An estimated 250 millions of Hb molecules are packed in a red blood cell. During intravascular hemolysis, the destruction of RBCs results in release of billions of Hb molecules in circulation. The cell-free Hb in flowing blood can contribute towards many cytotoxic effects including tissue damage and organ dysfunction. Cell-free Hb has been shown to be associated with the pathogenesis of endothelial dysfunction, intravascular thrombosis, altered immune responses along with acute inflammations, and lung and renal injury in different disease conditions such as Paroxysmal Nocturnal Hemoglobinuria (PNH) and Sickle Cell Disease (SCD) [1,2]. A number of iatrogenic diseased states are resulted due to excessive release of cell-free Hb during hemodialysis, cardiac bypass, and transfusion therapies.
Extensive studies have shown that the accumulation of cell-free Hb causes great deal of cytotoxicity [3,4]. The oxidation products of Hb are the most potent mediator that manifests the Hb toxicity via free radicals. Heme, one of the degradation products of Hb, induces oxidative damages via ROS generation. Heme triggers production of inflammatory mediators through activation of selective signaling pathways in immune cells. Under normal conditions this potential source of oxidative hazard is minimized by Hp and Hpx, which bind free Hb and heme respectively and facilitate their removal from blood. However, during the hemolytic disease conditions, the oxidized Hb or Hb derivatives can increase many of the above clinical consequences including inflammations. Overwhelmed with the excessive amount of cell-free Hb in circulations, the scavenging system fails to work adequately further exacerbating the oxidative stress. The prevailing pro-oxidant conditions may lead to the development of pro-inflammatory symptoms, which promotes tissue injuries and organ damages. The present review focuses on the pro-oxidant properties of cell-free Hb and its general consequences on immune components in hemolytic disorders.
Normally the removal of senescent RBCs from the circulation leads to certain degree of intravascular hemolysis, which is readily cleared up by the conventional Hb- scavenging mechanisms. However, in patients with hemolytic disorders such as PNH and SCD, the scavenging systems are overwhelmed with excess of cell-free Hb. The breakdown products of Hb such as heme exhibit intrinsic toxicity. Heme interferes with the oxidant and anti-oxidant balance in the tissue . The cell-free Hb produces harmful reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide (H2O2) . Furthermore, in presence of ROS the free-Hb is oxidized into methhemoglobin (MetHb), chracterized by the change in the oxidation state of Iron (Fe) from ferrous (Fe+2) to ferric (Fe+3). The pro-oxidant effects of cell-free Hb are mostly related to conformational change observed in the Hb molecule (related to oxidation state of iron) which also affects its ability to interact with the cellular receptors .
Hb exists in a dynamic equilibrium state of tetramer and αβ-unit heterodimer where the dimer state predominates at low plasma Hb concentration. Dimers being small in size (~32kDa) have an easy access to tissues and cells and can even be translocated across endothelial barrier thereby increasing cellular susceptibility of oxidant-mediated cell injury [8,9]. After tissue extravasation, the cell-free Hb is oxidised in presence of ROS leading to formation of MetHb and superoxide . Being unstable, MetHb readily releases heme. Once released, free heme causes oxidative damage and inflammation via Fenton chemistry, thus acting as a prototypic damage-associated molecular pattern (DAMP) [11,12]. These pro-oxidant effects are specific to MetHb than OxyHb through oxidation via H2O2 or oxygen radicals .
Dismutaion of superoxide anions formed during autoxidation of Hb dimers results in H2O2 formation , which is further involved in the secondary oxidative reactions with Hb . Cell-free Hb is highly reactive with H2O2 gaining pseudo-peroxidase properties . Fe2+ of Hb reacts with H2O2 via Fenton reaction generating highly reactive hydroxyl radical (.OH), which can further amplify the toxicity and the damage induce by free Hb . H2O2 produced oxidizes ferrous and ferric Hb to Fe (Fe4+)-ferryl Hb and Oxyferryl Hb, respectively. Ferryl-Hb further reacts with H2O2 producing heme degradation products and free iron, which is responsible for various pathophysiological conditions . An increase in these heme degradation products indicates an increased potential for oxidative stress, though the specific reactions involving these products remains elusive . Besides auto-oxidation, Hb may also remove Nitric Oxide (NO) and react with inorganic and organic peroxides and lipids, leading to the generation of oxygen-, heme-, and globin based radicals as well as other mediators .
In recent years, a great deal of interest has been directed towards the reaction of NO with Hb . Interaction of Hb with NO is among the most studied aspect of Hb induced toxicity. The consumption of NO and subsequent oxidation of Hb occur through mechanisms either by NO dioxygenation of oxy-Hb, which generates nitrate (NO3-) and ferric Hb (Hb-Fe3+) or by nitrosylation of iron in deoxy-Hb, which occurs when NO directly binds to iron in non-liganded ferrous form . The Hb-Fe3+ formed in the blood vessels can readily accumulate in tissues as well as in circulation inducing oxidative stress . Free Hb in plasma scavanges NO thereby reducing the availability of NO for various other essential physiological functions especially regulation of vascular tone and vasodilatation [22,23], relaxation of smooth muscle, neutrophil adhesion to endothelial cells and platelet activation etc. . Moreover, reaction of superoxide to NO results in generation of peroxynitrite which can oxidize oxy-Hb forming ferryl-Hb . Hemolysis also results in loss of erythrocyte arginase, an enzyme that metabolizes l-arginine. Reduction in the amount of l-arginine available for conversion to NO, further contributes to endothelial dysfunction [26,27]. Besides, the effects of NO on the oxidation and nitrate generation by free-Hb are also considered to be the inducer for cytotoxicity.
The free Hb interacts with NO to generate Hb-Fe3+ in tissues, which further promotes release of heme and its transfer to other proteins/lipids leading to secondary toxicity. Heme transfers its reactive porphyrin to membranes or soluble plasma and cell proteins and lipids resulting in formation of heme-albumin or heme lipid complexes. Free heme acts as a ligand for molecular signalling and interactions [8,28]. Under specific physiological conditions free-iron protoporphyrin of heme can act as an intermediate to transform recipient molecule into a reactive end product. The most identifiable toxic end product of heme in plasma is the oxidized low density lipoprotein (oxLDL) . LDL oxidation and associated inflammatory and cytotoxic effects mediated by cell-free Hb contributes significantly to the vascular injury [29,30]. Heme can alter cell activation state and physiology by selectively binding to several receptors, transcription factors and enzymes. Interaction of heme with Bach-1 (a transcriptional repressor of heme-oxygenase 1 or HO- 1) is well characterized and seems crucial for clearance of increased level of intravascular Hb . Heme is also reported as a ligand for nuclear receptor REV-ERB-αβ thereby playing important roles in regulation of circadian rhythm, adipogenesis and metabolism . Reversible inhibition of proteosome activity by heme has also been reported [33,34]. These interactions of heme might outline several new roles for heme in cell-free Hb induced toxicity in biological systems.
Body harbors a repertoire of scavenger proteins, receptors, and enzymes that accomplishes mechanisms related to clearance and detoxification of cell-free Hb and heme. The clearance pathway primarily involves the transport of cell-free Hb to the liver or macrophages, breakdown of the porphyrin by the HO-1 system into bilirubin and carbon monoxide and ultimately the recovery of the iron for de novo erythropoiesis. The Hb scavenger system comprises of several soluble plasma proteins viz. haptoglobin (Hp) and hemopexin (Hpx) along with the cellular receptors that bind protein-Hb, or protein-heme complexes and provide protection against systemic Hb and heme toxicity. The most studied pathway involved the plasma Hb scavenger Hp and the monocyte/macrophage Hb-Hp scavenger receptor CD163. Endocytosis of the Hb-Hp complex results in release of heme and up-regulation of hemoxygenase-1 (HO-1) thereby providing evidence for HO-1 dependent macrophage mediated clearance of heme. The scavenging of free-Hb by these phagocytes can be modulated by anti-inflammatory cytokines such as IL-4 and IL- 10. Recently Du et al. identified an Hb interactome comprising of Hb, Hp and lipid-free apolipoprotein A-I (apoAI). Apolipoprotein A-I, acts as a secondary antioxidant that interacts with Hb and quench the redox activity of Hb. It facilitates the uptake of Hb by interacting with a scavenger receptor class B type 1 (SR-B1) exhibited on macrophages and hepatocytes suggesting a novel receptor-mediated mechanism for clearance of cytotoxic Hb from plasma.
Hpx, the primary scavenger for free heme complements the plasma Hb-binding capacity provided by Hp. Mainly expressed in the liver Hpx is classified as class I acute phase protein induced by proinflammatory cytokines (i.e., IL-1 and IL-6). Interaction of free heme with Hpx is reported to be the strongest among the plasma hemeprotein interaction. Heme-Hpx binding and its subsequent uptake by the macrophage-associated LRP-1 receptor removes the heme from the circulation. The relevance of Hpx in clearance of cell-free Hb was highlighted in Hp/Hpx double knockout mice that are susceptible to severe liver inflammation, splenomegaly, and Hb induced oxidative damages. The Hb:Hp and heme:Hpx complexes are finally cleared by their respective endocytosis receptors CD163 and CD91/LRP before they are degraded by lysosomal proteases.
Being an oxygen carrier, Hb may act as a signalling molecule, activating oxygen sensitive transcription factors and target genes. Nevertheless, excessive cell-free Hb pose a severe pathological risk for vital systems, including renal, cardiovascular, gastrointestinal, neural, immunologic, coagulation related and many others [20,35]. Vasculature, particularly the endothelial cells due to their direct contact with plasma borne Hb, is among the most sensitive targets of the oxidation products generated by cell-free Hb. Cell-free Hb induced ROS increases pulmonary micro vascular endothelial permeability through an oxidant-dependent, mitochondrialmediated pathway . Interactions of Hb with hydrogen peroxide are known to alter thiol levels that might modulate endothelial cell survival . Accumulation of Hb dimers in kidney tubules results in acute renal dysfunction which can be attributed to Hb associated oxidative reactions . Studies explain that the Hb derivative, heme stimulate the secretion of chemokine Monocyte Chemo-Attractant Protein-1 (MCP-1) and Transforming Growth Factor b1 isoform1 (TGF beta-1) via NF-κB dependent pathway [39,40]. Also MetHb has been reported to stimulate lung epithelial cell to secrete chemokines via NF-κB and MAPK dependent pathway  suggesting a crucial pro-inflammatory role of cell-free Hb in development of acute lung injury.
At cellular level, heme and the ferryl radical can damage DNA and oxidize lipids and proteins, and thus perturb the cellular integrity. The intrinsic peroxidase activity of Hb also contribute to the oxidation of LDL and Polyunsaturated Fatty Acids (PUFAs) present in cell membrane, and Induces Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1) . It also impairs the activity of cytosolic enzymes such as glucose- 6-phosphate dehydrogenase and glutathione reductase. Heme is also known to induce activation of cell-damaging enzymes such as caspases and cathepsins [43,44]. Apart from the above mentioned functions, the oxidized Hb potentially modulates immune cell functions . The free heme increases number of regulatory T cells (Treg) and promotes neutrophil activation, which further leads to inflammation and tissue extravasation . The free Hb increases the survivability of neutrophils and prevents apoptosis via HO-1 and ferretin dependent mechanism. Recent report from our laboratory describes that the circulating monocytes are transformed into proinflammatory subsets, when engulf Hb activated platelets .
Cell-free Hb acts as host-derived Danger-Associated Molecular Patterns (DAMPs) that interacts with the components of innate immune system either directly or via binding to Pathogen-Associated Molecular Patterns (PAMPs). PAMPs (LPS and LTA) trigger the pseudo-peroxidase activity of Hb resulting in superoxide anion production which enhances the inflammatory potential of cellfree Hb . The components of innate immune system (dendritic cells/DC, macrophages, monocytes and neutrophils) have been reported to exhibit altered phenotypes and functional responses in hemolytic conditions. Reports also highlight the effect of cell-free Hb in altering function of T and B lymphocytes . Among Hb degradation products, heme has been reported to exhibit several pro-inflammatory activities related to leukocyte activation, and migration, expression of adhesion molecules and induction of cytokines and acute phase response proteins . Interestingly, mammalian cells have been reported to express surface protein that can bind heme . Neutrophils among the immune cells are first to respond to infection or damage. The migration of neutrophils to the site of inflammation is mediated by PAMPs or DAMPs derived from disrupted host cells. Heme has been shown to induce ROS and stimulate neutrophil migration utilizing the signature signalling pathways characteristic of chemoattractant molecules suggesting its role in amplification of inflammatory responses . Oxygen/ nitrogen based reactive species are also known to regulate Toll-like receptor (TLR)-mediated signalling [51,52]. Heme has been shown to utilize pattern recognition receptors like TLRs (Toll like Receptors) to mediate activation of the cells of innate immune system. Heme also induces oxidative burst, neutrophil recruitment using TLR4 signalling mechanisms . Similarly, the MetHb also behaves as DAMPs, but unlike heme it signals through TLR2 mediated NF-kB dependent pathway to stimulate neutrophil ROS production and induce pro-inflammatory cytokines and neutrophil apoptosis . Heme induces assembly of p62/SQTM1 ubiquitinated proteins known as Aggresome-Like Induced Structures (ALIS). ALIS formation is particularly mediated by ROS generated in response to Hb degradation products through activation of the transcription factor NRF2 thereby modulating heme induced inflammation . Recent study from Vinchi and colleagues showed that heme and its iron moiety, brings about phenotype change in macrophages leading them toward M1-like proinflammatory phenotype, a response that is specifically mediated by TLR4 signalling and ROS . Heme via modulation of HO-1 also regulates DC maturation and function by modulating the p38 MAPK-CREB/ATF1 signalling . Also there are reports that suggest that ROS induced by heme is responsible for necrotic cell death of macrophages via TNF-α dependent mechanism . Several other studies have shown that Hb treatment with multiple TLR ligands or PAMPs synergistically induces cytokines, such as IL-1b, IL-6, IL-8, and TNF-α in macrophages [58,59]. The heme induced levels of ROS apart from inducing cell death might prime the responsiveness of cells of the innate immune system . Taken together, all these reports collectively suggest a high likelihood of degradation products of Hb binding to certain DAMPs/PAMPs thereby inducing ROS that can further enhance the pro-inflammatory signalling in the immune cells.
The pro-oxidative properties the free Hb also triggers inflammatory signaling  which further impact the functional state and responsiveness of immune cells and acts as a causal agent for cell damage. Heme induced ROS has been shown to induce expression of pro-inflammatory adhesion molecules on endothelium and blood cells and increase vascular permeability leading to greater leukocyte influx . It also interacts with the cells of innate immune with unknown mechanism and contributes to the pathogenesis of several diseases [21,62]. Neutrophils in response to heme have been shown to generate ROS through NADPH oxidase-dependent Protein Kinase C (PKC) activity leading to cytoskeleton reorganization . Heme has potential role in activation and acceleration of inflammation in both sterile and infectious condition  and triggers inflammasome activation playing active role in pathogenesis of hemolytic disorders . Inflammasome, an innate immune component, act as receptor and sensor for PAMPs and DAMPs and regulate the caspase-1 activation which facilitate chronic inflammation . It consists of several Pattern Recognition Receptors (PRRs) including NODlike Receptors, (NLRs) and the Absent in Melanoma 2 (AIM)-like Receptors (ALRs) which oligomerize to form caspase-1-activating scaffold and cleave the IL-1 family cytokines to generate active form of IL1b and IL-18. The continuous presence of these cytokine leads to chronic inflammation and cell death (pyrotopsis) [62,65,66]. The general and common molecules which oligomerise to form inflammasome in cytosol are Apoptosis-associated Speck-like protein (ASC) and Caspase Activation and Recruitment Domain (CARD). ASC multimers interact with CARD and upstream sensor molecule which act as scaffold in activation of procaspase-1 and initiate the chronic inflammatory cascade . Depending upon the nature of interacting receptors in inflammasome complex it is broadly categorized into two major forms, canonical and noncanonical inflammasome. NLRP1, NLRP3, NLRC4, AIM2 and pyrin which comprises of Nucleotide Oligomerization Domain (NOD), Leucine-Rcich Repeat (LRR)-containing proteins comes under canonical forms. Non-canonical inflammasome however, are not well characterized but complement the function of canonical inflammasome in both mice and human by targeting caspase-11 and caspase-4 and/or caspase-5 respectively [66,68].
Heme, an auto-oxidation product of Hb induces the activation of NLRP3 inflammasome. Heme stimulates spleen tyrosine kinase (syk) via an unknown receptor involved in direct alterations of lipid rafts leading to mitochondrial ROS generation and NLRP3 activation . The hemolysis increases the cell-free Hb which has inflammatory effects and act as one of the cause of cell death. The activation of inflammatory cascade by heme specifically in macrophages primed with LPS enhances the IL1b processing via nucleotide-binding domain and Leucine Rich Repeat (LRR) containing family, pyrin domain containing 3 (NLRP3). The activation of NLRP3 via heme in macrophages depends on spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K+ efflux. This finding suggests the potential role of heme in the induction of hemolysis induced-lethality .
The pro-apoptotic roles of free Hb have been suggested promoting various inflammatory impacts on immune cells. Apoptosis is a process, which is required for maintenance of cellular homeostasis. Besides having beneficial role, dysregulation in this cellular process can lead to tissue damage, chronic inflammation and can also promote cancer . The free-Hb initiates inflammatory responses and the persistence inflammations may leads to apoptosis and cell death . It is well documented that cell-free Hb leads to platelet activation and its apoptosis. Hb binds to platelets via GP1bα, which leads to its activation and enhanced expression of pro-apoptotic proteins Bak, Bax, cytochrome C and activated caspase-9 and caspase-3 . The direct effect of cell-free Hb on immune cell apoptosis is yet to be explored. However, the impact of cell-free Hb on innate immune cells majorly on monocytes which is one of the important phagocytic cells of immune system has been explored by our group. Our study reported that the monocytes in hemolytic diseases such as PNH and SCD, show the elevation in expression of phosphatidylserine (PS). This finding was correlated with cell-free Hb and platelets activated with cell-free Hb in the in vitro study where we have treated the monocytes with Hb and Hb activated platelets and observed the same . The finding of the other independent study suggest that cell-free Hb and its component i.e. heme has anti-apoptotic role in neutrophils, which is the innate immune cells and classically known as the first cell which migrates at the site of inflammation. Heme modulates expression of pro- and anti-apoptotic Bcl-2 family members. Heme induced Bcl- XL synthesis and promoted Bad degradation. Other study has shown that heme preserves mitochondria stability and shifts the Bcl- XL/ Bad ratio in a ROS-dependent manner . Heme has been shown to delay apoptosis in neutrophils. The effect is heme oxygenase and ROS dependent which protect the neutrophil apoptosis via ERK, PI3K and NF-kB dependent pathways. The delay in apoptosis or the increased life-span of neutrophil may have impact on the trigger of inflammatory response in the hemolytic conditions . Heme induced ROS has been reported to destabilize macrophage membrane leading to necrosis. Moreover, heme activated TNFR1 and TLR4/ MyD88-dependent production of tumor necrosis factor (TNF) has been suggested to induce macrophage necrosis .
The cell-free Hb as well as the derivaties of its oxidative products such as heme is a hallmark of hemolysis that may laed to various clinical consequences in diseases such as SCD, PNH, polycythemia, and drug-induced hemolytic anemia. Extensive survey of literature suggests that pro-oxidant, cytotoxic and inflammatory attributes of the Hb derivatives play crucial role in the pathogenesis of various cellular functions including immune responses. However, a clear insight into the mechanisms of the patho-physiology of oxidative damages induced by the derivatives of Hb is challanging. Although several studies have described the mechanism of inflammasome activation and immune modulation by the Hb/heme induced oxidative stress but the crucial gaps for developing targeted therapeutics to counteract these clinical consequences in hemolytic conditions remains unfilled.
Several studies have explored the use of scavenger proteins like haptoglobin (Hp) and hemopexin (Hpx) as therapeutic agents to abrogate the cytotoxic effect of cell-free Hb and heme. Apart from using scavenger protein as a part of treatment regime, therapeutic strategies should be developed to utilize anti-oxidants or other drugs exhibiting anti-oxidant and anti-inflammatory properties to attenuate the damaging effects of cell-free Hb induced ROS. Considering the anti-inflammatory potential of steroids, use of glucocorticoids is being explored for treatment of hemolysis induced inflammations in PNH and SCD. Furthermore, Vallelian et al. reported that glucocorticoid therapy in patients polarized monocytes into a M2/alternatively activated phenotype with higher expression of Hb-scavenger receptor (CD163) further enhancing its Hb clearance capacity . The synthetic anti-oxidants like carbazole and its derivatives as therapeutics can be potential but are of limited use because of its toxicity . In addition, anti-oxidants such as vitamin E and polyphenols when given together with iron chelators, may provide a substantial improvement in the pathophysiology of hemolytic anaemias and particularly in thalassemia and may also prove important in ameliorating oxidative stress parameters . Flavonoids (like kaempferol, quercetin, morin and rutin) can be used as natural antioxidants for the treatment and prevention of pathophysiological conditions which occurs as a result of oxidative stress during red blood cell hemolysis [76,77] has been used in the treatment of anemia. All these reports culminate to conclusion that more extensive investigations are needed to understand the pathways that can be therapeutically targeted for treating pathological conditions associated with intravascular hemolysis.
Authors thank UNESCO-Regional Centre for Biotechnology, Department of Biotechnology, New Delhi, India for research facilities and financial support. Financial support in form of NPDF from Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India is gratefully acknowledged.
Citation: Gupta S, Bhasym A, Ojha A and Guchhait P. Role of Free Radicals in the Alterations of Immune Responses in Hemolytic Disorders. Ann Clin Exp Immunol. 2017; 1(1): 1001.