Pathology, organ distribution, and immune response after single and repeated intravenous injection of rats with clinical-grade parvovirus H1.

Parvovirus H1 (H1PV) is an autonomous parvovirus that is transmitted in rodent populations. Its natural host is rats. H1PV infection is nonpathogenic except in rat and hamster fetuses and newborns. H1PV infection of human cancer cells caused strong oncolytic effects in preclinical models. For a clinical trial of H1PV in patients with brain tumors, clinical-grade H1PV was produced according to Good Manufacturing Practices. This report focuses on results obtained after a single high-dose intravenous injection of highly purified H1PV in 30 rats and multiple (n = 17) intravenous injections at 3 dose levels in 223 rats. In both studies, no virus-related mortality or macroscopic organ changes related to H1PV occurred. Histopathology after multiple virus injections revealed minimal diffuse bile duct hyperplasia in livers of animals of the highest dose group and germinal center development in spleens of animals from the high-dose group. Liver changes were reversible within a 2-wk recovery period after the last injection. Hematology, blood chemistry, and coagulation analyses did not reveal significant toxicologic changes due to H1PV. Virus injection stimulated the production of IgG antibodies but did not alter mononuclear cell function or induce cytokine release. PCR analysis showed dose-dependent levels of viral genomes in all organs tested. The virus was excreted primarily through feces. These data provide important information regarding H1PV infection in its natural host. Due to the confirmation of the favorable safety profile of H1PV in a permissive animal model, a phase I/IIa clinical trial of H1PV in brain tumor patients could be initiated.

Structural and functional analysis of a nuclear localization signal in SOCS1.

Suppressor of cytokine signaling 1 (SOCS1) belongs to a family of genes involved in inducible feedback inhibition of janus kinases (JAKs) and signal transducers and activators of transcription (STATs) signaling pathway. Recently, we were able to show that SOCS1 surprisingly translocates to the nucleus due to the presence of a functional nuclear localization signal (NLS). However, the precise nature of the NLS remained ill-defined. Here we investigated further details of the SOCS1 NLS and analyzed its functional importance. We show that nuclear transport of SOCS1 particularly depends on the second cluster of basic amino acid residues within the NLS. Neither the first nor a nearby identified third cluster of basic amino acids were sufficient for mediating nuclear localization of SOCS1. Altering the subcellular localization of SOCS1 by mutating clusters of arginine residues within the NLS did not affect the inhibition of interferon mediated STAT1 tyrosine-phosphorylation, but surprisingly led to impaired inhibitory activity of STAT mediated reporter gene induction and IFN-gamma induced CD54 regulation. A SOCS-box deletion mutant (E176X) also had reduced inhibitory activity. In contrast, nuclear factor kappaB (NFkappaB) signaling was not affected by SOCS1 wt or mutants. Thus, SOCS1 may accomplish its inhibitory function in the IFN-pathway in part through nuclear localization.

Identification of a nuclear localization signal in suppressor of cytokine signaling 1.

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of janus kinase and signal transducer and activators of transcription signaling pathways. In addition, SOCS1 has been identified to regulate stability of nuclear NF-kappaB subunits. However, details about the regulation of the nuclear pool of SOCS1 are unknown. Using different experimental approaches, we observed that SOCS1 but no further SOCS family members localized to the nucleus when expressed in various cell lines. Nuclear transport was confirmed for endogenous SOCS1 in macrophages stimulated with IFN-gamma. Sequence analysis revealed a bipartite nuclear localization signal (NLS) located between the src-homology 2 (SH2) domain and the SOCS box of SOCS1. Deletion of this region, introduction of a series of R/A point mutations, or substitution of this sequence with the respective region of SOCS3 resulted in loss of nuclear localization. Fusion of the SOCS1-NLS to cytokine-inducible SH2 region containing protein (CIS) resulted in nuclear localization of this otherwise cytoplasmic protein. SOCS1 mutants with loss of nuclear localization were still effective in suppressing IFN-alpha-mediated STAT1 tyrosine phosphorylation. However, they showed decreased inhibition of IFN-gamma-mediated induction of CD54. The results identify a hitherto unknown transport of SOCS1 into the nucleus which extends the spectrum of SOCS1 inhibitory activity.

Regulation of innate immunity by suppressor of cytokine signaling (SOCS) proteins.

Innate immunity represents the first line of defense against invading pathogens. Toll-like receptors (TLRs) are important for activation of innate immunity. Moreover, cytokines mediate communication of cells and are necessary to mount an appropriately regulated immune response. However, activation of innate immunity has to be tightly controlled to avoid overshooting immune reactions. Suppressor of cytokine signaling (SOCS) proteins have been identified as inducible feedback inhibitors of cytokine receptors and have been shown to be of crucial importance for the limitation of inflammatory responses. In this review, we describe the role of SOCS proteins in macrophages and dendritic cells (DCs). Based on our own findings, we show that SOCS proteins are directly induced by stimulation of TLRs. However, SOCS proteins do not interfere with direct TLR signaling, but avoid overshooting activation by regulating paracrine IFN-beta signaling. In addition, SOCS proteins in macrophages and DCs regulate the sensitivity towards IFN-gamma and GM-CSF, thereby modulating anti-microbial activity of macrophages and differentiation of DCs. We discuss that SOCS induction can also be used by microbes to evade immune defense, and this is exemplified by the parasite Toxoplasma gondii which induces SOCS1 to inhibit IFN-gamma-mediated macrophage activation. Taken together, the findings indicate that SOCS proteins play an important role in the balanced activation of innate immunity during infectious encounter.

Microbial immune evasion employing suppressor of cytokine signaling (SOCS) proteins.

Cytokines mediate communication between cells of the immune system and are of crucial importance to induce an appropriately regulated immune response to invading pathogens. Cytokine receptor signaling has to be tightly controlled to balance anti-microbial and tissue-destructive effects, both of which are inherently associated with cytokine-mediated inflammation. Suppressor of cytokine signaling (SOCS) proteins have been identified as intracellular, inducible feedback inhibitors which limit the signal magnitude of cytokines employing Janus kinase (Jak) and signal transducer and activator of transcription (STAT) pathways. Interfering with cytokine receptor signaling has been shown to be a promising strategy used by various microbial pathogens to evade otherwise detrimental immune responses. To this, microbes make use of a variety of different means. Recent reports now indicate that certain bacteria, viruses and parasites have also learned to use the host's inhibitory SOCS proteins for manipulating cytokine receptor signaling, especially to circumvent the actions of interferon. Progress in the field of microbial immune evasion mediated by SOCS proteins is discussed in this review. Modulating the host's SOCS system therefore could also be a promising new approach for molecular therapeutic strategies.

Involvement of suppressors of cytokine signaling in toll-like receptor-mediated block of dendritic cell differentiation.

Dendritic cells (DCs) are important sentinels within innate immunity, monitoring the presence of infectious microorganisms. They operate in 2 different maturation stages, with transition from immature to mature DCs being induced by activation of toll-like receptors (TLRs). However, TLRs are also expressed on precursor cells of DCs. Here we analyzed the effects of TLR stimulation during the process of granulocyte-macrophage-colony-stimulating factor (GM-CSF)-mediated in vitro generation of immature DCs from precursor cells. We show that TLR triggering deviated phenotypic and functional differentiation from CD14+ monocytes to CD1a+ DCs. Similar results were obtained when differentiation of murine myeloid DCs from bone marrow cells was analyzed. The inhibitory effects were independent of soluble factors. TLR stimulation in DC precursor cells induced proteins of the suppressor of cytokine signaling family (SOCS), which correlated with loss of sensitivity to GM-CSF. Overexpression of SOCS-1 abolished GM-CSF signal transduction. Moreover, forced SOCS-1 expression in DC precursors mimicked the inhibitory effects on DC generation observed for TLR stimulation. The results indicate that TLR stimulation during the period of DC generation interferes with and deviates DC differentiation and that these effects are mediated particularly by SOCS-1.

Suppressor of cytokine signaling (SOCS) proteins indirectly regulate toll-like receptor signaling in innate immune cells.

Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-3 and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I interferon. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-3, or CIS on prototypical TLR target genes such as tumor necrosis factor-alpha. However, we found that TLR-2, TLR-3, TLR-4, and TLR-9 stimulation induced interferon-beta (IFN-beta), which is able to exert auto- and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-3 and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NF kappa B signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NF kappa B pathway.