The model of BLM-induced lung fibrosis represents the most commonly applied experimental model. BLM is a chemotherapeutic antibiotic that has been identified as a pro-fibrotic agent when lymphoma patients developed pulmonary fibrosis after intravenous administration of BLM. It has been used in multiple species including mice, rats, guinea pigs, hamsters, dogs, and primates; yet, mice are most common (21). A sheep model is also currently under development (22). A recent ATS workshop report confirmed that there is a consensus view of the intratracheal BLM model as “the best-characterized animal model available for preclinical testing” (23).

Silica administration into murine lungs leads to the development of fibrotic nodules that resemble lesions that develop in humans following exposure to mineral fibers and particulate aerosols (45). Silica delivery presents with many variations including aerosolization, intratracheal, or orotracheal instillation (46–50). The fibrotic response is strain dependent with C57BL/6 mice found to be more susceptible than CBA/J mice after intratracheal delivery of silica fibers. Nodules develop around silica deposits and silica fibers are easily identified both by histology and polarization microscopy (47). The fibrotic response is associated with limited inflammation and enhanced fibrotic lesions mediated by increased production of pro-fibrotic growth factors and cytokines including PDGF, TGF-β, TNFa, and IL-10 (51–53). Kinetics of injury is highly heterogeneous and dependent upon route of administration, dose regimens, and formulations of silica particles (45). In particular, intratracheal models are easier, shorter (fibrosis develops within 14–28 days), and cost-efficient, while aerosolized route of delivery takes longer to produce fibrotic lesions (40–120 days) (45). Heating preparation procedures before instillation are mandatory in order to inactivate any trace endotoxin (45). The greatest advantage of the silica model of lung fibrosis is the persistence of fibrotic lesions due to diminished clearance of silica particles from the lungs (51, 52, 54). However, the model presents with major caveats including problematic and highly expensive equipment for aerosolized delivery, prolonged waiting periods until development of fibrosis (4–16 weeks), lack of reproducibility of fibrotic pattern, and absence of characteristic UIP-like lesions such as fibroblastic foci, regional heterogeneity, and hyperplastic epithelium. These have severely limited its widespread applicability in the preclinical setting (20).

Another model that recapitulates an important form of human lung fibrosis is that of asbestos exposure. Asbestos-induced model of lung fibrosis is clearly distinguished from IPF by several histologic findings including asbestos bodies embedded within the fibrous tissue, fewer myofibroblasts foci and bronchial wall fibrosis. In some cases, the pattern of UIP can be also present (55, 56). Some of these features are recapitulated in inhalation models in animals and have helped us understand the pathogenesis of both asbestosis and IPF (57–61). A single intratracheal administration of asbestos fibers mediates development of fibrosis; however, the model presents with several caveats since fibrosis that tends to be central rather than subpleural and is quite often unevenly distributed between lungs. Inhalation models develop a more peripheral pattern; yet, disease development can be prolonged, especially if using chrysotile fibers. The intratracheal animal models with amphibole fibers follow the kinetics of BLM models, with fibrosis development at day 7 and peak at day 14. Inhalation models may take up to a month for establishment of fibrotic injuries. Mechanistically, the deposition of asbestos fibers triggers fibrosis through alveolar epithelial cell apoptosis, M2 polarization of macrophages, and overproduction of pro-fibrotic cytokines by activated T lymphocytes, all events leading to myofibroblast differentiation and extracellular matrix production (57–61).

IPF is an age-related disease paradigm, and more recently, it has been proposed that many of the hallmarks of aging including genomic instability, telomere attrition, epigenetic alterations, deregulated cellular bioenergetics, and cellular senescence, can be considered characteristics of the fibrotic lung (62–64). Studies have shown that older mice are more susceptible than younger mice to pro-fibrotic stimuli including BLM (26). This is of particular interest given that IPF is predominant in older individuals. Transgenic deletion of senescence-related genes including RAGE, and relaxin has been associated with spontaneous age-dependent development of lung fibrosis indicating the cardinal role of aging in disease susceptibility (65–67). On the other hand, the role of “virome” as a pro-fibrotic mediator has been further dissected in the context of aged-related development of lung fibrosis by demonstrating that only aged mice (>15 months) develop γ-herpesvirus-68-induced lung fibrosis through a mechanism that involved alveolar epithelial cell reprogramming to produce pro-fibrotic factors and enhanced TGF-β signaling in lung fibroblasts (68). In addition, Torres-Gonzalez et al. (69) reported that aging mice receiving gamma herpesvirus responded with endoplasmic reticulum stress, apoptosis of type II lung epithelial cells, and activation of profibrotic pathways.

This evidence could be reminiscent to the presence of herpes viral genomes within IPF lungs and the epidemiological association between viral infections and IPF acute exacerbations (70).

During the past two decades, more sophisticated and advanced methods have been widely used to study the features of lung fibrosis on an experimental setting. Both gene transfer via adenoviral or lentiviral vectors and transgenic approaches have been used to overexpress pro-fibrotic cytokines including TGF-β, TNF-α, IL-1β, and IL-13 and promote fibrotic phenotypes by dissecting downstream signaling pathways that are highly relevant to human lung fibrosis (71–73). The overexpression of TGF-β can be produced via adenoviral intranasal delivery or doxycycline-induced transgenic overexpression in CC-10-positive lung epithelial cells. Both models are strain dependent with C57BL/6 mice being more susceptible that BALB/c. In the doxycycline-inducible Clara cell (CC10)-promoter driven model of TGF-β-induced lung fibrosis, addition of doxycycline to the water of animals leads to release of the tetracycline-controlled transcriptional suppressor allowing the reverse tetracycline transactivator to bind to the transgene (TGF-β) and promote its acute expression even 12 h after treatment with doxycycline (74). That leads to alveolar epithelial apoptosis and myofibroblast accumulation leading to airway and parenchymal fibrotic response starting at day 7 and peaking at days 14–21. Fibrosis may persist and progress over the duration of doxycycline exposure for up to 2 months (74). Similar kinetics are also observed with adenoviral delivery of TGF-β through the intranasal route leading to epithelial cell apoptosis (day 1), mononuclear cell infiltration (days 3–7), and fibrotic scarring that tends to be more persistent than those produced by BLM exposure and thus tend to mimic better human disease features (71). Nevertheless, both models quite often produce highly variable and heterogeneous kinetics of injury with regards to severity and extent of lesions and lack of major reproducibility. A similar concept has been also applied for adenoviral-mediated gene transfer of IL-1β (75) and TNF-α (76, 77) or lung-specific transgenic overexpression of IL-13 (78), thereby resulting in an early inflammatory response and later collagen deposition through activation of TGF-β signaling pathway. Nonetheless, these models are not well established and thus can only be used to dissect relevant pathogenic pathways and not on a general basis to recapitulate the complexity of human disease.

Fluorescent isothiocyanate (FITC) is another chemical compound used to induce experimental lung fibrosis (79). Fluorescein acts as a hapten and binds to airway proteins, thus acting as a depot for prolonged exposure to the injurious stimulus leading to fibrotic responses within 2–4 weeks that persist up to 24 weeks (79, 80). The model produces relatively reproducible and persistent fibrotic phenotypes in both BALB/c and C57BL/6 mice. Disadvantages include absence of representative UIP findings and predominant inflammatory infiltrates that precede fibrosis (80). The model is mostly dependent on Th2 cytokines (IL-13) and was seminally discovered to explore the relationship between the chemokine signaling receptor 2 (CCR2) and its ligand CCL12 for recruitment of fibrocytes during progression of fibrosis (81). It offers the advantage of easily trackable fluorescence-labeled fibrotic tissues. Nevertheless, model robustness is largely dependent on technical issues that are highly variable including the batch of FITC and the size of the particles formulated through sonication (20). Smaller particle sizes due to prolonged sonication may lead to acute toxicity and death (20). Finally and most important, FITC is an artificial chemical compound with limited human relevance since this type of injurious stimulus has never been described in humans (20).

Radiation-induced fibrosis represents a human relevant injury that leads to development of fibrosis which is strain dependent (C57BL/6 are the most susceptible) and can be local or systemic if other organs are not shielded (82–87). It is a relatively slow procedure that results in mature fibrosis after 24 weeks; yet, fibrosis is majorly dependent on inflammation and free-radical-mediated DNA damage and less on TGF-β (84).

Familial models of IPF have been also used to study the contribution of the disease genetic background that significantly altered our perspective regarding disease pathogenesis and treatment response. Mice with targeted deletion of shelterin, a six-protein complex that binds and preserves telomeric repeats, from type alveolar epithelial cells, have been shown to develop spontaneous fibrosis (88, 89).

Mutations in the telomere and telomerase genes have been associated with familial IPF (90). Telomere dysfunction results in alveolar epithelial stem cell senescence, which is sufficient to drive lung remodeling and recruit inflammation. Telomerase reverse transcriptase has been reported to be transiently increased in BLM, hypoxic, or silica-induced lung injury (91–93). On the other hand, telomerase-deficient mice, despite significant telomere shortening, did not present with enhanced BLM-induced fibrotic responses (94).

Although mutations resulting in SP-C deficiencies are linked to a small subset of spontaneous and familial cases of interstitial lung disease (ILD) and interstitial pulmonary fibrosis (95, 96), SP-C-deficient mice do not fully recapitulate familial interstitial pulmonary fibrosis (97) as they develop mild ILD and an emphysematous phenotype. It is more than evident that these mutations may generate a susceptible phenotype; yet, a second hit of environmental origin is needful to partially recapitulate human phenotype.

Finally, humanized models of lung fibrosis involving the intravenous instillation of human IPF lung fibroblasts into immunodeficient non-obese diabetic mice (NOD/SCID) have recently garnered much attention (98–100). This model allows for cell trafficking during different stages of fibrosis development and progression, offers unique insights into different fibroblast populations that reflect IPF heterogeneity, and dissects the contribution of epithelial–fibroblast crosstalk into the disease pathogenesis considering the absence of immune cells (99). Nevertheless, the latter is not representative of human disease where immune cells appear to play cardinal role. Another major disadvantage that limits its widespread applicability is the high cost and the specialized housing that is required (101). The use of animals with humanized immune system may also provide unique insights and fully recapitulate features of IPF (101).

Each experimental model presents with its own kinetics of fibrotic injury; however, investigators have applied standard operating procedures for reproducible evaluations of lung fibrosis. In view of the pathologic hallmarks of IPF, appropriate read-out assays include assessment of collagen deposition, alveolar epithelial cell apoptosis, and BALF complemented by survival analysis and respiratory mechanics. These are achieved with the following modalities: (1) histological analysis with Masson trichrome and H&E staining coupled with Aschroft score that quantifies extent of fibrotic changes, (2) hydroxyproline or total collagen content for quantification of lung collagen deposition, (3) TUNEL assay for the identification of apoptotic cells, (4) BALF analysis to assess changes in differential cell count and levels of inflammatory and fibrotic markers, (5) survival analysis with Kaplan–Meier plots, (6) in vivo lung function measurements (elastance and compliance) using the Flexi-vent ventilator, and (7) micro-CT imaging which provides state-of-the art multidimensional imaging of the injured lung that is reminiscent of HRCT applied for IPF diagnosis (19).

The past 35 years more than 500 experimental studies have been performed describing therapeutic efficacy of novel compounds in the BLM model. Unfortunately, less than 5% have applied a therapeutic protocol indicated by drug administration at >7 days following BLM challenge (18, 114). Even day 7 in most of the experimental models represents a stage of inflammation or early fibrosis, evidence that comes in contrast to the clinical situation in which treatment is initiated after onset of symptoms and when fibrosis has already been established. Intriguingly, pirfenidone and nintedanib received approval to proceed to clinical trials based on preventive protocols or even therapeutic protocols targeting the inflammatory or the early-fibrotic phase of the BLM model (115–117). In addition, most of the therapeutic compounds have been preclinically tested in young animals while it has been clearly shown that aged mice are more susceptible to fibrotic injury (26), which is in accordance with patients with IPF. Importantly, preclinical efficacy of the majority of anti-fibrotic agents was tested in a single model, majorly the BLM-induced model, and based on histologic end points, such as collagen deposition that are not clinically relevant, at least to the extent of lung function tests or survival analysis. Moreover, many of the therapeutic outcomes were subject to evaluation bias considering that most of the preclinical studies were not blinded and the investigator was aware of the animals’ treatment. Reproducibility issues arising from different experimental settings between different labs could also account for discrepancies in treatment effects and lack of generalizable results. Finally, it is worth mentioning that animal size needs to be balanced with the statistical power needed to generate robust data and that insufficient reporting of experimental animal data or unpublished negative therapeutic results severely hamper the validity of experimental studies.

An animal model is a simple representation of a complex biology system. Critics focusing on the reasons why an animal model cannot fully reproduce human disease are not helpful and do not elicit a solution. The role of an animal model is to recapitulate specific aspects of a disease. Consequently, animal models should be carefully selected, designed, and conducted in order to bridge translational gaps between bench and bedside. Currently, the BLM model of lung fibrosis represents the cheapest, easiest, fastest, most reproducible, and thus most extensively used animal model of IPF; advantages that overcome the handicap of minimal representation of human disease. So far, it has provided us with invaluable insights into IPF pathogenesis, prognosis, and treatment. We recommend the use of the male aged BLM mouse model as the first-line animal model to test safety and efficacy of a therapeutic compound administered during the stage of established fibrosis. Nevertheless, efficacy preclinical studies should be enriched with two or even three animal models including clinically relevant end points such as lung function mechanics, survival analysis as well as biomarkers (28). Collaboration between veterinary and human clinical researchers must be encouraged in order to establish and solidify a common language, and common diagnostic criteria and nomenclature, thus strengthening the opportunity for advancements toward a cure for lung fibrosis in both animal and humans. Humanization of animal models, spontaneous fibroses animals and application of high-throughput “omics” tools may help us improve the clinical translation in the near future.