Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Substance interaction with the lung cell membrane leading to lung fibrosis||KeyEvent|
Key Event Description
How does this KE work
Naïve CD4+ T cells differentiate into four types of Th cells – Th1, Th2, Th17 and inducible regulatory T cells following exposure to infectious agents. The differentiation process begins when antigen presenting cells (APCs) come in contact with toxic substances and is mainly driven by cytokines that make up the microenvironment. For example, increased concentrations of IL-12 secreted by APCs in the environment may be biased towards Th1 type and increased IL-6 or IL-4 in the environment may commit to Th2 type differentiation. Th1 cytokines IFNg and IL-12 induce inflammation, aid in clearance of toxic substances, induce tissue damage and control the fibrotic responses. IFNg has suppressive effects on the production of extracellular matrix proteins including collagen and fibronectin. The Th2 response suppresses Th1 mediated response, which results in decreased Th1 cell-mediated tissue damage but at the same time contributing to the persistence of toxic substances leading to perpetuation of tissue damage, triggering uncontrolled healing response. The major sources of Th2 cytokines are Th2 cells themselves; however, mast cells, macrophages, epithelial cells and activated fibroblasts have shown to produce IL-4, IL-13 and IL-10 upon appropriate stimulation. Th2 cytokines IL-4 and IL-13 regulate wound healing.
KE4 associative event - Macrophage polarisation
Depending on the lung microenvironment (damaged cells, microbial products, activated lymphocytes), the precursor monocytes differentiate into distinct types of macrophages. Classically activated (M1) macrophages and alternatively activated (M2) macrophages are the important ones to consider in the context of this AOP. The M1 macrophages produce high levels of pro-inflammatory cytokines, mediate resistance to pathogens, induce generation of high levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and T helper (Th) 1 type responses. M1 macrophages produce IL-1, IL-12, IL-23 and induce Th1 cell infiltration and activation. The M2 macrophages secrete anti-inflammatory mediators, by which they play a role in regulation of inflammation. The M2 polarisation is mediated by Th2 cytokines such as IL-4 and IL-13, which in turn, promotes M2 activation. M2 macrophages express immunosuppressive molecules such as IL-10, Arginase-1 and -2 (Arg-1, Arg-2), which suppress the induction of Th1 cells that produce the anti-fibrotic cytokine IFNg. The activity of M2 is associated with tissue remodelling, immune regulation, tumour promotion, tissue regeneration and effective phagocytic activity. During chronic inflammation, the phenotype of infiltrating macrophages is suggested to resemble that of M2, which is suggested to play a role in lung fibrosis.
Evidence for its perturbation
For fibroplasia or fibrosis, the type of CD4+ T cell response that develops is crucial. Studies conducted in mice that do not express Th2 cytokines IL-4, IL-5 and IL-13 show complete attenuation of fibrosis despite the highly active Th1 response. Th2 cytokines IL-4 and IL-13 are elevated in fibrotic lungs; IL-13 activates TGFb1 and initiates fibroblast proliferation and differentiation in lung fibrosis (Lee, 2001). Overexpression of IL-13 induces sub-epithelial airway fibrosis in mice in the absence of any other external pro-inflammatory or pro-fibrotic stimulus (Zhu, 1999). Both MWCNTs and SWCNTs induce elevated expression of IL-4 and IL-13 in BALF of mouse lungs (Park, 2011), and increased levels of IL-25 and IL-33 in BALF and mouse lungs exposed to MWCNTs (Dong, 2016). In a rare human study, increased levels IL-4 and IL-5 were observed in the sputum of humans exposed to MWCNTs at an occupational setting (Fatkhutdinova, 2016). Overexpression of IL-10 increases IL-4 and IL-13 production and lung fibrosis following exposure to silica (Barbarin, 2005). Alveolar macrophages from asbestosis patients (a form of lung fibrosis) exhibit M2 phenotype (Chao et al., 2013). Ex vivo culture of alveolar macrophages obtained from BALF of patients suffering from IPF with collagen type I showed enhanced levels of M2 macrophage markers CCL-18, CCL-2 and CD204 (Stahl, 2013). Th2 response associated expression of IL-33 cytokine enhances polarisation of M2 macrophages and inducing M2-mediated expression of IL-13 and TGFb1 in mice (Dong, 2014). Cigarette smoke induces expression of genes associated with M2 sub-phenotypes, which is further enhanced in smokers presenting with COPD (Shaykhiev, 2009).
How It Is Measured or Detected
Targeted enzyme-linked immunosorbent assays (ELISA) or real-time quantitative polymerase chain reaction (qRT-PCR) (routinely used and recommended)
The ELISA and qRT-PCR are routinely used to assess the levels of protein and mRNA of several Th1 and Th2 cytokines including IL-4, IL-5, IL-13, IL-10, IL-12, IFNg. In addition, the levels of Transforming growth factor b (TGFb) is also assessed, expression of which is increased following induction of IL-13 synthesis. The other genes of relevance to Th2 response and eventual pro-fibrotic response include Arg-1 and Arg-2. BALF supernatant collected from lungs of animals exposed to toxic substances or human patients is used. Tissue homogenates or cell pellets can also be used. Expression of these genes and proteins can be assessed in in vitro cell cultures exposed to pro-fibrotic stimulus.
Apart from assaying single protein or gene at a time, cytokine bead arrays or cytokine PCR arrays can be used to detect a whole panel of Th1 and/or Th2 cytokines using a multiplex method. This method is quantitative and especially advantageous when the sample amount available for testing is scarce.
The details of ELISA and qRT-PCR are described under MIE. The details of BALF sample collection is described under KE2.
Domain of Applicability
- Barbarin, V., Xing, Z., Delos, M., Lison, D. and Huaux, F. (2005). Pulmonary overexpression of IL-10 augments lung fibrosis and Th2 responses induced by silica particles. American Journal of Physiology-Lung Cellular and Molecular Physiology, 288(5), pp.L841-L848.
- Dong, J. and Ma, Q. (2016). In vivo activation of a T helper 2-driven innate immune response in lung fibrosis induced by multi-walled carbon nanotubes. Archives of Toxicology, 90(9), pp.2231-2248.
- Fatkhutdinova, L., Khaliullin, T., Vasil'yeva, O., Zalyalov, R., Mustafin, I., Kisin, E., Birch, M., Yanamala, N. and Shvedova, A. (2016). Fibrosis biomarkers in workers exposed to MWCNTs. Toxicology and Applied Pharmacology, 299, pp.125-131.
- He, C., Ryan, A., Murthy, S. and Carter, A. (2013). Accelerated Development of Pulmonary Fibrosis via Cu,Zn-superoxide Dismutase-induced Alternative Activation of Macrophages. Journal of Biological Chemistry, 288(28), pp.20745-20757.
- Huaux, F., Liu, T., McGarry, B., Ullenbruch, M., Xing, Z. and Phan, S. (2003). Eosinophils and T Lymphocytes Possess Distinct Roles in Bleomycin-Induced Lung Injury and Fibrosis. The Journal of Immunology, 171(10), pp.5470-5481.
- Lee, C., Homer, R., Zhu, Z., Lanone, S., Wang, X., Koteliansky, V., Shipley, J., Gotwals, P., Noble, P., Chen, Q., Senior, R. and Elias, J. (2001). Interleukin-13 Induces Tissue Fibrosis by Selectively Stimulating and Activating Transforming Growth Factor β1. The Journal of Experimental Medicine, 194(6), pp.809-822.
- Li, D., Guabiraba, R., Besnard, A., Komai-Koma, M., Jabir, M., Zhang, L., Graham, G., Kurowska-Stolarska, M., Liew, F., McSharry, C. and Xu, D. (2014). IL-33 promotes ST2-dependent lung fibrosis by the induction of alternatively activated macrophages and innate lymphoid cells in mice. Journal of Allergy and Clinical Immunology, 134(6), pp.1422-1432.e11.
- Park, E., Roh, J., Kim, S., Kang, M., Han, Y., Kim, Y., Hong, J. and Choi, K. (2011). A single intratracheal instillation of single-walled carbon nanotubes induced early lung fibrosis and subchronic tissue damage in mice. Archives of Toxicology, 85(9), pp.1121-1131.
- Shaykhiev, R., Krause, A., Salit, J., Strulovici-Barel, Y., Harvey, B., O'Connor, T. and Crystal, R. (2009). Smoking-Dependent Reprogramming of Alveolar Macrophage Polarization: Implication for Pathogenesis of Chronic Obstructive Pulmonary Disease. The Journal of Immunology, 183(4), pp.2867-2883.
- Stahl, M., Schupp, J., Jäger, B., Schmid, M., Zissel, G., Müller-Quernheim, J. and Prasse, A. (2013). Lung Collagens Perpetuate Pulmonary Fibrosis via CD204 and M2 Macrophage Activation. PLoS ONE, 8(11), p.e81382.
- Tao, B., Jin, W., Xu, J., Liang, Z., Yao, J., Zhang, Y., Wang, K., Cheng, H., Zhang, X. and Ke, Y. (2014). Myeloid-Specific Disruption of Tyrosine Phosphatase Shp2 Promotes Alternative Activation of Macrophages and Predisposes Mice to Pulmonary Fibrosis. The Journal of Immunology, 193(6), pp.2801-2811.
- Zhu, Z., Homer, R., Wang, Z., Chen, Q., Geba, G., Wang, J., Zhang, Y. and Elias, J. (1999). Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. Journal of Clinical Investigation, 103(6), pp.779-788.