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

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

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

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

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

⁴Physicians Committee for Responsible Medicine, Washington, DC.

*Point of contact

Sabina Halappanavar, PhD

Research Scientist, Genomics, Nanotoxicology and Alternative Methods Laboratory

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

Sir Frederick G Banting Research Centre,

251 Sir Frederick Banting Driveway, Building 22

Ottawa, ON, Canada, K1A 0K9

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

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

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

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

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.

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

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

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.

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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