Neutrophil-TLR9 is a therapeutic target for acute chest syndrome in sickle cell disease Free

Introduction: Acute chest syndrome (ACS), a severe form of acute lung injury, is a leading cause of morbidity and mortality in patients with sickle cell disease (SCD). The current therapy for ACS is primarily supportive and the etiological mechanism underlying lung injury remains incompletely understood. Neutrophil extracellular traps (NETs) composed of extracellular chromatin decorated with neutrophil proteases act as potent damage-associated molecular patterns (DAMPs) that promote pulmonary vaso-occlusion and ACS. Recent evidence suggests that type I interferon (IFN-I) signaling in neutrophils promotes gasdermin D (GSDMD)-dependent NETs formation, leading to lung injury in SCD mice. However, the mechanisms responsible for IFN-α/β (type I IFNs) production in neutrophils, and whether this pathway can be therapeutically targeted to prevent NETs generation and ACS remains unknown. Neutrophils are the only circulating leukocytes that abundantly express the nucleic acid receptor toll-like receptor 9 (TLR9) on their surface (rather than in endosomes), enabling direct recognition and binding of circulating DNA, whose levels are known to be elevated in the plasma of SCD patients during ACS. Despite these observations, the role of surface TLR9 in driving NETs generation and promoting lung injury in SCD has never been investigated.

Methods Townes knock-in humanized control (AS) and SCD (SS) mice were intravenously (IV) challenged with 10 μmol/kg oxyhemoglobin (oxy-Hb) ± TLR9 blocking Ab to induce vaso-occlusive crisis and lung injury. Lung injury was assessed using histology, lung vaso-occlusion was assessed using intravital lung microscopy, and peripheral blood neutrophils were isolated using a negative selection for omics and biochemical analysis. Fresh peripheral blood was collected in sodium citrate from SCD patients (steady state or hospitalized with ACS) and race-matched healthy controls and used either untreated or treated with 20μM hemin ± TLR9 antagonist, and neutrophils were subsequently isolated by density gradient method. Isolated neutrophils were used for gene expression analysis, immunoprecipitation, western blotting, and ELISA to assess IFN-I signaling and NETs-associated pathways. Imaging flow cytometry was used to assess circulating DNA such as NETs in the human or mice plasma.

Results: IV oxy-Hb challenge led to lung injury (histology) and occlusion of pulmonary arterioles by neutrophil-platelet-erythrocyte aggregates (intravital lung microscopy) in SCD but not control mice, which was prevented by pretreatment with anti-TLR9 function blocking Ab. In addition, imaging flow cytometry of plasma revealed significantly higher levels of circulating fragments of NETs (cNETs triple positive for DNA, neutrophil elastase and histones) in IV oxyHb challenged SCD mice and SCD patients hospitalized with ACS, which were significantly attenuated following TLR9 inhibition. Flow cytometry revealed higher functional TLR9 surface expression in neutrophils of IV oxy-Hb challenged SCD than control mice. ELISA, transcriptomics and western blot analysis±co-IP revealed significantly higher cleavage of TLR9, TLR9-dependent phosphorylation of IRF7 (pIRF7), which drove IFN-α production in neutrophils of IV oxy-Hb challenged SCD but not control mice. Elevated IFN-I responses were accompanied by increased expression of key IFN-I and NETs-related transcripts, including Ifnar, Jak-Stat, Caspase-11, Gsdmd, and neutrophil elastase (Elne), along with enhanced formation of TLR9-dependent downstream signaling complex IRAK4-TRAF6-IRF7. Consistently, TLR9 blockade reduced IRF7 phosphorylation and downstream IFN-I responses. Ex vivo studies revealed that pIRF7 levels were also elevated in neutrophils of SCD patient but not control human neutrophils. Collectively, these findings highlight a critical role for neutrophil-intrinsic TLR9 signaling in driving IFN-I–mediated NETs generation and lung injury in SCD.

Conclusion: This study provides the first evidence that TLR9-dependent IFN-I production in neutrophils drives NETs generation in SCD. Activation of the TLR9–IRF7–IFN-I signaling pathway emerges as a central mechanism promoting lung injury during ACS. Currently, studies in SCD patient blood and humanized SCD mouse models are underway to determine whether therapeutic TLR9 inhibition can mitigate interferonopathy and prevent ACS.