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2493 DNA Sensing By Platelets Promotes Acute Chest Syndrome in Sickle Cell Disease

Program: Oral and Poster Abstracts
Session: 113. Sickle Cell Disease, Sickle Cell Trait, and Other Hemoglobinopathies, Excluding Thalassemias: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Sickle Cell Disease, Translational Research, Genetic Disorders, Hemoglobinopathies, Diseases, Immune mechanism, Biological Processes, Pathogenesis
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Rikesh K Dubey, PhD1, Omika Katoch, PhD1*, Nicholas Swendrowski, MS1* and Prithu Sundd, PhD1,2

1Thrombosis and Hemostasis Program, VERSITI Blood Research Institute, Milwaukee, WI
2Medical College of Wisconsin, Division of Hematology and Oncology, Milwaukee, WI

Introduction: Sickle cell disease (SCD) is an autosomal recessive genetic disorder affecting millions of people worldwide. Vaso-occlusion leading to sterile acute lung injury (clinically referred as acute chest syndrome) is one of the primary reasons of mortality among SCD patients. Recent studies have highlighted the role of circulating cell free DNA in triggering vaso-occlusion dependent acute lung injury in SCD. Although platelets express a functional DNA-binding toll-like-receptor 9 (TLR-9) on the surface, the role of platelet-TLR9 in promoting pulmonary thrombo-inflammation and lung injury in SCD has never been investigated.

Aim: To determine whether and how platelet TLR9 promotes acute chest syndrome in SCD.

Methods: Townes humanized knock-in SS and AS mice were used as SCD and control mice, respectively. Fresh blood samples were collected from SCD or race matched control human subjects in sodium citrate and processed to generate platelet rich plasma (PRP) or platelet poor plasma (PPP) within 2 hours of blood draw. Mice were intravenously (IV) treated with 10 mmol/kg oxy-hemoglobin (oxy-Hb) to trigger vaso-occlusive crisis as described elsewhere (Dubey et al, Haematologica 2023). Intravital lung microscopy was used to assess thrombo-inflammation in the lung and histology was used to assess lung injury. Imaging flow cytometry was used to assess circulating DNA such as neutrophil extracellular traps (NETs) in the human or mice plasma. Circulating platelets were isolated from mice or human blood and used for western blotting ± immuno-precipitation or ELISA.

Results: Intravital (in vivo) lung microscopy in live mice and imaging flow cytometry (in vitro) of plasma revealed for the first time that lung vaso-occlusion and elevated levels of circulating NETs in SCD mice challenged with oxy-Hb was associated with significant upregulation of TLR9 surface expression in platelets. Notably, blocking TLR9 function with an antibody reduced the frequency and size of lung vaso-occlusions in SCD mice by approximately six- and four-fold, respectively. Identical to SCD mice, TLR9 surface expression was also significantly higher in SCD than control human platelets, and the expression was further upregulated following treatment of SCD patient platelet-rich-plasma (PRP) with oxy-Hb, which was associated with elevated phosphorylation of tank-binding kinase-1 (TBK-1) and transcription factor IRF3, a key player in the interferon-1 response pathway. Furthermore, platelet phospho-proteomics revealed altered phosphorylation of IRF3 and several other components of TLR9 pathway in platelets of SCD than control mice challenged with IV oxy-Hb, suggestive of TLR9-dependent activation of platelets in SCD, leading to lung vaso-occlusion by neutrophil-platelet aggregates.

Conclusion: Our current findings suggest for the first time that activation of nucleic acid receptor TLR9 on the surface of platelets by cell free circulating DNA contributes platelet activation in SCD. Therapeutic inhibition of platelet TLR9 prevents lung vaso-occlusion and development of lung injury in SCD.

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

*signifies non-member of ASH