Guanylate Binding Protein-1 Research Articles

Ubiquitination and degradation of GBPs by a Shigella effector to suppress host defence.

Interferon-inducible guanylate-binding proteins (GBPs) mediate cell-autonomous antimicrobial defences. Shigella flexneri, a Gram-negative cytoplasmic free-living bacterium that causes bacillary dysentery, encodes a repertoire of highly similar type III secretion system effectors called invasion plasmid antigen Hs (IpaHs). IpaHs represent a large family of bacterial ubiquitin-ligases, but their function is poorly understood. Here we show that S. flexneri infection induces rapid proteasomal degradation of human guanylate binding protein-1 (hGBP1). We performed a transposon screen to identify a mutation in the S. flexneri gene ipaH9.8 that prevented hGBP1 degradation. IpaH9.8 targets hGBP1 for degradation via Lys48-linked ubiquitination. IpaH9.8 targets multiple GBPs in the cytoplasm independently of their nucleotide-bound states and their differential function in antibacterial defence, promoting S. flexneri replication and resulting in the death of infected mice. In the absence of IpaH9.8, or when binding of GBPs to IpaH9.8 was disrupted, GBPs such as hGBP1 and mouse GBP2 (mGBP2) translocated to intracellular S. flexneri and inhibited bacterial replication. Like wild-type mice, mutant mice deficient in GBP1-3, 5 and 7 succumbed to S. flexneri infection, but unlike wild-type mice, mice deficient in these GBPs were also susceptible to S. flexneri lacking ipaH9.8. The mode of IpaH9.8 action highlights the functional importance of GBPs in antibacterial defences. IpaH9.8 and S. flexneri provide a unique system for dissecting GBP-mediated immunity.

Interplay of GTPases and Cytoskeleton in Cellular Barrier Defects during Gut Inflammation.

An essential role of the intestine is to build and maintain a barrier preventing the luminal gut microbiota from invading the host. This involves two coordinated physical and immunological barriers formed by single layers of intestinal epithelial and endothelial cells, which avoid the activation of local immune responses or the systemic dissemination of microbial agents, and preserve tissue homeostasis. Accordingly, alterations of epithelial and endothelial barrier functions have been associated with gut inflammation, for example during inflammatory bowel disease (IBD). The discriminative control of nutriment uptake and sealing toward potentially pathological microorganisms requires a profound regulation of para- and transcellular permeability. On the subcellular level, the cytoskeleton exerts key regulatory functions in the maintenance of cellular barriers. Increased epithelial/endothelial permeability occurs primarily as a result of a reorganization of cytoskeletal–junctional complexes. Pro-inflammatory mediators such as cytokines can induce cytoskeletal rearrangements, causing inflammation-dependent defects in gut barrier function. In this context, small GTPases of the Rho family and large GTPases from the Dynamin superfamily appear as major cellular switches regulating the interaction between intercellular junctions and actomyosin complexes, and in turn cytoskeleton plasticity. Strikingly, some of these proteins, such as RhoA or guanylate-binding protein-1 (GBP-1) have been associated with gut inflammation and IBD. In this review, we will summarize the role of small and large GTPases for cytoskeleton plasticity and epithelial/endothelial barrier in the context of gut inflammation.

Guanylate-Binding Protein 1 Inhibits Nuclear Delivery of Kaposi's Sarcoma-Associated Herpesvirus Virions by Disrupting Formation of Actin Filament.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a typical gammaherpesvirus that establishes persistent lifelong infection in host cells. In order to establish successful infection, KSHV has evolved numerous immune evasion strategies to bypass or hijack the host immune system. However, host cells still produce immune cytokines abundantly during primary KSHV infection. Whether the immune effectors produced are able to inhibit viral infection and how KSHV successfully conquers these immune effectors remain largely unknown. The guanylate-binding protein 1 (GBP1) gene is an interferon-stimulated gene and exerts antiviral functions on several RNA viruses; however, its function in DNA virus infection is less well understood. In this study, we found that KSHV infection increases both the transcriptional and protein levels of GBP1 at the early stage of primary infection by activating the NF-κB pathway. The overexpression of GBP1 significantly inhibited KSHV infection, while the knockdown of GBP1 promoted KSHV infection. The GTPase activity and dimerization of GBP1 were demonstrated to be responsible for its anti-KSHV activity. Furthermore, we found that GBP1 inhibited the nuclear delivery of KSHV virions by disrupting the formation of actin filaments. Finally, we demonstrated that replication and transcription activator (RTA) promotes the degradation of GBP1 through a proteasome pathway. Taken together, these results provide a new understanding of the antiviral mechanism of GBP1, which possesses potent anti-KSHV activity, and suggest the critical role of RTA in the evasion of the innate immune response during primary infection by KSHV.IMPORTANCE GBP1 can be induced by various cytokines and exerts antiviral activities against several RNA viruses. Our study demonstrated that GBP1 can exert anti-KSHV function by inhibiting the nuclear delivery of KSHV virions via the disruption of actin filaments. Moreover, we found that KSHV RTA can promote the degradation of GBP1 through a proteasome-mediated pathway. Taken together, our results elucidate a novel mechanism of GBP1 anti-KSHV activity and emphasize the critical role of RTA in KSHV evasion of the host immune system during primary infection.

P 28 Plasma Guanylate-Binding Protein-1 concentrations during pregnancy and preeclampsia

Introduction During preeclampsia, the leading cause of maternal death in developed countries, the levels of proinflammatory cytokines, like TNF-α and IFN-γ, are increased. These proinflammatory cytokines have the potential to induce the secretion of Guanylate-Binding Protein-1 (GBP-1), a factor regulating angiogenesis, by endothelial cells. We hypothesized that the plasma levels of GBP-1 would be increased during pregnancy and even further during preeclampsia (PE). Objective The objective of this study was te measure GBP-1 in pregnancy and preeclampsia. Methods GBP-1 was measured in peripheral plasma samples by ELISA. These plasma samples were collected from 6 severe late-onset PE and 48 severe early-onset PE pregnancies, 42 gestational age matched healthy pregnant women (25 for the early-onset and 17 for the late-onset PE) and 22 non-pregnant women. The levels of GBP-1 mRNA were determined in biopsies of the chorionic villi from 13 healthy pregnancies, 9 severe early-onset PE and 6 severe late-onset PE pregnancies using qPCR. Results The GBP-1 concentration of severe early-onset PE pregnancies (median 31 pg/ml; 95%CI 12 – 72 pg/ml) and those with severe late-onset PE (median 50 pg/ml; 42 – 68 pg/ml) were comparable (Mann Whitney, p = 0.518), but both were significantly decreased as compared to their gestational matched healthy pregnancies (respective medians 94 pg/ml (95%CI 77 – 160 pg/ml) and 117 pg/ml (95%CI 103 – 234 pg/ml) and Mann Whitney respectively p Conclusions In line with our hypothesis, GBP-1 plasma levels were increased during pregnancy. In contrast to our hypothesis we observed decreased GBP-1 plasma levels during PE as compared to healthy pregnancy. Although the placenta may be the source of increased GBP-1 during pregnancy, the reason why GBP-1 is decreased in preeclampsia remains to be established. More research is needed to get insight into the GBP-1 production during pregnancy and preeclampsia.

Abstract 5007: Role of the interferon-gamma response pathway in immune escape of colorectal carcinoma

Abstract The presence of an interferon-dominated Th1 immune response in colorectal carcinoma (CRC) has been associated with improved clinical outcome. We have previously identified the guanylate-binding protein 1 (GBP-1), one of the major IFN-γ-induced proteins, as a marker for the Th1 immune response and an independent positive prognostic factor in CRC. We also found that GBP-1 is a necessary mediator of the anti-tumorigenic effects of IFNγ. Interestingly, the expression of GBP-1 in CRC was observed more frequently in the desmoplastic stroma than in the directly adjacent tumor cells, suggesting that CRC tumor cells might become resistant to IFN-γ in the course of tumorigenesis. Indeed, we found that several colorectal carcinoma cell lines failed to express GBP-1 or other interferon-stimulated genes after treatment with IFN-γ, and were resistant to IFN-γ-induced apoptosis or proliferation inhibition. In these cell lines, the loss of IFN-γresponsiveness correlated either with the down-regulation of the IFN-γ receptor alpha chain (IFNγRα) or with the presence of a mis-glycosylated form of IFNγRα, which displayed an aberrant intracellular localization. We further validated these findings at the clinical level by showing that the expression of IFNγRα is decreased in CRC compared to normal tissue. In addition, the down-regulation of IFNγRα expression in CRC correlated with a reduced cancer-related survival together with a higher rate of distant metastasis. In addition, in-vivo colon tumorigenesis models using conditional knock-out mice revealed that the absence of IFNγRα expression by intestinal epithelial cells fosters tumor growth. Altogether, our data suggest that the loss of IFN-γ responsiveness is a common event in CRC and protects tumor cells against the anti-tumorigenic effects of IFN-γ. Citation Format: Nathalie Britzen-Laurent, Julia Straube, Philipp Tripal, Maximilian Waldner, Christoph Becker, Susanne Merkel, Roland S. Croner, Michael Sturzl. Role of the interferon-gamma response pathway in immune escape of colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5007. doi:10.1158/1538-7445.AM2017-5007

Processing and secretion of guanylate binding protein-1 depend on inflammatory caspase activity.

Human guanylate binding protein‐1 (GBP‐1) belongs to the family of large GTPases. The expression of GBP‐1 is inducible by inflammatory cytokines, and the protein is involved in inflammatory processes and host defence against cellular pathogens. GBP‐1 is the first GTPase which was described to be secreted by eukaryotic cells. Here, we report that precipitation of GBP‐1 with GMP‐agarose from cell culture supernatants co‐purified a 47‐kD fragment of GBP‐1 (p47‐GBP‐1) in addition to the 67‐kD full‐length form. MALDI‐TOF sequencing revealed that p47‐GBP‐1 corresponds to the C‐terminal helical part of GBP‐1 and lacks most of the globular GTPase domain. In silico analyses of protease target sites, together with cleavage experiments in vitro and in vivo, showed that p67‐GBP‐1 is cleaved by the inflammatory caspases 1 and 5, leading to the formation of p47‐GBP‐1. Furthermore, the secretion of p47‐GBP‐1 was found to occur via a non‐classical secretion pathway and to be dependent on caspase‐1 activity but independent of inflammasome activation. Finally, we showed that p47‐GBP‐1 represents the predominant form of secreted GBP‐1, both in cell culture supernatants and, in vivo, in the cerebrospinal fluid of patients with bacterial meningitis, indicating that it may represent the biologically active form of extracellular GBP‐1. These findings confirm the involvement of caspase‐1 in non‐classical secretion mechanisms and open novel perspectives for the extracellular function of secreted GBP‐1.

Guanylate-binding protein 1 (GBP1) contributes to the immunity of human mesenchymal stromal cells against Toxoplasma gondii

Mesenchymal stromal cells (MSCs) have recently been shown to play important roles in mammalian host defenses against intracellular pathogens, but the molecular mechanism still needs to be clarified. We confirmed that human MSCs (hMSCs) prestimulated with IFN-γ showed a significant and dose-dependent ability to inhibit the growth of two types of Toxoplasma gondii [type I RH strain with green fluorescent proteins (RH/GFP) or type II PLK strain with red fluorescent proteins (PLK/RED)]. However, in contrast to previous reports, the anti-T. gondii activity of hMSCs was not mediated by indoleamine 2,3-dioxygenase (IDO). Genome-wide RNA sequencing (RNA-seq) analysis revealed that IFN-γ increased the expression of the p65 family of human guanylate-binding proteins (hGBPs) in hMSCs, especially hGBP1. To analyze the functional role of hGBPs, stable knockdowns of hGBP1, -2, and -5 in hMSCs were established using a lentiviral transfection system. hGBP1 knockdown in hMSCs resulted in a significant loss of the anti-T. gondii host defense property, compared with hMSCs infected with nontargeted control sequences. hGBP2 and -5 knockdowns had no effect. Moreover, the hGBP1 accumulation on the parasitophorous vacuole (PV) membranes of IFN-γ-stimulated hMSCs might protect against T. gondii infection. Taken together, our results suggest that hGBP1 plays a pivotal role in anti-T. gondii protection of hMSCs and may shed new light on clarifying the mechanism of host defense properties of hMSCs.

Guanylate-Binding Protein-1 protects ovarian cancer cell lines but not breast cancer cell lines from killing by paclitaxel

Forced expression of the cytokine-induced large GTPase, human Guanylate-Binding Protein-1 (hGBP-1), in ovarian cancer cell lines increases resistance to paclitaxel. Elevated hGBP-1 RNA in ovarian tumors correlates with shorter recurrence-free survival. In contract, hGBP-1 is part of a gene signature predicting improved prognosis in all subtypes of breast cancers. hGBP-1 does not confer paclitaxel resistance on MCF-7 and TMX2-28 breast cancer cells. Expression of the isotype of the hGBP-1-interacting protein, PIM1, which may contribute to paclitaxel resistance when associated with hGBP-1, is different in breast and ovarian cancer cell lines. Breast cancer cell lines express the 44 kDa isoform of PIM-1, and ovarian cancer cell lines express the 33 kDa isoform.

Tetrameric assembly of hGBP1 is crucial for both stimulated GMP formation and antiviral activity.

Interferon-γ inducible human guanylate binding protein-1 (hGBP1) shows a unique characteristic that hydrolyses GTP to a mixture of GDP and GMP through successive cleavages, with GMP being the major product. Like other large GTPases, hGBP1 undergoes oligomerization upon substrate hydrolysis, which is essential for the stimulation of activity. It also exhibits antiviral activity against many viruses including hepatitis C. However, which oligomeric form is responsible for the stimulated activity leading to enhanced GMP formation and its influence on antiviral activity, are not properly understood. Using mutant and truncated proteins, our data indicate that transition-state-induced tetramerization is associated with higher rate of GMP formation. This is supported by chimaeras that are defective in both tetramerization and enhanced GMP formation. Unlike wild-type protein, chimaeras did not show allosteric interactions, indicating that tetramerization and enhanced GMP formation are allosterically coupled. Hence, we propose that after the cleavage of the first phosphoanhydride bond GDP·Pi-bound protein dimers transiently associate to form a tetramer that acts as an allosteric switch for higher rate of GMP formation. Biochemical and biophysical studies reveal that sequential conformational changes and interdomain communications regulate tetramer formation via dimer. Our studies also show that overexpression of the mutants, defective in tetramer formation in Rep2a cells do not inhibit proliferation of hepatitis C virus, indicating critical role of a tetramer in the antiviral activity. Thus, the present study not only highlights the importance of hGBP1 tetramer in stimulated GMP formation, but also demonstrates its role in the antiviral activity against hepatitis C virus.

Invited review: Mechanisms of GTP hydrolysis and conformational transitions in the dynamin superfamily.

ABSTRACTDynamin superfamily proteins are multidomain mechano‐chemical GTPases which are implicated in nucleotide‐dependent membrane remodeling events. A prominent feature of these proteins is their assembly‐ stimulated mechanism of GTP hydrolysis. The molecular basis for this reaction has been initially clarified for the dynamin‐related guanylate binding protein 1 (GBP1) and involves the transient dimerization of the GTPase domains in a parallel head‐to‐head fashion. A catalytic arginine finger from the phosphate binding (P‐) loop is repositioned toward the nucleotide of the same molecule to stabilize the transition state of GTP hydrolysis. Dynamin uses a related dimerization‐dependent mechanism, but instead of the catalytic arginine, a monovalent cation is involved in catalysis. Still another variation of the GTP hydrolysis mechanism has been revealed for the dynamin‐like Irga6 which bears a glycine at the corresponding position in the P‐loop. Here, we highlight conserved and divergent features of GTP hydrolysis in dynamin superfamily proteins and show how nucleotide binding and hydrolysis are converted into mechano‐chemical movements. We also describe models how the energy of GTP hydrolysis can be harnessed for diverse membrane remodeling events, such as membrane fission or fusion. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 580–593, 2016.

Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity

ABSTRACTMany viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity.IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV, including interferon-stimulated genes (ISGs), have been characterized. Using a minilibrary of porcine ISGs, we identify porcine guanylate-binding protein 1 (GBP1) as a potent antiviral ISG against CSFV. We further show that the anti-CSFV action of GBP1 depends on its GTPase activity. The K51 of GBP1, critical for its GTPase activity, is essential for the antiviral action of GBP1 against CSFV replication, and the binding of the NS5A protein to GBP1 antagonizes the GTPase activity and thus the antiviral effect. This study will facilitate the development of anti-CSFV therapeutic agents by targeting host factors and may provide a new strategy for the control of CSF.

Guanylate binding protein-1 mediates EGFRvIII and promotes glioblastoma growth in vivo but not in vitro.

Glioblastoma multiforme (GBM) is the most common and deadly primary brain tumor in adults. Epidermal growth factor receptor (EGFR) is frequently amplified and mutated in GBM. We previously reported that Guanylate binding protein-1 (GBP1) is a novel transcriptional target gene of EGFR and plays a role in GBM invasion. Here we demonstrate that GBP1 can also be induced by EGFRvIII at the transcriptional level through the p38 MAPK/Yin Yang 1 (YY1) signaling pathway. Silencing of GBP1 by RNA interference significantly inhibits EGFRvIII-mediated GBM cell proliferation in vitro and in a mouse model. Overexpression of GBP1 has no obvious effect on glioblastoma cell proliferation in vitro. In contrast, in an orthotopic glioma mouse model GBP1 overexpression significantly promotes glioma growth and reduces survival rate of glioma-bearing mice by increasing cell proliferation and decreasing cell apoptosis in tumor. Clinically, GBP1 expression is elevated in human GBM tumors and positively correlates with EGFRvIII status in GBM specimens, and its expression is inversely correlated with the survival rate of GBM patients. Taken together, these results reveal that GBP1 may serve as a potential therapeutic target for GBMs with EGFRvIII mutation.

Effect of polymorphisms in the GBP1, Mx1 and CD163 genes on host responses to PRRSV infection in pigs

Porcine reproductive and respiratory syndrome (PRRS) is the most economically important disease to the swine industry, and effective prevention strategy for this disease is still required. Guanylate-binding protein 1 (GBP1) and myxovirus resistance protein 1 (Mx1) are two important proteins belonging to the GTPase superfamily that have been previously described to show antiviral effects. CD163 is considered the most important receptor for PRRSV attachment and internalization. Therefore, the aim of the present study was to evaluate the effects of these genes on host resistance against PRRSV infection in conjunction with the host immune response following PRRSV challenge. The results showed that pigs with AG genotype for the GBP1 exon2 exhibited a significantly higher average daily weight gain (ADWG) and lower average viremia than AA or GG genotype. Furthermore, pigs harbouring the AG genotype for the GBP1 gene presented greater CD4+CD25+ and CD8+CD25+ T cell populations at 4 and 18 days post challenge (dpc), respectively, as compared with other genotypes whereas pigs with CC genotype for the CD163 gene displayed significantly higher nucleocapsid-specific antibody titers at 11dpc. However, pigs with a single 11-bp deletion or insertion in the Mx1 gene did not show significant differences in either weight gain or viremia. Based on these results, we concluded that GBP1 is most significantly associated with resistance against PRRSV infection and efficient T cell activation in pigs.

Interferon Gamma Counteracts the Angiogenic Switch and Induces Vascular Permeability in Dextran Sulfate Sodium Colitis in Mice.

Interferon (IFN)-γ is a central pathogenesis factor in inflammatory bowel disease (IBD) with pleiotropic effects on many different cell types. However, as yet, the immune modulatory functions of IFN-γ in IBD have been predominantly investigated. Based on previous studies showing that IFN-γ acts antiangiogenic in colorectal carcinoma, we investigated the effects of IFN-γ on the vascular system in IBD. Colon tissues of patients with IBD and dextran sulfate sodium-induced colitis in mice were subjected to immunohistochemistry, quantitative real-time polymerase chain reactions, and in situ hybridization to quantify cell activation, angiogenesis, and immune responses. Vascular structure and permeability in mice were analyzed by ultramicroscopy and in vivo confocal laser endomicroscopy. We showed a significantly increased blood vessel density in IBD and dextran sulfate sodium colitis. In mice, this was associated with a disorganized blood vessel structure and profound vascular leakage. As compared with genes associated with angiogenesis, genes associated with inflammatory cell activation including IFN-γ were more strongly upregulated in colitis tissues. IFN-γ exerted direct effects on endothelial cells in IBD tissues in vivo, as indicated by the expression of IFN-γ-induced guanylate binding protein 1 (GBP-1). Neutralization of IFN-γ in the acute dextran sulfate sodium colitis model demonstrated that this cytokine exerts endogenous angiostatic activity in IBD and contributes to increased vascular permeability. The dissection of the pleiotropic activities of IFN-γ in IBD provides new insights to the pathological functions of this cytokine and may be of high relevance for the optimization of combination therapy approaches.

Role of PDZ binding kinase (PBK) associated to guanylate binding protein 1 (GBP1) for drug resistance in ovarian cancer

Role of PDZ binding kinase (PBK) associated to guanylate binding protein 1 (GBP1) for drug resistance in ovarian cancer

Promotion of expression of interferon-stimulated genes in U937 monocytic cells by HIV RNAs, measured using stable isotope labeling with amino acids in cell culture (SILAC).

Type I interferon (IFN) exerts strong antiviral activity, particularly against human immunodeficiency virus (HIV), and although several viral proteins have been shown to deregulate IFN induction, little is known about the induction of type I IFNs by HIV RNAs. In the present study, we used the stable isotope labeling with amino acids in cell culture (SILAC) method to determine the proteomic profile in U937 monocytic cells after transfection with viral RNA of HIV. We then used a western blot assay to validate the proteomic results. It was revealed by the SILAC method that there were 1624 non-redundant peptides with quantitative information and 281 proteins with quantitative information in the HIV-RNA-transfected U937 cells when compared to cells transfected with control RNA. In particular, 6, 8 or 12 hours post-transfection, HIV RNA transfection promoted the expression of such interferon stimulated genes (ISGs) as interferon-induced proteins with tetratricopeptide repeats (IFITs), interferon-induced transmembrane proteins (IFITMs), interferon-induced gene 15 protein (ISG15), myxovirus (influenza virus) resistance protein 1 (MX1), and interferon-induced guanylate-binding protein 1 (GBP1), and this was confirmed by western blot assay. In conclusion, HIV RNA is a strong stimulator of IFNs, promoting the expression of such ISGs as IFITs, IFITMs, ISG15, MX1 and GBP1.

Rapid Regulation of Depression-Associated Genes in a New Mouse Model Mimicking Interferon-α-Related Depression in Hepatitis C Virus Infection.

Major depression is a serious side effect of interferon-α (IFN-α), which is used in the therapy of hepatitis C virus (HCV) infection. Due to the lack of reproducible animal models, the mechanisms underlying IFN-α-related depression are largely unknown. We herein established a mouse model, in which murine IFN-α (250 IU/day) and polyinosinic/polycytidylic acid (poly(I:C); 1 μg/day), a toll-like receptor-3 (TLR3) agonist that mimics the effect of HCV double-strand RNA, were continuously infused into the lateral ventricle via miniosmotic pumps over up to 14 days. The delivery of IFN-α and poly(I:C), but not of IFN-α or poly(I:C) alone, resulted in a reproducible depression-like state that was characterized by reduced exploration behavior in open-field tests, increased immobility in tail suspension and forced swimming tests, and a moderate loss of body weight. In the hippocampus and prefrontal cortex, the pro-inflammatory genes TNF-α, IL-6, tissue inhibitor of metalloproteinases-1 (Timp-1), CXC motif ligand-1 (Cxcl1), Cxcl10, and CC motif ligand-5 (Ccl5) were synergistically induced by IFN-α and poly(I:C), most pronounced after 14-day exposure. In comparison, the interferon-inducible genes of signal transducer and activator of transcription-1 (Stat1), guanylate binding protein-1 (Gbp1), proteasome subunit-β type-9 (Psmb9), ubiquitin-conjugating enzyme E2L-6 (Ube2l6), receptor transporter protein-4 (Rtp4), and GTP cyclohydrolase-1 (Gch1), which had previously been elevated in the blood of IFN-α-treated patients developing depression, in the brains of suicidal individuals, and in primary neurons exposed to IFN-α and poly(I:C), were induced even earlier, reaching maximum levels mostly after 24 hours. We propose that interferon-inducible genes might be useful markers of imminent depression.

Guanylate Binding Protein 1–Mediated Interaction of T Cell Antigen Receptor Signaling with the Cytoskeleton

GTPases act as important switches in many signaling events in cells. Although small and heterotrimeric G proteins are subjects of intensive studies, little is known about the large IFN-inducible GTPases. In this article, we show that the IFN-γ-inducible guanylate binding protein 1 (GBP-1) is a regulator of T cell activation. Silencing of GBP-1 leads to enhanced activation of early T cell Ag receptor/CD3 signaling molecules, including Lck, that is translated to higher IL-2 production. Mass spectrometry analyses showed that regulatory cytoskeletal proteins, like plastin-2 that bundles actin fibers and spectrin β-chain, brain 1 that links the plasma membrane to the actin cytoskeleton, are binding partners of GBP-1. The spectrin cytoskeleton influences cell spreading and surface expression of TCR/CD3 and the leukocyte phosphatase CD45. We found higher cell spreading and enhanced surface expression of TCR/CD3 and CD45 in GBP-1 silenced T cells that explain their enhanced TCR/CD3 signaling. We conclude that GBP-1 is a downstream processor of IFN-γ via which T cells regulate cytoskeleton-dependent cell functions.

IFN-γ–Driven Intratumoral Microenvironment Exhibits Superior Prognostic Effect Compared with an IFN-α–Driven Microenvironment in Patients with Colon Carcinoma

Interferon (IFN)-α and IFN-γ are cytokines with potent immunomodulating and anti-tumor activities. It is unknown which of the two IFNs may be more potent in the regulation of an anti-tumorigenic response in colorectal carcinoma or whether both cytokines cooperate. We, therefore, established human myxovirus resistance protein A and human guanylate-binding protein-1 as markers for the differential detection of IFN-α- and IFN-γ-driven tumor micromilieus, respectively. In vitro studies with different cultures of tumor cells from colorectal carcinoma and stroma cells showed that the expression of myxovirus resistance protein A was exclusively induced by IFN-α, whereas guanylate-binding protein-1 was strongly induced by IFN-γ and only weakly by IFN-α. This expression pattern was used to distinguish cell activation caused by the two cytokines in a clinical cohort of patients with colon carcinoma (n = 378). Patients with primary tumors expressing only guanylate-binding protein-1 exhibited the highest cancer-specific 5-year survival (94.0%, P = 0.006) compared with those expressing both factors (90.3%, P = 0.006), myxovirus resistance protein A alone (83.5%, P = 0.096), or none (72.8%). Our study describes a successful proof-of-principle approach that complex cytokine interaction networks can be dissected in human tissues and demonstrates that an IFN-γ-driven tumor microenvironment exhibits a superior prognostic effect compared with an IFN-α-driven tumor microenvironment in colon carcinoma.

Abstract 1521: Role of the guanylate-binding-protein 1 (GBP-1) in immunoediting of colorectal carcinoma.

Abstract The human guanylate-binding protein 1 (GBP-1) is among the proteins the most highly induced by IFN-γ in every cell type investigated as yet. In vivo, GBP-1 expression is associated with the presence of inflammation and has been observed in autoimmune diseases, inflammatory bowel diseases (IBD) and cancer. In colorectal carcinoma (CRC), we previously reported that the expression of GBP-1 in the cells of the desmoplastic stroma correlates with the presence of an IFN-γ-dominated Th1 microenvironment and with an increased cancer-related 5-year survival. Moreover, GBP-1 expression in the blood vessels of CRC tissue was associated with a reduced angiogenesis. However, the expression and the role of GBP-1 in the tumor cells of CRC remained unknown. In the present study, the analysis of GBP-1 expression in a series of 185 CRCs by immunohistochemistry confirmed that GBP-1 is expressed in stroma cells of CRCs and revealed a significantly less frequent expression in tumor cells, which was contradictory with the broad inducibility of GBP-1. Furthermore, three of six CRC cell lines treated with IFN-γ were unable to express GBP-1 indicating a reduced inducibility in colorectal tumor cells. On the contrary, non-transformed colon epithelial cells strongly expressed GBP-1 in vitro in presence of IFN-γ and in vivo in IBD. Reconstitution of GBP-1 expression in a negative CRC cell line inhibited cell proliferation, migration, invasion and anchorage-independent growth. Using RNA interference, we showed that GBP-1 mediates the anti-proliferative, anti-migratory, anti-invasive and anti-clonogenic effects of IFN-γ in CRC cells. In addition, GBP-1 was able to inhibit tumor growth in vivo. Altogether these results suggested that GBP-1 acts directly as a tumor suppressor in CRC by mediating the effects of IFN-γ. In addition, the loss of GBP-1 expression in tumor cells indicated that tumor evasion from the IFN-γ-dominated Th1 immune response might have occured. This was confirmed by showing, first, that the absence of GBP-1 inducibility in CRC cell lines correlates with resistance to the anti-proliferative and pro-apoptotic effects of IFN-γ and, second, that the resistant cell lines exhibit defects in the IFN-γ signaling pathway. Citation Format: Nathalie Britzen-Laurent, Karoline Lipnik, Matthias Ocker, Elisabeth Naschberger, Vera Schellerer, Roland Croner, Michael Vieth, Maximilian Waldner, Pablo Steinberg, Christine Hohenadl, Michael Stürzl. Role of the guanylate-binding-protein 1 (GBP-1) in immunoediting of colorectal carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1521. doi:10.1158/1538-7445.AM2013-1521