Chromogenic substrate is chemically converted by the enzyme coupled to the detection antibody, resulting in colour change. The amount of colour detected is directly proportional to the amount of cytokine in the sample that is bound to the capture antibody. The results are read using a spectrophotometer and compared to the levels of cytokine in control samples where cytokine is not expected to be secreted or to the samples containing known recombinant cytokine levels.

Interleukin (IL)-1α and -1β is activated or secreted into the cytosol following stimulus (Di Paolo and Shayakhmetov, 2016). Targeted ELISA can be used to quantify IL-1α  or IL-1β that is released in the culture supernatant of the cells exposed to toxicants, in bronchoalveolar lavage fluid and serum of exposed animals. The assay is also applicable to human serum, cerebrospinal fluid, and peritoneal fluids.

Similarly, other alarmins can also be quantified by ELISA. Western blot is another method that can be used to quantify the release of various alarmins using specific antibodies. ELISA or real-time reverse transcription-polymerase chain reaction (qRT-PCR) assays can also be used to quantify the expression of genes or proteins that are regulated by the receptor binding – e.g. downstream of TLR binding.

Frustrated phagocytosis and cellular uptake of NMs:

In vitro, interaction of NMs with the cellular membrane is investigated by assessing their uptake by lysosomes (Chen et al., 2013; Nel et al., 2009; Varela et al., 2012). Immunohistochemistry methods targeting lysosome specific proteins are regularly employed for this purpose. In co-localisation experiments, lysosomal marker Lysosomal-associated membrane protein 1 (LAMP1) antibody is used to detect particle co-localisation with lysosomes. A combination of Cytoviva hyperspectral microscope and immunolocalisation (Decan et al., 2016) or confocal microscopy to visualise co-localisation of fluorescence labelled nanoparticles with lysosomal markers have been used.

Frustrated phagocytosis is assessed using microscopic techniques such as time-lapse microscopy, backscatter electron microscopy and others (Donaldson et al., 2010; Murphy et al., 2012; Padmore et al., 2017; Pascolo et al., 2013; Schinwald et al., 2012). In addition, MIE 1668 of AOP303 notes other indirect methods for measuring frustrated phagocytosis.  

Cellular co-culture models of the pulmonary epithelium:

Complex co-culture systems, such as those containing epithelial cells and immune cells, better model the environment of the lung epithelium and can be used to study the interaction of potentially pro-fibrotic fibres and particles with resident lung cells. This type of model has been used, alongside electron microscopy, to study lung cell interactions with CNTs following 24 h in vitro exposure (Clift et al., 2014). More recently, the EpiAlveolar model, which contains primary human alveolar epithelial cells, endothelial cells, as well as fibroblasts was assessed for its ability to predict fibrosis induced by CNTs (Barasova et al., 2020). Using laser scanning, fluorescence, and enhanced darkfield microscopy, CNT interaction with the resident cells of the model was shown, and this interaction induced the formation of holes in the epithelial model (Barasova et al., 2020). While new co-culture models are a better recapitulation of the native lung environment as compared to traditional mono-cultures, the increased complexity necessitates enhanced expertise in tissue culture techniques, and can make them less practical as compared to submerged mono culture methods. 

Ex vivo model of the lung – Precision cut lung slices (PCLS):

Even closer to the in vivo condition than co-culture models, PCLS techniques capture the native lung architecture, cell-cell communication and cellularity of the lung. Advancement in culturing and cryopreservation techniques has increased accessibility and use of PCLS for longer term studies (Bai et al., 2016, Neuhaus et al., 2017). These slices can be cultured ex vivo for up to a week with minimal reduction in viability, and the technique has recently been assessed for its applicability to assess nanomaterial induced fibrosis ex vivo (Rahman et al., 2020). Using multi-walled carbon nanotubes (MWCNTs) and darkfield microscopy, interaction between the nanofibers and the lung epithelium could be determined. The main downside of this technique is the animal requirement, which precludes their use in a first-pass screening context for the MIE.

References

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4. Behzadi S, Serpooshan V, Tao W, Hamaly MA, Alkawareek MY, Dreaden EC, Brown D, Alkilany AM, Farokhzad OC, Mahmoudi M. Cellular uptake of nanoparticles: journey inside the cell. Chem Soc Rev. 2017 Jul 17;46(14):4218-4244. doi: 10.1039/c6cs00636a. 

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11. Decan N, Wu D, Williams A, Bernatchez S, Johnston M, Hill M, Halappanavar S. Characterization of in vitro genotoxic, cytotoxic and transcriptomic responses following exposures to amorphous silica of different sizes. Mutat Res Genet Toxicol Environ Mutagen. 2016 Jan 15;796:8-22. doi: 10.1016/j.mrgentox.2015.11.011.

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14. Di Paolo NC, Shayakhmetov DM. Interleukin 1α and the inflammatory process. Nat Immunol. 2016 Jul 19;17(8):906-13. doi: 10.1038/ni.3503.

15. Dinis-Oliveira RJ, Duarte JA, Sánchez-Navarro A, Remião F, Bastos ML, Carvalho F. Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment. Crit Rev Toxicol. 2008;38(1):13-71. doi: 10.1080/10408440701669959.

16. Donaldson K, Murphy FA, Duffin R, Poland CA. Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma. Part Fibre Toxicol. 2010 Mar 22;7:5. doi: 10.1186/1743-8977-7-5.

17. Dörger M, Münzing S, Allmeling AM, Messmer K, Krombach F. Differential responses of rat alveolar and peritoneal macrophages to man-made vitreous fibers in vitro. Environ Res. 2001 Mar;85(3):207-14. doi: 10.1006/enrs.2001.4234.

18. Franks TJ, Colby TV, Travis WD, Tuder RM, Reynolds HY, Brody AR, Cardoso WV, Crystal RG, Drake CJ, Engelhardt J, Frid M, Herzog E, Mason R, Phan SH, Randell SH, Rose MC, Stevens T, Serge J, Sunday ME, Voynow JA, Weinstein BM, Whitsett J, Williams MC. Resident cellular components of the human lung: current knowledge and goals for research on cell phenotyping and function. Proc Am Thorac Soc. 2008 Sep 15;5(7):763-6. doi: 10.1513/pats.200803-025HR.

19. Kierdorf K, Prinz M, Geissmann F, Gomez Perdiguero E. Development and function of tissue resident macrophages in mice. Semin Immunol. 2015 Dec;27(6):369-78. doi: 10.1016/j.smim.2016.03.017.

20. Kim JE, Lim HT, Minai-Tehrani A, Kwon JT, Shin JY, Woo CG, Choi M, Baek J, Jeong DH, Ha YC, Chae CH, Song KS, Ahn KH, Lee JH, Sung HJ, Yu IJ, Beck GR Jr, Cho MH. Toxicity and clearance of intratracheally administered multiwalled carbon nanotubes from murine lung. J Toxicol Environ Health A. 2010;73(21-22):1530-43. doi: 10.1080/15287394.2010.511578. 

21. Luettich K, Sharma M, Yepiskoposyan H, Breheny D, Lowe FJ. An Adverse Outcome Pathway for Decreased Lung Function Focusing on Mechanisms of Impaired Mucociliary Clearance Following Inhalation Exposure. Front Toxicol. 2021 Dec 14;3:750254. doi: 10.3389/ftox.2021.750254.

22. Li XX, Jiang DY, Huang XX, Guo SL, Yuan W, Dai HP. Toll-like receptor 4 promotes fibrosis in bleomycin-induced lung injury in mice. Genet Mol Res. 2015 Dec 21;14(4):17391-8. doi: 10.4238/2015.

23. Matzinger P. The danger model: a renewed sense of self. Science. 2002 Apr 12;296(5566):301-5. doi: 10.1126/science.1071059.

24. Mossman BT, Churg A. Mechanisms in the pathogenesis of asbestosis and silicosis. Am J Respir Crit Care Med. 1998 May;157(5 Pt 1):1666-80. doi: 10.1164/ajrccm.157.5.9707141.

25. Murphy FA, Schinwald A, Poland CA, Donaldson K. The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells. Part Fibre Toxicol. 2012 Apr 3;9:8. doi: 10.1186/1743-8977-9-8. 

26. Murthy S, Larson-Casey JL, Ryan AJ, He C, Kobzik L, Carter AB. Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure. FASEB J. 2015 Aug;29(8):3527-36. doi: 10.1096/fj.15-271304.

27. Nakayama M. Macrophage Recognition of Crystals and Nanoparticles. Front Immunol. 2018 Jan 29;9:103. doi: 10.3389/fimmu.2018.00103. 

28. Nel AE, Mädler L, Velegol D, Xia T, Hoek EM, Somasundaran P, Klaessig F, Castranova V, Thompson M. Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater. 2009 Jul;8(7):543-57. doi: 10.1038/nmat2442. 

29. Neuhaus V, Schaudien D, Golovina T, Temann UA, Thompson C, Lippmann T, Bersch C, Pfennig O, Jonigk D, Braubach P, Fieguth HG, Warnecke G, Yusibov V, Sewald K, Braun A. Assessment of long-term cultivated human precision-cut lung slices as an ex vivo system for evaluation of chronic cytotoxicity and functionality. J Occup Med Toxicol. 2017 May 26;12:13. doi: 10.1186/s12995-017-0158-5.

30. Nikota J, Banville A, Goodwin LR, Wu D, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. 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. Part Fibre Toxicol. 2017 Sep 13;14(1):37. doi: 10.1186/s12989-017-0218-0.

31. Padmore T, Stark C, Turkevich LA, Champion JA. Quantitative analysis of the role of fiber length on phagocytosis and inflammatory response by alveolar macrophages. Biochim Biophys Acta Gen Subj. 2017 Feb;1861(2):58-67. doi: 10.1016/j.bbagen.2016.09.031.

32. Pascolo L, Gianoncelli A, Schneider G, Salomé M, Schneider M, Calligaro C, Kiskinova M, Melato M, Rizzardi C. The interaction of asbestos and iron in lung tissue revealed by synchrotron-based scanning X-ray microscopy. Sci Rep. 2013;3:1123. doi: 10.1038/srep01123. 

33. Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, Stone V, Brown S, Macnee W, Donaldson K. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 2008 Jul;3(7):423-8. doi: 10.1038/nnano.2008.111.

34. Rabolli V, Badissi AA, Devosse R, Uwambayinema F, Yakoub Y, Palmai-Pallag M, Lebrun A, De Gussem V, Couillin I, Ryffel B, Marbaix E, Lison D, Huaux F. The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles. Part Fibre Toxicol. 2014 Dec 13;11:69. doi: 10.1186/s12989-014-0069-x.

35. Rahman L, Williams A, Gelda K, Nikota J, Wu D, Vogel U, Halappanavar S. 21st Century Tools for Nanotoxicology: Transcriptomic Biomarker Panel and Precision-Cut Lung Slice Organ Mimic System for the Assessment of Nanomaterial-Induced Lung Fibrosis. Small. 2020 Sep;16(36):e2000272. doi: 10.1002/smll.202000272.

36. Schinwald A, Donaldson K. Use of back-scatter electron signals to visualise cell/nanowires interactions in vitro and in vivo; frustrated phagocytosis of long fibres in macrophages and compartmentalisation in mesothelial cells in vivo. Part Fibre Toxicol. 2012 Aug 28;9:34. doi: 10.1186/1743-8977-9-34. 

37. Suwara MI, Green NJ, Borthwick LA, Mann J, Mayer-Barber KD, Barron L, Corris PA, Farrow SN, Wynn TA, Fisher AJ, Mann DA. IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts. Mucosal Immunol. 2014 May;7(3):684-93. doi: 10.1038/mi.2013.87.

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List of Key Events in the AOP

Event: 1496: Increased, secretion of proinflammatory mediators

Short Name: Increased proinflammatory mediators

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Human, mouse, rat

Cytokines are the common pro-inflammatory mediators secreted following inflammogenic stimuli. Cytokines can be defined as a diverse group of signaling protein molecules. They are secreted by different cell types in different tissues and in all mammalian species, irrespective of gender, age or sex. A lot of literature is available to support cross species, gender and developmental stage application for this KE. The challenge is the specificity; most cytokines exhibit redundant functions and many are pleotropic.

Key Event Description

Pro-inflammatory mediators are the chemical and biological molecules that initiate and regulate inflammatory reactions. Pro-inflammatory mediators are secreted following exposure to an inflammogen in a gender/sex or developmental stage independent manner. They are secreted during inflammation in all species. Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). Cell-derived pro-inflammatory mediators include cytokines, chemokines, and growth factors. Blood derived pro-inflammatory mediators include vasoactive amines, complement activation products and others. These modulators can be grouped based on the cell type that secrete them, their cellular localisation and also based on the type of immune response they trigger. For example, members of the interleukin (IL) family including IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-3, IL-5 and Granulocyte-macrophage colony stimulating factor (GM-CSF) are involved in the adaptive immune responses. The pro-inflammatory cytokines include IL-1 family (IL-1α , IL-1β, IL-1rα, IL-18, IL-36α, IL-36β, IL-36γ, IL-36Rα, IL-37), IL-6 family, Tumor necrosis factor (TNF) family, IL-17, and Interferon gamma (IFN-γ) (Turner et al., 2014). While IL-4 and IL-5 are considered T helper (Th) cell type 2 response, IFN-γ is suggested to be Th1 type response.

Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). However, IL-1 family cytokines, IL-4, IL-5, IL-6, TNF-α, IFN-γ are the commonly measured mediators in experimental animals and in humans. Similar gene expression patterns involving inflammation and matrix remodelling are observed in human patients of pulmonary fibrosis and mouse lungs exposed to bleomycin (Kaminski, 2002).

Literature evidence for its perturbation:

Several studies show increased proinflammatory mediators in rodent lungs and bronchoalveolar lavage fluid, and in cell culture supernatants following exposure to a variety of carbon nanotube (CNT) types and other materials. Poland et al., 2008 showed that long and thin CNTs (>5 µm) can elicit asbestos-like pathogenicity through the continual release of pro-inflammatory cytokines and reactive oxygen species. Exposure to crystalline silica induces release of inflammatory cytokines (TNF-α, IL-1, IL-6), transcription factors (Nuclear factor kappa B [NF-κB], Activator protein-1 [AP-1]) and kinase signalling pathways in mice that contain NF-κB luciferase reporter (Hubbard et al., 2002). Boyles et al., 2015 found that lung responses to long multi-walled carbon nanotubes (MWCNTs) included high expression levels of pro-inflammatory mediators Monocyte chemoattractant protein 1 (MCP-1), Transforming growth factor beta 1 (TGF-β1), and TNF-α (Boyles et al., 2015). Bleomycin administration in rodents induces lung inflammation and increased expression of pro-inflammatory mediators (Park et al., 2019). Inflammation induced by bleomycin, paraquat and CNTs is characterised by the altered expression of pro-inflammatory mediators. A large number of nanomaterials induce expression of cytokines and chemokines in lungs of rodents exposed via inhalation (Halappanavar et al., 2011; Husain et al., 2015a). Similarities are observed in gene programs involving pro-inflammatory event is observed in both humans and experimental mice (Zuo et al., 2002).

How it is Measured or Detected

The selection of pro-inflammatory mediators for investigation varies based on the expertise of the lab, cell types studied and the availability of the specific antibodies.

Real-time reverse transcription-polymerase chain reaction (qRT-PCR) – will measure the abundance of cytokine mRNA in a given sample. The method involves three steps: conversion of RNA into cDNA by reverse transcription method, amplification of cDNA using the PCR, and the real-time detection and quantification of amplified products (amplicons) (Nolan et al., 2006). Amplicons are detected using fluorescence, increase in which is directly proportional to the amplified PCR product. The number of cycles required per sample to reach a certain threshold of fluorescence (set by the user – usually set in the linear phase of the amplification, and the observed difference in samples to cross the set threshold reflects the initial amount available for amplification) is used to quantify the relative amount in the samples. The amplified products are detected by the DNA intercalating minor groove-binding fluorophore SYBR green, which produces a signal when incorporated into double-stranded amplicons. Since the cDNA is single stranded, the dye does not bind enhancing the specificity of the results. There are other methods such as nested fluorescent probes for detection, but SYBR green is widely used. RT-PCR primers specific to several pro-inflammatory mediators in several species including mouse, rat and humans, are readily available commercially.

Enzyme-linked immunosorbent assays (ELISA) – permit quantitative measurement of antigens in biological samples. The method is the same as described for the MIE. Both ELISA and qRT-PCR assays are used in vivo and are readily applicable to in vitro cell culture models, where cell culture supernatants or whole cell homogenates are used for ELISA or mRNA assays. Both assays are straight forward, quantitative and require relatively a small amount of input sample.

Apart from assaying single protein or gene at a time, cytokine bead arrays or cytokine PCR arrays can also be used to detect a whole panel of inflammatory mediators in a multiplex method (Husain et al., 2015b). This method is quantitative and especially advantageous when the sample amount available for testing is scarce. Lastly, immunohistochemistry can also be used to detect specific immune cell types producing the pro-inflammatory mediators and its downstream effectors in any given tissue (Costa et al., 2017). Immunohistochemistry results can be used as weight of evidence; however, the technique is not quantitative and depending on the specific antibodies used, the assay sensitivity may also become an issue (Amsen and De Visser, 2009).

Cell models - of varying complexity have been used to assess the expression of pro-inflammatory mediators. Two dimensional submerged monocultures of the main fibrotic effector cells – lung epithelial cells, macrophages, and fibroblasts – have routinely been used in vitro due to the large literature base, and ease of use, but do not adequately mimic the in vivo condition (Sharma et al., 2016; Sundarakrishnan et al., 2018). Recently, the EpiAlveolar in vitro lung model (containing epithelial cells, endothelial cells, and fibroblasts) was used to predict the fibrotic potential of MWCNTs, and researchers noted increases in the pro-inflammatory molecules TNF-α, IL-1β, and the pro-fibrotic TGF-β using ELISA (Barasova et al., 2020). A similar, but less complicated co-culture model of immortalized human alveolar epithelial cells and idiopathic pulmonary fibrosis patient derived fibroblasts was used to assess pro-fibrotic signalling, and noted enhanced secretion of Platelet derived growth factor (PDGF) and Basic fibroblast growth factor (bFGF), as well as evidence for epithelial to mesenchymal transition of epithelial cells in this system (Prasad et al., 2014). Models such as these better capitulate the in vivo pulmonary alveolar capillary, but have lower reproducibility as compared to traditional submerged mono-culture experiments.

References

1. Amsen D, de Visser KE, Town T. Approaches to determine expression of inflammatory cytokines. Methods Mol Biol. 2009;511:107-42. doi: 10.1007/978-1-59745-447-6_5. 

2. Barosova H, Maione AG, Septiadi D, Sharma M, Haeni L, Balog S, O'Connell O, Jackson GR, Brown D, Clippinger AJ, Hayden P, Petri-Fink A, Stone V, Rothen-Rutishauser B. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020 Apr 28;14(4):3941-3956. doi: 10.1021/acsnano.9b06860. 

3. Boyles MS, Young L, Brown DM, MacCalman L, Cowie H, Moisala A, Smail F, Smith PJ, Proudfoot L, Windle AH, Stone V. Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos. Toxicol In Vitro. 2015 Oct;29(7):1513-28. doi: 10.1016/j.tiv.2015.06.012. 

4. Costa PM, Gosens I, Williams A, Farcal L, Pantano D, Brown DM, Stone V, Cassee FR, Halappanavar S, Fadeel B. Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles. J Appl Toxicol. 2018 Mar;38(3):385-397. doi: 10.1002/jat.3548.

5. Halappanavar S, Jackson P, Williams A, Jensen KA, Hougaard KS, Vogel U, Yauk CL, Wallin H. Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study. Environ Mol Mutagen. 2011 Jul;52(6):425-39. doi: 10.1002/em.20639. 

6. Hubbard AK, Timblin CR, Shukla A, Rincón M, Mossman BT. Activation of NF-kappaB-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol. 2002 May;282(5):L968-75. doi: 10.1152/ajplung.00327.2001.

7. Husain M, Kyjovska ZO, Bourdon-Lacombe J, Saber AT, Jensen KA, Jacobsen NR, Williams A, Wallin H, Halappanavar S, Vogel U, Yauk CL. Carbon black nanoparticles induce biphasic gene expression changes associated with inflammatory responses in the lungs of C57BL/6 mice following a single intratracheal instillation. Toxicol Appl Pharmacol. 2015a Dec 15;289(3):573-88. doi: 10.1016/j.taap.2015.11.003.

8. Husain M, Wu D, Saber AT, Decan N, Jacobsen NR, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice. Nanotoxicology. 2015b;9(8):1013-22. doi: 10.3109/17435390.2014.996192.

9. Kaminski N. Microarray analysis of idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol. 2003 Sep;29(3 Suppl):S32-6.

10. Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol. 2004 Mar 1;172(5):2731-8. doi: 10.4049/jimmunol.172.5.2731.

11. Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1(3):1559-82. doi: 10.1038/nprot.2006.236.

12. Park SJ, Im DS. Deficiency of Sphingosine-1-Phosphate Receptor 2 (S1P₂) Attenuates Bleomycin-Induced Pulmonary Fibrosis. Biomol Ther (Seoul). 2019 May 1;27(3):318-326. doi: 10.4062/biomolther.2018.131.

13. Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, Stone V, Brown S, Macnee W, Donaldson K. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 2008 Jul;3(7):423-8. doi: 10.1038/nnano.2008.111.

14. Prasad S, Hogaboam CM, Jarai G. Deficient repair response of IPF fibroblasts in a co-culture model of epithelial injury and repair. Fibrogenesis Tissue Repair. 2014 Apr 29;7:7. doi: 10.1186/1755-1536-7-7. 

15. Sharma M, Nikota J, Halappanavar S, Castranova V, Rothen-Rutishauser B, Clippinger AJ. Predicting pulmonary fibrosis in humans after exposure to multi-walled carbon nanotubes (MWCNTs). Arch Toxicol. 2016 Jul;90(7):1605-22. doi: 10.1007/s00204-016-1742-7. 

16. Sundarakrishnan A, Chen Y, Black LD, Aldridge BB, Kaplan DL. Engineered cell and tissue models of pulmonary fibrosis. Adv Drug Deliv Rev. 2018 Apr;129:78-94. doi: 10.1016/j.addr.2017.12.013.

17. Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim Biophys Acta. 2014 Nov;1843(11):2563-2582. doi: 10.1016/j.bbamcr.2014.05.014. 

18. Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Lollini L, Morris D, Kim Y, DeLustro B, Sheppard D, Pardo A, Selman M, Heller RA. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6292-7. doi: 10.1073/pnas.092134099.

Event: 1497: Increased, recruitment of inflammatory cells

Short Name: Recruitment of inflammatory cells

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Human, mouse, rat

Key Event Description

Pro-inflammatory cells originate in bone marrow and are recruited to the site of infection or injury via circulation following specific pro-inflammatory mediator (cytokine and chemokine) signalling. Pro-inflammatory cells are recruited to lungs to clear the invading pathogen or the toxic substance. Monocytes (dendritic cells, macrophages, and neutrophils) are subsets of circulating white blood cells that are involved in the immune responses to pathogen or toxicant stimuli (Kolaczkowska and Kubes, 2013; Kopf et al., 2015). They are derived from the bone marrow. They can differentiate into different macrophage types and dendritic cells. They can be categorised based on their size, the type of cell surface receptors and their ability to differentiate following external or internal stimulus such as increased expression of cytokines. Monocytes participate in tissue healing, clearance of toxic substance or pathogens, and in the initiation of adaptive immunity. Recruited monocytes can also influence pathogenesis (Ingersoll et al., 2011). Sensing or recognition of pathogens and harmful substances results in the recruitment of monocytes to lungs (Shi and Pamer, 2011). Activated immune cells secrete a variety of pro-inflammatory mediators, the purpose of which is to propagate the immune signalling and response, which when not controlled, leads to chronic inflammation, cell death and tissue injury. Thus, Event 1496 and Event 1497 act in a positive feedback loop mechanism and propagate the proinflammatory environment.

Literature evidence for its perturbation:

Macrophages accumulate in bronchoalveolar fluid (BALF) post-exposure to bleomycin (Phan et al., 1980; Smith et al., 1995). Nanomaterial (NM)-induced inflammation is predominantly neutrophilic (Poulsen et al., 2015; Rahman L et al., 2017a; Rahman et al., 2017b; Shvedova et al., 2005). An increased number of neutrophils (Reynolds et al., 1977) is observed in the BALF of patients with idiopathic pulmonary fibrosis. Eosinophils are a type of white blood cells and a type of granulocytes (contain granules and enzymes) that are recruited following exposure to allergens, during allergic reactions such as asthma or during fibrosis (Reynolds et al., 1977). Multi-walled carbon nanotubes (MWCNTs) induce increased eosinophil count in lungs (Købler C et al., 2015). MWCNTs act as allergens and induce lung infiltration of eosinophils and cause airway hypersensitivity (Beamer et al., 2013).

It is important to note that the stressor-induced Event 1495, Event 1496, and Event 1497 are part of the functional changes that we collectively consider as inflammation, and together, they mark the initiation of acute inflammatory phase. Event 1495 and Event 1496 occur at the cellular level. Event 1497 occurs at the tissue level.

How it is Measured or Detected

In vivo, recruitment of pro-inflammatory cells is measured using BALF cellularity assay. The fluid lining the lung epithelium is lavaged (BALF) and its composition is assessed as marker of lung immune response to the toxic substances or pathogens. BALF is assessed quantitatively for types of infiltrating cells, levels and types of cytokines and chemokines. Thus, BALF assessment can aid in developing dose-response of a substance, to rank a substances’ potency and to set up no effect level of exposure for the regulatory decision making. For NMs, in vivo BALF assessment is recommended as a mandatory test (discussed in ENV/JM/MONO(2012)40 and also in OECD inhalation test guideline for NMs). Temporal changes in the BALF composition can be prognostic of initiation and progression of lung immune disease (Cho et al., 2010).

In vitro, it is difficult to assess the recruitment of pro-inflammatory cells. Thus, a suit of pro-inflammatory mediators specific to cell types are assessed using the same techniques mentioned above (real-time reverse transcription-polymerase chain reaction [qRT-PCR], enzyme-linked immunosorbent assays [ELISA], immunohistochemistry) in cell culture models, as indicative of recruitment of cells into the lungs. Alternatively, the use of precision cut lung slices can allow for limited assessment of recruitment of tissue resident inflammatory cells, based on the repertoire of cells remaining in the specific slice following harvesting. This method was used to show that there is a histological increase in inflammatory foci following treatment with bleomycin and MWCNTs (Rahman et al., 2020). Finally, more complicated microfluidic lung-on-a-chip devices can be used to assess the migration of select immune cells and fibroblasts toward a simulated epithelium following treatment with a pro-fibrotic compound (He et al., 2017). However, this method is limited to two cell types, and it lacks the reservoirs of immune cells present in the body in vivo.

References

1. Beamer CA, Girtsman TA, Seaver BP, Finsaas KJ, Migliaccio CT, Perry VK, Rottman JB, Smith DE, Holian A. IL-33 mediates multi-walled carbon nanotube (MWCNT)-induced airway hyper-reactivity via the mobilization of innate helper cells in the lung. Nanotoxicology. 2013 Sep;7(6):1070-81. doi: 10.3109/17435390.2012.702230.

2. Cho WS, Duffin R, Poland CA, Howie SE, MacNee W, Bradley M, Megson IL, Donaldson K. Metal oxide nanoparticles induce unique inflammatory footprints in the lung: important implications for nanoparticle testing. Environ Health Perspect. 2010 Dec;118(12):1699-706. doi: 10.1289/ehp.1002201. 

3. He J, Chen W, Deng S, Xie L, Feng J, Geng J, et al. Modeling alveolar injury using microfluidic co-cultures for monitoring bleomycin-induced epithelial/fibroblastic cross-talk disorder. RSC Advances. 2017 7(68):42738-49. doi: 10.1039/C7RA06752F.

4. Ingersoll MA, Platt AM, Potteaux S, Randolph GJ. Monocyte trafficking in acute and chronic inflammation. Trends Immunol. 2011 Oct;32(10):470-7. doi: 10.1016/j.it.2011.05.001.

5. Købler C, Poulsen SS, Saber AT, Jacobsen NR, Wallin H, Yauk CL, Halappanavar S, Vogel U, Qvortrup K, Mølhave K. Time-dependent subcellular distribution and effects of carbon nanotubes in lungs of mice. PLoS One. 2015 Jan 23;10(1):e0116481. doi: 10.1371/journal.pone.0116481. 

6. Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol. 2013 Mar;13(3):159-75. doi: 10.1038/nri3399. 

7. Kopf M, Schneider C, Nobs SP. The development and function of lung-resident macrophages and dendritic cells. Nat Immunol. 2015 Jan;16(1):36-44. doi: 10.1038/ni.3052.

8. Phan SH, Thrall RS, Ward PA. Bleomycin-induced pulmonary fibrosis in rats: biochemical demonstration of increased rate of collagen synthesis. Am Rev Respir Dis. 1980 Mar;121(3):501-6. doi: 10.1164/arrd.1980.121.3.501. 

9. Poulsen SS, Saber AT, Williams A, Andersen O, Købler C, Atluri R, Pozzebon ME, Mucelli SP, Simion M, Rickerby D, Mortensen A, Jackson P, Kyjovska ZO, Mølhave K, Jacobsen NR, Jensen KA, Yauk CL, Wallin H, Halappanavar S, Vogel U. MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. Toxicol Appl Pharmacol. 2015 Apr 1;284(1):16-32. doi: 10.1016/j.taap.2014.12.011. 

10. Rahman L, Wu D, Johnston M, William A, Halappanavar S. Toxicogenomics analysis of mouse lung responses following exposure to titanium dioxide nanomaterials reveal their disease potential at high doses. Mutagenesis. 2017a Jan;32(1):59-76. doi: 10.1093/mutage/gew048. 

11. Rahman L, Jacobsen NR, Aziz SA, Wu D, Williams A, Yauk CL, White P, Wallin H, Vogel U, Halappanavar S. Multi-walled carbon nanotube-induced genotoxic, inflammatory and pro-fibrotic responses in mice: Investigating the mechanisms of pulmonary carcinogenesis. Mutat Res Genet Toxicol Environ Mutagen. 2017b Nov;823:28-44. doi: 10.1016/j.mrgentox.2017.08.005. 

12. Rahman L, Williams A, Gelda K, Nikota J, Wu D, Vogel U, Halappanavar S. 21st Century Tools for Nanotoxicology: Transcriptomic Biomarker Panel and Precision-Cut Lung Slice Organ Mimic System for the Assessment of Nanomaterial-Induced Lung Fibrosis. Small. 2020 Sep;16(36):e2000272. doi: 10.1002/smll.202000272. 

13. Reynolds HY, Fulmer JD, Kazmierowski JA, Roberts WC, Frank MM, Crystal RG. Analysis of cellular and protein content of broncho-alveolar lavage fluid from patients with idiopathic pulmonary fibrosis and chronic hypersensitivity pneumonitis. J Clin Invest. 1977 Jan;59(1):165-75. doi: 10.1172/JCI108615.

14. Shi C, Pamer EG. Monocyte recruitment during infection and inflammation. Nat Rev Immunol. 2011 Oct 10;11(11):762-74. doi: 10.1038/nri3070. 

15. Shvedova AA, Kisin ER, Mercer R, Murray AR, Johnson VJ, Potapovich AI, Tyurina YY, Gorelik O, Arepalli S, Schwegler-Berry D, Hubbs AF, Antonini J, Evans DE, Ku BK, Ramsey D, Maynard A, Kagan VE, Castranova V, Baron P. Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. Am J Physiol Lung Cell Mol Physiol. 2005 Nov;289(5):L698-708. doi: 10.1152/ajplung.00084.2005.  

16. Smith RE, Strieter RM, Zhang K, Phan SH, Standiford TJ, Lukacs NW, Kunkel SL. A role for C-C chemokines in fibrotic lung disease. J Leukoc Biol. 1995 May;57(5):782-7. doi: 10.1002/jlb.57.5.782. 

Event: 1498: Loss of alveolar capillary membrane integrity

Short Name: Loss of alveolar capillary membrane integrity

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Key Event Description

The alveolar-capillary membrane (ACM) is the gas exchange surface of the lungs that is only ~0.3µm thick and is the largest surface area within the lung that separates the interior of the body from the environment. It is comprised of the microvascular endothelium, interstitium, and alveolar epithelium. As a consequence of its anatomical position, and the large surface area, it is the first point of contact for any inhaled pathogen, particles or toxic substances. Thus, ACM is subjected to injury constantly and rapidly repaired following the external insults without formation of fibrosis or scar tissue. The extent of ACM injury or how rapidly its integrity is restored is a pivotal determinant of whether the lung restores its normal functioning following an injury or is replaced by fibrotic lesion or scar tissue (Fukuda et al., 1987; Schwarz et al., 2001). Significant loss of endothelium and epithelium of the ACM results in loss of the barrier and membrane integrity. Increased membrane permeability leading to efflux of protein-rich fluid into the peribronchovascular interstitium and the distal airspaces of the lung, disruption of normal fluid transport via downregulated Na⁺ channels or malfunctioning Na⁺/K⁺ATPase pumps, loss of surfactant production, increased expression of epithelial or endothelial cell markers such as Intercellular adhesion molecule-1 (ICAM-1) or decreased expression of surfactant protein D (SP-D) are few of the markers of decreasing lung compliance arising from the lost integrity of ACM (Johnson and Matthay, 2010).

Literature evidence for its perturbation:

Bleomycin exposure causes alveolar barrier dysfunction (Miyoshi et al., 2013). Cigarette smoke impairs tight junction proteins and leads to altered permeability of the epithelial barrier (Schamberger et al., 2014). Exposure to bleomycin destroys the structural architecture of tight junctions, increases permeability, epithelial death and loss of specialised repair proteins such as claudins. Thoracic radiation and bleomycin induced lung injury results in decreased expression of E-cadherin and Aquaporin-5 (AQP5) expression (Almeida et al., 2013; Gabazza et al., 2004).

Repeated exposure to biopersistent toxic substances, pathogens or lung irritants initiate non-resolving inflammation and ACM injury (Costabel et al., 2012). Chronic inflammation mediated by overexpression of cytokines such as Interleukine (IL)-1 (Kolb et al., 2001), Tumor necrosis factor alpha (TNF-α) (Sime et al., 1998), T helper type 2 cytokine IL-13 or exposure to specific proteinases initiate ACM injury, leading to significant loss of the epithelium and endothelium of the ACM resulting loss of barrier integrity. In patients diagnosed with idiopathic pulmonary fibrosis (IPF), both type 1 pneumocyte & endothelial cell injury with ACM barrier loss is observed.

Bleomycin and silica exposure generate persistent inflammation and lung damage (Chua et al., 2005; Thrall and Scaliso, 1995). Exposure to single-walled carbon nanotubes (SWCNTs) induces persistent inflammation, granuloma formation and diffuse intestinal fibrosis in mice after pharyngeal aspiration (Shvedova et al., 2005). Multi-walled carbon nanotubes act as allergens and induce lung infiltration of eosinophils and cause airway hypersensitivity (Beamer et al., 2013). Inhaled particles induce chronic inflammation (Ernst et al., 2002; Hamilton et al., 2008; Thakur et al., 2008). Increased numbers of alveolar macrophages, neutrophils and eosinophils are observed in the bronchoalveolar lavage fluid (BALF) of patients suffering from IPF and chronic inflammation is associated with decreased survival (Parra et al., 2007; Schwartz et al., 1991; Yasuoka et al., 1985).

The BALF of patients diagnosed with interstitial diseases contains increased levels of 8-isoprostane (Psathakis et al., 2006) and carbonyl-modified proteins (Lenz et al., 1996), markers of oxidative modification of lipids and proteins. In vivo, increased reactive oxygen species (ROS) levels in rodents (Ghio et al., 1998) and enzymatic production of nitric oxide in rat alveolar macrophages is observed after asbestos exposure (Quinlan et al., 1998). Some nanoparticles induce oxidative stress that contributes to cellular toxicity (Shi et al., 2012). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase derived ROS is a critical determinant of the pulmonary response to SWCNTs in mice (Shvedova et al., 2008). Oxidative lipidomics analysis of the lungs of carbon nanotube (CNT)-exposed mice showed, phospholipid oxidation (Tyurina et al., 2011). ROS synthesis is suggested to be important for inflammosome activation involving NLR-related protein 3 complex, activated caspase-1 and IL-1β, which is observed following exposure to a variety of pro-inflammatory stimuli including, asbestos and crystalline silica (Cassel et al., 2008; Dostert et al., 2008) and long needle-like CNTs. In the case of asbestos, frustrated phagocytosis triggered ROS synthesis leads to inflammosome activation, which is associated with asbestos induced pathology (Dostert et al., 2008).

How it is Measured or Detected

Proteinosis, BALF protein content:

Compromised ACM barrier integrity in vivo can be measured by measuring total protein or total albumin content in the BALF derived from experimental animals exposed to lung toxicants or in human patients suffering from lung fibrosis. In addition to albumin, the total urea in BALF is also a good indicator of the ACM integrity loss (Schmekel et al., 1992).

Cell type considerations:

ACM loss is a tissue level event. In vitro, assays with human cells are desired; however, the use of cells derived from experimental animals including alveolar macrophages, dendritic cells, epithelial cells, and neutrophils are routinely used. Primary cells are preferred over immortalised cell types that are in culture for a long period of time. In vitro, studies often assess the altered expression of pro-inflammatory mediators, increased ROS synthesis or oxidative stress and cytotoxicity events, an interplay between these three biological events occurring following exposure to stressors, is suggested to induce cell injury, which is reflective of tissue injury or loss of ACM (Halappanavar et al., 2019) in vivo.

Cytotoxicity assessment:

Cellular viability or cytotoxicity assays are the most commonly used endpoints to assess the leaky or compromised cell membrane. The most commonly employed method is the trypan blue exclusion assay – a dye exclusion assay where cells with intact membrane do not permit entry of the dye into cells and thus remain clear, whereas the dye diffuses into cells with damaged membrane turning them to blue colour. Other high throughput assays that use fluorescent DNA stains such as ethidium bromide or propidium iodide can also be used and cells that have incorporated the dye can be scored using flow cytometry.

Lactate dehydrogenase (LDH) release assay is a very sensitive cytotoxicity assay that measures the amount of LDH released in the media following membrane injury. The assay is based on measuring the reduction of nicotinamide adenine dinucleotide (NAD) and conversion of a tetrazolium dye that is measured at a wavelength of 490 nm.

The Calcein AM assay depends on the hydrolysis of calcein AM (a non-fluorescent hydrophobic compound that permeates live cells by simple diffusion) by non-specific intracellular esterases resulting in production of calcein, a hydrophilic and strongly fluorescent compound that is readily released into the cell culture media by the damaged cells.

Although the above mentioned assays work for almost all chemicals, insoluble substances such as nanomaterials can confound the assay by inhibiting the enzyme activity or interfering with the absorbance reading. Thus, care must be taken to include appropriate controls in the assays.

Transepithelial/transendothelial electrical resistance (TEER):

TEER is an accepted quantitative technique that measures the integrity of tight junctions in cell culture models of endothelial and epithelial cell monolayers. They are based on measuring ohmic resistance or measuring impedance across a wide range of frequencies.

Other:

The other methods include targeted reverse transcription polymerase chain reaction (RT-PCR) or enzyme-linked immunosorbent assays (ELISA) for tight junction proteins, cell adhesion molecules and inflammatory mediators such as Interferon gamma (IFN-γ), IL-10, and IL-13. Advanced in vitro co-culture models, like the EpiAlveolar model system, and other similar systems present an intact capillary membrane that can be used to assess loss in the membrane integrity (via TEER) after exposure to pro-fibrotic stressors like crystalline silica and Transforming growth factor beta (TGF-β) (Barasova et al., 2020, Kasper et al., 2011).

References

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2. Barosova H, Maione AG, Septiadi D, Sharma M, Haeni L, Balog S, O'Connell O, Jackson GR, Brown D, Clippinger AJ, Hayden P, Petri-Fink A, Stone V, Rothen-Rutishauser B. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020 Apr 28;14(4):3941-3956. doi: 10.1021/acsnano.9b06860. 

3. Beamer CA, Girtsman TA, Seaver BP, Finsaas KJ, Migliaccio CT, Perry VK, Rottman JB, Smith DE, Holian A. IL-33 mediates multi-walled carbon nanotube (MWCNT)-induced airway hyper-reactivity via the mobilization of innate helper cells in the lung. Nanotoxicology. 2013 Sep;7(6):1070-81. doi: 10.3109/17435390.2012.702230. 

4. Cassel SL, Eisenbarth SC, Iyer SS, Sadler JJ, Colegio OR, Tephly LA, Carter AB, Rothman PB, Flavell RA, Sutterwala FS. The Nalp3 inflammasome is essential for the development of silicosis. Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9035-40. doi: 10.1073/pnas.0803933105.

5. Chua F, Gauldie J, Laurent GJ. Pulmonary fibrosis: searching for model answers. Am J Respir Cell Mol Biol. 2005 Jul;33(1):9-13. doi: 10.1165/rcmb.2005-0062TR.

6. Costabel U, Bonella F, Guzman J. Chronic hypersensitivity pneumonitis. Clin Chest Med. 2012 Mar;33(1):151-63. doi: 10.1016/j.ccm.2011.12.004.

7. Dostert C, Pétrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science. 2008 May 2;320(5876):674-7. doi: 10.1126/science.1156995.

8. Ernst H, Rittinghausen S, Bartsch W, Creutzenberg O, Dasenbrock C, Görlitz BD, Hecht M, Kairies U, Muhle H, Müller M, Heinrich U, Pott F. Pulmonary inflammation in rats after intratracheal instillation of quartz, amorphous SiO2, carbon black, and coal dust and the influence of poly-2-vinylpyridine-N-oxide (PVNO). Exp Toxicol Pathol. 2002 Aug;54(2):109-26. doi: 10.1078/0940-2993-00241. 

9. Fukuda Y, Ishizaki M, Masuda Y, Kimura G, Kawanami O, Masugi Y. The role of intraalveolar fibrosis in the process of pulmonary structural remodeling in patients with diffuse alveolar damage. Am J Pathol. 1987 Jan;126(1):171-82.

10. Gabazza EC, Kasper M, Ohta K, Keane M, D'Alessandro-Gabazza C, Fujimoto H, Nishii Y, Nakahara H, Takagi T, Menon AG, Adachi Y, Suzuki K, Taguchi O. Decreased expression of aquaporin-5 in bleomycin-induced lung fibrosis in the mouse. Pathol Int. 2004 Oct;54(10):774-80. doi: 10.1111/j.1440-1827.2004.01754.x.

11. Ghio AJ, Kadiiska MB, Xiang QH, Mason RP. In vivo evidence of free radical formation after asbestos instillation: an ESR spin trapping investigation. Free Radic Biol Med. 1998 Jan 1;24(1):11-7. doi: 10.1016/s0891-5849(97)00063-4. 

12. Halappanavar S, van den Brule S, Nymark P, Gaté L, Seidel C, Valentino S, Zhernovkov V, Høgh Danielsen P, De Vizcaya A, Wolff H, Stöger T, Boyadziev A, Poulsen SS, Sørli JB, Vogel U. Adverse outcome pathways as a tool for the design of testing strategies to support the safety assessment of emerging advanced materials at the nanoscale. Part Fibre Toxicol. 2020 May 25;17(1):16. doi: 10.1186/s12989-020-00344-4.

13. Hamilton RF Jr, Thakur SA, Holian A. Silica binding and toxicity in alveolar macrophages. Free Radic Biol Med. 2008 Apr 1;44(7):1246-58. doi: 10.1016/j.freeradbiomed.2007.12.027.

14. Johnson ER, Matthay MA. Acute lung injury: epidemiology, pathogenesis, and treatment. J Aerosol Med Pulm Drug Deliv. 2010 Aug;23(4):243-52. doi: 10.1089/jamp.2009.0775.

15. Kasper J, Hermanns MI, Bantz C, Maskos M, Stauber R, Pohl C, Unger RE, Kirkpatrick JC. Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: comparison with conventional monocultures. Part Fibre Toxicol. 2011 Jan 27;8(1):6. doi: 10.1186/1743-8977-8-6. 

16. Kolb M, Margetts PJ, Anthony DC, Pitossi F, Gauldie J. Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest. 2001 Jun;107(12):1529-36. doi: 10.1172/JCI12568.

17. Lenz AG, Costabel U, Maier KL. Oxidized BAL fluid proteins in patients with interstitial lung diseases. Eur Respir J. 1996 Feb;9(2):307-12. doi: 10.1183/09031936.96.09020307. 

18. Miyoshi K, Yanagi S, Kawahara K, Nishio M, Tsubouchi H, Imazu Y, Koshida R, Matsumoto N, Taguchi A, Yamashita S, Suzuki A, Nakazato M. Epithelial Pten controls acute lung injury and fibrosis by regulating alveolar epithelial cell integrity. Am J Respir Crit Care Med. 2013 Feb 1;187(3):262-75. doi: 10.1164/rccm.201205-0851OC. 

19. Parra ER, Kairalla RA, Ribeiro de Carvalho CR, Eher E, Capelozzi VL. Inflammatory cell phenotyping of the pulmonary interstitium in idiopathic interstitial pneumonia. Respiration. 2007;74(2):159-69. doi: 10.1159/000097133.

20. Psathakis K, Mermigkis D, Papatheodorou G, Loukides S, Panagou P, Polychronopoulos V, Siafakas NM, Bouros D. Exhaled markers of oxidative stress in idiopathic pulmonary fibrosis. Eur J Clin Invest. 2006 May;36(5):362-7. doi: 10.1111/j.1365-2362.2006.01636.x.

21. Quinlan TR, BeruBe KA, Hacker MP, Taatjes DJ, Timblin CR, Goldberg J, Kimberley P, O'Shaughnessy P, Hemenway D, Torino J, Jimenez LA, Mossman BT. Mechanisms of asbestos-induced nitric oxide production by rat alveolar macrophages in inhalation and in vitro models. Free Radic Biol Med. 1998 Mar 15;24(5):778-88. doi: 10.1016/s0891-5849(97)00357-2.

22. Schamberger AC, Mise N, Jia J, Genoyer E, Yildirim AÖ, Meiners S, Eickelberg O. Cigarette smoke-induced disruption of bronchial epithelial tight junctions is prevented by transforming growth factor-β. Am J Respir Cell Mol Biol. 2014 Jun;50(6):1040-52. doi: 10.1165/rcmb.2013-0090OC. 

23. Schmekel B, Bos JA, Khan AR, Wohlfart B, Lachmann B, Wollmer P. Integrity of the alveolar-capillary barrier and alveolar surfactant system in smokers. Thorax. 1992 Aug;47(8):603-8. doi: 10.1136/thx.47.8.603. 

24. Schwartz DA, Helmers RA, Dayton CS, Merchant RK, Hunninghake GW. Determinants of bronchoalveolar lavage cellularity in idiopathic pulmonary fibrosis. J Appl Physiol (1985). 1991 Nov;71(5):1688-93. doi: 10.1152/jappl.1991.71.5.1688. 

25. Schwarz MA. Acute lung injury: cellular mechanisms and derangements. Paediatr Respir Rev. 2001 Mar;2(1):3-9. doi: 10.1053/prrv.2000.0095.

26. Shi J, Karlsson HL, Johansson K, Gogvadze V, Xiao L, Li J, Burks T, Garcia-Bennett A, Uheida A, Muhammed M, Mathur S, Morgenstern R, Kagan VE, Fadeel B. Microsomal glutathione transferase 1 protects against toxicity induced by silica nanoparticles but not by zinc oxide nanoparticles. ACS Nano. 2012 Mar 27;6(3):1925-38. doi: 10.1021/nn2021056. 

27. Shvedova AA, Kisin ER, Mercer R, Murray AR, Johnson VJ, Potapovich AI, Tyurina YY, Gorelik O, Arepalli S, Schwegler-Berry D, Hubbs AF, Antonini J, Evans DE, Ku BK, Ramsey D, Maynard A, Kagan VE, Castranova V, Baron P. Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. Am J Physiol Lung Cell Mol Physiol. 2005 Nov;289(5):L698-708. doi: 10.1152/ajplung.00084.2005. 

28. Shvedova AA, Kisin ER, Murray AR, Kommineni C, Castranova V, Fadeel B, Kagan VE. Increased accumulation of neutrophils and decreased fibrosis in the lung of NADPH oxidase-deficient C57BL/6 mice exposed to carbon nanotubes. Toxicol Appl Pharmacol. 2008 Sep 1;231(2):235-40. doi: 10.1016/j.taap.2008.04.018. 

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

Short Name: Activation of Th2 cells

AOPs Including This Key Event

Biological Context

Key Event Description

Naïve CD4+ T cells differentiate into four types of T helper (Th) cells – Th1, Th2, Th17 and inducible regulatory T cells following exposure to infectious agents. The differentiation process begins when antigen presenting cells (APCs) come in contact with toxic substances and is mainly driven by cytokines that make up the microenvironment. For example, increased concentrations of Interleukin (IL)-12 secreted by APCs in the environment may be biased towards Th1 type and increased IL-6 or IL-4 in the environment may commit to Th2 type differentiation. Th1 cytokines, Interferon gamma (IFN-γ) and IL-12 induce inflammation, aid in clearance of toxic substances, induce tissue damage and control the fibrotic responses. IFN-γ has suppressive effects on the production of extracellular matrix proteins including collagen and fibronectin. The Th2 response suppresses Th1 mediated response, which results in decreased Th1 cell-mediated tissue damage but at the same time contributing to the persistence of toxic substances leading to perpetuation of tissue damage, triggering uncontrolled healing response. The major sources of Th2 cytokines are Th2 cells themselves; however, mast cells, macrophages, epithelial cells and activated fibroblasts have shown to produce IL-4, IL-13 and IL-10 upon appropriate stimulation. Th2 cytokines IL-4 and IL-13 regulate wound healing.

Literature evidence for its perturbation in the context of pulmonary fibrosis:

For fibroplasia or fibrosis, the type of CD4+ T cell response that develops is crucial. Studies conducted in mice that do not express Th2 cytokines IL-4, IL-5 and IL-13 show complete attenuation of fibrosis despite the highly active Th1 response. Th2 cytokines IL-4 and IL-13 are elevated in fibrotic lungs; IL-13 activates Transforming growth factor beta 1 (TGF-β1) and initiates fibroblast proliferation and differentiation in lung fibrosis (Lee et al., 2001). Overexpression of IL-13 induces sub-epithelial airway fibrosis in mice in the absence of any other external pro-inflammatory or pro-fibrotic stimulus (Zhu et al., 1999). Both multi-walled carbon nanotubes (MWCNTs) and single-walled carbon nanotubes induce elevated expression of IL-4 and IL-13 in bronchoalveolar lavage fluid (BALF) of mouse lungs (Park et al., 2011), and increased levels of IL-25 and IL- 33 in BALF and mouse lungs exposed to MWCNTs (Dong and Ma, 2018). In a rare human study, increased levels IL-4 and IL-5 were observed in the sputum of humans exposed to MWCNTs at an occupational setting (Fatkhutdinova et al., 2016). Overexpression of IL-10 increases IL-4 and IL-13 production and lung fibrosis following exposure to silica (Barbarin et al., 2005). Alveolar macrophages from asbestosis patients (a form of lung fibrosis) exhibit M2 phenotype (He et al., 2013). Ex vivo culture of alveolar macrophages obtained from BALF of patients suffering from idiopathic pulmonary fibrosis with collagen type I showed enhanced levels of M2 macrophage markers C-C motif chemokine ligand (CCL)-18, CCL-2 and CD204 (Stahl et al., 2013). Th2 response associated expression of IL-33 cytokine enhances polarisation of M2 macrophages and inducing M2-mediated expression of IL-13 and TGF-β1 in mice (Dong and Ma, 2018). Cigarette smoke induces expression of genes associated with M2 sub-phenotypes, which is further enhanced in smokers presenting with chronic obstructive pulmonary disease (Shaykhiev et al., 2009).

How it is Measured or Detected

Targeted enzyme-linked immunosorbent assays (ELISA) or real-time quantitative polymerase chain reaction (qRT-PCR) (routinely used and recommended):

The ELISA and qRT-PCR are routinely used to assess the levels of protein and mRNA of several Th1 and Th2 cytokines including IL-4, IL-5, IL- 13, IL-10, IL-12, IFN-γ. In addition, the levels of TGF-β is also assessed, expression of which is increased following induction of IL-13 synthesis. The other genes of relevance to Th2 response and eventual pro-fibrotic response include Arginine (Arg)-1 and Arg-2. BALF supernatant collected from lungs of animals exposed to toxic substances or human patients is used. Tissue homogenates or cell pellets can also be used. Expression of these genes and proteins can be assessed in in vitro cell cultures exposed to pro-fibrotic stimulus. Apart from assaying single protein or gene at a time, cytokine bead arrays or cytokine PCR arrays can be used to detect a whole panel of Th1 and/or Th2 cytokines using a multiplex method. This method is quantitative and especially advantageous when the sample amount available for testing is scarce. The details of ELISA and qRT-PCR are described under Event 1495. The details of BALF sample collection is described under Event 1497.

References

1. Barbarin V, Xing Z, Delos M, Lison D, Huaux F. Pulmonary overexpression of IL-10 augments lung fibrosis and Th2 responses induced by silica particles. Am J Physiol Lung Cell Mol Physiol. 2005 May;288(5):L841-8. doi: 10.1152/ajplung.00329.2004.

2. Dong J, Ma Q. Type 2 Immune Mechanisms in Carbon Nanotube-Induced Lung Fibrosis. Front Immunol. 2018 May 22;9:1120. doi: 10.3389/fimmu.2018.01120.  

3. Fatkhutdinova LM, Khaliullin TO, Vasil'yeva OL, Zalyalov RR, Mustafin IG, Kisin ER, Birch ME, Yanamala N, Shvedova AA. Fibrosis biomarkers in workers exposed to MWCNTs. Toxicol Appl Pharmacol. 2016 May 15;299:125-31. doi: 10.1016/j.taap.2016.02.016. 

4. He C, Ryan AJ, Murthy S, Carter AB. Accelerated development of pulmonary fibrosis via Cu,Zn-superoxide dismutase-induced alternative activation of macrophages. J Biol Chem. 2013 Jul 12;288(28):20745-57. doi: 10.1074/jbc.M112.410720. 

5. Lee CG, Homer RJ, Zhu Z, Lanone S, Wang X, Koteliansky V, Shipley JM, Gotwals P, Noble P, Chen Q, Senior RM, Elias JA. Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1). J Exp Med. 2001 Sep 17;194(6):809-21. doi: 10.1084/jem.194.6.809.

6. Park EJ, Roh J, Kim SN, Kang MS, Han YA, Kim Y, Hong JT, Choi K. A single intratracheal instillation of single-walled carbon nanotubes induced early lung fibrosis and subchronic tissue damage in mice. Arch Toxicol. 2011 Sep;85(9):1121-31. doi: 10.1007/s00204-011-0655-8. 

7. Shaykhiev R, Krause A, Salit J, Strulovici-Barel Y, Harvey BG, O'Connor TP, Crystal RG. Smoking-dependent reprogramming of alveolar macrophage polarization: implication for pathogenesis of chronic obstructive pulmonary disease. J Immunol. 2009 Aug 15;183(4):2867-83. doi: 10.4049/jimmunol.0900473.

8. Stahl M, Schupp J, Jäger B, Schmid M, Zissel G, Müller-Quernheim J, Prasse A. Lung collagens perpetuate pulmonary fibrosis via CD204 and M2 macrophage activation. PLoS One. 2013 Nov 20;8(11):e81382. doi: 10.1371/journal.pone.0081382.

9. Zhu Z, Homer RJ, Wang Z, Chen Q, Geba GP, Wang J, Zhang Y, Elias JA. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest. 1999 Mar;103(6):779-88. doi: 10.1172/JCI5909. 

Event: 1500: Increased, fibroblast proliferation and myofibroblast differentiation

Short Name: Increased cellular proliferation and differentiation

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Key Event Description

Fibroblasts are non-hematopoietic, non-epithelial and non-endothelial cells. In steady state conditions, they are distributed throughout the mesenchyme. During the wound healing process, fibroblasts are rapidly recruited from mesenchymal cells or in case of exaggerated repair, and they can also be derived from fibrocytes in the bone marrow. They are not terminally differentiated. They synthesise structural proteins (fibrous collagen, elastin), adhesive proteins (laminin and fibronectins) and ground substance (glycosaminoglycans – hyaluronan and glycoproteins) proteins of the extracellular matrix (ECM) that provide structural support to tissue architecture and function. Fibroblasts play an important role in ECM maintenance and turnover, wound healing, inflammation and angiogenesis. They provide structural integrity to the newly formed wound. Fibroblasts with Alpha smooth muscle actin (α-SMA) expression are called myofibroblasts. It is thought that differentiating fibroblasts residing in the lung are the primary source of myofibroblast (CD45 Col I α-SMA) cells (Hashimoto et al., 2001; Serini and Gabbiani, 1999). Myofibroblasts can also originate from epithelial-mesenchymal transition (EMT) (Kim et al., 2006). The other sources of fibroblasts include fibrocytes that likely originate in the bone marrow and migrate to the site of injury upon cytokine signaling. Fibrocytes are capable of differentiating into fibroblasts or myofibroblasts, and comprise less than 1% of the circulating pool of leukocytes and express chemokines C-C motif chemokine receptor (CCR)2, C-X-C chemokine receptor (CXCR)4 and CCR7 in addition to a characteristic pattern of biomarkers, including collagen I and III, CD34, CD43 and CD45 (Abe et al., 2001; Bucala et al., 1994; Chesney et al., 1998). In bleomycin-induced lung fibrosis model, human CD34 CD45 collagen I CXCR4 cells (fibrocytes) are shown to migrate to the lungs in response to both bleomycin and C-X-C motif chemokine ligand (CXCL)12 (which is the only chemokine known to bind to CXCR4) (Phillips et al., 2004). 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. 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.

Literature evidence for its perturbation in the context of pulmonary fibrosis:

Idiopathic pulmonary fibrosis is characterised by progressive fibroblast and myofibroblast proliferation and excessive deposition of ECM (Kuhn and McDonald., 1991). High levels of α -SMA protein and increased number of α-SMA positive cells were observed in mouse lungs treated with multi-walled carbon nanotubes (MWCNTs) as early as day 1 post-exposure (Dong and Ma, 2016). Fibrotic lesions observed in mice treated with asbestos show proliferating fibroblasts and collagen deposition. The same study also demonstrated that bronchoalveolar lavage fluid supernatant derived from asbestos exposed lungs was sufficient to stimulate fibroblast proliferation in vitro (Lemaire et al., 1986). Fibrotic foci developed in rat lungs following exposure to bleomycin show α-SMA expressing myofibroblasts (Vyalov et al., 1993). Several in vitro studies have shown fibroblast proliferation following carbon nanotube treatment (Hussain et al., 2014; Wang et al., 2010a; Wang et al., 2010b).

How it is Measured or Detected

Immunohistochemistry (routinely used and recommended):

Proliferation of fibroblasts and activation of myofibroblasts is normally detected using individual antibodies against vimentin, procollagen 1 and α-SMA, specific markers of fibroblasts and myofibroblasts (Zhang, 1994). It is recommended to use more than one marker to confirm the activation of fibroblasts. The species-specific antibodies for all the markers are commercially available and the technique works in both in vitro and in vivo models as well as in human specimens. Immunohistochemistry is performed using immunoperoxidase technique. Formalin fixed and paraffin embedded lung sections are sliced in 3-5µm thin slices and reacted with diluted H₂O₂ for 10 min to block the endogenous peroxidase activity. The slices are then incubated with appropriate dilutions of primary antibody against the individual markers followed by incubation with the secondary antibody that is biotinylated. The slices are incubated for additional 30 minutes for avidin-biotin amplification and reacted with substrate 3’3’ diaminobenzidine before visualising the cells under the light microscope. Although only semiquantitative, morphometric analysis of the lung slices can be conducted to quantify the total number of cells expressing the markers against the control lung sections where expression of specific markers is expected to be low or nil. For the morphometric analysis, using ocular grids, images of 20-25 non-overlapping squares (0.25 mm) from 2-3 random lung section are taken under 20x magnification. Minimum of three animals per treatment group are assessed. Some researchers include only those cells that are positive for both procollagen I and α-SMA markers.

The limitation of the technique is that the antibodies have to be of high quality and specific. Background noise due to non-specific reactions can yield false-positive results.

In vitro, expression of type-1 collagen, Thy-1 cell surface antigen (Thy-1), cyclooxygenase-2 (COX2) and caveolin-1 (CAV1) are used as markers of homogeneous population of fibroblasts. Increased expression of Transforming growth factor beta (TGF-β) and α-SMA is used as markers of differentiated myofibroblasts. Transcription factor SMAD family member 3 (Smad3) is the other marker measured in vitro to assess the fibroblast proliferation and differentiation. Several in vitro studies using lung epithelial cells (e.g. A549 cells) have shown that asbestos induces markers of EMT (Tamminen et al., 2012), which is mediated by the activation of TGF-β-p-Smad2 (Kim et al., 2006).

Hydrogels:

Hydrogels are water-swollen crosslinked polymer networks. They are used to mimic the original ECM. Hydrogels consist of collagen, fibrin, hyaluronic acid or synthetic materials such as polyacrylamide enriched with ECM proteins, etc. Hydrogels can be prepared to express inherent biological signals, mechanical properties (e.g., modulus) and biochemical properties (e.g., proteins) of the ECM. Fibroblasts are usually cultured in fibrin and type-1 collagen that represent the matrix of the wound healing. Thus, the well-constructed hydrogel can be used to assess cell proliferation, activation and matrix synthesis as reflective of fibroblast activation. For naturally derived hydrogen scaffolds, cells derived directly from animal or human tissues can be used (Smithmyer et al., 2014).

Fibroblast proliferation assay:

Several primary and immortalised fibroblast types can be used for the assay. Proliferation assays such as water-soluble tetrazolium salts (WST)-1 and propidium iodide staining of cells have been used to show dose-dependent increase in MWCNT-induced increase in fibroblast proliferation that is in alignment with in vivo mouse fibrogenic response (Azad et al., 2013; Vietti et al., 2013) to the same material.

Advanced co-culture models (myofibroblast differentiation):

Co-culture models that mimic the alveolar capillary membrane (such as those listed for Event 1496 & Event 1498) can be used to assess myofibroblast differentiation in response to pro-fibrotic stressors using immunofluorescent staining for α-SMA. More complex in vitro microfluidic lung-on-a-chip models (such as the one listed for Event 1497) can be used to assess myofibroblast differentiation in the same stead. These provide a more realistic exposure model as opposed to a submerged monoculture of fibroblasts, however they require a higher degree of technical skill and advanced fabrication which may not be suitable for all labs.

References

1. Abe R, Donnelly SC, Peng T, Bucala R, Metz CN. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol. 2001 Jun 15;166(12):7556-62. doi: 10.4049/jimmunol.166.12.7556. 

2. Azad N, Iyer AK, Wang L, Liu Y, Lu Y, Rojanasakul Y. Reactive oxygen species-mediated p38 MAPK regulates carbon nanotube-induced fibrogenic and angiogenic responses. Nanotoxicology. 2013 Mar;7(2):157-68. doi: 10.3109/17435390.2011.647929.

3. Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med. 1994 Nov;1(1):71-81.

4. Chesney J, Metz C, Stavitsky AB, Bacher M, Bucala R. Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes. J Immunol. 1998 Jan 1;160(1):419-25.

5. Dong J, Ma Q. Myofibroblasts and lung fibrosis induced by carbon nanotube exposure. Part Fibre Toxicol. 2016 Nov 4;13(1):60. doi: 10.1186/s12989-016-0172-2.  

6. Hashimoto S, Gon Y, Takeshita I, Maruoka S, Horie T. IL-4 and IL-13 induce myofibroblastic phenotype of human lung fibroblasts through c-Jun NH2-terminal kinase-dependent pathway. J Allergy Clin Immunol. 2001 Jun;107(6):1001-8. doi: 10.1067/mai.2001.114702.

7. Hussain S, Sangtian S, Anderson SM, Snyder RJ, Marshburn JD, Rice AB, Bonner JC, Garantziotis S. Inflammasome activation in airway epithelial cells after multi-walled carbon nanotube exposure mediates a profibrotic response in lung fibroblasts. Part Fibre Toxicol. 2014 Jun 10;11:28. doi: 10.1186/1743-8977-11-28. 

8. Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN, Sheppard D, Chapman HA. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A. 2006 Aug 29;103(35):13180-5. doi: 10.1073/pnas.0605669103.

9. Kuhn C, McDonald JA. The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. Am J Pathol. 1991 May;138(5):1257-65. 

10. Lemaire I, Beaudoin H, Massé S, Grondin C. Alveolar macrophage stimulation of lung fibroblast growth in asbestos-induced pulmonary fibrosis. Am J Pathol. 1986 Feb;122(2):205-11.

11. Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, Belperio JA, Keane MP, Strieter RM. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest. 2004 Aug;114(3):438-46. doi: 10.1172/JCI20997. 

12. Serini G, Gabbiani G. Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res. 1999 Aug 1;250(2):273-83. doi: 10.1006/excr.1999.4543.

13. Smithmyer ME, Sawicki LA, Kloxin AM. Hydrogel scaffolds as in vitro models to study fibroblast activation in wound healing and disease. Biomater Sci. 2014 May 1;2(5):634-650. doi: 10.1039/C3BM60319A. 

14. Tamminen JA, Myllärniemi M, Hyytiäinen M, Keski-Oja J, Koli K. Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling. J Cell Biochem. 2012 Jul;113(7):2234-47. doi: 10.1002/jcb.24094.

15. Vietti G, Ibouraadaten S, Palmai-Pallag M, Yakoub Y, Bailly C, Fenoglio I, Marbaix E, Lison D, van den Brule S. Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay. Part Fibre Toxicol. 2013 Oct 10;10:52. doi: 10.1186/1743-8977-10-52. 

16. Vyalov SL, Gabbiani G, Kapanci Y. Rat alveolar myofibroblasts acquire alpha-smooth muscle actin expression during bleomycin-induced pulmonary fibrosis. Am J Pathol. 1993 Dec;143(6):1754-65. 

17. Wang L, Mercer RR, Rojanasakul Y, Qiu A, Lu Y, Scabilloni JF, Wu N, Castranova V. Direct fibrogenic effects of dispersed single-walled carbon nanotubes on human lung fibroblasts. J Toxicol Environ Health A. 2010a;73(5):410-22. doi: 10.1080/15287390903486550. 

18. Wang X, Xia T, Ntim SA, Ji Z, George S, Meng H, Zhang H, Castranova V, Mitra S, Nel AE. Quantitative techniques for assessing and controlling the dispersion and biological effects of multiwalled carbon nanotubes in mammalian tissue culture cells. ACS Nano. 2010b Dec 28;4(12):7241-52. doi: 10.1021/nn102112b. 

19. Zhang K, Rekhter MD, Gordon D, Phan SH. Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohistochemical and in situ hybridization study. Am J Pathol. 1994 Jul;145(1):114-25. 

Event: 68: Accumulation, Collagen

Short Name: Accumulation, Collagen

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Humans: Bataller and  Brenner, 2005; Decaris et al., 2015.  

Mice: Dalton et al., 2009; Leung et al., 2008; Nan et al., 2013.

Rats: Hamdy and El-Demerdash, 2012; Li et al., 2012; Luckey and Petersen, 2001; Natajaran et al., 2006.

 

Key Event Description

Collagen is mostly found in fibrous tissues such as tendons, ligaments and skin. It is also abundant in corneas, cartilage, bones, blood vessels, the gut, intervertebral discs, and the dentin in teeth. In muscle tissue, it serves as a major component of the endomysium. Collagen is the main structural protein in the extracellular space in the various connective tissues, making up from 25% to 35% of the whole-body protein content. In normal tissues, collagen provides strength, integrity, and structure. When tissues are disrupted following injury, collagen is needed to repair the defect. If too much collagen is deposited, normal anatomical structure is lost, function is compromised, and fibrosis results.

The fibroblast is the most common collagen producing cell. Collagen-producing cells may also arise from the process of transition of differentiated epithelial cells into mesenchymal cells. This has been observed e.g. during renal fibrosis (transformation of tubular epithelial cells into fibroblasts) and in liver injury (transdifferentiation of hepatocytes and cholangiocytes into fibroblasts) (Henderson and Iredale, 2007)^.

There are close to 20 different types of collagen found with the predominant form being type I collagen. This fibrillar form of collagen represents over 90 percent of our total collagen and is composed of three very long protein chains which are wrapped around each other to form a triple helical structure called a collagen monomer. Collagen is produced initially as a larger precursor molecule called procollagen. As the procollagen is secreted from the cell, procollagen proteinases remove the extension peptides from the ends of the molecule. The processed molecule is referred to as collagen and is involved in fiber formation. In the extracellular spaces the triple helical collagen molecules line up and begin to form fibrils and then fibers. Formation of stable crosslinks within and between the molecules is promoted by the enzyme lysyl oxidase and gives the collagen fibers tremendous strength (Diegelmann,2001)^. The overall amount of collagen deposited by fibroblasts is a regulated balance between collagen synthesis and collagen catabolism. Disturbance of this balance leads to changes in the amount and composition of collagen. Changes in the composition of the extracellular matrix initiate positive feedback pathways that increase collagen production.

Normally, collagen in connective tissues has a slow turn over; degradating enzymes are collagenases, belonging to the family of matrix metalloproteinases. Other cells that can synthesize and release collagenase are macrophages, neutrophils, osteoclasts, and tumor cells (Di Lullo et al., 2002; Kivirikko and Risteli, 1976; Miller and Gay, 1987; Prockop and Kivirikko, 1995).

 

How it is Measured or Detected

Determination of the amount of collagen produced in vitro can be done in a variety of ways ranging from simple colorimetric assays to elaborate chromatographic procedures using radioactive and non-radioactive material. What most of these procedures have in common is the need to destroy the cell layer to obtain solubilized collagen from the pericellular matrix. Rishikof et al. describe several methods to assess the in vitro production of type I collagen: Western immunoblotting of intact alpha1(I) collagen using antibodies directed to alpha1(I) collagen amino and carboxyl propeptides, the measurement of alpha1(I) collagen mRNA levels using real-time polymerase chain reaction, and methods to determine the transcriptional regulation of alpha1(I) collagen using a nuclear run-on assay (Rishikof et al., 2005). 

Histological staining with stains such as Masson Trichrome, Picro-sirius red are used to identify the tissue/cellular distribution of collagen, which can be quantified using morphometric analysis both in vivo and in vitro. The assays are routinely used and are quantitative.

Sircol Collagen Assay for collagen quantification:

The Serius dye has been used for many decades to detect collagen in histology samples. The Serius Red F3BA selectively binds to collagen and the signal can be read at 540 nm (Chen and Raghunath, 2009; Nikota et al., 2017).

Hydroxyproline assay:

Hydroxyproline is a non-proteinogenic amino acid formed by the prolyl-4-hydroxylase. Hydroxyproline is only found in collagen and thus, it serves as a direct measure of the amount of collagen present in cells or tissues. Colorimetric methods are readily available and have been extensively used to quantify collagen using this assay (Chen and Raghunath, 2009; Nikota et al., 2017).

Ex vivo precision cut tissue slices

Precision cut tissue slices mimic the whole organ response and allow histological assessment, an endpoint of interest in regulatory decision making. While this technique uses animals, the number of animals required to conduct a dose-response study can be reduced to 1/4^th of what will be used in whole animal exposure studies (Rahman et al., 2020). 

 

References

1. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005 Feb;115(2):209-18. doi: 10.1172/JCI24282. 

2. Chen CZ, Raghunath M. Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis state of the art. Fibrogenesis Tissue Repair. 2009 Dec 15;2:7. doi: 10.1186/1755-1536-2-7. 

3. Dalton SR, Lee SM, King RN, Nanji AA, Kharbanda KK, Casey CA, McVicker BL. Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice. Biochem Pharmacol. 2009 Apr 1;77(7):1283-90. doi: 10.1016/j.bcp.2008.12.023. 

4. Decaris ML, Emson CL, Li K, Gatmaitan M, Luo F, Cattin J, Nakamura C, Holmes WE, Angel TE, Peters MG, Turner SM, Hellerstein MK. Turnover rates of hepatic collagen and circulating collagen-associated proteins in humans with chronic liver disease. PLoS One. 2015 Apr 24;10(4):e0123311. doi: 10.1371/journal.pone.0123311.

5. Di Lullo GA, Sweeney SM, Korkko J, Ala-Kokko L, San Antonio JD. Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem. 2002 Feb 8;277(6):4223-31. doi: 10.1074/jbc.M110709200.

6. Diegelmann R. Collagen Metabolism. Wounds. 2001;13:177-82. Available at www.medscape.com/viewarticle/423231 (accessed on 20 January 2016).

7. Hamdy N, El-Demerdash E. New therapeutic aspect for carvedilol: antifibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage. Toxicol Appl Pharmacol. 2012 Jun 15;261(3):292-9. doi: 10.1016/j.taap.2012.04.012. 

8. Henderson NC, Iredale JP. Liver fibrosis: cellular mechanisms of progression and resolution. Clin Sci (Lond). 2007 Mar;112(5):265-80. doi: 10.1042/CS20060242.

9. Kivirikko KI, Risteli L. Biosynthesis of collagen and its alterations in pathological states. Med Biol. 1976 Jun;54(3):159-86.

10. Leung TM, Tipoe GL, Liong EC, Lau TY, Fung ML, Nanji AA. Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis. Int J Exp Pathol. 2008 Aug;89(4):241-50. doi: 10.1111/j.1365-2613.2008.00590.x. 

11. Li L, Hu Z, Li W, Hu M, Ran J, Chen P, Sun Q. Establishment of a standardized liver fibrosis model with different pathological stages in rats. Gastroenterol Res Pract. 2012;2012:560345. doi: 10.1155/2012/560345. 

12. Luckey SW, Petersen DR. Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats. Exp Mol Pathol. 2001 Dec;71(3):226-40. doi: 10.1006/exmp.2001.2399.

13. Miller EJ, Gay S. The collagens: an overview and update. Methods Enzymol. 1987;144:3-41. doi: 10.1016/0076-6879(87)44170-0. 

14. Nan YM, Kong LB, Ren WG, Wang RQ, Du JH, Li WC, Zhao SX, Zhang YG, Wu WJ, Di HL, Li Y, Yu J. Activation of peroxisome proliferator activated receptor alpha ameliorates ethanol mediated liver fibrosis in mice. Lipids Health Dis. 2013 Feb 6;12:11. doi: 10.1186/1476-511X-12-11.

15. Natarajan SK, Thomas S, Ramamoorthy P, Basivireddy J, Pulimood AB, Ramachandran A, Balasubramanian KA. Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models. J Gastroenterol Hepatol. 2006 Jun;21(6):947-57. doi: 10.1111/j.1440-1746.2006.04231.x.

16. Nikota J, Banville A, Goodwin LR, Wu D, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. 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. Part Fibre Toxicol. 2017 Sep 13;14(1):37. doi: 10.1186/s12989-017-0218-0. 

17. Prockop DJ, Kivirikko KI. Collagens: molecular biology, diseases, and potentials for therapy. Annu Rev Biochem. 1995;64:403-34. doi: 10.1146/annurev.bi.64.070195.002155. 

18. Rahman L, Williams A, Gelda K, Nikota J, Wu D, Vogel U, Halappanavar S. 21st Century Tools for Nanotoxicology: Transcriptomic Biomarker Panel and Precision-Cut Lung Slice Organ Mimic System for the Assessment of Nanomaterial-Induced Lung Fibrosis. Small. 2020 Sep;16(36):e2000272. doi: 10.1002/smll.202000272.

19. Rishikof DC, Kuang PP, Subramanian M, Goldstein RH. Methods for measuring type I collagen synthesis in vitro. Methods Mol Med. 2005;117:129-40. doi: 10.1385/1-59259-940-0:129. 

List of Adverse Outcomes in this AOP

Event: 1458: Pulmonary fibrosis

Short Name: Pulmonary fibrosis

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Evidence for Perturbation by Stressor

Bleomycin

Bleomycin is a potent anti-tumour drug, routinely used for treating various types of human cancers (Umezawa H et al., 1967; Adamson IY, 1976). Lung injury and lung fibrosis are the major adverse effects of this drug in humans (Hay J et al., 1991). Bleomycin is shown to induce lung fibrosis in animals – such as dogs (Fleischman RW et al., 1971), mice (Adamson IY and Bowden DH, 1974), and hamsters (Snider GL et al., 1978) and is widely used as a model to study the mechanisms of fibrosis (reviewed in Moeller A et al., 2008; Gilhodes J-C et al., 2017).

1. Umezawa H, Ishizuka M, Maeda K, Takeuchi T. Studies on bleomycin. Cancer. 1967 May;20(5):891-5.
2. Adamson IY. Pulmonary toxicity of bleomycin. Environ Health Perspect. 1976 Aug;16:119-26.
3. Hay J, Shahzeidi S, Laurent G  Mechanisms of bleomycin induced lung damage. 1991 Arch Toxicol 65:81–94.
4. Fleischman RW, Baker JR, Thompson GR, et al. Bleomycin-induced interstitial pneumonia in dogs. Thorax. 1971;26(6):675-682.
5. Adamson IYR, Bowden DH. The Pathogenesis of Bleomycin-Induced Pulmonary Fibrosis in Mice. The American Journal of Pathology. 1974;77(2):185-198.
6. Snider GL, Celli BR, Goldstein RH, O'Brien JJ, Lucey EC. Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Lung volumes, volume-pressure relations, carbon monoxide uptake, and arterial blood gas studied. Am Rev Respir Dis. 1978 Feb; 117(2):289-97.
7. Moeller A, Ask K, Warburton D, Gauldie J, Kolb M. The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis? The international journal of biochemistry & cell biology. 2008;40(3):362-382.
8. Gilhodes J-C, Julé Y, Kreuz S, Stierstorfer B, Stiller D, Wollin L (2017) Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis. PLoS ONE 12(1): e0170561.

Carbon nanotubes, Multi-walled carbon nanotubes, single-walled carbon nanotubes, carbon nanofibres

Carbon nanotubes (CNTs) are allotropes of carbon, are made of rolled up sheet of graphene (single-walled carbon nanotubes) and are tubular in shape. A multi-walled carbon nanotube (MWCNT) is a multi-layered concentric cylinder of graphene sheets stacked one inside the other (N. Saifuddin et al., 2013). CNTs exhibit a combination of unique mechanical, thermal, and electronic properties and are highly desired commercially. They are light weight but their tensile strength is 50 times higher than that of steel, and they are stable chemically as well as in the environment. Consequently, they are produced in massive amounts and are increasingly incorporated in several industrial products.

CNTs are high aspect ratio materials and are shown to cause lung fibrosis in animals (Muller J et al., 2005; Porter DW et al., 2010; Dong and Ma 2016; Vietti, et al., 2016). In an intelligence bulletin published by NIOSH on ‘Occupational exposure to carbon nanotubes and nanofibers’, NIOSH reviewed 54 individual animal studies investigating the pulmonary toxicity induced by CNTs and reported that half of those studies consistently showed lung fibrosis (NIOSH bulletin, 2013). However, the evidence is inconsistent and the occurrence of fibrotic pathology is influenced by the specific physical-chemical properties of CNTs (i.e. length, rigidity), their dispersion in exposure vehicle, and the mode of exposure (Duke and Bonner 2018).

1. Dong, J., & Ma, Q. (2016). Myofibroblasts and lung fibrosis induced by carbon nanotube exposure. Particle and fibre toxicology, 13(1), 60.

2. Duke, K. S., & Bonner, J. C. (2018). Mechanisms of carbon nanotube-induced pulmonary fibrosis: a physicochemical characteristic perspective. Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology, 10(3), e1498.

3. Muller, J., Huaux, F., Moreau, N., Misson, P., Heilier, J. F., Delos, M., Arras, M., Fonseca, A., Nagy, J. B., & Lison, D. (2005). Respiratory toxicity of multi-wall carbon nanotubes. Toxicology and applied pharmacology, 207(3), 221–231.

4. NIOSH: Occupational exposure to carbon nanotubes and nanofibers: current intelligence bulletin 65. 2013.

5. Porter, D. W., Hubbs, A. F., Mercer, R. R., Wu, N., Wolfarth, M. G., Sriram, K., Leonard, S., Battelli, L., Schwegler-Berry, D., Friend, S., Andrew, M., Chen, B. T., Tsuruoka, S., Endo, M., & Castranova, V. (2010). Mouse pulmonary dose- and time course-responses induced by exposure to multi-walled carbon nanotubes. Toxicology, 269(2-3), 136–147.

6. N. Saifuddin, A. Z. Raziah, and A. R. Junizah. Carbon Nanotubes: A Review on Structure and Their Interaction with Proteins. Journal of Chemistry, vol. 2013, Article ID 676815, 18 pages, 2013.

7. Vietti, G., Lison, D., & van den Brule, S. (2016). Mechanisms of lung fibrosis induced by carbon nanotubes: towards an Adverse Outcome Pathway (AOP). Particle and fibre toxicology, 13, 11.

 

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Key Event Description

Pulmonary fibrosis is broadly defined as the thickening or scarring of lung tissue, due to excessive deposition of extracellular matrix. In the normal human lung, the nasopharynx and the conducting airways are mainly covered by epithelium composed of ciliated, mucous secreting cells in direct contact with the basement membrane with submucosal glands containing goblet, duct, and serous cells also contributing to the fluid balance and mucous production (Koval and Sidhaye, 2017). Within this epithelium, basal cells are found which are stimulated to proliferate and differentiate in response to injury (Koval and Sidhaye, 2017). Further down the lung, in the terminal bronchiole region, the epithelium does not contain submucosal glands, but instead contains club cells which produce pulmonary surfactant and can differentiate into bronchiolar or alveolar epithelial cells (AECs). Finally, in the terminal airspaces, the epithelium is made up entirely of type I and type II AECs. In between the two adjacent alveoli are two layers of alveolar epithelium resting on basement membrane, which consists of interstitial space, pulmonary capillaries, elastin and collagen fibres. Thus, the alveolar capillary membrane (ACM), where gas exchange takes place, is made up of the alveolar epithelium and alveolar endothelium (Gracey et al, 1968). In pulmonary fibrosis, damage to the pulmonary epithelium results in excessive deposition of collagen by constitutively activated myofibroblasts during the wound healing response. This causes a pronounced decrease in the number of capillaries within the alveolar septa with asymmetric deposition of collagen and cells between part of the surface of a capillary and the nearby alveolar lining. In areas where capillaries are not present, the ACM is occupied with collagen and cells.

How it is Measured or Detected

In vivo, histopathological analysis is used for assessing fibrotic lung disease. Morphometric analysis of the diseased area versus total lung area is used to quantitatively stage the fibrotic disease. Although, some inconsistencies can be introduced during the analysis due to the experience of the individual scoring the disease, the histological stain, etc., a numerical scale with grades from 0 to 8, originally developed by Ashcroft et al., 1988 is assigned to indicate the amount of fibrotic tissue in histological samples. This scale is applied to diagnose lung fibrosis in both human and animal samples. Modifications to this scoring system were proposed (Hubner et al., 2008), which enables morphological distinctions thus enabling a better grading of the disease. Using the modified scoring system, bleomycin induced lung fibrosis in rats was scored as follows: Grade 0 – normal lung, Grade 1 – isolated alveolar septa with gentle fibrotic changes, Grade 2 – knot like formation in fibrotic areas in alveolar septa, Grade 3 – contiguous fibrotic walls of alveolar septa, Grade 4 – single fibrotic masses, Grade 5 – confluent fibrotic masses, Grade 6 – large contiguous fibrotic masses, Grade 7 – air bubbles and Grade 8 – fibrotic obliteration. Further morphometric analysis can be conducted to quantify the total disease area (Nikota et al., 2017).

Lungs are formalin fixed and paraffin embedded such that an entire cross section of lung can be presented on a slide. The entire cross section is captured in a series of images using wide field light microscope. Areas of alveolar epithelium thickening and consolidated air space are identified. ImageJ software (freely available) is used to trace the total area (green line) and the diseased area (red line) imaged and quantified. The diseased area is equal to disease area/total area (Nikota et al., 2017).

In vitro, there is no single assay that can measure the alveolar thickness. However, a combination of assays spanning various KEs described above provide a measure of the extent of fibrogenesis potential of tested substances. Real-time reverse transcription-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assays (ELISA) measuring increased collagen, Transforming growth factor beta 1 (TGF-β1) and various pro-inflammatory mediators are used as sensitive markers of potential of substances to induce the adverse outcome of lung fibrosis.

References

1. Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988 Apr;41(4):467-70. doi: 10.1136/jcp.41.4.467.

2. Gracey DR, Divertie MB, Brown AL Jr. Alveolar-capillary membrane in idiopathic interstitial pulmonary fibrosis. Electron microscopic study of 14 cases. Am Rev Respir Dis. 1968 Jul;98(1):16-21. doi: 10.1164/arrd.1968.98.1.16.

3. Hübner RH, Gitter W, El Mokhtari NE, Mathiak M, Both M, Bolte H, Freitag-Wolf S, Bewig B. Standardized quantification of pulmonary fibrosis in histological samples. Biotechniques. 2008 Apr;44(4):507-11, 514-7. doi: 10.2144/000112729. 

4. Koval M, Sidhaye VK. Introduction: The Lung Epithelium. In: Sidhaye VK, Koval M, editors. Lung Epithelial Biology in the Pathogenesis of Pulmonary Disease. Boston: Academic Press; 2017. p. xiii-xviii. Elsevier.

5. Nikota J, Banville A, Goodwin LR, Wu D, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. 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. Part Fibre Toxicol. 2017 Sep 13;14(1):37. doi: 10.1186/s12989-017-0218-0. 

Appendix 2

List of Key Event Relationships in the AOP