Expression of PD-1/PD-L1 axis in mediastinal lymph nodes and lung tissue of human and experimental lung fibrosis indicates a potential therapeutic target for idiopathic pulmonary fibrosis.

BACKGROUND: Mediastinal lymph node enlargement is prevalent in patients with idiopathic pulmonary fibrosis (IPF). Studies investigating whether this phenomenon reflects specific immunologic activation are lacking. METHODS: Programmed cell death-1 (PD-1)/ programmed cell death ligand-1 (PD-L1) expression in mediastinal lymph nodes and lung tissues was analyzed. PD-1, PD-L1 mRNA expression was measured in tracheobronchial lymph nodes of mice following bleomycin-induced injury on day 14. Finally, the effect of the PD-1 inhibitor, pembrolizumab, in bleomycin-induced pulmonary fibrosis was investigated. RESULTS: We analyzed mediastinal lymph nodes of thirty-three patients (n = 33, IPF: n = 14, lung cancer: n = 10, concomitant IPF and lung cancer: n = 9) and lung tissues of two hundred nineteen patients (n = 219, IPF: 123, controls: 96). PD-1 expression was increased, while PD-L1 expression was decreased, in mediastinal lymph nodes of patients with IPF compared to lung cancer and in IPF lungs compared to control lungs. Tracheobronchial lymph nodes isolated on day 14 from bleomycin-treated mice exhibited increased size and higher PD-1, PD-L1 mRNA levels compared to saline-treated animals. Pembrolizumab blunted bleomycin-induced lung fibrosis, as indicated by reduction in Ashcroft score and improvement in respiratory mechanics. CONCLUSIONS: Mediastinal lymph nodes of patients with IPF exhibit differential expression profiles than those of patients with lung cancer indicating distinct immune-mediated pathways regulating fibrogenesis and carcinogenesis. PD-1 expression in mediastinal lymph nodes is in line with lung tissue expression. Lower doses of pembrolizumab might exert antifibrotic effects. Clinical trials aiming to endotype patients based on mediastinal lymph node profiling and accordingly implement targeted therapies such as PD-1 inhibitors are greatly anticipated.

Autotaxin and chronic inflammatory diseases.

Autotaxin (ATX) is a secreted glycoprotein, widely present in biological fluids including blood. ATX catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a growth factor-like, signaling phospholipid. LPA exerts pleiotropic effects mediated by its G-protein-coupled receptors that are widely expressed and exhibit overlapping specificities. Although ATX also possesses matricellular properties, the majority of ATX reported functions in adulthood are thought to be mediated through the extracellular production of LPA. ATX-mediated LPA synthesis is likely localized at the cell surface through the possible interaction of ATX with integrins or other molecules, while LPA levels are further controlled by a group of membrane-associated lipid-phosphate phosphatases. ATX expression was shown to be necessary for embryonic development, and ATX deficient embryos exhibit defective vascular homeostasis and aberrant neuronal system development. In adult life, ATX is highly expressed in the adipose tissue and has been implicated in diet-induced obesity and glucose homeostasis with multiple implications in metabolic disorders. Additionally, LPA has been shown to affect multiple cell types, including stromal and immune cells in various ways. Therefore, LPA participates in many processes that are intricately involved in the pathogenesis of different chronic inflammatory diseases such as vascular homeostasis, skeletal and stromal remodeling, lymphocyte trafficking and immune regulation. Accordingly, increased ATX and LPA levels have been detected, locally and/or systemically, in patients with chronic inflammatory diseases, most notably idiopathic pulmonary fibrosis (IPF), chronic liver diseases, and rheumatoid arthritis. Genetic and pharmacological studies in mice have confirmed a pathogenetic role for ATX expression and LPA signaling in chronic inflammatory diseases, and provided the proof of principle for therapeutic interventions, as exemplified by the ongoing clinical trials for IPF.

IAP antagonists Birinapant and AT-406 efficiently synergise with either TRAIL, BRAF, or BCL-2 inhibitors to sensitise BRAFV600E colorectal tumour cells to apoptosis.

BACKGROUND: High expression levels of Inhibitors of Apoptosis Proteins (IAPs) have been correlated with poor cancer prognosis and block the cell death pathway by interfering with caspase activation. SMAC-mimetics are small-molecule inhibitors of IAPs that mimic the endogenous SMAC and promote the induction of cell death by neutralizing IAPs. METHODS: In this study, anti-tumour activity of new SMAC-mimetics Birinapant and AT-406 is evaluated against colorectal adenocarcinoma cells and IAP cross-talk with either oncogenic BRAF or BCL-2, or with the TRAIL are further exploited towards rational combined protocols. RESULTS: It is shown that pre-treatment of SMAC-mimetics followed by their combined treatment with BRAF inhibitors can decrease cell viability, migration and can very efficiently sensitize colorectal tumour cells to apoptosis. Moreover, co-treatment of TRAIL with SMAC-mimetics can efficiently sensitize resistant tumour cells to apoptosis synergistically, as shown by median effect analysis. Finally, Birinapant and AT-406 can synergise with BCL-2 inhibitor ABT-199 to reduce viability of adenocarcinoma cells with high BCL-2 expression. CONCLUSIONS: Proposed synergistic rational anticancer combined protocols of IAP antagonists Birinapant and AT-406 in 2D and 3D cultures can be later further exploited in vivo, from precision tumour biology to precision medical oncology.

Increased lipocalin-2 expression in pulmonary inflammation and fibrosis.

Introduction: Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive interstitial lung disease with dismal prognosis. The underlying pathogenic mechanisms are poorly understood, resulting in a lack of effective treatments. However, recurrent epithelial damage is considered critical for disease initiation and perpetuation, via the secretion of soluble factors that amplify inflammation and lead to fibroblast activation and exuberant deposition of ECM components. Lipocalin-2 (LCN2) is a neutrophil gelatinase-associated lipocalin (NGAL) that has been suggested as a biomarker of kidney damage. LCN2 has been reported to modulate innate immunity, including the recruitment of neutrophils, and to protect against bacterial infections by sequestering iron. Methods: In silico analysis of publicly available transcriptomic datasets; ELISAs on human IPF patients' bronchoalveolar lavage fluids (BALFs); bleomycin (BLM)-induced pulmonary inflammation and fibrosis and LPS-induced acute lung injury (ALI) in mice: pulmonary function tests, histology, Q-RT-PCR, western blot, and FACS analysis. Results and discussion: Increased LCN2 mRNA expression was detected in the lung tissue of IPF patients negatively correlating with respiratory functions, as also shown for BALF LCN2 protein levels in a cohort of IPF patients. Increased Lcn2 expression was also detected upon BLM-induced pulmonary inflammation and fibrosis, especially at the acute phase correlating with neutrophilic infiltration, as well as upon LPS-induced ALI, an animal model characterized by neutrophilic infiltration. Surprisingly, and non withstanding the limitations of the study and the observed trends, Lcn2-/- mice were found to still develop BLM- or LPS-induced pulmonary inflammation and fibrosis, thus questioning a major pathogenic role for Lcn2 in mice. However, LCN2 qualifies as a surrogate biomarker of pulmonary inflammation and a possible indicator of compromised pulmonary functions, urging for larger studies.

SH2 Domain-Containing Phosphatase-SHP2 Attenuates Fibrotic Responses through Negative Regulation of Mitochondrial Metabolism in Lung Fibroblasts.

BACKGROUND: We have previously shown that SHP2 downregulation may predispose fibroblasts to differentiate into myofibroblasts and proposed a role for SHP2 downregulation in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Recent data have shown that SHP2 localizes to the mitochondrial intercristae, and its overexpression enhances mitochondrial metabolism leading to oxidative stress and senescence. OBJECTIVE: To determine the effect of SHP2 on fibrotic responses. METHODS AND RESULTS: Primary mouse lung fibroblasts derived from mice carrying a conditional knock-in mutation (D61G/+), rendering the SHP2 catalytic domain constitutively active, had reduced proliferation (1.6-fold, p < 0.05), migration (2-fold, p < 0.05), as well as reduced responsiveness of TGFB-1 induced fibroblasts-to-myofibroblasts differentiation, compared to wild-type ones. Electron microscope analysis revealed that SHP2 D61G/+ mouse lung fibroblasts were characterized by mitochondrial abnormalities, including swollen mitochondria with disrupted electron-lucent cristae and an increased number of autophagosomes compared to wild-type ones. SHP2 D61G/+ MLFs exhibited increased protein levels of autophagy markers, including LC3B-II and p-62, evidence that was confirmed by immunofluorescence analysis. Mitochondrial function analysis revealed that stable (genotype D61G/+) overexpression of SHP2 led to impaired mitochondrial function, as assessed by decreased mitochondrial membrane potential (1.29-fold, p < 0.05), coupling efficiency (1.82 fold, p < 0.05), oxygen consumption rate (1.9-fold, p < 0.05), and increased reactive oxygen species production both at baseline (1.75-fold, p < 0.05) and following H2O2 stimulation (1.63-fold, p < 0.05) compared to wild-type ones (SHP2+/+). SHP2 D61G/+ mouse lung fibroblasts showed enhanced AMPK activity, as well as decreased activation of the mTORC1 signaling pathway, potentially leading to ineffective mitochondrial metabolism and increased autophagy. CONCLUSIONS: SHP2 attenuates fibrotic responses in fibroblast cell lines through negative regulation of mitochondrial metabolism and induction of autophagy. SHP2 activation may represent a promising therapeutic strategy for patients with fibrotic lung diseases.

SRC and TKS5 mediated podosome formation in fibroblasts promotes extracellular matrix invasion and pulmonary fibrosis.

The activation and accumulation of lung fibroblasts resulting in aberrant deposition of extracellular matrix components, is a pathogenic hallmark of Idiopathic Pulmonary Fibrosis, a lethal and incurable disease. In this report, increased expression of TKS5, a scaffold protein essential for the formation of podosomes, was detected in the lung tissue of Idiopathic Pulmonary Fibrosis patients and bleomycin-treated mice. Τhe profibrotic milieu is found to induce TKS5 expression and the formation of prominent podosome rosettes in lung fibroblasts, that are retained ex vivo, culminating in increased extracellular matrix invasion. Tks5+/- mice are found resistant to bleomycin-induced pulmonary fibrosis, largely attributed to diminished podosome formation in fibroblasts and decreased extracellular matrix invasion. As computationally predicted, inhibition of src kinase is shown to potently attenuate podosome formation in lung fibroblasts and extracellular matrix invasion, and bleomycin-induced pulmonary fibrosis, suggesting pharmacological targeting of podosomes as a very promising therapeutic option in pulmonary fibrosis.

Obesity Reshapes the Microbial Population Structure along the Gut-Liver-Lung Axis in Mice.

The microbiome is emerging as a major player in tissue homeostasis in health and disease. Gut microbiome dysbiosis correlates with several autoimmune and metabolic diseases, while high-fat diets and ensuing obesity are known to affect the complexity and diversity of the microbiome, thus modulating pathophysiology. Moreover, the existence of a gut-liver microbial axis has been proposed, which may extend to the lung. In this context, we systematically compared the microbiomes of the gut, liver, and lung of mice fed a high-fat diet to those of littermates fed a matched control diet. We carried out deep sequencing of seven hypervariable regions of the 16S rRNA microbial gene to examine microbial diversity in the tissues of interest. Comparison of the local microbiomes indicated that lung tissue has the least diverse microbiome under healthy conditions, while microbial diversity in the healthy liver clustered closer to the gut. Obesity increased microbial complexity in all three tissues, with lung microbial diversity being the most modified. Obesity promoted the expansion of Firmicutes along the gut-liver-lung axis, highlighting staphylococcus as a possible pathologic link between obesity and systemic pathophysiology, especially in the lungs.

Collagen 1a1 Expression by Airway Macrophages Increases In Fibrotic ILDs and Is Associated With FVC Decline and Increased Mortality.

Within the Interstitial Lung Diseases (ILD), patients with idiopathic pulmonary fibrosis (IPF) and a subset of those with non-IPF fibrotic ILD have a distinct clinical phenotype of progression despite management. This group of patients has been collectively termed the progressive fibrotic phenotype (PFP). Their early recognition may facilitate access to antifibrotic therapies to prevent or slow progression. Macrophages/monocytes within the lung orchestrate the progression and maintenance of fibrosis. A novel role for monocyte-derived macrophages during tissue damage and wound healing is the expression of collagens. We examined Collagen 1a1 expression in airway macrophages from ILD patients at diagnosis. COL1A1 mRNA levels from BAL cells were elevated in IPF and Non-IPF patients. The presence of a UIP pattern and a subsequent progressive phenotype were significantly associated with the higher BAL COL1A1 levels. In Non-IPF patients, higher COL1A1 levels were associated with a more than twofold increase in mortality. The intracellular localisation of COL1A1 in airway macrophages was demonstrated by confocal microscopy in CD45 and CD163 co-staining assays. Additionally, airway macrophages co-expressed COL1A1 with the profibrotic SPP1 gene product osteopontin. The levels of SPP1 mRNA and OPN in the BAL were significantly higher in IPF and Non-IPF patients relative to healthy. Our results suggest that profibrotic airway macrophages are increased in the BAL of patients with IPF and other ILDs and co-express COL1A1 and OPN. Importantly, COL1A1 expression by pro-fibrotic airway macrophages could be a marker of disease progression and poor survival in ILDs.