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

Joan D. Beckman, Fuad Abdullah, Chunsheng Chen, Rachel Kirchner, Dormarie Rivera-Rodriguez, Zachary M. Kiser, Aithanh Nguyen, Ping Zhang, Julia Nguyen, Robert P. Hebbel, John D. Belcher, Gregory M. Vercellotti

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21 Scopus citations


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.

Original languageEnglish (US)
Article number613278
JournalFrontiers in immunology
StatePublished - Jan 19 2021

Bibliographical note

Funding Information:
Conflict of Interest: JBec receives funding from Bayer not related to work herein. JBel and GV receive research funding from CSL Behring and Mitobridge (Astellas).

Funding Information:
The authors would like to thank Elena Aronovich, Jacob Lloyd, and Kristine Nachbor for assistance with laboratory work.

Funding Information:
Research funding from NIH 5R01HL114567. ZK was supported by the Hematology Research Training Grant T32 L007062/HL/ NHLBI. RK and DR-R received funding from University of Minnesota Life Sciences Undergraduate Research Program (LSSURP) 5R25HL088728-14.

Publisher Copyright:
© Copyright © 2021 Beckman, Abdullah, Chen, Kirchner, Rivera-Rodriguez, Kiser, Nguyen, Zhang, Nguyen, Hebbel, Belcher and Vercellotti.


  • Toll-like receptor 4
  • endothelium
  • heme
  • sickle cell disease
  • vaso-occlusive events


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