SNAPSHOT

Evidence for Perturbation by Stressor

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Overview for Molecular Initiating Event

Evidence for MIE perturbation

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

Key Event Description

 

How this MIE works

Background

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 K 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. 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 to 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, 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 (Matzinger, 2002).

How it is Measured or Detected

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 (Nelson et al., 2016; 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).

 

References

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. Matzinger, P. (2002). The Danger Model: A Renewed Sense of Self. Science, 296(5566), pp.301-305.
9. 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.
10. 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.
11. Nakayama, M. (2018). Macrophage Recognition of Crystals and Nanoparticles. Frontiers in Immunology, 9.
12. Bianchi, M. (2006). DAMPs, PAMPs and alarmins: all we need to know about danger. Journal of Leukocyte Biology, 81(1), pp.1-5.
13. 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.
14. 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.
15. 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.
16. Denholm, E. and Phan, S. (1990). Bleomycin Binding Sites on Alveolar Macrophages. Journal of Leukocyte Biology, 48(6), pp.519-523.
17. Di Paolo, N. and Shayakhmetov, D. (2016). Interleukin 1α and the inflammatory process. Nature Immunology, 17(8), pp.906-913.
18. 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.
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. 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.
21. 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.
22. 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.
23. 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).
24. 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).
25. 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.
26. Rabolli, V., Badissi, A., Devosse, R., Uwambayinema, F., Yakoub, Y., Palmai-Pallag, M., Lebrun, A., De Gussem, V., Couillin, I., Ryffel, B., Marbaix, E., Lison, D. and Huaux, F. (2014). The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles. Particle and Fibre Toxicology, 11(1).
27. 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.
28. 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.
29. Decan, N., Wu, D., Williams, A., Bernatchez, S., Johnston, M., Hill, M. and 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, pp.8-22.
30. Di Paolo, N. and Shayakhmetov, D. (2016). Interleukin 1α and the inflammatory process. Nature Immunology, 17(8), pp.906-913.
31. 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.
32. 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).
33. Rabolli, V., Badissi, A., Devosse, R., Uwambayinema, F., Yakoub, Y., Palmai-Pallag, M., Lebrun, A., De Gussem, V., Couillin, I., Ryffel, B., Marbaix, E., Lison, D. and Huaux, F. (2014). The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles. Particle and Fibre Toxicology, 11(1).
34. 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.
35. 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.

Domain of Applicability

Key Event Description

How this KE works

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 GM-CSF are involved in the adaptive immune responses. The pro-inflammatory cytokines include IL-1 family (IL-1a, IL-1b, IL-1ra, IL-18, IL-36a, IL-36b, IL-36g, IL-36Ra, IL-37), IL-6 family, TNF family, IL-17, and IFNg (Turner et al., 2014). While IL-4 and IL-5 are considered T helper (Th) cell type 2 response, IFNg 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, TNFa, IFNg 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).

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 CNT types and other fibrogenic 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 ROS (reactive oxygen species). Exposure to crystalline silica induces release of inflammatory cytokines (TNFa, IL-1, IL-6), transcription factors (NF-kb, AP-1) and kinase signalling pathways in mice that contain NFkB luciferase reporter (Hubbard et al., 2002). Boyles et al., 2015 found that lung responses to long MWCNTs included high expression levels of pro-inflammatory mediators MCP-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 NMs induce expression of cytokines and chemokines in lungs of rodents exposed via inhalation (Halappanavar et al., 2010; Husain et al., 2015). 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 proinflammatory mediators for investigation varies based on the expertise of the lab, cell type studied and availability of the specific antibodies.  

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.

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 T 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 flourophore 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.

ELISA assays – 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., 2015). 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 et al., 2009).

References

1. Alberts, D., Chen, H., Woolfenden, J., Moon, T., Chang, S., Hall, J., Himmelstein, K., Gross, J. and Salmon, S. (1979). Pharmacokinetics of bleomycin in man. Cancer Chemotherapy and Pharmacology, 3(1).
2. Brömme, D., Rossi, A., Smeekens, S., Anderson, D. and Payan, D. (1996). Human Bleomycin Hydrolase:  Molecular Cloning, Sequencing, Functional Expression, and Enzymatic Characterization. Biochemistry, 35(21), pp.6706-6714.
3. Canellos, G., Anderson, J., Propert, K., Nissen, N., Cooper, M., Henderson, E., Green, M., Gottlieb, A. and Peterson, B. (1992). Chemotherapy of Advanced Hodgkin's Disease with MOPP, ABVD, or MOPP Alternating with ABVD. New England Journal of Medicine, 327(21), pp.1478-1484.
4. Claussen, C. and Long, E. (1999). Nucleic Acid Recognition by Metal Complexes of Bleomycin. Chemical Reviews, 99(9), pp.2797-2816.
5. Forn-Cuní, G., Varela, M., Pereiro, P., Novoa, B. and Figueras, A. (2017). Conserved gene regulation during acute inflammation between zebrafish and mammals. Scientific Reports, 7(1).
6. Froudarakis, M., Hatzimichael, E., Kyriazopoulou, L., Lagos, K., Pappas, P., Tzakos, A., Karavasilis, V., Daliani, D., Papandreou, C. and Briasoulis, E. (2013). Revisiting bleomycin from pathophysiology to safe clinical use. Critical Reviews in Oncology/Hematology, 87(1), pp.90-100.
7. Hay, J., Shahzeidi, S. and Laurent, G. (1991). Mechanisms of bleomycin-induced lung damage. Archives of Toxicology, 65(2), pp.81-94.
8. Hubbard, A., Timblin, C., Shukla, A., Rincón, M. and Mossman, B. (2002). Activation of NF-κB-dependent gene expression by  silica in lungs of luciferase reporter mice. American Journal of Physiology-Lung Cellular and Molecular Physiology, 282(5), pp.L968-L975.
9. Ohnuma, T., Holland, J., Masuda, H., Waligunda, J. and Goldberg, G. (1974). Microbiological assay of bleomycin: Inactivation, tissue distribution, and clearance. Cancer, 33(5), pp.1230-1238.
10. Sleijfer, S. (2001). Bleomycin-Induced Pneumonitis. Chest, 120(2), pp.617-624.
11. Tashiro, J., Rubio, G., Limper, A., Williams, K., Elliot, S., Ninou, I., Aidinis, V., Tzouvelekis, A. and Glassberg, M. (2017). Exploring Animal Models That Resemble Idiopathic Pulmonary Fibrosis. Frontiers in Medicine, 4.
12. Twentyman, P. (1983). Bleomycin—mode of action with particular reference to the cell cycle. Pharmacology & Therapeutics, 23(3), pp.417-441.
13. Umezawa, H., Maeda, K., Takeuchi, T. and Okami, Y. (1966). NEW ANTIBIOTICS, BLEOMYCIN A AND B. The Journal of Antibiotics, XIX(5), pp.200-209.
14. Umezawa, H., Suhara, Y., Takita, T. and Maeda, K. (2019). PURIFICATION OF BLEOMYCINS. Journal of Antibiotics, XIX(5), pp.210-215.
15. Yu, Z., Schmaltz, R., Bozeman, T., Paul, R., Rishel, M., Tsosie, K. and Hecht, S. (2013). Selective Tumor Cell Targeting by the Disaccharide Moiety of Bleomycin. Journal of the American Chemical Society, 135(8), pp.2883-2886.

Key Event Description

Increased, recruitment of pro-inflammatory cells

How it works

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. 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 MA et al., 2011). Sensing or recognition of pathogens and harmful substances results in the recruitment of monocytes to lungs (Shi C and Pamer EG, 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, KE1 and KE2 act in a positive feedback loop mechanism and propagate the pro-inflammatory environment. All pro-fibrotic agents induce leukocyte infiltration in a dose and time-dependent manner.

Evidence for its perturbation

Macrophages acuumulate in bronchoalveolar fluid (BALF) post-exposure to fibrogenic bleomycin (Phan, 1980; Smith, 1995). NM-induced inflammation is predominantly neutrophilic (Shevedova, 2005; Rahman L, 2017; Rahman, 2017; Poulsen 2015). Increased number of pro-inflammatory cells (Zuo, 2002), neutrophils (Reynolds 1997) is observed in the BALF of IPF patients. 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, 1997). MWCNTs induce increased eosinophil count in lungs (Købler C, 2015). MWCNTs act as allergens and induce lung infiltration of eosinophils and cause airway hypersensitivity (Beamer, 2013).

It is important to note that the stressor-induced MIE, KE1 and KE2 are part of the functional changes that we collectively consider as inflammation, and together, they mark the initiation of acute inflammatory phase. MIE and KE1 occur at the cellular level. KE2 occurs at the tissue level.

How it is Measured or Detected

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 (BALF) is lavaged 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 TG 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 (qRT-PCR, ELISA, immunohistochemistry) in cell culture models, as indicative of recruitment of cells into the lungs. Details of in vitro methods are described under KE2.

References

1. Cho, W., Duffin, R., Poland, C., Howie, S., MacNee, W., Bradley, M., Megson, I. and Donaldson, K. (2010). Metal Oxide Nanoparticles Induce Unique Inflammatory Footprints in the Lung: Important Implications for Nanoparticle Testing. Environmental Health Perspectives, 118(12), pp.1699-1706.
2. Ingersoll, M., Platt, A., Potteaux, S. and Randolph, G. (2011). Monocyte trafficking in acute and chronic inflammation. Trends in Immunology, 32(10), pp.470-477.
3. Købler, C., Poulsen, S., Saber, A., Jacobsen, N., Wallin, H., Yauk, C., Halappanavar, S., Vogel, U., Qvortrup, K. and Mølhave, K. (2015). Time-Dependent Subcellular Distribution and Effects of Carbon Nanotubes in Lungs of Mice. PLOS ONE, 10(1), p.e0116481.
4. Kolaczkowska, E. and Kubes, P. (2013). Neutrophil recruitment and function in health and inflammation. Nature Reviews Immunology, 13(3), pp.159-175.
5. Kopf, M., Schneider, C. and Nobs, S. (2014). The development and function of lung-resident macrophages and dendritic cells. Nature Immunology, 16(1), pp.36-44.
6. Phan, S., Thrall, R. and Ward, P. (1980). Bleomycin-induced Pulmonary Fibrosis in Rats: Biochemical Demonstration of Increased Rate of Collagen Synthesis1,2. American Review of Respiratory Disease, 121(3), pp.501-506.
7. Poulsen, S., Saber, A., Williams, A., Andersen, O., Købler, C., Atluri, R., Pozzebon, M., Mucelli, S., Simion, M., Rickerby, D., Mortensen, A., Jackson, P., Kyjovska, Z., Mølhave, K., Jacobsen, N., Jensen, K., Yauk, C., Wallin, H., Halappanavar, S. and Vogel, U. (2015). MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. Toxicology and Applied Pharmacology, 284(1), pp.16-32.
8. Rahman, L., Jacobsen, N., Aziz, S., Wu, D., Williams, A., Yauk, C., White, P., Wallin, H., Vogel, U. and Halappanavar, S. (2017). Multi-walled carbon nanotube-induced genotoxic, inflammatory and pro-fibrotic responses in mice: Investigating the mechanisms of pulmonary carcinogenesis. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 823, pp.28-44.
9. Rahman, L., Wu, D., Johnston, M., Williams, A. and Halappanavar, S. (2016). Toxicogenomics analysis of mouse lung responses following exposure to titanium dioxide nanomaterials reveal their disease potential at high doses. Mutagenesis, 32(1), pp.59-76.
10. Reynolds, H., Fulmer, J., Kazmierowski, J., Roberts, W., Frank, M. and Crystal, R. (1977). Analysis of cellular and protein content of broncho-alveolar lavage fluid from patients with idiopathic pulmonary fibrosis and chronic hypersensitivity pneumonitis. Journal of Clinical Investigation, 59(1), pp.165-175.
11. Shi, C. and Pamer, E. (2011). Monocyte recruitment during infection and inflammation. Nature Reviews Immunology, 11(11), pp.762-774.
12. Shvedova, A., Kisin, E., Mercer, R., Murray, A., Johnson, V., Potapovich, A., Tyurina, Y., Gorelik, O., Arepalli, S., Schwegler-Berry, D., Hubbs, A., Antonini, J., Evans, D., Ku, B., Ramsey, D., Maynard, A., Kagan, V., Castranova, V. and Baron, P. (2005). Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. American Journal of Physiology-Lung Cellular and Molecular Physiology, 289(5), pp.L698-L708.
13. Smith, R., Stricter, R., Zhang, K., Phan, S., Standiford, T., Lukacs, N. and Kunkel, S. (1995). A role for C-C chemokines in fibrotic lung disease. Journal of Leukocyte Biology, 57(5), pp.782-787.

Domain of Applicability

Key Event Description

Loss of alveolar capillary membrane integrity

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 (Fakuda et al., 1987; Shwarz 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 D are few of the markers of decreasing lung compliance arising from the lost integrity of ACM (Johnson and Matthay, 2010).

KE3 associative event 1 - Chronic inflammation

In the presence of continuous stimulus (e.g., presence of biopersistent toxic fibres such as asbestos, MWCNTs) or following repeated stimulus (e.g., repeated exposure to silica or coal dust), the ensuing cell injury fuels the inflammatory mechanisms leading to accumulation of immune cells, prolonged inflammation and aggravated tissue damage. This sustained and perpetuated immunological response is termed as chronic inflammation. During this phase, active inflammation, tissue injury and destruction, and tissue repair processes proceed in tandem. Thus, the causative substance must contain unique physico-chemical properties that grant the material biopersistance in the pulmonary environment or the pulmonary system has to be repeatedly exposed to the same substance that perpetuates the tissue injury leading to loss of ACM. Although, increases in number of neutrophils are observed during chronic inflammation, mononuclear phagocytes (circulating monocytes, tissue macrophages) and lymphoid cells mark this phase. The macrophages, components of mononuclear phagocyte system, are the predominant cells in chronic inflammation. Activated macrophages release a variety of cytokines, chemokines, growth factors, which when uncontrolled, lead to extensive tissue injury, cellular death and necrosis, the other characteristics of chronic inflammation, leading to ACM loss. The other types of inflammatory cells found in chronic inflammation include eosinophils in allergen induced lung fibrosis, lymphocytes and epithelial cells.

KE3 associative event 2 - Oxidative stress  

Superoxide anion (O2-) and the hydroxyl radical (OH) are the common ROS found in the biological systems that are unstable due to unpaired electrons. As such, all tissues including lung have efficient antioxidant system to counteract the ROS induced oxidation. The antioxidant enzymes including superoxide dismutase, catalase and glutathione peroxidase act directly to inactivate ROS and associated reactions. In addition, phase II detoxifying enzymes, including glutathione-S-transferase, NADPH quinone oxidoreductase and glutamate-cysteine ligase catalytic act as indirect antioxidant enzymes. However, when the balance between oxidant and antioxidants is tipped towards oxidants, oxidative stress occurs. ROS, when interacted with biomolecules, initiate their oxidation. During the process, proteins, DNA and lipids are oxidized. Oxidative stress modulates the cellular signalling processes and contributes to oxidative stress-induced tissue injury. It also plays a role in the tissue injury caused by inflammatory processes in lungs. The infiltrating neutrophils and macrophages generate superoxide anion which is converted to hydrogen peroxide by superoxide dismutase enzyme. OH is formed by a secondary reaction in the presence of Fe2+. ROS can also be produced by NADPH oxidase present in phagocytes. The other enzyme that contributes to ROS synthesis during inflammatory processes is the myeloperoxidase from neutrophils.  In a self-perpetuating loop, inflammatory cells generate oxidative stress leading to increased airspace epithelial permeability, increased cell death and increased expression of pro-inflammatory genes, all of which lead to secretion of inflammatory cytokines/chemokines leading to prolonged and chronic inflammation.

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 pro-fibrotic drug bleomycin destroys structural architecture of the 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 expression (Almeida et al., 2013; Gabazza et al., 2004).

Epithelium and basement membrane injury is a prerequisite for the development of fibrotic lesion (Brody et al., 1981). Repeated exposure to or 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 IL-1 (Kolb et al., 2001), TNFa (Sime et al., 1998), T-helper type 2 cytokine IL-13 or exposure to specific proteinases initiate ACM injury, significant loss of the epithelium and endothelium of the ACM resulting in loss of the barrier. In patients diagnosed with idiopathic pulmonary fibrosis, both type 1 pneumocyte and endothelial cell injury with the ACM barrier loss is observed.

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

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

How it is Measured or Detected

Proteinosis, BAL fluid protein content

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

Cel 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, 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.

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 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 NMs 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.

The other methods include targeted RT-PCR or ELISA assays for tight junction proteins, cell adhesion molecules and inflammatory mediators such as IFNg, IL-10, and IL-13.   

References

1. Almeida, C., Nagarajan, D., Tian, J., Leal, S., Wheeler, K., Munley, M., Blackstock, W. and Zhao, W. (2013). The Role of Alveolar Epithelium in Radiation-Induced Lung Injury. PLoS ONE, 8(1), p.e53628.
2. Beamer, C., Girtsman, T., Seaver, B., Finsaas, K., Migliaccio, C., Perry, V., Rottman, J., Smith, D. and Holian, A. (2012). IL-33 mediates multi-walled carbon nanotube (MWCNT)-induced airway hyper-reactivity via the mobilization of innate helper cells in the lung. Nanotoxicology, 7(6), pp.1070-1081.
3. Brody, A., Soler, P., Basset, F., Haschek, W. and Witschi, H. (1981). Epithelial-Mesenchymal Associations of Cells in Human Pulmonary Fibrosis and in BHT-Oxygen-Induced Fibrosis in Mice. Experimental Lung Research, 2(3), pp.207-220.
4. Cassel, S., Eisenbarth, S., Iyer, S., Sadler, J., Colegio, O., Tephly, L., Carter, A., Rothman, P., Flavell, R. and Sutterwala, F. (2008). The Nalp3 inflammasome is essential for the development of silicosis. Proceedings of the National Academy of Sciences, 105(26), pp.9035-9040.
5. Chua, F., Gauldie, J. and Laurent, G. (2005). Pulmonary Fibrosis. American Journal of Respiratory Cell and Molecular Biology, 33(1), pp.9-13.
6. Costabel, U., Bonella, F. and Guzman, J. (2012). Chronic Hypersensitivity Pneumonitis. Clinics in Chest Medicine, 33(1), pp.151-163.
7. Dostert, C., Petrilli, V., Van Bruggen, R., Steele, C., Mossman, B. and Tschopp, J. (2008). Innate Immune Activation Through Nalp3 Inflammasome Sensing of Asbestos and Silica. Science, 320(5876), pp.674-677.
8. Ernst, H., Rittinghausen, S., Bartsch, W., Creutzenberg, O., Dasenbrock, C., Görlitz, B., Hecht, M., Kairies, U., Muhle, H., Müller, M., Heinrich, U. and Pott, F. (2002). 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). Experimental and Toxicologic Pathology, 54(2), pp.109-126.
9. Gabazza, E., Kasper, M., Ohta, K., Keane, M., D'Alessandro-Gabazza, C., Fujimoto, H., Nishii, Y., Nakahara, H., Takagi, T., Menon, A., Adachi, Y., Suzuki, K. and Taguchi, O. (2004). Decreased expression of aquaporin-5 in bleomycin-induced lung fibrosis in the mouse. Pathology International, 54(10), pp.774-780.
10. Ghio, A., Kadiiska, M., Xiang, Q. and Mason, R. (1998). In Vivo Evidence of Free Radical Formation After Asbestos Instillation. Free Radical Biology and Medicine, 24(1), pp.11-17.
11. Hamilton, R., Thakur, S. and Holian, A. (2008). Silica binding and toxicity in alveolar macrophages. Free Radical Biology and Medicine, 44(7), pp.1246-1258.
12. He, C., Murthy, S., McCormick, M., Spitz, D., Ryan, A. and Carter, A. (2011). Mitochondrial Cu,Zn-Superoxide Dismutase Mediates Pulmonary Fibrosis by Augmenting H₂O₂ Generation. Journal of Biological Chemistry, 286(17), pp.15597-15607.
13. Johnson, E. and Matthay, M. (2010). Acute Lung Injury: Epidemiology, Pathogenesis, and Treatment. Journal of Aerosol Medicine and Pulmonary Drug Delivery, 23(4), pp.243-252.
14. Kolb, M., Margetts, P., Anthony, D., Pitossi, F. and Gauldie, J. (2001). Transient expression of IL-1β induces acute lung injury and chronic repair leading to pulmonary fibrosis. Journal of Clinical Investigation, 107(12), pp.1529-1536.
15. Kulkarni, T., de Andrade, J., Zhou, Y., Luckhardt, T. and Thannickal, V. (2016). Alveolar epithelial disintegrity in pulmonary fibrosis. American Journal of Physiology-Lung Cellular and Molecular Physiology, 311(2), pp.L185-L191.
16. Lenz, A., Costabel, U. and Maier, K. (1996). Oxidized BAL fluid proteins in patients with interstitial lung diseases. European Respiratory Journal, 9(2), pp.307-312.
17. Miyoshi, K., Yanagi, S., Kawahara, K., Nishio, M., Tsubouchi, H., Imazu, Y., Koshida, R., Matsumoto, N., Taguchi, A., Yamashita, S., Suzuki, A. and Nakazato, M. (2013). Epithelial Pten Controls Acute Lung Injury and Fibrosis by Regulating Alveolar Epithelial Cell Integrity. American Journal of Respiratory and Critical Care Medicine, 187(3), pp.262-275.
18. Parra, E., Kairalla, R., Ribeiro de Carvalho, C., Eher, E. and Capelozzi, V. (2006). Inflammatory Cell Phenotyping of the Pulmonary Interstitium in Idiopathic Interstitial Pneumonia. Respiration, 74(2), pp.159-169.
19. Psathakis, K., Mermigkis, D., Papatheodorou, G., Loukides, S., Panagou, P., Polychronopoulos, V., Siafakas, N. and Bouros, D. (2006). Exhaled markers of oxidative stress in idiopathic pulmonary fibrosis. European Journal of Clinical Investigation, 36(5), pp.362-367.
20. Quinlan, T., BeruBe, K., Hacker, M., Taatjes, D., Timblin, C., Goldberg, J., Kimberley, P., O’Shaughnessy, P., Hemenway, D., Torino, J., Jimenez, L. and Mossman, B. (1998). Mechanisms of Asbestos-induced Nitric Oxide Production by Rat Alveolar Macrophages in Inhalation and in vitro Models. Free Radical Biology and Medicine, 24(5), pp.778-788.
21. Schamberger, A., Mise, N., Jia, J., Genoyer, E., Yildirim, A., Meiners, S. and Eickelberg, O. (2014). Cigarette Smoke–Induced Disruption of Bronchial Epithelial Tight Junctions Is Prevented by Transforming Growth Factor-β. American Journal of Respiratory Cell and Molecular Biology, 50(6), pp.1040-1052.
22. Schmekel, B., Bos, J., Khan, A., Wohlfart, B., Lachmann, B. and Wollmer, P. (1992). Integrity of the alveolar-capillary barrier and alveolar surfactant system in smokers. Thorax, 47(8), pp.603-608.
23. Schwartz, D., Helmers, R., Dayton, C., Merchant, R. and Hunninghake, G. (1991). Determinants of bronchoalveolar lavage cellularity in idiopathic pulmonary fibrosis. Journal of Applied Physiology, 71(5), pp.1688-1693.
24. Schwarz, M. (2001). Acute lung injury: cellular mechanisms and derangements. Paediatric Respiratory Reviews, 2(1), pp.3-9.
25. Shi, J., Karlsson, H., Johansson, K., Gogvadze, V., Xiao, L., Li, J., Burks, T., Garcia-Bennett, A., Uheida, A., Muhammed, M., Mathur, S., Morgenstern, R., Kagan, V. and Fadeel, B. (2012). Microsomal Glutathione Transferase 1 Protects Against Toxicity Induced by Silica Nanoparticles but Not by Zinc Oxide Nanoparticles. ACS Nano, 6(3), pp.1925-1938.
26. Shvedova, A., Kisin, E., Mercer, R., Murray, A., Johnson, V., Potapovich, A., Tyurina, Y., Gorelik, O., Arepalli, S., Schwegler-Berry, D., Hubbs, A., Antonini, J., Evans, D., Ku, B., Ramsey, D., Maynard, A., Kagan, V., Castranova, V. and Baron, P. (2005). Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. American Journal of Physiology-Lung Cellular and Molecular Physiology, 289(5), pp.L698-L708.
27. Shvedova, A., Kisin, E., Murray, A., Kommineni, C., Castranova, V., Fadeel, B. and Kagan, V. (2008). Increased accumulation of neutrophils and decreased fibrosis in the lung of NADPH oxidase-deficient C57BL/6 mice exposed to carbon nanotubes. Toxicology and Applied Pharmacology, 231(2), pp.235-240.
28. Sime, P., Marr, R., Gauldie, D., Xing, Z., Hewlett, B., Graham, F. and Gauldie, J. (1998). Transfer of Tumor Necrosis Factor-α to Rat Lung Induces Severe Pulmonary Inflammation and Patchy Interstitial Fibrogenesis with Induction of Transforming Growth Factor-β1 and Myofibroblasts. The American Journal of Pathology, 153(3), pp.825-832.
29. Thakur, S., Hamilton, R. and Holian, A. (2008). Role of Scavenger Receptor A Family in Lung Inflammation from Exposure to Environmental Particles. Journal of Immunotoxicology, 5(2), pp.151-157.
30. Tyurina, Y., Kisin, E., Murray, A., Tyurin, V., Kapralova, V., Sparvero, L., Amoscato, A., Samhan-Arias, A., Swedin, L., Lahesmaa, R., Fadeel, B., Shvedova, A. and Kagan, V. (2011). Global Phospholipidomics Analysis Reveals Selective Pulmonary Peroxidation Profiles upon Inhalation of Single-Walled Carbon Nanotubes. ACS Nano, 5(9), pp.7342-7353.
31. YASUOKA, S., NAKAYAMA, T., KAWANO, T., OGUSHI, F., DOI, H., HAYASHI, H. and TSUBURA, E. (1985). Comparison of cell profiles on bronchial and bronchoalveolar lavage fluids between normal subjects and patient with idiopathic pulmonary fibrosis. The Tohoku Journal of Experimental Medicine, 146(1), pp.33-45.

Key Event Description

How does this KE work

Naïve CD4+ T cells differentiate into four types of 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 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 IFNg and IL-12 induce inflammation, aid in clearance of toxic substances, induce tissue damage and control the fibrotic responses. IFNg 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.

KE4 associative event - Macrophage polarisation

Depending on the lung microenvironment (damaged cells, microbial products, activated lymphocytes), the precursor monocytes differentiate into distinct types of macrophages. Classically activated (M1) macrophages and alternatively activated (M2) macrophages are the important ones to consider in the context of this AOP. The M1 macrophages produce high levels of pro-inflammatory cytokines, mediate resistance to pathogens, induce generation of high levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and  T helper (Th) 1 type responses. M1 macrophages produce IL-1, IL-12, IL-23 and induce Th1 cell infiltration and activation. The M2 macrophages secrete anti-inflammatory mediators, by which they play a role in regulation of inflammation. The M2 polarisation is mediated by Th2 cytokines such as IL-4 and IL-13, which in turn, promotes M2 activation. M2 macrophages express immunosuppressive molecules such as IL-10, Arginase-1 and -2 (Arg-1, Arg-2), which suppress the induction of Th1 cells that produce the anti-fibrotic cytokine IFNg. The activity of M2 is associated with tissue remodelling, immune regulation, tumour promotion, tissue regeneration and effective phagocytic activity. During chronic inflammation, the phenotype of infiltrating macrophages is suggested to resemble that of M2, which is suggested to play a role in lung fibrosis.

Evidence for its perturbation

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 TGFb1 and initiates fibroblast proliferation and differentiation in lung fibrosis (Lee, 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, 1999). Both MWCNTs and SWCNTs induce elevated expression of IL-4 and IL-13 in BALF of mouse lungs (Park, 2011), and increased levels of IL-25 and IL-33 in BALF and mouse lungs exposed to MWCNTs (Dong, 2016). 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, 2016). Overexpression of IL-10 increases IL-4 and IL-13 production and lung fibrosis following exposure to silica (Barbarin, 2005). Alveolar macrophages from asbestosis patients (a form of lung fibrosis) exhibit M2 phenotype (Chao et al., 2013). Ex vivo culture of alveolar macrophages obtained from BALF of patients suffering from IPF with collagen type I showed enhanced levels of M2 macrophage markers CCL-18, CCL-2 and CD204 (Stahl, 2013). Th2 response associated expression of IL-33 cytokine enhances polarisation of M2 macrophages and inducing M2-mediated expression of IL-13 and TGFb1 in mice (Dong, 2014). Cigarette smoke induces expression of genes associated with M2 sub-phenotypes, which is further enhanced in smokers presenting with COPD (Shaykhiev, 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, IFNg. In addition, the levels of Transforming growth factor b (TGFb) 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 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 MIE. The details of BALF sample collection is described under KE2.

References

1. Barbarin, V., Xing, Z., Delos, M., Lison, D. and Huaux, F. (2005). Pulmonary overexpression of IL-10 augments lung fibrosis and Th2 responses induced by silica particles. American Journal of Physiology-Lung Cellular and Molecular Physiology, 288(5), pp.L841-L848.
2. Dong, J. and Ma, Q. (2016). In vivo activation of a T helper 2-driven innate immune response in lung fibrosis induced by multi-walled carbon nanotubes. Archives of Toxicology, 90(9), pp.2231-2248.
3. Fatkhutdinova, L., Khaliullin, T., Vasil'yeva, O., Zalyalov, R., Mustafin, I., Kisin, E., Birch, M., Yanamala, N. and Shvedova, A. (2016). Fibrosis biomarkers in workers exposed to MWCNTs. Toxicology and Applied Pharmacology, 299, pp.125-131.
4. He, C., Ryan, A., Murthy, S. and Carter, A. (2013). Accelerated Development of Pulmonary Fibrosis via Cu,Zn-superoxide Dismutase-induced Alternative Activation of Macrophages. Journal of Biological Chemistry, 288(28), pp.20745-20757.
5. Huaux, F., Liu, T., McGarry, B., Ullenbruch, M., Xing, Z. and Phan, S. (2003). Eosinophils and T Lymphocytes Possess Distinct Roles in Bleomycin-Induced Lung Injury and Fibrosis. The Journal of Immunology, 171(10), pp.5470-5481.
6. Lee, C., Homer, R., Zhu, Z., Lanone, S., Wang, X., Koteliansky, V., Shipley, J., Gotwals, P., Noble, P., Chen, Q., Senior, R. and Elias, J. (2001). Interleukin-13 Induces Tissue Fibrosis by Selectively Stimulating and Activating Transforming Growth Factor β1. The Journal of Experimental Medicine, 194(6), pp.809-822.
7. Li, D., Guabiraba, R., Besnard, A., Komai-Koma, M., Jabir, M., Zhang, L., Graham, G., Kurowska-Stolarska, M., Liew, F., McSharry, C. and Xu, D. (2014). IL-33 promotes ST2-dependent lung fibrosis by the induction of alternatively activated macrophages and innate lymphoid cells in mice. Journal of Allergy and Clinical Immunology, 134(6), pp.1422-1432.e11.
8. Park, E., Roh, J., Kim, S., Kang, M., Han, Y., Kim, Y., Hong, J. and Choi, K. (2011). A single intratracheal instillation of single-walled carbon nanotubes induced early lung fibrosis and subchronic tissue damage in mice. Archives of Toxicology, 85(9), pp.1121-1131.
9. Shaykhiev, R., Krause, A., Salit, J., Strulovici-Barel, Y., Harvey, B., O'Connor, T. and Crystal, R. (2009). Smoking-Dependent Reprogramming of Alveolar Macrophage Polarization: Implication for Pathogenesis of Chronic Obstructive Pulmonary Disease. The Journal of Immunology, 183(4), pp.2867-2883.
10. Stahl, M., Schupp, J., Jäger, B., Schmid, M., Zissel, G., Müller-Quernheim, J. and Prasse, A. (2013). Lung Collagens Perpetuate Pulmonary Fibrosis via CD204 and M2 Macrophage Activation. PLoS ONE, 8(11), p.e81382.
11. Tao, B., Jin, W., Xu, J., Liang, Z., Yao, J., Zhang, Y., Wang, K., Cheng, H., Zhang, X. and Ke, Y. (2014). Myeloid-Specific Disruption of Tyrosine Phosphatase Shp2 Promotes Alternative Activation of Macrophages and Predisposes Mice to Pulmonary Fibrosis. The Journal of Immunology, 193(6), pp.2801-2811.
12. Zhu, Z., Homer, R., Wang, Z., Chen, Q., Geba, G., Wang, J., Zhang, Y. and Elias, J. (1999). Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. Journal of Clinical Investigation, 103(6), pp.779-788.

Domain of 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 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 a-smooth muscle actin 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 (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 CCR2, CXCR4 and CCR7 in addition to a characteristic pattern of biomarkers, including collagen I and III, CD34, CD43 and CD45 (Bucala, 1994; Chesney et al., 1998; Abe et al., 2001).  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 CXCL12 (which is the only chemokine known to bind to CXCR4) (Phillip 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.

Evidence for its perturbation

IPF is characterised by progressive fibroblast and myofibroblast proliferation and excessive deposition of extracellular matrix (Kuhn et al., 1991). High levels of a-SMA protein and increased number of a-SMA positive cells were observed in mouse lungs treated with MWCNTs as early as day 1 post-exposure (Dong et al., 2015). Fibrotic lesions observed in mice treated with asbestos show proliferating fibroblasts and collagen deposition. The same study also demonstrated that BALF 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 a-SMA expressing myofibroblasts (Vyalov et al., 1993). Several in vitro studies have shown fibroblast proliferation following CNT treatment (Wang et al., 2010; Wang et al., 2010; Hussain et al., 2014).

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 alpha-smooth muscle actin, specific markers of fibroblasts and myofibroblasts (Kai, 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 semi-quantitative, 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 mm²) 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 alpha smooth muscle 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, cyclooxygenase-2 and vaeolin-1 are used as markers of homogeneous population of fibroblasts. Increased expression of TGF-b and a-smooth muscle actin is used as markers of differentiated myofibroblasts. Transcription factor 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 epithelial-mesenchymal transition (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 extracellular matrix (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 (PI) 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 (Vietti et al., 2013; Azad et al., 2013) to the same material.

 

References

1. Abe, R., Donnelly, S., Peng, T., Bucala, R. and Metz, C. (2001). Peripheral Blood Fibrocytes: Differentiation Pathway and Migration to Wound Sites. The Journal of Immunology, 166(12), pp.7556-7562.
2. Azad N, Iyer A.K.V., Lu Y., Wang L., Rojanasakul Y. (2013). P38/MAPK Regulates Single-walled Carbon Nanotube-Induced Fibroblast Proliferation and Collagen Production. Nanotoxicology, 7(2), 157–168.
3. Bucala, R., Spiegel, L., Chesney, J., Hogan, M. and Cerami, A. (1994). Circulating Fibrocytes Define a New Leukocyte Subpopulation That Mediates Tissue Repair. Molecular Medicine, 1(1), pp.71-81.
4. Chesney J, Metz C, Stavitsky A-B, et al. (1998) Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes. J Immunol 160:419–425.
5. Dong, J., Porter, D., Batteli, L., Wolfarth, M., Richardson, D. and Ma, Q. (2014). Pathologic and molecular profiling of rapid-onset fibrosis and inflammation induced by multi-walled carbon nanotubes. Archives of Toxicology, 89(4), pp.621-633.
6. Hashimoto, S., Gon, Y., Takeshita, I., Maruoka, S. and Horie, T. (2001). IL-4 and IL-13 induce myofibroblastic phenotype of human lung fibroblasts through c-Jun NH2-terminal kinase–dependent pathway. Journal of Allergy and Clinical Immunology, 107(6), pp.1001-1008.
7. Hussain, S., Sangtian, S., Anderson, S., Snyder, R., Marshburn, J., Rice, A., Bonner, J. and Garantziotis, S. (2014). Inflammasome activation in airway epithelial cells after multi-walled carbon nanotube exposure mediates a profibrotic response in lung fibroblasts. Particle and Fibre Toxicology, 11(1), p.28.
8. Kim, K., Kugler, M., Wolters, P., Robillard, L., Galvez, M., Brumwell, A., Sheppard, D. and Chapman, H. (2006). Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proceedings of the National Academy of Sciences, 103(35), pp.13180-13185.
9. Lemaire I, Beaudoin H, Massé S, Grondin C. (1986). Alveolar macrophage stimulation of lung fibroblast growth in asbestos-induced pulmonary fibrosis. Am J Pathol. 122(2):205–211.
10. Phillips, R., Burdick, M., Hong, K., Lutz, M., Murray, L., Xue, Y., Belperio, J., Keane, M. and Strieter, R. (2004). Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. Journal of Clinical Investigation, 114(3), pp.438-446.
11. Serini, G. and Gabbiani, G. (1999). Mechanisms of Myofibroblast Activity and Phenotypic Modulation. Experimental Cell Research, 250(2), pp.273-283.
12. Smithmyer, M., Sawicki, L. and Kloxin, A. (2014). Hydrogel scaffolds asin vitromodels to study fibroblast activation in wound healing and disease. Biomater. Sci., 2(5), pp.634-650.
13. Tamminen, J., Myllärniemi, M., Hyytiäinen, M., Keski-Oja, J. and Koli, K. (2012). Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling. Journal of Cellular Biochemistry, 113(7), pp.2234-2247.
14. Vietti, G., Ibouraadaten, S., Palmai-Pallag, M., Yakoub, Y., Bailly, C., Fenoglio, I., Marbaix, E., Lison, D. and van den Brule, S. (2013). Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay. Particle and Fibre Toxicology, 10(1), p.52.
15. Vyalov SL, Gabbiani G, Kapanci Y. (1993). Rat alveolar myofibroblasts acquire alpha-smooth muscle actin expression during bleomycin-induced pulmonary fibrosis. Am J Pathol. 143(6):1754–1765.
16. Wang, L., Mercer, R., Rojanasakul, Y., Qiu, A., Lu, Y., Scabilloni, J., Wu, N. and Castranova, V. (2010). Direct Fibrogenic Effects of Dispersed Single-Walled Carbon Nanotubes on Human Lung Fibroblasts. Journal of Toxicology and Environmental Health, Part A, 73(5-6), pp.410-422.
17. Wang, X., Xia, T., Ntim, S., Ji, Z., George, S., Meng, H., Zhang, H., Castranova, V., Mitra, S. and Nel, A. (2010). Quantitative Techniques for Assessing and Controlling the Dispersion and Biological Effects of Multiwalled Carbon Nanotubes in Mammalian Tissue Culture Cells. ACS Nano, 4(12), pp.7241-7252.
18. Zhang K. (1994). Myofibroblasts and Their Role in Lung Collagen Gene Expression during Pulmonary Fibrosis. American Journal of Pathology, Vol. 145, No. 1.

Key Event Description

ECM is a macromolecular structure that provides physical support to tissues and is essential for organ function. The composition of ECM is tissue specific and consists mainly of fibrous proteins, glycoproteins, and proteoglycans. The ECM in lung is compartmentalised to basement membrane and the interstitial space. Fibroblasts found in the interstitial space are the main sources of ECM in lung (White, 2015). Altered composition of ECM is observed in several lung diseases of inflammatory origin in humans including Chronic Obstructive Pulmonary Disease, asthma and idiopathic lung fibrosis. The composition and architecture of the ECM determines 1) the open sites of attachment that are available to cells, 2) the mechanical properties of the ECM and 3) the mechanical loading (breathing) experienced by the cells. Thus, changes in the ECM composition during the exaggerated wound healing process determines if an organism commits to fibrotic process or completes the wound healing (Blaubouer et al., 2014).

In lung fibrosis, an exaggerated amount of ECM is distributed in the alveolar parenchyma in a non-heterogenous manner, leading to lower spirometry readings implying occlusion of alveolar regions and reduced gas exchange. Collagen 1 and Collagen III are suggested to be the main components of the ECM in the thickened alveolar septa in fibrosis with other constituents such as fibronectin, elastin and tenacin C (Zhang et al., 1994; Hinz, 2006; Kuhn and McDonald, 1991; Crabb et al., 2006; Bensadoun, 1996; Klingberg et al., 2013; McKleroy et al., 2013). It is suggested that ECM composition dramatically changes during the fibrotic process. The early fibrotic process is characterised by collagen III deposition and collagen 1 predominates the later stages of the fibrosis. Excessive collagen production by myofibroblasts is necessary for the development of fibrosis (scarred tissue), with established areas of scar formation containing almost exclusively Type I collagen (Bateman et al., 1981; McKleroy et al., 2013; Zhang et al., 1994). Studies have demonstrated that while total collagen increases in IPF, there is also a shift toward the less elastic type I collagen, which contributes to the stiffness of the scar tissue within the lung (Nimni, 1983; Rozin et al., 2005; McKleroy, Lee and Atabai, 2013).

The fibrotic ECM contains characteristic accumulation of fibroblasts and myofibroblasts, which are the major contributors of ECM synthesised. The proliferation of fibroblasts and their differentiation into myofibroblasts is, in turn, guided by the composition and structure of the ECM. For example, Studies have demonstrated that cytokines secreted in response to inflammation are capable of activating fibroblasts, and that these changes could cause alterations in the fibroblasts that lead to excessive proliferation and ECM deposition (Sivakumar, 2012; Wynn, T.A., 2011).

 

How it is Measured or Detected

How it is measured

The qRT-PCR, ELISA, and immunohistochemistry are routinely used to assess the levels of protein and mRNA levels. The various genes and proteins that are assessed include, collagen I, collagen III, elastin and tenacin C. 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^TM 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).

References

1. Bateman, E., Turner-Warwick, M. and Adelmann-Grill, B. (1981). Immunohistochemical study of collagen types in human foetal lung and fibrotic lung disease. Thorax, 36(9), pp.645-653.
2. Bensadoun, E., Burke, A., Hogg, J. and Roberts, C. (1996). Proteoglycan deposition in pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine, 154(6), pp.1819-1828.
3. Chen, C. and Raghunath, M. (2009). Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis _ state of the art. Fibrogenesis & Tissue Repair, 2(1).
4. Crabb, R., Chau, E., Decoteau, D. and Hubel, A. (2006). Microstructural Characteristics of Extracellular Matrix Produced by Stromal Fibroblasts. Annals of Biomedical Engineering, 34(10), pp.1615-1627.
5. HINZ, B. (2006). Masters and servants of the force: The role of matrix adhesions in myofibroblast force perception and transmission. European Journal of Cell Biology, 85(3-4), pp.175-181.
6. 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;138(5):1257–1265.
7. Klingberg, F., Hinz, B. and White, E. (2012). The myofibroblast matrix: implications for tissue repair and fibrosis. The Journal of Pathology, 229(2), pp.298-309.
8. McKleroy, W., Lee, T. and Atabai, K. (2013). Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis. American Journal of Physiology-Lung Cellular and Molecular Physiology, 304(11), pp.L709-L721.
9. 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).
10. Nimni, M. (1983). Collagen: Structure, function, and metabolism in normal and fibrotic tissues. Seminars in Arthritis and Rheumatism, 13(1), pp.1-86.
11. Rozin, G., Gomes, M., Parra, E., Kairalla, R., de Carvalho, C. and Capelozzi, V. (2005). Collagen and elastic system in the remodelling process of major types of idiopathic interstitial pneumonias (IIP). Histopathology, 46(4), pp.413-421.
12. Sivakumar, P., Ntolios, P., Jenkins, G. and Laurent, G. (2012). Into the matrix. Current Opinion in Pulmonary Medicine, 18(5), pp.462-469.
13. White, E. (2015). Lung Extracellular Matrix and Fibroblast Function. Annals of the American Thoracic Society, 12(Supplement 1), pp.S30-S33.
14. Wynn, T. (2011). Integrating mechanisms of pulmonary fibrosis. The Journal of Experimental Medicine, 208(7), pp.1339-1350.
15. 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;145(1):114–125.

Evidence for Perturbation by Stressor

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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). 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.

1. 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.
2. Julie Muller, Franc¸ois Huaux, Nicolas Moreau, Pierre Misson, Jean-Francois Heilier,Monique Delos, Mohammed Arras, Antonio Fonseca, Janos B. Nagy, Dominique Lison Respiratory toxicity of multi-wall carbon nanotubes. Toxicology and Applied Pharmacology 207 (2005) 221–231.
3. Dale W. Porter, Ann F. Hubbs, Robert R. Mercer, Nianqiang Wu, Michael G. Wolfarth, Krishnan Sriram, Stephen Leonard, Lori Battelli, Diane Schwegler-Berry, Sherry Friend, Michael Andrew, Bean T. Chen, Shuji Tsuruoka, Morinobu Endo, Vincent Castranova, Mouse pulmonary dose- and time course-responses induced by exposure to multi-walled carbon nanotubes. Toxicology, Volume 269, Issues 2–3, 2010, Pages 136-147.
4. NIOSH: Occupational exposure to carbon nanotubes and nanofibers: current intelligence bulletin 65. 2013.

 

Domain of Applicability

Key Event Description

Lung/pulmonary fibrosis – thickened alveolar septa

Fibrosis or scarring is a fixed end result of damage in a tissue not capable of regeneration with no possibility of restoring the original tissue architecture. In normal lung, an individual alveolus spans about 0.2mm in diameter and there are 300 million alveoli in an adult male lung. 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, where gas exchange takes place, is made up of the alveolar epithelium and alveolar endothelium (Gracey, 1968). In fibrotic disease however, a pronounced decrease in the number of capillaries within the alveolar septa is found 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 alveolar capillary membrane is occupied with collagen and cells.

 

How it is Measured or Detected

How it is measured

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, 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. qRT-PCR and ELISA assays measuring increased collagen, TGFβ1 and various pro-inflammatory mediators are used as sensitive markers of potential of substances to induce the adverse outcome of lung fibrosis.

 

References

1. Gracey DR, Divertie MB and Brown Jr. AL. Alveolar-Capillary Membrane in Idiopathic Interstitial Pulmonary Fibrosis. 1968. American Review of Respiratory Disease, 98(1), pp. 16–21.
2. Ashcroft, T., J.M. Simpson, and V. Timbrell. 1988. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J. Clin. Pathol. 41:467-470.
3. Ralf-Harto Hübner, Wolfram Gitter, Nour Eddine El Mokhtari, Micaela Mathiak, Marcus Both,Hendrik Bolte, Sandra Freitag-Wolf, and Burkhard Bewig. 2008. BioTechniques 44:507-517.
4. 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.

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List of Adjacent Key Event Relationships