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BACKGROUND AND PURPOSE The Arp2/3 multiprotein complex regulates branched polymerisation of the actin cytoskeleton and may contribute to collagen synthesis and fibrogenesis in the lung. EXPERIMENTAL APPROACH Expression of Arp2/3 components was assessed in human lung fibroblasts and in the bleomycin-induced pulmonary fibrosis model in mice. The Arp2/3 complex was repressed with the allosteric inhibitor CK666 and with interfering RNAs targeting the ARP2, ARP3 and ARPC2 subunits (siARP2, siARP3 and siARPC2) in CCD-16Lu human lung fibroblasts in vitro. Mice received daily intraperitoneal injections of CK666 from the 7th to the 14th day after tracheal bleomycin instillation. KEY RESULTS Expression of Arp2/3 complex subunits mRNAs was increased in fibroblasts treated with TGF-β1 and in the lungs of bleomycin-treated mice compared with controls. In vitro, CK666 and siARPC2 inhibited cell growth and TGF-β1-induced α-smooth muscle actin (ACTA2) and collagen-1 (COL1) expression. CK666 also decreased ACTA2 and COL1 expression in unstimulated cells. CK666 reduced Akt phosphorylation and repressed phospho-GSK3β, β-catenin and MRTF-A levels in unstimulated fibroblasts. In vivo, CK666 reduced levels of both procollagen-1 and insoluble collagen in bleomycin-treated mice. CONCLUSION AND IMPLICATIONS Expression of the Arp2/3 complex was increased in profibrotic environments in vitro and in vivo. Inhibition of the Arp2/3 complex repressed ACTA2 and COL1 expression and repressed an Akt/phospho-GSK3β/β-catenin/MRTF-A pathway in lung fibroblasts. CK666 exerted antifibrotic properties in the lung in vivo. Inhibition of the Arp2/3 complex could represent an interesting new therapy for idiopathic pulmonary fibrosis and other fibrotic interstitial lung diseases.

International audience; IPF is a devastating lung disease with limited therapeutic possibilities. FGF19, an endocrine FGF, was recently shown to decrease liver fibrosis. To ask whether FGF19 had anti-fibrotic properties in the lung and decipher its effects on common features associated with lung fibrogenesis. We assessed, by Elisa, FGF19 levels in plasma and bronchoalveolar lavage fluids (BALF)obtained from controls and IPF patients. In vivo, using an intravenously administered adeno11 associated virus (AAV), we overexpressed FGF19 at the fibrotic phase of two experimental models of murine lung fibrosis and assessed its effect on lung morphology, lung collagen content, fibrosis markers and pro fibrotic mediator expression, at mRNA and protein levels. In vitro, we investigated whether FGF19 could modulate the TGFβ-induced differentiation of primary human lung fibroblast into myofibroblast and the apoptosis of murine alveolar type II cell. While FGF19 was not detected in BALF, FGF19 concentration was decreased in the plasma of IPF patients compared to controls. In vivo, the overexpression of FGF19 was associated with a marked decrease of lung fibrosis and fibrosis markers, with a decrease of pro fibrotic mediator expression and lung collagen content. In vitro, FGF19 decreased alveolar type 2 epithelial cell apoptosis through the decrease of the proapoptotic BIM protein expression and prevented TGF-ß induced myofibroblast differentiation through the inhibition of JNK phosphorylation. Altogether these data identify FGF19 as an anti-fibrotic molecule with a potential therapeutic interest in fibrotic lung disorders.

Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix production and remodeling could be a promising avenue for IPF treatment. Analysis of public transcriptomic database identified paired-related homeobox protein-1 (PRRX1) as an upregulated mesenchymal transcription factor (TF) in IPF. We confirmed that PRRX1 isoforms were upregulated in IPF lung tissue and strongly expressed by lung fibroblasts. In vitro, PRRX1 expression was up-regulated by cues associated with proliferative and anti-fibrotic properties in lung fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 TFs expression in a PDGFR dependent manner in control ones. Meanwhile, signals promoting myofibroblastic differentiation decreased PRRX1 TF. We demonstrated that PRRX1 inhibition decreased fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-{beta} driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to a TGF-{beta} response global decrease. Finally, targeted inhibition of Prrx1 TFs attenuated fibrotic remodeling both in vivo with intra-tracheal antisense oligonucleotides in the bleomycin mice model of lung fibrosis and ex vivo using mouse and Human precision-cut lung slices stimulated with fibrosis cytokine cocktail. Altogether, our results identified PRRX1 as a mesenchymal transcription factor driving myofibroblastic phenotype and lung fibrogenesis. Brief SummaryInhibition of a single fibroblast-associated transcription factor, namely paired-related homeobox protein 1, is sufficient to dampen lung fibrogenesis.

Interstitial lung fibroblast activation coupled with extracellular matrix production is a pathological signature of pulmonary fibrosis, and is governed by transforming growth factor (TGF)-β1/Smad signalling. TGF-β1 and oxidative stress cooperate to drive fibrosis. Cells can produce reactive oxygen species through activation and/or induction of NADPH oxidases, such as dual oxidase (DUOX1/2). Since DUOX enzymes, as extracellular hydrogen peroxide (H2O2­­)-generating systems, are involved in extracellular matrix formation and in wound healing in different experimental models, we hypothesised that DUOX-based NADPH oxidase plays a role in the pathophysiology of pulmonary fibrosis.Our in vivo data (idiopathic pulmonary fibrosis patients and mouse models of lung fibrosis) showed that the NADPH oxidase DUOX1 is induced in response to lung injury. DUOX1-deficient mice (DUOX1+/− and DUOX1−/−) had an attenuated fibrotic phenotype. In addition to being highly expressed at the epithelial surface of airways, DUOX1 appears to be well expressed in the fibroblastic foci of remodelled lungs. By using primary human and mouse lung fibroblasts, we showed that TGF-β1 upregulates DUOX1 and its maturation factor DUOXA1 and that DUOX1-derived H2O2 promoted the duration of TGF-β1-activated Smad3 phosphorylation by preventing phospho-Smad3 degradation. Analysis of the mechanism revealed that DUOX1 inhibited the interaction between phospho-Smad3 and the ubiquitin ligase NEDD4L, preventing NEDD4L-mediated ubiquitination of phospho-Smad3 and its targeting for degradation.These findings highlight a role for DUOX1-derived H2O2 in a positive feedback that amplifies the signalling output of the TGF-β1 pathway and identify DUOX1 as a new therapeutic target in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by the deposition of excessive extracellular matrix and the destruction of lung parenchyma, resulting from an aberrant wound-healing response. Although IPF is often associated with an imbalance in protease activity, the mechanisms underlying the sustained repair mechanisms are not fully understood. Here, we addressed the role of the recently identified, membrane-anchored serine protease human airway trypsin-like protease (HAT). In the present study, we show that both HAT expression and activity were up-regulated in human IPF specimens. Next, adenoviral overexpression of HAT before bleomycin challenge attenuated lung injury as well as extracellular matrix deposition in the bleomycin-induced pulmonary fibrosis model. In vitro, HAT prevented specific fibrosis-associated responses in primary human pulmonary fibroblasts and induced the expression of mediators associated with the prostaglandin E2 pathway. Altogether, our findings suggested that HAT could have a protective role in IPF and other fibrotic lung disorders.-Menou, A., Flajolet, P., Duitmen, J., Justet, A., Moog, S., Jaillet, M., Tabèze, L., Solhonne, B., Garnier, M., Mal, H., Mordant, P., Castier, Y., Cazes, A., Sallenave, J.-M., Mailleux, A. A., Crestani, B. Human airway trypsin-like protease exerts potent, antifibrotic action in pulmonary fibrosis.

International audience; Fibroblast growth factor 9 (FGF9) is necessary for fetal lung development and is expressed by epithelium and mesothelium. We evaluated the role of FGF9 overexpression on adenoviral-induced pleural injury in vivo and determined the biological effects of FGF9 on mesothelial cells in vitro. We assessed the expression of FGF9 and FGF receptors by mesothelial cells in both human and mouse lungs. Intrapleural injection of an adenovirus expressing human FGF9 (AdFGF9) or a control adenovirus (AdCont) was performed. Mice were euthanized at days 3, 5, and 14 Expression of FGF9 and markers of inflammation and myofibroblastic differentiation was studied by qPCR and immunohistochemistry. In vitro, rat mesothelial cells were stimulated with FGF9 (20 ng/ml), and we assessed its effect on proliferation, survival, migration, and differentiation. FGF9 was expressed by mesothelial cells in human idiopathic pulmonary fibrosis. FGF receptors, mainly FGFR3, were expressed by mesothelial cells in vivo in humans and mice. AdCont instillation induced diffuse pleural thickening appearing at day 5, maximal at day 14 The altered pleura cells strongly expressed α-smooth muscle actin and collagen. AdFGF9 injection induced maximal FGF9 expression at day 5 that lasted until day 14 FGF9 overexpression prevented pleural thickening, collagen and fibronectin accumulation, and myofibroblastic differentiation of mesothelial cells. In vitro, FGF9 decreased mesothelial cell migration and inhibited the differentiating effect of transforming growth factor-β1. We conclude that FGF9 has a potential antifibrotic effect on mesothelial cells.

Introduction: IPF is characterized by a reactivation of signaling pathways involved in lung development, such as Fibroblast Growth Factors (FGF). FGFR4 is a FGF receptor activated by endocrine FGF like FGF19. FGFR4 is expressed in the normal and fibrotic lung. Recent evidence indicates that overexpression of FGF19 have antifibrotic properties in experimental liver fibrosis. The aim of this study was to determine the lung anti fibrotic properties of FGF19 in vitro and in vivo Material and Methods: We assessed FGF19 in plasma by Elisa from controls and IPF patients. Human lung fibroblasts from IPF and control patients were stimulated by TGF s (1ng/mL) with or without FGF19 (20 ng/mL) during 30 minutes or 48 hours. Adeno-associated virus-FGF19 (AAV-FGF19) or a control (AAV-GFP) (6x1010 viral genomes) was IV administered in C56/Bl6 male mice at day 0. Intratracheal administration of bleomycine was realized at day 14. ELISA studied mice at day 28. We quantified plasma FGF-19 levels. We assessed inflammation and pro fibrotic markers by qPCR, and western blot. Results: Compared to controls, FGF19 level plasma was significantly decreased in plasma from IPF patients. In vitro, FGF19 prevented the TGFs induced myofibroblast differentiation by decreasing αSMA, Collagen1, fibronectin expression (mRNA and protein levels.) and JNK phosphorylation. In vivo, the AAV-FGF19 group showed significantly less lung fibrosis and a decrease of fibrosis marker at mRNA and protein levels 14 days after bleomycine exposure. Conclusion and Perspective: FGF19 have anti-fibrotic properties in the lung and could be an innovative therapeutic target in IPF. Further studies are needed to determine the implication of FGFR4 in FGF19 anti fibrotic properties.

Background Idiopathic pulmonary fibrosis is a devastating fibrotic diffuse parenchymal lung disorder that remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. CCAAT/enhancer binding protein delta (C/EBPδ) is a transcription factor that mediates critical cellular functions in pathophysiology and which was recently suggested to be a key regulatory component in IPF. The purpose of this study was to prove or refute the importance of C/EBPδ in pulmonary fibrosis. Methods Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and C/EBPδ deficient mice. At different time intervals after bleomycin instillation, fibrosis was assessed by hydroxyproline analysis, histochemistry and q-PCR for fibrotic marker expression. Results C/EBPδ deficient mice developed pulmonary fibrosis to a similar degree as wildtype mice as evident from similar Ashcroft scores, hydroxyproline levels and expression levels of collagen, fibronectin and α-smooth muscle actin at both 14 and 21 days after bleomycin instillation. The resolution of fibrosis, assessed at 48 days after bleomycin instillation, was also similar in wildtype and C/EBPδ deficient mice. In line with the lack of effect of C/EBPδ on fibrosis progression/resolution, macrophage recruitment and/or differentiation were also not different in wildtype or C/EBPδ deficient mice. Conclusions Overall, C/EBPδ does not seem to affect bleomycin-induced experimental pulmonary fibrosis and we challenge the importance of C/EBPδ in pulmonary fibrosis. Relevance for patients This study shows that the transcription factor C/EBPδ does not play a major role in the development of pulmonary fibrosis. Pharmacological targeting of C/EBPδ is therefore not likely to have a beneficial effect for patients suffering from pulmonary fibrosis.

Regulator of telomere length 1 (RTEL1) mutations have been evidenced in 5–9% of familial pulmonary fibrosis; however, the phenotype of patients with interstitial lung disease (ILD) and RTEL1 mutations is poorly understood.Whole exome sequencing was performed in 252 probands with ILD and we included all patients with ILD and RTEL1 mutation. RTEL1 expression was evaluated by immunochemistry in the lungs of controls, as well as in RTEL1 and telomerase reverse transcriptase (TERT) mutation carriers.We identified 35 subjects from 17 families. Median age at diagnosis of ILD was 53.1 years (range 28.0–80.6). The most frequent pulmonary diagnoses were idiopathic pulmonary fibrosis (n=20, 57%), secondary ILD (n=7, 20%) and unclassifiable fibrosis or interstitial pneumonia with autoimmune features (n=7, 20%). The median transplant-free and overall survival periods were 39.2 months and 45.3 months, respectively. Forced vital capacity at diagnosis was the only factor associated with decreased transplant-free survival. Extra-pulmonary manifestations were less frequent as compared to other telomere-related gene mutation carriers. A systematic analysis of the literature identified 110 patients with ILD and RTEL1 mutations (including this series) and confirmed the heterogeneity of the pulmonary phenotype, the prevalence of non-idiopathic diseases and the low prevalence of extra-pulmonary manifestations.Immunohistochemistry showed that RTEL1 was expressed by bronchial and alveolar epithelial cells, as well as by alveolar macrophages and lymphocytes, but not by fibroblasts.

Introduction Au cours de la fibrose pulmonaire idiopathique (FPI) les voies de signalisation du developpement telles que la voie des Fibroblast Growth Factors (FGF) sont reactivees. Le recepteur FGFR4 a ete suggere comme possible modulateur de la fibrogenese. L’invalidation de Fgfr4 favoriserait le developpement de la fibrose hepatique apres exposition au tetrachloride carbone chez la souris. Nous nous sommes interesses a l’implication de FGFR4 dans la fibrogenese pulmonaire. Methodes L’expression de FGFR4 a ete determinee dans des extraits de poumons totaux de FPI ou de temoins (Western blot, RT-PCR). Le developpement de la fibrose pulmonaire a ete evalue dans un modele murin apres injection intra-tracheale unique de bleomycine (40 μg) chez des souris Fgfr4 -/- et des souris Wild type (WT) littermates, et apres inhibition pharmacologique specifique de FGFR4 par l’inhibiteur Z chez des souris WT par rapport a un groupe controle recevant le solvant seul. L’inhibiteur Z a ete administre par gavage selon un modele preventif de J-1, veille de l’injection de bleomycine, a J14, et selon un modele therapeutique de J7 a J14. La morphologie pulmonaire, le contenu pulmonaire en hydroxyproline et l’expression en RT-PCR et Western blot de marqueurs de fibrose (collagene, fibronectine, l’α-actine du muscle lisse) ont ete evalues. Resultat L’expression de FGFR4 etait diminuee dans des extraits pulmonaires de patients FPI par rapport aux temoins, au niveau proteique et en ARNm. Les souris Fgfr4-/- ne presentaient pas de difference par rapport aux WT concernant le score de fibrose apres injection de bleomycine. Le niveau d’expression proteique du collagene etait diminue chez les souris Fgfr4-/-, cependant le contenu en hydroyproline n’etait pas modifie. Une augmentation de l’α-actine du muscle lisse etait observee au niveau proteique, sans difference significative pour la fibronectine. L’inhibition de FGFR4 par l’inhibiteur Z, n’induisait aucune difference phenotypique par rapport aux souris controles. Conclusion L’expression pulmonaire de FGFR4 etait diminuee au cours de la FPI. Cependant, l’invalidation de Fgfr4 et l’inhibition pharmacologique de FGFR4 ne modifiaient pas le developpement de la fibrose bleomycine-induite chez la souris. Ces donnees ne soutiennent pas l’hypothese d’une implication de FGFR4 dans la physiopathologie de la FPI.

Introduction Le cancer pulmonaire est une complication frequente des patients atteints de fibrose pulmonaire idiopathique (FPI). Notre hypothese est que le microenvironnement du tissu fibreux favorise la croissance tumorale. Notre objectif etait de determiner si la matrice extra-cellulaire (MEC) provenant de patients atteints de FPI favorisait la transition epithelio-mesenchymateuse (TEM) de cellules epitheliales porteuses ou non d’une mutation oncogenique par rapport a une MEC de poumon temoin (T). Methodes Des echantillons provenant de poumons sains (sujets temoins) ou de poumons de FPI recueillis lors de la transplantation pulmonaire etaient congeles a −80 °C et des sections de 500 μm etaient obtenues au cryostat, et decellularisees selon Parker et al. [1] . 3,105 cellules HBEC 3 KT, controles ou mutees pour l’oncogene RASV12, etaient cultivees en presence de sections decellularisees. Au terme de 72H de culture, nous avons evalue l’expression des marqueurs de TEM par PCR quantitative. Les resultats etaient exprimes en % de la condition basale (cellules cultivees sur plastique) et compares par un test de Mann Whitney. Resultats La culture sur MEC, T ou FPI, s’accompagnait d’une augmentation de l’ARNm de SNAIL et ZEB1 (2 facteurs de transcriptions impliques dans la TEM), de l’α-actine du muscle lisse (αSMA) et du collagene (A1COL1) dans les cellules HBEC 3 KT. Il n’y avait pas de difference entre les matrices T ou FPI. L’expression de la cadherine 11, de la vimentine et de la fibronectine augmentait seulement sur MEC FPI. En presence de la mutation de RAS, l’expression de αSMA, fibronectine et collagene est augmentee tandis que l’expression de SNAIL, ZEB1, de la vimentine et de la cadherine 11 n’est pas modifiee par rapport aux conditions basales. L’expression de la E-cadherine etait augmentee dans toutes les conditions de culture et dans les deux types cellulaires. Conclusion Lorsqu’elles sont cultivees sur une MEC de poumon, les cellules HBEC surexpriment certains marqueurs mesenchymateux et des composants de la MEC (collagene, fibronectine). L’effet est plus marque pour les MEC de FPI. La presence de la mutation de RAS n’amplifie pas l’effet de la MEC.

Introduction La fibrose pulmonaire idiopathique (FPI) est une pathologie fibrosante pulmonaire associee a des agressions repetees de l’epithelium alveolaire pulmonaire âge et la reactivation de voies de developpement embryonnaire, conduisant ainsi a la migration et l’accumulation de cellules mesenchymateuses, en particulier les myofibroblastes, aux sites de de lesion. Ainsi, PRRX1 est un facteur de transcription mesenchymateux retrouve augmente dans les fibroblastes de poumons de patients atteints de FPI, et dans le modele de fibrose pulmonaire murin apres instillation de bleomycine. Ce caractere profibrosant a ete confirme au laboratoire par l’utilisation d’un oligonucleotide anti-sens dirige contre Prrx1, induisant une diminution drastique des lesions fibrosantes induites par la bleomycine. L’objectif de ce projet est de determiner le lignage cellulaire des cellules exprimant Prrx1 afin de mieux comprendre leur origine et devenir au cours du developpement de la fibrose. Methodes Les souris Prrx1:CreERT2-IRES-GFP;Rosa26iTomato permettent de marquer de maniere inductible, par la proteine fluorescente tdTomato, les cellules exprimant Prrx1 en utilisant du tamoxifene. Un lobe est fixe pour realiser des immunohistochimies et le reste digere pour une etude ciblee par cytometrie en flux du lignage mesenchymateux (Tomatopos, CD45neg, EPCAMneg, CD31neg). Resultats Les premieres donnees de FACS ont mis en evidence une augmentation du nombre de cellules tdTomato-positives chez les souris instillees a la bleomycine, comparees aux souris naives avec une proportion importante de cellules mesenchymateuses. Sur les coupes de poumons, on retrouve egalement un marquage tdTomato-positif assez diffus, surtout dans les zones de fibrose, et aucun dans les souris naives. Conclusion Nos resultats suggerent que les cellules Prrx1 positives representent une population peu presente au niveau basal, qui va ensuite croitre et donner naissance essentiellement a des cellules mesenchymateuses lors du developpement de la fibrose.

Introduction L’atteinte pulmonaire de l’infection a SARS-CoV-2, responsable du COVID-19, est grave dans 15–20% des cas, avec une pneumopathie bilaterale presentant les caracteristiques histologiques d’un dommage alveolaire diffus. Les fibrocytes circulants, precurseurs des fibroblastes impliques dans la reparation tissulaire, sont associes a un mauvais pronostic dans les maladies fibrosantes pulmonaires et le syndrome de detresse respiratoire aigue. Notre objectif etait de quantifier les fibrocytes circulants chez les patients atteints de COVID et de determiner leur valeur pronostique. Methodes Les fibrocytes circulants (CD45 + /CD15 -/CD34 + /Collagene-1 +) ont ete quantifies en cytometrie de flux chez 57 patients (42 hommes, âge moyen 60 ans [3–87]) hospitalises pour pneumopathie bilaterale hypoxemiante et 15 sujets sains apparies pour l’âge et le sexe. L’infection a SARS-CoV-2 etait confirmee par une PCR naso-pharyngee. L’etendue scannographique des lesions a ete quantifiee. Resultats Le taux median de fibrocytes circulants etait significativement augmente chez les patients par rapport aux temoins (2,49% vs 1,82%, p 75%: 0,57%). Le % de fibrocytes etait diminue lorsqu’une corticotherapie systemique etait deja en place au moment du prelevement (2,28% vs 2,82%, p = 0,04). Un taux de fibrocytes significativement plus bas (1,25%) etait observe chez les patients decedes (6/57) par rapport aux patients rentres a domicile a J28 (2,52%, p = 0,03). Concernant l’evolution scannographique a 3 mois, une regression complete des lesions (13/32) etait associee a un % initial de fibrocytes significativement plus eleve par rapport aux patients presentant une regression partielle ou une stabilite (2,95% vs 2,18%, p = 0,03). Conclusion Le taux de fibrocytes circulants etait significativement augmente chez les patients hospitalises pour une pneumopathie hypoxemiante COVID-19 par rapport a des sujets sains. Les patients ayant l’atteinte la plus severe avaient des % de fibrocytes plus bas, alors qu’un % de fibrocytes plus eleve etait associe a une regression complete des lesions scannographique a 3 mois.

IPF is characterized by a reactivation of signaling pathways involved in lung development, such as Fibroblast Growth Factors (FGF). FGFR4 is a FGF receptor activated by endocrine FGFs, such as FGF19, in the presence of the co-receptor s klotho. Recent evidence indicates that complete deletion of FGFR4 and Fgf15 (murine orthologue of FGF19) leads to increased liver fibrosis suggesting that the FGFR4/FGF19 axis has some antifibrotic properties. The aim of this work was to study the regulation of FGFR4 expression and to assess the effect of its activation/inibition on the lung fibroblast phenotype. Material and Methods: We assessed FGFR4, s klotho and FGF19 expression by immunohistochemistry RT-PCR in lung tissue from controls and IPF patients. Human lung fibroblasts from IPF and control patients were stimulated in vitro with pro- or anti-fibrotic factors with or without FGF19 (20 ng/mL) during 48 hours. We studied the expression of FGFR4 at mRNA level and assessed the effect of FGF19 on fibroblast differentiation, proliferation and migration. Results: FGFR4 and s klotho were expressed by alveolar epithelial cells and by lung fibroblast in IPF lung, while FGF19 was not detected. FGFR4 mRNA expression by control and IPF fibroblasts was downregulated by pro fibrotic factors (TGFβ, CTGF, ET-1) and up-regulated by HGF. FGF9 prevented the TGFs- induced myofibroblast differentiation by significantly decreasing αSMA, Collagen1 , fibronectin, PAI-1 and CTGF at mRNA and protein levels. FGF19 increased fibroblast migration but didn9t influence proliferation. Conclusion: FGF19 inhibits TGFβ-induced differentiation in vitro. FGF19 may have anti-fibrotic properties in the lung which need to be documented in vivo.

Regulator of telomere length 1 (

IF 12.784 (2016); International audience; Periplakin is a component of the desmosomes that acts as a cytolinker between intermediate filament scaffolding and the desmosomal plaque. Periplakin is strongly expressed by epithelial cells in the lung and is a target antigen for autoimmunity in idiopathic pulmonary fibrosis. The aim of this study was to determine the role of periplakin during lung injury and remodeling in a mouse model of lung fibrosis induced by bleomycin. We found that periplakin expression was downregulated in the whole lung and in alveolar epithelial cells following bleomycin-induced injury. Deletion of the Ppl gene in mice improved survival and reduced lung fibrosis development after bleomycin-induced injury. Notably, Ppl deletion promoted an antiinflammatory alveolar environment linked to profound changes in type 2 alveolar epithelial cells, including overexpression of antiinflammatory cytokines, decreased expression of profibrotic mediators, and altered cell signaling with a reduced response to TGF-β1. These results identify periplakin as a previously unidentified regulator of the response to injury in the lung.

International audience

Background Idiopathic pulmonary fibrosis is a devastating fibrotic diffuse parenchymal lung disorder that remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. CCAAT/enhancer binding protein delta (C/EBPδ) is a transcription factor that mediates critical cellular functions in pathophysiology and which was recently suggested to be a key regulatory component in IPF. The purpose of this study was to prove or refute the importance of C/EBPδ in pulmonary fibrosis. Methods Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and C/EBPδ deficient mice. At different time intervals after bleomycin instillation, fibrosis was assessed by hydroxyproline analysis, histochemistry and q-PCR for fibrotic marker expression. Results C/EBPδ deficient mice developed pulmonary fibrosis to a similar degree as wildtype mice as evident from similar Ashcroft scores, hydroxyproline levels and expression levels of collagen, fibronectin and α-smooth muscle actin at both 14 and 21 days after bleomycin instillation. The resolution of fibrosis, assessed at 48 days after bleomycin instillation, was also similar in wildtype and C/EBPδ deficient mice. In line with the lack of effect of C/EBPδ on fibrosis progression/resolution, macrophage recruitment and/or differentiation were also not different in wildtype or C/EBPδ deficient mice. Conclusions Overall, C/EBPδ does not seem to affect bleomycin-induced experimental pulmonary fibrosis and we challenge the importance of C/EBPδ in pulmonary fibrosis. Relevance for patients This study shows that the transcription factor C/EBPδ does not play a major role in the development of pulmonary fibrosis. Pharmacological targeting of C/EBPδ is therefore not likely to have a beneficial effect for patients suffering from pulmonary fibrosis.

International audience; It has become more and more evident that the BCL-2 family proteins mediate a wide range of non-apoptotic functions. The pro-apoptotic BAX protein has been reported in interphasic nuclei. Whether the nuclear form of BAX could be involved in non-apoptotic function is still unknown. Our study showed for the first time that BAX was associated with chromatin in vitro. Next, we used gain and loss of function approaches to decipher the potential role of nuclear BAX in non-apoptotic cells. In vitro, nuclear BAX promoted cell proliferation in lung epithelial cells and primary human lung fibroblasts by modulating CDKN1A expression. Interestingly, BAX occupancy of CDKN1A promoter was specifically enriched close to the transcription-starting site. Nuclear BAX also modulated the basal myofibroblastic differentiation and migration of primary human lung fibroblasts. Finally, BAX nuclear localization was associated in vivo with the remodelling of lung parenchyma during development, tumorigenesis as well as fibrosis compared to control adult human lungs. Hence, our study established for the first time, a strong link between the nuclear localization of the pro-apoptotic BAX protein and key basic cellular functions in the non-apoptotic setting.

Introduction La Fibrose Pulmonaire Idiopathique (FPI) se caracterise par une reactivation des voies de signalisation impliquees dans le developpement pulmonaire, telles que les facteurs de croissance des fibroblastes (FGF). FGFR4 est un recepteur du FGF active par les FGF endocriniens, tels que le FGF19, en presence du co-recepteur s klotho. Ce recepteur est seul FGFR qui n’est pas cible par le nintedanib, un traitement anti-fibrosant qui a demontre son efficacite dans la FPI. Des donnees recentes indiquent que la deletion complete de FGFR4 et Fgf15 (orthologue murin du FGF19) entraine une augmentation de la fibrose hepatique, ce qui suggere que l’axe FGFR4/FGF19 possede certaines proprietes anti-fibrotiques. Le but de cette etude etait d’etudier la regulation de l’expression de FGFR4 et d’evaluer l’effet de son activation/inhibition sur le phenotype des fibroblastes du poumon. Methodes Nous avons evalue l’expression de FGFR4, s klotho et FGF19 par immunohistochimie RT-PCR dans le tissu pulmonaire de temoins et de patients atteints de FPI. Les fibroblastes du poumon humain provenant de la FPI et des patients temoins ont ete stimules in vitro avec des facteurs pro- ou anti-fibrotiques avec ou sans FGF19 (20 ng/mL) pendant 48 heures. Nous avons etudie l’effet de l’activation de FGFR4 par le FGF19 sur la differenciation, la proliferation et la migration des fibroblastes pulmonaires temoins ou FPI. Resultats FGFR4 et s klotho sont exprimes par les cellules epitheliales alveolaires et par les fibroblastes pulmonaire dans le poumon normal et fibreux. Le FGF19 n’est pas detecte au niveau pulmonaire, en revanche la concentration plasmatique du FGF19 est significativement diminuee chez les patients atteints de FPI par rapport aux controles. La stimulation des fibroblastes pulmonaires par des facteurs profibrotiques (TGFβ, CTGF, ET-1) induit une diminution du contenu en ARNM du FGFR4 alors qu’une stimulation de ces cellules induit une augmentation du contenu en ARN messager de FGFR4. Le FGF19 previent la differenciation des myofibroblastes induite par le TGFs en diminuant significativement contenus proteiques et en ARNm d’αSMA, de collagene 1, de fibronectine, de PAI-1 et de CTGF aux niveaux de l’ARNm et des proteines. Le FGF19 augmente la migration des fibroblastes mais n’influence pas la proliferation. Conclusion L’axe FGFR4/FGF19 a des proprietes anti-fibrotiques in vitro qui doivent etre confirmees in vivo. Cet axe pourrait constituer dans le futur une cible therapeutique innovante dans la FPI.

Key developmental lung signaling pathways are reactivated in IPF. FGF9 and FGF18 are involved in epithelial–mesenchymal interactions and are critical for lung development. We evaluated the expression of FGF9, FGF18 and FGFR in lung tissue from controls and IPF patients and assessed their effect on proliferation, survival, migration and differentiation of control and IPF human lung fibroblasts (HLF). HLF were cultured with FGF9 and FGF18. FAS-ligand induced apoptosis was assayed by measuring cleaved-PARP expression. Expression of FGFR mRNA, α-smooth muscle actin, collagen I and MMPs was analyzed with qPCR, western blot and zymography. Migratory capacities were evaluated in a modified Boyden chamber. siRNA were used to test FGFR3 implication. FGF9, FGF18 and all FGFR were present in the remodeled alveolar epithelium close to the fibroblast foci. Only FGFR3 was expressed in fibroblast foci. In vitro , FGF9 did not affect fibroblasts proliferation. FGF9/FGF18 decreased Fas-ligand induced apoptosis in control but not in IPF fibroblasts. FGF9 prevented basal and TGF-β1–induced myofibroblast differentiation. FGF9/FGF18 increased the migratory capacities of HLF, and FGF9 actively modulated MMPs activity. HLF mostly expressed FGFR1c and FGFR3b. FGFR3 inhibition prevented pERK activation by FGF9/FGF18 and partially inhibited their effect on differentiation and migration. These results identify FGF9 as an anti-apoptotic, anti-differentation and pro-migratory growth factor. FGF18 was a less potent molecule. FGFR3 drives part of the action of FGF9/FGF18. Both growth factors likely contribute to the fibrotic process. This abstract has been presented previously at the European Respiratory Society9s Lung Science Conference in March 2016.

Introduction Fibroblast differentiation in myofibroblasts is a key feature of idiopathic pulmonary fibrosis (IPF). Paired-related homeobox (PRRX) transcription factors (TF) regulate mesenchymal cells during development and are key TF at the center of a network coordinating fibroblast differentiation. We hypothesized that PRRX1 modulates fibroblast phenotype in IPF. Material and methods Immunohistochemistry (IHC) and real-time quantitative PCR for PRRX were performed on human lung samples and primary human lung fibroblasts (HLF) cultured from IPF and controls. PRRX1 overexpression was obtained in HLF with plasmid transfection. Functional experiments were performed by seeding HLF on dishes with an elastic modulus (EM) of 1.5 or 28 kPa or on a rigid dish. The effects of the ROCK and PTGS2 inhibitors Fasudil and NS398 on PRRX1 expression were evaluated. Results PRRX1 was upregulated in IPF lung and HLF as compared to controls and was mostly detected in fibroblast foci. PRRX1 expression was controlled by soluble factors and by the extracellular matrix stiffness: Profibrotic factors TGF-β and ET-1 downregulated PRRX1 expression in HLF, while PDGF-AA and PGE2 increased PRRX1 expression only in HLF from IPF patients. Soft matrix (EM 1.5 kPa) enhanced PRRX1 expression in HLF through the activation of PTGS2, while a rigid matrix inhibited PRRX1 expression through ROCK activation. PRRX1 overexpression inhibited PDGF-dependent migration, increased the expression of phosphorylated ERK and promoted control and IPF HLF survival after serum deprivation or FAS-L activation. Conclusion These results indicate that PRRX1 is regulated by the fibrotic environment in the lung and controls lung fibroblast migration and survival.

Idiopathic pulmonary fibrosis (IPF) is characterized by an accumulation of extracellular matrix proteins and fibroblasts in the distal airways. Key developmental lung signaling pathways are reactivated in IPF. For instance, fibroblast growth factor 9 (FGF9) and FGF18, involved in epithelial-mesenchymal interactions, are critical for lung development. We evaluated the expression of FGF9, FGF18, and FGF receptors (FGFRs) in lung tissue from controls and IPF patients and assessed their effect on proliferation, survival, migration, and differentiation of control and IPF human lung fibroblasts (HLFs). FGF9, FGF18, and all FGFRs were present in the remodeled alveolar epithelium close to the fibroblast foci in IPF lungs. FGFR3 was generally detected in fibroblast foci by immunohistochemistry. In vitro, HLFs mainly expressed mesenchyme-associated FGFR isoforms (FGFR1c and FGFR3c) and FGFR4. FGF9 did not affect fibroblast proliferation, whereas FGF18 inhibited cell growth in control fibroblasts. FGF9 and FGF18 decreased Fas-ligand-induced apoptosis in control but not in IPF fibroblasts. FGF9 prevented transforming growth factor β1-induced myofibroblast differentiation. FGF9 and FGF18 increased the migratory capacities of HLF, and FGF9 actively modulated matrix metalloproteinase activity. In addition, FGFR3 inhibition by small interfering RNA impacted p-ERK activation by FGF9 and FGF18 and their effects on differentiation and migration. These results identify FGF9 as an antiapoptotic and promigratory growth factor on HLF, maintaining fibroblasts in an undifferentiated state. The biological effects of FGF9 and FGF18 were partially driven by FGFR3. FGF18 was a less potent molecule. Both growth factors likely contribute to the fibrotic process in vivo.

Introduction La fibrose pulmonaire idiopathique (FPI) est une maladie progressive mortelle, de physiopathologie est incertaine. Le fibroblaste, cellule centrale de la reparation alveolaire normale, semble jouer un role cle dans la fibrogenese en particulier par la re-activation aberrante de facteurs de transcription du developpement. PRRX1 est un facteur de transcription du developpement mesenchymateux. Il apparait comme etant surexprime dans les poumons totaux et des fibroblastes de FPI par rapport aux temoins. Nos objectifs etaient de determiner l’effet de facteurs pro- et anti-fibrosants sur son expression et de determiner son effet sur le phenotype des fibroblastes in vitro par une approche de gain de fonction, afin d’etablir son implication dans la FPI. Methodes Nous avons utilise des cultures primaires de fibroblastes humains de temoins et de FPI. Les cellules ont ete stimulees par differents agents pro-fibrosants (TGFβ, endotheline-1), et anti-fibrosants (PGE2, HGF) pendant 48 h. Le niveau d’expression de l’ARNm de PRRX1 a ete mesure par qPCR. PRRX1 a ete surexprime par transfection de plasmide afin d’analyser de la proliferation par comptage cellulaire, la migration en chambre de Boyden modifiee, et la differenciation par analyse de l’expression de marqueurs myofibroblastiques (αSMA, collagene 1, fibronectine 1). Resultats Le TGFβ et l’endotheline 1, agents pro-fibrosants, diminuent son expression alors que la PGE2, agent anti-fibrosant, l’augmente, au niveau de l’ARNm. Sur le plan phenotypique apres surexpression de PRRX1, on observait une augmentation de la proliferation des fibroblastes temoins et de fibrose. Il n’existait pas d’augmentation de la migration, ni de la differenciation. Conclusion PRRX1 module le phenotype des fibroblastes de temoins et de FPI en favorisant leur proliferation et en consequence le developpement de la FPI. En perspective, une approche par perte de fonctions (siRNA) permettra de mieux comprendre et etudier la modulation du phenotype des fibroblastes par PRRX1.

Elevated kallikrein 5 expression is sufficient to trigger the majority of the clinical hallmarks of Netherton syndrome., Netherton syndrome (NS) is a severe genetic skin disease in which absence of a key protease inhibitor causes congenital exfoliative erythroderma, eczematous-like lesions, and atopic manifestations. Several proteases are overactive in NS, including kallikrein-related peptidase (KLK) 5, KLK7, and elastase-2 (ELA2), which are suggested to be part of a proteolytic cascade initiated by KLK5. To address the role of KLK5 in NS, we have generated a new transgenic murine model expressing human KLK5 in the granular layer of the epidermis (Tg-KLK5). Transgene expression resulted in increased proteolytic activity attributable to KLK5 and its downstream targets KLK7, KLK14, and ELA2. Tg-KLK5 mice developed an exfoliative erythroderma with scaling, growth delay, and hair abnormalities. The skin barrier was defective and the stratum corneum was detached through desmosomal cleavage. Importantly, Tg-KLK5 mice displayed cutaneous and systemic hallmarks of severe inflammation and allergy with pruritus. The skin showed enhanced expression of inflammatory cytokines and chemokines, infiltration of immune cells, and markers of Th2/Th17/Th22 T cell responses. Moreover, serum IgE and Tslp levels were elevated. Our study identifies KLK5 as an important contributor to the NS proteolytic cascade and provides a new and viable model for the evaluation of future targeted therapies for NS or related diseases such as atopic dermatitis.