Quaternary ammonium iminofullerenes improve root growth of oxidative-stress maize through ASA-GSH cycle modulating redox homeostasis of roots and ROS-mediated root-hair elongation

Background Various environmental factors are capable of oxidative stress to result in limiting plant development and agricultural production. Fullerene-based carbon nanomaterials can enable radical scavenging and positively regulate plant growth. Even so, to date, our knowledge about the mechanism of fullerene-based carbon nanomaterials on plant growth and response to oxidative stress is still unclear. Results 20 or 50 mg/L quaternary ammonium iminofullerenes (IFQA) rescued the reduction in root lengths and root-hair densities and lengths of Arabidopsis and maize induced by accumulation of endogenous hydrogen peroxide (H2O2) under 3-amino-1,2,4-triazole or exogenous H2O2 treatment, as well as the root active absorption area and root activity under exogenous H2O2 treatment. Meanwhile, the downregulated contents of ascorbate acid (ASA) and glutathione (GSH) and the upregulated contents of dehydroascorbic acid (DHA), oxidized glutathione (GSSG), malondialdehyde (MDA), and H2O2 indicated that the exogenous H2O2 treatment induced oxidative stress of maize. Nonetheless, application of IFQA can increase the ratios of ASA/DHA and GSH/GSSG, as well as the activities of glutathione reductase, and ascorbate peroxidase, and decrease the contents of H2O2 and MDA. Moreover, the root lengths were inhibited by buthionine sulfoximine, a specific inhibitor of GSH biosynthesis, and subsequently rescued after addition of IFQA. The results suggested that IFQA could alleviate exogenous-H2O2-induced oxidative stress on maize by regulating the ASA-GSH cycle. Furthermore, IFQA reduced the excess accumulation of ROS in root hairs, as well as the NADPH oxidase activity under H2O2 treatment. The transcript levels of genes affecting ROS-mediated root-hair development, such as RBOH B, RBOH C, PFT1, and PRX59, were significantly induced by H2O2 treatment and then decreased after addition of IFQA. Conclusion The positive effect of fullerene-based carbon nanomaterials on maize-root-hair growth under the induced oxidative stress was discovered. Application IFQA can ameliorate oxidative stress to promote maize-root growth through decreasing NADPH-oxidase activity, improving the scavenging of ROS by ASA-GSH cycle, and regulating the expressions of genes affecting maize-root-hair development. It will enrich more understanding the actual mechanism of fullerene-based nanoelicitors responsible for plant growth promotion and protection from oxidative stress. Graphical Abstract

Trimethylamine N-Oxide Promotes Autoimmunity and a Loss of Vascular Function in Toll-like Receptor 7-Driven Lupus Mice

Plasma levels of trimethylamine N-oxide (TMAO) are elevated in lupus patients. We analyzed the implication of TMAO in autoimmunity and vascular dysfunction of the murine model of systemic lupus erythematosus (SLE) induced by the activation of the Toll-like receptor (TLR)7 with imiquimod (IMQ). Female BALB/c mice were randomly divided into four groups: untreated control mice, control mice treated with the trimethylamine lyase inhibitor 3,3-dimethyl-1-butanol (DMB), IMQ mice, and IMQ mice treated with DMB. The DMB-treated groups were administered the substance in their drinking water for 8 weeks. Treatment with DMB reduced plasma levels of TMAO in mice with IMQ-induced lupus. DMB prevents the development of hypertension, reduces disease progression (plasma levels of anti-dsDNA autoantibodies, splenomegaly, and proteinuria), reduces polarization of T lymphocytes towards Th17/Th1 in secondary lymph organs, and improves endothelial function in mice with IMQ-induced lupus. The deleterious vascular effects caused by TMAO appear to be associated with an increase in vascular oxidative stress generated by increased NADPH oxidase activity, derived in part from the vascular infiltration of Th17/Th1 lymphocytes, and reduced nrf2-driven antioxidant defense. In conclusion, our findings identified the bacterial-derived TMAO as a regulator of immune system, allowing for the development of autoimmunity and endothelial dysfunction in SLE mice.

Heightened turnover and failed maturation of monocyte-derived macrophages in murine Chronic Granulomatous Disease

Loss of NADPH oxidase activity leads to altered phagocyte responses and exaggerated inflammation in Chronic Granulomatous Disease (CGD). We sought to assess the effects of Nox2 absence on monocyte-derived macrophages (MoMacs) in gp91phox-/y mice during zymosan-induced peritonitis. MoMacs from CGD and wild type (WT) peritonea were characterized over time after zymosan injection. Though numbers lavaged from both genotypes were virtually identical, there were marked differences in maturation: newly recruited WT MoMacs rapidly enlarged and matured, losing Ly6C and gaining MHCII, CD206 and CD36, while CGD MoMacs remained small and were mostly Ly6C+MHCII-. RNAseq analyses showed few intrinsic differences between genotypes in newly recruited MoMacs but significant differences with time. WT MoMacs demonstrated changes in metabolism, adhesion and reparative functions, while CGD MoMacs remained inflammatory. PKH dye labeling demonstrated that while WT MoMacs were mostly recruited within the first 24h and remained in the peritoneum while maturing and enlarging, CGD monocytes streamed into the peritoneum for days with many migrating to the diaphragm where they were found in fibrin(ogen) clots surrounding clusters of neutrophils in nascent pyogranulomata. Importantly, these observations appeared to be driven by milieu: adoptive transfer of CGD MoMacs into inflamed peritonea of WT mice resulted in immunophenotypic maturation and normal behavior, whereas altered maturation/behavior of WT MoMacs resulted from transfer into inflamed peritonea of CGD mice. Additionally, Nox2-deficient MoMacs behaved similarly to their Nox2-sufficient counterparts within the largely WT milieu of mixed bone marrow chimeras. These data demonstrate persistent recruitment with fundamental failure of MoMac maturation in CGD.

Synergistic inhibition of platelet function by NADPH oxidase inhibitors and clopidogrel

Previous studies have shown platelets play an important role in prothrombotic complications due to several factors such as hyperglycemia, oxidative stress, and hypercholesterolemia, which affected platelets reactivity. Platelets activation involves ADP stimulation via P2Y12 receptor, whereas reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide (H2O2) produced by NADPH oxidase (Nox) act as the second messenger, which involved in platelets activation and may contribute to thrombus formation. The aim of the present study was to investigate the influence of Nox on the purinergic receptor (P2Y12 receptor) in activation of human platelets function stimulated by platelets agonist. This research explored the effects of Nox inhibitors and clopidogrel either alone or in combination, on various agonist-stimulated human platelets, including platelets aggregation and adhesion measured by modified LTA, expression of platelets activation markers, and calcium mobilization using flow cytometry, and ROS formation, NADPH oxidase activity, as well as cAMP levels by chemiluminescence assay. Taken together, findings from these experiments suggest that the combination of clopidogrel and Nox inhibitors synergistically reduced platelets aggregation, platelets adhesion, and expression of platelets activation marker during late activation, ROS formation, NADPH oxidase activity, calcium mobilization and increased cAMP levels in vitro. This combination showed that P2Y12receptor reactivity was influenced by the activation of NADPH oxidase. Thus, these data demonstrated a potential combination therapy to reduce the risk of thrombosis formation.

The inflammation influence on pathological remodeling of pulmonary arteries in monocrotaline-induced pulmonary hypertension in rats

Aim To investigate the inflammation role on pathological remodeling of pulmonary arteries (PA) in monocrotaline-induced pulmonary hypertension (mPAH) in rats with joint assessment serum and tissue inflammatory biomarkers and the morphological arteries changes. Methods The mPAH was induced by a subcutaneous monocrotaline injection (60 mg/kg) in male rats and control group received a single saline solution. Baseline and every 2, 4, 6, 8 weeks after the serum concentrations of interleukin-6 (IL-6), interleukin-10 (IL-10) were measured by enzyme-linked immunosorbent assay; matrix metalloproteinase-9 (MMP-9), interleukin-1β (IL-1β), collagen type 1 and 3, smooth muscle actin α (SMA-α) in lung tissue were investigated immunohistochemically and quantitative measurements of intima and media thickness were done by planimetry. The functional activity neutrophil changes measured by chemiluminescence and fluorescent methods. Results The IL-10 increased after 2 weeks of mPAH (5,9 vs 0,6 pg/ml, p<0,05) vs control and then it decreased to initial values by 8 weeks (0,06 vs 0,62 pg/ml, p>0,05). The increasing IL-6 (30,3 vs 0,01 pg/ml, p<0,05) and the maximum expression of IL-1β in the lung tissue (0,119 vs 0,099 index of expression (IE), p<0,05) we observed 4 weeks after mPAH. The SMA-α (29,4 vs 40,2 IE, p<0,05) and MMP-9 (1,6 vs 0,8 IE, p<0,05) expression level significantly raised 4 weeks later vs control and remained in a high level until 8 week. A significant increase of type 1 collagen expression was observed at all phases of the experiment, and high level type 3 collagen expression was observed from 4 to 8 weeks (7,0 vs 10,4 IE, p<0,05). The histological characteristics of remodeling these were a thickening of the media (30,6 vs 56,0 μm, p<0,05) and the subintimal layer (1,6 vs 10,9 μm, p<0,05) of the PA. 2 weeks after mPAH cell priming occurred which manifested by modification ROS generation systems, decrease NADPH oxidase activity, increase of myeloperoxidase secretion (MPO), enhance of unbound cytosolic calcium ions, mitochondrial potential reduction. From 4 to 8 weeks an increase NADPH oxidase activity and MPO secretion was revealed into the extracellular environment which leads to overproduction of hypochlorous acid. This functional activity reprogramming of circulating neutrophils indicate associated with time-development of mPAH. Conclusion The inflammation is the most important mediator of pathological remodeling processes in mPA. The monocrotaline launches a neutrophil reaction at an early stage of PAH with changes their functional activity which leads to immune cells recruitment into the lung tissue, producing inflammation and proliferation biomarkers. The hyperplasia of smooth muscle cells and reconstruction of the extracellular matrix are the result of this process and leads to increase intima and media thickness. The high MMP-9, SMA-α, IL-6 activity in 6–8 weeks reflects maintenance local inflammatory potential. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Basic and applied sciences - medicine, subprogram “Diagnostics and therapy of diseases” on the assignment “To establish the molecular-cellular mechanisms of the development of irreversible remodeling of pulmonary vessels in pulmonary arterial hypertension in an experiment in vivo.”

Late Onset of Chronic Granulomatous Disease Revealed by Paecilomyces lilacinus Cutaneous Infection

Chronic granulomatous disease (CGD) is an inherited immunodeficiency due to defective leukocyte NADPH responsible for recurrent infections and aberrant inflammation. Mutations in the CYBB gene are responsible for the X-linked CGD and account for approximately 70% of the cases. CGD is diagnosed during childhood in males. Female carriers may have biased X-inactivation and may present with clinical manifestations depending on the level of residual NADPH oxidase activity. We report the case of a previously asymptomatic female carrier who was diagnosed at age 67 with a skin infection with the rare fungus Paecilomyces lilacinus as the first manifestation of CGD. Dihydrorhodamine 123 (DHR) activity was below 10%. Next-generation sequencing (NGS) revealed mutations in DNMT3A, ASXL1, and STAG2 suggesting that clonal hematopoiesis could be responsible for a progressive loss of NADPH oxidase activity and the late onset of X-linked CGD in this patient. Long-term follow-up of asymptomatic carrier women seems to be essential after 50 years old.

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro

The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding effector protein AvrBs3 from the bacterial pathogen Xanthomonas. Expression of Bs3 in yeast inhibited proliferation, demonstrating that Bs3 function is not restricted to the plant kingdom. The Bs3 sequence shows striking similarity to flavin monooxygenases (FMOs), an FAD- and NADPH-containing enzyme class that is known for the oxygenation of a wide range of substrates and their potential to produce H2O2. Since H2O2 is a hallmark metabolite in plant immunity, we analyzed the role of H2O2 during Bs3 HR. We purified recombinant Bs3 protein from E. coli and confirmed the FMO function of Bs3 with FAD binding and NADPH oxidase activity in vitro. Translational fusion of Bs3 to the redox reporter roGFP2 indicated that the Bs3-dependent HR induces an increase of the intracellular oxidation state in planta. To test if the NADPH oxidation and putative H2O2 production of Bs3 is sufficient to induce HR, we adapted previous studies which have uncovered mutations in the NADPH binding site of FMOs that result in higher NADPH oxidase activity. In vitro studies demonstrated that recombinant Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3. Translational fusions to roGFP2 showed that Bs3S211A also increased the intracellular oxidation state in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta, ultimately revealing that H2O2 produced by Bs3 on its own is not sufficient to trigger HR.

Regulation of NADPH Oxidase-Mediated Superoxide Production by Acetylation and Deacetylation

Oral treatment of apolipoprotein E-knockout (ApoE-KO) mice with the putative sirtuin 1 (SIRT1) activator resveratrol led to a reduction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the heart. In contrast, the SIRT1 inhibitor EX527 enhanced the superoxide production in isolated human polymorphonuclear granulocytes. In human monocytic THP-1 cells, phorbol ester-stimulated superoxide production was enhanced by inhibitors of histone deacetylases (HDACs; including quisinostat, trichostatin A (TSA), PCI34051, and tubastatin A) and decreased by inhibitors of histone acetyltransferases [such as garcinol, curcumin, and histone acetyltransferase (HAT) Inhibitor II]. These results indicate that protein acetylation and deacetylation may represent crucial mechanisms regulating NADPH oxidase-mediated superoxide production. In cell-free systems, incubation of recombinant Rac1 with SIRT1 resulted in decreased Rac1 acetylation. Mass spectrometry analyses identified lysine 166 (K166) in Rac1 as a residue targeted by SIRT1. Deacetylation of Rac1 by SIRT1 markedly reduced the interaction of Rac1 with p67phox in in vitro assays. Computational modeling analyses revealed that K166 deacetylation of Rac1 led to a 5-fold reduction in its binding affinity to guanosine-5'-triphosphate, and a 21-fold decrease in its binding potential to p67phox. The latter is crucial for Rac1-mediated recruitment of p67phox to the membrane and for p67phox activation. In conclusion, both SIRT1 and non-sirtuin deacetylases play a role in regulating NADPH oxidase activity. Rac1 can be directly deacetylated by SIRT1 in a cell-free system, leading to an inhibition of Rac1-p67phox interaction. The downstream targets of non-sirtuin deacetylases are still unknown. The in vivo significance of these findings needs to be investigated in future studies.

Inhibition of Rac1 GTPase Decreases Vascular Oxidative Stress, Improves Endothelial Function, and Attenuates Atherosclerosis Development in Mice

Aims: Oxidative stress and inflammation contribute to atherogenesis. Rac1 GTPase regulates pro-oxidant NADPH oxidase activity, reactive oxygen species (ROS) formation, actin cytoskeleton organization and monocyte adhesion. We investigated the vascular effects of pharmacological inhibition of Rac1 GTPase in mice.Methods and Results: We treated wild-type and apolipoprotein E-deficient (ApoE−/−) mice with Clostridium sordellii lethal toxin (LT), a Rac1 inhibitor, and assessed vascular oxidative stress, expression and activity of involved proteins, endothelial function, macrophage infiltration, and atherosclerosis development. LT-treated wild-type mice displayed decreased vascular NADPH oxidase activity and ROS production. Therapeutic LT doses had no impact on behavior, food intake, body weight, heart rate, blood pressure, vascular and myocardial function, differential blood count, and vascular permeability. ApoE−/− mice were fed a cholesterol-rich diet and were treated with LT or vehicle. LT treatment led to decreased aortic Rac1 GTPase activity, NADPH oxidase activity and ROS production, but had no impact on expression and membrane translocation of NADPH oxidase subunits and RhoA GTPase activity. LT-treated mice showed improved aortic endothelium-dependent vasodilation, attenuated atherosclerotic lesion formation and reduced macrophage infiltration of atherosclerotic plaques. Concomitant treatment of cholesterol-fed ApoE−/− mice with LT, the specific synthetic Rac1 inhibitor NSC 23766 or simvastatin comparably reduced aortic Rac1 activity, NADPH oxidase activity, oxidative stress, endothelial dysfunction, atherosclerosis development, and macrophage infiltration.Conclusions: These findings identify an important role of the small GTPase Rac1 in atherogenesis and provide a potential target for anti-atherosclerotic therapy.