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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL114567-06
Application #
9418078
Study Section
Molecular and Cellular Hematology Study Section (MCH)
Program Officer
Mitchell, Phyllis
Project Start
2017-02-01
Project End
2021-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
6
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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
Nath, Karl A; Belcher, John D; Nath, Meryl C et al. (2017) The Role of TLR4 Signaling in the Nephrotoxicity of Heme and Heme Proteins. Am J Physiol Renal Physiol :ajprenal.00432.2017
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
Shi, Patricia A; Choi, Erika; Chintagari, Narendranath R et al. (2016) Sustained treatment of sickle cell mice with haptoglobin increases HO-1 and H-ferritin expression and decreases iron deposition in the kidney without improvement in kidney function. Br J Haematol 175:714-723
Vercellotti, Gregory M; Zhang, Ping; Nguyen, Julia et al. (2016) Hepatic Overexpression of Hemopexin Inhibits Inflammation and Vascular Stasis in Murine Models of Sickle Cell Disease. Mol Med 22:
Belcher, John D; Chen, Chunsheng; Nguyen, Julia et al. (2015) The fucosylation inhibitor, 2-fluorofucose, inhibits vaso-occlusion, leukocyte-endothelium interactions and NF-?B activation in transgenic sickle mice. PLoS One 10:e0117772
Chintagari, Narendranath Reddy; Nguyen, Julia; Belcher, John D et al. (2015) Haptoglobin attenuates hemoglobin-induced heme oxygenase-1 in renal proximal tubule cells and kidneys of a mouse model of sickle cell disease. Blood Cells Mol Dis 54:302-6
Belcher, John D; Chen, Chunsheng; Nguyen, Julia et al. (2014) Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease. Blood 123:377-90
Vercellotti, Gregory M; Belcher, John D (2014) Not simply misshapen red cells: multimolecular and cellular events in sickle vaso-occlusion. J Clin Invest 124:1462-5
Vercellotti, Gregory M; Khan, Fatima B; Nguyen, Julia et al. (2014) H-ferritin ferroxidase induces cytoprotective pathways and inhibits microvascular stasis in transgenic sickle mice. Front Pharmacol 5:79

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