The NLRP3 inflammasome fires up heme-induced inflammation in hemolytic conditions

Abstract

Overactive inflammatory responses are central to the pathophysiology of many hemolytic conditions including sickle cell disease. Excessive hemolysis leads to elevated serum levels of heme due to saturation of heme scavenging mechanisms. Extracellular heme has been shown to activate the NLRP3 inflammasome, leading to activation of caspase-1 and release of pro-inflammatory cytokines IL-1β and IL-18. Heme also activates the non-canonical inflammasome pathway, which may contribute to NLRP3 inflammasome formation and leads to pyroptosis, a type of inflammatory cell death. Some clinical studies indicate there is a benefit to blocking the NLRP3 inflammasome pathway in patients with sickle cell disease and other hemolytic conditions. However, a thorough understanding of the mechanisms of heme-induced inflammasome activation is needed to fully leverage this pathway for clinical benefit. This review will explore the mechanisms of heme-induced NLRP3 inflammasome activation and the role of this pathway in hemolytic conditions including sickle cell disease.

Figure 1.. Heme production and catabolism.

Upon erythrocyte lysis, extracellular tetrameric Hb dissociates into dimers that exist in a dynamic equilibrium with the tetramers. These dimers bind tightly to haptoglobin (Hp) leading to CD163-mediated clearance of the Hb-Hp complex from circulation by macrophages. Depletion of Hp leads to the accumulation and oxidation of extracellular Hb, which results in the release of heme. This heme binds to albumin and is subsquently sequestered by tight binding to hemopexin (Hpx). The heme-Hpx complex binds to the CD91 receptor on macrophages promoting internalization to clear heme from circulation. After uptake, the Hb-Hp and the heme-Hpx complexes are degraded releasing heme. Heme is then catabolized by heme oxygenase-1 (HO-1), producing carbon monoxide (CO), ferrous iron (Fe(II)), biliverdin, and its reduced form bilirubin. The resulting Fe(II) is sequestered by ferritin and recycled back into circulation.

Figure 2.. Proposed mechanism of heme-induced NLRP3 activation.

Signal 1 is initiated by heme binding to RAGE or the MD-2/TLR4 complex to activate the NF-kB pathway. NF-κB induces expression of pro-IL-1β, pro-IL-18 and NLRP3. S100A8 amplifies heme-induced priming. This pathway constitutes Signal 1. In Signal 2, heme induces mitochondrial reactive oxygen species (mtROS) generation via spleen tyrosine kinase (Syk). The pathway most likely involves Syk activation of NADPH oxidase-2 (NOX2) via protein kinase C (PKC) or phosphoinositide 3 kinase (PI3K) signaling. Active NOX2 then triggers the production of mtROS. Mitochondrial ROS and potassium (K+) efflux induce the assembly of the NLRP3 inflammasome and recruitment of caspase-1. Caspase-1 then cleaves pro-IL-1β and pro-IL-18. In Signal 3, heme activates caspase-4 and caspase-5. Caspase-4 induces cleavage of GSDMD which forms the GSDMD pore and allows for release of active IL-1β and IL-18. Formation of GSDMD pores also facilitates the efflux of potassium ions (K⁺) that in turn can lead to the activation of the NLRP3 inflammasome and influx of ions inducing pyroptosis. Caspase-5 is required for IL-1β cleavage independent of GSDMD cleavage and may induce NLRP3 activation.

Figure 3.. Mechanisms of heme-induced vaso-occlusion.

A) Activation of the endothelium. Heme triggers TLR4 signaling leading to endothelial cell activation and expression of endothelial adhesion molecules B) Activation of the TLR4 pathway in macrophages. Heme induces tumor necrosis factor-α (TNFα) (green circles) secretion in macrophages through the TLR4 signaling pathway. TNFα triggers endothelial cell activation and expression of adhesion factors. C) Activation of the NLRP3 pathway in macrophages. Heme acts both as a priming and as an intracellular signal that triggers NLRP3 inflammasome activation to produce the mature cytokines IL-1β and IL-18 (pink and purple circles respectively). These cytokines induce inflammatory activation, neutrophil infiltration, and adhesion factors expression. D) Cytokine-induced recruitment and binding of neutrophils. Neutrophil recruitment and activation leads to neutrophil adhesion to the endothelium and sickled erythrocytes E) Platelet activation and formation of platelet-neutrophil aggregates. NLRP3-inflammasome activation in platelets leads to the generation of extracellular vesicles (EVs) that carry IL-1β. These platelet extracellular vesicles promote the generation of platelet-neutrophil aggregates in a caspase-1 and IL-1β dependent manner