Inhibition of the Complement Alternative Pathway Attenuates Hemolysis and Preserves Renal Function in a Mouse Model of Sickle Cell Disease

Introduction: the alternative pathway (AP) of complement activation plays a significant role in the pathophysiology of sickle cell disease (SCD), contributing to hemolysis and subsequent organ damage with aging (unpublished data from our group). Intravascular hemolysis in SCD leads to the release of hemoglobin, which activates the complement system, exacerbating vascular and renal injury. This study investigates the effects of AP complement inhibition on hemolysis and renal function in a chronic mouse model of aged SCD.

Methods: we used Townes HbSS mice, a well-established SCD model, to evaluate the therapeutic potential of inhibiting AP complement components. HbSS mice (8-week-old) were administered an antisense oligonucleotide (ASO) targeting factor B (100 mg/kg), the anti-C5 antibody BB5.1 (30 mg/kg), or a combination of both, over a 12-week period. Samples were collected from 20 weeks-old HbSS mice, when AP complement activation is elevated (unpublished data, please refer to our abstract on complement and aging in SCD). Plasma levels of factor B, C3b fragment, C5a, and C5b9 were measured using ELISA. Red blood cell (RBC) opsonization by C3/C3b was assessed through flow cytometry, alongside the expression of complement regulators CD55 and CD59. Hemolysis was quantified by measuring plasma lactate dehydrogenase (LDH) levels, plasma-free hemoglobin (PFH), and total bilirubin through biochemical assays. Complete blood count was performed using Procyte Dx. Renal function was evaluated by urinary levels of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), established markers of renal injury.

Results: ASO against factor B significantly reduced plasma levels of factor B and C3b (p<0.01), while all treatments decreased plasma C5a and C5b9 levels (p<0.05). Notably, ASO factor B (alone or in combination) markedly decreased RBC opsonization by C3/C3b (p<0.001). Treatment with BB5.1 (alone or in combination) led to an increase in CD55 and CD59 expression on RBCs (p<0.05). All therapeutic interventions resulted in significant reductions in LDH, PFH, and bilirubin levels, indicating attenuated hemolysis. Importantly, no impairment in renal function was observed across all treatment groups, as evidenced by stable urinary KIM-1 and NGAL levels (reaching levels similar to HbAA mice at the same age).

Conclusion: The results highlight the pivotal role of AP complement activation in SCD-associated hemolysis and renal injury. Inhibition of factor B and C5 effectively reduced complement-mediated RBC lysis and improved hematologic parameters. C3b opsonization of RBCs facilitates their lysis, and the reduction of CD55 and CD59 expression further influences complement activation on RBCs. ASO fB significantly decreased plasma fB and C3b levels, blocking RBC C3b opsonization. BB5.1 therapy significantly decreased sC5b9 and increased RBC CD55 and CD59. Combination of both showed decrease in markers of hemolysis and preservation of renal function, emphasizing the therapeutic potential of complement modulation in SCD. Complement activation is a key contributor to intravascular hemolysis, and its inhibition can prevent complement-mediated renal injury. Improvement in renal function is particularly significant, as renal impairment is a common and severe complication in SCD, impacting patient morbidity and mortality. These findings support further exploration of complement-targeted therapies to ameliorate hemolysis and preserve organ function in SCD patients.

Trovati Maciel:Alexion Pharmaceuticals: Research Funding; LGD France: Research Funding; Bristol-Myers Squibb: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Imara Inc.: Research Funding. Brochier:Institut Roche: Current Employment. Kiialainen:F. Hoffmann-La Roche Ltd: Current Employment. David:F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company; Cerus Corporation: Current equity holder in publicly-traded company. Hermine:BMS: Research Funding; Inatherys: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding; Roche: Research Funding; Alexion: Research Funding; AB Science: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding; MSD Avenir: Research Funding.