Evaluating the Mitochondrial Activity and Inflammatory State of Dimethyl Sulfoxide Differentiated PLB‐985 Cells
Authors: Jougleux Jean‐Luc , Léger Jacob L. , Roy Patrick , Petga Marie‐Ange Djeungoue , Chatelain Étienne Hébert , Boudreau Luc H. 
Published: June 2021

INTRODUCTIONNeutrophils play a key role as the first line of defense against pathogens. Their ability to generate and release inflammatory mediators (eicosanoids or cytokines) helps promote the inflammatory response and consequently restore the hemostasis. While active participants in several steps of the normal inflammatory response, neutrophils are also involved in chronic inflammatory diseases such as asthma, atherosclerosis and arthritis. Given their dual role in the modulation of inflammation, regulating the inflammatory response of neutrophils has been an important therapeutic approach pursued by numerous researchers. The neutrophil's lifespan is relatively short (8–12h), which can be problematic for some in vitro experiments. To address this issue, researchers used the human monomyelocyte cell line PLB‐985 as an alternative for some of their in vitro experiments. PLB‐985 cells can be differentiated into a more neutrophil‐like phenotype upon exposure to several agonists, including dimethyl sulfoxide (DMSO). Whether this differentiation of PLB‐985 affects important features related to neutrophil's normal functions (i.e. mitochondrial activity, eicosanoid production) remains elusive and characterizing these changes will be the focal point of this project.OBJECTIVESWe assessed and compared the mitochondrial activity and inflammatory state of PLB‐985 cells following differentiation.METHODSDifferentiation of PLB‐985 cells was performed using 1% DMSO for 6 days and was confirmed by immunofluorescence microscopy and flow cytometry using an anti‐CD11b‐PE cell surface antibody. Proliferation and apoptosis assays were performed by flow cytometry using the eFluor 670 and 7‐aminoactinomycin D labelers respectively. The mitochondrial respiratory assay was determined by high‐resolution O2 respirometry (Oroboros Instrument). The inflammatory state of the cells was evaluated by transcriptomic, proteomic and lipidomic approaches.RESULTSDifferentiation affected the proliferation of PLB‐985 cells (P<0.0001), without inducing apoptosis. A significant decrease in the mitochondrial respiration was observed in differentiated PLB‐985 cells (P<0.0005). However, the overall mitochondria content was not affected. Immunoblotting with mitochondrial antibodies revealed a strong modulation of the succinate dehydrogenase A (P<0.0001), superoxide dismutase 2 (P<0.0001), ubiquinol‐cytochrome c reductase core protein 2 (P<0.05) and ATP synthase subunit α (P<0.01) in differentiated PLB‐985 cells. Finally, eicosanoids (leukotriene B4, 12‐hydroxyheptadecatrienoic and 15‐hydroxyeicosatetraenoic acids) production was significantly increased in differentiated cells (P<0.01).CONCLUSIONSOur data demonstrate that the differentiation process of PLB‐985 cells does not impact their viability despite a reduced respiratory state of the cells. This process is also accompanied by modulations of the inflammatory state of the cell. Finally, our data suggests that PLB‐985 cells could be a suitable in vitro candidate to study mitochondrial‐related dysfunctions in inflammatory diseases.Support or Funding InformationCIHR, NBHRF, NBIFThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.