EFFECT OF LARIFAN ON MONOCYTES OF AGED C57BL/6 AND BALB/C MICE IN VITRO

The iron chelator deferasirox (DFX) prevents complications related to transfusional iron overload in several haematological disorders characterized by marrow failure. It is also able to induce haematological responses in a percentage of treated patients, particularly in those affected by myelodysplastic syndromes. The underlying mechanisms responsible for this feature, however, are still poorly understood. In this study, we investigated the effect of DFX-treatment in human haematopoietic/progenitor stem cells, focussing on its impact on the redox balance, which proved to control the interplay between stemness maintenance, self-renewal and differentiation priming. Here we show, for the first time, that DFX treatment induces a significant diphenyleneiodonium-sensitive reactive oxygen species (ROS) production that leads to the activation of POU5F1 (OCT4), SOX2 and SOX17 gene expression, relevant in reprogramming processes, and the reduction of the haematopoietic regulatory proteins CTNNB1 (β-Catenin) and BMI1. These DFX-mediated events were accompanied by decreased CD34 expression, increased mitochondrial mass and up-regulation of the erythropoietic marker CD71 (TFRC) and were compound-specific, dissimilar to deferoxamine. Our findings would suggest a novel mechanism by which DFX, probably independently on its iron-chelating property but through ROS signalling activation, may influence key factors involved in self-renewal/differentiation of haematopoietic stem cells.

Circulating levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) play an important role in the development and progression of atherosclerosis. We hypothesized that the inflammatory marker C-reactive protein (CRP) might stimulate sLOX-1 release by activating tumor necrosis factor-α converting enzyme (TACE). Macrophages differentiated from THP-1 cells were stimulated with TNF-α and further treated with CRP in the absence or presence of specific inhibitors or small interfering RNA (siRNA). Our results showed that CRP increased sLOX-1 release from activated macrophages in a dose-dependent manner and that these effects were regulated by Fc γ receptor II (FcγRII)-mediated p47(phox) phosphorylation, reactive oxygen species (ROS) production, and TACE activation. CRP also enhanced sLOX-1 release from macrophages derived from peripheral blood mononuclear cells (PBMC) of patients with acute coronary syndrome (ACS). Pretreatment with antibody against FcγRII or with CD32 siRNA, p47(phox) siRNA, apocynin, N-acetylcysteine, tumor necrosis factor-α protease inhibitor 1 (TAPI-1) or TACE siRNA attenuated sLOX-1 release induced by CRP. CRP also elevated serum sLOX-1 levels in a rabbit model of atherosclerosis. Thus, CRP might stimulate sLOX-1 release, and the underlying mechanisms possibly involved FcγRII-mediated p47(phox) phosphorylation, ROS production, and TACE activation.

Role of Phagocyte Oxidase in UVA-Induced Oxidative Stress and Apoptosis in Keratinocytes

The calcium-ROS-pH triangle and mitochondrial permeability transition: challenges to mimic cardiac ischemia-reperfusion

Sonosensitized Aggregation-Induced Emission Dots with Capacities of Immunogenic Cell Death Induction and Multivalent Blocking of Programmed Cell Death-Ligand 1 for Amplified Antitumor Immunotherapy

Vascular permeability is a complex process involving the coordinated regulation of multiple signaling pathways in the endothelial cell. It has long been documented that vascular endothelial growth factor (VEGF) greatly enhances microvascular permeability; however, the molecular mechanisms controlling VEGF-induced permeability remain unknown. Treatment of microvascular endothelial cells with VEGF led to an increase in reactive oxygen species (ROS) production. ROS are required for VEGF-induced permeability as treatment with the free radical scavenger, N-acetylcysteine, inhibited this effect. Additionally, treatment with VEGF caused ROS-dependent tyrosine phosphorylation of both vascular-endothelial (VE)-cadherin and beta-catenin. Rac1 was required for the VEGF-induced increase in permeability and adherens junction protein phosphorylation. Knockdown of Rac1 inhibited VEGF-induced ROS production consistent with Rac lying upstream of ROS in this pathway. Collectively, these data suggest that VEGF leads to a Rac-mediated generation of ROS, which, in turn, elevates the tyrosine phosphorylation of VE-cadherin and beta-catenin, ultimately regulating adherens junction integrity.

The finding that upon neuronal activation glutamate is transported postsynaptically from synaptic clefts and increased lactate availability for neurons suggest that brain mitochondria (BM) utilize a mixture of substrates, namely pyruvate, glutamate, and the tricarboxylic acid cycle metabolites. We studied how glutamate affected oxidative phosphorylation and reactive oxygen species (ROS) production in rat BM oxidizing pyruvate + malate or succinate. Simultaneous oxidation of glutamate + pyruvate + malate increased state 3 and uncoupled respiration by 52 and 71%, respectively. The state 4 ROS generation increased 100% over BM oxidizing pyruvate + malate and 900% over that of BM oxidizing glutamate + malate. Up to 70% of ROS generation was associated with reverse electron transport. These effects of pyruvate + glutamate + malate were observed only with BM and not with liver or heart mitochondria. The effects of glutamate + pyruvate on succinate-supported respiration and ROS generation were not organ-specific and depended only on whether mitochondria were isolated with or without bovine serum albumin. With the non-bovine serum albumin brain and heart mitochondria oxidizing succinate, the addition of pyruvate and glutamate abrogated inhibition of Complex II by oxaloacetate. We conclude that (i) during neuronal activation, simultaneous oxidation of glutamate + pyruvate temporarily enhances neuronal mitochondrial ATP production, and (ii) intrinsic inhibition of Complex II by oxaloacetate is an inherent mechanism that protects against ROS generation during reverse electron transport.

UVB Radiation Induces Apoptosis in Keratinocytes by Activating a Pathway Linked to “BLT2-Reactive Oxygen Species”

Recent investigations suggest that neutrophils may play an important role in the late-phase allergen-induced inflammation in allergic airway diseases. The aim of this study was to evaluate neutrophil chemotaxis, phagocytic activity, and reactive oxygen species (ROS) production in patients with allergic rhinitis and asthma challenged with inhaled Dermatophagoides pteronyssinus. Eighteen patients with allergic rhinitis and 14 with allergic asthma, all sensitized to D. pteronyssinus, as well as 15 healthy individuals underwent bronchial challenge with D. pteronyssinus. Peripheral blood collection and neutrophil isolation were performed 24 h before as well as 7 and 24 h after bronchial challenge. Neutrophils chemotaxis, phagocytic activity, and ROS production were analyzed by flow cytometer. Neutrophil chemotaxis and ROS production were increased, while phagocytic activity was decreased 24 h before challenge in patient groups compared with healthy individuals. After challenge, neutrophil chemotaxis and phagocytic activity increased after 7 and 24 h, when ROS production, only after 24 h. Bronchial allergen challenge had no influence for neutrophils activity in healthy subjects. Activated chemotaxis, phagocytic activity, and ROS production of peripheral blood neutrophils after challenge with D. pteronyssinus reflect an enhanced systemic inflammation in allergic rhinitis and asthma patients with induced late-phase airway inflammation.

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

Background. Blood monocytes play a crucial role in immunity as effector cells of innate immunity. However, they can also promote hyperinflammation, as was described in COVID-19. Many viral infections trigger hyperinflammation by inhibiting type I interferon synthesis, necessitating search of interferon-based or interferonogenic treatments like Larifan – bacteriophagederived dsRNA with interferonogenic and immunomodulatory properties. Global statistics indicate that viral infections, including SARS-CoV-2, as well as hyperinflammation occur more frequently in males, especially in the older age group, and significantly depends on genetically determined profile of immune reactivity. The aim of this study was a comparative assessment of the impact of Larifan on the metabolic profile of peripheral blood monocytes from aged male C57BL/6 and BALB/c mice in vitro. Methods. Male aged C57BL/6 and BALB/c mice were used in this study. Blood samples were collected from facial vein and treated with Larifan in vitro. Phagocytic activity, ROS production, and expression of phenotypic markers were assessed by flow cytometry. Only live monocytes were gated and included in the analysis. Data are presented as median and interquartile range (IQR). Statistical differences were calculated using Kruskal–Wallis test, with significance set at p < 0.05. Results. BALB/c mice showed a lower baseline phagocytic index than C57Bl/6, but phagocytosis percentages were comparable. Treatment with Larifan reduced the phagocytosis percentage in both strains, yet the phagocytic index rose in BALB/c mice after dsRNA exposure. ROS production was higher in C57Bl/6 mice, with Larifan reducing ROS levels significantly in both strains. CD80 baseline expression levels were higher in BALB/c, and dsRNA increased CD80-positive cells as well as decreased expression level of CD80 in BALB/c mice only. CD206 expression was lower in BALB/c but unaffected by Larifan, while dsRNA reduced both number of CD206-positive cells and CD206 levels in C57Bl/6 mice. Conclusions. The metabolic profile of monocytes differs between Th1-dominant C57Bl/6 and Th2-biased BALB/c mice, with higher baseline indicators in C57Bl/6 mice. Larifan treatment exerts anti-inflammatory effects by reducing ROS synthesis in both strains, with BALB/c mice also displaying increased phagocytosis and reduced antigen-presenting capability

To investigate the influence of pregnancy on the production of reactive oxygen species (ROS) from mouse peripheral blood neutrophils (PMN), the levels of NO and cytokines from serum, the activation of T lymphocytes, and initially find the immune regulation effects of pregnancy on the mouse peripheral blood lymphocytes. Take the BALB/c mice which were at the mid trimester of pregnancy (day 14) as the object, full blood staining using ROS probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) combing with flow cytometry was used to test the levels of ROS from PMN. The production of NO from peripheral blood serum were analyzed by Griess kit while the soluble cytokines interleukin (IL) 6, IL-10, monocyte chemotactic protein 1 (MCP-1), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and IL-12 were detected by liquid protein quantitative technology cytometric bead array (CBA) using flow cytometry. The activation of peripheral blood lymphocytes at early, middle and later phases which marked with CD69, CD25 and CD71 respectively were tested by flow cytometry and two-color fluorescent staining. Comparing to the normal non-pregnant mouse, pregnancy obviously promoted the production of ROS from PMN (101.1±2.2 versus 134.5±10.3, P<0.05). Comparing to the normal non-pregnant mouse, pregnancy obviously promoted the secretion of NO [(22.7±0.7) versus (36.3±1.2) µmol/L, P<0.01]. In normal non-pregnant mouse, the serum levels of IL-6, IL-10, MCP-1, IFN-γ, TNF-α and IL-12 were (9.3±0.5), (26.7±0.9), (21.2±1.6), (14.5±1.8), (22.6±1.6) and (8.4±1.2) pg/ml, while in pregnancy group the levels were (26.5±1.0), (40.4±2.5), (25.1±0.7), (457.4±17.9), (93.2±4.3) and (7.5±0.9) pg/ml correspondingly; the levels of IL-6, IFN-γ, TNF-α from peripheral blood serum (P<0.01), while had no effects on the production of IL-10 and MCP-1 (P>0.05). About the CD+3 T lymphocytes activation, in normal non-pregnant mouse, the CD69, CD25 and CD71 expression rate were (0.43±0.15)%, (5.13±0.25)% and (0.37±0.11)%, while in pregnancy group the CD69, CD25 and CD71 expression rate were (0.40±0.10)%, (6.17±0.40)% and (6.10±0.31)%. The levels of middle and later phases markers as CD25 and CD71 were highly up-regulated (P<0.05), while the early phase action CD69 had no obvious variation (P>0.05). The mid trimester of pregnancy promoted the production of ROS from PMN, the levels of NO, IL-6, IFN-γ, TNF-α from peripheral blood serum, and the middle- and later-phase activation of T lymphocytes.

Reactive Oxygen Species Are Differentially Regulated in Chronic Myeloid Leukemia Versus Philadelphia Negative Myeloproliferative Neoplasms

Growth-arrested 3T3-L1 preadipocytes rapidly express CCAAT/enhancer-binding protein-beta (C/EBPbeta) upon hormonal induction of differentiation. However, the DNA binding activity of C/EBPbeta is not activated until the cells synchronously reenter S phase during the mitotic clonal expansion (MCE) phase of differentiation. In this period, C/EBPbeta is sequentially phosphorylated by MAPK and glycogen synthase kinase-3beta, inducing C/EBPbeta DNA binding activity and transcription of its target genes. Because the DNA binding activity of C/EBPbeta is further enhanced by oxidation in vitro, we investigated how redox state affects C/EBPbeta DNA binding and MCE during adipogenesis. When 3T3-L1 cells were treated with H(2)O(2) and hormonal stimuli, differentiation was accelerated with increased expression of peroxisome proliferator-activated receptor gamma. Interestingly, cell cycle progression (S to G(2)/M phase) was markedly enhanced by H(2)O(2), whereas antioxidants caused an S phase arrest during the MCE. H(2)O(2) treatment resulted in the early appearance of a punctate pattern observed by immunofluorescent staining of C/EBPbeta, which is a hallmark for C/EBPbeta binding to regulatory elements, whereas a short antioxidant treatment rapidly dispersed the centromeric localization of C/EBPbeta. Consistently, reactive oxygen species production was increased during 3T3-L1 differentiation. Our results indicate that redox-induced C/EBPbeta DNA binding activity, along with the dual phosphorylation of C/EBPbeta, is required for the MCE and terminal differentiation of adipocytes.

Elevated Reactive Oxygen Species Production In Sickle Erythrocytes Is Modulated by a Pathway Involving Endothelin-1, TGFβ1, PKC, and Rac GTPases