Whole Exome Sequencing Reveals Novel Candidate Genes in Familial Forms of Glaucomatous Neurodegeneration.

Glaucoma is the largest cause of irreversible blindness with a multifactorial genetic etiology. This study explores novel genes and gene networks in familial forms of primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) to identify rare mutations with high penetrance. Thirty-one samples from nine MYOC-negative families (five POAG and four PACG) underwent whole-exome sequencing and analysis. A set of prioritized genes and variations were screened in an independent validation cohort of 1536 samples and the whole-exome data from 20 sporadic patients. The expression profiles of the candidate genes were analyzed in 17 publicly available expression datasets from ocular tissues and single cells. Rare, deleterious SNVs in AQP5, SRFBP1, CDH6 and FOXM1 from POAG families and in ACACB, RGL3 and LAMA2 from PACG families were found exclusively in glaucoma cases. AQP5, SRFBP1 and CDH6 also revealed significant altered expression in glaucoma in expression datasets. Single-cell expression analysis revealed enrichment of identified candidate genes in retinal ganglion cells and corneal epithelial cells in POAG; whereas for PACG families, retinal ganglion cells and Schwalbe's Line showed enriched expression. Through an unbiased exome-wide search followed by validation, we identified novel candidate genes for familial cases of POAG and PACG. The SRFBP1 gene found in a POAG family is located within the GLC1M locus on Chr5q. Pathway analysis of candidate genes revealed enrichment of extracellular matrix organization in both POAG and PACG.

Regulation of cardiovascular health and disease by visceral adipose tissue-derived metabolic hormones.

Visceral adipose tissue (VAT) is a metabolic organ known to regulate fat mass, and glucose and nutrient homeostasis. VAT is an active endocrine gland that synthesizes and secretes numerous bioactive mediators called 'adipocytokines/adipokines' into systemic circulation. These adipocytokines act on organs of metabolic importance like the liver and skeletal muscle. Multiple preclinical and in vitro studies showed strong evidence of the roles of adipocytokines in the regulation of metabolic disorders like diabetes, obesity and insulin resistance. Adipocytokines, such as adiponectin and omentin, are anti-inflammatory and have been shown to prevent atherogenesis by increasing nitric oxide (NO) production by the endothelium, suppressing endothelium-derived inflammation and decreasing foam cell formation. By inhibiting differentiation of vascular smooth muscle cells (VSMC) into osteoblasts, adiponectin and omentin prevent vascular calcification. On the other hand, adipocytokines like leptin and resistin induce inflammation and endothelial dysfunction that leads to vasoconstriction. By promoting VSMC migration and proliferation, extracellular matrix degradation and inflammatory polarization of macrophages, leptin and resistin increase the risk of atherosclerotic plaque vulnerability and rupture. Additionally, the plasma concentrations of these adipocytokines alter in ageing, rendering older humans vulnerable to cardiovascular disease. The disturbances in the normal physiological concentrations of these adipocytokines secreted by VAT under pathological conditions impede the normal functions of various organs and affect cardiovascular health. These adipokines could be used for both diagnostic and therapeutic purposes in cardiovascular disease.

Modulations in human neutrophil metabolome and S-glutathionylation of glycolytic pathway enzymes during the course of extracellular trap formation.

Neutrophil extracellular trap formation (NETosis) has been irrefutably referred to as a distinct and unique form of active cell death with the purpose to counteract invading pathogens or augmenting the inflammatory cascade. Since the discovery, consistent efforts have been made to understand the various aspects of the initiation and sustenance of NETosis. In this study, using a global metabolomics approach during the phorbol 12-myristate 13-acetate (PMA) induced NETosis in human neutrophils, various metabolic pathways were found to be altered which includes intermediates related to, carbohydrate metabolism, and redox related metabolites, nucleic acid metabolism, and amino acids metabolism. Enrichment analysis of the metabolite sets highlighted the importance of the pentose phosphate pathway (PPP) and glutathione metabolism PMA-induced NETotic neutrophils. Further, analysis of the glutathyniolation status of neutrophil proteins by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) indicated six different glutathionylated proteins: among them, two metabolically important proteins were α-enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with MALDI score 166 and 70 respectively. Other proteins were lactoferrin, ß-actin, c-myc promoter-binding protein, and uracil DNA glycosylase with MALDI scores of 96, 167, 104, and 68 respectively. Besides, activation of signalling proteins involved in metabolic regulation is also correlated with NETosis. Altogether, a balance between reactive oxygen species-glutathione metabolism seems to regulate the activity of glycolytic enzymes such as GAPDH and α-enolase during PMA-induced NETosis in a time-dependent manner.

nNOS induction and NOSIP interaction impact granulopoiesis and neutrophil differentiation by modulating nitric oxide generation.

Nitric oxide (NO), a versatile free radical and a signalling molecule, plays an important role in the haematopoiesis, inflammation and infection. Impaired proliferation and differentiation of myeloid cells lead to malignancies and Hematopoietic deficiencies. This study was aimed to define the role of nNOS derived NO in neutrophil differentiation (in-vitro) and granulopoiesis (in-vivo) using multipronged approaches. The results obtained from nNOS over-expressing K562 cells revealed induction in C/EBPα derived neutrophil differentiation as evident by an increase in the expression of neutrophil specific cell surface markers, genes, transcription factors and functionality. nNOS mediated response also involved G-CSFR-STAT-3 axis during differentiation. Consistent increase in NO generation was observed during neutrophil differentiation of mice and human CD34+ HSPCs. Furthermore, granulopoiesis was abrogated in the nNOS inhibitor treated mice, depicting a decrease in the numbers of BM mature and progenitor neutrophils. Likewise, in vitro inhibition of nNOS in human CD34+ HSPCs indicated an indispensable role of nNOS in neutrophil differentiation. Expression of nNOS inhibitory protein, NOSIP was significantly and consistently decreased during the final stage of differentiation and was linked with the augmentation in NO release. Moreover, neutrophils from CML patients had more NOSIP and less NO generation as compared to the PMNs from healthy individuals. The present study thus indicates a critical role of nNOS, and its interaction with NOSIP during neutrophil differentiation. The study also highlights the importance of nNOS in the neutrophil progenitor proliferation and differentiation warranting investigations to assess its role in the haematopoiesis-related disorders.

Pyroptotic and apoptotic cell death in iNOS and nNOS overexpressing K562 cells: A mechanistic insight.

Previous studies from this lab and others have demonstrated that nitric oxide (NO) in a concentration dependent manner, modulated neutrophil and leukemic cell survival. Subsequent studies delineated importance of iNOS in neutrophil differentiation and leukemic cell death. On the contrary, role of nNOS in survival of these cells remains least understood. Present study was therefore undertaken to assess and compare the role of iNOS and nNOS in the survival of NOS overexpressing myelocytic K562 cells. Cells with almost similar iNOS and nNOS activities displayed comparable cell cycle perturbation, Annexin V positivity, mitochondrial dysfunction, augmented DCF fluorescence, and also attenuated expression of antioxidants. Moreover, induction in cell death was also accompanied by the activation of pJNK/p38MAPK/Erk1/2 and reduction in PI3K/Akt/mTOR signaling. Treatment of NOS isoform overexpressing K562 cells with NAC, a potent free radical scavenger prevented cell death and also the modulations in the signaling proteins. In addition, enhanced expression of CASP1 and CASP4 genes, along with increased Caspase-1 cleavage and increased IL-1ß release were significantly more in K562iNOS cells, which indicate priming of these cells for pyroptotic cell death. On the other hand, K562nNOS cells, displayed much enhanced CASP3 gene expression, Caspase-3 cleavage and Caspase-3 activity. Results obtained indicate that similar level of iNOS or nNOS activation in K562 cells, preferred pyroptotic and apoptotic cell death respectively.

Catalase S-Glutathionylation by NOX2 and Mitochondrial-Derived ROS Adversely Affects Mice and Human Neutrophil Survival.

Neutrophil survival and oxidative stress during inflammatory conditions are linked to tissue damage. The present study explores less understood role of catalase, the enzyme catalysing hydrogen peroxide decomposition, in neutrophil survival/death. Importantly, inhibition of catalase activity following S-glutathionylation in the PMA, NO, or zymosan-activated neutrophils or treatment with catalase inhibitor led to neutrophil death. On the contrary, introducing reducing environment by TCEP rescued catalase activity and significantly improved neutrophil survival. Furthermore, augmentation in ROS generation by NOX-2 activation or induction of mitochondrial ROS by Antimycin-A induced catalase S-glutathionylation and cell death, which was prevented in the neutrophil cytosolic factor1 (NCF-1-/-) cells or was rescued by MitoTEMPO, a mitochondrial ROS scavenger, thus, suggesting a correlation between catalase S-glutathionylation/activity inhibition and reduced neutrophil survival. Altogether, enhanced NOX2 activation/mitochondrial dysfunction led to reduced survival of human and mice neutrophils, due to H2O2 accumulation, S-glutathionylation of catalase, and reduction in its enzymatic activity. The present study thus demonstrated mitigation of catalase activity under oxidative stress-impacted neutrophil survival.

Glycolysis dependent lactate formation in neutrophils: A metabolic link between NOX-dependent and independent NETosis.

Neutrophil extracellular traps (NETs) play a pivotal role in the innate immune defense, as well as in the pathophysiology of various inflammatory disease conditions. Two major types of NETosis have been described - NOX-dependent and independent. The present study was undertaken to assess metabolic requirements of NETs formation by using PMA and A23187 as the inducers of NOX-dependent and NOX-independent NETosis respectively. Both these inducers caused an increase in ECAR, lactate dehydrogenase (LDH) activity, PKM2 dimerization and reduction in pyruvate kinase M2 (PKM2) activity, promoting lactate formation through Warburg effect. Interestingly exogenous treatment with lactate also induced NETs formation in human neutrophils, while inhibition of LDH activity significantly reduced NETosis by both the pathways. Moreover, NETosis and lactate accumulation during LPS induced sepsis in mice was inhibited by sodium oxamate, LDH inhibitor, demonstrating the importance of lactate in an experimental model of NETosis. Present study thus confirms importance of glycolysis in NETosis and also reveals role of lactate in NETs formation. It also reports sharing of the common metabolic pathway by NOX-dependent and independent NETosis.

Augmentation of iNOS expression in myeloid progenitor cells expedites neutrophil differentiation.

Neutrophils play important role in immunity and inflammation through diverse mechanisms. Reports from this lab and others have demonstrated involvement of NO in neutrophil adhesion, chemotaxis, bacterial killing, reactive oxygen species generation, neutrophil extracellular traps' formation, and apoptosis. Constitutive expression of iNOS in human neutrophils has also been documented. The role of NO-iNOS in neutrophil differentiation however remains ill-defined. The present study was undertaken to understand the role of NO generated from iNOS in the neutrophil differentiation by using iNOS-overexpressing K562 cells (K562iNOS ) and iNOS-deficient murine progenitor cells (lineage negative cells; lin-ve ). We observed that iNOS overexpression led to increased neutrophilic differentiation in K562 cells; more specifically an early and accelerated neutrophilic differentiation was spotted in K562iNOS . These observations were further validated using iNOS knockout lin-ve cells or hematopoietic progenitor cells that exhibited delayed neutrophil differentiation in comparison to its wild-type counterpart. In addition, a significant increase in the gene expression of iNOS during neutrophilic differentiation of CD34+ hematopoietic stem and progenitor cells derived from human bone marrow further substantiates importance of iNOS in neutrophil differentiation. Moreover, a significant increase in NO generation during neutrophil differentiation was observed and enhanced neutrophil differentiation with NO donor was also observed, implying the importance of NO in neutrophil differentiation. Collectively, using alternative approaches, we demonstrated that neutrophil differentiation is significantly influenced by iNOS or NO, suggesting the possibility of exploiting this novel link for therapeutic aspects of NO generated from iNOS and neutrophil differentiation in hematopoiesis-related disorders.

L-Arginine and tetrahydrobiopterin supported nitric oxide production is crucial for the microbicidal activity of neutrophils.

Recent report from this lab has shown role of Rac2 in the translocation of inducible nitric oxide synthase (iNOS) to the phagosomal compartment of polymorphonuclear leukocytes (PMNs) following phagocytosis of beads. This study was undertaken to further assess the status and role of tetrahydrobiopterin (BH4), a redox-sensitive cofactor, L-arginine, and the substrate of nitric oxide synthase (NOS) in sustained nitric oxide (˙NO) production in killing of phagocytosed microbes (Escherichia coli) by human PMNs. Time-dependent study revealed consistent NO and reactive oxygen species (ROS) production in the PMNs following phagocytosis of beads. In addition, levels of L-arginine and BH4 were maintained or increased simultaneously to support the enzymatic activity of NOS in the bead activated PMNs. Moreover, translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits along with iNOS was reconfirmed in the isolated phagosomes. We demonstrate that increase in the level of NO was supported by L-arginine and BH4 to kill E. coli, by using PMNs from NOS2-/- mice, human PMNs treated with biopterin inhibitor, N-acetyl serotonin (NAS), or by suspending human PMNs in L-arginine deficient medium. Altogether, this study demonstrates that following phagocytosis, sustained. NO production in the PMNs was well-maintained by redox sensitive cofactor, BH4 and substrate, and L-arginine to enable microbial killing. Further results suggest NO production in the human PMNs, along with ROS and myeloperoxidase (MPO) is important to execute antimicrobial activity.