The macrophage heme-heme oxygenase-1 system and its role in inflammation

Accepted Manuscript Review The macrophage heme-heme oxygenase-1 system and its role in inflammation Vijith Vijayan, Frank A.D.T.G. Wagener, Stephan Immenschuh PII: DOI: Reference:

S0006-2952(18)30067-4 https://doi.org/10.1016/j.bcp.2018.02.010 BCP 13055

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

7 December 2017 12 February 2018

Please cite this article as: V. Vijayan, F.A.D. Wagener, S. Immenschuh, The macrophage heme-heme oxygenase-1 system and its role in inflammation, Biochemical Pharmacology (2018), doi: https://doi.org/10.1016/j.bcp. 2018.02.010

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The macrophage heme-heme oxygenase-1 system and its role in inflammation Vijith Vijayan 1, Frank A.D.T.G. Wagener 2, Stephan Immenschuh 1# 1

Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany 2 Department of Orthodontics and Craniofacial Biology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, PO-Box 9101, 6500 HB Nijmegen, The Netherlands

#

Corresponding author Stephan Immenschuh, MD Institute for Transfusion Medicine Hannover Medical School Carl-Neuberg-Str. 1, 30625 Hannover, Germany Tel.: +49-511-532-6704/ Fax: +49-511-532-2079 e-mail: [email protected]

Abstract Heme oxygenase (HO)-1, the inducible isoform of the heme-degrading enzyme HO, plays a critical role in inflammation and iron homeostasis. Regulatory functions of HO-1 are mediated via the catalytic breakdown of heme, which is an iron-containing tetrapyrrole complex with potential pro-oxidant and pro-inflammatory effects. The HO reaction produces the antioxidant and anti-inflammatory compounds carbon monoxide (CO) and biliverdin, subsequently converted into bilirubin, along with iron, which is reutilized for erythropoiesis. HO-1 is up-regulated by a plethora of stimuli and injuries in most cell types and tissues and provides salutary effects by restoring physiological homeostasis. Notably, HO-1 exhibits critical immuno-modulatory functions in macrophages, which are a major cell population of the mononuclear phagocyte system. Macrophages play a key role as sentinels and regulators of the immune system and HO-1 in these cells appears to be of critical importance for driving resolution of inflammatory responses. In this review, the complex functions and regulatory mechanisms of HO-1 in macrophages will be high-lighted. A particular focus will be the intricate interactions of HO-1 with its substrate heme, which play a contradictory role in distinct physiological and pathophysiological settings. The therapeutic potential of targeted modulation of the macrophage heme-HO-1 system will be discussed in the context of inflammatory disorders.

I - Introduction

Macrophages are an integral part of the mononuclear phagocyte system, which also encompasses monocytes, dendritic cells and osteoclasts (1). Mononuclear phagocytes share the common function of phagocytosis, but serve distinct roles in different

microenvironments

and

conditions.

Macrophages

are

a

highly

heterogeneous cell population and their regulatory functions are directed by distinct factors such as tissue localization (2). For example, stromal macrophages in the bone marrow support erythrocyte differentiation, whereas macrophages in the brain (microglia) promote neurogenesis. Extensive reviews on tissue-specific functions of macrophages have been given by others (1-3). Along with its tissue-specific functions, macrophages are principal regulators of immune homeostasis. They can either promote inflammation (M1-polarized macrophages) via a Th1 response by the production of pro-inflammatory cytokines and reactive oxygen species (ROS) or inhibit inflammation (M2-polarized macrophages) via a Th2 response by the production of anti-inflammatory cytokines (1-3). Although the M1/M2 concept has been

coined

to

distinguish

various

immunologically

functional

states

of

macrophages, which is defined by the expression of specific marker subsets, the situation in vivo is much more complex and only incompletely reflected by this categorization. For example, in many pathological conditions macrophages express mixed phenotypes or unique features that cannot be explained with the classical concept of M1/M2 polarization. Importantly, macrophages exhibit high plasticity and can adapt to various local needs and conditions that result in specific phenotypes and subsets (3, 4) to ultimately restore homeostasis after inflammation. In various experimental animal models of

inflammatory diseases,

fine-tuning of

the

phenotypical characteristics in macrophages has been shown to promote resolution of inflammation (5, 6). Heme oxygenase (HO)-1 belongs to the multitude of antiinflammatory genes expressed by macrophages, whose primary function is to provide heme homeostasis and to protect against free heme-induced toxicity (7). Interestingly, HO-1 induction in macrophages has been shown to functionally switch these cells to an anti-inflammatory phenotype (8). In this review, we cover the functional role of macrophage-specific HO-1 in physiology and pathophysiology with a particular focus on the complex interactions with its substrate heme. Moreover, we

discuss the possibilities of HO-1-mediated therapeutic strategies to fine-tune macrophage response in inflammatory disorders.

II Heme and the HO system Heme, a complex of iron and protoporphyrin IX, has versatile functions, which are critical for the survival of all aerobic organisms including mammalians and bacteria (9). The central iron in heme facilitates six ligand binding sites (10), four of which are occupied by nitrogen atoms of the tetrapyrrole ring. The remaining two binding sites can be occupied by specific amino acids of proteins (11) or by gases such as oxygen, nitric oxide and carbon monoxide (CO) (12). Heme as the prosthetic group of hemoglobin (Hb) and myoglobin is crucially involved in transport and storage of oxygen, respectively. Moreover, heme serves critical functions in numerous other hemoproteins to mediate fundamental cellular processes such as electron transfer of the respiratory chain, drug metabolism, oxidase and peroxidase enzyme reactions (13, 14). It has been proposed that most mammalian cells may contain a ‘committed’ heme pool where heme is covalently or non-covalently bound to hemoproteins, and a labile ‘regulatory’ heme pool, which is available for trafficking within the cell (15). Thus, specific heme transporters and chaperones are necessary to harness the oxidative properties, while distributing heme to the various cellular apo-hemoproteins (16, 17). Intracellular heme can be acquired via de novo synthesis from glycine and succinyl CoA by multi-enzymatic conversion (18). Because of its redox capability heme is crucial as a prosthetic group in mitochondrial respiratory chain complexes. Alternatively, heme can be exported via the mitochondrial heme exporter feline leukemia virus subgroup C receptor (Flvcr) 1b (19), which allows trafficking within the cell to virtually all other subcellular compartments and organelles via distinct cellular routes (15, 17). Moreover, heme can also be exported from the cell via breast cancer resistance protein and Flvcr1a (16, 20). Heme up-take

from the extracellular

compartment is mediated by heme importers, such as heme carrier protein 1 (HCP1) and Flvcr2. Extracellular heme can be derived from the diet, which is taken up by enterocytes in the intestines via specific transporter molecules, including the heme importer HCP1 (21). A small portion of this heme will be exported from the cell (17). Hemopexin and, to a lesser extent, albumin binds heme and transfers it to other cells and tissues. There are many different heme importers and exporters underscoring the importance of intra- and extra-cellular heme trafficking (16, 17).

In human, approximately 80% of heme is synthesized in erythrocytes, 15% in liver and 5% in non-erythroid cells of other tissues (22). Heme of erythrocytes is ultimately recycled in the reticuloendothelial system of the spleen and liver (23, 24). Notably, HO, which enzymatically degrades heme into equimolar amounts of CO, iron and biliverdin, plays a critical role in this process (25). Biliverdin, in turn is enzymatically converted to bilirubin via biliverdin reductase (26). In physiological conditions the highest HO-activity is detected in cells and tissues, which are involved in the removal of senescent erythrocytes (27-29). Two different isoforms of HO are known: an inducible isoform, HO-1, and a constitutive isoform, HO-2 (30, 31). Interestingly, heme synthesis is intimately intertwined with heme degradation. To meet the high demand of iron for Hb synthesis, erythrocytes are able to utilize iron from HOdegraded heme (32).

III The heme-HO system in macrophages 1. Role of HO-1 in macrophages for heme detoxification and iron recycling Initial evidence for a functional role of HO-1 in iron recycling has come from a HO-1 knockout mouse model, which exhibited signs of anemia and iron overload (33). Subsequently, similar findings have also been reported for a patient with genetic HO1 deficiency (34). Remarkably, HO has been shown to exhibit constitutively high levels of activity and gene expression in liver (35-37) and spleen (29, 38) tissue macrophages under physiological conditions. More detailed studies in genetically HO-1 deficient mice indicated that lack of HO-1 renders macrophages sensitive to cell death following erythrocyte exposure in vitro, which may explain the loss of tissue macrophages in liver and spleen of these animals (39). Consistent with this notion, bone marrow transplantation led to a repopulation of these tissues with wildtype macrophages, particularly in the liver and rescued iron reutilization defects (40). Furthermore, heme-induced HO-1 activity in monocytes has also been shown to regulate the iron exporter ferroportin, which plays a pivotal role for iron recycling (41). Hence, HO-1 activity in tissue-specific macrophages appears to play a fundamental role in iron recycling and erythropoiesis. In pathophysiological conditions, high levels of free heme released from cell-free Hb, myoglobin or other intracellular hemoproteins, can have detrimental effects. The central iron atom of the heme molecule can cause excess ROS production via Fenton chemistry. Moreover, heme has potential cytotoxic effects via lipid peroxidation, protein cross-linking and DNA damage (14, 42). In particular, the endothelial layer of blood vessels is highly sensitive to pro-inflammatory activation (18, 43) and toxicity (44) by heme. Mammalians possess specific protective mechanisms to counteract the toxicity of Hb and heme via the serum proteins haptoglobin (Hp) and hemopexin (Hx) (7, 45-47). Hp binds cell-free Hb, which is released from hemolytic erythrocytes and after exhaustion of Hp binding capacity, excess free heme is scavenged by Hx (48). Interestingly, plasma levels of Hp and Hx have been shown to inversely correlate with mortality in sepsis patients (49, 50).

HO-1 in macrophages appears to play an important role for the clearance of Hb-Hp and heme-Hx complexes (51). Hb-Hp complexes are specifically taken up by monocytes (52) and macrophages via a CD163-mediated receptor endocytosisdependent mechanism (53-55) (depicted in Figure 2). By contrast, heme-Hx complexes are taken up via LRP1/CD91, the expression of which is not restricted to macrophages (56). In turn, sequestered heme is enzymatically degraded via HO-1 in macrophages into the protective molecules CO and biliverdin/bilirubin (51). Moreover, Hb-Hp complex-dependent up-regulation of HO-1 activity is linked to an increased expression of ferritin in macrophages (53) to protect against iron-induced toxicity. A previous report, which showed that increased expression of Hp and Hx in liver and kidney (39) was not sufficient to protect HO-1 knockout mice against hemeinduced toxicity, might also be in line with this notion. In summary, HO-1 in macrophages may act as a heme degrading funnel, which is utilized by the serum proteins Hp and Hx to remove free Hb and heme from the circulation whilst mediating beneficial effects with the heme degradation products. 2. Immunomodulatory role of HO-1 in myeloid cells In 1996 Willis et al, reported that induction of HO enzyme activity protects against inflammation,

whereas

inhibition

of

HO

activity

exacerbated

experimental

inflammation in an animal model of carrageenin-induced pleurisy (57). Observations in HO-1 knockout mice (33) and in patients with human genetic HO-1 deficiency (34, 58, 59) corroborated a major immunomodulatory role for the inducible HO isoform, HO-1. Subsequently, specific immuno-regulatory functions of HO-1 have been demonstrated in vitro in cell cultures of HO-1 deficient splenocytes, which exhibited increased

secretion

of

pro-inflammatory

cytokines

upon

activation

with

lipopolysaccharide (LPS) (60) and in human peripheral blood mononuclear cells containing a loss-of-function mutation of HO-1 (61). The importance of myeloidspecific HO-1 expression has also been shown in mice with conditional ablation of HO-1 in myeloid cells (62). These animals were prone to viral attacks and exacerbated autoimmune responses due to lack of interferon-β-mediated antiviral responses (62). Interestingly, various studies have reported that the antiinflammatory effects of IL-10 in macrophages appear to be mediated via induction of HO-1 (51, 63). However, the lack of tools to detect HO-1 enzyme activity in real-time has made it difficult to understand the importance of enzyme-dependent and -

independent functions of HO-1. The majority of the immunomodulatory properties of HO-1 protein can be mimicked by using the enzymatic byproducts CO and bilirubin (29, 64), which would implicate a critical role for HO-1 activity. Enzymatically inactive HO-1 can still mediate protection against hydrogen peroxide-induced toxicity (65) suggesting enzyme-independent functions of HO-1. HO-1 can be localized in the nucleus, where it does not seem to exhibit enzymatic activity, but can mediate transactivation of protective pathways (66, 67). Finally, independent reports have indicated that HO-1 induction polarizes macrophages into an anti-inflammatory phenotype (M2 polarization (reviewed in (8)). Collectively, these findings suggest that HO-1 is a critical mediator of the innate immune response and that the therapeutic potential of HO-1 may depend on both the enzymatic activity and protein expression of HO-1. It is important to note that up-regulation HO-1 in macrophages may not always be beneficial. For example, conditional deletion of HO-1 in myeloid cells has been shown to be associated with protection against insulin resistance in mice (68). Similarly, the effects of HO-1 induction in infectious diseases are conflicting and are only incompletely understood. On the one hand, HO-1 derived CO appears to provide protection via promoting bacterial clearance by increased macrophage bactericidal activity against bacterial species such as Eschericia coli, Enterococcus faecalis and Salmonella typhimurium (69) and to also promote phagocytosis of some strains of bacteria (70). Although bilirubin suppresses killing of bacteria by reducing the neutrophil burst via its antioxidant activity (71), it also reduces the viability of gram-positive bacteria by disrupting its membrane (72). Moreover, in polymicrobial sepsis HO-1 provides disease tolerance by promoting host survival without restricting the bacterial load (73). On the other hand, HO-1 induction has been linked to intracellular survival of bacterial pathogens such as Salmonella typhimurium (74), Mycobacterium abscessus (75) via its ROS-diminishing properties or reduced inflammatory cytokine production as in the case of Mycobacterium tuberculosis (76). Similarly, HO-1 activity can increase the availability of iron, which blocks the phagolysosomal fusion needed for pathogen killing and may also serve as a nutrient for bacterial growth (75, 77). A dual nature of HO-1 in infectious disorders has also been shown in malaria, in which HO-1 induction may promote infection in the liver stage (78), but may also provide tolerance in the blood stage of the disease (79, 80).

Anti-inflammatory effects of HO-1 induction may also be detrimental in infections with pathogens that favor an M2 environment such as Fasciola hepatica (81). Moreover, the role of myeloid HO-1 in tumors appears to be contradictory and further studies are necessary (82) 3. Heme-dependent immunomodulatory changes in macrophages The ideal mode to induce both enzymatic activity and protein expression of HO-1 appears to be the application of its substrate heme. However, exposure of macrophages to heme can activate these cells and the functional phenotype of activated macrophages is ambiguous. Reportedly, free heme may act via toll-like receptor (TLR)-4 to induce expression of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α (83, 84), but, details of the underlying mechanism are not clear (85). In bone marrow-derived murine macrophages heme amplifies LPSdependent inflammatory responses (86) and activates the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome (87, 88). Heme- and irondependent pro-inflammatory effects in macrophages are mediated via ROS, which lead to a pro-inflammatory (M1) phenotype (84). Interestingly, scavenging of heme by Hx reverts proinflammatory activation of macrophages by this molecule in a murine sickle cell disease model (84). Recently, it was also shown that heme can potently inhibit macrophage phagocytosis of bacteria by disrupting cytoskeletal dynamics (89) and that it can inhibit interferon-γ responses in THP-1 monocytic cells (90), which disturbs the immune effector functions of these cells. By contrast, it has also been demonstrated that exposure of macrophages to heme can lead to an antiinflammatory (M2) phenotype. Specifically, heme may also induce the specialized anti-inflammatory phenotype M-hem in human peripheral-blood derived primary macrophages, which is distinct from the classical M2 phenotype (91, 92). M-hem macrophages are characterized by increased intracellular iron levels, up-regulated HO-1 and IL-10 expression along with decreased inflammatory activation and have been proposed to be protective against atherosclerosis (91). Similarly, hemeactivated murine macrophages appear to have functional anti-inflammatory features such as increased phagocytosis, decreased NO production and increased arginase activity (93). Importantly, such heme-associated anti-inflammatory properties are dependent on the enzymatic activity of HO-1. These contradictory observations may be due to different experimental settings such as the presence/absence and

concentration of the serum proteins Hx and albumin that can modulate the effects of heme (73) (94). In summary, in vitro findings on heme-mediated activation of macrophages are contradictory and further studies are required to clarify these ambiguities. Noteworthy, Hb-Hp complexes have been shown to induce an antiinflammatory phenotype in macrophages (53). This may indicate that abrogation of pro-oxidant properties of heme in heme-protein complexes may serve as a novel therapeutic strategy to induce HO activity.

IV Regulation of HO-1 gene expression HO-1 is transcriptionally up-regulated by a plethora of stimuli, which activate numerous signaling cascades and transcriptional factors (TFs). TFs such as AP-1 and NF-kB, which are critical for stress- and inflammation-dependent gene induction, along with a number of other nuclear factors have been shown to activate HO-1 gene expression (95-100). The complex mechanisms how these TFs act in concert to up-regulate HO-1 for a given stimulus have been reviewed elsewhere (29, 101103). Second messengers such as nitric oxide (NO) (104) and the prostaglandin 15deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) can also induce HO-1 gene expression in macrophages (105). In the following, a brief summary on the basic principles of HO-1 gene regulation via its substrate heme and the specific role of the mutual interplay of the nuclear factor NFE2-related factor 2 (NRF2) and BTB and CNC homology 1 (BACH1) will be given. However, it should be pointed out that hememediated induction of HO-1 in macrophages is not restricted to the NRF2/BACH1 system, but the activating transcriptional factor 1 (ATF1) has also been implicated in this regulation (91). Heme activates the master regulator of the anti-oxidant stress response, NRF2, which mediates the up-regulation of a battery of phase II detoxifying genes (106). Remarkably, HO-1 induction by NRF2 is regulated via interplay with the transcriptional repressor BACH1 at the Maf recognition element (MARE) in the HO-1 promoter. Heme binds to cysteine-proline motifs of BACH1, which then leads to its nuclear export and proteasomal degradation (107, 108). In parallel, Nrf2 translocates into the nucleus and binds to the BACH1 free MARE to induce transcription. Notably, inactivation of BACH1 is sufficient to up-regulate HO-1 gene expression (109-111) and inactivation of BACH1 by heme also modulates expression of the iron-regulatory

genes ferritin and ferroportin (112, 113). Similarly, heme-protein complexes such as heme-Hx have been shown to induce HO-1 in macrophages via BACH1 regulation (114). Mechanisms that control BACH1 inactivation by non-heme inducers of HO-1 are less well understood, but HO-1 inducers such as arsenite and cadmium have been shown to regulate the nuclear export of BACH1 (111, 115). It is plausible that mechanisms of BACH1 export independent of heme binding are involved in this regulation (116-118). In conclusion, HO-1 is specifically regulated via inactivation of the heme-dependent nuclear repressor BACH1. The potency of putative selective pharmacological HO-1 inducers may be tested for their ability to inactivate BACH1mediated repression.

V Species-specific differences in HO-1 gene regulation Species-specific differences are known for the regulation of various TLR4-dependent inflammatory genes in mouse and human macrophages (119). Similarly, interspecies differences also apply for the regulation of HO-1 in response to various stimuli such as heat-shock (120), interferon- (121), hypoxia (122) and LPS (123) in mouse and human, which may be critical for the translation of experimental findings in mice to clinical applications. The complexity of HO-1 regulation has been further increased by cell-type-specific regulatory HO-1 gene expression patterns in humans (124, 125) Heme-mediated induction of HO-1 requires two binding sites in the distal promoters of the mouse and human genes, but the human promoter requires an additional enhancer element for the up-regulation of HO-1 gene (126, 127). Notably, a major factor for potential therapeutic strategies involving HO-1 is the presence of a microsatellite polymorphism of (GT)n repeats in human, but not rodent HO-1 promoter (128). Longer (GT)n repeats are associated with reduced up-regulation of HO-1 and increased susceptibility to various clinically relevant conditions, particularly in cardiovascular diseases (129). Interestingly, heme arginate infusion has been shown to induce HO-1 protein expression in human volunteers irrespective of the (GT)n polymorphism (130). Similarly, injection of heme-albumin complexes also upregulated HO-1 activity in healthy human volunteers, although the (GT)n

polymorphism was not considered in this study (131). Hence, a potentially ideal pharmacological HO-1 inducer could be its substrate heme.

VI Heme-HO-1 system in macrophages as a target for therapeutic applications Inflammation and oxidative stress play a critical role in the pathogenesis of numerous pathological conditions including cardiovascular or neurological disorders such as atherosclerosis and Alzheimer’s disease. Therefore, anti-inflammatory and antioxidant strategies appear to hold major promise as therapeutic applications in such conditions. It should be noted, however, that numerous strategies, which seemed to be favorable in animal models, could not be translated into the clinic. For example, treatment with antioxidant vitamins turned out to be inefficient in clinicial trials (132, 133) and it is conceivable that antioxidant molecules may not reach their indicated target site (134). Due to the critical role for macrophages in the regulation of inflammation and the major anti-inflammatory potential of HO-1 in these cells, various approaches that apply the heme-HO system in macrophages as a therapeutic target will be discussed in the following. Macrophage-based cell therapy Autologous cell therapies with macrophages are intended to generate specific effector cell populations ex vivo from different sources, which are then readministered to the host (Figure 1) (135). Clinical trials using autologous cells appear to be promising, because only few side effects have been reported (135). Macrophage-based cell therapies, in which HO-1 expression has been specifically modulated, could be attractive for treatment of disorders with exacerbated or chronic inflammation. To this end, heme-based preparations such as heme arginate and heme particles have been applied for specific induction of HO-1 (136-138). Moreover, pharmacological compounds such as adiponectin through an IL-10dependent mechanism or 4-2(2-aminoethyl)-benzenesulfonyl fluoride have been shown to induce macrophage HO-1 in a cell-type specific manner (139, 140). Independently, phytochemicals including curcumin and quercetin up-regulate HO-1 and the effects of such compounds have been studied in detail for potential clinical

applications (134, 141). A limitation of these pharmacological approaches could be the sustainability and duration of HO-1 up-regulation in vivo, as previously demonstrated for the application of heme arginate (137). Alternatively, HO-1 may be targeted via gene therapy. Although in vivo gene therapy may only become feasible on a large scale when tissue/cell specific HO-1 promoters will be available such approaches may be feasible ex vivo in specified cell populations. Accordingly, adoptive transfer of ex vivo modified HO-1-overexpressing macrophages has been shown to prevent inflammation in a model of ischemia/reperfusion injury in wild-type and Nrf2 knockout mice (142, 143). Independently, HO-1 can be specifically induced in macrophages in vivo. Given the current limitations of our understanding on the complex regulation and effector functions of HO-1, it is likely that both enzyme-dependent and -independent functions are responsible for the beneficial effects of this enzyme. Thus, application of (a) heme-based carrier(s) for specific HO-1 induction rather than a non-heme HO1 inducer might be more feasible for therapeutic applications. Along this line, heminmediated induction of HO-1 in macrophages has been shown to have salutary effects in an experimental mouse model of pancreatitis (144). Remarkably, however, administration of free heme may have harmful side effects and is not targeted to a specific cell-type. Thus, heme preparations with specific proteins or liposomes might be a feasible approach for targeted induction of HO-1 in macrophages and various strategies are discussed in the following. Hb-Hp complex CD163, which is a scavenger receptor for Hb-Hp, is also known as a biomarker for anti-inflammatory macrophages. Specific HO-1 induction in such macrophages can be achieved via administration of the Hb-Hp complex, mechanistic-details of which are discusses previously (Figure 2). It is remarkable that Hb bound Hp does not exert pro-oxidant properties (145) and has been shown to induce the expression of HO-1 and related anti-inflammatory genes (53, 146, 147). Heme-Hx complex Up-take of the heme-Hx complex can induce HO-1 expression in both cultured monocytes (56) and THP1-derived macrophages (114)

by receptor-mediated

endocytosis as discussed above (Figure 2). Additionally, Hx unlike Hp is not

degraded after its uptake (45) and Hx alone has been shown to exert antiinflammatory effects in macrophages (148, 149). Hence, heme-Hx therapy might be beneficial via both heme-induced HO-1 activity and anti-inflammatory effects of Hx. Liposomal-mediated delivery of heme Drug delivery systems that apply liposomes as carriers have been approved by the US Food and Drug Administration (FDA), and might become applicable for targeted therapies with heme. In a recent study, Ben-Mordechai et al, tested the efficacy of such an approach using heme in an experimental model of myocardial infarction. A hyaluronan lipid-based heme carrier targeted macrophages of the affected heart tissue and caused phenotypical switching of these cells into an anti-inflammatory phenotype, thereby improving ventricular remodeling and infarct repair. Although, this lipid-based heme carrier induced HO-1 in macrophages in vitro, the specific role for HO-1 in the protective effects has not been shown in this study (138). Finally, it is important to note that targeted up-regulation of HO-1 failed to exert beneficial effects if induced after the onset of the disease in experimental animal models of inflammation (144, 150). Therefore, the therapeutic potential of HO-1 to mediate resolution of inflammation needs further scrutiny in clinical settings and additional studies will be required. Clearly, targeted induction of HO-1 might have its greatest therapeutic potential in clinical situations such as solid organ transplantation (151), in which a prophylactic approach is required.

Acknowledgments ° Work in SI’s laboratory is supported by grant IM 20/4-1 from the Deutsche Forschungsgemeinschaft, Bonn (Germany) and grant EKFS 2012_A309 from the Else Kröner Fresenius Stiftung, Bad Homburg (Germany). FW’s group is supported by the Dr Vaillant Foundation and the Dutch Burns Foundation (P16.03)

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Figure legends Figure 1: HO-1 modulated anti-inflammatory macrophages as therapeutic approach Macrophages obtained from the indicated sources can be treated ex vivo with the various pharmacological inducers of HO-1 or can be genetically engineered to overexpress HO-1 leading to the polarization of these cells into anti-inflammatory macrophages. These cells by virtue of their regenerative capacity and IL-10 secretion can restore homeostasis.

Figure 2: Specific induction of HO-1 in macrophages via application of haptoglobin (Hb) and hemopexin (Hx) Application of heme-Hx and/or Hb-Hp complexes may be applicable to treat nonhemolytic conditions in which HO-1 up-regulation is desired. Hb-Hp complexes specifically target CD163 positive macrophages in organs such as spleen, liver and kidney. These macrophages take up Hb-Hp complex via receptor-mediated endocytosis and heme in Hb is provided for specific degradation via HO-1. Heme-Hx may also be incorportated into macrophages via CD91/LRP1 receptor-mediated degradation. This mechanism, however, is not specific because various other celltypes also express CD91/LRP1. The application of heme-based carriers efficiently blocks free heme-dependent generation of ROS and induce HO-1 expression, which in turn, leads to polarization of these macrophages to M2. Moreover, the enzymatic degradation of heme by HO-1 produces CO and bilirubin which exerts additional anti-inflammatory and anti-oxidant properties, respectively.