Vassilis Aidinis

Modeling pulmonary fibrosis with bleomycin.

Purpose of review Idiopathic pulmonary fibrosis is a chronic pulmonary disease of unknown origin ultimately leading to death. No treatment exists yet and animal models have been employed in order to elucidate its etiopathogenesis. Here, we summarize the characteristics of the bleomycin animal model, the most commonly used model of pulmonary fibrosis, highlighting recent advances it has led us to despite its disadvantages. Recent findings Repetitive intratracheal administration of bleomycin more effectively mimics the chronic aspect of pulmonary fibrosis, as well as other characteristics including the presence of hyperplastic alveolar epithelial cells. Epithelial-to-mesenchymal transition seems to be a major contributor to the lung fibroblast population. There is growing evidence that macrophages, as well as fibrocytes, are largely involved in disease progression by mediating fibroblast and myofibroblast activation. In addition, molecules involved in phospholipid homeostasis are now becoming more appealing as potential therapeutic targets. Summary Despite its disadvantages, the bleomycin animal model remains the best available experimental tool for studying disease pathogenesis and testing of novel pharmaceutical compounds. Two such compounds currently showing some promise in clinical trials have emerged through the bleomycin model and preliminary results of basic research using this model have shown that more candidate compounds may follow in the near future.

The RNA-Binding Protein Elavl1/HuR Is Essential for Placental Branching Morphogenesis and Embryonic Development

ABSTRACT HuR is an RNA-binding protein implicated in a diverse array of pathophysiological processes due to its effects on the posttranscriptional regulation of AU- and U-rich mRNAs. Here we reveal HuRs requirement in embryonic development through its genetic ablation. Obligatory HuR-null embryos exhibited a stage retardation phenotype and failed to survive beyond midgestation. By means of conditional transgenesis, we restricted HuRs mutation in either embryonic or endothelial compartments to demonstrate that embryonic lethality is consequent to defects in extraembryonic placenta. HuRs absence impaired the invagination of allantoic capillaries into the chorionic trophoblast layer and the differentiation of syncytiotrophoblast cells that control the morphogenesis and vascularization of the placental labyrinth and fetal support. HuR-null embryos rescued from these placental defects proceeded to subsequent developmental stages but displayed defects in skeletal ossification, fusions in limb elements, and asplenia. By coupling gene expression measurements, data meta-analysis, and HuR-RNA association assays, we identified transcription and growth factor mRNAs controlled by HuR, primarily at the posttranscriptional level, to guide morphogenesis, specification, and patterning. Collectively, our data demonstrate the dominant role of HuR in organizing gene expression programs guiding placental labyrinth morphogenesis, skeletal specification patterns, and splenic ontogeny.

The RAG1 Homeodomain Recruits HMG1 and HMG2 To Facilitate Recombination Signal Sequence Binding and To Enhance the Intrinsic DNA-Bending Activity of RAG1-RAG2

ABSTRACT V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.

Global Impairment of CD4+CD25+FOXP3+ Regulatory T Cells in Idiopathic Pulmonary Fibrosis

RATIONALE The implication of T cells in the pathogenesis of idiopathic pulmonary fibrosis (IPF) is controversial. CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) are pivotal in maintaining immune homeostasis, but their role in IPF pathophysiology has not yet been studied. OBJECTIVES To explore Treg dynamics and function in IPF. METHODS Treg levels and dynamics were analyzed by flow cytometry in the peripheral blood (PB) and bronchoalveolar lavage (BAL) of 21 patients with IPF, 35 patients with lung diseases other than IPF (patients without IPF), 20 patients with collagen vascular diseases with pulmonary parenchymal involvement (CVD-IP), and 28 healthy volunteers. The suppression of autologous CD4(+)CD25(-) cell-proliferative responses and cytokine release by magnetic bead-isolated Tregs was evaluated by proliferation assays and cytometric bead array. Correlations of Treg function and levels with lung function parameters were also performed. MEASUREMENTS AND MAIN RESULTS In patients with IPF, both BAL and PB Tregs were reduced compared with those of healthy volunteers and patients without IPF, although not always significantly. Treg levels were not affected by the administration of low-dose prednisone in four nonresponding patients. The suppressor potential of BAL and PB Tregs was compromised in patients with IPF and patients with CVD-IP, compared with healthy volunteers and patients without IPF. Similarly, the Treg-induced suppression of helper T-cell type 1 and 2 cytokine secretion was impaired in the BAL of patients with IPF and patients with CVD-IP. Moreover, the defective function of BAL Tregs correlated highly with parameters of disease severity. CONCLUSIONS This study provides the first evidence of global Treg impairment in IPF that strongly correlates with disease severity, suggesting a role for Tregs in the fibrotic process.

Lysoglycerophospholipids in chronic inflammatory disorders: The PLA2/LPC and ATX/LPA axes ☆

Lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA), the most prominent lysoglycerophospholipids, are emerging as a novel class of inflammatory lipids, joining thromboxanes, leukotrienes and prostaglandins with which they share metabolic pathways and regulatory mechanisms. Enzymes that participate in LPC and LPA metabolism, such as the phospholipase A(2) superfamily (PLA(2)) and autotaxin (ATX, ENPP2), play central roles in regulating LPC and LPA levels and consequently their actions. LPC/LPA biosynthetic pathways will be briefly presented and LPC/LPA signaling properties and their possible functions in the regulation of the immune system and chronic inflammation will be reviewed. Furthermore, implications of exacerbated LPC and/or LPA signaling in the context of chronic inflammatory diseases, namely rheumatoid arthritis, multiple sclerosis, pulmonary fibrosis and hepatitis, will be discussed. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Pulmonary Autotaxin Expression Contributes to the Pathogenesis of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic form of diffuse lung disease occurring mainly in older adults. Increased lysophosphatidic acid (LPA) concentrations have been reported in the alveolar space of both idiopathic pulmonary fibrosis patients and a corresponding animal model, whereas the genetic deletion or pharmacological inhibition of LPA receptor 1 attenuated the development of the modeled disease, suggesting a direct involvement of LPA in disease pathogenesis. In this report, increased concentrations of autotaxin (ATX; ENPP2), the enzyme largely responsible for extracellular LPA production, were detected in both murine and human fibrotic lungs. The genetic deletion of ATX from bronchial epithelial cells or macrophages attenuated disease severity, establishing ATX as a novel player in IPF pathogenesis. Furthermore, the pharmacological inhibition of ATX attenuated the development of the modeled disease, suggesting that ATX is a possible therapeutic target in IPF.

ATX expression and LPA signalling are vital for the development of the nervous system

Autotaxin (ATX) is a secreted glycoprotein widely present in biological fluids, originally isolated from the supernatant of melanoma cells as an autocrine motility stimulation factor. Its enzymatic product, lysophosphatidic acid (LPA), is a phospholipid mediator that evokes growth-factor-like responses in almost all cell types through G-protein coupled receptors. To assess the role of ATX and LPA signalling in pathophysiology, a conditional knockout mouse was created. Ubiquitous, obligatory deletion resulted to embryonic lethality most likely due to aberrant vascular branching morphogenesis and chorio-allantoic fusion. Moreover, the observed phenotype was shown to be entirely depended on embryonic, but not extraembryonic or maternal ATX expression. In addition, E9.5 ATX null mutants exhibited a failure of neural tube closure, most likely independent of the circulatory failure, which correlated with decreased cell proliferation and increased cell death. More importantly, neurite outgrowth in embryo explants was severely compromised in mutant embryos but could be rescued upon the addition of LPA, thus confirming a role for ATX and LPA signalling in the development of the nervous system. Finally, expression profiling of mutant embryos revealed attenuated embryonic expression of HIF-1a in the absence of ATX, suggesting a novel effector pathway of ATX/LPA.

Autotaxin expression from synovial fibroblasts is essential for the pathogenesis of modeled arthritis

Synovial fibroblasts from patients and mice with arthritis express autotaxin, and ablation of autotaxin in fibroblasts ameliorates disease.

Soluble TNF Mediates the Transition from Pulmonary Inflammation to Fibrosis

Background Fibrosis, the replacement of functional tissue with excessive fibrous tissue, can occur in all the main tissues and organ systems, resulting in various pathological disorders. Idiopathic Pulmonary Fibrosis is a prototype fibrotic disease involving abnormal wound healing in response to multiple sites of ongoing alveolar epithelial injury. Methodology/Principal Findings To decipher the role of TNF and TNF-mediated inflammation in the development of fibrosis, we have utilized the bleomycin-induced animal model of Pulmonary Fibrosis and a series of genetically modified mice lacking components of TNF signaling. Transmembrane TNF expression is shown to be sufficient to elicit an inflammatory response, but inadequate for the transition to the fibrotic phase of the disease. Soluble TNF expression is shown to be crucial for lymphocyte recruitment, a prerequisite for TGF-b1 expression and the development of fibrotic lesions. Moreover, through a series of bone marrow transfers, the necessary TNF expression is shown to originate from the non-hematopoietic compartment further localized in apoptosing epithelial cells. Conclusions These results suggest a primary detrimental role of soluble TNF in the pathologic cascade, separating it from the beneficial role of transmembrane TNF, and indicate the importance of assessing the efficacy of soluble TNF antagonists in the treatment of Idiopathic Pulmonary Fibrosis.

Toll-like Receptor 7-triggered Immune Response in the Lung Mediates Acute and Long-Lasting Suppression of Experimental Asthma

RATIONALE Toll-like receptor (TLR) 7/8 ligands are promising candidate drugs for the treatment of allergic asthma and rhinitis. Although their clinical application depends on the development of strategies for topical administration to the lung, this has not been explored in preclinical disease models. OBJECTIVES To examine the therapeutic effectiveness, persistence of effect, and mode of action of intranasal TLR7 ligand administration in allergic airway disease. METHODS Wild-type, IFN-alpha receptor (IFN-alphaR)(-/-), IFN-gamma(-/-), CD8(-/-), TLR7(-/-), and radiation-induced chimeric mice deficient in hematopoietic TLR7 expression were subjected to an established model of allergic airway disease. R-848, a specific TLR7 agonist in mice, was administered prophylactically or therapeutically and effects of treatment on helper T-cell type 2 (Th2) responses, eosinophilia, goblet cell metaplasia, and airway hyperresponsiveness were assessed. MEASUREMENTS AND MAIN RESULTS Intranasal R-848 administration induced a transient immune response characterized by type I interferon production and infiltration of innate immune cells into the lung. This conferred long-term suppression of allergic airway disease via two complementary molecular processes, one mediated by type I interferons and providing acute protection by directly inhibiting effector Th2 responses, and one mediated by immunoregulatory CD8(+) T cells and inducing long-lasting protection by suppressing Th2 responses in an IFN-gamma-dependent manner. CONCLUSIONS Intranasal R-848 administration is an effective treatment for allergic airway disease. It hijacks an otherwise proinflammatory immune process triggered by TLR7 to mediate long-lasting disease suppression. This provides important insight into the efficacy and mode of action of TLR7 ligands in murine models of allergic airway disease and paves the way for their clinical application in humans.