Heme Oxygenase-1 in Macrophages Drives Septic Cardiac Dysfunction via Suppressing Lysosomal Degradation of Inducible Nitric Oxide SynthaseNovelty and Significance
Rationale: To date, our understanding of the role of HO-1 (heme oxygenase-1) in inflammatory diseases has mostly been limited to its catalytic function and the potential for its heme-related catabolic products to suppress inflammation and oxidative stress. Whether and how HO-1 in macrophages plays a role in the development of septic cardiac dysfunction has never been explored.
Objective: Here, we investigated the role of macrophage-derived HO-1 in septic cardiac dysfunction.
Methods and Results: Intraperitoneal injection of lipopolysaccharide significantly activated HO-1 expression in cardiac infiltrated macrophages. Surprisingly, we found that myeloid conditional HO-1 deletion in mice evoked resistance to lipopolysaccharide-triggered septic cardiac dysfunction and lethality in vivo, which was accompanied by reduced cardiomyocyte apoptosis in the septic hearts and decreased peroxynitrite production and iNOS (inducible NO synthase) in the cardiac infiltrated macrophages, whereas proinflammatory cytokine production and macrophage infiltration were unaltered. We further demonstrated that HO-1 suppression abolished the lipopolysaccharide-induced iNOS protein rather than mRNA expression in macrophages. Moreover, we confirmed that the inhibition of HO-1 promoted iNOS degradation through a lysosomal rather than proteasomal pathway in macrophages. Suppression of the lysosomal degradation of iNOS by bafilomycin A1 drove septic cardiac dysfunction in myeloid HO-1–deficient mice. Mechanistically, we demonstrated that HO-1 interacted with iNOS at the flavin mononucleotide domain, which further prevented iNOS conjugation with LC3 (light chain 3) and subsequent lysosomal degradation in macrophages. These effects were independent of HO-1’s catabolic products: ferrous ion, carbon monoxide, and bilirubin.
Conclusions: Our results indicate that HO-1 in macrophages drives septic cardiac dysfunction. The mechanistic insights provide potential therapeutic targets to treat septic cardiac dysfunction.
Sepsis is a systemic inflammatory response that is accompanied by excessive production of inflammatory cytokines, oxidative stress, and multiple organ dysfunction.1–3 Lipopolysaccharide (LPS)—a Gram-negative bacterial endotoxin—is known to be a central mediator of sepsis and sepsis-related multiple organ dysfunction or death.4–6 Cardiac dysfunction is a common complication of sepsis.7,8 The incidence of cardiac dysfunction is as high as 60% during the first 3 days of admission for septic shock and is associated with high mortality in patients.9
Meet the First Author, see p 1480
Increasing evidence suggests that septic cardiac dysfunction is mediated by multiple proinflammatory mediators that act directly or indirectly to cause cardiac injury.3 During sepsis, macrophages are prominent immune cells in the response to LPS. These cells play important roles in the initiation and resolution of inflammation.10,11 In addition to the proinflammatory cytokines, iNOS (inducible NO synthase) is activated and highly expressed in activated macrophages, in which it generates high amounts of NO. iNOS-derived NO reacts with another free radical, superoxide, to form peroxynitrite, which leads to severe oxidative stress and inflammatory response.12,13
HO-1 (heme oxygenase-1)—an enzyme that catalyzes the degradation of heme—is ubiquitously expressed in various cell types, including macrophages and cardiovascular cells, and has traditionally been considered to be protective against inflammatory response and oxidative injury in the past decades.14 It has been shown that LPS induces HO-1 expression, which limits the cytokine production by macrophages.15 HO-1 induction or administration of heme catabolites has been found to have antioxidant effects in cardiovascular disease states, such as ischemia–reperfusion injury, neointimal hyperplasia, and spontaneous hypertension in rats.16 However, a recent study reported that the specific deletion of HO-1 in macrophages limited the inflammatory responses in the liver and fat tissues, thereby mitigating insulin resistance in mice fed a high-fat diet.17
Because macrophages are central mediators of septic cardiac dysfunction, we aimed to directly investigate the role of HO-1 in these cells. Using conditional mouse genetics, we found that, contrary to our expectations, HO-1 loss of function in macrophages led to resistance to LPS-induced cardiac dysfunction. The results indicate that HO-1 in macrophages is necessary for the development of septic cardiac dysfunction and call for a reevaluation of numerous findings in the field.
The authors declare that all supporting data are available within the article and its Online Data Supplement.
The detailed methods are available in the Online Data Supplement.
HO-1 Is Activated in Murine Septic Hearts
To evaluate the potential role of HO-1 in septic cardiac dysfunction, we investigated the levels of HO-1 and the infiltration of macrophage in LPS-induced cardiac dysfunction. We injected the WT (wild type) mice intraperitoneally with either LPS or an equal volume of PBS. The survival rate was monitored every 3 hours for a 60-hour period, and cardiac function was assessed by echocardiography 12 hours after injection. As expected, LPS injection lead to a significant decrease of cardiac function and caused a significantly lower survival rate in mice (Figure 1A through 1C).
We further investigated the levels of HO-1 in the hearts. Western blot detection and immunofluorescence staining revealed increased levels of HO-1 expression and macrophage infiltration in the LPS-induced failing hearts (Figure 1D and 1E), which paralleled the increased HO-1 activity in the hearts of the LPS-treated animals (Online Figure IA). Of note, HO-1 was particularly evident in the inflammatory macrophages rather than in the cardiomyocytes (Figure 1E; Online Figure IIA). These data suggested that myeloid HO-1 is an effector of LPS-induced septic cardiac dysfunction.
Myeloid Cell Deficiency of HO-1 Resists Septic Cardiac Dysfunction
On the basis of the observed increases in HO-1 in the macrophages in LPS-induced cardiac dysfunction and the central role of macrophages in sepsis, we next tested the contribution of macrophage HO-1 toward septic cardiac dysfunction. To this end, we crossed conditional HO-1fl/fl mice to LysM-Cre transgenic mice, creating myeloid cell-specific HO-1 knockout mice (HO-1ΔMΦ). Littermates not carrying the LysM-Cre transgene (HO-1fl/fl) served as controls. The deletion efficiency was confirmed in peritoneal macrophages (Online Figure XIVB and XIVC).
Interestingly, and in contrast to our initial expectations, we observed markedly improved cardiac function in the HO-1ΔMΦ mice compared with the HO-1fl/fl mice 12 hours after the LPS injection (Figure 2A and 2B; Online Table II). Furthermore, the HO-1ΔMΦ mice were more resistant to the LPS-caused lethality compared with the controls (Figure 2C). Cardiac function and survival rates were equally unremarkable after PBS injection.
Because calcium homeostasis is important to the maintenance of cardiac function, we next examined the expression of SERCA2a (sarco-endoplasmic reticulum calcium adenosine triphosphatase 2a) and RyR2 (ryanodine receptor 2) mRNA, which are 2 genes that are important in calcium homeostasis.18,19 Quantitative real-time polymerase chain reaction analysis showed a significant reduction in the SERCA2a and RyR2 mRNA expression in the hearts of the LPS-challenged mice. However, HO-1 deletion in the myeloid cells did not restore the levels of SERCA2a and RyR2 mRNA under LPS challenge compared with the HO-1fl/fl mice (Online Figure IIIA and IIIB). ATP production is one of the essential functions of mitochondria, and mitochondrial dysfunction is associated with many cardiac pathologies, including heart failure.20 We, therefore, assayed the intracellular ATP levels to evaluate the bioenergenic activities of mitochondria in hearts. The result showed that HO-1 deletion in the myeloid cells significantly ameliorated the reduced levels of intracellular ATP in the LPS-challenged heart tissues (Online Figure IIIC). We also measured the NF-kB (nuclear factor-kappa B) and JNK (c-Jun N-terminal kinase) signaling activation in heart tissues. The expression of P-P65 (phosphorylated-P65) and P-JNK (phosphorylated-JNK) was significantly enhanced in hearts of LPS-challenged HO-1ΔMΦ mice. Myeloid cell-specific HO-1 deficiency markedly suppressed the P-P65 activated by LPS but had no effect on the induction of P-JNK by LPS (Online Figure IVA).
We also evaluated the blood pressure using hemodynamic measurements. LPS treatment significantly reduced the systolic arterial pressure and diastolic arterial pressure in the HO-1fl/fl mice (Online Figure VA and VB). However, compared with the LPS-challenged HO-1fl/fl mice, both systolic arterial pressure and diastolic arterial pressure were unaltered in the LPS-injected HO-1ΔMΦ mice (Online Figure VA and VB). The data suggest that the protective effect of myeloid HO-1 deficiency on LPS-induced septic cardiac dysfunction is blood pressure independent. Thus, HO-1 deletion in myeloid cells prevented LPS-induced cardiac dysfunction and subsequent lethality.
Protective Effect of HO-1 Deletion in Myeloid Cells on Septic Cardiac Dysfunction Is Independent of Alterations in the Macrophage Infiltration and Inflammatory Cytokine Production
LPS induces a proinflammatory state and the accumulation of macrophages in systemic circulation and regional heart tissues, which results in impaired cardiac function.5,21 We determined whether myeloid HO-1 ablation altered cardiac macrophage infiltration on septic cardiac dysfunction. Immunofluorescence and flow cytometric analyses of the macrophage markers showed no significant difference between the LPS-challenged HO-1ΔMΦ and HO-1fl/fl mice in the numbers of the infiltrating macrophages in hearts (Figure 3F and 3G; Online Figure VIA and VIB).
We further assayed the proinflammatory cytokine expression. The analyses revealed that the levels of TNF-α (tumor necrosis factor-α), IL (interleukin)-6, IL-1β, and IL-12b mRNA in the hearts were not significantly different between the LPS-challenged HO-1ΔMΦ and HO-1fl/fl mice, despite a slight decrease in the expression of IL-12a mRNA in the LPS-challenged HO-1ΔMΦ mice (Online Figure VIIA through VIIE). Similarly, the analyses revealed no significant differences in the levels of TNF-α, IL-6, IL-1β, IL-12a, and IL-12b mRNA in the peritoneal macrophages (Online Figure VIIIA through VIIIE). Moreover, the ELISA assay showed no significant differences in the TNF-α, IL-6, IL-1β, and IL-12 serum cytokine levels between the LPS-challenged HO-1ΔMΦ and HO-1fl/fl mice (Online Figure IXA through IXD). Thus, the protective effect of HO-1 myeloid deficiency on septic cardiac dysfunction was independent of the regulation of the macrophage infiltration and proinflammatory cytokine expression.
Decreased Cardiac Peroxynitrite Production and Cardiomyocyte Apoptosis in Septic Myeloid HO-1–Deficient Mice
The general literature would have predicted enhanced oxidative stress on HO-1 deletion.22–25 Because no significant differences in the levels of proinflammatory cytokines between LPS-challenged HO-1ΔMΦ and control mice, we next assayed the levels of superoxide and peroxynitrite, which is a powerful oxidant that is formed by the reaction between superoxide with the free radical NO. LPS injection led to a significant production of cardiac superoxide as indicated by dihydroethidium staining (Online Figure XA and XB) and 3-nitrotyrosine, which is a stable detectable molecule in hearts as indicated by immunofluorescence staining and Western blot (Figure 3A and 3B). In strong contrast to our expectation, the LPS-challenged HO-1ΔMΦ mice exhibited a significantly lower level of 3-nitrotyrosine in the hearts than the HO-1fl/fl mice (Figure 3A and 3B). The dihydroethidium staining was not significantly different between the LPS-treated HO-1ΔMΦ and HO-1fl/fl mice (Online Figure XA and XB).
Excessive peroxynitrite mediates the NO-dependent oxidative stress and cell damage.13,26 Consistent with the reduced cardiac nitrotyrosine in the HO-1ΔMΦ mice, the expression of cleaved caspase-3 protein in these mice was significantly lower than that of the controls (Figure 3E), which indicated attenuated myocardial apoptosis. This effect was further confirmed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling) staining, which showed that the HO-1ΔMΦ mice showed a robust reduction of cardiomyocyte apoptosis (Figure 3C and 3D). Thus, HO-1 in myeloid cells exaggerated the septic myocardial damage by promoting peroxynitrite but not superoxide production in vivo.
Myeloid Cell Ablation of HO-1 Limits the Induction of iNOS Protein Rather Than Its mRNA in Septic Hearts
Next, we asked how HO-1 elicits peroxynitrite generation. It has been reported that iNOS is activated and generates a massive amount of NO in macrophages during sepsis.27,28 We, therefore, measured the levels of iNOS in septic hearts. As indicated by immunofluorescence staining and Western blotting, iNOS was significantly augmented in LPS-induced failing hearts in the HO-1fl/fl mice (Figure 3F and 3I). However, consistent with the decreased level of 3-nitrotyrosine, we observed a profound reduction of the cardiac iNOS protein induction in the HO-1ΔMΦ mice compared with the HO-1fl/fl mice (Figure 3F and 3I). Furthermore, double immunofluorescent staining of CD68 and iNOS indicated that iNOS was predominantly expressed in the infiltrated macrophages rather than in the cardiomyocytes (Figure 3F and 3H; Online Figure XIA). Hence, the reduction of iNOS in macrophages was responsible for the decrease in the peroxynitrite level in HO-1ΔMΦ mice. We further measured the mRNA levels of iNOS, as well as eNOS (endothelial NO synthase) and nNOS (neuronal NO synthase), which are 2 additional forms of NOS that can generate NO in hearts. As indicated in Online Figure XIIA, LPS significantly induced iNOS mRNA expression but had no effect on eNOS. nNOS was not detectable in hearts. In strong contrast to our expectation, unlike the dramatic reduction in protein levels, quantitative real-time polymerase chain reaction analysis of heart tissues revealed no significant difference in the mRNA expression of iNOS between the LPS-challenged HO-1ΔMΦ and HO-1fl/fl mice (Online Figure XIVA). Collectively, myeloid cell-specific HO-1 knockout blunted the LPS-induced iNOS protein but not mRNA expression in the cardiac infiltrated macrophages in vivo.
iNOS Inhibition Alleviates Septic Cardiac Dysfunction and Myocardial Damage In Vivo and In Vitro
We applied S-methylisothiourea sulfate—an inhibitor of iNOS—to confirm the role of iNOS in the LPS-induced septic cardiac dysfunction. As shown in Online Figure XIIIA through XIIIC, LPS significantly decreased the cardiac systolic function and induced the expression of cardiac cleaved caspase-3. These effects could be abolished by S-methylisothiourea sulfate treatment.
We further examined whether iNOS in macrophages was crucial for cardiomyocytes damage in vitro. LPS did not significantly induce cleaved caspase-3 expression in H9c2 cells (Online Figure XIIID). However, when cocultured with bone marrow-derived macrophages (BMDM), LPS markedly increased cleaved caspase-3 expression in H9c2 cells, indicating that macrophages are essential in the mediation of LPS-induced cardiomyocyte injury (Online Figure XIIID). We further found that S-methylisothiourea sulfate pretreatment of the BMDM could significantly suppress the LPS-triggered cleaved caspase-3 expression in the H9c2 cells in our BMDM/H9c2 coculture system (Online Figure XIIIE). In summary, we demonstrated that iNOS inhibition in macrophages could alleviate septic cardiac dysfunction and myocardial damage.
HO-1 Suppression Inhibits LPS-Induced iNOS Protein but Not mRNA Expression in Macrophages
Because LPS-challenged HO-1ΔMΦ mice exhibited reduced iNOS protein but not mRNA levels in the cardiac infiltrated macrophages, we further assayed this phenomenon in peripheral macrophages. We collected the peritoneal macrophages from the LPS-injected mice. Consistent with the results in septic hearts, we observed significantly decreased levels of the protein rather than the mRNA levels of iNOS in the peritoneal macrophages of the HO-1ΔMΦ mice compared with the HO-1fl/fl mice (Online Figure XIVB through XIVD). Moreover, ablation of HO-1 in myeloid cells remarkably suppressed the levels of nitrite, which indicated lower levels of NO generation, in the sera of the LPS-exposed HO-1ΔMΦ mice than those of the HO-1fl/fl mice (Online Figure XIVE).
Next, we attempted to confirm our in vivo findings in vitro. As expected, HO-1 silencing significantly decreased iNOS protein expression in BMDM that were treated with LPS compared with a nontargeted scrambled siRNA (Figure 4A and 4B). Consistent with the iNOS protein expression, the NO production indicated by the nitrite levels in cell culture media was significantly decreased in the HO-1–silenced group compared with the scrambled siRNA-transfected group after LPS stimulation (Figure 4D). However, HO-1 silencing did not significantly reduce the LPS-induced iNOS mRNA expression in BMDM (Figure 4C). Additionally, we verified those findings using a known chemical inhibitor of HO-1, zinc protoporphyrin IX (ZnPPIX). Similarly, LPS-induced iNOS protein levels and nitrite levels, rather than mRNA expression, were significantly decreased after incubation of ZnPPIX (Figure 4E through 4H). In summary, we demonstrated that HO-1 suppression inhibited the LPS-induced iNOS protein expression independent of alterations in the iNOS mRNA expression in macrophages.
HO-1 Inhibition Leads to iNOS Protein Instability and Lysosomal Degradation in Macrophages
As described above, HO-1 regulated the LPS-stimulated iNOS protein but not mRNA expression in macrophages. These results suggested that post-translational mechanisms may be involved. We examined whether HO-1 affected the protein stability of iNOS. LPS-stimulated RAW 264.7 cells were treated with the protein synthesis inhibitor cycloheximide in the presence or absence of ZnPPIX. As depicted in Figure 5A, ZnPPIX significantly accelerated the reduction in the iNOS levels, which suggested that HO-1 inhibition markedly reduced the iNOS stability.
Next, we tested whether the decrease in the iNOS protein levels that was associated with HO-1 suppression was because of an enhanced degradation of iNOS. The iNOS reduction after ZnPPIX treatment or HO-1 silencing in LPS-induced RAW 264.7 cells was not blocked by the proteasome inhibitors, MG132 and PR-11, but was suppressed by the lysosome inhibitors, chloroquine and bafilomycin A1 (BafA1; Figure 5B through 5E; Online Figure XVA), which indicated that HO-1 inhibition enhanced the iNOS lysosomal degradation in macrophages. We further performed confocal microscopy and found that iNOS colocalized with the lysosomal marker LAMP-1 in LPS-incubated RAW 264.7 cells after ZnPPIX treatment (Figure 5F). Collectively, these data implied that HO-1 suppression mediated the lysosome-dependent degradation of iNOS.
HO-1–Regulated iNOS Protein Expression Is Not Through Its Metabolites Ferrous Ion, Carbon Monoxide, and Bilirubin in Macrophages
HO-1 catalyzes the degradation of heme with the release of ferrous ion, carbon monoxide, and bilirubin. We questioned whether these metabolites of HO-1 catalysis might be responsible for the regulation of the iNOS protein by HO-1. To test this, we exposed RAW 264.7 cells to ferric ammonium citrate as a ferrous ion source, the carbon monoxide-releasing molecule-3 or bilirubin, and examined the iNOS protein expression. We observed that supplementation with ferrous ion, carbon monoxide-releasing molecule-3, or bilirubin did not significantly restore the ZnPPIX-reduced iNOS protein expression in LPS-induced RAW 264.7 cells (Online Figure XVIA through XVIC). These data indicated that the regulation of LPS-induced iNOS by HO-1 was independent of the HO-1 metabolites in macrophages.
HO-1 Binds iNOS at the Flavin Mononucleotide Domain
These observations prompted us to test whether HO-1 binds directly to iNOS. We coexpressed HO-1 and iNOS plasmids in HEK293T cells. Immunoprecipitation assays revealed an interaction between HO-1 and iNOS (Figure 6A and 6B). This interaction was further confirmed in LPS-induced BMDM (Online Figure XVIIA). However, this interaction was dramatically blocked by supplementation with ZnPPIX, which competitively binded with HO-1 (Online Figure XVIIIA). Mammalian iNOS is composed of 2 principal domains: an N-terminal oxidase domain and a C-terminal reductase domain (Figure 6C), which are connected by an intervening CaM (calmodulin)-binding region. The N-terminal oxidase domain contains the heme domain. The reductase domain is divided into the flavin mononucleotide (FMN), flavin adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate subdomains.29,30 To determine which of these domains was responsible for protein interaction between iNOS and HO-1, we constructed various truncated mutations of iNOS and transfected them into HEK293T cells. The iNOS 66 to 709 amino acid (aa) and 499 to 1144 aa truncation variants failed to bind with HO-1 whereas the 66 to 498 aa and 709 to 1144 aa truncation still showed combination with HO-1 (Figure 6D through 6F). These results indicated that the 499 to 708 aa of iNOS is required for the interaction with HO-1. Further, the 503 to 523 aa (CaM-binding region) and 2 to 535 aa truncated forms of iNOS bound to HO-1 (Figure 6G and 6H), whereas the 535 to 709 aa (FMN domain) truncation failed to bind to HO-1 (Figure 6I). These results implied that HO-1 interacted with iNOS at the FMN domain.
Interaction Between HO-1 and iNOS Inhibits iNOS Association With LC3
Next, we asked whether the HO-1/iNOS interaction regulated the iNOS lysosomal degradation. LC3 (light chain 3) is linked to the autophagic membrane from initiation until fusion with the lysosome, which results in degradation of the protein.31 We investigated whether LC3 mediated the iNOS degradation. Immunoprecipitation analysis of whole cell lysates of HEK293T cells that overexpressed iNOS and LC3 confirmed the binding between iNOS and LC3 (Figure 7A). Further, this iNOS/LC3 interaction was confirmed in LPS-induced BMDM (Online Figure XVIIB). We next assayed whether HO-1 inhibition influenced the interaction between iNOS and LC3. ZnPPIX markedly induced colocalization of iNOS and LC3 in LPS-induced RAW 264.7 cells, and this colocalization was almost invisible 6 hours after the ZnPPIX supplementation (Figure 7B).
To further confirm the involvement of the HO-1/iNOS interaction in the regulation of the iNOS association with LC3, we tested whether the mutations in iNOS that blocked the interaction with HO-1 led to an augmented binding to LC3. The WT, 66 to 709 aa truncation (lacks binding with HO-1) and 66 to 498 aa truncation (exists binding with HO-1) forms of iNOS were individually coexpressed with LC3 plasmids in HEK293T cells. As expected, the 66 to 709 aa truncation of iNOS combined with much more LC3 compared with the WT and 66 to 498 aa truncated iNOS, which served as controls (Figure 7C). The result was further confirmed by confocal microscopic detection, which showed that more LC3 and 66 to 709 aa truncated iNOS colocalization was observed compared with WT and 66 to 498 aa truncated iNOS in RAW 264.7 cells (Figure 7D). In summary, these data implied that the interaction between HO-1 and iNOS was essential to prevent iNOS association with LC3 and subsequent lysosomal degradation.
BafA1 Prevents iNOS Degradation and Reverses the Protective Effect of Myeloid HO-1 Deficiency on Septic Cardiac Dysfunction In Vivo
Because our in vitro data suggested that HO-1 prevented iNOS lysosomal degradation in macrophages, the final question we asked was whether the lysosome inhibitor, BafA1, would abolish the protective effect of HO-1 deletion in the myeloid cells on septic cardiac dysfunction in vivo. To this end, mice were pretreated with dimethyl sulfoxide or BafA1 and then challenged with LPS or PBS. As shown in Figure 8A through 8C, LPS led to a significant cardiac dysfunction and caused a significant induction of iNOS expression. These effects could be restored by HO-1 ablation in myeloid cells. However, BafA1 treatment, which blocked the decrease in iNOS that was caused by HO-1 ablation in the myeloid cells, resulted in exaggerated cardiac dysfunction compared with the LPS-treated myeloid HO-1–deficient mice. These data indicated that myeloid HO-1 deficiency prevented septic cardiac dysfunction through iNOS lysosomal degradation.
In the present study, we provide the first evidence that HO-1 serves as a positive regulator of iNOS and NO-derived oxidative stress in macrophages during septic cardiac dysfunction. HO-1 deficiency in myeloid cells has a protective effect on LPS-induced cardiac dysfunction and survival in vivo, which is associated with an attenuation in the expression of iNOS protein rather than its mRNA expression and reduced peroxynitrite production in macrophages. Mechanistically, we identify that HO-1 interacts with iNOS at its FMN domain, which further prevents the lysosomal degradation of the iNOS protein by inhibiting its binding with LC3 (Figure 8D).
In our study, one of the most important findings is that myeloid-derived HO-1 is identified as a critical instigator of NO-derived oxidative stress and assigned an unexpected harmful role in triggering LPS-induced cardiac dysfunction in vivo. Our conclusion is supported by several observations. First, genetic or pharmacological HO-1 inhibition in macrophages suppressed iNOS protein expression and NO generation. Second, HO-1 is primarily induced in myocardial infiltrated macrophages, and HO-1 ablation in myeloid cells attenuates the induction of iNOS and peroxynitrite production in septic hearts. Third, myeloid-specific HO-1 deficiency evokes resistance to LPS-induced septic cardiac dysfunction and lethality in vivo. These effects are independent of alterations in the infiltration of myocardial macrophages. Most importantly, the lysosome inhibitor BafA1, which restored iNOS protein expression, reversed the protective effect of the myeloid HO-1 deletion on septic cardiac dysfunction. We, therefore, conclude that ablating HO-1 in macrophages has a marked protective effect against septic cardiac dysfunction.
This finding may seem surprising considering the general assumption that HO-1 acts as an antioxidative stress and anti-inflammatory molecule. A recent report showed that cardiac-specific deletion of HO-1 exacerbated oxidative stress-induced cardiac dysfunction.32 However, there are several differences between their study and ours. First, the fundamental cause for the different results lies in the different cell-specific HO-1 gene knockout mice and animal models. In their study, cardiomyocyte-specific HO-1 knockout mice and the control mice were exposed to the oxidative stress of 100% O2, whereas in our study, myeloid cell-specific HO-1 knockout mice and their littermates were challenged with LPS to induce cardiac dysfunction. Second, the mechanism of mitochondria-mediated oxidative stress in their study is different from peroxynitrite/nitrotyrosine-mediated oxidative injury and cardiac dysfunction in ours. Third, our data show HO-1 is markedly increased and distributed in the infiltrated macrophages rather than the cardiomyocytes in the LPS-induced septic cardiac dysfunction in mice. In fact, inhibition of myeloid-derived HO-1 has been reported to be protective against Listeria monocytogenes infection, metabolic inflammation, and insulin resistance.17,33
Another important and surprising finding is that HO-1 prevents iNOS protein degradation and augments the NO-mediated oxidative stress in macrophages. To date, the role of HO-1 in inflammatory disease is believed to be restricted to the ability of its heme-related catabolic products ferrous ion, carbon monoxide, and bilirubin to exert conventional antiapoptotic, antioxidative, and anti-inflammatory effects.14,34–37 Our current results add another dimension to the roles of HO-1 by providing the first evidence that this protein can augment NO-derived oxidative stress in macrophages independent of its catalytic effect. In the current work, we show that HO-1 binds with iNOS at its FMN domain. This binding prevents iNOS degradation through the lysosomal pathway.
It is also interesting that the HO-1–regulated iNOS degradation is lysosome-dependent in macrophages, which extends the current knowledge of the post-translational regulation of iNOS.38 Our study confirmed the interaction between iNOS and LC3. This process facilitates iNOS trafficking into the lysosome. Loss of conjugation between HO-1 and iNOS, either by pharmacological inhibitor or by gene truncation, augments the interaction between iNOS and LC3 and leads to subsequent lysosomal degradation. In the previous studies, iNOS was reported to be degraded by the proteasomes.38 However, in these studies, LPS was used to induce iNOS expression, which would also trigger HO-1 induction. Thus, the degradation of iNOS is mediated by both lysosomes and proteasomes, depending on various conditions and mechanisms.
A recent study demonstrated that heme inhibits phagocytosis and drives bacterial infections and that HO-1 deficiency in macrophages results in an increased susceptibility to Escherichia coli sepsis.39 It should be noted that although the peritonitis model is more relevant to sepsis in human,5 the septic cardiac dysfunction is primarily mediated by LPS-induced endotoxemia, which triggers infiltration of inflammatory cells and subsequent myocardial injury.8 Our study suggests that HO-1 plays diverse roles in sepsis. Organ-specific intervention, or after sufficient antibiotic treatment, the ablation of HO-1 in macrophages may further improve the outcomes of septic cardiac dysfunction.
It should be noted that we used the LysM-Cre system to explore the role of HO-1 in myeloid cells in septic cardiac dysfunction in vivo. Previous literature reported that the LysM-Cre system not only targets the monocyte/macrophages but also neutrophils.40 Our data showed that HO-1 is particularly expressed in the infiltrated macrophages in septic hearts and that macrophages are essential to mediate LPS-induced cardiac injury.5,21 It is reasonable that macrophage-derived HO-1 acts as a critical instigator in triggering LPS-induced cardiac dysfunction in vivo. However, no macrophage-specific transgenic model is currently available. This is a limitation of the study.
Taken together, our findings reveal an unexpected effect of HO-1 in macrophages on the innate immune response in septic cardiac dysfunction and pave the way toward future therapeutic approaches. This may provide a basis for investigations of the role of HO-1 signaling in macrophages under conditions in which macrophage-mediated inflammation is also of critical importance, such as atherosclerosis, lung injury, and inflammatory bowel disease.
Harald Esterbauer (Clinical Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna) and Anupam Agarwal (Division of Nephrology, Department of Medicine, University of Alabama at Birmingham) are acknowledged for providing the HO-1fl/fl mice. Sicong Chen (Clinical Research Center, The Second Affiliated Hospital, Zhejiang University College of Medicine) is acknowledged for providing technical supports for plasmid construction.
Sources of Funding
This work was supported by the National Natural Science Foundation of China (81470384 and 81670259 to M. Xiang and 81670390 to Z. Cai).
In March 2018, the average time from submission to first decision for all original research papers submitted to Circulation Research was 10.69 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.118.312910/-/DC1.
- Nonstandard Abbreviations and Acronyms
- amino acid
- bafilomycin A1
- bone marrow-derived macrophages
- flavin mononucleotide
- heme oxygenase-1
- inducible NO synthase
- light chain 3
- ryanodine receptor 2
- sarco-endoplasmic reticulum calcium adenosine triphosphatase 2a
- tumor necrosis factor-α
- wild type
- zinc protoporphyrin IX
- Received February 14, 2018.
- Revision received April 2, 2018.
- Accepted April 17, 2018.
- © 2018 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Catabolic products of HO-1 (heme oxygenase-1) suppress inflammation and oxidative stress.
Septic cardiac dysfunction is mediated by multiple proinflammatory mediators and oxidative stress.
iNOS (inducible NO synthase)-derived NO reacts with superoxide to form peroxynitrite, which leads to severe oxidative stress.
Macrophages are central mediators of septic cardiac dysfunction.
What New Information Does This Article Contribute?
Myeloid-specific HO-1 deficiency evokes resistance to lipopolysaccharide-induced septic cardiac dysfunction and lethality in vivo.
HO-1 deletion in myeloid cells attenuates iNOS induction and peroxynitrite production in septic hearts in vivo.
Genetic or pharmacological HO-1 inhibition in macrophages suppresses iNOS protein expression and NO generation.
HO-1 interacts with iNOS at its flavin mononucleotide domain, which further prevents iNOS protein lysosomal degradation by inhibiting its binding with LC3 (light chain 3).
Administration of the lysosome inhibitor bafilomycin A1 reverses the protective effect of myeloid HO-1 deletion on septic cardiac dysfunction.
HO-1has mostly been considered to suppress inflammation and oxidative stress by its catalytic function and the potential for its heme-related catabolic products. However, the role of macrophage-derived HO-1 in inflammatory diseases remains unclear. Here, we report that myeloid conditional HO-1 deletion in mice evoked resistance to lipopolysaccharide-triggered septic cardiac dysfunction in vivo, which was accompanied by decreased peroxynitrite production and iNOS in the cardiac infiltrated macrophages, independent of proinflammatory cytokine production and macrophage infiltration. Mechanistically, we demonstrate that HO-1 interacted with iNOS at the flavin mononucleotide domain, which further prevented iNOS conjugation with LC3 and subsequent lysosomal degradation in macrophages. These effects were independent of HO-1’s catabolic products: ferrous ion, carbon monoxide, and bilirubin. Moreover, suppression of the lysosomal degradation of iNOS by bafilomycin A1 drove septic cardiac dysfunction in myeloid HO-1–deficient mice in vivo. These findings illustrate that HO-1 in macrophages is necessary for the development of septic cardiac dysfunction.