Hemolysis impairs hepatic regeneration in sickle cell disease via altered kupffer cell dynamics and inflammation

Abstract Sickle Cell Disease (SCD) is characterized by the release of hemoglobin and heme into the circulation, saturation of related plasma scavengers, and heme/iron loading of macrophages. By acting as a damage-associated molecular pattern, free heme drives a pro-inflammatory activation program in macrophages, stimulating cell cytokine production and inducing sterile inflammation in SCD. Overall, this mechanism contributes to the inflammatory landscape and complications typically associated with this disease. In this context, heme/iron-activated Kupffer macrophages (KC) have emerged as key mediators of hepatic inflammation, perpetuating oxidative stress, driving tissue damage, and exacerbating hepatic fibrosis. While previous studies addressed heme ability to induce inflammatory cytokine production by KCs and alter their functional properties, heme role in hepatic regeneration via macrophage activation remain unexplored. Indeed, KC-derived inflammatory mediators play a key role in liver regeneration upon tissue damage by stimulating hepatocyte dedifferentiation and proliferation. Here, we asked whether heme-induced chronic cytokine release affects hepatic regeneration and contributes to SCD hepatopathy, and how heme-altered KC population dynamics influence this process.Using mouse model of heme overload, SCD, and macrophage depletion or preservation, we investigated the impact of heme on KC dynamics and inflammation, and their implications for liver regeneration in SCD. KC dynamics were significantly altered in sickle HbS mice, with decreased embryonic (EmKC) and increased monocyte-derived KCs (MoKC), phenotype recapitulated by in vivo heme and iron treatments. HbS EmKCs exhibited apoptosis and impaired self-renewal, evidenced by cell senescence, G0/G1 arrest, and reduced levels of KI67, IL-6R and CSF-1R, critical for proliferation. Interestingly, MoKCs displayed more severe inflammatory skewing than EmKCs in HbS mice, associated with senescence and higher TLR4 levels as well as IL-6 and ROS production, indicating that these cells are the major mediators of heme-driven inflammation. Hx treatment in HbS mice mitigated KC inflammation by both diminishing TLR4 activation and preventing KC dynamics alteration. Ultimately, inflammatory MoKCs in HbS mice were associated with the presence of hepatic regenerative processes, including bile duct hyperplasia and proliferating KI67/Ccnd1+ hepatocytes, exhibiting reduced mature (ASGPR,HNF4a,C/EBPa) and increased fetal (SOX9,EpCAM,Taz) markers, features recapitulated by in vivo heme treatment. While strategies that increase MokC repopulation exacerbated heme-driven accumulation of fetal-like proliferative hepatocytes, those that preserve EmKCs improved liver regeneration through effective hepatocyte maturation, implicating heme-altered KC dynamics and inflammation in excessive hepatocyte dedifferentiation and loss of adult hepatocyte functions. Our findings show for the first time that heme alters KC dynamics in SCD via EmKC senescence and selectively drives MoKC activation, leading to immature hepatocyte accumulation, with detrimental consequences for liver regeneration. We propose that targeted therapies aimed at expanding EmKCs or limiting MoKC response likely alleviate hepatic inflammation and ameliorate liver regeneration and functions, with benefit for SCD patients with hepatopathy.