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Thank you for attending THSNA 2026. The virtual meeting is now closed.
Thank you for attending THSNA 2026. The virtual meeting is now closed.
Presentation Details
| TLR9 PROTMOTES NEUTROPHIL EXTRACELLULAR TRAPS GENERATION IN SICKLE CELL DISEASE(THSNA �Travel Awardee) AYYANAR SIVANANTHAM1, SHANE HOWE2, NICHOLAS SWENDROWSKI1, JOSHUA J FIELD2, 3, PRITHU SUNDD1, 4. 1Thrombosis and Hemostasis Program, Versiti Blood Research Institute and Blood Center of Wisconsin, Milwaukee, WI, USA.2Translational Hematology Program, Versiti Blood Research Institute, Milwaukee, WI, USA.3Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, USA.4Department of Biomedical Engineering, Medical College of Wisconsin, Milwaukee, WI, USA |
Abstract
Background: Sickle cell disease (SCD) is a monogenic disorder, which affects 8 million people worldwide. Acute chest syndrome (ACS) is a primary reason for mortality among SCD patients. There are no preventive or rescue therapies available to treat ACS. Recent evidence suggests that type I interferon (IFN-I) response in neutrophils promotes gasdermin D (GSDMD)-dependent NETs formation, leading to ACS in SCD mice (Vats et al. Blood 2022). However, the mechanisms for IFN-I production in neutrophils remain unknown. Neutrophils uniquely express TLR9 on their surface, unlike other leukocytes where it is confined to endosomes. Chronic hemolysis and ischemia-reperfusion injury releases circulating cell free DNAs and HMGB1, which may serve as ligand for the neutrophil surface TLR9. Despite these observations, the role of TLR9 in driving NETs formation and promoting ACS in SCD has never been investigated. Objective: To determine whether TLR9 activation promotes type-I IFN response by neutrophils in SCD. Method: Townes humanized knock-in AS and SS mice were used as control and SCD mice, respectively. Intravenous challenged with 10 μmol/kg oxyhemoglobin (oxy-Hb) to induce vaso-occlusive crisis and lung injury. SCD mice were IV administered oxy-Hb without or with pretreatment with TLR9 blocking Ab and lung vaso-occlusion was assessed using intravital lung microscopy. Also, Peripheral blood neutrophils were isolated using a negative selection. For ex vivo human studies, fresh peripheral blood was collected in sodium citrate from SCD patients and race-matched healthy controls. Whole blood was pretreated with a TLR9 antagonist, stimulated with 20μM hemin, 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. Result: oxy-Hb induced ACS, causing vaso-occlusion of pulmonary arterioles by neutrophil-platelet aggregates and NETs in the lung of SCD but not control mice. Neutrophils from oxy-Hb challenged SCD mice exhibited cleaved (active) TLR9 and robust IRF7 phosphorylation, resulting in increased IFN-α production. This IFN-I response was associated with increased expression of IFN-I–responsive and NET-related genes. Importantly, functional TLR9 blockade significantly reduced lung vaso-occlusion, IFN-α production, expression of IFN-I–responsive and NET-related genes, and IRF7 phosphorylation in oxy-Hb–challenged SCD mice. For ex vivo human studies, fresh peripheral blood was collected in sodium citrate from SCD patients and race-matched healthy controls. Whole blood was pretreated with a TLR9 antagonist ± 20μM hemin. Neutrophils isolated from SCD patient blood revealed that pIRF7 and IFN- α production levels were elevated, with further increase after hemin stimulation. TLR9 inhibition reduced IRF7 phosphorylation and IFN-I responses. Conclusion: These findings provide the first direct evidence that TLR9-dependent IFN-I production in neutrophils drive NETs formation in SCD. Activation of the TLR9–IRF7–IFN-I pathway contributes to ACS. Currently, experiments are underway to determine whether therapeutic targeting of TLR9 can mitigate interferonopathy and prevent ACS in SCD mice.
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