Pig BVDV-2 non-structural protein (Npro) links to cellular antiviral response in vitro.

In this study, we constructed for the first time a full-length cDNA clone of pig-original bovine viral diarrhea virus 2 (BVDV-2) strain SH-28, modified the cDNA clone (pASH28) for mutant pASHΔNpro and derived virus strain vASHΔNpro by deleting the genomic region encoding the Npro polypeptide, and examined significance of protein Npro for antiviral responses in vitro. Data showed that Npro-deletion mutant virus vASHΔNpro led to significant overexpression of oligo adenylate synthetase (OAS), myxovirus-resistant protein 1 (Mx1), and ubiquitin-like protein 15 (ISG15). Data also revealed that overexpression of Npro, but not NS2 and NS3 proteins, resulted in significant down-regulation of OAS, Mx1, and ISG15 production (p ≤ 0.05) in bovine cells as well as porcine cells transfected with Npro recombinant eukaryotic expression plasmids. Npro (but not NS2 and NS3) was also found to inhibit poly(IC) from inducing production of type I interferon (IFN-I). These results indicated that protein Npro may play multiple roles in regulating antiviral response in host cells interfered by pig BVDV-2 strain, and provided useful information to understand better the mechanism of BVDV-2 persistent infection in pigs.

A Rapid and Versatile Method for Generating Proteins with Defined Ubiquitin Chains.

Ubiquitin and polyubiquitin chains target proteins for a wide variety of cellular processes. Ubiquitin-mediated targeting is regulated by the lysine through which the ubiquitins are linked as well as the broader ubiquitin landscape on the protein. The mechanisms of this regulation are not fully understood. For example, the canonical proteasome targeting signal is a lysine 48-linked polyubiquitin chain, and the canonical endocytosis signal is a lysine 63-linked polyubiquitin chain. However, lysine 63-linked polyubiquitin chains can also target substrates for degradation. Biochemical studies of ubiquitinated proteins have been limited by the difficulty of building proteins with well-defined polyubiquitin chains. Here we describe an efficient and versatile method for synthesizing ubiquitin chains of defined linkage and length. The synthesized ubiquitin chains are then attached to any protein containing a ubiquitin moiety. These proteins can be used to study ubiquitin targeting in in vitro assays in the tightly controlled manner required for biochemical studies.

Increased Ubqln2 expression causes neuron death in transgenic rats.

Pathogenic mutation of ubiquilin 2 (UBQLN2) causes neurodegeneration in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. How UBQLN2 mutations cause the diseases is not clear. While over-expression of UBQLN2 with pathogenic mutation causes neuron death in rodent models, deletion of the Ubqln2 in rats has no effect on neuronal function. Previous findings in animal models suggest that UBQLN2 mutations cause the diseases mainly through a gain rather than a loss of functions. To examine whether the toxic gain in UBQLN2 mutation is related to the enhancement of UBQLN2 functions, we created new transgenic rats over-expressing wild-type human UBQLN2. Considering that human UBQLN2 may not function properly in the rat genome, we also created transgenic rats over-expressing rat's own Ubqln2. When over-expressed in rats, both human and rat wild-type Ubqln2 caused neuronal death and spatial learning deficits, the pathologies that were indistinguishable from those observed in mutant UBQLN2 transgenic rats. Over-expressed wild-type UBQLN2 formed protein inclusions attracting the autophagy substrate sequestosome-1 and the proteasome component 26S proteasome regulatory subunit 7. These findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that the enhancement of UBQLN2 functions is involved in UBQLN2 pathogenesis. Pathogenic mutation in ubiquilin 2 (UBQLN2) causes neurodegeneration in ALS and FTLD. Studies in rodent models suggest a gain of toxic function in mutant UBQLN2. We created new transgenic rats as a relevant model and examined whether enhancing wild-type UBQLN2 expression is implicated in the pathogenesis of mutant UBQLN2. We observed that over-expression of human or rat wild-type Ubqln2 caused protein aggregation and neuronal death in transgenic rats. Our findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that uncontrolled enhancement of UBQLN2 function is involved in UBQLN2 pathogenesis. Read the Editorial Highlight for this article on page 159.

Biology of Zika Virus Infection in Human Skin Cells.

UNLABELLED: Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family, which includes dengue, West Nile, yellow fever, and Japanese encephalitis viruses, that causes a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here we examine the importance of human skin in the entry of ZIKV and its contribution to the induction of antiviral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, including DC-SIGN, AXL, Tyro3, and, to a lesser extent, TIM-1, permitted ZIKV entry, with a major role for the TAM receptor AXL. The ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing antibody and specific RNA silencing. ZIKV induced the transcription of Toll-like receptor 3 (TLR3), RIG-I, and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15, and MX1, characterized by strongly enhanced beta interferon gene expression. ZIKV was found to be sensitive to the antiviral effects of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes, whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy, and the specific autophagy inhibitor 3-methyladenine. The results presented herein permit us to gain further insight into the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging flavivirus. IMPORTANCE: Zika virus (ZIKV) is an arbovirus belonging to the Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor and for cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide the first general insights into the interaction between ZIKV and its mammalian host.

Hepatic expression of proteasome subunit alpha type-6 is upregulated during viral hepatitis and putatively regulates the expression of ISG15 ubiquitin-like modifier, a proviral host gene in hepatitis C virus infection.

The interferon-stimulated gene 15 (ISG15) plays an important role in the pathogenesis of hepatitis C virus (HCV) infection. ISG15-regulated proteins have previously been identified that putatively affect this proviral interaction. The present observational study aimed to elucidate the relation between ISG15 and these host factors during HCV infection. Transcriptomic and proteomic analyses were performed using liver samples of HCV-infected (n = 54) and uninfected (n = 10) or HBV-infected controls (n = 23). Primary human hepatocytes (PHH) were treated with Toll-like receptor ligands, interferons and kinase inhibitors. Expression of ISG15 and proteasome subunit alpha type-6 (PSMA6) was suppressed in subgenomic HCV replicon cell lines using specific siRNAs. Comparison of hepatic expression patterns revealed significantly increased signals for ISG15, IFIT1, HNRNPK and PSMA6 on the protein level as well as ISG15, IFIT1 and PSMA6 on the mRNA level in HCV-infected patients. In contrast to interferon-stimulated genes, PSMA6 expression occurred independent of HCV load and genotype. In PHH, the expression of ISG15 and PSMA6 was distinctly induced by poly(I:C), depending on IRF3 activation or PI3K/AKT signalling, respectively. Suppression of PSMA6 in HCV replicon cells led to significant induction of ISG15 expression, thus combined knock-down of both genes abrogated the antiviral effect induced by the separate suppression of ISG15. These data indicate that hepatic expression of PSMA6, which is upregulated during viral hepatitis, likely depends on TLR3 activation. PSMA6 affects the expression of immunoregulatory ISG15, a proviral factor in the pathogenesis of HCV infection. Therefore, the proteasome might be involved in the enigmatic interaction between ISG15 and HCV.

Interferon-stimulated gene, 15 kDa (ISG15) in ovarian high-grade serous carcinoma: prognostic impact and link to NF-κB pathway.

The ubiquitin-like protein interferon-stimulated gene, 15 kDa (ISG15) plays an ambiguous role in the progression and response to chemotherapy of solid cancers. We aimed to investigate the prognostic impact of ISG15 and its link to the nuclear factor κB pathway in ovarian high-grade serous carcinoma. Immunohistochemistry was performed in a cohort of 128 primary ovarian high-grade serous carcinomas treated with standard surgery and adjuvant chemotherapy using tissue microarrays. In addition, 28 matched relapsed carcinomas were investigated. ISG15 protein expression was significantly increased in relapsed carcinomas as compared to primary tumors (P=0.027). In primary carcinoma, ISG15 was positively associated with total inhibitor of κB α (IκBα) (P=0.001) as well as nuclear and cytoplasmic phospho-IκBα (p-IκBα) expression (P=0.039 and P=0.002, respectively). Patients with ISG15-positive carcinomas had a significantly longer overall survival in univariate analysis (P=0.002), and in multivariate analysis [hazard ratio=0.35 (95% confidence interval, 0.14-0.84, P=0.019)]. ISG15 is a potential prognostic marker in high-grade serous carcinoma of the ovary. Its impact on survival might be explained by its tight link to the nuclear factor κB pathway, and the further evaluation of the interplay between ISGylation machinery and nuclear factor κB, particularly with regard to response to chemotherapy, would be desirable.

Interferon stimulated gene 15 has an anti-apoptotic effect on MIN6 cells.

Type 1 diabetes, one of two major forms of diabetes, results from the complete destruction of pancreatic beta cells. Viral infection has been suggested to be a trigger of beta cell destruction, the pathogenesis of type 1 diabetes. The aim of this study was to clarify the role of the protein encoded by intherferon stimulated gene (ISG) 15, an antiviral effector, in the development of this clinical entity. We used the mouse beta cell line MIN6 to investigate the role of ISG15 and paid special attention to apoptosis. Although not detected in native MIN6 cells, free ISG15 and ISG15 conjugated proteins were both present in dose-dependently increased amounts following stimulation with interferon alpha. As assessed both by caspase 3/7 activity and an annexin V assay, the percentage of apoptotic MIN6 cells (after exposure to the inflammatory cytokines of interleukin-1beta plus interferon gamma or tumor necrosis factor alpha) was decreased by pretreatment with adenovirus-expressing ISG15 and increased by expressing a short hairpin RNA directed against ISG15. In conclusion, ISG15 has an anti-apoptotic effect on MIN6 cells. Thus, promoting ISG15 expression in the pancreatic beta cells could be a potential therapeutic approach for patients with type 1 diabetes.

Recombinant production of the antimicrobial peptide NZ17074 in Pichia pastoris using SUMO3 as a fusion partner.

UNLABELLED: The antimicrobial peptide NZ17074, which is derived from arenicin-3 isolated from Arenicola marina, displayed high activity against a broad range of pathogenic bacteria and fungi. However, NZ17074 has not been produced using fermentation technology. The aim of this work was to study the expression of difficult-to-express NZ17074 in Pichia pastoris by fusing with SUMO3. The DNA fragments of NZ17074 and SUMO3 were fused into SUMO3-NZ17074 using overlap PCR and cloned into the pPICZαA vector to construct the pPICZ-SUMO3-NZ17074 expression vector. The rSUMO3-NZ17074 fusion protein, purified by Ni(2) (+) -chelating affinity chromatography, was cleaved by 50% formic acid at 50°C for 28 h to release recombinant NZ17074 (rNZ17074). After purification with second affinity column, 4·1 mg rNZ17074 peptide with the purity over 90% was obtained from per litre fermentation culture. The rNZ17074 peptide exhibited the significant inhibition activity against Gram-negative bacteria: its minimal inhibitory concentrations (MICs) against Escherichia coli, Salmonella enteritidis and Pseudomonas aeruginosa were 2-4, 2 and 8-16 µg ml(-1) , respectively, which indicated that SUMO3 is a good fusion partner for the expression of the toxic peptide. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant active NZ17074 was produced with Pichia pastoris by using high-density fermentation technology for the first time. Our findings demonstrated the usefulness of SUMO-fusion technology as an effective expression strategy for synthesizing peptides in yeast. This SUMO3 expression system with a lower cost would likely be widely used for the production of other cytotoxic proteins including antimicrobial peptides.

Gene expression changes in initiation and progression of oral squamous cell carcinomas revealed by laser microdissection and oligonucleotide microarray analysis.

Oral carcinogenesis is a complex process involving multiple genes. However, the genetic changes involved in this process are not apparent in identical oral squamous cell carcinomas (OSCCs). According to pathological characteristics, samples of normal tissue, oral dysplastic lesions (ODLs), and invasive cancers were obtained from identical OSCCs using laser microdissection (LMD). Large-scale gene expression profiling was carried out on 33 samples derived from 11 OSCCs. We analyzed genes differentially expressed in normal tissues vs. ODLs and in ODLs vs. invasive tumors and identified 15 candidate genes with continuously increasing or decreasing expression during oral carcinogenesis. One of these genes, ISG15, was chosen for further characterization. Real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that ISG15 expression consistently increased during oral tumorigenesis. An ISG15 high-expression level was significantly associated with poor prognosis (p = 0.027). In addition, patients with high-expression tumors had a poorer 5-year survival rate than patients with low expression levels (p = 0.019). In conclusion, we identified 15 genes with continuously increasing or decreasing expression during oral carcinogenesis. One of these, ISG15, is likely to be associated with both dysgenesis and tumorigenesis and may be a potential prognostic marker for oral cancer.

Hepatitis C virus reveals a novel early control in acute immune response.

Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response.

Hepatitis C virus load and expression of a unique subset of cellular genes in circulating lymphoid cells differentiate non-responders from responders to pegylated interferon alpha-ribavirin treatment.

Based on investigations of liver biopsy material, certain cellular genes have been implicated as correlates of success or failure to interferon alpha-ribavirin (IFN/RBV) therapy against hepatitis C. The current study aimed at determining whether expression of host genes thought to be relevant to HCV replication in the liver would be correlated with HCV infection status in peripheral blood mononuclear cells (PBMCs) and also with patient responsiveness to IFN/RBV treatment. Therefore, PBMCs from patients with chronic hepatitis C responding (n = 35) or not (n = 49) to IFN/RBV and from healthy controls (n = 15) were evaluated for HCV RNA load and cellular gene expression. Non-responders had 3- to 10-fold higher basal levels of interleukin (IL)-8, IFN-stimulated gene 15 (ISG15), 2',5'-oligoadenylate synthetase (OAS), and Toll-like receptors (TLR)-4, -5, and -7 compared to responders. Non-responders with similar post-treatment follow-ups as responders persistently expressed 6- to 20-fold greater levels of IL-8, ISG15, and OAS after therapy. Higher expression of IFN-α, IFN-γ, and IFN-λ was found in PBMCs of individuals achieving sustained virological response, either before or after therapy. Pre-treatment HCV RNA loads in PBMCs of non-responders were significantly higher (P = 0.016) than those of responders. In conclusion, the data indicate that immune cells of responders and non-responders to IFN/RBV therapy exhibited significantly different virological and host gene expression profiles. Elevated baseline HCV loads and TLR-4, -5, and -7 levels, and persistently high levels of IL-8, ISG15, and OAS were correlated with IFN non-responsiveness. The results warrant further investigations on the utilization of PBMCs for predicting success or failure to IFN-based therapies.

Nuclear localization or inclusion body formation of ataxin-2 are not necessary for SCA2 pathogenesis in mouse or human.

Instability of CAG DNA trinucleotide repeats is the mutational mechanism for several neurodegenerative diseases resulting in the expansion of a polyglutamine (polyQ) tract. Proteins with long polyQ tracts have an increased tendency to aggregate, often as truncated fragments forming ubiquitinated intranuclear inclusion bodies. We examined whether similar features define spinocerebellar ataxia type 2 (SCA2) pathogenesis using cultured cells, human brains and transgenic mouse lines. In SCA2 brains, we found cytoplasmic, but not nuclear, microaggregates. Mice expressing ataxin-2 with Q58 showed progressive functional deficits accompanied by loss of the Purkinje cell dendritic arbor and finally loss of Purkinje cells. Despite similar functional deficits and anatomical changes observed in ataxin-1[Q80] transgenic lines, ataxin-2[Q58] remained cytoplasmic without detectable ubiquitination.

Up-regulation of interferon-stimulated gene15 and its conjugates by tumor necrosis factor-α via type I interferon-dependent and -independent pathways.

Interferon-stimulated gene15 (ISG15) is the first characterized ubiquitin-like protein, which is strongly induced by type I interferons (IFN-α/ß), bacterial endotoxin, and cellular stress. Up-regulation of ISG15 is observed in several cancer cell types and is associated with cancer progression. As many cytokines can influence all stages of tumorigenesis, the elevated expression of ISG15 system may be regulated in cancer cells by inflammatory cytokines. In this study, we showed that TNF-α, but not TGF-ß and IL-6, up-regulates levels of both ISG15 and its conjugates in human lung carcinoma A549 and human squamous carcinoma HSC4 cell lines. Induction of ISG15 and its conjugates by TNF-α was dose-dependent and required mediation of p38 MAP kinase and Jak1 through up-regulation of endogenous type I interferon expression. SB202190 (p38 MAPK inhibitor) and Jak1 inhibitor suppressed TNF-α-induced expression of ISG15 and its conjugates. However, only SB202190 inhibited the expression of type I interferons by TNF-α. Although B18R, a soluble type I interferon receptor, totally abolished the effect of exogenous IFN-ß, it was unable to inhibit completely the TNF-α-induced ISG15 production. In addition, the initiation of ISG15 induction by TNF-α was detected earlier than that of IFN-ß induction. Taken together, TNF-α elicits the induction of ISG15 and ISG15 conjugates not only via the autocrine stimulation of type I interferon expression, but also through a type I interferon-independent pathway. These data provide a possible link between inflammatory response and cancer progression.

Effect of laparoscopic splenectomy in patients with Hepatitis C and cirrhosis carrying IL28B minor genotype.

BACKGROUND: IL28B and ITPA genetic variants are associated with the outcome of pegylated-interferon and ribavirin (PEG-IFN/RBV) therapy. However, the significance of these genetic variants in cirrhotic patients following splenectomy has not been determined. METHODS: Thirty-seven patients with HCV-induced cirrhosis who underwent laparoscopic splenectomy (Spx group) and 90 who did not (non-Spx group) were genotyped for IL28B and ITPA. The outcome or adverse effects were compared in each group. Interferon-stimulated gene 15 (ISG15) and protein kinase R expression in the spleen was measured using total RNA extracted from exenterate spleen. RESULTS: Sustained virological response (SVR) rate was higher in patients carrying IL28B major genotype following splenectomy (50% vs 27.3%) and in patients carrying minor genotype in the Spx group compared to non-Spx group (27.3% vs 3.6%, P < 0.05). Pretreatment splenic ISG expression was higher in patients carrying IL28B major. There was no difference in progression of anemia or thrombocytopenia between patients carrying each ITPA genotype in the Spx group. Although splenectomy did not increase hemoglobin (Hb) level, Hb decline tended to be greater in the non-Spx group. In contrast, splenectomy significantly increased platelet count (61.1 × 103/µl vs 168.7 × 103/µl, P < 0.01), which was maintained during the course of PEG-IFN/RBV therapy. CONCLUSIONS: IL28B genetic variants correlated with response to PEG-IFN/RBV following splenectomy. Splenectomy improved SVR rate among patients carrying IL28B minor genotype and protected against anemia and thrombocytopenia during the course of PEG-IFN/RBV therapy regardless of ITPA genotype.

Differential expression pattern of ISG15 in different tissue explants and cells induced by various interferons.

Interferon stimulated gene 15 (ISG15), an ubiquitin cross-reactive protein, can conjugate to target proteins. Unlike ubiquitination, protein modification by ISG15 does not target protein for degradation, but enhances the cellular response to interferon (IFN), which plays a key role in antiviral responses. In this study, Western blot and/or immunocytochemistry were performed to explore the ISG15 expression patterns in explants of bovine endometrium, mammary gland and kidney, as well as Madin-Darby bovine kidney (MDBK), endometrial and mammary cells stimulated by IFN-α, -ß, and -τ. Western blot indicated that there are differential minimum antiviral units among recombinant bovine interferon-α (rbIFN-α, 10(2) IU/mL), rbIFN-ß (10(3) IU/mL) and rbIFN-τ (10(4) IU/mL) in regard to stimulating saturation expression of free and ISG15-conjugated proteins by MDBK cells and endometrial and mammary explants. These results were further confirmed through immunocytochemical analysis of MDBK, endometrial and mammary cells. For the first time it has been shown that the expression pattern of ISG15-conjugated proteins occurs in a tissue-specific manner. Furthermore, the present findings provide the first evidence of 10- to 100-fold differences in minimum antiviral units of rbIFN-α, rbIFN-ß, and rbIFN-τ in regard to stimulating saturation expression of ISG15.

Genome-wide analysis of oral squamous cell carcinomas revealed over expression of ISG15, Nestin and WNT11.

BACKGROUND: Multistep pathways and mechanisms are involved in the development of oral cancer. Chromosomal alterations are one of such key mechanisms implicated oral carcinogenesis. Therefore, this study aims to determine the genomic copy number alterations (CNAs) in oral squamous cell carcinoma (OSCC) using array comparative genomic hybridization (aCGH) and in addition attempt to correlate CNAs with modified gene expression. MATERIALS AND METHODS: Genome-wide screening was performed on 15 OSCCs using high-density aCGH. On the basis of pathway analysis, three genes (ISG15, Nestin and WNT11) which mapped to CNA regions were selected for further evaluation of their mRNA expression using quantitative reverse transcriptase PCR (qRT-PCR). RESULTS: Copy number alterations were observed on multiple genomic regions, including amplifications on 1p, 3q, 5p, 6p, 7p, 8q, 9q, 11q, 12q, 16p, 18p and deletions on 3p, 7q, 8p, 11q, 19q and 20q. Among the three selected genes, ISG15 had the highest mRNA expression level with a 22.5-fold increase, followed by Nestin with a 4.5-fold increase and WNT11 with a 2.5-fold increase. CONCLUSIONS: This study has identified several major CNAs in oral cancer genomes and indicated that this correlates with over expression of the ISG15, WNT11, and Nestin genes.

Rickettsia conorii infection stimulates the expression of ISG15 and ISG15 protease UBP43 in human microvascular endothelial cells.

Rickettsia conorii, an obligate intracellular bacterium and the causative agent of Mediterranean spotted fever, preferentially infects microvascular endothelial cells of the mammalian hosts leading to onset of innate immune responses, characterized by the activation of intracellular signaling mechanisms, release of pro-inflammatory cytokines and chemokines, and killing of intracellular rickettsiae. Our recent studies have shown that interferon (IFN)-ß, a cytokine traditionally considered to be involved in antiviral immunity, plays an important role in the autocrine/paracrine regulation of host defense mechanisms and control of R. conorii growth in the host endothelial cells. Here, we show that R. conorii infection induces the expression of ISG15 (an interferon-stimulated gene coding a protein of 17kD) and UBP43 (an ISG15-specific protease) at the levels of mRNA and protein and report the evidence of ISGylation of as yet unidentified target proteins in cultured human microvascular endothelium. Infection-induced expression of ISG15 and UBP43 requires intracellular replication of rickettsiae and production of IFN-ß, because treatment with tetracycline and presence of an antibody capable of neutralizing IFN-ß activity resulted in near complete attenuation of both responses. Inhibition of R. conorii-induced ISG15 by RNA interference results in significant increase in the extent of rickettsial replication, whereas UBP43 knockdown yields a reciprocal inhibitory effect. In tandem, these results demonstrate the stimulation of interferon-ß-mediated innate immune mechanisms capable of perturbing the growth and replication of pathogenic rickettsiae and provide first evidence for ISG15-mediated post-translational modification of host cellular proteins during infection with an intracellular bacterium.

Infection of in vivo differentiated human mast cells with hantaviruses.

Increased vascular permeability is a key feature of the pathological symptoms caused by hantaviruses. Here, we analysed the interaction between hantaviruses and mast cells, which regulate vascular homeostasis. In highly purified human skin mast cells increasing amounts of Hantaan (HTNV) and, to a lower extent, Prospect Hill (PHV) virions were produced. Replication was confirmed by the production of viral plus-strand RNA as determined by a virus strand-specific RT-PCR. PHV but not HTNV elicited early expression of beta interferon, MxA, ISG15 and CCL5 consistent to studies with other cell types. The data demonstrate that mature mast cells are permissive to infection with hantaviruses. This interaction might contribute to the development of vascular leakage syndrome.

The Overexpression of CD80 and ISG15 Are Associated with the Progression and Metastasis of Breast Cancer by a Meta-Analysis Integrating Three Microarray Datasets.

Breast cancer is a common cancer and could result in a substantial mortality. The study aimed to screen gene signatures associated with the development and metastasis of breast cancer and explore their regulation mechanisms. Three datasets of GSE10797, GSE8977 and GSE3744 were downloaded from GEO (Gene Expression Omnibus) database, containing 55 breast cancer samples and 27 normal samples. After data preprocessing using limma software and RMA (robust multi-array average) algorithm, DEGs (differentially expressed genes) between breast tumor and normal tissues in three individual experiments were identified using MADAM package. Function and pathway enrichment analyses were performed for the DEGs. Transcription factors and TAGs (tumor associated genes) among the DEGs were recognized and the PPI (protein-protein-interaction) network for the DEGs was constructed using Cytoscape software. The mRNA expression was analyzed via real-time quantitative PCR and protein expression was measured by western blotting. Totally, 100 DEGs were identified, including 33 up-regulated genes and 67 down-regulated genes. Among them, up-regulated DEGs such as CD80 was enriched in toll-like receptor (TLR) interaction pathway and the TAG, ISG15 was related to RIG-I-like receptor signaling pathway, while CXCL10 was involved in both of the two pathways. Whereas, the down-regulated DEG, CXCL12 was significantly associated with axon guidance pathway. Additionally, these DEGs were also pivotal nodes in the PPI network with high degrees. Besides, CXCL10 and CD80 were both interacted with IFNG. The mRNA expression of ISG15 was obviously enhanced in human breast cancer cells MCF-7, while no significant difference of CXCL10 mRNA level was found between MCF10A and MCF-7 cells. Moreover, the proteins expression levels of CD80 and ISG15 were significantly increased in MCF-7, MDA-MB-468 and MDA-MB-231 breast cancer cells than in normal MCF10A cells. CD80 might be responsible for the breast cancer's progression and metastasis via regulating innate immune system. In addition, ISG15 is identified as a crucial gene signature associated with breast cancer development and metastasis via RIG-I-like receptor signaling pathway.

IFNγ potentiates TNFα/TNFR1 signaling to induce FAT10 expression in macrophages.

INTRODUCTION: The tight regulation of the cytokine network during macrophage activation is of prime importance to enable a fast and potent innate immune response against exogenous pathogens. The inflammation mediating ubiquitin-like protein HLA-F adjacent transcript number 10 (FAT10) was shown to be transcriptionally regulated by and also regulate the nuclear factor-κB (NFκB) signaling pathway. However, very little is known about the regulation of FAT10 gene expression during macrophage activation. RESULTS: RNA sequencing of interferon (IFN)γ-stimulated mouse peritoneal macrophages analyzed by ingenuity pathway analysis revealed significant involvement of tumor necrosis factor receptor 1 (TNFR1) signaling in addition to IFNγ signaling. Subsequently, IFNγ robustly upregulated FAT10 expression compared to a milder induction seen with TNFα or lipopolysaccharide (LPS) stimulation. While low dose IFNγ with TNFα synergistically elevated FAT10 expression, preincubation of macrophages with IFNγ strongly augmented TNFα-induced FAT10 expression. Moreover, a short preincubation with IFNγ, which did not elevate FAT10, was sufficient to potentiate the induction of FAT10 by TNFα. A double augmentation mechanism of TNFα signaling was demonstrated, where IFNγ rapidly induced the expression of TNFα and TNFR1, which further augmented the induction of TNFα and TNFR1 expression by TNFα. Importantly, the induction of FAT10 by IFNγ in macrophages from TNFα-deficient or TNFR1-deficient mice was completely inhibited compared to macrophages from wild type (WT) mice. Finally, we show that TNFα-induced FAT10 expression is dependent on NFκB signaling. CONCLUSION: IFNγ potentiates the TNFα/TNFR1 signaling pathway to induce FAT10 expression in mouse macrophages, mediated through NFκB network.