Identification of a Heme Activation Site on the MD-2/TLR4 Complex

Belcher, John D.; Zhang, Ping; Nguyen, Julia; Kiser, Zachary Monroe; Nath, Karl A.; Hu, Jianjun; Trent, John O.; Vercellotti, Gregory M.

doi:10.3389/fimmu.2020.01370

Cited by 27 publications

(39 citation statements)

References 30 publications

(60 reference statements)

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“…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. Heme is a DAMP that can activate the innate immune pattern recognition receptor complex of CD14, MD-2 and TLR4 (8-10); this process promotes a pro-inflammatory and pro-adhesive phenotype, which ultimately leads to VOC (1,(8)(9)(10). Herein, we demonstrate that loss of TLR4 signaling in SCD leads to decreased VOC stimulated by numerous agonists, including heme, LPS and H/R.…”

Section: Discussionmentioning

confidence: 83%

“…Recently, the role of the innate immune system in perpetuating SCD inflammation and vaso-occlusive physiology has been recognized (1)(2)(3)(4)(5)(6)(7)(8)(9). Specifically, 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). Heme mediates pain via TLR4 in SCD mice and blockade or knockout of TLR4 attenuates hyperalgesia suggesting heme -induced microglial activation via TLR4 in the central nervous system contributes to the initiation and maintenance of sickle pain (12).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Discussionmentioning

confidence: 83%

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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“…TLR4 signaling induced by heme or its canonical ligand, lipopolysaccharide (LPS), requires cofactors CD14 and MD-2 (3,8,(11)(12)(13)(14)(15). The transduction mechanism for TLR4 activation is well characterized for LPS from gram negative bacteria.…”

Section: Introductionmentioning

confidence: 99%

“…In addition to being a required co-factor for TLR4 responses to LPS, MD-2 is also a required cofactor for TLR4 responses to heme ( 15 ). Heme binds to MD-2 at amino acids W23 and Y34 on MD-2, at a site independent of the LPS-binding site, to activate TLR4 pro-inflammatory signaling ( 15 ).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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Recent evidence indicates that hemolysis in sickle cell disease (SCD) promotes inflammation via innate immune signaling through toll-like receptor 4 (TLR4). Free heme released by hemolyzed red blood cells can bind to myeloid differentiation factor-2 (MD-2) and activate TLR4 pro-inflammatory signaling on endothelium to promote vaso-occlusion and acute chest syndrome in murine models of SCD. MD-2 is co-expressed with TLR4 on cell membranes, but in inflammatory conditions, soluble MD-2 (sMD-2) is elevated in plasma. sMD-2 levels were significantly increased in human and murine sickle (SS) plasma as compared to normal (AA) plasma. Human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells incubated with human SS plasma had significant increases in pro-inflammatory IL-8, IL-6, and soluble VCAM-1 secretion compared to endothelial cells incubated with AA plasma. The increase in HUVEC IL-8 secretion was blocked by depletion of sMD-2 from SS plasma and enhanced by the addition of sMD-2 to AA plasma. The TLR4 signaling inhibitor, TAK-242, inhibited HUVEC IL-8 secretion in response to SS plasma by 85%. Heme-agarose pull-down assays and UV/Vis spectroscopy demonstrated that heme binds to sMD-2. Hemopexin, a high affinity heme-binding protein, inhibited HUVEC IL-8 secretion induced by SS plasma or SS and AA plasma supplemented with sMD-2. These data suggest that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD.

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“…As efficient TLR4-dependent cell activation by LPS requires the complex interplay of TLR4 with CD14, myeloid differentiation protein-2 (MD-2) and the serum protein lipopolysaccharide binding protein (LBP) ( 29 ) it is likely that cooperation of these proteins is also critically involved in heme-dependent TLR4 signaling ( Figure 1 ). Notably, a heme activation site has recently been identified in human MD-2 which appears to play a critical regulatory role in TLR4 signaling by heme ( 30 ).…”

Section: Direct Activation Of Tlr4 Signaling By Hemementioning

confidence: 99%

TLR4 Signaling by Heme and the Role of Heme-Binding Blood Proteins

2020

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Toll-like receptors (TLRs), also known as pattern recognition receptors, respond to exogenous pathogens and to intrinsic danger signals released from damaged cells and tissues. The tetrapyrrole heme has been suggested to be an agonist for TLR4, the receptor for the pro-inflammatory bacterial component lipopolysaccharide (LPS), synonymous with endotoxin. Heme is a double-edged sword with contradictory functions. On the one hand, it has vital cellular functions as the prosthetic group of hemoproteins including hemoglobin, myoglobin, and cytochromes. On the other hand, if released from destabilized hemoproteins, non-protein bound or “free” heme can have pro-oxidant and pro-inflammatory effects, the mechanisms of which are not fully understood. In this review, the complex interactions between heme and TLR4 are discussed with a particular focus on the role of heme-binding serum proteins in handling extracellular heme and its impact on TLR4 signaling. Moreover, the role of heme as a direct and indirect trigger of TLR4 activation and species-specific differences in the regulation of heme-dependent TLR4 signaling are highlighted.

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Identification of a Heme Activation Site on the MD-2/TLR4 Complex

Cited by 27 publications

References 30 publications

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

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

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

TLR4 Signaling by Heme and the Role of Heme-Binding Blood Proteins

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