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Background In the INBUILD trial in patients with progressive fibrosing interstitial lung diseases (ILDs), nintedanib reduced the rate of decline in forced vital capacity compared with placebo, with side-effects that were manageable for most patients. We used data from the INBUILD trial to characterize further the safety and tolerability of nintedanib. Methods Patients with fibrosing ILDs other than idiopathic pulmonary fibrosis (IPF), who had experienced progression of ILD within the 24 months before screening despite management deemed appropriate in clinical practice, were randomized to receive nintedanib 150 mg twice daily or placebo. To manage adverse events, treatment could be interrupted or the dose reduced to 100 mg twice daily. We assessed adverse events and dose adjustments over the whole trial. Results A total of 332 patients received nintedanib and 331 received placebo. Median exposure to trial drug was 17.4 months in both treatment groups. Adverse events led to treatment discontinuation in 22.0% of patients treated with nintedanib and 14.5% of patients who received placebo. The most frequent adverse event was diarrhea, reported in 72.3% of patients in the nintedanib group and 25.7% of patients in the placebo group. Diarrhea led to treatment discontinuation in 6.3% of patients in the nintedanib group and 0.3% of the placebo group. In the nintedanib and placebo groups, respectively, 48.2% and 15.7% of patients had ≥ 1 dose reduction and/or treatment interruption. Serious adverse events were reported in 44.3% of patients in the nintedanib group and 49.5% of patients in the placebo group. The adverse event profile of nintedanib was generally consistent across subgroups based on age, sex, race and weight, but nausea, vomiting and dose reductions were more common among female than male patients. Conclusions The adverse event profile of nintedanib in patients with progressive fibrosing ILDs other than IPF is consistent with its established safety and tolerability profile in patients with IPF and characterized mainly by gastrointestinal events, particularly diarrhea. Management of adverse events using symptomatic therapies and dose adjustment is important to minimize the impact of adverse events and help patients remain on therapy. Trial registration Registered 21 December 2016, https://clinicaltrials.gov/ct2/show/NCT02999178 Graphical Abstract A video abstract summarizing the key results presented in this manuscript is available at: https://www.globalmedcomms.com/respiratory/cottin/INBUILDsafety .

Idiopathic pulmonary fibrosis (IPF) is a fatal and irreversible disease with few effective treatments. Alveolar macrophages (AMs) are involved in the development of IPF from the initial stages due to direct exposure to air and respond to external oxidative damage (a major inducement of pulmonary fibrosis). Oxidative stress in AMs plays an indispensable role in promoting fibrosis development. The oligopeptide histidine transporter SLC15A3, mainly expressed on the lysosomal membrane of macrophages and highly expressed in the lung, has proved to be involved in innate immune and antiviral signaling pathways. In this study, we demonstrated that during bleomycin (BLM)- or radiation-induced pulmonary fibrosis, the recruitment of macrophages induced an increase of SLC15A3 in the lung, and the deficiency of SLC15A3 protected mice from pulmonary fibrosis and maintained the homeostasis of the pulmonary microenvironment. Mechanistically, deficiency of SLC15A3 resisted oxidative stress in macrophages, and SLC15A3 interacted with the scaffold protein p62 to regulate its expression and phosphorylation activation, thereby regulating p62-nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant stress pathway protein, which is related to the production of reactive oxygen species (ROS). Overall, our data provided a novel mechanism for targeting SLC15A3 to regulate oxidative stress in macrophages, supporting the therapeutic potential of inhibiting or silencing SLC15A3 for the precautions and treatment of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that leads to respiratory decline caused by scarring and thickening of lung tissues. Multiple pathways contribute to the fibrotic process in this disease, such as inflammation, epithelial-to-mesenchymal transition, and oxidative stress. The Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway is a key regulator of profibrotic signaling, as it affects the organization of actin-myosin and the remodeling of the extracellular matrix. ROCK1/2, a downstream effector of RhoA, is overexpressed in patients with IPF and is a promising target for IPF therapy. However, because of the hypotensive side effects of ROCK1/2 inhibitors, selective ROCK2 compounds are being explored. In this study, we report the discovery of GNS-3595, a potent and selective ROCK2 inhibitor that has ∼80-fold selectivity over ROCK1 at physiological concentrations of ATP. GNS-3595 effectively inhibited ROCK2-mediated phosphorylation of myosin light chain and reduced the expression of fibrosis-related proteins (e.g., collagen, fibronectin, and α-smooth muscle actin) in various cellular models. GNS-3595 also prevented transforming growth factor β-induced fibroblast-to-myofibroblast transition. In addition, in a bleomycin-induced mouse model of pulmonary fibrosis, therapeutic exposure to GNS-3595, suppressed lung fibrosis, stabilized body weight loss, and prevented fibrosis-induced lung weight gain. Transcriptome and protein expression analysis from lung tissues showed that GNS-3595 can revert the fibrosis-related gene expression induced by bleomycin. These results indicate that GNS-3595 is a highly potent, selective, and orally active ROCK2 inhibitor with promising therapeutic efficacy against pulmonary fibrosis.

This study explored the therapeutic potential of Number 2 Feibi Recipe (FBR2), a traditional Chinese medicine prescription, in the treatment of idiopathic pulmonary fibrosis (IPF). Specifically, it investigated whether FBR2 mediated its effects by modulating miR-199a-5p expression, activating the SIRT1/AMPK/mTOR pathway and then promoting autophagy. Eighty-four mice were divided into seven groups: Control, bleomycin (BLM), FBR2, self-complementary adeno-associated virus expressing miR-199a-5p (scAAV-miR-199a-5p), scAAV-negative control (scAAV-NC), FBR2 + BLM + scAAV-miR-199a-5p, and FBR2 + BLM + scAAV-NC. Fluorescence staining, miR-199a-5p expression levels, histopathological changes, protein expression and autophagy activity were assessed using various techniques, including RT-qPCR, histopathological staining, Western blotting, Elisa and transmission electron microscopy (TEM). FBR2 treatment significantly reduced miR-199a-5p expression elevated by BLM and improved histopathology, reduced levels of α-smooth muscle actin (α-SMA) and collagen-III, and increased E-cadherin expression. FBR2 also enhanced autophagy, as evidenced by elevated LC3-II and Beclin-1 levels and reduced p62 expression. Additionally, it activated the SIRT1/AMPK/mTOR pathway. Finally, network pharmacology identified 227 compounds in FBR2, among which Kaempferol and Quercetin (two major shared constituents) showed strong binding to miR-199a-5p in molecular docking, suggesting their potential as key active components. This study showed FBR2 attenuated BLM-induced pulmonary fibrosis in mice by reducing miR-199a-5p expression and promoting autophagy which may be achieved by modulating the SIRT1/AMPK/mTOR pathway. These findings provide novel insights into the mechanism of FBR2 and its potential as a therapeutic approach for IPF.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by excessive extracellular matrix deposition and limited therapeutic options. Conventional preclinical models, such as the bleomycin (BLM)-induced lung fibrosis (BILF) model in immunocompetent mice, fail to replicate the complexity of the human immune environment and are thus unsuitable for evaluating human-specific antibody and cell therapies. To overcome this limitation, we developed two humanized mouse models with functional human immune components: one engrafted with peripheral blood mononuclear cells (PBMCs) and the other with hematopoietic stem cells (HSCs). Pulmonary fibrosis was induced using BLM in both humanized models. In the PBMC-humanized model, human T cells accounted for 79.9% ± 9.7% of CD45 + cells; however, this model was limited by high incidence of graft-versus-host disease (GvHD). In contrast, the HSC-humanized model demonstrated stable multilineage engraftment (T cells: 20.1% ± 28.3%; B cells: 56.8% ± 25.4%), absence of GvHD, and consistent fibrotic pathology. Both models exhibited increased inflammatory infiltration, fibroblast activation, and collagen deposition following BLM induction. Nintedanib treatment significantly ameliorated fibrotic changes in both models. These findings establish the HSC-humanized BILF model as a clinically relevant platform for investigating human immune responses in IPF and for preclinical evaluation of human-targeted therapies.

Idiopathic pulmonary fibrosis (IPF) is a prevalent interstitial lung disease with high mortality. CD38 is a main enzyme for intracellular nicotinamide adenine dinucleotide (NAD + ) degradation in mammals. It has been reported that CD38 participated in pulmonary fibrosis through promoting alveolar epithelial cells senescence. However, the roles of endothelial CD38 in pulmonary fibrosis remain unknown. In the present study, we observed that the elevated expression of CD38 was related to endothelial-to-mesenchymal transition (EndMT) of lung tissues in IPF patients and bleomycin (BLM)-induced pulmonary fibrosis mice and also in human umbilical vein endothelial cells (HUVECs) treated with BLM. Micro-computed tomography (MCT) and histopathological staining showed that endothelial cell-specific CD38 knockout (CD38 EndKO ) remarkably attenuated BLM-induced pulmonary fibrosis. In addition, CD38 EndKO significantly inhibited TGFβ-Smad3 pathway-mediated excessive extracellular matrix (ECM), reduced Toll-like receptor4-Myeloid differentiation factor88-Mitogen-activated protein kinases (TLR4-MyD88-MAPK) pathway-mediated endothelial inflammation and suppressed nicotinamide adenine dinucleotide phosphate oxidases1 (NOX1)-mediated oxidative stress. Furthermore, we demonstrated that 3-TYP, a SIRT3-specific inhibitor, markedly reversed the protective effect of HUVECs CD38KD cells and 78 C, a CD38-specific inhibitor, on BLM-induced EndMT in HUVECs. Therefore, we concluded that CD38 EndKO significantly ameliorated BLM-induced pulmonary fibrosis through inhibiting ECM, endothelial inflammation and oxidative stress, further alleviating EndMT in mice. Our findings suggest that endothelial CD38 may be a new therapeutic target for the prevention and treatment of pulmonary fibrosis clinically. Graphical Abstract Protective role and the underlying mechanism of endothelial CD38 in bleomycin-induced pulmonary fibrosis in mice. Endothelial cells-specific CD38 knockout (CD38 EndKO ) inhibited TGFβ-Smad3-mediated ECM, ROS-mediated oxidative stress and TLR4-mediated inflammation, and in turn, suppressed endothelial-to-mesenchymal transition (EndMT), eventually, alleviated pulmonary fibrosis induced by bleomycin in mice, suggesting endothelial CD38 may be a therapeutic target for the prevention and the treatment of pulmonary fibrosis clinically

Idiopathic Pulmonary Fibrosis (IPF) is a debilitating and fatal lung disease characterized by the excessive formation of scar tissue and decline of lung function. Despite extensive research, only two FDA-approved drugs exist for IPF, with limited efficacy and relevant side effects. Thus, there is an urgent need for new effective therapies, whose discovery strongly relies on IPF animal models. Despite some limitations, the Bleomycin (BLM)-induced lung fibrosis mouse model is widely used for antifibrotic drug discovery and for investigating disease pathogenesis. The initial acute inflammation triggered by BLM instillation and the spontaneous fibrosis resolution that occurs after 3 weeks are the major drawbacks of this system. In the present study, we applied micro-CT technology to a longer-lasting, triple BLM administration fibrosis mouse model to define the best time-window for Nintedanib (NINT) treatment. Two different treatment regimens were examined, with a daily NINT administration from day 7 to 28 (NINT 7–28), and from day 14 to 28 (NINT 14–28). For the first time, we automatically derived both morphological and functional readouts from longitudinal micro-CT. NINT 14–28 showed significant effects on morphological parameters after just 1 week of treatment, while no modulations of these biomarkers were observed during the preceding 7–14-days period, likely due to persistent inflammation. Micro-CT morphological data evaluated on day 28 were confirmed by lung histology and bronchoalveolar lavage fluid (BALF) cells; Once again, the NINT 7–21 regimen did not provide substantial benefits over the NINT 14–28. Interestingly, both NINT treatments failed to improve micro-CT-derived functional parameters. Altogether, our findings support the need for optimized protocols in preclinical studies to expedite the drug discovery process for antifibrotic agents. This study represents a significant advancement in pulmonary fibrosis animal modeling and antifibrotic treatment understanding, with the potential for improved translatability through the concurrent structural–functional analysis offered by longitudinal micro-CT.

Background Transcriptome profiling by RNA sequencing (RNAseq) can provide insightful information on the molecular processes underlying disease development and progression. Although fresh tissue represents the preferred source material for RNAseq, here, we investigated the feasibility of applying RNAseq analysis to single 10 μm thick formalin-fixed and paraffin-embedded (FFPE) lung slides from the lungs of control and bleomycin (BLM)-treated mice. This approach aims at providing spatial-oriented transcriptomic data, that can be integrated with in vivo and ex vivo readouts obtained on the same sample, as a way to enhance the mechanistic information and biomarker/target discovery potential of preclinical models of fibrotic lung diseases. Methods RNAseq analysis was conducted on individual FFPE slides from the lungs of both controls and BLM-treated mice. The results were initially validated by comparison with publicly available bulk data from fresh-frozen (FF) mouse tissues, both untreated and BLM-treated, as well as human idiopathic pulmonary fibrosis (IPF) biopsies. Unsupervised cluster analysis was performed on Differentially Expressed Genes (DEGs) distinguishing untreated and BLM-treated fibrotic lung samples. For each sample, Pearson correlation analysis was used to compare expression levels of individual gene clusters with Ashcroft Scores and aeration compartments quantitatively assessed on the matched 2D micro-CT coronal slice. Results Over 90% of annotated genes within the FFPE dataset were shared with gene signatures retrieved from FF bulk datasets. Differentially modulated gene clusters were mainly found to be associated with extracellular matrix (ECM) organization, tissue remodeling, and inflammatory response pathways. For each sample, expression levels of individual gene clusters were highly correlated with 2D histology readouts and aeration compartments determined on matched 2D coronal slices by micro-CT imaging. Conclusions FFPE lung tissue represents a valuable alternative to fresh tissue for RNAseq analysis, allowing to achieve a more precise, spatially oriented picture of pulmonary disease development. This approach is thus instrumental to a better characterization of the molecular changes associated to each sample. It can also contribute to a more informed interpretation of histology and micro-CT imaging data, paving the way to the identification of translationally relevant biomarkers as well as novel candidate targets for the development of more effective therapeutic interventions.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease with limited effective therapies. Interstitial macrophages (IMs), especially those derived from monocytes, play an unknown role in IPF pathogenesis. By using single-cell RNA sequencing (scRNA-seq), bleomycin (BLM)-induced pulmonary fibrosis mouse lungs were analyzed to characterize the cellular landscape and heterogeneity of macrophages in this model. scRNA-seq was used to identify distinct interstitial macrophage subpopulations in fibrotic lungs, with monocyte-derived macrophages exhibiting a pro-fibrotic gene expression profile enriched in wound healing, extracellular matrix (ECM) remodeling, and pro-fibrotic cytokine production functions. A pseudotime analysis revealed that IMs originated from monocytes and differentiated along a specific trajectory. A cell–cell communication analysis demonstrated strong interactions between monocyte-derived interstitial macrophages (Mo-IMs) and fibroblasts through the transforming growth factor beta (TGFβ), secreted phosphoprotein 1 (SPP1), and platelet-derived growth factor (PDGF) signaling pathways. Flow cytometry validated the presence and expansion of Mo-IMs subpopulations in BLM-treated mice. This study reveals the cellular heterogeneity and developmental trajectory of lung macrophages in early BLM-induced pulmonary fibrosis, highlighting the crucial role of Mo-IMs with a pro-fibrotic phenotype in IPF pathogenesis via interactions with fibroblasts. Targeting these specific macrophage subpopulations and associated signaling pathways may provide novel therapeutic strategies for IPF.

Background Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by a constant incidence rate. Unfortunately, effective pharmacological treatments for this condition are lacking and the identification of novel therapeutic approaches and underlying pathological mechanisms are required. This study investigated the potential of quercetin in alleviating pulmonary fibrosis by promoting autophagy and activation of the SIRT1/AMPK pathway. Methods Mouse models of IPF were divided into four treatment groups: control, bleomycin (BLM), quercetin (Q), and quercetin + EX-527 (Q + E) treatment. Pulmonary fibrosis was induced in the mouse models through intratracheal instillation of BLM. Various indexes were identified through histological staining, Western blotting analysis, enzyme-linked immunosorbent assay, immunohistochemistry, and transmission electron microscopy. Results Quercetin treatment ameliorated the pathology of BLM-induced pulmonary fibrosis of mice by reducing α-smooth muscle actin (α-SMA), collagen I (Col I), and collagen III (Col III) levels, and also improved the level of E-cadherin in lung tissue. Furthermore, Quercetin significantly enhanced LC3II/LC3I levels, decreased P62 expression, and increased the number of autophagosomes in lung tissue. These effects were accompanied by the activation of the SIRT1/AMPK pathway. Treatment with EX-527, an inhibitor for SIRT1, reversed all effects induced by quercetin. Conclusion This study showed that quercetin could alleviate pulmonary fibrosis and improve epithelial-mesenchymal transition by acting on the SIRT1/AMPK signaling pathway, which may be achieved by regulating the level of autophagy.