Bleomycin-induced Lung Injury Research Articles

Mitoquinone alleviates bleomycin-induced acute lung injury via inhibiting mitochondrial ROS-dependent pulmonary epithelial ferroptosis.

Numerous studies demonstrated that bleomycin (BLM) caused acute lung injury (ALI). This study explored the role of mitochondrial reactive oxygen species (ROS) on BLM-induced ALI and pulmonary epithelial ferroptosis. Male C57BL/6J mice were intratracheally injected with BLM (3.0mg/kg). BEAS-2B cells, human bronchial epithelial cells, were cultured with BLM (10μg/ml). Pulmonary MDA and 4-HNE, two markers of lipid peroxidation, were elevated in BLM-exposed mice. Oxidized lipids were upregulated in BLM-exposed BEAS-2B cells. Ferroptosis-characteristic ultrastructure, mainly disappearance of mitochondrial bilayer membrane structure and cristae, was observed in BLM-exposed pulmonary epithelium. Ferrostatin-1, a specific inhibitor of ferroptosis, attenuated BLM-evoked pulmonary lipid peroxidation, ferroptosis-characteristic mitochondrial ultrastructure and pulmonary epithelial death. The in vitro experiments showed that mitochondrial membrane potentials (MMPs) were decreased and mitochondrial ROS were increased in BLM-exposed BEAS-2B cells. Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, prevented BLM-induced MMP reduction and mitochondrial ROS elevation in BEAS-2B cells. The in vivo experiment found that MitoQ attenuated BLM-evoked GSH depletion and lipid peroxidation in mouse lungs. Moreover, MitoQ prevented BLM-induced ferroptosis-characteristic mitochondrial changes, pulmonary epithelial death and ALI. In conclusion, mitochondrial ROS are an initiator of BLM-induced pulmonary epithelial ferroptosis. Mitochondria-targeted antioxidants may be used as potential therapeutic agents for BLM-induced ALI.

A Non-Coding Variant in LOC105371512 Confers Susceptibility to Bleomycin-Induced Lung Injury

Background: Bleomycin has been used as a cornerstone in the treatment of a variety of cancer types including Hodgkin Lymphoma (HL) and Germ Cell Tumor. Unfortunately, bleomycin has its complications, such as Bleomycin-induced pneumonitis (BIP) occurring in ~20% of patients and is fatal in 4-5%. In addition, there are no trials guiding effective treatment for BIP. While brentuximab as an alternative for bleomycin has shown good results in HL, it is still not considered as a standard first line treatment for most of the world. We aimed to investigate the significance of known genetic associations [(HFE rs1799945 (H63D) and BLMH rs1050565 (I443V)] in Asian patients, and identify previously uncharacterized genetic biomarkers associated with the predisposition to BIP. Through functional studies, we also sought to investigate the mechanisms in which these genetic biomarkers led to BIP. Methods: We analyzed 133 HL patients treated with bleomycin in Singapore. Clinical data was extracted from electronic medical records, BIP was characterized and graded by CTCAEv4. Genomic DNA was genotyped using Infinium Global Screening Array-24 v1.0 for 700,078 markers. Alt-R® CRISPR-Cas9 was used for the gene-editing experiment. Results: A total of 21.43% of patients developed clinically significant BIP, defined as CTCAEv4 ≥ Grade 2. Cumulative bleomycin dose was significantly lower in the BIP cases, as BIP patients had bleomycin discontinued from future therapy. All other clinical features were not significantly different between the groups. We replicated an association of HFE rs1799945 (H63D) with BIP and identified 4 new potential genetic predictors. LOC105371512 rs189773 was ranked the top loci. We genotyped 4 lung cancer cell lines. Comparing rs189773 CC and TT genotypes, CC had lower expression of pro-fibrotic markers, while higher expression of anti-fibrotic ones. We next used CRISPR/Cas9 to generate cells with either CC or TT genotype at rs189773 locus using the same cell line (A459). Interestingly, the BLMH protein expression level showed a reduction in A549(TT) compared to A549(CC), with a more remarkable effect in the presence of bleomycin. Screening the fibrotic biomarkers further revealed that growth factors and pro-fibrotic interleukins were up-regulated in A549(TT) compared to A549(CC) cells especially when the cells were treated with bleomycin. Next, we found that this SNP is located in a transcriptionally active region, with a higher activity level for the presence of T at the position compared to C. The rs189773-flanking region was subsequently shown to enhance the transcription level of distal genes with consistent effects on the profibrotic growth factors and cytokines. The qPCR study demonstrated that BLMH was not regulated at the mRNA level. We thus treated the cells with bleomycin and a 26S proteasome inhibitor, bortezomib. Interestingly, the bleomycin-induced down-regulation of BLMH protein was rescued upon co-treating the cells with both bleomycin and bortezomib. This proteasome inhibitor drug consistently diminished the heightened levels of profibrotic markers induced by bleomycin. Conclusion: In conclusion, in the first pharmacogenomics-GWAS study on BIP in Asians, rs189773 was ranked the top likely loci, with functional studies showing that this variant is in an enhancer region, modulating the cellular response to bleomycin via regulation of a distal locus, BLMH, and profibrotic growth factors. At the post-translational level, inhibition of the 26S proteasome by bortezomib restores BLMH expression and suppresses profibrotic growth factors. This study provides insights into future screening targets and potential new treatment options for BIP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Salt-inducible kinase 2 regulates fibrosis during bleomycin-induced lung injury

Idiopathic pulmonary fibrosis is a progressive and normally fatal disease with limited treatment options. The tyrosine kinase inhibitor nintedanib has recently been approved for the treatment of idiopathic pulmonary fibrosis, and its effectiveness has been linked to its ability to inhibit a number of receptor tyrosine kinases including the platelet-derived growth factor, vascular endothelial growth factor, and fibroblast growth factor receptors. We show here that nintedanib also inhibits salt-inducible kinase 2 (SIK2), with a similar IC50 to its reported tyrosine kinase targets. Nintedanib also inhibited the related kinases SIK1 and SIK3, although with 12-fold and 72-fold higher IC50s, respectively. To investigate if the inhibition of SIK2 may contribute to the effectiveness of nintedanib in treating lung fibrosis, mice with kinase-inactive knockin mutations were tested using a model of bleomycin-induced lung fibrosis. We found that loss of SIK2 activity protects against bleomycin-induced fibrosis, as judged by collagen deposition and histological scoring. Loss of both SIK1 and SIK2 activity had a similar effect to loss of SIK2 activity. Total SIK3 knockout mice have a developmental phenotype making them unsuitable for analysis in this model; however, we determined that conditional knockout of SIK3 in the immune system did not affect bleomycin-induced lung fibrosis. Together, these results suggest that SIK2 is a potential drug target for the treatment of lung fibrosis.

CNP-miR146a Mitigates Methicillin-Resistant Staphylococcus aureus–Induced Acute Lung Injury by Decreasing Inflammation

INTRODUCTION: ARDS has a nearly 40% in-hospital mortality, and current treatment is largely limited to supportive care. Pneumonia and sepsis are the underlying causes responsible for ARDS in a majority of cases with inflammation as a central pathophysiologic feature. We have previously shown that CNP-miR146a, a radical scavenging cerium oxide nanoparticle (CNP) conjugated to the anti-inflammatory microRNA (miR)-146a, reduces bleomycin-induced acute lung injury by decreasing inflammation and oxidative stress. We therefore hypothesized that CNP-miR146a would decrease inflammation in methicillin-resistant Staphylococcus aureus (MRSA)–induced acute lung injury. METHODS: C57BL/6 mice (n = 46) were given IT MRSA or saline (control) followed 4 hours later by 1 ng/50 μL IT CNP-miR146a (MRSA + CNP-miR146a) or saline (control, MRSA). At 24 hours after infection, bronchoalveolar lavage fluid was analyzed by ELISA for proinflammatory cytokines. Whole lungs were processed for total RNA extraction, and quantitative polymerase chain reaction analysis was performed to evaluate proinflammatory gene expression. Statistical significance was determined by 1-way ANOVA (p < 0.05 significant). RESULTS: MRSA-injured lungs had significantly higher relative gene expression of proinflammatory IL-8 (p = 0.0056), IL-1β (p = 0.0023), and TNFα (p = 0.0024) compared with controls. Treatment with CNP-miR146a significantly lowered the expression of IL-6 (p = 0.0271), IL-8 (p = 0.0232), IL-1β (p = 0.0499), and TNFα (p = 0.0121). MRSA injury also significantly increased bronchoalveolar lavage fluid IL-6 protein concentration (p = 0.0011) compared with control, and treatment with CNP-miR146a returned this to control levels (p = 0.0227; Figure).Figure.: CNP-miR146a, a radical scavenging cerium oxide nanoparticle conjugated to the anti-inflammatory microRNA-146a; MRSA, methicillin-resistant Staphylococcus aureus.CONCLUSION: CNP-miR146a rescues the MRSA-injured lung by decreasing inflammation and bronchoalveolar proinflammatory protein leak, and therefore has significant promise as a potential therapeutic for ARDS.

Intratracheal Administration of Acyl Coenzyme A Acyltransferase-1 Inhibitor K-604 Reduces Pulmonary Inflammation Following Bleomycin-Induced Lung Injury.

Acute lung injury (ALI) is characterized by epithelial damage, barrier dysfunction, and pulmonary edema. Macrophage activation and failure to resolve play a role in ALI; thus, macrophage phenotype modulation is a rational target for therapeutic intervention. Large, lipid-laden macrophages have been observed in various injury models, including intratracheal bleomycin (ITB), suggesting that lipid storage may play a role in ALI severity. The endoplasmic reticulum-associated enzyme acyl coenzyme A acyltransferase-1 (Acat-1/Soat1) is highly expressed in macrophages, where it catalyzes the esterification of cholesterol, leading to intracellular lipid accumulation. We hypothesize that inhibition of Acat-1 will reduce macrophage activation and improve outcomes of lung injury in ITB. K-604, a selective inhibitor of Acat-1, was used to reduce cholesterol esterification and hence lipid accumulation in response to ITB. Male and female C57BL6/J mice (n = 16-21/group) were administered control, control + K-604, ITB, or ITB + K-604 on d0, control or K-604 on d3, and were sacrificed on day 7. ITB caused significant body weight loss and an increase in cholesterol accumulation in bronchoalveolar lavage cells. These changes were mitigated by Acat-1 inhibition. K-604 also significantly reduced ITB-induced alveolar thickening. Surfactant composition was normalized as indicated by a significant decrease in phospholipid: SP-B ratio in ITB+K-604 compared with ITB. K-604 administration preserved mature alveolar macrophages, decreased activation in response to ITB, and decreased the percentage mature and pro-fibrotic interstitial macrophages. These results show that inhibition of Acat-1 in the lung is associated with reduced inflammatory response to ITB-mediated lung injury.SIGNIFICANCE STATEMENTAcyl coenzyme A acyltransferase-1 (Acat-1) is critical to lipid droplet formation, and thus inhibition of Acat-1 presents as a pharmacological target. Intratracheal administration of K-604, an Acat-1 inhibitor, reduces intracellular cholesterol ester accumulation in lung macrophages, attenuates inflammation and macrophage activation, and normalizes mediators of surface-active function after intratracheal bleomycin administration in a rodent model. The data presented within suggest that inhibition of Acat-1 in the lung improves acute lung injury outcomes.

Histopathological, physiological and biochemical assessment of resveratrol nanocapsules efficacy in bleomycin-induced acute and chronic lung injury in rats

Acute lung injury (ALI) is a life-threatening illness which may progress to chronic pulmonary fibrosis (CPF). Resveratrol (RSV), a natural polyphenol, is known to exert several pharmacological effects on lung injury. However, its physicochemical properties and pharmacokinetic profile limit its clinical applications. In this study, RSV was loaded into lipid nanocapsules (LNCs) aiming to overcome these limitations. RSV-LNCs were prepared by phase inversion method and showed small uniform particle size (∼55 nm, PdI 0.04) with high entrapment efficiency >99%. The efficacy of RSV-LNCs in the prophylaxis against ALI and treatment of CPF was investigated in bleomycin-induced lung injury. For assessment of ALI, rats were administered a single oral dose of RSV (10 mg/kg) either free or as RSV-LNCs 4 h before bleomycin and euthanized 3 days later. For CPF, treatments in the same dose were given daily from days 10–20 after bleomycin and rats were euthanized on day-21. Results showed enhanced beneficial role for RSV-LNCs, compared to RSV, in the prevention of ALI as demonstrated by preservation of pulmonary microscopic and ultrastructural architecture and improvement of pulmonary functions. Analysis of BALF revealed reduction in oxidative stress markers, IL-6 level, leukocytosis and neutrophilia. iNOS and c-caspase 3 immunohistochemical expression and CD68+ cells immunofluorescence were inhibited. However, RSV-LNCs failed to show any improvement in oxidative stress, chronic inflammation, apoptosis and collagen deposition in CPF. In conclusion, RSV-LNCs are promising nanoplatforms for mitigating ALI detrimental effects. Future research investigating higher doses and longer durations of treatment is recommended to evaluate RSV-LNCs anti-fibrotic potential in CPF.

Time and phenotype-dependent transcriptome analysis in AAV-TGFβ1 and Bleomycin-induced lung fibrosis models

We have previously established a novel mouse model of lung fibrosis based on Adeno-associated virus (AAV)-mediated pulmonary overexpression of TGFβ1. Here, we provide an in-depth characterization of phenotypic and transcriptomic changes (mRNA and miRNA) in a head-to-head comparison with Bleomycin-induced lung injury over a 4-week disease course. The analyses delineate the temporal state of model-specific and commonly altered pathways, thereby providing detailed insights into the processes underlying disease development. They further guide appropriate model selection as well as interventional study design. Overall, Bleomycin-induced fibrosis resembles a biphasic process of acute inflammation and subsequent transition into fibrosis (with partial resolution), whereas the TGFβ1-driven model is characterized by pronounced and persistent fibrosis with concomitant inflammation and an equally complex disease phenotype as observed upon Bleomycin instillation. Finally, based on an integrative approach combining lung function data, mRNA/miRNA profiles, their correlation and miRNA target predictions, we identify putative drug targets and miRNAs to be explored as therapeutic candidates for fibrotic diseases. Taken together, we provide a comprehensive analysis and rich data resource based on RNA-sequencing, along with a strategy for transcriptome-phenotype coupling. The results will be of value for TGFβ research, drug discovery and biomarker identification in progressive fibrosing interstitial lung diseases.

Pomegranate juice ameliorates pulmonary fibrosis by regulating inflammatory response and epithelial mesenchymal transformation

POMJ could ameliorate pulmonary fibrosis in vivo and in vitro . Based on the analysis of the bioactive ingredients and the anti-oxidation ability of POMJ, we explored the potential role and underlying mechanisms of POMJ against PF both in in vivo and in vitro experimental model. POMJ could improve BLM-induced PF by alleviating the inflammatory response and the progression of EMT, and also could ameliorate EMT process in TGF-β1 stimulated cell model by inhibiting the activation of NF-κB pathway. • The potential bioactive ingredients in POMJ were analyzed using LC-HR/MS. • POMJ prevents bleomycin-induced lung injury by reducing inflammation response. • POMJ alleviated EMT in vivo and in vitro . • POMJ suppressed TGF‐β1‐induced NF‐κB pathway activation in EMT. Pulmonary fibrosis (PF) is a chronic, progressive interstitial pneumonia characterized by abnormal epithelial -mesenchymal transition (EMT) and persistent inflammatory injury. Pomegranate has multiple biological activities, including antioxidant, anti-inflammatory and anticancer activities. In this study, we evaluated the effect of pomegranate juice (POMJ) in ameliorating PF and explored the underlying mechanisms in vivo and in vitro . Compared to the Vehicle group, POMJ attenuated bleomycin (BLM)-induced histopathological changes and collagen deposition in the lungs and alleviated the inflammatory response. Western blot and immunohistochemistry results revealed that POMJ treatment reduced the expression of Collagen I and Vimentin and increased the expression of E-cadherin. In addition, in TGF-β1-treated A549 cells, it was further confirmed, which may be associated with inhibition of NF-κB pathway activation. Taken together, these findings suggest that POMJ ameliorates lung injury by regulating the inflammatory response and the process of EMT, exhibiting great potential to ameliorate pulmonary fibrosis.

Pulmonary Delivery of Hydroxychloroquine Nanostructured Lipid Carrier as a Potential Treatment of COVID-19.

Coronavirus Disease 2019 (COVID-19) is a pandemic caused by severe acute respiratory syndrome coronavirus 2. Pneumonia is considered the most severe and long-term complication of COVID-19. Among other drugs, hydroxychloroquine (HCQ) was repurposed for the management of COVID-19; however, low efficacy and cardiac toxicity of the conventional dosage form limited its use in COVID-19. Therefore, utilizing nanotechnology, a pulmonary delivery system of HCQ was investigated to overcome these limitations. HCQ was formulated in nanostructured lipid carriers (HCQ-NLCs) using the hot emulsification–ultrasonication method. Furthermore, the prepared formulation was evaluated in vitro. Moreover, the efficacy was tested in vivo in a bleomycin-induced acute lung injury mice model. Intriguingly, nanoformulations were given by the intratracheal route for 6 days. HCQ-NLCs showed a mean particle size of 277 nm and a good drug release profile. Remarkably, acute lung injury induced by bleomycin was associated with a marked elevation of inflammatory markers and histological alterations in lung tissues. Astoundingly, all these changes were significantly attenuated with HCQ-NLCs. The pulmonary delivery of HCQ-NLCs likely provided adequate targeting to lung tissues. Nevertheless, there is hope that this novel strategy will eventually lead to the improved effectiveness and diminished probability of alarming adverse drug reactions.

A Case of Progressive Bleomycin Lung Toxicity Refractory to Steroid Therapy

Bleomycin is a common chemotherapy agent used to treat germinative tumors. Bleomycin-induced lung injury (BILI) is an uncommon but devastating adverse effect of its use. It occurs in 10-20% of patients receiving bleomycin, and the initial diagnosis is usually made by new-onset respiratory symptoms and reduced diffusing capacity for carbon monoxide (DLCO). Mainstay treatment includes discontinuing bleomycin, corticosteroids, and supplemental oxygen if needed. We present a case of a 38-year-old male who was found to have a severe presentation of bleomycin-induced lung injury after chemotherapy for metastatic mixed germ cell testicular cancer. During his course, he was treated with the standard of care regimen of corticosteroids and salvage therapy with infliximab but ultimately died from complications of his illness. This case report is noteworthy because our patient had progressive bleomycin-induced lung injury, despite discontinuing bleomycin many months prior, consistent high-dose corticosteroid treatment, and even salvage therapy. In all patients on bleomycin, pulmonary function monitoring is essential, and any complaints of dyspnea should prompt concern for bleomycin-induced lung injury. If initial treatment does not improve their condition, more aggressive measures may be necessary.

Endocannabinoid signalling/cannabinoid receptor 2 is involved in icariin-mediated protective effects against bleomycin-induced pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease of unknown aetiology with limited effective treatment options. It is important to explore novel therapeutic targets and develop potential drugs for IPF. PurposeThe aim of the present study was to analyse nontargeted plasma metabolites in patients with IPF and investigate whether cannabinoid receptor (CB2) activation mediates the antifibrotic effect of icariin (ICA). MethodsWe used an untargeted metabolomics method to detect the global metabolic profiles in the plasma of stable IPF patients and patients with stable chronic obstructive pulmonary disease (COPD), as well as healthy subjects. The untargeted liquid chromatography-mass spectrometry (LC-MS) analysis revealed that IPF showed differential metabolites and perturbed signalling pathways. ICA is pharmacologically bioactive and possesses extensive therapeutic capacities such as osteoprotective, neuroprotective, cardiovascular protective, anti-cancer, anti-inflammation and reproductive function. Therefore, ICA was administered to a pulmonary fibrosis rat model for 4 weeks and then the effect of ICA on pulmonary fibrosis was examined by dissection and histology. ResultsThe metabolites in the plasma were determined by untargeted LC-MS. An unsupervised principal component analysis (PCA) was used to observe the distribution of each sample, and a supervised partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) results showed that there was significant separation between any two groups. ROC curve analyses revealed that 8 metabolites with high AUCs above 0.7 between the three groups of plasma samples. Pathway enrichment analysis revealed that 3 metabolites are involved in retrograde endocannabinoid signalling. Meanwhile, Retrograde endocannabinoid signalling was identified significantly different in IPF group from other groups by Kyoto encyclopedia of Genes and Genomes (KEGG) pathway analysis, and then we further confirmed the endocannabinoid signalling by detecting the expression of the main receptors in bleomycin-induced pulmonary fibrosis, COPD rat model and normal rats. Consistent with previous studies, we found that the elevation of CB1 and CB2 in the lung tissues could be a signature of the pulmonary fibrosis rat model. Importantly, ICA may alleviate bleomycin-induced lung injury by decreasing CB1 and CB2 expression in the bleomycin-induced rat model. ConclusionTaken together, we measured the global metabolic profile of IPF patients and identified CB2 as a novel potential target. ICA treatment demonstrated outstanding therapeutic effects on bleomycin-induced pulmonary fibrosis and targeting on CB2 may be the main underlying mechanism. ICA is a promising drug candidate to cure pulmonary fibrosis and mediate antagonists of the CB2 receptor.

Influenza A Virus Causes Shedding of the Alveolar Epithelial Glycocalyx through Activation of Sheddases

ObjectiveThe healthy alveolar epithelium is protected by a heparan sulfate rich, glycosaminoglycan layer called the epithelial glycocalyx. Our group found that the epithelial glycocalyx is shed in patients with acute respiratory distress syndrome (ARDS). In murine models of LPS‐ or bleomycin‐induced acute lung injury, sheddases (membrane‐bound enzymes that cleave extracellular potions of transmembrane proteins) are upregulated and associated with glycocalyx shedding and increased lung permeability. ARDS is commonly caused by viral infections including influenza A (IAV). In murine models, IAV causes massive and persistent glycocalyx shedding into the airspace but the mechanisms by which this occurs are unknown. The objective of this work is to determine the molecular processes underlying IAV‐induced shedding of the glycocalyx.HypothesisWe hypothesize that IAV causes glycocalyx shedding through induction of host sheddases.MethodsWe examined the literature and curated a list of sheddases associated with IAV with potential to cleave the glycocalyx (MMP‐7, ‐2, ‐9 and their inhibitors TIMP‐1 and ‐2). C57BL/6 mice were infected intranasally with A/PR/8/34 (H1N1) at 30,000 PFU/mouse and bronchoalveolar lavage and lung tissue were collected at day 1, 3, and 7 post infection. Sheddase expression was assessed by RT‐qPCR and RNAscope was used to localize lung sheddase expression in infected and uninfected lungs. MLE‐12 mouse lung epithelial cells were infected with viable or heat‐inactivated (56C for 30 min) A/PR/8/34 (H1N1) at a MOI of 1 and sheddase expression measured by RT‐qPCR.ResultsMice infected with IAV develop significant lung inflammation (increased BAL inflammatory cells), lung permeability (increased BAL protein), and increased glycocalyx shedding. MMP‐7 is upregulated in infected vs. uninfected lungs at day 1 and 3 post infection, then returns to baseline levels by day 7. MMP‐7 is only expressed in cells that are directly infected by IAV. Expression of the MMP‐7 inhibitor TIMP‐1 is similar to uninfected lungs on day 1, but increases 50‐fold on day 3. In contrast, MMP‐2 and MMP‐9, as well as their inhibitor TIMP‐2 are not upregulated in the first 7 days after IAV infection. Preliminary studies in lung epithelial cells suggest that heat‐inactivated IAV fails to upregulate MMP‐7.ConclusionsTogether, these data suggest that localized IAV infection increases MMP‐7 in a murine model of IAV infection, but has no effect on several other sheddases. This suggests that MMP‐7 may modulate IAV‐induced glycocalyx shedding. Future studies will explore the mechanisms of IAV induced glycocalyx shedding which could provide molecular targets for clinical intervention in IAV‐ARDS pathogenesis.

Extracellular superoxide dismutase (EC-SOD) R213G variant reduces mitochondrial ROS and preserves mitochondrial function in bleomycin-induced lung injury: EC-SOD R213G variant and intracellular redox regulation

Extracellular superoxide dismutase (EC-SOD) is highly expressed in the lung and vasculature. A common human single nucleotide polymorphism (SNP) in the matrix binding region of EC-SOD leads to a single amino acid substitution, R213G, and alters EC-SOD tissue binding affinity. The change in tissue binding affinity redistributes EC-SOD from tissue to extracellular fluids. Mice (R213G mice) expressing a knock-in of this EC-SOD SNP exhibit elevated plasma and reduced lung EC-SOD content and activity and are protected against bleomycin-induced lung injury and inflammation. It is unknown how the redistribution of EC-SOD alters site-specific redox-regulated molecules relevant for protection. In this study, we tested the hypothesis that the change in the local EC-SOD content would influence not only the extracellular redox microenvironment where EC-SOD is localized but also protect the intracellular redox status of the lung. Mice were treated with bleomycin and harvested 7 days post-treatment. Superoxide levels, measured by electron paramagnetic resonance (EPR), were lower in plasma and Bronchoalveolar lavage fluid (BALF) cells in R213G mice compared to wild-type (WT) mice, while lung cellular superoxide levels in R213G mice were not elevated post-bleomycin compared to WT mice despite low lung EC-SOD levels. Lung glutathione redox potential (EhGSSG), determined by HPLC and fluorescence, was more oxidized in WT compared to R213G mice. In R213G mice, lung mitochondrial oxidative stress was reduced shown by mitochondrial superoxide level measured by EPR in lung and the resistance to bleomycin-induced cardiolipin oxidation. Bleomycin treatment suppressed mitochondrial respiration in WT mice. Mitochondrial function was impaired at baseline in R213G mice but did not exhibit further suppression in respiration post-bleomycin. Collectively, the results indicate that R213G variant preserves intracellular redox state and protects mitochondrial function in the setting of bleomycin-induced inflammation.

L-plastin enhances NLRP3 inflammasome assembly and bleomycin-induced lung fibrosis.

SUMMARYMacrophage adhesion and stretching have been shown to induce interleukin (IL)-1β production, but the mechanism of this mechanotransduction remains unclear. Here we specify the molecular link between mechanical tension on tissue-resident macrophages and activation of the NLRP3 inflammasome, which governs IL-1β production. NLRP3 activation enhances antimicrobial defense, but excessive NLRP3 activity causes inflammatory tissue damage in conditions such as pulmonary fibrosis and acute respiratory distress syndrome. We find that the actin-bundling protein L-plastin (LPL) significantly enhances NLRP3 assembly. Specifically, LPL enables apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) oligomerization during NLRP3 assembly by stabilizing ASC interactions with the kinase Pyk2, a component of cell-surface adhesive structures called podosomes. Upon treatment with exogenous NLRP3 activators, lung-resident alveolar macrophages (AMs) lacking LPL exhibit reduced caspase-1 activity, IL-1β cleavage, and gasdermin-D processing. LPL−/− mice display resistance to bleomycin-induced lung injury and fibrosis. These findings identify the LPL-Pyk2-ASC pathway as a target for modulation in NLRP3-mediated inflammatory conditions.

Effect of lung pericyte-like cell ablation on the bleomycin model of injury and repair.

We previously showed that pericyte-like cells derived from the FoxD1-lineage contribute to myofibroblasts following bleomycin-induced lung injury. However, their functional significance in lung fibrosis remains unknown. In this study, we used a model of lung pericyte-like cell ablation to test the hypothesis that pericyte-like cell ablation attenuates lung fibrosis in bleomycin-induced lung injury. Lung fibrosis was induced by intratracheal instillation of bleomycin. To ablate pericyte-like cells in the lung, diphtheria toxin (DT) was administered to Foxd1-Cre;Rosa26-iDTR mice at two different phases of bleomycin-induced lung injury. For early ablation, we coadministered bleomycin with DT and harvested mice at days 7 and 21. To test the effect of ablation after acute injury, we delivered DT 7 days after bleomycin administration. We assessed fibrosis by lung hydroxyproline content and semiquantitative analysis of picrosirius red staining. We performed bronchoalveolar lavage to determine cell count and differential. We also interrogated mRNA expression of fibrosis-related genes in whole lung RNA. Compared with DT-insensitive littermates where pericyte-like cells were not ablated, DT-sensitive animals exhibited no difference in fibrosis at day 21 both in the early and late pericyte ablation models. However, early ablation of pericytes reduced acute lung inflammation, as indicated by decreased inflammatory cells. Our data confirm a role for pericytes in regulating pulmonary inflammation in early lung injury.

Syndecan-2 regulates PAD2 to exert antifibrotic effects on RA-ILD fibroblasts

Rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD) is the most common pulmonary complication of RA, increasing morbidity and mortality. Anti-citrullinated protein antibodies have been associated with the development and progression of both RA and fibrotic lung disease; however, the role of protein citrullination in RA-ILD remains unclear. Here, we demonstrate that the expression of peptidylarginine deiminase 2 (PAD2), an enzyme that catalyzes protein citrullination, is increased in lung homogenates from subjects with RA-ILD and their lung fibroblasts. Chemical inhibition or genetic knockdown of PAD2 in RA-ILD fibroblasts attenuated their activation, marked by decreased myofibroblast differentiation, gel contraction, and extracellular matrix gene expression. Treatment of RA-ILD fibroblasts with the proteoglycan syndecan-2 (SDC2) yielded similar antifibrotic effects through regulation of PAD2 expression, phosphoinositide 3-kinase/Akt signaling, and Sp1 activation in a CD148-dependent manner. Furthermore, SDC2-transgenic mice exposed to bleomycin-induced lung injury in an inflammatory arthritis model expressed lower levels of PAD2 and were protected from the development of pulmonary fibrosis. Together, our results support a SDC2-sensitive profibrotic role for PAD2 in RA-ILD fibroblasts and identify PAD2 as a promising therapeutic target of RA-ILD.

Time-dependent effect of inhaled cigarette smoke exposure in the bleomycin-induced lung injury rat model.

Cigarette smoke (CS) substances are known to induce diverse ailments such as cancer, decreased immunity, and lung diseases. Although some studies have been actively conducted to evaluate cigarette toxicity, the current animal exposure methods, that is, exposure of 28- or 90-days, require considerable research cost and lead to obscure results of the CS effects. In a previous study, we compared the effects of CS in a rat model of bleomycin (BLM) and lipopolysaccharide (LPS) induced lung disease. We determined that compared to the LPS-induced rat model, the BLM-induced rat model was more sensitive to alterations in secreting cytokines and total cell number. In the current study, we further confirmed the time-point of effective inhalation exposure by CS in the BLM-induced lung injury rat model. Using an automatic video instillator, rats were administered a single dose of 2.5mg/kg BLM (day 1), and subsequently exposed to CS via inhalation (nose-only) 4h/day, for 1, 2, 3, and 4 weeks. The bronchoalveolar lavage fluid (BALF) was obtained from the right lung lobes, total cell numbers were counted, and chemokine and cytokine expressions were evaluated using Enzyme-Linked Immunosorbent Assay. For the 1-week exposure, we observed a greater increase of neutrophils in the BLM + CS 300 μg/L group than in the BLM or CS 300 μg/L groups. Exposure of CS in the BLM-induced lung injury rat model enhanced the secretions of chemokines and cytokines, such as CCL2/MCP-1, CXCL2/MIP-2 and TNF-α, at 1week. Immunohistochemistry and Hematoxylin and Eosin staining of lungs at 1-2 weeks after exposure clearly confirmed this tendency in the increased levels of CCL2/MCP-1 and TNF-α. Taken together, these results indicate that the rat model of BLM-induced lung injury is more sensitive to CS exposure than other rat models, and may be an appropriate model to evaluate the effect of CS exposure at 1-2 weeks.

Extracellular Superoxide Dismutase (EC-SOD) R213G variant reduces mitochondrial ROS and preserves mitochondrial function in bleomycin-induced lung injury

Extracellular Superoxide Dismutase (EC-SOD) R213G variant reduces mitochondrial ROS and preserves mitochondrial function in bleomycin-induced lung injury

Timeline of Multi-Organ Plasma Extravasation After Bleomycin-Induced Acute Lung Injury.

Acute lung injury (ALI) is characterized by the abrupt onset of clinically significant hypoxemia in the context of non-hydrostatic pulmonary edema. Acute lung injury is associated with cytokine release and plasma extravasation (PEx) that can cause pulmonary edema and subsequently acute respiratory distress syndrome (ARDS). Therefore, it is critical we understand the relationship between ALI and lung PEx. In addition, it is also important to assess PEx in the lungs and other organs post-ALI since ALI/ARDS often causes multi-organ failure. We hypothesized that ALI induces time-dependent lung PEx, which promotes extravasation in the heart, liver, kidney, spleen, pancreas, and gastrointestinal (GI) tract, in a time-dependent manner. To test our hypothesis, we administered bleomycin or saline via tracheal intubation in 8-week-old Sprague Dawley rats. At the terminal experiments, Evans Blue was injected (IV) through the femoral vein to allow for the visualization of PEx. Plasma extravasation of desired organs was evaluated at 3-, 7-, 14-, 21-, and 28-days after bleomycin or saline treatment by evaluating Evans Blue concentrations calorimetrically at fluorescence excitation wavelength of 620 nm (bandwidth 10 nm) and an emission wavelength of 680 nm (bandwidth 40 nm). Data show that ALI induces lung PEx beginning at day 3 and peaking between 7 and 21 days. Extravasation was also seen in all organs at varying degrees beginning at day 3 and peaking between days 7 and 14. Resolution appears to start after day 21 and continues past day 28. We conclude that ALI caused by bleomycin incites a time-dependent PEx of the lungs and multiple other organs.

Thrombospondin-1 Plays a Major Pathogenic Role in Experimental and Human Bronchopulmonary Dysplasia.

Rationale: Extremely preterm infants develop bronchopulmonary dysplasia (BPD), a chronic lung injury that lacks effective treatment. TSP-1 (thrombospondin-1) is an antiangiogenic protein that activates TGF-β1 (transforming growth factor-β1), a cytokine strongly linked to both experimental and human BPD. Objectives:1) To examine effects of inhibiting TSP-1-mediated TGF-β1 activation (LSKL [leucine-serine-lysine-leucine]) in neonatal rats with bleomycin-induced lung injury; 2) to examine effects of a TSP-1 mimic (ABT-510) on lung morphology; and 3) to determine whether TSP-1 and related signaling peptides are increased in lungs of human preterm infants at risk for BPD. Methods: From Postnatal Days 1 to 14, rat pups received daily intraperitoneal bleomycin (1 mg/kg) or vehicle and were treated with daily subcutaneous LSKL (20 mg/kg) or vehicle alone. Separate animals were treated with vehicle or ABT-510 (30 mg/kg/d). Paraffin-embedded lung tissues from 47 autopsies (controls; death <28 d, n = 30 and BPD at risk; death ⩾28 d, n = 17) performed on infants born <29 completed weeks' gestation were semiquantified for injury markers (collagen, macrophages, and 3-nitrotyrosine), TSP-1, and TGF-β1. Measurements and Main Results: Bleomycin or ABT-510 increased lung TGF-β1 activity and macrophage influx, caused pulmonary hypertension, and led to alveolar and microvascular hypoplasia. Treatment with LSKL partially prevented abnormal lung morphology secondary to bleomycin. Lungs from human infants at risk for BPD had increased contents of TSP-1 and TGF-β1 when compared with controls. TGF-β1 content correlated with markers of lung injury. Conclusions: TSP-1 inhibits alveologenesis in neonatal rats, in part via the upregulated activity of TGF-β1. Observations in human lungs suggest a similar pathogenic role for TSP-1 in infants at risk for BPD.