Bleomycin-induced Lung Injury Research Articles

Lipocalin-2 as a prognostic marker in patients with acute exacerbation of idiopathic pulmonary fibrosis

BackgroundLipocalin-2 (LCN2) is a secretory glycoprotein upregulated by oxidative stress; moreover, patients with idiopathic pulmonary fibrosis (IPF) have shown increased LCN2 levels in bronchoalveolar lavage fluid (BALF). This study aimed to determine whether circulatory LCN2 could be a systemic biomarker in patients with IPF and to investigate the role of LCN2 in a bleomycin-induced lung injury mouse model.MethodsWe measured serum LCN2 levels in 99 patients with stable IPF, 27 patients with acute exacerbation (AE) of IPF, 51 patients with chronic hypersensitivity pneumonitis, and 67 healthy controls. Further, LCN2 expression in lung tissue was evaluated in a bleomycin-induced lung injury mouse model, and the role of LCN2 was investigated using LCN2-knockout (LCN2 -/-) mice.ResultsSerum levels of LCN2 were significantly higher in patients with AE-IPF than in the other groups. The multivariate Cox proportional hazards model showed that elevated serum LCN2 level was an independent predictor of poor survival in patients with AE-IPF. In the bleomycin-induced lung injury mouse model, a higher dose of bleomycin resulted in higher LCN2 levels and shorter survival. Bleomycin-treated LCN2 -/- mice exhibited increased BALF cell and protein levels as well as hydroxyproline content. Moreover, compared with wild-type mice, LCN2-/- mice showed higher levels of circulatory 8-isoprostane as well as lower Nrf-2, GCLC, and NQO1 expression levels in lung tissue following bleomycin administration.ConclusionsOur findings demonstrate that serum LCN2 might be a potential prognostic marker of AE-IPF. Moreover, LCN2 expression levels may reflect the severity of lung injury, and LCN2 may be a protective factor against bleomycin-induced acute lung injury and oxidative stress.

Bleomycin induces senescence and repression of DNA repair via downregulation of Rad51

BackgroundBleomycin, a potent antitumor agent, is limited in clinical use due to the potential for fatal pulmonary toxicity. The accelerated DNA damage and senescence in alveolar epithelial cells (AECs) is considered a key factor in the development of lung pathology. Understanding the mechanisms for bleomycin-induced lung injury is crucial for mitigating its adverse effects.MethodsHuman lung epithelial (A549) cells were exposed to bleomycin and subsequently assessed for cellular senescence, DNA damage, and double-strand break (DSB) repair. The impact of Rad51 overexpression on DSB repair and senescence in AECs was evaluated in vitro. Additionally, bleomycin was intratracheally administered in C57BL/6 mice to establish a pulmonary fibrosis model.ResultsBleomycin exposure induced dose- and time-dependent accumulation of senescence hallmarks and DNA lesions in AECs. These effects are probably due to the inhibition of Rad51 expression, consequently suppressing homologous recombination (HR) repair. Mechanistic studies revealed that bleomycin-mediated transcriptional inhibition of Rad51 might primarily result from E2F1 depletion. Furthermore, the genetic supplement of Rad51 substantially mitigated bleomycin-mediated effects on DSB repair and senescence in AECs. Notably, decreased Rad51 expression was also observed in the bleomycin‐induced mouse pulmonary fibrosis model.ConclusionsOur works suggest that the inhibition of Rad51 plays a pivotal role in bleomycin-induced AECs senescence and lung injury, offering potential strategies to alleviate the pulmonary toxicity of bleomycin.

Evaluation of interleukin-1 and interleukin-6 receptor antagonists in a murine model of acute lung injury.

The acute exudative phase of acute respiratory distress syndrome (ARDS), a severe form of respiratory failure, is characterized by alveolar damage, pulmonary oedema, and an exacerbated inflammatory response. There is no effective treatment for this condition, but based on the major contribution of inflammation, anti-inflammatory strategies have been evaluated in animal models and clinical trials, with conflicting results. In COVID-19 ARDS patients, interleukin (IL)-1 and IL-6 receptor antagonists (IL-1Ra and IL-6Ra, kineret and tocilizumab, respectively) have shown some efficacy. Moreover, we have previously developed novel peptides modulating IL-1R and IL-6R activity (rytvela and HSJ633, respectively) while preserving immune vigilance and cytoprotective pathways. We aimed to assess the efficacy of these novel IL-1Ra and IL-6Ra, compared to commercially available drugs (kineret, tocilizumab) during the exudative phase (day 7) of bleomycin-induced acute lung injury (ALI) in mice. Our results first showed that none of the IL-1Ra and IL-6Ra compounds attenuated bleomycin-induced weight loss and venous increase. Histological analyses and lung water content measurements also showed that these drugs did not improve lung injury scores or pulmonary oedema, after the bleomycin challenge. Finally, IL-1Ra and IL-6Ra failed to alleviate the inflammatory status of the mice, as indicated by cytokine levels and alveolar neutrophil infiltration. Altogether, these results indicate a lack of beneficial effects of IL-1R and IL-6R antagonists on key parameters of ALI in the bleomycin mouse model.

Antrodia cinnamomea extract alleviates bleomycin-induced pulmonary fibrosis in mice by inhibiting the mTOR pathway

BackgroundPulmonary fibrosis is a progressive diffuse parenchymal lung disorder with a high mortality rate. Studies have indicated that injured lung tissues release various pro-inflammatory factors, and produce a large amount of nitric oxide. There is also accumulation of collagen and oxidative stress-induced injury, collectively leading to pulmonary fibrosis. Antrodia cinnamomea is an endemic fungal growth in Taiwan, and its fermented extracts exert anti-inflammatory effects to alleviate liver damages. Hence, we hypothesized and tested the feasibility of using A. cinnamomea extracts for treatment of pulmonary fibrosis. MethodsThe TGF-β1-induced human lung fibroblast cells (MRC-5) in vitro cell assay were used to evaluate the effects of A. cinnamomea extracts on the collagen production in MRC-5. Eight-week-old ICR mice were intratracheally administered bleomycin and then fed with an A. cinnamomea extract on day 3 post-administration of bleomycin. At day 21 post-bleomycin administration, the pulmonary functional test, the expression level of inflammation- and fibrosis-related genes in the lung tissue, and the histopathological change were examined. ResultsThe A. cinnamomea extract significantly attenuated the expression level of collagen in the TGF-β1-induced MRC-5 cells. In the A. cinnamome-treated bleomycin-induced lung fibrotic mice, the bodyweight increased, pulmonary functions improved, the lung tissues expression level of inflammatory factor and the fibrotic indicator were decreased, and the histopathological results showed the reduction of thickening of the inter-alveolar septa. ConclusionsThe Antrodia cinnamomea extract significant protects mice against bleomycin-induced lung injuries through improvement of body weight gain and lung functions, and attenuation of expression of inflammatory and fibrotic indicators.

SPAUTIN-1 alleviates LPS-induced acute lung injury by inhibiting NF-κB pathway in neutrophils

BackgroundAcute lung injury (ALI) is an inflammatory condition characterized by acute damage to lung tissue. SPAUTIN-1, recognized as a small molecule drug targeting autophagy and USP10/13, has been reported for its potential to inhibit oxidative stress damage in various tissue injuries. However, the role and mechanism of SPAUTIN-1 in ALI remain unclear. This study aims to elucidate the protective effects of SPAUTIN-1 on ALI, with a particular focus on its role and mechanism in pulmonary inflammatory responses. MethodsLipopolysaccharides (LPS) were employed to induce inflammation-mediated ALI. Bleomycin was used to induce non-inflammation-mediated ALI. The mechanism of SPAUTIN-1 action was identified through RNA-Sequencing and subsequently validated in mouse primary cells. Tert-butyl hydroperoxide (TBHP) was utilized to create an in vitro model of lung epithelial cell oxidative stress with MLE-12 cells. ResultsSPAUTIN-1 significantly mitigated LPS-induced lung injury and inflammatory responses, attenuated necroptosis and apoptosis in lung epithelial cells, and inhibited autophagy in leukocytes and epithelial cells. However, SPAUTIN-1 exhibited no significant effect on bleomycin-induced lung injury. RNA-sequencing results demonstrated that SPAUTIN-1 significantly inhibited the NF-κB signaling pathway in leukocytes, a finding consistently confirmed by mouse primary cell assays. In vitro experiments further revealed that SPAUTIN-1 effectively mitigated oxidative stress injury in MLE-12 cells induced by TBHP. ConclusionSPAUTIN-1 alleviated LPS-induced inflammatory injury by inhibiting the NF-κB pathway in leukocytes and protected epithelial cells from oxidative damage, positioning it as a potential therapeutic candidate for ALI.

Capsaicin ameliorate pulmonary fibrosis via antioxidant Nrf-2/ PPAR- γ pathway activation and inflammatory TGF-β1/ NF-κB/COX II pathway inhibition.

Bleomycin is an effective antibiotic with a significant anticancer properties, but its use is limited due to its potential to induce dose-dependent pulmonary fibrosis. Therefore, this study aimed to assess the therapeutic potential of Capsaicin as an additional treatment to enhance patient tolerance to Bleomycin compared to the antifibrotic drug Pirfenidone. Pulmonary fibrosis was induced in rats through by a single intratracheal Bleomycin administration in day zero, followed by either Capsaicin or Pirfenidone treatment for 7days. After the animals were sacrificed, their lungs were dissected and examined using various stains for macroscopic and histopathological evaluation. Additionally, the study assessed various antioxidant, anti-inflammatory, and antifibrotic parameters were assessed. Rats exposed to Bleomycin exhibited visible signs of fibrosis, histopathological alterations, increased collagen deposition, and elevated mucin content. Bleomycin also led to heightened increased inflammatory cells infiltration in the bronchoalveolar lavage, elevated fibrosis biomarkers such as hydroxyproline, alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta (TGF-β1), increased inflammatory markers including tumor necrosis factor-alpha (TNF-α), interlukine-6 (Il-6), interlukine-1β (Il-1β) nuclear factor-kappa B (NF-κB), and Cyclooxygenase-2 (COX-2), and transforming growth factor-beta (TGF-β1),. Furthermore, it reduced the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ), increased oxidative stress biomarkers like nitric oxide (NO), malondialdehyde (MDA), myeloperoxidase (MPO) and protein carbonyl. Bleomycin also decreased the expression of nuclear factor erythroid 2-related factor 2 (Nrf-2), reduced glutathione (GSH), total antioxidant capacity, and the activities of catalase and superoxide dismutase (SOD). Treating the animals with Capsaicin and Pirfenidone following Bleomycin exposure resulted in improved lung macroscopic and microscopic characteristics, reduced collagen deposition (collagen I and collagen III) and mucin content, decreased inflammatory cell infiltration, lowered levels of hydroxyproline, α-SMA, and TGF-β1, decreased TNF-α, Il-6, Il-1β, NF-κB, and COX-2, increased PPAR-γ and Nrf-2 expression, and improvement improved in all oxidative stress biomarkers. In summary, Capsaicin demonstrates significant antifibrotic activity against Bleomycin-induced lung injury that may be attributed, at least in part, to the antioxidant and anti-inflammatory activities of Capsaicin mediated by upregulation of PPAR-γ and Nrf-2 expression and decreasing. TGF-β1, NF-κB and COX II proteins concentrations.

Function of KvLQT1 potassium channels in a mouse model of bleomycin-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) is characterized by an exacerbated inflammatory response, severe damage to the alveolar-capillary barrier and a secondary infiltration of protein-rich fluid into the airspaces, ultimately leading to respiratory failure. Resolution of ARDS depends on the ability of the alveolar epithelium to reabsorb lung fluid through active transepithelial ion transport, to control the inflammatory response, and to restore a cohesive and functional epithelium through effective repair processes. Interestingly, several lines of evidence have demonstrated the important role of potassium (K+) channels in the regulation of epithelial repair processes. Furthermore, these channels have previously been shown to be involved in sodium/fluid absorption across alveolar epithelial cells, and we have recently demonstrated the contribution of KvLQT1 channels to the resolution of thiourea-induced pulmonary edema in vivo. The aim of our study was to investigate the role of the KCNQ1 pore-forming subunit of KvLQT1 channels in the outcome of ARDS parameters in a model of acute lung injury (ALI). We used a molecular approach with KvLQT1-KO mice challenged with bleomycin, a well-established ALI model that mimics the key features of the exudative phase of ARDS on day 7. Our data showed that KvLQT1 deletion exacerbated the negative outcome of bleomycin on lung function (resistance, elastance and compliance). An alteration in the profile of infiltrating immune cells was also observed in KvLQT1-KO mice while histological analysis showed less interstitial and/or alveolar inflammatory response induced by bleomycin in KvLQT1-KO mice. Finally, a reduced repair rate of KvLQT1-KO alveolar cells after injury was observed. This work highlights the complex contribution of KvLQT1 in the development and resolution of ARDS parameters in a model of ALI.

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-β 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-β 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 IL1β 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.

Bleomycin-induced modulations of PARP 1 activity, NAD+ and PARG content in rat lung nuclei

Bleomycin-induced lung pathology in rodents is a well recognized animal model widely used for evaluation of new therapeutic approaches in treatment of lung inflammation and fibrotic diseases. It is documented that poly(ADP-ribose)polymerase 1 activity has a significant role in development of inflammatory processes in the heart, liver and brain. Herein, we used biochemical and immunochemical methods for estimation of poly(ADP-ribose)polymerase 1 (PARP 1) activity, NAD+ and poly(ADP-ribose)glycohydrolase (PARG) protein content in rat lung nuclei during the inflammatory phase in a bleomycin-induced one-hit rat model. To evaluate the influence of bleomycin – induced alterations in DNA structure on regulation of poly(ADP-ribose)polymerase 1 activation pathways, we isolated DNA from nuclei of lung tissues in the phase of acute lung inflammation induced by bleomycin, and DNA melting profiles were investigated. In the present study we investigated whether naturally occurring water-soluble polyphenol tannic acid with widely accepted anti-fibrotic and anti-inflammatory effects can influence poly(ADP-ribose)polymerase 1 activity, NAD+ and poly(ADP-ribose)glycohydrolase protein content in nuclear fraction isolated from rat lung tissues in a bleomycin-induced acute lung injury model. It was demonstrated that NAD+ level and poly(ADP-ribose)glycohydrolase protein content decreased in rat lung nuclei during the inflammatory phase in the bleomycin-induced acute lung injury model. Treatment of rats with tannic acid enhanced the effects displayed by bleomycin in lung nuclei, thus indicating synergistic interaction with the drug in the field covering PARP 1 activity, poly(ADP-ribose)glycohydrolase (PARG) protein and NAD+ content in lung nuclei. We observed PARP 1 inhibition in nuclei of lung tissue during the inflammatory phase in the bleomycin-induced acute lung injury rat model, which could be coupled with the drop of NAD+ level in nuclei. In the present study we highlighted that bleomycin (BLM) can induce DNA destabilization in lung nuclei. It was proposed that bleomycin-induced modulations in DNA structure could hamper PARP 1 binding with DNA and down-regulate the enzyme activating pathway in lung nuclei. The role of poly(ADP-ribose)glycohydrolase depletion in lung nuclei and sequential accumulation of poly(ADP-ribose)polymers in lung cells, which triggers their destruction and tissues damage, was proposed. It is suggested that in the light of synergistic interaction between bleomycin and tannic acid (TA) the anti-inflammatory role of tannic acid should be repurposed.

Inhibition of sphingosine 1-phosphate receptor 3 ameliorates bleomycin-induced pulmonary fibrosis by suppressing macrophage M2 polarization

Pulmonary fibrosis is a devastating lung disease without effective treatment options. Sphingosine-1-phosphate receptor 3 (S1pr3), a receptor for the lipid signaling molecule sphingosine-1-phosphate, has been shown to mediate the development of pulmonary fibrosis, although the underlying mechanism is not fully understood. Here, we found increased expression of S1pr3 in the lung during the process of bleomycin-induced pulmonary fibrosis in mice and specific overexpression of S1pr3 in the infiltrated M2 macrophages. We constructed LysM-Cre+/S1pr3flox/flox mice, in which S1pr3 was conditionally depleted in myeloid cells, and this depletion protected mice from bleomycin-induced lung injury and fibrosis, with reduced M2 macrophage accumulation in the lung. Increased S1pr3 expression was found in bone marrow-derived macrophages after alternatively activated by IL4 ex vivo, while loss of S1pr3 attenuated IL-4-induced M2 polarization in bone marrow-derived macrophages by repressing the PI3K/Akt-Stat3 signaling pathway. Moreover, the S1pr3 inhibitors CAY10444 and TY52156 exerted protective effects on pulmonary fibrosis in mice. Taken together, our research showed that inhibition of S1pr3 ameliorates bleomycin-induced pulmonary fibrosis by reducing macrophage M2 polarization via the PI3K/Akt-Stat3 signaling pathway, indicating that S1pr3 may be a potential target for pulmonary fibrosis treatment.

Role of lung ornithine aminotransferase in idiopathic pulmonary fibrosis: regulation of mitochondrial ROS generation and TGF-β1 activity

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung remodeling and the excessive accumulation of extracellular matrix (ECM) proteins. In a previous study, we found that the levels of ornithine aminotransferase (OAT), a principal enzyme in the proline metabolism pathway, were increased in the lungs of patients with IPF. However, the precise role played by OAT in the pathogenesis of IPF is not yet clear. The mechanism by which OAT affects fibrogenesis was assessed in vitro using OAT-overexpressing and OAT-knockdown lung fibroblasts. The therapeutic effects of OAT inhibition were assessed in the lungs of bleomycin-treated mice. OAT expression was increased in fibrotic areas, principally in interstitial fibroblasts, of lungs affected by IPF. OAT levels in the bronchoalveolar lavage fluid of IPF patients were inversely correlated with lung function. The survival rate was significantly lower in the group with an OAT level >75.659 ng/mL than in the group with an OAT level ≤75.659 ng/mL (HR, 29.53; p = 0.0008). OAT overexpression and knockdown increased and decreased ECM component production by lung fibroblasts, respectively. OAT knockdown also inhibited transforming growth factor-β1 (TGF)-β1 activity and TGF-β1 pathway signaling. OAT overexpression increased the generation of mitochondrial reactive oxygen species (ROS) by activating proline dehydrogenase. The OAT inhibitor L-canaline significantly attenuated bleomycin-induced lung injury and fibrosis. In conclusion, increased OAT levels in lungs affected by IPF contribute to the progression of fibrosis by promoting excessive mitochondrial ROS production, which in turn activates TGF-β1 signaling. OAT may be a useful target for treating patients with fibrotic lung diseases, including IPF.

Innate Immune Training Initiates Efferocytosis to Protect against Lung Injury.

Innate immune training involves myelopoiesis, dynamic gene modulation, and functional reprogramming of myeloid cells in response to secondary heterologous challenges. The present study evaluates whether systemic innate immune training can protect tissues from local injury. Systemic pretreatment of mice with β-glucan, a trained immunity agonist, reduces the mortality rate of mice with bleomycin-induced lung injury and fibrosis, as well as decreasing collagen deposition in the lungs. β-Glucan pretreatment induces neutrophil accumulation in the lungs and enhances efferocytosis. Training of mice with β-glucan results in histone modification in both alveolar macrophages (AMs) and neighboring lung epithelial cells. Training also increases the production of RvD1 and soluble mediators by AMs and efferocytes. Efferocytosis increases trained immunity in AMs by stimulating RvD1 release, thus inducing SIRT1 expression in neighboring lung epithelial cells. Elevated epithelial SIRT1 expression is associated with decreased epithelial cell apoptosis after lung injury, attenuating tissue damage. Further, neutrophil depletion dampens the effects of β-glucan on macrophage accumulation, epigenetic modification in lung macrophages, epithelial SIRT1 expression, and injury-mediated fibrosis in the lung. These findings provide mechanistic insights into innate immune training and clues to the potential ability of centrally trained immunity to protect peripheral organs against injury-mediated disorders.

Hypoxia represses FOXF1 in lung endothelial cells through HIF-1α.

Introduction: Forkhead Box F1 (FOXF1) transcription factor plays a critical role in lung angiogenesis during embryonic development and lung repair after injury. FOXF1 expression is decreased in endothelial cells after lung injury; however, molecular mechanisms responsible for the FOXF1 transcript changes in injured lung endothelium remain unknown. Methods: We used immunostaining of injured mouse lung tissues, FACS-sorted lung endothelial cells from hypoxia-treated mice, and data from patients diagnosed with hypoxemic respiratory failure to demonstrate that hypoxia is associated with decreased FOXF1 expression. Endothelial cell cultures were used to induce hypoxia in vitro and identify the upstream molecular mechanism through which hypoxia inhibits FOXF1 gene expression. Results: Bleomycin-induced lung injury induced hypoxia in the mouse lung tissue which was associated with decreased Foxf1 expression. Human FOXF1 mRNA was decreased in the lungs of patients diagnosed with hypoxemic respiratory failure. Mice exposed to hypoxia exhibited reduced Foxf1 expression in the lung tissue and FACS-sorted lung endothelial cells. In vitro, hypoxia (1% of O2) or treatment with cobalt (II) chloride increased HIF-1α protein levels but inhibited FOXF1 expression in three endothelial cell lines. Overexpression of HIF-1α in cultured endothelial cells was sufficient to inhibit Foxf1 expression. siRNA-mediated depletion of HIF-1α prevented the downregulation of Foxf1 gene expression after hypoxia or cobalt (II) chloride treatment. Conclusion: Hypoxia inhibits FOXF1 expression in endothelial cells in a HIF-1α dependent manner. Our data suggest that endothelial cell-specific inhibition of HIF-1α via gene therapy can be considered to restore FOXF1 and improve lung repair in patients with severe lung injury.

Thrombocyte-derived Dickkopf1 promotes macrophage polarization in the Bleomycin-induced lung injury model.

Immune responses are crucial to maintaining tissue homeostasis upon tissue injury. Upon various types of challenges, macrophages play a central role in regulating inflammation and tissue repair processes. While an immunomodulatory role of Wnt antagonist Dickkopf1 (DKK1) has been implicated, the role of Wnt antagonist DKK1 in regulating macrophage polarization in inflammation and the tissue repair process remains elusive. Here we found that DKK1 induces gene expression profiles to promote inflammation and tissue repair in macrophages. Importantly, DKK1 induced various genes, including inflammation and tissue repair, via JNK (c-jun N-terminal kinase) in macrophages. Furthermore, DKK1 potentiated IL-13-mediated macrophage polarization and activation. The co-inhibition of JNK and STAT6 markedly decreased gene expressions relevant to inflammation and fibrosis by DKK1 and IL-13. Interestingly, thrombocyte-specific deletion of DKK1 in mice reduced collagen deposition and decreased Arg1, CD206, HIF1α, and IL1β protein expressions in monocyte-derived alveolar macrophages in the acute sterile bleomycin (BLM)-induced lung injury model. These data suggested that thrombocytes communicate with macrophages via DKK1 to orchestrate inflammation and repair in this model. Taken together, our study demonstrates DKK1's role as an important regulatory ligand for macrophage polarization in the injury-induced inflammation and repair process in the lung.

IL-22 Binding Protein Controls IL-22–Driven Bleomycin-Induced Lung Injury

The high mortality rates of acute lung injury and acute respiratory distress syndrome challenge the field to identify biomarkers and factors that can be exploited for therapeutic approaches. IL-22 is a cytokine that has antibacterial and reparative properties in the lung. However, it also can exacerbate inflammation and requires tight control by the extracellular inhibitory protein known as IL-22 binding protein (IL-22BP) (Il22ra2). This study showed the necessity of IL-22BP in controlling and preventing acute lung injury using IL-22BP knockout mice (Il22ra2-/-) in the bleomycin model of acute lung injury/acute respiratory distress syndrome. Il22ra2-/- mice had greater sensitivity (weight loss and death) and pulmonary inflammation in the acute phase (first 7 days) of the injury compared with wild-type C57Bl/6 controls. The inflammation was driven by excess IL-22 production, inducing the influx of pathogenic IL-17A+ γδ T cells to the lung. Interestingly, this inflammation was initiated in part by the noncanonical IL-22 signaling to macrophages, which express the IL-22 receptor (Il22ra1) invivo after bleomycin challenge. This study further showed that IL-22 receptor alpha-1+ macrophages can be stimulated by IL-22 to produce a number of IL-17-inducing cytokines such as IL-1β, IL-6, and transforming growth factor-β1. Together, the results suggest that IL-22BP prevents IL-22 signaling to macrophages and reduces bleomycin-mediated lung injury.

Qualitative Histopathological Analysis of Bleomycin-Induced Lung Injury

Recent evidence has suggested that the accumulation of cholesterol-laden “fatty macrophages” in the lung may play a key role in the presentation of ALI 1, a condition that affects nearly 200,000 people per year in the United States. Studying the pathways of these cells suggests that preventing the formation and build-up of these macrophages could prevent many of the characteristic symptoms of acute lung injury. Specifically, this experiment aims to explore the effect that ACAT1 inhibition by K604 has on fatty macrophage formation and outcomes of ALI. To test this, different groups of mice were administered either PBS (as control), ITB (intratracheal bleomycin), PBS + K604, or ITB + K604. After the mice were sacrificed, the left lung lobes were extracted, inflated, embedded, and sectioned for microscopic scanning and analysis with the ImageJ software. The lung tissue was characterized using 3 unbiased parameters: alveolar wall thickness (µm), cell infiltration (number of nuclei), and percent white space. Simultaneously, a cholesterol assay was performed using cells extracted from mouse bronchoalveolar lavage (BAL). The results show that K604 was successful at preventing ITB-induced increases in cell infiltration, consolidation, and alveolar thickening as well as lipid accumulation in macrophages by limiting the formation of cholesterol esters through ACAT1 inhibition. In this respect, this experiment shows that K604 might be a viable pharmaceutical target in the treatment of ALI.

Lung mechanics showing sex-based differences and circadian time-of-day response to bleomycin-induced lung injury in mice.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that impairs lung mechanical properties due to dysregulated extracellular matrix remodeling. Lung function assessment is an important physiological endpoint in the mouse model of pulmonary fibrosis (PF) that has gained a broader scientific acceptance in the field. IPF pathophysiology shows sex-based differences, disproportionately affecting more men compared to women. Prior reports suggest that the circadian clock is perturbed during the pathogenesis of PF. We have comprehensively assessed the sex-based differences and time-of-day response (at Zeitgeber time: ZT0/6:00 a.m. or ZT12/6 p.m.) in lung mechanics among sham (control) versus bleomycin (BLM)-challenged female and male (C57BL/6: WT) mice using Flexi-vent. BLM challenge altered lung function significantly in males in both total lung (reduced dynamic compliance, and increased resistance and elastance) as well as lung tissue-specific parameters (increased tissue elastance and tissue damping) but less pronounced in females. BLM-challenged mice showed a time-of-day response in lung function at ZT0 versus ZT12, which was pronounced in the ZT0 BLM group. Overall, these findings provide a comprehensive analysis of altered lung function in female and male mice and the time-of-day difference in lung function parameters following BLM-induced lung fibrosis.

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.

Ficolin B secreted by alveolar macrophage exosomes exacerbates bleomycin-induced lung injury via ferroptosis through the cGAS-STING signaling pathway

Pathogenesis exploration and timely intervention of lung injury is quite necessary as it has harmed human health worldwide for years. Ficolin B (Fcn B) is a recognition molecule that can recognize a variety of ligands and play an important role in mediating the cell cycle, immune response, and tissue homeostasis in the lung. However, the role of Fcn B in bleomycin (BLM)-induced lung injury is obscure. This study aims to investigate the sources of Fcn B and its mechanism in BLM-induced lung injury. WT, Fcna-/-, and Fcnb-/- mice were selected to construct the BLM-induced lung injury model. Lung epithelial cells were utilized to construct the BLM-induced cell model. Exosomes that were secreted from alveolar macrophages (AMs) were applied for intervention by transporting Fcn B. Clinical data suggested M-ficolin (homologous of Fcn B) was raised in plasma of interstitial lung disease (ILD) patients. In the mouse model, macrophage-derived Fcn B aggravated BLM-induced lung injury and fibrosis. Fcn B further promoted the development of autophagy and ferroptosis. Remarkably, cell experiment results revealed that Fcn B transported by BLM-induced AMs exosomes accelerated autophagy and ferroptosis in lung epithelial cells through the activation of the cGAS-STING pathway. In contrast, the application of 3-Methyladenine (3-MA) reversed the promotion effect of Fcn B from BLM-induced AMs exosomes on lung epithelial cell damage by inhibiting autophagy-dependent ferroptosis. Meanwhile, in the BLM-induced mice model, the intervention of Fcn B secreted from BLM-induced AMs exosomes facilitated lung injury and fibrosis via ferroptosis. In summary, this study demonstrated that Fcn B transported by exosomes from AMs exacerbated BLM-induced lung injury by promoting lung epithelial cells ferroptosis through the cGAS-STING signaling pathway.

Atractylodinol prevents pulmonary fibrosis through inhibiting TGF-β receptor 1 recycling by stabilizing vimentin

Pirfenidone and nintedanib are only anti-pulmonary fibrosis (PF) drugs approved by the FDA. However, they are not target specific, and unable to modify the disease status. Therefore, it is still desirable to discover more effective agents against PF. Vimentin (VIM) plays key roles in tissue regeneration and wound healing, but its molecular mechanism remains unknown. In this work, we demonstrated that atractylodinol (ATD) significantly inhibits TGF-β1-induced epithelial-mesenchymal transition and fibroblast-to-myofibroblast transition invitro. ATD also reduces bleomycin-induced lung injury and fibrosis in mice models. Mechanistically, ATD inhibited TGF-β receptor I recycling by binding to VIM (KD= 454nM) and inducing the formation of filamentous aggregates. In conclusion, we proved that ATD (derived from Atractylodes lancea) modified PF by targeting VIM and inhibiting the TGF-β/Smad signaling pathway. Therefore, VIM is a druggable target and ATD is a proper drug candidate against PF. We prove a novel VIM function that TGF-β receptor I recycling. These findings paved the way to develop new targeted therapeutics against PF.