Interferon-stimulated Gene 15 Expression Research Articles

Identification and validation of interferon-stimulated gene 15 as a biomarker for dermatomyositis by integrated bioinformatics analysis and machine learning.

Dermatomyositis (DM) is an autoimmune disease that primarily affects the skin and muscles. It can lead to increased mortality, particularly when patients develop associated malignancies or experience fatal complications such as pulmonary fibrosis. Identifying reliable biomarkers is essential for the early diagnosis and treatment of DM. This study aims to identify and validate pivotal diagnostic biomarker for DM through integrated bioinformatics analysis and clinical sample validation. Gene expression datasets GSE46239 and GSE142807 from the Gene Expression Omnibus (GEO) database were merged for analysis. Differentially expressed genes (DEGs) were identified and subjected to enrichment analysis. Advanced machine learning methods were utilized to further pinpoint hub genes. Weighted gene co-expression network analysis (WGCNA) was also conducted to discover key gene modules. Subsequently, we derived intersection gene from these methods. The diagnostic performance of the candidate biomarker was evaluated using analysis with dataset GSE128314 and confirmed by immunohistochemistry (IHC) in skin lesion biopsy specimens. The CIBERSORT algorithm was used to analyze immune cell infiltration patterns in DM, then the association between the hub gene and immune cells was investigated. Gene set enrichment analysis (GSEA) was performed to understand the biomarker's biological functions. Finally, the drug-gene interactions were predicted using the DrugRep server. Interferon-stimulated gene 15 (ISG15) was identified by intersecting DEGs, advanced machine learning-selected genes and key module genes from WGCNA. ROC analysis showed ISG15 had a high Area under the curve (AUC) of 0.950. IHC findings confirmed uniformly positive expression of ISG15, particularly in perivascular regions and lymphocytes, contrasting with universally negative expression in controls. Further analysis revealed that ISG15 is involved in abnormalities in various immune cells and inflammation-related pathways. We also predicted three drugs targeting ISG15, supported by molecular docking studies. Our study identifies ISG15 as a highly specific diagnostic biomarker for DM, ISG15 may be closely related to the pathogenesis of DM, demonstrating promising potential for clinical application.

Increased expression of Interferon-stimulated gene 15 (ISG15) in cervical cells on day 14 of pregnancy in Holstein heifers

In cattle, expression of interferon (IFN)-stimulated genes in the female reproductive tract has been reported as an early pregnancy diagnostic tool, as early as d 17 of pregnancy. The hypothesis of this study was that expression of ISG15 in the cervix of pregnant heifers is increased on d 14 of pregnancy. The objective was to compare the expression of ISG15 in cervical cells between pregnant and cyclic heifers (control, sham-inseminated) on d 14, 16, and 18 after insemination (d 0; D0). Holstein heifers were submitted to an estrus synchronization protocol and inseminated with extender only (“control,” n = 6), or with regular semen (n = 15). Heifers were classified as pregnant (n = 10) by ultrasound at D30 through the detection of a viable embryo with a heartbeat. Blood samples from the coccygeal vein were collected to determine serum progesterone concentrations on D14, D16, and D18. The expression of ISG15 and PGR in cervical cells collected through cytobrush was measured on D14, D16, and D18. A receiver operating characteristic (ROC) curve was calculated to quantify the pregnancy diagnostic accuracy of ISG15 and PGR expression. The expression levels of ISG15 in cervical cells were significantly greater in pregnant compared with control heifers on D14, and remained greater on D16 and D18, while differences in PGR were observed only on D18. Scatter plots and ROC analyses showed the most accurate prediction of pregnancy for ISG15 on D16. In conclusion, cervical cells express greater ISG15 mRNA in pregnant vs control heifers as early as D14 post-insemination, with the best accuracy on D16.

Chlamydia-driven ISG15 expression dampens the immune response of epithelial cells independently of ISGylation.

Excessive inflammation upon Chlamydia trachomatis infection can cause severe damages in the female genital tract. This obligate intracellular bacterium develops mainly in epithelial cells, whose innate response contributes to the overall inflammatory response to infection. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) stimulates interferon γ (IFNγ) production and is required for bacterial clearance in several infectious contexts. Here, we describe and investigate the consequences of the increase in ISG15 expression by epithelial cells infected with C. trachomatis. Infection of HeLa cells and primary ecto-cervical epithelial cells resulted in a transcriptional upregulation of ISG15 expression. This did not involve the canonical type I interferon (IFN-I) signaling pathway and depended instead on the activation of the STING/TBK1/IRF3 pathway. The absence or reduction of ISG15 synthesis led to increased production of several cytokines and chemokines, including interleukin (IL) 6 and IL8. This implicates that ISG15 normally dampens the immune response induced by C. trachomatis infection in epithelial cells. ISG15 exerted its control from an intracellular location, but without involving ISGylation. Finally, higher levels of inflammation and delayed bacterial clearance were observed in the genital tracts of ISG15-KO mice infected by C. trachomatis compared with wild-type animals; however, IFNγ production was unchanged. Altogether, our data show that ISG15 expression acts as a brake on the immune response to C. trachomatis infection in epithelial cells and limits bacterial burden and inflammation in mice.IMPORTANCEInfection of epithelial cells by Chlamydia trachomatis elicits an innate immune response by these cells. The signaling pathways involved, and their outcomes, are still very poorly understood. In this paper, we described how Chlamydia infection triggered the expression of ISG15, a small molecule normally associated to type I interferon (IFN-I) signaling and control of INF-γ production. ISG15 synthesis by epithelial cells attenuated their immune response to Chlamydia infection. In mice, we observed that ISG15 displayed a marginal role in modulating the production of IFN-γ, a key component of the host immune response to infection, but facilitated bacterial clearance. Overall, our study strengthens the importance of ISG15 not only in the resolution of viral but also of bacterial infection and document its role of "immune brake" in the context of Chlamydia infection.

Dopamine Inhibits the Expression of Hepatitis B Virus Surface and e Antigens by Activating the JAK/STAT Pathway and Upregulating Interferon-stimulated Gene 15 Expression.

Hepatitis B virus (HBV) infection is a major risk factor for cirrhosis and liver cancer, and its treatment continues to be difficult. We previously demonstrated that a dopamine analog inhibited the packaging of pregenomic RNA into capsids. The present study aimed to determine the effect of dopamine on the expressions of hepatitis B virus surface and e antigens (HBsAg and HBeAg, respectively) and to elucidate the underlying mechanism. We used dopamine-treated HBV-infected HepG2.2.15 and NTCP-G2 cells to monitor HBsAg and HBeAg expression levels. We analyzed interferon-stimulated gene 15 (ISG15) expression in dopamine-treated cells. We knocked down ISG15 and then monitored HBsAg and HBeAg expression levels. We analyzed the expression of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway factors in dopamine-treated cells. We used dopamine hydrochloride-treated adeno-associated virus/HBV-infected mouse model to evaluate HBV DNA, HBsAg, and HBeAg expression. HBV virus was collected from HepAD38.7 cell culture medium. Dopamine inhibited HBsAg and HBeAg expression and upregulated ISG15 expression in HepG2.2.15 and HepG2-NTCP cell lines. ISG15 knockdown increased HBsAg and HBeAg expression in HepG2.2.15 cells. Dopamine-treated cells activated the JAK/STAT pathway, which upregulated ISG15 expression. In the adeno-associated virus-HBV murine infection model, dopamine downregulated HBsAg and HBeAg expression and activated the JAK-STAT/ISG15 axis. Dopamine inhibits the expression of HBsAg and HBeAg by activating the JAK/STAT pathway and upregulating ISG15 expression.

ISG15 mediates the function of extracellular vesicles in promoting ovarian cancer progression and metastasis

AbstractThe interferon stimulated gene 15 (ISG15), a ubiquitin like protein and its conjugates have been implicated in various human malignancies. However, its role in ovarian cancer progression and metastasis is largely unknown. In high grade serous ovarian cancer (HGSOC), ascites is the major contributor to peritoneal metastasis. In this study, we identified significantly elevated ISG15 protein expression in HGSOC patient ascites, ascites derived primary ovarian cancer cells (POCCs), POCC small extracellular vesicles (sEVs) as well as metastatic tissue. Our results demonstrates that ISG15 increases exocytosis in ascites‐derived POCCs by decreasing the endosome‐lysosomal fusion, indicating a key role in sEV secretion. Further, knockdown (KD) of ISG15 resulted in a significant decrease in vesicles secretion from HGSOC cells and in vivo mouse models, leading to reduced HGSOC cell migration and invasion. Furthermore, our pre‐clinical mouse model studies revealed the influence of vesicular ISG15 on disease progression and metastasis. In addition, knockdown of ISG15 or using the ISG15 inhibitor, DAP5, in combination therapy with carboplatin showed to improve the platinum sensitivity in‐vitro and reduce tumour burden in‐vivo. We also found that ISG15 expression within sEV represents a promising prognostic marker for HGSOC patients. Our findings suggest that ISG15 is a potential therapeutic target for inhibiting progression and metastasis in HGSOC and that vesicular ISG15 expression could be a promising biomarker in the clinical management of ovarian cancer.Significance: High‐grade serous ovarian cancer (HGSOC) has high morbidity and mortality rates, but its progression and metastasis are still poorly understood, and there is an urgent need for early detection and targeted therapies. Our study presents novel findings that implicate ISG15‐mediated vesicular proteins in the advancement and spread of HGSOC. These results offer pre‐clinical evidence of potential new molecular targets, prognostic markers and therapeutic strategies for HGSOC that could ultimately enhance patient survival.

Abstract C052: Effect of ginger extracts on hepatocellular carcinoma cell lines-derived from Caucasian, Asian, and African American patients

Abstract Hepatocellular Carcinoma (HCC) is a highly fatal disease with mortality running parallel to its incidence. For HCC, race/ethnicity plays a vital role in determining incidence, mortality, and survival rates. The incidence of HCC is highest in Asia and Africa. Furthermore, there is a statistically significant increase in incidence and mortality and a decrease in 5-year survival rates in African American (AA)/Black patients compared to non-Hispanic white patients. There is a knowledge gap in our understanding of the molecular mechanism underlying the HCC racial disparity between AA/Black, White, and Asian patients. To understand the underlying cause, we performed bioinformatics on existing gene expression data. We found that type I Interferon (IFN-I) signaling pathway showed statistically significant activation in AA/Black patients compared to white patients. Over 60% of Gen Xers/Millennials are taking a more holistic approach to their diet to prevent chronic diseases, including cancer. We hypothesized that dietary compounds exert anticancer effects on HCC, and because of their anti-inflammatory property, they might modulate the IFN-I signaling pathway. We tested ginger extracts on HCC derived from white (HepG2), Black (Hep3B and O/20), and Asian (Hu7) cancer patients. A dose-response of these extracts was used to determine IC50s on these cell lines using an MTT cell proliferation assay. Activation of INF-1-mediated downstream signaling proteins, including JAK1, TYK2, STAT1, and STAT2 phosphorylation status, was determined on a Western blot analysis using phospho-specific primary antibodies. The expression of Interferon Signaling Genes (ISGs), including Myxovirus resistance gene 1 (MX1), 2′,5′-oligoadenylate synthetase (OAS1), Interferon-alpha inducible protein 6 (IFI6), and Interferon stimulated gene 15 (ISG15) was assayed using a quantitative Real-Time Polymerase Chain Reaction (RT-PCR). Ginger has a significantly (P<0.05) lower IC50s (mg/ml) on cell lines from Black patients (Hep3B=156 ± 3, and O/20=162 ± 3) than cell lines from white (HepG2=176±5) or Asian (Hu7=174 ± 5) patients. Our data show that untreated cells exhibited phosphorylation/activation of IFN-1 downstream mediators, including JAK1, TYK2, STAT1, and STAT2 proteins. Ginger treatment reduced the phosphorylation of these IFN downstream mediators in all HCC cell lines. Furthermore, expression of MX1, ISG15, IF16, and OAS1 was also reduced in all HCC cells lines in a dose-dependent manner; however, the effects on gene expression were more sensitive to a lower concentration of ginger extract in Black patients derived HCC cell lines than other HCC cell lines. Currently, research is underway to identify the active chemical agent in ginger and test it on other patient-derived HCC lines and in an in vivo model. In conclusion, our data suggest that ginger can potentially attenuate IFN-mediated signaling pathways in HCC, and cells from Black HCC patients may be more sensitive to ginger (Funded: P20 CA264068-01). Citation Format: Sadia Kanwal, Eva Davis, Seung Lee, Milton Omar Faison, Devanand Sarkar, Rafat Ali Siddiqui. Effect of ginger extracts on hepatocellular carcinoma cell lines-derived from Caucasian, Asian, and African American patients [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C052.

Increase of ISG15 in psoriasis lesions and its promotion of keratinocyte proliferation via the Hif-1α signalling pathway.

Psoriasis is a frequent chronic, recurrent and immune-mediated inflammatory skin disease, whose pathogenesis remains unclear at present. The role of antiviral protein in the pathogenesis of psoriasis is the focus of current research. Interferon stimulated gene 15 (ISG15) is an important antiviral protein. In this study, the expression of ISG15 saw a significant increase through the immunohistochemical detection of imiquimod (IMQ)-induced mice. In the psoriasis cell model, a remarkable increase also occurred in the expression of ISG15. In this study, it was found that the cell cycle was blocked in G1/S conversion, and a reduction took place in the proliferation of keratinocytes and the expression of a cell cycle-related protein-cyclin D1 after the knockout of ISG15 in the psoriasis cell model. After that, messenger ribonucleic acid (mRNA) sequencing and Gene Ontology/Kyoto Encyclopedia of Genes and Genomes (GO/KEGG) analysis indicated its close association with the hypoxia inducible factor-1α (HIF-1α) signalling pathway. Western blot showed a decrease in the expression of HIF-1α and vascular endothelial growth factor C (VEGFC) after the knockout of the ISG15 gene. The rescue experiment verified that ISG15 promotes the proliferation of keratinocytes by regulating the HIF-1α signalling pathway. It was concluded that psoriasis cells and mouse models witnessed the increased expression of ISG15. In psoriasis, knocking out ISG15 inhibits the proliferation of keratinocytes and blocks the cell cycle. Besides, ISG15 promotes the proliferation of keratinocytes through the HIF-1α signalling pathway.

ISG15–USP18 Dysregulation by Oxidative Stress Promotes IFN-γ Secretion from CD8+ T Cells in Vitiligo

Excessive oxidative stress is thought to play pathologic roles in cellular senescence and autoimmune disorders by inducing inflammation and breaking down immune tolerance. In this study, we sought to identify the factors linking oxidative stress to autoimmunity and cellular senescence in vitiligo, where elevated oxidative stress plays an important role. RNA sequencing analysis of hydrogen peroxide-treated melanocytes revealed upregulation of ISG15. The upregulation of ISG15 was observed in vitiligo skin tissues as well as in the blood of patients with vitiligo, whereas USP18 downregulation was observed in vitiligo melanocytes and vitiligo skin tissues. Oxidative stress induced hypermethylation of the USP18 promoter region in keratinocytes and melanocytes, and USP18 promoter hypermethylation was also confirmed in vitiligo skin tissues. Our results indicate that USP18 promoter hypermethylation caused by oxidative stress increases ISG15 expression in keratinocytes and melanocytes along with senescence changes, leading CD8+ T cells to produce IFN-γ, the main pathogenic cytokine in vitiligo. Therefore, the ISG15-USP18 network may be important in oxidative stress-induced autoimmunity and cellular senescence in vitiligo pathogenesis.

Abstract LB156: Identified the ISG15 mediated extracellular vesicles drives ovarian cancer progression and metastasis: extracellular vesicular ISG15 is a potential biomarker and therapeutic target

Abstract Background & Objective: High-grade serous ovarian cancer (HGSOC) accounts for over 80% of all epithelial ovarian cancer (OC) diagnoses, and the majority of patients with HGSOC are diagnosed with advanced metastatic disease. Aberrant expression of interferon stimulated gene 15 (ISG15) has been demonstrated in human malignancies, and we have identified that ISG15 is highly expressed in HGSOC tumors and peritoneal ascites. This study seeks to determine whether ISG15 contributes to HGSOC progression and metastasis through extracellular vesicle (EVs) secretion. Method: ISG15 expression was analyzed in ascites samples and primary ovarian cancer cells (POCC) from different patients by ELISA and WB. Cell surface biotinylation assay was done to show the modulation of endo and exocytosis by ISG15 and STAT3. Immunoprecipitation pull down assay was done to demonstrate the interaction of ISG15 with activated STAT3. Confocal microscopy showed the co-localization of STAT3 with the endosome marker TSG101. In-vivo studies were done using bio-luminescence imaging in orthotopic ovarian tumor mouse models to measure tumor progression and metastasis. Results: ISG15 was found to be significantly elevated in HGSOC metastases (pelvic, mesenteric and diaphragmatic samples) as compared to primary ovarian tumors or benign samples. We observed that ISGylation was increased in POCC cells derived from ascites with increased USP18 expression. Our results confirmed a significant decrease in EV’s secretion in ISG15 KD POCC cells. Mice injected with ISG15 OE -POCC cells showed increased tumor burden when compared to the ISG15Kd cells in mice. Furthermore, we observed a significant reduction of ovarian tumor growth and metastasis in an orthotopic mouse model treated with a small molecule inhibitor-DAP5 that targeted ISG15 or exosome blocker (Amiloride) compared to untreated mice. In addition, we found the expression of ISG15 within the EVs represents a promising development in elucidating prognostic markers for HGSOC patients. Conclusion: Based on our results, ISG15 expression is elevated in HGSOC patient ascites and metastatic disease sites. The aberrant expression of ISG15 in patient ascites plays a key role in the secretion of EVs carrying ISG15, which contributes to HGSOC progression and metastases. Our study provides the pre-clinical evidence regarding new molecular targets, novel prognostic markers, and innovative therapeutic strategies for HGSOC, aiming to ultimately improve the survival of patients suffering from this disease. Citation Format: Kalpana Deepa Priya Dorayappan, Vincent Wagner, Dongju Park, Meghan M. Newcomer, Michelle DS Lightfoot, Deepika Kalaiyarasan, Takahiko Sakaue, Wafa Khadraoui, Casey Cosgrove, Larry J. Maxwell, Qi-En Wang Wang, David O’Malley, Raphael E. Pollock, David E. Cohn, Selvendiran Karuppaiyah. Identified the ISG15 mediated extracellular vesicles drives ovarian cancer progression and metastasis: extracellular vesicular ISG15 is a potential biomarker and therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB156.

Abstract LB201: A novel combination of Listeria-based immunotherapy and repurposed drug combats colorectal cancer

Abstract Background: Colorectal cancer (CRC) has been a difficult-to-treat cancer with a significant proportion of microsatellite stable (MSS-CRC) patients who do not respond to immunotherapy. Currently, various strategies are being investigated to sensitize MSS-CRC tumors by converting them to immunologically “hot” tumors and improve responsiveness to immunotherapy. In this study, we demonstrate a novel strategy of combining a repurposed FDA-approved compound, Pimavanserin tartrate (PVT), with a Listeria monocytogenes (Lm)-based vaccine targeting Interferon-Stimulated Gene 15(ISG15), Lm-LLO-ISG15, against MSS-CRC tumors. Our findings demonstrate that both agents synergize to produce CD8+ T cell-dependent therapeutic efficacy in immunotherapy-resistant MSS-CRC tumors. Methods: The direct killing effect of PVT in vitro was confirmed by cytotoxic SRB assay. BALB/c mice bearing CT26, a murine model of MSS-CRC, were randomly assigned to receive either PBS, PVT, Lm-LLO-ISG15, or PVT+Lm-LLO-ISG15. Mice were monitored for tumor growth kinetics to study the anti-cancer effect of each monotherapy and the combination therapy. Changes in immune-related gene and protein expression were analyzed by qPCR and Western blot, respectively. Various immune cell subsets in the tumors and spleens were evaluated by multi-color flow cytometry and immunohistochemistry. In addition, organs such as the liver, kidney, heart, lungs, spleen, brain, and pancreas were collected for safety analysis. Results: PVT efficiently induced apoptosis and directly killed both human and mouse MSS-CRC cell lines in vitro. Further, PVT treatment enhanced T-cell activation and induced ISG15 expression, thus, sensitizing the tumor cells to targeting by the CTL-mediated immunotherapy, Lm-LLO-ISG15. While each monotherapy failed to demonstrate anti-tumor efficacy, treatment with the combination therapy (PVT + Lm-LLO-ISG15) significantly controlled tumor burden and extended the median survival. The therapeutic efficacy of the combination approach was associated with a higher infiltration of CD8+ T cells in the TME and an increased population of effector memory T cells (CD4+CD44hiCD62Llo) in the spleen. The anti-tumor efficacy of combination therapy on subcutaneous CT26 tumor growth was completely abrogated after the depletion of CD8+ T cells. Moreover, the safety profile of dual therapy was comparable to that of each monotherapy and PBS. Conclusions: MSS-CRCs are considered “immunologically cold” tumors that represent great challenges for any standalone therapies. In this study, we evaluated, for the first time, the therapeutic efficacy of a novel combination of PVT and Lm-LLO-ISG15 in an aggressive MSS-CRC mouse model. Combination therapy demonstrated a synergistic effect that led to a significant reduction of tumor burden and an extension of median survival. While the underlying mechanism for synergism requires further examination, our findings strongly suggest that combining PVT and Lm-LLO-ISG15 could be a promising approach for MSS-CRC patients. Citation Format: Shreyas Ramchandra Gaikwad, Hong My Nguyen, Sanjay K. Srivastava, Laurence Wood. A novel combination of Listeria-based immunotherapy and repurposed drug combats colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB201.

The role of interferon-stimulated gene 15 in the occurence and progression of cervical squamous cell carcinoma.

To identify molecular markers for early diagnosis and new targets for treatment of cervical squamous cell carcinoma. Our study involved 52 carcinoma tissues that were confirmed pathologically as cervical squamous cell carcinoma (CSCC) at the Fourth Hospital of Hebei Medical University in 2021. We obtained 36 control specimens from patients who had undergone hysterectomy for benign uterine diseases in 2021, with no cervical lesions as confirmed by pathology. Total RNA was extracted from all the samples. Reverse transcription and quantitative real-time PCR were performed. Immunohistochemical staining for interferon-stimulated gene 15 (ISG15) protein was performed. Descriptive analyses including mean and standard deviation were used to compare different groups. For data that do not conform to normal distribution, we use Wilcox rank sum test to make statistics to compare different groups with the median and interquartile. Mann Whitney U test was used to compare non-parametric continuous data, and categorical variables were analyzed using chi-square test. Receiver operating characteristic (ROC) curve was used to evaluate the possibility of using ISG15 as a new biomarker for cervical squamous cell carcinoma. Compared with normal cervical tissues, mRNA expression of ISG15 in cervical cancer tissues was significantly lower (P<0.01); mRNA expression was significantly lower in patients with nerve invasion (P<0.05). Difference in ISG15 protein expression was statistically significant (no expression/low expression) in the cancer samples compared to normal tissues (P<0.01). The area under ROC curve was 0.810 (P<0.001) and the sensitivity and specificity were 75% and 54%, respectively. Spearman's correlation analysis showed that ISG15 mRNA was positively correlated with protein expression (r=0.358, P=0.001). Deficiency of ISG15 may be associated with the occurrence and progression of CSCC. It could be used as a potential tumor marker in research and treatment of CSCC.

The role of interferon-stimulated gene 15 in the occurence and progression of cervical squamous cell carcinoma.

To identify molecular markers for early diagnosis and new targets for treatment of cervical squamous cell carcinoma. Our study involved 52 carcinoma tissues that were confirmed pathologically as cervical squamous cell carcinoma (CSCC) at the Fourth Hospital of Hebei Medical University in 2021. We obtained 36 control specimens from patients who had undergone hysterectomy for benign uterine diseases in 2021, with no cervical lesions as confirmed by pathology. Total RNA was extracted from all the samples. Reverse transcription and quantitative real-time PCR were performed. Immunohistochemical staining for interferon-stimulated gene 15 (ISG15) protein was performed. Descriptive analyses including mean and standard deviation were used to compare different groups. For data that do not conform to normal distribution, we use Wilcox rank sum test to make statistics to compare different groups with the median and interquartile. Mann Whitney U test was used to compare non-parametric continuous data, and categorical variables were analyzed using chi-square test. Receiver operating characteristic (ROC) curve was used to evaluate the possibility of using ISG15 as a new biomarker for cervical squamous cell carcinoma. Compared with normal cervical tissues, mRNA expression of ISG15 in cervical cancer tissues was significantly lower (P<0.01); mRNA expression was significantly lower in patients with nerve invasion (P<0.05). Difference in ISG15 protein expression was statistically significant (no expression/low expression) in the cancer samples compared to normal tissues (P<0.01). The area under ROC curve was 0.810 (P<0.001) and the sensitivity and specificity were 75% and 54%, respectively. Spearman's correlation analysis showed that ISG15 mRNA was positively correlated with protein expression (r=0.358, P=0.001). Deficiency of ISG15 may be associated with the occurrence and progression of CSCC. It could be used as a potential tumor marker in research and treatment of CSCC.

A Listeria-based vaccine targeting ISG15 exerts anti-tumor efficacy in renal cell carcinoma.

Renal cell carcinoma (RCC) is the deadliest form of urological cancer and is projected to be the fourth most common neoplasm in the USA in males by 2040. In addition to the current poor prognosis with 5-year survival rates hardly reaching 15%, the prevalence of resistance to currently available systemic therapies has also established an urgent need to develop new treatment regimen(s) for advanced RCC. Interferon-stimulated gene 15 (ISG15) is the first identified ubiquitin-like modifier and has been intensively studied for its central role in innate immunity against intracellular pathogens. However, in this study, we identified ISG15 as a novel tumor-associated antigen and prognostic marker in RCC. Further, we therapeutically targeted elevated ISG15 expression by means of a Listeria monocytogenes (Lm)-based vaccine, designated Lm-LLO-ISG15, in both subcutaneous and orthotopic RCC mouse models. Treatment with Lm-LLO-ISG15 resulted in an influx of tumor-infiltrating effector T cells and significant anti-tumor efficacy in both subcutaneous and orthotopic RCC tumor models. Treatment with Lm-LLO-ISG15 also generated a robust interferon-gamma response and attracted a larger pool of polyfunctional T cells into the tumor microenvironment. Importantly, the therapeutic efficacy of Lm-LLO-ISG15 in RCC is comparable to that of anti-PD-1 and sunitinib, the current frontline therapies for RCC patients. Collectively, our work illustrates that targeting ISG15 in RCC with a CTL-based immunotherapy such as Lm-LLO-ISG15 is a promising and potentially translatable therapeutic strategy to enhance survival in RCC patients.

Free ISG15 inhibits Pseudorabies virus infection by positively regulating type I IFN signaling.

Interferon-stimulated gene 15 (ISG15) is strongly upregulated during viral infections and exerts pro-viral or antiviral actions. While many viruses combat host antiviral defenses by limiting ISG expression, PRV infection notably increases expression of ISG15. However, studies on the viral strategies to regulate ISG15-mediated antiviral responses are limited. Here, we demonstrate that PRV-induced free ISG15 and conjugated proteins accumulation require viral gene expression. Conjugation inhibition assays showed that ISG15 imposes its antiviral effects via unconjugated (free) ISG15 and restricts the viral release. Knockout of ISG15 in PK15 cells interferes with IFN-β production by blocking IRF3 activation and promotes PRV replication. Mechanistically, ISG15 facilitates IFNα-mediated antiviral activity against PRV by accelerating the activation and nuclear translocation of STAT1 and STAT2. Furthermore, ISG15 facilitated STAT1/STAT2/IRF9 (ISGF3) formation and ISGF3-induced IFN-stimulated response elements (ISRE) activity for efficient gene transcription by directly interacting with STAT2. Significantly, ISG15 knockout mice displayed enhanced susceptibility to PRV, as evidenced by increased mortality and viral loads, as well as more severe pathology caused by excessive production of the inflammatory cytokines. Our studies establish the importance of free ISG15 in IFNα-induced antiviral immunity and in the control of viral infections.

Listeria-Based Vaccines Targeting Interferon-Stimulated Gene 15 (ISG15) for Renal Cell Carcinoma and Colorectal Cancer

<h3>Purpose/Objective(s)</h3> Interferon-stimulated gene 15 (ISG15) is the first identified ubiquitin-like modifier and has been implicated for its central role in innate immunity against intracellular pathogens. Since cancer cells oftentimes have defects in type I IFN signaling pathways, there is a growing body of evidence that demonstrated the role of ISG15 in cancer progression. Here, we investigate to understand if ISG15 could be another novel cancer-associated antigen in renal cell carcinomas (RCC) and colorectal cancer (CRC). Our research objectives include (i) to identify if the elevated expression of ISG15 in RCC and CRC has any prognosis signature, (ii) to explore if ISG15 can be targeted by mean of Listeria-based vaccines, and (iii) to analyze how Listeria-based vaccines targeting ISG15 change the tumor microenvironment of RCC and CRC in syngeneic mouse models. <h3>Materials/Methods</h3> Data on RNA and protein expression of ISG15 and the Kaplan–Meier survival analysis was obtained from Human Protein Atlas, The Cancer Genome Atlas, and from the University of Alabama at Birmingham, Comprehensive Cancer Center (UALCAN). RNA was extracted from tissue or cells and converted to cDNA. The cDNA was then subjected to qPCR analysis with primers specific to ISG15. All mouse experiments were performed in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) at the TTUHSC. <h3>Results</h3> We found that the elevated expression of ISG15 in RCC and CRC is significantly associated with an unfavorable prognosis. Similarly, RCC and CRC murine cell lines also expressed a higher level of ISG15 compared to the normal fibroblast cell line. In subcutaneous syngeneic mouse models of RCC and CRC, vaccination with a Listeria-based vaccine targeting ISG15, designated Lm-LLO-ISG15, significantly controls primary tumor burden and extends median survival compared to control groups. In both models, treatment with Lm-LLO-ISG15 inflames the tumor microenvironment, enhances the influx of immune cells, and induces the expression of programmed cell death protein 1 (PD-1). <h3>Conclusion</h3> ISG15 is a novel cancer-associated antigen and a potential target for active immunotherapies such as Listeria-based vaccines. Treatment with Lm-LLO-ISG15 significantly controls tumor burden in both RCC and CRC syngeneic mouse models and sensitizes the tumors with anti-PD-1 therapies.

Interferon-stimulated gene 15 and ISGylation are upregulated in glioblastoma

Interferon stimulated gene 15 (ISG15) encodes a 15-kDa ubiquitin-like protein that acts as a posttranslational modifier of target proteins via ISGylation, a catalytic process similar to ubiquitination. Protein ISGylation is associated with the modulation of protein stability and protein-protein interactions. Furthermore, non-conjugated ISG15 (free ISG15) is secreted to act as a cytokine-like protein in some cellular contexts. The expression of ISG15 in some cancer types is dysregulated, but its expression status in glioblastoma, a malignant brain tumor highly aggressive and invasive, requires more studies. To explore the potential of ISG15 as a biomarker for glioblastoma, we first evaluated the ISG15 levels in glioblastoma cell lines and the effect of IFN-γ treatment on protein levels and localization of ISG15. In addition, we analyzed the ISG15 levels in glioblastoma samples compared to healthy brain tissue. Our results indicate that ISG15 levels are increased in glioblastoma and are upregulated in response to IFN-γ stimulus, suggesting that ISG15 and ISGylation may play a central role in glioblastoma progression. Thus, ISG15/ISGyaltion may be useful as biomarkers of this type of malignant brain tumors.

Preeclampsia is Associated With Reduced ISG15 Levels Impairing Extravillous Trophoblast Invasion.

Among several interleukin (IL)-6 family members, only IL-6 and IL-11 require a gp130 protein homodimer for intracellular signaling due to lack of intracellular signaling domain in the IL-6 receptor (IL-6R) and IL-11R. We previously reported enhanced decidual IL-6 and IL-11 levels at the maternal-fetal interface with significantly higher peri-membranous IL-6 immunostaining in adjacent interstitial trophoblasts in preeclampsia (PE) vs. gestational age (GA)-matched controls. This led us to hypothesize that competitive binding of these cytokines to the gp130 impairs extravillous trophoblast (EVT) differentiation, proliferation and/or invasion. Using global microarray analysis, the current study identified inhibition of interferon-stimulated gene 15 (ISG15) as the only gene affected by both IL-6 plus IL-11 vs. control or IL-6 or IL-11 treatment of primary human cytotrophoblast cultures. ISG15 immunostaining was specific to EVTs among other trophoblast types in the first and third trimester placental specimens, and significantly lower ISG15 levels were observed in EVT from PE vs. GA-matched control placentae (p = 0.006). Induction of primary trophoblastic stem cell cultures toward EVT linage increased ISG15 mRNA levels by 7.8-fold (p = 0.004). ISG15 silencing in HTR8/SVneo cultures, a first trimester EVT cell line, inhibited invasion, proliferation, expression of ITGB1 (a cell migration receptor) and filamentous actin while increasing expression of ITGB4 (a receptor for hemi-desmosomal adhesion). Moreover, ISG15 silencing further enhanced levels of IL-1β-induced pro-inflammatory cytokines (CXCL8, IL-6 and CCL2) in HTR8/SVneo cells. Collectively, these results indicate that ISG15 acts as a critical regulator of EVT morphology and function and that diminished ISG15 expression is associated with PE, potentially mediating reduced interstitial trophoblast invasion and enhancing local inflammation at the maternal-fetal interface. Thus, agents inducing ISG15 expression may provide a novel therapeutic approach in PE.

Interferon‑stimulated gene 15 promotes progression of endometrial carcinoma and weakens antitumor immune response.

Endometrial carcinoma (EC) is one of the most common gynecological cancers with a poor prognosis. Therefore, clarifying the details of the molecular mechanisms is of great importance for EC diagnosis and clinical management. Interferon-stimulated gene 15 (ISG15) plays an important role in the development of various cancers. However, its role in EC remains unclear. High ISG15 expression was observed in EC, which was associated with poor clinical outcomes and pathological stage of patients with EC, thus representing a promising marker for EC progression. Further exploratory analysis revealed that the elevated ISG15 levels in EC were driven by aberrant DNA methylation, independent of copy number variation and specific transcription factor aberrations. Accordingly, knockdown of ISG15 by small interfering RNA attenuated the malignant cellular phenotype of EC cell lines, including proliferation and colony formation in vitro. Finally, investigation of the molecular mechanisms indicated that ISG15 promoted the cell cycle G1/S transition in EC. Furthermore, ISG15 promoted EC progression by activating the MYC proto-oncogene protein signaling pathway. Moreover, ECs with high levels of ISG15 harbored a more vital immune escape ability, evidenced not only by significantly less invasive CD8+ T cells, but also higher expression of T cell inhibitory factors, such as programmed death-ligand 1. These results suggest a tumor-promoting role of ISG15 in EC, which may be a promising marker for diagnosis, prognosis and therapeutic immunity.

Interferon-stimulated gene 15 pathway is a novel mediator of endothelial dysfunction and aneurysms development in angiotensin II infused mice through increased oxidative stress.

Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that induces a reversible post-translational modification (ISGylation) and can also be secreted as a free form. ISG15 plays an essential role as host-defence response to microbial infection; however, its contribution to vascular damage associated with hypertension is unknown. Bioinformatics identified ISG15 as a mediator of hypertension-associated vascular damage. ISG15 expression positively correlated with systolic and diastolic blood pressure and carotid intima-media thickness in human peripheral blood mononuclear cells. Consistently, Isg15 expression was enhanced in aorta from hypertension models and in angiotensin II (AngII)-treated vascular cells and macrophages. Proteomics revealed differential expression of proteins implicated in cardiovascular function, extracellular matrix and remodelling, and vascular redox state in aorta from AngII-infused ISG15-/- mice. Moreover, ISG15-/- mice were protected against AngII-induced hypertension, vascular stiffness, elastin remodelling, endothelial dysfunction, and expression of inflammatory and oxidative stress markers. Conversely, mice with excessive ISGylation (USP18C61A) show enhanced AngII-induced hypertension, vascular fibrosis, inflammation and reactive oxygen species (ROS) generation along with elastin breaks, aortic dilation, and rupture. Accordingly, human and murine abdominal aortic aneurysms showed augmented ISG15 expression. Mechanistically, ISG15 induces vascular ROS production, while antioxidant treatment prevented ISG15-induced endothelial dysfunction and vascular remodelling. ISG15 is a novel mediator of vascular damage in hypertension through oxidative stress and inflammation.

Streptococcus pneumoniae autolysin LytA inhibits ISG15 and ISGylation through decreasing bacterial DNA abnormally accumulated in the cytoplasm of macrophages

Interferon stimulated gene 15 (ISG15) is one of the most robustly upregulated interferon stimulated genes (ISGs) and also a ubiquitin-like modifier which has been reported to play an important role in host defense against pathogens. Cytosolic nucleic acids detected by DNA sensors induce type Ⅰ interferons (IFN-Ⅰs) and ISGs in host cells. Streptococcus pneumoniae (S. pn) autolysin LytA triggers bacterial lysis and then S. pn-derived genomic DNA (hereafter referred to as S. pn-DNA) can be released and accumulates in the cytoplasm of host cells. However, it remains elusive whether LytA-mediated S. pn-DNA release is involved in ISG15 induction. Here we verified that ISG15 conjugation system can be widely activated by S. pn and cytosolic S. pn-DNA in host cells. Moreover, the phagocytosis of macrophages to the mutant strain S. pn D39 ΔlytA was enhanced when compared to S. pn D39, which in turn increased S. pn-DNA uptake into macrophages and augmented ISG15 expression. ISG15 might upregulate proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in macrophages and further promoted the clearance of S. pn in the absence of LytA. These results indicate that S. pn autolysis blunts ISG15 induction through preventing bacteria internalization and reducing cytosolic S. pn-DNA accumulation in macrophages, revealing a new strategy of S. pn for avoiding elimination. This study will help us to further understand the role of ISG15 during S. pn infection as well as the regulatory mechanisms of immune responses mediated by bacterial autolysis and bacterial DNA.