Experimental Model Of Lung Fibrosis Research Articles

Updates on the controversial roles of regulatory lymphoid cells in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and severe form of pulmonary fibrosis, characterized by scar formation in the lung interstitium. Transforming growth factor beta (TGF-β) is known as a key mediator in the fibrotic process, acting on fibroblasts and mediating their proliferation and differentiation into myofibroblasts. Although the immune system is not considered responsible for the initiation of IPF, markers of tolerogenic immunity define the pro-fibrotic microenvironment in the lungs. In homeostatic conditions, regulatory T cells (Tregs) constitute the main lymphoid population responsible for maintaining peripheral tolerance. Similar to Tregs, regulatory B cells (Bregs) represent a recently described subset of B lymphocytes with immunosuppressive functions. In the context of IPF, numerous studies have suggested a role for Tregs in enhancing fibrosis, mainly via the secretion of TGF-β. In humans, most studies show increased percentages of Tregs associated with the severity of IPF, although their exact role remains unclear. In mice, the most commonly used model involves triggering acute lung inflammation with bleomycin, leading to a subsequent fibrotic process. Consequently, data are still conflicting, as Tregs may play a protective role during the inflammatory phase and a deleterious role during the fibrotic phase. Bregs have been less studied in the context of IPF, but their role appears to be protective in experimental models of lung fibrosis. This review presents the latest updates on studies exploring the implication of regulatory lymphoid cells in IPF and compares the different approaches to better understand the origins of conflicting findings.

Vascular-Parenchymal Cross-Talk Promotes Lung Fibrosis through BMPR2 Signaling.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive lung scarring. IPF-related pulmonary vascular remodeling and pulmonary hypertension (PH) result in a particularly poor prognosis. Objectives: To study the pathogenesis of vascular remodeling in fibrotic lungs and its contribution to progression of fibrosis. Methods: We used an experimental model of lung fibrosis associated with PH by transient overexpression of active TGF-β1 (transforming growth factor-β1). Samples from patients with fibrotic lung diseases were analyzed in depth using immunostaining, gene expression, and gene mutations. Measurements and Main Results: We found a reduction in endothelial cells (ECs) and activation of vascular smooth muscle cells (VSMCs) in fibrotic lungs. Coculturing fibroblasts with VSMCs or ECs from fibrotic lungs induced fibrotic phenotypes in fibroblasts. IPF fibroblasts induced EC death and activation of VSMCs in coculture systems. Decreased concentrations of BMPR2 (bone morphogenic protein receptor 2) and its signaling were observed in ECs and VSMCs from fibrotic lungs in both rats and humans. On fibroblasts treated with media from VSMCs, BMPR2 suppression in VSMCs led to fibrogenic effects. Tacrolimus activated BMPR2 signaling and attenuated fibrosis and PH in rodent lungs. Whole-exome sequencing revealed rare mutations in PH-related genes, including BMPR2, in patients with IPF undergoing transplantation. A unique missense BMPR2 mutation (p.Q721R) was discovered to have dysfunctional effects on BMPR2 signaling. Conclusions: Endothelial dysfunction and vascular remodeling in PH secondary to pulmonary fibrosis enhance fibrogenesis through impaired BMPR2 signaling. Tacrolimus may have value as a treatment of advanced IPF and concomitant PH. Genetic abnormalities may determine the development of PH in advanced IPF.

B Cells Are Not Involved in the Regulation of Adenoviral TGF-β1- or Bleomycin-Induced Lung Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is an irreversible, age-related diffuse parenchymal lung disease of poorly defined etiology. Many patients with IPF demonstrate distinctive lymphocytic interstitial infiltrations within remodeled lung tissue with uncertain pathogenetic relevance. Histopathological examination of explant lung tissue of patients with IPF revealed accentuated lymphoplasmacellular accumulations in close vicinity to, or even infiltrating, remodeled lung tissue. Similarly, we found significant accumulations of B cells interfused with T cells within remodeled lung tissue in two murine models of adenoviral TGF-β1 or bleomycin (BLM)-induced lung fibrosis. Such B cell accumulations coincided with significantly increased lung collagen deposition, lung histopathology, and worsened lung function in wild-type (WT) mice. Surprisingly, B cell-deficient µMT knockout mice exhibited similar lung tissue remodeling and worsened lung function upon either AdTGF-β1 or BLM as for WT mice. Comparative transcriptomic profiling of sorted B cells collected from lungs of AdTGF-β1- and BLM-exposed WT mice identified a large set of commonly regulated genes, but with significant enrichment observed for Gene Ontology terms apparently not related to lung fibrogenesis. Collectively, although we observed B cell accumulations in lungs of IPF patients as well as two experimental models of lung fibrosis, comparative profiling of characteristic features of lung fibrosis between WT and B cell-deficient mice did not support a major involvement of B cells in lung fibrogenesis in mice.

PTX3 Regulation of Inflammation, Hemostatic Response, Tissue Repair, and Resolution of Fibrosis Favors a Role in Limiting Idiopathic Pulmonary Fibrosis.

PTX3 is a soluble pattern recognition molecule (PRM) belonging to the humoral innate immune system, rapidly produced at inflammatory sites by phagocytes and stromal cells in response to infection or tissue injury. PTX3 interacts with microbial moieties and selected pathogens, with molecules of the complement and hemostatic systems, and with extracellular matrix (ECM) components. In wound sites, PTX3 interacts with fibrin and plasminogen and favors a timely removal of fibrin-rich ECM for an efficient tissue repair. Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown origin, associated with excessive ECM deposition affecting tissue architecture, with irreversible loss of lung function and impact on the patient’s life quality. Maccarinelli et al. recently demonstrated a protective role of PTX3 using the bleomycin (BLM)-induced experimental model of lung fibrosis, in line with the reported role of PTX3 in tissue repair. However, the mechanisms and therapeutic potential of PTX3 in IPF remained to be investigated. Herein, we provide new insights on the possible role of PTX3 in the development of IPF and BLM-induced lung fibrosis. In mice, PTX3-deficiency was associated with worsening of the disease and with impaired fibrin removal and subsequently increased collagen deposition. In IPF patients, microarray data indicated a down-regulation of PTX3 expression, thus suggesting a potential rational underlying the development of disease. Therefore, we provide new insights for considering PTX3 as a possible target molecule underlying therapeutic intervention in IPF.

Beta Adrenoceptor Ligands for the Treatment of Group 3 Pulmonary Hypertension and Cor Pulmonale: A Novel Therapeutic Target?

Pulmonary hypertension is a common complication of idiopathic pulmonary fibrosis (IPF), which leads to cor pulmonale and right-heart failure. β-blockers have been used extensively for the treatment of left heart disease, yet their use for right-heart failure is controversial. Recent studies have shown that β-blockers may be beneficial for the treatment of pulmonary arterial hypertension. However, whether β-receptors ligands are effective in PH and cor pulmonale associated with lung fibrosis has not been evaluated. Here we tested the effects of β adrenergic receptors (βAR) ligands in an experimental model of lung fibrosis and PH where fibrotic injury and cardiovascular complications co-exist. Using lung tissues isolated from explanted lungs from patients with IPF (with and without a diagnosis of PH), we determined βAR gene expression levels. Next, the capacity of either carvedilol (a βAR blocker) or clenbuterol (a β2AR agonist) to improve bleomycin (BLM)-induced alterations in cardiovascular physiology or lung function were assessed. These experiments were performed using lung function and transthoracic Echocardiography (Fig 1). After completion of these physiological readouts, the lungs were excised for assessment of fibrotic injury by RT-PCR, immunoblots and histology. Our experiments demonstrate reduced expression levels of the β2AR, but not β1AR or β3AR in IPF lung vs controls. We also report a significant correlation between elevated mPAP and reduced β2AR expression. Remarkably, our results using carvedilol or clenbuterol revealed that these agents improved right-heart function without impacting lung function. Taken together, these results demonstrate that β-receptor ligands are able to treat PH and cor pulmonale associated with lung fibrosis without altering fibrotic matrix deposition. These results are significant since PH is the single most significant predictor of mortality in IPF where limited therapies exist.

Alterations in cardiovascular function in an experimental model of lung fibrosis and pulmonary hypertension.

What is the central question of this study? We have evaluated changes in cardiovascular physiology using echocardiography in an experimental model of lung fibrosis. What is the main finding and its importance? Remarkably, we report changes in cardiovascular function as early as day7, concomitant with evidence of vascular remodelling. We also report that isolated pulmonary arteries were hypercontractile in response to a thromboxane A2 agonist. These findings are significant because the development of pulmonary hypertension is one of the most significant predictors of mortality in patients with lung fibrosis, where there are no available therapies and a lack of animal models. Group III pulmonary hypertension is observed in patients with chronic lung diseases such as chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis. Pulmonary hypertension (PH) develops as a result of extensive pulmonary vascular remodelling and resultant changes in vascular tone that can lead to right ventricle hypertrophy. This eventually leads to right heart failure, which is the leading indicator of mortality in patients with idiopathic pulmonary fibrosis. Treatments for groupIII PH are not available, in part owing to a lack of viable animal models. Here, we have evaluated the cardiovascular changes in a model of lung fibrosis and PH. Data obtained from this study indicated that structural alterations in the right heart, such as right ventricular wall hypertrophy, occurred as early as day14, and similar increases in right ventricle chamber size were seen between days21 and 28. These structural changes were correlated with decreases in the systolic function of the right ventricle and right ventricular cardiac output, which also occurred between the same time points. Characterization of pulmonary artery dynamics also highlighted that PH might be occurring as early as day21, indicated by reductions in the velocity-time integral; however, evidence for PH is apparent as early as day 7, indicated by the significant reduction in pulmonary acceleration time values. These changes are consistent with evidence of vascular remodelling observed histologically starting on day 7. In addition, we report hyperactivity of bleomycin-exposed pulmonary arteries to a thromboxaneA2 receptor (Tbxa2r) agonist.

Comparative study of the effects of mesenchymal stem cells with different delivery methods in an experimental model of lung fibrosis

Background: Transplantation of mesenchymal stem cells (MSCs) is one of the most promising directions in the treatment of idiopathic pulmonary fibrosis. In experimental small animal studies, intravenous and endobronchial (installation) techniques are used for the cell preparation delivery, while in humans inhalation of drugs is the simplest and most available method.
 Aim: The aim of the study was to determine the optimal type of a nebulizer for viability of MSCs during nebulization, followed by a comparison of the effects of inhalation and intravenous delivery methods in a standard model of bleomycin pulmonary fibrosis in rabbits.
 Methods: At the first stage, the survival of MSCs was assessed ex vivo after 10 minutes of compressor, ultrasound and mesh nebulization. Subsequently we used a nebulizer, which showed the best result in the cells, viability. At the next stage аfter bronchoscopic installation of bleomycin, 5 rabbits received intravenous transplantation of 2×106 allogeneic BMMSCs, other 5 rabbits — 2×107 MSCs inhaled via a compressor nebulizer; the control healthy and bleomycin groups included 5 animals each.
 Results: The highest degree of viability of MSC was maintained after passing via the compressor nebulizer (72%), a significantly lower survival rate was observed in ultrasonic nebulization (20%) and no live cells were detected after mesh nebulization. Both groups treated with MSC had a significantly lower fibrosis index on the Ashcroft morphometric scale than the control group of bleomycin fibrosis. Collagen expression in the lung tissue was significantly higher in all the groups with bleomycin injury, but in animals which underwent MSC inhalation, it was significantly different (0.51 point) from the bleomycin group without treatment (2.1 points). The level of neutrophils in the BAL fluid was significantly lower in animals which received the intravenous MSC therapy. The levels of TNF-α and TGF-β1 in the BAL fluids tended to decrease in the treatment groups, but did not differ significantly from control.
 Conclusions: The highest survival rate of MSCs is observed when using a compressor nebulizer, which apparently should be considered as the best way for delivering cells to the respiratory tract. Both inhalation and intravenous administration of MSCs cause similar effects of inhibiting the development of bleomycin-induced pulmonary fibrosis, which indicates the possibility of using both ways of cell delivery without loss of effectiveness.

12 - Oxidation state of peroxiredoxin 4 in lungs from patients with pulmonary fibrosis and mouse models of fibrosis

Rationale Every year 34,000 people in the United States are diagnosed with Idiopathic Pulmonary Fibrosis (IPF). The average survival time for these patients is only 3-5 years after diagnosis. Recently we have shown that the overexpression of the ER localized peroxidase, Peroxiredoxin 4 (Prdx4) attenuates murine type II alveolar cells cell death and decreases collagen deposition in mouse models of pulmonary fibrosis. The goal of this study is to understand the redox status of Prdx4 in IPF and experimental models of lung fibrosis. Methods C57B6 mice were oropharyngeal instilled with bleomycin. After the establishment of pulmonary fibrosis Prdx4 was overexpressed by instillation of Adeno-Peroxiredoxin 4 (AdPrdx4). Mouse lung tissue as well as samples from IPF patients were analyzed for Prdx4 content and redox status. Results The overexpression of Prdx4 in mice with bleomycin-induced fibrosis resulted in a significant decrease in collagen content in mouse lungs overexpressing Prdx4 compared to mice receiving AdCtrl. Lung tissue from mice with bleomycin-induced fibrosis and patients with IPF showed altered Prdx4 oxidation states, with a shift towards high molecular weight complexes. A dramatic increase in only high molecular weight Prdx4 protein was observed in the bronchoaveolar lavage fluid from mice with bleomycin-induced fibrosis, along with increases in overoxidized Prdx (Prdx-SO3), suggesting that overoxidized high molecular weight Prdx4 complexes are selectively released from cells in models of pulmonary fibrosis. Conclusion This data reveals that an altered redox status of Prdx4 occurs in IPF and mouse models of pulmonary fibrosis and that augmentation of Prdx4 has a potential positive therapeutic relevance in pulmonary fibrosis.

Low‐dose administration of bleomycin leads to early alterations in lung mechanics

What is the central question of this study? When do alterations in pulmonary mechanics occur following chronic low-dose administration of bleomycin? What is the main finding and its importance? Remarkably, we report changes in lung mechanics as early as day7 that corresponded to parameters determined from single-frequency forced oscillation manoeuvres and pressure-volume loops. These changes preceded substantial histological changes or changes in gene expression levels. These findings are significant to refine drug discovery in idiopathic pulmonary fibrosis, where preclinical studies using lung function parameters would enhance the translational potential of drug candidates where lung function readouts are routinely performed in the clinic. Idiopathic pulmonary fibrosis (IPF) is the most widespread form of interstitial lung disease and, currently, there are only limited treatment options available. In preclinical animal models of lung fibrosis, the effectiveness of experimental therapeutics is often deemed successful via reductions in collagen deposition and expression of profibrotic genes in the lung. However, in clinical studies, improvements in lung function are primarily used to gauge the success of therapeutics directed towards IPF. Therefore, we examined whether changes in respiratory system mechanics in the early stages of an experimental model of lung fibrosis can be used to refine drug discovery approaches for IPF. C57BL/6J mice were administered bleomycin (BLM) or a vehicle control i.p. twice a week for 4weeks. At 7, 14, 21, 28 and 33days into the BLM treatment regimen, indices of respiratory system mechanics and pressure-volume relationships were measured. Concomitant with these measurements, histological and gene analyses relevant to lung fibrosis were performed. Alterations in respiratory system mechanics and pressure-volume relationships were observed as early as 7days after the start of BLM administration. Changes in respiratory system mechanics preceded the appearance of histological and molecular indices of lung fibrosis. Administration of BLM leads to early changes in respiratory system mechanics that coincide with the appearance of representative histological and molecular indices of lung fibrosis. Consequently, these data suggest that dampening the early changes in respiratory system mechanics might be used to assess the effectiveness of experimental therapeutics in preclinical animal models of lung fibrosis.

Phosphatidylethanolamine Induces an Antifibrotic Phenotype in Normal Human Lung Fibroblasts and Ameliorates Bleomycin-Induced Lung Fibrosis in Mice

Lung surfactant is a complex mixture of phospholipids and specific proteins but its role in the pathogenesis of interstitial lung diseases is not established. Herein, we analyzed the effects of three representative phospholipid components, that is, dipalmitoilphosphatidylcoline (DPPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), on collagen expression, apoptosis and Ca2+ signaling in normal human lung fibroblasts (NHLF) and probed their effect in an experimental model of lung fibrosis. Collagen expression was measured with RT-PCR, apoptosis was measured by using either the APOPercentage assay kit (Biocolor Ltd., Northern Ireland, UK) or the Caspase-Glo 3/7 assay (Promega, Madison, WI, USA) and Ca2+ signaling by conventional epifluorescence imaging. The effect in vivo was tested in bleomycin-induced lung fibrosis in mice. DPPC and PG did not affect collagen expression, which was downregulated by PE. Furthermore, PE promoted apoptosis and induced a dose-dependent Ca2+ signal. PE-induced Ca2+ signal and apoptosis were both blocked by phospholipase C, endoplasmic reticulum pump and store-operated Ca2+ entry inhibition. PE-induced decrease in collagen expression was attenuated by blocking phospholipase C. Finally, surfactant enriched with PE and PE itself attenuated bleomycin-induced lung fibrosis and decreased the soluble collagen concentration in mice lungs. This study demonstrates that PE strongly contributes to the surfactant-induced inhibition of collagen expression in NHLF through a Ca2+ signal and that early administration of Beractant enriched with PE diminishes lung fibrosis in vivo.

Role of Peroxiredoxin 4 in Collagen Deposition in Mouse Models of Fibrosis

Rationale: Every year 34,000 people in the USA are diagnosed with Idiopathic Pulmonary Fibrosis (IPF). The average survival time for these patients is only 3-5 years after diagnosis. Lung epithelial cell injury via the Fas cell death pathway has been shown to be important in the progression of IPF. Fas s-glutathionylation enhances the apoptotic signal in murine type II alveolar cells. Recently we have shown that the overexpression of the ER localized peroxidase, Peroxiredoxin 4 (Prdx4) attenuates Fas s-glutathionylation and cell death in murine type II alveolar cells. The goal of this study is to understand the role of Prdx4 in experimental models of lung fibrosis. C57B6 mice were oropharyngeal instilled with Bleomycin or AdTGFb1. After the establishment of pulmonary fibrosis Prdx4 was overexpressed by instillation of Adeno-Peroxiredoxin 4 (AdPrdx4). One week after the administration of AdPrdx4 mice lungs were harvested to determine extent of fibrosis and oxidation state of Fas. The overexpression of Prdx4 was confirmed in mouse lung homogenates by qRT-PCR, western blot and immunohistochemistry. Hydroxyproline analysis of mouse lungs showed a significant decrease in collagen content in mouse lungs overexpressing Prdx4 compared to mice receiving AdCtrl. Similarly, expression of alpha smooth muscle actin, a marker for fibroblast activation, was attenuated following administration of AdPrdx4. This data reveals that augmentation of Prdx4 has a potential positive therapeutic relevance in pulmonary fibrosis.

Impaired anti-fibrotic effect of bone marrow-derived mesenchymal stem cell in a mouse model of pulmonary paracoccidioidomycosis.

Bone marrow-derived mesenchymal stem cells (BMMSCs) have been consider as a promising therapy in fibrotic diseases. Experimental models suggest that BMMSCs may be used as an alternative therapy to treat chemical- or physical-induced pulmonary fibrosis. We investigated the anti-fibrotic potential of BMMSCs in an experimental model of lung fibrosis by infection with Paracoccidioides brasiliensis. BMMSCs were isolated and purified from BALB/c mice using standardized methods. BALB/c male mice were inoculated by intranasal infection of 1.5x106 P. brasiliensis yeasts. Then, 1x106 BMMSCs were administered intra venous at 8th week post-infection (p.i.). An additional group of mice was treated with itraconazole (ITC) two weeks before BMMSCs administration. Animals were sacrificed at 12th week p.i. Histopathological examination, fibrocytes counts, soluble collagen and fibrosis-related genes expression in lungs were evaluated. Additionally, human fibroblasts were treated with homogenized lung supernatants (HLS) to determine induction of collagen expression. Histological analysis showed an increase of granulomatous inflammatory areas in BMMSCs-treated mice. A significant increase of fibrocytes count, soluble collagen and collagen-3α1, TGF-β3, MMP-8 and MMP-15 genes expression were also observed in those mice. Interestingly, when combined therapy BMMSCs/ITC was used there is a decrease of TIMP-1 and MMP-13 gene expression in infected mice. Finally, human fibroblasts stimulated with HLS from infected and BMMSCs-transplanted mice showed a higher expression of collagen I. In conclusion, our findings indicate that late infusion of BMMSCs into mice infected with P. brasiliensis does not have any anti-fibrotic effect; possibly because their interaction with the fungus promotes collagen expression and tissue remodeling.

Inhibition of hyaluronan synthesis attenuates pulmonary hypertension associated with lung fibrosis.

Group III pulmonary hypertension (PH) is a highly lethal and widespread lung disorder that is a common complication in idiopathic pulmonary fibrosis (IPF) where it is considered to be the single most significant predictor of mortality. While increased levels of hyaluronan have been observed in IPF patients, hyaluronan-mediated vascular remodelling and the hyaluronan-mediated mechanisms promoting PH associated with IPF are not fully understood. Explanted lung tissue from patients with IPF with and without a diagnosis of PH was used to identify increased levels of hyaluronan. In addition, an experimental model of lung fibrosis and PH was used to test the capacity of 4-methylumbeliferone (4MU), a hyaluronan synthase inhibitor to attenuate PH. Human pulmonary artery smooth muscle cells (PASMC) were used to identify the hyaluronan-specific mechanisms that lead to the development of PH associated with lung fibrosis. In patients with IPF and PH, increased levels of hyaluronan and expression of hyaluronan synthase genes are present. Interestingly, we also report increased levels of hyaluronidases in patients with IPF and IPF with PH. Remarkably, our data also show that 4MU is able to inhibit PH in our model either prophylactically or therapeutically, without affecting fibrosis. Studies to determine the hyaluronan-specific mechanisms revealed that hyaluronan fragments result in increased PASMC stiffness and proliferation but reduced cell motility in a RhoA-dependent manner. Taken together, our results show evidence of a unique mechanism contributing to PH in the context of lung fibrosis.

Photobiomodulation therapy improves both inflammatory and fibrotic parameters in experimental model of lung fibrosis in mice.

Lung fibrosis (LF) is a chronic and progressive lung disease characterized by pulmonary parenchyma progressive lesion, inflammatory infiltration, and interstitial fibrosis. It is developed by excessive collagen deposition and other cellular matrix components, resulting in severe changes in the alveolar architecture. Considering the absence of effective treatment, the aim of this study was to investigate the effect of photobiomodulation therapy (PBMT) on the development of PF. For this purpose, we used C57BL6 mice subjected to induction of LF by bleomycin administration (1.5U/kg) by orotracheal route and, after 14days of the induction, mice were treated with PBMT applied to the thorax 1×/day for 8days (wavelength 660±20nm, power 100mW, radiant exposure 5J/cm2, irradiance 33.3mW/cm2, spot size 2.8cm2, total energy 15J, time of irradiation: 150s) and inflammatory and fibrotic parameters were evaluated with or without PBMT. Our results showed that PBMT significantly reduced the number of inflammatory cells in the alveolar space, collagen production, interstitial thickening, and static and dynamic pulmonary elastance. In addition, we observed reduced levels of IL-6 e CXCL1/KC released by pneumocytes in culture as well as reduced level of CXCL1/KC released by fibroblasts in culture. We can conclude that the PBMT improves both inflammatory and fibrotic parameters showing a promising therapy which is economical and has no side effects.

TMPRSS4: a novel serine protease involved in IPF development?

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal lung disease characterized by the expansion of the fibroblast population and excessive extracellular matrix accumulation with the subsequent destruction of the lung architecture. Its pathogenesis is incompletely understood but evidence suggests an epithelial pathway in which IPF may result from epithelial microinjuries and activation and abnormal wound healing. TMPRSS4 is a novel type II transmembrane serine protease found at the cell surface. Although their biological functions are unknown it has been suggested that TMPRSS4 promotes migration and facilitates the epithelial to mesenchymal transition (EMT), two critical processes in the pathogenesis of IPF. Thus, we hypothesized that overexpression of TMPRSS4 in the lung could promote the initiation and/or progression of IPF.AimTo determine the role of TMPRSS4 in IPF.MethodsWe first examined the lung expression and localization of TMPRSS4 in IPF and normal lungs by qPCR and immunohistochemistry. Additionally, we measured the expression of TMPRSS4 in human lung fibroblasts and one alveolar and one bronchial epithelial cell lines. Then we explored the role of this protease in an experimental model of lung fibrosis. TMPRSS4 haplodeficient and wild type mice received an intratracheal instillation of bleomycin and were studied at 28 days post‐instillation.ResultsWe found that this protease is not expressed in normal human lungs while variable amounts of the mRNA were found in IPF lungs (2−DCT 1.1×10−2 ± 9.4×10−5). TMPRSS4 was primarily expressed by epithelial and mast cells. The protease was not found in lung fibroblasts but different levels of mRNA were revealed in alveolar (2−DCT 7.5×10−7 ± 2×10−7) and bronchial (2−DCT 4.2×10−5 ± 3.6×10−6) epithelial cells. The lung fibrotic response, measured by hydroxyproline ( 178.9 ± 12.4 versus 110.7 ± 13.8 mg/lung; p<0.05) and morphology (40% versus 15% extent of the lesions) was significantly attenuated in the haplodeficient mice.ConclusionsOur findings indicate that TMPRSS4 may have a profibrotic role in IPF and other fibrotic lung disorders.Support or Funding InformationPosgrado en Ciencias Biológicas, UNAM; CONACYT SALUD‐2014‐01‐234088

Antifibrotic and Anti-inflammatory Activity of the Tyrosine Kinase Inhibitor Nintedanib in Experimental Models of Lung Fibrosis

The tyrosine kinase inhibitor nintedanib (BIBF 1120) is in clinical development for the treatment of idiopathic pulmonary fibrosis. To explore its mode of action, nintedanib was tested in human lung fibroblasts and mouse models of lung fibrosis. Human lung fibroblasts expressing platelet-derived growth factor (PDGF) receptor-α and -β were stimulated with platelet-derived growth factor BB (homodimer) (PDGF-BB). Receptor activation was assessed by autophosphorylation and cell proliferation by bromodeoxyuridine incorporation. Transforming growth factor β (TGFβ)-induced fibroblast to myofibroblast transformation was determined by α-smooth muscle actin (αSMA) mRNA analysis. Lung fibrosis was induced in mice by intratracheal bleomycin or silica particle administration. Nintedanib was administered every day by gavage at 30, 60, or 100 mg/kg. Preventive nintedanib treatment regimen started on the day that bleomycin was administered. Therapeutic treatment regimen started at various times after the induction of lung fibrosis. Bleomycin caused increased macrophages and lymphocytes in the bronchoalveolar lavage (BAL) and elevated interleukin-1β (IL-1β), tissue inhibitor of metalloproteinase-1 (TIMP-1), and collagen in lung tissue. Histology revealed chronic inflammation and fibrosis. Silica-induced lung pathology additionally showed elevated BAL neutrophils, keratinocyte chemoattractant (KC) levels, and granuloma formation. Nintedanib inhibited PDGF receptor activation, fibroblast proliferation, and fibroblast to myofibroblast transformation. Nintedanib significantly reduced BAL lymphocytes and neutrophils but not macrophages. Furthermore, interleukin-1β, KC, TIMP-1, and lung collagen were significantly reduced. Histologic analysis showed significantly diminished lung inflammation, granuloma formation, and fibrosis. The therapeutic effect was dependent on treatment start and duration. Nintedanib inhibited receptor tyrosine kinase activation and the proliferation and transformation of human lung fibroblasts and showed antifibrotic and anti-inflammatory activity in two animal models of pulmonary fibrosis. These results suggest that nintedanib may impact the progressive course of fibrotic lung diseases such as idiopathic pulmonary fibrosis.

An Assessment of Epithelial and Mesenchymal Phenotypes in Experimental and Clinical Pulmonary Fibrosis

Epithelial injury has been implicated as a driving factor for the pathogenesis of idiopathic pulmonary fibrosis (IPF). In this study we investigated changes in epithelial and mesenchymal markers in experimental models of fibrosis and associated this with IPF. TGFβ1induced an epithelial to mesenchymal transition (EMT) phenotype in A549 cells and normal human bronchial epithelial cells, with A549 cells exhibiting a more profound transition to a mesenchymal phenotype. TGFβ1overexpression in the lungs of mice resulted in an early increase in mesenchymal cell markers and apoptotic genes that preceded collagen deposition, suggesting an early epithelial injury triggers the downstream fibrotic response. In contrast, bleomycin had a gradual increase in mesenchymal cell marker and a decrease in E-cadherin expression that correlated with collagen protein deposition. Finally, we compared normal healthy lung tissue with surgical lung biopsies from IPF patients and observed alterations in epithelial and mesenchymal cell markers, as well as an increase in the apoptotic marker GSK3β. Interestingly, the mesenchymal changes were more profound in rapidly progressive patients in comparison to IPF patients with slowing progressing disease. In summary, this study provides evidence of alterations in epithelial and mesenchymal markers in experimental models of lung fibrosis and how these findings are relevant to clinical disease.

Chronic inflammation and lung fibrosis: pleotropic syndromes but limited distinct phenotypes.

Experimental models of lung fibrosis have been disappointing in predicting therapeutic responses to a wide variety of interventions in clinical fibrosing lung diseases. There are multiple potential reasons, but this fundamentally calls into question the validity of the models and their fidelity to clinical syndromes. We propose that the clinical diseases associated with pulmonary fibrosis, although manifesting a broad array of widely different clinical presentations and features, result in essentially two distinct phenotypes of fibrosis that we will describe. The most common and problematic of these are not effectively modeled experimentally. In this review, we present several clinical entities as examples of the phenotypic distinctions. The first two represent the extremes: postinflammatory fibrosis observed in hypersensitivity pneumonitis (HP) and dysregulated matrix deposition as observed in idiopathic pulmonary fibrosis (IPF). We also present a third clinical entity, that of lung disease associated with rheumatoid arthritis (rheumatoid lung), representing a condition that can manifest as either phenotype, and offering a potential opportunity to explore the mechanisms underlying the pathogenesis of the two distinct fibrotic phenotypes.

Chemokine (C-C motif) ligand 2 mediates direct and indirect fibrotic responses in human and murine cultured fibrocytes

BackgroundFibrocytes are a population of circulating bone-marrow-derived cells that express surface markers for leukocytes and mesenchymal cells, and are capable of differentiating into myofibroblasts. They have been observed at sites of active fibrosis and increased circulating numbers correlate with mortality in idiopathic pulmonary fibrosis (IPF). Inhibition of chemokine (C-C motif) receptor 2 (CCR2) during experimental models of lung fibrosis reduces lung collagen deposition, as well as reducing lung fibrocyte accumulation. The aim of the present study was to determine whether human and mouse fibrocytes express functional CCR2.ResultsFollowing optimized and identical human and murine fibrocyte isolation, both cell sources were shown to be positive for CCR2 by flow cytometry and this expression colocalized with collagen I and CD45. Human blood fibrocytes stimulated with the CCR2 ligand chemokine (C-C motif) ligand 2 (CCL2), demonstrated increased proliferation (P < 0.005) and differentiation into myofibroblasts (P < 0.001), as well as a chemotactic response (P < 0.05). Murine fibrocytes also responded to CCR2 stimulation, with CCL12 being more potent than CCL2.ConclusionsThis study directly compares the functional responses of human and murine fibrocytes to CCR2 ligands, and following comparable isolation techniques. We have shown comparable biological effects, strengthening the translatability of the murine models to human disease with respect to targeting the CCR2 axis to ameliorate disease in IPF patients.

Oxidant–Antioxidant Imbalance as a Potential Contributor to the Progression of Human Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia. IPF is a disease with poor prognosis and an aggressive nature, and poses major challenges to clinicians. Thus, a large part of research in the area has focused on the pathogenesis on IPF. Characteristic features in IPF include fibrotic lesions devoid of inflammatory cell infiltrates. There are experimental models of lung fibrosis (e.g., bleomycin-induced fibrosis), but they typically contain a prominent inflammatory pattern in the lung, which leads to relatively diffuse lung fibrosis. Nonetheless, experimental models have provided important information about the progression and pathways contributing to the lung fibrosis, including activation of transforming growth factor beta (TGF-beta). Both patient material and experimental models of lung fibrosis have displayed marked elevation of several markers of oxidant burden and signs for disturbed antioxidant/oxidant balance. Several studies also suggest that reactive oxygen species can cause activation of growth-regulatory cytokines, including TGF-beta. In addition, there are indications that endogenous and exogenous antioxidants/redox modulators can influence fibrogenesis, protect the lung against fibrosis, and prevent its progression. Factors that restore the antioxidant capacity and prevent sustained activation of growth-regulatory cytokines may have a therapeutic role in IPF.