Role of the ERK Pathway for Oxidant-Induced Parthanatos in Human Lymphocytes

<div><p>Reactive oxygen species (ROS) are formed by myeloid cells as a defense strategy against microorganisms. ROS however also trigger poly(ADP-ribose) polymerase 1- (PARP-1) dependent cell death (parthanatos) in adjacent lymphocytes, which has been forwarded as a mechanism of immune escape in several forms of cancer. The present study assessed the role of mitogen-activated protein kinases (MAPKs), in particular the extracellular signal-regulated kinase (ERK), in ROS-induced signal transduction leading to lymphocyte parthanatos. We report that inhibitors of ERK1/2 phosphorylation upheld natural killer (NK) cell-mediated cytotoxicity under conditions of oxidative stress and rescued NK cells and CD8⁺ T lymphocytes from cell death induced by ROS-producing monocytes. ERK1/2 phosphorylation inhibition also protected lymphocytes from cell death induced by exogenous hydrogen peroxide (H₂O₂) and from ROS generated by xanthine oxidase or glucose oxidase. Phosphorylation of ERK1/2 was observed in lymphocytes shortly after exposure to ROS. ROS-generating myeloid cells and exogenous H₂O₂ triggered PARP 1-dependent accumulation of poly ADP-ribose (PAR), which was prevented by ERK pathway inhibitors. ERK1/2 phosphorylation was induced by ROS independently of PARP-1. Our findings are suggestive of a role for ERK1/2 in ROS-induced lymphocyte parthanatos, and that the ERK axis may provide a therapeutic target for the protection of lymphocytes against oxidative stress.</p></div

Inhibition of ERK phosphorylation in lymphocytes exposed to oxygen radicals by ERK pathway inhibitor but not by PARP inhibitor.

<p>PBMCs or NK cells were preincubated in presence or absence of ERK1/2 inhibitor PD98059 (25 µM) or PARP-1 inhibitor PJ34 (2 µM) for 1 h at 37°C. (<b>A</b>) A Representative dot plots of pERK⁺ NK cells after 10 min exposure to PMA-stimulated monocytes_.is shown. (<b>B</b>) Mean ± SEM of pERK positive cells in gated lymphocytes after 10 min exposure to H₂O₂. (<b>C</b>) Mean ± SEM of pERK positive NK cells after 10 min exposure to PMA-stimulated monocytes. <b>B–C</b>: mean ± SEM of 4–6 experiments. *P<0.05, **P<0.01 and ***P<0.001.</p

Retained cytotoxicity of NK cells rescued from ROS induced cell death by an ERK1/2 inhibitor.

<p>NK cells were co-incubated with monocytes at Mo:NK ratios of 0.25∶1, 0.5∶1 and 1∶1 overnight in the presence or absence of the ERK1/2 inhibitor PD98059 (25 μM). Anti-CD20 (rituximab) and CFSE-labeled 221 target cells were subsequently added at an effector to target cell ratio of 1∶1 as per the NK cell count prior to incubation. Lysis of 221 cells by ADCC was estimated in a 4 hour assay using the Live/Dead Fixable Far Red Dead Cell Stain kit and flow cytometry. Panel (<b>A</b>) shows contour density plots from a representative experiment displaying an inverse relationship between NK cell and target cell viability. Panel (<b>B</b>) shows NK cell cytotoxicity (mean ± SEM) of seven donors using the lowest Mo:NK cell ratio in which PD98059 rescued NK cells from apoptosis. *P<0.05, **P<0.01 and ***P<0.001.</p

Oxidant-induced poly ADP-ribose accumulation: role of the ERK pathway.

<p>(<b>A–B</b>) Lymphocytes were preincubated with or without the ERK1/2 inhibitor PD98059 (25 µM) or the PARP-1 inhibitor PJ34 (2 µM) for 1 hr at 37°C before exposure to 500 µM H₂O₂ for 20 min. PAR accumulation was analyzed in whole cell lysates. (<b>A</b>) Representative Western blot and (<b>B</b>) mean ± SEM of results from 4 donors (O.D., optical density). β-tubulin was utilized as loading control and H₂O₂-treated HELA cells served as positive controls. (<b>C</b>) Flow cytometry analysis of PAR accumulation following exposure to H₂O₂ (500 µM, filled circle) or PBS (open square). (<b>D</b>) Inhibition of PAR formation in lymphocytes after preincubation with PD98059 (25 µM) or PJ34 (2 µM). PAR accumulation was measured after 20 min exposure to 500 µM H₂O₂ by flow cytometry. Data are the MFI of PAR in gated lymphocytes (mean ± SEM of results obtained in 4–6 donors) *P<0.05, **P<0.01 and ***P<0.001).</p

Protection of lymphocytes from ROS-induced apoptosis by an ERK pathway inhibitor.

<p>MACS-purified human CD8⁺ T cells or NK cells were preincubated with the ERK1/2 inhibitor PD98059 (25 µM) (filled triangle) for 1 h at 37°C. The T cells and NK cells were then incubated overnight in the presence of PD98059 with H₂O₂ at indicated concentrations (<b>A–B</b>) or with ROS-producing monocytes (MØ) at indicated MØ:NK ratios (<b>C–D</b>). Lymphocyte viability was assessed using the Live/Dead Fixable Violet Dead Cell Stain kit. ERK inhibitor-equivalent concentrations of DMSO were used as control (open square). Results obtained using DMSO did not significantly differ from PBS. Data are the mean ± SEM of results obtained using blood from 3–7 donors. *P<0.05, **P<0.01 and ***P<0.001.</p

Data_Sheet_1_Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2.PDF

<p>In patients with acute myeloid leukemia (AML), treatment with histamine dihydrochloride (HDC) and low-dose IL-2 (HDC/IL-2) in the post-chemotherapy phase has been shown to reduce the incidence of leukemic relapse. The clinical benefit of HDC/IL-2 is pronounced in monocytic forms of AML, where the leukemic cells express histamine type 2 receptors (H₂R) and the NAPDH oxidase-2 (NOX2). HDC ligates to H₂Rs to inhibit NOX2-derived formation of reactive oxygen species, but details regarding the anti-leukemic actions of HDC remain to be elucidated. Here, we report that human NOX2⁺ myelomonocytic/monocytic AML cell lines showed increased expression of maturation markers along with reduced leukemic cell proliferation after exposure to HDC in vitro. These effects of HDC were absent in corresponding leukemic cells genetically depleted of NOX2 (NOX2^−/−). We also observed that exposure to HDC altered the expression of genes involved in differentiation and cell cycle progression in AML cells and that these effects required the presence of NOX2. HDC promoted the differentiation also of primary monocytic, but not non-monocytic, AML cells in vitro. In a xenograft model, immunodeficient NOG mice were inoculated with wild-type or NOX2^−/− human monocytic AML cells and treated with HDC in vivo. The administration of HDC reduced the in vivo expansion of NOX2^+/+, but not of NOX2^−/− human monocytic AML cells. We propose that NOX2 may be a conceivable target in the treatment of monocytic AML.</p

Impact of <i>IL28B</i> (<i>rs12979860</i>) on normalized ALT and AST to platelet ratio index (APRI) among HCV genotype 2 and 3 infected patients.

<p>Box plots displaying the 10^th, 25^th, 50^th, 75^th, and 90^th percentiles and p-values obtained using Kruskal-Wallis test.</p

Association between <i>IL28B</i> (<i>rs12979860</i>) and portal inflammation grade (Ishak protocol) and steatosis grade among HCV genotype 2 and 3 infected patients.

<p>Histogram displaying the proportion of patients, and p-value obtained using Chi-squared test.</p

Baseline Characteristics with Patients Grouped According to HCV genotype and <i>IL28B</i> Genetic Variations (<i>rs12979860</i>).

<p>Data presented as median (25^th–75th percentile)^a or n^b.</p

Impact of <i>IL28B</i> (<i>rs12979860</i>) on baseline viral load and decline in mean HCV RNA day 0–3 among HCV genotype 2 and 3 infected patients.

<p>Box plots displaying the 10^th, 25^th, 50^th, 75^th, and 90^th percentiles, and p-values obtained using Kruskal-Wallis test.</p