SNAPSHOT

Evidence for Perturbation by Stressor

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Overview for Molecular Initiating Event

Evidence for MIE perturbation

Not many studies investigate the exact mode of interaction between the toxic substance and the cellular membrane components. Rather, the consequences of such interaction such as, the release of intracellular contents (alarmins), increases in genes or protein synthesis downstream of receptor binding or cellular uptake are measured as indicative of occurrence of such interactions (Nel AE et al., 2009; Cheng L-C et al., 2013). Because of the physical –chemical properties such as surface charge, ENMs 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 2011 a; Pascolo L et al., 2013). Intratracheally instilled crystalline silica interacts with lung epithelial cells and alveolar macrophages and induces pro-inflammatory cascade via NF-κb signaling pathway (Hubbard AK, 2002).

Exposure to MWCNTs results in increased IL-1a protein levels in the bronchoalveolar lavage fluid (BALF) of lungs in mice (Nikota J et al., 2017) and lung epithelial cells exposed to silica show lysosomal accumulation of silica in a size-dependent manner (Decan N et al., 2016).

Key Event Description

Background

The human lung with the surface area of ~100 m² is ventilated by 10-20,000 litres of air per day. Thus, it is the largest organ of the human body that is exposed to the environment contaminated with pathogens, organic and inorganic materials. The gas exchange between blood and the inhaled air takes place across a membrane that is only 1-2 µm thick, making the lung an easy target for foreign invasion and toxic chemicals in the environment. To combat the constant attack by the pathogens and chemicals, the lung is equipped with several defence mechanisms; a layer of epithelial cells acting as physical barrier to limit entry of pathogens and specialised cells of the immune system with defence functions (reviewed in Weitnauer M et al., 2016). The human lung consists of approximately 40 different resident cell types that play different roles during homeostasis, injury, repair, and disease states (Franks TJ et al., 2008). Of these cells, resident airway epithelial cells, alveolar 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.

Receptor-mediated interactions

The chemicals or pathogens interact with cellular membrane to gain access to the organisms’ interior. This interaction between the pathogens/substances and lung cells can occur via binding of the pathogens or pathogen associated molecular patterns (PAMPs, microbial molecules) to the receptors present on the surfaces of the resident cells. For example, the airway epithelium and the mucosal layer form a physical barrier and contribute to the first line of defence. The resident epithelial cells, by default, under the normal homeostatic conditions, are adjusted to the local microbial burden and therefore, are less responsive to microbial stimulation. The resident epithelial cell surfaces express receptors that recognise and sense the presence of pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) resulting in epithelial cell activation. PRRs are also present on other resident cells including alveolar macrophages. PRRs can be activated by interaction with debris from dying cells (cellular fragments) as a result of interaction with toxic substances. Engagement of PRRs and consequent cell activation is observed in various organisms including flies and mammals (Matzinger, 2002).

Opsonisation driven interactions

In case of non-pathogens such as insoluble particles or engineered nanomaterials (ENMs), interaction with cellular membrane can occur via the process of opsonisation. For example, the surfaces of ENMs can be adsorbed with opsonins (protein corona) such as immunoglobulins, complement proteins, or serum proteins that are then recognised by the phagocytes (macrophages). Opsonised ENMs attach to the cellular membrane of phagocytes via ligand-receptor interactions. Some of the receptors on the cell surface that are engaged by the opsonins include Fc receptors and complement protein receptors (reviewed in Behzadi S et al., 2017). The endocytosis processes such as phagocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, and micropinocytosis define the interactions between cell membrane and the particles (airborne or ENMs).

Physical or mechanical interactions

The other type of interaction can involve physical damage to cells. High aspect ratio (HAR) materials such as asbestos or CNTs can pierce the cellular membrane of epithelial cells or resident macrophages resulting in cell injury or non-programmed cellular death. The resident macrophages are present in all tissues, and in a steady state, 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. Resident macrophages in the lung, bone, brain, liver, spleen, skin, and in the intestine are known as alveolar macrophages, osteoclasts, microglia, Kupffer cells, splenic macrophages, Langerhans cells and intestinal macrophages, respectively (Kierdorf K et al., 2015). It has been shown that alveolar macrophages trying to engulf HARs including asbestos and CNTs that are long and stiff undergo frustrated phagocytosis because of their inability to engulf the piercing fibres and subsequently lead to cell injury (Mossman BT et al., 1998; Donaldson K et al., 2010).

 

 

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 or activation of TLR receptors - indicative of binding of PAMPs to 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. DAMPs, also called as alarmins are endogenous molecules that are released by stressed cells. A few of the putative alarmins that can be effectively measured in biological samples including cultured cells are High Mobility Group Binding 1 (HMGB1) protein, Heat Shock proteins (HSPs), uric acid, annexins, S100 proteins and IL-1α (Bianchi ME, 2007).

Of all, IL-1a is the most commonly measured alarmin. IL-1a is the principal pro-inflammatory moiety and is a designated ‘alarmin’ in the cell that alerts the host to injury or damage (Di Paolo NC, 2016). It is shown that administration of necrotic cells to mice results in neutrophilic inflammation that was entirely mediated by IL-1a released from the dying or necrosed cells and consequent activation of IL-1 Receptor 1 (IL-1R1) signalling (Suwaraa MI et al., 2014). IL-1a is released following exposure to MWCNTs (Nikota J et al., 2017) and silica (Rabolli V et al., 2014). IL-1a can be cleaved from its precursor form to the active form; however, it is not a prerequisite since both precursor and cleaved forms of IL-1a are active that bind and activate IL-1R1 signaling.

The release of DAMPs can be measured by the techniques listed below.

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 D et al., 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 is activated or secreted into the cytosol following stimulus. Targeted ELISA for IL-1a can be used to quantify IL-1a 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.

Measuring cellular uptake of ENMs

Lysosomal localisation – In vitro, interaction of ENMs with the cellular membrane can be investigated by assessing their uptake by the lysosomes (Varela JA 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 N et al., 2016) or confocal microscopy to visualise co-localisation of fluorescence labelled nanoparticles with lysosomal markers have been used.

References

1. Weitnauer M, Mijosek V, Dalpke AH. Control of local immunity by airway epithelial cells. Mucosal Immunology (2016) 9, 287–29.
2. Franks TJ, Colby TV, Travis WD, Tuder RM, ....Williams MC. Resident cellular components of the human lung: current knowledge and goals for research on cell phenotyping and function.  Proc Am Thorac SocBeh.. 2008 Sep 15;5(7):763-6.
3. Polly Matzinger. The Danger Model: A Renewed Sense of Self Science 2002 Apr 12;296(5566):301-5.
4. Behzadi S, Serpooshan V, Tao W, Hamaly MA, ....Mahmoudi M. Cellular uptake of nanoparticles: journey inside the cell. Chem. Soc. Rev., 2017 Jul 17;46(14):4218-4244
5. Kierdorf K, Prinz M, Geissmann F, Perdiguero EG. Development and function of tissue resident macrophages in mice. Seminars in immunology. 2015;27(6):369-378.
6. Ken Donaldson, Fiona A Murphy, Rodger Duffin, Craig A Poland. Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma. Particle and Fibre Toxicology 2010, 7:5
7. Brooke T. Mossman and Andrew Churg. Mechanisms in the Pathogenesis of Asbestosis and Silicosis. Am J Respir Crit Care Med. 1998 Vol 157. pp 1666–1680.

8. Marco E. Bianchi. DAMPs, PAMPs and alarmins: all we need to know about danger. Journal of Leukocyte Biology 2007 Jan;81(1):1-5.

9. Nelson C Di Paolo & Dmitry M Shayakhmetov. Interleukin 1α and the inflammatory process. Nat Immunol. 2016 Jul 19;17(8):906-13.

10. Suwaraa MI, Greena NJ, Borthwicka LA, ..... and DA mann. IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts. Mucosal Immunology (2014) 7, 684–693.

11. Nikota J, Banville A, .......and 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; 14: 37.

12. Rabolli V, Badissi AA, Devosse R, .......and Huaux F. The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles. Part Fibre Toxicol. 2014; 11: 69.

13. Amsen D, de Visser KE, Town T. Approaches to Determine Expression of Inflammatory Cytokines. Methods in molecular biology (Clifton, NJ). 2009;511:107-142.

14. Juan A Varela, Mariana G Bexiga, Christoffer Åberg, Jeremy C Simpson and Kenneth A Dawson. Quantifying size-dependent interactions between fluorescently labeled polystyrene nanoparticles and mammalian cells. Journal of Nanobiotechnology 2012, 10:39.

15. Nathalie Decan, Dongmei Wu, Andrew Williams, Stéphane Bernatchez, Michael Johnston, Myriam Hill, Sabina Halappanavar, Characterization of in vitro genotoxic, cytotoxic and transcriptomic responses following exposures to amorphous silica of different sizes, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Volume 796, 2016, Pages 8-22.

16. Nel AE, …Klaessiga F, Castranova V and Thompson M. Understanding biophysicochemical interactions at the nano–bio interface. Nature Materials 8, 543–557 (2009).

17. Liang-Chien Cheng, Xiumei Jiang, Jing Wang, Chunying Chen and Ru-Shi Liu. Nano–bio effects: interaction of nanomaterials with cells. Nanoscale, 2013, 5, 3547-3569.

18. National Institute of Occupational Safety and Health. 2011a. Current Intelligence Bulletin 62. Asbestos fibers and other elongate mineral particles: state of the science and roadmap for research. Department of Health and Human Services, Centers for Disease Control and Prevention, DHHS (NIOSH) Publication Number 2011-159.　

19. Lorella Pascolo, Alessandra Gianoncelli, Giulia Schneider, Murielle Salome, Manuela Schneider,Carla Calligaro, Maya Kiskinova, Mauro Melato & Clara Rizzardi. The interaction of asbestos and iron in lung tissue revealed by synchrotron basedscanning X-ray microscopy. Scientific Reports 3, 2013. Article number: 1123.

20. Hubbard, Andrea K., Cynthia R. Timblin, Arti Shukla, Mercedes Rinco´n, and Brooke T. Mossman. Activation of NF-_B-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol 282: L968–L975, 2002; 10.1152.

21. Nikota J, Banville A, .......and 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; 14: 37.

22. Rabolli V, Badissi A, Devosse R, .......and Huaux F. The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles. Part Fibre Toxicol. 2014; 11: 69.

     

Key Event Description

Proinflammatory mediators are the chemical and biological molecules that initiate and regulate inflammatory reactions. Cell-derived proinflammatory mediators include cytokines, chemokines, and growth factors. Blood derived proinflammatory 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 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. Some of the proinflammatory 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 MD 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 proinflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas J et al., 2004). However, IL-1 family cytokines, IL-4, IL-5, IL-6, TNFa, IFNg are the commonly measured mediators in experimental animal models and in humans. Proinflammatory mediators are secreted following exposure to an inflammogen in a gender/sex or developmental stage independent manner.

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. 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 AK, 2002). Long and thin CNTs (>5 µm) can elicit asbestos-like pathogenicity through the continual release of pro-inflammatory cytokines and ROS (Poland CA, 2008). Lung responses to long MWCNTs included high expression levels of MCP-1, TGF-β1, and TNF-α (Boyles MSP, 2015). In vitro, CNTs have been shown to induce increased secretion of cytokines and chemokines (He X, 2011; Herano S, 2010).

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.

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 proinflammatory mediators in several species including mouse, rat and humans, are readily available commercially.

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 D et al., 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.

Both ELISA and qRT-PCR assays 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 M 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 proinflammatory mediators and its downstream effectors in any given tissue (Costa PM 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 D et al., 2009).

References

23. Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochimica et Biophysica Acta 1843 (2014) 2563–2582.
24. Javier Mestas and Christopher C. W. Hughes. Of mice and not men: differences between mouse and human immunology. J Immunol 2004; 172:2731-2738.
25. Hubbard, Andrea K., Cynthia R. Timblin, Arti Shukla, Mercedes Rinco´n, and Brooke T. Mossman. Activation of NF-_B-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol 282: L968–L975, 2002; 10.1152
26. Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, et al. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 2008; 3:423–428.
27. Matthew S.P. Boyles, Lesley Young, David M. Brown, Laura MacCalman, Hilary Cowie, Anna Moisala, Fiona Smail, Paula J.W. Smith, Lorna Proudfoot, Alan H. Windle, Vicki Stone, Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos, In Toxicology in Vitro, Volume 29, Issue 7, 2015, Pages 1513-1528.
28. He X, Young SH, Schwegler-Berry D, Chisholm WP, Fernback JE, Ma Q. Multiwalled carbon nanotubes induce a fibrogenic response by stimulating reactive oxygen species production, activating NF-kappaB signaling, and promoting fibroblast-to-myofibroblast transformation. Chem Res Toxicol. 2011;24:2237–2248.
29. Seishiro Hirano, Yuji Fujitani, Akiko Furuyama, Sanae Kanno, Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells, In Toxicology and Applied Pharmacology, Volume 249, Issue 1, 2010, Pages 8-15.
30. Tania Nolan, Rebecca E Hands & Stephen A Bustin. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1(3):1559-82.
31. Derk Amsen, Karin E. de Visser, and Terrence Town. Approaches to determine expression of inflammatory cytokines. Methods Mol Biol. 2009; 511: 107–142.
32. Mainul Husain, Dongmei Wu, Anne T. Saber, Nathalie Decan, Nicklas R. Jacobsen, Andrew Williams, Carole L. Yauk, Hakan Wallin, Ulla Vogel and Sabina Halappanavar. Intratracheally Instilled Titanium Dioxide Nanoparticles Translocate to Heart and Liver, and Activate Complement Cascade in the Heart of C57BL/6 Mice. Nanotoxicology. 2015;9(8):1013-22.
33. Costa PM, Gosens I, Williams A, et al. Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles. J Appl Toxicol. 2017;1–13.

Key Event Description

Proinflammatory cells originate in bone marrow and are recruited to the site of infection or injury via circulation following specific proinflammatory mediator (cytokine and chemokine) signalling. Proinflammatory 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).

Dendritic cells are antigen-presenting cells (APCs) and they stimulate naïve T-cell proliferation. They are found in the airway epithelium, the alveolar septa, pulmonary capillaries and airway spaces. APCs identify, ingest and process an antigen and present the antigen to resident T-cells in the lymph node initiating the immune response (Kopf M et al., 2015).

Macrophages are the components of mononuclear phagocyte system; they phagocytose pathogens, nanoparticles and other cellular debris. Mononuclear phagocytes originate in bone marrow as blood monocytes, which migrate to different tissues where they differentiate into tissue macrophages. At least three types of macrophages exist in lung: bronchial macrophages, interstitial macrophages and alveolar macrophages. They are found in the air space of the alveoli, parenchymal space (interstitial space) between adjacent alveoli. Lung macrophages can be characterised by their expression of surface markers. Activated macrophages release a variety of cytokines, chemokines, and growth factors (Kopf M et al., 2015).

Neutrophils are a type of polymorphonuclear leukocytes that are among the first to be recruited to an injured or inflamed site and play a major role in acute inflammation. They are the fundamental effectors of the host immune system and their basic role is to clear the infection or exogenous agents causing tissue injury. Neutrophils are continuously generated in the bone marrow and can be found in spleen, liver and lung. The lung holds a large reservoir of mature, terminally differentiated neutrophils, which are patrolling within the lung vasculature during steady state conditions (Kolaczkowska E and Kubes P, 2013). Neutrophil recruitment is initiated by the changes in the endothelium surface following stimulation by alarmin, DAMPs or inflammatory mediators released from resident leukocytes when they come in contact with pathogens or injurious external stimuli.

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 allergen induced fibrosis.

Activated immune cells secrete a variety of proinflammatory mediators, the purpose of which is to propagate the immune signalling and response, which when not controlled, lead to chronic inflammation, extensive cellular death and tissue injury.

It is important to note that the stressor-induced MIE, KE1 and KE2 are part of the functional changes that we collectively considered as inflammation, and together, they mark the initiation of acute inflammation phase.

How it is Measured or Detected

In vivo, recruitment of proinflammatory cells is measured using bronchoalveolar lavage fluid (BALF) cellularity assay.

The airway epithelium is the largest surface that is targeted by inhaled substances including ENMs. The fluid lining the epithelium (BAL fluid) is lavaged and its composition is assessed as marker of lung immune response to the toxic substances or pathogens. BAL is assessed quantitatively for types of infiltrating cells, levels and types of cytokines and chemokines. Thus, BAL fluid 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 regulatory purposes. For ENMs, in vivo BAL assessment is recommended as a mandatory test (discussed in ENV/JM/MONO(2012)40 and also in OECD inhalation TG for ENMs). Temporal changes in the BAL composition can be prognostic of initiation and progression of lung immune disease (Cho W-S et al., 2010).

In vitro, it is difficult to assess the recruitment of proinflammatory cells. Thus, a suit of proinflammatory 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.

References

34. Molly A. Ingersoll, Andrew M. Platt, Stephane Potteaux, and Gwendalyn J. Randolph. Monocyte trafficking in acute and chronic inflammation. Trends Immunol. 2011 Oct; 32(10): 470–477.
35. Chao Shi and Eric G. Pamer. Monocyte recruitment during infection and inflammation. Nat Rev Immunol. 2011 Oct 10; 11(11): 762–774.
36. Manfred Kopf, Christoph Schneider and Samuel P Nobs. The development and function of lung-resident macrophages and dendritic cells. Nature Immunology 16, 36–44 (2015).
37. Elzbieta Kolaczkowska and Paul Kubes. Neutrophil recruitment and function in health and inflammation. Nature Reviews Immunology 2013. 13, 159-175.
38. Cho W-S, Duffin R, Poland CA, et al. Metal Oxide Nanoparticles Induce Unique Inflammatory Footprints in the Lung: Important Implications for Nanoparticle Testing. Environmental Health Perspectives. 2010;118(12):1699-1706.

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. The cellular composition of ACM includes the type 1 alveolar epithelial cells, the capillary endothelial cells, and the endothelial and epithelial basement membranes. As a consequence of its anatomical position, and the large surface area, it is the first point of contact for any inhaled pathogens, particles or toxic substances. Thus, ACM is subjected to injury 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. Repeated exposure to or biopersistent toxic substances, pathogens or lung irritants leading to non-resolving inflammation contribute to the ACM injury. Chronic inflammation mediated by overexpression of cytokines such as IL-1, TGFb, T-helper type 2 cytokine IL-13 or exposure to specific proteinases initiate ACM injury leading to significant loss of the epithelium and endothelium of the ACM resulting 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. In addition, in normal conditions, the epithelial barrier consisting of alveolar epithelial type 1 and type 2 cells establish close contacts with the neighbouring cells via cell-cell adhesions through the intercellular junction complexes such as tight junctions, adherens junction and desmosomes (Vareille M et al., 2011; Kulkarni T et al., 2016). The various proteins found at the epithelial cell surface play a role in maintaining the cellular adhesions and intercellular junction complexes, which are critical for maintaining the integrity of alveolar epithelium. Exposure to pro-fibrotic drug bleomycin destroys structural architecture of the tight junctions leading to increased  permeability, epithelial death and loss of specialised proteins such as caludins required for repair and restoration of alveolar epithelial barrier. Thoracic radiation and bleomycin-induced lung injury result in decreased expression of E-cadherin and Aquaporin-5 expression – proteins involved in adherens and in transgenic mice lacking aquaporin-5, increased lung fibrosis is observed. 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 ER et al., 2010).

Alveolar capillary membrane integrity loss is also associated with other events such as chronic inflammation and Reactive Oxygen Species (ROS) synthesis. Chronic inflammation and ROS synthesis are described below as associative events to ACM loss.

Chronic inflammation as an associative event in the loss of ACM

Chronic inflammation, by definition is associated with tissue injury (Wallace WA et al., 2007). In the presence of continuous stimulus (e.g., presence of biopersistent toxic fibres such as asbestos, multi walled carbon nanotubes) or following repeated stimulus (e.g., repeated exposure to silica or coal dust), the ensuing tissue 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. The chronicity of the inflammatory process occurs after prolonged acute inflammation and is a crucial component of the lung fibrotic response. During the chronic inflammatory 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.

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. Macrophages are the key inflammatory cells linking inflammation with repair and fibrosis. 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. The other types of inflammatory cells found in chronic inflammation include eosinophils in allergen induced lung fibrosis, lymphocytes and epithelial cells.

Oxidative stress as an associative event in the ACM loss

Oxidative stress is an important event that influences the extent of lung injury (reviewed in MacNee W, 2001). Superoxide anion (O2-) and the hydroxyl radical (OH) are the common ROS found in the biological systems that are unstable due to unpaired electrons. ROS, when interacted with biomolecules, initiate their oxidation. 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. 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 proinflammatory genes, all of which lead to secretion of inflammatory cytokines/chemokines leading to prolonged and chronic inflammation. Nrf2, a member of the cap’n’collar basic leucine zipper transcription factors is suggested to play an important role in orchestrating the antioxidant defence against ROS via antioxidant response element. Nrf2 is shown to be protective against pulmonary inflammation and fibrosis induced by oxidants (Kikuchi N et al., 2010).

Human idiopathic pulmonary fibrosis is suggested to result from increased ROS synthesis and imbalances in oxidant/antioxidant levels in distal alveolar space. Antioxidant treatment of mice attenuates bleomycin-induced oxidative stress and subsequent lung fibrosis (Wang HD, 2002). Several ENMs have been shown to induce oxidative stress (a state of redox disequilibrium), an event associated with their in vivo toxicity (Xia T, 2008). Bleomycin induced lung pathology involves oxidative stress. In mice treated with antioxidants, bleomycin-induced inflammation and pathology is attenuated (Kelly C, 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 B et al., 1992).

The physical-chemical properties of chemicals, their intended applications and exposure levels must be considered as important factors influencing the extent of lung injury. Although not quantitative, in case of ENMs, the physical presence of ENMs (biopersistence) must be confirmed in lungs following exposure, using Transmission Electron Microscopy or Cytoviva Nanoscale Hyperspectral Microscope. High aspect ratio materials including ENMs injure the ACM in rodent models.

Cell type considerations

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.

Cellular damage

Upon interaction with toxic substances, phagocytic cells internalize toxic substances leading to respiratory burst and release of ROS. A fluorimetric assay that relies on the intracellular oxidation of 5- and 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (carboxy H2DCFDA) (Molecular Probes) has been used to detect ROS release in cells in vitro (Decan et al., 2016); however, it is important to note that the results are not specific to the types of radicals detected. In addition, lipid peroxidation, protein oxidation, and protein carbonylation can be measured as indicative of oxidative stress using proteomics techniques (Riebeling et al., 2001). Measurement of intracellular glutathione levels using the ThiolTracker™ Violet assay (Decan et al., 2016) or glutathionylation of proteins is also used. Oxidative stress can also be measured by assessing the relevant genes and proteins associated with antioxidant pathways (Riebeling et al., 2001). Other biomolecule modifications such as nitrosylation, reflective of oxidative stress, can be measured by measuring nitrosylated tissue proteins, or increase in NO production, and nitrate/nitrite ratio in BAL. In addition to tissue analysis, acellular glutathione levels, antioxidants and NO production in BAL supernatant can be used to assess ROS synthesis. These methods have been routinely used to measure ROS release following exposures to several toxic substances including ENMs.

Cytotoxicity assessment

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

Lactate dehydrogenase (LDH) release assay is a very sensitive cytotoxicity assay that measures the amount of LDH released in the media following membrane injury. The assay is based on measuring the reduction of 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 ENMs can confound the assays by inhibiting the enzyme activity or interfering with the absorbance reading. Thus, care must be taken to include appropriate controls in the assay.

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 qRT-PCR or ELISA assays for tight junction proteins, cell adhesion molecules and inflammatory mediators such as IFNg, IL-10, and IL-13.  

 

References

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40. Tejaswini Kulkarni, Joao de Andrade, Yong Zhou, Tracy Luckhardt, and Victor J. Thannickal. Alveolar epithelial disintegrity in pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 311: L185–L191, 2016.
41. Johnson ER, Matthay MA. Acute Lung Injury: Epidemiology, Pathogenesis, and Treatment. Journal of Aerosol Medicine and Pulmonary Drug Delivery. 2010;23(4):243-252.
42. Wallace WA, Fitch PM, Simpson AJ, Howie SE. Inflammation-associated remodelling and fibrosis in the lung – a process and an end point. International Journal of Experimental Pathology. 2007;88(2):103-110. doi:10.1111/j.1365-2613.2006.00515.x.

43. MacNee William. Oxidative stress and lung inflammation in airways diseases. European Journal of Pharmacology. 2001 429(1-3):195-207.

44. Norihiro Kikuchi, Yukio IshiiEmail, Yuko Morishima, Yuichi Yageta, Norihiro Haraguchi, Ken Itoh, Masayuki Yamamoto and Nobuyuki Hizawa. Nrf2 protects against pulmonary fibrosis by regulating the lung oxidant level and Th1/Th2 balance. Respiratory Research 2010 11:31
45. Wang HD, Yamaya M, Okinaga S, Jia YX, Kamanaka M, Takahashi H, Guo LY, Ohrui T, Sasaki H. Bilirubin ameliorates bleomycin-induced pulmonary fibrosis in rats. Am J Respir Crit Care Med. 2002;165:406–411
46. Xia T, Kovochich M, Liong M, et al. Comparison of the Mechanism of Toxicity of Zinc Oxide and Cerium Oxide Nanoparticles Based on Dissolution and Oxidative Stress Properties. ACS nano. 2008;2(10):2121-2134. doi:10.1021/nn800511k.
47. Tian Xia, Michael Kovochich, Jonathan Brant, Matt Hotze, Joan Semp, Terry Oberley, Constantinos Sioutas, Joanne I. Yeh, Mark R. Wiesner, and Andre E. Nel. Comparison of the Abilities of Ambient and Manufactured Nanoparticles To Induce Cellular Toxicity According to an Oxidative Stress Paradigm. Nano Letters 2006 Vol. 6, No. 8 1794-1807.
48. Kelly C. Teixeira, Fernanda S. Soares, Luís G.C. Rocha, Paulo C.L. Silveira, Luciano A. Silva, Samuel S. Valença, Felipe Dal Pizzol, Emilio L. Streck, Ricardo A. Pinho, Attenuation of bleomycin-induced lung injury and oxidative stress by N-acetylcysteine plus deferoxamine, In Pulmonary Pharmacology & Therapeutics, Volume 21, Issue 2, 2008, Pages 309-316.
49. B Schmekel, J A H Bos, A R Khan, B Wohlfart, B Lachmann, P Wollmer. Integrity of the alveolar-capillary barrier and alveolar surfactant system in smokers. Thorax 1992;47:603-608.
50. Nathalie Decan, Dongmei Wu, Andrew Williams, Stéphane Bernatchez, Michael Johnston, Myriam Hill, Sabina Halappanavar, Characterization of in vitro genotoxic, cytotoxic and transcriptomic responses following exposures to amorphous silica of different sizes, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Volume 796, 2016, Pages 8-22.
51. Riebeling C, Wiemann M, Schnekenburger J, Kuhlbusch TA, Wohlleben W, Luch A, Haase A. Toxicol Appl Pharmacol. 2016 May 15;299:24-9.

Key Event Description

How does this KE work

The loss of ACM involving epithelial cell injury engages the adaptive immune system resulting in the activation of CD4+ T cells.  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 (Kidd P, 2003). As described above, 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 (Lukacs NW. et al., 2001). 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 have shown complete attenuation of fibrosis despite the highly active Th1 response, clearly demonstrating the link between Th2 response and fibrosis. 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. In animal models of fibrosis, overexpression of IFNg negatively regulates fibrotic process. On the other hand, Th2 cytokines IL-4 and IL-13 regulate wound healing and contribute to the development and extension of fibrosis. IL-4 and IL-13 are suggested to stimulate the production of extracellular matrix proteins by activated fibroblasts; overexpression of IL-4 or IL-13 in fibroblasts increases ECM deposition. 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.  Neutrophils recruitment during acute inflammation initiates Th2 immune response and secretion of Th2 type cytokines and chemokines (Lekkerkerker N, 2014). The members of the FIZZ (resistin-like molecules, RELM) proteins is induced in lung airway and epithelial cells following exposure to fibrogenic bleomycin (Liu T, 2004; Liu T, 2014).  The expression of FIZZ is shown to be mediated by Th2 signalling and is involved in recruitment of inflammatory cells to lungs (Nair 2003; Munitz 2008; Angelini 2010; Madala 2012).

Macrophage polarisation as an associative event in the activation of Th2 cells

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 induce T helper (Th) 1 type responses. M1 macrophages produce IL-1, IL-12, IL-23 and induce Th1 cell infiltration and activation. M1 macrophages are associated with antigen presentation, microbiocidal and antitumour activities. 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-1 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 (Martinez FO and Gordon S, 2014). 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.

Pharyngeal aspiration of MWCNT for 7 days in mice induced lung fibrosis via activation of Th2 cells and Th2- mediated immune response, and included increased expression of Th2 cytokines such as IL-4, IL-13 and other genes. In addition, activation of Th2-mediated signaling pathway involving STAT6 transcription factor was also observed (Dong J and Ma Q, 2016). Meta-analysis of gene expression data derived from lungs of mice exposed to MWCNTs showed that the MWCNT-induced gene expression profiles are similar to the expression profiles induced in Th2 signalling-mediated lung fibrosis (Nikota J, 2016). In another study, mice deficient in STAT6 transcription factor showed attenuated lung fibrosis following exposure MWCNT, that was accompanied by reduced expression of Th2 cytokines and chemokines (Nikota J, 2017). Polarisation to Th2 and M2 phenotypes is observed in bleomycin-induced lung fibrosis (Li D, 2014).

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 FIZZ-1, 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

52. Parris Kidd. Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev 2003;8(3):223-246.
53. Nicholas W. Lukacs, Cory Hogaboam, Stephen W. Chensue, Kate Blease and Steven L. Kunkel. Type 1/Type 2 Cytokine Paradigm and the Progression of Pulmonary Fibrosis. CHEST 2001; 120;5s-8s.
54. N. Lekkerkerker, Annemarie; Aarbiou, Jamil; van Es, Thomas; A.J. Janssen, Richard. Cellular players in lung fibrosis. Current Pharmaceutical Design, Volume 18, Number 27, September 2012, pp. 4093-4102(10).
55. Liu, T. J., H. Jin, M. Ullenbruch, B. Hu, N. Hashimoto, B. Moore, A. McKenzie,. N. W. Lukacs, and S. H. Phan. 2004. Regulation of found in inflammatory zone 1 expression in bleomycin-induced lung fibrosis: role of IL-4/IL-13 and mediation via STAT-6. J. Immunol. 173: 3425–3431.
56. Liu T, Yu H, Ullenbruch M, et al. The In Vivo Fibrotic Role of FIZZ1 in Pulmonary Fibrosis. Fu J, ed. PLoS ONE. 2014;9(2):e88362.
57. Meera G Nair, Daniel W Cochrane, Judith E Allen, Macrophages in chronic type 2 inflammation have a novel phenotype characterized by the abundant expression of Ym1 and Fizz1 that can be partly replicated in vitro, In Immunology Letters, Volume 85, Issue 2, 2003, Pages 173-180.
58. Munitz A, Waddell A, Seidu L, et al. Resistin-like molecule α enhances myeloid cell activation and promotes colitis. The Journal of allergy and clinical immunology. 2008;122(6):1200-1207.e1.
59. Angelini DJ, Su Q, Kolosova IA, Fan C, Skinner JT, Yamaji-Kegan K, et al. (2010) Hypoxia-Induced Mitogenic Factor (HIMF/FIZZ1/RELMα) Recruits Bone Marrow-Derived Cells to the Murine Pulmonary Vasculature. PLoS ONE 5(6): e11251
60. Madala SK, Edukulla R, Davis KR, et al. Resistin-like molecule alpha1 (Fizz1) recruits lung dendritic cells without causing pulmonary fibrosis. Respiratory Research. 2012;13(1):51. doi:10.1186/1465-9921-13-51.
61. Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Reports. 2014;6:13. doi:10.12703/P6-13.
62. Dong J, Ma Q. In vivo activation of a T helper 2-driven innate immune response in lung fibrosis induced by multi-walled carbon nanotubes. Archives of toxicology. 2016;90(9):2231-2248.
63. Nikota J, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Meta-analysis of transcriptomic responses as a means to identify pulmonary disease outcomes for engineered nanomaterials. Part Fibre Toxicol. 2016 May 11;13(1):25.
64. 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.
65. Dong Li, Rodrigo Guabiraba, Anne-Gaëlle Besnard, Mousa Komai-Koma, Majid S. Jabir, Li Zhang, Gerard J. Graham, Mariola Kurowska-Stolarska, Foo Y. Liew, Charles McSharry, Damo Xu, IL-33 promotes ST2-dependent lung fibrosis by the induction of alternatively activated macrophages and innate lymphoid cells in mice, In Journal of Allergy and Clinical Immunology, Volume 134, Issue 6, 2014, Pages 1422-1432.e11.

Key Event Description

Fibroblasts are non-hematopoietic, non-epithelial and non-endothelial cells. In steady state conditions, they are distributed throughout the mesenchyme (Phan SH, 2008). 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. Fibroblasts are the workhorse of the connective tissue that joins and holds other tissues together in the body. 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. In the context of lung fibrosis, fibroblasts play an important role in ECM maintenance and turnover, wound healing, inflammation and angiogenesis (Kendall RT, 2014). 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 et al 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 (CXCL12 binds 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[SH1] . 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.

Epithelial-mesenchymal transition is a process by which epithelial cells lose their original phenotype, acquire fibroblast-like properties, and display reduced cell adhesion (which is crucial for the detachment of epithelial cells prior to migration to the site of injury) and increased motility (Lekkerkerker et al 2012). During this process, epithelial cells also lose typical markers such as E-cadherin and zona occludens-1 (ZO-1) and acquire mesenchymal markers such as fibroblast-specific protein-1, vimentin and α-SMA (Grunert et al 2003; Zeisberg and Neilson 2009). 

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 (Zhang 1994). It is recommended to use more than one marker to confirm the activation of fibroblasts. The species-specific antibodies for all the markers are commercially available and the technique works in both in vitro and in vivo models as well as in human specimens. Immunohistochemistry is performed using immunoperoxidase technique. Formalin fixed and paraffin embedded lung sections are sliced in 3-5µm thin slices and reacted with diluted H₂O₂ for 10 min to block the endogenous peroxidase activity. The slices are then incubated with appropriate dilutions of primary antibody against the individual markers followed by incubation with the secondary antibody that is biotinylated. The slices are incubated for additional 30 minutes for avidin-biotin amplification and reacted with substrate 3’3’ diaminobenzidine before visualising the cells under the light microscope. Although only 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 (Megan 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 to the same material (Vietti 2013; Azad 2013)

References

66. Phan SH. Biology of Fibroblasts and Myofibroblasts. Proceedings of the American Thoracic Society. 2008;5(3):334-337. doi:10.1513/pats.200708-146DR.
67. Kendall RT, Feghali-Bostwick CA. Fibroblasts in fibrosis: novel roles and mediators. Frontiers in Pharmacology. 2014;5:123. doi:10.3389/fphar.2014.00123.
68. Shu Hashimoto, Yasuhiro Gon, Ikuko Takeshita, Shuichiro Maruoka, and Takashi Horie. 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 , Volume 107 , Issue 6 , 1001 – 1008.
69. Serini G, Gabbiani G. Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res. 1999 Aug 1;250(2):273–283.
70. Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN, Sheppard D, Chapman HA. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci USA 2006;103:13180–13185Kim et al 2006. Proc. Natl. Acad. Sci. USA 103: 13180-13185.
71. Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med. 1994 Nov;1(1):71-81.
72. 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.
73. Riichiro Abe, Seamas C. Donnelly, Tina Peng, Richard Bucala and Christine N. Metz. J Peripheral Blood Fibrocytes: Differentiation Pathway and Migration to Wound Sites Immunol  June 15, 2001,  166  (12)  7556-7562.
74. N. Lekkerkerker, Annemarie; Aarbiou, Jamil; van Es, Thomas; A.J. Janssen, Richard. Cellular Players in Lung Fibrosis Current Pharmaceutical Design, Volume 18, Number 27, September 2012, pp. 4093-4102(10).
75. Grünert S, Jechlinger M, Beug H. Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis. 2003. Nat. Rev. Mol. Cell Biol. 4: 657-665.
76. Zeisberg M, Neilson EG. Biomarkers for epithelial-mesenchymal transitions. The Journal of Clinical Investigation. 2009;119(6):1429-1437.
77. Kai Zhang, Mark D. Rekhter, David Gordon,and Sem H. Phan. Myofibroblasts and Their Role in Lung Collagen Gene Expression during Pulmonary Fibrosis A Combined Immunohistochemical and in Situ Hybridization Study. American Journal of Pathology, Vol. 145, No. 1, July 1994
78. Tamminen, J. A., Myllärniemi, M., Hyytiäinen, M., Keski-Oja, J. and Koli, K. (2012), Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling. J. Cell. Biochem., 113: 2234–2247.
79. Kim KK, Kugler MC, Wolters PJ, et al. 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 of the United States of America. 2006;103(35):13180-13185.
80. Megan E. Smithmyer, Lisa A. Sawicki and April M. Kloxin. Hydrogel scaffolds as in vitro models to study fibroblast activation in wound healing and disease Biomater. Sci., 2014, 2, 634.
81. Vietti G, Ibouraadaten S, Palmai-Pallag M, et al. Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay. Particle and Fibre Toxicology. 2013;10:52. doi:10.1186/1743-8977-10-52.
82. Azad N, Iyer A.K.V., Lu Y., Wang L., Rojanasakul Y. P38/MAPK Regulates Single-walled Carbon Nanotube-Induced Fibroblast Proliferation and Collagen Production. Nanotoxicology (2013),7(2), 157–168.

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. 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 (reviewed in White 2015).  

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. 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, 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 (Blaauboer ME, 2014). 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, P., 2012; Wynn, T.A., 2011).

In lung fibrosis, an exaggerated amount of ECM is distributed in the alveolar parenchyma in a non-heterogenous manner, occluding alveolar regions leading to 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, K., 1994; Hinz, B., 2006; Kuhn, C., 1991; Crabb, R.A., 2006; Bensadoun, E.S., 1996; Klingberg, F., 2013; McKleroy, W., 2013). Excessive collagen production by myofibroblasts forms the basis of scar formation containing almost exclusively Type I collagen (Bateman, ED, 1981; McKleroy, W., 2013; Zhang, K., 1994). Several types of collagen exist, with differences based on their tissue localisation and function. However, type I collagen is the most abundant throughout the body, as well as in lung scar tissue. 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, M.E.1983; Rozin, G.F., 2005; McKleroy, W., 2013).

How it is Measured or Detected

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 (Clarice ZC, 2009).

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 (Clarice ZC, 2009).

References

83. Eric S. White. Lung Extracellular Matrix and Fibroblast Function. Ann Am Thorac Soc. 2015 Vol 12, Supplement 1, pp S30–S33.
84. Marjolein E. Blaauboer, Fee R. Boeijen, Claire L. Emson, Scott M. Turner, Behrouz Zandieh-Doulabi, Roeland Hanemaaijer, Theo H. Smit, Reinout Stoop, Vincent Everts, Extracellular matrix proteins: A positive feedback loop in lung fibrosis?, In Matrix Biology, Volume 34, 2014, Pages 170-178, ISSN 0945-053X,
85. Sivakumar, Pitchumania; Ntolios, Paschalisb; Jenkins, Gislic; Laurent, Geoffreyb. Into the matrix: targeting fibroblasts in pulmonary fibrosis. Current Opinion in Pulmonary Medicine: September 2012 - Volume 18 - Issue 5 - p 462–469.
86. Thomas A. Wynn. Integrating mechanisms of lung fibrosis. Journal of Experimental Medicine  Jul 2011,  208  (7)  1339-1350.
87. Zhang, K., Rekhter, M.D., Gordon, D., and Phan, S.H.: Co-Expression of o-smooth muscle actin and type I collagen in fibroblast-like cells of rat lungs with bleomycininduced pulmonary fibrosis: a combined immuno-histochemical and in situ hybridization study. Am. J. Pathol. 1994; 145:114-125
88. 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, 175-181.
89. 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;138:1257–1265.
90. Crabb RA, EP Chau, DM Decoteau, A Hubel. Multifunctional polymeric microfibers with prolonged drug delivery and structural support capabilities. Annals of Biomedical Engineering 34 (10), 1615-1627, 2006. 19, 2006.
91. Bensadoun E, Burke AK, Hogg JC, Roberts CR. Proteoglycan deposition in pulmonary fibrosis. Am J. Respir. Crit. Care Med. 1996;154:1819–1828.
92. Klingberg F, Hinz B, White ES. The myofibroblast matrix: implications for tissue repair and fibrosis. The Journal of pathology. 2013;229(2):298-309.
93. McKleroy W, Lee TH, Atabai K.Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis. Am J Physiol Lung Cell Mol Physiol 304:L709-L721.
94. Bateman, E., Turner-Warwick, M., and Adelmann-Grill, B. C. Immunohistochemical study of collagen types in human foetal lung and fibrotic lung disease. Thorax 1981 ;36:645-653.
95. Zhang, K., Rekhter, M.D., Gordon, D., and Phan, S.H.: Co-Expression of o-smooth muscle actin and type I collagen in fibroblast-like cells of rat lungs with bleomycininduced pulmonary fibrosis: a combined immuno-histochemical and in situ hybridization study. Am. J. Pathol. 1994; 145:114-125.
96. Nimni ME. Collagen: Structure, function, and metabolism in normal and fibrotic tissues Seminars in arthritis and rheumatism; 1983 Aug ; 13(1) 1-86.
97. Rozin GF, Gomes MM, Parra ER, Kairalla RA, de Carvalho CR, Capelozzi VL. Collagen and elastic system in the remodelling process of major types of idiopathic interstitial pneumonias (IIP) Histopathology. 2005;46:413–421.
98. Clarice ZC Chen and Michael Raghunath. Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis - state of the art. Fibrogenesis & Tissue Repair 2009, 2:7.

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 DR, 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

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 R-H, 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 as shown in (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 J, 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