Human Lung Biopsies Research Articles

Pseudomonas aeruginosa Infection and Inflammation in Cystic Fibrosis: A Pilot Study With Lung Explants and a Novel Histopathology Scoring System.

Pseudomonas aeruginosa is the predominant bacterial pathogen colonizing the cystic fibrosis (CF) lung. Mixed populations of nonmucoid and mucoid variants of P. aeruginosa have been isolated from the CF airway. While the association between mucoid variants and pulmonary function decline is well-established, their impact on inflammation and tissue damage in advanced CF lung disease remains unclear. This pilot study utilized 1 non-CF and 3 CF lung explants to examine lobar distribution, inflammation, and histopathology related to nonmucoid and mucoid P. aeruginosa infection. To study tissue damage, we developed a novel lung histopathology scoring system, the first applied to human CF lung biopsies, which is comprised of five indicators: bronchiolar epithelial infiltrate, luminal inflammation, peribronchial/bronchiolar infiltrate, peribronchiolar fibrosis, and alveolar involvement. Mucoid P. aeruginosa variants were distributed throughout the CF lung but associated with greater concentrations of proinflammatory cytokines, IL-1β, TNF-α, IL-6, IL-8, and IFN-γ, and one anti-inflammatory cytokine, IL-10, compared to nonmucoid variants. CF lung explants exhibited higher histopathology scores compared to a non-CF lung control. In mixed-variant infection, nonmucoid constituents associated with increased bronchiolar epithelial infiltration, one indicator of histopathology. This pilot study suggests ongoing interplay between host and bacterial elements in late-stage CF pulmonary disease. Mucoid P. aeruginosa infection correlates with inflammation regardless of lung lobe, whereas nonmucoid P. aeruginosa is associated with increased inflammatory cell infiltration. The development of a novel lung histopathology scoring system lays the groundwork for future large-cohort investigations.

Liproxstatin-1 Alleviates Lung Transplantation-induced Cold Ischemia-Reperfusion Injury by Inhibiting Ferroptosis.

Primary graft dysfunction, which is directly related to cold ischemia-reperfusion (CI/R) injury, is a major obstacle in lung transplantation (LTx). Ferroptosis, a novel mode of cell death elicited by iron-dependent lipid peroxidation, has been implicated in ischemic events. This study aimed to investigate the role of ferroptosis in LTx-CI/R injury and the effectiveness of liproxstatin-1 (Lip-1), a ferroptosis inhibitor, in alleviating LTx-CI/R injury. LTx-CI/R-induced signal pathway alterations, tissue injury, cell death, inflammatory responses, and ferroptotic features were examined in human lung biopsies, the human bronchial epithelial (BEAS-2B) cells, and the mouse LTx-CI/R model (24-h CI/4-h R). The therapeutic efficacy of Lip-1 was explored and validated both in vitro and in vivo. In human lung tissues, LTx-CI/R activated ferroptosis-related signaling pathway, increased the tissue iron content and lipid peroxidation accumulation, and altered key protein (GPX4, COX2, Nrf2, and SLC7A11) expression and mitochondrial morphology. In BEAS-2B cells, the hallmarks of ferroptosis were significantly evidenced at the setting of both CI and CI/R compared with the control, and the effect of adding Lip-1 only during CI was much better than that of only during reperfusion by Cell Counting Kit-8. Furthermore, Lip-1 administration during CI markedly relieved LTx-CI/R injury in mice, as indicated by significant improvement in lung pathological changes, pulmonary function, inflammation, and ferroptosis. This study revealed the existence of ferroptosis in the pathophysiology of LTx-CI/R injury. Using Lip-1 to inhibit ferroptosis during CI could ameliorate LTx-CI/R injury, suggesting that Lip-1 administration might be proposed as a new strategy for organ preservation.

Shedding LIGHT and TL1A on Pulmonary Fibrosis

Abstract Currently, anti-inflammatory drugs fail to reduce pulmonary fibrosis (PF) and tissue remodeling in the clinic. Despite the advancements made in understanding the molecular pathways driving PF, there is still an unmet need to develop innovative and effective anti-fibrotic drugs. Our lab has identified two novel mediators of fibrosis belonging to the tumor necrosis factor superfamily (TNFSF), LIGHT (TNFSF14) and TL1A (TNFSF15). Using a well characterized mouse model of PF driven by bleomycin, we showed that blocking LIGHT or TL1A signaling in isolation, by genetic ablation or antibody-mediated neutralization, significantly reduces PF. In gain of function studies, the in vivo administration of LIGHT or TL1A in isolation recapitulated fibrosis symptoms reminiscent of human disease. Furthermore, our data suggest that LIGHT promotes thymic stromal lymphopoietin (TLSP) expression by epithelial cells, whereas TL1A activates fibroblasts to promote periostin expression. Both TSLP and periostin are drivers of PF. Importantly, we observed an upregulation of LIGHT and DR3 (the receptor for TL1A) in human lung biopsies of patients suffering SSc and IPF. We have found that LIGHT and TL1A can drive fibrosis independently of one another and that the in vivo co-administration of these TNFSF members synergize to maximize inflammation and collagen deposition. Using antagonistic fusion proteins to neutralize LIGHT and TL1A signaling, we will monitor PF in vivo and check whether greater efficacy is achieved when LIGHT and TL1A are targeted concomitantly post-disease onset. The relevance of this work on PF associated with ILDs in humans is tremendous.

Role of pulmonary epithelial arginase-II in activation of fibroblasts and lung inflammaging.

Elevated arginases including type-I (Arg-I) and type-II isoenzyme (Arg-II) are reported to play a role in aging, age-associated organ inflammaging, and fibrosis. A role of arginase in pulmonary aging and underlying mechanisms are not explored. Our present study shows increased Arg-II levels in aging lung of female mice, which is detected in bronchial ciliated epithelium, club cells, alveolar type 2 (AT2) pneumocytes, and fibroblasts (but not vascular endothelial and smooth muscle cells). Similar cellular localization of Arg-II is also observed in human lung biopsies. The age-associated increase in lung fibrosis and inflammatory cytokines, including IL-1β and TGF-β1 that are highly expressed in bronchial epithelium, AT2 cells, and fibroblasts, are ameliorated in arg-ii deficient (arg-ii-/- ) mice. The effects of arg-ii-/- on lung inflammaging are weaker in male as compared to female animals. Conditioned medium (CM) from human Arg-II-positive bronchial and alveolar epithelial cells, but not that from arg-ii-/- cells, activates fibroblasts to produce various cytokines including TGF-β1 and collagen, which is abolished by IL-1β receptor antagonist or TGF-β type I receptor blocker. Conversely, TGF-β1 or IL-1β also increases Arg-II expression. In the mouse models, we confirmed the age-associated increase in IL-1β and TGF-β1 in epithelial cells and activation of fibroblasts, which is inhibited in arg-ii-/- mice. Taken together, our study demonstrates a critical role of epithelial Arg-II in activation of pulmonary fibroblasts via paracrine release of IL-1β and TGF-β1, contributing to pulmonary inflammaging and fibrosis. The results provide a novel mechanistic insight in the role of Arg-II in pulmonary aging.

Mapping of functional SARS-CoV-2 receptors in human lungs establishes differences in variant binding and SLC1A5 as a viral entry modulator of hACE2.

The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated invitro, the relationship between hACE2 expression and invivo infection is less understood. Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both invitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. We propose a method for mapping functional SARS-CoV-2 receptors invivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. This work was supported by anunrestricted grant from"Fondazione Romeo ed Enrica Invernizzi" to Stefano Biffo and byAIRC under MFAG 2021 - ID. 26178 project - P.I. Manfrini Nicola.

HIF1A-dependent induction of alveolar epithelial PFKFB3 dampens acute lung injury.

Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us toward a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxP/loxP SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid instillation. Studies in genetic models linked Pfkfb3 expression and function to Hif1a. Not only did intratracheal pyruvate instillation reconstitute Pfkfb3loxP/loxP or Hif1aloxP/loxP SPC-ER-Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies demonstrated increased PFKFB3 staining in injured lungs and colocalized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counterbalancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI.

Comprehensive Analysis of Fibroblast Activation Protein Expression in Interstitial Lung Diseases.

Rationale: Sustained activation of lung fibroblasts and the resulting oversynthesis of the extracellular matrix are detrimental events for patients with interstitial lung diseases (ILDs). Lung biopsy is a primary evaluation technique for the fibrotic status of ILDs, and is also a major risk factor for triggering acute deterioration. Fibroblast activation protein (FAP) is a long-known surface biomarker of activated fibroblasts, but its expression pattern and diagnostic implications in ILDs are poorly defined. Objectives: The present study aims to comprehensively investigate whether the expression intensity of FAP could be used as a potential readout to estimate or measure the amounts of activated fibroblasts in ILD lungs quantitatively. Methods: FAP expression in human primary lung fibroblasts as well as in clinical lung specimens was first tested using multiple experimental methods, including real-time quantitative PCR (qPCR), Western blot, immunofluorescence staining, deep learning measurement of whole slide immunohistochemistry, as well as single-cell sequencing. In addition, FAP-targeted positron emission tomography/computed tomography imaging PET/CT was applied to various types of patients with ILD, and the correlation between the uptake of FAP tracer and pulmonary function parameters was analyzed. Measurements and Main Results: Here, it was revealed, for the first time, FAP expression was upregulated significantly in the early phase of lung fibroblast activation event in response to a low dose of profibrotic cytokine. Single-cell sequencing data further indicate that nearly all FAP-positive cells in ILD lungs were collagen-producing fibroblasts. Immunohistochemical analysis validated that FAP expression level was closely correlated with the abundance of fibroblastic foci on human lung biopsy sections from patients with ILDs. We found that the total standard uptake value (SUV) of FAP inhibitor (FAPI) PET (SUVtotal) was significantly related to lung function decline in patients with ILD. Conclusions: Our results strongly support that invitro and invivo detection of FAP can assess the profibrotic activity of ILDs, which may aid in early diagnosis and the selection of an appropriate therapeutic window.

Integration and Reanalysis of Four RNA-Seq Datasets Including BALF, Nasopharyngeal Swabs, Lung Biopsy, and Mouse Models Reveals Common Immune Features of COVID-19.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), has spread over the world causing a pandemic which is still ongoing since its emergence in late 2019. A great amount of effort has been devoted to understanding the pathogenesis of COVID-19 with the hope of developing better therapeutic strategies. Transcriptome analysis using technologies such as RNA sequencing became a commonly used approach in study of host immune responses to SARS-CoV-2. Although substantial amount of information can be gathered from transcriptome analysis, different analysis tools used in these studies may lead to conclusions that differ dramatically from each other. Here, we re-analyzed four RNA-sequencing datasets of COVID-19 samples including human bronchoalveolar lavage fluid, nasopharyngeal swabs, lung biopsy and hACE2 transgenic mice using the same standardized method. The results showed that common features of COVID-19 include upregulation of chemokines including CCL2, CXCL1, and CXCL10, inflammatory cytokine IL-1β and alarmin S100A8/S100A9, which are associated with dysregulated innate immunity marked by abundant neutrophil and mast cell accumulation. Downregulation of chemokine receptor genes that are associated with impaired adaptive immunity such as lymphopenia is another common feather of COVID-19 observed. In addition, a few interferon-stimulated genes but no type I IFN genes were identified to be enriched in COVID-19 samples compared to their respective control in these datasets. These features are in line with results from single-cell RNA sequencing studies in the field. Therefore, our re-analysis of the RNA-seq datasets revealed common features of dysregulated immune responses to SARS-CoV-2 and shed light to the pathogenesis of COVID-19.

Genome-wide spatial expression profiling in formalin-fixed tissues

Formalin-fixed paraffin embedding (FFPE) is the most widespread long-term tissue preservation approach. Here, we report a procedure to perform genome-wide spatial analysis of mRNA in FFPE-fixed tissue sections, using well-established, commercially available methods for imaging and spatial barcoding using slides spotted with barcoded oligo(dT) probes to capture the 3' end of mRNA molecules in tissue sections. We applied this method for expression profiling and cell type mapping in coronal sections from the mouse brain to demonstrate the method's capability to delineate anatomical regions from a molecular perspective. We also profiled the spatial composition of transcriptomic signatures in two ovarian carcinosarcoma samples, exemplifying the method's potential to elucidate molecular mechanisms in heterogeneous clinical samples. Finally, we demonstrate the applicability of the assay to characterize human lung and kidney organoids and a human lung biopsy specimen infected with SARS-CoV-2. We anticipate that genome-wide spatial gene expression profiling in FFPE biospecimens will be used for retrospective analysis of biobank samples, which will facilitate longitudinal studies of biological processes and biomarker discovery.

Spns2 Transporter Contributes to the Accumulation of S1P in Cystic Fibrosis Human Bronchial Epithelial Cells.

The role of S1P in Cystic Fibrosis (CF) has been investigated since 2001, when it was first described that the CFTR channel regulates the inward transport of S1P. From then on, various studies have associated F508del CFTR, the most frequent mutation in CF patients, with altered S1P expression in tissue and plasma. We found that human bronchial epithelial immortalized and primary cells from CF patients express more S1P than the control cells, as evidenced by mass spectrometry analysis. S1P accumulation relies on two- to four-fold transcriptional up-regulation of SphK1 and simultaneous halving of SGPL1 in CF vs. control cells. The reduction of SGPL1 transcription protects S1P from irreversible degradation, but the excessive accumulation is partially prevented by the action of the two phosphatases that are up-regulated compared to control cells. For the first time in CF, we describe that Spns2, a non-ATP dependent transporter that normally extrudes S1P out of the cells, shows deficient transcriptional and protein expression, thus impairing S1P accrual dissipation. The in vitro data on CF human bronchial epithelia correlates with the impaired expression of Spns2 observed in CF human lung biopsies compared to healthy control.

Induction of alveolar and bronchiolar phenotypes in human lung organoids.

Patient‐derived organoids have revolutionized biomedical research and therapies by "transferring the patient into the Petri dish". In vitro access to human lung organoids representing distal lung tissue, i.e. alveolar organoids, would facilitate research pertaining to a wide range of medical conditions and might open for a future approach to individualized treatment.We propose a protocol to derive a single human lung biopsy towards both alveolar and bronchiolar organoids. By modulating Wnt pathway, we obtained a differential gene expression of the main markers for both subtypes, such as a higher expression of surfactant protein C in alveolar organoids or a higher expression of mucine 5AC in bronchiolar organoids. Although the specific cell enrichment was not complete, the differentiation was observed as early as passage 1 based on morphology, and confirmed by QPCR and histology at passage 2. These results are consistent with a functional specification of lung epithelium towards both alveoli‐ and bronchi‐enriched organoids from first passages

0062 Improved Circadian Data Ordering in the Presence of Biological and Technical Confounds

Abstract Introduction We recently used unsupervised machine learning to order genome scale data along a circadian cycle. CYCLOPS (Anafi et al PNAS 2017) encodes high dimensional genomic data onto an ellipse and offers the potential to identify circadian patterns in large data-sets. This approach requires many samples from a wide range of circadian phases. Individual data-sets often lack sufficient samples. Composite expression repositories vastly increase the available data. However, these agglomerated datasets also introduce technical (e.g. processing site) and biological (e.g. age or disease) confounders that may hamper circadian ordering. Methods Using the FLUX machine learning library we expanded the CYCLOPS network. We incorporated additional encoding and decoding layers that model the influence of labeled confounding variables. These layers feed into a fully connected autoencoder with a circular bottleneck, encoding the estimated phase of each sample. The expanded network simultaneously estimates the influence of confounding variables along with circadian phase. We compared the performance of the original and expanded networks using both real and simulated expression data. In a first test, we used time-labeled data from a single-center describing human cortical samples obtained at autopsy. To generate a second, idealized processing center, we introduced gene specific biases in expression along with a bias in sample collection time. In a second test, we combined human lung biopsy data from two medical centers. Results The performance of the original CYCLOPS network degraded with the introduction of increasing, non-circadian confounds. The expanded network was able to more accurately assess circadian phase over a wider range of confounding influences. Conclusion The addition of labeled confounding variables into the network architecture improves circadian data ordering. The use of the expanded network should facilitate the application of CYCLOPS to multi-center data and expand the data available for circadian analysis. Support This work was supported by the National Cancer Institute (1R01CA227485-01)

RGS4 promotes allergen- and aspirin-associated airway hyperresponsiveness by inhibiting PGE2 biosynthesis

Allergens elicit host production of mediators acting on G-protein-coupled receptors to regulate airway tone. Among these is prostaglandin E2 (PGE2), which, in addition to its role as a bronchodilator, has anti-inflammatory actions. Some patients with asthma develop bronchospasm after the ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, a disorder termed aspirin-exacerbated respiratory disease. This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2. We sought to understand the functions of regulator of G protein signaling 4 (RGS4), a cytoplasmic protein expressed in airway smooth muscle and bronchial epithelium that regulates the activity of G-protein-coupled receptors, in asthma. We examined RGS4 expression in human lung biopsies by immunohistochemistry. We assessed airways hyperresponsiveness (AHR) and lung inflammation in germline and airway smooth muscle-specific Rgs4-/- mice and in mice treated with an RGS4 antagonist after challenge with Aspergillus fumigatus. We examined the role of RGS4 in nonsteroidal anti-inflammatory drug-associated bronchoconstriction by challenging aspirin-exacerbated respiratory disease-like (ptges1-/-) mice with aspirin. RGS4 expression in respiratory epithelium is increased in subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in Rgs4-/- mice, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen-induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation. The RGS4 antagonist CCG203769 attenuated AHR induced by allergen or aspirin challenge of wild-type or ptges1-/- mice, respectively, in association with increased airway PGE2 levels. RGS4 may contribute to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma.

X-ray Micro-Computed Tomography for Nondestructive Three-Dimensional (3D) X-ray Histology

Historically, micro-computed tomography (μCT) has been considered unsuitable for histologic analysis of unstained formalin-fixed, paraffin-embedded soft tissue biopsy specimens because of a lack of image contrast between the tissue and the paraffin. However, we recently demonstrated that μCT can successfully resolve microstructural detail in routinely prepared tissue specimens. Herein, we illustrate how μCT imaging of standard formalin-fixed, paraffin-embedded biopsy specimens can be seamlessly integrated into conventional histology workflows, enabling nondestructive three-dimensional (3D) X-ray histology, the use and benefits of which we showcase for the exemplar of human lung biopsy specimens. This technology advancement was achieved through manufacturing a first-of-kind μCT scanner for X-ray histology and developing optimized imaging protocols, which do not require any additional sample preparation. 3D X-ray histology allows for nondestructive 3D imaging of tissue microstructure, resolving structural connectivity and heterogeneity of complex tissue networks, such as the vascular network or the respiratory tract. We also demonstrate that 3D X-ray histology can yield consistent and reproducible image quality, enabling quantitative assessment of a tissue's 3D microstructures, which is inaccessible to conventional two-dimensional histology. Being nondestructive, the technique does not interfere with histology workflows, permitting subsequent tissue characterization by means of conventional light microscopy–based histology, immunohistochemistry, and immunofluorescence. 3D X-ray histology can be readily applied to a plethora of archival materials, yielding unprecedented opportunities in diagnosis and research of disease.

LEM domain–containing protein 3 antagonizes TGFβ–SMAD2/3 signaling in a stiffness-dependent manner in both the nucleus and cytosol

Transforming growth factor-β (TGFβ) signaling through SMAD2/3 is an important driver of pathological fibrosis in multiple organ systems. TGFβ signaling and extracellular matrix (ECM) stiffness form an unvirtuous pathological circuit in which matrix stiffness drives activation of latent TGFβ, and TGFβ signaling then drives cellular stress and ECM synthesis. Moreover, ECM stiffness also appears to sensitize cells to exogenously activated TGFβ through unknown mechanisms. Here, using human fibroblasts, we explored the effect of ECM stiffness on a putative inner nuclear membrane protein, LEM domain-containing protein 3 (LEMD3), which is physically connected to the cell's actin cytoskeleton and inhibits TGFβ signaling. We showed that LEMD3-SMAD2/3 interactions are inversely correlated with ECM stiffness and TGFβ-driven luciferase activity and that LEMD3 expression is correlated with the mechanical response of the TGFβ-driven luciferase reporter. We found that actin polymerization but not cellular stress or LEMD3-nuclear-cytoplasmic couplings were necessary for LEMD3-SMAD2/3 interactions. Intriguingly, LEMD3 and SMAD2/3 frequently interacted in the cytosol, and we discovered LEMD3 was proteolytically cleaved into protein fragments. We confirmed that a consensus C-terminal LEMD3 fragment binds SMAD2/3 in a stiffness-dependent manner throughout the cell and is sufficient for antagonizing SMAD2/3 signaling. Using human lung biopsies, we observed that these nuclear and cytosolic interactions are also present in tissue and found that fibrotic tissues exhibit locally diminished and cytoplasmically shifted LEMD3-SMAD2/3 interactions, as noted in vitro Our work reveals novel LEMD3 biology and stiffness-dependent regulation of TGFβ by LEMD3, providing a novel target to antagonize pathological TGFβ signaling.

FOXF1 Inhibits Pulmonary Fibrosis by Preventing CDH2-CDH11 Cadherin Switch in Myofibroblasts

SUMMARYIdiopathic pulmonary fibrosis (IPF) is characterized by aberrant accumulation of collagen-secreting myofibroblasts. Development of effective therapies is limited due to incomplete understanding of molecular mechanisms regulating myofibroblast expansion. FOXF1 transcription factor is expressed in resident lung fibroblasts, but its role in lung fibrosis remains unknown due to the lack of genetic mouse models. Through comprehensive analysis of human IPF genomics data, lung biopsies, and transgenic mice with fibroblast-specific inactivation of FOXF1, we show that FOXF1 inhibits pulmonary fibrosis. FOXF1 deletion increases myofibroblast invasion and collagen secretion and promotes a switch from N-cadherin (CDH2) to Cadherin-11 (CDH11), which is a critical step in the acquisition of the profibrotic phenotype. FOXF1 directly binds to Cdh2 and Cdh11 promoters and differentially regulates transcription of these genes. Re-expression of CDH2 or inhibition of CDH11 in FOXF1-deficient cells reduces myofibroblast invasion in vitro. FOXF1 inhibits pulmonary fibrosis by regulating a switch from CDH2 to CDH11 in lung myofibroblasts.

CSF1R inhibition prevents radiation pulmonary fibrosis by depletion of interstitial macrophages.

Radiation-induced lung fibrosis (RIF) is a delayed side-effect of chest radiotherapy, frequently associated with macrophage infiltration.We aimed to characterise the role of pulmonary macrophages in RIF using human lung biopsies from patients receiving radiotherapy for thorax malignancies and a RIF model developed in C57BL/6 mice after 16-Gy thorax irradiation.High numbers of macrophages (both interstitial and alveolar) were detected in clinical and preclinical RIF. In the preclinical model, upregulation of T-helper (Th)2 cytokines was measured, whereas Th1 cytokines were downregulated in RIF tissue lysate. Bronchoalveolar lavage demonstrated upregulation of both types of cytokines. At steady state, tissue-infiltrating macrophages (IMs) expressed 10-fold more arginase (Arg)-1 than alveolar macrophages (AMs), and a 40-fold upregulation of Arg-1 was found in IMs isolated from RIF. IMs, but not AMs, were able to induce myofibroblast activation in vitro In addition, whereas depletion of AMs using Clodrosome didn't affect RIF score, depletion of IMs using a clinically available colony-stimulating factor receptor-1 (CSF1R) neutralising antibody was antifibrotic.These findings suggest differential contributions of alveolar versus interstitial macrophages in RIF, highlighting the fibrogenic role of IMs. The CSF1/CSF1R pathway was identified as a new therapeutic target to inhibit RIF.

Parenchymal Airspace Profiling: Sensitive Quantification and Characterization of Lung Structure Evaluating Parenchymal Destruction.

Lung morphometry was introduced over 50 years ago to provide quantitative evaluation of the lung structure. The existing parameters, such as mean linear intercept and destructive index, suffer from simplistic data interpretation and a subjective data acquisition process. To overcome these existing shortcomings, parenchymal airspace profiling (PAP) was developed to provide a more detailed and unbiased quantitative method. Following the standard protocols of fixation, embedding, and sectioning, lung micrographs were: (1) marked with nonparenchymal area, preprocessed, and binarized under the researcher's supervision; (2) analyzed with a statistical learning method, Gaussian mixture model, to provide an unbiased categorization of parenchymal airspace compartments, corresponding to a single alveolus, alveolar sac, and ductal/destructive airspace; and (3) further quantified into morphometric parameters, including reference volume, alveolar count, and ductal/destructive fraction (DF) based on stereological principles. PAP was performed on hematoxylin and eosin-stained lung sections from mice and rabbits. Unbiased categorization revealed differences in alveolar size among several mouse strains (NZW/LacJ<AKR/J<A/J<C57BL/6J) and across species (mouse<rabbit). Further quantification indicates that parenchymal destruction, modeled in mouse lungs with 1-month smoke exposure, resulted in decreased alveolar count, increased DF, but no significant differences in mean linear intercept. DF also provides a robust measurement that is not biased by processing artifacts, magnification, or reference volume, which are common limitations in human lung biopsies or data obtained from different laboratories. PAP is a novel approach to lung morphometry that offers more detailed characterization of the lung structure, sensitivity, and robustness than presently used methods for evaluating parenchymal destruction.

Cancer biomarker sensing using packaged plasmonic optical fiber gratings: Towards in vivo diagnosis

This work presents the development of an innovative plasmonic optical fiber (OF) immunosensor for the detection of cytokeratin 17 (CK17), a biomarker of interest for lung cancer diagnosis. The development of this sensing platform is such that it can be assessed in non-liquid environments, demonstrating that a surface plasmon resonance (SPR) can be excited in this case. For this purpose, detections have been first carried out on CK17 encapsulated in gel matrix in the aim of mimicking tissue samples. Gold-coated OF immunosensors were embedded in a specifically designed packaging providing enough stiffness to penetrate into soft matters. Resulting reflected spectra have revealed, for the first time, the presence of a stable SPR signal recorded in soft matters. Experiments conducted to detect CK17 trapped in a porous polyacrylamide gel matrix have highlighted the specific and selective biosensor response towards the target protein. Finally, the packaged OF immunosensor has been validated by a preliminary test on human lung biopsy, which has confirmed the ex-vivo CK17 detection. Consequently, this work represents an important milestone towards the detection of biomarkers in tissues, which is still a clinical challenge for minimally-invasive in vivo medical diagnosis.

Human Lung Spheroids as In Vitro Niches of Lung Progenitor Cells with Distinctive Paracrine and Plasticity Properties.

Basic and translational research on lung biology has discovered multiple progenitor cell types, specialized or facultative, responsible for turnover, renewal, and repair. Isolation of populations of resident lung progenitor cells (LPCs) has been described by multiple protocols, and some have been successfully applied to healthy human lung tissue. We aimed at understanding how different cell culture conditions may affect, in vitro, the phenotype of LPCs to create an ideal niche‐like microenvironment. The influence of different substrates (i.e., fibronectin, gelatin, laminin) and the impact of a three‐dimensional/two‐dimensional (3D/2D) culture switch on the biology of LPCs isolated as lung spheroids (LSs) from normal adult human lung biopsy specimens were investigated. We applied a spheroid culture system as the selective/inductive step for progenitor cell culture, as described in many biological systems. The data showed a niche‐like proepithelial microenvironment inside the LS, highly sensitive to the 3D culture system and significantly affecting the phenotype of adult LPCs more than culture substrate. LSs favor epithelial phenotypes and LPC maintenance and contain cells more responsive to specific commitment stimuli than 2D monolayer cultures, while secreting a distinctive set of paracrine factors. We have shown for the first time, to our knowledge, how culture as 3D LSs can affect LPC epithelial phenotype and produce strong paracrine signals with a distinctive secretomic profile compared with 2D monolayer conditions. These findings suggest novel approaches to maintain ex vivo LPCs for basic and translational studies. Stem Cells Translational Medicine 2017;6:767–777