Nitric oxide-mediated apoptosis of neutrophils through caspase-8 and caspase-3-dependent mechanism

Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway.Neutrophils are the most abundant terminally differentiated white blood cells. Although in a normal healthy human, 1–2 × 10¹¹ neutrophils are produced daily but hardly a few survive for more than 10 h in circulation.^{1, 2} Neutrophil phagocytose invading pathogens and kill them by producing reactive oxygen intermediates and/or by proteolytic enzymes. Besides pathogen clearance, neutrophils are also detrimental in a number of inflammatory diseases.³ Spontaneous apoptosis is thus crucial for neutrophil homeostasis and resolution of inflammation. Neutrophil apoptosis is controlled by apoptotic and survival pathways, which are modulated by pro- and anti-inflammatory cytokines, caspases and calpains. Moreover, a critical balance between reactive oxygen species (ROS) and anti-oxidants is required for cell survival. In neutrophils, ROS is largely produced by the enzyme NADPH oxidase (NOX) which adversely affects their survival.^{4, 5, 6} Yan et al.⁷ have recently demonstrated that NOX4 derived ROS following TGF-β stimulation induced apoptosis in endothelial cells.Nitric oxide (NO), a gaseous signalling molecule synthesized by NO synthase (NOS) from l-arginine, regulates several cellular functions such as vasodilation, migration, proliferation, differentiation and apoptosis. Cell death is induced following enhanced levels of NO from inducible nitric oxide synthase (iNOS) during inflammation, ischaemia/reperfusion or by NO donors such as DETA-NO, sodium nitroprusside and S-nitroso-N-acetyl-penicillamine.^{8, 9, 10} Our previous work has demonstrated a dose-dependent pro- and anti-apoptotic effect of NO on promyelocytic cell line HL-60.¹¹ Two isoforms of NOS-iNOS and nNOS are constitutively expressed in human and mice PMNs¹² but their regulation and interplay in neutrophil apoptosis is still enigmatic.Caspases having a crucial role in the modulation of apoptosis and apoptotic pathways have two components; caspase-8, an initiator caspase¹³ which mediates Fas induced death pathway, and caspase-9, which is vital for the mitochondrial mediated death. Opening of the mitochondrial membrane transition pore leads to cytochrome c release into the cytosol-forming apoptosis protease activating factor-1 (Apaf-1), a multimeric complex known as apoptosome which then activate pro-caspase-9. On the other hand, caspase-8 cleaves BID to tBID which translocate to mitochondria and release cytochrome c.⁵ Caspase-3, the effector caspase, is important for both extrinsic and intrinsic pathway with well documented role in the regulation of neutrophil apoptosis.¹⁴ It was shown that the anti-apoptotic effect of NO was related to the inhibition of caspase-3 activation through cGMP-dependent and independent mechanisms.¹⁵ S-glutathionylation is a redox-based regulatory mechanism which regulates caspase cleavage and its activation. Caspase-3 undergoes glutathionylation at Cys (163, 184 and 220) which prevents its cleavage and activation.¹⁶ In endothelial cells, TNF-α induced caspase-3 cleavage and apoptosis are regulated by caspase-3 glutathionylation/deglutathionylation cycles.¹⁷The present study demonstrates the crucial role of NO/iNOS in neutrophil survival. NO-induced ROS generation in human PMNs and mice bone marrow derived neutrophils (BMDN) led to caspase-8 cleavage, activation of BID and initiation of the mitochondrial death pathway. Augmented ROS production and apoptosis in NO pre-treated cells were attenuated in neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice BMDN or VAS-2870 treated human PMNs suggesting role of NOX in NO mediated initiation of apoptosis. NO-induced deglutathionylation of caspase-3 and -8 suggest redox mediated modulation of neutrophil apoptosis. Moreover, spontaneous apoptosis of BMDN was reduced in iNOS KO mice, iNOS silenced or iNOS inhibitor treated human PMNs, implying the importance of iNOS in neutrophil apoptosis. Altogether, these findings demonstrate the role of caspase-3, -8 and -9 in NO/iNOS induced neutrophil apoptosis.     

Variation of swimming speed enhances the chemotactic migration of Escherichia coli

Studies on chemotaxis of Escherichia coli have shown that modulation of tumble frequency causes a net drift up the gradient of attractants. Recently, it has been demonstrated that the bacteria is also capable of varying its runs speed in uniform concentration of attractant. In this study, we investigate the role of swimming speed on the chemotactic migration of bacteria. To this end, cells are exposed to gradients of a non-metabolizable analogue of glucose which are sensed via the Trg sensor. When exposed to a gradient, the cells modulate their tumble duration, which is accompanied with variation in swimming speed leading to drift velocities that are much higher than those achieved through the modulation of the tumble duration alone. We use an existing intra-cellular model developed for the Tar receptor and incorporate the variation of the swimming speed along with modulation of tumble frequency to predict drift velocities close to the measured values. The main implication of our study is that E. coli not only modulates the tumble frequency, but may also vary the swimming speed to affect chemotaxis and thereby efficiently sample its nutritionally rich environment.

Electronic supplementary material

The online version of this article (doi:10.1007/s11693-015-9174-x) contains supplementary material, which is available to authorized users.     

Vanadium in Biosphere and Its Role in Biological Processes

Ultra-trace elements or occasionally beneficial elements (OBE) are the new categories of minerals including vanadium (V). The importance of V is attributed due to its multifaceted biological roles, i.e., glucose and lipid metabolism as an insulin-mimetic, antilipemic and a potent stress alleviating agent in diabetes when vanadium is administered at lower doses. It competes with iron for transferrin (binding site for transportation) and with lactoferrin as it is secreted in milk also. The intracellular enzyme protein tyrosine phosphatase, causing the dephosphorylation at beta subunit of the insulin receptor, is inhibited by vanadium, thus facilitating the uptake of glucose inside the cell but only in the presence of insulin. Vanadium could be useful as a potential immune-stimulating agent and also as an antiinflammatory therapeutic metallodrug targeting various diseases. Physiological state and dose of vanadium compounds hold importance in causing toxicity also. Research has been carried out mostly on laboratory animals but evidence for vanadium importance as a therapeutic agent are available in humans and large animals also. This review examines the potential biochemical and molecular role, possible kinetics and distribution, essentiality, immunity, and toxicity-related study of vanadium in a biological system.