Heme oxygenase-1 is an equid alphaherpesvirus 8 replication restriction host protein and suppresses viral replication via the PKCß/ERK1/ERK2 and NO/cGMP/PKG pathway.

Equid alphaherpesvirus 8 (EqHV-8) is one of the most economically important viruses that is known to cause severe respiratory disease, abortion, and neurological syndromes in equines. However, no effective vaccines or therapeutic agents are available to control EqHV-8 infection. Heme oxygenase-1 (HO-1) is an antioxidant defense enzyme that displays significant cytoprotective effects against different viral infections. However, the literature on the function of HO-1 during EqHV-8 infection is little. We explored the effects of HO-1 on EqHV-8 infection and revealed its potential mechanisms. Our results demonstrated that HO-1 induced by cobalt-protoporphyrin (CoPP) or HO-1 overexpression inhibited EqHV-8 replication in susceptible cells. In contrast, HO-1 inhibitor (zinc protoporphyria) or siRNA targeting HO-1 reversed the anti-EqHV-8 activity. Furthermore, biliverdin, a metabolic product of HO-1, mediated the anti-EqHV-8 effect of HO-1 via both the protein kinase C (PKC)ß/extracellular signal-regulated kinase (ERK)1/ERK2 and nitric oxide (NO)-dependent cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling pathways. In addition, CoPP protected the mice by reducing the EqHV-8 infection in the lungs. Altogether, these results indicated that HO-1 can be developed as a promising therapeutic strategy to control EqHV-8 infection.IMPORTANCEEqHV-8 infections have threatened continuously donkey and horse industry worldwide, which induces huge economic losses every year. However, no effective vaccination strategies or drug against EqHV-8 infection until now. Our present study found that one host protien HO-1 restrict EqHV-8 replication in vitro and in vivo. Furthermore, we demonstrate that HO-1 and its metabolite biliverdin suppress EqHV-8 relication via the PKCß/ERK1/ERK2 and NO/cGMP/PKG pathways. Hence, we believe that HO-1 can be developed as a promising therapeutic strategy to control EqHV-8 infection.

The effects of biliverdin on pressure-induced unfolding of apomyoglobin: The specific role of Zn2+ ions.

Apomyoglobin (apoMb), a model protein in biochemistry, exhibits a strong propensity to bind various ligands, which makes it a good candidate as a carrier of bioactive hydrophobic drugs. The stability of its hydrophobic pocket determines its potential as a carrier of bioactive compounds. High pressure (HP) is a potent tool for studying protein stability, revealing the specific role of hydrophobic cavities in unfolding. We probed the effects of biliverdin (BV) binding and its complex with Zn2+ ions on the structure and HP stability of apoMb. CD spectroscopy and SAXS measurements revealed that BV and BV-Zn2+ complexes make the apoMb structure more compact with higher α-helical content. We performed in situ HP measurements of apoMb intrinsic fluorescence to demonstrate the ability of BV to stabilise apoMb structure at HP conditions. Furthermore, the presence of Zn2+ within the apoMb-BV complex significantly enhances the BV stabilisation effect. In situ visible absorption study of BV chromophore confirmed the ability of Zn2+ to increase the stability of apoMb-BV complex under HP: the onset of complex dissociation is shifted by ∼100 MPa in presence of Zn2+. By combining HP-fluorescence and HP-visible absorption spectroscopy, our strategy highlights the crucial role of tetrapyrrole-metal complexes in stabilising apoMb hydrophobic pocket.

Heme Oxygenase-1 and Its Metabolites Carbon Monoxide and Biliverdin, but Not Iron, Exert Antiviral Activity against Porcine Circovirus Type 3.

Porcine circovirus type 3 (PCV3) is a newly discovered pathogen that causes porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, multisystemic inflammation, and reproductive failure. Heme oxygenase-1 (HO-1), a stress-inducible enzyme, exerts protective functions by converting heme into carbon monoxide (CO), biliverdin (BV), and iron. However, the effects of HO-1 and its metabolites on PCV3 replication remain unknown. In this study, experiments involving specific inhibitors, lentivirus transduction, and small interfering RNA (siRNA) transfection revealed that active PCV3 infection reduced HO-1 expression and that the expression of HO-1 negatively regulated virus replication in cultured cells, depending on its enzymatic activity. Subsequently, the effects of the HO-1 metabolites (CO, BV, and iron) on PCV3 infection were investigated. The CO inducers (cobalt protoporphyrin IX [CoPP] or tricarbonyl dichloro ruthenium [II] dimer [CORM-2]) mediate PCV3 inhibition by generating CO, and this inhibition is reversed by hemoglobin (Hb; a CO scavenger). The inhibition of PCV3 replication by BV depended on BV-mediated reactive oxygen species (ROS) reduction, as N-acetyl-l-cysteine affected PCV3 replication while reducing ROS production. The reduction product of BV, bilirubin (BR), specifically promoted nitric oxide (NO) generation and further activated the cyclic GMP/protein kinase G (cGMP/PKG) pathway to attenuate PCV3 infection. Both the iron provided by FeCl3 and the iron chelated by deferoxamine (DFO) with CoPP treatment failed to affect PCV3 replication. Our data demonstrate that the HO-1-CO-cGMP/PKG, HO-1-BV-ROS, and HO-1-BV-BR-NO-cGMP/PKG pathways contribute crucially to the inhibition of PCV3 replication. These results provide important insights regarding preventing and controlling PCV3 infection. IMPORTANCE The regulation of host protein expression by virus infection is the key to facilitating self-replication. As an important emerging pathogen of swine, clarification of the interaction between PCV3 infection and the host enables us to understand the viral life cycle and pathogenesis better. Heme oxygenase-1 (HO-1) and its metabolites carbon monoxide (CO), biliverdin (BV), and iron have been demonstrated to involve a wealth of viral replications. Here, we, for the first time, demonstrated that HO-1 expression decreases in PCV3-infected cells and negatively regulates PCV3 replication and that the HO-1 metabolic products CO and BV inhibit PCV3 replication by the CO- or BV/BR/NO-dependent cGMP/PKG pathway or BV-mediated ROS reduction, but the iron (the third metabolic product) does not. Specifically, PCV3 infection maintains normal proliferation by downregulating HO-1 expression. These findings clarify the mechanism by which HO-1 modulates PCV3 replication in cells and provide important targets for preventing and controlling PCV3 infection.

Pre-conditioning of gingival epithelial cells with sub-apoptotic concentrations of curcumin prevents pro-inflammatory cytokine release.

BACKGROUND AND OBJECTIVE: Plaque-induced gingival inflammation (gingivitis) is ubiquitous in humans. The epithelial barrier reacts to the presence of oral bacteria and induces inflammatory cascades. The objective of this study was to investigate the mechanism by which the small molecule micronutrient curcumin could decrease inflammatory response in vitro to oral bacterium heat-killed Fusobacterium nucleatum as curcumin could be a useful compound for combatting gingivitis already consumed by humans. METHODS: H400 oral epithelial cell line was pre-conditioned with curcumin and the production of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and translocation of transcription factors was used to monitor inflammatory responses. Haem oxygenase (HO-1) expression and molecules that HO-1 releases were evaluated for their potential to reduce the quantity of cytokine production. Immunofluorescence microscopy and Western blotting were used to evaluate changes in transcription factor and enzyme location. RESULTS: Pre-conditioning of H400 cells with a sub-apoptotic concentration of curcumin (20 µM) attenuated secretion of Granulocyte-Macrophage - Colony-Stimulating Factor (GM-CSF) and reduced NFkB nuclear translocation. This pre-conditioning caused an increase in nuclear Nrf2; an initial drop (at 8 h) followed by an adaptive increase (at 24 h) in glutathione; and an increase in haem oxygenase (HO-1) expression. Inhibition of HO-1 by SnPPIX prevented the curcumin-induced attenuation of GM-CSF production. HO-1 catalyses the breakdown of haem to carbon monoxide, free iron and biliverdin: the HO-1/CO anti-inflammatory pathway. Elevations in carbon monoxide, achieved using carbon monoxide releasing molecule-2 (CORM2) treatment alone abrogated F. nucleatum-induced cytokine production. Biliverdin is converted to bilirubin by biliverdin reductase (BVR). This pleiotropic protein was found to increase in cell membrane expression upon curcumin treatment. CONCLUSION: Curcumin decreased inflammatory cytokine production induced by Fusobacterium nucleatum in H400 oral epithelial cells. The mechanism of action appears to be driven by the increase of haem oxygenase and the production of carbon monoxide.

Enterococcus faecalis promotes the progression of colorectal cancer via its metabolite: biliverdin.

BACKGROUND: Enterococcus faecalis (Efa) has been shown to be a "driver bacteria" in the occurrence and development of colorectal cancer (CRC). This study aims to explore the effect of specific metabolites of Efa on CRC. METHODS: The pro-tumor effects of Efa were assessed in colonic epithelial cells. The tumor-stimulating molecule produced by Efa was identified using liquid chromatography mass spectrometry (LC-MS). The proliferative effect of metabolites on CRC cells in vitro was assayed as well. The concentration of vascular endothelial growth factor A (VEGFA) and interleukin-8 (IL-8) was determined using enzyme-linked immunosorbent assay (ELISA). Tubular formation assay of human umbilical vein endothelial cells (HUVEC) and cell migration assay were applied to study angiogenesis. Additionally, western blot analysis was used to investigate key regulatory proteins involved in the angiogenesis pathway. Tumor growth was assessed using mouse models with two CRC cells and human colon cancer organoid. RESULTS: Co-incubation with the conditioned medium of Efa increased the proliferation of cultured CRC cells. Biliverdin (BV) was determined as the key metabolite produced by Efa using LC-MS screening. BV promoted colony formation and cell proliferation and inhibited cell cycle arrest of cultured CRC cells. BV significantly increased the expression level of IL-8 and VEGFA by regulating the PI3K/AKT/mTOR signaling pathway, leading to the acceleration of angiogenesis in CRC. The up-regulation of proliferation and angiogenesis by BV were also confirmed in mice. CONCLUSION: In conclusion, BV, as the tumor-stimulating metabolite of Efa, generates proliferative and angiogenic effects on CRC, which is mainly mediated by the activation of PI3K/AKT/mTOR.

Biliverdin/Bilirubin Redox Pair Protects Lens Epithelial Cells against Oxidative Stress in Age-Related Cataract by Regulating NF-κB/iNOS and Nrf2/HO-1 Pathways.

Age-related cataract (ARC) is the leading cause of vision impairment globally. It has been widely accepted that excessive reactive oxygen species (ROS) accumulation in lens epithelial cells (LECs) is a critical risk factor for ARC formation. Biliverdin (BV)/bilirubin (BR) redox pair is the active by-product of heme degradation with robust antioxidative stress and antiapoptotic effects. Thus, we purpose that BV and BR may have a therapeutic effect on ARC. In the present study, we determine the expression levels of enzymes regulating BV and BR generation in human lens anterior capsule samples. The therapeutic effect of BV/BR redox pair on ARC was assessed in hydrogen peroxide (H2O2)-damaged mouse LECs in vitro. The NF-κB/inducible nitric oxide synthase (iNOS) and nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathways were evaluated to illustrate the molecular mechanism. The results revealed that the mRNA expressions of Nrf2, HO-1, and biliverdin reductase A (BVRA) were all decreased in human samples of age-related nuclear cataract. BV/BR redox pair pretreatment protected LECs against H2O2 damage by prohibiting NF-κB p65 nuclear trafficking, ameliorating iNOS expression, reducing intracellular and mitochondrial ROS levels, and restoring glutathione (GSH) and superoxide dismutase (SOD) levels. BV and BR pretreatment also regulated the expression of apoptotic molecules (Bax, Bcl-2, and cleaved caspase-3), thus decreasing the apoptosis of LECs. In addition, BV/BR pair promoted Nrf2 nuclear accumulation and HO-1 induction, whereas the knockdown of BVRA counteracted the effect of BV on activating Nrf2/HO-1 pathway and antiapoptosis. These findings implicated that BV/BR redox pair protects LECs against H2O2-induced apoptosis by regulating NF-κB/iNOS and Nrf2/HO-1 pathways. Moreover, BVRA is responsible for BV-mediated cytoprotection by reductive conversion of BV to BR. This trial is registered with ChiCTR2000036059.

GAF-CaMP3-sfGFP, An Enhanced Version of the Near-Infrared Genetically Encoded Positive Phytochrome-Based Calcium Indicator for the Visualization of Neuronal Activity.

The first generation of near-infrared, genetically encoded calcium indicators (NIR-GECIs) was developed from bacterial phytochrome-based fluorescent proteins that utilize biliverdin (BV) as the chromophore moiety. However, NIR-GECIs have some main drawbacks such as either an inverted response to calcium ions (in the case of NIR-GECO1) or a limited dynamic range and a lack of data about their application in neurons (in the case of GAF-CaMP2-superfolder green fluorescent protein (sfGFP)). Here, we developed an enhanced version of the GAF-CaMP2-sfGFP indicator, named GAF-CaMP3-sfGFP. The GAF-CaMP3-sfGFP demonstrated spectral characteristics, molecular brightness, and a calcium affinity similar to the respective characteristics for its progenitor, but a 2.9-fold larger DF/F response to calcium ions. As compared to GAF-CaMP2-sfGFP, in cultured HeLa cells, GAF-CaMP3-sfGFP had similar brightness but a 1.9-fold larger DF/F response to the elevation of calcium ions levels. Finally, we successfully utilized the GAF-CaMP3-sfGFP for the monitoring of the spontaneous and stimulated activity of neuronal cultures and compared its performance with the R-GECO1 indicator using two-color confocal imaging. In the cultured neurons, GAF-CaMP3-sfGFP showed a linear DF/F response in the range of 0-20 APs and in this range demonstrated a 1.4-fold larger DF/F response but a 1.3- and 2.4-fold slower rise and decay kinetics, respectively, as compared to the same parameters for the R-GECO1 indicator.

Biliverdin administration regulates the microRNA-mRNA expressional network associated with neuroprotection in cerebral ischemia reperfusion injury in rats.

Inflammatory response has an important role in the outcome of cerebral ischemia reperfusion injury (CIR). Biliverdin (BV) administration can relieve CIR in rats, but the mechanism remains unknown. The aim of the present study was to explore the expressional network of microRNA (miRNA)­mRNA in CIR rats following BV administration. A rat middle cerebral artery occlusion model with BV treatment was established. After neurobehavior was evaluated by neurological severity scores (NSS), miRNA and mRNA expressional profiles were analyzed by microarray technology from the cerebral cortex subjected to ischemia and BV administration. Then, bioinformatics prediction was used to screen the correlation between miRNA and mRNA, and 20 candidate miRNAs and 33 candidate mRNAs were verified by reverse transcription­quantitative polymerase chain reaction. Furthermore, the regulation relationship between ETS proto­oncogene 1 (Ets1) and miRNA204­5p was examined by luciferase assay. A total of 86 miRNAs were differentially expressed in the BV group compared with the other groups. A total of 10 miRNAs and 26 candidate genes were identified as a core 'microRNA­mRNA' regulatory network that was linked with the functional improvement of BV administration in CIR rats. Lastly, the luciferase assay results confirmed that miRNA204­5p directly targeted Ets1. The present findings suggest that BV administration may regulate multiple miRNAs and mRNAs to improve neurobehavior in CIR rats, by influencing cell proliferation, apoptosis, maintaining ATP homeostasis, and angiogenesis.

Heme Oxygenase-1 inhibits spring viremia of carp virus replication through carbon monoxide mediated cyclic GMP/Protein kinase G signaling pathway.

Spring viremia of carp virus (SVCV) is the etiological agent of spring viremia of carp (SVC) and causes mass mortality in common carp (Cyprinus carpio). Currently, no effective treatments or commercial vaccines against SVCV are available. Heme oxygenase-1 (HO-1), an enzyme that catalyzes the degradation of heme to produce carbon monoxide (CO), biliverdin and ferrous iron (Fe2+), exerts anti-oxidant, antiinflammatory and anti-apoptotic properties. Previous studies demonstrated that nuclear factor-erythroid 2 related factor 2 (Nrf2) functions as an important upstream regulator of HO-1 and exhibits robust activity against SVCV infection. In this study, we further examined the antiviral activity of HO-1 against SVCV infection. The elevated expression of HO-1 was induced upon cobalt protoporphyrin (CoPP) treatment in EPC cells without affecting cell viability and thus inhibited SVCV replication in a dose dependent manner. Knocking down of HO-1 rescued SVCV replication. Thereby, the antiviral activity of ROS/Nrf2/HO-1 axis was confirmed in EPC cells. Furthermore, HO-1 enzymatic products CO, but not biliverdin, markedly inhibited SVCV replication via the activation of cyclic GMP/protein kinase G signaling pathway. Collectively, these findings suggest potential drug or therapy that induced the Nrf2/HO-1/CO/cGMP/PKG signaling pathway as a promising strategy for treating SVC.

Heme oxygenase-1 is a potent inhibitor of placental ischemia-mediated endothelin-1 production in cultured human glomerular endothelial cells.

Preeclampsia is a pregnancy-specific disorder of maternal hypertension and reduced renal hemodynamics linked to reduced endothelial function. Placental ischemia is thought to be the culprit of this disease, as it causes the release of factors like tumor necrosis factor (TNF)-α that induce vascular endothelin-1 (ET-1) production. Interestingly, placental ischemia-induced hypertension in rats [reduced uterine perfusion pressure (RUPP) model] is abolished by ETA receptor blockade, suggesting a critical role for ET-1. Although it has been found that systemic induction of heme oxygenase (HO)-1 is associated with reduced ET-1 production and attenuated hypertension, it is unclear whether HO-1 directly modulates the increased ET-1 response to placental factors. We tested the hypothesis that HO-1 or its metabolites inhibit ET-1 production in human glomerular endothelial cells induced by serum of RUPP rats or TNF-α. Serum (5%) from RUPP hypertensive (mean arterial blood pressure 119 ± 9 mmHg) vs. normotensive pregnant (NP, 101 ± 6 mmHg, P < 0.001) rats increased ET-1 production (RUPP 168.8 ± 18.1 pg/ml, NP 80.3 ± 22.7 pg/ml, P < 0.001, n = 12/group). HO-1 induction [25 µM cobalt photoporphyrin (CoPP)] abolished RUPP serum-induced ET-1 production (1.6 ± 0.8 pg/ml, P < 0.001), whereas bilirubin (10 µM) significantly attenuated ET-1 release (125.3 ± 5.2 pg/ml, P = 0.005). Furthermore, TNF-α-induced ET-1 production (TNF-α 31.0 ± 8.4 vs. untreated 7.5 ± 0.4 pg/ml, P < 0.001) was reduced by CoPP (1.5 ± 0.8 pg/ml, P < 0.001) and bilirubin (10.5 ± 4.3 pg/ml, P < 0.001). These results suggest that circulating factors released during placental ischemia target the maternal glomerular endothelium to increase ET-1, and that pharmacological induction of HO-1 or bilirubin could be a treatment strategy to block this prohypertensive pathway in preeclampsia.

Carbon monoxide and biliverdin suppress bovine viral diarrhoea virus replication.

Bovine viral diarrhoea virus (BVDV) causes significant economic losses to the cattle industry worldwide. Previously, we demonstrated that heme oxygenase-1 (HO-1) can inhibit BVDV replication via an unknown molecular mechanism. To elucidate the mechanism involved, we assess whether the HO-1 downstream metabolites carbon monoxide (CO), biliverdin (BV) and iron affect BVDV replication. We treated Madin-Darby bovine kidney (MDBK) cells with an exogenous CO donor, CORM-2. We found that CORM-2 but not its inactive form (iCORM-2) inhibited BVDV replication in a dose-dependent and time duration-dependent manner, suggesting a CO-specific mediation of the CORM-2 antiviral effect. Direct incubation of BVDV with high-dose CORM-2 reduced virus titres, suggesting that CORM-2 attenuates BVDV growth by both physically inactivating virus particles in the extracellular environment and affecting intracellular BVDV replication, but mainly via an intracellular mechanism. Exogenous BV treatment, both post-infection and co-incubation with BVDV, inhibited BVDV replication in a dose-dependent manner, indicating that BV has potent antiviral activity against BVDV. Direct incubation of BVDV with BV had no significant effect on virus titres, indicating that BV is not virucidal and attenuates BVDV growth by affecting intracellular BVDV replication. Furthermore, BV was found to affect BVDV penetration but not attachment. However, increased iron via addition of FeCl3 did not interfere with BVDV replication. Collectively, the results of the present study demonstrate that the HO-1 metabolites BV and CO, but not iron, inhibit BVDV replication. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of BVDV replication but also suggest potential new control measures for future BVDV infection.

Cell growth of immortalized arachnoid cells in the presence of fibroblasts and blood products.

OBJECT: The pathophysiology of non-obstructive hydrocephalus involves alteration in cerebrospinal fluid (CSF) pathways. The exact mechanism is unknown, but as arachnoid CSF egress is a major route of CSF removal, damage or alteration to the growth of arachnoid cells may influence the rate of CSF absorption. We investigated the effect of soluble factors secreted by fibroblasts and the presence of blood products on arachnoid cell growth. METHODS: An immortalized arachnoid cell line was developed and cells were grown on semipermeable membranes in a culture chamber. Arachnoid cells were plated in Transwells®, with fibroblasts separated from the arachnoid cells. Cell phenotype was analyzed and cell growth rates were determined by manual counts. Similar experiments were conducted with biliverdin, bilirubin, as well as fibroblast challenge. DNA content in the cell cultures was then determined as corroborative data. Cell counts for the additional arachnoid cell lines were calculated at each day and represented the controls. RESULTS: Cell counts increased with each time point. Arachnoid cells in the three experimental conditions showed a statistically significant decrease in cell counts for each day when compared to the control group. Post hoc analysis showed differences between the control and experimental conditions but no significant difference between groups. The DNA content for each experimental condition was reduced at all time points when compared to the control arachnoid cells, but only became statistically significant at day 7. CONCLUSION: Inflammation and hemorrhage are two common conditions associated with the development of hydrocephalus. The arachnoid membrane is exposed to fibroblasts and blood products (bilirubin, biliverdin) in these conditions, and their effect on arachnoid cell growth was studied. We have shown that arachnoid cell growth decreases in the presence of fibroblasts, bilirubin, and biliverdin. Given its intimate relationship with CSF, it is possible that the decreased growth of arachnoid cells may affect absorption and thus contribute to the development of hydrocephalus.

Haem oxygenase-1 polymorphisms can affect HCV replication and treatment responses with different efficacy in humanized mice.

BACKGROUND & AIMS: Enhancement of host anti-oxidant enzymes, such as haemoxygenase-1, may attenuate virus-mediated hepatocyte injury, while the induction of HO-1 by cobalt-protoporphyrin-IX (CoPP) administration, as the application of its haem degradation product biliverdin (BV), was shown to hinder HCV replication in vitro. In addition, (GT)n -repeats length in the polymorphic region of the HO-1 promoter may affect HO-1 expression and responsiveness to infection and disease severity. Aim of this study was to investigate the antiviral and hepatoprotective effects of CoPP-mediated HO-1 induction, alone or in combination with interferon alpha (peg-IFNα), in HCV-infected mice harbouring hepatocytes from donors with different HO-1-promoter polymorphisms. METHODS: Upon establishment of HCV infection, CoPP, BV and peg-IFNα were given alone or in combination. Viraemia changes and intrahepatic human gene expression were determined by qRT-PCR and immunohistochemistry. RESULTS: CoPP administration increased human HO-1 expression and significantly reduced viraemia, although changes correlated with promoter length (Δ0.5log and Δ2log reduction with medium- and short-polymorphism respectively). Polymorphisms did not influence BV-mediated antiviral effects (Δ1log). Notably, HO-1 induction attenuated basal HCV-driven enhancement of interferon genes and pro-inflammatory cytokines, both in cells with short- or medium-polymorphisms. Moreover, simultaneous administration of CoPP and peg-IFNα reduced viraemia even stronger (median 3log), whereas 1log viraemia reduction was determined in mice receiving peg-IFNα monotherapy. CONCLUSIONS: Although the protective function of HO-1 could be elicited in vivo with both host polymorphisms, the strength of HO-1 induction and suppression of HCV occurred in a polymorphism-dependent manner, indicating that host-genetic determinants may affect disease progression and infection outcome.

Human heme oxygenase 1 is a potential host cell factor against dengue virus replication.

Dengue virus (DENV) infection and replication induces oxidative stress, which further contributes to the progression and pathogenesis of the DENV infection. Modulation of host antioxidant molecules may be a useful strategy for interfering with DENV replication. In this study, we showed that induction or exogenous overexpression of heme oxygenase-1 (HO-1), an antioxidant enzyme, effectively inhibited DENV replication in DENV-infected Huh-7 cells. This antiviral effect of HO-1 was attenuated by its inhibitor tin protoporphyrin (SnPP), suggesting that HO-1 was an important cellular factor against DENV replication. Biliverdin but not carbon monoxide and ferrous ions, which are products of the HO-1 on heme, mediated the HO-1-induced anti-DENV effect by non-competitively inhibiting DENV protease, with an inhibition constant (Ki) of 8.55 ± 0.38 µM. Moreover, HO-1 induction or its exogenous overexpression, rescued DENV-suppressed antiviral interferon response. Moreover, we showed that HO-1 induction by cobalt protoporphyrin (CoPP) and andrographolide, a natural product, as evidenced by a significant delay in the onset of disease and mortality, and virus load in the infected mice's brains. These findings clearly revealed that a drug or therapy that induced the HO-1 signal pathway was a promising strategy for treating DENV infection.

Suppression of human alloreactive T cells by linear tetrapyrroles; relevance for transplantation.

The main limitation to successful transplantation is the antigraft response developed by the recipient immune system, and the adverse side effects of immunosuppressive agents which are associated with significant toxicity and counter indications such as infection and cancer. Furthermore, immunosuppressants do little to prevent ischemia-reperfusion injury during the transplantation procedure itself hence there is a growing need to develop novel immunosuppressive drugs specifically aimed at prolonging graft survival. Linear tetrapyrroles derived from the breakdown of mammalian heme have been shown in numerous studies to play a protective role in allograft transplantation and ischemia-reperfusion injury; however, commercial sources of these products have not been approved for use in humans. Plants and algae produce equivalent linear tetrapyrroles called bilins that serve as chromophores in light-sensing. One such marine-derived tetrapyrrole, phycocyanobilin (PCB), shows significant structural similarity to mammalian biliverdin (BV) and may prove to be a safer alternative for use in the clinic if it can exert direct effects on human immune cells. Using a mixed lymphocyte reaction, we quantified the allogeneic responses of recipient cells to donor cells and found that PCB, like BV, effectively suppressed proliferation and proinflammatory cytokine production. In addition, we found that BV and PCB can directly downregulate the proinflammatory responses of both innate dendritic cells and adaptive T cells. We therefore propose that PCB may be an effective therapeutic drug in the clinical setting of transplantation and may also have wider applications in regulating inappropriate inflammation.

Protective role of biliverdin against bile acid-induced oxidative stress in liver cells.

The accumulation of bile acids affects mitochondria causing oxidative stress. Antioxidant defense is accepted to include biotransformation of biliverdin (BV) into bilirubin (BR) through BV reductase α (BVRα). The mutation (c.214C>A) in BLVRA results in a non-functional enzyme (mutBVRα). Consequently, homozygous carriers suffering from cholestasis develop green jaundice. Whether BVRα deficiency reduces BV-dependent protection against bile acids is a relevant question because a screening of the mut-BLVRA allele (a) in 311 individuals in Greenland revealed that this SNP was relatively frequent in the Inuit population studied (1% a/a and 4.5% A/a). In three human liver cell lines an inverse correlation between BVRα expression (HepG2>Alexander>HuH-7) and basal reactive oxygen species (ROS) levels was found, however the ability of BV to reduce oxidative stress and cell death induced by deoxycholic acid (DCA) or potassium dichromate (PDC) was similar in these cells. The transduction of BVRα or mutBVRα in human placenta JAr cells with negligible BVRα expression or the silencing of endogenous BVRα expression in liver cells had no effect on DCA-induced oxidative stress and cell death or BV-mediated cytoprotection. DCA stimulated both superoxide anion and hydrogen peroxide production, whereas BV only inhibited the latter. DCA and other dihydroxy-bile acids, but not PDC, induced up-regulation of both BVRα and heme oxygenase-1 (HO-1) in liver cells through a FXR independent and BV insensitive mechanism. In conclusion, BV exerts direct and BVRα-independent antioxidant and cytoprotective effects, whereas bile acid accumulation in cholestasis stimulates the expression of enzymes favoring the heme biotransformation into BV and BR.

Biliverdin targets enolase and eukaryotic initiation factor 2 (eIF2α) to reduce the growth of intraerythrocytic development of the malaria parasite Plasmodium falciparum.

In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identified P. falciparum enolase (Pf enolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-length Plasmodium enolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV to P. falciparum enolase. Furthermore, addition of BV and haem targets the phosphorylation of Plasmodium falciparum eIF2α factor, an eukaryotic initiation factor phosphorylated by eIF2α kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2α phosphorylation pattern as a molecular mechanism for its action.

Biliverdin protects against cisplatin-induced apoptosis of renal tubular epithelial cells.

Biliverdin (BV) has long been thought to be a cytotoxic metabolic waste product. It has also been demonstrated to have important cytoprotective functions during oxidative stress. The present study aimed to examine the cytoprotective effect of BV on NRK-52E cells, a proximal tubular cell line derived from rat kidney. Cells were treated with 50 µmol/L cisplatin for 24 h (cisplatin group) or pre-treated with BV for 30 min, then with 50 µmol/L cisplatin for 24 h (cisplatin+BV group). Those given no treatment served as a control. Cell apoptosis was evaluated by flow cytometry and cell viability by Cell Counting Kit-8 (CCK-8). The protein expressions of cleaved caspase3, Bax and Bcl-2 were assessed by Western blotting. Reactive oxygen species (ROS) levels were measured using carboxydichlorodihydrofluorescein diacetate (H2DCF). The results showed that cisplatin induced the apoptosis of NRK-52E cells, decreased cell viability, and increased the formation of ROS by upregulating the expression of cleaved caspase3 and Bax and decreasing Bcl-2 protein expression. These effects could be significantly reversed by pretreatment with BV. It was concluded that BV can protect against cisplatin-induced cell apoptosis through the anti-oxidative effects.

The Effects of Heme Oxygenase By-Products on the Proliferation and Invasion of HUVECs, HTR-8/SVneo Cells, 3A(tPA 30-1) Cells, and HESCs Under Varying Oxygen Concentrations.

OBJECTIVE: Abnormal spiral artery remodeling during early pregnancy leads to preeclampsia. The proliferation and invasion of trophoblasts in pregnancy are important for spiral artery remodeling. This study examined whether heme oxygenase (HO) by-products (carbon monoxide biliverdin, and iron) play roles in regulating the restoration of proliferation and invasion of human umbilical vein endothelial cells (HUVECs), HTR-8/SV-neo cells originating from first-trimester trophoblasts, 3A(tPA 30-1) obtained from term trophoblasts, and human endometrial stromal cells (HESCs) inhibited by zinc protoporphyrin IX (Znpp-9). STUDY DESIGN: We explored whether HO by-products restored the proliferation and invasion of HUVECs, HTR-8/SVneo cells, 3A(tPA 30-1) cells, and HESCs inhibited by Znpp-9 depending on the oxygen concentration. RESULTS: Bilirubin promoted proliferation of HUVECs, HTR-8/SVneo cells, 3A(tPA-30-1) cells, and HESCs under both hypoxic and normoxic conditions. Biliverdin also promoted invasion of HUVECs, HTR-8/SVneo cells, 3A(tPA30-1) cells, and HESCs under both hypoxic and normoxic conditions. Carbon monoxide-releasing molecule 2 promoted the proliferation and invasion of specific cell types depending on the oxygen concentration. CONCLUSION: Our data suggest that HO by-products differentially stimulate the proliferation and invasion of cells involved in pregnancy maintenance. When HO by-products are considered to be stimulants during the invasion and proliferation of such cells, both target cells and the gestational period should be considered.

Induction of heme oxygenase-1 contributes to survival of Mycobacterium abscessus in human macrophages-like THP-1 cells.

Mycobacterium abscessus (M.abs) is a rapidly growing mycobacterial species that infects macrophages, and is an important pathogen in patients with cystic fibrosis. We studied the early stages of M.abs infection of macrophages, with emphasis on the role of heme-oxygenase-1 (HO-1) in this infection. THP-1 cells were activated using TPA into macrophage-like cells and infected with M.abs for different time points. M.abs infection robustly induced HO-1 expression in the THP-1 cells. Production of HO-1 was p38 MAPK-dependent, as p38 inhibitors suppressed HO-1 induction. Pretreatment with HO-1 inhibitors tin-protoporphyrin (SnPP) significantly inhibited M.abs growth inside macrophages. Furthermore, inhibiting HO-1 using HO-1 siRNA or the HO-1 upstream signaling molecule; Nrf2 using Nrf2 siRNA resulted in similar inhibition of M.abs. In contrast, inducing HO-1 did not increase M.abs intracellular growth above control. Products of HO-1 metabolism of heme are bilirubin, biliverdin, carbon monoxide (CO) and iron. The addition of either bilirubin or biliverdin, but not CO, completely restored the SnPP inhibitory effect and partially that with HO-1 siRNA. To understand the mechanisms, we used Syto-62 labeled M.abs to infect macrophages. Interestingly, HO-1 inhibition promoted M.abs-containing phagosome fusion with lysosomes, which should enhance M.abs killing. M.abs infection enhanced THP-1 ROS production as demonstrated by increased DHE, DCF fluorescence, and EPR signal. HO-1 inhibition further increased ROS production in infected macrophages. Our results indicate that HO-1 induction is important for M.abs growth during the early stages of infection, and that the HO-1 products bilirubin and biliverdin, perhaps through modulation of intracellular ROS levels, may be involved.