SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

IFIT1 + neutrophil is a causative factor of immunosuppressive features of poorly cohesive carcinoma (PCC).

The importance of the immune microenvironment in poorly cohesive carcinoma (PCC) has been highlighted due to its limited response rate to conventional therapy and emerging treatment resistance. A combination of clinical cohorts, bioinformatics analyses, and functional/molecular experiments revealed that high infiltration of Interferon Induced Protein with Tetratricopeptide Repeats 1 (IFIT1) + tumor-associated neutrophils (TANs) is a distinguishing feature of PCC patients. Upregulation of IFIT1 + TANs promote migration and invasion of gastric cancer (GC) cell lines (MKN45 and MKN74) and stimulates the growth of cell-derived xenograft models. Besides, by promoting macrophage secreted phosphoprotein 1 (SPP1) expression and facilitating cancer-associated fibroblast and endothelial cell recruitment and activation through TANs, IFIT1 promotes a mesenchymal phenotype, which is associated with a poor prognosis. Importantly, compared to non-PCC (NPCC), PCC tumors is more immunosuppressive. Mechanistically, IFIT1 can be stimulated by IFN-γ and contributes to the expression of Programmed Cell Death 1 Ligand (PDL1) in TANs. We demonstrated in mouse models that IFIT1 + PDL1 + TANs can induce acquired resistance to anti-PD-1 immunotherapy, which may be responsible for the difficulty of PCC patients to benefit from immunotherapy. This work highlights the role of IFIT1 + TANs in mediating the remodeling of the tumor immune microenvironment and immunotherapeutic resistance and introduces IFIT1 + TANs as a promising target for precision therapy of PCC.

Nsp16 shields SARS-CoV-2 from efficient MDA5 sensing and IFIT1-mediated restriction.

Methylation of the mRNA 5' cap by cellular methyltransferases enables efficient translation and avoids recognition by innate immune factors. Coronaviruses encode viral 2'-O-methyltransferases to shield their RNA from host factors. Here, we generate recombinant SARS-CoV-2 harboring a catalytically inactive 2'-O-methyltransferase Nsp16, Nsp16mut, and analyze viral replication in human lung epithelial cells. Although replication is only slightly attenuated, we find SARS-CoV-2 Nsp16mut to be highly immunogenic, resulting in a strongly enhanced release of type I interferon upon infection. The elevated immunogenicity of Nsp16mut is absent in cells lacking the RNA sensor MDA5. In addition, we report that Nsp16mut is highly sensitive to type I IFN treatment and demonstrate that this strong antiviral effect of type I IFN is mediated by the restriction factor IFIT1. Together, we describe a dual role for the 2'-O-methyltransferase Nsp16 during SARS-CoV-2 replication in avoiding efficient recognition by MDA5 and in shielding its RNA from interferon-induced antiviral responses, thereby identifying Nsp16 as a promising target for generating attenuated and highly immunogenic SARS-CoV-2 strains and as a potential candidate for therapeutic intervention.

Characterization of an African swine fever virus outbreak in India and comparative analysis of immune genes in infected and surviving crossbreed vs. indigenous Doom pigs.

Since 2020, African swine fever (ASF) has affected all pig breeds in Northeast India except Doom pigs, a unique indigenous breed from Assam and the closest relatives of Indian wild pigs. ASF outbreaks result in significant economic losses for pig farmers in the region. Based on sequencing and phylogenetic analysis of the B646L (p72) gene, it has been determined that ASFV genotype II is responsible for outbreaks in this region. Recent studies have shown that MYD88, LDHB, and IFIT1, which are important genes of the immune system, are involved in the pathogenesis of ASFV. The differential expression patterns of these genes in surviving ASFV-infected and healthy Doom breed pigs were compared to healthy controls at different stages of infection. The ability of Doom pigs to withstand common pig diseases, along with their genetic resemblance to wild pigs, make them ideal candidates for studying tolerance to ASFV infection. In the present study, we investigated the natural resistance to ASF in Doom pigs from an endemic area in Northeast India. The results of this study provide important molecular insights into the regulation of ASFV tolerance genes.

Diagnosis of pulmonary tuberculosis via identification of core genes and pathways utilizing blood transcriptional signatures: a multicohort analysis.

BACKGROUND: Blood transcriptomics can be used for confirmation of tuberculosis diagnosis or sputumless triage, and a comparison of their practical diagnostic accuracy is needed to assess their usefulness. In this study, we investigated potential biomarkers to improve our understanding of the pathogenesis of active pulmonary tuberculosis (PTB) using bioinformatics methods. METHODS: Differentially expressed genes (DEGs) were analyzed between PTB and healthy controls (HCs) based on two microarray datasets. Pathways and functional annotation of DEGs were identified and ten hub genes were selected. They were further analyzed and selected, then verified with an independent sample set. Finally, their diagnostic power was further evaluated between PTB and HCs or other diseases. RESULTS: 62 DEGs mostly related to type I IFN pathway, IFN-γ-mediated pathway, etc. in GO term and immune process, and especially RIG-I-like receptor pathway were acquired. Among them, OAS1, IFIT1 and IFIT3 were upregulated and were the main risk factors for predicting PTB, with adjusted risk ratios of 1.36, 3.10, and 1.32, respectively. These results further verified that peripheral blood mRNA expression levels of OAS1, IFIT1 and IFIT3 were significantly higher in PTB patients than HCs (all P < 0.01). The performance of a combination of these three genes (three-gene set) had exceeded that of all pairwise combinations of them in discriminating TB from HCs, with mean AUC reaching as high as 0.975 with a sensitivity of 94.4% and a specificity of 100%. The good discernibility capacity was evaluated d via 7 independent datasets with an AUC of 0.902, as well as mean sensitivity of 87.9% and mean specificity of 90.2%. In regards to discriminating PTB from other diseases (i.e., initially considered to be possible TB, but rejected in differential diagnosis), the three-gene set equally exhibited an overall strong ability to separate PTB from other diseases with an AUC of 0.999 (sensitivity: 99.0%; specificity: 100%) in the training set, and 0.974 with a sensitivity of 96.4% and a specificity of 98.6% in the test set. CONCLUSION: The described commonalities and unique signatures in the blood profiles of PTB and the other control samples have considerable implications for PTB biosignature design and future diagnosis, and provide insights into the biological processes underlying PTB.

circ_0086296 induced atherosclerotic lesions via the IFIT1/STAT1 feedback loop by sponging miR-576-3p.

Extensive inflammation of endothelial cells (ECs) facilitates atherosclerotic lesion formation. Circular RNA (circRNA) participates in atherosclerosis (AS)-related inflammation responses; however, whether and how circ_0086296 regulates atherosclerotic inflammation and lesions have not been investigated. Microarray analysis, quantitative real-time polymerase chain reaction, and fluorescence in situ hybridization assay were performed to detect the expression and location of hsa_circ_0086296 in human carotid artery plaques, aorta of atherosclerotic mice, and human umbilical vein endothelial cells (HUVECs). Sanger sequencing was used to verify the loop structure of circ_0086296. The relationship among circ_0086296, miR-576-3p, IFIT1, STAT1, and EIF4A3 was validated using bioinformatics, luciferase assay, RNA pull-down assay, and RNA immunoprecipitation. The atherosclerosis mouse model was used to evaluate the function of circ_0086296 in vivo. circ_0086296 expression was significantly upregulated in human carotid artery plaques, oxidized low-density lipoprotein (ox-LDL)-treated HUVECs, and the aorta of atherosclerotic mice. Functional analysis indicated that circ_0086296 promotes ECs injury in vitro and atherosclerosis progression in vivo. The mechanism analysis indicated that circ_0086296 sponged miR-576-3p to promote IFIT1-STAT1 expression. Moreover, STAT1 upregulated circ_0086296 expression, forming the circ_0086296/miR-576-3p/IFIT1/STAT1 feedback loop. Notably, inhibition of the circ_0086296/miR-576-3p/IFIT1 axis could block atherosclerotic lesion formation both in vivo and in vitro. Finally, circ_0086296 was overexpressed in exosomes of patients with atherosclerosis and exosomes of ox-LDL-treated ECs. Therefore, the circ_0086296/miR-576-3p/IFIT1/STAT1 feedback loop participates in atherosclerosis progression and contributes to the high circ_0086296 expression observed in the exosomes of serum of patients with atherosclerosis. This study sought to provide a deep understanding of the mechanisms underlying the aberrant EC phenotype in AS.

IFIT Proteins Are Involved in CXCL10 Expression in Human Glomerular Endothelial Cells Treated with a Toll-Like Receptor 3 Agonist.

INTRODUCTION: Various viruses including a novel coronavirus (SARS-CoV-2) can infect the kidney. When viruses invade the glomeruli from the bloodstream, glomerular endothelial cells (GECs) initiate the innate immune reactions. We investigated the expression of interferon (IFN)-induced protein with tetratricopeptide repeats (IFIT) 1/2/3, antiviral molecules, in human GECs treated with a toll-like receptor (TLR) 3 agonist. Role of IFIT1/2/3 in the expression of C-X-C motif chemokine ligand 10 (CXCL10) was also examined. METHODS: Human GECs were cultured and stimulated with polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 agonist. Real-time qPCR, Western blotting, and ELISA were used to examine the expression of IFIT1/2/3, IFN-ß, and CXCL10. RNA interference against IFN-ß or IFIT1/2/3 was also performed. RESULTS: Expression of IFIT1/2/3 and CXCL10 was induced by poly IC in GECs. The inductions were inhibited by RNA interfering of IFN-ß. Knockdown of IFIT1/2/3 decreased the CXCL10 expression. Knockdown of IFIT3 decreased the expression of IFIT1 and IFIT2 proteins. CONCLUSION: IFIT1/2/3 and CXCL10 were induced by poly IC via IFN-ß in GECs. IFIT1/2/3 may increase the expression of CXCL10 which induces lymphocyte chemotaxis and may inhibit the replication of infected viruses. These molecules may play a role in GEC innate immune reactions in response to viruses.

Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2'-O methylations.

IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2'-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-Å complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations (syn and anti) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2'-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self- versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA.

Hepatitis E virus polymerase binds to IFIT1 to protect the viral RNA from IFIT1-mediated translation inhibition.

Hepatitis E virus (HEV) induces interferons and regulates the induction of interferon-stimulated genes (ISGs) in the host cell. HEV infection has been shown to promote the expression of different ISGs, such as ISG15, IFIT1, MX1, RSAD2/Viperin and CxCL10, in cell culture and animal models. Interferon-induced protein with tetratricopeptide repeat 1 (IFIT1) is an ISG-encoded protein that inhibits the translation of viral RNA, having 5'-triphosphate or the mRNA lacking 2'-O-methylation on the 5'cap. In this study, we found that IFIT1 binds to HEV RNA to inhibit its translation. HEV replication is also restricted in hepatoma cells with overexpressed IFIT1. However, despite this binding of IFIT1 to HEV RNA, HEV successfully replicates in hepatoma cells in the infection scenario. In an effort to identify the underlying mechanism, we found that HEV RNA-dependent RNA polymerase (RdRp) binds to IFIT1, thereby protecting the viral RNA from IFIT1-mediated translation inhibition. RdRp sequesters IFIT1, resulting in the successful progression of viral replication in the infected cells. Thus, we discovered a distinct pro-viral role of HEV RdRp that is crucial for successful infection in the host, and propose a unique mechanism developed by HEV to overcome IFIT1-mediated host immune response.

Grouper IFIT1 inhibits iridovirus and nodavirus infection by positively regulating interferon response.

Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), one of the interferon stimulated genes (ISGs), is strongly induced by type I interferon (IFN), double-stranded RNAs and virus infection. To investigate the actions of fish IFIT1 in response to virus infection, we cloned an IFIT1 homolog from orange spotted grouper (EcIFIT1) and clarified its function in this study. The full-length cDNA of EcIFIT1 is 1839 bp, which is composed of 436 amino acid (aa) residues, with 77.8% and 22.8% identity to IFIT1 homolog of yellow perch (Perca flavescens) and humans (homo sapiens), respectively. Sequence alignment analysis showed that EcIFIT1 contained three tetratricopeptide repeats (TPRs). Tissue distribution analysis indicated that EcIFIT1 was abundant in intestine, spleen, liver, and heart. Moreover, EcIFIT1 was significantly up-regulated by Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) infection, and polyinosinic-polycytidylic acid (poly I:C) or lipopolysaccharide (LPS) treatment in vitro. Under fluorescence microscopy, EcIFIT1 was found to localize throughout the cytoplasm in transfected cells. EcIFIT1 overexpression significantly suppressed the replication of SGIV and RGNNV, demonstrated by decreasing the cytopathic effect (CPE) severity, viral gene transcription and the virus titers. Further studies showed that the ectopic expression of EcIFIT1 increased the transcription level of IFN related molecules, including IFN regulatory factor (IRF) 3, IRF7, IFN stimulated gene (ISG) 15 and myxovirus resistance gene (MX) I. Meanwhile, the expression levels of pro-inflammation cytokines were differently regulated by the ectopic expression of EcIFIT1. In addition, flow cytometry analysis suggested that EcIFIT1 overexpression affected cell cycle progression by mediating S/G2 transition. Taken together, our results indicated that EcIFIT1 might exert antiviral function against fish virus by up-regulating interferon response or affecting cell cycle.

Proteomic Identification of Interferon-Induced Proteins with Tetratricopeptide Repeats as Markers of M1 Macrophage Polarization.

Macrophages, which accumulate in tissues during inflammation, may be polarized toward pro-inflammatory (M1) or tissue reparative (M2) phenotypes. The balance between these phenotypes can have a substantial influence on the outcome of inflammatory diseases such as atherosclerosis. Improved biomarkers of M1 and M2 macrophages would be beneficial for research, diagnosis, and monitoring the effects of trial therapeutics in such diseases. To identify novel biomarkers, we have characterized the global proteomes of THP-1 macrophages polarized to M1 and M2 states in comparison with unpolarized (M0) macrophages. M1 polarization resulted in increased expression of numerous pro-inflammatory proteins including the products of 31 genes under the transcriptional control of interferon regulatory factor 1 (IRF-1). In contrast, M2 polarization identified proteins regulated by components of the transcription factor AP-1. Among the most highly upregulated proteins under M1 conditions were the three interferon-induced proteins with tetratricopeptide repeats (IFITs: IFIT1, IFIT2, and IFIT3), which function in antiviral defense. Moreover, IFIT1, IFIT2, and IFIT3 mRNA were strongly upregulated in M1 polarized human primary macrophages and IFIT1 was also expressed in a subset of macrophages in aortic sinus and brachiocephalic artery sections from atherosclerotic ApoE-/- mice. On the basis of these results, we propose that IFITs may serve as useful markers of atherosclerosis and potentially other inflammatory diseases.

Ifit1 Protects Against Lipopolysaccharide and D-galactosamine-Induced Fatal Hepatitis by Inhibiting Activation of the JNK Pathway.

Treatment of mice with lipopolysaccharide (LPS) and the liver-specific transcriptional inhibitor D-(+)-galactosamine (GalN) induces fatal hepatitis, which is mediated by tumor necrosis factor α (TNF-α) and characterized by massive hepatic apoptosis. Previous studies suggest that GalN increases the sensitivity to LPS/TNF-α, probably by blocking the transcription of protective factors, but the identity of most of these factors is still unclear. Here, we report that Ifit1 protects against LPS/GalN-induced fatal hepatitis. Forced expression of Ifit1 in hepatocytes significantly diminished TNF-α-mediated apoptosis. Moreover, targeted expression of Ifit1 in the liver by recombinant adeno-associated virus serotype 8 protected mice from LPS/GalN-induced lethal hepatitis, which was associated with the inhibition of TNF-α-mediated activation of the c-Jun N-terminal kinase (JNK)-Bim cascade. Furthermore, Ifit1 bound to a scaffolding protein Axin and inhibited its function to mediate JNK activation. Together, our data demonstrate that Ifit1 is a novel protective factor that inhibits LPS/GalN-induced (TNF-α-mediated) fatal hepatitis, suggesting that Ifit1 is a potential therapeutic target for treatment of inflammatory liver diseases.

The Role of Interferon-Induced Proteins with Tetratricopeptide Repeats 1 and 2 in Sepsis-Induced Acute Liver Injury.

Background: Sepsis refers to a life-threatening organ dysfunction which can be resulted from the infection-induced dysregulated host response. A large number of inflammatory cytokines are released to act on the liver, making the liver one of the common target organs for the development of multiple organ dysfunction syndrome (MODS) in patients with sepsis. Sepsis-induced acute liver injury (SALI) can aggravate systemic disease. As a result, it is of great clinical significance to comprehend the molecular biological mechanism of SALI and to identify the markers for evaluating SALI. Interferon-induced proteins with tetratricopeptide repeats 1 and 2 (IFIT1, IFIT2) have been recognized as the anti-inflammatory factors that are widely expressed in various organs. The present study was aimed at clarifying the roles of IFIT1 and IFIT2 in the development of SALI. Methods: A two-sample Mendelian randomization (MR) analysis was employed. Summary statistics datas were obtained from GWAS for inflammatory factors [tumor necrosis factor (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6)], IFIT2, and sepsis as well as liver injury. Independent SNPs were selected as instrumental variables (IVs). Inverse variance weighted (IVW) in the MR analysis was adopted as the primary method for estimating the causal associations of inflammatory factors and IFIT2 with two diseases, and the associations of inflammatory factors with IFIT2. Additionally, weighted median method, MR-Egger and sensitivity analyses were applied in assessing the robustness of the results and ensure the result reliability. Subsequently, 119 healthy volunteers, 116 patients with sepsis and 116 SALI patients were recruited. The ELISA method was employed to quantify the expression levels of TNF-α, IL-1ß, and IL-6. Additionally, qRT-PCR was conducted to measure the expression of IFIT1 and IFIT2. Furthermore, the correlations of IFIT1 and IFIT2 with inflammatory factors, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were explored. Results: As shown by the MR analysis, the genetically predisposed sepsis was significantly associated with the risk of IL-1ß, with an odds ratio (OR) of 1.069 (95% confidence interval (CI), 1.015-1.127, p = 0.0119), and negatively associated with the risk of IL-6, with an OR of 0.880 (95% CI: 0.792-0.979, p= 0.0184). Meanwhile, there were positive causal effects of IL-6 (OR = 1.269, 95% CI: 1.032-1.561, p= 0.0238), IL-1ß (OR = 1.106, 95% CI: 1.010-1.211, p = 0.0299) and IFIT2 (OR = 1.191, 95% CI: 1.045-1.359, p = 0.0090) on liver injury. Additionally, there was a positive causal effect of IFIT2 (OR = 1.164, 95% CI: 1.035-1.309, p= 0.0110) on IL-1ß. Upon sensitivity analyses, there was weak evidence of such effects, indicating that the findings of this study were robust and reliable. Our results revealed the elevated levels of TNF-α, IL-1ß, and IL-6 in the blood samples of sepsis and SALI patients (p < 0.0001). Conversely, IFIT1 and IFIT2 demonstrated the significantly decreased levels in peripheral blood mononuclear cells (PBMCs) of SALI patients (p < 0.0001). Furthermore, the expression levels of IFIT1 and IFIT2 were both negatively correlated with ALT activity (r = -0.3426, p = 0.0002; r = -0.3069, p = 0.0008) and AST activity (r = -0.2483, p = 0.0072; r = -0.3261, p = 0.0004), respectively. Moreover, the expression of IFIT1 and IFIT2 was both negatively related to the levels of TNF-α (r = -0.5027, p < 0.0001; r = -0.4218, p < 0.0001), IL-1ß (r = -0.3349, p = 0.0002; r = -0.4070, p < 0.0001) and IL-6 (r = -0.2734, p = 0.0030; r = -0.3536, p < 0.0001), respectively. Conclusion: IFIT1 and IFIT2 can serve as the diagnostic markers for sepsis-related liver injury, and IFIT1 and IFIT2 may participate in the pathological process of sepsis-related liver injury by regulating inflammation and liver function.

IFIT1 modulates the proliferation, migration and invasion of pancreatic cancer cells via Wnt/ß-catenin signaling.

OBJECTIVES: Previously, Interferon-induced Protein with Tetratricopeptide Repeats 1 (IFIT1) has been shown to promote cancer development. Here, we aimed to explore the role of IFIT1 in the development and progression of pancreatic cancer, including the underlying mechanisms. METHODS: We explored IFIT1 expression in pancreatic cancer samples using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Cell Counting Kit-8 (CCK8), colony formation, scratch wound-healing and Transwell assays were performed to assess the proliferation, migration and invasion abilities of pancreatic cancer cells. Gene Set Enrichment Analysis (GSEA) and Western blotting were performed to assess the regulatory effect of IFIT1 on the Wnt/ß-catenin pathway. RESULTS: We found that upregulation of IFIT1 expression is common in pancreatic cancer and is negatively associated with overall patient survival. Knockdown of IFIT1 expression led to decreased proliferation, migration and invasion of pancreatic cancer cells. We also found that IFIT1 could regulate Wnt/ß-catenin signaling, and that a Wnt/ß-catenin agonist could reverse this effect. In addition, we found that IFIT1 can promote epithelial-mesenchymal transition (EMT) of pancreatic cancer cells. CONCLUSIONS: Our data indicate that IFIT1 increases pancreatic cancer cell proliferation, migration and invasion by activating the Wnt/ß-catenin pathway. In addition, we found that EMT could be regulated by IFIT1. IFIT1 may serve as a potential therapeutic target for pancreatic cancer.

Single-cell transcriptomics of blood identified IFIT1+ neutrophil subcluster expansion in NTM-PD patients.

OBJECTIVE: Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is caused by an imbalance between pathogens and impaired host immune responses. Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are the two major pathogens that cause NTM-PD. In this study, we sought to dissect the transcriptomes of peripheral blood immune cells at the single-cell resolution in NTM-PD patients and explore potential clinical markers for NTM-PD diagnosis and treatment. METHODS: Peripheral blood samples were collected from six NTM-PD patients, including three MAB-PD patients, three MAC-PD patients, and two healthy controls. We employed single-cell RNA sequencing (scRNA-seq) to define the transcriptomic landscape at a single-cell resolution. A comprehensive scRNA-seq analysis was performed, and flow cytometry was conducted to validate the results of scRNA-seq. RESULTS: A total of 27,898 cells were analyzed. Nine T-cells, six mononuclear phagocytes (MPs), and four neutrophil subclusters were defined. During NTM infection, naïve T-cells were reduced, and effector T-cells increased. High cytotoxic activities were shown in T-cells of NTM-PD patients. The proportion of inflammatory and activated MPs subclusters was enriched in NTM-PD patients. Among neutrophil subclusters, an IFIT1+ neutrophil subcluster was expanded in NTM-PD compared to healthy controls. This suggests that IFIT1+ neutrophil subcluster might play an important role in host defense against NTM. Functional enrichment analysis of this subcluster suggested that it is related to interferon response. Cell-cell interaction analysis revealed enhanced CXCL8-CXCR1/2 interactions between the IFIT1+ neutrophil subcluster and NK cells, NKT cells, classical mononuclear phagocytes subcluster 1 (classical Mo1), classical mononuclear phagocytes subcluster 2 (classical Mo2) in NTM-PD patients compared to healthy controls. CONCLUSIONS: Our data revealed disease-specific immune cell subclusters and provided potential new targets of NTM-PD. Specific expansion of IFIT1+ neutrophil subclusters and the CXCL8-CXCR1/2 axis may be involved in the pathogenesis of NTM-PD. These insights may have implications for the diagnosis and treatment of NTM-PD.

Independent, parallel pathways to CXCL10 induction in HCV-infected hepatocytes.

BACKGROUND & AIMS: The pro-inflammatory chemokine CXCL10 is induced by HCV infection in vitro and in vivo, and is associated with outcome of IFN (interferon)-based therapy. We studied how hepatocyte sensing of early HCV infection via TLR3 (Toll-like receptor 3) and RIG-I (retinoic acid inducible gene I) led to expression of CXCL10. METHODS: CXCL10, type I IFN, and type III IFN mRNAs and proteins were measured in PHH (primary human hepatocytes) and hepatocyte lines harboring functional or non-functional TLR3 and RIG-I pathways following HCV infection or exposure to receptor-specific stimuli. RESULTS: HuH7 human hepatoma cells expressing both TLR3 and RIG-I produced maximal CXCL10 during early HCV infection. Neutralization of type I and type III IFNs had no impact on virus-induced CXCL10 expression in TLR3+/RIG-I+ HuH7 cells, but reduced CXCL10 expression in PHH. PHH cultures were positive for monocyte, macrophage, and dendritic cell mRNAs. Immunodepletion of non-parenchymal cells (NPCs) eliminated marker expression in PHH cultures, which then showed no IFN requirement for CXCL10 induction during HCV infection. Immunofluorescence studies also revealed a positive correlation between intracellular HCV Core and CXCL10 protein expression (r(2) = 0.88, p ≤ 0.001). CONCLUSIONS: While CXCL10 induction in hepatocytes during the initial phase of HCV infection is independent of hepatocyte-derived type I and type III IFNs, NPC-derived IFNs contribute to CXCL10 induction during HCV infection in PHH cultures.

Japanese encephalitis virus infection modulates the expression of suppressors of cytokine signaling (SOCS) in macrophages: implications for the hosts' innate immune response.

Viruses have evolved various mechanisms to subvert the host's immune system and one of them is preventing the infected cells from sending out chemotactic signals to activate the adaptive immune response. Japanese encephalitis virus (JEV) is a neuropathologic flavivirus that is responsible for significant number of child mortalities in various parts of South-East Asia. In this study we show that JEV modulates suppressors of cytokine signaling (SOCS)1 and 3 expression in macrophages to bring about changes in the JAK-STAT signaling cascade, so as to inhibit proinflammatory cyto/chemokine release. Using real time PCR, immunoblotting and immunofluorescent staining, we show that the expression of type 1 interferons and intracellular expression of viral genes are also affected over time. Also, following the initial activation of SOCS1 and 3, there is production of interferon-inducible anti-viral proteins in the cells which may be responsible for inhibiting viral replication. However, even at later time points, viral genes were still detected from the macrophages, albeit at lesser quantities, than earlier time points, indicative of intracellular persistence of the virus in a latent form. On knocking down SOCS1 and SOCS3 we found a significant decrease in viral gene expression at an early time point, indicating the dysregulation of the signaling cascade leading to increased production of interferon-inducible anti-viral proteins. Taken together, our study provides an insight into the role of JEV infection in modulating the JAK-STAT pathway with the help of SOCS leading to the generation of an antiviral innate immune response.

Kruppel-like factor 13 acts as a tumor suppressor in thyroid carcinoma by downregulating IFIT1.

BACKGROUND: Kruppel-like factor 13 (KLF13) is a transcription factor and plays an important role in carcinogenesis. However, the significance of KLF13 in thyroid carcinoma (THCA) is underdetermined. In this study, we aimed to explore the clinical relevance and function of KLF13 in the progress of THCA. METHODS: The expression of KLF13 in thyroid carcinoma and normal tissue was investigated by qPCR and IHC assay. The expression of KLF13 and IFIT1 in cell samples was investigated with Western blot assay. Cell proliferation ability was detected with CCK8 and colony formation assay. Cell growth in vivo with or without KLF13 overexpression was evaluated on a xenograft model. Cell migration ability was measured with Transwell assay. Cell cycle was detected with flow cytometer. The downstream genes of KLF13 were screened using RNA-seq assay. Luciferase activity was employed to assess the transcriptional regulation of KLF13 on IFIT1 promoter. RESULTS: KLF13 expression was downregulated in THCA samples. KLF13 knockdown and overexpression promoted and inhibited the proliferation and migration of THCA cells, respectively. The RNA-seq, RT-qPCR and immunoblotting data showed that KLF13 knockdown significantly potentiated IFIT1 expression at both mRNA and protein levels. Luciferase assays showed that KLF13 suppressed the transcription activity of IFIT1 promoter. Besides, IFIT1 upregulation was critical for the proliferation and migration of THCA cell lines. Lastly, silencing of IFIT1 greatly reversed the proliferation and migration induced by KLF13 knockdown. CONCLUSIONS: In conclusion, KLF13 may function as an anti-tumor protein in THCA by regulating the expression of IFIT1 and offer a theoretical foundation for treating thyroid carcinoma.

Fusobacterium nucleatum stimulates cell proliferation and promotes PD-L1 expression via IFIT1-related signal in colorectal cancer.

Fusobacterium nucleatum (F. nucleatum) is enriched in colorectal cancer (CRC) tissues and a high amount of F. nucleatum was associated with an immunosuppressive tumor environment. PD-L1 is an important immune checkpoint expressed on tumor cells and promotes tumor immune escape. Whether PD-L1 is regulated by F. nucleatum is still unclear. We demonstrated that F. nucleatum promoted CRC progression and upregulated PD-L1 protein expression in CRC cell lines. Combined m6A-seq and RNA-seq identified m6A-modified IFIT1 mediating F. nucleatum induced PD-L1 upregulation. IFIT1 mRNA was modified with m6A modifications in 3'UTR and the m6A levels were altered by F. nucleatum treatment. Our results also indicated that IFIT1 served as a potential oncogene in CRC and regulated PD-L1 protein levels through altering PD-L1 ubiquitination. Clinical CRC data confirmed the correlation among F. nucleatum abundance, IFIT1 and PD-L1 expressions. Our work highlighted the function of F. nucleatum in stimulating PD-L1 expression through m6A-modified IFIT1 and provided new aspects for understanding F. nucleatum mediated immune escape.

Overexpression of IFIT1 protects against LPS-induced acute lung injury via regulating CCL5-p65NF-κB signaling.

Acute lung injury (ALI) is featured by intensive inflammatory responses causing significant morbidity and mortality. Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), induced by interferon (IFN), has been discovered to modulate viral infection and cell apoptosis and inhibit the production of pro-inflammatory cytokines. However, it's role and mechanism in ALI remain unclear and need to be explored furtherly. Here, we discovered that IFIT1 decreased the expression of TNF-α, IL-1ß and IL-6 in mouse-derived macrophage cells (MH-S) and alleviated apoptosis of murine lung epithelial cells (MLE-12) induced by MH-S cell supernatant, contributing to anti-inflammatory and antiapoptotic effects in vitro and in vivo. Moreover, RNA sequencing analysis (RNA-seq) showed that inflammatory chemokine CC motif chemokine ligand 5 (CCL5) partially eliminated the protective effects of IFIT1 and promoted the expression of inflammatory cytokines TNF-α, IL-1ß and IL-6 by CCL5-p65NF-κB signaling pathway. This study demonstrated that IFIT1 attenuated ALI-associated inflammation and cell apoptosis by regulating the CCL5-p65NF-κB signaling pathway. These findings are of great significance for the treatment of lung injury.