Origins of pathological myofibroblasts in lung fibrosis: insights from lineage tracing mouse models in the single-cell RNA sequencing era.

Idiopathic pulmonary fibrosis (IPF) is a rare interstitial lung disease with a poor prognosis. Chronic microinjuries, mainly caused by environmental factors to an aging alveolar epithelium, would lead to the aberrant differentiation and accumulation of aberrant mesenchymal cells with a contractile phenotype, known as fibrosis-associated myofibroblasts, which trigger abnormal extracellular matrix accumulation and fibrosis. The origin of those pathological myofibroblasts in pulmonary fibrosis is not fully understood to date. Lineage tracing methods using mouse models have opened new avenues for studying cell fate in a pathological context. This review aims to present a nonexhaustive list of different potential sources of those harmful myofibroblasts during lung fibrosis, based on these in vivo approaches, and considering the normal and fibrotic lung cellular atlas recently established by single-cell RNA sequencing.

Human airway trypsin-like protease exerts potent, antifibrotic action 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.

Macrophage Polarization Favors Epithelial Repair During Acute Respiratory Distress Syndrome.

OBJECTIVES: Alveolar macrophage polarization and role on alveolar repair during human acute respiratory distress syndrome remain unclear. This study aimed to determine during human acute respiratory distress syndrome: the alveolar macrophage polarization, the effect of alveolar environment on macrophage polarization, and the role of polarized macrophages on epithelial repair. DESIGN: Experimental ex vivo and in vitro investigations. SETTING: Four ICUs in three teaching hospitals. PATIENTS: Thirty-three patients with early moderate-to-severe acute respiratory distress syndrome were enrolled for assessment of the polarization of alveolar macrophages. INTERVENTIONS: Polarization of acute respiratory distress syndrome macrophages was studied by flow cytometry and quantitative polymerase chain reaction. Modulation of macrophage polarization was studied in vitro using phenotypic and functional readouts. Macrophage effect on repair was studied using alveolar epithelial cells in wound healing models. MEASUREMENTS AND MAIN RESULTS: Ex vivo, alveolar macrophages from early acute respiratory distress syndrome patients exhibited anti-inflammatory characteristics with high CD163 expression and interleukin-10 production. Accordingly, early acute respiratory distress syndrome-bronchoalveolar lavage fluid drives an acute respiratory distress syndrome-specific anti-inflammatory macrophage polarization in vitro, close to that induced by recombinant interleukin-10. Culture supernatants from macrophages polarized in vitro with acute respiratory distress syndrome-bronchoalveolar lavage fluid or interleukin-10 and ex vivo acute respiratory distress syndrome alveolar macrophages specifically promoted lung epithelial repair. Inhibition of the hepatocyte growth factor pathway in epithelial cells and hepatocyte growth factor production in macrophages both reversed this effect. Finally, hepatocyte growth factor and soluble form of CD163 concentrations expressed relatively to macrophage count were higher in bronchoalveolar lavage fluid from acute respiratory distress syndrome survivors. CONCLUSIONS: Early acute respiratory distress syndrome alveolar environment drives an anti-inflammatory macrophage polarization favoring epithelial repair through activation of the hepatocyte growth factor pathway. These results suggest that macrophage polarization may be an important step for epithelial repair and acute respiratory distress syndrome recovery.

FGF9 prevents pleural fibrosis induced by intrapleural adenovirus injection in mice.

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-ß1. We conclude that FGF9 has a potential antifibrotic effect on mesothelial cells.

FGF9 and FGF18 in idiopathic pulmonary fibrosis promote survival and migration and inhibit myofibroblast differentiation of human lung fibroblasts in vitro.

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 ß1-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.

Serum Amyloid P Contained in Alveolar Fluid From Patients With Acute Respiratory Distress Syndrome Mediates the Inhibition of Monocyte Differentiation into Fibrocyte.

OBJECTIVE: Alveolar fibrocytes are monocyte-derived mesenchymal cells associated with poor prognosis in patients with acute respiratory distress syndrome. Our aims were to determine the following: 1) the ability of monocytes from acute respiratory distress syndrome patients to differentiate into fibrocytes; 2) the influence of the acute respiratory distress syndrome alveolar environment on fibrocyte differentiation; and 3) mediators involved in this modulation, focusing on serum amyloid P. DESIGN: Experimental in vitro investigation. SETTING: Two ICUs of a teaching hospital. PATIENTS: Twenty-five patients (19 mild-to-severe acute respiratory distress syndrome and six matched ventilated controls without acute respiratory distress syndrome) were enrolled. Six healthy volunteers served as non-ventilated controls. INTERVENTIONS: Peripheral blood mononuclear cells were isolated from acute respiratory distress syndrome, ventilated controls, and non-ventilated controls blood and cultured in vitro. Fibrocytes were counted at basal condition and after culture with broncho-alveolar lavage fluid. Plasma and broncho-alveolar lavage fluid serum amyloid P contents were determined by western blot and enzyme-linked immunosorbent assay. Serum amyloid P was located in normal and acute respiratory distress syndrome lung by immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: Acute respiratory distress syndrome peripheral blood mononuclear cells had a three-fold increased ability to differentiate into fibrocytes compared to ventilated controls or non-ventilated controls. Acute respiratory distress syndrome broncho-alveolar lavage fluid inhibited by 71% (55-94) fibrocyte differentiation compared to saline control. Ventilated controls' broncho-alveolar lavage fluid was a less potent inhibitor (51% [23-66%] of inhibition), whereas non-ventilated controls' broncho-alveolar lavage fluid had no effect on fibrocyte differentiation. Serum amyloid P concentration was decreased in plasma and dramatically increased in broncho-alveolar lavage fluid during acute respiratory distress syndrome. Alveolar serum amyloid P originated, in part, from the release of serum amyloid P associated with lung connective tissue during acute respiratory distress syndrome. Serum amyloid P depletion decreased the inhibitory effect of acute respiratory distress syndrome broncho-alveolar lavage fluid by 60%, whereas serum amyloid P replenishment of serum amyloid P-depleted acute respiratory distress syndrome broncho-alveolar lavage fluid restored their full inhibitory effect. CONCLUSIONS: The presence of fibrocytes in the lung during acute respiratory distress syndrome could result in a balance between higher ability of monocytes to differentiate into fibrocytes and the inhibitory effect of the alveolar environment, mainly dependent on serum amyloid P.

Targeting the hedgehog-glioma-associated oncogene homolog pathway inhibits bleomycin-induced lung fibrosis in mice.

Idiopathic pulmonary fibrosis has been associated with the reactivation of developmental pathways, notably the Hedgehog-Glioma-associated oncogene homolog (GLI) pathway. In this study, we determined whether the Hedgehog pathway was activated in bleomycin-induced lung injury in mice, and whether targeting the Hedgehog-Gli pathway could decrease bleomycin-induced lung fibrosis. After intratracheal injection of bleomycin on Day 0, C57Bl6 mice received GDC-0449 (an inhibitor of Smoothened, the transducer of the pathway), or 2,2'-[[Dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N dimethylbenzenamine (GANT61; an inhibitor of GLI transcription factors in the nucleus), from Day 7 to Day 13. At Day 14, whole-lung homogenates were obtained for morphological analysis, assessment of cell apoptosis and proliferation, collagen quantification, and evaluation of profibrotic (transforming growth factor-ß, connective tissue growth factor, plasminogen activator inhibitor 1, vascular endothelial growth factor-A) and proinflammatory mediators (IL-1ß) expression. We showed that the Hedgehog pathway was activated in bleomycin-induced lung fibrosis on Day 14 after injury, with an increased lung expression of the ligand, Sonic Hedgehog, and with increased messenger RNA expression and nuclear localization of GLI1 and GLI2. Inhibition of Smoothened with GDC-0449 did not influence the development of bleomycin-induced lung fibrosis. By contrast, the inhibition of GLI activity with GANT61 decreased lung fibrosis and lung collagen accumulation, and promoted an antifibrotic and anti-inflammatory environment. Our results identify the hedgehog-Gli pathway as a profibrotic pathway in experimental fibrosis. Inhibition of the Hedgehog-Gli pathway at the level of GLI transcriptional activity could be a therapeutic option in fibrotic lung diseases.

The endocrine FGFs axis: A systemic anti-fibrotic response that could prevent pulmonary fibrogenesis?

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease for which therapeutic options are limited, with an unmet need to identify new therapeutic targets. IPF is thought to be the consequence of repeated microlesions of the alveolar epithelium, leading to aberrant epithelial-mesenchymal communication and the accumulation of extracellular matrix proteins. The reactivation of developmental pathways, such as Fibroblast Growth Factors (FGFs), is a well-described mechanism during lung fibrogenesis. Secreted FGFs with local paracrine effects can either exert an anti-fibrotic or a pro-fibrotic action during this pathological process through their FGF receptors (FGFRs) and heparan sulfate residues as co-receptors. Among FGFs, endocrine FGFs (FGF29, FGF21, and FGF23) play a central role in the control of metabolism and tissue homeostasis. They are characterized by a low affinity for heparan sulfate, present in the cell vicinity, allowing them to have endocrine activity. Nevertheless, their interaction with FGFRs requires the presence of mandatory co-receptors, alpha and beta Klotho proteins (KLA and KLB). Endocrine FGFs are of growing interest for their anti-fibrotic action during liver, kidney, or myocardial fibrosis. Innovative therapies based on FGF19 or FGF21 analogs are currently being studied in humans during liver fibrosis. Recent data report a similar anti-fibrotic action of endocrine FGFs in the lung, suggesting a systemic regulation of the pulmonary fibrotic process. In this review, we summarize the current knowledge on the protective effect of endocrine FGFs during the fibrotic processes, with a focus on pulmonary fibrosis.

The hedgehog system machinery controls transforming growth factor-ß-dependent myofibroblastic differentiation in humans: involvement in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown cause. Key signaling developmental pathways are aberrantly expressed in IPF. The hedgehog pathway plays a key role during fetal lung development and may be involved in lung fibrogenesis. We determined the expression pattern of several Sonic hedgehog (SHH) pathway members in normal and IPF human lung biopsies and primary fibroblasts. The effect of hedgehog pathway inhibition was assayed by lung fibroblast proliferation and differentiation with and without transforming growth factor (TGF)-ß1. We showed that the hedgehog pathway was reactivated in the IPF lung. Importantly, we deciphered the cross talk between the hedgehog and TGF-ß pathway in human lung fibroblasts. TGF-ß1 modulated the expression of key components of the hedgehog pathway independent of Smoothened, the obligatory signal transducer of the pathway. Smoothened was required for TGF-ß1-induced myofibroblastic differentiation of control fibroblasts, but differentiation of IPF fibroblasts was partially resistant to Smoothened inhibition. Furthermore, functional hedgehog pathway machinery from the primary cilium, as well as GLI-dependent transcription in the nucleus, was required for the TGF-ß1 effects on normal and IPF fibroblasts during myofibroblastic differentiation. These data identify the GLI transcription factors as potential therapeutic targets in lung fibrosis.

Identification of Paired-related Homeobox Protein 1 as a key mesenchymal transcription factor in pulmonary fibrosis.

Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF. PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-ß/PGE2 balance in vitro in control and IPF human lung fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR-dependent manner in control ones. PRRX1 inhibition decreased human lung fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-ß driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-ß response global decrease. Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using human and mouse precision-cut lung slices. Our results identified PRRX1 as a key mesenchymal transcription factor during lung fibrogenesis.

ERS International Congress 2022: highlights from the Basic and Translational Science Assembly.

In this review, the Basic and Translational Science Assembly of the European Respiratory Society provides an overview of the 2022 International Congress highlights. We discuss the consequences of respiratory events from birth until old age regarding climate change related alterations in air quality due to pollution caused by increased ozone, pollen, wildfires and fuel combustion as well as the increasing presence of microplastic and microfibres. Early life events such as the effect of hyperoxia in the context of bronchopulmonary dysplasia and crucial effects of the intrauterine environment in the context of pre-eclampsia were discussed. The Human Lung Cell Atlas (HLCA) was put forward as a new point of reference for healthy human lungs. The combination of single-cell RNA sequencing and spatial data in the HLCA has enabled the discovery of new cell types/states and niches, and served as a platform that facilitates further investigation of mechanistic perturbations. The role of cell death modalities in regulating the onset and progression of chronic lung diseases and its potential as a therapeutic target was also discussed. Translational studies identified novel therapeutic targets and immunoregulatory mechanisms in asthma. Lastly, it was highlighted that the choice of regenerative therapy depends on disease severity, ranging from transplantation to cell therapies and regenerative pharmacology.

BIM regulates apoptosis during mammary ductal morphogenesis, and its absence reveals alternative cell death mechanisms.

The adult, virgin mammary gland is a highly organized tree-like structure formed by ducts with hollowed lumen. Although lumen formation during pubertal development appears to involve apoptosis, the molecular mechanisms that regulate this process are not known. Here, we demonstrate that disruption of the BH3-only proapoptotic factor Bim in mice prevents induction of apoptosis in and clearing of the lumen in terminal end buds during puberty. However, cells that fill the presumptive luminal space are eventually cleared from the adjacent ducts by a caspase-independent death process. Within the filled Bim(-/-) ducts, epithelial cells are deprived of matrix attachment and undergo squamous differentiation prior to clearing. Similarly, we also detect squamous differentiation in vitro when immortalized mammary epithelial cells are detached from the matrix. These data provide important mechanistic information on the processes involved in sculpting the mammary gland and demonstrate that BIM is a critical regulator of apoptosis in vivo.

Anti-parietal cell autoimmunity is associated with an accelerated decline of lung function in IPF patients.

BACKGROUND: Autoantibodies against lung epithelial antigens are often detected in patients with Idiopathic Pulmonary Fibrosis (IPF). Anti-Parietal Cell Antibodies (APCA) target the H+/K+ATPase (proton pump). APCA prevalence and lung H+/K+ATPase expression was never studied in IPF patients. METHODS: We retrospectively collected clinical, lung function and imaging data from APCA positive patients (APCA+IPF) and compared them with APCA negative IPF patients matched on the date of diagnostic assessment. H+/K+ATPase expression was assessed with immunohistochemistry and PCR. RESULTS: Among 138 IPF patients diagnosed between 2007 and 2014 and tested for APCA, 19 (13.7%) APCA+ patients were identified. APCA+IPF patients were 16 men and 3 women, mean age 71 years. The median titer of APCA was 1:160. A pernicious anemia was present in 5 patients and preceded the fibrosis in 3 cases. With a mean follow up of 31 months, 2 patients had an exacerbation and 7 patients died. As compared with 19 APCA- IPF patients, APCA+IPF patients had a less severe disease with better DLCO (57% vs 43% predicted), preserved PaO2 (85 ± 8 mmHg vs 74 ± 11 mmHg), a lower rate of honeycombing on HRCT (58% vs 89%), but they experienced an accelerated decline of FVC (difference 61.4 ml/year; p = .0002). The H+/K+ATPase was strongly expressed by hyperplastic alveolar epithelial cells in the fibrotic lung. CONCLUSION: Anti-parietal cell autoimmunity is detected in some IPF patients and is associated with an accelerated decline of lung function. Anti-parietal cell autoimmunity may promote lung fibrosis progression.

The pro-apoptotic BAX protein influences cell growth and differentiation from the nucleus in healthy interphasic cells.

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.