Endothelial TLR4 Expression Mediates Vaso-Occlusive Crisis in Sickle Cell Disease

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.

Soluble MD-2 and Heme in Sickle Cell Disease Plasma Promote Pro-Inflammatory Signaling in Endothelial Cells

“…Using immunohistochemical analysis, Wolfs et al found MD-2 is expressed by endothelial cells and inflammatory cells in the livers and lungs of septic patients and suggested these cells are a source of the enhanced circulating sMD-2 levels during acute systemic inflammatory diseases such as endotoxemia and sepsis (22). In a recently published study, we found both MD-2 and MIP-2a (a murine IL-8 homologue) mRNA levels were increased in the livers of Townes SS mice compared to control AA mice, these differences are consistent with markedly increased hemolysis in Townes SS mice (32). In this study, we showed that HUVEC secreted sMD-2 in response to heme or LPS.…”

“…The characteristics of human control and SCD subjects are summarized in Supplementary Data Table 1. Complete blood counts and serum chemistries of Townes AA and SS mice were published in a recent report from our lab (32).…”

An Analysis of the Multifaceted Roles of Heme in the Pathogenesis of Cancer and Related Diseases

“…Similarly, another study found an association between heme from hemolysis and TLR4 signaling on inflammatory and ECs [257]. Labile heme acts as a damage-associated molecular pattern (DAMP) and binds cofactor soluble myeloid differentiation factor-2 (sDM2), activating endothelial TLR4 and causing activation of the endothelium and vasoocclusion in a SCD mouse model [258].…”

Significance of Heme and Heme Degradation in the Pathogenesis of Acute Lung and Inflammatory Disorders

“…Heme can play a pro-inflammatory role in the context of hemolytic disorders such as SCD, malaria, and propagate neuronal injury during intracerebral hemorrhage [ 25 , 28 , 119 ]. Heme-dependent TLR4 activation was linked to pathological effects in SCD, including endothelial cell activation and vaso-occlusion [ 25 , 120 ]. Finally, heme induced neuroinflammation may promote the pathology of neurodegenerative disorders [ 121 ].…”