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Key Event Title
Substance interaction with the lung resident cell membrane components
|Level of Biological Organization|
Key Event Components
|pattern recognition receptor signaling pathway||increased|
|toll-like receptor signaling pathway||Toll-like receptor||increased|
|toll-like receptor 4 signaling pathway||Toll-like receptor 4||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Substance interaction with the lung cell membrane leading to lung fibrosis||MolecularInitiatingEvent||Sabina Halappanavar (send email)||Under development: Not open for comment. Do not cite||EAGMST Under Review|
|Interaction with lung cells leads to lung cancer||MolecularInitiatingEvent||Penny Nymark (send email)||Under development: Not open for comment. Do not cite|
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). 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).
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. 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 High Aspect Ratio (HARs) fibres that are long and stiff undergo frustrated phagocytosis because of their inability to engulf the piercing fibres and subsequently lead to cell injury (Mossman and Churg, 1998; Donaldson K et al., 2010). 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 receptor 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, Ndimethyl-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 (Matzinger, 2002).
How It Is Measured or Detected
Detection of Danger Associated Molecular Patterns (DAMPs) or homeostasis-altering molecular processes (HAMPs):
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 (Suwara et al., 2014; Nikota et al., 2017; Rabolli et al., 2014).
Targeted enzyme-linked immunosorbent assays (ELISA) (routinely used and recommended):
ELISA assays – permit 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.
IL-1a and IL-1b is activated or secreted into the cytosol following stimulus. Targeted ELISA can be used to quantify IL-1a or IL1b 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. Westernblot is another method that can be used to quantify the release of various alarmins using specific antibodies. qRT-PCR or ELISA assays can also be used to quantify 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 (Varela et al., 2012). Immunohistochemistry methods targeting lysosome specific proteins are regularly employed for this purpose. In co-localisation experiments, lysosomal marker 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 (Donaldson et al., 2010).
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 Hr 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:
Even closer to the in vivo condition than co-culture models, precision cut lung slice (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 MWCNT 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.
Domain of 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 IPF; however, anti-inflammatory drugs have proven ineffective for treating IPF. Danger signalling axis including uric acid, ATP and IL-33/ST2 has been proven to promote lung fibrosis in animals.
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.
1. Amsen, D. and De Visser, K. (2009). Approaches to Determine Expression of Inflammatory Cytokines. Methods in molecular biology.. 511th ed. (Clifton, NJ), pp.107-142.
2. Barosova, H., Maione, A. G., Septiadi, D., Sharma, M., Haeni, L., Balog, S., O'Connell, O., Jackson, G. R., Brown, D., Clippinger, A. J., Hayden, P., Petri-Fink, A., Stone, V., & Rothen-Rutishauser, B. (2020). Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS nano, 14(4), 3941–3956.
3. Bai Y, Krishnamoorthy N, Patel KR, Rosas I, Sanderson MJ, Ai X. (2016). Cryopreserved Human Precision-Cut Lung Slices as a Bioassay for Live Tissue Banking. A Viability Study of Bronchodilation with Bitter-Taste Receptor Agonists. Am J Respir Cell Mol Biol. 2016 May;54(5):656-63.
4. Behzadi, S., Serpooshan, V., Tao, W., Hamaly, M., Alkawareek, M., Dreaden, E., Brown, D., Alkilany, A., Farokhzad, O. and Mahmoudi, M. (2017). Cellular uptake of nanoparticles: journey inside the cell. Chemical Society Reviews, 46(14), pp.4218-4244.
5. Bianchi, M. (2006). DAMPs, PAMPs and alarmins: all we need to know about danger. Journal of Leukocyte Biology, 81(1), pp.1-5.
6. Boyles, M., Young, L., Brown, D., MacCalman, L., Cowie, H., Moisala, A., Smail, F., Smith, P., Proudfoot, L., Windle, A. and Stone, V. (2015). Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos. Toxicology in Vitro, 29(7), pp.1513-1528.
7. Brown, D., Kinloch, I., Bangert, U., Windle, A., Walter, D., Walker, G., Scotchford, C., Donaldson, K. and Stone, V. (2007). An in vitro study of the potential of carbon nanotubes and nanofibres to induce inflammatory mediators and frustrated phagocytosis. Carbon, 45(9), pp.1743-1756.
8. Cheng, L., Jiang, X., Wang, J., Chen, C. and Liu, R. (2013). Nano–bio effects: interaction of nanomaterials with cells. Nanoscale, 5(9), p.3547.
9. Clift, M. J., Endes, C., Vanhecke, D., Wick, P., Gehr, P., Schins, R. P., Petri-Fink, A., & Rothen-Rutishauser, B. (2014). A comparative study of different in vitro lung cell culture systems to assess the most beneficial tool for screening the potential adverse effects of carbon nanotubes. Toxicological sciences : an official journal of the Society of Toxicology, 137(1), 55–64.
10. Decan, N., Wu, D., Williams, A., Bernatchez, S., Johnston, M., Hill, M., & Halappanavar, S. (2016). Characterization of in vitro genotoxic, cytotoxic and transcriptomic responses following exposures to amorphous silica of different sizes. Mutation research. Genetic toxicology and environmental mutagenesis, 796, 8–22.
11. Denholm, E. and Phan, S. (1990). Bleomycin Binding Sites on Alveolar Macrophages. Journal of Leukocyte Biology, 48(6), pp.519-523.
12. Desai, O., Winkler, J., Minasyan, M. and Herzog, E. (2018). The Role of Immune and Inflammatory Cells in Idiopathic Pulmonary Fibrosis. Frontiers in Medicine, 5.
13. Di Paolo, N. and Shayakhmetov, D. (2016). Interleukin 1α and the inflammatory process. Nature Immunology, 17(8), pp.906-913.
14. Dinis-Oliveira, R., Duarte, J., Sánchez-Navarro, A., Remião, F., Bastos, M. and Carvalho, F. (2008). Paraquat Poisonings: Mechanisms of Lung Toxicity, Clinical Features, and Treatment. Critical Reviews in Toxicology, 38(1), pp.13-71.
15. Donaldson, K., Murphy, F., Duffin, R. and Poland, C. (2010). Asbestos, carbon nanotubes and the pleural mesothelium: a review and the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma. Particle and Fibre Toxicology, 7(1), p.5.
16. Dörger, M., Münzing, S., Allmeling, A., Messmer, K. and Krombach, F. (2001). Differential Responses of Rat Alveolar and Peritoneal Macrophages to Man-Made Vitreous Fibers in Vitro. Environmental Research, 85(3), pp.207-214.
17. Franks, T., Colby, T., Travis, W., Tuder, R., Reynolds, H., Brody, A., Cardoso, W., Crystal, R., Drake, C., Engelhardt, J., Frid, M., Herzog, E., Mason, R., Phan, S., Randell, S., Rose, M., Stevens, T., Serge, J., Sunday, M., Voynow, J., Weinstein, B., Whitsett, J. and Williams, M. (2008). Resident Cellular Components of the Human Lung: Current Knowledge and Goals for Research on Cell Phenotyping and Function. Proceedings of the American Thoracic Society, 5(7), pp.763-766.
18. Kierdorf, K., Prinz, M., Geissmann, F. and Gomez Perdiguero, E. (2015). Development and function of tissue resident macrophages in mice. Seminars in Immunology, 27(6), pp.369-378.
19. Kim, J., Lim, H., Minai-Tehrani, A., Kwon, J., Shin, J., Woo, C., Choi, M., Baek, J., Jeong, D., Ha, Y., Chae, C., Song, K., Ahn, K., Lee, J., Sung, H., Yu, I., Beck, G. and Cho, M. (2010). Toxicity and Clearance of Intratracheally Administered Multiwalled Carbon Nanotubes from Murine Lung. Journal of Toxicology and Environmental Health, Part A, 73(21-22), pp.1530-1543.
20. Li, X., Jiang, D., Huang, X., Guo, S., Yuan, W. and Dai, H. (2015). Toll-like receptor 4 promotes fibrosis in bleomycin-induced lung injury in mice. Genetics and Molecular Research, 14(4), pp.17391-17398.
21. Matzinger, P. (2002). The Danger Model: A Renewed Sense of Self. Science, 296(5566), pp.301-305.
22. MOSSMAN, B. and CHURG, A. (1998). Mechanisms in the Pathogenesis of Asbestosis and Silicosis. American Journal of Respiratory and Critical Care Medicine, 157(5), pp.1666-1680.
23. Murthy, S., Larson-Casey, J., Ryan, A., He, C., Kobzik, L. and Carter, A. (2015). Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure. The FASEB Journal, 29(8), pp.3527-3536.
24. Nakayama, M. (2018). Macrophage Recognition of Crystals and Nanoparticles. Frontiers in Immunology, 9.
25. National Institute of Occupational Safety and Health (NIOSH) (2011). Asbestos fibers and other elongate mineral particles: state of the science and roadmap for research.. pp.Current Intelligence Bulletin 62. Publication Number 2011-159.
26. Nel, A., Mädler, L., Velegol, D., Xia, T., Hoek, E., Somasundaran, P., Klaessig, F., Castranova, V. and Thompson, M. (2009). Understanding biophysicochemical interactions at the nano–bio interface. Nature Materials, 8(7), pp.543-557.
27. 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.
28. 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).
29. Pascolo, L., Gianoncelli, A., Schneider, G., Salomé, M., Schneider, M., Calligaro, C., Kiskinova, M., Melato, M. and Rizzardi, C. (2013). The interaction of asbestos and iron in lung tissue revealed by synchrotron-based scanning X-ray microscopy. Scientific Reports, 3(1).
30. Poland, C., Duffin, R., Kinloch, I., Maynard, A., Wallace, W., Seaton, A., Stone, V., Brown, S., MacNee, W. and Donaldson, K. (2008). Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nature Nanotechnology, 3(7), pp.423-428.
31. Rabolli, V., Badissi, A., Devosse, R., Uwambayinema, F., Yakoub, Y., Palmai-Pallag, M., Lebrun, A., De Gussem, V., Couillin, I., Ryffel, B., Marbaix, E., Lison, D. and Huaux, F. (2014). The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica microand nanoparticles. Particle and Fibre Toxicology, 11(1).
32. Rahman, L., Williams, A., Gelda, K., Nikota, J., Wu, D., Vogel, U., & Halappanavar, S. (2020). 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, 16(36), e2000272.
33. Suwara, M., Green, N., Borthwick, L., Mann, J., Mayer-Barber, K., Barron, L., Corris, P., Farrow, S., Wynn, T., Fisher, A. and Mann, D. (2013). IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts. Mucosal Immunology, 7(3), pp.684-693.
34. Varela, J., Bexiga, M., Åberg, C., Simpson, J. and Dawson, K. (2012). Quantifying size-dependent interactions between fluorescently labeled polystyrene nanoparticles and mammalian cells. Journal of Nanobiotechnology, 10(1), p.39.