All Publications

  • NK-like CD8+ γδ T cells are expanded in persistent Mycobacterium tuberculosis infection. Science immunology Roy Chowdhury, R., Valainis, J. R., Dubey, M., von Boehmer, L., Sola, E., Wilhelmy, J., Guo, J., Kask, O., Ohanyan, M., Sun, M., Huang, H., Huang, X., Nguyen, P. K., Scriba, T. J., Davis, M. M., Bendall, S. C., Chien, Y. H. 2023; 8 (81): eade3525


    The response of gamma delta (γδ) T cells in the acute versus chronic phases of the same infection is unclear. How γδ T cells function in acute Mycobacterium tuberculosis (Mtb) infection is well characterized, but their response during persistent Mtb infection is not well understood, even though most infections with Mtb manifest as a chronic, clinically asymptomatic state. Here, we analyze peripheral blood γδ T cells from a South African adolescent cohort and show that a unique CD8+ γδ T cell subset with features of "memory inflation" expands in chronic Mtb infection. These cells are hyporesponsive to T cell receptor (TCR)-mediated signaling but, like NK cells, can mount robust CD16-mediated cytotoxic responses. These CD8+ γδ T cells comprise a highly focused TCR repertoire, with clonotypes that are Mycobacterium specific but not phosphoantigen reactive. Using multiparametric single-cell pseudo-time trajectory analysis, we identified the differentiation paths that these CD8+ γδ T cells follow to develop into effectors in this infection state. Last, we found that circulating CD8+ γδ T cells also expand in other chronic inflammatory conditions, including cardiovascular disease and cancer, suggesting that persistent antigenic exposure may drive similar γδ T cell effector programs and differentiation fates.

    View details for DOI 10.1126/sciimmunol.ade3525

    View details for PubMedID 37000856

  • Early non-neutralizing, afucosylated antibody responses are associated with COVID-19 severity. Science translational medicine Chakraborty, S., Gonzalez, J. C., Sievers, B. L., Mallajosyula, V., Chakraborty, S., Dubey, M., Ashraf, U., Cheng, B. Y., Kathale, N., Tran, K. Q., Scallan, C., Sinnott, A., Cassidy, A., Chen, S. T., Gelbart, T., Gao, F., Golan, Y., Ji, X., Kim-Schulze, S., Prahl, M., Gaw, S. L., Gnjatic, S., Marron, T. U., Merad, M., Arunachalam, P. S., Boyd, S. D., Davis, M. M., Holubar, M., Khosla, C., Maecker, H. T., Maldonado, Y., Mellins, E. D., Nadeau, K. C., Pulendran, B., Singh, U., Subramanian, A., Utz, P. J., Sherwood, R., Zhang, S., Jagannathan, P., Tan, G. S., Wang, T. T. 1800: eabm7853


    A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated IgG antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by mRNA SARS-CoV-2 vaccines were instead highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. To study the biology afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG-Fc gamma receptor (FcgammaR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from COVID-19 patients induced inflammatory cytokine production and robust infiltration of the lung by immune cells. By contrast, vaccine-elicited IgG did not promote an inflammatory lung response. Together, these results show that IgG-FcgammaR interactions are able to regulate inflammation in the lung and may define distinct lung activities associated with the IgG that are associated with severe COVID-19 and protection against infection with SARS-CoV-2.

    View details for DOI 10.1126/scitranslmed.abm7853

    View details for PubMedID 35040666

  • Expression of inducible NOS is indispensable for the antiproliferative and proapoptotic effect of imatinib in BCR-ABL positive cells JOURNAL OF LEUKOCYTE BIOLOGY Singh, A., Awasthi, D., Dubey, M., Nagarkoti, S., Chandra, T., Barthwal, M., Tripathi, A., Dikshit, M. 2021; 110 (5): 853-866


    Chronic myeloid leukemia (CML) is characterized by constitutive BCR-ABL kinase activity, an aggressive proliferation of immature cells, and reduced differentiation. Targeting tyrosine kinase activity of BCR-ABL with imatinib is an effective therapy for the newly diagnosed CML patients; however, 20%-30% of the patients initially treated with imatinib eventually experience treatment failure. Therefore, early identification of these patients is of high clinical relevance. In the present study, we by undertaking a direct comparison of inducible NOS (iNOS) status in neutrophils from healthy volunteers, newly diagnosed, imatinib responder, and resistant CML patients as well as by conducting in vitro studies in K562 cells demonstrated that inhibition of BCR-ABL by imatinib or siRNA significantly enhanced NO generation and iNOS expression. Indeed, patients exhibiting treatment failure or imatinib resistance were less likely to induce NO generation/iNOS expression. Our findings further demonstrated that imatinib mediated antiproliferative and proapoptotic effect in BCR-ABL+ cells associated with enhanced iNOS expression, and it was significantly prevented in the presence of L-NAME, 1400W, or iNOS siRNA. Overexpression of iNOS in K562 cells expectedly enhanced imatinib sensitivity on cytostasis and apoptosis, even at lower concentration (0.1 μM) of imatinib. Mechanistically, imatinib or BCR-ABL siRNA following deglutathionylation of NF-κB, enhanced its binding to iNOS promoter and induced iNOS transcription. Deglutathionylation of procaspase-3 however associated with increased caspase-3 activity and cell apoptosis. Taken together, results obtained suggest that monitoring NO/iNOS level could be useful to identify patients likely to be responsive or resistant to imatinib and can be used to personalized alternative therapy.

    View details for DOI 10.1002/JLB.1A0820-514R

    View details for Web of Science ID 000613666400001

    View details for PubMedID 33527482

  • Catalase S-Glutathionylation by NOX2 and Mitochondrial-Derived ROS Adversely Affects Mice and Human Neutrophil Survival. Inflammation Nagarkoti, S., Dubey, M., Sadaf, S., Awasthi, D., Chandra, T., Jagavelu, K., Kumar, S., Dikshit, M. 2019; 42 (6): 2286-2296


    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.

    View details for DOI 10.1007/s10753-019-01093-z

    View details for PubMedID 31646444

  • S-Glutathionylation of p47phox sustains superoxide generation in activated neutrophils. Biochimica et biophysica acta. Molecular cell research Nagarkoti, S., Dubey, M., Awasthi, D., Kumar, V., Chandra, T., Kumar, S., Dikshit, M. 2018; 1865 (2): 444-454


    Post-translational modifications (PTMs) induced conformational changes of proteins can cause their activation or inactivation. Neutrophils clear pathogen through phagocytosis and oxidative burst generation, while participate in inflammation through sustained and uncontrolled generation of ROS. In activated PMNs, cytosolic NOX-2 subunit p47phox following phosphorylation interacts with p67phox, p40phox and along with Rac2 translocate to the membrane. Phosphorylation of p47phox subunit occurs in both short spurts as well as sustained ROS generation, suggesting towards the unidentified molecular mechanism(s) driving these two diverse outcomes by various stimuli. The present study demonstrates that in PMA or NO treated neutrophils a subunit of NOX2, p47phox gets glutathionylated to sustain ROS generation along with a decrease in catalase, Grx-1 activity and change in GSH/GSSG ratio. Surprisingly, fMLP treated cells neither showed sustained ROS production nor glutathionylation of p47phox. S-Glutathionylation was always secondary to phosphorylation of p47phox and inhibition of glutathionylation did not alter phosphorylation but specifically impaired sustained ROS production. Interestingly, forced S-glutathionylation of p47phox converted the fMLP induced ROS generation into sustained release of ROS. We then identified the glutathionylation susceptible cysteine residues of p47phox by LC-MS/MS with IAM switch mapping. Site-directed mutagenesis of cysteine residues further mitigated p47phox S-glutathionylation. Thus, we demonstrate that p47phox S-glutathionylation plays an essential key role in the sustained ROS generation by human neutrophils.

    View details for DOI 10.1016/j.bbamcr.2017.11.014

    View details for PubMedID 29195919

  • Nitric oxide-mediated apoptosis of neutrophils through caspase-8 and caspase-3-dependent mechanism CELL DEATH & DISEASE Dubey, M., Nagarkoti, S., Awasthi, D., Singh, A. K., Chandra, T., Kumaravelu, J., Barthwal, M. K., Dikshit, M. 2016; 7


    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.

    View details for DOI 10.1038/cddis.2016.248

    View details for Web of Science ID 000382326800010

    View details for PubMedID 27584786

    View details for PubMedCentralID PMC5059853

  • High oxidative stress adversely affects NFκB mediated induction of inducible nitric oxide synthase in human neutrophils: Implications in chronic myeloid leukemia. Nitric oxide : biology and chemistry Singh, A. K., Awasthi, D., Dubey, M., Nagarkoti, S., Kumar, A., Chandra, T., Barthwal, M. K., Tripathi, A. K., Dikshit, M. 2016; 58: 28-41


    Increasing evidence support bimodal action of nitric oxide (NO) both as a promoter and as an impeder of oxygen free radicals in neutrophils (PMNs), however impact of high oxidative stress on NO generation is less explored. In the present study, we comprehensively investigated the effect of high oxidative stress on inducible nitric oxide synthase (iNOS) expression and NO generation in human PMNs. Our findings suggest that PMA or diamide induced oxidative stress in PMNs from healthy volunteers, and high endogenous ROS in PMNs of chronic myeloid leukemia (CML) patients attenuate basal as well as LPS/cytokines induced NO generation and iNOS expression in human PMNs. Mechanistically, we found that under high oxidative stress condition, S-glutathionylation of NFκB (p50 and p65 subunits) severely limits iNOS expression due to its reduced binding to iNOS promoter, which was reversed in presence of DTT. Furthermore, by using pharmacological inhibitors, scavengers and molecular approaches, we identified that enhanced ROS generation via NOX2 and mitochondria, reduced Grx1/2 expression and GSH level associated with NFκB S-glutathionylation in PMNs from CML patients. Altogether data obtained suggest that oxidative status act as an important regulator of NO generation/iNOS expression, and under enhanced oxidative stress condition, NOX2-mtROS-NFκB S-glutathionylation is a feed forward loop, which attenuate NO generation and iNOS expression in human PMNs.

    View details for DOI 10.1016/j.niox.2016.06.002

    View details for PubMedID 27264783

  • Oxidized LDL induced extracellular trap formation in human neutrophils via TLR-PKC-IRAK-MAPK and NADPH-oxidase activation FREE RADICAL BIOLOGY AND MEDICINE Awasthi, D., Nagarkoti, S., Kumar, A., Dubey, M., Singh, A. K., Pathak, P., Chandra, T., Barthwal, M. K., Dikshit, M. 2016; 93: 190-203


    Neutrophil extracellular traps (NETs) formation was initially linked with host defence and extracellular killing of pathogens. However, recent studies have highlighted their inflammatory potential. Oxidized low density lipoprotein (oxLDL) has been implicated as an independent risk factor in various acute or chronic inflammatory diseases including systemic inflammatory response syndrome (SIRS). In the present study we investigated effect of oxLDL on NETs formation and elucidated the underlying signalling mechanism. Treatment of oxLDL to adhered PMNs led to a time and concentration dependent ROS generation and NETs formation. OxLDL induced free radical formation and NETs release were significantly prevented in presence of NADPH oxidase (NOX) inhibitors suggesting role of NOX activation in oxLDL induced NETs release. Blocking of both toll like receptor (TLR)-2 and 6 significantly reduced oxLDL induced NETs formation indicating requirement of both the receptors. We further identified Protein kinase C (PKC), Interleukin-1 receptor associated kinase (IRAKs), mitogen-activated protein kinase (MAPK) pathway as downstream intracellular signalling mediators involved in oxLDL induced NETs formation. OxLDL components such as oxidized phospholipids (lysophosphatidylcholine (LPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC)) were most potent NETs inducers and might be crucial for oxLDL mediating NETs release. Other components like, oxysterols, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were however less potent as compared to oxidized phospholipids. This study thus demonstrates for the first time that treatment of human PMNs with oxLDL or its various oxidized phopholipid component mediated NETs release, implying their role in the pathogenesis of inflammatory diseases such as SIRS.

    View details for DOI 10.1016/j.freeradbiomed.2016.01.004

    View details for Web of Science ID 000371219400018

    View details for PubMedID 26774674

  • L-Plastin S-glutathionylation promotes reduced binding to beta-actin and affects neutrophil functions FREE RADICAL BIOLOGY AND MEDICINE Dubey, M., Singh, A. K., Awasthi, D., Nagarkoti, S., Kumar, S., Ali, W., Chandra, T., Kumar, V., Barthwal, M. K., Jagavelu, K., Sanchez-Gomez, F. J., Lamas, S., Dikshit, M. 2015; 86: 1-15


    Posttranslational modifications (PTMs) of cytoskeleton proteins due to oxidative stress associated with several pathological conditions often lead to alterations in cell function. The current 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 β-actin promotes reduced chemotaxis, polarization, bactericidal activity, and phagocytosis. We identified Cys-206, Cys-283, and Cys-460as S-thiolated residues in the β-actin-binding domain of LPL, where cys-460 had 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 PMN functions. Thus, enhanced nitroxidative stress may results 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.

    View details for DOI 10.1016/j.freeradbiomed.2015.04.008

    View details for Web of Science ID 000360569700001

    View details for PubMedID 25881549

  • Interaction of Inducible Nitric Oxide Synthase with Rac2 Regulates Reactive Oxygen and Nitrogen Species Generation in the Human Neutrophil Phagosomes: Implication in Microbial Killing ANTIOXIDANTS & REDOX SIGNALING Jyoti, A., Singh, A. K., Dubey, M., Kumar, S., Saluja, R., Keshari, R. S., Verma, A., Chandra, T., Kumar, A., Bajpai, V. K., Barthwal, M. K., Dikshit, M. 2014; 20 (3): 417-431


    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.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.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.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.

    View details for DOI 10.1089/ars.2012.4970

    View details for Web of Science ID 000330804800003

    View details for PubMedID 23875749

  • S-glutathionylation: relevance in diabetes and potential role as a biomarker BIOLOGICAL CHEMISTRY Sanchez-Gomez, F. J., Espinosa-Diez, C., Dubey, M., Dikshit, M., Lamas, S. 2013; 394 (10): 1263-1280


    Glutathione is considered the main regulator of redox balance in the cellular milieu due to its capacity for detoxifying deleterious molecules. The oxidative stress induced as a result of a variety of stimuli promotes protein oxidation, usually at cysteine residues, leading to changes in their activity. Mild oxidative stress, which may take place in physiological conditions, induces the reversible oxidation of cysteines to sulfenic acid form, while pathological conditions are associated with higher rates of reactive oxygen species production, inducing the irreversible oxidation of cysteines. Among these, neurodegenerative disorders, cardiovascular diseases and diabetes have been proposed to be pathogenetically linked to this state. In diabetes-associated vascular complications, lower levels of glutathione and increased oxidative stress have been reported. S-glutathionylation has been proposed as a posttranslational modification able to protect proteins from over-oxidizing environments. S-glutathionylation has been identified in proteins involved in diabetic models both in vitro and in vivo. In all of them, S-glutathionylation represents a mechanism that regulates the response to diabetic conditions, and has been described to occur in erythrocytes and neutrophils from diabetic patients. However, additional studies are necessary to discern whether this modification represents a biomarker for the early onset of diabetic vascular complications.

    View details for DOI 10.1515/hsz-2013-0150

    View details for Web of Science ID 000324050400003

    View details for PubMedID 24002664

  • Cytokines Induced Neutrophil Extracellular Traps Formation: Implication for the Inflammatory Disease Condition PLOS ONE Keshari, R. S., Jyoti, A., Dubey, M., Kothari, N., Kohli, M., Bogra, J., Barthwal, M. K., Dikshit, M. 2012; 7 (10)


    Neutrophils (PMNs) and cytokines have a critical role to play in host defense and systemic inflammatory response syndrome (SIRS). Neutrophil extracellular traps (NETs) have been shown to extracellularly kill pathogens, and inflammatory potential of NETs has been shown. Microbial killing inside the phagosomes or by NETs is mediated by reactive oxygen and nitrogen species (ROS/RNS). The present study was undertaken to assess circulating NETs contents and frequency of NETs generation by isolated PMNs 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. Plasma obtained from SIRS patients, if added to the PMNs isolated from healthy subjects, enhanced NETs release and free radical formation. Expressions of inflammatory cytokines (IL-1β, TNFα and IL-8) in the PMNs as well as their circulating levels were significantly augmented in SIRS subjects. Treatment of neutrophils from healthy subjects with TNFα, IL-1β, or IL-8 enhanced free radicals generation and NETs formation, which was mediated through the activation of NADPH oxidase and MPO. Pre-incubation of plasma from SIRS with TNFα, IL-1β, or IL-8 antibodies reduced the NETs release. Role of IL-1β, TNFα and IL-8 thus seems to be involved in the enhanced release of NETs in SIRS subjects.

    View details for DOI 10.1371/journal.pone.0048111

    View details for Web of Science ID 000310262500037

    View details for PubMedID 23110185

  • Neutrophil extracellular traps contain mitochondrial as well as nuclear DNA and exhibit inflammatory potential. Cytometry. Part A : the journal of the International Society for Analytical Cytology Keshari, R. S., Jyoti, A., Kumar, S., Dubey, M., Verma, A., Srinag, B. S., Krishnamurthy, H., Barthwal, M. K., Dikshit, M. 2012; 81 (3): 238-247


    Neutrophils expel extracellular traps (NETs) to entrap and exterminate the invaded micro-organisms. Acute/chronic inflammatory disorders are often observed with aberrantly enhanced NETs formation and high nitric oxide (NO) availability. Recent study from this laboratory demonstrated release of NETs from human neutrophils following treatment with SNP or SNAP. This study is an extension of our previous finding 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. NO-mediated NETs exhibited extracellular bacterial killing as determined by colony forming units. NO-mediated NETs formation was due to the activation of NADPH oxidase and myeloperoxidase. NO- or PMA-mediated NETs were positive for both nuclear and mitochondrial DNA as well as proteolytic enzymes. Incubation of NETs with human platelets enhanced the release of IL-1β and IL-8, while with THP-1 cells, release of IL-1β, IL-8, and TNFα was observed. This study demonstrates 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 proinflammatory cytokines.

    View details for DOI 10.1002/cyto.a.21178

    View details for PubMedID 22170804