Circulating Extracellular Vesicles Enable IFN-I Response in Neutrophils of Sickle Cell Disease Mice

Introduction: Acute chest syndrome, a type of acute lung injury, is a major cause of mortality in Sickle Cell Disease (SCD). The current therapy for acute chest syndrome (ACS) is primarily supportive, and the etiological mechanism underlying lung injury remains incompletely understood. Neutrophil extracellular traps (NETs), composed of externalized chromatin decorated with proteases, are potent neutrophil-derived DAMPs that promote ACS. New evidence suggests that type I interferon (IFN-I) signaling in neutrophils promotes gasdermin-D (GSDMD)-dependent dependent NETs generation, leading to lung injury in SCD mice (Vats et al, Blood 2022). However, the mechanism underlying the activation of IFN-I signaling in the neutrophils of SCD mice or patients remains elusive. Importantly, circulating extracellular vesicles (EVs) are significantly upregulated in the plasma of SCD patients at baseline, further upregulated during ACS, and promote lung injury (Vats et al, AJRCCM 2020), but whether EVs promote NETs generation in SCD remains unknown.

Methods: Townes knock-in humanized control (AS) and SCD (SS) mice were intravenously challenged with physiological saline or 10 μmol/kg of oxy-hemoglobin (oxy-Hb) to trigger a vaso-occlusive crisis, leading to lung injury as described recently (Dubey et al, Haematologica 2023). Neutrophils and EVs were isolated from the peripheral blood using the negative selection method and size exclusion chromatography, respectively, as described in our previous studies (Vats et al, Blood 2022; Vats et al, AJRCCM 2022). Neutrophils were lysed and used in western blotting, RNAseq, and ELISA. EVs were quantified using nano-particle-tracking analysis and used in mass spectrometry analysis. Bioinformatics analysis of the EV-proteome was conducted using Perseus. Gene set enrichment analysis (GSEA) were performed using Ingenuity Pathway Analysis (IPA), Reactome, KEGG, and GO databases with Cluster Profiler.

Results: IFN-α protein expression and gene expression of downstream IFN-I signaling molecules were significantly upregulated in neutrophils of IV oxy-Hb treated SCD compared to control mice. Next, in silico analysis was conducted to assess how EV uptake by neutrophils can regulate gene expression in neutrophils. Remarkably, pathway analysis revealed 47 proteins that were differentially expressed in IV oxy-Hb treated SCD compared to control mice EVs, and potentially regulated IFN-I (α and β) production, IFN-I pathway activation, upregulation of IFN-I stimulated genes (ISGs), and genes associated with NETs generation, metabolism, and adhesion to the endothelium following EV uptake by neutrophils.

Conclusion: These findings suggest for the first time that neutrophils produce IFN-I in SCD mice, and the uptake of circulating EVs by neutrophils may contribute to IFN-I signaling and NETs generation in SCD. Currently, in vitro studies in SCD patient blood and in vivo studies in SCD mice are underway to identify major IFN-I synthesis pathways in neutrophils that are regulated by EVs, and how this interferonopathy can be regulated therapeutically to prevent lung injury in SCD.

Sundd:Novartis AG: Research Funding; CSL Behring Inc:: Research Funding; IHP Therapeutics: Research Funding.