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ObjectiveDiabetic cardiomyopathy (DCM) is a common complication of type 2 diabetes, which is characterised by dysfunction of the coronary microvascular endothelium as an important determinant of adverse cardiac remodeling. Whilst multiple signaling pathways are linked with DCM, specific mediators remain poorly understood. This study focused on interferon-stimulated gene 15 (ISG15) as a novel driver of endothelial dysfunction in experimental DCM and potential therapeutic target.Methods and ResultsIngenuity Pathway Analysis (IPA) was performed on data obtained from both bulk and single nuclei RNAseq analyses of mouse DCM tissue using R and Partek. Data sets met the criteria of an adjusted p-value of 0.05 and a log2 fold change ranging from -1.5 to 1.5, focusing on differentially expressed genes with endothelial cell enrichment. Interferon signalling and activation of interferon-related factors were identified as the most significantly altered canonical pathways linked with endothelial dysfunction, with expression of several component genes found to be significantly upregulated within the dataset, including ISG15 which was validated by real time RT-PCR analysis of DCM mouse tissue. Parallel in vitro studies established treatment of human coronary microvascular endothelial cells (HCMECs) with 25 mmol/L D-glucose for 14 days (versus L-glucose osmotic control) as a reliable model of experimental diabetes, characterised by barrier dysfunction (increased FITC-Dextran transfer, reduced expression of e.g. ZO-1, claudin-5, β-catenin) and significantly increased ISG15 expression. ISG15 siRNA knockdown restored high glucose-induced HCMEC barrier dysfunction and expression of the key adherens junction protein, β-catenin, but had no impact on expression of tight junction proteins, ZO-1 and claudin-5.ConclusionTaken together, these data highlight ISG15 is an important driver of coronary microvascular endothelial dysfunction in experimental diabetes which may represent a potential therapeutic target to reduce progression of adverse cardiovascular remodelling.Conflict of Interestnone

ISG15 deficiency is a rare disease caused by autosomal recessive variants in the ISG15 gene, which encodes the ISG15 protein. The ISG15 protein plays a dual role in both the type I and II interferon (IFN) immune pathways. Extracellularly, the ISG15 protein is essential for IFN-γ-dependent anti-mycobacterial immunity, while intracellularly, ISG15 is necessary for USP18-mediated downregulation of IFN-α/β signalling. Due to this dual role, ISG15 deficiency can present with various clinical phenotypes, ranging from susceptibility to mycobacterial infection to autoinflammation characterised by necrotising skin lesions, intracerebral calcification, and pulmonary involvement. In this report, we describe novel variants found in two different families that result in complete ISG15 deficiency and severe skin ulceration. Whole exome sequencing identified a heterozygous missense p.Q16X ISG15 variant and a heterozygous multigene 1p36.33 deletion in the proband from the first family. In the second family, a homozygous total ISG15 gene deletion was detected in two siblings. We also conducted further analysis, including characterisation of cytokine dysregulation, interferon-stimulated gene expression, and p-STAT1 activation in lymphocytes and lesional tissue. Finally, we demonstrate the complete and rapid resolution of clinical symptoms associated with ISG15 deficiency in one sibling from the second family following treatment with the Janus kinase (JAK) inhibitor baricitinib.

Increased number of tumor-infiltrating CD8+ lymphocytes is associated with improved survival in patients with advanced stage high grade serous ovarian cancer (HGSOC) but the underlying molecular mechanism has not been thoroughly explored. Using transcriptome profiling of microdissected HGSOC tissue with high and low CD8+ lymphocyte count and subsequent validation studies, we demonstrated that significantly increased ISG15 (Interferon-stimulated gene 15) expression in HGSOC was associated with high CD8+ lymphocyte count and with the improvement in median overall survival in both univariate and multivariate analyses. Further functional studies showed that endogenous and exogenous ISG15 suppressed ovarian cancer progression through ISGylation of ERK in HGSOC, and activation of NK cells and CD8+ T lymphocytes. These data suggest that the development of treatment strategies based on up-regulating ISG15 in ovarian cancer cells or increased circulating ISG15 in ovarian cancer patients is warranted.

Abstract Veterans who have served in the military are at a nearly 60% greater risk of being diagnosed with amyotrophic lateral sclerosis (ALS). Literature reports suggest that a history of traumatic brain injury (TBI) may be a risk factor for ALS in veterans. However, no diagnostic biomarkers are available for identifying ALS risk/development in TBI-exposed veterans. Here, using a Wes assay, we show that ISGylation, a conjugated form of interferon-stimulated gene 15 protein, is significantly elevated in the lumbar spinal cords (SC-Ls) of TBI-ALS compared with ALS veterans without a previous history of TBI (nonTBI-ALS). Although not as striking as in TBI-ALS veterans, ISGylation is also increased in nonTBI-ALS compared with normal veterans. Notably, no changes in ISGylation were seen in occipital lobe samples obtained from the same patients, suggesting that elevated ISGylation is distinct to ALS disease-specific SC-Ls. Moreover, we detected increased ISGylation in cerebral spinal fluid samples of TBI-ALS veterans. Other results using cultured lymphocyte cell lines show a similar trend of increased ISGylation in ALS patients from the general population. Together, these data suggest that ISGylation could serve as a diagnostic biomarker for TBI-ALS veterans, nonTBI-ALS veterans, and nonveterans affected by ALS.

Pregnancy loss (PL) in lactating dairy cows disrupts reproductive and productive efficiency. We evaluated the expression of interferon-stimulated genes (ISG) in blood leukocytes, vaginal and cervical epithelial cells, luteolysis-related genes, progesterone, and pregnancy-associated glycoprotein (PAG) profiles in lactating dairy cows (n = 86) to gain insight about PL. Expression of ISG on d17, d19, and d21 was greater in cows that maintained the pregnancy (P33) compared to nonpregnant with no PL (NP). Greater ISG differences between groups were observed in the cervix (96.7-fold) than vagina (31.0-fold), and least in blood leukocytes (5.6-fold). Based on individual profiles of ISG and PAG, PL was determined to occur either before (∼13%) or after (∼25%) d22. For cows with PL before d22, ISG expression was similar on d17 but by d21 was lower and OXTR was greater than P33 cows and similar to NP; timing of luteolysis was similar compared to NP cows suggesting embryonic failure to promote luteal maintenance and to attach to the endometrium (no increase in PAG). For cows with PL after d22, ISG expression was similar to P33 cows on d17, d19, and d21 and luteolysis, when it occurred, was later than NP cows; delayed increase in PAG suggested later or inadequate embryonic attachment. In conclusion, PL before d22 occurred due to embryonic demise/failure to signal for luteal maintenance, as reflected in reduced ISG expression by d21. Alternatively, embryos with PL between d22 and 33 adequately signaled for luteal maintenance (ISG) but had delayed/inadequate embryonic attachment and/or inappropriate luteolysis causing PL. Summary Sentence Evaluation of interferon-stimulated genes in multiple tissues showed that the cervix had the greatest expression and revealed that ∼13% of pregnancy loss occurred between d17 and 22, whereas ∼25% of pregnancy loss occurred between d22 and 33. Graphical Abstract

In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the molecular basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.

Vogt–Koyanagi–Harada (VKH) disease is a systemic autoimmune disorder affecting multiple organs, including eyes, skin, and central nervous system. It is known that monocytes significantly contribute to the development of autoimmune disease. However, the subset heterogeneity with unique functions and signatures in human circulating monocytes and the identity of disease-specific monocytic populations remain largely unknown. Here, we employed an advanced single-cell RNA sequencing technology to systematically analyze 11,259 human circulating monocytes and genetically defined their subpopulations. We constructed a precise atlas of human blood monocytes, identified six subpopulations—including S100A12, HLA, CD16, proinflammatory, megakaryocyte-like, and NK-like monocyte subsets—and uncovered two previously unidentified subsets: HLA and megakaryocyte-like monocyte subsets. Relative to healthy individuals, cellular composition, gene expression signatures, and activation states were markedly alternated in VKH patients utilizing cell type-specific programs, especially the CD16 and proinflammatory monocyte subpopulations. Notably, we discovered a diseaserelevant subgroup, proinflammatory monocytes, which showed a discriminative gene expression signature indicative of inflammation, antiviral activity, and pathologic activation, and converted into a pathologic activation state implicating the active inflammation during VKH disease. Additionally, we found the cell type-specific transcriptional signature of proinflammatory monocytes, ISG15, whose production might reflect the treatment response. Taken together, in this study, we present discoveries on accurate classification, molecular markers, and signaling pathways for VKH disease-associated monocytes. Therapeutically targeting this proinflammatory monocyte subpopulation would provide an attractive approach for treating VKH, as well as other autoimmune diseases.

Pancreatic cancer (PC) is one of the most aggressive and devastating types of cancer owing to its poor prognosis and deadly characteristics. It is well established that aberrations in the expression of key regulatory genes, namely tumor suppressors and oncogenes, predispose patients to progression and metastasis of PC. Upregulation of Williams-Beuren syndrome chromosomal region 22 (WBSCR22) expression, a ribosomal biogenesis factor, has been reported in multiple types of human cancer. However, the role of WBSCR22 and its underlying mechanism in PC have not been well investigated. In the present study, the tumor suppressive role of WBSCR22 was reported in PC for the first time; the results indicated that WBSCR22 overexpression (OE) significantly suppressed cellular proliferation, migration, invasion and tumorigenesis in vivo and in vitro. RNA-sequencing analysis revealed that WBSCR22 negatively regulated the transcription of interferon-stimulated gene 15 (ISG15) downstream, which is a ubiquitin-like modifier protein involved in metabolic and proteasome degradation pathways, while the antitumor function of WBSCR22-OE could be rescued by ISG15 OE. In addition, the oncogenic role of ISG15 was further confirmed in PC; its upregulation promoted the proliferation, migration, invasion and tumorigenesis of PC. Furthermore, WBSCR22 and its cofactor tRNA methyltransferase activator subunit 11-2 (TRMT112) functioned synergistically in PC, and concurrent ectopic OE of WBSCR22 and TRMT112 further promoted the tumor suppressive potential of WBSCR22 in PC. Collectively, the findings indicated that WBSCR22 played an important role in PC development and that the WBSCR22/ISG15 axis may provide a novel therapeutic strategy for PC treatment.

The protein ISG15 encoded by interferon-stimulated gene (ISG) 15 is the first identified member of the ubiquitin-like protein family and exists in the form of monomers and conjugated complexes. Like ubiquitin, ISG15 can mediate an ubiquitin-like modification by covalently modifying other proteins, known as ISGylation. There is growing evidence showing that both the free and conjugated ISG15 are involved in multiple key cellular processes, including autophagy, exosome secretion, DNA repair, immune regulation, and cancer occurrence and progression. In this review, we aim to further clarify the function of ISG15 and ISGylation in cancer, demonstrate the important relationship between ISG15/ISGylation and cancer, and emphasize new insights into the different roles of ISG15/ISGylation in cancer progression. This review may contribute to therapeutic intervention in cancer. However, due to the limitations of current research, the regulation of ISG15/ISGylation on cancer progression is not completely clear, thus further comprehensive and sufficient correlation studies are still needed.

BackgroundLymphovascular invasion (LVI) is a prognostic factor in early-stage invasive breast cancer (BC). Through bioinformatics, data analyses of multiple BC cohorts revealed the positive association between interferon-stimulated gene 15 (ISG15) LVI status. Thus, we explored the prognostic significance of ISG15 in BC.MethodsThe prognostic significance of ISG15 mRNA was assessed in METABRIC (n = 1980), TCGA (n = 854) and Kaplan–Meier Plotter (n = 3951). ISG15 protein was evaluated using immunohistochemistry (n = 859) in early-stage invasive BC patients with long-term follow-up. The associations between ISG15 expression and clinicopathological features, expression of immune cell markers and patient outcome data were evaluated.ResultsHigh mRNA and protein ISG15 expression were associated with LVI, higher histological grade, larger tumour size, hormonal receptor negativity, HER2 positivity, p53 and Ki67. High ISG15 protein expression was associated with HER2-enriched BC subtypes and immune markers (CD8, FOXP3 and CD68). High ISG15 mRNA and ISG15 expressions were associated with poor patient outcome. Cox proportional multivariate analysis revealed that the elevated ISG15 expression was an independent prognostic factor of shorter BC-specific survival.ConclusionThis study provides evidence for the role of ISG15 in LVI development and BC prognosis. Further functional studies in BC are warranted to evaluate the therapeutic potential of ISG15.