Abstract
Heme, released from red blood cells in sickle cell disease (SCD), interacts with toll-like receptor 4 (TLR4) to activate NF-κB leading to the production of cytokines and adhesion molecules which promote inflammation, pain, and vaso-occlusion. In SCD, TLR4 inhibition has been shown to modulate heme-induced microvascular stasis and lung injury. We sought to delineate the role of endothelial verses hematopoietic TLR4 in SCD by developing a TLR4 null transgenic sickle mouse. We bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. SS-Tlr4-/- had similar complete blood counts and serum chemistries as SS-Tlr4+/+ mice. However, SS-Tlr4-/- mice developed significantly less microvascular stasis in dorsal skin fold chambers than SS-Tlr4+/+ mice in response to challenges with heme, lipopolysaccharide (LPS), and hypoxia/reoxygenation (H/R). To define a potential mechanism for decreased microvascular stasis in SS-Tlr4-/- mice, we measured pro-inflammatory NF-κB and adhesion molecules in livers post-heme challenge. Compared to heme-challenged SS-Tlr4+/+ livers, SS-Tlr4-/- livers had lower adhesion molecule and cytokine mRNAs, NF-κB phospho-p65, and adhesion molecule protein expression. Furthermore, lung P-selectin and von Willebrand factor immunostaining was reduced. Next, to establish if endothelial or hematopoietic cell TLR4 signaling is critical to vaso-occlusive physiology, we created chimeric mice by transplanting SS-Tlr4-/- or SS-Tlr4+/+ bone marrow into AA-Tlr4-/- or AA-Tlr4+/+ recipients. Hemin-stimulated microvascular stasis was significantly decreased when the recipient was AA-Tlr4-/-. These data demonstrate that endothelial, but not hematopoietic, TLR4 expression is necessary to initiate vaso-occlusive physiology in SS mice.
Highlights
Sickle cell disease (SCD), which is caused by a single point mutation in the b-globin gene of hemoglobin, manifests with chronic intra- and extravascular hemolysis, oxidative stress, inflammation, and vaso-occlusive crisis (VOC)
Male and female heterozygous offspring were bred together and pups expressing exclusively human alpha- and beta-globins and at least one deleted Tlr4 gene were selected for backcrossing 9 generations with Townes-AA Tlr4+/+ mice from our colony with AA mice heterozygous for Tlr4 knockout selected for breeding with AATlr4+/+ mice at each new generation
Intravascular hemolysis of sickle red blood cells release hemoglobin S (HbS) into the plasma which is promptly oxidized to methemoglobin, which readily releases free heme
Summary
Sickle cell disease (SCD), which is caused by a single point mutation in the b-globin gene of hemoglobin, manifests with chronic intra- and extravascular hemolysis, oxidative stress, inflammation, and vaso-occlusive crisis (VOC). Heme, which is released during intravascular hemolysis, is able to serve as a damage-associated molecular pattern (DAMP) to stimulate TLR4 signaling on blood cells and the vasculature leading to vaso-occlusion and pulmonary injury [8,9,10,11]. Work done in drug-induced hemolysis models suggests that TLR4-mediated P-selectin release increases complement activation to further drive endothelial activation [13]. These studies have raised speculation that TLR4 and complement-targeted therapies may reduce severity of VOC in SCD
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