Human Idiopathic Pulmonary Fibrosis Research Articles

Identification of CFH and FHL2 as biomarkers for idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology with a poor prognosis, characterized by a lack of effective diagnostic and therapeutic interventions. The role of immunity in the pathogenesis of IPF is significant, yet remains inadequately understood. This study aimed to identify potential key genes in IPF and their relationship with immune cells by integrated bioinformatics analysis and verify by in vivo and in vitro experiments.MethodsGene microarray data were obtained from the Gene Expression Omnibus (GEO) for differential expression analysis. The differentially expressed genes (DEGs) were identified and subjected to functional enrichment analysis. By utilizing a combination of three machine learning algorithms, specific genes associated with idiopathic pulmonary fibrosis (IPF) were pinpointed. Then their diagnostic significance and potential co-regulators were elucidated. We further analyzed the correlation between key genes and immune infiltrating cells via single-sample gene set enrichment analysis (ssGSEA). Subsequently, a single-cell RNA sequencing data (scRNA-seq) was used to explore which cell types expressed key genes in IPF samples. Finally, a series of in vivo and in vitro experiments were conducted to validate the expression of candidate genes by western blot (WB), quantitative real-time PCR (qRT-PCR), and immunohistochemistry (IHC) analysis.ResultsA total of 647 DEGs of IPF were identified based on two datasets, including 225 downregulated genes and 422 upregulated genes. They are closely related to biological functions such as cell migration, structural organization, immune cell chemotaxis, and extracellular matrix. CFH and FHL2 were identified as key genes with diagnostic accuracy for IPF by three machine learning algorithms. Analysis using ssGSEA revealed a significant association of both CFH and FHL2 with diverse immune cells, such as B cells and NK cells. Further scRNA-seq analysis indicated CFH and FHL2 were specifically upregulated in human IPF tissues, which was confirmed by in vitro and in vivo experiments.ConclusionIn this study, CFH and FHL2 have been identified as novel potential biomarkers for IPF, with potential diagnostic utility in future clinical applications. Subsequent investigations into the functions of these genes in IPF and their interactions with immune cells may enhance comprehension of the disease’s pathogenesis and facilitate the identification of therapeutic targets.

[64Cu]Cu-PEG-FUD peptide for noninvasive and sensitive detection of murine pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease resulting in irreversible scarring within the lungs. However, the lack of biomarkers that enable real-time assessment of disease activity remains a challenge in providing efficient clinical decision-making and optimal patient care in IPF. Fibronectin (FN) is highly expressed in fibroblastic foci of the IPF lung where active extracellular matrix (ECM) deposition occurs. Functional upstream domain (FUD) tightly binds the N-terminal 70-kilodalton domain of FN that is crucial for FN assembly. In this study, we first demonstrate the capacity of PEGylated FUD (PEG-FUD) to target FN deposition in human IPF tissue ex vivo. We subsequently radiolabeled PEG-FUD with 64Cu and monitored its spatiotemporal biodistribution via μPET/CT imaging in mice using the bleomycin-induced model of pulmonary injury and fibrosis. We demonstrated [64Cu]Cu-PEG-FUD uptake 3 and 11 days following bleomycin treatment, suggesting that radiolabeled PEG-FUD holds promise as an imaging probe in aiding the assessment of fibrotic lung disease activity.

Insights into Disease Progression of Translational Preclinical Rat Model of Interstitial Pulmonary Fibrosis through Endpoint Analysis.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease characterized by the relentless deposition of extracellular matrix (ECM), causing lung distortions and dysfunction. Animal models of human IPF can provide great insight into the mechanistic pathways underlying disease progression and a means for evaluating novel therapeutic approaches. In this study, we describe the effect of bleomycin concentration on disease progression in the classical rat bleomycin model. In a dose-response study (1.5, 2, 2.5 U/kg i.t), we characterized lung fibrosis at day 14 after bleomycin challenge using endpoints including clinical signs, inflammatory cell infiltration, collagen content, and bronchoalveolar lavage fluid-soluble profibrotic mediators. Furthermore, we investigated fibrotic disease progression after 2 U/kg i.t. bleomycin administration at days 3, 7, and 14 by quantifying the expression of clinically relevant signaling molecules and pathways, epithelial mesenchymal transition (EMT) biomarkers, ECM components, and histopathology of the lung. A single bleomycin challenge resulted in a progressive fibrotic response in rat lung tissue over 14 days based on lung collagen content, histopathological changes, and modified Ashcroft score. The early fibrogenesis phase (days 3 to 7) is associated with an increase in profibrotic mediators including TGFβ1, IL6, TNFα, IL1β, CINC1, WISP1, VEGF, and TIMP1. In the mid and late fibrotic stages, the TGFβ/Smad and PDGF/AKT signaling pathways are involved, and clinically relevant proteins targeting galectin-3, LPA1, transglutaminase-2, and lysyl oxidase 2 are upregulated on days 7 and 14. Between days 7 and 14, the expressions of vimentin and α-SMA proteins increase, which is a sign of EMT activation. We confirmed ECM formation by increased expressions of procollagen-1Aα, procollagen-3Aα, fibronectin, and CTGF in the lung on days 7 and 14. Our data provide insights on a complex network of several soluble mediators, clinically relevant signaling pathways, and target proteins that contribute to drive the progressive fibrotic phenotype from the early to late phase (active) in the rat bleomycin model. The framework of endpoints of our study highlights the translational value for pharmacological interventions and mechanistic studies using this model.

Brg1/PRMT5 nuclear complex epigenetically regulates FOXO1 in IPF mesenchymal progenitor cells.

We have discovered intrinsically fibrogenic mesenchymal progenitor cells (MPCs) in the human idiopathic pulmonary fibrosis (IPF) lung. IPF MPCs display a durably distinct transcriptome, suggesting that they have undergone epigenetic modifications. Prior studies indicate that the chromatin remodeler Brg1 associates with the arginine methyltransferase PRMT5 to epigenetically regulate transcription factors. We hypothesize that a Brg1/PRMT5 nuclear complex epigenetically regulates critical nodes in IPF MPC self-renewal signaling networks. IPF and control MPCs were isolated from primary mesenchymal cell lines established from IPF and control patients. RNA-sequencing identified increased expression of the FOXO1 transcription factor in IPF MPCs compared with controls, a result we confirmed by Q-PCR and Western blot analysis. Immunoprecipitation identified a CD44/Brg1/PRMT5 nuclear complex in IPF MPCs. Chromatin immunoprecipitation assays showed that PRMT5 and its methylation mark H3R2me2 are enriched on the FOXO1 promoter. We show that loss of Brg1 and PRMT5 function decreases FOXO1 expression and impairs IPF MPC self-renewal, and that loss of FOXO1 function decreases IPF MPC self-renewal and expression of the SOX2 and OCT4 stemness markers. Our findings indicate that the FOXO1 gene is overexpressed in IPF MPCs in a CD44/Brg1/PRMT5 nuclear complex-dependent manner. Our data suggest that Brg1 alters chromatin accessibility, enriching PRMT5 occupancy on the FOXO1 promoter, and PRMT5 methylates histone H3 arginine 2 (H3R2) on the FOXO1 promoter, increasing its expression. Our data are in accord with the concept that this coordinated interplay is responsible for promoting IPF MPC self-renewal and maintaining a critical pool of fibrogenic MPCs that drive IPF progression.NEW & NOTEWORTHY Our research offers valuable understanding regarding the epigenetic control of IPF MPC. The data we obtained strongly support the idea that the coordination between chromatin remodeling and histone methylation plays a key role in regulating transcription factors. Specifically, our findings indicate that FOXO1, an essential transcription factor, likely governs the self-renewal of IPF MPC, which is crucial for maintaining a critical pool of fibrogenic MPCs. This interplay could be an important therapeutic target.

Toosendanin Restrains Idiopathic Pulmonary Fibrosis by Inhibiting ZEB1/CTBP1 Interaction.

Extensive deposition of extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) is due to hyperactivation and proliferation of pulmonary fibroblasts. However, the exact mechanism is not clear. This study focused on the role of CTBP1 in lung fibroblast function, elaborated its regulation mechanism, and analyzed the relationship between CTBP1 and ZEB1. Meanwhile, the antipulmonary fibrosis effect and its molecular mechanism of Toosendanin were studied. Human IPF fibroblast cell lines (LL-97A and LL-29) and normal fibroblast cell lines (LL-24) were cultured in vitro. The cells were stimulated with FCS, PDGF-BB, IGF-1, and TGF-β1, respectively. BrdU detected cell proliferation. The mRNA expression of CTBP1 and ZEB1 was detected by QRT-PCR. Western blotting was used to detect the expression of COL1A1, COL3A1, LN, FN, and α-SMA proteins. An animal model of pulmonary fibrosis was established to analyze the effects of CTBP1 silencing on pulmonary fibrosis and lung function in mice. CTBP1 was up-regulated in IPF lung fibroblasts. Silencing CTBP1 inhibits growth factor-driven proliferation and activation of lung fibroblasts. Overexpression of CTBP1 promotes growth factor-driven proliferation and activation of lung fibroblasts. Silencing CTBP1 reduced the degree of pulmonary fibrosis in mice with pulmonary fibrosis. Western blot, CO-IP, and BrdU assays confirmed that CTBP1 interacts with ZEB1 and promotes the activation of lung fibroblasts. Toosendanin can inhibit the ZEB1/CTBP1protein interaction and further inhibit the progression of pulmonary fibrosis. CTBP1 can promote the activation and proliferation of lung fibroblasts through ZEB1. CTBP1 promotes lung fibroblast activation through ZEB1, thereby increasing excessive deposition of ECM and aggravating IPF. Toosendanin may be a potential treatment for pulmonary fibrosis. The results of this study provide a new basis for clarifying the molecular mechanism of pulmonary fibrosis and developing new therapeutic targets.

Regulation of epithelial transitional states in murine and human pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease arising from impaired regeneration of the alveolar epithelium after injury. During regeneration, type 2 alveolar epithelial cells (AEC2s) assume a transitional state that upregulates multiple keratins and ultimately differentiate into AEC1s. In IPF, transitional AECs accumulate with ineffectual AEC1 differentiation. However, whether and how transitional cells cause fibrosis, whether keratins regulate transitional cell accumulation and fibrosis, and why transitional AECs and fibrosis resolve in mouse models but accumulate in IPF are unclear. Here, we show that human keratin 8 (KRT8) genetic variants were associated with IPF. Krt8-/- mice were protected from fibrosis and accumulation of the transitional state. Keratin 8 (K8) regulated the expression of macrophage chemokines and macrophage recruitment. Profibrotic macrophages and myofibroblasts promoted the accumulation of transitional AECs, establishing a K8-dependent positive feedback loop driving fibrogenesis. Finally, rare murine transitional AECs were highly senescent and basaloid and may not differentiate into AEC1s, recapitulating the aberrant basaloid state in human IPF. We conclude that transitional AECs induced and were maintained by fibrosis in a K8-dependent manner; in mice, most transitional cells and fibrosis resolved, whereas in human IPF, transitional AECs evolved into an aberrant basaloid state that persisted with progressive fibrosis.

Genetic deficiency of the transcription factor NFAT1 confers protection against fibrogenic responses independent of immune influx.

Idiopathic pulmonary fibrosis (IPF) is marked by unremitting matrix deposition and architectural distortion. Multiple profibrotic pathways contribute to the persistent activation of mesenchymal cells (MCs) in fibrosis, highlighting the need to identify and target common signaling pathways. The transcription factor nuclear factor of activated T cells 1 (NFAT1) lies downstream of second messenger calcium signaling and has been recently shown to regulate key profibrotic mediator autotaxin (ATX) in lung MCs. Herein, we investigate the role of NFAT1 in regulating fibroproliferative responses during the development of lung fibrosis. Nfat1-/--deficient mice subjected to bleomycin injury demonstrated improved survival and protection from lung fibrosis and collagen deposition as compared with bleomycin-injured wild-type (WT) mice. Chimera mice, generated by reconstituting bone marrow cells from WT or Nfat1-/- mice into irradiated WT mice (WT→WT and Nfat1-/-→WT), demonstrated no difference in bleomycin-induced fibrosis, suggesting immune influx-independent fibroprotection in Nfat1-/- mice. Examination of lung tissue and flow sorted lineageneg/platelet-derived growth factor receptor alpha (PDGFRα)pos MCs demonstrated decreased MC numbers, proliferation [↓ cyclin D1 and 5-ethynyl-2'-deoxyuridine (EdU) incorporation], myofibroblast differentiation [↓ α-smooth muscle actin (α-SMA)], and survival (↓ Birc5) in Nfat1-/- mice. Nfat1 deficiency abrogated ATX expression in response to bleomycin in vivo and MCs derived from Nfat1-/- mice demonstrated decreased ATX expression and migration in vitro. Human IPF MCs demonstrated constitutive NFAT1 activation, and regulation of ATX in these cells by NFAT1 was confirmed using pharmacological and genetic inhibition. Our findings identify NFAT1 as a critical mediator of profibrotic processes, contributing to dysregulated lung remodeling and suggest its targeting in MCs as a potential therapeutic strategy in IPF.NEW & NOTEWORTHY Idiopathic pulmonary fibrosis (IPF) is a fatal disease with hallmarks of fibroblastic foci and exuberant matrix deposition, unknown etiology, and ineffective therapies. Several profibrotic/proinflammatory pathways are implicated in accelerating tissue remodeling toward a honeycombed end-stage disease. NFAT1 is a transcriptional factor activated in IPF tissues. Nfat1-deficient mice subjected to chronic injury are protected against fibrosis independent of immune influxes, with suppression of profibrotic mesenchymal phenotypes including proliferation, differentiation, resistance to apoptosis, and autotaxin-related migration.

In vivo study for Therapeutic Potential of Bioactive Compounds from Curcuma longa and Tinospora cordifolia against Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with no known cause. It is a severe condition characterized by the accumulation of excessive extracellular matrix (ECM) in the lungs, leading to difficulty in oxygen intake. In this study, the researchers aimed to investigate the potential therapeutic effects of Curcuma longa and Tinospora cordifolia extracts in a mouse model of bleomycin-induced pulmonary fibrosis. The mice were divided into groups and treated with either the extracts, pirfenidone (a commonly used IPF treatment), or a control. The effects of these treatments on lung edema, body weight, antioxidant enzyme activity, collagen content and myeloperoxidase (MPO) activity were assessed. The results showed that treatment with the extracts and pirfenidone led to improvements in lung edema and body weight compared to the bleomycin group. The extracts also exhibited antioxidant and anti-inflammatory effects by increasing the activity of catalase and superoxide dismutase enzymes while reducing MPO activity. Furthermore, the extracts reduced collagen content, indicating a potential anti-fibrotic effect. Histological analysis of lung tissue confirmed the beneficial effects of the extracts, showing reduced lung damage, inflammatory cell infiltration and collagen deposition. These findings suggest that Curcuma longa and Tinospora cordifolia extracts may have therapeutic potential in treating IPF. In conclusion, this study demonstrates the potential anti-fibrotic and anti-inflammatory effects of Curcuma longa and Tinospora cordifolia extracts in a bleomycin-induced mouse model of pulmonary fibrosis. Further research is warranted to elucidate the underlying mechanisms and to determine their efficacy in human IPF patients. These findings highlight the potential of traditional medicines as alternative or complementary treatments for IPF, offering advantages such as long-term duration, low adverse effects and stable therapeutic impact.

Dynamic atlas of immune cells reveals multiple functional features of macrophages associated with progression of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease with a high mortality rate and unclarified aetiology. Immune response is elaborately regulated during the progression of IPF, but immune cells subsets are complicated which has not been detailed described during IPF progression. Therefore, in the current study, we sought to investigate the role of immune regulation by elaborately characterize the heterogeneous of immune cells during the progression of IPF. To this end, we performed single-cell profiling of lung immune cells isolated from four stages of bleomycin-induced pulmonary fibrosis-a classical mouse model that mimics human IPF. The results revealed distinct components of immune cells in different phases of pulmonary fibrosis and close communication between macrophages and other immune cells along with pulmonary fibrosis progression. Enriched signals of SPP1, CCL5 and CXCL2 were found between macrophages and other immune cells. The more detailed definition of the subpopulations of macrophages defined alveolar macrophages (AMs) and monocyte-derived macrophages (mo-Macs)-the two major types of primary lung macrophages-exhibited the highest heterogeneity and dynamic changes in expression of profibrotic genes during disease progression. Our analysis suggested that Gpnmb and Trem2 were both upregulated in macrophages and may play important roles in pulmonary fibrosis progression. Additionally, the metabolic status of AMs and mo-Macs varied with disease progression. In line with the published data on human IPF, macrophages in the mouse model shared some features regarding gene expression and metabolic status with that of macrophages in IPF patients. Our study provides new insights into the pathological features of profibrotic macrophages in the lung that will facilitate the identification of new targets for disease intervention and treatment of IPF.

MicroRNA-124-3p alleviates repetitive bleomycin induced idiopathic pulmonary fibrosis in mouse by repressing Wnt/β-catenin signaling component AXIN1

Background
 MicroRNA (miR)-124-3p is a crucial player in the transforming growth factor β1-induced in vitro fibrogenic differentiation of mesenchymal stem cells. In the current study we aimed to further verify the in vivo role of miR-124-3p in a mouse model of idiopathic pulmonary fibrosis (IPF).
 Methods
 Mouse IPF model was established using repetitive intratracheal bleomycin (BLM) dosing, followed by in vivo delivery of miR-124-3p. Masson’s trichrome staining, Hematoxyline-eosin (H&E), as well as modified Ashcroft score, were performed on lung tissues to assess extent of pulmonary fibrosis. Collagen deposition was examined using hydroxyproline assay. Inflammatory cell counts were evaluated in the bronchoalveolar Lavage (BAL) fluid. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to assess apoptosis in lung tissues.
 Results
 Repetitive BLM injection induced elevated Fibrosis score and collagen deposition, elevated numbers of inflammatory cells in the BAL fluid, and promoted in lung tissues of mice. MiR-124-3p, to a considerable extent, reversed the BLM-induced IPF symptoms in terms of fibrosis score and collagen deposition in the lung tissues, reduced the BLM-elevated inflammatory cells in the BAL fluid and the percentage of BLM-induced apoptotic cells in lung tissues. AXIN1, a pivotal component in Wnt signaling, was also significantly inhibited by miR-124-3p in the experimental mice.
 Conclusion
 MiR-124-3p serves as a therapeutic target in the mouse model of IPF by repressing Wnt/β-catenin signaling component AXIN1, and holds great clinical potential in molecular therapies to treat human IPF patients.

Increased lipocalin-2 expression in pulmonary inflammation and fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive interstitial lung disease with dismal prognosis. The underlying pathogenic mechanisms are poorly understood, resulting in a lack of effective treatments. However, recurrent epithelial damage is considered critical for disease initiation and perpetuation, via the secretion of soluble factors that amplify inflammation and lead to fibroblast activation and exuberant deposition of ECM components. Lipocalin-2 (LCN2) is a neutrophil gelatinase-associated lipocalin (NGAL) that has been suggested as a biomarker of kidney damage. LCN2 has been reported to modulate innate immunity, including the recruitment of neutrophils, and to protect against bacterial infections by sequestering iron. In silico analysis of publicly available transcriptomic datasets; ELISAs on human IPF patients' bronchoalveolar lavage fluids (BALFs); bleomycin (BLM)-induced pulmonary inflammation and fibrosis and LPS-induced acute lung injury (ALI) in mice: pulmonary function tests, histology, Q-RT-PCR, western blot, and FACS analysis. Increased LCN2 mRNA expression was detected in the lung tissue of IPF patients negatively correlating with respiratory functions, as also shown for BALF LCN2 protein levels in a cohort of IPF patients. Increased Lcn2 expression was also detected upon BLM-induced pulmonary inflammation and fibrosis, especially at the acute phase correlating with neutrophilic infiltration, as well as upon LPS-induced ALI, an animal model characterized by neutrophilic infiltration. Surprisingly, and non withstanding the limitations of the study and the observed trends, Lcn2-/- mice were found to still develop BLM- or LPS-induced pulmonary inflammation and fibrosis, thus questioning a major pathogenic role for Lcn2 in mice. However, LCN2 qualifies as a surrogate biomarker of pulmonary inflammation and a possible indicator of compromised pulmonary functions, urging for larger studies.

The Regenerative Power of Stem Cells: Treating Bleomycin-Induced Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with no known cure, characterized by the formation of scar tissue in the lungs, leading to respiratory failure. Although the exact cause of IPF remains unclear, the condition is thought to result from a combination of genetic and environmental factors. One of the most widely used animal models to study IPF is the bleomycin-induced lung injury model in mice. In this model, the administration of the chemotherapeutic agent bleomycin causes pulmonary inflammation and fibrosis, which closely mimics the pathological features of human IPF. Numerous recent investigations have explored the functions of various categories of stem cells in the healing process of lung injury induced by bleomycin in mice, documenting the beneficial effects and challenges of this approach. Differentiation of stem cells into various cell types and their ability to modulate tissue microenvironment is an emerging aspect of the regenerative therapies. This review article aims to provide a comprehensive overview of the role of stem cells in repairing bleomycin-induced lung injury. It delves into the mechanisms through which various types of stem cells, including mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, and lung resident stem cells, exert their therapeutic effects in this specific model. We have also discussed the unique set of intermediate markers and signaling factors that can influence the proliferation and differentiation of alveolar epithelial cells both during lung repair and homeostasis. Finally, we highlight the challenges and opportunities associated with translating stem cell therapy to the clinic for IPF patients. The novelty and implications of this review extend beyond the understanding of the potential of stem cells in treating IPF to the broader field of regenerative medicine. We believe that the review paves the way for further advancements in stem cell therapies, offering hope for patients suffering from this debilitating and currently incurable disease.

Investigating the role of platelets and platelet-derived transforming growth factor-β in idiopathic pulmonary fibrosis.

Transforming growth factor-β1 (TGFβ1) is the key profibrotic cytokine in idiopathic pulmonary fibrosis (IPF), but the primary source of this cytokine in this disease is unknown. Platelets have abundant stores of TGFβ1, although the role of these cells in IPF is ill-defined. In this study, we investigated whether platelets, and specifically platelet-derived TGFβ1, mediate IPF disease progression. Patients with IPF and non-IPF patients were recruited to determine platelet reactivity, and separate cohorts of patients with IPF were followed for mortality. To study whether platelet-derived TGFβ1 modulates pulmonary fibrosis (PF), mice with a targeted deletion of TGFβ1 in megakaryocytes and platelets (TGFβ1fl/fl.PF4-Cre) were used in the well-characterized bleomycin-induced pulmonary fibrosis (PF) animal model. In a discovery cohort, we found significantly higher mortality in patients with IPF who had elevated platelet counts within the normal range. However, our validation cohort did not confirm this observation, despite significantly increased platelets, neutrophils, active TGFβ1, and CCL5, a chemokine produced by inflammatory cells, in the blood, lung, and bronchoalveolar lavage (BAL) of patients with IPF. In vivo, we showed that despite platelets being readily detected within the lungs of bleomycin-treated mice, neither the degree of pulmonary inflammation nor fibrosis was significantly different between TGFβ1fl/fl.PF4-Cre and control mice. Our results demonstrate for the first time that platelet-derived TGFβ1 does not significantly mediate inflammation or fibrosis in a PF animal model. Furthermore, our human studies revealed blood platelet counts do not consistently predict mortality in IPF but other platelet-derived mediators, such as C-C chemokine ligand 5 (CCL5), may promote neutrophil recruitment and human IPF.NEW & NOTEWORTHY Platelets are a rich source of profibrotic TGFβ; however, the role of platelets in idiopathic pulmonary fibrosis (IPF) is unclear. We identified that patients with IPF have significantly more platelets, neutrophils, and active TGFβ in their airways than control patients. Using an animal model of IPF, we demonstrated that platelet-derived TGFβ does not significantly drive lung fibrosis or inflammation. Our findings offer a better understanding of platelets in both human and animal studies of IPF.

Inhibition of tyrosine kinase Fgr prevents radiation-induced pulmonary fibrosis (RIPF)

Cellular senescence is involved in the development of pulmonary fibrosis as well as in lung tissue repair and regeneration. Therefore, a strategy of removal of senescent cells by senolytic drugs may not produce the desired therapeutic result. Previously we reported that tyrosine kinase Fgr is upregulated in ionizing irradiation-induced senescent cells. Inhibition of Fgr reduces the production of profibrotic proteins by radiation-induced senescent cells in vitro; however, a mechanistic relationship between senescent cells and radiation-induced pulmonary fibrosis (RIPF) has not been established. We now report that senescent cells from the lungs of mice with RIPF, release profibrotic proteins for target cells and secrete chemotactic proteins for marrow cells. The Fgr inhibitor TL02-59, reduces this release of profibrotic chemokines from the lungs of RIPF mice, without reducing numbers of senescent cells. In vitro studies demonstrated that TL02-59 abrogates the upregulation of profibrotic genes in target cells in transwell cultures. Also, protein arrays using lung fibroblasts demonstrated that TL02-59 inhibits the production of chemokines involved in the migration of macrophages to the lung. In thoracic-irradiated mice, TL02-59 prevents RIPF, significantly reduces levels of expression of fibrotic gene products, and significantly reduces the recruitment of CD11b+ macrophages to the lungs. Bronchoalveolar lavage (BAL) cells from RIPF mice show increased Fgr and other senescent cell markers including p16. In human idiopathic pulmonary fibrosis (IPF) and in RIPF, Fgr, and other senescent cell biomarkers are increased. In both mouse and human RIPF, there is an accumulation of Fgr-positive proinflammatory CD11b+ macrophages in the lungs. Thus, elevated levels of Fgr in lung senescent cells upregulate profibrotic gene products, and chemokines that might be responsible for macrophage infiltration into lungs. The detection of Fgr in senescent cells that are obtained from BAL during the development of RIPF may help predict the onset and facilitate the delivery of medical countermeasures.

Nicotinamide N-methyltransferase mediates lipofibroblast–myofibroblast transition and apoptosis resistance

Metabolism controls cellular phenotype and fate. In this report, we demonstrate that nicotinamide N-methyltransferase (NNMT), a metabolic enzyme that regulates developmental stem cell transitions and tumor progression, is highly expressed in human idiopathic pulmonary fibrosis (IPF) lungs, and is induced by the pro-fibrotic cytokine, transforming growth factor-β1 (TGF-β1) in lung fibroblasts. NNMT silencing reduces the expression of extracellular matrix proteins, both constitutively and in response to TGF-β1. Furthermore, NNMT controls the phenotypic transition from homeostatic, pro-regenerative lipofibroblasts to pro-fibrotic myofibroblasts. This effect of NNMT is mediated, in part, by the downregulation of lipogenic transcription factors, TCF21 and PPARγ, and the induction of a less proliferative but more differentiated myofibroblast phenotype. NNMT confers an apoptosis-resistant phenotype to myofibroblasts that is associated with the downregulation of pro-apoptotic members of the Bcl-2 family, including Bim and PUMA. Together, these studies indicate a critical role for NNMT in the metabolic reprogramming of fibroblasts to a pro-fibrotic and apoptosis-resistant phenotype and support the concept that targeting this enzyme may promote regenerative responses in chronic fibrotic disorders such as IPF.

AAV9-Tspyl2 gene therapy retards bleomycin-induced pulmonary fibrosis by modulating downstream TGF-β signaling in mice

Idiopathic pulmonary fibrosis (IPF) is a devastating fibrotic lung disease characterized by scarring and destruction of the lung architecture, with limited treatment options. Targeted gene therapy to restore cell division autoantigen-1 (CDA1) expression may be a potential treatment approach to delay the progression of pulmonary fibrosis (PF). Here, we focused on CDA1, which was significantly decreased in human IPF, in a mouse model of bleomycin (BLM)-induced PF, and in transforming growth factor (TGF-β)-challenged lung fibroblasts. In vitro, CDA1 overexpression by lentivirus infection in human embryonic lung fibroblasts (HFL1 cells) inhibited the production of pro-fibrotic and pro-inflammatory cytokines, lung fibroblast-to-myofibroblast transition, and extracellular matrix protein expression induced by exogenous TGF‐β1 treatment, whereas CDA1 knockdown with small interfering RNA promoted this effect. CDA1 overexpression also inhibited cell proliferation and migration. In a mouse model of BLM-induced PF, we provided novel evidence that the intratracheal delivery of adeno-associated virus serotype 9 carrying the mouse Tspyl2 gene reduced lung tissue inflammation and fibrosis. Mechanistically, CDA1, as a transcription regulator, could repress the TGF-β signal transduction in vivo and in vitro. In conclusion, our results show that Tspyl2 gene therapy plays an antifibrotic role by inhibiting the lung fibroblast-to-myofibroblast transition and downstream TGF-β/Smad3 signaling transduction in BLM-induced PF in mice, suggesting that CDA1 is an appropriate and promising therapeutic target for PF.

Pulmonary fibrosis: A short- or long-term sequelae of severe COVID-19?

The pandemic of coronavirus disease 2019 (COVID‑19), caused by a novel severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2), has caused an enormous impact on the global healthcare. SARS-CoV-2 infection primarily targets the respiratory system. Although most individuals testing positive for SARS-CoV-2 present mild or no upper respiratory tract symptoms, patients with severe COVID-19 can rapidly progress to acute respiratory distress syndrome (ARDS). ARDS-related pulmonary fibrosis is a recognized sequelae of COVID-19. Whether post-COVID-19 lung fibrosis is resolvable, persistent, or even becomes progressive as seen in human idiopathic pulmonary fibrosis (IPF) is currently not known and remains a matter of debate. With the emergence of effective vaccines and treatments against COVID-19, it is now important to build our understanding of the long-term sequela of SARS-CoV-2 infection, to identify COVID-19 survivors who are at risk of developing chronic pulmonary fibrosis, and to develop effective anti-fibrotic therapies. The current review aims to summarize the pathogenesis of COVID-19 in the respiratory system and highlights ARDS-related lung fibrosis in severe COVID-19 and the potential mechanisms. It envisions the long-term fibrotic lung complication in COVID-19 survivors, in particular in the aged population. The early identification of patients at risk of developing chronic lung fibrosis and the development of anti-fibrotic therapies are discussed.

Early Detection and Staging of Lung Fibrosis Enabled by Collagen-Targeted MRI Protein Contrast Agent.

Chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), are major leading causes of death worldwide and are generally associated with poor prognoses. The heterogeneous distribution of collagen, mainly type I collagen associated with excessive collagen deposition, plays a pivotal role in the progressive remodeling of the lung parenchyma to chronic exertional dyspnea for both IPF and COPD. To address the pressing need for noninvasive early diagnosis and drug treatment monitoring of pulmonary fibrosis, we report the development of human collagen-targeted protein MRI contrast agent (hProCA32.collagen) to specifically bind to collagen I overexpressed in multiple lung diseases. When compared to clinically approved Gd3+ contrast agents, hProCA32.collagen exhibits significantly better r1 and r2 relaxivity values, strong metal binding affinity and selectivity, and transmetalation resistance. Here, we report the robust detection of early and late-stage lung fibrosis with stage-dependent MRI signal-to-noise ratio (SNR) increase, with good sensitivity and specificity, using a progressive bleomycin-induced IPF mouse model. Spatial heterogeneous mapping of usual interstitial pneumonia (UIP) patterns with key features closely mimicking human IPF, including cystic clustering, honeycombing, and traction bronchiectasis, were noninvasively detected by multiple MR imaging techniques and verified by histological correlation. We further report the detection of fibrosis in the lung airway of an electronic cigarette-induced COPD mouse model, using hProCA32.collagen-enabled precision MRI (pMRI), and validated by histological analysis. The developed hProCA32.collagen is expected to have strong translational potential for the noninvasive detection and staging of lung diseases, and facilitating effective treatment to halt further chronic lung disease progression.

Lung endothelial cells regulate pulmonary fibrosis through FOXF1/R-Ras signaling

Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF.

Interleukin-11 disrupts alveolar epithelial progenitor function

Interleukin-11 (IL-11) is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF), since IL-11 induces myofibroblast differentiation and stimulates their excessive collagen deposition in the lung. In IPF there is disrupted alveolar structural architecture, yet the effect of IL-11 on the dysregulated alveolar repair remains to be elucidated. We hypothesised that epithelial-fibroblast communication associated with lung repair is disrupted by IL-11. Thus, we studied whether IL-11 affects the repair responses of alveolar lung epithelium using mouse lung organoids and precision-cut lung slices (PCLS). Additionally, we assessed the anatomical distribution of IL-11 and IL-11 receptor (IL-11R) in human control and IPF lungs using immunohistochemistry. IL-11 protein was observed in airway epithelium, macrophages and in IPF lungs, also in areas of alveolar type 2 (AT2) cell hyperplasia. IL-11R staining was predominantly present in smooth muscle and macrophages. In mouse organoid co-cultures of epithelial cells with lung fibroblasts, IL-11 decreased organoid number and reduced the fraction of Prosurfactant Protein C-expressing organoids, indicating dysfunctional regeneration initiated by epithelial progenitors. In mouse PCLS exposed to IL-11, ciliated cell markers were increased. The response of primary human fibroblasts to IL-11 on gene expression level was minimal, though bulk RNA-sequencing revealed IL-11 modulated various processes which are associated with IPF, including unfolded protein response, glycolysis and Notch signalling. IL-11 disrupts alveolar epithelial regeneration by inhibiting progenitor activation and suppressing the formation of mature alveolar epithelial cells. Evidence for a contribution of dysregulated fibroblast-epithelial communication to this process is limited.