Early IFN-α signatures and persistent dysfunction are distinguishing features of NK cells in severe COVID-19.

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.

The deficiency of DNASE1L3 does not affect systemic sclerosis pathogenesis in two inducible murine models of the disease.

We induced systemic sclerosis (SSc)-like disease in both wild-type and Dnase1l3-deficient mice using two distinct approaches involving bleomycin and hypochlorous acid injections. Our observations revealed that the deficiency in DNASE1L3 did not affect tissue fibrosis or inflammation caused by these treatments. Despite the association of single nucleotide polymorphisms in humans with SSc pathogenesis, our study demonstrates that DNASE1L3 is dispensable in two inducible murine models of SSc-like pathogenesis.

Gene therapy with AAV9-SGPL1 in an animal model of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-ß signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-ß signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1ß as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

CasRx-based Wnt activation promotes alveolar regeneration while ameliorating pulmonary fibrosis in a mouse model of lung injury.

Wnt/ß-catenin signaling is an attractive target for regenerative medicine. A powerful driver of stem cell activity and hence tissue regeneration, Wnt signaling can promote fibroblast proliferation and activation, leading to fibrosis, while prolonged Wnt signaling is potentially carcinogenic. Thus, to harness its therapeutic potential, the activation of Wnt signaling must be transient, reversible, and tissue specific. In the lung, Wnt signaling is essential for alveolar stem cell activity and alveolar regeneration, which is impaired in lung fibrosis. Activation of Wnt/ß-catenin signaling in lung epithelium may have anti-fibrotic effects. Here, we used intratracheal adeno-associated virus 6 injection to selectively deliver CasRx into the lung epithelium, where it reversibly activates Wnt signaling by simultaneously degrading mRNAs encoding Axin1 and Axin2, negative regulators of Wnt/ß-catenin signaling. Interestingly, CasRx-mediated Wnt activation specifically in lung epithelium not only promotes alveolar type II cell proliferation and alveolar regeneration but also inhibits lung fibrosis resulted from bleomycin-induced injury, relevant in both preventive and therapeutic settings. Our study offers an attractive strategy for treating pulmonary fibrosis, with general implications for regenerative medicine.

Nanoparticle targeting of de novo profibrotic macrophages mitigates lung fibrosis.

The pathogenesis of lung fibrosis involves hyperactivation of innate and adaptive immune pathways that release inflammatory cytokines and growth factors such as tumor growth factor (TGF)ß1 and induce aberrant extracellular matrix protein production. During the genesis of pulmonary fibrosis, resident alveolar macrophages are replaced by a population of newly arrived monocyte-derived interstitial macrophages that subsequently transition into alveolar macrophages (Mo-AMs). These transitioning cells initiate fibrosis by releasing profibrotic cytokines and remodeling the matrix. Here, we describe a strategy for leveraging the up-regulation of the mannose receptor CD206 in interstitial macrophages and Mo-AM to treat lung fibrosis. We engineered mannosylated albumin nanoparticles, which were found to be internalized by fibrogenic CD206+ monocyte derived macrophages (Mo-Macs). Mannosylated albumin nanoparticles incorporating TGFß1 small-interfering RNA (siRNA) targeted the profibrotic subpopulation of CD206+ macrophages and prevented lung fibrosis. The findings point to the potential utility of mannosylated albumin nanoparticles in delivering TGFß-siRNA into CD206+ profibrotic macrophages as an antilung fibrosis strategy.

COVID-19 and pulmonary fibrosis: A potential role for lung epithelial cells and fibroblasts.

The COVID-19 pandemic rapidly spread around the world following the first reports in Wuhan City, China in late 2019. The disease, caused by the novel SARS-CoV-2 virus, is primarily a respiratory condition that can affect numerous other bodily systems including the cardiovascular and gastrointestinal systems. The disease ranges in severity from asymptomatic through to severe acute respiratory distress requiring intensive care treatment and mechanical ventilation, which can lead to respiratory failure and death. It has rapidly become evident that COVID-19 patients can develop features of interstitial pulmonary fibrosis, which in many cases persist for as long as we have thus far been able to follow the patients. Many questions remain about how such fibrotic changes occur within the lung of COVID-19 patients, whether the changes will persist long term or are capable of resolving, and whether post-COVID-19 pulmonary fibrosis has the potential to become progressive, as in other fibrotic lung diseases. This review brings together our existing knowledge on both COVID-19 and pulmonary fibrosis, with a particular focus on lung epithelial cells and fibroblasts, in order to discuss common pathways and processes that may be implicated as we try to answer these important questions in the months and years to come.

Mechanical Stretch Induces Senescence of Lung Epithelial Cells and Drives Fibroblast Activation by Paracrine Mechanisms.

Severe lung injury requiring mechanical ventilation may lead to secondary fibrosis. Senescence, a cell response characterized by cell cycle arrest and a shift towards a proinflammatory/profibrotic phenotype, is one of the involved mechanisms. Here, we explore the contribution of mechanical stretch as trigger of senescence of the respiratory epithelium and its link with fibrosis. Human lung epithelial cells and fibroblasts were exposed in vitro to mechanical stretch, and senescence assessed. In addition, fibroblasts were exposed to culture media preconditioned by senescent epithelial cells and their activation was studied. Transcriptomic profiles from stretched, senescent epithelial cells and activated fibroblasts were combined to identify potential activated pathways. Finally, the senolytic effects of digoxin were tested in these models. Mechanical stretch induced senescence in lung epithelial cells, but not in fibroblasts. This stretch-induced senescence has specific features compared to senescence induced by doxorubicin. Fibroblasts were activated after exposure to supernatants conditioned by epithelial senescent cells. Transcriptomic analyses revealed notch signaling as a potential responsible for the epithelial-mesenchymal crosstalk, as blockade of this pathway inhibits fibroblast activation. Treatment with digoxin reduced the percentage of senescent cells after stretch and ameliorated the fibroblast response to preconditioned media. These results suggest that lung fibrosis in response to mechanical stretch may be caused by the paracrine effects of senescent cells. This pathogenetic mechanism can be pharmacologically manipulated to improve lung repair.

Lung Fibrosis Is Linked to Increased Endothelial Cell Activation and Dysfunctional Vascular Barrier Integrity.

Pulmonary fibrosis (PF) can be a fatal disease characterized by progressive lung scarring. It is still poorly understood how the pulmonary endothelium is involved in the disease pathogenesis. Differences of the pulmonary vasculature between patients and donors were analyzed using transmission electron microscopy, immunohistochemistry, and single-cell RNA sequencing. Vascular barrier resistance, endothelial-immune cell adhesion, and sensitivity to an inflammatory milieu were studied in vitro. Integrity and activation markers were measured by ELISA in human plasma. Transmission electron microscopy demonstrated abnormally swollen endothelial cells (ECs) in fibrotic lungs compared with donors. A more intense CD31 and von Willebrand Factor (vWF) and patchy vascular endothelial (VE)-Cadherin staining in fibrotic lungs supported the presence of a dysregulated endothelium. Integrity markers CD31, VE-Cadherin, Thrombomodulin, and VEGFR-2 (vascular endothelial growth factor receptor-2) and activation marker vWF gene expression was increased in different endothelial subpopulations (e.g., arterial, venous, general capillary, aerocytes) in PF. This was associated with a heightened sensitivity of fibrotic ECs to TNF-α or IFN-γ and elevated immune cell adhesion. The barrier strength was overall reduced in ECs from fibrotic lungs. vWF and IL-8 were increased in the plasma of patients, whereas VE-Cadherin, Thrombomodulin, and VEGFR-2 were decreased. VE-Cadherin staining was also patchy in biopsy tissue and was decreased in plasma samples of patients with PF 6 months after the initial diagnosis. Our data demonstrate highly abnormal ECs in PF. The vascular compartment is characterized by hyperactivation and increased immune cell adhesion, as well as dysfunctional endothelial barrier function. Reestablishing EC homeostasis and function might represent a new therapeutic option for fibrotic lung diseases.

Iron-laden macrophage-mediated paracrine profibrotic signaling induces lung fibroblast activation.

Idiopathic pulmonary fibrosis (IPF) is a devastating condition characterized by progressive lung scarring and uncontrolled fibroblast proliferation, inevitably leading to organ dysfunction and mortality. Although elevated iron levels have been observed in patients and animal models of lung fibrosis, the mechanisms linking iron dysregulation to lung fibrosis pathogenesis, particularly the role of macrophages in orchestrating this process, remain poorly elucidated. Here we evaluate iron metabolism in macrophages during pulmonary fibrosis using both in vivo and in vitro approaches. In murine bleomycin- and amiodarone-induced pulmonary fibrosis models, we observed significant iron deposition and lipid peroxidation in pulmonary macrophages. Intriguingly, the ferroptosis regulator glutathione peroxidase 4 (GPX4) was upregulated in pulmonary macrophages following bleomycin instillation, a finding corroborated by single-cell RNA sequencing analysis. Moreover, macrophages isolated from fibrotic mouse lungs exhibited increased transforming growth factor (TGF)-ß1 expression that correlated with lipid peroxidation. In vitro, iron overload in bone marrow-derived macrophages triggered lipid peroxidation and TGF-ß1 upregulation, which was effectively suppressed by ferroptosis inhibitors. When cocultured with iron-overloaded macrophages, lung fibroblasts exhibited heightened activation, evidenced by increased α-smooth muscle actin and fibronectin expression. Importantly, this profibrotic effect was attenuated by treating macrophages with a ferroptosis inhibitor or blocking TGF-ß receptor signaling in fibroblasts. Collectively, our study elucidates a novel mechanistic paradigm in which the accumulation of iron within macrophages initiates lipid peroxidation, thereby amplifying TGF-ß1 production, subsequently instigating fibroblast activation through paracrine signaling. Thus, inhibiting iron overload and lipid peroxidation warrants further exploration as a strategy to suppress fibrotic stimulation by disease-associated macrophages. NEW & NOTEWORTHY This study investigates the role of iron in pulmonary fibrosis, specifically focusing on macrophage-mediated mechanisms. Iron accumulation in fibrotic lung macrophages triggers lipid peroxidation and an upregulation of transforming growth factor (TGF)-ß1 expression. Coculturing iron-laden macrophages activates lung fibroblasts in a TGF-ß1-dependent manner, which can be mitigated by ferroptosis inhibitors. These findings underscore the potential of targeting iron overload and lipid peroxidation as a promising strategy to alleviate fibrotic stimulation provoked by disease-associated macrophages.

O-GlcNAc regulates anti-fibrotic genes in lung fibroblasts through EZH2.

Epigenetic modifications are involved in fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and contribute to the silencing of anti-fibrotic genes. H3K27me3, a key repressive histone mark, is catalysed by the methyltransferase enhancer of Zeste homologue 2 (EZH2), which is regulated by the post-translational modification, O-linked N-Acetylglucosamine (O-GlcNAc). In this study, we explored the effects of O-GlcNAc and EZH2 on the expression of antifibrotic genes, cyclooxygenase-2 (Cox2) and Heme Oxygenase (Homx1). The expression of Cox2 and Hmox1 was examined in primary IPF or non-IPF lung fibroblasts with or without EZH2 inhibitor EZP6438, O-GlcNAc transferase (OGT) inhibitor (OSMI-1) or O-GlcNAcase (OGA) inhibitor (thiamet G). Non-IPF cells were also subjected to TGF-ß1 with or without OGT inhibition. The reduced expression of Cox2 and Hmox1 in IPF lung fibroblasts is restored by OGT inhibition. In non-IPF fibroblasts, TGF-ß1 treatment reduces Cox2 and Hmox1 expression, which was restored by OGT inhibition. ChIP assays demonstrated that the association of H3K27me3 is reduced at the Cox2 and Hmox1 promoter regions following OGT or EZH2 inhibition. EZH2 levels and stability were decreased by reducing O-GlcNAc. Our study provided a novel mechanism of O-GlcNAc modification in regulating anti-fibrotic genes in lung fibroblasts and in the pathogenesis of IPF.

Evolutionary genetics of pulmonary anatomical adaptations in deep-diving cetaceans.

BACKGROUND: Cetaceans, having experienced prolonged adaptation to aquatic environments, have undergone evolutionary changes in their respiratory systems. This process of evolution has resulted in the emergence of distinctive phenotypic traits, notably the abundance of elastic fibers and thickened alveolar walls in their lungs, which may facilitate alveolar collapse during diving. This structure helps selective exchange of oxygen and carbon dioxide, while minimizing nitrogen exchange, thereby reducing the risk of DCS. Nevertheless, the scientific inquiry into the mechanisms through which these unique phenotypic characteristics govern the diving behavior of marine mammals, including cetaceans, remains unresolved. RESULTS: This study entails an evolutionary analysis of 42 genes associated with pulmonary fibrosis across 45 mammalian species. Twenty-one genes in cetaceans exhibited accelerated evolution, featuring specific amino acid substitutions in 14 of them. Primarily linked to the development of the respiratory system and lung morphological construction, these genes play a crucial role. Moreover, among marine mammals, we identified eight genes undergoing positive selection, and the evolutionary rates of three genes significantly correlated with diving depth. Specifically, the SFTPC gene exhibited convergent amino acid substitutions. Through in vitro cellular experiments, we illustrated that convergent amino acid site mutations in SFTPC contribute positively to pulmonary fibrosis in marine mammals, and the presence of this phenotype can induce deep alveolar collapse during diving, thereby reducing the risk of DCS during diving. CONCLUSIONS: The study unveils pivotal genetic signals in cetaceans and other marine mammals, arising through evolution. These genetic signals may influence lung characteristics in marine mammals and have been linked to a reduced risk of developing DCS. Moreover, the research serves as a valuable reference for delving deeper into human diving physiology.

The effect of adrenalectomy on bleomycin-induced pulmonary fibrosis in mice.

Progressive lung fibrosis is often fatal and has limited treatment options. Though the mechanisms are poorly understood, fibrosis is increasingly linked with catecholamines such as adrenaline (AD) and noradrenaline (NA), and hormones such as aldosterone (ALD). The essential functions of the adrenal glands include the production of catecholamines and numerous hormones, but the contribution of adrenal glands to lung fibrosis remains less well studied. Here, we characterized the impact of surgical adrenal ablation in the bleomycin model of lung fibrosis. Wild type mice underwent surgical adrenalectomy or sham surgery followed by bleomycin administration. We found that while bleomycin-induced collagen over-deposition in the lung was not affected by adrenalectomy, histologic indices of lung remodeling were ameliorated. These findings were accompanied by a decrease of lymphocytes in bronchoalveolar lavage (BAL) and macrophages in lung tissues, along with concomitant reductions in alpha smooth muscle actin (⍺SMA) and fibronectin. Surgical adrenalectomy completely abrogated AD, not NA, detection in all compartments. Systemic ALD levels were reduced after adrenalectomy while ALD levels in lung tissues remained unaffected. Taken together, these results support the presence of a pulmonary-adrenal axis in lung fibrosis and suggest that adrenalectomy is protective in this disease. Further investigation will be needed to better understand this observation and aid in the development of novel therapeutic strategies.

Pulmonary siRNA Delivery with Sophisticated Amphiphilic Poly(Spermine Acrylamides) for the Treatment of Lung Fibrosis.

RNA interference (RNAi) is an efficient strategy to post-transcriptionally silence gene expression. While all siRNA drugs on the market target the liver, the lung offers a variety of currently undruggable targets, which can potentially be treated with RNA therapeutics. To achieve this goal, the synthesis of poly(spermine acrylamides) (P(SpAA) is reported herein. Polymers are prepared via polymerization of N-acryloxysuccinimide (NAS) and afterward this active ester is converted into spermine-based pendant groups. Copolymerizations with decylacrylamide are employed to increase the hydrophobicity of the polymers. After deprotection, polymers show excellent siRNA encapsulation to obtain perfectly sized polyplexes at very low polymer/RNA ratios. In vitro 2D and 3D cell culture, ex vivo and in vivo experiments reveal superior properties of amphiphilic spermine-copolymers with respect to delivery of siRNA to lung cells in comparison to commonly used lipid-based transfection agents. In line with the in vitro results, siRNA delivery to human lung explants confirm more efficient gene silencing of protease-activated receptor 2 (PAR2), a G protein-coupled receptor involved in fibrosis. This study reveals the importance of the balance between efficient polyplex formation, cellular uptake, gene knockdown, and toxicity for efficient siRNA delivery in vitro, in vivo, and in fibrotic human lung tissue ex vivo.

Macrophages as determinants and regulators of systemic sclerosis-related interstitial lung disease.

BACKGROUND: Interstitial lung disease (ILD) is the primary cause of mortality in systemic sclerosis (SSc), an autoimmune disease characterized by tissue fibrosis. SSc-related ILD (SSc-ILD) occurs more frequently in females aged 30-55 years, whereas idiopathic pulmonary fibrosis (IPF) is more prevalent in males aged 60-75 years. SSc-ILD occurs earlier than IPF and progresses rapidly. FCN1, FABP4, and SPP1 macrophages are involved in the pathogenesis of lung fibrosis; SPP1 macrophages demonstrate upregulated expression in both SSc-ILD and IPF. To identify the differences between SSc-ILD and IPF using single-cell analysis, clarify their distinct pathogeneses, and propose directions for prevention and treatment. METHODS: We performed single-cell RNA sequencing on NCBI Gene Expression Omnibus (GEO) databases GSE159354 and GSE212109, and analyzed lung tissue samples across healthy controls, IPF, and SSc-ILD. The primary measures were the filtered genes integrated with batch correction and annotated cell types for distinguishing patients with SSc-ILD from healthy controls. We proposed an SSc-ILD pathogenesis using cell-cell interaction inferences, and predicted transcription factors regulating target genes using SCENIC. Drug target prediction of the TF gene was performed using Drug Bank Online. RESULTS: A subset of macrophages activates the MAPK signaling pathway under oxidative stress. Owing to the lack of inhibitory feedback from ANNEXIN and the autoimmune characteristics, this leads to an earlier onset of lung fibrosis compared to IPF. During initial lung injury, fibroblasts begin to activate the IL6 pathway under the influence of SPP1 alveolar macrophages, but IL6 appears unrelated to other inflammatory and immune cells. This may explain why tocilizumab (an anti-IL6-receptor antibody) only preserves lung function in patients with early SSc-ILD. Finally, we identified BCLAF1 and NFE2L2 as influencers of MAPK activation in macrophages. Metformin downregulates NFE2L2 and could serve as a repurposed drug candidate. CONCLUSIONS: SPP1 alveolar macrophages play a role in the profibrotic activity of IPF and SSc-ILD. However, SSc-ILD is influenced by autoimmunity and oxidative stress, leading to the continuous activation of MAPK in macrophages. This may result in an earlier onset of lung fibrosis than in IPF. Such differences could serve as potential research directions for early prevention and treatment.

Anti-carbamylated protein antibodies drive AEC II toward a profibrotic phenotype by interacting with carbamylated TLR5.

OBJECTIVES: This study looked at the role of anti-carbamylated protein (anti-CarP) antibodies in contributing to lung fibrosis in CTD-associated interstitial lung disease (ILD) in an autoantigen-dependent manner. METHODS: ELISA was used to test serum samples, including 89 from the CTD-ILD group and 170 from the non-CTD-ILD group, for anti-CarP levels. Male C57BL/6 mice were used for the pulmonary fibrosis model and anti-CarP treatment in vivo (n = 5) and patient serum-derived or commercialized anti-CarP was used for cell treatment. We identified the carbamylated membrane protein via immunofluorescence (IF) and co-immunoprecipitation followed by mass spectrometry (MS) analysis. Quantitative RT-PCR, IF and western blot were performed to explore the antigen-dependent role of anti-CarP. A native electrophoretic mobility shift assay and MS analysis were used to verify direct interaction and carbamylation sites. RESULTS: A significantly higher serum anti-CarP level was observed in CTD with ILD than without ILD. In vivo, intrapulmonary delivery of anti-CarP induces epithelial-mesenchymal transition (EMT) and microfibrotic foci. Carbamylation was enriched in type II alveolar epithelial cells (AEC II). A novel carbamylated membrane receptor, specifically recognized by anti-CarP, was identified as toll-like receptor 5 (TLR5). We found anti-CarP induces the nuclear translocation of NF-κB and downstream events, including EMT and expression of inflammatory cytokines in AEC II, which were reversed by TLR5 blocking or TLR5 knockdown. Moreover, up to 12 lysine carbamylation sites were found in TLR5 ectodomain, allowing the interaction of anti-CarP with carbamylated TLR5. CONCLUSIONS: Overall, we found anti-CarP drives aberrant AEC II activation by interacting with carbamylated TLR5 to promote ILD progression.

Loss of interferon regulatory factor-1 prevents lung fibrosis by upregulation of pon1 expression.

BACKGROUND: Interferon regulatory factor-1 (IRF1) is a transcription factor that plays a significant role in various biological processes, including inflammatory injury, viral infection, cell death, and immune responses, and it has been extensively studied in the context of different lung diseases. However, the mechanism underlying its involvement in lung fibrosis remains largely unknown. METHODS: Wild type (WT) mice, IRF1 global-null mice (Irf1-/-) were subjected to a bleomycin-induced lung fibrosis model to enable examination of the role of IRF1 in lung fibrosis. Proteomic analysis of lung tissue from WT and Irf1-/- mice treated with saline or bleomycin was performed to explore the mechanism of IRF1 in regulating lung fibrosis. RESULTS: In the bleomycin-induced fibrosis mouse model, increased expression of IRF1 was observed. Irf1 knockout mice displayed decreased lung fibrosis relative to WT mice following treatment with bleomycin. The protein expression of fibronectin, as assessed by the Western blot analysis of lung tissues, was downregulated in Irf1-/- mice. We observed a similar reduction in collagen content using hydroxyproline detection. Histologically, there was less collagen deposition in the lungs of Irf1-/- mice compared with WT mice. Proteomics data revealed that IRF1 may be involved in lung fibrosis via the regulation of ferroptosis. We determined that paraoxonase 1(PON1), a poorly characterized protein in lung fibrosis, was upregulated in Irf1-/- mice following exposure to bleomycin. In vitro experiments revealed that IRF1 could regulate the level of GSH and MDA through PON1. We also determined that PON1 levels were lower in the plasma of IPF patients compared with healthy controls. CONCLUSION: Our data highlight the importance of IRF1 in the fibrotic process, and PON1 may be a potential mediator of IRF1 in the progression of lung fibrosis.

Quantitative micro-CT-derived biomarkers elucidate age-related lung fibrosis in elder mice.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF), prevalently affecting individuals over 60 years of age, has been mainly studied in young mouse models. The limited efficacy of current treatments underscores the need for animal models that better mimic an aged patient population. We addressed this by inducing pulmonary fibrosis in aged mice, using longitudinal micro-CT imaging as primary readout, with special attention to animal welfare. METHODS: A double bleomycin dose was administered to 18-24 months-old male C57Bl/6j mice to induce pulmonary fibrosis. Bleomycin dosage was reduced to as low as 75% compared to that commonly administered to young (8-12 weeks-old) mice, resulting in long-term lung fibrosis without mortality, complying with animal welfare guidelines. After fibrosis induction, animals received Nintedanib once-daily for two weeks and longitudinally monitored by micro-CT, which provided structural and functional biomarkers, followed by post-mortem histological analysis as terminal endpoint. RESULTS: Compared to young mice, aged animals displayed increased volume, reduced tissue density and function, and marked inflammation. This increased vulnerability imposed a bleomycin dosage reduction to the lowest tested level (2.5 µg/mouse), inducing a milder, yet persistent, fibrosis, while preserving animal welfare. Nintedanib treatment reduced fibrotic lesions and improved pulmonary function. CONCLUSIONS: Our data identify a downsized bleomycin treatment that allows to achieve the best trade-off between fibrosis induction and animal welfare, a requirement for antifibrotic drug testing in aged lungs. Nintedanib displayed significant efficacy in this lower-severity disease model, suggesting potential patient stratification strategies. Lung pathology was quantitatively assessed by micro-CT, pointing to the value of longitudinal endpoints in clinical trials.

Phœnix dactylifera, L. seed oil alleviates Bleomycin-induced pulmonary fibrosis and oxidative stress in Wistar rats.

OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is the most serious form of interstitial lung disease. We aimed to investigate the effect of Phœnix dactylifera, L. seed oil (DSO) on a murine model of IPF induced by bleomycin (BLM). METHODS: Male Wistar rats were treated with a single intra-tracheal injection of BLM (4 mg/kg) and a daily intraperitoneal injection of DSO (75, 150 and 300 mg/kg) for 4 weeks. RESULTS: Our phytochemical results showed that DSO has an important antioxidant activity with a high content of polyphenols and flavonoids. High-Performance Liquid Chromatography (HPLC) and Gas chromatography/mass spectrometry (GC-MS) analysis revealed a high amount of oleic and lauric acids and a large quantity of vitamins. Histological examination showed a significant reduction in fibrosis score and collagen bands in the group of rats treated with 75 mg/kg of DSO compared to the BLM group. DSO (75 mg/kg) reversed also the increase in catalase and malondialdehyde (MDA) levels while higher doses (150 and 300 mg/kg) are ineffective against the deleterious effects of BLM. We revealed also that DSO has no renal or hepatic cytotoxic effects. CONCLUSION: DSO can play antioxidant and antifibrotic effects on rat models of pulmonary fibrosis at the lowest dose administered.

Ablation of Stage I-II Non-Small Cell Lung Cancer in Patients With Interstitial Lung Disease: A Multicenter Retrospective Study.

BACKGROUND. Treatment options for patients with interstitial lung disease (ILD) who develop stage I-II non-small cell lung cancer (NSCLC) are severely limited, given that surgical resection, radiation, and systemic therapy are associated with significant morbidity and mortality. OBJECTIVE. The aim of this study was to evaluate the safety and efficacy of percutaneous ablation of stage I-II NSCLC in patients with ILD. METHODS. This retrospective study included patients with ILD and stage I-II NSCLC treated with percutaneous ablation in three health systems between October 2004 and February 2023. At each site, a single thoracic radiologist, blinded to clinical outcomes, reviewed preprocedural chest CT examinations for the presence and type of ILD according to 2018 criteria proposed by the American Thoracic Society, European Respiratory Society, Japanese Respiratory Society, and Latin American Thoracic Society. The primary outcome was 90-day major (grade ≥ 3) adverse events, based on Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. Secondary outcomes were hospital length of stay (HLOS), local tumor control, and overall survival (OS). RESULTS. The study included 33 patients (19 men, 14 women; median age, 78 years; 16 patients with Eastern Cooperative Oncology Group performance status ≤ 1) with ILD who underwent 42 percutaneous ablation sessions (21 cryoablations, 11 radiofrequency ablations, 10 microwave ablations) of 43 NSCLC tumors ((median tumor size, 1.6 cm; IQR, 1.4-2.5 cm; range, 0.7-5.4 cm; 37 stage I, six stage II). The extent of lung fibrosis was 20% or less in 24 patients; 17 patients had imaging findings of definite or probable usual interstitial pneumonia. The 90-day major adverse event rate was 14% (6/42), including one CTCAE grade 4 event. No acute ILD exacerbation or death occurred within 90 days after ablation. The median HLOS was 1 day (IQR, 0-2 days). Median imaging follow-up for local tumor control was 17 months (IQR, 11-32 months). Median imaging or clinical follow-up for OS was 16 months (IQR, 6-26 months). Local tumor control and OS were 78% and 77%, respectively, at 1 year and 73% and 46% at 2 years. CONCLUSION. Percutaneous ablation appears to be a safe and effective treatment option for stage I-II NSCLC in the setting of ILD after multidisciplinary selection. CLINICAL IMPACT. Patients with ILD and stage I-II NSCLC should be considered for percutaneous ablation given that they are frequently ineligible for surgical resection, radiation, and systemic therapy.

Effects of home-based telerehabilitation-assisted inspiratory muscle training in patients with idiopathic pulmonary fibrosis: A randomized controlled trial.

BACKGROUND AND OBJECTIVE: There are few studies that have used inspiratory muscle training (IMT) as an intervention for patients with isolated idiopathic pulmonary fibrosis (IPF). This study aimed to investigate and interpret the effects of home-based telerehabilitation-assisted IMT in patients with IPF. METHODS: Twenty-eight participants with IPF took part in the study. Lung function tests, functional exercise capacity by 6-min walk distance (6MWD), dyspnoea perception by modified medical research council dyspnoea scale (mMRC), and inspiratory muscle strength by maximal inspiratory pressure (MIP) were assessed. IMT was performed twice a day, 7 days/week, for 8 weeks. The intervention group (n = 14) performed IMT at 50% of their baseline MIP while the control group (n = 14) performed IMT without applied resistance. Loading intensity was progressed by keeping the load at 4-6 on a modified Borg scale for the highest tolerable perceived respiratory effort for each patient. RESULTS: Dyspnoea based on mMRC score (p < 0.001, η2 effect size = 0.48) significantly decreased within the intervention group compared with the control group. There were significant increases in the intervention group compared to the control group based on 6MWD (p < 0.001, η2 effect size = 0.43), MIP (p = 0.006, η2 effect size = 0.25) and MIP % predicted (p = 0.008, η2 effect size = 0.25). CONCLUSION: The findings of this study suggest that an 8-week home-based telerehabilitation-assisted IMT intervention produced improvements in inspiratory muscle strength, leading to improvements in functional exercise capacity and dyspnoea.