ABSTRACT
Title
Bilirubin: an endogenous molecule with antiviral activity in vitro.
Authors
R. Santangelo1*, C. Mancuso2*, S. Marchetti1, E. Barone2, R. Siciliano2 and G. Fadda1
Institutes of 1Microbiology and 2Pharmacology, Catholic University School of Medicine, Rome, Italy.
*Equally contributed
Institutes of 1Microbiology and 2Pharmacology, Catholic University School of Medicine, Rome, Italy.
*Equally contributed
Abstract
The heme oxygenase/biliverdin reductase (HO/BVR) axis is the main metabolic pathway by which heme is degraded. The combined action of these enzymes converts heme into ferrous iron (FeII), carbon monoxide, and biliverdin-IX-alpha (biliverdin), which is the precursor of bilirubin-IX-alpha (bilirubin). For several years, the by-products of the HO/BVR axis were considered mere waste products, but in the past 2 decades, a number of investigators have focused their attention on both HO/BVR and their products in an attempt to elucidate their true biological functions. In 1987 the antioxidant properties of bilirubin were described and 6 years later carbon monoxide was proposed as an endogenous neuromodulator. Carbon monoxide and bilirubin also help maintain the cell’s redox equilibrium by activating important pro-survival signalling pathways, such as those involving the proto-oncogene Akt or the mitogen-activated protein kinase family, and by scavenging reactive oxygen and nitrogen species. These findings suggest that upregulation of the HO/BVR axis could be a useful mechanism for improving cells’ responses to stress. In addition, the up-regulation of the HO/BVR system was shown to play an important role against bacterial and viral infections. The overexpression of the inducible isoform of HO (HO-1) displayed a significant antiviral activity against hepatitis B and C virus (HBV and HCV, respectively) infections. With regard to the by-products of the HO/BVR system involved in this antiviral activity, biliverdin was shown to interfere with HCV and human herpes virus (HHV)-6 replication as well as to reduce the cytopathic effect of human immunodeficiency type 1 virus (HIV) in MT-4 cells. In addition, carbon monoxide inhibited the growth of Escherichia coli as Staphylococcus aureus and iron almost completely decreased HCV core mRNA and protein. Taken together these results underlined a major role for the HO/BVR system in antiviral and antibacterial defense. That said, it is noteworthy to mention that no data are available to date about the potential antiviral activity of bilirubin, the final product of heme metabolism through the HO/BVR system.
The aim of this work was to explore the antiviral activity of bilirubin in vitro. Since intracellular bilirubin exists in the free form whereas in the bloodstream the bile pigment is bound to human serum albumin the antiviral activity of both bilirubin and albumin-bound bilirubin were studied. The viral species against which bilirubin, alone and complexed with albumin, were tested are the human herpes simplex type 1 virus (HSV-1), a DNA virus which belong to the Herpesviridae family, and an Enterovirus which belong to the Picornaviridae family (RNA viruses). All these viral strains are responsible for important acute and chronic diseases in humans.
Bilirubin, at concentrations 1-10 µM, close to those found in blood and tissues, significantly reduced HSV-1 (1x105 plaque forming units) and Enterovirus (1x105 plaque forming units) replication in Hep-2 and Vero cell lines, respectively. Bilirubin inhibited viral replication when given cells 2 hours before, concomitantly and 2 hours after viral infection. Furthermore, bilirubin retained its antiviral activity even complexed with a saturating concentration of human serum-albumin (20 µM). Moreover, 10 µM bilirubin increased the formation of nitric oxide and the phosphorylation of JNK in Vero and Hep-2 cell lines, respectively, thus implying a role of these two pathways in the mechanism of antiviral activity of the bile pigment.
In conclusion, these results support the antiviral effect of bilirubin against HSV-1 and Enterovirus in vitro, and put the basis for further basic and clinical studies to understand the real role of bilirubin as an endogenous antiviral molecule.
The aim of this work was to explore the antiviral activity of bilirubin in vitro. Since intracellular bilirubin exists in the free form whereas in the bloodstream the bile pigment is bound to human serum albumin the antiviral activity of both bilirubin and albumin-bound bilirubin were studied. The viral species against which bilirubin, alone and complexed with albumin, were tested are the human herpes simplex type 1 virus (HSV-1), a DNA virus which belong to the Herpesviridae family, and an Enterovirus which belong to the Picornaviridae family (RNA viruses). All these viral strains are responsible for important acute and chronic diseases in humans.
Bilirubin, at concentrations 1-10 µM, close to those found in blood and tissues, significantly reduced HSV-1 (1x105 plaque forming units) and Enterovirus (1x105 plaque forming units) replication in Hep-2 and Vero cell lines, respectively. Bilirubin inhibited viral replication when given cells 2 hours before, concomitantly and 2 hours after viral infection. Furthermore, bilirubin retained its antiviral activity even complexed with a saturating concentration of human serum-albumin (20 µM). Moreover, 10 µM bilirubin increased the formation of nitric oxide and the phosphorylation of JNK in Vero and Hep-2 cell lines, respectively, thus implying a role of these two pathways in the mechanism of antiviral activity of the bile pigment.
In conclusion, these results support the antiviral effect of bilirubin against HSV-1 and Enterovirus in vitro, and put the basis for further basic and clinical studies to understand the real role of bilirubin as an endogenous antiviral molecule.