Peripheral Blood Macrophages (PBMCs) from Adults with Sickle Cell Disease Have a Unique Phenotype
Authors: Lisk Christina , Cendali Francesca I , Swindle Delany , George Gemlyn , Hassell Kathryn Louise , Nuss Rachelle , Buehler Paul , D'Alessandro Angelo , Irwin David 
Published: December 2024

Introduction: Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis resulting in oxidative stress and chronic inflammation. It has been suggested that up two thirds of hemolysis occurs through erythrophagocytosis, which may iron overload macrophages driving a unique phenotype supporting systemic inflammation. The remaining one third of hemolysis occurs through lysis of RBC, releasing hemoglobin, heme, and iron, all of which are known to be pro-inflammatory mediators. Nitric Oxide availability and oxidative stress through excessive production of reactive oxygen species (ROS), has been shown to affects the endothelium, the alternate complement pathway, and Neutrophil Extracellular Traps (NETs). It is well known that heme binds to Toll-like-receptor 4 (TLR4) on macrophages polarizing them to an M1 state. Thus, circulating macrophages, which may have been both iron-loaded and heme activated, could by a primary driver in SCD inflammation. Hypothesis: We hypothesized that peripheral blood mononuclear cells (PBMCs) in SCD patients are both iron overloaded and phenotypically different than those of healthy individuals. Methods: PBMCs were isolated from eighteen (n=18) healthy adult controls (HC) and twenty-six (n=26) SCD adults to determine iron content and proteomic and metabolomic differences. Results: Our data shows PBMCs from SCD patients have significantly higher iron concentrations compared to healthy adults. Proteomic analysis shows that out of 6,487 proteins analyzed, 2870 were significantly different between healthy controls and SCD (p-value £ 0.05). The top 100 differentiated proteins (82 of which were downregulated) were recorded and used for network pathway analysis that revealed “regulation of catalytic activity” as one of the most downregulated pathways. We then analyzed the proteomic data based on cellular and molecular pathways. Pathways related to iron had 326 proteins within it and ~14% of the proteins were significantly altered (28 increased and 18 decreased in SCD PBMCs). Of the 200 inflammatory proteins analyzed, 33 were significantly changed between HC and SCD. Lastly, the complement pathway included 672 proteins of which 118 differed significantly. Interestingly, when metabolites were analyzed, the glycine/serine metabolism and ammonia recycling pathways were shown to be significantly increased in SCD when compared to healthy controls. Amino sugar metabolism was downregulated in PBMCs from SCD. Together, these data emphasize an altered immune state in SCD patients when compared to healthy controls. Conclusion: Our data shows that PBMCs in SCD patients are characterized by greater iron concentrations and are phenotypically different than those in healthy adults. The data demonstrate PBMCs from adults with SCD express a proinflammatory phenotype and supports our hypothesis that circulating PBMCs may be a critical driver in SCD-associated chronic inflammation.