Hemolysis and the intravascular release of hemoglobin S are central to the pathophysiology of sickle cell disease (SCD). During the current funding period, we showed that heme derived from sickle red blood cells acts as a damage-associated molecular pattern that can activate toll-like receptor 4 (TLR4) of the innate immune system, independently of its cognate ligand lipopolysaccharide, leading to oxidant production, rapid P-selectin and von Willebrand factor expression on endothelium, and vaso-occlusion (VO) in SCD mice. We hypothesize that the innate immune system, including TLR4 and complement, is fundamental to understanding hemolysis-driven inflammation, coagulation, VO, and the cumulative organ pathology in SCD.
The first aim of our proposal is to identify the heme- binding site on the myeloid differentiation factor 2 (MD-2)/TLR4 complex. To date, no studies have examined the heme-binding site on the MD-2/TLR4 complex. Identifying the heme-binding site on MD-2/TLR4 is vital for developing therapies to interrupt heme-driven inflammation and VO.
The second aim will examine the impact of global Tlr4 deficiency and the specific contribution of TLR4 in leukocytes, platelets, and the vessel wall to SCD pathogenesis. Recent studies by our group and others used Tlr4-/-, non-sickle cell mice transplanted with Tlr4+/+ sickle bone marrow to underscore the importance of TLR4 in the vessel wall in promoting VO and acute chest syndrome in SCD. However, the impact of Tlr4 deficiency on inflammation, coagulation, and cumulative organ pathology has not been tested in a global Tlr4-/- SCD mouse model. We will test the hypothesis that TLR4 is a key signaling pathway that translates hemolysis into inflammation, VO, and organ pathology in SCD.
Our third aim will examine the role of complement activation in SCD hemolysis, inflammation, and VO. The alternative complement pathway is abnormally activated in SCD and is amplified by phosphatidylserine on the outer leaflet of sickle red blood cells and microparticles. We will test the hypothesis that complement activation on the surface of sickle red blood cells and microparticles stimulates inflammation, coagulation, hemolysis, and VO in SCD. Using state-of-the-art molecular techniques including site-directed mutagenesis, bone marrow transplants, cellular/biochemical studies, breeding a unique sickle/Tlr4-deficient mouse model, and in vivo vascular imaging in murine models of SCD, we will provide the foundation for the development of new therapies targeting multiple pathways to interrupt SCD pathophysiology. Understanding TLR4 and complement activation in SCD is expected to lead to therapies targeting the heme-binding site on the MD-2/TLR4 complex and complement inhibitors that will improve the quality of life of SCD patients.

Public Health Relevance

Despite remarkable improvements in the lifespan and quality of life of patients with sickle cell disease, devastating organ dysfunction, painful crises, and early death remain all too prevalent. The proposed studies proposed here will examine how heme-mediated inflammation and activation of the innate immune system affect the blood vessels and organs of sickle cell patients. New therapies are proposed that target and limit the adverse vascular effects of heme and inflammation.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Molecular and Cellular Hematology Study Section (MCH)
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Qasba, Pankaj
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University of Minnesota Twin Cities
Internal Medicine/Medicine
Schools of Medicine
United States
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Belcher, John D; Chen, Chunsheng; Nguyen, Julia et al. (2018) Haptoglobin and hemopexin inhibit vaso-occlusion and inflammation in murine sickle cell disease: Role of heme oxygenase-1 induction. PLoS One 13:e0196455
Nath, Karl A; Belcher, John D; Nath, Meryl C et al. (2018) Role of TLR4 signaling in the nephrotoxicity of heme and heme proteins. Am J Physiol Renal Physiol 314:F906-F914
Belcher, John D; Gomperts, Edward; Nguyen, Julia et al. (2018) Oral carbon monoxide therapy in murine sickle cell disease: Beneficial effects on vaso-occlusion, inflammation and anemia. PLoS One 13:e0205194
Conran, Nicola; Belcher, John D (2018) Inflammation in sickle cell disease. Clin Hemorheol Microcirc 68:263-299
Santiago, Rayra Pereira; Guarda, Caroline Conceição; Figueiredo, Camylla Vilas Boas et al. (2018) Serum haptoglobin and hemopexin levels are depleted in pediatric sickle cell disease patients. Blood Cells Mol Dis 72:34-36
Cataldo, Giuseppe; Lunzer, Mary M; Olson, Julie K et al. (2018) Bivalent ligand MCC22 potently attenuates nociception in a murine model of sickle cell disease. Pain 159:1382-1391
Jana, Sirsendu; Strader, Michael Brad; Meng, Fantao et al. (2018) Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles. JCI Insight 3:
Solovey, Anna; Somani, Arif; Belcher, John D et al. (2017) A monocyte-TNF-endothelial activation axis in sickle transgenic mice: Therapeutic benefit from TNF blockade. Am J Hematol 92:1119-1130
Gomperts, Edward; Belcher, John D; Otterbein, Leo E et al. (2017) The role of carbon monoxide and heme oxygenase in the prevention of sickle cell disease vaso-occlusive crises. Am J Hematol 92:569-582
Belcher, John D; Chen, Chunsheng; Nguyen, Julia et al. (2017) Control of Oxidative Stress and Inflammation in Sickle Cell Disease with the Nrf2 Activator Dimethyl Fumarate. Antioxid Redox Signal 26:748-762

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