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Key Event Title
Increased, secretion of proinflammatory mediators
|Level of Biological Organization|
Key Event Components
|cytokine production involved in inflammatory response||Cytokine||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Substance interaction with the lung cell membrane leading to lung fibrosis||KeyEvent||Sabina Halappanavar (send email)||Under development: Not open for comment. Do not cite||EAGMST Under Review|
|SARS-CoV-2 leads to acute respiratory distress||KeyEvent||Young Jun Kim (send email)||Open for comment. Do not cite||Under Development|
|AT1R, lung fibrosis||KeyEvent||Young Jun Kim (send email)||Under development: Not open for comment. Do not cite||Under Development|
|Dysregulated fibrinolysis/bradykinin leading to hyperinflammation||KeyEvent||Penny Nymark (send email)||Under development: Not open for comment. Do not cite||Under Development|
|Frustrated phagocytosis leads to malignant mesothelioma||KeyEvent||Penny Nymark (send email)||Under development: Not open for comment. Do not cite|
|TLR9 activation leading to Multi Organ Failure and ARDS||KeyEvent||Gillina Bezemer (send email)||Under development: Not open for comment. Do not cite|
|Covalent binding to proteins leads to Respiratory Sensitisation/Sensitization/Allergy||KeyEvent||Kristie Sullivan (send email)||Under Development: Contributions and Comments Welcome||Under Development|
|Binding to ACE2 leads to lung fibrosis||KeyEvent||Young Jun Kim (send email)||Open for comment. Do not cite||Under Development|
|Interaction with lung cells leads to lung cancer||KeyEvent||Penny Nymark (send email)||Under development: Not open for comment. Do not cite|
Key Event Description
Pro-inflammatory mediators are the chemical and biological molecules that initiate and regulate inflammatory reactions. Pro-inflammatory mediators are secreted following exposure to an inflammogen in a gender/sex or developmental stage independent manner. They are secreted during inflammation in all species. Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). Cell-derived pro-inflammatory mediators include cytokines, chemokines, and growth factors. Blood derived pro-inflammatory mediators include vasoactive amines, complement activation products and others. These modulators can be grouped based on the cell type that secrete them, their cellular localisation and also based on the type of immune response they trigger. For example, members of the interleukin (IL) family including IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-3, IL-5 and 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).
Literature evidence for its perturbation:
Several studies show increased proinflammatory mediators in rodent lungs and bronchoalveolar lavage fluid, and in cell culture supernatants following exposure to a variety of CNT types and other materials. Poland et al., 2008 showed that long and thin CNTs (>5 µm) can elicit asbestos-like pathogenicity through the continual release of pro-inflammatory cytokines and 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., 2011; Husain et al., 2015a). Similarities are observed in gene programs involving pro-inflammatory event is observed in both humans and experimental mice (Zuo et al., 2002).
How It Is Measured or Detected
The selection of pro-inflammatory mediators for investigation varies based on the expertise of the lab, cell types studied and the availability of the specific antibodies.
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 fluorophore SYBR green, which produces a signal when incorporated into double-stranded amplicons. Since the cDNA is single stranded, the dye does not bind enhancing the specificity of the results. There are other methods such as nested fluorescent probes for detection but SYBR green is widely used. RT-PCR primers specific to several pro-inflammatory mediators in several species including mouse, rat and humans, are readily available commercially.
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., 2015b). This method is quantitative and especially advantageous when the sample amount available for testing is scarce. Lastly, immunohistochemistry can also be used to detect specific immune cell types producing the pro-inflammatory mediators and its downstream effectors in any given tissue (Costa et al., 2017). Immunohistochemistry results can be used as weight of evidence; however, the technique is not quantitative and depending on the specific antibodies used, the assay sensitivity may also become an issue (Amsen and De Visser, 2009).
Cell models - of varying complexity have been used to assess the expression of pro-inflammatory mediators. Two dimensional submerged monocultures of the main fibrotic effector cells – lung epithelial cells, macrophages, and fibroblasts – have routinely been used in vitro due to the large literature base, and ease of use, but do not adequately mimic the in vivo condition (Sundarakrishnan et al., 2018, Sharma et al., 2016). Recently, the EpiAlveolar in vitro lung model (containing epithelial cells, endothelial cells, and fibroblasts) was used to predict the fibrotic potential of MWCNT, and researchers noted increases in the pro-inflammatory molecules TNF-a, IL-1b, and the pro-fibrotic TGF-b using ELISA assays (Barasova et al., 2020). A similar, but less complicated co-culture model of immortalized human alveolar epithelial cells and IPF patient derived fibroblasts was used to assess pro-fibrotic signalling, and noted enhanced secretion of PDGF and bFGF (basic fibroblast growth factor), as well as evidence for epithelial to mesenchymal transition of epithelial cells in this system (Prasad et al., 2014). Models such as these better capitulate the in vivo pulmonary alveolar capillary, but have lower reproducibility as compared to traditional submerged mono-culture experiments.
Domain of Applicability
Human, mouse, rat
Cytokines are the common pro-inflammatory mediators secreted following inflammogenic stimuli. Cytokines can be defined as diverse group of signaling protein molecules. They are secreted by different cell types in different tissues and in all mammalian species, irrespective of gender, age or sex. A lot of literature is available to support cross species, gender and developmental stage application for this KE. The challenge is the specificity; most cytokines exhibit redundant functions and many are pleotropic.
1. Amsen, D. and De Visser, K. (2009). Approaches to Determine Expression of Inflammatory Cytokines. Methods in molecular biology.. 511th ed. (Clifton, NJ), pp.107-142.
2. Barosova, H., Maione, A. G., Septiadi, D., Sharma, M., Haeni, L., Balog, S., O'Connell, O., Jackson, G. R., Brown, D., Clippinger, A. J., Hayden, P., Petri-Fink, A., Stone, V., & Rothen-Rutishauser, B. (2020). Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS nano, 14(4), 3941–3956.
3. Boyles, M. S., Young, L., Brown, D. M., MacCalman, L., Cowie, H., Moisala, A., Smail, F., Smith, P. J., Proudfoot, L., Windle, A. H., & 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 : an international journal published in association with BIBRA, 29(7), 1513–1528.
4. Costa, P. M., Gosens, I., Williams, A., Farcal, L., Pantano, D., Brown, D. M., Stone, V., Cassee, F. R., Halappanavar, S., & Fadeel, B. (2018). Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles. Journal of applied toxicology : JAT, 38(3), 385–397.
5. Halappanavar, S., Jackson, P., Williams, A., Jensen, K. A., Hougaard, K. S., Vogel, U., Yauk, C. L., & Wallin, H. (2011). Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study. Environmental and molecular mutagenesis, 52(6), 425–439.
6. 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.
7. Husain, M., Kyjovska, Z. O., Bourdon-Lacombe, J., Saber, A. T., Jensen, K. A., Jacobsen, N. R., Williams, A., Wallin, H., Halappanavar, S., Vogel, U., & Yauk, C. L. (2015a). Carbon black nanoparticles induce biphasic gene expression changes associated with inflammatory responses in the lungs of C57BL/6 mice following a single intratracheal instillation. Toxicology and applied pharmacology, 289(3), 573–588.
8. Husain, M., Wu, D., Saber, A. T., Decan, N., Jacobsen, N. R., Williams, A., Yauk, C. L., Wallin, H., Vogel, U., & Halappanavar, S. (2015b). Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice. Nanotoxicology, 9(8), 1013–1022.
9. Kaminski N. (2003). Microarray analysis of idiopathic pulmonary fibrosis. American journal of respiratory cell and molecular biology, 29(3 Suppl), S32–S36.
10. Mestas, J., & Hughes, C. C. (2004). Of mice and not men: differences between mouse and human immunology. Journal of immunology (Baltimore, Md. : 1950), 172(5), 2731–2738.
11. Nolan, T., Hands, R. E., & Bustin, S. A. (2006). Quantification of mRNA using real-time RT-PCR. Nature protocols, 1(3), 1559–1582.
12. Park, S. J., & Im, D. S. (2019). Deficiency of Sphingosine-1-Phosphate Receptor 2 (S1P2) Attenuates Bleomycin-Induced Pulmonary Fibrosis. Biomolecules & therapeutics, 27(3), 318–326. https://doi.org/10.4062/biomolther.2018.131
13. Poland, C. A., Duffin, R., Kinloch, I., Maynard, A., Wallace, W. A., Seaton, A., Stone, V., Brown, S., Macnee, W., & Donaldson, K. (2008). Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nature nanotechnology, 3(7), 423–428.
14. Prasad, S., Hogaboam, C. M., & Jarai, G. (2014). Deficient repair response of IPF fibroblasts in a co-culture model of epithelial injury and repair. Fibrogenesis & tissue repair, 7, 7.
15. Sharma, M., Nikota, J., Halappanavar, S., Castranova, V., Rothen-Rutishauser, B., & Clippinger, A. J. (2016). Predicting pulmonary fibrosis in humans after exposure to multi-walled carbon nanotubes (MWCNTs). Archives of toxicology, 90(7), 1605–1622.
16. Sundarakrishnan, A., Chen, Y., Black, L. D., Aldridge, B. B., & Kaplan, D. L. (2018). Engineered cell and tissue models of pulmonary fibrosis. Advanced drug delivery reviews, 129, 78–94.
17. Turner, M. D., Nedjai, B., Hurst, T., & Pennington, D. J. (2014). Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochimica et biophysica acta, 1843(11), 2563–2582.
18. Zuo, F., Kaminski, N., Eugui, E., Allard, J., Yakhini, Z., Ben-Dor, A., Lollini, L., Morris, D., Kim, Y., DeLustro, B., Sheppard, D., Pardo, A., Selman, M., & Heller, R. A. (2002). Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proceedings of the National Academy of Sciences of the United States of America, 99(9), 6292–6297.