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.

The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5′-monophosphate–activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

Aims: Present study explores importance of inducible nitric oxide synthase (iNOS) and its interaction with Rac2 in reactive oxygen species (ROS)/reactive nitrogen species (RNS) generation, protein-nitration and in microbial killing by neutrophils. Results: The iNOS transcript and protein were constitutively present in human as well as in mice neutrophils. iNOS protein was found in cytosol, granules containing elastase and gelatinase, and in other subcellular organelles in resting human neutrophils. After phagocytosis of bovine serum albumin (BSA) coated beads, both human and mice neutrophils showed significant elevation in superoxide radicals, nitric oxide (NO), ROS/RNS and consequent BSA nitration. These responses were significantly reduced in presence of iNOS, NADPH oxidase (NOX), myeloperoxidase or Rac inhibitors, as well as in iNOS, Nox2 and Rac2 silenced human or iNOS-knockout mice neutrophils. Complex formed on interaction of iNOS with Rac2 coprecipitated with anti-Rac2, predominantly in cytosol in resting human neutrophils, while iNOS–Rac2 complex translocated to phagosomes after phagocytosis. This was accompanied by generation of superoxide radicals, NO, ROS/RNS and consequent BSA-nitration. Importance of Rac2 in iNOS mediated NO formation and microbial killing was confirmed by pretreatment of mice with Rac inhibitor, NSC23766 that significantly abrogated NO release and microbial killing in vivo. Innovation: Present study highlights 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. Conclusions: Altogether results obtained demonstrate the role of iNOS in NO and ROS/RNS generation, after phagocytosis of coated latex beads by human polymorphonuclear neutrophils. These studies imply functional importance of iNOS and its interaction with Rac2 in pathogen killing by the neutrophils. Antioxid. Redox Signal. 20, 417–431.

Bacterial and fungal infections are a major cause of morbidity and mortality in neutropenic patients. Donor-derived neutrophil transfusions have been used for prophylaxis or treatment for infection in neutropenic patients. However, the short half-life and the limited availability of large numbers of donor-derived neutrophils for transfusion remain a significant hurdle in the implementation of neutrophil transfusion therapy. Here, we investigate the in vitro and in vivo activity of neutrophils generated from human induced pluripotent stem cells (iPSC), a potentially unlimited resource to produce neutrophils for transfusion. Phenotypic analysis of iPSC-derived neutrophils reveal reactive oxygen species production at similar or slightly higher than normal peripheral blood neutrophils, but have an ∼50%–70% reduced Escherichia coli phagocytosis and phorbol 12-myristate 13-acetate induced formation of neutrophil extracellular traps (NET). Signaling of granulocytic precursors identified impaired AKT activation, but not ERK or STAT3, in agonist-stimulated iPSC-derived neutrophils. Expression of a constitutively activated AKT in iPSC-derived neutrophils restores most phagocytic activity and NET formation. In a model of bacterial induced peritonitis in immunodeficient mice, iPSC-derived neutrophils, with or without corrected AKT activation, migrate similarly to the peritoneal fluid as peripheral blood neutrophils, whereas the expression of activated AKT significantly improves their phagocytic activity in vivo. Stem Cells Translational Medicine 2019;8:557–567

Introduction: The role of increased oxidative stress and alterations to the nitric oxide (NO) pathway have been implicated in major depressive disorder (MDD). The two pathways interact closely with each other but have not been studied simultaneously in MDD. This study aimed to assess and compare the levels of oxidative and nitrosative stress in the neutrophils (PMNs) of drug-naive MDD patients and their first-degree relatives. Methods: 29 drug-naive MDD patients and 27 healthy first-degree relatives and healthy controls aged 18–45 years were included in this study. An assessment of the levels of reactive oxygen species (ROS), nitrites, neuronal NO synthase (nNOS), and myeloperoxidase in PMNs, and cortisol in serum was carried out. Results: Compared to healthy controls, the generation of free radicals, myeloperoxidase activity, and nNOS mRNA expression in PMNs, and cortisol level in serum were significantly higher in drug-naive depression patients. Indeed, increased levels of myeloperoxidase and serum cortisol were also noted in first-degree relatives. The total nitrite content in the PMNs and plasma however was significantly lower in both patients and first-degree relatives. Interestingly, a positive correlation was established in the ROS levels in the PMNs, plasma and neutrophil nitrite, and the serum cortisol level between MDD patients and their first-degree relatives. Conclusion: The results of this study contribute towards a better understanding of the familial association of depressive disorder, and demonstrate for the first time that neutrophil ROS/RNS, plasma nitrite, and serum cortisol levels are positively correlated between MDD patients and their first-degree relatives. However, further studies in larger, more diverse samples are needed to extend these pathways as potential biomarkers to identify persons at high risk for psychopathology at an early stage.