Interferon-stimulated Gene Transcripts Research Articles

Interferon Inducible Pseudouridine Modification in Human Transcriptome by Quantitative Nanopore Sequencing

Pseudouridine (Ψ) is an abundant mRNA modification in the mammalian transcriptome, but its functions have remained elusive due to the difficulty of transcriptome‐wide mapping. The Ψ derivative, N1‐methyl‐Ψ, substitutes every U residue in the Pfizer/BioNTech and Moderna COVID mRNA vaccines. Previous research revealed that Ψ increases translation of reporter genes and this effect depends on Protein kinase R (PKR), an interferon inducible gene. Thus, we hypothesize that endogenous Ψ modification in mRNA may be associated with interferon induced cellular response. In order to map Ψ in the human transcriptome, we developed a nanopore direct RNA sequencing method for quantitative Ψ mapping (NanoPsu, https://github.com/sihaohuanguc/Nanopore_psU) that utilizes native content training on 1329 Ψ and U sites, machine learning modeling and prediction, and single read linkage analysis. We validated our method using quantitative RT‐PCR of Ψ sites in specific mRNAs and through the observation of global Ψ level decrease upon knockdown of TRUB1, a known Ψ writer for mRNA. We apply the nanopore direct RNA sequencing method to interferon β and γ treated human cells and reveal a widespread differential distribution of Ψ in the human transcriptome. Biologically we find interferon inducible Ψ modifications in interferon stimulated gene transcripts which is consistent with a role of Ψ in enabling efficacy of mRNA vaccines. Single read analysis of the nanopore data reveals that multiple Ψ sites within the same transcript can be linked with each other. In summary, our results reveal a potential role of mRNA Ψ modification in interferon induced response and provide a new aspect in the research of mechanism of immune response.

The circadian clock component BMAL1 regulates SARS-CoV-2 entry and replication in lung epithelial cells

SummaryThe coronavirus disease 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract via spike glycoprotein binding to angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organism's response to its environment and can regulate host susceptibility to virus infection. We demonstrate that silencing the circadian regulator Bmal1 or treating lung epithelial cells with the REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry and replication. Importantly, treating infected cells with SR9009 limits SARS-CoV-2 replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced interferon-stimulated gene transcripts in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to limit SARS-CoV-2 infection. Our study highlights alternative approaches to understand and improve therapeutic targeting of SARS-CoV-2.

Autosomal-dominant early-onset spastic paraparesis with brain calcification due to IFIH1 gain-of-function.

We describe progressive spastic paraparesis in two male siblings and the daughter of one of these individuals. Onset of disease occurred within the first decade, with stiffness and gait difficulties. Brisk deep tendon reflexes and extensor plantar responses were present, in the absence of intellectual disability or dermatological manifestations. Cerebral imaging identified intracranial calcification in all symptomatic family members. A marked upregulation of interferon-stimulated gene transcripts was recorded in all three affected individuals and in two clinically unaffected relatives. A heterozygous IFIH1 c.2544T>G missense variant (p.Asp848Glu) segregated with interferon status. Although not highly conserved (CADD score 10.08 vs. MSC-CADD score of 19.33) and predicted as benign by in silico algorithms, this variant is not present on publically available databases of control alleles, and expression of the D848E construct in HEK293T cells indicated that it confers a gain-of-function. This report illustrates, for the first time, the occurrence of autosomal-dominant spastic paraplegia with intracranial calcifications due to an IFIH1-related type 1 interferonopathy.

Severe combined immunodeficiency in stimulator of interferon genes (STING) V154M/wild-type mice

Autosomal dominant gain-of-function mutations in human stimulator of interferon genes (STING) lead to a severe autoinflammatory disease called STING-associated vasculopathy with onset in infancy that is associated with enhanced expression of interferon-stimulated gene transcripts. The goal of this study was to analyze the phenotype of a new mouse model of STING hyperactivation and the role of type I interferons in this system. We generated a knock-in model carrying an amino acid substitution (V154M) in mouse STING, corresponding to a recurrent mutation seen in human patients with STING-associated vasculopathy with onset in infancy. Hematopoietic development and tissue histology were analyzed. Lymphocyte activation and proliferation were assessed invitro. STING V154M/wild-type (WT) mice were crossed to IFN-α/β receptor (IFNAR) knockout mice to evaluate the type I interferon dependence of the mutant Sting phenotype recorded. In STING V154M/WT mice we detected variable expression of inflammatory infiltrates in the lungs and kidneys. These mice showed a marked decrease in survival and developed a severe combined immunodeficiency disease (SCID) affecting B, T, and natural killer cells, with an almost complete lack of antibodies and a significant expansion of monocytes and granulocytes. The blockade in B- and T-cell development was present from early immature stages in bone marrow and thymus. In addition, invitro experiments revealed an intrinsic proliferative defect of mature T cells. Although the V154M/WT mutant demonstrated increased expression of interferon-stimulated genes, the SCID phenotype was not reversed in STING V154M/WT IFNAR knockout mice. However, the antiproliferative defect in T cells was rescued partially by IFNAR deficiency. STING gain-of-function mice developed an interferon-independent SCID phenotype with a T-cell, B-cell, and natural killer cell developmental defect and hypogammaglobulinemia that is associated with signs of inflammation in lungs and kidneys. Only the intrinsic proliferative defect of T cells was partially interferon dependent.

Genetic, Phenotypic, and Interferon Biomarker Status in ADAR1-Related Neurological Disease.

We investigated the genetic, phenotypic, and interferon status of 46 patients from 37 families with neurological disease due to mutations in ADAR1. The clinicoradiological phenotype encompassed a spectrum of Aicardi-Goutières syndrome, isolated bilateral striatal necrosis, spastic paraparesis with normal neuroimaging, a progressive spastic dystonic motor disorder, and adult-onset psychological difficulties with intracranial calcification. Homozygous missense mutations were recorded in five families. We observed a p.Pro193Ala variant in the heterozygous state in 22 of 23 families with compound heterozygous mutations. We also ascertained 11 cases from nine families with a p.Gly1007Arg dominant-negative mutation, which occurred de novo in four patients, and was inherited in three families in association with marked phenotypic variability. In 50 of 52 samples from 34 patients, we identified a marked upregulation of type I interferon-stimulated gene transcripts in peripheral blood, with a median interferon score of 16.99 (interquartile range [IQR]: 10.64-25.71) compared with controls (median: 0.93, IQR: 0.57-1.30). Thus, mutations in ADAR1 are associated with a variety of clinically distinct neurological phenotypes presenting from early infancy to adulthood, inherited either as an autosomal recessive or dominant trait. Testing for an interferon signature in blood represents a useful biomarker in this context.

Hepatology highlights

Hepatology highlights

Characterization of human disease phenotypes associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, and IFIH1.

Aicardi-Goutières syndrome is an inflammatory disease occurring due to mutations in any of TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR or IFIH1. We report on 374 patients from 299 families with mutations in these seven genes. Most patients conformed to one of two fairly stereotyped clinical profiles; either exhibiting an in utero disease-onset (74 patients; 22.8% of all patients where data were available), or a post-natal presentation, usually within the first year of life (223 patients; 68.6%), characterized by a sub-acute encephalopathy and a loss of previously acquired skills. Other clinically distinct phenotypes were also observed; particularly, bilateral striatal necrosis (13 patients; 3.6%) and non-syndromic spastic paraparesis (12 patients; 3.4%). We recorded 69 deaths (19.3% of patients with follow-up data). Of 285 patients for whom data were available, 210 (73.7%) were profoundly disabled, with no useful motor, speech and intellectual function. Chilblains, glaucoma, hypothyroidism, cardiomyopathy, intracerebral vasculitis, peripheral neuropathy, bowel inflammation and systemic lupus erythematosus were seen frequently enough to be confirmed as real associations with the Aicardi-Goutieres syndrome phenotype. We observed a robust relationship between mutations in all seven genes with increased type I interferon activity in cerebrospinal fluid and serum, and the increased expression of interferon-stimulated gene transcripts in peripheral blood. We recorded a positive correlation between the level of cerebrospinal fluid interferon activity assayed within one year of disease presentation and the degree of subsequent disability. Interferon-stimulated gene transcripts remained high in most patients, indicating an ongoing disease process. On the basis of substantial morbidity and mortality, our data highlight the urgent need to define coherent treatment strategies for the phenotypes associated with mutations in the Aicardi-Goutières syndrome-related genes. Our findings also make it clear that a window of therapeutic opportunity exists relevant to the majority of affected patients and indicate that the assessment of type I interferon activity might serve as a useful biomarker in future clinical trials.

Conceptus-induced changes in the gene expression of blood immune cells and the ultrasound-accessed luteal function in beef cattle: how early can we detect pregnancy?

We aimed to identify the functional characteristics of the corpus luteum (CL) by color Doppler ultrasonography and changes in interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells (PBMCs) during early pregnancy in beef cows. We then aimed to use these features to establish earlier pregnancy diagnosis methods. In experiment 1, the CL size and blood flow were accessed by Doppler ultrasonography, and the PBMCs were isolated on Days 8, 12, 15, 18, 20, 22, 25, 30, 45, and 60 post-timed artificial insemination (TAI) from pregnant (n = 10) and nonpregnant cows (n = 12). The abundance of ISG (OAS1, MX1, MX2, and ISG15) transcripts was measured by quantitative PCR. Analyses of OAS1 and MX2 expression in isolated PBMCs (ISG-PBMC method) and Doppler imaging of CL (Doppler-US method) were performed to test the accuracy of these methods for the diagnosis of pregnancy on Day 20 post-TAI (n = 110; experiment 2). In experiment 1, the luteal volume and blood flow were reduced in nonpregnant cows during the first weeks post-TAI, but an evaluation of CL vascularization and size was efficient in identifying nonpregnant cows on Day 20 post-TAI. The expression of ISGs in PBMCs can be stimulated by the presence of a viable conceptus between Days 15 and 22 post-TAI, and the expression of these genes reaches a peak on Day 20. In experiment 2, the Doppler-US and ISG-PBMC methods resulted in similar specificity (85.5 and 87.7%, respectively). However, only the Doppler-US method resulted in 100% sensitivity. In conclusion, the greatest abundance of ISGs in PBMCs and a high detection of luteolysis by Doppler imaging on Day 20 post-TAI can be feasibly used for the earlier detection of nonpregnant cows in reproductive programs. The level of accuracy for our described pregnancy methods is high on Day 20 (80%-91%), but only the Doppler imaging method results in an absence of false-negative diagnoses.

MicroRNA-122 Abundance in Hepatocellular Carcinoma and Non-Tumor Liver Tissue from Japanese Patients with Persistent HCV versus HBV Infection

Mechanisms of hepatic carcinogenesis in chronic hepatitis B and hepatitis C are incompletely defined but often assumed to be similar and related to immune-mediated inflammation. Despite this, several studies hint at differences in expression of miR-122, a liver-specific microRNA with tumor suppressor properties, in hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) versus hepatitis C virus (HCV) infection. Differences in the expression of miR-122 in these cancers would be of interest, as miR-122 is an essential host factor for HCV but not HBV replication. To determine whether the abundance of miR-122 in cancer tissue is influenced by the nature of the underlying virus infection, we measured miR-122 by qRT-PCR in paired tumor and non-tumor tissues from cohorts of HBV- and HCV-infected Japanese patients. miR-122 abundance was significantly reduced from normal in HBV-associated HCC, but not in liver cancer associated with HCV infection. This difference was independent of the degree of differentiation of the liver cancer. Surprisingly, we also found significant differences in miR-122 expression in non-tumor tissue, with miR-122 abundance reduced from normal in HCV- but not HBV-infected liver. Similar differences were observed in HCV- vs. HBV-infected chimpanzees. Among HCV-infected Japanese subjects, reductions in miR-122 abundance in non-tumor tissue were associated with a single nucleotide polymorphism near the IL28B gene that predicts poor response to interferon-based therapy (TG vs. TT genotype at rs8099917), and correlated negatively with the abundance of multiple interferon-stimulated gene transcripts. Reduced levels of miR-122 in chronic hepatitis C thus appear to be associated with endogenous interferon responses to the virus, while differences in miR-122 expression in HCV- versus HBV-associated HCC likely reflect virus-specific mechanisms contributing to carcinogenesis. The continued expression of miR-122 in HCV-associated HCC may signify an important role for HCV replication late in the progression to malignancy.

Temporal Dynamics of the Transcriptional Response to Dengue Virus Infection in Nicaraguan Children

Dengue is the most prevalent mosquito-borne human illness worldwide. The ability to predict disease severity during the earliest days of the illness is a long-sought, but unachieved goal.We examined human genome-wide transcript abundance patterns in daily peripheral blood mononuclear cell (PBMC) samples from 41 children hospitalized with dengue virus (DENV) infection in Nicaragua, as well as 8 healthy control subjects. Nine patients had primary dengue fever (DF1), 11 had dengue fever with serologic evidence of prior DENV infection, i.e., secondary dengue fever (DF2), 12 had dengue hemorrhagic fever (DHF), and 9 had dengue shock syndrome (DSS). We identified 2,092 genes for which transcript abundance differed significantly between patients on days 3–6 of fever and healthy subjects (FDR<1%). Prior DENV infection explained the greatest amount of variation in gene expression among patients. The number of differentially expressed genes was greatest on fever day 3 in patients with DF1, while the number in patients with DF2 or DHF/DSS was greatest on day 5. Genes associated with the mitotic cell cycle and B cell differentiation were expressed at higher levels, and genes associated with signal transduction and cell adhesion were expressed at lower levels, in patients versus healthy controls. On fever day 3, a set of interferon-stimulated gene transcripts was less abundant in patients who subsequently developed DSS than in other patient groups (p<0.05, ranksum). Patients who later developed DSS also had higher levels of transcripts on day 3 associated with mitochondrial function (p<0.01, ranksum). These day 3 transcript abundance findings were not evident on subsequent fever days.In conclusion, we identified differences in the timing and magnitude of human gene transcript abundance changes in DENV patients that were associated with serologic evidence of prior infection and with disease severity. Some of these differential features may predict the outcome of DENV infection.

Diminished production of monocyte proinflammatory cytokines during human immunodeficiency virus viremia is mediated by type I interferons.

The effect of human immunodeficiency virus (HIV) infection and high-level HIV replication on the function of monocytes was investigated. HIV-positive patients had elevated levels of spontaneous production of some or all of the monocyte proinflammatory cytokines measured (interleukin-1beta [IL-1beta], IL-6, and tumor necrosis factor alpha [TNF-alpha]) compared to uninfected controls. In patients on therapy with high frequencies of monocytes producing proinflammatory cytokines, this frequency was diminished in the context of viremia during an interruption of therapy. Diminished production of proinflammatory cytokines during viremia was restored by culture with autologous CD4(+) T cells or monocytes from an on-therapy time point or lipopolysaccharide (LPS). Microarray analysis demonstrated that diminished monocyte production of proinflammatory cytokines was correlated with elevated type I interferon-stimulated gene transcripts. The addition of exogenous alpha 2A interferon diminished the spontaneous production of IL-1beta, IL-6, and TNF-alpha but did not affect responses to LPS, recapitulating the changes observed for HIV-viremic patients. These results suggest that monocyte function is diminished during high-level HIV viremia and that this effect is mediated by chronic stimulation by type I interferons. This effect on monocytes during viremia may play a role in diminished innate or adaptive immune system functions in HIV-infected patients. In addition, the restoration of these functions may also play a role in some immune reconstitution syndromes observed during initiation of therapy.