Neutrophil serine proteases in cystic fibrosis: role in disease pathogenesis and rationale as a therapeutic target.

Chronic airway inflammation is a central feature in the pathogenesis of bronchiectasis (BE), which can be caused by cystic fibrosis (CFBE; hereafter referred to as CF lung disease) and non-CF-related conditions (NCFBE). Inflammation in both CF lung disease and NCFBE is predominantly driven by neutrophils, which release proinflammatory cytokines and granule proteins, including neutrophil serine proteases (NSPs). NSPs include neutrophil elastase, proteinase 3 and cathepsin G. An imbalance between NSPs and their antiproteases has been observed in people with CF lung disease and people with NCFBE. While the role of the protease/antiprotease imbalance is well established in both CF lung disease and NCFBE, effective therapies targeting NSPs are lacking. In recent years, the introduction of CF transmembrane conductance regulator (CFTR) modulator therapy has immensely improved outcomes in many people with CF (pwCF). Despite this, evidence suggests that airway inflammation persists, even in pwCF treated with CFTR modulator therapy. In this review, we summarise current data on neutrophilic inflammation in CF lung disease to assess whether neutrophilic inflammation and high, uncontrolled NSP levels play similar roles in CF lung disease and in NCFBE. We discuss similarities between the neutrophilic inflammatory profiles of people with CF lung disease and NCFBE, potentially supporting a similar therapeutic approach. Additionally, we present evidence suggesting that neutrophilic inflammation persists in pwCF treated with CFTR modulator therapy, at levels similar to those in people with NCFBE. Collectively, these findings highlight the ongoing need for new treatment strategies targeting neutrophilic inflammation in CF lung disease.

Understanding how methyltransferase-like 3 functions in lung diseases: From pathogenesis to clinical application.

Lung diseases have complex pathogenesis and treatment challenges, showing an obvious increase in the rate of diagnosis and death every year. Therefore, elucidating the mechanism for their pathogenesis and treatment ineffective from novel views is essential and urgent. Methyltransferase-like 3 (METTL3) is a novel post-transcriptional regulator for gene expression that has been implicated in regulating lung diseases, including that observed in chronic conditions such as pulmonary fibrosis (PF), pulmonary arterial hypertension (PAH), and chronic obstructive pulmonary disease (COPD), as well as acute conditions such as pneumonia, severe acute respiratory syndrome coronavirus 2 infection, and sepsis-induced acute respiratory distress syndrome. Notably, a comprehensive summary and analysis of findings from these studies might help understand lung diseases from the novel view of METTL3-regulated mechanism, however, such a review is still lacking. Therefore, this review aims to bridge such shortage by summarising the roles of METTL3 in lung diseases, establishing their interrelationships, and elucidating the potential applications of METTL3 regarding diagnosis, treatment, and prognosis. The analysis collectively suggests METTL3 is contributable to the onset and progression of these lung diseases, thereby prospecting METTL3 as a valuable biomarker for their diagnosis, treatment, and prognosis. In conclusion, this review offers elucidation into the correlation between METTL3 and lung diseases in both research and clinical settings and highlights potential avenues for exploring the roles of METTL3 in the respiratory system.

Anti-astmatic effect of ROCK inhibitor, GSK429286 A, in experimentally induced allergic airway inflammation.

BACKGROUND: Allergic asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness, inflammation and remodeling. ROCK inhibitors have now been shown to have the potential to alleviate these symptoms, although the specific effects of a new ROCK inhibitor, GSK429286 A, remain underexplored. OBJECTIVE: The aim of this study was to evaluate the therapeutic effects of a novel ROCK inhibitor, GSK429286 A, which exhibits a high affinity for both ROCK1 and ROCK2 isoforms, on allergic asthma in a guinea pig model, focusing on its effects on airway hyperresponsiveness, inflammation, and remodeling. METHODS: To induce allergic asthma, guinea pigs were sensitized with ovalbumin for 28 days, and in the middle of sensitization they were treated with different doses of the RoCK inhibitor, GSK429286 A. The study evaluated the effect of the administered doses on the reduction of airway hyperresponsiveness, by measuring specific airway resistance (sRaw), and the number of coughs after citric acid inhalation. We also monitored the anti-inflammatory effect by measuring levels of inflammatory cytokines, IL-2, IL-4, IL-5, IL-13, and remodeling markers, such as collagen deposition, and goblet cell hyperplasia. In addition, we monitored the possible anti-remodeling effect of GSK429286 A by histopathological examination. RESULTS: The ROCK inhibitor, GSK429286 A, showed an effect on suppressing airway hyperresponsiveness by reducing sRaw and the number of coughs in treated guinea pigs compared to controls. Our investigated drug suppressed the release of key mediators of inflammation, including IL-2, IL-4, and IL-5, thus demonstrating the effect of this ROCK inhibitor on the suppression of inflammation in the airways. Finally, GSK429286 A reduced markers of airway remodeling such as collagen deposition and goblet cell hyperplasia. CONCLUSION: GSK429286 A, an inhibitor of the ROCK pathway, exhibits significant anti-inflammatory and antiremodeling effects in a guinea pig model of allergic asthma. Indeed, we demonstrate its effect on suppressing airway hyperreactivity and reducing cough frequency. These findings suggest that GSK429286 A may be a promising therapeutic agent for allergic asthma, although further studies are needed to investigate its long-term efficacy, underlying mechanisms, and optimal dosing strategy.

The pH-sensitive translocation of V-ATPase in the small airway of cystic fibrosis pigs.

In contrast to pig large airways, the pH of airway surface liquid (ASL) in pig small airways is regulated by CFTR-mediated HCO-3 secretion and the vacuolar-type H+ ATPase (V-ATPase) proton secretion. We hypothesized that, in cystic fibrosis (CF), the ASL pH of small airways is acidic, and the V-ATPase is internalized. We quantified proton secretion during the addition of an alkaline test solution by measuring changes in a pH-dependent fluorescent dye generated by porcine small airway epithelia in the absence and presence of bafilomycin A1. The pH-dependent translocation of V-ATPase in ex vivo and in vivo preparations was measured using immunolocalization of V-ATPase. We found that bafilomycin-sensitive proton secretion stopped when the ASL pH was less than 7.10. In non-CF pigs and mice, we found that V-ATPase was localized in the apical membrane, and internalized when the lungs were instilled with a pH 6.8 solution. Studies in which we immediately fixed lungs from pigs revealed apical V-ATPase detection in non-CF piglets and less apical detection in CF piglets. Our data suggest that V-ATPase in small airways is internalized when the ASL pH is acidic. The decrease in apical localization of V-ATPase in CF pigs is consistent with an acidic ASL pH.NEW & NOTEWORTHY In this study, we describe that vacuolar-type H+ ATPase (V-ATPase) internalizes when the airway surface liquid (ASL) pH in pig small airways is less than 7.10. Furthermore, we found that V-ATPase is not localized to the apical membrane in the small airways of newborn cystic fibrosis pigs.

Effective-Component Compatibility of Bufei Yishen Formula III Suppresses Mitochondrial Oxidative Damage in COPD: Via Pkm2/Nrf2 Pathway.

Purpose: The main objective of this study was to explore the mechanism of effective component compatibility of Bufei Yishen formula III (ECC-BYF III) in inhibiting mitochondrial oxidative stress in a rat model of chronic obstructive pulmonary disease (COPD). Methods: A549 cells exposed to cigarette smoke extract (CSE) were used to establish a model of mitochondrial oxidative damage. The cells were treated with the plasmid encoding Pkm2 and the enzymes and proteins involved in oxidative stress and mitochondrial function were measured. A rat model of COPD was established using CS and bacteria. Two different treatments were established, ECC-BYF III (5.5 mg/kg/d) and N-acetylcysteine (54 mg/kg/day). Animals were tested for pulmonary function (Vt, PEF, FVC, FEV0.1s and Cdyn) after eight weeks of therapy and were sacrificed. Pulmonary H&E staining was performed, and the total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content were measured. The mitochondrial function was also examined. Furthermore, the Pkm2/Nrf2 signaling pathway was evaluated. Results: Overexpression of Pkm2 dramatically ameliorated the CS-induced mitochondrial oxidative damage. Further studies indicated that ECC-BYF III significantly improved mitochondrial function and inhibited oxidative stress in the lung tissues of COPD rats. Moreover, it can upregulate mitochondrial respiratory chain enzyme activity. ECC-BYF III also decreased the MDA content and increased T-SOD, GSH-Px, and T-AOC expression to facilitate oxidative homeostasis. Finally, our results indicated that the Pkm2/Nrf2 pathway is regulated by ECC-BYF III in A549 cells and lung tissue. Conclusion: These results indicate that ECC-BYF III exerts a strong effective therapeutic effect against cigarette smoke combined with bacteria-induced COPD in rats by activating the Pkm2/Nrf2 signaling pathway and restoring mitochondrial oxidative stress. Although more in vivo animal model research is needed to confirm these findings, this study contributes new data to support the conventional usage of ECC-BYF III.

Decreased Liver Kinase B1 Expression and Impaired Angiogenesis in a Murine Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is characterized by impaired lung alveolar and vascular growth. We investigated the hypothesis that neonatal exposure to hyperoxia leads to persistent BPD phenotype caused by decreased expression of liver kinase B1 (LKB1), a key regulator of mitochondrial function. We exposed mouse pups from Postnatal Day (P)1 through P10 to 21% or 75% oxygen. Half of the pups in each group received metformin or saline intraperitoneally from P1 to P10. Pups were killed at P4 or P10 or recovered in 21% O2 until euthanasia at P21. Lung histology and morphometry, immunofluorescence, and immunoblots were performed to detect changes in lung structure and expression of LKB1; downstream targets AMPK, PGC-1α, and electron transport chain (ETC) complexes; and Notch ligands Jagged 1 and delta-like 4. LKB1 signaling and in vitro angiogenesis were assessed in human pulmonary artery endothelial cells (exposed to 21% or 95% O2 for 36 hours. Levels of LKB1, phosphorylated AMPK, PGC-1α, and ETC complexes were decreased in lungs at P10 and P21 in hyperoxia. Metformin increased LKB1, phosphorylated AMPK, PGC-1α, and ETC complexes at P10 and P21 in pups exposed to hyperoxia. Radial alveolar count was decreased, and mean linear intercept increased in pups exposed to hyperoxia at P10 and P21; these were improved by metformin. Lung capillary density was decreased in hyperoxia at P10 and P21 and was increased by metformin. In vitro angiogenesis was decreased in human pulmonary artery endothelial cells by 95% O2 and was improved by metformin. Decreased LKB1 signaling may contribute to decreased alveolar and vascular growth in a mouse model of BPD.

Endothelial Chromatin-Remodeling Enzymes Regulate the Production of Critical ECM Components During Murine Lung Development.

BACKGROUND: The chromatin-remodeling enzymes BRG1 (brahma-related gene 1) and CHD4 (chromodomain helicase DNA-binding protein 4) independently regulate the transcription of genes critical for vascular development, but their coordinated impact on vessels in late-stage embryos has not been explored. METHODS: In this study, we genetically deleted endothelial Brg1 and Chd4 in mixed background mice (Brg1fl/fl;Chd4fl/fl;VE-Cadherin-Cre), and littermates that were negative for Cre recombinase were used as controls. Tissues were analyzed by immunostaining, immunoblot, and flow cytometry. Quantitative reverse transcription polymerase chain reaction was used to determine gene expression, and chromatin immunoprecipitation revealed gene targets of BRG1 and CHD4 in cultured endothelial cells. RESULTS: We found Brg1/Chd4 double mutants grew normally but died soon after birth with small and compact lungs. Despite having normal cellular composition, distal air sacs of the mutant lungs displayed diminished ECM (extracellular matrix) components and TGFß (transforming growth factor-ß) signaling, which typically promotes ECM synthesis. Transcripts for collagen- and elastin-related genes and the TGFß ligand Tgfb1 were decreased in mutant lung endothelial cells, but genetic deletion of endothelial Tgfb1 failed to recapitulate the small lungs and ECM defects seen in Brg1/Chd4 mutants. We instead found several ECM genes to be direct targets of BRG1 and CHD4 in cultured endothelial cells. CONCLUSIONS: Collectively, our data highlight essential roles for endothelial chromatin-remodeling enzymes in promoting ECM deposition in the distal lung tissue during the saccular stage of embryonic lung development.

New insight on porcine carboxylesterases expression and activity in lung tissues.

Over the course of the last twenty years, there has been a growing recognition of the pig's potential as a valuable model for studying human drug metabolism. This study aimed to investigate the expression, enzymatic activity, inhibitory susceptibility, and cellular localization of carboxylesterases (CES) in porcine lung tissue not yet explored. Our results showed that CESs hydrolysis activity followed Michaelis-Menten kinetics in both cytosolic and microsomal fractions of porcine lung tissues (N = 8), with comparable hydrolysis rates for tested substrates, namely 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD). We also determined the CESs hydrolysis activity in a representative sample of the porcine liver that, as expected, displayed higher activity than the lung ones. The study demonstrated variable levels of enzyme activities and interindividual variability in both porcine lung fractions. Inhibition studies used to assess the CESs' involvement in the hydrolysis of pNPA, 4-MUA, and FD suggested that CESs may be the enzymes primarily involved in the metabolism of ester compounds in the pig lung tissue. Overall, this study provides insight into the distribution and diversity of CES isoforms involved in substrate hydrolysis across different cellular fractions (cytosol and microsomes) in porcine lungs.

JAK2 inhibitors improve RA combined with pulmonary fibrosis in rats by downregulating SMAD3 phosphorylation.

BACKGROUND: JAK inhibitors are well known for the treatment of rheumatoid arthritis (RA), but whether they can be used to treat pulmonary fibrosis, a common extra-articular disease of RA, remains to be clarified. METHODS: A jak2 inhibitor, CEP33779 (CEP), was administered to a rat model of RA-associated interstitial lung disease to observe the degree of improvement in both joint swelling and pulmonary fibrosis. HFL1 cells were stimulated with TGF-ß1 to observe the expression of p-JAK2. Then, different concentrations of related gene inhibitors (JAK2, TGFß-R1/2, and p-STAT3) or silencers (STAT3, JAK2) were administered to HFL1 cells, and the expression levels of related proteins were detected to explore the underlying mechanisms of action. RESULTS: CEP not only reduced the degree of joint swelling and inflammation in rats but also improved lung function, inhibited the pro-inflammatory factors IL-1ß and IL-6, reduced lung inflammation and collagen deposition, and alleviated lung fibrosis. CEP decreased the expression levels of TGFß-R2, p-SMAD, p-STAT3, and ECM proteins in rat lung tissues. TGF-ß1 induced HFL1 cells to highly express p-JAK2, with the most pronounced expression at 48 h. The levels of p-STAT3, p-SMAD3, and ECM-related proteins were significantly reduced after inhibition of either JAK2 or STAT3. CONCLUSION: JAK2 inhibitors may be an important and novel immunotherapeutic drug that can improve RA symptoms while also delaying or blocking the development of associated pulmonary fibrotic disease. The mechanism may be related to the downregulation of p-STAT3 protein via inhibition of the JAK2/STAT signaling pathway, which affects the phosphorylation of SMAD3.

PI3Kγδ inhibition suppresses key disease features in a rat model of asthma.

BACKGROUND: Two isoforms of Phosphoinositide 3-kinase (PI3K), p110γ and p110δ, are predominantly expressed in leukocytes and represent attractive therapeutic targets for the treatment of allergic asthma. The study aim was to assess the impact of administration of an inhaled PI3Kγδ inhibitor (AZD8154) in a rat model of asthma. METHODS: Firstly, we checked that the tool compound, AZD8154, inhibited rat PI3K γ & δ kinases using rat cell-based assays. Subsequently, a time-course study was conducted in a rat model of asthma to assess PI3K activity in the lung and how it is temporally associated with other key transcription pathways and asthma like features of the model. Finally, the impact on lung dosed AZD8154 on target engagement, pathway specificity, airway inflammation and lung function changes was assessed. RESULTS: Data showed that AZD8154 could inhibit rat PI3K γ & δ isoforms and, in a rat model of allergic asthma the PI3K pathway was activated in the lung. Intratracheal administration of AZD8154 caused a dose related suppression PI3K pathway activation (reduction in pAkt) and unlike after budesonide treatment, STAT and NF-κB pathways were not affected by AZD8154. The suppression of the PI3K pathway led to a marked inhibition of airway inflammation and reduction in changes in lung function. CONCLUSION: These data show that a dual PI3Kγδ inhibitor suppress key features of disease in a rat model of asthma to a similar degree as budesonide and indicate that dual PI3Kγδ inhibition may be an effective treatment for people suffering from allergic asthma.

Treatment with bestatin (the exogenous synthetic inhibitor of metalloproteinases) reduces the activity of metalloproteinase 2 and 12 in the spleen and lung tissues of rats in a model of lipopolysaccharide-induced sepsis.

Sepsis is caused by an inadequate or dysregulated host response to infection. Enzymes causing cellular degradation are matrix metalloproteinases (MMPs). Lipopolysaccharide (LPS) is used in models of sepsis in laboratory settings The aim of the study was to measure MMP 2 and 12 concentrations in spleen and lungs in rats in which septic shock was induced by LPS. The experiment was carried out on 40 male Wistar rats (5 groups of 8): 0. controls 1. administered LPS 2. administered bestatin 3. LPS and bestatin 4.bestatin and after 6 hours LPS Animals were decapitated. Lungs and spleens were collected. Concentrations of MMP-2 and MMP-12 were determined using immunoenzymatic methods. Mean (±SD) MMP-2 in the controls was 43.57 ± 20.53 ng/ml in the lungs and 1.7 ± 0.72 ng/ml in the spleen; Group 1: 31.28 ± 13.13 ng/ml, 0.83 ± 0.8 ng/ml; Group 2: 44.24 ± 22.75 ng /ml, 1.01 ± 0.32 ng/ml; Group 3: 35.94 ± 15.13 ng/ml, 0.41 ± 0.03 ng/ml; Group 4:79.42 ± 44.70 ng/ml, 0.45 ± 0.15, respectively. Mean MMP-12 in controls was 19.79 ± 10.01 ng/ml in lungs and 41.13 ± 15.99 ng/ml in the spleen; Group 1:27.97 ± 15.1 ng/ml; 40.44 ± 11.2 ng/ml; Group 2: 37.93 ± 25.38 ng/ml 41.05 ± 18.08 ng/ml; Group 3: 40.59 ± 11.46 ng/ml, 35.16 ± 12.89 ng/ml; Group 4: 39.4 ± 17.83 ng/ml, 42.04 ± 12.35 ng/ml, respectively. CONCLUSIONS: 1. Bestatin reduces MMP 2 and 12 levels in spleen and lungs. 2. Treatment with bestatin minimizes the effect of LPS.

Role of lung ornithine aminotransferase in idiopathic pulmonary fibrosis: regulation of mitochondrial ROS generation and TGF-ß1 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-ß1 (TGF)-ß1 activity and TGF-ß1 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-ß1 signaling. OAT may be a useful target for treating patients with fibrotic lung diseases, including IPF.

WNT/RYK signaling functions as an antiinflammatory modulator in the lung mesenchyme.

A number of inflammatory lung diseases, including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and pneumonia, are modulated by WNT/ß-catenin signaling. However, the underlying molecular mechanisms remain unclear. Here, starting with a forward genetic screen in mouse, we identify the WNT coreceptor Related to receptor tyrosine kinase (RYK) acting in mesenchymal tissues as a cell survival and antiinflammatory modulator. Ryk mutant mice exhibit lung hypoplasia and inflammation as well as alveolar simplification due to defective secondary septation, and deletion of Ryk specifically in mesenchymal cells also leads to these phenotypes. By analyzing the transcriptome of wild-type and mutant lungs, we observed the up-regulation of proapoptotic and inflammatory genes whose expression can be repressed by WNT/RYK signaling in vitro. Moreover, mesenchymal Ryk deletion at postnatal and adult stages can also lead to lung inflammation, thus indicating a continued role for WNT/RYK signaling in homeostasis. Our results indicate that RYK signaling through ß-catenin and Nuclear Factor kappa B (NF-κB) is part of a safeguard mechanism against mesenchymal cell death, excessive inflammatory cytokine production, and inflammatory cell recruitment and accumulation. Notably, RYK expression is down-regulated in the stromal cells of pneumonitis patient lungs. Altogether, our data reveal that RYK signaling plays critical roles as an antiinflammatory modulator during lung development and homeostasis and provide an animal model to further investigate the etiology of, and therapeutic approaches to, inflammatory lung diseases.

Automated high-throughput in vitro assays to identify metabolic hotspots and protease stability of structurally diverse, pharmacologically active peptides for inhalation.

The inhalation of peptides comes with the advantage of directly targeting the lung as tissue of interest. However, peptides are often rapidly metabolized in lung tissue through proteolytic cleavage. We have developed an assay workflow to obtain half-life and metabolite ID data for peptides incubated with four proteases abundant in lungs of asthma and COPD patients. The assay system has been validated using 28 structurally diverse linear and cyclic peptides with a molecular weight between 708 and 5808 Da. Experimental conditions for incubation, sample preparation, chromatography, data acquisition and analysis are compatible with the required throughput in early stage peptide projects. Together with co-crystal structures and Ala scans, we are using the described assay workflow to guide the first chemical modifications of peptide hits in early respiratory drug discovery projects.

Identification of targetable kinases in idiopathic pulmonary fibrosis.

BACKGROUND: Tyrosine kinase activation plays an important role in the progression of pulmonary fibrosis. In this study, we analyzed the expression of 612 kinase-coding and cancer-related genes using next-generation sequencing to identify potential therapeutic targets for idiopathic pulmonary fibrosis (IPF). METHODS: Thirteen samples from five patients with IPF (Cases 1-5) and eight samples from four patients without IPF (control) were included in this study. Six of the thirteen samples were obtained from different lung segments of a single patient who underwent bilateral pneumonectomy. Gene expression analysis of IPF lung tissue samples (n = 13) and control samples (n = 8) was performed using SureSelect RNA Human Kinome Kit. The expression of the selected genes was further confirmed at the protein level by immunohistochemistry (IHC). RESULTS: Gene expression analysis revealed a correlation between the gene expression signatures and the degree of fibrosis, as assessed by Ashcroft score. In addition, the expression analysis indicated a stronger heterogeneity among the IPF lung samples than among the control lung samples. In the integrated analysis of the 21 samples, DCLK1 and STK33 were found to be upregulated in IPF lung samples compared to control lung samples. However, the top most upregulated genes were distinct in individual cases. DCLK1, PDK4, and ERBB4 were upregulated in IPF case 1, whereas STK33, PIM2, and SYK were upregulated in IPF case 2. IHC revealed that these proteins were expressed in the epithelial layer of the fibrotic lesions. CONCLUSIONS: We performed a comprehensive kinase expression analysis to explore the potential therapeutic targets for IPF. We found that DCLK1 and STK33 may serve as potential candidate targets for molecular targeted therapy of IPF. In addition, PDK4, ERBB4, PIM2, and SYK might also serve as personalized therapeutic targets of IPF. Additional large-scale studies are warranted to develop personalized therapies for patients with IPF.

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

Cyclooxygenase-2 deficiency attenuates lipopolysaccharide-induced inflammation, apoptosis, and acute lung injury in adult mice.

Many lung diseases are caused by an excessive inflammatory response, and inflammatory lung diseases are often modeled using lipopolysaccharide (LPS) in mice. Cyclooxygenase-2 (COX-2) encoded by the Ptgs2 gene is induced in response to inflammatory stimuli including LPS. The objective of this study was to test the hypothesis that mice deficient in COX-2 (Ptgs2-/-) will be protected from LPS-induced lung injury. Wild-type (WT; CD1 mice) and Ptgs2-/- mice (on a CD1 background) were treated with LPS or vehicle for 24 h. LPS treatment resulted in histological evidence of lung injury, which was attenuated in the Ptgs2-/- mice. LPS treatment increased the mRNA levels for tumor necrosis factor-α, interleukin-10, and monocyte chemoattractant protein-1 in the lungs of WT mice, and the LPS-induced increases in these levels were attenuated in the Ptgs2-/- mice. The protein levels of active caspase-3 and caspase-9 were lower in the LPS-treated lungs of Ptgs2-/- mice than in LPS-treated WT mice, as were the number of terminal deoxynucleotide transferase dUTP nick end labeling-positive cells in lung sections. LPS exposure resulted in a greater lung wet-to-dry weight ratio (W/D) in WT mice, suggestive of pulmonary edema, while in LPS-treated Ptgs2-/- mice, the W/D was not different from controls and less than in LPS-treated WT mice. These results demonstrate that COX-2 is involved in the inflammatory response to LPS and suggest that COX-2 not only acts as a downstream participant in the inflammatory response, but also acts as a regulator of the inflammatory response likely through a feed-forward mechanism following LPS stimulation.

Lysyl oxidase regulation and protein aldehydes in the injured newborn lung.

During newborn lung injury, excessive activity of lysyl oxidases (LOXs) disrupts extracellular matrix (ECM) formation. Previous studies indicate that TGFß activation in the O2-injured mouse pup lung increases lysyl oxidase (LOX) expression. But how TGFß regulates this, and whether the LOXs generate excess pulmonary aldehydes are unknown. First, we determined that O2-mediated lung injury increases LOX protein expression in TGFß-stimulated pup lung interstitial fibroblasts. This regulation appeared to be direct; this is because TGFß treatment also increased LOX protein expression in isolated pup lung fibroblasts. Then using a fibroblast cell line, we determined that TGFß stimulates LOX expression at a transcriptional level via Smad2/3-dependent signaling. LOX is translated as a pro-protein that requires secretion and extracellular cleavage before assuming amine oxidase activity and, in some cells, reuptake with nuclear localization. We found that pro-LOX is processed in the newborn mouse pup lung. Also, O2-mediated injury was determined to increase pro-LOX secretion and nuclear LOX immunoreactivity particularly in areas populated with interstitial fibroblasts and exhibiting malformed ECM. Then, using molecular probes, we detected increased aldehyde levels in vivo in O2-injured pup lungs, which mapped to areas of increased pro-LOX secretion in lung sections. Increased activity of LOXs plays a critical role in the aldehyde generation; an inhibitor of LOXs prevented the elevation of aldehydes in the O2-injured pup lung. These results reveal new mechanisms of TGFß and LOX in newborn lung disease and suggest that aldehyde-reactive probes might have utility in sensing the activation of LOXs in vivo during lung injury.

Indoleamine 2,3-dioxygenase (IDO)-1 and IDO-2 activity and severe course of COVID-19.

COVID-19 is a pandemic with high morbidity and mortality. In an autopsy cohort of COVID-19 patients, we found extensive accumulation of the tryptophan degradation products 3-hydroxy-anthranilic acid and quinolinic acid in the lungs, heart, and brain. This was not related to the expression of the tryptophan-catabolizing indoleamine 2,3-dioxygenase (IDO)-1, but rather to that of its isoform IDO-2, which otherwise is expressed rarely. Bioavailability of tryptophan is an absolute requirement for proper cell functioning and synthesis of hormones, whereas its degradation products can cause cell death. Markers of apoptosis and severe cellular stress were associated with IDO-2 expression in large areas of lung and heart tissue, whereas affected areas in brain were more restricted. Analyses of tissue, cerebrospinal fluid, and sequential plasma samples indicate early initiation of the kynurenine/aryl-hydrocarbon receptor/IDO-2 axis as a positive feedback loop, potentially leading to severe COVID-19 pathology. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Exosomal miR-150 partially attenuated acute lung injury by mediating microvascular endothelial cells and MAPK pathway.

BACKGROUND: Acute lung injury (ALI) is a respiratory disease with high morbidity and mortality rates. Currently, there is no effective treatment to complement mechanical ventilation. Exosomes and microRNAs (miRNAs) are promising agents for the management of this disease. METHODS: Exosomes were isolated from mouse bone marrow stromal stem cells (BMSCs). The levels of two miRNAs, miR-542-3P and miR-150, in exosomes were determined using RT-PCR, and miR-150 was selected for further study. ALI model was established in mice using lipopolysaccharides, and then, they were treated with saline, exosomes, miRNA agomirs, or miRNA antagomirs. The concentrations of TNF-α, IL-6, and IL-1ß and the number of neutrophils and macrophages in the bronchoalveolar lavage fluid were measured. The wet/dry weight ratio of the lung tissue was calculated, and tissue pathology and apoptosis were observed using hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. CD34 and VE-cadherin expression was detected using immunofluorescence. Proteins associated with apoptosis and MAPK signaling were detected using Western blotting, and miR-150 expression in lung tissue was evaluated using RT-PCR. RESULTS: We successfully isolated BMSCs and exosomes and showed that the level of miR-150 was significantly higher than that of miR-542-3p. Exosomes and miR-150 reduced inflammation and lung edema while maintaining the integrity of the alveolar structure. They also mitigated microvascular endothelial cell injury by regulating the caspase-3, Bax/Bcl-2, and MAPK signaling. CONCLUSIONS: Exosomal miR-150 attenuates lipopolysaccharide-induced ALI through the MAPK pathway.