Lung Explants Research Articles

The microbiota plays a critical role in the reactivity of lung immune components to innate ligands.

The gut microbiota contributes to shaping efficient and safe immune defenses in the gut. However, little is known about the role of the gut and/or lung microbiota in the education of pulmonary innate immune responses. Here, we tested whether the endogenous microbiota in general can modulate the reactivity of pulmonary tissue to pathogen stimuli by comparing the response of specific-pathogen-free (SPF) and germ-free (GF) mice. Thus, we observed earlier and greater inflammation in the pulmonary compartment of GF mice than that of SPF mice after intranasal instillation to lipopolysaccharide (LPS), a component of Gram-negative bacteria. Toll-like receptor 4 (TLR4) was more abundantly expressed in the lungs of GF mice than those of SPF mice at steady state, which could predispose the innate immunity of GF mice to strongly react to the environmental stimuli. Lung explants were stimulated with different TLR agonists or infected with the human airways pathogen, respiratory syncytial virus (RSV), resulting in greater inflammation under almost all conditions for the GF explants. Finally, alveolar macrophages (AM) from GF mice presented a higher innate immune response upon RSV infection than those of SPF mice. Overall, these data suggest that the presence of microbiota in SPF mice induced a process of innate immune tolerance in the lungs by a mechanism which remains to be elucidated. Our study represents a step forward to establishing the link between the microbiota and the immune reactivity of the lungs.

Natural history and mechanisms of COPD

ABSTRACTThe natural history of COPD is complex, and the disease is best understood as a syndrome resulting from numerous interacting factors throughout the life cycle with smoking being the strongest inciting feature. Unfortunately, diagnosis is often delayed with several longitudinal cohort studies shedding light on the long ‘preclinical’ period of COPD. It is now accepted that individuals presenting with different COPD phenotypes may experience varying natural history of their disease. This includes its inception, early stages and progression to established disease. Several scenarios regarding lung function course are possible, but it may conceptually be helpful to distinguish between individuals with normal maximally attained lung function in their early adulthood who thereafter experience faster than normal FEV1 decline, and those who may achieve a lower than normal maximally attained lung function. This may be the main mechanism behind COPD in the latter group, as the decline in FEV1 during their adult life may be normal or only slightly faster than normal. Regardless of the FEV1 trajectory, continuous smoking is strongly associated with disease progression, development of structural lung disease and poor prognosis. In developing countries, factors such as exposure to biomass and sequelae after tuberculosis may lead to a more airway‐centred COPD phenotype than seen in smokers. Mechanistically, COPD is characterized by a combination of structural and inflammatory changes. It is unlikely that all patients share the same individual or combined mechanisms given the heterogeneity of resultant phenotypes. Lung explants, bronchial biopsies and other tissue studies have revealed important features. At the small airway level, progression of COPD is clinically imperceptible, and the pathological course of the disease is poorly described. Asthmatic features can further add confusion. However, the small airway epithelium is likely to represent a key focus of the disease, combining impaired subepithelial crosstalk and structural/inflammatory changes. Insufficient resolution of inflammatory processes may facilitate these changes. Pathologically, epithelial metaplasia, inversion of the goblet to ciliated cell ratio, enlargement of the submucosal glands and neutrophil and CD8‐T‐cell infiltration can be detected. Evidence of type 2 inflammation is gaining interest in the light of new therapeutic agents. Alarmin biology is a promising area that may permit control of inflammation and partial reversal of structural changes in COPD. Here, we review the latest work describing the development and progression of COPD with a focus on lung function trajectories, exacerbations and survival. We also review mechanisms focusing on epithelial changes associated with COPD and lack of resolution characterizing the underlying inflammatory processes.

The RNA-binding protein Quaking regulates multiciliated and basal cell abundance in the developing lung.

RNA-binding proteins (RBPs) form complexes with RNA, changing how the RNA is processed and thereby regulating gene expression. RBPs are important sources of gene regulation during organogenesis, including the development of lungs. The RBP called Quaking (QK) is critical for embryogenesis, yet it has not been studied in the developing lung. Here, we show that QK is widely expressed during rat lung development and into adulthood. The QK isoforms QK5 and QK7 colocalize to the nuclei of nearly all lung cells. QK6 is present in the nuclei and cytoplasm of mesenchymal cells and is only present in the epithelium during branching morphogenesis. QK knockdown in embryonic lung explants caused a greater number of multiciliated cells to appear in the airways, at the expense of basal cells. The mRNA of multiciliated cell genes and the abundance of FOXJ1/SOX2+ cells increased after knockdown, whereas P63/SOX2+ cells decreased. The cytokine IL-6, a known regulator of multiciliated cell differentiation, had increased mRNA levels after QK knockdown, although protein levels remained unchanged. Further studies are necessary to confirm whether QK acts as a blocker for the IL-6-induced differentiation of basal cells into multiciliated cells, and a conditional QK knockout would likely lead to additional discoveries on QK's role during lung development.

Phospholipid dynamics in ex vivo lung cancer and normal lung explants

In cancer cells, metabolic pathways are reprogrammed to promote cell proliferation and growth. While the rewiring of central biosynthetic pathways is being extensively studied, the dynamics of phospholipids in cancer cells are still poorly understood. In our study, we sought to evaluate de novo biosynthesis of glycerophospholipids (GPLs) in ex vivo lung cancer explants and corresponding normal lung tissue from six patients by utilizing a stable isotopic labeling approach. Incorporation of fully 13C-labeled glucose into the backbone of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinositol (PI) was analyzed by liquid chromatography/mass spectrometry. Lung cancer tissue showed significantly elevated isotopic enrichment within the glycerol backbone of PE, normalized to its incorporation into PI, compared to that in normal lung tissue; however, the size of the PE pool normalized to the size of the PI pool was smaller in tumor tissue. These findings indicate enhanced PE turnover in lung cancer tissue. Elevated biosynthesis of PE in lung cancer tissue was supported by enhanced expression of the PE biosynthesis genes ETNK2 and EPT1 and decreased expression of the PC and PI biosynthesis genes CHPT1 and CDS2, respectively, in different subtypes of lung cancer in publicly available datasets. Our study demonstrates that incorporation of glucose-derived carbons into the glycerol backbone of GPLs can be monitored to study phospholipid dynamics in tumor explants and shows that PE turnover is elevated in lung cancer tissue compared to normal lung tissue.

The Development of Integrin Alpha-8 Deficient Lungs Shows Reduced and Altered Branching and a Correction of the Phenotype During Alveolarization.

Lung development involves epithelial–mesenchymal interactions and integrins represent one of the key elements. These extracellular matrix receptors form hetero-dimers of alpha and beta subunits. The integrin α8β1 is highly expressed in mouse tissues, including lung. It forms a cellular receptor for fibronectin, vitronectin, osteopontin, nephronectin, and tenascin-C. This study aims to investigate the role of the integrin α8-subunit (α8) during lung development. Wild type and α8-deficient lungs were explanted at embryonic days 11.5/12.5. After 24–73 h in culture α8-deficient lung explants displayed reduced growth, reduced branching, enlarged endbuds, altered branching patterns, and faster spontaneous contractions of the airways as compared to wild type. Postnatally, a stereological investigation revealed that lung volume, alveolar surface area, and the length of the free septal edge were significantly reduced in α8-deficient lungs at postnatal days P4 and P7. An increased formation of new septa in α8-deficient lungs rescued the phenotype. At day P90 α8-deficient lungs were comparable to wild type. We conclude that α8β1 takes not only part in the control of branching, but also possesses a morphogenic effect on the pattern and size of the future airways. Furthermore, we conclude that the phenotype observed at day P4 is caused by reduced branching and is rescued by a pronounced formation of the new septa throughout alveolarization. More studies are needed to understand the mechanism responsible for the formation of new septa in the absence of α8β1 in order to be of potential therapeutic benefit for patients suffering from structural lung diseases.

Paracrine stimulation of perinatal lung functional and structural maturation by mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) were shown to harbor therapeutic potential in models of respiratory diseases, such as bronchopulmonary dysplasia (BPD), the most common sequel of preterm birth. In these studies, cells or animals were challenged with hyperoxia or other injury-inducing agents. However, little is known about the effect of MSCs on immature fetal lungs and whether MSCs are able to improve lung maturity, which may alleviate lung developmental arrest in BPD.MethodsWe aimed to determine if the conditioned medium (CM) of MSCs stimulates functional and structural lung maturation. As a measure of functional maturation, Na+ transport in primary fetal distal lung epithelial cells (FDLE) was studied in Ussing chambers. Na+ transporter and surfactant protein mRNA expression was determined by qRT-PCR. Structural maturation was assessed by microscopy in fetal rat lung explants.ResultsMSC-CM strongly increased the activity of the epithelial Na+ channel (ENaC) and the Na,K-ATPase as well as their mRNA expression. Branching and growth of fetal lung explants and surfactant protein mRNA expression were enhanced by MSC-CM. Epithelial integrity and metabolic activity of FDLE cells were not influenced by MSC-CM. Since MSC’s actions are mainly attributed to paracrine signaling, prominent lung growth factors were blocked. None of the tested growth factors (VEGF, BMP, PDGF, EGF, TGF-β, FGF, HGF) contributed to the MSC-induced increase of Na+ transport. In contrast, inhibition of PI3-K/AKT and Rac1 signaling reduced MSC-CM efficacy, suggesting an involvement of these pathways in the MSC-CM-induced Na+ transport.ConclusionThe results demonstrate that MSC-CM strongly stimulated functional and structural maturation of the fetal lungs. These effects were at least partially mediated by the PI3-K/AKT and Rac1 signaling pathway. Thus, MSCs not only repair a deleterious tissue environment, but also target lung cellular immaturity itself.

ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

BackgroundCharacterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2+) and distal (SOX9+) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance.MethodsThe expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and β-Catenin were quantified by Western blot.ResultsExperimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho β-Catenin, and BMP4.ConclusionsThese studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

CCR2 Mediates Chronic LPS-Induced Pulmonary Inflammation and Hypoalveolarization in a Murine Model of Bronchopulmonary Dysplasia.

The histopathology of bronchopulmonary dysplasia (BPD) includes hypoalveolarization and interstitial thickening due to abnormal myofibroblast accumulation. Chorioamnionitis and sepsis are major risk factors for BPD development. The cellular mechanisms leading to these lung structural abnormalities are poorly understood. We used an animal model with repeated lipopolysaccharide (LPS) administration into the airways of immature mice to simulate prolonged airway exposure to gram-negative bacteria, focusing on the role of C-C chemokine receptor type 2-positive (CCR2+) exudative macrophages (ExMf). Repetitive LPS exposure of immature mice induced persistent hypoalveolarization observed at 4 and 18 days after the last LPS administration. LPS upregulated the expression of lung pro-inflammatory cytokines (TNF-α, IL-17a, IL-6, IL-1β) and chemokines (CCL2, CCL7, CXCL1, and CXCL2), while the expression of genes involved in lung alveolar and mesenchymal cell development (PDGFR-α, FGF7, FGF10, and SPRY1) was decreased. LPS induced recruitment of ExMf, including CCR2+ ExMf, as well as other myeloid cells like DCs and neutrophils. Lungs of LPS-exposed CCR2−/− mice showed preserved alveolar structure and normal patterns of α-actin and PDGFRα expression at the tips of the secondary alveolar crests. Compared to wild type mice, a significantly lower number of ExMf, including TNF-α+ ExMf were recruited to the lungs of CCR2−/− mice following repetitive LPS exposure. Further, pharmacological inhibition of TLR4 with TAK-242 also blocked the effect of LPS on alveolarization, α-SMA and PDGFRα expression. TNF-α and IL-17a induced α-smooth muscle actin expression in the distal airspaces of E16 fetal mouse lung explants. In human preterm lung mesenchymal stromal cells, TNF-α reduced mRNA and protein expression of PDGFR-α and decreased mRNA expression of WNT2, FOXF2, and SPRY1. Collectively, our findings demonstrate that in immature mice repetitive LPS exposure, through TLR4 signaling increases lung inflammation and impairs lung alveolar growth in a CCR2-dependent manner.

REGIONAL INFECTION AND INFLAMMATION IN CYSTIC FIBROSIS: A PILOT STUDY WITH LUNG EXPLANTS AND A NOVEL HISTOPATHOLOGY GRADING SYSTEM

SESSION TITLE: Genetic and Developmental Disorders Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: The objective of this study was to use lung explants to determine the influence of nonmucoid and mucoid Pseudomonas aeruginosa (P.a.) variants on lobar inflammation and tissue damage in cystic fibrosis (CF). METHODS: We obtained three lung explants from adult CF patients undergoing lung transplantation. 1cm^3 superficial (parenchymal) and deep (airway) biopsies were obtained from all lung lobes in each explant (n=35 biopsies). Each biopsy was split into two fractions. One was homogenized to be plated on P.a.-isolation media (for growth of nonmucoid/mucoid P.a.) and also applied to a multiplex cytokine array. The other tissue fraction was formalin-fixed and prepared for hematoxylin and eosin (H&E) staining. A pathologist blindly assessed H&E slides for five indicators of tissue damage via a novel histopathology scoring scheme for CF lung tissue. RESULTS: All biopsies were categorized as infected with no P.a., nonmucoid P.a. only, mucoid P.a. only, or mixed mucoid and nonmucoid P.a. Mucoid and nonmucoid variants were distributed throughout the CF lung explants, without preference for specific lobes. Independent of infection, there were no statistically significant differences in inflammatory cytokines or histopathology among all lung lobes. However, mucoid P.a. infection, in single- or mixed-variant populations, was associated with greater concentrations of IL-1β, TNF-α, and IFN-γ compared to nonmucoid P.a. infection (p<0.01). Additionally, as mucoid P.a. percentage increased within a mixed-variant infection, the concentrations of all pro-inflammatory cytokines tested also increased: IL-1β, TNF-α, IL-6, IL-8, and IFN-γ (p<0.05). Despite the association between mucoid P.a. infection and cytokine concentrations, there was no correlation between P.a. variants and histopathology in this cohort. CONCLUSIONS: In CF lung explants, there were no differences in inflammation or tissue damage among lung lobes. Independent of lobe, mucoid P.a. infection was associated with greater regional inflammation. There were no significant differences in histopathology of the lung tissue when comparing mucoid or nonmucoid P.a. infection. CLINICAL IMPLICATIONS: In this pilot study, we demonstrated that mucoid P.a. infection was associated with acute-on-chronic inflammation in lung explants. As such, current/novel antimicrobials should target mucoid P.a. isolates throughout CF patients’ lifetimes. Furthermore, correlations between P.a. infection and inflammation within this cohort suggest that cytokines merit further study as long-term biomarkers in CF. Finally, although P.a. infection was not associated with tissue damage, we applied a detailed histopathology scoring system to CF patient lung tissue for the first time. This method can be utilized in future large-cohort studies investigating host-bacterial relationships in situ. DISCLOSURES: No relevant relationships by Don Hayes, source=Web Response No relevant relationships by Sankalp Malhotra, source=Web Response No relevant relationships by Daniel Wozniak, source=Web Response No relevant relationships by Ching Yang, source=Web Response

PATHOLOGY OF ALL ASPECTS OF END-STAGE PULMONARY SARCOIDOSIS: A RETROSPECTIVE ANALYSIS OF LUNG EXPLANTS FROM A HIGH-VOLUME LUNG TRANSPLANTATION CENTER

SESSION TITLE: Late-breaking Abstract Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: Sarcoidosis is a multisystem disease of unknown etiology with variable prognosis; 10-30% of patients develop advanced fibrosis progressing to end stage pulmonary sarcoidosis (ESPS). ESPS carries a poor prognosis and lung transplantation becomes the last resort for some of these patients. Sarcoidosis can involve all parts of the respiratory system. Explanted lungs provide a unique opportunity to examine the underlying pathophysiology and patterns of fibrosis and other architectural involvement in ESPS. METHODS: We retrospectively evaluated histologic sections from twenty-four explants from patients transplanted for ESPS diagnosed clinically or by biopsy. Explants were evaluated for all components, including lung parenchyma, airways, vasculature, lymph nodes and pleura. The extent of fibrosis was scored categorized as: 1 = 1% to 25%; 2 = 26% to 50%; 3 = 51% to 75%; 4 = 76% to 100% of lung parenchyma. RESULTS: Twenty-four patients underwent lung transplantation at our institution between March 2012 to January 2020. All explants had extensive fibrosis with the majority (70.1%) found in a patchy pattern along a central and peribronchovascular distribution; twelve explants had a fibrosis score of 3 or 4. Presence of architectural distortion in the form of honeycombing was identified in four patients, two of which showed upper lobe predominant changes. Rare fibroblast foci were present in seven patients with an average of 1/10 high power fields. Parenchymal granulomas were present in all but five patients. Airway examination showed granulomatous involvement in eight patients. All explants had histologic signs consistent with a clinical impression of pulmonary hypertension including five with granulomas in vasculature; none had definitive findings of pulmonary veno-occlusive disease. Three explants had granulomatous involvement of pleura while fourteen had evidence of chronic pleuritis. Lymph nodes were evaluated in twenty-one explants; eight of which had granulomatous involvement. CONCLUSIONS: In this large analysis of lung explants from patients with ESPS, all parts of the respiratory system were examined to assess the extensive involvement in sarcoidosis. We indeed found a widespread but variable involvement of these with granulomatous inflammation and fibrosis. This might be one of the contributory factors in the variability of patient presentation and prognosis in pulmonary sarcoidosis. These will be correlated with clinical, radiological, physiological and hemodynamic features as well as outcomes. CLINICAL IMPLICATIONS: Understanding the underlying histologic pattern(s) is important to target therapy for not only patients with ESPS but also hopefully advance the field towards preventing end stage disease in patients with pulmonary sarcoidosis. DISCLOSURES: No relevant relationships by Amandeep Aneja, source=Web Response No relevant relationships by Francis Cordova, source=Web Response no disclosure on file for Gerard Criner; Human Factors Study Participant relationship with Nuvaira Inc Please note: $1001 - $5000 Added 07/29/2020 by Andrew Gangemi, source=Web Response, value=Honoraria No relevant relationships by Rohit Gupta, source=Web Response No relevant relationships by Nirag Jhala, source=Web Response no disclosure on file for A. Mamary; no disclosure on file for Kriti Mittal; No relevant relationships by Catherine Myers, source=Web Response No relevant relationships by Stephanie Tittaferrante, source=Web Response No relevant relationships by Matthew Zheng, source=Web Response

COMPARISON OF PRE- VS POST-LUNG TRANSPLANT CAUSE OF INTERSTITIAL LUNG DISEASE

SESSION TITLE: Transplantation Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: The aim of this study is to compare the clinical diagnosis of interstitial lung disease to that determined on post-explant evaluation. METHODS: Retrospective study conducted through chart review of 50 consecutive lung transplants performed in patients with interstitial lung disease (ILD) at The University of Health Sciences Center at San Antonio. All charts of patients over 18 years of age who underwent lung transplantation with the diagnosis of ILD were carefully reviewed to determine the clinical pre-transplant diagnosis. Studies included high-resolution computed tomography (HRCT), serologic evaluation of connective tissue disease in all patients. Data from transbronchial biopsy or open lung biopsy were reviewed in determining the pre-transplant diagnosis. We compared the pre-lung transplant diagnosis to the post-transplant, pathologic diagnosis determined from the explanted lungs. RESULTS: A total of 50 patients with ILD who had either single or bilateral lung transplant were analyzed. Thirty-three patients (66%) had pre-transplant (pre-tx) diagnosis of usual interstitial pneumonia (UIP), eight patients (16%) had pre-tx diagnosis of nonspecific interstitial pneumonia (NSIP), six patients (12%) had a pre-tx diagnosis of connective tissue disease – ILD (CTD-ILD), one patient (2%) with pre-tx diagnosis of hypersensitivity pneumonitis (HSP) while four patients (8%) had other diagnoses, including indeterminate end stage lung disease (ESLD), type I congenital cystic adenomatoid malformation (CCAM) and constrictive bronchiolitis with fibrosis. After lung explant biopsies were reviewed, seventeen (34%) cases were reclassified compared to the pre-tx diagnoses. However, out of these seventeen cases, only six cases (12%) were clinically significant. Out of the six clinically significant misdiagnoses, three had a final diagnosis of HSP and three had a final diagnosis of NSIP. The remaining discrepancies in diagnosis were disorders t hat are known to progress to UIP. CONCLUSIONS: Almost two-thirds (66%) of all cases had consistent pre-transplant diagnoses when compared to post- transplant diagnoses. Most of the remaining cases (22%) had disorders known to progress to UIP. However, in 12% of cases, the diagnosis changed from a disorder with a progressive course to one that carries a different prognosis and might have responded to alternative therapies (NSIP and HSP). CLINICAL IMPLICATIONS: Definitive diagnosis in patients with ILD is both difficult and challenging. However, it is imperative to be cautious and observant when searching for disorders that are more responsive to environmental changes or responsive to immunotherapy. Early diagnosis of accurate ILD along with the use of multi-disciplinary discussion (MDD) can improve appropriate therapy in patients with ILD. DISCLOSURES: No relevant relationships by Sarah Hackman, source=Web Response No relevant relationships by Tasnim Islam, source=Web Response No relevant relationships by Jay Peters, source=Web Response

Lung immunoglobulin A immunity dysregulation in cystic fibrosis.

BackgroundIn cystic fibrosis (CF), recurrent infections suggest impaired mucosal immunity but whether production of secretory immunoglobulin A (S-IgA) is impaired remains elusive. S-IgA is generated following polymeric immunoglobulin receptor (pIgR)-mediated transepithelial transport of dimeric (d-)IgA and represents a major defence through neutralisation of inhaled pathogens like Pseudomonas aeruginosa (Pa).MethodsHuman lung tissue (n = 74), human sputum (n = 118), primary human bronchial epithelial cells (HBEC) (cultured in air-liquid interface) (n = 19) and mouse lung tissue and bronchoalveolar lavage were studied for pIgR expression, IgA secretion and regulation.FindingsIncreased epithelial pIgR immunostaining was observed in CF lung explants, associated with more IgA-producing plasma cells, sputum and serum IgA, especially Pa-specific IgA. In contrast, pIgR and IgA transport were downregulated in F508del mice, CFTR-inhibited HBEC, and CF HBEC. Moreover, the unfolded protein response (UPR) due to F508del mutation, inhibited IgA transport in Calu-3 cells. Conversely, pIgR expression and IgA secretion were strongly upregulated following Pa lung infection in control and F508del mice, through an inflammatory host response involving interleukin-17.InterpretationA complex regulation of IgA secretion occurs in the CF lung, UPR induced by CFTR mutation/dysfunction inhibiting d-IgA transcytosis, and Pa infection unexpectedly unleashing this secretory defence mechanism.FundingThis work was supported by the Forton's grant of the King Baudouin's Foundation, Belgium, the Fondazione Ricerca Fibrosi Cistica, Italy, and the Fonds National de la Recherche Scientifique, Belgium.

Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing.

SummaryThe emergence of the novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to require at least 12-18 months, and the typical timeline for approval of a novel antiviral therapeutic can exceed 10 years. Thus, repurposing of known drugs could significantly accelerate the deployment of novel therapies for COVID-19. Towards this end, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. We report the identification of 100 molecules that inhibit viral replication, including 21 known drugs that exhibit dose response relationships. Of these, thirteen were found to harbor effective concentrations likely commensurate with achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2–4, and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334. Notably, MDL-28170, ONO 5334, and apilimod were found to antagonize viral replication in human iPSC-derived pneumocyte-like cells, and the PIKfyve inhibitor also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, the known pharmacological and human safety profiles of these compounds will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.

Abstract A31: Podxl as therapeutic target for metastasis

Abstract Osteosarcoma (OS) is the most common bone tumor in pediatric patients. Metastasis is a major cause of mortality and morbidity. The rarity of this disease coupled with the challenges of drug development for metastatic cancers have slowed the delivery of improvements in long-term outcomes for these patients. Recently, we found that high podocalyxin (gene name PODXL, protein name Podxl) expression is associated with high metastatic profile in OS cell lines. Consistent with this data, we found a significant association between high Podxl expression and poor outcome in pediatric OS patients. Podxl is a cell surface transmembrane glycoprotein belonging to the CD34 family. The functional role of Podxl in tumorigenesis is largely unknown, but it has been demonstrated to promote cancer cell invasion and migration and to enhance metastatic potential. Podxl is upregulated on a variety of human tumors, facilitates disease progression, and is a promising target for immunotherapy as an approach to blocking metastatic disease. Accordingly, in collaboration for the Centre for Drug Research and Development, Kelly McNagny and his collaborators have developed a novel panel of monoclonal antibodies to human Podxl and have explored their utility in suppressing tumor cell growth in vitro and in vivo. To define the relevance of Podxl in the biology of metastasis, we examined Podxl expression in different OS cell lines with different metastatic profiles. Then, we studied the importance of Podxl in migration and invasion processes and in in vitro 3D cell system formation. To assess Podxl’s role in metastatic progression we performed the Pulmonary Metastasis Assay (PuMA). PuMA is an ex vivo lung explant and closed cell culture system that permits to study the biology of lung colonization by fluorescence microscopy. With PuMA model, we assessed the effects of antimetastatic therapeutics over time. In the present study, we found that suppression of Podxl profoundly impairs cell proliferation, migration, invasion properties, and tumorsphere formation in OS cells in vitro. With PuMA model, we found that Podxl is required for the progression of metastasis disease. These data tend to validate podocalyxin as a regulator of tumor progression and a novel therapeutic target in OS. It is necessary to perform in vivo experiments to further explore the functions of Podxl and the effect of the podocalyxin-specific monoclonal antibody (coupled or not to a toxic payload) to suppress the metastasis progression in our model. Citation Format: Anne-Chloe Dhez, Irene Paassen, Poul Sorensen. Podxl as therapeutic target for metastasis [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A31.

Abstract A19: Immunologic dysfunctions of NSG mice confer higher engraftment levels of xenograft Ewing sarcoma metastasis in the PuMA model compared to NOD-SCID mice

Abstract Introduction: Ewing sarcoma is characterized by expression of EWS-ETS fusion proteins generated by chromosomal translocations and comprises bone and/or soft tissue tumors affecting mainly children and young adults. The majority of patients respond well to conventional therapy, but 5-year overall survival of patients with pulmonary metastasis at diagnosis or patients with recurrence remains dismal. The pulmonary metastasis assay (PuMA) is an ex vivo lung explant system developed to study the biology of lung colonization in osteosarcoma (OS), using fluorescence microscopy. The most significant among several advantage of the PuMA model over conventional assays for metastasis is that it recapitulates the initial stages of lung colonization (detachment from primary tumor, intravasation to blood circulation, and arrest to the secondary site) and allows the assessment of antimetastatic effects of a given intervention (candidate gene knockdown or drug treatment) in a 3D microenvironment. For unknown reasons, Ewing sarcoma cell lines do not survive lung colonization using NOD-SCID mice in the PuMA model. NSG mice are generated by crossing NOD-SCID with IL2γ receptor null mice (NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ). These further immunocompromised mice have emerged as successful models to study breast cancer and melanoma metastasis. We herein compared metastatic engraftment of Ewing sarcoma A673 cells in NOD-SCID versus NSG mice in the PUMA model. Methods: Metastatic tumor growth was accessed using PuMA. Briefly, 1 × 106 viable A673 dTomato labeled tumor cells were injected into NOD-SCID or NSG mice via tail veins. Mice were then euthanized, chest cavities exposed, and tracheas cannulated, and PneumaCult-ALI medium (Stem Cell Tech) mixed with 1.2% agarose was infused into the lung. Lung slices of ~1-2mm from each lung lobe were incubated in 6 well plates with PneumaCult-ALI (day 0). Lung slice images were captured on days 0, 3, 7, and 14 using a Leica Stereo Microscope to evaluate tumor growth and H&amp;E staining. Results: In NOD-SCID mice, A673 cell did not form metastatic lesions 3 days after successful injection. In NSG mice, tumor cells were able to successfully colonize the lungs (fold change of tumor burden = 1, 1.5, and 3.2 on day 0, 3, and 7, respectively). Conclusion: Our results show that NSG mice confer higher engraftment levels of xenografted A673 cells in PuMA assays compared to NOD-SCID. NSG and NOD-SCID mice share T- and B-cell depletion and loss of C5 complement, but NSG mice present extremely low NK activity and innate immunity. NOD-SCID may rely on functionally immature macrophages and residual NK activity to target Ewing sarcoma cells. Better understanding specific immunologic response in Ewing sarcoma patients and the mechanisms by which Ewing tumor cells evade immune system may help the development of novel therapy able to prevent advanced disease. Citation Format: Renata Scopim-Ribeiro, Michael M. Lizardo, Anne-Chloe Dhez, Amal M. El-Naggar, Poul H. Sorensen. Immunologic dysfunctions of NSG mice confer higher engraftment levels of xenograft Ewing sarcoma metastasis in the PuMA model compared to NOD-SCID mice [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A19.

Rare Pulmonary Connective Tissue Type Mast Cells Regulate Lung Endothelial Cell Angiogenesis

Within the human lung, mast cells typically reside adjacent to the conducting airway and assume a mucosal phenotype (MCT). In rare pathologic conditions, connective tissue phenotype mast cells (MCTCs) can be found in the lung parenchyma. MCTCs accumulate in the lungs of infants with severe bronchopulmonary dysplasia, a chronic lung disease associated with preterm birth, which is characterized by pulmonary vascular dysmorphia. The human mast cell line (LUVA) was used to model MCTCs or MCTs. The ability of MCTCs to affect vascular organization during fetal lung development was tested in mouse lung explant cultures. The effect of MCTCs on invitro tube formation and barrier function was studied using primary fetal human pulmonary microvascular endothelial cells. The mechanistic role of MCTC proteases was tested using inhibitors. MCTCLUVA but not MCTLUVA was associated with vascular dysmorphia in lung explants. Invitro, the addition of MCTCLUVA potentiated fetal human pulmonary microvascular endothelial cell interactions, inhibited tube stability, and disrupted endothelial cell junctions. Protease inhibitors ameliorated the ability of MCTCLUVA to alter endothelial cell angiogenic activities invitro and exvivo. These data indicate that MCTCs may directly contribute to disrupted angiogenesis in bronchopulmonary dysplasia. A better understanding of factors that regulate mast cell subtype and their different effector functions is essential.

Chloroquine Inhibits the Release of Inflammatory Cytokines by Human Lung Explants.

On human lung parenchymal explants, chloroquine concentration clinically achievable in the lung (100 µM) inhibited the lipopolysaccharide-induced release of TNF-ɑ (by 76%), IL-6 (by 68%), CCL2 (by 72%), and CCL3 (by 67%). Besides its antiviral activity, chloroquine might also mitigate the cytokine storm associated with severe pneumonia caused by coronaviruses.

Mitochondrial Uncoupling Protein‐2 Drives Fibroblast Senescence in Age‐Related Lung Fibrosis by Altering Bioenergetics and Reactive Oxygen Species

Background Mitochondrial dysfunction is a potential link between aging and age‐related pathology, including lung fibrosis (idiopathic pulmonary fibrosis, IPF). (Myo)Fibroblasts are the effector cells in fibrosis. Transcriptomic analyses revealed high expression of mitochondrial uncoupling protein‐2 (UCP2) in lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis, compared to healthy controls. Our objective was to examine the role of UCP2 in mediating mitochondrial dysfunction, fibroblast senescence and myofibroblast differentiation in age‐related pathologic fibrosis.MethodsWe studied tissues and fibroblasts obtained from lung explants of IPF patients and utilized the murine model of lung fibrosis and delayed resolution following bleomycin‐induced injury in aged mice. We silenced UCP2 expression using siRNA in vitro and in vivo. Specific endpoints were analyzed by RT‐PCR, SDS‐PAGE western blotting, immunofluorescence, flow cytometry, mass spectrometry and extracellular flux analysis.ResultsUCP2 gene expression was increased in lungs (whole lung lysates as well as fibroblasts) of IPF patients compared to those without IPF; in particular, mesenchymal stromal cells derived from bronchoalveolar lavage of patients with rapidly progressive clinical course had higher UCP2 expression compared to those with a slower course. Lung fibroblasts from aged mice expressed higher UCP2 than younger counterparts, more pronounced after bleomycin‐induced lung injury. The lungs as well as fibroblasts isolated from IPF patients in addition, demonstrated lower steady state ATP levels than those from non‐IPF controls. UCP2 silencing in IPF fibroblasts restored ATP levels, decreased pro‐fibrotic markers such as α‐SMA and collagen; this was associated with enhanced mitochondrial biogenesis, decreased mitochondrial ROS (superoxide and hydrogen peroxide), increased ATP‐linked oxygen consumption and decreased aerobic glycolysis. In addition, UCP2 silencing induced a decrease in senescence markers and decreased expression of senescence‐associated secretory phenotype (SASP) genes. In aged mice after bleomycin injury, therapeutic oro‐tracheal administration of UCP2 siRNA resulted in decreased lung collagen and fibrotic remodeling, indicating enhanced resolution capacity.ConclusionOur results suggest that expression of mitochondrial UCP2 in lung fibroblasts increases with age. UCP2 is highly expressed in lung fibroblasts of patients with IPF, more so in those with rapidly progressive clinical course; UCP2 drives a senescent fibroblast phenotype involving lower ATP levels and impairs resolution of bleomycin injury‐induced fibrosis in aged mice. Further studies are warranted to study the link between cellular aging and UCP2, and the therapeutic potential of targeting UCP2 in age‐related fibrotic disorders such as IPF.Support or Funding InformationNIH grants K08 HL135399 (SR); P01 HL114470 and R01 AG046210 (VJT), VA Merit Award I01BX003056 (VJT)Schematic depicting the putative role played by UCP2 in aging fibroblast phenotypes.Figure 1

Decreased Immune Activation in Cystic Fibrosis Airway Epithelium Following Lung Transplant

End-stage cystic fibrosis (CF) can be managed by lung transplantation (LT), but chronic allograft dysfunction (CLAD) limits long-term survival. While Pseudomonas aeruginosa airway infection (PsA) is a risk factor for CLAD for most lung allograft recipients, this association does not hold for CF recipients. We hypothesized that differences in host epithelial immune responses and the composition of airway microbial communities could contribute to protection from CLAD in CF recipients with PsA. We compared transcriptomes from clinical airway brushes following LT for 28 subjects, grouped by CF status and PsA culture result (CF+PsA+, n=8; CF+PsA-, n=6; CF-PsA+, n=5; and CF-PsA-, n=9) using DEseq2. For in vitro analysis of immune responses, airway epithelial cells (AEC) were isolated from lung explants (pre-LT) or surveillance airway brushes (post-LT) and grown at air-liquid interface until polarized and differentiated. Gene expression differences and DNA methylation were compared by CF status using quantitative PCR and DNA methylation array. For subjects with a positive PsA culture following LT, there were 193 differentially expressed genes between CF and non-CF airways. Pathway analysis demonstrated suppression of interferon response in CF airways. Relative to non-CF referents, CF AEC in vitro transitioned from increased expression of neutrophil-associated genes (Cxcl8 and Lcn2) pre-LT, to decreased expression of interferon-associated genes (Mx1 and Oas1) post-LT. DNA methylation analysis showed hypermethylation of promoters in type I interferon responsive genes (p = 0.002). All CF+PsA+ subjects had mucoid PsA strains while 72% of non-CF subjects had non-mucoid PsA (p <0.001). The combination of attenuated interferon responses and prevalence of mucoid strains may explain the differential outcomes in CF LT recipients with PsA.

The RNFT2/IL-3Rα axis regulates IL-3 signaling and innate immunity.

Interleukin-3 (IL-3) receptor α (IL-3Rα) is the α subunit of the ligand-specific IL-3R and initiates intracellular signaling in response to IL-3. IL-3 amplifies proinflammatory signaling and cytokine storm in murine sepsis models. Here we found that RNFT2 (RING finger transmembrane-domain containing protein 2, also TMEM118), a previously uncharacterized RING finger ubiquitin E3 ligase, negatively regulated IL-3-dependent cellular responses through IL-3Rα ubiquitination and degradation in the proteasome. In vitro, IL-3 stimulation promoted IL-3Rα proteasomal degradation dependent on RNFT2, and we identified IL-3Rα lysine 357 as a ubiquitin acceptor site. We determined that LPS priming reduces RNFT2 abundance, extends IL-3Rα half-life, and sensitizes cells to the effects of IL-3, acting synergistically to increase proinflammatory signaling. In vivo, IL-3 synergized with LPS to exacerbate lung inflammation in LPS and Pseudomonas aeruginosa-challenged mice; conversely, IL-3 neutralization reduced LPS-induced lung injury. Further, RNFT2 overexpression reduced lung inflammation and injury, whereas Rnft2 knockdown exacerbated inflammatory responses in LPS-induced murine lung injury. Last, we examined RNFT2 and IL-3Rα in human lung explants from patients with cystic fibrosis and also showed that IL-3 is elevated in mechanically ventilated critically ill humans at risk for acute respiratory distress syndrome. These results identify RNFT2 as a negative regulator of IL-3Rα and show a potential role for the RNFT2/IL-3Rα/IL-3 axis in regulating innate immune responses in the lung.