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Radiation‐induced lung injury (RILI) mainly contributes to the complications of thoracic radiotherapy. RILI can be divided into radiation pneumonia (RP) and radiation‐induced lung fibrosis (RILF). Once RILF occurs, patients will eventually develop irreversible respiratory failure; thus, a new treatment strategy to prevent RILI is urgently needed. This study explored the therapeutic effect of pirfenidone (PFD), a Food and Drug Administration (FDA)‐approved drug for (IPF) treatment, and its mechanism in the treatment of RILF. In vivo, C57BL/6 mice received a 50 Gy dose of X‐ray radiation to the whole thorax with or without the administration of PFD. Collagen deposition and fibrosis in the lung were reversed by PFD treatment, which was associated with reduced M2 macrophage infiltration and inhibition of the transforming growth factor‐β1 (TGF‐β1)/Drosophila mothers against the decapentaplegic 3 (Smad3) signalling pathway. Moreover, PFD treatment decreased the radiation‐induced expression of TGF‐β1 and phosphorylation of Smad3 in alveolar epithelial cells (AECs) and vascular endothelial cells (VECs). Furthermore, IL‐4–induced M2 macrophage polarization and IL‐13–induced M2 macrophage polarization were suppressed by PFD treatment in vitro, resulting in reductions in the release of arginase‐1 (ARG‐1), chitinase 3‐like 3 (YM‐1) and TGF‐β1. Notably, the PFD‐induced inhibitory effects on M2 macrophage polarization were associated with downregulation of nuclear factor kappa‐B (NF‐κB) p50 activity. Additionally, PFD could significantly inhibit ionizing radiation‐induced chemokine secretion in MLE‐12 cells and consequently impair the migration of RAW264.7 cells. PFD could also eliminate TGF‐β1 from M2 macrophages by attenuating the activation of TGF‐β1/Smad3. In conclusion, PFD is a potential therapeutic agent to ameliorate fibrosis in RILF by reducing M2 macrophage infiltration and inhibiting the activation of TGF‐β1/Smad3.

Alveolar epithelial cells (AECs) participate in the pathogenesis of pulmonary fibrosis, producing pro‐inflammatory mediators and undergoing epithelial‐to‐mesenchymal transition (EMT). Herein, we demonstrated the critical role of Forkhead Box M1 (Foxm1) transcription factor in radiation‐induced pulmonary fibrosis. Foxm1 was induced in AECs following lung irradiation. Transgenic expression of an activated Foxm1 transcript in AECs enhanced radiation‐induced pneumonitis and pulmonary fibrosis, and increased the expression of IL‐1 β, Ccl2 , Cxcl5 , Snail1 , Zeb1 , Zeb2 and Foxf1 . Conditional deletion of Foxm1 from respiratory epithelial cells decreased radiation‐induced pulmonary fibrosis and prevented the increase in EMT‐associated gene expression. siRNA‐mediated inhibition of Foxm1 prevented TGF‐β‐induced EMT in vitro . Foxm1 bound to and increased promoter activity of the Snail1 gene, a critical transcriptional regulator of EMT. Expression of Snail1 restored TGF‐β‐induced loss of E‐cadherin in Foxm1‐deficient cells in vitro . Lineage‐tracing studies demonstrated that Foxm1 increased EMT during radiation‐induced pulmonary fibrosis in vivo . Foxm1 is required for radiation‐induced pulmonary fibrosis by enhancing the expression of genes critical for lung inflammation and EMT. This study establishes the in vivo relevance of FoxM1 in the context of radiation‐induced fibrosis. FoxM1 ablation in the respiratory epithelium supports a regulatory role during EMT and pulmonary inflammation that could become of therapeutic relevance.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease, characterized by damage of lung epithelial cells, excessive deposition of extracellular matrix in the lung interstitium, and enhanced activation and proliferation of fibroblasts. S100a4, also termed FSP-1 (fibroblast-specific protein-1), was previously considered as a marker of fibroblasts but recent findings in renal and liver fibrosis indicated that M2 macrophages are an important cellular source of S100a4. Thus, we hypothesized that also in pulmonary fibrosis, M2 macrophages produce and secrete S100a4, and that secreted S100a4 induces the proliferation and activation of fibroblasts. To prove this hypothesis, we comprehensively characterized two established mouse models of lung fibrosis: infection of IFN-γR mice with MHV-68 and intratracheal application of bleomycin to C57BL/6 mice. We further provide data using primary macrophages and fibroblasts to investigate the mechanism by which S100A4 exerts its effects. Finally, we inhibit S100a4 in the bleomycin-induced lung fibrosis model by treatment with niclosamide. Our data suggest that S100a4 is produced and secreted by M2 polarized alveolar macrophages and enhances the proliferation and activation of lung fibroblasts. Inhibition of S100a4 might represent a potential therapeutic strategy for pulmonary fibrosis.

Bleomycin‐induced lung fibrosis is a debilitating disease, linked to high morbidity and mortality in chemotherapy patients. The MRTF/SRF transcription pathway has been proposed as a potential therapeutic target, as it is critical for myofibroblast differentiation, a hallmark of fibrosis. In human lung fibroblasts, the MRTF/SRF pathway inhibitor, CCG‐257081, effectively decreased mRNA levels of downstream genes: smooth muscle actin and connective tissue growth factor, with IC50s of 4 and 15 μM, respectively. The ability of CCG‐257081 to prevent inflammation and fibrosis, measured via pulmonary collagen content and histopathology, was tested in a murine model of bleomycin‐induced lung fibrosis. Animals were given intraperitoneal bleomycin for 4 weeks and concurrently dosed with CCG‐257081 (0, 10, 30, and 100 mg/kg PO), a clinical anti‐fibrotic (nintedanib) or the clinical standard of care (prednisolone). Mice treated with 100 mg/kg CCG‐257081 gained weight vs. vehicle‐treated control mice, while those receiving nintedanib and prednisolone lost significant weight. Hydroxyproline content and histological findings in tissue of animals on 100 mg/kg CCG‐257081 were not significantly different from naive tissue, indicating successful prevention. Measures of tissue fibrosis were comparable between CCG‐257081 and nintedanib, but only the MRTF/SRF inhibitor decreased plasminogen activator inhibitor‐1 (PAI‐1), a marker linked to fibrosis, in bronchoalveolar lavage fluid. In contrast, prednisolone led to marked increases in lung fibrosis by all metrics. This study demonstrates the potential use of MRTF/SRF inhibitors to prevent bleomycin‐induced lung fibrosis in a clinically relevant model of the disease. MRTF/SRF transcription pathway inhibitors prevent the transcription of hallmark genes associated with lung fibrosis, making them attractive options for the prevention of drug‐induced lung fibrosis.

Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-β) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-β release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivo lung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-β activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in vivo.

Activated stellate cells play a role in fibrosis development in the liver, pancreas, and kidneys. The fusion protein R-III, which consists of retinol-binding protein and albumin domain III, has been demonstrated to attenuate liver and renal fibrosis by suppressing stellate cell activation. In this study, we investigated the efficacy of R-III against bleomycin-induced lung fibrosis in mice. R-III reduced lung fibrosis and primarily localized in autofluorescent cells in the lung tissue. Furthermore, we isolated lung stellate cells (LSCs) from rat lungs using the isolation protocol employed for hepatic stellate cells (HSCs). LSCs shared many characteristics with HSCs, including the presence of vitamin A-containing lipid droplets and the expression of alpha-smooth muscle actin and collagen type I, markers for activated HSCs/myofibroblasts. LSCs spontaneously transdifferentiated into myofibroblasts in in vitro culture, which was inhibited by R-III. These findings suggest that R-III may reduce lung fibrosis by inactivating LSCs and could be a promising treatment for extrahepatic fibrosis.

The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. Flow cytometry is a mainstream tool for studying the immune system. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells (DCs) accurately and consistently in the normal mouse lung has not been described. Here we developed a panel of surface markers and an analysis strategy that accurately identify all known populations of macrophages and DCs, and their precursors in the lung during steady-state conditions and bleomycin-induced injury. Using this panel, we assessed the polarization of lung macrophages during the course of bleomycin-induced lung injury. Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycin-induced lung injury, whereas markers of classically activated macrophages were expressed only during the acute phase. Taken together, these data suggest that this flow cytometric panel is very helpful in identifying macrophage and DC populations and their state of activation in normal, injured, and fibrotic lungs.

ObjectiveTo assess the safety and efficacy of rituximab in systemic sclerosis (SSc) in clinical practice.MethodsWe performed a prospective study including patients with SSc from the European Scleroderma Trials and Research (EUSTAR) network treated with rituximab and matched with untreated patients with SSc. The main outcomes measures were adverse events, skin fibrosis improvement, lung fibrosis worsening and steroids use among propensity score-matched patients treated or not with rituximab.Results254 patients were treated with rituximab, in 58% for lung and in 32% for skin involvement. After a median follow-up of 2 years, about 70% of the patients had no side effect. Comparison of treated patients with 9575 propensity-score matched patients showed that patients treated with rituximab were more likely to have skin fibrosis improvement (22.7 vs 14.03 events per 100 person-years; OR: 2.79 [1.47–5.32]; p=0.002). Treated patients did not have significantly different rates of decrease in forced vital capacity (FVC)>10% (OR: 1.03 [0.55–1.94]; p=0.93) nor in carbon monoxide diffusing capacity (DLCO) decrease. Patients having received rituximab were more prone to stop or decrease steroids (OR: 2.34 [1.56–3.53], p<0.0001). Patients treated concomitantly with mycophenolate mofetil had a trend for better outcomes as compared with patients receiving rituximab alone (delta FVC: 5.22 [0.83–9.62]; p=0.019 as compared with controls vs 3 [0.66–5.35]; p=0.012).ConclusionRituximab use was associated with a good safety profile in this large SSc-cohort. Significant change was observed on skin fibrosis, but not on lung. However, the limitation is the observational design. The potential stabilisation of lung fibrosis by rituximab has to be addressed by a randomised trial.

Dysregulation of cellular metabolism has been shown to participate in several pathologic processes. However, the role of metabolic reprogramming is not well appreciated in the pathogenesis of organ fibrosis. To determine if glycolytic reprogramming participates in the pathogenesis of lung fibrosis and assess the therapeutic potential of glycolytic inhibition in treating lung fibrosis. A cell metabolism assay was performed to determine glycolytic flux and mitochondrial respiration. Lactate levels were measured to assess glycolysis in fibroblasts and lungs. Glycolytic inhibition by genetic and pharmacologic approaches was used to demonstrate the critical role of glycolysis in lung fibrosis. Augmentation of glycolysis is an early and sustained event during myofibroblast differentiation, which is dependent on the increased expression of critical glycolytic enzymes, in particular, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Augmented glycolysis contributes to the stabilization of hypoxia-inducible factor 1-α, a master regulator of glycolytic enzymes implicated in organ fibrosis, by increasing cellular levels of tricarboxylic acid cycle intermediate succinate in lung myofibroblasts. Inhibition of glycolysis by the PFKFB3 inhibitor 3PO or genomic disruption of the PFKFB3 gene blunted the differentiation of lung fibroblasts into myofibroblasts, and attenuated profibrotic phenotypes in myofibroblasts isolated from the lungs of patients with idiopathic pulmonary fibrosis. Inhibition of glycolysis by 3PO demonstrates therapeutic benefit in bleomycin-induced and transforming growth factor-β1-induced lung fibrosis in mice. Our data support the novel concept of glycolytic reprogramming in the pathogenesis of lung fibrosis and provide proof-of-concept that targeting this pathway may be efficacious in treating fibrotic disorders, such as idiopathic pulmonary fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a devastating condition leading to respiratory failure and >3000 deaths/year in the UK. Therapeutic approaches are limited and there is an urgent need to better understand mechanisms driving pulmonary fibrosis to support development of new anti-fibrotics. There is spatial and temporal heterogeneity of pathological changes within IPF tissue, which may correlate to changes in pathophysiological mediators of disease and clinical progression. Here, we utilise an intrapatient approach for target identification and validation by comparing histologically distinct regions of tissue from within the same IPF lung.MethodsMacroscopically ‘normal’, ‘intermediate’ and end-stage ‘fibrotic’ tissue was sampled under pathology guidance from the upper left lobe of explant IPF lungs (n=8) collected from patients undergoing lung transplantation. Histological assessment of the regions confirmed distinct pathology before samples were subject to unbiased proteomics assessment alongside aged-matched non-diseased unused donor (UD) lungs (n=10). Ingenuity Pathway Analysis (IPA) was performed to identify novel upstream regulators of fibrosis, from which inhibitory compounds targeting these regulators were selected and anti-fibrotic efficacy was assessed in IPF-derived precision cut slices (PCS).ResultsPrincipal component analysis showed IPF samples clustered based on region of tissue and became less similar to UD controls in correlation with disease severity. We identified markers/pathways significantly modulated in the intermediate region compared to other regions of the IPF lung. The intermediate region is the site of active tissue remodelling and therefore the region that needs to be targeted therapeutically to limit disease progression. Validation of PCS from these distinct regions showed that only intermediate-derived PCS increased collagen-1α1 secretion spontaneously throughout culture, suggesting enhanced disease progression in these PCS. A total of n=18 candidate compounds targeting upregulated markers/pathways modulated in the IPF intermediate region were assessed, of which n=10 exhibited robust anti-fibrotic effects in IPF-derived PCS.ConclusionWe have identified distinct patterns of protein expression that are modulated in line with changes in disease severity. Interrogation of protein heterogeneity identified novel targets that have been validated in the PCS system via inhibitors, confirming involvement in disease pathogenesis.Please refer to page A286 for declarations of interest related to this abstract.