Exploring alveolar recruitability using positive end-expiratory pressure in mice overexpressing TGF-ß1: a structure-function analysis.

Pre-injured lungs are prone to injury progression in response to mechanical ventilation. Heterogeneous ventilation due to (micro)atelectases imparts injurious strains on open alveoli (known as volutrauma). Hence, recruitment of (micro)atelectases by positive end-expiratory pressure (PEEP) is necessary to interrupt this vicious circle of injury but needs to be balanced against acinar overdistension. In this study, the lung-protective potential of alveolar recruitment was investigated and balanced against overdistension in pre-injured lungs. Mice, treated with empty vector (AdCl) or adenoviral active TGF-ß1 (AdTGF-ß1) were subjected to lung mechanical measurements during descending PEEP ventilation from 12 to 0 cmH2O. At each PEEP level, recruitability tests consisting of two recruitment maneuvers followed by repetitive forced oscillation perturbations to determine tissue elastance (H) and damping (G) were performed. Finally, lungs were fixed by vascular perfusion at end-expiratory airway opening pressures (Pao) of 20, 10, 5 and 2 cmH2O after a recruitment maneuver, and processed for design-based stereology to quantify derecruitment and distension. H and G were significantly elevated in AdTGF-ß1 compared to AdCl across PEEP levels. H was minimized at PEEP = 5-8 cmH2O and increased at lower and higher PEEP in both groups. These findings correlated with increasing septal wall folding (= derecruitment) and reduced density of alveolar number and surface area (= distension), respectively. In AdTGF-ß1 exposed mice, 27% of alveoli remained derecruited at Pao = 20 cmH2O. A further decrease in Pao down to 2 cmH2O showed derecruitment of an additional 1.1 million alveoli (48%), which was linked with an increase in alveolar size heterogeneity at Pao = 2-5 cmH2O. In AdCl, decreased Pao resulted in septal folding with virtually no alveolar collapse. In essence, in healthy mice alveoli do not derecruit at low PEEP ventilation. The potential of alveolar recruitability in AdTGF-ß1 exposed mice is high. H is optimized at PEEP 5-8 cmH2O. Lower PEEP folds and larger PEEP stretches septa which results in higher H and is more pronounced in AdTGF-ß1 than in AdCl. The increased alveolar size heterogeneity at Pao = 5 cmH2O argues for the use of PEEP = 8 cmH2O for lung protective mechanical ventilation in this animal model.

Ectopic expression of C-type lectin Mincle renders mice susceptible to staphylococcal pneumonia.

Staphylococcus aureus is a prevalent pathogen in pneumonia and harbors glycolipids which may serve as molecular patterns in Mincle (Macrophage inducible C-type lectin) dependent pathogen recognition. We examined the role of Mincle in lung defense against S. aureus in WT, Mincle KO and Mincle transgenic (tg) mice. Two glycolipids, glucosyl-diacylglycerol (Glc-DAG) and diglucosyl-diacylglycerol (Glc2-DAG) were purified, of which only Glc-DAG triggered Mincle reporter cell activation and professional phagocyte responses. Proteomic profiling revealed that Glc2-DAG blocked Glc-DAG-induced cytokine responses, thereby acting as inhibitor of Glc-DAG/Mincle-signaling. WT mice responded to S. aureus with a similar lung pathology as Mincle KO mice, most likely due to Glc2-DAG-dependent inhibition of Glc-DAG/Mincle-signaling. In contrast, ectopic Mincle expression caused severe lung pathology in S. aureus-infected mice characterized by bacterial outgrowth and fatal pneumonia. Collectively, Glc2-DAG inhibits Glc-DAG/Mincle-dependent responses in WT mice, whereas sustained Mincle expression overrides Glc2-DAG-mediated inhibitory effects, conferring increased host susceptibility to S. aureus.

Inflammation-inducible promoters to overexpress immune inhibitory factors by MSCs.

BACKGROUND: Mesenchymal stromal cells (MSCs) are excessively investigated in the context of inflammation-driven diseases, but the clinical results are often moderate. MSCs are naturally activated by inflammatory signals, which lead to the secretion of immune inhibitory factors in inflamed tissues. Many work groups try to improve the therapeutic outcome of MSCs by genetic modification and the constitutive overexpression of immune modulatory transgenes. However, the ectopic secretion of immune inhibitory transgenes increases the chances of infections, and constitutive transgene expression is not necessary for chronic diseases undergoing different inflammatory stages. METHODS: We designed and tested inflammation-induced promoters to control transgene expression from integrating lentiviral vectors in human umbilical cord MSCs. Therefore, we investigated different combinations of general transcription factor elements to achieve a minimal promoter with low basal activity. The best candidates were combined with interferon-induced GAS or ISRE DNA motifs. The constructs with the highest transgene expression upon addition of pro-inflammatory cytokines were compared to vectorized promoters from inflammation-induced genes (CD317, CXCL9, CXCL10, CXCL11 and IDO1). Finally, we investigated IL10 as a potential immune inhibitory transgene by transcriptome analyses, ELISA and in an acute lung injury mouse model. RESULTS: The synthetic promoters achieved a high and specific transgene expression upon IFN-γ addition. However, the CXCL11 promoter showed synergistic activity upon IFN-γ, TNF-α and IL1-ß treatment and surpassed the transgene expression height of all tested promoters in the study. We observed in transcriptome analyses that IL10 has no effect on MSCs and in ELISA that IL10 is only secreted by our genetically modified and activated CXCL11-IL10-MSCs. Finally, transplanted CXCL11-IL10-MSCs increased CD19+ and CD4+ lymphoid cells, and decreased CD11b+ Ly6g myeloid cells in an ALI mouse model. CONCLUSION: These results provide new insights into MSC inflammatory activation and the subsequent translation into a tool for a tailored expression of transgenes in inflammatory microenvironments. The newly developed promoter elements are potentially interesting for other inflamed tissues, and can be combined with other elements or used in other cell types.

S100A9 is indispensable for survival of pneumococcal pneumonia in mice.

S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn2+ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia. Trial registration: Clinical Trials register (DRKS00000620).

Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA.

BACKGROUND: As circulating DNA (cirDNA) is mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood, apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA. METHODS: We studied (1) the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; (2) the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; and (3) the in vitro NET degradation in serum; for this, we exploited the synergistic analytical information provided by specifically quantifying DNA by qPCR, and used shallow WGS and capillary electrophoresis to perform fragment size analysis. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET-associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N = 28; systemic lupus erythematosus, N = 10; metastatic colorectal cancer, N = 10; and from healthy individuals, N = 114). RESULTS: Our studies reveal that gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24 h incubation in serum corresponded to mono-N DNA); "ex vivo" NET formation, as demonstrated by a concurrent 5-, 5-, and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2 and 41% of the remaining DNA after 2 h in serum corresponded to 1-10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8 h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO, and cirDNA was detected in plasma samples from lupus, COVID-19, and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI. CONCLUSIONS: Our work describes the mechanisms by which NET and cirDNA are linked. In doing so, we demonstrate that NET are a major source of mono-N cirDNA independent of apoptosis and establish a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions. We also demonstrate a link between immune response and cirDNA.

Neutrophilic Pleuritis Is a Severe Complication of Klebsiella pneumoniae Pneumonia in Old Mice.

The pathomechanisms underlying the frequently observed fatal outcome of Klebsiella pneumoniae pneumonia in elderly patients are understudied. In this study, we examined the early antibacterial immune response in young mice (age 2-3 mo) as compared with old mice (age 18-19 mo) postinfection with K. pneumoniae. Old mice exhibited significantly higher bacterial loads in lungs and bacteremia as early as 24 h postinfection compared with young mice, with neutrophilic pleuritis nearly exclusively developing in old but not young mice. Moreover, we observed heavily increased cytokine responses in lungs and pleural spaces along with increased mortality in old mice. Mechanistically, Nlrp3 inflammasome activation and caspase-1-dependent IL-1ß secretion contributed to the observed hyperinflammation, which decreased upon caspase-1 inhibitor treatment of K. pneumoniae-infected old mice. Irradiated old mice transplanted with the bone marrow of young mice did not show hyperinflammation or early bacteremia in response to K. pneumoniae. Collectively, the accentuated lung pathology observed in K. pneumoniae-infected old mice appears to be due to regulatory defects of the bone marrow but not the lung, while involving dysregulated activation of the Nlrp3/caspase-1/IL-1ß axis.

Loss of Serpina1 in Mice Leads to Altered Gene Expression in Inflammatory and Metabolic Pathways.

The SERPINA1 gene encodes alpha1-antitrypsin (AAT), an acute phase glycoprotein and serine protease inhibitor that is mainly (80-90%) produced in the liver. Point mutations in the SERPINA1 gene can lead to the misfolding, intracellular accumulation, and deficiency of circulating AAT protein, increasing the risk of developing chronic liver diseases or chronic obstructive pulmonary disease. Currently, siRNA technology can knock down the SERPINA1 gene and limit defective AAT production. How this latter affects other liver genes is unknown. Livers were taken from age- and sex-matched C57BL/6 wild-type (WT) and Serpina1 knockout mice (KO) aged from 8 to 14 weeks, all lacking the five serpin A1a-e paralogues. Total RNA was isolated and RNA sequencing, and transcriptome analysis was performed. The knockout of the Serpina1 gene in mice changed inflammatory, lipid metabolism, and cholesterol metabolism-related gene expression in the liver. Independent single-cell sequencing data of WT mice verified the involvement of Serpina1 in cholesterol metabolism. Our results from mice livers suggested that designing therapeutic strategies for the knockout of the SERPINA1 gene in humans must account for potential perturbations of key metabolic pathways and consequent mitigation of side effects.

Role of matrix metalloprotease-2 and MMP-9 in experimental lung fibrosis in mice.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a diffuse parenchymal lung disease characterized by exuberant deposition of extracellular matrix (ECM) proteins in the lung interstitium, which contributes to substantial morbidity and mortality in IPF patients. Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopeptidases, many of which have been implicated in the regulation of ECM degradation in lung fibrosis. However, the roles of MMP-2 and -9 (also termed gelatinases A and B) have not yet been explored in lung fibrosis in detail. METHODS: AdTGF-ß1 was applied via orotracheal routes to the lungs of WT, MMP-2 KO, MMP-9 KO and MMP-2/-9 dKO mice on day 0 to induce lung fibrosis. Using hydroxyproline assay, FlexiVent based lung function measurement, histopathology, western blot and ELISA techniques, we analyzed MMP-2 and MMP-9 levels in BAL fluid and lung, collagen contents in lung and lung function in mice on day 14 and 21 post-treatment. RESULT: IPF lung homogenates exhibited significantly increased levels of MMP-2 and MMP-9, relative to disease controls. Enzymatically active MMP-2 and MMP-9 was increased in lungs of mice exposed to adenoviral TGF-ß1, suggesting a role for these metalloproteinases in lung fibrogenesis. However, we found that neither MMP-2 or MMP-9 nor combined MMP-2/-9 deletion had any effect on experimental lung fibrosis in mice. CONCLUSION: Together, our data strongly suggest that both gelatinases MMP-2 and MMP-9 play only a subordinate role in experimental lung fibrosis in mice.

B Cells Are Not Involved in the Regulation of Adenoviral TGF-ß1- or Bleomycin-Induced Lung Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is an irreversible, age-related diffuse parenchymal lung disease of poorly defined etiology. Many patients with IPF demonstrate distinctive lymphocytic interstitial infiltrations within remodeled lung tissue with uncertain pathogenetic relevance. Histopathological examination of explant lung tissue of patients with IPF revealed accentuated lymphoplasmacellular accumulations in close vicinity to, or even infiltrating, remodeled lung tissue. Similarly, we found significant accumulations of B cells interfused with T cells within remodeled lung tissue in two murine models of adenoviral TGF-ß1 or bleomycin (BLM)-induced lung fibrosis. Such B cell accumulations coincided with significantly increased lung collagen deposition, lung histopathology, and worsened lung function in wild-type (WT) mice. Surprisingly, B cell-deficient µMT knockout mice exhibited similar lung tissue remodeling and worsened lung function upon either AdTGF-ß1 or BLM as for WT mice. Comparative transcriptomic profiling of sorted B cells collected from lungs of AdTGF-ß1- and BLM-exposed WT mice identified a large set of commonly regulated genes, but with significant enrichment observed for Gene Ontology terms apparently not related to lung fibrogenesis. Collectively, although we observed B cell accumulations in lungs of IPF patients as well as two experimental models of lung fibrosis, comparative profiling of characteristic features of lung fibrosis between WT and B cell-deficient mice did not support a major involvement of B cells in lung fibrogenesis in mice.

Intracellularly Released Cholesterol from Polymer-Based Delivery Systems Alters Cellular Responses to Pneumolysin and Promotes Cell Survival.

Cholesterol is highly abundant within all human body cells and modulates critical cellular functions related to cellular plasticity, metabolism, and survival. The cholesterol-binding toxin pneumolysin represents an essential virulence factor of Streptococcus pneumoniae in establishing pneumonia and other pneumococcal infections. Thus, cholesterol scavenging of pneumolysin is a promising strategy to reduce S. pneumoniae induced lung damage. There may also be a second cholesterol-dependent mechanism whereby pneumococcal infection and the presence of pneumolysin increase hepatic sterol biosynthesis. Here we investigated a library of polymer particles varying in size and composition that allow for the cellular delivery of cholesterol and their effects on cell survival mechanisms following pneumolysin exposure. Intracellular delivery of cholesterol by nanocarriers composed of Eudragit E100-PLGA rescued pneumolysin-induced alterations of lipid homeostasis and enhanced cell survival irrespective of neutralization of pneumolysin.

Alpha-1 antitrypsin deficiency impairs lung antibacterial immunity in mice.

Alpha-1 antitrypsin (AAT) is a major inhibitor of serine proteases in mammals. Therefore, its deficiency leads to protease-antiprotease imbalance and a risk for developing lung emphysema. Although therapy with human plasma-purified AAT attenuates AAT deficiency-related emphysema, its impact on lung antibacterial immunity is poorly defined. Here, we examined the effect of AAT therapy on lung protective immunity in AAT-deficient (KO) mice challenged with Streptococcus pneumoniae. AAT-KO mice were highly susceptible to S. pneumoniae, as determined by severe lobar pneumonia and early mortality. Mechanistically, we found that neutrophil-derived elastase (NE) degraded the opsonophagocytically important collectins, surfactant protein A (SP-A) and D (SP-D), which was accompanied by significantly impaired lung bacterial clearance in S. pneumoniae-infected AAT-KO mice. Treatment of S. pneumoniae-infected AAT-KO mice with human AAT protected SP-A and SP-D from NE-mediated degradation and corrected the pulmonary pathology observed in these mice. Likewise, treatment with Sivelestat, a specific inhibitor of NE, also protected collectins from degradation and significantly decreased bacterial loads in S. pneumoniae-infected AAT-KO mice. Our findings show that NE is responsible for the degradation of lung SP-A and SP-D in AAT-KO mice affecting lung protective immunity in AAT deficiency.

Role of the COX2-PGE2 axis in S. pneumoniae-induced exacerbation of experimental fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease (ILD) associated with high morbidity and mortality. Patients with ILD frequently develop an acute exacerbation of their disease, which may be triggered by viral and/or bacterial infections. Prostaglandin E2 (PGE2) is an eicosanoid released in a cyclooxygenase-2 (COX2)-dependent manner and is considered to contribute to regulation of lung fibrosis. However, its role in infection-induced exacerbation of lung fibrosis is poorly defined. We found significantly increased levels of PGE2 in lung tissue of patients with ILD. Increased levels of PGE2 were also found in lung tissue of mice with AdTGF-ß1-induced lung fibrosis and even more so in Streptococcus pneumoniae exacerbated lung fibrosis. Type II alveolar epithelial cells (AT II cells) and alveolar macrophages (AM) contributed to PGE2 release during exacerbating fibrosis. Application of parecoxib to inhibit PGE2 synthesis ameliorated lung fibrosis, whereas intratracheal application of PGE2 worsened lung fibrosis in mice. Both interventions had no effect on S. pneumoniae-exacerbated lung fibrosis. Together, we found that the COX2-PGE2 axis has dual roles in fibrosis that may offset each other: PGE2 helps resolve infection/attenuate inflammation in fibrosis exacerbation but accentuates TGF-ß/AT II cell-mediated fibrosis. These data support the efficacy of COX/PGE2 interventions in the setting of non-exacerbating lung fibrosis.

Overexpression of Macrophage-Inducible C-Type Lectin Mincle Aggravates Proinflammatory Responses to Streptococcus pneumoniae with Fatal Outcome in Mice.

Macrophage-inducible C-type lectin (Mincle)-dependent sensing of pathogens triggers proinflammatory immune responses in professional phagocytes that contribute to protecting the host against pathogen invasion. In this study, we examined whether overexpression of Mincle that is designed to improve early pathogen sensing by professional phagocytes would improve lung-protective immunity against Streptococcus pneumoniae in mice. Proteomic profiling of alveolar macrophages of Mincle transgenic (tg) mice stimulated with the Mincle-specific pneumococcal ligand glucosyl-diacylglycerol (Glc-DAG) revealed increased Nlrp3 inflammasome activation and downstream IL-1ß cytokine release that was not observed in Glc-DAG-stimulated Mincle knockout or Nlrp3 knockout macrophages. Along this line, Mincle tg mice also responded with a stronger Nlrp3 expression and early proinflammatory cytokine release after challenge with S. pneumoniae, ultimately leading to fatal pneumonia in the Mincle tg mice. Importantly, Nlrp3 inhibitor treatment of Mincle tg mice significantly mitigated the observed hyperinflammatory response to pneumococcal challenge. Together, we show that overexpression of the pattern recognition receptor Mincle triggers increased Glc-DAG-dependent Nlrp3 inflammasome activation in professional phagocytes leading to fatal pneumococcal pneumonia in mice that is amenable to Nlrp3 inhibitor treatment. These data show that ectopic expression of the Mincle receptor confers increased susceptibility rather than resistance to S. pneumoniae in mice, thus highlighting the importance of an inducible Mincle receptor expression in response to microbial challenge.

Regulatory T Cells Limit Pneumococcus-Induced Exacerbation of Lung Fibrosis in Mice.

Patients with idiopathic pulmonary fibrosis (IPF) can experience life-threatening episodes of acute worsening of their disease, termed acute exacerbation of IPF, which may be caused by bacterial and/or viral infections. The potential for regulatory T cells (Tregs) to limit disease progression in bacterially triggered fibrosis exacerbation has not been explored so far. In the current study, we show that the number of Tregs was significantly increased in mice with established AdTGF-ß1-induced lung fibrosis and further increased in mice with pneumococcal infection-induced lung fibrosis exacerbation. Diphtheria toxin-induced depletion of Tregs significantly worsened infection-induced fibrosis exacerbation as determined by increased lung collagen deposition, lung histology, and elevated pulmonary Th1/Th2 cytokine levels. Conversely, IL-2 complex-induced Treg expansion in wild-type mice with established lung fibrosis completely inhibited pneumococcal infection-induced fibrosis exacerbation as efficaciously as antibiotic treatment while preserving lung antibacterial immunity in mice. Collectively, these findings demonstrate the efficacy of Tregs as "silencers," suppressing infection-induced exacerbation of lung fibrosis in mice, and their expansion may offer a novel adjunctive treatment to limit acute exacerbations in patients with IPF.

Synthesis of α-Glucosyl Diacylglycerides as potential adjuvants for Streptococcus pneumoniae vaccines.

α-Glucosyl diacylglycerols (αGlc-DAGs) play an important role in providing protective immunity against Streptococcus pneumoniae infection through the engagement of the Macrophage inducible C-type lectin (Mincle). Herein, we efficiently synthesised αGlc-DAGs containing C12, C14, C16 and C18 acyl chains in 7 steps and 44-47% overall yields, and demonstrated that Mincle signaling was dependent on lipid length using mMincle and hMincle NFAT-GFP reporter cells. The greatest production of GFP in both cell types was elicited by C14 αGlc-DAG. Accordingly, C14 αGlc-DAG has potential to act as an adjuvant to augment the immune response against S. pneumoniae antigens.

A comparison of curated gene sets versus transcriptomics-derived gene signatures for detecting pathway activation in immune cells.

BACKGROUND: Despite the significant contribution of transcriptomics to the fields of biological and biomedical research, interpreting long lists of significantly differentially expressed genes remains a challenging step in the analysis process. Gene set enrichment analysis is a standard approach for summarizing differentially expressed genes into pathways or other gene groupings. Here, we explore an alternative approach to utilizing gene sets from curated databases. We examine the method of deriving custom gene sets which may be relevant to a given experiment using reference data sets from previous transcriptomics studies. We call these data-derived gene sets, "gene signatures" for the biological process tested in the previous study. We focus on the feasibility of this approach in analyzing immune-related processes, which are complicated in their nature but play an important role in the medical research. RESULTS: We evaluate several statistical approaches to detecting the activity of a gene signature in a target data set. We compare the performance of the data-derived gene signature approach with comparable GO term gene sets across all of the statistical tests. A total of 61 differential expression comparisons generated from 26 transcriptome experiments were included in the analysis. These experiments covered eight immunological processes in eight types of leukocytes. The data-derived signatures were used to detect the presence of immunological processes in the test data with modest accuracy (AUC = 0.67). The performance for GO and literature based gene sets was worse (AUC = 0.59). Both approaches were plagued by poor specificity. CONCLUSIONS: When investigators seek to test specific hypotheses, the data-derived signature approach can perform as well, if not better than standard gene-set based approaches for immunological signatures. Furthermore, the data-derived signatures can be generated in the cases that well-defined gene sets are lacking from pathway databases and also offer the opportunity for defining signatures in a cell-type specific manner. However, neither the data-derived signatures nor standard gene-sets can be demonstrated to reliably provide negative predictions for negative cases. We conclude that the data-derived signature approach is a useful and sometimes necessary tool, but analysts should be weary of false positives.

Four hours of veno-venous extracorporeal membrane oxygenation using bi-caval cannulation affects kidney function and induces moderate lung damage in a mouse model.

BACKGROUND: Improvement of single site cannulation for extracorporeal membrane oxygenation (ECMO) therapy is pivotal for reduction of patient morbidity and mortality in respiratory failure. To further improve the cardiopulmonary outcomes and reduce end organ damage, we established a murine model for single site cannulation with a double lumen cannula. RESULTS: We created a hemodynamically stable double lumen cannula and successfully implanted it through the jugular vein into the upper and lower vena cava. This allowed adequate drainage of the blood. Blood gas analysis showed excellent oxygenation and CO2 reduction. There was no excessive bleeding. No signs of right heart congestion were present which was confirmed in the histological analysis of the liver. Histology demonstrated moderate lung damage and mild acute kidney injury. Neutrophil infiltration was similar in ECMO and sham kidneys. CONCLUSIONS: Veno-venous extracorporeal circulation deteriorates kidney function and promotes moderate pulmonary damage.

The FMS-like tyrosine kinase-3 ligand/lung dendritic cell axis contributes to regulation of pulmonary fibrosis.

RATIONALE: Dendritic cells (DC) accumulate in the lungs of patients with idiopathic lung fibrosis, but their pathogenetic relevance is poorly defined. OBJECTIVES: To assess the role of the FMS-like tyrosine kinase-3 ligand (Flt3L)-lung dendritic cell axis in lung fibrosis. MEASUREMENTS AND MAIN RESULTS: We demonstrate in a model of adenoviral gene transfer of active TGF-ß1 that established lung fibrosis was accompanied by elevated serum Flt3L levels and subsequent accumulation of CD11bpos DC in the lungs of mice. Patients with idiopathic pulmonary fibrosis also demonstrated increased levels of Flt3L protein in serum and lung tissue and accumulation of lung DC in explant subpleural lung tissue specimen. Mice lacking Flt3L showed significantly reduced lung DC along with worsened lung fibrosis and reduced lung function relative to wild-type (WT) mice, which could be inhibited by administration of recombinant Flt3L. Moreover, therapeutic Flt3L increased numbers of CD11bpos DC and improved lung fibrosis in WT mice exposed to AdTGF-ß1. In this line, RNA-sequencing analysis of CD11bpos DC revealed significantly enriched differentially expressed genes within extracellular matrix degrading enzyme and matrix metalloprotease gene clusters. In contrast, the CD103pos DC subset did not appear to be involved in pulmonary fibrogenesis. CONCLUSIONS: We show that Flt3L protein and numbers of lung DC are upregulated in mice and humans during pulmonary fibrogenesis, and increased mobilisation of lung CD11bpos DC limits the severity of lung fibrosis in mice. The current study helps to inform the development of DC-based immunotherapy as a novel intervention against lung fibrosis in humans.

Surfactant dysfunction and alveolar collapse are linked with fibrotic septal wall remodeling in the TGF-ß1-induced mouse model of pulmonary fibrosis.

In human idiopathic pulmonary fibrosis (IPF), collapse of distal airspaces occurs in areas of the lung not (yet) remodeled. Mice lungs overexpressing transforming growth factor-ß1 (TGF-ß1) recapitulate this abnormality: surfactant dysfunction results in alveolar collapse preceding fibrosis and loss of alveolar epithelial type II (AE2) cells' apical membrane surface area. Here we examined whether surfactant dysfunction-related alveolar collapse due to TGF-ß1 overexpression is linked to septal wall remodeling and AE2 cell abnormalities. Three and 6 days after gene transfer of TGF-ß1, mice received either intratracheal surfactant (Surf-groups: Curosurf®, 100 mg/kg bodyweight) or 0.9% NaCl (Saline-groups). On days 7 (D7) and 14 (D14), lung mechanics were assessed followed by design-based stereology at light and electron microscopic level to quantify structures. Compared with Saline, Surf showed significantly improved tissue elastance, increased numbers of open alveoli, as well as reduced alveolar size heterogeneity on D7. Deterioration in lung mechanics was highly correlated to the loss of open alveoli. On D14, lung mechanics, number of open alveoli, and alveolar size heterogeneity remained significantly improved in the Surf-group. Volumes of extracellular matrix and collagen fibrils in septal walls were significantly reduced, whereas the apical membrane surface area of AE2 cells was increased in Surf compared with Saline. In remodeled tissue with collapsed alveoli, three-dimensional reconstruction of AE2 cells based on scanning electron microscopy array tomography revealed that AE2 cells were trapped without contact to airspaces in the TGF-ß1 mouse model. Similar observations were made in human IPF. Based on correlation analyses, the number of open alveoli and of alveolar size heterogeneity were highly linked with the loss of apical membrane surface area of AE2 cells and deposition of collagen fibrils in septal walls on D14. In conclusion, surfactant replacement therapy stabilizes alveoli and prevents extracellular matrix deposition in septal walls in the TGF-ß1 model.