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 cell membrane leading to lung fibrosis||KeyEvent|
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.
Domain of Applicability
- Parris Kidd. Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev 2003;8(3):223-246.
- 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.
- 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).
- 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.
- 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.
- 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.
- 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.
- 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
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.