Anantha Harijith

Reactive oxygen species at the crossroads of inflammasome and inflammation

Inflammasomes form a crucial part of the innate immune system. These are multi-protein oligomer platforms that are composed of intracellular sensors which are coupled with caspase and interleukin activating systems. Nod-like receptor protein (NLRP) 3, and 6 and NLRC4 and AIM2 are the prominent members of the inflammasome family. Inflammasome activation leads to pyroptosis, a process of programmed cell death distinct from apoptosis through activation of Caspase and further downstream targets such as IL-1β and IL-18 leading to activation of inflammatory cascade. Reactive oxygen species (ROS) serves as important inflammasome activating signals. ROS activates inflammasome through mitogen-activated protein kinases (MAPK) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Dysregulation of inflammasome plays a significant role in various pathological processes. Viral infections such as Dengue and Respiratory syncytial virus activate inflammasomes. Crystal compounds in silicosis and gout also activate ROS. In diabetes, inhibition of autophagy with resultant accumulation of dysfunctional mitochondria leads to enhanced ROS production activating inflammasomes. Activation of inflammasomes can be dampened by antioxidants such as SIRT-1. Inflammasome and related cascade could serve as future therapeutic targets for various pathological conditions.

Lysophosphatidic Acid Receptor–2 Deficiency Confers Protection against Bleomycin-Induced Lung Injury and Fibrosis in Mice

Idiopathic pulmonary fibrosis is a devastating disease characterized by alveolar epithelial cell injury, the accumulation of fibroblasts/myofibroblasts, and the deposition of extracellular matrix proteins. Lysophosphatidic acid (LPA) signaling through its G protein-coupled receptors is critical for its various biological functions. Recently, LPA and LPA receptor 1 were implicated in lung fibrogenesis. However, the role of other LPA receptors in fibrosis remains unclear. Here, we use a bleomycin-induced pulmonary fibrosis model to investigate the roles of LPA2 in pulmonary fibrogenesis. In the present study, we found that LPA2 knockout (Lpar2(-/-)) mice were protected against bleomycin-induced lung injury, fibrosis, and mortality, compared with wild-type control mice. Furthermore, LPA2 deficiency attenuated the bleomycin-induced expression of fibronectin (FN), α-smooth muscle actin (α-SMA), and collagen in lung tissue, as well as levels of IL-6, transforming growth factor-β (TGF-β), and total protein in bronchoalveolar lavage fluid. In human lung fibroblasts, the knockdown of LPA2 attenuated the LPA-induced expression of TGF-β1 and the differentiation of lung fibroblasts to myofibroblasts, resulting in the decreased expression of FN, α-SMA, and collagen, as well as decreased activation of extracellular regulated kinase 1/2, Akt, Smad3, and p38 mitogen-activated protein kinase. Moreover, the knockdown of LPA2 with small interfering RNA also mitigated the TGF-β1-induced differentiation of lung fibroblasts. In addition, LPA2 deficiency significantly attenuated the bleomycin-induced apoptosis of alveolar and bronchial epithelial cells in the mouse lung. Together, our data indicate that the knockdown of LPA2 attenuated bleomycin-induced lung injury and pulmonary fibrosis, and this may be related to an inhibition of the LPA-induced expression of TGF-β and the activation and differentiation of fibroblasts.

Sphingosine-1-phosphate lyase is an endogenous suppressor of pulmonary fibrosis: role of S1P signalling and autophagy

Introduction Idiopathic pulmonary fibrosis (IPF) is characterised by accumulation of fibroblasts and myofibroblasts and deposition of extracellular matrix proteins. Sphingosine-1-phosphate (S1P) signalling plays a critical role in pulmonary fibrosis. Methods S1P lyase (S1PL) expression in peripheral blood mononuclear cells (PBMCs) was correlated with pulmonary functions and overall survival; used a murine model to check the role of S1PL on the fibrogenesis and a cell culture system to study the effect of S1PL expression on transforming growth factor (TGF)-β- and S1P-induced fibroblast differentiation. Results S1PL expression was upregulated in fibrotic lung tissues and primary lung fibroblasts isolated from patients with IPF and bleomycin-challenged mice. TGF-β increased the expression of S1PL in human lung fibroblasts via activation and binding of Smad3 transcription factor to Sgpl1 promoter. Overexpression of S1PL attenuated TGF-β-induced and S1P-induced differentiation of human lung fibroblasts through regulation of the expression of LC3 and beclin 1. Knockdown of S1PL (Sgpl1+/−) in mice augmented bleomycin-induced pulmonary fibrosis, and patients with IPF reduced Sgpl1 mRNA expression in PBMCs exhibited higher severity of fibrosis and lower survival rate. Conclusion These studies suggest that S1PL is a novel endogenous suppressor of pulmonary fibrosis in human IPF and animal models.

Sphingosine Kinase 1 Deficiency Confers Protection against Hyperoxia-Induced Bronchopulmonary Dysplasia in a Murine Model: Role of S1P Signaling and Nox Proteins

Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1(-/-) (Sphk1(-/-)), sphingosine kinase 2(-/-) (Sphk2(-/-)), and S1P lyase(+/-) (Sgpl1(+/-)) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1(-/-), but not Sphk2(-/-) or Sgpl1(+/-), mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling-regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia.

The Mitochondrial Cardiolipin Remodeling Enzyme Lysocardiolipin Acyltransferase Is a Novel Target in Pulmonary Fibrosis

RATIONALE Lysocardiolipin acyltransferase (LYCAT), a cardiolipin-remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, is necessary for maintaining normal mitochondrial function and vascular development. We hypothesized that modulation of LYCAT expression in lung epithelium regulates development of pulmonary fibrosis. OBJECTIVES To define a role for LYCAT in human and murine models of pulmonary fibrosis. METHODS We analyzed the correlation of LYCAT expression in peripheral blood mononuclear cells (PBMCs) with the outcomes of pulmonary functions and overall survival, and used the murine models to establish the role of LYCAT in fibrogenesis. We studied the LYCAT action on cardiolipin remodeling, mitochondrial reactive oxygen species generation, and apoptosis of alveolar epithelial cells under bleomycin challenge. MEASUREMENTS AND MAIN RESULTS LYCAT expression was significantly altered in PBMCs and lung tissues from patients with idiopathic pulmonary fibrosis (IPF), which was confirmed in two preclinical murine models of IPF, bleomycin- and radiation-induced pulmonary fibrosis. LYCAT mRNA expression in PBMCs directly and significantly correlated with carbon monoxide diffusion capacity, pulmonary function outcomes, and overall survival. In both bleomycin- and radiation-induced pulmonary fibrosis murine models, hLYCAT overexpression reduced several indices of lung fibrosis, whereas down-regulation of native LYCAT expression by siRNA accentuated fibrogenesis. In vitro studies demonstrated that LYCAT modulated bleomycin-induced cardiolipin remodeling, mitochondrial membrane potential, reactive oxygen species generation, and apoptosis of alveolar epithelial cells, potential mechanisms of LYCAT-mediated lung protection. CONCLUSIONS This study is the first to identify modulation of LYCAT expression in fibrotic lungs and offers a novel therapeutic approach for ameliorating lung inflammation and pulmonary fibrosis.

c-Abl mediated tyrosine phosphorylation of paxillin regulates LPS-induced endothelial dysfunction and lung injury

Paxillin is phosphorylated at multiple residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and acute lung injury (ALI) remains unclear. We used siRNA and site-specific nonphosphorylable mutants of paxillin to abrogate the function of paxillin to determine its role in lung endothelial permeability and ALI. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (HLMVECs) resulted in enhanced tyrosine phosphorylation of paxillin at Y31 and Y118 with no significant change in Y181 and significant barrier dysfunction. Knockdown of paxillin with siRNA attenuated LPS-induced endothelial barrier dysfunction and destabilization of VE-cadherin. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase, but not by Src and focal adhesion kinase. c-Abl siRNA significantly reduced LPS-induced endothelial barrier dysfunction. Transfection of HLMVECs with paxillin Y31F, Y118F, and Y31/118F double mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization. In vivo, the c-Abl inhibitor AG957 attenuated LPS-induced pulmonary permeability in mice. Together, these results suggest that c-Abl mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury.

Coronin 1B Regulates S1P-Induced Human Lung Endothelial Cell Chemotaxis: Role of PLD2, Protein Kinase C and Rac1 Signal Transduction

Coronins are a highly conserved family of actin binding proteins that regulate actin-dependent processes such as cell motility and endocytosis. We found that treatment of human pulmonary artery endothelial cells (HPAECs) with the bioactive lipid, sphingosine-1-phosphate (S1P) rapidly stimulates coronin 1B translocation to lamellipodia at the cell leading edge, which is required for S1P-induced chemotaxis. Further, S1P-induced chemotaxis of HPAECs was attenuated by pretreatment with small interfering RNA (siRNA) targeting coronin 1B (∼36%), PLD2 (∼45%) or Rac1 (∼50%) compared to scrambled siRNA controls. Down regulation PLD2 expression by siRNA also attenuated S1P-induced coronin 1B translocation to the leading edge of the cell periphery while PLD1 silencing had no effect. Also, S1P-induced coronin 1B redistribution to cell periphery and chemotaxis was attenuated by inhibition of Rac1 and over-expression of dominant negative PKC δ, ε and ζ isoforms in HPAECs. These results demonstrate that S1P activation of PLD2, PKC and Rac1 is part of the signaling cascade that regulates coronin 1B translocation to the cell periphery and the ensuing cell chemotaxis.

Role of sphingosine kinase 1 and S1P transporter Spns2 in HGF-mediated lamellipodia formation in lung endothelium

Hepatocyte growth factor (HGF) signaling via c-Met is known to promote endothelial cell motility and angiogenesis. We have previously reported that HGF stimulates lamellipodia formation and motility of human lung microvascular endothelial cells (HLMVECs) via PI3K/Akt signal transduction and reactive oxygen species generation. Here, we report a role for HGF-induced intracellular sphingosine-1-phosphate (S1P) generation catalyzed by sphingosine kinase 1 (SphK1), S1P transporter, spinster homolog 2 (Spns2), and S1P receptor, S1P1, in lamellipodia formation and perhaps motility of HLMVECs. HGF stimulated SphK1 phosphorylation and enhanced intracellular S1P levels in HLMVECs, which was blocked by inhibition of SphK1. HGF enhanced co-localization of SphK1/p-SphK1 with actin/cortactin in lamellipodia and down-regulation or inhibition of SphK1 attenuated HGF-induced lamellipodia formation in HLMVECs. In addition, down-regulation of Spns2 also suppressed HGF-induced lamellipodia formation, suggesting a key role for inside-out S1P signaling. The HGF-mediated phosphorylation of SphK1 and its localization in lamellipodia was dependent on c-Met and ERK1/2 signaling, but not the PI3K/Akt pathway; however, blocking PI3K/Akt signaling attenuated HGF-mediated phosphorylation of Spns2. Down-regulation of S1P1, but not S1P2 or S1P3, with specific siRNA attenuated HGF-induced lamellipodia formation. Further, HGF enhanced association of Spns2 with S1P1 that was blocked by inhibiting SphK1 activity with PF-543. Moreover, HGF-induced migration of HLMVECs was attenuated by down-regulation of Spns2. Taken together, these results suggest that HGF/c-Met-mediated lamellipodia formation, and perhaps motility is dependent on intracellular generation of S1P via activation and localization of SphK1 to cell periphery and Spns2-mediated extracellular transportation of S1P and its inside-out signaling via S1P1.

Hyperoxia- induced p47phox activation and ROS generation is mediated through S1P transporter Spns2, and S1P/S1P1&2 signaling axis in lung endothelium.

Hyperoxia-induced lung injury adversely affects ICU patients and neonates on ventilator assisted breathing. The underlying culprit appears to be reactive oxygen species (ROS)-induced lung damage. The major contributor of hyperoxia-induced ROS is activation of the multiprotein enzyme complex NADPH oxidase. Sphingosine-1-phosphate (S1P) signaling is known to be involved in hyperoxia-mediated ROS generation; however, the mechanism(s) of S1P-induced NADPH oxidase activation is unclear. Here, we investigated various steps in the S1P signaling pathway mediating ROS production in response to hyperoxia in lung endothelium. Of the two closely related sphingosine kinases (SphKs)1 and 2, which synthesize S1P from sphingosine, only Sphk1(-/-) mice conferred protection against hyperoxia-induced lung injury. S1P is metabolized predominantly by S1P lyase and partial deletion of Sgpl1 (Sgpl1(+/-)) in mice accentuated lung injury. Hyperoxia stimulated S1P accumulation in human lung microvascular endothelial cells (HLMVECs), and downregulation of S1P transporter spinster homolog 2 (Spns2) or S1P receptors S1P1&2, but not S1P3, using specific siRNA attenuated hyperoxia-induced p47(phox) translocation to cell periphery and ROS generation in HLMVECs. These results suggest a role for Spns2 and S1P1&2 in hyperoxia-mediated ROS generation. In addition, p47(phox) (phox:phagocyte oxidase) activation and ROS generation was also reduced by PF543, a specific SphK1 inhibitor in HLMVECs. Our data indicate a novel role for Spns2 and S1P1&2 in the activation of p47(phox) and production of ROS involved in hyperoxia-mediated lung injury in neonatal and adult mice.

Expression profiling of genes regulated by sphingosine kinase1 signaling in a murine model of hyperoxia induced neonatal bronchopulmonary dysplasia

BackgroundSphingosine- 1-Phosphate (S1P) is a bioactive lipid and an intracellular as well as an extracellular signaling molecule. S1P ligand specifically binds to five related cell surface G-protein-coupled receptors (S1P1-5). S1P levels are tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases, lipid phosphate phosphatases and S1P lyase. We previously reported that knock down of SphK1 (Sphk1−/−) in a neonatal mouse BPD model conferred significant protection against hyperoxia induced lung injury. To better understand the underlying molecular mechanisms, genome-wide gene expression profiling was performed on mouse lung tissue using Affymetrix MoGene 2.0 array.ResultsTwo-way ANOVA analysis was performed and differentially expressed genes under hyperoxia were identified using Sphk1−/− mice and their wild type (WT) equivalents. Pathway (PW) enrichment analyses identified several signaling pathways that are likely to play a key role in hyperoxia induced lung injury in the neonates. These included signaling pathways that were anticipated such as those involved in lipid signaling, cell cycle regulation, DNA damage/apoptosis, inflammation/immune response, and cell adhesion/extracellular matrix (ECM) remodeling. We noted hyperoxia induced downregulation of the expression of genes related to mitotic spindle formation in the WT which was not observed in Sphk1−/− neonates. Our data clearly suggests a role for SphK1 in neonatal hyperoxic lung injury through elevated inflammation and apoptosis in lung tissue. Further, validation by RT-PCR on 24 differentially expressed genes showed 83% concordance both in terms of fold change and vectorial changes. Our findings are in agreement with previously reported human BPD microarray data and completely support our published in vivo findings. In addition, the data also revealed a significant role for additional unanticipitated signaling pathways involving Wnt and GADD45.ConclusionUsing SphK1 knockout mice and differential gene expression analysis, we have shown here that S1P/SphK1 signaling plays a key role in promoting hyperoxia induced DNA damage, inflammation, apoptosis and ECM remodeling in neonatal lungs. It also appears to suppress pro-survival cellular responses involved in normal lung development. We therefore propose SphK1 as a therapeutic target for the development drugs to combat BPD.