Cardiotonic Steroids Stimulate Macrophage Inflammatory Responses Through a Pathway Involving CD36, TLR4, and Na/K-ATPase

Abstract

Objective Circulating levels of cardiotonic steroids (CTS) are elevated in various chronic inflammatory conditions, but the role of CTS in inflammation remains largely unknown. We have previously shown that the CTS ouabain stimulates pro-inflammatory responses in murine macrophages. In this study, we aim to explore the mechanism how CTS induce pro-inflammatory responses in primary murine and human macrophages. Approach and Results Using both murine peritoneal macrophages and human monocyte-derived macrophages, we demonstrated that ouabain activated NF-κB, leading to pro-inflammatory cytokine (e.g. MCP-1, TNF-α, IL-1β and IL-6) production. By applying siRNA techniques as well as murine peritoneal macrophages isolated from genetically modified mice, we showed that macrophages partially deficient in Na/K-ATPase, the receptor for CTS or fully deficient in the scavenger receptor CD36 or TLR4 were resistant to ouabain-induced NF-κB activation, suggesting an indispensible role of these three receptors in this pathway. Mechanistically, this effect of ouabain was independent of the ion transport function of the Na/K-ATPase. Instead, ouabain stimulated a signaling complex including Na/K-ATPase, CD36 and TLR4. Subsequently, TLR4 recruited MyD88 adaptor protein for NF-κB activation. Furthermore, intraperitoneal injection of ouabain into mice specifically recruited Ly6C+CCR2+ monocyte subtypes to the peritoneal cavities, indicating that the CTS ouabain triggers inflammation in vivo. Conclusions CTS activate NF-κB leading to pro-inflammatory cytokine production in primary macrophages through a signaling complex including CD36, TLR4 and Na/K-ATPase. These findings warrant further studies on endogenous CTS in chronic inflammatory diseases such as atherosclerosis.

Cardiotonic Steroids and the Sodium Trade Balance: New Insights into Trade-Off Mechanisms Mediated by the Na+/K+-ATPase

“…Over the last five years, many studies have suggested a relationship between CTS and inflammation. There is some evidence that circulating levels of CTS are elevated during the inflammatory response [ 16 ] and many studies have linked specific cardiac glycosides to a proinflammatory response. Others have shown evidence of CTS inducing an anti-inflammatory response.…”

“…This article will also highlight new developments in what is known about molecular partners of the NKA which help mediate these trade-off pathways. Further, while NKA ligands, such as CTS were first recognized as regulators of renal sodium transport and arterial pressure [ 14 , 15 ], recent findings have highlighted mechanistic links by which CTS modulate interactions of molecular partners with the NKA, especially as this pertains to modulation of immunity, inflammation, and fibrosis [ 16 , 17 , 18 ]. The objective of the present review is to examine the molecular mechanisms of CTS as they relate to these inflammatory and fibrotic processes.…”

The Na/K-ATPase Signaling: From Specific Ligands to General Reactive Oxygen Species

“…In various chronic inflammatory conditions, circulating cardiotonic steroids are elevated that are capable of stimulating a proinflammatory response in murine and human macrophages. This process involves ouabain-stimulated activation of NF-κB through a signaling complex of Na/K-ATPase, CD36, and TLR4, leading to increases in proinflammatory cytokines MCP-1, TNF-α, IL-1β, and IL-6 [ 49 , 50 , 51 ].…”

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

“…Although CGs also decrease the intracellular K + , their exact role in macrophage activation and death is more controversial, presumably because of the distinct sensitivity of murine Na + ,K + -ATPase to CGs (Perne et al, 2009;Price and Lingrel, 1988) and the potential implication of Na +, K + -ATPase in signalling events independently of ion transport (Cavalcante-Silva et al, 2017). 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).…”