A3AR antagonism mitigates metabolic dysfunction-associated steatotic liver disease by exploiting monocyte-derived Kupffer cell necroptosis and inflammation resolution

Metabolic dysfunction-associated steatotic liver (MASLD) progression is driven by chronic inflammation and fibrosis, largely influenced by Kupffer cell (KC) dynamics, particularly replenishment of pro-inflammatory monocyte-derived KCs (MoKCs) due to increased death of embryo-derived KCs. Adenosine A3 receptor (A3AR) plays a key role in regulating metabolism and immune responses, making it a promising therapeutic target. This study aimed to investigate the impact of selective A3AR antagonism for regulation of replenished MoKCs, thereby improving MASLD. A3AR expression was significantly elevated in KCs from both patients with MASLD and fast-food diet (FFD)-fed mice. A3AR knockout (KO) mice displayed marked improvements in hepatic inflammation and fibrosis along with a reduction in CLEC4F-positive KCs. The spatial transcriptomics of these KCs revealed disrupted mitochondrial integrity, increased oxidative stress, and enhanced cell death due to A3AR deletion. Similarly, in vivo FM101 treatment, a highly potent and selective antagonist of A3AR with a truncated 4′-thioadenosine structure, mitigated FFD-induced MASLD in mice. Mechanistically, FM101 induces β-arrestin2-mediated A3AR degradation, leading to mitochondrial dysfunction-mediated necroptosis in KCs. Consistently, A3AR was highly expressed in monocyte-derived macrophages in MASLD patients, with strong correlations with macrophage activation and monocyte chemoattractant gene sets. Thus, FM101 induced necroptosis in pro-inflammatory MoKCs, facilitating anti-inflammatory effects. This study demonstrated that inhibiting A3AR via FM101 or genetic deletion alleviates MASLD by inducing mitochondrial dysfunction and subsequent necroptosis in MoKCs, establishing FM101 as a promising therapeutic strategy for MASLD. Targeting A3AR in Kupffer cells through FM101, a potent A3AR inhibitor, mitigates liver inflammation and fibrosis in MASLD by inducing mitochondrial dysfunction and necroptosis, especially in monocyte-derived Kupffer cells. [Display omitted] •A3AR expression correlates with macrophage activation and monocyte chemoattractant expression in MASLD patients.•Elevated A3AR expression is observed predominantly in pro-inflammatory monocyte-derived Kupffer cells (MoKCs) in MASLD patients.•A3AR deletion or FM101, an oral A3AR antagonist, reduces hepatic inflammation and fibrosis in MASLD.•FM101 induces mitochondrial stress-mediated necroptosis in MoKCs, mitigating inflammation and fibrosis in MASLD.