Mild respiratory COVID can cause multi-lineage neural cell and myelin dysregulation.

COVID survivors frequently experience lingering neurological symptoms that resemble cancer-therapy-related cognitive impairment, a syndrome for which white matter microglial reactivity and consequent neural dysregulation is central. Here, we explored the neurobiological effects of respiratory SARS-CoV-2 infection and found white-matter-selective microglial reactivity in mice and humans. Following mild respiratory COVID in mice, persistently impaired hippocampal neurogenesis, decreased oligodendrocytes, and myelin loss were evident together with elevated CSF cytokines/chemokines including CCL11. Systemic CCL11 administration specifically caused hippocampal microglial reactivity and impaired neurogenesis. Concordantly, humans with lasting cognitive symptoms post-COVID exhibit elevated CCL11 levels. Compared with SARS-CoV-2, mild respiratory influenza in mice caused similar patterns of white-matter-selective microglial reactivity, oligodendrocyte loss, impaired neurogenesis, and elevated CCL11 at early time points, but after influenza, only elevated CCL11 and hippocampal pathology persisted. These findings illustrate similar neuropathophysiology after cancer therapy and respiratory SARS-CoV-2 infection which may contribute to cognitive impairment following even mild COVID.

IFN-γ Is Protective in Cytokine Release Syndrome-associated Extrapulmonary Acute Lung Injury.

The mechanisms by which excessive systemic activation of adaptive T lymphocytes, as in cytokine release syndrome (CRS), leads to innate immune cell-mediated acute lung injury (ALI) or acute respiratory distress syndrome, often in the absence of any infection, remains unknown. Here, we investigated the roles of IFN-γ and IL-17A, key T-cell cytokines significantly elevated in patients with CRS, in the immunopathogenesis of CRS-induced extrapulmonary ALI. CRS was induced in wild-type (WT), IL-17A- and IFN-γ knockout (KO) human leukocyte antigen-DR3 transgenic mice with 10 µg of the superantigen, staphylococcal enterotoxin B, given intraperitoneally. Several ALI parameters, including gene expression profiling in the lungs, were studied 4, 24, or 48 hours later. Systemic T-cell activation with staphylococcal enterotoxin B resulted in robust upregulation of several chemokines, S100A8/A9, matrix metalloproteases, and other molecules implicated in tissue damage, granulocyte as well as agranulocyte adhesion, and diapedesis in the lungs as early as 4 hours, which was accompanied by subsequent neutrophil/eosinophil lung infiltration and severe ALI in IFN-γ KO mice. These pathways were significantly underexpressed in IL-17A KO mice, which manifested mildest ALI and intermediate in WT mice. Neutralization of IFN-γ worsened ALI in WT and IL-17A KO mice, whereas neutralizing IL-17A did not mitigate lung injury in IFN-γ KO mice, suggesting a dominant protective role for IFN-γ in ALI and that IL-17A is dispensable. Ruxolitinib, a Janus kinase inhibitor, increased ALI severity in WT mice. Thus, our study identified novel mechanisms of ALI in CRS and its differential modulation by IFN-γ and IL-17A.

Thymus-derived B cell clones persist in the circulation after thymectomy in myasthenia gravis.

Myasthenia gravis (MG) is a neuromuscular, autoimmune disease caused by autoantibodies that target postsynaptic proteins, primarily the acetylcholine receptor (AChR) and inhibit signaling at the neuromuscular junction. The majority of patients under 50 y with AChR autoantibody MG have thymic lymphofollicular hyperplasia. The MG thymus is a reservoir of plasma cells that secrete disease-causing AChR autoantibodies and although thymectomy improves clinical scores, many patients fail to achieve complete stable remission without additional immunosuppressive treatments. We speculate that thymus-associated B cells and plasma cells persist in the circulation after thymectomy and that their persistence could explain incomplete responses to resection. We studied patients enrolled in a randomized clinical trial and used complementary modalities of B cell repertoire sequencing to characterize the thymus B cell repertoire and identify B cell clones that resided in the thymus and circulation before and 12 mo after thymectomy. Thymus-associated B cell clones were detected in the circulation by both mRNA-based and genomic DNA-based sequencing. These antigen-experienced B cells persisted in the circulation after thymectomy. Many circulating thymus-associated B cell clones were inferred to have originated and initially matured in the thymus before emigration from the thymus to the circulation. The persistence of thymus-associated B cells correlated with less favorable changes in clinical symptom measures, steroid dose required to manage symptoms, and marginal changes in AChR autoantibody titer. This investigation indicates that the diminished clinical response to thymectomy is related to persistent circulating thymus-associated B cell clones.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

Integrated Single-Cell Atlas of Endothelial Cells of the Human Lung.

BACKGROUND: Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. METHODS: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. RESULTS: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1, and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic-venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). CONCLUSIONS: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

Development of an immunohistochemical assay for Siglec-15.

Siglec-15, a member of sialic-acid binding immunoglobulin type lectins, is normally expressed by myeloid cells and upregulated in some human cancers and represents a promising new target for immunotherapy. While PD-L1 blockade is an important strategy for immunotherapy, its effectiveness is limited. The expression of Siglec-15 has been demonstrated to be predominantly mutually exclusive to PD-L1 in certain cancer histologies. Thus, there is significant opportunity for Siglec-15 as an immunotherapeutic target for patients that do not respond to PD-1/PD-L1 inhibition. The aim of this study was to prospectively develop an immunohistochemical (IHC) assay for Siglec-15 to be used as a companion diagnostic for future clinical trials. Here, we create and validate an IHC assay with a novel recombinant antibody to the cytoplasmic domain of Siglec-15. To find an enriched target, this antibody was first used in a quantitative fluorescence (QIF) assay to screen a broad range of tumor histologies to determine tumor types where Siglec-15 demonstrated high expression. Based on this and previous data, we focused on development of a chromogenic IHC assay for lung cancer. Then we developed a scoring system for this assay that has high concordance amongst pathologist readers. We then use this chromogenic IHC assay to test the expression of Siglec-15 in two cohorts of NSCLC. We found that this assay shows a higher level of staining in both tumor and immune cells compared to previous QIF assays utilizing a polyclonal antibody. However, similar to that study, only a small percentage of positive Siglec-15 cases showed high expression for PD-L1. This validated assay for Siglec-15 expression may support development of a companion diagnostic assay to enrich for patients expressing the Siglec-15 target for therapy.

Low ambient humidity impairs barrier function and innate resistance against influenza infection.

In the temperate regions, seasonal influenza virus outbreaks correlate closely with decreases in humidity. While low ambient humidity is known to enhance viral transmission, its impact on host response to influenza virus infection and disease outcome remains unclear. Here, we showed that housing Mx1 congenic mice in low relative humidity makes mice more susceptible to severe disease following respiratory challenge with influenza A virus. We find that inhalation of dry air impairs mucociliary clearance, innate antiviral defense, and tissue repair. Moreover, disease exacerbated by low relative humidity was ameliorated in caspase-1/11-deficient Mx1 mice, independent of viral burden. Single-cell RNA sequencing revealed that induction of IFN-stimulated genes in response to viral infection was diminished in multiple cell types in the lung of mice housed in low humidity condition. These results indicate that exposure to dry air impairs host defense against influenza infection, reduces tissue repair, and inflicts caspase-dependent disease pathology.

Effectiveness of Thermal Ablation and Stereotactic Radiotherapy Based on Stage I Lung Cancer Histology.

PURPOSE: To assess whether the effectiveness of thermal ablation (TA) and stereotactic body radiotherapy (SBRT) as initial treatments for stage I lung cancer varies depending on the histological subtype. MATERIALS AND METHODS: The 2004-2016 National Cancer Database was queried for patients with American Joint Committee on Cancer stage I lung cancer treated with TA or SBRT. Patients <18 years, those treated with surgery or chemotherapy, or those with unknown survival and follow-up were excluded. TA and SBRT patients were 1:5 propensity score matched separately for each histological subtype to adjust for confounders. Overall survival (OS) was assessed using Cox models. RESULTS: A total of 28,425 patients were included (SBRT, n = 27,478; TA, n = 947). TA was more likely to be used in Caucasian patients, those with more comorbidities and smaller neuroendocrine tumors (NETs) of the lower lobe, and those whose treatment had taken place in the northeastern United States. After propensity score matching, a cohort with 4,085 SBRT and 817 TA patients with balanced confounders was obtained. In this cohort, OS for TA and SBRT was comparable (hazard ratio = 1.07; 95% confidence interval,0.98-1.18; P = .13), although it varied by histological subtypes: higher OS for TA was observed in patients with non-small cell NETs (vs SBRT hazard ratio = 0.48; 95% confidence interval, 0.24-0.95; P = .04). No significant OS differences between TA and SBRT were noted for adenocarcinomas, squamous cell carcinomas, small cell carcinomas, and non-neuroendocrine large cell carcinomas (each, P > .1). CONCLUSIONS: OS following TA and SBRT for stage I lung cancer is comparable for most histological subtypes, except that OS is longer after TA in non-small cell NETs.

Reliability of histopathologic diagnosis of fibrotic interstitial lung disease: an international collaborative standardization project.

BACKGROUND: Current interstitial lung disease (ILD) diagnostic guidelines assess criteria across clinical, radiologic and pathologic domains. Significant interobserver variation in histopathologic evaluation has previously been shown but the specific source of these discrepancies is poorly documented. We sought to document specific areas of difficulty and develop improved criteria that would reduce overall interobserver variation. METHODS: Using an internet-based approach, we reviewed selected images of specific diagnostic features of ILD histopathology and whole slide images of fibrotic ILD. After an initial round of review, we confirmed the presence of interobserver variation among our group. We then developed refined criteria and reviewed a second set of cases. RESULTS: The initial round reproduced the existing literature on interobserver variation in diagnosis of ILD. Cases which were pre-selected as inconsistent with usual interstitial pneumonia/idiopathic pulmonary fibrosis (UIP/IPF) were confirmed as such by multi-observer review. Cases which were thought to be in the spectrum of chronic fibrotic ILD for which UIP/IPF were in the differential showed marked variation in nearly all aspects of ILD evaluation including extent of inflammation and extent and pattern of fibrosis. A proposed set of more explicit criteria had only modest effects on this outcome. While we were only modestly successful in reducing interobserver variation, we did identify specific reasons that current histopathologic criteria of fibrotic ILD are not well defined in practice. CONCLUSIONS: Any additional classification scheme must address interobserver variation in histopathologic diagnosis of fibrotic ILD order to remain clinically relevant. Improvements to tissue-based diagnostics may require substantial resources such as larger datasets or novel technologies to improve reproducibility. Benchmarks should be established for expected outcomes among clinically defined subgroups as a quality metric.

False-positive pathology: improving reproducibility with the next generation of pathologists.

The external validity of the scientific literature has recently come into question, popularly referred to as the "reproducibility crisis." It is now generally acknowledged that too many false positive or non-reproducible results are being published throughout the biomedical and social science literature due to misaligned incentives and poor methodology. Pathology is likely no exception to this problem, and may be especially prone to false positives due to common observational methodologies used in our research. Spurious findings in pathology contribute inefficiency to the scientific literature and detrimentally influence patient care. In particular, false positives in pathology affect patients through biomarker development, prognostic classification, and cancer overdiagnosis. We discuss possible sources of non-reproducible pathology studies and describe practical ways our field can improve research habits, especially among trainees.

Reanalysis of the NCCN PD-L1 companion diagnostic assay study for lung cancer in the context of PD-L1 expression findings in triple-negative breast cancer.

The companion diagnostic test for checkpoint inhibitor immune therapy is an immunohistochemical test for PD-L1. The test has been shown to be reproducible for expression in tumor cells, but not in immune cells. Immune cells were used in the IMpassion130 trial which showed PD-L1 expression was associated with a better outcome. Two large studies have been done assessing immune cell PD-L1 expression in lung cancer. Here, we reanalyze one of those studies, to show that, even with an easier scoring method, there is still only poor agreement between assays and pathologist for immune cell PD-L1 expression.

SH2 Domain-Containing Phosphatase-2 Is a Novel Antifibrotic Regulator in Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease with dismal prognosis and no cure. The potential role of the ubiquitously expressed SH2 domain-containing tyrosine phosphatase-2 (SHP2) as a therapeutic target has not been studied in IPF. OBJECTIVES: To determine the expression, mechanistic role, and potential therapeutic usefulness of SHP2 in pulmonary fibrosis. METHODS: The effects of SHP2 overexpression and inhibition on fibroblast response to profibrotic stimuli were analyzed in vitro in primary human and mouse lung fibroblasts. In vivo therapeutic effects were assessed in the bleomycin model of lung fibrosis by SHP2-lentiviral administration and transgenic mice carrying a constitutively active SHP2 mutation. MEASUREMENTS AND MAIN RESULTS: SHP2 was down-regulated in lungs and lung fibroblasts obtained from patients with IPF. Immunolocalization studies revealed that SHP2 was absent within fibroblastic foci. Loss of SHP2 expression or activity was sufficient to induce fibroblast-to-myofibroblast differentiation in primary human lung fibroblasts. Overexpression of constitutively active SHP2 reduced the responsiveness of fibroblasts to profibrotic stimuli, including significant reductions in cell survival and myofibroblast differentiation. SHP2 effects were mediated through deactivation of fibrosis-relevant tyrosine kinase and serine/threonine kinase signaling pathways. Mice carrying the Noonan syndrome-associated gain-of-function SHP2 mutation (SHP2D61G/+) were resistant to bleomycin-induced pulmonary fibrosis. Restoration of SHP2 levels in vivo through lentiviral delivery blunted bleomycin-induced pulmonary fibrosis. CONCLUSIONS: Our data suggest that SHP2 is an important regulator of fibroblast differentiation, and its loss as observed in IPF facilitates profibrotic phenotypic changes. Augmentation of SHP2 activity or expression should be investigated as a novel therapeutic strategy for IPF.

Plexin C1 deficiency permits synaptotagmin 7-mediated macrophage migration and enhances mammalian lung fibrosis.

Pulmonary fibrosis is a progressive and often fatal condition that is believed to be partially orchestrated by macrophages. Mechanisms that control migration of these cells into and within the lung remain undefined. We evaluated the contributions of the semaphorin receptor, plexin C1 (PLXNC1), and the exocytic calcium sensor, synaptotagmin 7 (Syt7), in these processes. We evaluated the role of PLXNC1 in macrophage migration by using Boyden chambers and scratch tests, characterized its contribution to experimentally induced lung fibrosis in mice, and defined the mechanism for our observations. Our findings reveal that relative to control participants, patients with idiopathic pulmonary fibrosis demonstrate excessive monocyte migration and underexpression of PLXNC1 in the lungs and circulation, a finding that is recapitulated in the setting of scleroderma-related interstitial lung disease. Relative to wild type, PLXNC1-/- mouse macrophages are excessively migratory, and PLXNC1-/- mice show exacerbated collagen accumulation in response to either inhaled bleomycin or inducible lung targeted TGF-ß1 overexpression. These findings are ameliorated by replacement of PLXNC1 on bone marrow-derived cells or by genetic deletion of Syt7. These data demonstrate the previously unrecognized observation that PLXNC1 deficiency permits Syt7-mediated macrophage migration and enhances mammalian lung fibrosis.-Peng, X., Moore, M., Mathur, A., Zhou, Y., Sun, H., Gan, Y., Herazo-Maya, J. D., Kaminski, N., Hu, X., Pan, H., Ryu, C., Osafo-Addo, A., Homer, R. J., Feghali-Bostwick, C., Fares, W. H., Gulati, M., Hu, B., Lee, C.-G., Elias, J. A., Herzog, E. L. Plexin C1 deficiency permits synaptotagmin 7-mediated macrophage migration and enhances mammalian lung fibrosis.

Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues. RESULTS: We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four. CONCLUSIONS: Our results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.

Inhibition of Regulatory-Associated Protein of Mechanistic Target of Rapamycin Prevents Hyperoxia-Induced Lung Injury by Enhancing Autophagy and Reducing Apoptosis in Neonatal Mice.

Administration of supplemental oxygen remains a critical clinical intervention for survival of preterm infants with respiratory failure. However, prolonged exposure to hyperoxia can augment pulmonary damage, resulting in developmental lung diseases embodied as hyperoxia-induced acute lung injury and bronchopulmonary dysplasia (BPD). We sought to investigate the role of autophagy in hyperoxia-induced apoptotic cell death in developing lungs. We identified increased autophagy signaling in hyperoxia-exposed mouse lung epithelial-12 cells, freshly isolated fetal type II alveolar epithelial cells, lungs of newborn wild-type mice, and human newborns with respiratory distress syndrome and evolving and established BPD. We found that hyperoxia exposure induces autophagy in a Trp53-dependent manner in mouse lung epithelial-12 cells and in neonatal mouse lungs. Using pharmacological inhibitors and gene silencing techniques, we found that the activation of autophagy, upon hyperoxia exposure, demonstrated a protective role with an antiapoptotic response. Specifically, inhibiting regulatory-associated protein of mechanistic target of rapamycin (RPTOR) in hyperoxia settings, as evidenced by wild-type mice treated with torin2 or mice administered (Rptor) silencing RNA via intranasal delivery or Rptor+/-, limited lung injury by increased autophagy, decreased apoptosis, improved lung architecture, and increased survival. Furthermore, we identified increased protein expression of phospho-beclin1, light chain-3-II and lysosomal-associated membrane protein 1, suggesting altered autophagic flux in the lungs of human neonates with established BPD. Collectively, our study unveils a novel demonstration of enhancing autophagy and antiapoptotic effects, specifically through the inhibition of RPTOR as a potentially useful therapeutic target for the treatment of hyperoxia-induced acute lung injury and BPD in developing lungs.

Increased susceptibility of Cftr-/- mice to LPS-induced lung remodeling.

Cystic fibrosis (CF) is caused by homozygous mutations of the CF transmembrane conductance regulator (CFTR) Cl(-) channel, which result in chronic pulmonary infection and inflammation, the major cause of morbidity and mortality. Although these processes are clearly related to each other, each is likely to contribute to the pathology differently. Understanding the contribution of each of these processes to the overall pathology has been difficult, because they are usually so intimately connected. Various CF mouse models have demonstrated abnormal immune responses compared with wild-type (WT) littermates when challenged with live bacteria or bacterial products acutely. However, these studies have not investigated the consequences of persistent inflammation on lung tissue in CF mice, which may better model the lung pathology in patients. We characterized the lung pathology and immune response of Cftr(-/-) (CF) and Cftr(+/+) (WT) mice to chronic administration of Pseudomonas aeruginosa lipopolysaccharide (LPS). We show that, after long-term repeated LPS exposure, CF mice develop an abnormal and persistent immune response, which is associated with more robust structural changes in the lung than those observed in WT mice. Although CF mice and their WT littermates develop lung pathology after chronic exposure to LPS, the inflammation and damage resolve in WT mice. However, CF mice do not recover efficiently, and, as a consequence of their chronic inflammation, CF mice are more susceptible to morphological changes and lung remodeling. This study shows that chronic inflammation alone contributes significantly to aspects of CF lung pathology.

Cathepsin E promotes pulmonary emphysema via mitochondrial fission.

Emphysema is characterized by loss of lung elasticity and irreversible air space enlargement, usually in the later decades of life. The molecular mechanisms of emphysema remain poorly defined. We identified a role for a novel cathepsin, cathepsin E, in promoting emphysema by inducing mitochondrial fission. Unlike previously reported cysteine cathepsins, which have been implicated in cigarette smoke-induced lung disease, cathepsin E is a nonlysosomal intracellular aspartic protease whose function has been described only in antigen processing. We examined lung tissue sections of persons with chronic obstructive pulmonary disease, a clinical entity that includes emphysematous change. Human chronic obstructive pulmonary disease lungs had markedly increased cathepsin E protein in the lung epithelium. We generated lung epithelial-targeted transgenic cathepsin E mice and found that they develop emphysema. Overexpression of cathepsin E resulted in increased E3 ubiquitin ligase parkin, mitochondrial fission protein dynamin-related protein 1, caspase activation/apoptosis, and ultimately loss of lung parenchyma resembling emphysema. Inhibiting dynamin-related protein 1, using a small molecule inhibitor in vitro or in vivo, inhibited cathepsin E-induced apoptosis and emphysema. To the best of our knowledge, our study is the first to identify links between cathepsin E, mitochondrial fission, and caspase activation/apoptosis in the pathogenesis of pulmonary emphysema. Our data expand the current understanding of molecular mechanisms of emphysema development and may provide new therapeutic targets.

Conditional overexpression of TGFß1 promotes pulmonary inflammation, apoptosis and mortality via TGFßR2 in the developing mouse lung.

BACKGROUND: Earlier studies have reported that transforming growth factor beta 1(TGFß1) is a critical mediator of hyperoxia-induced acute lung injury (HALI) in developing lungs, leading to impaired alveolarization and a pulmonary phenotype of bronchopulmonary dysplasia (BPD). However, the mechanisms responsible for the TGFß1-induced inflammatory signals that lead to cell death and abnormal alveolarization are poorly understood. We hypothesized that TGFß1 signaling via TGFßR2 is necessary for the pathogenesis of the BPD pulmonary phenotype resulting from HALI. METHODS: We utilized lung epithelial cell-specific TGFß1 overexpressing transgenic and TGFßR2 null mutant mice to evaluate the effects on neonatal mortality as well as pulmonary inflammation and apoptosis in developing lungs. Lung morphometry was performed to determine the impaired alveolarization and multicolor flow cytometry studies were performed to detect inflammatory macrophages and monocytes in lungs. Apoptotic cell death was measured with TUNEL assay, immunohistochemistry and western blotting and protein expression of angiogenic mediators were also analyzed. RESULTS: Our data reveals that increased TGFß1 expression in newborn mice lungs leads to increased mortality, macrophage and immature monocyte infiltration, apoptotic cell death specifically in Type II alveolar epithelial cells (AECs), impaired alveolarization, and dysregulated angiogenic molecular markers. CONCLUSIONS: Our study has demonstrated the potential role of inhibition of TGFß1 signaling via TGFßR2 for improved survival, reduced inflammation and apoptosis that may provide insights for the development of potential therapeutic strategies targeted against HALI and BPD.