Inhibition by Teriflunomide of Erythrocyte Cell Membrane Scrambling Following Energy Depletion, Oxidative Stress and Ionomycin

Abstract

Background/Aims: Teriflunomide, an inhibitor of pyrimidine synthesis and thus proliferation of activated T and B lymphocytes, is successfully used for treatment of inflammatory disease. Teriflunomide has further been shown to trigger apoptosis of tumor cells and has thus been considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Triggers of cell membrane scrambling include energy depletion, oxidative stress and increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). The present study explored whether teriflunomide modifies eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, and [Ca²⁺]_i from Fluo3 fluorescence. Results: Oxidative stress (60 min exposure to 0.3 mM tert-butylhydroperoxide), energy depletion (removal of glucose for 48 hours), and exposure to the Ca²⁺ ionophore ionomycin (1 µM, 60 min) all increased annexin-V-binding, decreased forward scatter and enhanced Fluo3 fluorescence. Teriflunomide (5 µg/ml) did not significantly influence Fluo3 fluorescence, forward scatter and annexin-V-binding under control conditions but significantly blunted the increase of annexin-V-binding following oxidative stress, energy depletion and ionomycin exposure. Teriflunomide further blunted the increase of Fluo3 fluorescence following energy depletion, but did not significantly interfere with increase of Fluo3 fluorescence following oxidative stress and ionomycin exposure. Conclusion: Teriflunomide is a novel inhibitor of suicidal erythrocyte death.

Stimulation of Erythrocyte Cell Membrane Scrambling by C-Reactive Protein

“…The effect is at least partially due to increase C-reactive protein, which increases cytosolic Ca 2+ activity and ceramide formation in erythrocytes. As several inhibitors of eryptosis are available [2-4, 93, 94], eryptosis and subsequent development of anemia may be prevented.…”

Oxidative stress, eryptosis and anemia: a pivotal mechanistic nexus in systemic diseases

“…To the extent that stimulated eryptosis may lead to anemia and thrombosis, it may be desirable to inhibit eryptosis. As a matter of fact, a wide variety of compounds such as blebbistatin, phlorhizin, vitamin C, resveratrol, reversine, hydroxytyrosol, volasertib, ASP3026 and teriflunomide have previously been shown to inhibit eryptosis in vitro . Some of these substances have previously been shown to counteract oxidative stress and may potentially be effective in vivo and could, therefore, be considered for the treatment of eryptosis‐induced anemia in different clinical conditions.…”

Teriflunomide preserves peripheral nerve mitochondria from oxidative stress-mediated alterations

“… 29 , 31 Moreover, a study assessing the effect of TFN on eryptosis indicated that concentrations ranging from 3.7 to 37 µM TFN might compensate oxidative stress-mediated erythrocyte changes in vitro . 32 In rats, it has been shown that after one single injection of 10 µg/g TFN, approximately 2–4% of the blood concentration was found in the brain (~2.5–4.1 µM). 30 Although an extrapolation to the human reality is not exact, we could suppose in treated patients a TFN concentration within the nervous system of about 3–7 µM.…”