Resistance to Intrinsic Apoptosis Stimuli in Lymphocytes : A Role for Mitochondrial Dysfunction

Background: Detection of human immunotoxicity is relevant to predictive and regulatory toxicology but is usually complex, while nodal steps may provide simple and suitable endpoints. NF-kB activation is both a promising therapeutic target and an important immunotoxicity endpoint. Conventional methods employed to determine nuclear translocation of NF-kB lack statistical robustness (microscopy) or the ability to discern heterogeneity within the sampled populations (Western blotting and gel shift assays). Previously, we developed an in vitro assay of immunotoxicity based on quantitative measurement by Multispectral Image-in-Flow Cytometry (MsIFC) of NF-kB translocation in a monocytic cell line. MsIFC combines the high image content information of microscopy with the high throughput and multiparametric analysis of flow cytometry which overcomes the aforementioned limitations of conventional assays. In this study, we have adapted and validated this assay for human peripheral whole-blood samples. Materials and Methods: The degree of NF-kB nuclear translocation was quantified in U937 cells and in peripheral blood samples after treating samples with different compounds affecting in vitro NF-kB-dependent immune functions, including activators (LPS and PMA), inhibitors (PDTC and Wedelolactone) and six test immunotoxicants. Negative controls were cell cultures treated with appropriate solution vehicles. To analyze the effects of NF-kB inhibitors, samples were pre-incubated with or without them for 1h and then treated with activating or pro-oxidant agents. After treatment, surface staining was performed for 100 μl whole blood samples using PC5 antiCD14, erythrocytes were lysed by addition of 2 ml BD FACS Lysing Solution 1X (Becton Dickinson). Then cells were fixed and permeabilized with 0.1 % Triton-4% PFA and stained with antiNF-kB p50 conjugated to Alexa Fluor 488 (Biolegend). Afterwards, cells were washed and counterstained with 7-AAD (Molecular Probes). MsIFC data were obtained for at least 15,000 events per sample using an ImageStream100 system (Amnis). Results: LPSor PMA-induced NF-kB nuclear translocation was quantified in U937 cells and peripheral blood samples after 2 and 24 h of treatment with different compounds affecting in vitro NFkB-dependent immune functions, including test immunotoxicants such as lindane, diazepam, hexachlorobenzene, tbutylhydroperoxide, verapamil and mercury chloride. Our results show that MsIFC allows to quantify the effect of xenobiotics and biological regulators on NF-kB nuclear translocation in peripheral bood samples. Automated image processing algorithms allowed to calculate the percentage of lymphocytes and monocytes showing NF-kB nuclear translocation in each condition. IC50 values could be derived, thus classifying the immunotoxic potency of test compounds and allowing comparison with general cytotoxicity, as measured by PI assay or Microplate Alamar Blue assay. Conclusions: This assay is a fast and simple method for detecting immunotoxicity on processes associated with this molecular target. This approach is not limited to NF-kB translocation and may be used for any transcription factor with action related to its intracellular location. Supported by Ministerio de Ciencia e Innovacion (BIO2010-19870) and Conselleria de Educacion de la Generalitat Valenciana (ACOMP2013/102).