Heme Augments Toll-like Receptor 4 Signalling in Sickle Cell and Healthy Control Monocytes
Authors: Dagur Pradeep K , McCoy J. Philip , Nichols J , Mendelsohn Laurel , Seamon C , Kato Gregory J , van Beers Eduard J 
Published: October 2019

Abstract
Introduction
Inflammation is increased and related to early mortality in patients with sickle cell disease.(1) This inflammation is associated with upregulation of Toll-like receptor 4 and iron regulated genes in human sickle cell peripheral blood mononuclear cells. In sickle cell mice, heme released during intravascular hemolysis augments pro-inflammatory Toll-like receptor 4 signalling and this results in subsequent organ damage and death.
In this study we evaluated whether heme in human sickle cell monocytes is associated with increased Toll-like receptor 4 mediated pro-inflammatory cytokine production.
Methods
Fresh whole blood from patients (n=10) and controls (n=10) was used for a calcein assay to measure intracellular iron or was incubated with combinations of vehicle, a Toll-like receptor 4 ligand (lipopolysaccharide, 100 ng/ml) and/or an iron chelator (0.1 mM deferasirox (Exjade)). After three hours, the percentage of monocytes with detectable levels of intracellular interleukin-6 and tumor necrosis factor-alpha was quantified by flow cytometry. In an additional experiment fresh whole blood of patients (n=8) was incubated with combinations of vehicle, a Toll-like receptor 4 ligand (lipopolysaccharide, 1 ng/ml) and/or 20uM heme.
Results
Intracellular monocyte iron was correlated (R-spearman and P-value) positively with plasma levels of C-reactive protein in patients and controls (R=0.454, P=0.044), confirming that high intracellular iron in monocytes is associated with a pro-inflammatory state in vivo.
Compared to incubation with lipopolysaccharide alone, co-incubation of fresh human sickle cell blood with lipopolysaccharide and heme increased the absolute percentage of monocytes producing interleukin-6 with a median 8.5% (interquartile range -5.6-22.1, p=0.17) and tumor necrosis factor-alpha with 15.2% (2.0-21.2, p=0.025). Incubation of fresh sickle cell monocytes with heme alone did not increase interleukin-6 and tumor necrosis factor-alpha production significantly (respectively 0.1% and 0.0%).
Compared to incubation with lipopolysaccharide alone, co-incubation of lipopolysaccharide with the iron chelator deferasirox significantly decreased the absolute percentage of interleukin-6 producing monocytes with 20.4% (15.2-26.3) (P=0.004), further supporting the involvement of intracellular monocyte iron in Toll-like receptor 4 response.
Conclusion
We show that levels of intracellular monocyte iron correlate to markers of inflammation in human sickle cell patients. In an additional ex vivo experiment we show that the same monocytes have an increased Toll-like receptor 4 mediated inflammatory response when exposed to heme and a decreased inflammatory response when treated with an iron chelator.
We suggest that heme bound iron which is released during intravascular hemolysis and scavenged by monocytes, is a cause of monocyte activation and pro-inflammatory state in sickle cell disease, by augmenting Toll-like receptor 4 signaling. Iron chelation might be an interesting therapeutic option to decrease this pro-inflammatory effect of heme.
Figure Monocyte Toll-like receptor 4 dependent pro-inflammatory cytokine production is augmented by heme and inhibited by iron chelation.
(A) Compared to incubation of fresh human sickle cell blood with the Toll-like receptor 4 ligand lipopolysaccharide alone, co-incubation of lipopolysaccharide together with the iron chelator deferasirox significantly decreased the absolute percentage of interleukin-6 producing monocytes with 20.4% (15.2-26.3) (P=0.004) (B) In contrast, compared to incubation with lipopolysaccharide alone, co-incubation lipopolysaccharide together with heme increased the absolute percentage of monocytes producing interleukin-6 with a median 8.5% (interquartile range -5.6-22.1, p=0.17) and tumor necrosis factor-alpha with 15.2% (2.0-21.2, p=0.025). *** p<0.005 *p<0.05
1. van Beers EJ, Yang Y, Raghavachari N, Tian X, Allen DT, Nichols JS, e.a. Iron, inflammation, and early death in adults with sickle cell disease. Circ Res. 16 januari 2015;116(2):298-306.
Figure 1. Figure 1.

Disclosures
van Beers: Novartis: Research Funding.