Clemastine fumarate accelerates accumulation of disability in progressive multiple sclerosis by enhancing pyroptosis
Joanna Kocot, Peter Kosa, Shinji Ashida, Nicolette A. Pirjanian, Raphaela Goldbach-Mansky, Karin Peterson, Valentina Fossati, Steven M. Holland, Bibiana Bielekova
Joanna Kocot, Peter Kosa, Shinji Ashida, Nicolette A. Pirjanian, Raphaela Goldbach-Mansky, Karin Peterson, Valentina Fossati, Steven M. Holland, Bibiana Bielekova
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Research Article Immunology Neuroscience

Clemastine fumarate accelerates accumulation of disability in progressive multiple sclerosis by enhancing pyroptosis

Abstract

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS. Clemastine fumarate, the over-the-counter antihistamine and muscarinic receptor blocker, has remyelinating potential in MS. A clemastine arm was added to an ongoing platform clinical trial, targeting residual activity by precision, biomarker-guided combination therapies of multiple sclerosis (TRAP-MS) (ClinicalTrials.gov NCT03109288), to identify a cerebrospinal fluid (CSF) remyelination signature and to collect safety data on clemastine in patients progressing independently of relapse activity (PIRA). The clemastine arm was stopped per protocol-defined criteria when 3 of 9 patients triggered individual safety stopping criteria. Clemastine-treated patients had significantly higher treatment-induced disability progression slopes compared with the remaining TRAP-MS participants. Quantification of approximately 7,000 proteins in CSF samples collected before and after clemastine treatment showed significant increases in purinergic signaling and pyroptosis. Mechanistic studies showed that clemastine with sublytic doses of extracellular adenosine triphosphate (ATP) activates inflammasome and induces pyroptotic cell death in macrophages. Clemastine with ATP also caused pyroptosis of induced pluripotent stem cell–derived human oligodendrocytes. Antagonist of the purinergic channel P2RX7, which is strongly expressed in oligodendrocytes and myeloid cells, blocked these toxic effects of clemastine. Finally, reanalysis of published single-nucleus RNA-Seq (snRNA-Seq) studies revealed increased P2RX7 expression and pyroptosis transcriptional signature in microglia and oligodendrocytes in the MS brain, especially in chronic active lesions. The CSF proteomic pyroptosis score was increased in untreated MS patients, was higher in patients with progressive than relapsing-remitting disease, and correlated significantly with the rates of MS progression. Collectively, this identifies pyroptosis as a likely mechanism of CNS injury underlying PIRA even outside of clemastine toxicity.

Authors

Joanna Kocot, Peter Kosa, Shinji Ashida, Nicolette A. Pirjanian, Raphaela Goldbach-Mansky, Karin Peterson, Valentina Fossati, Steven M. Holland, Bibiana Bielekova

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Figure 2

CLM potentiation effect on ATP-induced inflammasome activation and GSDMD-driven pyroptotic cell death in THP-1 cells.

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CLM potentiation effect on ATP-induced inflammasome activation and GSDMD...
The THP-1 cells were primed with 200 ng/mL LPS overnight and then treated either with medium + DMSO (negative control, Ctrl), 10 μg/mL CLM, 2 mM ATP ± 10 μg/mL CLM, or 2.5 μM nigericin (positive control) for 3 hours, 6 hours, and 18 hours. Release of the proinflammatory cytokine IL-1β into the culture medium of THP-1 cells (A) and THP-1 GSDMD–/– cells (B), determined by ELISA assay. Activity of caspase-1 in the culture medium of THP-1 cells (C) and THP-1 GSDMD–/– cells (D), determined using bioluminescent assay. Lytic cell death of THP-1 cells (E) and THP-1 GSDMD–/– cells (F), determined by LDH activity released into the culture medium. Cell viability of THP-1 cells (G) and THP-1 GSDMD–/– cells (H) evaluated by the MTS assay. (A–H) Data are presented as mean ± SD of 3 independent experiments, each performed in duplicate (n = 6). One-way ANOVA followed by Dunnett’s multiple-comparisons test was used to compare the testing groups with control group (Ctrl). One-way ANOVA test followed by Holm-Šidák’s multiple comparison was used to compare ATP and ATP+CLM group (to assess the potentiation effect of CLM). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Panel A, ATP+CLM at 3 hours (n = 1), nigericin at 3 hours (n = 3), 6 hours (n = 5), and 18 hours (n = 5) — above detection limit of the assay. Panel B, Ctrl at 3 hours (n = 5) and 6 hours (n = 3) — below detection limit of the assay. Nigericin at 18 hours (n = 6) — above detection limit of the assay. (I) Flow cytometry analysis of cell uptake of SYTOX Green in THP-1 cells primed with 200 ng/mL LPS overnight and then treated either with medium + DMSO (control), 10 μg/mL CLM, or 2 mM ATP ± 10 μg/mL CLM for 90 minutes. Representative plot of 2 independent experiments.