Activation of Th2 cells leads to Increased cellular proliferation and differentiation

The biological plausibility for this KER is high. There is a widely understood functional relationship between Th2 response related mediators, and their ability to induce proliferation and differentiation of fibroblasts (Shao et al., 2008; Wynn, 2004; Wynn, 2012).

The empirical support for this KER is high. There is a plethora of dose and time response evidence, which show that Th2 cytokines induce the activation and proliferation of fibroblasts (Hashimoto et al., 2001; Lee et al., 2001; additional references can be found in Table 1).

A majority of the weight of evidence studies assess collagen synthesis as a proxy to fibroblast proliferation and myofibroblast differentiation. A few studies have shown that Th2 cytokine IL-4 stimulates fibroblast proliferation (Sempowski et al., 1994) and production of ECM components (Postlethwaite et al., 1992). In human studies, the progression of idiopathic pulmonary fibrosis (IPF) is also associated with a sustained IL-4 production (Ando et al., 1999; Wallace and Howie, 1999). Th2 cytokines induce expression and activity of TGF-β1, levels of which are elevated in bronchoalveolar lavage fluid (BALF) of patients suffering from lung interstitial diseases, is a potent inducer of myofibroblast differentiation and collagen synthesis (Kurosaka et al., 1998; Redington et al., 1997). Exposure of Signal transducer and activator of transcription 6 (STAT6) deficient mice to multi-walled carbon nanotubes (MWCNTs), suppressed acute lung inflammation, expression of Th2-mediated gene expression, reduced vimentin positive cells (marker of fibroblasts), levels of collagen synthesis and reduced the overall fibrotic response to MWCNTs (Nikota et al., 2017). Mice deficient in IL-33r (St2, Th2 response cytokine) or mice treated with anti-IL33 antibody, showed reduced lung inflammation, reduced collagen production and fibrotic pathology induced by bleomycin. IL-33 deficient mice treated with bleomycin showed reduced levels of IL-1 and other pro-inflammatory cytokines. Mice administered exogenously with mature IL-33 enhanced bleomycin-induced lung inflammation, collagen synthesis and fibrotic lesions (Li et al., 2014).

Dose-Response Relationship:

In vivo and in vitro studies have demonstrated a dose-response relationship, at the higher dose of the stressor, Th2 cells leads to increased, fibroblast proliferation, and myofibroblast differentiation.

Lo Re et al. (2011) evaluated the role of regulatory T cells (Treg cells) in a mouse model of lung fibrosis induced by silica (SiO₂) particles. SiO₂ particles administered 2.5 mg per mouse by pharyngeal instillation induced an increase in the levels of CD4⁺Foxp3⁺ regulatory T lymphocytes in lungs after 3 and 15 days of administration. Treg cells, purified from Foxp3-GFP transgenic mice administered with SiO₂, stimulated lung fibroblast proliferation in vitro by producing Platelet derived growth factor subunit B (PDGF-B) and Transforming growth factor beta (TGF-b) in a dose-dependent manner. Moreover, these results indicated that the activation of Th2 response (KE4; Event 1499) was needed to activate fibroblast proliferation (KE5; Event 1500). They determined that effector T cells purified from SiO₂-treated mice, in the absence of Treg cells, induced fibrosis by producing IL-4, suggesting that many T cell pathways lead to the fibroproliferative process.

Liu et al. (2011) investigated the role of Found in inflammatory zone 2 (FIZZ2) in pulmonary fibrosis in a rodent bleomycin model and the potential role of FIZZ2 in human fibrotic lung disease. FIZZ2 has been found in pulmonary fibrosis after 14 days of exposure to bleomycin in mice (2U and 10 U/Kg) and in lung tissue from patients with IPF and nonspecific interstitial pneumonia. The expression was localized mainly to airway epithelial cells and alveolar epithelial cells (AECs), and to a lesser extent in alveolar macrophages and smooth muscle and endothelial cells. AECs were isolated from rats and humans, and they were exposed to 10 ng/ml rIL-4, rIL-13, rIL-17 and Interferon gamma (INF-g). After 4 h, rIL-4 and rIL-13, induced FIZZ2 mRNA expression in rat lungs. After 8 h, rIL-13 increased FIZZ2 mRNA expression in rat lungs, and rIL-4 and IL-13 induced an expression of FIZZ2 MRNA in human lungs. These results indicate that FIZZ2 mRNA expression is driven by Th2-type cytokines. Mouse lung fibroblasts were isolated and treated with recombinant mouse FIZZ2 at different concentrations. Collagen I deposition was observed at 10 and 25 ng/ml, and a-smooth muscle actin (a-SMA) was induced at 25, 50, and 200 ng/ml; meanwhile, cell proliferation was observed at 10, 25, and 50 ng/ml. These results suggested that FIZZ2 had direct profibrogenic activity. Furthermore, FIZZ2 acts as a chemoattractant for bone marrow cells, especially BM-derived CD11^c+ dendritic cells. In knockout mice treated with bleomycin, a decrease in the FIZZ2 expression was seen, and the adverse effects produced by FIZZ2 decreased. The authors concluded that FIZZ2 is a Th2-associated multifunctional mediator which plays a role in fibroblast proliferation mediated via STAT6 signaling.

Temporal Relationship:

In vitro and in vivo studies have demonstrated that Th2 cells are activated prior to fibroblast proliferation and myofibroblast differentiation.

Dong et al. (2016) demonstrated that MWCNTs activated Th2 immune responses. Male C57BL/6J mice were administered with 40 mg/mouse MWCNTs by pharyngeal aspiration for 1, 3, and 7 days.  On days 1, 3, and 7, an increase in the expression of Th2 cytokines (IL-4 and IL-3), as well as an induction of STAT6 and GATA Binding Protein 3 (GATA-3) was seen. At day 7, the presence of collagen I fibers was evident.

In another study, male Wistar rats were administered with 5 mg/kg bleomycin by intratracheal instillation. The inflammatory response was evaluated after 7, 14, and 28 days of exposure. Bleomycin increased hydroxyproline levels, total cell counts, and the expression of Nuclear Factor Kappa B (NF-κB) p65 in lung tissue. Collagen type I increased in a time-dependent manner. At day 7, the Th type 1 (Th1) response was suppressed, based on a decrease of IFN-g and an increase of IL-4 levels. Mice treated with hydrogen sulfide showed less intense effects than mice treated with bleomycin (Cao et al., 2014).

Yin et al. (2013) studied the role of IL-33 in cutaneous wound healing. Male BALB/c mice were injured on the dorsal skin and administered with murine recombinant IL-33 (1.0 mg/mouse). After 5 days of injury, M2 macrophages accumulation and mRNA expression of M2-associated genes increased (fibronectin and collagen). Collagen deposition increased in a time-dependent manner, and the percentage of wound closure and re-epithelization increased over 14 days. This study indicates that macrophage polarization, which is associated with KE4 (Event 1499) preceded the KE5 (Event 1500: fibroblast proliferation). IL-33 is essential for homeostasis and wound healing.

Wynes et al. (2004) evaluated the proliferative response associated with Th2 activation. Insulin-like growth factor-I (IGF-I) is a fibroblast growth and survival factor that has been implicated in the pathogenesis of IPF. The authors observed that mouse bone marrow-derived macrophages from C3H/HeJ mice treated with IL-4 2 ng/ml for 26 h released IGF-I. CCL39 myofibroblasts, cultured with conditioned media from IL-4-treated macrophages, consumed IGF-1 and avoided apoptosis (caspase-3 activity reduced and pro-survival kinases Protein kinase B [Akt] and extracellular signal-regulated kinase [ERK] were activated). The survival effect was lost when IGF was immunodepleted from macrophage-condition media with IGF-I-specific antibodies. These results indicate that a Th2 response conditions macrophages to release mediators which induce persistence of fibroblasts in a fibrotic setting.

Meziani et al. (2018) studied the role of large doses of radiation in promoting M2 macrophage polarization. In patients with thoracic malignancies and preoperative radiotherapy between 25 and 60 Gray (Gy), an infiltration of CD163+ macrophages was found in fibrotic areas. The pulmonary infiltration was characterized during radiation-induced lung fibrosis in a murine model. Female C57BL/6J mice were locally irradiated at the thorax with a dose rate of 1.08 Gy min^-1. A single dose of 16 Gy was locally administered to the whole thorax. Infiltrating macrophages (IMs) and alveolar macrophages were isolated post-irradiation. They observed the number of Icam1+ IMs transiently increased at day 6, and an increase number of CD206+ IMs at week 20 post-irradiation. At this time, Th1 cytokines decreased, and Metalloproteinase inhibitor 1 (TIMP-1) increased. Moreover, IMs express high levels of Arg-1. IMs were co-cultured with normal fibroblasts and increased the expression of a-SMA and TGF-b1. They found that IMs isolated from normal mouse lungs, activated in vitro with IL-13 and IL-4 for 24 h, were able to increase a-SMA levels. After 20 weeks, irradiation induced collagen deposition and an increase in the expression of TGF-b1, Plasminogen activator inhibitor 1 (PAI-1), and SMAD family member (Smad)2/3 phosphorylation in lung tissue. Depletion of tissue IMs by anti-Colony stimulating factor 1 receptor (CSF1R) after thoracic irradiation, blocks the observed effects.

Gibbons et al. (2011) studied the role of circulating monocytes and lung macrophages in the pathogenesis of lung fibrosis and the importance of M2 macrophages and Ly6Chi monocyte phenotype. Female C57Bl/6 mice were given 0.05 U bleomycin or 1X10⁸ plaque forming unit (pfu) Adenoviral TGF-b (AdTGF-b) intratracheally. Bleomycin induced early fibrosis at day 18, progressive fibrosis at day 32, or resolving fibrosis at day 56. Bleomycin increased the expression of a-SMA and Collagen type I alpha 1 chain (Col1A1) at day 25. At this time point, they also found an increase in the level of Arg activity and Ym1, and at day 32 an increase in the number of cells per field (markers of M2 macrophages). Macrophages isolated from patients with IPF showed CD163, a human marker of M2 macrophages. AdTGF-b induced an increase in BALF TGF-b at day 5, and collagen deposition at day 14. The administration of liposomal clodronate intratracheally (100 ml) at 10, 21-23 after the exposure to bleomycin or AdTGF-b decreased fibrosis, collagen deposition and M2 macrophages. The depletion of circulating monocytes reduced fibrosis and M2 macrophages. It was found that Ly6Chi inflammatory monocytes were the direct precursor of the M2 lung macrophages.

Due to multifarious functions of several cytokines involved in the process of inflammation and repair, the timing of when a pathway is intervened in an experiment is important in the assessment of the KER studies. For example, exposure to pro-fibrotic bleomycin stimulates IL-4 production during the acute inflammatory phase, which is suggested to limit the recruitment of T lymphocytes and production of damaging cytokines such as Tumor necrosis factor alpha (TNF-α), IFN-γ, and nitric oxide, playing a tissue protective role. However, production of IL- 4 during the chronic phase of tissue repair and healing, favors fibrosis manifestation. Treatment of IL4 -/- mice with low doses of bleomycin induced fewer fibrotic lesions compared to IL-4 +/+ mice. However, treatment of high doses of bleomycin induced more lethality in IL-4 -/- mice compared to the wild type mice (Huaux et al., 2003). Moreover, the KEs represented in AOP 173 can function in parallel in a positive feedback loop, perpetuating and magnifying the response at each stage. The resulting microenvironment may contain the same molecules in different proportions exhibiting different functions. Thus, the complexity of the process and the functional heterogeneity of the molecular players involved, makes it nearly impossible to establish KERs using a targeted deletion of one single gene or a pathway in a study, which is how most of the studies are designed.