Argyrios Tzouvelekis

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.

Epigenetics in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal chronic lung disorder with no effective treatment and a prognosis worse than that of lung cancer. Despite extensive research efforts, its etiology and pathogenesis still remain largely unknown. Current experimental evidence has shifted the disease paradigm from chronic inflammation towards the premise of abnormal epithelial wound repair in response to repeated epigenetic injurious stimuli in genetically predisposed individuals. Epigenetics is defined as the study of heritable changes in gene function by factors other than an individuals DNA sequence, providing valuable information regarding adaption of genes to environmental changes. Although cancer is the most studied disease with relevance to epigenetic modifications, recent data support the idea that epigenomic alterations may lead to variable disease phenotypes, including fibroproliferative lung disorders such as IPF. This review article summarizes the latest experimental and translational epigenetic studies in the research field of chronic lung disorders, mainly focusing on IPF, highlights current methodology limitations, and underlines future directions and perspectives.

Validation of a 52-gene risk profile for outcome prediction in patients with idiopathic pulmonary fibrosis: an international, multicentre, cohort study

Background The clinical course of Idiopathic Pulmonary Fibrosis (IPF) is unpredictable. Clinical prediction tools are not accurate enough to predict disease outcomes. Methods All-comers with Idiopathic Pulmonary Fibrosis diagnosis were enrolled in a six-cohort study. Peripheral blood mononuclear cells or whole blood was collected at baseline from 425 participants and during follow up from 98 patients. The 52-gene signature was measured by the nCounter® analysis system in four cohorts and extracted from microarray data in two others. The Scoring Algorithm for Molecular Subphenotypes (SAMS) was used to classify patients into low or high risk groups based on a 52-gene signature. Mortality and transplant-free survival were studied using Competing risk and Cox proportional-hazard models, respectively. Time course data and response to anti-fibrotic drugs were analyzed using linear mixed-effect models. Findings The application of SAMS to the 52-gene signature identified two groups of IPF patients (low and high risk) with significant differences in mortality or transplant-free survival in each of the six cohorts (HR 2·03–4·37). Pooled data revealed similar results for mortality (HR:2·18, 95%CI:1·53–3·09, P<0·0001) or transplant-free survival (HR:2·04, 95%CI: 1·52–2·74, P<0·0001). Adding 52-gene risk profiles to the Gender, Age and Physiology (GAP) index significantly improved its mortality predictive accuracy. Temporal changes in SAMS scores were associated with changes in forced vital capacity (FVC) in two cohorts. Untreated patients did not shift their risk profile over time. A simultaneous increase in up score and decrease in down score was predictive of transplant-free survival (HR:3·18· 95%CI 1·16, 8·76, P=0·025) in the Pittsburgh cohort. A simultaneous decrease in up score and increase in down score after initiation of anti-fibrotic drugs was associated with a significant (P=0·005) improvement in FVC in the Yale cohort. Interpretation The peripheral blood 52-gene expression signature is predictive of outcome in patients with IPF. The potential value of the 52-gene signature in predicting response to therapy should be determined in prospective studies.

Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis

Rationale: Idiopathic pulmonary fibrosis (IPF) involves the accumulation of &agr;‐smooth muscle actin‐expressing myofibroblasts arising from interactions with soluble mediators such as transforming growth factor‐&bgr;1 (TGF‐&bgr;1) and mechanical influences such as local tissue stiffness. Whereas IPF fibroblasts are enriched for aerobic glycolysis and innate immune receptor activation, innate immune ligands related to mitochondrial injury, such as extracellular mitochondrial DNA (mtDNA), have not been identified in IPF. Objectives: We aimed to define an association between mtDNA and fibroblast responses in IPF. Methods: We evaluated the response of normal human lung fibroblasts (NHLFs) to stimulation with mtDNA and determined whether the glycolytic reprogramming that occurs in response to TGF‐&bgr;1 stimulation and direct contact with stiff substrates, and spontaneously in IPF fibroblasts, is associated with excessive levels of mtDNA. We measured mtDNA concentrations in bronchoalveolar lavage (BAL) from subjects with and without IPF, as well as in plasma samples from two longitudinal IPF cohorts and demographically matched control subjects. Measurements and Main Results: Exposure to mtDNA augments &agr;‐smooth muscle actin expression in NHLFs. The metabolic changes in NHLFs that are induced by interactions with TGF‐&bgr;1 or stiff hydrogels are accompanied by the accumulation of extracellular mtDNA. These findings replicate the spontaneous phenotype of IPF fibroblasts. mtDNA concentrations are increased in IPF BAL and plasma, and in the latter compartment, they display robust associations with disease progression and reduced event‐free survival. Conclusions: These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.

Exploring Animal Models That Resemble Idiopathic Pulmonary Fibrosis

Large multicenter clinical trials have led to two recently approved drugs for patients with idiopathic pulmonary fibrosis (IPF); yet, both of these therapies only slow disease progression and do not provide a definitive cure. Traditionally, preclinical trials have utilized mouse models of bleomycin (BLM)-induced pulmonary fibrosis—though several limitations prevent direct translation to human IPF. Spontaneous pulmonary fibrosis occurs in other animal species, including dogs, horses, donkeys, and cats. While the fibrotic lungs of these animals share many characteristics with lungs of patients with IPF, current veterinary classifications of fibrotic lung disease are not entirely equivalent. Additional studies that profile these examples of spontaneous fibroses in animals for similarities to human IPF should prove useful for both human and animal investigators. In the meantime, studies of BLM-induced fibrosis in aged male mice remain the most clinically relevant model for preclinical study for human IPF. Addressing issues such as time course of treatment, animal size and characteristics, clinically irrelevant treatment endpoints, and reproducibility of therapeutic outcomes will improve the current status of preclinical studies. Elucidating the mechanisms responsible for the development of fibrosis and disrepair associated with aging through a collaborative approach between researchers will promote the development of models that more accurately represent the realm of interstitial lung diseases in humans.

Extracellular matrix remodeling in idiopathic pulmonary fibrosis. It is the ‘bed’ that counts and not ‘the sleepers’

ABSTRACT Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterized by irreversible fibrosis. Current disease pathogenesis assumes an aberrant wound healing process in response to repetitive injurious stimuli leading to apoptosis of epithelial cells, activation of fibroblasts and accumulation of extracellular matrix (ECM). Particularly, lung ECM is a highly dynamic structure that lies at the core of several physiological and developmental pathways. The scope of this review article is to summarize current knowledge on the role of ECM in the pathogenesis of IPF, unravel novel mechanistic data and identify future more effective therapeutic targets. Areas covered: The exact mechanisms through which lung microenvironment activates fibroblasts and inflammatory cells, regulates profibrotic signaling cascades through growth factors, integrins and degradation enzymes ultimately leading to excessive matrix deposition are discussed. Furthermore, the potential therapeutic usefulness of specific inhibitors of matrix deposition or activators of matrix degradation pathways are also presented. Expert commentary: With a gradually increasing worldwide incidence IPF still present a major challenge in clinical research due to its unknown etiopathogenesis and current ineffective treatment approaches. Today, there is an amenable need for more effective therapeutic targets and ECM components may represent one.

Cardiovascular Risk Assessment in a Cohort of Newly Diagnosed Patients with Obstructive Sleep Apnea Syndrome

Objectives Obstructive sleep apnea syndrome (OSAS) is associated with increased cardiovascular morbidity and mortality. The aim of this study was to assess whether the 10-year risk for cardiovascular disease in newly diagnosed patients with OSAS is increased. Materials and Methods Recently diagnosed, with polysomnography, consecutive OSAS patients were included. The Systematic Coronary Risk Evaluation (SCORE) and the Framingham Risk Score (FRS) were used to estimate the 10-year risk for cardiovascular disease. Results Totally, 393 individuals (73.3% males), scheduled to undergo a polysomnographic study with symptoms indicative of OSAS, were enrolled. According to apnea-hypopnea index (AHI), subjects were divided in four groups: mild OSAS (AHI 5–14.9/h) was diagnosed in 91 patients (23.2%), moderate OSAS (AHI 15–29.9/h) in 58 patients (14.8%), severe OSAS (AHI > 30/h) in 167 patients (42.5%), while 77 individuals (19.6%) had an AHI < 5/h and served as controls. Increased severity of OSAS was associated with increased SCORE (p < 0.001) and FRS values (p < 0.001). More specifically, a significant correlation was observed both between AHI and SCORE (r=0.251,  p < 0.001) and AHI and FRS values (r=0.291,  p < 0.001). Furthermore, a negative correlation was observed between FRS values and sleep efficiency (r=−0.224,  p=0.006). Conclusions The 10-year risk for cardiovascular morbidity and mortality seems to increase with severity of OSAS. Physicians should bear this finding in mind, in order to seek for and consecutively eliminate risk factors for cardiovascular disease and to prevent future cardiovascular events in OSAS patients.

S100A12 as a marker of worse cardiac output and mortality in pulmonary hypertension: S100A12 in pulmonary hypertension

Molecular biomarkers are needed to refine prognostication and phenotyping of pulmonary hypertension (PH) patients. S100A12 is an emerging biomarker of various inflammatory diseases. This study aims to determine the prognostic value of S100A12 in PH.

Corrigendum: Longitudinal “Real-World” Outcomes of Pirfenidone in Idiopathic Pulmonary Fibrosis in Greece

[This corrects the article on p. 213 in vol. 4, PMID: 29238708.].

Bleomycin Revisited: A Direct Comparison of the Intratracheal Micro-Spraying and the Oropharyngeal Aspiration Routes of Bleomycin Administration in Mice

Idiopathic Pulmonary Fibrosis (IPF) is a fatal disease characterized by exuberant deposition of extracellular matrix components, deterioration of lung architecture and impairment of lung functions. Its etiopathogenesis remains incompletely understood, as reflected in the lack of an appropriate therapy. Modeling the human disease in mice via the administration of bleomycin (BLM), despite the inherent limitations, has provided valuable insights into the underlying pathogenetic mechanisms, and has been instrumental for the development and validation of new pharmacologic interventions. Here we have directly compared the, most widely used, intratracheal (IT) route of administration with oropharyngeal aspiration (OA). Our results suggest that the OA route of BLM-administration can be used as a safe and effective alternative, minimizing peri-operative and experimental mortality, while preserving a solid fibrotic profile, as assessed with a plethora of standardized readout assays.