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Abstract We describe an immunosuppressive peptide corresponding to the kinase inhibitory region (KIR) of the intracellular checkpoint protein suppressor of cytokine signaling 1 (SOCS-1) that binds to the phospho-tyrosine containing regions of the tyrosine kinases JAK2 and TYK2 and the adaptor protein MAL, and thereby inhibits signaling downstream from these signaling mediators. The peptide, SOCS1-KIR, is thus capable of downregulating overactive JAK/STAT or NF-kB signaling in somatic cells, including those in many compartments of the eye. Attachment of poly-arginine to this peptide (R9-SOCS1-KIR) allows it to penetrate the plasma membrane in aqueous media. R9-SOCS1-KIR was tested in ARPE-19 cells and was found to attenuate mediators of inflammation by blocking the inflammatory effects of IFNγ, TNFα, or IL-17A. R9-SOCS1-KIR and also protected against TNFα or IL-17A mediated damage to the barrier properties of ARPE-19 cells, as evidenced by immunostaining with the tight junction protein, zona occludin 1 (ZO-1), and measurement of transepithelial electrical resistance (TEER). Experimental autoimmune uveitis (EAU) was generated in B10. RIII mice using a peptide of interphotoreceptor retinal binding protein (IRBP161−180) as immunogen. Topical administration of R9-SOCS1-KIR, 2 days before (prophylactic), or 7 days after immunization (therapeutic) protected ocular structure and function as seen by fundoscopy, optical coherence tomography (OCT), and electroretinography (ERG). The ability R9-SOCS1-KIR to suppress ocular inflammation and preserve barrier properties of retinal pigment epithelium makes it a potential candidate for treatment of autoimmune uveitis. Highlights • The kinase inhibitory region of SOCS-1 linked to poly-arginine (R9-SOCS1-KIR) and its inactive control peptide were synthesized. • R9-SOCS1-KIR attenuated pro-inflammatory effects of IFNγ, TNFα, and IL-17 in ARPE-19 cells, thus showing a simultaneous inhibition Th1 and Th17 cell functions. • Damage to barrier properties of ARPE-19 cells caused by TNFα or IL-17 was prevented in the presence of R9-SOCS1-KIR. • Topical delivery of R9-SOCS1-KIR, 2 days before or 7 days after immunization prevented damage in a mouse model of experimental autoimmune uveitis. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have suggested that activated wild-type and mutant Janus kinase JAK2 play a role in the epigenetics of histone modification, where it phosphorylates histone H3 on tyrosine 41(H3pY41). We showed that type I IFN signaling involves activated TYK2 in the nucleus. ChIP-PCR demonstrated the presence of receptor subunits IFNAR1 and IFNAR2 along with TYK2, STAT1, and H3pY41 specifically at the promoter of the OAS1 gene in IFN treated cells. A complex of IFNAR1, TYK2, and STAT1α was also shown in the nucleus by immunoprecipitation. IFN treatment was required for TYK2 activation in the nucleus. The presence of IFNAR1, IFNAR2, and activated STAT1 and STAT2, as well as the type I IFN in the nucleus of treated cells was confirmed by the combination of Western blotting and confocal microscopy. Trimethylated histone H3 lysine 9 underwent demethylation and subsequent acetylation specifically in the region of the OAS1 promoter. Resultant N-terminal truncated IFN mimetics functioned intracellularly as antivirals as well as therapeutics against experimental allergic encephalomyelitis without the undesirable side effects that limit the therapeutic efficacy of IFNβ in treatment of multiple sclerosis. The findings indicate that IFN signaling is complex like that of steroid signaling. [Copyright &y& Elsevier]

The maintenance of immune homeostasis requires regulatory T cells (Treg cells). Here we found that Treg cell-specific ablation of Ubc13, a Lys63 (K63)-specific ubiquitin-conjugating enzyme, caused aberrant T cell activation and autoimmunity. Although Ubc13 deficiency did not affect the survival of Treg cells or expression of the transcription factor Foxp3, it impaired the in vivo suppressive function of Treg cells and rendered them sensitive to the acquisition of T helper type 1 (TH1) cell- and interleukin 17 (IL-17)-producing helper T (TH17) cell-like effector phenotypes. This function of Ubc13 involved its downstream target, the kinase IKK. The Ubc13-IKK signaling axis controlled the expression of specific Treg cell effector molecules, including IL-10 and SOCS1. Collectively, our findings suggest that the Ubc13-IKK signaling axis regulates the molecular program that maintains Treg cell function and prevents Treg cells from acquiring inflammatory phenotypes. [ABSTRACT FROM AUTHOR]

Suppressor of cytokine signaling 1-deficient (SOCS1-/-) mice, which are lymphopenic, die <3 wk after birth of a T cell-mediated autoimmune inflammatory disease characterized by leukocyte infiltration and destruction of vital organs. Notably, Foxp3+ regulatory T cells (Tregs) have been shown to be particularly potent in inhibiting inflammation-associated autoimmune diseases. We observed that SOCS1-/- mice were deficient in peripheral Tregs despite enhanced thymic development. The adoptive transfer of SOCS1-sufficient Tregs, CD4+ T lymphocytes, or administration of SOCS1 kinase inhibitory region (KIR), a peptide that partially restores SOCS1 function, mediated a statistically significant but short-term survival of SOCS1-/- mice. However, the adoptive transfer of SOCS1-sufficient CD4+ T lymphocytes, combined with the administration of SOCS1-KIR, resulted in a significant increase in the survival of SOCS1-/- mice both short and long term, where 100% death occurred by day 18 in the absence of treatment. Moreover, the CD4+/SOCS1-KIR combined therapy resulted in decreased leukocytic organ infiltration, reduction of serum IFN-γ, and enhanced peripheral accumulation of Foxp3+ Tregs in treated mice. These data show that CD4+/SOCS1-KIR combined treatment can synergistically promote the long-term survival of perinatal lethal SOCS1-/- mice. In addition, these results strongly suggest that SOCS1 contributes to the stability of the Foxp3+ Treg peripheral population under conditions of strong proinflammatory environments. [ABSTRACT FROM AUTHOR]

Abstract: We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The β-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The results suggest a novel role for activated JAKs in epigenetic events for specific gene activation. [Copyright &y& Elsevier]

Abstract: Suppressors of cytokine signaling (SOCS) negatively regulate the immune response, primarily by interfering with the JAK/STAT pathway. We have developed a small peptide corresponding to the kinase inhibitory region (KIR) sequence of SOCS-1, SOCS1-KIR, which inhibits kinase activity by binding to the activation loop of tyrosine kinases such as JAK2 and TYK2. Treatment of SJL/J mice with SOCS1-KIR beginning 12days post-immunization with myelin basic protein (MBP) resulted in minimal symptoms of EAE, while most control treated mice developed paraplegia. SOCS1-KIR treatment suppressed interleukin-17A (IL-17A) production by MBP-specific lymphocytes, as well as MBP-induced lymphocyte proliferation. When treated with IL-23, a key cytokine in the terminal differentiation of IL-17-producing cells, MBP-sensitized cells produced IL-17A and IFNγ; SOCS1-KIR was able to inhibit the production of these cytokines. SOCS1-KIR also blocked IL-23 and IL-17A activation of STAT3. There is a deficiency of SOCS-1 and SOCS-3 mRNA expression in CD4+ T cells that infiltrate the CNS, reflecting a deficiency in regulation. Consistent with therapeutic efficacy, SOCS1-KIR reversed the cellular infiltration of the CNS that is associated with EAE. We have shown here that a SOCS-1 like effect can be obtained with a small functional region of the SOCS-1 protein that is easily produced. [Copyright &y& Elsevier]

The suppressor of cytokine signaling 1 (SOCS-1) protein modulates cytokine signaling by binding to and inhibiting the function of Janus kinases (JAKs), ErbB, and other tyrosine kinases. We have developed a small tyrosine kinase inhibitor peptide (Tkip) that binds to the autophosphorylation site of tyrosine kinases and inhibits activation of STAT transcription factors. We have also shown that a peptide corresponding to the kinase-inhibitory region of SOCS-1, SOCS1-KIR, similarly interacts with the activation loop of JAK2 and blocks STAT activation. Poxviruses activate cellular tyrosine kinases, such as ErbB-1 and JAK2, in the infection of cells. We used the pathogenesis of vaccinia virus in C57BL/6 mice to determine the ability of the SOCS-1 mimetics to protect mice against lethal vaccinia virus infection. Injection of mice intraperitoneally with Tkip or SOCS1-KIR containing a palmitate for cell penetration, before and at the time of intranasal challenge with 2 × 106 PFU of vaccinia virus, resulted in complete protection at 100 μg. Initiation of treatment 1 day postinfection resulted in 80% survival. Administration of SOCS-1 mimetics by the oral route also protected mice against lethal effects of the virus. Both SOCS1-KIR and Tkip inhibited vaccinia virus transcription and replication at early and possibly later stages of infection. Vaccinia virus-induced phosphorylation of ErbB-1 and JAK2 was inhibited by the mimetics. Protected mice mounted a strong humoral and cellular response to vaccinia virus. The use of SOCS-1 mimetics in the treatment of poxvirus infections reveals an endogenous regulatory system that previously was not known to have an antiviral function. [ABSTRACT FROM AUTHOR]

Abstract: Interferon (IFN) γ’s ability to localise in the nucleus and function in gene activation has been known for some time, although the role of the conventional nuclear transporting importin molecules is unclear. Here, we demonstrate for the first time the direct recognition of IFNγ and an IFNγ mimetic peptide by IMPα and the IMPα/β heterodimer, where the IFNγ mimetic shows higher affinity. Significantly, this correlates well both with in vivo ability to target green fluorescent protein to the nucleus in transfected cells as determined by quantitative confocal laser scanning microscopy, as well as GAS promoter activity of a luciferase reporter. This has important implications for IFNγ’s anti-viral action, and the potential use of the IFNγ mimetic in antiviral therapies. Structured summary: MINT-6478573, MINT-6478550, MINT-6478516: IFN gamma (uniprotkb:P01580) directly interacts (MI:0407) with IMP beta (uniprotkb:Q6GTI5) and IMP alpha (uniprotkb:P52293) by enzyme linked immunosorbent assay (MI:0411) MINT-6478503, MINT-6478490: IFN gamma (uniprotkb:P01580) directly interacts (MI:0407) with IMP alpha (uniprotkb:P52293) by enzyme linked immunosorbent assay (MI:0411) MINT-6478601: T-ag (uniprotkb:P03070) directly interacts (MI:0407) with IMP beta (uniprotkb:Q6GTI5) and IMP alpha (uniprotkb:P52293) by enzyme linked immunosorbent assay (MI:0411) MINT-6478541: T-ag (uniprotkb:P03070) directly interacts (MI:0407) with IMP alpha (uniprotkb:P52293) by enzyme linked immunosorbent assay (MI:0411) [Copyright &y& Elsevier]

The staphylococcal enterotoxin superantigens are among the most potent T cell stimulators known. They have been shown to alter the course of disease in experimental allergic encephalomyelitis, an animal model for multiple sclerosis (MS). We have previously demonstrated that two of the staphylococcal enterotoxins, SEA and SEB, are able to reactivate paralysis in PL/J mice which had been immunized with myelin basic protein (MBP) and resolved an initial episode of paralysis. In PL/J mice, Ac1–11 is the dominant encephalitogenic determinant of MBP. We hypothesized that superantigen reactivation of experimental allergic encephalomyelitis (EAE) may result in the spreading of T cell specificities for other epitopes of MBP. PL/J mice which had resolved an initial episode of EAE were treated with SEA and developed a second episode of paralysis. At the onset of symptoms, mice were sacrificed and splenocytes were stimulated in vitro with a panel of MBP peptides. EAE reactivation by SEA resulted in the spreading of T cell specificites to residues 100 to 120 of MBP. While intramolecular spreading did occur, spreading to other antigens did not, as evidenced by the lack of response to a proteolipid protein (PLP) peptide and heat shock protein 60 (hsp 60). To further characterize the epitope MBP 100–120, PL/J mice were immunized with MBP 100–120. No initial development of disease was observed. However, administration of SEA 2 weeks after MBP 100–120 immunization resulted in the onset of paralysis. In addition to a proliferative response to MBP 100–120, these mice also exhibited a proliferative response to the flanking MBP peptides 81–100 and 120–140. Thus, SEA is able to induce intramolecular epitope spreading in PL/J mice after reactivation of EAE. [Copyright &y& Elsevier]

Several blinding diseases affecting the retina and optic nerve are exacerbated by or caused by dysregulated inflammation and oxidative stress. These diseases include uveitis, age related macular degeneration, diabetic retinopathy and glaucoma. Consequently, despite their divergent symptoms, treatments that reduce oxidative stress and suppress inflammation may be therapeutic. The production of inflammatory cytokines and their activities are regulated by a class of proteins termed Suppressors of Cytokine Signaling (SOCS). SOCS1 and SOCS3 are known to dampen signaling via pathways employing Janus kinases and signal transducer and activator of transcription proteins (JAK/STAT), Toll-like Receptors (TLR), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen activated kinase (MAPK) and NLR family pyrin domain containing 3 (NLRP3). We have developed cell-penetrating peptides from the kinase inhibitory region of the SOCS1 and SOCS3 (denoted as R9-SOCS1-KIR and R9-SOCS3-KIR) and tested them in retinal pigment epithelium (RPE) cells and in macrophage cell lines. SOCS-KIR peptides exhibited anti-inflammatory, anti-oxidant and anti-angiogenic properties. In cell culture, both Th1 and Th17 cells were suppressed together with the inhibition of other inflammatory markers. We also observed a decrease in oxidants and a simultaneous rise in neuroprotective and anti-oxidant effectors. In addition, treatment prevented the loss of gap junction proteins and the ensuing drop in transepithelial electrical resistance in RPE cells. When tested in mouse models by eye drop instillation, they showed protection against autoimmune uveitis, as a prophylactic as well as a therapeutic. Mice with endotoxin-induced uveitis were protected by eye drop administration as well. R9-SOCS3-KIR was particularly effective against the pathways acting through STAT3, e.g. IL-6 and VEGF-A mediated responses that lead to macular degeneration. Eye drop administration of R9-SOCS3-KIR stimulated production of antioxidant effectors and reduced clinical symptoms in mouse model of oxidative stress that replicates the RPE injury occurring in AMD. Because these peptides suppress multiple pathogenic stimuli and because they can be delivered topically to the cornea, they are attractive candidates for therapeutics for uveitis, macular degeneration, diabetic retinopathy and glaucoma., Competing Interests: CA and HJ are listed as inventors in a patent held by the University of Florida for the technology involving R9-SOCS3-KIR peptide. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ahmed, Johnson and Lewin.)

Purpose: Inflammation and oxidative stress contribute to age-related macular degeneration (AMD) and other retinal diseases. We tested a cell-penetrating peptide from the kinase inhibitory region of an intracellular checkpoint inhibitor suppressor of cytokine signaling 3 (R9-SOCS3-KIR) peptide for its ability to blunt the inflammatory or oxidative pathways leading to AMD., Methods: We used anaphylatoxin C5a to mimic the effect of activated complement, lipopolysaccharide (LPS), and tumor necrosis factor alpha (TNFα) to stimulate inflammation and paraquat to induce mitochondrial oxidative stress. We used a human retinal pigment epithelium (RPE) cell line (ARPE-19) as proliferating cells and a mouse macrophage cell line (J774A.1) to follow cell propagation using microscopy or cell titer assays. We evaluated inflammatory pathways by monitoring the nuclear translocation of NF-κB p65 and mitogen-activated protein kinase p38. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the induction of inflammatory markers. In differentiated ARPE-19 monolayers, we evaluated the integrity of tight junction proteins through microscopy and the measurement of transepithelial electrical resistance (TEER). We used intraperitoneal injection of sodium iodate in mice to test the ability of R9-SOC3-KIR to prevent RPE and retinal injury, as assessed by fundoscopy, optical coherence tomography, and histology., Results: R9-SOCS3-KIR treatment suppressed C5a-induced nuclear translocation of the NF-kB activation domain p65 in undifferentiated ARPE-19 cells. TNF-mediated damage to tight junction proteins in RPE, and the loss of TEER was prevented in the presence of R9-SOCS3-KIR. Treatment with the R9-SOCS3-KIR peptide blocked the C5a-induced expression of inflammatory genes. The R9-SOCS3-KIR treatment also blocked the LPS-induced expression of interleukin-6, MCP1, cyclooxygenase 2, and interleukin-1 beta. R9-SOCS3-KIR prevented paraquat-mediated cell death and enhanced the levels of antioxidant effectors. Daily eye drop treatment with R9-SOCS3-KIR protected against retinal injury caused by i.p. administration of sodium iodate., Conclusions: R9-SOCS3-KIR blocks the induction of inflammatory signaling in cell culture and reduces retinal damage in a widely used RPE/retinal oxidative injury model. As this peptide can be administered through corneal instillation, this treatment may offer a convenient way to slow down the progression of ocular diseases arising from inflammation and chronic oxidative stress., (Copyright © 2023 Molecular Vision.)

The field of antiviral therapeutics is fixated on COVID19 and rightly so as the fatalities at the height of the pandemic in the United States were almost 1,000,000 in a twelve month period spanning parts of 2020/2021. A coronavirus called SARS-CoV2 is the causative virus. Development of a vaccine through molecular biology approaches with mRNA as the inducer of virus spike protein has played a major role in driving down mortality and morbidity. Antivirals have been of marginal value in established infections at the level of hospitalization. Thus, the current focus is on early symptomatic infection of about the first five days. The Pfizer drug paxlovid which is composed of nirmatrelvir, a peptidomimetic protease inhibitor of SARS-CoV2 Mpro enzyme, and ritonavir to retard degradation of nirmatrelvir, is the current FDA recommended treatment of early COVID19. There is no evidence of broad antiviral activity of paxlovid against other diverse viruses such as the influenza virus, poxviruses, as well as a host of respiratory viruses. Although type I interferons (IFNs) are effective against SARS-CoV2 in cell cultures and in early COVID19 infections, they have not been broadly recommended as therapeutics for COVID19. We have developed stable peptidomimetics of both types I and II IFNs based on our noncanonical model of IFN signaling involving the C-terminus of the IFNs. We have also identified two members of intracellular checkpoint inhibitors called suppressors of cytokine signaling (SOCS), SOCS1 and SOCS3 (SOCS1/3), and shown that they are virus induced intrinsic virulence proteins with activity against IFN signaling enzymes JAK2 and TYK2. We developed a peptidomimetic antagonist, based on JAK2 activation loop, against SOCS1/3 and showed that it synergizes with the IFN mimetics for potent broad spectrum antiviral activity without the toxicity of intact IFN molecules. IFN mimetics and the SOCS1/3 antagonist should have an advantage over currently used antivirals in terms of safety and potency against a broad spectrum of viruses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Johnson and Ahmed.)

Suppressors of Cytokine Signaling (SOCS) are intracellular proteins that negatively regulate the induction of cytokines. Amongst these, SOCS1 and SOCS3 are particularly involved in inhibition of various interferons. Several viruses have hijacked this regulatory pathway: by inducing SOCS1and 3 early in infection, they suppress the host immune response. Within the cell, SOCS1/3 binds and inhibits tyrosine kinases, such as JAK2 and TYK2. We have developed a cell penetrating peptide from the activation loop of the tyrosine kinase, JAK2 (residues 1001-1013), denoted as pJAK2 that acts as a decoy and suppresses SOCS1 and 3 activity. This peptide thereby protects against several viruses in cell culture and mouse models. Herein, we show that treatment with pJAK2 inhibited the replication and release of the beta coronavirus HuCoV-OC43 and reduced production of the viral RNA, as measured by RT-qPCR, Western blot and by immunohistochemistry. We confirmed induction of SOCS1 and 3 in rhabdomyosarcoma (RD) cells, and this induction was suppressed by pJAK2 peptide. A peptide derived from the C-terminus of IFNα (IFNα-C) also inhibited replication of OC43. Furthermore, IFNα-C plus pJAK2 provided more potent inhibition than either peptide alone. To extend this study to a pandemic beta-coronavirus, we determined that treatment of cells with pJAK2 inhibited replication and release of SARS-CoV-2 in Calu-3 cells. We propose that these peptides offer a new approach to therapy against the rapidly evolving strains of beta-coronaviruses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ahmed, Grams, Bloom, Johnson and Lewin.)

The staphylococcal enterotoxins (SEs) are classified as superantigens due to their potent stimulation of the immune system resulting in T cell activation and prodigious cytokine production and toxicity. This study examined the ability of superantigens to induce prophylactic antiviral activity in vivo and in vitro and evaluated potential superantigen mimetic peptides. Prophylactic treatment of mice in vivo with intraperitoneal injections of SE superantigens SEA and SEB (both at 20  μ g/day for 3 days) prevented encephalomyocarditis virus (EMCV)-induced lethality in 100% and 80% of mice, respectively, as compared with control saline-treated groups in which EMCV was lethal to all mice. Furthermore, SEA (2  μ g/mL) and SEB (1  μ g/mL) induced antiviral activity in mouse splenocytes to produce an antiviral factor since their supernatant prevented EMCV lysis of L929 cells in tissue culture. It was found that superantigens do not directly prevent EMCV infection, but rather indirectly through inducing interferon gamma (IFN γ ) production in cells as the antiviral factor. Evaluation of various superantigen mimetic peptides showed that one peptide (SEA3) had superantigen-like activity by inducing IFN γ production in cells but without the cellular proliferation, as associated with superantigens. However, the induction of IFN γ activation by the SEA3 peptide was not as pronounced, and took a much higher peptide concentration, when compared with the parent superantigen. If the negative side effects of superantigens can be eliminated, their beneficial properties can be harnessed for prophylactic treatment of viral infections and other pathologies requiring a robust immune response.

Purpose: Uveitis is an ocular inflammation that can affect individuals of all ages and is a major cause of blindness. We have tested the therapeutic efficacy of a cell penetrating peptide from the kinase inhibitory region of suppressor of cytokine signaling 1, denoted as R9-SOCS1-KIR., Methods: We stimulated J774A.1 cells with lipopolysaccharide (LPS) in the presence of R9-SOCS1-KIR or its inactive control peptide. Effect on inflammatory pathways was followed by the nuclear translocation of nuclear factor κB p65 subunit and phosphorylated-p38. Synthesis of inflammatory markers induced by LPS was tested by reverse transcriptase polymerase chain reaction, Western blot analysis, and ELISA of cell supernatants. We monitored effects on the barrier properties of a differentiated ARPE-19 monolayer treated with LPS. We treated C57BL/6 mice topically with either R9-SOCS1-KIR or vehicle and injected their eyes intravitreally with LPS. Eyes were analyzed by fundoscopy, fluorescein angiography, optical coherence tomography, histology, Western blotting, multiplex enzyme-linked immunosorbent assay, and flow cytometry., Results: Treatment with R9-SOCS1-KIR resulted in suppression of signaling through nuclear factor κB and p-p38 pathways. R9-SOCS1-KIR suppressed the expression of inflammatory genes, the secretion of inflammatory makers such as nitric oxide, and IL-1β induced by LPS. Increased permeability of retinal pigment epithelial cell monolayers was prevented. Corneal administration of R9-SOCS1-KIR blocked the acute inflammation observed in LPS-injected mouse eyes., Conclusions: Treatment with R9-SOCS1-KIR alleviated the inflammatory responses in cell culture. Topical delivery of this peptide on mouse eyes protected against LPS-induced damage., Translational Relevance: Topical delivery of R9-SOCS1-KIR peptide allows the patient to self-administer the drug, while preventing any systemic effects on unrelated organs.

The suppressor of cytokine signaling (SOCS) family of intracellular checkpoint inhibitors has received little recognition compared to other checkpoint inhibitors. Two members of this family, SOCS1 and SOCS3, are indispensable, since SOCS1 knockout in mice results in neonatal death due to interferon gamma (IFNγ) induced inflammatory disease, and SOCS3 knockout leads to embryonic lethality. We have shown that SOCS1 and SOCS3 (SOCS1/3) function as virus induced intrinsic virulence factors for influenza A virus, EMC virus, herpes simplex virus 1 (HSV-1), and vaccinia virus infections. Other viruses such as pathogenic pig enteric coronavirus and coronavirus induced severe acute respiratory syndrome (SARS) spike protein also induce SOCS virus intrinsic virulence factors. SOCS1/3 exert their viral virulence effect via inhibition of type I and type II interferon (IFN) function. Specifically, the SOCS bind to the activation loop of receptor-associated tyrosine kinases JAK2 and TYK2 through the SOCS kinase inhibitory region (KIR), which inhibits STAT transcription factor activation by the kinases. Activated STATs are required for IFN function. We have developed a small peptide antagonist of SOCS1/3 that blocks SOCS1/3 inhibitory activity and prevents virus pathogenesis. The antagonist, pJAK2(1001-1013), is comprised of the JAK2 activation loop, phosphorylated at tyrosine 1007 with a palmitate for cell penetration. The remarkable thing about SOCS1/3 is that it serves as a broad, simple tool of perhaps most pathogenic viruses to avoid innate host IFN defense. We suggest in this Perspective that SOCS1/3 antagonist is a simple counter measure to SOCS1/3 and should be an effective mechanism as a prophylactic and/or therapeutic against the COVID-19 pandemic that is caused by coronavirus SARS-CoV2., (Copyright © 2020 Johnson, Lewin and Ahmed.)

A small group of only seven transcription factors known as STATs (signal transducer and activator of transcription) are considered to be canonical determinants of specific gene activation for a plethora of ligand/receptor systems. The activation of STATs involves a family of four tyrosine kinases called JAK kinases. JAK1 and JAK2 activate STAT1 in the cytoplasm at the heterodimeric gamma interferon (IFN γ ) receptor, while JAK1 and TYK2 activate STAT1 and STAT2 at the type I IFN heterodimeric receptor. The same STATs and JAKs are also involved in signaling by functionally different cytokines, growth factors, and hormones. Related to this, IFN γ -activated STAT1 binds to the IFN γ -activated sequence (GAS) element, but so do other STATs that are not involved in IFN γ signaling. Activated JAKs such as JAK2 and TYK2 are also involved in the epigenetics of nucleosome unwrapping for exposure of DNA to transcription. Furthermore, activated JAKs and STATs appear to function coordinately for specific gene activation. These complex events have not been addressed in canonical STAT signaling. Additionally, the function of noncoding enhancer RNAs, including their role in enhancer/promoter interaction is not addressed in the canonical STAT signaling model. In this perspective, we show that JAK/STAT signaling, involving membrane receptors, is essentially a variation of cytoplasmic nuclear receptor signaling. Focusing on IFN signaling, we showed that ligand, IFN receptor, the JAKs, and the STATs all undergo endocytosis and ATP-dependent nuclear translocation to promoters of genes specifically activated by IFNs. We argue here that the vacuolar ATPase (V-ATPase) proton pump probably plays a key role in endosomal membrane crossing by IFNs for receptor cytoplasmic binding. Signaling of nuclear receptors such as those of estrogen and dihydrotestosterone provides templates for making sense of the specificity of gene activation by closely related cytokines, which has implications for lymphocyte phenotypes.

The canonical model of cytokine signaling via the JAK/STAT pathway dominates our view of signal transduction but provides no insight into the significance of the simultaneous presence of activated JAKs and STATs in the nucleus of cells treated with cytokines. Such a mechanistic shortcoming challenges the usefulness of the model in its present form. Focusing on the interferon (IFN) cytokines, we have developed a noncanonical model of IFN signaling that naturally connects activated JAKs and STATs at or near response elements of genes that are activated by the IFNs. Specifically, cells treated with IFN γ showed association of activated STAT1 α and JAK2 at the GAS element of genes activated by IFN γ . For IFN α treated cells, the association involved activated STAT1 α and TYK2 JAK kinase at the ISRE promoter. The power of the noncanonical model is that it provides mechanistic insight into specific gene activation at the level of the associated epigenetics, akin to that of steroid/steroid receptor signaling., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

We have developed an antagonist to suppressor of cytokine signaling 1 (SOCS1), pJAK2(1001-1013), which corresponds to the activation loop of the Janus kinase JAK2, which is the binding site for the kinase inhibitory region (KIR) of SOCS1. Internalized pJAK2(1001-1013) inhibits SOCS1 and SOCS3. SOCS1 has been shown to be an influenza virus-induced virulence factor that enhances infection of cells. The antagonist was protective in cell culture and in influenza virus PR8 lethally infected C57BL/6 mice. The SOCS antagonist also prevented adverse morbidity as assessed by parameters, such as weight loss and drop in body temperature, and showed potent induction of both the cellular and humoral immune responses to the influenza virus candidate universal antigen matrix protein 2 (M2e). The SOCS antagonist, thus, protected mice against lethal influenza virus infection and possessed potent adjuvancy against the M2e candidate influenza virus universal vaccine antigen.

Suppressors of cytokine signaling (SOCS) are inducible intracellular proteins that play essential regulatory roles in both immune and non-immune function. Of the eight known members, SOCS1 and SOCS3 in conjunction with regulatory T cells play key roles in regulation of the immune system. Molecular tools such as gene transfections and siRNA have played a major role in our functional understanding of the SOCS proteins where a key functional domain of 12-amino acid residues called the kinase inhibitory region (KIR) has been identified on SOCS1 and SOCS3. KIR plays a key role in inhibition of the JAK2 tyrosine kinase, which in turn plays a key role in cytokine signaling. A peptide corresponding to KIR (SOCS1-KIR) bound to the activation loop of JAK2 and inhibited tyrosine phosphorylation of STAT1α transcription factor by JAK2. Cell internalized SOCS1-KIR is a potent therapeutic in the experimental allergic encephalomyelitis (EAE) mouse model of multiple sclerosis and showed promise in a psoriasis model and a model of diabetes-associated cardiovascular disease. By contrast, a peptide, pJAK2(1001-1013), that corresponds to the activation loop of JAK2 is a SOCS1 antagonist. The antagonist enhanced innate and adaptive immune response against a broad range of viruses including herpes simplex virus, vaccinia virus, and an EMC picornavirus. SOCS mimetics and antagonists are thus potential therapeutics for negative and positive regulation of the immune system.

The classical canonical model of interferon (IFN) signaling focuses solely on the activation of STAT transcription factors, which limits the model in terms of specific gene activation, associated epigenetic events, and IFN mimetic development. Accordingly, we have developed a noncanonical model of IFN signaling and report the development of short type I IFN peptide mimetic peptides based on the model. The mimetics, human IFNα1(152-189), human IFNβ(150-187), and ovine IFNτ(156-195) are derived from the C-terminus of the parent IFNs and function intracellularly based on the noncanonical model. Vaccinia virus produces a decoy IFN receptor (B18R) that inhibits type I IFN, but the IFN mimetics bypass B18R for effective antiviral activity. By contrast, both parent IFNs and mimetics inhibited vesicular stomatitis virus. The mimetics also possessed anti-tumor activity against murine melanoma B16 tumor cells in culture and in mice, including synergizing with suppressor of cytokine signaling 1 antagonist. Finally, the mimetics were potent therapeutics against experimental allergic encephalomyelitis, a mouse model of multiple sclerosis. The mimetics lack toxic side effects of the parent IFNs and, thus, are a potent therapeutic replacement of IFNs as therapeutics.

The canonical model of interferon (IFN) signaling focuses solely on the activation of STAT transcription factors which, according to the model, are initiated by the singular event of cross-linkage of the receptor extracellular domain by the IFN. The IFN has no further function beyond this. The model thus provides no approach to circumventing poxviruses decoy receptors that compete with the IFN receptors for IFNs. This simple event has allowed smallpox virus to decimate human populations throughout the ages. We have developed a noncanonical model of IFN signaling that has resulted in the development of small peptide mimetics to both types I and II IFNs. In this report, we focus on a type I IFN mimetic at positions 152 to 189, IFN-α1(152-189), which corresponds to the C terminus of human IFN-α1. This mimetic functions intracellularly and is thus not recognized by the B18R vaccinia virus decoy receptor. Mimetic synthesized with an attached palmitate (lipo-) for cell penetration protects mice from a lethal dose of vaccinia virus, while the parent IFN-α1 is ineffective. Unlike IFN-α1, the mimetic does not bind to the B18R decoy receptor. It further differs from the parent IFN in that it lacks the toxicity of weight loss and bone marrow suppression in mice while at the same time possessing a strong adjuvant effect on the immune system. The mimetic is thus an innate and adaptive immune regulator that is evidence of the dynamic nature of the noncanonical model of IFN signaling, in stark contrast to the canonical or classical model of signaling., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Regulatory T cells (Tregs) play an indispensable role in the prevention of autoimmune disease, as interferon gamma (IFNγ) mediated, lethal auto-immunity occurs (in both mice and humans) in their absence. In addition, Tregs have been implicated in preventing the onset of autoimmune and auto-inflammatory conditions associated with aberrant IFNγ signaling such as type 1 diabetes, lupus, and lipopolysaccharide (LPS) mediated endotoxemia. Notably, suppressor of cytokine signaling-1 deficient (SOCS1(-/-)) mice also succumb to a lethal auto-inflammatory disease, dominated by excessive IFNγ signaling and bearing similar disease course kinetics to Treg deficient mice. Moreover SOCS1 deficiency has been implicated in lupus progression, and increased susceptibility to LPS mediated endotoxemia. Although it has been established that Tregs and SOCS1 play a critical role in the regulation of IFNγ signaling, and the prevention of lethal auto-inflammatory disease, the role of Treg/SOCS1 cross-talk in the regulation of IFNγ signaling has been essentially unexplored. This is especially pertinent as recent publications have implicated a role of SOCS1 in the stability of peripheral Tregs. This review will examine the emerging research findings implicating a critical role of the intersection of the SOCS1 and Treg regulatory pathways in the control of IFN gamma signaling and immune system function.

Signaling by cytokines such as the interferons (IFNs) involves Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) transcription factors. The beauty of the classical model of JAK-STAT signaling is its simplicity in that JAK-activated STATs in the nucleus are responsible for specific gene activation. The fact that many ligands, growth factors, and hormones use the same STAT transcription factors, but exert different functions at the level of the cell, tissue, and organ would suggest significant shortcomings in the classical model. Our studies have resulted in the development of a non-canonical, more complex model of IFN signaling that bears a striking resemblance to that of steroid hormone (SH)/steroid receptor (SR) signaling. Thus, both types I and II IFN signaling involves nuclear translocation of complexed ligand, receptor, activated JAKs, and activated STATs to the promoters of the genes that are specifically activated by the IFNs, where they are involved in specific gene activation and epigenetic remodeling. Receptor intracellular domains play an important role in binding the C-terminus of the IFNs, which is the basis for our development of IFN mimetics. The IFN mimetics are not recognized by poxvirus decoy receptors, since the decoy receptors compete for extracellular binding and not intracellular binding. Further, the type I IFN mimetics provide therapeutic protection against experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis, without the side effects. Extracellular receptor binding by intact IFN is the primary reason for undesirable side effects of flu-like symptoms, bone-marrow suppression, and weight loss. The non-canonical model of IFN signaling thus provides insight into the specificity of such signaling and a mechanism for development of IFN mimetics. It is our contention that this model applies to other cytokines.

The classical model of cytokine signaling dominates our view of specific gene activation by cytokines such as the interferons (IFNs). The importance of the model extends beyond cytokines and applies to hormones such as growth hormone (GH) and insulin, and growth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FGF). According to this model, ligand activates the cell via interaction with the extracellular domain of the receptor. This results in activation of receptor or receptor-associated tyrosine kinases, primarily of the Janus activated kinase (JAK) family, phosphorylation and dimerization of the signal transducer and activator of transcription (STAT) transcription factors, which dissociate from the receptor cytoplasmic domain and translocate to the nucleus. This view ascribes no further role to the ligand, JAK kinase, or receptor in either specific gene activation or the associated epigenetic events. The presence of dimeric STATs in the nucleus essentially explains it all. Our studies have resulted in the development of a non-canonical, more complex model of IFNγ signaling that is akin to that of steroid hormone (SH)/steroid receptor (SR) signaling. We have shown that ligand, receptor, activated JAKs, and STATs are associated with specific gene activation, where the receptor subunit IFNGR1 functions as a co-transcription factor and the JAKs are involved in associated epigenetic events. We found that the type I IFN system functions similarly. The fact that GH receptor, insulin receptor, EGF receptor, and FGF receptor undergo nuclear translocation upon ligand binding suggests that they may also function similarly. The SH/SR nature of type I and II IFN signaling provides insight into the specificity of signaling by members of cytokine families. The non-canonical model could also provide better understanding to more complex cytokine families such as those of IL-2 and IL-12, whose members often use the same JAKs and STATs, but also have different functions and properties.

Many cytokines, hormones and growth factors use the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway for cell signalling and specific gene activation. In the classical model, ligand is said to interact solely with the receptor extracellular domain, which triggers JAK activation of STATs at the receptor cytoplasmic domain. Activated STATs are then said to carry out nuclear events of specific gene activation. Given the limited number of STATs (seven) and the activation of the same STATs by cytokines with different functions, the mechanism of the specificity of their signalling is not obvious. Focusing on IFNγ (interferon γ), we have shown that ligand, receptor and activated JAKs are involved in nuclear events that are associated with specific gene activation, where the receptor subunit IFNGR1 (IFNγ receptor 1) functions as a transcription/co-transcription factor and the JAKs are involved in key epigenetic events. RTKs (receptor tyrosine kinases) such as EGFR [EGF (epidermal growth factor) receptor] and FGFR [FGF (fibroblast growth factor) receptor] also undergo nuclear translocation in association with their respective ligands. EGFR and FGFR, like IFNGR1, have been shown to function as transcription/co-transcription factors. The RTKs also regulate other kinases that have epigenetic effects. Our IFNγ model, as well as the RTKs EGFR and FGFR, have similarities to that of steroid receptor signalling. These systems consist of ligand-receptor-co-activator complexes at the genes that they activate. The co-activators consist of transcription factors and kinases, of which the latter play an important role in the associated epigenetics. It is our view that signalling by cytokines such as IFNγ is but a variation of specific gene activation by steroid hormones.

The mechanism of specific gene activation by cytokines that use JAK/STAT signalling pathway is unknown. There are four different types of JAKs and seven different types of STATs. In the classical model of signaling, ligand interacts solely with the receptor extracellular domain, which triggers JAK activation at the receptor cytoplasmic domain. Activated STATs are then said to carry out nuclear events of specific gene activation, including associated epigenetic changes that cause heterochromatin destabilization. Ligand, receptor, and JAKs play no further role in the classical model. Given the limited number of STATs and the activation of the same STATs by cytokines with different functions, the mechanism of the specificity of their signalling is not obvious. Focusing on gamma interferon (IFNγ), we have shown that ligand, receptor, and activated JAKs are involved in nuclear events that are associated with specific gene activation. In this model, receptor subunit IFNGR1 functions as a transcription/cotranscription factor and the JAKs are involved in key epigenetic events that are required for specific gene activation. The model has implications for gene activation in cancer as well as stem cell differentiation.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated neuroinflammatory disease that is often used as a model of multiple sclerosis. EAE can follow either relapsing-remitting (RR) or chronic (CH) courses, yet the factors responsible for differentially inducing these forms of disease remain largely unknown. Proinflammatory cytokines play an important role in EAE, and signaling by these cytokines can be negatively regulated by the suppressor of cytokine signaling 1 protein (SOCS1). We assessed if differential expression of SOCS1 could contribute to the clinical course of RR and CH forms of EAE induced in the same mouse strain (C57BL/6) using the same myelin antigen (myelin oligodendrocyte glycoprotein). We show that SOCS1 mRNA levels are significantly elevated in the spinal cord in early stages of both RR- and CH-EAE. SOCS1 protein is highly expressed in immune cells in EAE lesions in the spinal cord, with expression predominantly localized within macrophages. Importantly, the number of Mac-1(+) macrophages expressing SOCS1 at the peak stage of RR disease is three-fold greater than in CH-EAE. Furthermore, the macrophage effector molecule iNOS, whose expression is regulated by SOCS1, is significantly reduced at the peak of RR- versus CH-EAE. Finally, the administration of a SOCS1-mimetic peptide reduces disease severity in the CH-EAE model. Thus, the differential expression of SOCS1 may contribute to the development of RR and CH forms of EAE., (© 2010 Wiley-Liss, Inc.)

Suppressors of cytokine signaling (SOCSs) are negative regulators of both innate and adaptive immunity via inhibition of signaling by cytokines such as type I and type II IFNs. We have developed a small peptide antagonist of SOCS-1 that corresponds to the activation loop of JAK2. SOCS-1 inhibits both type I and type II IFN activities by binding to the kinase activation loop via the kinase inhibitory region of the SOCS. The antagonist, pJAK2(1001-1013), inhibited the replication of vaccinia virus and encephalomyocarditis virus in cell culture, suggesting that it possesses broad antiviral activity. In addition, pJAK2(1001-1013) protected mice against lethal vaccinia and encephalomyocarditis virus infection. pJAK2(1001-1013) increased the intracellular level of the constitutive IFN-beta, which may play a role in the antagonist antiviral effect at the cellular level. Ab neutralization suggests that constitutive IFN-beta may act intracellularly, consistent with recent findings on IFN-gamma intracellular signaling. pJAK2(1001-1013) also synergizes with IFNs as per IFN-gamma mimetic to exert a multiplicative antiviral effect at the level of transcription, the cell, and protection of mice against lethal viral infection. pJAK2(1001-1013) binds to the kinase inhibitory region of both SOCS-1 and SOCS-3 and blocks their inhibitory effects on the IFN-gamma activation site promoter. In addition to a direct antiviral effect and synergism with IFN, the SOCS antagonist also exhibits adjuvant effects on humoral and cellular immunity as well as an enhancement of polyinosinic-polycytidylic acid activation of TLR3. The SOCS antagonist thus presents a novel and effective approach to enhancement of host defense against viruses.

Keratinocytes are important for the acute phase of HSV-1 infection and subsequent persistence in sensory nervous tissue. In this study, we showed that keratinocytes (HEL-30) were refractory to IFN-gamma induction of an antiviral state to HSV-1 infection, while IFN-gamma did induce an antiviral state in fibroblasts (L929). This led us to examine the possible role of suppressor of cytokine signaling-1 (SOCS-1) in this refractiveness. RT-PCR analysis of SOCS-1 mRNA expression in HSV-1-infected cells showed a 4-fold increase for keratinocytes while having a negligible effect on fibroblasts. A similar pattern was observed at the level of SOCS-1 protein induction. Activation of STAT1alpha in keratinocytes was inhibited by HSV-1 infection. A direct effect of HSV-1 on the SOCS-1 promoter was shown in a luciferase reporter gene assay. We have developed a small peptide antagonist of SOCS-1, pJAK2(1001-1013), that had both an antiviral effect in keratinocytes against HSV-1 as well as a synergistic effect on IFN-gamma induction of an antiviral state. HSV-1 ICP0 mutant was inhibited by IFN-gamma in HEL-30 cells and was less effective than wild-type virus in induction of SOCS-1 promoter. We conclude that SOCS-1 plays an important role in the inhibition of the antiviral effect of IFN-gamma in keratinocytes infected with HSV-1. The use of SOCS-1 antagonist to abrogate this refractiveness could have a transformational effect on therapy against viral infections.

Suppressor of cytokine signaling (SOCS)-1 protein modulates signaling by IFN-gamma by binding to the autophosphorylation site of JAK2 and by targeting bound JAK2 to the proteosome for degradation. We have developed a small tyrosine kinase inhibitor peptide (Tkip) that is a SOCS-1 mimetic. Tkip is compared in this study with the kinase inhibitory region (KIR) of SOCS-1 for JAK2 recognition, inhibition of kinase activity, and regulation of IFN-gamma-induced biological activity. Tkip and a peptide corresponding to the KIR of SOCS-1, ((53))DTHFRTFRSHSDYRRI((68)) (SOCS1-KIR), both bound similarly to the autophosphorylation site of JAK2, JAK2(1001-1013). The peptides also bound to JAK2 peptide phosphorylated at Tyr(1007), pJAK2(1001-1013). Dose-response competitions suggest that Tkip and SOCS1-KIR similarly recognize the autophosphorylation site of JAK2, but probably not precisely the same way. Although Tkip inhibited JAK2 autophosphorylation as well as IFN-gamma-induced STAT1-alpha phosphorylation, SOCS1-KIR, like SOCS-1, did not inhibit JAK2 autophosphorylation but inhibited STAT1-alpha activation. Both Tkip and SOCS1-KIR inhibited IFN-gamma activation of Raw 264.7 murine macrophages and inhibited Ag-specific splenocyte proliferation. The fact that SOCS1-KIR binds to pJAK2(1001-1013) suggests that the JAK2 peptide could function as an antagonist of SOCS-1. Thus, pJAK2(1001-1013) enhanced suboptimal IFN-gamma activity, blocked SOCS-1-induced inhibition of STAT3 phosphorylation in IL-6-treated cells, enhanced IFN-gamma activation site promoter activity, and enhanced Ag-specific proliferation. Furthermore, SOCS-1 competed with SOCS1-KIR for pJAK2(1001-1013). Thus, the KIR region of SOCS-1 binds directly to the autophosphorylation site of JAK2 and a peptide corresponding to this site can function as an antagonist of SOCS-1.

We have developed small peptide mimetics of IFN-gamma that can bypass the poxvirus virulence factor B8R protein, which binds to intact IFN-gamma and prevents its interaction with receptor extracellular domain. Thus, these peptides inhibit vaccinia virus replication in cell culture where intact IFN-gamma is ineffective. We demonstrate here that the mouse IFN-gamma-mimetic peptide, IFN-gamma(95-132), protects C57BL/6 mice against overwhelming lethal vaccinia virus infection. The mimetic peptide was synthesized with an attached lipophilic group for penetration of cell plasma membrane. Injection of mimetic i.p. before and at the time of intranasal (10(6) PFU) or i.p. (10(7) PFU) challenge with virus resulted in complete protection at 200 microg of mimetic and 40-60% protection at 5 microg of mimetic. Initiation of treatment of mice with IFN-gamma mimetic up to 2 days postinfection resulted in complete protection against death, whereas initiation of treatment at 6 days postinfection resulted in 40% protection. Administration of mimetic by the oral route also completely protected mice against the intranasal route of a lethal dose of vaccinia virus challenge. In addition to its direct antiviral effect, the mimetic also possessed adjuvant effects in boosting humoral and cellular immunity to vaccinia virus. The combination of antiviral and adjuvant effects by the IFN mimetic probably plays a role in its potent anti-vaccinia virus properties. These results suggest an effective therapeutic against ongoing, lethal poxvirus infections that taps into innate and adaptive host defenses.

We have demonstrated previously that the C-terminal gamma interferon (IFN-gamma) mimetic peptide consisting of residues 95 to 133 [IFN-gamma(95-133)], which contains the crucial IFN-gamma nuclear localization sequence (NLS), has antiviral activity in tissue culture. Here we evaluate the efficacy of this peptide and its derivatives first in vitro and then in an animal model of lethal viral infection with the encephalomyocarditis (EMC) virus. Deletion of the NLS region from the IFN-gamma mimetic peptide IFN-gamma(95-133) resulted in loss of antiviral activity. However, the NLS region does not have antiviral activity in itself. Replacing the NLS region of IFN-gamma(95-133) with the NLS region of the simian virus 40 large T antigen retains the antiviral activity in tissue culture. IFN-gamma(95-133) prevented EMC virus-induced lethality in mice in a dose-dependent manner compared to controls. Mice treated with IFN-gamma(95-133) had no or low EMC virus titers in their internal organs, whereas control mice had consistently high viral titers, especially in the heart tissues. Injection of B8R protein, which is encoded by poxviruses as a defense mechanism to neutralize host IFN-gamma, did not inhibit IFN-gamma(95-133) protection against a lethal dose of EMC virus, whereas mice treated with rat IFN-gamma were not protected. The data presented here show that the IFN-gamma mimetic peptide IFN-gamma(95-133) prevents EMC virus infection in vivo and in vitro and may have potential against other lethal viruses, such as the smallpox virus, which encodes the B8R protein.

We have shown previously that IFN-gamma and one of its receptor subunits, IFNGR1, are translocated to the nucleus, together with STAT1alpha as one macromolecular complex, via the classical importin-dependent pathway. In this study, we have identified the nuclear targets of IFN-gamma and IFNGR1. By chromatin immunoprecipitation followed by PCR, IFN-gamma, its receptor subunit IFNGR1, and STAT1alpha were found to be associated with the IFN-gamma-activated sequence (GAS) in the promoter of two of the genes stimulated by IFN-gamma. Immunoprecipitated chromatin also showed the association of the IFN-gamma, IFNGR1, and STAT1alpha on the same DNA sequence. Examination of nuclear extracts from WISH cells treated with IFN-gamma revealed the specific binding of IFN-gamma, IFNGR1, and STAT1alpha to biotinylated GAS nucleotide sequence. Association of IFN-gamma, IFNGR1, and STAT1alpha with the GAS promoter was also demonstrated by EMSA. Transfection with a GAS-luciferase gene together with the IFNGR1 and nonsecreted IFN-gamma resulted in enhanced reporter activity. In addition, IFNGR1 fused to the yeast GAL4 DNA binding domain resulted in enhanced transcription from a GAL4 response element, suggesting the presence of a trans activation domain in IFNGR1. Our observations put IFN-gamma and its receptor subunit, IFNGR1, in direct contact with the promoter region of IFN-gamma-activated genes with associated increased activity, thus suggesting a transcriptional/cotranscriptional role for IFN-gamma/IFNGR1 as well as a possible role in determining the specificity of IFN-gamma action.

Interaction of ligands such as epidermal growth factor and interferon-gamma with the extracellular domains of their plasma membrane receptors results in internalization followed by translocation into the nucleus of the ligand and/or receptor. There has been reluctance, however, to ascribe signaling importance to this, the focus instead being on second messenger pathways, including mobilization of kinases and inducible transcription factors (TFs). The latter, however, fails to explain the fact that so many ligands stimulate the same second messenger cascades/TFs, and yet show distinct gene activation profiles. This is particularly apt in the case of the seven STAT TFs that are held to be the mediators of the distinct cellular functions of over 60 ligands. The current review focuses on five representative nuclear localizing ligands for which there is documentation of translocation into the cytosol and nucleus through well-characterized pathways, in addition to a role in gene activation by ligand/receptor in the nucleus.

Interferons exhibit strong antiproliferative, antiviral and immunomodulatory properties. A combination of these properties makes them ideal candidates for the treatment of cancer and viral infections. However, their use as a protein therapeutic is limited due to their short serum half-life and certain undesirable side-effects. Some of these limitations are likely to be overcome by using interferons in a gene therapy modality. Gene therapy offers the advantage of generating higher sustained local concentration of this cytokine. In addition, it is possible to target the desired tissues and/or tumors by using specific promoters and to modulate the levels of the cytokine by using regulatable promoters. Results from studies in experimental tumor models of gene therapy for cancer and for certain viral infections have been encouraging and suggest that this may be a feasible and attractive alternative to interferon protein therapy.

IFN-tau, a novel type I IFN that possesses immunomodulatory properties, lacks toxicity normally associated with other type I IFNs. We examined the effects of oral IFN-tau alone and in combination with oral glatiramer acetate in experimental allergic encephalomyelitis (EAE). By comparison of oral administration of IFN-alpha, -beta, and -tau to myelin basic protein-specific TCR-transgenic mice, we demonstrate these type I IFNs promote secretion of the Th2 cytokine IL-10 with similar efficiency. Whereas IFN-alpha and -beta induced IFN-gamma secretion, a Th1 cytokine, IFN-tau did not. Oral IFN-tau alone suppressed EAE. When suboptimal doses were administered orally in combination to wild-type mice, IFN-tau and glatiramer acetate had a synergistic beneficial effect in suppression of EAE. This combination was associated with TGF-beta secretion and enhanced IL-10 production. Thus, IFN-tau is a potential candidate for use as a single agent or in combination therapy for multiple sclerosis.

IFN-gamma contains a nuclear localization sequence that may play a role in the nuclear transport of activated STAT1alpha via a complex of IFN-gamma/IFN-gamma receptor (IFNGR)-1/STAT1alpha with the nuclear importer nucleoprotein interactor 1. In this study, we examine the mechanism of endocytosis of IFNGR-1 and the relationship of its nuclear translocation to that of STAT1alpha. In untreated WISH cells, both IFNGR-1 and IFNGR-2 were constitutively localized within caveolae-like microdomains isolated from plasma membrane. However, treatment of cells with IFN-gamma resulted in rapid migration of IFNGR-1, but not IFNGR-2, from these microdomains. Filipin pretreatment, which specifically inhibits endocytosis from caveolae-like microdomains, inhibited the nuclear translocation of IFN-gamma and IFNGR-1 as well as the tyrosine phosphorylation and nuclear translocation of STAT1alpha, but did not affect the binding of IFN-gamma to these cells. In the Jurkat T lymphocyte cell line, which does not express caveolin-1, nuclear translocation of IFNGR-1 and STAT1alpha were similarly inhibited by filipin pretreatment. Isolation of lipid microdomains from Jurkat cells showed that both IFNGR-1 and IFNGR-2 were associated with lipid microdomains only after stimulation with IFN-gamma, suggesting that the IFNGR subunits are recruited to lipid microdomains by IFN-gamma binding in lymphocytes (Jurkat) in contrast to their constitutive presence in epithelial (WISH) cells. In contrast, treatments that block clathrin-dependent endocytosis did not inhibit either activation or nuclear translocation of STAT1alpha or the nuclear translocation of IFN-gamma or IFNGR-1. Thus, membrane lipid microdomains play an important role in IFN-gamma-initiated endocytic events involving IFNGR-1, and the nuclear translocation of IFN-gamma, IFNGR-1, and STAT1alpha.