Expression of IFN-Inducible Genes with Antiviral Function OAS1 and MX1 in Health and under Conditions of Recurrent Herpes Simplex Infection

We studied the expression of IFN-inducible genes OAS1 and Mx1 in lysates of peripheral blood mononuclear cells from patients suffering from recurrent Herpes simplex infections in comparison with healthy people. To induce the expression of the studied genes, blood mononuclears were incubated with recombinant IFN-α2b in concentrations of 1, 10, and 100 U/ml for 3 h and then the content of the studied transcripts was evaluated. Relative expression of OAS1 and Mx1 in patients with recurrent forms of Herpes simplex both during the acute stage and clinical remission did not differ significantly from that in healthy people after stimulation with IFN-α2b in a concentration of 1 U/ml and in higher concentrations (10 and 100 U/ml). It was concluded that intracellular signal transduction in IFN-α-activated cells in vitro was not disturbed in patients with recurrent forms of Herpes simplex infection. Thus, the reported phenomenon of IFN-signalling distortion by Herpes simplex virus proteins observed in experiments on model cell lines infected with Herpes simplex virus was not confirmed in our experiments on peripheral blood mononuclear cells from patients with Herpes simplex infection.

Thirty years passed from the synthesis of the first representative of acyclic nucleosides that remain the standard treatment in infections caused by the Herpes simplex virus (HSV), but the problem of recurrent HSV infections remains not solved. High frequency of recurrences and secondary immunodeficiency that results from long-term virus persistence actualize studies of the mechanisms of viral reactivation and HSV escape from immune surveillance [1,2,[10][11][12][13][14]. Experiments on model cell lines showed that HSV proteins in infected cells inhibit transfer of the activation signal from IFN-α/β receptor to the nucleus, which leads to suppression of the cell response to IFN-α. This effect is associated, in particular, with disorders of Jak/ STAT signal pathway activation [3,4,6,8]. Insufficient production of type I IFN by mononuclear blood cells in patients with recurrent forms of HSV infection was reported not once [5,7,9]. However, clinicians know the cases of "clinical-laboratory discrepancy" phenomenon, i.e. severe course of Herpes simplex infection with high frequency of recurrencies against the background of not only normal, but even elevated parameters of interferon status. The causes of the ineffectiveness of IFN preparations and IFN inducers in some patients and their excellent therapeutic effect in others remain unclear. There are hundreds of type I IFN-inducible genes, but in the context of antiviral protection, genes of serine/threonine protein kinase R, Mx proteins (IFN-induced GTPases of the dynamin superfamily), and genes encoding 2'5'-oligonucleotide synthase. Based on our technical capacities we chose genes OAS1 and Mx1.
The aim of this study was to assess the integrity of IFN-signaling or to detect possible disorders in it for the expression of two IFN-inducible genes with antiviral function, OAS1 and Mx1, in mononuclear cells of patients suffering from recurrent forms of Herpes simplex infection in comparison with healthy people.

MATERIALS AND METHODS
The study included 15 subjects (age 19 to 64 years) with verified diagnosis of Herpes simplex on outpatient and hospital treatment at the Department of Immunotherapy and Allergology, Institute of Immunology, Federal Medical-Biological Agency of Russia. Inclusion criteria were the presence of 6 or more recurrences per year (i.e., frequent HSV reactivations) and the absence of other active inflammatory or infectious diseases at the time of the study. Venous blood was taken from all patients during the period of recurrence (no later than 48 h after appearance of the first vesicles) and during remission. The control group consisted of 15 healthy people aged 22 to 75 years. Criteria for inclusion into the control group were the absence of herpetic infection for at least the last year, the absence of acute infections for at least 1 month until the time of blood collection, and the absence of chronic inflammatory and autoimmune diseases (according to questionnaire survey). All patients and healthy volunteers included in the study gave written consent to participate in the study; the consent form was approved by the Interuniversity Ethics Committee of the Pharmaceutical and Medical Association. Venous heparinized blood samples (15 ml) were obtained from all participants.
Mononuclear cells (MNC) were isolated in a Ficoll-Urographin density gradient. For induction of OAS1 and Mx1 genes, the isolated MNC were cultured in 1.5-ml Eppendorf tubes (0.5×10 6 /ml; 0.5 ml per tube) with recombinant human IFN-α2b (Invitrogen) in concentrations of 1, 10, and 100 U/ml for 3 h in a CO 2 incubator (5% CO 2 , 37 o C). A control aliquot of the cell suspension was cultured without IFN-α. After incubation, the tubes were centrifuged, the supernatant was removed; the precipitate was resuspended and lysed with 500 μl TRI Reagent. The lysates were stored at -70 o C.
Total RNA was isolated from the lysate (500 μl) by phenol-chloroform extraction. Reverse transcription was performed using RevertAid First Strand cDNA Synthesis Kit #k1621 kit (Fermentas Life Sci-ences) according to manufacturer's instructions. The resulting cDNA was amplified by real-time PCR. Four samples were obtained from each patient and healthy donor: before stimulation and after stimulation with 1, 10, and 100 U/ml IFN-α. In each sample, the content of OAS1 and Mx1 mRNA was measured. β-Actin gene mRNA was used for standardization of the results. Samples of cDNA (1 μl) were added to wells of the plate (control wells contained 1 μl of MilliQ water) and mixed with 19 μl reagent solution containing 1 μl 20× mixture of primers and TaqMan probes, 10 μl Statistical processing of the results was conducted using SPSS 11.5 and Microsoft Excel software. The data was presented as the median (10th-90th percentile). Two groups were compared using the nonparametric Mann-Whitney U test.

RESULTS
In patients with recurrent forms of HSV infection during the exacerbation stage, the median of relative OAS1 mRNA level in MNC lysates after 3 h incubation with recombinant IFN-α in concentrations of 1, 10, and 100 U/ml was 33.9, 48, and 44.8, respectively. At the stage of clinical remission, this parameter tended to increase to 40.1, 56.6, and 81.7, respectively, but the changes did not reach statistical significance, probably due to broad scatter of the results. Neither during exacerbation, nor during clinical remission, the relative OAS1 mRNA content in IFN-α-activated MNK from patients differed significantly from that in healthy individuals. Significant differences were absent both in samples stimulated by recombinant IFN-α at a concentration of 1 U/ml and in samples stimulated by higher concentrations ( Table 1).
Analysis of Mx1 gene expression yielded similar results. In patients with recurrent forms of HSV infection during exacerbation stage, the relative level of Mx1 mRNA tended to decrease in comparison with that during remission, which also did not reach statistical significance, probably due to broad scatter of the results. However, neither during exacerbation, nor du ring clinical remission, the relative level of Mx1 mRNA in lysates of IFN-α-activated MNC from patients did not significantly differ from that in healthy people. No significant differences were found for all tested IFN-α concentrations (Table 2).
Thus, it can be concluded that intracellular signal transduction in IFN-α-activated cells is preserved in patients with recurrent forms of Herpes simplex, under the conditions of in vitro simulation. Our study carried out on peripheral blood MNC from patients with HSV infection does not confirm the reported phenomenon of IFN-signal distortion by HSV proteins observed in experiments on model cell lines infected with HSV. Our findings suggest that administration of IFN-α preparations and IFN inductors is advisable in complex therapy of recurrent forms of HSV infection, because IFN-signalling in this group of patients is preserved.