Critical role for the oligoadenylate synthetase/RNase L pathway in response to IFN-beta during acute ocular herpes simplex virus type 1 infection

We previously demonstrated that IFN-beta transgene treatment protects mouse trigeminal ganglia (TG) cells from acute HSV-1 infection in vitro. However, IFN-alpha6 transgene treatment does not provide protection against acute HSV-1 infection in vitro, even though equivalent levels of IFN are expressed with both transgene treatments. In the present study we show that IFN-beta transgene treatment before acute ocular HSV-1 infection protects mice from HSV-1-mediated mortality, whereas IFN-alpha6 transgene treatment does not reduce mortality. Treatment with the IFN-beta and IFN-alpha6 transgenes was associated with increased expression of oligoadenylate synthetase (OAS)1a mRNA in the eye. However, protein kinase R mRNA was not up-regulated in the eye. In TG, only IFN-beta transgene treatment reduced infectious virus levels. Furthermore, in the absence of a functional OAS pathway, corneal HSV-1 Ag expression was more widespread, and the ability of IFN-beta transgene treatment to reduce infectious HSV-1 in eyes and TG was lost. Along with selective up-regulation of OAS1a mRNA expression in TG from IFN-beta transgene-treated mice, we found increased levels of phospho-STAT1. Likewise, p38 MAPK phosphorylation was increased in TG from IFN-beta transgene-treated mice, compared with both IFN-alpha6 and vector-treated mice. We also observed a time-dependent increase in JNK phosphorylation in TG from IFN-beta transgene-treated vs IFN-alpha6 and vector-treated mice. Our results demonstrate that IFN-beta is a potent antiviral cytokine that exerts protection against ocular HSV-1 infection via selective up-regulation of OAS1a mRNA in TG and by altering the phosphorylation of proteins in antiviral signaling cascades.     

Distinctive roles for 2',5'-oligoadenylate synthetases and double-stranded RNA-dependent protein kinase R in the in vivo antiviral effect of an adenoviral vector expressing murine IFN-beta

To evaluate the anti-HSV-1 mechanisms of murine IFN-beta in ocular infection, mice were transduced with an adenoviral vector expressing murine IFN-beta (Ad:IFN-beta). Ocular transduction with Ad:IFN-beta resulted in enhanced survival following infection with HSV-1. The protective effect was associated with a reduction in 1) viral titer, 2) viral gene expression, 3) IFN-gamma levels, and 4) the percentage of CD8(+) T lymphocyte and NK cell infiltration in infected tissue. Expression of IFN-beta resulted in an elevation of the IFN-induced antiviral gene 2',5'-oligoadenylate synthetase (OAS1a) but not dsRNA-dependent protein kinase R (PKR) in the cornea and trigeminal ganglion (TG). Mice deficient in the downstream effector molecule of the OAS pathway, RNase L, were no more sensitive to ocular HSV-1 compared with wild-type controls in the TG based on measurements of viral titer. However, the efficacy of Ad:IFN-beta was transiently lost in the eyes of RNase L mice. By comparison, PKR-deficient mice were more susceptible to ocular HSV-1 infection, and the antiviral efficacy following transduction with Ad:IFN-beta was significantly diminished in the eye and TG. These results suggest that PKR is central in controlling ocular HSV-1 infection in the absence of exogenous IFN, whereas the OAS pathway appears to respond to exogenous IFN, contributing to the establishment of an antiviral environment in a tissue-restricted manner.     

Genetic control directed toward spontaneous IFN-alpha/IFN-beta responses and downstream IFN-gamma expression influences the pathogenesis of a murine psoriasis-like skin disease

Psoriasis is an inflammatory skin disease, onset and severity of which are controlled by multiple genetic factors; aberrant expression of and responses to several cytokines including IFN-alpha/IFN-beta and IFN-gamma are associated with this "type 1" disease. However, it remains unclear whether genetic regulation influences these cytokine-related abnormalities. Mice deficient for IFN regulatory factor-2 (IRF-2) on the C57BL/6 background (IRF-2(-/-)BN mice) exhibited accelerated IFN-alpha/IFN-beta responses leading to a psoriasis-like skin inflammation. In this study, we found that this skin phenotype disappeared in IRF-2(-/-) mice with the BALB/c or BALB/c x C57BL/6 F(1) backgrounds. Genome-wide scan revealed two major quantitative trait loci controlled the skin disease severity. Interestingly, these loci were different from that for the defect in CD4(+) dendritic cells, another IFN-alpha/IFN-beta-dependent phenotype of the mice. Notably, IFN-gamma expression as well as spontaneous IFN-alpha/IFN-beta responses were up-regulated several fold spontaneously in the skin in IRF-2(-/-)BN mice but not in IRF-2(-/-) mice with "resistant" backgrounds. The absence of such IFN-gamma up-regulation in IRF-2(-/-)BN mice lacking the IFN-alpha/IFN-beta receptor or beta(2)-microglobulin indicated that accelerated IFN-alpha/IFN-beta signals augmented IFN-gamma expression by CD8(+) T cells in the skin. IFN-gamma indeed played pathogenic roles as skin inflammation was delayed and was much more infrequent when IRF-2(-/-)BN mice lacked the IFN-gamma receptor. Our current study thus revealed a novel genetic mechanism that kept the skin immune system under control and prevented skin inflammation through regulating the magnitude of IFN-alpha/IFN-beta responses and downstream IFN-gamma production, independently of CD4(+) dendritic cells.     

Interferon-beta from melanoma cells suppresses the proliferations of melanoma cells in an autocrine manner

Interferon (IFN)-alpha and IFN-beta have been utilized in the treatment of melanoma as a form of cytokine therapy. While previous studies have demonstrated that melanocytes and melanoma cells produce a number of cytokines, it remains unclear whether or not melanocytes and melanoma cells per se produce IFN-alpha or IFN-beta. In the present study, we investigated the expression of IFN-alpha or IFN-beta in human melanocytes and five melanoma cell lines: G-361, C32TG, MMAc, MEWO and VMRC-MELG at both mRNA and protein levels. Both IFN-alpha and IFN-beta mRNA were detected in normal human melanocytes. Likewise, IFN-alpha mRNA was detected in all five melanoma cell lines. However, IFN-beta mRNA was only detected in one melanoma cell line, VMRC-MELG. When melanocytes and melanoma cells were treated with a potent IFN inducer, polyinosinic:polycytidylic acid (poly I:C), the mRNA expression of both IFN-alpha and IFN-beta was significantly upregulated. Poly I:C was not able to induce melanocytes or melanoma cells to produce detectable amounts of IFN-alpha protein, but able to induce a significant amount of IFN-beta in melanocytes and two of the melanoma cell lines: MMAc and VMRC-MELG. Moreover, similar to exogenous IFN-alpha and IFN-beta, poly I:C significantly inhibited the proliferation of all five melanoma cell lines. This suppressive effect was partially blocked by anti-IFN-beta antibody treatment in the IFN-beta-producing melanoma cell lines: MMAc and VMRC-MELG, but not in the non-IFN-beta-producing cell lines: G-361, C32TG and MEWO. Collectively, these studies have demonstrated for the first time that human melanocytes and melanoma cells produce IFN-beta. Furthermore, melanoma cells are capable of suppressing their own proliferation via secretion of endogenous IFN-beta. This finding may have important implications for melanoma therapy.