Mitochondrial Generated Redox Stress Differently Affects the Endoplasmic Reticulum of Circulating Lymphocytes and Monocytes in Treatment-Naïve Hodgkin's Lymphoma

Abstract

Background: The redox stress caused by Hodgkin's lymphoma (HL) also involves the peripheral blood mononucleated cells (PBMCs) even before chemotherapy. Here, we tested whether lymphocytes and monocytes show a different response to the increased mitochondrial generation of reactive oxygen species (ROS).

Methods: PBMCs, isolated from the blood of treatment-naïve HL patients and control subjects, underwent assessment of malondialdehyde content and enzymatic activity of both hexose- and glucose-6P dehydrogenase (H6PD and G6PD) as well as flow cytometric analysis of mitochondrial ROS content. These data were complemented by evaluating the uptake of the fluorescent glucose analogue 2-NBDG that is selectively stored within the endoplasmic reticulum (ER).

Results: Malondialdehyde content was increased in the whole population of HL PBMCs. The oxidative damage matched an increased activity of G6PD, and even more of H6PD, that trigger the cytosolic and ER pentose phosphate pathways, respectively. At flow cytometry, the number of recovered viable cells was selectively decreased in HL lymphocytes that also showed a more pronounced increase in mitochondrial ROS generation and 2-NBDG uptake, with respect to monocytes.

Conclusions: PBMCs of HL patients display a selective mitochondrial and ER redox stress most evident in lymphocytes already before the exposure to chemotherapy toxicity.

Keywords: 2-NBDG; cancer; endoplasmic reticulum; lymphoma; mitochondria; pentose phosphate pathway; redox stress.

Figure 1

PBMC cell culture. Representative flow cytometry dot plots of forward- and side-scatter features (FSC, x-axis, and SSC, y-axis) of PBMCs showing the recovery of lymphocytes and monocytes in the expected gates of controls (A) and HL patients (B). Percentage of PBMCs (C), lymphocytes (D) and monocytes (E) sampled from controls (green) and HL patients (red). Graphs display individual data and mean ± SD. *** = p < 0.002, **** = p < 0.001.

Figure 2

PBMCs oxidative stress and antioxidant response. (A) Malondialdehyde content and (B) total antioxidant capacity evaluated in lysed PBMCs. (C) Mean fluorescence intensity (MFI) of MitoSOX evaluated in all PBMCs of controls (green) and HL patients (red). (D) Correlation between MitoSOX MFI in monocytes (x-axis) and lymphocytes (y-axis) of controls (green) and HL patients (red). Simple linear regression (continuous line) and 95% confidence bands of the best-fit line (dashed line) of controls (green) and HL patients (red). MitoSOX signal measured as mean fluorescence intensity (MFI) in lymphocytes (E) and in monocytes (F) of controls (green) and HL patients (red). Graphs display individual data and mean ± SD. * = p < 0.05, ** = p < 0.01, *** = p < 0.009.

Figure 3

Mitochondrial energetic function in PBMCs. (A) Typical trace from the Seahorse XFp of oxygen consumption rate (OCR) over time (x-axis) measured on intact PBMCs (10 × 10⁵ cells/well) collected from controls (green) and HL patients (red). Injections of ATP-synthase inhibitor Oligomycin A, and rotenone/antimycin A (Rot/AA) are indicated with black lines. Average and SD of at least 3 replicate wells are plotted. (B) Basal OCR measured in presence of glucose (11 mM) and (C) after injection of Oligomycin A expressed as % of corresponding basal OCR. (D) Extramitochondrial OCR after the inhibition of Complexes I, III, and V after sequential injection of Oligomycin A and Rot/AA, expressed as % of corresponding basal OCR. (E) Typical trace from the Seahorse XFp of extracellular acidification rate (ECAR) over time (x-axis) measured on intact PBMCs (10 × 10⁵ cells/well) collected from controls (green) and HL patients (red). Injections of Oligomycin A is indicated with black lines. Average and SD of at least 3 replicate wells are plotted. (F) Lactate dehydrogenase (LDH) activity, (G) basal ECAR measured in presence of glucose (11 mM) and (H) maximal ECAR after injection of Oligomycin A. Graphs display individual data and mean ± SD. ** = p < 0.01.

Figure 4

ER- and cytosolic- PPP activity. Catalytic function of H6PD (A) and G6PD (B), measured by enzymatic assay in lysed PBMCs sampled from controls (green) and HL patients (red). 2-NBDG uptake measured as mean fluorescence intensity (MFI) in all PBMCs (C), in lymphocytes (D) and in monocytes (E) of controls (green) and HL patients (red). Graphs display individual data and mean ± SD. ** = p < 0.01, *** = p < 0.002, **** = p < 0.001.

Figure 5

Correlation between ER function and glycolysis or mitochondria redox stress. Panel (A) displays the correlation between 2-NBDG MFI of monocytes (x-axis) and lymphocytes (y-axis) sampled from controls (green) and HL patients (red). Simple linear regression (continuous line) and 95% confidence bands of the best-fit line (dashed line) of controls (green) and HL patients (red). Shifting to the analysis of the whole PBMC population, panel (B) displays the absent proportionality between 2-NBDG and extracellular acidification rate (ECAR). By contrast, panel (C) reports the direct relation between 2-NBDG and MitoSOX fluorescence. The role of ER in 2-NBDG uptake is confirmed in panel (D) by the direct correlations between the fluorescence of this glucose analogue and that of the ERTracker glibenclamide (ERT). The relevance of ER-PPP in the response to the increase in mitochondrial ROS generation is indicated by the inverse correlation between MitoSOX fluorescence (x-axis) and H6PD activity (y-axis) reported in panel (E). This same correlation did not involve G6PD activity (F). Continuous lines represent the identified function while dashed lines represent the 95% confidence bands of the best-fit in both controls (green) and HL patients (red).