Inflammation-induced alterations in maternal-fetal Heme Oxygenase (HO) are associated with sustained innate immune cell dysregulation in mouse offspring.

Heme oxygenase-1 (HO-1) is an evolutionarily conserved stress response enzyme and important in pregnancy maintenance, fetal and neonatal outcomes, and a variety of pathologic conditions. Here, we investigated the effects of an exposure to systemic inflammation late in gestation [embryonic day (E)15.5] on wild-type (Wt) and HO-1 heterozygous (Het, HO-1+/-) mothers, fetuses, and offspring. We show that alterations in fetal liver and spleen HO homeostasis during inflammation late in gestation can lead to a sustained dysregulation of innate immune cell populations and intracellular myeloid HO-1 expression in the spleen through young adolescence [postnatal day 25] in mice.

Skeletal muscle heme oxygenase-1 activity regulates aerobic capacity.

Physical exercise has profound effects on quality of life and susceptibility to chronic disease; however, the regulation of skeletal muscle function at the molecular level after exercise remains unclear. We tested the hypothesis that the benefits of exercise on muscle function are linked partly to microtraumatic events that result in accumulation of circulating heme. Effective metabolism of heme is controlled by Heme Oxygenase-1 (HO-1, Hmox1), and we find that mouse skeletal muscle-specific HO-1 deletion (Tam-Cre-HSA-Hmox1fl/fl) shifts the proportion of muscle fibers from type IIA to type IIB concomitant with a disruption in mitochondrial content and function. In addition to a significant impairment in running performance and response to exercise training, Tam-Cre-HSA-Hmox1fl/fl mice show remarkable muscle atrophy compared to Hmox1fl/fl controls. Collectively, these data define a role for heme and HO-1 as central regulators in the physiologic response of skeletal muscle to exercise.

Heme Oxygenase-1 Deficiency and Oxidative Stress: A Review of 9 Independent Human Cases and Animal Models.

Since Yachie et al. reported the first description of human heme oxygenase (HO)-1 deficiency more than 20 years ago, few additional human cases have been reported in the literature. A detailed analysis of the first human case of HO-1 deficiency revealed that HO-1 is involved in the protection of multiple tissues and organs from oxidative stress and excessive inflammatory reactions, through the release of multiple molecules with anti-oxidative stress and anti-inflammatory functions. HO-1 production is induced in vivo within selected cell types, including renal tubular epithelium, hepatic Kupffer cells, vascular endothelium, and monocytes/macrophages, suggesting that HO-1 plays critical roles in these cells. In vivo and in vitro studies have indicated that impaired HO-1 production results in progressive monocyte dysfunction, unregulated macrophage activation and endothelial cell dysfunction, leading to catastrophic systemic inflammatory response syndrome. Data from reported human cases of HO-1 deficiency and numerous studies using animal models suggest that HO-1 plays critical roles in various clinical settings involving excessive oxidative stress and inflammation. In this regard, therapy to induce HO-1 production by pharmacological intervention represents a promising novel strategy to control inflammatory diseases.

Hmox1 (Heme Oxygenase-1) Protects Against Ischemia-Mediated Injury via Stabilization of HIF-1α (Hypoxia-Inducible Factor-1α).

OBJECTIVE: Hmox1 (heme oxygenase-1) is a stress-induced enzyme that catalyzes the degradation of heme to carbon monoxide, iron, and biliverdin. Induction of Hmox1 and its products protect against cardiovascular disease, including ischemic injury. Hmox1 is also a downstream target of the transcription factor HIF-1α (hypoxia-inducible factor-1α), a key regulator of the body's response to hypoxia. However, the mechanisms by which Hmox1 confers protection against ischemia-mediated injury remain to be fully understood. Approach and Results: Hmox1 deficient (Hmox1-/-) mice had impaired blood flow recovery with severe tissue necrosis and autoamputation following unilateral hindlimb ischemia. Autoamputation preceded the return of blood flow, and bone marrow transfer from littermate wild-type mice failed to prevent tissue injury and autoamputation. In wild-type mice, ischemia-induced expression of Hmox1 in skeletal muscle occurred before stabilization of HIF-1α. Moreover, HIF-1α stabilization and glucose utilization were impaired in Hmox1-/- mice compared with wild-type mice. Experiments exposing dermal fibroblasts to hypoxia (1% O2) recapitulated these key findings. Metabolomics analyses indicated a failure of Hmox1-/- mice to adapt cellular energy reprogramming in response to ischemia. Prolyl-4-hydroxylase inhibition stabilized HIF-1α in Hmox1-/- fibroblasts and ischemic skeletal muscle, decreased tissue necrosis and autoamputation, and restored cellular metabolism to that of wild-type mice. Mechanistic studies showed that carbon monoxide stabilized HIF-1α in Hmox1-/- fibroblasts in response to hypoxia. CONCLUSIONS: Our findings suggest that Hmox1 acts both downstream and upstream of HIF-1α, and that stabilization of HIF-1α contributes to Hmox1's protection against ischemic injury independent of neovascularization.

Heme oxygenase-1 deficiency presenting with interstitial lung disease and hemophagocytic flares.

BACKGROUND: Heme oxygenase-1 (HMOX1) catalyzes the metabolism of heme into carbon monoxide, ferrous iron, and biliverdin. Through biliverdin reductase, biliverdin becomes bilirubin. HMOX1-deficiency is a rare autosomal recessive disorder with hallmark features of direct antibody negative hemolytic anemia with normal bilirubin, hyperinflammation and features similar to macrophage activation syndrome. Clinical findings have included asplenia, nephritis, hepatitis, and vasculitis. Pulmonary features and evaluation of the immune response have been limited. CASE PRESENTATION: We present a young boy who presented with chronic respiratory failure due to nonspecific interstitial pneumonia following a chronic history of infection-triggered recurrent hyperinflammatory flares. Episodes included hemolysis without hyperbilirubinemia, immunodeficiency, hepatomegaly with mild transaminitis, asplenia, leukocytosis, thrombocytosis, joint pain and features of macrophage activation with negative autoimmune serologies. Lung biopsy revealed cholesterol granulomas. He was found post-mortem by whole exome sequencing to have a compound heterozygous paternal frame shift a paternal frame shift HMOX1 c.264_269delCTGG (p.L89Sfs*24) and maternal splice donor HMOX1 (c.636 + 2 T > A) consistent with HMOX1 deficiency. Western blot analysis confirmed lack of HMOX1 protein upon oxidant stimulation of the patient cells. CONCLUSIONS: Here, we describe a phenotype expansion for HMOX1-deficiency to include not only asplenia and hepatomegaly, but also interstitial lung disease with cholesterol granulomas and inflammatory flares with hemophagocytosis present in the bone marrow.

Characterization of hepatic macrophages and evaluation of inflammatory response in heme oxygenase-1 deficient mice exposed to scAAV9 vectors.

Adeno-associated viral (AAV) vectors are characterised by low immunogenicity, although humoral and cellular responses may be triggered upon infection. Following systemic administration high levels of vector particles accumulate within the liver. Kupffer cells (KCs) are liver resident macrophages and an important part of the liver innate immune system. Decreased functional activity of KCs can contribute to exaggerated inflammatory response upon antigen exposure. Heme oxygenase-1 (HO-1) deficiency is associated with considerably reduced numbers of KCs. In this study we aimed to investigate the inflammatory responses in liver and to characterise two populations of hepatic macrophages in adult wild type (WT) and HO-1 knockout (KO) mice following systemic administration of one or two doses (separated by 3 months) of self-complementary (sc)AAV9 vectors. At steady state, the livers of HO-1 KO mice contained significantly higher numbers of monocyte-derived macrophages (MDMs), but significantly less KCs than their WT littermates. Three days after re-administration of scAAV9 we observed increased mRNA level of monocyte chemoattractant protein-1 (Mcp-1) in the livers of both WT and HO-1 KO mice, but the protein level and the macrophage infiltration were not affected. Three days after the 1st and 3 days after the 2nd vector dose the numbers of AAV genomes in the liver were comparable between both genotypes indicating similar transduction efficiency, but the percentage of transgene-expressing MDMs and KCs was higher in WT than in HO-1 KO mice. In the primary culture, KCs were able to internalize AAV9 particles without induction of TLR9-mediated immune responses, but no transgene expression was observed. In conclusion, in vivo and in vitro cultured KCs have different susceptibility to scAAV9 vectors. Regardless of the presence or absence of HO-1 and initial numbers of KCs in the liver, scAAV9 exhibits a low potential to stimulate inflammatory response at the analysed time points.

Exacerbation of Neonatal Hemolysis and Impaired Renal Iron Handling in Heme Oxygenase 1-Deficient Mice.

In most mammals, neonatal intravascular hemolysis is a benign and moderate disorder that usually does not lead to anemia. During the neonatal period, kidneys play a key role in detoxification and recirculation of iron species released from red blood cells (RBC) and filtered out by glomeruli to the primary urine. Activity of heme oxygenase 1 (HO1), a heme-degrading enzyme localized in epithelial cells of proximal tubules, seems to be of critical importance for both processes. We show that, in HO1 knockout mouse newborns, hemolysis was prolonged despite a transient state and exacerbated, which led to temporal deterioration of RBC status. In neonates lacking HO1, functioning of renal molecular machinery responsible for iron reabsorption from the primary urine (megalin/cubilin complex) and its transfer to the blood (ferroportin) was either shifted in time or impaired, respectively. Those abnormalities resulted in iron loss from the body (excreted in urine) and in iron retention in the renal epithelium. We postulate that, as a consequence of these abnormalities, a tight systemic iron balance of HO1 knockout neonates may be temporarily affected.

Using ultrasound to define the time point of intrauterine growth retardation in a mouse model of heme oxygenase-1 deficiency†.

The enzyme heme oxygenase-1 (HO-1), encoded by the HMOX1 gene, mediates heme catabolism by cleaving free heme. We have previously revealed the importance of HO-1 in pregnancy. Here, we determined the impact of maternal or paternal HO-1 deficiency on fetal growth and placental parameters throughout gestation. We mated Hmox1-sufficient (WT), partial (HET)-, or total (KO)-deficient BALB/c female mice with Hmox1-WT or -KO BALB/c males and performed ultrasound analysis to monitor placental and fetal growth. Doppler measurements were used to determine maternal blood flow parameters. Offspring weights and feto-placental indices (FPI) were also determined. We found a significantly increased number of underdeveloped fetuses at gd10 in HET females that were mated with WT males compared with WT × WT pairings. At the same gestational age, underdeveloped placentas could be detected in HET females mated with KO males. Many fetuses from the KO × KO combination died in utero between gd12 and gd14. At gd14, abnormal placental parameters were found in surviving fetuses, which had significant reduced weights. Moreover, only 3.11% female and 5.33% male KO pups resulted from 10 HET × HET breeding pairs over 1 year. Our results show that HO-1 from both maternal and paternal origins is important for proper placental and fetal growth. Placental growth restriction and occurrence of abortions in mice that were partially or totally deficient in HO-1 were recorded in vivo from gd10 onwards. Future studies will focus on elucidating the cellular and molecular mechanisms behind these observations.

Heme Oxygenase-1 Is a Pivotal Modulator of Bone Turnover and Remodeling: Molecular Implications for Prostate Cancer Bone Metastasis.

Aims: Bone is the most frequent site of prostate cancer (PCa) metastasis. Tumor cells interact with the bone microenvironment interrupting tissue balance. Heme oxygenase-1 (HO-1; encoded by Hmox1) appears as a potential target in PCa maintaining the cellular homeostasis. Our hypothesis is that HO-1 is implicated in bone physiology and modulates the communication with PCa cells. Here we aimed at (i) assessing the physiological impact of Hmox1 gene knockout (KO) on bone metabolism in vivo and (ii) determining the alterations of the transcriptional landscape associated with tumorigenesis and bone remodeling in cells growing in coculture (PCa cells with primary mouse osteoblasts [PMOs] from BALB/c Hmox1+/+, Hmox1+/-, and Hmox1-/- mice). Results: Histomorphometric analysis of Hmox1-/- mice bones exhibited significantly decreased bone density with reduced remodeling parameters. A positive correlation between Hmox1 expression and Runx2, Col1a1, Csf1, and Opg genes was observed in PMOs. Flow cytometry studies revealed two populations of PMOs with different reactive oxygen species (ROS) levels. The high ROS population was increased in PMOs Hmox1+/- compared with Hmox1+/+, but was significantly reduced in PMOs Hmox1-/-, suggesting restrained ROS tolerance in KO cells. Gene expression was altered in PMOs upon coculture with PCa cells, showing a pro-osteoclastic profile. Moreover, HO-1 induction in PCa cells growing in coculture with PMOs resulted in a significant modulation of key bone markers such as PTHrP and OPG. Innovation and Conclusion: We here demonstrate the direct implications of HO-1 expression in bone remodeling and how it participates in the alterations in the communication between bone and prostate tumor cells.

Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood.

Granulocyte colony-stimulating factor (G-CSF) is used in clinical practice to mobilize cells from the bone marrow to the blood; however, it is not always effective. We show that cobalt protoporphyrin IX (CoPP) increases plasma concentrations of G-CSF, IL-6, and MCP-1 in mice, triggering the mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC). Compared with recombinant G-CSF, CoPP mobilizes higher number of HSPC and mature granulocytes. In contrast to G-CSF, CoPP does not increase the number of circulating T cells. Transplantation of CoPP-mobilized peripheral blood mononuclear cells (PBMC) results in higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G-CSF. Although CoPP is used to activate Nrf2/HO-1 axis, the observed effects are Nrf2/HO-1 independent. Concluding, CoPP increases expression of mobilization-related cytokines and has superior mobilizing efficiency compared with recombinant G-CSF. This observation could lead to the development of new strategies for the treatment of neutropenia and HSPC transplantation.

Hepatocellular heme oxygenase 1 deficiency does not affect inflammatory hepcidin regulation in mice.

Hepcidin is an iron regulatory peptide hormone that is secreted from hepatocytes and inhibits iron efflux from tissues to plasma. Under inflammatory conditions, hepcidin is transcriptionally induced by IL-6/STAT3 signaling and promotes hypoferremia, an innate immune response to infection. If this pathway remains unresolved, chronic overexpression of hepcidin contributes to the anemia of inflammation, a common medical condition. Previous work showed that carbon monoxide (CO) releasing drugs (CORMs) can attenuate inflammatory induction of hepcidin. Because CO is physiologically generated during heme degradation by heme oxygenase 1 (HO-1), an IL-6-inducible enzyme with anti-inflammatory properties, we hypothesized that hepatocellular HO-1 may operate as a physiological feedback regulator of hepcidin that resolves inflammatory signaling. To address this, we generated and analyzed hepatocyte-specific HO-1 knockout (Hmox1Alb-Cre) mice. We show that these animals mount appropriate hepcidin-mediated hypoferremic response to LPS-induced inflammation, with kinetics similar to those of control Hmox1fl/fl mice. Likewise, primary hepatocytes from Hmox1Alb-Cre and Hmox1fl/fl mice exhibit similar degree and kinetics of hepcidin induction following IL-6 treatment. We conclude that hepatocellular HO-1 has no physiological function on hepcidin regulation by the inflammatory pathway.

Pterostilbene 4'-ß-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1.

Heme oxygenase-1 (HO-1) can exert anti-inflammatory and antioxidant effects. Acute lung injury (ALI) is associated with increased inflammation and influx of proinflammatory cells and mediators in the airspaces and lung parenchyma. In this study, we demonstrate that pterostilbene 4'-ß-glucoside (4-PG), the glycosylated form of the antioxidant pterostilbene (PTER), can protect against lipopolysaccharide- (LPS-) or Pseudomonas aeruginosa- (P. aeruginosa-) induced ALI when applied as a pretreatment or therapeutic post-treatment, via the induction of HO-1. To determine whether HO-1 mediates the antioxidant and anti-inflammatory effects of 4-PG, we subjected mice genetically deficient in Hmox-1 to LPS-induced ALI and evaluated histological changes, HO-1 expression, and proinflammatory cytokine levels in bronchoalveolar lavage (BAL) fluid. 4-PG exhibited protective effects on LPS- or P. aeruginosa-induced ALI by ameliorating pathological changes in lung tissue and decreasing proinflammatory cytokines. In addition, HO-1 expression was significantly increased by 4-PG in cells and in mouse lung tissues. The glycosylated form of pterostilbene (4-PG) was more effective than PTER in inducing HO-1 expression. Genetic deletion of Hmox-1 abolished the protective effects of 4-PG against LPS-induced inflammatory responses. Furthermore, we found that 4-PG decreased both intracellular ROS levels and mitochondrial (mt) ROS production in a manner dependent on HO-1. Pharmacological application of the HO-1 reaction product carbon monoxide (CO), but not biliverdin or iron, conferred protection in Hmox-1-deficient macrophages. Taken together, these results demonstrate that 4-PG can increase HO-1 expression, which plays a critical role in ameliorating intracellular and mitochondrial ROS production, as well as in downregulating inflammatory responses induced by LPS. Therefore, these findings strongly suggest that HO-1 mediates the antioxidant and anti-inflammatory effects of 4-PG.

Microanatomic Distribution of Myeloid Heme Oxygenase-1 Protects against Free Radical-Mediated Immunopathology in Human Tuberculosis.

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that controls inflammatory responses and redox homeostasis; however, its role during pulmonary tuberculosis (TB) remains unclear. Using freshly resected human TB lung tissue, we examined the role of HO-1 within the cellular and pathological spectrum of TB. Flow cytometry and histopathological analysis of human TB lung tissues showed that HO-1 is expressed primarily in myeloid cells and that HO-1 levels in these cells were directly proportional to cytoprotection. HO-1 mitigates TB pathophysiology by diminishing myeloid cell-mediated oxidative damage caused by reactive oxygen and/or nitrogen intermediates, which control granulocytic karyorrhexis to generate a zonal HO-1 response. Using whole-body or myeloid-specific HO-1-deficient mice, we demonstrate that HO-1 is required to control myeloid cell infiltration and inflammation to protect against TB progression. Overall, this study reveals that zonation of HO-1 in myeloid cells modulates free-radical-mediated stress, which regulates human TB immunopathology.

Infused wild-type macrophages reside and self-renew in the liver to rescue the hemolysis and anemia of Hmox1-deficient mice.

Heme oxygenase 1 (HMOX1), the inducible enzyme that catabolizes the degradation of heme into biliverdin, iron, and carbon monoxide, plays an essential role in the clearance of senescent and damaged red blood cells, systemic iron homeostasis, erythropoiesis, vascular hemostasis, and oxidative and inflammatory stress responses. In humans, HMOX1 deficiency causes a rare and lethal disease, characterized by severe anemia, intravascular hemolysis, as well as vascular and tissue damage. Hmox1 knockout (KO) mice recapitulated the phenotypes of HMOX1-deficiency patients and could be rescued by bone marrow (BM) transplantation that engrafted donor's hematopoietic stem cells into the recipient animals after myeloablation. To find better therapy and elucidate the contribution of macrophages to the pathogenesis of HMOX1-deficiency disease, we infused wild-type (WT) macrophages into Hmox1 KO mice. Results showed that WT macrophages engrafted and proliferated in the livers of Hmox1 KO mice, which corrected the microcytic anemia, rescued the intravascular hemolysis, restored iron homeostasis, eliminated kidney iron overload and tissue damage, and provided long-term protection. These results showed that a single macrophage infusion delivered a long-term curative effect in Hmox1 KO mice, obviating the need for BM transplantation, and suggested that the HMOX1 disease stems mainly from the loss of viable reticuloendothelial macrophages. Our work provides new insights into the etiology of HMOX1 deficiency and demonstrates the potential of infusion of WT macrophages to prevent disease in patients with HMOX1 deficiency and potentially other macrophage-related diseases.

Partial Inhibition of HO-1 Attenuates HMP-Induced Hepatic Regeneration against Liver Injury in Rats.

We found better liver graft regeneration with hypothermic machine perfusion (HMP) compared with static cold storage (SCS) for the first time in our pilot study, but the underlying mechanisms are unknown. Upregulated heme oxygenase- (HO-) 1 expression has been reported to play a pivotal role in promoting hepatocyte proliferation. Here, we evaluated the novel role of HO-1 in liver graft protection by HMP. Rats with a heterozygous knockout of HO-1 (HO-1+/-) were generated and subjected to 3 h of SCS or HMP pre-half-size liver transplantation (HSLT) in vivo or 6 h of SCS or HMP in vitro; control rats were subjected to the same conditions (HO-1+/+). We found that HSLT induced significant elevation of the HO-1 protein level in the regenerated liver and that HO-1 haplodeficiency resulted in decreased proliferation post-HSLT. Compared with SCS, HMP induced significant elevation of the HO-1 protein level along with better liver recovery, both of which were reduced by HO-1 haplodeficiency. HO-1 haplodeficiency-induced decreased proliferation was responsible for the attenuated regenerative ability of HMP. Mechanistically, HO-1 haploinsufficiency resulted in suppression of hepatocyte growth factor (HGF)/Akt activity. Our results suggest that inhibition of HO-1 mitigates HMP-induced liver recovery effects related to proliferation, in part, by downregulating the HGF-Akt axis.

Heme oxygenase-1 deficiency promotes severity of sepsis in a non-surgical preterm mouse model.

BACKGROUND: Sepsis in preterm infants is associated with systemic inflammatory responses. The stress-response protein heme oxygenase-1 (HO-1) has protective anti-inflammatory properties. Recently, we reported a protective role of HO-1 using our non-surgical cecal slurry (CS) model in wild-type (WT) mouse pups. Here, we extend these findings to investigate the association of HO-1 deficiency with sepsis severity. METHODS: Adapting the Wynn model, we induced sepsis in 4-day-old HO-1-deficient (HO-1+/-, Het) pups to determine if HO-1 deficiency affected survival rates at the LD40 (2.0 mg/g) of WT pups. To see if HO-1 induction affected sepsis severity, we gave 30-µmol heme/kg subcutaneously to 3-day-old mice 24 h prior to sepsis induction. RESULTS: Post-sepsis induction, Het pups had a mortality of 85.0% (n = 20) and increased expression of the pro-inflammatory gene in the livers and affected hematologic profiles. Heme treatment 24 h prior to sepsis induction significantly increased liver HO activity, reduced mortality to 24.5% (n = 17), attenuated inflammatory responses, reduced spleen bacterial counts, and significantly increased peripheral neutrophils. CONCLUSIONS: A partial deficiency in HO-1 increased the progression and mortality in sepsis. Furthermore, induction of HO-1 significantly reduced the mortality even in Het pups. Thus, we conclude that HO-1 plays an important role in the protection against preterm sepsis.

Heme Oxygenase-1 in Macrophages Drives Septic Cardiac Dysfunction via Suppressing Lysosomal Degradation of Inducible Nitric Oxide Synthase.

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.

Myeloid Heme Oxygenase-1 Regulates the Acute Inflammatory Response to Zymosan in the Mouse Air Pouch.

Heme oxygenase-1 (HO-1) is induced by many stimuli to modulate the activation and function of different cell types during innate immune responses. Although HO-1 has shown anti-inflammatory effects in different systems, there are few data on the contribution of myeloid HO-1 and its role in inflammatory processes is not well understood. To address this point, we have used HO-1M-KO mice with myeloid-restricted deletion of HO-1 to specifically investigate its influence on the acute inflammatory response to zymosan in vivo. In the mouse air pouch model, we have shown an exacerbated inflammation in HO-1M-KO mice with increased neutrophil infiltration accompanied by high levels of inflammatory mediators such as interleukin-1ß, tumor necrosis factor-α, and prostaglandin E2. The expression of the degradative enzyme matrix metalloproteinase-3 (MMP-3) was also enhanced. In addition, we observed higher levels of serum MMP-3 in HO-1M-KO mice compared with control mice, suggesting the presence of systemic inflammation. Altogether, these findings demonstrate that myeloid HO-1 plays an anti-inflammatory role in the acute response to zymosan in vivo and suggest the interest of this target to regulate inflammatory processes.

Heme oxygenase-1 deficiency results in splenic T-cell dysregulation in offspring of mothers exposed to late gestational inflammation.

PROBLEM: Infection during pregnancy can disrupt regulatory/effector immune system balance, resulting in adverse pregnancy and fetal-neonatal outcomes. Heme oxygenase-1 (HO-1) is a major regulatory enzyme in the immune system. We observed maternal immune response dysregulation during late gestational inflammation (LGI), which may be mediated by HO-1. Here, we extend these studies to examine the immune response of offspring. METHOD OF STUDY: Pregnant wild-type (Wt) and HO-1 heterozygote (Het) dams were treated with lipopolysaccharide (LPS) or vehicle at E15.5. Pups' splenic immune cells were characterized using flow cytometry. RESULTS: CD3+ CD4+ CD25+ (Tregs) and CD3+ CD8+ (Teffs) T cells in Wt and Het were similar in control neonates and increased with age. We showed not only age- but also genotype-specific and long-lasting T-cell dysregulation in pups after maternal LGI. The persistent immune dysregulation, mediated by HO-1 deficiency, was reflected as a decrease in Treg FoxP3 and CD3+ CD8+ T cells, and an increase in CD4+ /CD8+ T-cell and Treg/Teff ratios in Hets compared with Wt juvenile mice after maternal exposure to LGI. CONCLUSION: Maternal exposure to LGI can result in dysregulation of splenic T cells in offspring, especially in those with HO-1 deficiency. We speculate that these immune alterations are the basis of adverse outcomes in neonates from mothers exposed to low-grade (subclinical) infections.

Heme oxygenase-1 mitigates ferroptosis in renal proximal tubule cells.

Ferroptosis is an iron-dependent form of regulated nonapoptotic cell death, which contributes to damage in models of acute kidney injury (AKI). Heme oxygenase-1 (HO-1) is a cytoprotective enzyme induced in response to cellular stress, and is protective against AKI because of its antiapoptotic and anti-inflammatory properties. However, the role of HO-1 in regulating ferroptosis is unclear. The purpose of this study was to elucidate the role of HO-1 in regulating ferroptotic cell death in renal proximal tubule cells (PTCs). Immortalized PTCs obtained from HO-1+/+ and HO-1-/- mice were treated with erastin or RSL3, ferroptosis inducers, in the presence or absence of antioxidants, an iron source, or an iron chelator. Cells were assessed for changes in morphology and metabolic activity as an indicator of cell viability. Treatment of HO-1+/+ PTCs with erastin resulted in a time- and dose-dependent increase in HO-1 gene expression and protein levels compared with vehicle-treated controls. HO-1-/- cells showed increased dose-dependent erastin- or RSL3-induced cell death in comparison to HO-1+/+ PTCs. Iron supplementation with ferric ammonium citrate in erastin-treated cells decreased cell viability further in HO-1-/- PTCs compared with HO-1+/+ cells. Cotreatment with ferrostatin-1 (ferroptosis inhibitor), deferoxamine (iron chelator), or N-acetyl-l-cysteine (glutathione replenisher) significantly increased cell viability and attenuated erastin-induced ferroptosis in both HO-1+/+ and HO-1-/- PTCs. These results demonstrate an important antiferroptotic role of HO-1 in renal epithelial cells.