Complement Component C5 and TLR Molecule CD14 Mediate Heme-Induced Thromboinflammation in Human Blood

Abstract

Heme is a critical danger molecule liberated from hemeproteins in various conditions, including from hemoglobin in hemolytic diseases. Heme may cause thromboinflammatory damage by activating inflammatory and hemostatic pathways, such as complement, the TLRs, coagulation, and platelets. In this study, we explored the effect of single and dual inhibition of complement component C5 and TLR coreceptor CD14 on heme-induced thromboinflammation in an ex vivo human whole blood model. Heme induced a dose-dependent activation of complement via the alternative pathway. Single inhibition of C5 by eculizumab attenuated the release of IL-6, IL-8, TNF, MCP-1, MIP-1a, IFN-g, LTB-4, MMP-8 and-9, and IL-1Ra with more than 60% (p < 0.05 for all) reduced the upregulation of CD11b on granulocytes and monocytes by 59 and 40%, respectively (p < 0.05), and attenuated monocytic tissue factor expression by 33% (p < 0.001). Blocking CD14 attenuated IL-6 and TNF by more than 50% (p < 0.05). In contrast to single inhibition, combined C5 and CD14 was required for a significantly attenuated prothrombin cleavage (72%, p < 0.05). Markers of thromboinflammation were also quantified in two patients admitted to the hospital with sickle cell disease (SCD) crisis. Both SCD patients had pronounced hemolysis and depleted plasma hemopexin and haptoglobin. Plasma heme and complement activation was markedly increased in one patient, a coinciding observation as demonstrated ex vivo. In conclusion, heme-induced thromboinflammation was largely attenuated by C5 inhibition alone, with a beneficial effect of adding a CD14 inhibitor to attenuate prothrombin activation. Targeting C5 has the potential to reduce thromboinflammation in SCD crisis patients.

Pro-inflammatory Actions of Heme and Other Hemoglobin-Derived DAMPs

“…Recently the heme-induced thrombogenecity was studied in an ex vivo human whole blood model. Heme-induced thromboinflammation was attenuated by the inhibition of the complement component C5 and the TLR coreceptor CD14 (141). Inhibition of the thromboinflammatory loop can be a meaningful therapeutic target in hemolytic diseases (141).…”

Hemolysis Derived Products Toxicity and Endothelium: Model of the Second Hit

“…Heme, but not Hb, is a potent modulator of the innate immune complement system [9] (Figure 4A). It inhibits the target molecules’ recognition by the classical complement pathway initiating protein C1q (Roumenina et al 2011), but activates the alternative complement pathway [121,129,130,131,132]. The first evidence for heme-mediated complement activation came in 1978, when Wilson et al showed in vitro that the addition of hemoglobin solutions (prepared from erythrocyte lysates from healthy or sickle cell patients) to human serum triggered activation of the alternative complement pathway, revealed by the presence of C3 and persistent FB cleavage products.…”

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

“…For instance, the ability of LPS to activate TLR4 depends on CD14, a glycophosphatidylinositol-anchored protein and co-receptor of MD-2 for LPS recognition ( 45 ), which may also control internalization of heme via TLR4 ( 10 ). Interestingly, soluble TLR4 co-receptor CD14 has recently been reported to mediate pro-inflammatory effects of heme in a whole blood model ( 46 ).…”