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Cardiovascular pathologies are closely associated with intravascular thrombotic episodes, which is a complex phenomenon. Thrombosis involves continual interactions between platelets, plasma proteins and endothelial cells.

The work undertaken in my lab has been focused on these interactive participants of thrombosis. Neutrophils (PMNs), the cohabitant of platelets in blood, release several factors that alter platelet response.PMNs are one of the most imperative cells in innate immunity and are often referred to as foot soldiers of innate immune response. Their importance can be revealed by the fact that they comprise of almost 60% of total leukocyte population and are the first cells recruited to the site of infection.

Migration of PMNs from blood to specific tissues following pathological insult is a key feature of the host inflammatory response, which is often localized and protective.

These cells, however, have a very short lifespan of 6-7h; during this short life span they perform very crucial functions in host defence. Upon infection, they employ several effective antimicrobial strategies against a broad range of pathogens.

These include microbial killing, phagocytosis, degranulation, and even sacrifice themselves by process known as Neutrophil extracellular traps (NETs) release referred as NETosis. Neutrophils, the suicide killers, possess efficient oxidative and non-oxidative machineries to execute the engulfed pathogens.Activated PMNs generate highest amount of reactive oxygen species in human body through NADPH-oxidase (NOX-2) and myeloperoxidase (MPO), while resting and activated cells generate nitric oxide (NO) in appreciable amounts.

NO, a signalling molecule is a key mediator in a diverse number of physiological and pathophysiological processes as well as in the immune system. Research in our lab has been focused to understand that how this gaseous molecule strongly influences various functions of neutrophils.

Effect of NO on human and rodent neutrophils associated ROS/RNS generation

Neutrophils when added to platelet suspension significantly reduced platelet aggregation, characterization of this labile inhibitory factor in our lab, led to the identification of NO. Even though the biological functions of NO were the subject of much research, its role in PMNs functions was very less defined at that time. We found it incredibly interesting why nature has devised such a short lived cells to generate NO as well as reactive oxygen species (ROS). Investigations in our lab were initially focused to study the interaction of NO with superoxide radicals, and also examined the possibility of NO regulating generation of ROS in PMNs.

We demonstrated that NO modulates PMNs free radical generation in a biphasic manner, inhibiting NOX-2 activity and scavenging radicals at higher concentrations while, at lower concentrations NO augmented free radical generation. NO activated NOX-2 activity by facilitating the migration of p47phox to the neutrophil membranes, and interaction of NO with MPO generate nitrogen dioxide (.NO2) and nitryl chloride (NO2Cl). These findings have direct implications in medical science and suggest the role of NO in innate immunity as an important modulator of neutrophil functions.

Seth, P. et al.Blood. 1994;84(8):2741-8; Dikshit, M. et al. Brit J Pharmacol. 1996; 119(3): 578–582; Sethi, S. et al. Blood. 1999;93(1):333-40.; Sethi, S. et al.Thromb Res. 2000;98(5):445-50; Sharma, P. et al. J Leuko Biol. 2004;75(6):1070-8.; Patel, S. et al.Free Rad Res. 2009;43: 514-521; Kumar, S. et al.Cytometry Part A. 2010;77(5):467-77;

Biochemical and molecular characterization of NOS isoforms, their subcellular distribution and regulation in human and rodent leukocytes.

Large stores of ascorbic acid in PMNs, were found to prevent oxidation of tetrahydrobiopterin, a cofactor of NOS, to support the enzymatic synthesis of NO. Studies on PMNs from various species, including human, provided a convincing explanation as to why PMNs store exceptionally large amounts of ascorbate. It was evidenced that ascorbate deficiency led to a decline in both expression (both nNOS and iNOS expression) and catalysis of NOS in neutrophils among scorbutic guinea pigs. This was owing to instability of tetrahydrobiopterin a vital co-factor for NOS catalysis, and a rise in corticosterone levels as evidenced in scurvy, thereby interfering with iNOS expression among the scorbutics. These studies support the contention of Sir Pauling that vitamin C enhances immunity against infections. Rodents and human PMNs constitutively express iNOS&nNOS, and do not possess eNOS. Similar to neurons, neutrophils express full length PDZ domain containing nNOS. In resting human neutrophils, iNOS protein was distributed in the cytosol, granules containing elastase & gelatinase, and also in other subcellular organelles. Studies on bone marrow derived PMNs in rodents exhibited differential expression of nNOS and iNOS during maturation. Interestingly iNOS expression was consistently augmented during the neutrophil maturation in the bone marrow.

Sethi, S. et al. NO Biol. Chem. 2001; 5(5): 482-93; Sharma, P. et al. NO Biol. Chem. 2002;7(2):119-26; Sharma, P. et al. BBRC. 2003; 12;309(1):12-7; Sharma, P. et al. J Leuko Biol. 2004;75(6):1070-8; Saini, R. et al.J Leuko Biol. 2006;79(3):519-28; Chatterjee, M. et al. Free Rad Biol Med. 2008;45(8):1084-93; Kumar, S. et al. Cytometry Part. 2010;77(5):467-77; Saluja, R. et al. Cell & Tissue Res. 2010;340(2):381-8;Saluja, R. et al. BBA Mol Cell Res. 2011; 1813(10):1700-7

Effect of NO and nitrite on the cell proliferation and apoptosis

Studies on promyelocytic HL-60 cells in this lab systematically explored effect of NO donor (DETA-NO) and NO metabolite, nitrite on the cell cycle. NO in lower concentrations had proliferative effect, while addition of higher concentrations led to cytostasis, apoptosis, mitochondrial membrane potential loss, caspase-3 activation and dUTP nick end labelling. The proliferative effect of NO was redox sensitive and augmented expression of various cell cycle regulators such as Cdk2, cyclin B, cyclin E, which was blocked by roscovitine, a Cdk2 inhibitor. S-Nitrosylation of Cdk2 and an increase in the Cdk2 associated kinase activity was observed for the first time in NO/nitrite treated cells.

Having identified the strong influence of NO on myelocytic cell line, we were further intrigued its effect on neutrophil life span. Therefore, present studies ongoing in our lab are undertaken to decipher the importance of NO/NOS in the neutrophil apoptosis. As unlike ROS/NOX2, role of NO/NOS is not clearly defined in the apoptosis of neutrophils (PMNs). We found that prolonged treatment of human or mice PMNs with NO led to enhanced ROS generation, caspase-8/caspase 3 cleavage, reduced MMP, and finally cellular apoptosis. We further elucidated the signalling pathway in detail and found NO induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via Bid cleavage. NO mediated augmentation of caspase-8 and Bid cleavage were significantly prevented in PMNs from NCF KO mice, implying the involvement of NOX-2 in NO induced apoptosis of PMNs. Furthermore, ROS, NO generation and iNOS expression were enhanced in a time dependent manner in human and mice PMNs undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human and mice PMNs significantly reduced the levels of apoptosis but not in nNOS. Impaired apoptosis of PMNs from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, results obtained suggest crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway.

Kumar, S. et al.Free Rad Biol Med. 2010; 48(6):851-61; Kumar, S. et al.J PharmacolExpTher, 2011;337(3):812-21; Dubey, al.Cell Death and Disease, 2016

Effect of NO on Neutrophils extracellular trap formation

Since NO and PMNs are well established mediators of inflammation, we thought it logical to investigate effect of NO donors on human PMNs in NETs release and also explored the possible role of free radicals. NO also mediated release of neutrophil extracellular traps (NETs) which was regulated by ERK and MAP kinase signalling. We further elaborated this study to explore the extracellular bacterial killing, source of DNA in the expelled NETs, their ability to induce proinflammatory cytokines release from platelets/THP-1 cells, and assessment of NO-mediated free radical formation by using a consistent NO donor, DETA-NONOate. We found that NO by augmenting enzymatic free radical generation release NETs to promote extracellular bacterial killing. These NETs were made up of mitochondrial and nuclear DNA and potentiated release of pro-inflammatory cytokines.

Further studies were undertaken to assess circulating NETs contents and frequency of NETs generation in PMNs isolated from SIRS patients. These patients displayed significant augmentation in the circulating myeloperoxidase (MPO) activity and DNA content, while PMA stimulated PMNs from these patients, generated more free radicals and NETs. Expressions of inflammatory cytokines (IL-1b, TNF and IL-8) in the PMNs as well as their circulating levels were significantly augmented in SIRS subjects.

Elevated level of oxLDL and its inflammatory response have been observed in numerous pathological conditions and is responsible for severe innate immune response in sterile Systemic Inflammatory Response Syndrome (SIRS) in humans. So it seemed logical to investigate the effect of oxLDL on NETs formation and elucidate the underlying signalling mechanism. Due to complexity of oxLDL composition we further investigated which component of the oxLDL is predominantly responsible or has most pronounced effect in inducing NETs release from human neutrophils. Data obtained clearly showed that oxLDL stimulated NETs release from human neutrophil via NOX activation.

Most importantly results obtained indicate the importance of oxidized phospholipids as a possible critical component of the oxLDL involved in triggering NETs release. Moreover, both TLR2/6 receptors seem to be additively involved in oxLDL mediated NETs formation. As for signalling pathway we identified the Protein kinase C (PKC), Interleukin-1 receptor associated kinase (IRAKs), extracellular-signal-regulated kinases (ERK1/2), and p38 MAPK mediators to be involved in oxLDL induced ROS generation and NETosis.

Patel, S. et al.NO Biol. Chem. 2010;22(3):226-34;Keshari, RS. et al. PLOS One. 2012; 7(10):e48111;Keshari, al. Cytometry A.2012;81(3):238-47;Keshari, RS. et al. J Cell Biochem. 2013; 114(3):532-40;Awasthi, D. et al.Free Rad Biol Med. 2016; 93:190-203.

Role of NO and ROS/RNS generation on microbial killing by neutrophils via novel interaction of RAC2 with iNOS proteins in human PMNs (potential mechanism to carry iNOS to the phagosomes for the generation of both ROS/RNS to kill the phagocytosed bacteria)

These studies highlight previously undefined role of Rac2–iNOS interaction, in translocation of iNOS to phagosomal compartment, and consequent NO, superoxide radicals, ROS/ RNS generation, BSA nitration, and microbial killing. Altogether results obtained demonstrate the role of iNOS in NO and ROS/RNS generation, after phagocytosis of coated latex beads by human polymorphonuclear neutrophils. Our studies imply functional importance of iNOS and its interaction with Rac2 in pathogen killing by the neutrophils. MoreoverNFkB, mediated transcriptional regulation of iNOS was found to be prevented in human PMNs under high oxidative stress conditions.

PatelSaini, R. et al. J Leuko Biol. 2006;79(3):519-28; Jyoti, al. Blood. 2012; 124(21): 3131;Jyoti, al.Antiox Redox Signal. 2014;20(3):417-31; Singh, AK. et al. NO BiolChem 2016[Epub ahead of print]

Effect of NO on neutrophil chemotaxis, migration, polarization, phagocytosis and bacterial killing

Neutrophil adhesion, chemotaxis, bacterial killing and apoptosis are modulated by nitric oxide (NO) which along with hydrogen peroxide may act as intracellular signal transducers. The cellular redox status is maintained by a delicate balance between ROS and RNS (RONS) production and their effective scavenging by cellular enzymatic and non enzymatic antioxidants. A perturbation in this balance is thus detrimental for cellular homeostasis. Activated human PMNs are one major source of ROS generation and also synthesise NO. Several research groups, including ours, have shown the involvement of NADPH oxidase (NOX-2) in NO mediated augmentation of PMNs free radical generation.

Dubey, al.Free Rad Biol Med. 2016; 86:1-15

This study evaluates the effect of nitric oxide (DETA-NO) induced oxidative stress related S-glutathionylation of cytoskeleton proteins in human PMNs. By using in vitro and genetic approaches, we showed that S-glutathionylation of L-plastin (LPL) and -actin promotes reduced chemotaxis, polarization, bactericidal activity and phagocytosis. We identified Cys- 206, Cys-283 and Cys-460 as S-thiolated residues in the -actin-binding domain of LPL, where cys-460 was with the maximum score. Site directed mutagenesis of LPL Cys-460 further confirmed the role in the redox regulation of LPL. S-thiolation diminished binding as well as the bundling activity of LPL. The presence of S-thiolated LPL was detected in neutrophils from both diabetic patients and db/db mice with impaired PMNs function. Thus, enhanced nitroxidative stress may result in LPL S-glutathionylation leading to impaired chemotaxis, polarization and bactericidal activity of human PMNs providing a mechanistic basis for their impaired functions in diabetes mellitus.

Circulating NO levels, NOS activity, and /or NOS expression in the diverse animal models of pathology as well as in the neutrophils from patients of various diseases

Studies were simultaneously conducted to assess the differences in the circulating level of NO generation or NOS expression in the animal models of hypertension, dyslipidemia, atherosclerosis, thrombosis, ischemia/ hypoxia-reoxygenation, ulcers, neurodegeneration, blood brain permeability alterations as well as in the patents of Parkinson’s disease, depression, schizophrenia, migraine, sepsis, SIRS, diabetes with cardiometabolic disorders, neutrophelia. NOS found in PMNs resembles nNOS, we in a multi-centric study, investigated status of NOS in the CNS disorder patients (Parkinson’s disease, schizophrenia, depression and migraine in which role of NO was well documented). A novel finding from this lab was a significant increase in the basal NOS activity in the PMNs of Parkinson’s disease patients. Patients of schizophrenia and depression on the other hand exhibited a significant decrease in the PMNs nitrite content, while beta-adrenergic receptor binding was reduced only in the patients of depression. Migraine patients did not demonstrate change in the PMNs nitrite content but increase in the platelet NOS activity was reverted back to the normal level within few days after the migraine attack. Our studies have also shown that NO regulates free radical generation from PMNs during pathological conditions such as endotoxemia, and hypoxia-reoxygenation.

Dikshit, M. et al,J PharmacolExp Therap. 1993; 265(3):1369-73; Shukla, A. et al, Free Rad Biol Med. 1993; 15(1):97-100; Shukla, A. et al,Neuroreport. 1995; 6(12):1629-32; Hegde, al,Pharmacol Res. 1997; 36(2):109-14; Sethi, S. et al. Blood. 1999;93(1):333-40; Barthwal, al. ActaNeurol Scand. 1999; 100(5):300-4; Srivastava, al. Psychopharmacology. 2001; 158(2):140-5.; Shukla, R. et al. Headache. 2001;41(5):475-81; Srivastava, al. Affective Disorder. 2002; 72(1):45-52; Shukla, R. et al. Int J Neurosciences. 2003; 113(8):1043-54; Shukla, R. et al. Cephalgia. 2004; 24(1):37-43; Singh, S. et al. Redox Report. 2005; 10(2):103-9; Shukla, R. et al. Int J Neurosciences. 2006;116(12):1391-402; Singh, S. et al. Brain Res. 2007; 54(2):233-50; Husain, al. Neurochem Res. 2009; 34(8):1427-32; Patel, S. et al. Free Rad Res. 2009; 43: 514-521;Chatterjee, al. Cell Mol Biol. 2007; 53(1):84-93; Kothari, al. J Crit Care. 2011 ;26(4):435.e1-7; Kothari, al.J ActaAnaesthesiol Scand. 2012; 56(3):307-15;Keshari, RS. et al. PLOS One. 2012; 7(10):e48111;Jyoti, al. Blood. 2012; 124(21): 3131;

Effect of NO on mitochondrial oxidative stress

As in accordance with previous study we observed that enhanced ROS and reduced mitochondrial ROS generation seemed associated with NO production. We are presently investigating the relationship if any between NO and mitochondrial ROS generation. The on-going study might unravel role of mitochondria which otherwise have minimal role in energy production in the PMNs. Instead their importance is implied in neutrophil apoptosis however molecular mechanisms involved are also being investigated in our lab.

Contribution to the New Drug Development:

We have been actively involved in the New Drug Development Program of CDRI. I led the program on anti-thrombotics at CDRI, which resulted in identification of several selective inhibitors of collagen induced platelet activation, a new class of anti-platelet molecules. Two promising molecules as anti-platelet agents have been identified at CDRI as drug candidate and are under preclinical development.

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