Last update and review: July 24, 2020.
Short summary.
Interferons induce expression of hundreds of antiviral genes. Interferons notably stimulate inducible nitric oxide synthase (NOS), and, thus, the production of large amounts of nitrogen oxyde (NO). A 1993 study by Karupiah et al. (1), that appears to be still factually correct, found that activity of iNOS is both necessary and sufficient for a substantial antiviral effect of IFN-gamma in vitro.
In this article we share our notes and several curated citations from the paper by Karupiah et al., 1993 (1), and from several other articles.
Read et al., 2019 (2):
Interferons (IFNs) are immunostimulatory cytokines secreted from infected cells and nearby immune cells that induce the expression of hundreds of antiviral genes (called interferon-stimulated genes (ISGs)). They possess diverse roles including chemoattraction, immune cell activation, and direct antiviral activity.
Karupiah et al., 1993 (1):
Interferons (IFNs) induce antiviral activity in many cell types. The ability of IFN-gamma to inhibit replication of ectromelia, vaccinia, and herpes simplex-1 viruses in mouse macrophages correlated with the cells’ production of nitric oxide (NO). Viral replication was restored in IFN-gamma-treated macrophages exposed to inhibitors of NO synthase. Conversely, epithelial cells with no detectable NO synthesis restricted viral replication when transfected with a complementary DNA encoding inducible NO synthase or treated with organic compounds that generate NO. In mice, an inhibitor of NO synthase converted resolving ectromelia virus infection into fulminant mousepox. Thus, induction of NO synthase can be necessary and sufficient for a substantial antiviral effect of IFN-gamma.
Interferon-gamma is capable of inducing iNOS and, thus, the production of large amounts of nitrogen oxyde (NO).
Karupiah et al., 1993 (1):
Several cytokines, including interferons (IFN-alpha, -beta, -gamma, and -omega) and tumor necrosis factors (TNF-alpha and -beta) display antiviral activity. The mechanisms of action of IFNs remain incompletely understood (as of the date of the publication in 1994).
The antimicrobial and antiproliferative actions of cytokines such as IFN-gamma are undergoing reassessment in light of their ability to induce the expression of iNOS, a gene encoding an isoform of nitric oxide synthase (NOS) that produces large amounts of the radical gas, NO, from a guanidino nitrogen of L-arginine.The inducibility of high-output NO production among many other cell types, such as hepatocytes, smooth and cardiac myocytes, keratinocytes, endothelium, mesangial cells, tumor cells, and some fibroblasts (2, 5), prompted the hypothesis that the induction of iNOS might also defend the host against the one class of pathogens that can infect all nucleated cells – viruses.
Karupiah et al., 1993 (1):
In vitro, IFN-gamma inhibits ectromelia virus (EV) replication in mouse RAW 264.7 macrophage-like cells and in primary mouse macrophages but not in L929 mouse fibroblasts or 293 human renal epithelial cells. The differential antiviral effect of IFN-gamma in these four cell types correlated with their ability to produce NO, measured as the stable oxidation product, nitrite (NO2-), see “Figure 1”.
Karupiah et al., 1993 (1):
In macrophages, the antiviral effect of IFN-gamma was substantially reversed by inclusion in the cultures of N(omega)-methyl-L-arginine (L-NMA), a guanidino-N-substituted L-arginine analog that acts as a competitive inhibitor of iNOS. In control macrophages, EV, the closely related vaccinia virus (VV), and herpes simplex virus type-1 (HSV-1) produced between 2.0 and 2.5 logl0 progeny virus over a 24-hour period. Treatment of the macrophages with IFN-gamma severely restricted replication of all three viruses. In the presence of L-NMA, IFN-gamma-induced inhibition of viral replication was reversed by 73 to 100%.
Karupiah et al., 1993 (1):
Because inhibition of iNOS blocked antiviral activity, we next tested whether NO, a product of iNOS, could confer antiviral activity.
Karupiah et al., 1993 (1):
These results support two conclusions. First, activity of iNOS is both necessary and sufficient for a substantial antiviral effect of IFN-gamma in vitro. Second, although iNOS has many potential enzymatic actions-deple tion of L-arginine and NADPH (reduced form of nicotinamide adenine dinucleotide phosphate), oxidation of tetrahydrobio pterin, production of hydrogen peroxide and citrulline, and reduction of cytochrome P-450 (2)-provision of NO alone is suffi cient to inhibit viral replication.
Karupiah et al., 1993 (1):
Finally, we tested the effect of an NOS inhibitor on viral replication in vivo. Inoculation of the virulent Moscow strain of EV into the footpad of C57BL/6 mice reproduces a natural, self-limited infection with a well-characterized immune response and pathology.
Of EV-infected control mice, 95% survived through day 14, by which time virus had been cleared from all organs examined.
Treatment of 20 EV-infected mice with L-NMA led to 30% mortality (mean time of death was 8.3 days after infection), increased titers of virus in spleen and liver in mice killed at 5 days after infection, and persistence of virus in mice killed at 14 days after infection, all hallmarks of fulminant mousepox.
These findings establish another mechanism through which IFN-gamma can exert an antiviral effect. Induction of iNOS is likely to play a role in the antiviral action of other cytokines besides IFN-gamma, as IFN-alpha and -betta, and TNF-alpha and -beta can synergize with other stimuli to induce iNOS expression.
More recent research seems to confirm the synergistic action of interferon-gamma with interferon-alpha and -beta in drinving macrophage-derived iNOS.
Bachmann et al., 2017 (3):
Whereas a crucial role for IFNγ concerning iNOS induction is established in diverse cell types including hepatocytes, information on the role of immunoregulatory type I IFN (including IFNα/β) as cofactor for induction of hepatocyte iNOS is currently lacking. Of note, IFNα/β is reported to efficiently drive monocyte/macrophage-derived iNOS in humans and mice.
IFN-alpha and -beta inhibited EV replication in primary mouse macrophages nearly as well as IFN-gamma, and this inhibition was not accompanied by NO2- production.
Karupiah et al., 1993 (1):
These results do not discount the participation of previously defined antiviral mechanisms of IFN-gamma, nor can all unexplained antiviral effects of cytokines be ascribed to the action of iNOS. For example, IFN-alpha and -beta inhibited EV replication in primary mouse macrophages nearly as well as IFN-gamma, and this inhibition was not accompanied by NO2- production nor was it reversed by L-NMA.
Karupiah et al., 1993 (1):
Advantages of NO as an antiviral agent may include (i) its ability to pass readily into neighboring cells, like some viruses but unlike antibody and complement; (ii) its action independent of immune recognition of the infected cell, in contrast to that of antiviral lymphocytes; and (iii) the likely multiplicity of its viral and virally exploited cellular targets, which may limit the capacity of viruses to develop resistance
Selected references:
1. Karupiah G, Xie QW, Buller RM, Nathan C, Duarte C, MacMicking JD. Inhibition of viral replication by interferon-gamma-induced nitric oxide synthase. Science. 1993;261(5127):1445-1448.
2. Scott A Read, Stephanie Obeid, Chantelle Ahlenstiel, Golo Ahlenstiel, The Role of Zinc in Antiviral Immunity, Advances in Nutrition, Volume 10, Issue 4, July 2019, Pages 696–710.
3. Bachmann M, Waibler Z, Pleli T, Pfeilschifter J, Mühl H. Type I Interferon Supports Inducible Nitric Oxide Synthase in Murine Hepatoma Cells and Hepatocytes and during Experimental Acetaminophen-Induced Liver Damage.Front Immunol. 2017;8:890. Published 2017 Jul 31.
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