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

Abstract

Damage associated molecular patterns (DAMPs) are endogenous molecules originate from damaged cells and tissues with the ability to trigger and/or modify innate immune responses. Upon hemolysis hemoglobin (Hb) is released from red blood cells (RBCs) to the circulation and give a rise to the production of different Hb redox states and heme which can act as DAMPs. Heme is the best characterized Hb-derived DAMP that targets different immune and non-immune cells. Heme is a chemoattractant, activates the complement system, modulates host defense mechanisms through the activation of innate immune receptors and the heme oxygenase-1/ferritin system, and induces innate immune memory. The contribution of oxidized Hb forms is much less studied, but some evidence show that these species might play distinct roles in intravascular hemolysis-associated pathologies independently of heme release. This review aims to summarize our current knowledge about the formation and pro-inflammatory actions of heme and other Hb-derived DAMPs.

Keywords: DAMP; NLRP3; TLR4 (toll-like receptor 4); heme; hemoglobin; hemolysis (red blood cells).

Figure 1

Pro-oxidant and antioxidant mechanisms is RBCs. O₂ binding to Hb initiates Hb auto-oxidation in which process metHb (Fe³⁺) and superoxide anion (${\text{O}}_2^{•-}$) are formed. MetHb is reduced by metHb reductase, while ${\text{O}}_2^{•-}$ is converted to H₂O₂ by superoxide dismutase (SOD). In the presence of transition metals such as Fe²⁺ or Cu⁺ a reaction between ${\text{O}}_2^{•-}$ and H₂O₂ occurs yielding hydroxyl radical (OH^•) (Haber Weiss reaction). Catalase, glutathione peroxidases (GPx), and peroxiredoxins (PRXs) decompose H₂O₂. The antioxidant system is completed with non-enzymatic low molecular weight scavengers, such as glutathione, ascorbic acid, and vitamin E. SOD, superoxide dismutase; GPx, glutathione peroxidase; PRXs, peroxiredoxins; GSH, reduced glutathione; GSSG, glutathione disulfide; GSR, glutathione-disulfide reductase; NADP⁺, nicotinamide adenine dinucleotide phosphate; NADPH, reduced NADP; G6PDH, glucose-6-phosphate dehydrogenase; Trx(r), reduced thioredoxin; Trx(ox), oxidized thioredoxin; TrxR, thioredoxin reductase.

Figure 2

Formation of oxidized Hb forms and labile heme upon hemolysis. Hb tetramers (${\text{HbFe}}^{2+}{\alpha }_2{\beta }_2$) is released from RBCs following intra- or extravascular hemolysis. (A) Hb outside of RBCs dimerize and can undergo spontaneous auto-oxidation (reaction I) to metHb (HbFe³⁺αβ). (B) Two-electron oxidation (reactions II) of Hb and metHb by H₂O₂ or lipid hydroperoxides (L-OOH) lead to the formation of ferrylHb (HbFe⁴⁺αβ) or ferrylHb radicals, respectively. (C) FerrylHb get stabilized via intramolecular electron transfer (reaction III) between iron and the globin chain forming globin radicals. Globin radicals get stabilized via covalent crosslinking (reaction IV) producing covalently crosslinked Hb multimers. Oxidized Hb forms (metHb, ferrylHb, covalently crosslinked Hb) release their heme prosthetic group (reactions V).

Figure 3

Targets of Hb-derived DAMPs. (I) Labile heme and ferrylHb induces endothelial cell activation characterized by NF-κB activation, elevated ROS production, and increased expression of adhesion molecules and pro-inflammatory cytokines. (II) Heme activates neutrophils characterized by elevated ROS production through the activation of NOX, increased production of IL-8 and NET formation. (III) Heme and ferrylHb induces monocyte and neutrophil chemotaxis. (IV) Labile heme and ferrylHb induces ROS production, NLRP3 activation, and pro-inflammatory cytokine production in LPS-primed macrophages. (V) Heme induces innate immune training through triggering epigenetic changes, such as acetylation of H3 at lysine-27 in monocytes and macrophages in a Syk-dependent manner. (VI) Heme induces complement activation leading to the formation of C3a and C5a activation fragments and the assembly of MAC. NF-κB, nuclear factor kappa B; ROS, reactive oxygen species; NOX, NADPH oxidase; NET, neutrophil extracellular trap; TLR4, toll-like receptor 4, NLRP3, NLR family pyrin domain containing 3; LPS, lipopolysaccharide; Syk, Spleen tyrosine kinase; H3, histone 3, MAC, membrane attack complex.