Inflammasome inhibition blocks cardiac glycoside cell toxicity

Abstract

Chronic heart failure and cardiac arrhythmias have high morbidity and mortality, and drugs for the prevention and management of these diseases are a large part of the pharmaceutical market. Among these drugs are plant-derived cardiac glycosides, which have been used by various cultures over millennia as both medicines and poisons. We report that digoxin and related compounds activate the NLRP3 inflammasome in macrophages and cardiomyocytes at concentrations achievable during clinical use. Inflammasome activation initiates the maturation and release of the inflammatory cytokine IL-1β and the programmed cell death pathway pyroptosis in a caspase-1–dependent manner. Notably, the same fluxes of potassium and calcium cations that affect heart contraction also induce inflammasome activation in human but not murine cells. Pharmaceuticals that antagonize these fluxes, including glyburide and verapamil, also inhibit inflammasome activation by cardiac glycosides. Cardiac glycoside–induced cellular cytotoxicity and IL-1β signaling are likewise antagonized by inhibitors of the NLRP3 inflammasome or the IL-1 receptor–targeting biological agent anakinra. Our results inform on the molecular mechanism by which the inflammasome integrates the diverse signals that activate it through secondary signals like cation flux. Furthermore, this mechanism suggests a contribution of the inflammasome to the toxicity and adverse events associated with cardiac glycosides use in humans and that targeted anti-inflammatories could provide an additional adjunct therapeutic countermeasure.

Cardiac glycosides cause selective cytotoxicity in human macrophages and ameliorate white adipose tissue homeostasis

“…Indeed, the addition of KCl in the media showed an almost complete rescue of cell viability and intracellular levels of Na + and Ca 2+ . This also confirms that, similar to cardiomyocytes, macrophage survival is tightly dependent on potassium efflux and the sensitivity to CGs is more prominent in human macrophage-like cells when compared to murine ones (LaRock et al, 2019 (Guerrero et al, 2019;Lopez-Lazaro, 2007). This raises the possibility of phenotypic similarities between senescent cells and macrophages and a recent report shows that chemotherapy-induced senescent breast cancer cells are highly enriched for macrophage genes and can perform phagocytosis (Tonnessen-Murray et al, 2019).…”

“…Caspase-1 activation in THP1 macrophage-like cells (LaRock et al, 2019). We thus evaluated the inflammasome activity by measuring luminescence resulting from catalytically active caspase-1 in the culture media of hMDMs.…”

“…In human PBMCs, monocytes and macrophages, nanomolar concentrations of ouabain stimulates pro-inflammatory cytokine expression, including IL-1β and TNF through NF-κB (Chen et al, 2017;Foey et al, 1997;Matsumori et al, 1997;Teixeira and Rumjanek, 2014) but only recent reports linked CG-induced cytokine release to inflammasome-mediated cell death (i.e. pyroptosis) (Kobayashi et al, 2017;LaRock et al, 2019).…”

The Pseudomonas aeruginosa protease LasB directly activates IL-1β

“…Expression was examined in cells lysed with RIPA (Millipore). RNA was isolated (Qiagen), cDNA synthesized with SuperScript III and Oligo(dT)20 primers (Invitrogen), and qPCR performed with KAPA SYBR Fast (Kapa Biosystems) with primers for il1a and il1b and relative expression normalised to gapdh and compared by ΔΔCt as previously [37] . In vitro transcription/translation was performed with the corresponding primers in Table 1 using pET-pro-IL-1β as a template and following the manufacturer's recommendations in 10 μl reaction volumes (TNT Coupled Reticulocyte Lysate; Promega).…”

Role of Inflammasome-independent Activation of IL-1β by thePseudomonas aeruginosaProtease LasB

“…Expression was examined in cells lysed with RIPA (Millipore). RNA was isolated (Qiagen), cDNA synthesized with SuperScript III and Oligo(dT)20 primers (Invitrogen), and qPCR performed with KAPA SYBR Fast (Kapa Biosystems) with primers for il1b and relative expression normalized to gapdh and compared by ΔΔCt as previously50 . In vitro transcription/translation was performed with the corresponding primers inTable 1using pET-pro-IL-1β as a template and following the manufacturer's recommendations in 10 µl reaction volumes (TNT Coupled Reticulocyte Lysate; Promega).…”