SUMMARYType I interferons (IFNs) are produced early in response to viral infection and modulate adaptive immunity. Previously we demonstrated localized protection against murine cytomegalovirus (MCMV) infection in IFN DNA-inoculated mice. Here we examine the effect of seven IFN subtypes (IFNA1, A2, A4, A5, A6, A9 and B), administered by DNA inoculation, on systemic MCMV infection and myocarditis. IFN transgene expression altered the pathogenesis of MCMV infection with regard to virus titre and myocarditis. IFNA6 treatment reduced MCMV replication whilst IFNA5 and A2 enhanced virus replication. IFNA6, A9, and B treatment inhibited acute myocarditis. A T helper type 1-like, antibody and cytokine, response correlated with decreased virus titre and myocarditis. In addition, IFNA6 was able to reduce chronic cardiac inflammation. This research into the effectiveness of seven type I IFNs, using DNA gene therapy, highlights the need for correct subtype usage in the treatment of disease. We demonstrate effective subtypes for treatment in both the acute and chronic phases of MCMV infection and the resultant development of myocarditis.

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IntroductionErythroleukemias represent the highly malignant M6 subtype of acute myeloid leukemia. 1 The J2E cell line, immortalized at the proerythroblast/basophilic erythroblast stage of erythroid development, 2 induces a rapid and fatal erythroleukemia in mice. 3 However, while these cells are immortalized, they are still capable of normal signaling in response to erythropoietin (Epo) stimulation. They maintain all biologic responses to Epo by differentiating biochemically with hemoglobin production and undergoing cellular alterations with a percentage of cells enucleating to form mature reticulocytes. 2,4-7 J2E cells also display increased proliferation and enhanced viability in the absence of serum as a result of Epo stimulation. 2,4,5 Type I interferon (IFN) subtypes are clinically effective in the treatment of disease conditions such as hepatitis, hairy cell leukemia, condyloma acuminatum, multiple sclerosis, and Kaposi sarcoma. [8][9][10][11] These IFNs have also proven effective in the treatment of both myelogenous and metastatic tumors, 12,13 and IFN-␣ has the ability to normalize hemoglobin levels in anemic patients. 14 Subtype diversity for treatment is limited however to human IFN-␣2 (huIFN-␣2) 15 and huIFN-␤ (IFN-␤-1b; IFN-␤-1a).The type I IFNs have been attributed to multiple and diverse functions in the immune response. This multigene family has over 14 IFN-␣ subtypes in humans and 10 IFN-␣ subtypes in mice, with a single IFN-␤ subtype for each. The murine and human IFN gene families are highly analogous. 16 Induced early in the immune response, IFNs stimulate antiviral, 17,18 antiproliferative, 19 and apoptotic activity, 20 as well as have numerous immunomodulatory effects. Type I IFNs regulate dendritic cell major histocompatibility complex (MHC) expression and maturation, 21-24 activate natural killer (NK) cells, 25 induce bystander T-cell proliferation, 26 selectively induce clonal CD8 ϩ T-cell expansion, 26 and skew the immune response to a Th1-like response. 27,28 At the cellular level, type I IFN binds the IFN-␣ receptor 2 (IFNAR2) subunit 29 instigating its association with the IFNAR1 subunit. 30 The preassociated Jak kinases, Jak1 and Tyk2, are subsequently cross-phosphorylated. 31,32 Phosphorylation of signal transducers and activators of transcription 2 (STAT2) occurs, which forms a heterodimer with STAT1 and phosphorylation of the latter. Phosphorylation of other STATs in response to 33 STAT4, 34 STAT5, and STAT6. 35,36 Phosphorylation of STAT4 however is not generally reported in the murine system and is believed to be due to a microsatellite insertion in Stat2. 37 44 Alternatively, IFN-␣ activation of p42 MAPK is essential for cell proliferation. 45 Previously, J2E cells stimulated with mixed murine IFN-␣ (muIFN-␣), IFN-␣1, and IFN-␣4 differed in their antiproliferative capacity, 46 while levels of Epo-induced differentiation were maintained. 47 In this report, we investigated the differential antileukemic properties of a panel of 7 type I IFN subtypes (-␣1, -␣2, -␣4, -␣5, -␣6, -␣9,...

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The SOCS family of genes are negative regulators of cytokine signalling with SOCS-1 displaying tumor suppressor activity. SOCS-1, CIS and SOCS-3 have been implicated in the regulation of red blood cell production. In this study, a detailed examination was conducted on the expression patterns of these three SOCS family members in normal erythroid progenitors and a panel of erythroleukemic cell lines. Unexpectedly, differences in SOCS gene expression were observed during maturation of normal red cell progenitors, viz changes to CIS were inversely related to the alterations of SOCS-1 and SOCS-3. Similarly, these SOCS genes were differentially expressed in transformed erythoid cellserythroleukemic cells immortalized at an immature stage of differentiation expressed SOCS-1 and SOCS-3 mRNA constitutively, whereas in more mature cell lines SOCS-1 and CIS were induced only after exposure to erythropoietin (Epo). Significantly, when ectopic expression of the tyrosine kinase Lyn was used to promote differentiation of immature cell lines, constitutive expression of SOCS-1 and SOCS-3 was completely suppressed. Modulation of intracellular signalling via mutated Epo receptors in mature erythroleukemic lines also highlighted different responses by the three SOCS family members. Close scrutiny of SOCS-1 revealed that, despite large increases in mRNA levels, the activity of the promoter did not alter after erythropoietin stimulation; in addition, erythroid cells from SOCS-1À/À mice displayed increased sensitivity to Epo. These observations indicate complex, stagespecific regulation of SOCS genes during normal erythroid maturation and in erythroleukemic cells.

Summary Delivery of type I IFN transgenes by naked DNA immunization can protect against cytomegalovirus infection and myocarditis. Here, we investigate IFN transgene expression, antiviral efficacy, and immunomodulation of myocarditis using various treatment regimes in a mouse CMV model. In vivo expression of the IFN transgene was observed in the sera for 35 days post-DNA inoculation. Prophylactic IFN-A6 and IFN-B DNA treatment for 14 days prior to murine cytomegalovirus (MCMV) infection was more efficacious in significantly reducing viral titres, than 2 days prior to or 2 days post-virus infection. Similarly, IFN-A6 DNA treatment commencing 14 days prior to virus infection was superior in suppressing both acute and chronic myocarditis. Furthermore, reduction of autoantibody titres was more pronounced when IFN was administered 14 days prior to viral infection. Combinational IFN gene therapy was assessed for synergy between IFN subtypes. Combination treatment with either IFN-A6/A9 or IFN-A6/B greatly reduced spleen viral titres while IFN-A6/B and IFN-A9/B reduced virus replication in the liver. Only IFN-A6/A9 and IFN-A9/B reduced acute viral myocarditis, whereas IFNA6/B treatment was most efficacious for autoimmune chronic myocarditis. Finally, treatment with IFN-A6 DNA 2 weeks post-MCMV infection proved effective at inhibiting the development of chronic autoimmune myocarditis. These findings suggest that immunomodulation of both antiviral and autoimmune responses by IFN DNA immunization may be an avenue for improved viral immunotherapy.

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