Impaired Reactive Oxygen Species Research Articles

Multifaceted roles of NADPH oxidases in the growth and pathogenicity of Beauveria bassiana

ABSTRACT Reactive oxygen species (ROS), synthesized by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, are vital molecules in biological cells, influencing various physiological processes such as fungal growth, development, and virulence. Beauveria bassiana, an entomopathogenic fungus, is a promising biopesticide for agricultural, forestry, and urban pest control. This study focuses on the characterization of NADPH oxidases (Noxs) in B. bassiana. Gene expression profiles of Noxs in B. bassiana (BbNoxs) were analysed using RT-qPCR. Knockout strains of single BbNoxA, BbNoxB, BbNoxR, and double BbNoxA and BbNoxB were constructed via homologous recombination, and their phenotypic characteristics were examined. Fungal virulence was evaluated using Galleria mellonella larvae, and infection structures formation and penetration ability were assessed on cicada wings. ROS production and actin assembly during fungal growth and infection were detected using staining and marker methods. Expression analysis revealed significant upregulation of BbNoxs during fungal growth and infection. Compared to the wild-type strain, single knockouts (ΔBbNoxA/B/R) and double knockout (ΔBbNoxAB) of BbNoxs exhibited reduced conidial yields, accelerated conidial germination rates. Deletion of BbNoxB or BbNoxR decreased fungal virulence compared to the WT strain in topical inoculation experiments. Additionally, loss of BbNoxB or BbNoxR impaired infection structures formation, penetration ability, ROS production, and actin aggregation during fungal infection. BbNoxs are crucial for fungal growth, development, and virulence in B. bassiana, playing essential roles in infection structures formation, penetration, ROS production, and actin assembly. Understanding their functions provides insights into B. bassiana’s pathogenic mechanisms.

Suppression of ITPKB degradation by Trim25 confers TMZ resistance in glioblastoma through ROS homeostasis

Temozolomide (TMZ) represents a standard-of-care chemotherapeutic agent in glioblastoma (GBM). However, the development of drug resistance constitutes a significant hurdle in the treatment of malignant glioma. Although specific innovative approaches, such as immunotherapy, have shown favorable clinical outcomes, the inherent invasiveness of most gliomas continues to make them challenging to treat. Consequently, there is an urgent need to identify effective therapeutic targets for gliomas to overcome chemoresistance and facilitate drug development. This investigation used mass spectrometry to examine the proteomic profiles of six pairs of GBM patients who underwent standard-of-care treatment and surgery for both primary and recurrent tumors. A total of 648 proteins exhibiting significant differential expression were identified. Gene Set Enrichment Analysis (GSEA) unveiled notable alterations in pathways related to METABOLISM_OF_LIPIDS and BIOLOGICAL_OXIDATIONS between the primary and recurrent groups. Validation through glioma tissue arrays and the Xiangya cohort confirmed substantial upregulation of inositol 1,4,5-triphosphate (IP3) kinase B (ITPKB) in the recurrence group, correlating with poor survival in glioma patients. In TMZ-resistant cells, the depletion of ITPKB led to an increase in reactive oxygen species (ROS) related to NADPH oxidase (NOX) activity and restored cell sensitivity to TMZ. Mechanistically, the decreased phosphorylation of the E3 ligase Trim25 at the S100 position in recurrent GBM samples accounted for the weakened ITPKB ubiquitination. This, in turn, elevated ITPKB stability and impaired ROS production. Furthermore, ITPKB depletion or the ITPKB inhibitor GNF362 effectively overcome TMZ chemoresistance in a glioma xenograft mouse model. These findings reveal a novel mechanism underlying TMZ resistance and propose ITPKB as a promising therapeutic target for TMZ-resistant GBM.

A32 CATHELICIDIN REGULATES GOBLET CELL MUCUS SECRETION DURING CITROBACTER RODENTIUM-INDUCED COLITIS

Abstract Background Colonic goblet cells by secreting Muc2 mucin and specific proteins is critical for physically entrapping and expelling invading enteropathogens. Thus, is not surprising that Muc2-/- littermates exhibit increased susceptibility to attaching/effacing Citrobacter rodentium colonization. The colonic epithelium also secretes small cathelicidin peptide, which potentially interacts intimately with goblet cells and was presumed to accumulate within the sterile inner mucus layer as a simple antimicrobial peptide defense. Aims To determine the effects of cathelicidin on mucin secretion in goblet cells during C. rodentium-induced colitis and the impact on the mucus barrier defense. Methods We used cathelicidin-deficient (Camp-/-) mice, mouse colonoids and human colonic LS174T like-goblet epithelial cells to elucidate the mechanisms by which cathelicidin regulates goblet cell secretions. Results Camp -/- littermates infected with C. rodentium displayed increased fecal shedding and epithelial colonization. Camp-/- littermates at the peak of C. rodentium infection (7 dpi) showed a deficient mucin layer with fewer Alcian blue/PAS filled goblet cells and a reduction in fucose (UEA-1+) and N-acetylglucosamine (WGA+) glycoproteins. By transmission electron microscopy (TEM), goblet cells in Camp-/- colons were swollen and retained a large number of mucus granules during C. rodentium infection. C. rodentium infected Camp-/- littermates showed impaired reactive oxygen species (ROS) production and a transcriptomic profiling associated with decreased ROS biosynthesis and an increase in ROS negative regulators. In mucin producing LS174T colonic epithelial cells, human cathelicidin LL-37 promptly induced the secretion of goblet cell-associated TFF3 and RELMβ, via a ROS-dependent mechanism. Conclusions These findings revealed that mice lacking cathelicidin (Camp-/-) were more susceptible to C. rodentium colonization caused by defective goblet cell mucus and mucin-associated protein secretion via a ROS-dependent mechanism. Importantly, cathelicidin regulated mucus secretion revealing a non microbicidal action of this peptide with homeostatic properties on the colonic mucus barrier, critical in excluding luminal microbiota away from the epithelia to clear bacterial infections and restore gut homeostasis. Funding Agencies NSERC

Eosinophils preserve bone homeostasis by inhibiting excessive osteoclast formation and activity via eosinophil peroxidase

Eosinophils are involved in tissue homeostasis. Herein, we unveiled eosinophils as important regulators of bone homeostasis. Eosinophils are localized in proximity to bone-resorbing osteoclasts in the bone marrow. The absence of eosinophils in ΔdblGATA mice results in lower bone mass under steady-state conditions and amplified bone loss upon sex hormone deprivation and inflammatory arthritis. Conversely, increased numbers of eosinophils in IL-5 transgenic mice enhance bone mass under steady-state conditions and protect from hormone- and inflammation- mediated bone loss. Eosinophils strongly inhibit the differentiation and demineralization activity of osteoclasts and lead to profound changes in the transcriptional profile of osteoclasts. This osteoclast-suppressive effect of eosinophils is based on the release of eosinophil peroxidase causing impaired reactive oxygen species and mitogen-activated protein kinase induction in osteoclast precursors. In humans, the number and the activity of eosinophils correlates with bone mass in healthy participants and rheumatoid arthritis patients. Taken together, experimental and human data indicate a regulatory function of eosinophils on bone.

The antioxidant betulinic acid enhances porcine oocyte maturation through Nrf2/Keap1 signaling pathway modulation.

During in vitro maturation, excess levels of reactive oxygen species (ROS) are a major cause of developmental defects in embryos. Betulinic acid (BA) is a naturally produced antioxidant in white birch bark. Recent studies have shown that BA exhibits antioxidant properties in various cells through the activation of antioxidant genes. Therefore, we investigated the effect of BA treatment on porcine oocytes and its underlying mechanism during oocyte maturation. Treatment with 0.1 μM BA significantly increased the proportion of MII oocytes compared with controls, and BA-treated oocytes had significantly higher development rates, trophectoderm cell numbers, and cell survival rates than controls. These results demonstrate that BA treatment improved the developmental competence of oocytes. Following BA treatment, oocytes exhibited reduced ROS levels and elevated glutathione (GSH) levels, accompanied by the enhanced expression of antioxidant genes, compared with control oocytes. To evaluate the antioxidant effects of BA, oocytes were exposed to H2O2, a potent ROS activator. Impaired nuclear maturation, ROS levels, and GSH levels induced in oocytes by H2O2 exposure was restored by BA treatment. As these antioxidant genes are regulated by the Nrf2/Keap1 signaling pathway, which is involved in antioxidant responses, we applied the Nrf2 inhibitor brusatol to investigate the effects of BA on this pathway. The negative effects of brusatol on meiotic maturation and oocyte quality, including levels of ROS, GSH, and antioxidant-related gene expression, were mitigated by BA treatment. Our results suggested that BA plays an effective role as an antioxidant in porcine oocyte maturation through adjusting the Nrf2/Keap1 signaling pathway. This finding provides valuable insights into the mechanisms governing oocyte maturation and embryonic development.

Zebrafish nos2a benefits bacterial proliferation via suppressing ROS and inducing NO production to impair the expressions of inflammatory cytokines and antibacterial genes

The enzyme nitric oxide synthase 2 or inducible NOS (NOS2), reactive oxygen species (ROS) and nitric oxide (NO) are important participants in various inflammatory and immune responses. However, the functional significances of the correlations among piscine NOS2, ROS and NO during pathogen infection remain unclear. In teleost, there are two nos2 genes (nos2a and nos2b). It has been previously reported that zebrafish nos2a behaves as a classical inducible NOS, and nos2b exerts some functions similar to mammalian NOS3. In the present study, we reported the functional characterization of zebrafish nos2a during bacterial infection. We found that zebrafish nos2a promoted bacterial proliferation, accompanied by an increased susceptibility to Edwardsiella piscicida infection. The nagative regulation of zebrafish nos2a during E. piscicida infection was characterized by the impaired ROS levels, the induced NO production and the decreased expressions of proinflammatory cytokines, antibacterial genes and oxidant factors. Furthermore, although both inducing ROS and inhibiting NO production significantly inhibited bacterial proliferation, only inhibiting NO production but not inducing ROS significantly increased resistance to E. piscicida infection. More importantly, ROS supplementation and inhibition of NO completely abolished this detrimental consequence mediated by zebrafish nos2a during E. piscicida infection. All together, these results firstly demonstrate that the innate response mediated by zebrafish nos2a in promoting bacterial proliferation is dependent on the lower ROS level and higher NO production. The present study also reveals that inhibition of NO can be effective in the protection against E. piscicida infection.

Targeting Unc51-like Autophagy Activating Kinase 1 (ULK1) Overcomes Adaptive Drug Resistance in Acute Myelogenous Leukemia.

ULK1 drives a cell-intrinsic adaptive resistance in AML and targeting ULK1-mediated autophagy can synergize with existing and emerging AML therapies to overcome drug-resistance and induce apoptosis.

Platelet Redox Imbalance in Hypercholesterolemia: A Big Problem for a Small Cell.

The imbalance between reactive oxygen species (ROS) synthesis and their scavenging by anti-oxidant defences is the common soil of many disorders, including hypercholesterolemia. Platelets, the smallest blood cells, are deeply involved in the pathophysiology of occlusive arterial thrombi associated with myocardial infarction and stroke. A great deal of evidence shows that both increased intraplatelet ROS synthesis and impaired ROS neutralization are implicated in the thrombotic process. Hypercholesterolemia is recognized as cause of atherosclerosis, cerebro- and cardiovascular disease, and, closely related to this, is the widespread acceptance that it strongly contributes to platelet hyperreactivity via direct oxidized LDL (oxLDL)-platelet membrane interaction via scavenger receptors such as CD36 and signaling pathways including Src family kinases (SFK), mitogen-activated protein kinases (MAPK), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In turn, activated platelets contribute to oxLDL generation, which ends up propagating platelet activation and thrombus formation through a mechanism mediated by oxidative stress. When evaluating the effect of lipid-lowering therapies on thrombogenesis, a large body of evidence shows that the effects of statins and proprotein convertase subtilisin/kexin type 9 inhibitors are not limited to the reduction of LDL-C but also to the down-regulation of platelet reactivity mainly by mechanisms sensitive to intracellular redox balance. In this review, we will focus on the role of oxidative stress-related mechanisms as a cause of platelet hyperreactivity and the pathophysiological link of the pleiotropism of lipid-lowering agents to the beneficial effects on platelet function.

Superoxide dismutase 2 ameliorates mitochondrial dysfunction in skin fibroblasts of Leber's hereditary optic neuropathy patients.

BackgroundIn Leber’s hereditary optic neuropathy (LHON), mtDNA mutations mediate mitochondrial dysfunction and apoptosis of retinal ganglion cells. Mitochondrial superoxide dismutase 2 (SOD2) is a crucial antioxidase against reactive oxygen species (ROS). This study aims to investigate whether SOD2 could ameliorate mtDNA mutation mediated mitochondrial dysfunction in skin fibroblasts of LHON patients and explore the underlying mechanisms.MethodsThe skin of normal healthy subjects and severe LHON patients harboring m.11778G > A mutation was taken to prepare immortalized skin fibroblast cell lines (control-iFB and LHON-iFB). LHON-iFB cells were transfected with SOD2 plasmid or negative control plasmid, respectively. In addition, human neuroblastoma SH-SY5Y cells and human primary retinal pigmental epithelium (hRPE) cells were stimulated by H2O2 after gene transfection. The oxygen consumption rate (OCR) was measured with a Seahorse extracellular flux analyzer. The level of ATP production, mitochondrial membrane potential, ROS and malondialdehyde (MDA) were measured separately with the corresponding assay kits. The expression level of SOD2, inflammatory cytokines and p-IκBα/IκBα was evaluated by western-blot. Assessment of apoptosis was performed by TUNEL assay.ResultsLHON-iFB exhibited lower OCR, ATP production, mitochondrial membrane potential but higher level of ROS and MDA than control-iFB. Western-blot revealed a significantly increased expression of IL-6 and p-IκBα/IκBα in LHON-iFB. Compared with the negative control, SOD2 overexpression increased OCR, ATP production and elevated mitochondrial membrane potential, but impaired ROS and MDA production. Besides, western-blot demonstrated exogenous SOD2 reduced the protein level of IL-6 and p-IκBα/IκBα. TUNEL assays suggested SOD2 inhibited cells apoptosis. Analogously, in SH-SY5Y and hRPE cells, SOD2 overexpression increased ATP production and mitochondrial membrane potential, but decreased ROS, MDA levels and suppressed apoptosis.ConclusionSOD2 upregulation inhibited cells apoptosis through ameliorating mitochondrial dysfunction and reducing NF-κB associated inflammatory response. This study further support exogenous SOD2 may be a promising therapy for the treatment of LHON.

Ethylene signaling increases reactive oxygen species accumulation to drive root hair initiation in Arabidopsis.

Root hair initiation is a highly regulated aspect of root development. The plant hormone ethylene and its precursor, 1-amino-cyclopropane-1-carboxylic acid, induce formation and elongation of root hairs. Using confocal microscopy paired with redox biosensors and dyes, we demonstrated that treatments that elevate ethylene levels lead to increased hydrogen peroxide accumulation in hair cells prior to root hair formation. In the ethylene-insensitive receptor mutant, etr1-3, and the signaling double mutant, ein3eil1, the increase in root hair number or reactive oxygen species (ROS) accumulation after ACC and ethylene treatment was lost. Conversely, etr1-7, a constitutive ethylene signaling receptor mutant, has increased root hair formation and ROS accumulation, similar to ethylene-treated Col-0 seedlings. The caprice and werewolf transcription factor mutants have decreased and elevated ROS levels, respectively, which are correlated with levels of root hair initiation. The rhd2-6 mutant, with a defect in the gene encoding the ROS-synthesizing RESPIRATORY BURST OXIDASE HOMOLOG C (RBOHC), and the prx44-2 mutant, which is defective in a class III peroxidase, showed impaired ethylene-dependent ROS synthesis and root hair formation via EIN3EIL1-dependent transcriptional regulation. Together, these results indicate that ethylene increases ROS accumulation through RBOHC and PRX44 to drive root hair formation.

Neutrophil NOX2 is Required to Control Experimental Urinary Tract Infection

Abstract Urinary tract infections (UTIs) affect 150 million people worldwide. Uropathogenic Escherichia coli (UPEC) is the primary pathogen causing over 80% of UTIs. Systemic antibiotic treatment is the only available therapy for UTIs. Thus, novel non-antibiotic treatments for UTI are necessary. The NADPH oxidase complex 2 (NOX2) has an essential role in the antimicrobial response against many bacterial and fungal infections, but its role during UTIs remains unknown. Objective To study the role of NOX2 in bacterial killing during experimental UTI. Methods Female wild-type (WT) C57BL/6 mice, NOX2 knockout (KO) mice, and WT mice treated with a NOX2 inhibitor were transurethrally infected with UPEC (CFT073 strain). NOX2 protein expression was analyzed by Western blotting. Cellular sources of NOX2 were identified by immunofluorescence. Neutrophils were isolated from murine bone marrow. Human neutrophils were purified from peripheral blood of healthy donors. Purified neutrophils were infected with UPEC in vitro. UPEC burden was assessed by enumerating colony forming units (CFU). Reactive oxygen species (ROS) production was quantified using the Amplex™ Red kit. Results NOX2 KO mice, or mice treated with NOX2 inhibitor, were more susceptible to experimental UTI compared to WT mice. Immunofluorescence microscopy localized NOX2 primarily in infiltrating neutrophils. Neutrophils from NOX2 KO mice exhibited impaired extracellular ROS production and increased intracellular bacteria compared to WT controls, after challenged with UPEC in vitro. Similar effects occurred in human and murine neutrophils following chemical inhibition of NOX2 in vitro. Conclusion Neutrophil NOX2 serves a critical antimicrobial role during experimental UTI. Supported by grant from NIH (K01 DK128379-01)

Galacto-oligosaccharides directly attenuate lipopolysaccharides-induced inflammatory response, oxidative stress and barrier impairment in intestinal epithelium

• Lipopolysaccharides induce an impaired barrier function, inflammatory cytokines, and ROS release. • Galacto-oligosaccharides alleviate the impaired barrier function of lipopolysaccharides-challenged IPEC-J2 cells. • Galacto-oligosaccharides inhibited the release of inflammatory cytokines and ROS in lipopolysaccharides-challenged IPEC-J2 cells and piglets. • Galacto-oligosaccharides relieve the inflammatory response via NF-κB pathway in lipopolysaccharides-challenged IPEC-J2 cells. Recently, the indirect promotion of gut health of piglets by galacto-oligosaccharides (GOS) through regulating gut microbes has been well studied. However, whether the GOS had a direct protective effect on intestinal epithelial cells when occurring inflammatory response should be further studied. In this study, we investigated the effect of GOS on lipopolysaccharides (LPS)-induced inflammation in IPEC-J2 cells and suckling piglets. We discovered that LPS exposure increased paracellular permeability and decreased the expression of tight junction proteins, and caused inflammatory response and oxidative stress. In contrast, GOS relieved LPS-induced barrier damage, inflammation, and oxidative stress. We also found that GOS promoted the expression of tight junction proteins that were decreased by LPS stimulation. GOS inhibited LPS-induced nuclear factor-kappa B phosphorylation, leading to the reduction of proinflammatory cytokine and reactive oxygen species (ROS) generation. These results indicate that GOS have a directly protective effect on intestinal epithelial cells when occurring inflammatory response.

Cadmium enhances root hair elongation through reactive oxygen species in Arabidopsis

We previously reported that Arabidopsis shows enhanced root hair density and length in response to cadmium (Cd). To understand how Cd stimulates root hair elongation, we examined the role of reactive oxygen species (ROS) and root hair elongation-related genes in root hair elongation/tip growth. In response to Cd, Arabidopsis displayed enhanced length and ROS levels in the root hairs, indicating ROS involvement. Furthermore, in response to Cd, ROS generation mutants, including root hair defective 2 ( rhd2/rbohc ), rbohh , rbohj , per1 , per44 and per73, which have shorter root hairs because of impaired ROS generation, also showed increased root hair length and ROS levels, suggesting the involvement of these genes in ROS production in Cd-induced root hair elongation. Supporting the participation of ROS, N-acetyl- L -cysteine (ROS scavenger) treatment reduced root hair elongation and ROS levels. Additionally, the expression of proline-rich extension-like receptor kinase (PERK)5 , PERK8, and root hair-specific 10 ( RHS10 ) , which are negative regulators of root hair elongation, was reduced by Cd and ROS (H 2 O 2 ). Furthermore, the expression of expansin A7 ( EXPA7 ) and expansin A18 ( EXPA18 ), which are involved in root hair elongation, was remarkably induced by Cd and H 2 O 2 in elongating root hairs and tips. Additionally, COBL9 , which is involved in cell wall formation, was highly expressed in root hairs and tips in response to Cd and H 2 O 2 . Furthermore, changes in auxin and ethylene levels showed a similar pattern to that of root hair elongation and ROS at various concentrations of Cd, suggesting the involvement of auxin and ethylene in Cd/ROS-induced root hair elongation. Combined, it appears that Cd enhances root hair elongation/tip growth by elevating the expression of EXPA7 , EXPA18 and COBL9 and by inhibiting the expression of PERK5 , PERK8, and RHS10 , which are ROS-mediated in Arabidopsis. Change of root hair elongation, ROS, auxin and ethylene at various concentrations of Cd in Col-0 Arabidopsis • Cd promotes root hair elongation by enhancing ROS level in Arabidopsis. • RBOHC, RBOHH, RBOHJ, PER1, PER44, and PER73 participate in Cd-induced ROS generation. • Auxin and ethylene may be involved in Cd-enhanced ROS and root hair elongation. • Cd-induced ROS elevates expressions of EXPA7 , EXPA18 and COBL9 . • Cd-enhanced ROS inhibits expressions of PERK5 , PERK8, and RHS10 .

The receptor-like cytosolic kinase RIPK activates NADP-malic enzyme 2 to generate NADPH for fueling ROS production

Reactive oxygen species (ROS) production is a conserved immune response in Arabidopsis primarily mediated by respiratory burst oxidase homolog D (RBOHD), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase associated with the plasma membrane. A rapid increase in NADPH is necessary to fuel RBOHD proteins and thus maintain ROS production. However, the molecular mechanism by which NADPH is generated to fuel RBOHD remains unclear. In this study, we isolated a new mutant allele of FLAGELLIN-INSENSITIVE 4 (FIN4), which encodes the first enzyme in de novo NAD biosynthesis. fin4 mutants show reduced NADPH levels and impaired ROS production. However, FIN4 and other genes involved in NAD- and NADPH-generating pathways are not highly upregulated upon elicitor treatment, raising a possibility that a cytosolic NADP-linked dehydrogenase might be post-transcriptionally activated to maintain the NADPH supply close to RBOHD. To verify this possibility, we isolated the proteins associated with RPM1-INDUCED PROTEIN KINASE (RIPK), a receptor-like cytoplasmic kinase that regulates broad-spectrum ROS signaling in plant immunity, and identified NADP-malic enzyme 2 (NADP-ME2), an NADPH-generating enzyme. Compared with wild-type plants, nadp-me2 mutants display decreased NADP-ME activity, lower NADPH levels, and reduced ROS production in response to immune elicitors. Furthermore, we found that RIPK can directly phosphorylate NADP-ME2 and enhance its activity in vitro. The phosphorylation of the NADP-ME2 S371 residue contributes to ROS production upon immune elicitor treatment and susceptibility to the necrotrophic bacterium Pectobacterium carotovorum. Collectively, our study suggests that RIPK phosphorylates and activates NADP-ME2 to rapidly increase cytosolic NADPH, thus fueling RBOHD to sustain ROS production in plant immunity.

Cardiac fibroblasts display endurance to ischemia, high ROS control and elevated respiration regulated by the JAK2/STAT pathway.

Cardiovascular diseases are the leading cause of death globally and more than four out of five cases are due to ischemic events. Cardiac fibroblasts (CF) contribute to normal heart development and function, and produce the post-ischemic scar. Here, we characterize the biochemical and functional aspects related to CF endurance to ischemia-like conditions. Expression data mining showed that cultured human CF (HCF) express more BCL2 than pulmonary and dermal fibroblasts. In addition, gene set enrichment analysis showed overrepresentation of genes involved in the response to hypoxia and oxidative stress, respiration and Janus kinase (JAK)/Signal transducer and Activator of Transcription (STAT) signaling pathways in HCF. BCL2 sustained survival and proliferation of cultured rat CF, which also had higher respiration capacity and reactive oxygen species (ROS) production than pulmonary and dermal fibroblasts. This was associated with higher expression of the electron transport chain (ETC) and antioxidant enzymes. CF had high phosphorylation of JAK2 and its effectors STAT3 and STAT5, and their inhibition reduced viability and respiration, impaired ROS control and reduced the expression of BCL2, ETC complexes and antioxidant enzymes. Together, our results identify molecular and biochemical mechanisms conferring survival advantage to experimental ischemia in CF and show their control by the JAK2/STAT signaling pathway. The presented data point to potential targets for the regulation of cardiac fibrosis and also open the possibility of a general mechanism by which somatic cells required to acutely respond to ischemia are constitutively adapted to survive it.

Impaired formation of neutrophil extracellular traps in patients with MDS

AbstractNeutrophil extracellular traps (NETs) are networks of extracellular fibers primarily composed of DNA and histone proteins, which bind pathogens. We investigated NET formation in 12 patients with myelodysplastic syndrome (MDS) and 15 age-adjusted normal controls after stimulation with phorbol-12-myristate-13-acetate (PMA). Histones and neutrophil elastase were visualized by immunostaining. Since NET formation is triggered by reactive oxygen species (ROS), mainly produced by reduced NADP-oxidase and myeloperoxidase (MPO), ROS were analyzed by flow cytometry using hydroethidine, 3'-(p-aminophenyl) fluorescein, and 3'-(hydroxyphenyl) fluorescein. On fluorescence microscopy, PMA-stimulated MDS neutrophils generated fewer NETs than controls (stimulated increase from 17% to 67% vs 17% to 85%) (P = .02) and showed less cellular swelling (P = .04). The decrease in mean fluorescence intensity (MFI) of 4',6-diamidino-2-phenylindole, indicating chromatin decondensation, was significantly less in MDS neutrophils than controls (ΔMFI 3467 vs ΔMFI 4687, P = .03). In addition, the decrease in MFI for fluorescein isothiocyanate, indicating release of neutrophil elastase from cytoplasmic granules, was diminished in patients with MDS (P = .00002). On flow cytometry, less cell swelling after PMA (P = .02) and a smaller decrease in granularity after H2O2 stimulation (P = .002) were confirmed. PMA-stimulated ROS production and oxidative burst activity did not reveal significant differences between MDS and controls. However, inhibition of MPO activity was more easily achieved in patients with MDS (P = .01), corroborating the notion of a partial MPO defect. We conclude that NET formation is significantly impaired in MDS neutrophils. Although we found abnormalities of MPO-dependent generation of hypochloride, impaired ROS production may not be the only cause of deficient NETosis in MDS.

Increased Neutrophil Aging Contributes to T Cell Immune Suppression by PD-L1 and Arginase-1 in HIV-1 Treatment Naïve Patients.

Neutrophils are characterized by their heterogeneity. They fight against pathogens and are involved in tissue injury repair and immune system regulation. Neutrophils have an extremely short life span in the peripheral blood and undergo aging after being released from the bone marrow. The over-aggregation of aged neutrophils is associated with phenotypical and functional changes. Here, we aimed to investigate the dynamics of neutrophil aging and its relationship with T cell exhaustion in HIV-1 infection, as they are not well understood. In this study, we enrolled 23 treatment naïve (TN) patients, 23 individuals that had received antiretroviral therapy (ART), and 21 healthy controls (HC). In these cohorts, we measured the degree of neutrophil aging, and its possible correlation with T cell dysfunction. In TN patients, peripheral neutrophils showed a more distinct aging phenotype and were over-activated compared to those in ART-treated patients. The degree of neutrophil aging was positively correlated with HIV-1 RNA viral load and negatively correlated with CD4+ T cell count. Moreover, aged neutrophils had impaired reactive oxygen species (ROS) production after lipopolysaccharide (LPS) stimulation, and were characterized by increased PD-L1 and arginase-1 expression in a time-dependent manner. Aged neutrophils demonstrated an increased inhibition of IFN-γ and TNF-α secretion by CD8+ T cell compared to non-aged neutrophils. The inhibition effect could be partially reversed by blocking PD-L1 and arginase-1 in vitro, and LPS was identified as an important activator of neutrophil aging. These results provide evidence that dampening neutrophil aging may provide a novel approach to recover T cell dysfunction in patients with HIV-1 infection.

Thromboxane-prostanoid receptor activation blocks ATP-sensitive potassium channels in rat aortas.

K+ channel activation is one of the major mechanisms involved in vasodilation. Vasoconstrictor agonists such as angiotensin II promote ATP-dependent potassium channels (KATP ) dysfunction. This study evaluates whether thromboxane-prostanoid (TP receptor) activation by the agonist U46619 increases reactive oxygen species (ROS) production in rat aortas, which could contribute to KATP channel dysfunction and impaired NO-dependent vasodilation. TP receptor activation with the selective agonist U46619 increased ROS in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), but the TP receptor antagonist SQ29548 abolished this effect. ECs and VSMCs incubation with ROS scavengers like Tiron or PEG-Catalase impaired U46619-induced ROS production. U46619 at the concentrations of 0.1 and 1µmol/L induced contractions with similar amplitude. KATP channel activation with pinacidil-induced relaxation was lower for the contractions induced with 0.1 or 1µmol/L U46619 than with 10nmol/L U46619. Acetylcholine-induced relaxation provided similar results. In aortas pre-contracted with 10nmol/L U46619, neither Tiron (100µmol/L) nor catalase (300U/mL) affected pinacidil-induced relaxation. However, in aortas pre-contracted with 0.1µmol/L U46619, catalase potentiated pinacidil-induced relaxation. Pinacidil potentiated acetylcholine-induced relaxation in aortas pre-contracted with 0.1 and 1µmol/L U46619. Incubation with 10nmol/L U46619 increased NO concentration in ECs. Taken together, these results show that high concentrations of the TP receptor agonist U46619 impair KATP channels, which is probably due to ROS production. It is likely that hydrogen peroxide is the ROS.

Elucidating ROS signaling networks and physiological changes involved in nanoscale zero valent iron primed rice seed germination sensu stricto

Reactive oxygen species (ROS) play pivotal roles during seed dormancy and germination. Metabolically active cells of seeds generate ROS and successful germination is governed by internal ROS contents, maintained within an optimum “oxidative window” by several ROS scavengers. Although ROS was previously considered hazardous, optimum ROS generation in seeds can mediate early seed germination by acting as messengers for cell signaling involved in endosperm weakening, stored food mobilization, etc. Recent reports suggest that nanopriming can expedite seed germination rates and enhance seed quality and crop performances. However, nanoparticle-driven signal cascades involved during seed germination are still unknown. The present study is aimed to explore molecular mechanisms for promoting germination in nanoprimed seeds and to investigate the plausible role of nanoparticle-mediated ROS generation in this process. Here rice seeds were primed with 20 mg L −1nanoscale zero valent iron (nZVI) for 72 h and several biochemical and physiological changes were monitored at different time points (5, 10, 20, 40, 60, and 80 h). To gain insight into roles of ROS in germination rate enhancement, intercellular ROS inhibitor, diphenyleneiodonium (DPI) was taken as another priming agent. Seed priming with DPI impaired seed germination percentage, hydrolytic enzyme activities due to ROS imbalance. On the contrary, seeds primed with both DPI and nZVI could recover from deleterious consequences of DPI treatment. Although DPI impaired intercellular ROS generation, nZVI can generate ROS independently which was confirmed from ROS localization assay. In both nZVI and the DPI and nZVI co-primed sets, significant up-regulation in genes like OsGA3Ox2, OsGAMYB were observed which are responsible for regulating the activity of several hydrolases and mediates efficient mobilization of storage food reserves of seeds. Thus, nZVI priming has potential to regulate intracellular ROS levels and orchestrate all the metabolic activities which eventually up-regulates seed germination rate and seed vigour.

P058 Persistence of neutrophil abnormalities in COVID-19 convalescence

Background/Aims Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may develop acute respiratory inflammation, due to an exaggerated immune response and some develop chronic complications. Neutrophils play a major role in the pathology of inflammatory diseases and have been shown to contribute to lung and vascular damage in COVID-19. Our aim was to establish a relationship between neutrophil phenotype and disease severity and to determine whether neutrophil abnormalities persist in convalescent patients. Methods Peripheral blood samples were obtained from acute COVID-19 patients (n = 74), follow-up (FU) patients discharged following inpatient admission (n = 56), a median of 87 days after discharge, and healthy controls (HCs, n = 22). Patients were stratified by disease severity based on inspired oxygen (FiO2) and admission to intensive care (ICU). Neutrophils were isolated from whole blood by negative selection for phenotyping and functional analysis. PBMC Isolation Tubes were used to quantify and phenotype low density neutrophils (LDNs) within the PBMC fraction. For quantification of reactive oxygen species (ROS) production, isolated neutrophils were incubated with a ROS reactive dye, DHR-123 and stimulated with PMA. All samples were stained and fixed prior to analysis by flow cytometry. Results There was a marked increase in neutrophils expressing the activation and degranulation markers, CD64 (P < 0.0001) and CD63 (P < 0.0001) and a reduction in neutrophils expressing the maturity markers, CD10 (P < 0.0005) and CD101 (P < 0.0005) in patients with acute COVID-19 compared to HCs. Increased frequency of neutrophils expressing CD64 (P < 0.005), CD63 (P < 0.01) and expressing decreased CD101 (P < 0.0001) were also detected in FU patients compared to HCs. Notably, 42.3 ± 4.4% of neutrophils were CD101lo in FU patients, compared to 29.0 ± 3.7% in acute patients and 9.6 ± 4.1% in HCs. These changes were most apparent in FU patients recovering from severe COVID-19 compared to mild or moderate disease. The frequency of LDNs in PBMCs from acute patients was significantly higher than HCs (P < 0.0001), and correlated with disease severity. Similarly, the frequency of LDNs in FU patients was significantly higher than in HCs (P < 0.0005). We found a trend towards higher basal ROS production in acute and FU patients, but a blunted response to PMA stimulated ROS production in neutrophils from acute patients versus HCs (P < 0.0001). Impaired ROS production persisted in FU patients compared to HCs (P < 0.01). Conclusion Circulating neutrophils in acute COVID-19 have an altered phenotype and comprise immature and activated cells. This altered phenotype persisted in convalescence and may contribute to the persistence of symptoms and an increased susceptibility to subsequent infections. Future work will aim to investigate the functional implications of these findings. Disclosure T.O. Williams: None. V. Kästele: None. E.R. Mann: None. S.B. Knight: None. M. Menon: None. C. Jagger: None. S. Khan: None. J.E. Konkel: None. T.N. Shaw: None. M. Rattray: Consultancies; M.R. has a paid consultancy with AstraZeneca. L. Pearmain: None. A. Horsley: None. A. Ustianowski: None. I. Prise: None. N.D. Bakerly: None. P.M. Dark: None. G.M. Lord: Corporate appointments; G.M.L. is cofounder and scientific advisory board member of Gritstone Oncology Inc., which is a public company that develops therapeutic vaccines (primarily for the treatment of cancer). A. Simpson: None. T. Felton: None. L. Ho: None. M. Feldmann: None. I. Bruce: None. J.R. Grainger: None. T. Hussell: None.