Interstitial lung disease (ILD) comprises a large number of chronic lung disease characterized by varying degrees of inflammation and fibrosis. Mostly they are idiopathic including idiopathic pulmonary fibrosis (IPF), which is a specific disorder characterized by progressive fibrosis leading commonly to end-stage lung disease, respiratory failure, and fatal outcome. IPF and many of these fibrotic ILDs lack effective therapy despite recent approval of two drugs to slow progression in certain IPF patients. Because there are no natural models for IPF, the use of animal models that reproduce key known features of the disease is warranted. Thus, different animal models have been developed to investigate key mechanisms underlying pathogenesis of pulmonary fibrosis and identify potential therapeutic targets for IPF. While no animal model can recapitulate all features of human disease, several are available to address select features of IPF and other fibrotic ILDs. Historically, among the first to be developed and used widely is the bleomycin model, which is the best-characterized and currently most extensively used animal model due to its ability to reproduce many aspects of IPF and other fibrotic ILDs, good reproducibility, and ease of induction. Studies using the bleomycin model have identified many of the cellular and molecular mechanisms now recognized as being important in pathogenesis of IPF and other fibrotic ILDs, as well as novel therapies for these diseases, including two recent drugs approved for treatment of IPF. This chapter will describe commonly used techniques for induction of the model by endotracheal administration of bleomycin through surgical and nonsurgical (transoral instillation).

Amphiregulin (AREG), an epidermal growth factor receptor ligand, is implicated in tissue repair and fibrosis but its cellular source and role in regeneration vs. fibrosis remain unclear. In this study we hypothesize that AREG induced in bone marrow derived CD11c+ cells is essential for pulmonary fibrosis. Thus the objectives were to evaluate the importance and role of AREG in pulmonary fibrosis, identify the cellular source of AREG induction and analyze its regulation of fibroblast function and activation. The results showed that lung AREG expression was significantly induced in bleomycin-induced pulmonary fibrosis. AREG deficiency in knockout (KO) mice significantly diminished pulmonary fibrosis. Analysis of AREG expression in major lung cell types revealed induction in fibrotic lungs predominantly occurred in CD11c+ cells. Moreover depletion of bone marrow derived CD11c+ cells suppressed both induction of lung AREG expression and pulmonary fibrosis. Conversely, adoptive transfer of bone marrow-derived CD11c+ cells from BLM-treated donor mice exacerbated pulmonary fibrosis but not if the donor cells were made AREG-deficient prior to transfer. CD11c+ cell conditioned media or co-culture stimulated fibroblast proliferation, activation and myofibroblast differentiation in an AREG dependent manner. Furthermore recombinant AREG induced telomerase reverse transcriptase (TERT) which appeared to be essential for the proliferative effect. Finally AREG significantly enhanced fibroblast motility, which was associated with increased expression of α6 integrin. These findings suggested that induced AREG specifically in recruited bone marrow-derived CD11c+ cells promoted bleomycin induced pulmonary fibrosis by activation of fibroblast TERT dependent proliferation, motility and indirectly, myofibroblast differentiation.

Subcutaneous lipoatrophy characteristically accompanies dermal fibrosis with de novo emergence of myofibroblasts such as in systemic sclerosis or scleroderma. Recently dermal adipocytes were shown to have the capacity to differentiate to myofibroblasts in an animal model. Transforming growth factor b can induce this phenomenon in vitro; however its in vivo significance is unclear. Because found in inflammatory zone 1 (FIZZ1) is an inducer of myofibroblast differentiation but an inhibitor of adipocyte differentiation, we investigated its potential role in adipocyte transdifferentiation to myofibroblast in dermal fibrosis. FIZZ1 caused significant and rapid suppression of the expression of fatty acid binding protein 4 and peroxisome proliferator-activated receptor-g in adipocytes, consistent with dedifferentiation with loss of lipid and Oil Red O staining. The suppression was accompanied subsequently with stimulation of a-smooth muscle actin and type I collagen expression, indicative of myofibroblast differentiation. In vivo FIZZ1 expression was significantly elevated in the murine bleomycin-induced dermal fibrosis model, which was associated with significant reduction in adipocyte marker gene expression and subcutaneous lipoatrophy. Finally, FIZZ1 knockout mice exhibited significantly reduced bleomycin-induced dermal fibrosis with greater preservation of the subcutaneous fat than wild-type mice. These findings suggested that the FIZZ1 induction of adipocyte transdifferentiation to myofibroblast might be a key pathogenic mechanism for the accumulation of myofibroblasts in dermal fibrosis. (Am J Pathol 2015 http://dx

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Telomerase is typically expressed in cellular populations capable of extended replication, such as germ cells, tumor cells, and stem cells, but is also induced in tissue injury, repair and fibrosis. Its catalytic component, telomerase reverse transcriptase (TERT) is induced in lung fibroblasts from patients with fibrotic interstitial lung disease and in rodents with bleomycin-induced pulmonary fibrosis. To evaluate the fibroblast specific role of TERT in pulmonary fibrosis, transgenic mice bearing a floxed TERT allele were generated, and then crossed with an inducible collagen α2(I)-Cre mouse line to generate fibroblast specific TERT conditional knockout mice. TERT-specific deficiency in mesenchymal cells caused attenuation of pulmonary fibrosis as manifested by reduced lung hydroxyproline content, type I collagen and α-smooth muscle actin mRNA levels. The TERT-deficient mouse lung fibroblasts displayed decreased cell proliferative capacity and higher susceptibility to induced apoptosis compared with control cells. Additionally TERT deficiency was associated with heightened α-smooth muscle actin expression indicative of myofibroblast differentiation. However the impairment of cell proliferation and increased susceptibility to apoptosis would cause a reduction in the myofibroblast progenitor population necessary to mount a successful myofibroblast-dependent fibrotic response. These findings identified a key role for TERT in fibroblast proliferation and survival essential for pulmonary fibrosis.