Noninvasive assessment of the lung inflammation-fibrosis axis by targeted imaging of CMKLR1.

Precision management of fibrotic lung diseases is challenging due to their diverse clinical trajectories and lack of reliable biomarkers for risk stratification and therapeutic monitoring. Here, we validated the accuracy of CMKLR1 as an imaging biomarker of the lung inflammation-fibrosis axis. By analyzing single-cell RNA sequencing datasets, we demonstrated CMKLR1 expression as a transient signature of monocyte-derived macrophages (MDMφ) enriched in patients with idiopathic pulmonary fibrosis (IPF). Consistently, we identified MDMφ as the major driver of the uptake of CMKLR1-targeting peptides in a murine model of bleomycin-induced lung fibrosis. Furthermore, CMKLR1-targeted positron emission tomography in the murine model enabled quantification and spatial mapping of inflamed lung regions infiltrated by CMKLR1-expressing macrophages and emerged as a robust predictor of subsequent lung fibrosis. Last, high CMKLR1 expression by bronchoalveolar lavage cells identified an inflammatory endotype of IPF with poor survival. Our investigation supports the potential of CMKLR1 as an imaging biomarker for endotyping and risk stratification of fibrotic lung diseases.

Assessment of pulmonary fibrosis induced by paraquat using Al18F-NODA-FAPI-04 PET/CT.

The lack of a highly sensitive method to evaluate paraquat (PQ)-induced pulmonary fibrosis and predict disease progression remains an unresolved clinic issue. Fibroblast activation protein (FAP) may play an important role in the pathogenesis of PQ-induced pulmonary fibrosis. We aimed to evaluate the role of FAP in the PQ-induced pulmonary fibrosis and the utility of fibroblast activation protein inhibitor (FAPI) for positron emission tomography (PET) imaging in PQ-induced pulmonary fibrosis. In our study, two cases of PQ poisoning were presented and FAPI PET/CT was performed as a novel imaging technique. The uptake of FAPI increased in both cases of PQ poisoning. Animal experiments were then performed to validate the findings in the patients. Physiological FAPI lung uptake was higher in mice of the PQ group than in the control group. The results of histological analysis and Western blot were consistent with the findings of PET/CT imaging. The pulmonary fibrosis animal model was developed by intragastric gavage of PQ. PET/CT imaging was performed after injection of FAPI. Lung tissues of mice were collected for fibrosis assessment after imaging. Immunohistochemistry for FAP, histology and Western blot for collagen were performed to further validate the imaging findings. In conclusion, FAPI was involved in the pathogenesis of fibrosis induced by PQ, and PET/CT with FAPI could detect lung fibrogenesis, making it a promising tool to assess early disease activity and predict disease progression.

MicroRNA-205-5p targets E2F1 to promote autophagy and inhibit pulmonary fibrosis in silicosis through impairing SKP2-mediated Beclin1 ubiquitination.

Silicosis is an occupational disease characterized by extensive pulmonary fibrosis, and the underlying pathological process remains uncertain. Herein, we explored the molecular mechanism by which microRNA-205-5p (miR-205-5p) affects the autophagy of alveolar macrophages (AMs) and pulmonary fibrosis in mice with silicosis through the E2F transcription factor 1 (E2F1)/S-phase kinase-associated protein 2 (SKP2)/Beclin1 axis. Alveolar macrophages (MH-S cells) were exposed to crystalline silica (CS) to develop an in vitro model, and mice were treated with CS to establish an in vivo model. Decreased Beclin1 and increased SKP2 and E2F1 were identified in mice with silicosis. We silenced or overexpressed miR-205-5p, E2F1, SKP2 and Beclin1 to investigate their potential roles in pulmonary fibrosis in vivo and autophagy in vitro. Recombinant adenovirus mRFP-GFP-LC3 was transduced into the MH-S cells to assay autophagic flow. Knocking down Beclin1 promoted pulmonary fibrosis and suppressed the autophagy. Co-immunoprecipitation and ubiquitination assays suggested that SKP2 induced K48-linked ubiquitination of Beclin1. Furthermore, chromatin immunoprecipitation-PCR revealed the site where E2F1 bound to the SKP2 promoter between 1638 bp and 1645 bp. As shown by dual-luciferase reporter gene assay, the transfection with miR-205-5p mimic inhibited the luciferase activity of the wild-type E2F1 3'untranslated region, suggesting that miR-205-5p targeted E2F1. Additionally, miR-205-5p overexpression increased autophagy and reduced the pulmonary fibrosis, while overexpression of E2F1 or SKP2 or inhibition of Beclin1 could annul this effect. The current study elucidated that miR-205-5p targeted E2F1, thereby inhibiting SKP2-mediated Beclin1 ubiquitination to promote macrophage autophagy and inhibit pulmonary fibrosis in mice with silicosis.

Inhibition of EZH2 prevents acute respiratory distress syndrome (ARDS)-associated pulmonary fibrosis by regulating the macrophage polarization phenotype.

BACKGROUND: We recently reported histone methyltransferase enhancer of zeste homolog 2 (EZH2) as a key epigenetic regulator that contributes to the dysfunction of innate immune responses to sepsis and subsequent lung injury by mediating the imbalance of macrophage polarization. However, the role of EZH2 in acute respiratory distress syndrome (ARDS)-associated fibrosis remains poorly understood. METHODS: In this study, we investigated the role and mechanisms of EZH2 in pulmonary fibrosis in a murine model of LPS-induced ARDS and in ex-vivo cultured alveolar macrophages (MH-S) and mouse lung epithelial cell line (MLE-12) by using 3-deazaneplanocin A (3-DZNeP) and EZH2 the small interfering (si) RNA. RESULTS: We found that treatment with 3-DZNeP significantly ameliorated the LPS-induced direct lung injury and fibroproliferation by blocking EMT through TGF-ß1/Smad signaling pathway and regulating shift of macrophage phenotypes. In the ex-vivo polarized alveolar macrophages cells, treatment with EZH2 siRNA or 3-DZNeP suppressed the M1 while promoted the M2 macrophage differentiation through modulating the STAT/SOCS signaling pathway and activating PPAR-γ. Moreover, we identified that blockade of EZH2 with 3-DZNeP suppressed the epithelial to mesenchymal transition (EMT) in co-cultured bronchoalveolar lavage fluid (BALF) and mouse lung epithelial cell line through down-regulation of TGF-ß1, TGF-ßR1, Smad2 while up-regulation of Smad7 expression. CONCLUSIONS: These results indicate that EZH2 is involved in the pathological process of ARDS-associated pulmonary fibrosis. Targeting EZH2 may be a potential therapeutic strategy to prevent and treat pulmonary fibrosis post ARDS.

Assessment of mustard vesicant lung injury and anti-TNF-α efficacy in rodents using live-animal imaging.

Nitrogen mustard (NM) causes acute lung injury, which progresses to fibrosis. This is associated with a macrophage-dominant inflammatory response and the production of proinflammatory/profibrotic mediators, including tumor necrosis factor alpha (TNF-α). Herein, we refined magnetic resonance imaging (MRI) and computed tomography (CT) imaging methodologies to track the progression of NM-induced lung injury in rodents and assess the efficacy of anti-TNF-α antibody in mitigating toxicity. Anti-TNF-α antibody was administered to rats (15 mg/kg, every 8 days, intravenously) beginning 30 min after treatment with phosphate-buffered saline control or NM (0.125 mg/kg, intratracheally). Animals were imaged by MRI and CT prior to exposure and 1-28 days postexposure. Using MRI, we characterized acute lung injury and fibrosis by quantifying high-signal lung volume, which represents edema, inflammation, and tissue consolidation; these pathologies were found to persist for 28 days following NM exposure. CT scans were used to assess structural components of the lung and to register changes in tissue radiodensities. CT scans showed that in control animals, total lung volume increased with time. Treatment of rats with NM caused loss of lung volume; anti-TNF-α antibody mitigated this decrease. These studies demonstrate that MRI and CT can be used to monitor lung disease and the impact of therapeutic intervention.

Ionizing Radiation Promotes Epithelial-to-Mesenchymal Transition in Lung Epithelial Cells by TGF-ß-producing M2 Macrophages.

BACKGROUND/AIM: Ionizing radiation induces pulmonary fibrosis, which is a common dose-limiting complication in patients receiving radiotherapy. Fibrosis occurs through the accumulation of large amounts of ECM components, synthesized by myofibroblasts in damaged lung tissue. Epithelial cells serve as one of the cellular sources of myofibroblasts via the epithelial-to-mesenchymal transition (EMT) process. In this study, we investigated the role of TGF-ß-secreting M2 macrophages in association with ionizing radiation-induced EMT. MATERIALS AND METHODS: The lung epithelial cell line MLE12, was irradiated and the expression of EMT markers and chemokines was examined. Moreover, the mouse lung macrophage MH-S cell line was cultured with conditioned media from irradiated MLE12 cells, to examine the effects of the secreted factors on the migration ability of macrophages. For the murine pulmonary fibrosis model, mice were locally irradiated and the levels of M1 or M2 macrophage-related markers and cytokines were measured in bronchoalvelolar lavage (BAL) fluid and lung tissue. RESULTS: In MLE12 cells, irradiation directly induced expression of EMT-related markers and secretion of various chemokines, which lead to macrophage migration. Interestingly, the sub-population of macrophages recruited in the lung of mice after thoracic irradiation was M2 macrophages that expressed Arg-1 and CD206. M2 macrophages induced the MLE12 to undergo phenotypic conversion to form fibroblast-like cells, which leads to a down-regulation of epithelial markers and an up-regulation of new EMT-related markers. In thoracic irradiated mice, pro-inflammatory cytokines such as IL-1ß, IL-4 and IL-10 were increased at 2 weeks, but returned to normal levels from 16 weeks or 24 weeks after irradiation. However, thoracic irradiation led to a rapid increase of TGF-ß and IGF-1 levels, which lasted up to 24 weeks. It was confirmed that M2 macrophages secreted the high levels of TGF-ß. Moreover, the elimination of TGF-ß from M2 macrophages attenuated mesenchymal transition of MLE12. CONCLUSION: TGF-ß-secreting M2 macrophages play an important regulatory role in mesenchymal transition of epithelial cells in the lung of irradiated mice, thus contributing to radiation-induced pulmonary fibrosis.

Lung Defense through IL-8 Carries a Cost of Chronic Lung Remodeling and Impaired Function.

IL-8-dependent inflammation is a hallmark of host lung innate immunity to bacterial pathogens, yet in many human lung diseases, including chronic obstructive pulmonary disease, bronchiectasis, and pulmonary fibrosis, there are progressive, irreversible, pathological changes associated with elevated levels of IL-8 in the lung. To better understand the duality of IL-8-dependent host immunity to bacterial infection and lung pathology, we expressed human IL-8 transgenically in murine bronchial epithelium, and investigated the impact of overexpression on lung bacterial clearance, host immunity, and lung pathology and function. Persistent IL-8 expression in bronchial epithelium resulted in neutrophilia, neutrophil maturation and activation, and chemotaxis. There was enhanced protection against challenge with Pseudomonas aeruginosa, and significant changes in baseline expression of innate and adaptive immunity transcripts for Ccl5, Tlr6, IL-2, and Tlr1. There was increased expression of Tbet and Foxp3 in response to the Pseudomonas antigen OprF, indicating a regulatory T-cell phenotype. However, this enhanced bacterial immunity came at a high price of progressive lung remodeling, with increased inflammation, mucus hypersecretion, and fibrosis. There was increased expression of Ccl3 and reduced expression of Claudin 18 and F11r, with damage to epithelial organization leading to leaky tight junctions, all of which resulted in impaired lung function with reduced compliance, increased resistance, and bronchial hyperreactivity as measured by whole-body plethysmography. These results show that IL-8 overexpression in the bronchial epithelium benefits lung immunity to bacterial infection, but specifically drives lung damage through persistent inflammation, lung remodeling, and damaged tight junctions, leading to impaired lung function.

Use of a pro-fibrogenic mechanism-based predictive toxicological approach for tiered testing and decision analysis of carbonaceous nanomaterials.

Engineered carbonaceous nanomaterials (ECNs), including single-wall carbon nanotubes (SWCNTs), multiwall carbon nanotubes (MWCNTs), graphene, and graphene oxide (GO), are potentially hazardous to the lung. With incremental experience in the use of predictive toxicological approaches, seeking to relate ECN physicochemical properties to adverse outcome pathways (AOPs), it is logical to explore the existence of a common AOP that allows comparative analysis of broad ECN categories. We established an ECN library comprising three different types of SWCNTs, graphene, and graphene oxide (two sizes) for comparative analysis according to a cell-based AOP that also plays a role in the pathogenesis of pulmonary fibrosis. SWCNTs synthesized by Hipco, arc discharge and Co-Mo catalyst (CoMoCAT) methods were obtained in their as-prepared (AP) state, following which they were further purified (PD) or coated with Pluronic F108 (PF108) or bovine serum albumin (BSA) to improve dispersal and colloidal stability. GO was prepared as two sizes, GO-small (S) and GO-large (L), while the graphene samples were coated with BSA and PF108 to enable dispersion in aqueous solution. In vitro screening showed that AP- and PD-SWCNTs, irrespective of the method of synthesis, as well as graphene (BSA) and GO (S and L) could trigger interleukin-1ß (IL-1ß) and transforming growth factor-ß1 (TGF-ß1) production in myeloid (THP-1) and epithelial (BEAS-2B) cell lines, respectively. Oropharyngeal aspiration in mice confirmed that AP-Hipco tubes, graphene (BSA-dispersed), GO-S and GO-L could induce IL-1ß and TGF-ß1 production in the lung in parallel with lung fibrosis. Notably, GO-L was the most pro-fibrogenic material based on rapid kinetics of pulmonary injury. In contrast, PF108-dispersed SWCNTs and -graphene failed to exert fibrogenic effects. Collectively, these data indicate that the dispersal state and surface reactivity of ECNs play key roles in triggering a pro-fibrogenic AOP, which could prove helpful for hazard ranking and a proposed tiered testing approach for large ECN categories.

Pulmonary biocompatibility assessment of inhaled single-wall and multiwall carbon nanotubes in BALB/c mice.

With the widespread application of carbon nanotubes (CNTs) in diverse commercial processes, scientists are now concerned about the potential health risk of occupational exposures. In this study, CNT-induced pulmonary toxicity was investigated by exposing BALB/c mice to aerosolized single-wall (SW) CNT and multiwall (MW) CNT (5 µg/g of mice) for 7 consecutive days in a nose-only exposure system. Microscopic studies showed that inhaled CNTs were homogeneously distributed in the mouse lung. The total number of bronchoalveolar lavage polymorphonuclear leukocytes recovered from the mice exposed to SWCNT and MWCNT (1.2 × 10(6) ± 0.52 and 9.87 × 10(5) ± 1.45; respectively) was significantly greater than control mice (5.46 × 10(5) ± 0.78). Rapid development of pulmonary fibrosis in mice that inhaled CNT was also confirmed by significant increases in the collagen level. The lactate dehydrogenase levels were increased nearly 2- and 2.4-fold in mice that inhaled SWCNT and MWCNT, respectively, as compared with control mice. In addition, exposure of CNTs to mice showed a significant (p < 0.05) reduction of antioxidants (glutathione, superoxide dismutase, and catalase) and induction of oxidants (myloperoxidase, oxidative stress, and lipid peroxidation) compared with control. Apoptosis-related proteins such as caspase-3 and -8 activities were also significantly increased in mice that inhaled CNT than in control mice. Together, this study shows that inhaled CNTs induce inflammation, fibrosis, alteration of oxidant and antioxidant levels, and induction of apoptosis-related proteins in the lung tissues to trigger cell death.

A novel method for accurate collagen and biochemical assessment of pulmonary tissue utilizing one animal.

AIM: The purpose of this study was to develop an improved method for collagen and protein assessment of fibrotic lungs while decreasing animal use. METHODS: 8-10 week old, male C57BL/6 mice were given a single intratracheal instillation of crocidolite asbestos or control titanium dioxide. Lungs were collected on day 14 and dried as whole lung, or homogenized in CHAPS buffer, for hydroxyproline analysis. Insoluble and salt-soluble collagen content was also determined in lung homogenates using a modified Sirius red colorimetric 96-well plate assay. RESULTS: The hydroxyproline assay showed significant increases in collagen content in the lungs of asbestos-treated mice. Identical results were present between collagen content determined on dried whole lung or whole lung homogenates. The Sirius red plate assay showed a significant increase in collagen content in lung homogenates however, this assay grossly over-estimated the total amount of collagen and underestimated changes between control and fibrotic lungs, conclusions: The proposed method provides accurate quantification of collagen content in whole lungs and additional homogenate samples for biochemical analysis from a single animal. The Sirius-red colorimetric plate assay provides a complementary method for determination of the relative changes in lung collagen but the values tend to overestimate absolute values obtained by the gold standard hydroxyproline assay and underestimate the overall fibrotic injury.

Resting energy expenditure in cryptogenic fibrosing alveolitis.

A proportion of patients with chronic airflow limitation appear to have a raised resting energy expenditure (REE). This has been suggested as the reason for weight loss which may occur in these patients. A previous study found an increased REE in patients with interstitial lung disease of mixed aetiology. We were interested in studying REE in a more homogeneous group, with cryptogenic fibrosing alveolitis (CFA). Twenty patients with CFA were studied. They were compared with 18 controls matched for age, sex, weight and height. REE was measured by indirect calorimetry. Fat-free mass (FFM), was estimated by anthropometry. Patients had respiratory function tests performed, disability related to breathlessness was assessed by the activity section of the St George's Respiratory Questionnaire. Mean REE in the CFA group was not different from the control group: 5.20 (0.56) versus 5.12 (0.51) kj x h(-1) x kgFFM(-1). REE was elevated to greater than 110% of the value predicted by the Harris-Benedict equation in one CFA patient and in no control subjects. There was no correlation of REE with weight, pulmonary function tests, arterial oxygen saturation or activity score. The prevalence of a raised resting energy expenditure in cryptogenic fibrosing alveolitis patients with low transfer factor and relatively preserved vital capacity is low, and is less than that reported previously in a group of patients with interstitial lung disease of mixed aetiology.

Radionuclide imaging in interstitial lung disease.

The use of radionuclides to evaluate interstitial lung disease (ILD) has a long history. In the mid- and late 1970 s, it was appreciated that gallium uptake was seen in the lungs of patients with sarcoidosis or idiopathic pulmonary fibrosis. In both of these conditions, the amount of uptake seemed to correlate with other measures of inflammation in the lung. Gallium uptake was shown to predict response to therapy and persistent disease. However, several limitations to gallium scanning soon became apparent. The procedure is costly, it requires 48 to 72 hours for proper interpretation, and the uptake reverses quickly during corticosteroid therapy. The use of aerosol scanning with 99mTc-pentetic acid (DTPA) has also been shown to be useful in ILD. The clearance of 99mTc-DTPA aerosol is markedly increased in both sarcoidosis and other inflammatory lung diseases. However, the limitations of 99mTc-DTPA include the fact that cigarette smoking will also cause increased clearance. Therefore, the value of 99mTc-DTPA scanning value seems to be limited to nonsmokers. Several recent papers published on these and other techniques are reviewed here.

Resting energy expenditure in interstitial lung disease.

Because interstitial lung disease increases the work of breathing, the aim of this study was to determine if this condition is associated with increased energy requirements. A group of 12 clinically stable patients with interstitial lung disease was studied. Patients with a history of weight loss had significantly more severe lung volume restriction. Regression analysis showed that 42% of body weight variation was explained by vital capacity (p less than 0.025). Resting energy expenditure was measured by standard methods of indirect calorimetry. The measurements were performed with a ventilated hood during prolonged steady-state periods after an overnight fast. We found that resting energy expenditure was increased to 117.3 and 118.7% of the predicted basal metabolic rate, according to Fleisch and to Harris and Benedict reference values, respectively (p less than 0.001). Furthermore, resting energy expenditure was increased to 120.8% of the predicted value according to body fat-free mass (p less than 0.001). This extra energy expenditure in patients with interstitial lung disease is similar to that recently reported in patients with chronic obstructive pulmonary disease.