Transcriptomic Analysis Shows a Common Multi-Organ Inflammatory/Complement Signature in Hemolysis-Induced Damage

Introduction: Intravascular hemolysis is a major contributor to tissue injury in transfusion reactions, hemoglobinopathies such as sickle cell disease (SCD) and hemolytic anemias. The release of hemoglobin and heme, usually confined to red blood cells, leads to oxidative stress, endothelial activation, and a prothrombotic and pro-inflammatory phenotype. Nevertheless, the underlying mechanism is not fully understood.

Methods: To understand the organ injury in hemolytic conditions, we used RNAseq to explore the transcriptomic changes of lung, spleen, liver and kidney in a mouse model of hemolytic crisis triggered by phenylhydrazine injection. We analyzed immune cell influx by MCP counter and the impacted biological pathways by Gene Set Enrichment Analysis (GSEA). As complement was one of the common enriched pathways in all tested organs of the hemolytic mice accompanied by C5a elevation in plasma, we expressed and validated a recombinant version of the mouse C5 inhibiting antibody BB5.1 and tested it in the mouse model. The transcriptional response to complement inhibition was assessed by Quantigene (80-gene plex).

Results: Phenylhydrazine-induced hemolysis caused dysregulation of the renal (165 DEG, differentially expressed genes), pulmonary (521), hepatic (704) and splenic (1696) transcriptomes. GSEA analysis revealed an enrichment of gene sets linked to xenobiotic metabolism, reflecting heme degradation, cell death, cell cycle arrest, metabolic reprogramming and inflammatory pathways related to TNFa, IL6 and IL2, as well as the complement system. Indeed, C5a plasma levels in phenylhydrazine-treated mice were increased. Inhibition of C5 with BB5.1 (1g/mouse) reduced C5a release and attenuated the inflammatory response to hemolysis, particularly in the lung.

Conclusion: Here we provide the first transcriptomic signature atlas of mouse organs with intravascular hemolysis, underpinning a key role of inflammation, oxidative stress and metabolic reprogramming and complement activation. These findings support further exploration of complement-targeted therapies to ameliorate hemolysis and preserve organ function in SCD patients.

Brochier:Institut Roche: Current Employment. Guinot:Institut Roche: 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. Roumenina:Roche: Research Funding; CSL Behring: Research Funding; Commit Bio: Research Funding; Alexion: Consultancy; Commit Bio: Consultancy. Grunenwald:Astrazeneca: Honoraria.