STAT1 Isoforms Differentially Regulate NK Cell Maturation and Anti-tumor Activity.

Natural killer (NK) cells are important components of the innate immune defense against infections and cancers. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is essential for NK cell maturation and NK cell-dependent tumor surveillance. Two alternatively spliced isoforms of STAT1 exist: a full-length STAT1α and a C-terminally truncated STAT1ß isoform. Aberrant splicing is frequently observed in cancer cells and several anti-cancer drugs interfere with the cellular splicing machinery. To investigate whether NK cell-mediated tumor surveillance is affected by a switch in STAT1 splicing, we made use of knock-in mice expressing either only the STAT1α (Stat1α/α) or the STAT1ß (Stat1ß/ß ) isoform. NK cells from Stat1α/α mice matured normally and controlled transplanted tumor cells as efficiently as NK cells from wild-type mice. In contrast, NK cells from Stat1ß/ß mice showed impaired maturation and effector functions, albeit less severe than NK cells from mice that completely lack STAT1 (Stat1-/- ). Mechanistically, we show that NK cell maturation requires the presence of STAT1α in the niche rather than in NK cells themselves and that NK cell maturation depends on IFNγ signaling under homeostatic conditions. The impaired NK cell maturation in Stat1ß/ß mice was paralleled by decreased IL-15 receptor alpha (IL-15Rα) surface levels on dendritic cells, macrophages and monocytes. Treatment of Stat1ß/ß mice with exogenous IL-15/IL-15Rα complexes rescued NK cell maturation but not their effector functions. Collectively, our findings provide evidence that STAT1 isoforms are not functionally redundant in regulating NK cell activity and that the absence of STAT1α severely impairs, but does not abolish, NK cell-dependent tumor surveillance.

The effect of equine herpesvirus type 4 on type-I interferon signaling molecules.

Equine herpesvirus type 4 (EHV-4) is mildly pathogenic but is a common cause of respiratory disease in horses worldwide. We previously demonstrated that unlike EHV-1, EHV-4 is not a potent inducer of type-I IFN and does not suppress that IFN response, especially during late infection, when compared to EHV-1 infection in equine endothelial cells (EECs). Here, we investigated the impact of EHV-4 infection in EECs on type-I IFN signaling molecules at 3, 6, and 12 hpi. Findings from our study revealed that EHV-4 did not induce nor suppress TLR3 and TLR4 expression in EECs at all the studied time points. EHV-4 was able to induce variable amounts of IRF7 and IRF9 in EECs with no evidence of suppressive effect on these important transcription factors of IFN-α/ß induction. Intriguingly, EHV-4 did interfere with the phosphorylation of STAT1/STAT2 at 3 hpi and 6 hpi, less so at 12 hpi. An active EHV-4 viral gene expression was required for the suppressive effect of EHV-4 on STAT1/STAT2 phosphorylation during early infection. One or more early viral genes of EHV-4 are involved in the suppression of STAT1/STAT2 phosphorylation observed during early time points in EHV-4-infected EECs. The inability of EHV-4 to significantly down-regulate key molecules of type-I IFN signaling may be related to the lower severity of pathogenesis when compared with EHV-1. Harnessing this knowledge may prove useful in controlling future outbreaks of the disease.

The Fish-Specific Protein Kinase (PKZ) Initiates Innate Immune Responses via IRF3- and ISGF3-Like Mediated Pathways.

PKZ is a fish-specific protein kinase containing Zα domains. PKZ is known to induce apoptosis through phosphorylating eukaryotic initiation factor 2α kinase (eIF2α) in the same way as double-stranded RNA-dependent protein kinase (PKR), but its exact role in detecting pathogens remains to be fully elucidated. Herein, we have found that PKZ acts as a fish-specific DNA sensor by initiating IFN expression through IRF3- or ISGF3-like mediated pathways. The expression pattern of PKZ is similar to those of innate immunity mediators stimulated by poly (dA:dT) and poly (dG:dC). DNA-PKZ interaction can enhance PKZ phosphorylation and dimerization in vitro. These findings indicate that PKZ participates in cytoplasmic DNA-mediated signaling. Subcellular localization assays have also shown that PKZ is located in the cytoplasm, which suggests that PKZ acts as a cytoplasmic PRR. Meanwhile, co-IP assays have shown that PKZ can separately interact with IRF3, STING, ZDHHC1, eIF2α, IRF9, and STAT2. Further investigations have revealed that PKZ can activate IRF3 and STAT2; and that IRF3-dependent and ISGF3-like dependent mediators are critical for PKZ-induced IFN expression. These results demonstrate that PKZ acts as a special DNA pattern-recognition receptor, and that PKZ can trigger immune responses through IRF3-mediated or ISGF3-like mediated pathways in fish.

In vitro assessment of the biologic activity of interferon beta formulations used for the treatment of relapsing multiple sclerosis.

A new formulation (NF) of subcutaneous (sc) interferon (IFN) ß-1a was developed in an attempt to improve injection tolerability and immunogenicity. We compared antiviral and IFNß-stimulated gene (ISG) activities of IFNß-1a sc NF with IFNß-1a sc original formulation and IFNß-1b sc. When equivalent unit amounts were compared, the IFNß formulations demonstrated similar antiviral activity and induced similar levels of ISG mRNA. However, on a weight basis (ng/mL), significantly more IFNß-1b sc was needed to equal the antiviral activity of either IFNß-1a sc formulation, and both IFNß-1a sc formulations induced significantly higher levels of ISG mRNA than IFNß-1b sc.

Immunostimulatory effect of laminarin on RAW 264.7 mouse macrophages.

This study investigated the immunostimulatory effects of laminarin with respect to inflammatory mediators such as hydrogen peroxide, calcium, nitric oxide, various cytokines, transcription factors, and immune response gene in RAW 264.7 mouse macrophages. Laminarin did not reduce the cell proliferation of RAW 264.7 mouse macrophages at concentrations up to 500 µg/mL. Laminarin significantly increased the release of hydrogen peroxide, calcium, nitric oxide, monocyte chemotactic protein-1, vascular endothelial growth factor, leukemia inhibitory factor, and granulocyte-colony stimulating factor with enhancing expression of Signal Transducer and Activator of Transcription 1 (STAT1), STAT3, c-Jun, c-Fos, and cyclooxygenase-2 mRNA in RAW 264.7 cells. The results suggest that laminarin has immunostimulatory properties, strengthening immune reactions via the transcription factor pathway in macrophages.

Rotavirus and reovirus modulation of the interferon response.

The mammalian reoviruses and rotaviruses have evolved specific mechanisms to evade the Type I interferon (IFN) antiviral response. Rotavirus likely represses the IFN response by at least 4 mechanisms. First, the rotavirus protein NSP1, most likely functioning as an E3 ligase, can induce proteasome-dependent degradation of the transcription factors IRF3, IRF5, and IRF7 to prevent their induction of IFN. Second, NSP1 can induce proteasome-dependent degradation of the ubiquitin ligase complex protein beta-TrCP, resulting in stabilization of I kappaB and concomitant failure of virus to activate NF-kappaB for induction of IFN. Third, rotavirus may sequester NF-kappaB in viroplasms. And fourth, rotavirus can prevent STAT1 and STAT2 nuclear translocation. The predominant mechanism for rotavirus inhibition of the IFN response is likely both rotavirus strain-specific and cell type-specific. The mammalian reoviruses also display strain-specific differences in their modulation of the IFN response. Reovirus activates RIG-I and IPS-1 for phosphorylation of IRF3. Reovirus-induced activation of MDA5 also participates in induction if IFN-beta, perhaps through activation of NF-kappaB. Reovirus likely inhibits the IFN response by at least 3 virus strain-specific mechanisms. First, the reovirus mu2 protein can induce an unusual nuclear accumulation of IRF9 and repress IFN-stimulated gene (ISG) expression, most likely by disrupting IRF9 function as part of the heterotrimeric transcription factor complex, ISGF3. Second, the reovirus sigma 3 protein can bind dsRNA and prevent activation of the latent antiviral effector protein PKR. And third, genetic approaches have identified the reovirus lambda 2 and sigma 2 proteins in virus strain-specific modulation of the IFN response, but the significance remains unclear. In sum, members of the family Reoviridae have evolved a variety of mechanisms to subvert the host's innate protective response.

Calcineurin inhibitors down-regulate iNOS expression by destabilizing mRNA.

In inflammation, bacterial products and pro-inflammatory cytokines induce the expression of inducible nitric oxide synthase (iNOS) and formation of high amounts of nitric oxide (NO). In a number of inflammatory diseases NO has pro-inflammatory and cytotoxic effects. The aim of the present study was to investigate the effects of immunosuppressive drugs cyclosporin A (CsA), tacrolimus (FK-506) and pimecrolimus on NO production through iNOS pathway in activated macrophages and fibroblasts. Calcineurin inhibitors (CsA, FK-506 and pimecrolimus) inhibited NO production and iNOS expression in a concentration-dependent manner, CsA being more potent than FK-506 and pimecrolimus. No effect on the activation or activity of the transcription factors nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 1 (STAT-1) was found. CsA, FK-506 and pimecrolimus did not reduce iNOS mRNA levels when measured 6-8 h after the inflammatory stimulus, but significantly lower levels of iNOS mRNA were found after 24 h incubation. Also, in cells transfected with a luciferase gene under the control of 3' untranslated region (3'UTR) of iNOS, CsA reduced luciferase activity. In conclusion, the results suggest that calcineurin inhibitors cyclosporin A, tacrolimus (FK-506) and pimecrolimus inhibit iNOS expression and NO production in response to inflammatory stimuli by enhancing the decay of iNOS mRNA by a 3'UTR-dependent manner. The findings add our knowledge on the anti-inflammatory effects of CsA, FK-506 and pimecrolimus, and suggest that calcineurin may have a role in the regulation of iNOS expression.