COPD (chronic obstructive pulmonary disease) is caused by exposure to toxic gases and particles, most often CS (cigarette smoke), leading to emphysema, chronic bronchitis, mucus production and a ...subsequent decline in lung function. The disease pathogenesis is related to an abnormal CS-induced inflammatory response of the lungs. Similar to active (mainstream) smoking, second hand (sidestream) smoke exposure severely affects respiratory health. These processes can be studied in vivo in models of CS exposure of mice. We compared the acute inflammatory response of female C57BL/6 mice exposed to two concentrations 250 and 500 mg/m3 TPM (total particulate matter) of sidestream and mainstream CS for 3 days and interpreted the biological effects based on physico-chemical differences in the gas and particulate phase composition of CS. BAL (bronchoalveolar lavage fluid) was obtained to perform differential cell counts and to measure cytokine release. Lung tissue was used to determine mRNA and protein expression of proinflammatory genes and to assess tissue inflammation. A strong acute inflammatory response characterized by neutrophilic influx, increased cytokine secretion KC (keratinocyte chemoattractant), TNF-α (tumour necrosis factor α), MIP-2 (macrophage inflammatory protein 2), MIP-1α and MCP-1 (monocyte chemoattractant protein-1), pro-inflammatory gene expression KC, MIP-2 and MMP12 (matrix metalloproteinase 12) and up-regulated GM-CSF (granulocyte macrophage colony-stimulating factor) production was observed in the mainstream model. After sidestream exposure there was a dampened inflammatory reaction consisting only of macrophages and diminished GM-CSF levels, most likely caused by elevated CO concentrations. These results demonstrate that the composition of CS determines the dynamics of inflammatory cell recruitment in COPD mouse models. Different initial inflammatory processes might contribute to COPD pathogenesis in significantly varying ways, thereby determining the outcome of the studies.
Background and Purpose
Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/β‐catenin signalling is reduced in emphysema, and exogenous ...activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/β‐catenin signalling and assess its potential to activate lung epithelial cells and repair.
Experimental Approach
We screened 1216 human‐approved compounds for Wnt/β‐catenin signalling activation using luciferase reporter cells and selected candidates based on their computationally predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell‐derived lung organoids and in vivo using a murine elastase‐induced emphysema model.
Key Results
The primary screen identified 16 compounds that significantly induced Wnt/β‐catenin‐dependent luciferase activity. Selected compounds activated Wnt/β‐catenin signalling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/β‐catenin inhibition, confirming the Wnt/β‐catenin‐dependent mechanism of action. Amlexanox was used for in vivo evaluation, and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces pro‐regenerative signalling in emphysema.
Conclusion and Implications
Using a drug screen based on Wnt/β‐catenin activity, organoid assays and a murine emphysema model, amlexanox was identified as a novel potential therapeutic agent for emphysema.
Chronic obstructive lung disease determines morbidity and mortality of patients with cystic fibrosis (CF). CF airways are characterized by a nonresolving neutrophilic inflammation. After pathogen ...contact or prolonged activation, neutrophils release DNA fibres decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). NETs have been described to act in a beneficial way for innate host defense by bactericidal, fungicidal, and virucidal actions. On the other hand, excessive NET formation has been linked to the pathogenesis of autoinflammatory and autoimmune disease conditions. We quantified free DNA structures characteristic of NETs in airway fluids of CF patients and a mouse model with CF-like lung disease. Free DNA levels correlated with airflow obstruction, fungal colonization, and CXC chemokine levels in CF patients and CF-like mice. When viewed in combination, our results demonstrate that neutrophilic inflammation in CF airways is associated with abundant free DNA characteristic for NETosis, and suggest that free DNA may be implicated in lung function decline in patients with CF.
Chronic obstructive pulmonary disease (COPD) is characterized by a progressive decline in lung function, caused by exposure to exogenous particles, mainly cigarette smoke (CS). COPD is initiated and ...perpetuated by an abnormal CS-induced inflammatory response of the lungs, involving both innate and adaptive immunity. Specifically, B cells organized in iBALT structures and macrophages accumulate in the lungs and contribute to CS-induced emphysema, but the mechanisms thereof remain unclear. Here, we demonstrate that B cell-deficient mice are significantly protected against CS-induced emphysema. Chronic CS exposure led to an increased size and number of iBALT structures, and increased lung compliance and mean linear chord length in wild-type (WT) but not in B cell-deficient mice. The increased accumulation of lung resident macrophages around iBALT and in emphysematous alveolar areas in CS-exposed WT mice coincided with upregulated MMP12 expression. In vitro coculture experiments using B cells and macrophages demonstrated that B cell-derived IL-10 drives macrophage activation and MMP12 upregulation, which could be inhibited by an anti-IL-10 antibody. In summary, B cell function in iBALT formation seems necessary for macrophage activation and tissue destruction in CS-induced emphysema and possibly provides a new target for therapeutic intervention in COPD.
Transient receptor potential A1 (TRPA1) channels were originally characterized in neuronal tissues but also identified in lung epithelium by staining with fluorescently coupled TRPA1 antibodies. Its ...exact function in non-neuronal tissues, however, is elusive. TRPA1 is activated in vitro by hypoxia and hyperoxia and is therefore a promising TRP candidate for sensing hyperoxia in pulmonary epithelial cells and for inducing alveolar epithelial hyperplasia. Here, we isolated tracheal, bronchial, and alveolar epithelial cells and show low but detectable TRPA1 mRNA levels in all these cells as well as TRPA1 protein by Western blotting in alveolar type II (AT II) cells. We quantified changes in intracellular Ca
2+
(Ca
2+
i
) levels induced by application of hyperoxic solutions in primary tracheal epithelial, bronchial epithelial, and AT II cells isolated from wild-type (WT) and TRPA1-deficient (TRPA1−/−) mouse lungs. In all cell types, we detected hyperoxia-induced rises in Ca
2+
i
levels, which were not significantly different in TRPA1-deficient cells compared to WT cells. We also tested TRPA1 function in a mouse model for hyperoxia-induced alveolar epithelial hyperplasia. A characteristic significant increase in thickening of alveolar tissues was detected in mouse lungs after exposure to hyperoxia, but not in normoxic WT and TRPA1−/− controls. Quantification of changes in lung morphology in hyperoxic WT and TRPA1−/− mice, however, again revealed no significant changes. Therefore, TRPA1 expression does neither appear to be a key player for hyperoxia-induced changes in Ca
2+
i
levels in primary lung epithelial cells, nor being essential for the development of hyperoxia-induced alveolar epithelial hyperplasia.
Pulmonary fibrosis (PF) is a chronic progressive lung disease without effective medical treatment options leading to respiratory failure and death within 3–5years of diagnosis. The pathological ...process of PF is driven by aberrant wound-healing involving fibroblasts and myofibroblasts differentiated by secreted profibrotic transforming growth factor β (TGF-β1). Classical transient receptor potential 6 (TRPC6), a Na+- and Ca2+-permeable cation channel, is able to promote myofibroblast conversion of primary rat cardiac and human dermal fibroblasts and TRPC6-deficiency impaired wound healing after injury. To study a potential role of TRPC6 in the development of PF we analyzed lung function, gene and protein expression in wild-type (WT) and TRPC6-deficient (TRPC6−/−) lungs utilizing a bleomycin-induced PF-model. Fibrotic WT-mice showed a significant higher death rate while bleomycin-treated TRPC6-deficient mice were partly protected from fibrosis as a consequence of a lower production of collagen and an almost normal function of the respiratory system (reduced resistance and elastance compared to fibrotic WT-mice). On a molecular level TGF-β1 induced TRPC6 up-regulation, increased Ca2+ influx and nuclear NFAT localization in WT primary murine lung fibroblasts (PMLFs) resulting in higher stress fiber formation and accelerated contraction rates as compared to treated TRPC6-deficient fibroblasts. Therefore, we conclude that TRPC6 is an important determinant for TGF-β1-induced myofibroblast differentiation during fibrosis and specific channel inhibitors might be beneficial in a future treatment of PF.
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•TRPC6-/- mice show less collagen production and normal lung function in pulmonary fibrosis compared to wild-type controls.•TRPC6 expression is up-regulated in TGF-β1-induced lung fibroblast-myofibroblast transdifferentiation.•Lack of TRPC6 decreases cell contraction in lung fibroblasts.
We demonstrate the applicability of propagation-based X-ray phase-contrast imaging at a laser-assisted compact light source with known phantoms and the lungs and airways of a mouse. The Munich ...Compact Light Source provides a quasi-monochromatic beam with partial spatial coherence, and high flux relative to other non-synchrotron sources (up to 10
ph/s). In our study we observe significant edge-enhancement and quantitative phase-retrieval is successfully performed on the known phantom. Furthermore the images of a small animal show the potential for live bio-imaging research studies that capture biological function using short exposures.
Bronchiolitis obliterans syndrome (BOS) is a major complication after lung transplantation (LTx). BOS is characterised by massive peribronchial fibrosis, leading to air trapping-induced pulmonary ...dysfunction. Cathepsin B, a lysosomal cysteine protease, has been shown to enforce fibrotic pathways in several diseases. However, the relevance of cathepsin B in BOS progression has not yet been addressed. The aim of the study was to elucidate the function of cathepsin B in BOS pathogenesis.We determined cathepsin B levels in bronchoalveolar lavage fluid (BALF) and lung tissue from healthy donors (HD) and BOS LTx patients. Cathepsin B activity was assessed
a fluorescence resonance energy transfer-based assay and protein expression was determined using Western blotting, ELISA and immunostaining. To investigate the impact of cathepsin B in the pathophysiology of BOS, we used an
orthotopic left LTx mouse model. Mechanistic studies were performed
using macrophage and fibroblast cell lines.We found a significant increase of cathepsin B activity in BALF and lung tissue from BOS patients, as well as in our murine model of lymphocytic bronchiolitis. Moreover, cathepsin B activity was associated with increased biosynthesis of collagen and had a negative effect on lung function. We observed that cathepsin B was mainly expressed in macrophages that infiltrated areas characterised by a massive accumulation of collagen deposition. Mechanistically, macrophage-derived cathepsin B contributed to transforming growth factor-β1-dependent activation of fibroblasts, and its inhibition reversed the phenotype.Infiltrating macrophages release active cathepsin B, thereby promoting fibroblast activation and subsequent collagen deposition, which drive BOS. Cathepsin B represents a promising therapeutic target to prevent the progression of BOS.
Mechanical forces, which are crucial for most downstream signaling pathways in lung (patho-)physiology, may also regulate the efficacy of drugs. We investigated the role of mechanical forces on the ...effectiveness of inhaled and systemic (oral) administration of an anti-fibrosis drug. We established an induced triple coculture fibrosis model of a tight alveolar endothelial-epithelial barrier combined with pro-fibrotically stimulated primary fibroblasts derived from healthy donors and compared it to an analogous triple coculture model with fibroblasts from idiopathic pulmonary fibrosis (IPF) patients (innate IPF model). The 3D in vitro fibrosis models were established on a biomimetic, stretchable basement (BETA) membrane and cultured at an air-liquid interface (ALI). These fibrosis models were treated with an FDA-approved anti-fibrosis drug (oral and aerosolized application of Nintedanib) under static and dynamic culture conditions – including cyclic mechanical stretch and medium-flow induced shear stress – leveraging our advanced millifluidic CIVIC mini-lung technology. Fibrosis markers were characterized by protein and immunofluorescence analysis supplemented with real-time measurement of pulmonary compliance as a functional assay. Nintedanib shows more potent anti-inflammatory (IL1β, IL-6, and IL8) and anti-fibrotic (αSMA, soluble and deposited (type I) collagen, and compliance) effects on our IPF models under dynamic culture conditions for both delivery methods. Mechanotransduction enhanced the restoration of alveolar phenotypes after drug delivery, as indicated by surfactant protein C and Yes-associated protein levels. Our findings suggest that cyclic mechanical stretch plays a crucial role in the drug efficacy of Nintedanib. Albeit Nintedanib's anti-fibrotic and anti-inflammatory potency in both delivery routes is similar, the inhaled administration has a lower off-target dose fraction than oral application. Thus, inhaled Nintedanib showed a superior therapeutic index to systemic (oral) application.
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Chronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitis, small airway remodelling and emphysema. Emphysema is the destruction of alveolar structures, leading to enlarged ...airspaces and reduced surface area impairing the ability for gaseous exchange. To further understand the pathological mechanisms underlying progressive emphysema, we used MS-based approaches to quantify the lung, bronchoalveolar lavage fluid (BALF) and serum metabolome during emphysema progression in the established murine porcine pancreatic elastase (PPE) model on days 28, 56 and 161, compared with PBS controls. Partial least squares (PLS) analysis revealed greater changes in the metabolome of lung followed by BALF rather than serum during emphysema progression. Furthermore, we demonstrate for the first time that emphysema progression is associated with a reduction in lung-specific L-carnitine, a metabolite critical for transporting long-chain fatty acids into the mitochondria for their subsequent β-oxidation. In vitro, stimulation of the alveolar epithelial type II (ATII)-like LA4 cell line with L-carnitine diminished apoptosis induced by both PPE and H2O2. Moreover, PPE-treated mice demonstrated impaired lung function compared with PBS-treated controls (lung compliance; 0.067±0.008 ml/cmH20 compared with 0.035±0.005 ml/cmH20, P<0.0001), which improved following supplementation with L-carnitine (0.051±0.006, P<0.01) and was associated with a reduction in apoptosis. In summary, our results provide a new insight into the role of L-carnitine and, importantly, suggest therapeutic avenues for COPD.
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