Interferon-inducible Protein Research Articles

Double-stranded RNA-dependent protein kinase (PKR) in antiviral defence in fish and mammals

The interferon-inducible double-stranded RNA-dependent protein kinase (PKR) is one of the key antiviral arms of the innate immune system. Upon binding of viral double stranded RNA, a viral Pattern Associated Molecular Pattern (PAMP), PKR gets activated and phosphorylates the eukaryotic translation initiation factor 2α (eIF2α) resulting in a protein shut-down that limits viral replication. Since its discovery in the mid-seventies, PKR has been shown to be involved in multiple important cellular processes including apoptosis, proinflammatory and innate immune responses. Viral subversion mechanisms of PKR underline its importance in the antiviral response of the host. PKR activation pathways and its mechanisms of action were previously identified and characterised mostly in mammalian models. However, fish Pkr and fish-specific paralogue Z-DNA-dependent protein kinase (Pkz) also play key role in antiviral defence. This review gives an update on the current knowledge on fish Pkr/Pkz, their conditions of activation and their implication in the immune responses to viruses, in comparison to their mammalian counterparts.

Whole‐genome sequencing identifies interferon-induced protein IFI6/IFI27-like as a strong candidate gene for VNN resistance in European sea bass

BackgroundViral nervous necrosis (VNN) is a major disease that affects European sea bass, and understanding the biological mechanisms that underlie VNN resistance is important for the welfare of farmed fish and sustainability of production systems. The aim of this study was to identify genomic regions and genes that are associated with VNN resistance in sea bass.ResultsWe generated a dataset of 838,451 single nucleotide polymorphisms (SNPs) identified from whole-genome sequencing (WGS) in the parental generation of two commercial populations (A: 2371 individuals and B: 3428 individuals) of European sea bass with phenotypic records for binary survival in a VNN challenge. For each population, three cohorts were submitted to a red-spotted grouper nervous necrosis virus (RGNNV) challenge by immersion and genotyped on a 57K SNP chip. After imputation of WGS SNPs from their parents, quantitative trait loci (QTL) were mapped using a Bayesian sparse linear mixed model (BSLMM). We found several QTL regions that were specific to one of the populations on different linkage groups (LG), and one 127-kb QTL region on LG12 that was shared by both populations and included the genes ZDHHC14, which encodes a palmitoyltransferase, and IFI6/IFI27-like, which encodes an interferon-alpha induced protein. The most significant SNP in this QTL region was only 1.9 kb downstream of the coding sequence of the IFI6/IFI27-like gene. An unrelated population of four large families was used to validate the effect of the QTL. Survival rates of susceptible genotypes were 40.6% and 45.4% in populations A and B, respectively, while that of the resistant genotype was 66.2% in population B and 78% in population A.ConclusionsWe have identified a genomic region that carries a major QTL for resistance to VNN and includes the ZDHHC14 and IFI6/IFI27-like genes. The potential involvement of the interferon pathway, a well-known anti-viral defense mechanism in several organisms (chicken, human, or fish), in survival to VNN infection is of particular interest. Our results can lead to major improvements for sea bass breeding programs through marker-assisted genomic selection to obtain more resistant fish.

ROS-responsive nanomodulators downregulate IFITM3 expression and eliminate ROS for Alzheimer’s disease combination treatment

Neuronal damage caused by β-amyloid (Aβ) aggregates and excess reactive oxygen species (ROS) is a crucial pathogenic event in Alzheimer's disease (AD). However, current Aβ-targeting RNA interference (RNAi) treatments have shown limited therapeutic efficacy due to ineffective intracerebral siRNA delivery and overlooked crosstalk between excess ROS and Aβ aggregates in the brain. Herein, a ROS-responsive nanomodulator (NM/CM) was developed for the combinational treatment of RNAi and ROS elimination for AD. NM/CM was coated with 4T1 cell membranes, which endowed NM/CM with the capability to cross blood–brain barrier (BBB). After being internalized by neural cells, NM/CM releases curcumin (Cur) and siIFITM3 spontaneously into the cytoplasm. The released Cur can eliminate ROS, protecting neurons from oxidative damage and reducing the production of Aβ induced by ROS-related neuroinflammation. The released siIFITM3 can downregulate the expression of interferon-induced transmembrane protein 3 (IFITM3), thereby reducing the abnormal Aβ production mediated by IFITM3. As a result, NM/CM remarkably alleviated ROS- and Aβ aggregate-induced neurotoxicity in vitro, showing significant neuroprotective effects. This work demonstrates the potential of NM/CM in the development of novel and effective AD combination therapies.

Abstract 395: Interferon-induced Transmembrane Protein 3 (ifitm3) Mediates Platelet Functions During Inflammatory Stress

Background: Thrombosis, precipitated by dysregulated platelet responses, contributes to adverse clinical outcomes, including organ failure and death during systemic inflammation (e.g., sepsis). Previously, our lab identified interferon-induced transmembrane protein 3 (IFITM3) as a mediator of anti-viral responses in platelets. However, whether IFITM3 mediates platelet functions and subsequent thrombosis during systemic inflammation is unknown. Methods: Cecal-ligation and Puncture (CLP) model of sepsis and Interferon alpha (IFNα)-induced sterile inflammation model was performed on WT and platelet- and MK-specific IFITM3 knockout strain (IFITM3 fl/fl PF4-Cre +/- ). P-Selectin and CD63 expression, Platelet Factor 4 (PF4) release, platelet-leukocyte aggregates, plasma cytokines (TNF-α, IL-6, CXCL1), as well as survival outcomes, platelet deposition and aggregates within organs (e.g. lungs), plasma aspartate aminotransferase, lactate dehydrogenase, D-Dimer levels were measured by flow cytometry, enzyme-linked immunosorbent assays (ELISAs), and immunohistochemistry. In vivo collagen-epinephrine-induced pulmonary embolism model, and ex vivo thrombus formation under flow using microfluidic devices were performed. Results: CLP sepsis or IFNα-induced sterile inflammation upregulated IFITM3 in platelets which were associated with significantly enhanced secretion of P-Selectin, PF4, and CD63, and increased mortality in a collagen-epinephrine-induced murine pulmonary embolism model. Biochemical assays demonstrated IFITM3 interacts with VAMP-8, the primary v-SNARE that drives platelet secretion. Platelets lacking IFITM3 showed reduced granule secretion and platelet-leukocyte aggregate formation. Sepsis-induced mortality, platelet-leukocyte aggregates, platelet deposition within lungs, and markers of organ failure and thrombosis (e.g., aspartate aminotransferase, lactate dehydrogenase, D-Dimer), and thrombus formation under flow were all significantly reduced in the absence of IFITM3 in CLP sepsis. Conclusions: Collectively, our findings suggest that IFITM3 is a novel regulator of secretion and aggregation in platelets and contributes to thrombotic events during systemic inflammation.

ISG15 Deficiency: An expanding phenotype

IntroductionISG15 is a Type I IFN-inducible intracellular protein that interacts with and stabilizes USP18, a negative regulator of IFN-I pathway, and thus acts in a negative feedback loop. Lung damage caused by pulmonary fibrosis cannot be repaired, and can require lung transplantation; therefore, a better understanding of the causes is crucial. Here, we describe the case of a 13-year-old female presented with interstitial lung disease due to a mutation in ISG15. There is an increasing number of ISG15 deficient patients who present with lung fibrosis, but the significance of ISG15 in proper wound healing and fibrosis is unknown. We aim to investigate ISG15’s role in fibrosis by studying this mutant. MethodsWhole exome sequencing and molecular cloning was performed to identify the mutation and its expressivity. INFα stimulation of cells carrying the mutation to assess differential levels of apoptosis, necroptosis and pyroptosis compared to WT cells. Scratch wound assay to assess the mutant’s ability to effectively heal a wound. Fibrosis due to the overactivation of fibroblasts to myofibroblasts due to TGFβ and IFNα exposure. ResultsThe mutation is a duplication of a cysteine at position 463 of ISG15, causing a frameshift mutation at the very end of the gene which generates an mRNA transcript without a stop codon. Further analysis revealed that even though the mutant ISG15 mRNA gets transcribed, it does not get translated (ISG15 deficiency). In-vitro experiments revealed no changed in apoptosis, necroptosis nor pyroptosis when mutant cells were treated with IFNα, but they were more sensitive to the antiproliferation effects of IFNα. Therefore, these data suggest a possible mechanism by which ISG15 deficiency could aid to lung fibrosis due to the inability of the fibroblasts to properly proliferate and heal a wound.

Genetic polymorphism between the Sorani and Hawrami kurdish populations and COVID-19 outcome.

Coronavirus Disease 2019 (COVID-19) is a global pandemic, and mortality and clinical consequences vary across countries. One of the factors influencing COVID-19 outcomes is genetic polymorphism. Two Kurdish populations, Sorani and Hawrami, live in the Sulaimani province of the Kurdistan Region of Iraq. It seems Hawrami had a milder COVID-19 outcome. According to previous research conducted on various ethnic groups across the globe, single nucleotide polymorphisms (SNPs) in the interferon-induced transmembrane protein 3 (IFITM3) and interluken-6 (IL6) genes were associated with the severity of COVID-19 in those populations. We hypothesized that Hawrami may have protective SNPs. So, in this study, we used DNA sequencing to genotype three IFITM3 SNPs and nine IL6 SNPs by DNA sequencing to investigate the association of Sorani and Hawrami population polymorphisms. Genotype AA for the rs12252 SNP in IFITM3 was insignificantly more common in the Sorani group (54% vs. 44%). The Hawrami population showed a higher percentage of the CC genotype of the rs34481144 SNP in the IFITM3 gene (62% vs. 44.3%) and a higher proportion of the non-risky GG genotype of the rs1800795 SNP in the IL6 gene (53.4 vs. 43.3); however, the SNPs were insignificantly associated between the two populations. IFITM3 and IL6 SNPs have no statistically significant association between the two Kurdish populations. The decreased proportion of non-risk alleles at rs34481144 and rs1800795 in the Hawrami population may partially support the research hypothesis. However, contrary to our hypothesis, the Sorani group had an insignificantly higher protective variant of the rs12252 SNP.

Structural study of novel vaccinia virus E3L and dsRNA-dependent protein kinase complex

E3L (RNA-binding protein E3) is one of the key IFN resistance genes encoded by VV and consists of 190 amino acids with a highly conserved carboxy-terminal double-stranded RNA-binding domain (dsRBD). PKR (dsRNA-dependent protein kinase) is an IFN-induced protein involved in anti-cell and antiviral activity. PKR inhibits the initiation of translation through alpha subunit of the initiation factor eIF2 (eIF2α) and mediates several transcription factors such as NF-κB, p53 or STATs. Activated PKR also induces apoptosis in vaccinia virus infection. E3L is required for viral IFN resistance and directly binds to PKR to block activation of PKR. In this work, we determined the three-dimensional complex structure of E3L and PKR using cryo-EM and determined the important residues involved in the interaction. In addition, PKR peptide binds to E3L and can increase protein levels of phosphorus-PKR and phosphorus-eIF2α-induced cell apoptosis through upregulation of phosphorus-PKR in HEK293 cells. Taken together, structural insights into E3L and PKR will provide a new optimization and development of vaccinia virus drugs.

Mycoplasma fermentans infection induces human necrotic neuronal cell death via IFITM3-mediated amyloid-β (1–42) deposition

Mycoplasma fermentans is a proposed risk factor of several neurological diseases that has been detected in necrotic brain lesions of acquired immunodeficiency syndrome patients, implying brain invasiveness. However, the pathogenic roles of M. fermentans in neuronal cells have not been investigated. In this study, we found that M. fermentans can infect and replicate in human neuronal cells, inducing necrotic cell death. Necrotic neuronal cell death was accompanied by intracellular amyloid-β (1–42) deposition, and targeted depletion of amyloid precursor protein by a short hairpin RNA (shRNA) abolished necrotic neuronal cell death. Differential gene expression analysis by RNA sequencing (RNA-seq) showed that interferon-induced transmembrane protein 3 (IFITM3) was dramatically upregulated by M. fermentans infection, and knockdown of IFITM3 abolished both amyloid-β (1–42) deposition and necrotic cell death. A toll-like receptor 4 antagonist inhibited M. fermentans infection-mediated IFITM3 upregulation. M. fermentans infection also induced necrotic neuronal cell death in the brain organoid. Thus, neuronal cell infection by M. fermentans directly induces necrotic cell death through IFITM3-mediated amyloid-β deposition. Our results suggest that M. fermentans is involved in neurological disease development and progression through necrotic neuronal cell death.

Regulation of Staphylococcal Enterotoxin-Induced Inflammation in Spleen Cells from Diabetic Mice by Polyphenols.

Patients with diabetes are known to be more susceptible to infections following the establishment of Staphylococcus aureus in their nasal passages and on their skin. The present study evaluated the effects of staphylococcal enterotoxin A (SEA) on the immune responses of spleen cells derived from diabetic mice, and examined the effects of polyphenols, catechins, and nobiletin on inflammation-related gene expression associated with the immune response. (-)-Epigallocatechin gallate (EGCG), possessing hydroxyl groups, interacted with SEA, whereas nobiletin, possessing methyl groups, did not interact with SEA. The exposure of spleen cells derived from diabetic mice to SEA enhanced the expression of interferon gamma, suppressor of cytokine signaling 1, signal transducer and activator of transcription 3, interferon-induced transmembrane protein 3, Janus kinase 2, and interferon regulatory factor 3, suggesting that SEA sensitivity is variable in the development of diabetes. Both EGCG and nobiletin changed the expression of genes related to SEA-induced inflammation in spleen cells, suggesting that they inhibit inflammation through different mechanisms. These results may lead to a better understanding of the SEA-induced inflammatory response during diabetogenesis, and the establishment of methods to control these effects with polyphenols.

CD169 expression on monocytes as a marker for assessing type I interferon status in pediatric inflammatory diseases

BackgroundEvaluation of type I interferons (IFNs) in inflammatory or autoimmune diseases is challenging because of their rapid clearance in peripheral blood. The IFN gene expression signature has recently been used to evaluate the IFN status; however, this is often a labor-intensive and time-consuming procedure. Therefore, we assessed the feasibility of measuring expression of an IFN-inducible protein, CD169 (Siglec-1), on monocytes and circulating levels of soluble CD169 as alternative markers for type I IFN status in various pediatric inflammatory diseases. MethodsData from flow cytometric analysis of surface CD169 on monocytes and an enzyme-linked immunosorbent assay of soluble CD169 in peripheral blood were compared with serum IFN-α levels in 8 patients with viral infections, 5 with bacterial infections, 10 with systemic lupus erythematosus (SLE), 5 with Kikuchi-Fujimoto disease (KFD), 7 with Kawasaki disease (KD), and 8 with inflammatory bowel disease (IBD), and in 8 healthy controls. ResultsSurface CD169 expression was detected mainly on CD14+ monocytes and was significantly increased in patients with viral infections, SLE, and KFD, but not in patients with bacterial infections, KD, and IBD. There were similar trends for circulating soluble CD169; however, there was a significant increase only in patients with viral infections. Surface CD169 levels were significantly correlated with serum levels of IFN-α and soluble CD169. ConclusionAnalysis of CD169 expression on CD14+ monocytes may be useful for rapid assessment of type I IFN status for differentiation of pediatric inflammatory diseases from type 1 IFN-mediated diseases.

Interferon-λ3 alleviates intestinal epithelium injury induced by porcine rotavirus in mice

Interferons are a group of glycoproteins that are expressed in various cell types in their inflammatory responses to infections. In this study, we explored the protective effects of porcine interferon-λ3 (PIFN-λ3) on intestinal inflammation and injury in mice induced by porcine rotavirus (PRV). BALB/c mice were administrated by PIFN-λ3 or phosphate buffer solution (PBS) for three days prior to PRV infection. We show that PRV infection caused acute inflammatory responses in mice, as indicated by increases in serum concentrations of inflammatory cytokines such as the interlukin-1β (IL-1β), interlukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (P < 0.05). However, PIFN-λ3 administration not only decreased their concentrations but also elevated the concentrations of immunoglobulin (Ig) M and IgG in the PRV challenged mice (P < 0.05). PRV infection significantly decreased the jejunal villus height and the ratio of villus height to crypt depth (V/C); however, PIFN-λ3 treatment significantly elevated the villus height and the abundance of tight junction protein ZO-1 in the jejunum (P < 0.05). Moreover, PIFN-λ3 decreased the replication of PRV in the jejunal epithelium, but significantly increased the abundance of sIgA and the activities of maltase and sucrase in the PRV-challenged mice (P < 0.05). Interestingly, PIFN-λ3 elevated the expression levels of sodium/glucose cotransporter 1 (SGLT1) and mucin 2 (MUC2) in the PRV-challenged mice (P < 0.05). Moreover, PIFN-λ3 significantly increased the expression levels of IL-10, signal transducer and activator of transcription 1 (STAT1), and critical interferon-stimulated genes such as the 2′-5′ oligoadenylate synthetase-like 1 (OASL1), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) and radical S-adenosyl methionine domain containing 2 (RSAD2) in the jejunum upon PRV infection (P < 0.05). The anti-virus and anti-inflammatory effect of PIFN-λ3 should make it an attractive candidate to prevent various pathogen-induced bowel diseases.

ISG15 Is Required for the Dissemination of Vaccinia Virus Extracellular Virions.

Viruses have developed many different strategies to counteract immune responses, and Vaccinia virus (VACV) is one of a kind in this aspect. To ensure an efficient infection, VACV undergoes a complex morphogenetic process resulting in the production of two types of infective virions: intracellular mature virus (MV) and extracellular enveloped virus (EV), whose spread depends on different dissemination mechanisms. MVs disseminate after cell lysis, whereas EVs are released or propelled in actin tails from living cells. Here, we show that ISG15 participates in the control of VACV dissemination. Infection of Isg15-/- mouse embryonic fibroblasts with VACV International Health Department-J (IHD-J) strain resulted in decreased EV production, concomitant with reduced induction of actin tails and the abolition of comet-shaped plaque formation, compared to Isg15+/+ cells. Transmission electron microscopy revealed the accumulation of intracellular virus particles and a decrease in extracellular virus particles in the absence of interferon-stimulated gene 15 (ISG15), a finding consistent with altered virus egress. Immunoblot and quantitative proteomic analysis of sucrose gradient-purified virions from both genotypes reported differences in protein levels and composition of viral proteins present on virions, suggesting an ISG15-mediated control of viral proteome. Lastly, the generation of a recombinant IHD-J expressing V5-tagged ISG15 (IHD-J-ISG15) allowed us to identify several viral proteins as potential ISG15 targets, highlighting the proteins A34 and A36, which are essential for EV formation. Altogether, our results indicate that ISG15 is an important host factor in the regulation of VACV dissemination. IMPORTANCE Viral infections are a constant battle between the virus and the host. While the host's only goal is victory, the main purpose of the virus is to spread and conquer new territories at the expense of the host's resources. Along millions of years of incessant encounters, poxviruses have developed a unique strategy consisting in the production two specialized "troops": intracellular mature virions (MVs) and extracellular virions (EVs). MVs mediate transmission between hosts, and EVs ensure advance on the battlefield mediating the long-range dissemination. The mechanism by which the virus "decides" to shed from the primary site of infection and its significant impact in viral transmission is not yet fully established. Here, we demonstrate that this process is finely regulated by ISG15/ISGylation, an interferon-induced ubiquitin-like protein with broad antiviral activity. Studying the mechanism that viruses use during infection could result in new ways of understanding our perpetual war against disease and how we might win the next great battle.

Risk factors and early markers for echovirus type 11 associated haemorrhage-hepatitis syndrome in neonates, a retrospective cohort study.

Echovirus type 11(E-11) can cause fatal haemorrhage-hepatitis syndrome in neonates. This study aims to investigate clinical risk factors and early markers of E-11 associated neonatal haemorrhage-hepatitis syndrome. This is a multicentre retrospective cohort study of 105 neonates with E-11 infection in China. Patients with haemorrhage-hepatitis syndrome (the severe group) were compared with those with mild disease. Clinical risk factors and early markers of haemorrhage-hepatitis syndrome were analysed. In addition, cytokine analysis were performed in selective patients to explore the immune responses. In addition to prematurity, low birth weight, premature rupture of fetal membrane, total parenteral nutrition (PN) (OR, 28.7; 95% CI, 2.8-295.1) and partial PN (OR, 12.9; 95% CI, 2.2-77.5) prior to the onset of disease were identified as risk factors of developing haemorrhage-hepatitis syndrome. Progressive decrease in haemoglobin levels (per 10 g/L; OR, 1.5; 95% CI, 1.1-2.0) and platelet (PLT) < 140 × 10⁹/L at early stage of illness (OR, 17.7; 95% CI, 1.4-221.5) were associated with the development of haemorrhage-hepatitis syndrome. Immunological workup revealed significantly increased interferon-inducible protein-10(IP-10) (P < 0.0005) but decreased IFN-α (P < 0.05) in peripheral blood in severe patients compared with the mild cases. PN may potentiate the development of E-11 associated haemorrhage-hepatitis syndrome. Early onset of thrombocytopenia and decreased haemoglobin could be helpful in early identification of neonates with the disease. The low level of IFN-α and elevated expression of IP-10 may promote the progression of haemorrhage-hepatitis syndrome.

Wei Chang An pill alleviates 2,4,6-trinitro-benzenesulfonic acid-induced ulcerative colitis by inhibiting epithelial-mesenchymal transition process

Objective: To investigate the inhibitory effect and mechanism of Wei Chang An pill (WCA) on ulcerative colitis (UC). Methods: A TNBS-induced UC model was established, and WCA was administered orally for one week. Body weight, colon length, disease activity index (DAI) score, and colon mucosa damage index (CMDI) score were recorded. Cytokine expression in lipopolysaccharide (LPS)-stimulated THP-1 cells was evaluated to determine the anti-inflammatory effects of WCA and its active ingredients. Immunohistochemistry and immunofluorescence were performed to detect the expression of epithelial-mesenchymal transition (EMT) markers E-cadherin and vimentin in rat UC and WCA groups, and in Caco-2 cells stimulated with conditioned medium (CM) from THP-1 cells, with or without LPS or WCA. Results: WCA significantly inhibited body weight loss, decreased DAI and CMDI scores, blocked colon length shortening, and improved histological damage in UC rats. Furthermore, both myeloperoxidase (MPO) activities and cytokine expression in UC tissues were significantly suppressed by WCA as well. In THP-1 cells, the mRNA expression of interferon-inducible protein (IP)-10, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and NF-κB inhibitor α (IkBα) was significantly suppressed by WCA and its active ingredients. E-cadherin expression in UC rats and CM-stimulated Caco-2 cells was downregulated and vimentin expression was upregulated, whereas both were blocked when administered with WCA. Conclusion: Our data showed that WCA alleviated UC progression by inhibiting inflammation-induced EMT progression.

IFITM3 blocks influenza virus entry by sorting lipids and stabilizing hemifusion

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically affects virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells.

Smooth Muscle Cell Phenotypic Switch Induced by Traditional Cigarette Smoke Condensate: A Holistic Overview.

Cigarette smoke (CS) is a risk factor for inflammatory diseases, such as atherosclerosis. CS condensate (CSC) contains lipophilic components that may represent a systemic cardiac risk factor. To better understand CSC effects, we incubated mouse and human aortic smooth muscle cells (SMCs) with CSC. We evaluated specific markers for contractile [i.e., actin, aortic smooth muscle (ACTA2), calponin-1 (CNN1), the Kruppel-like factor 4 (KLF4), and myocardin (MYOCD) genes] and inflammatory [i.e., IL-1β, and IL-6, IL-8, and galectin-3 (LGALS-3) genes] phenotypes. CSC increased the expression of inflammatory markers and reduced the contractile ones in both cell types, with KLF4 modulating the SMC phenotypic switch. Next, we performed a mass spectrometry-based differential proteomic approach on human SMCs and could show 11 proteins were significantly affected by exposition to CSC (FC ≥ 2.7, p ≤ 0.05). These proteins are active in signaling pathways related to expression of pro-inflammatory cytokines and IFN, inflammasome assembly and activation, cytoskeleton regulation and SMC contraction, mitochondrial integrity and cellular response to oxidative stress, proteostasis control via ubiquitination, and cell proliferation and epithelial-to-mesenchymal transition. Through specific bioinformatics resources, we showed their tight functional correlation in a close interaction niche mainly orchestrated by the interferon-induced double-stranded RNA-activated protein kinase (alternative name: protein kinase RNA-activated; PKR) (EIF2AK2/PKR). Finally, by combining gene expression and protein abundance data we obtained a hybrid network showing reciprocal integration of the CSC-deregulated factors and indicating KLF4 and PKR as the most relevant factors.

IFITM2 Presents Antiviral Response through Enhancing Type I IFN Signaling Pathway.

Interferon (IFN) helps cells fight viral infections by further inducing the expression of many downstream IFN-stimulated genes (ISGs). Human interferon-inducible transmembrane proteins (IFITM) are one of these ISGs. The antiviral function of human IFITM1, IFITM2, and IFITM3 are well known. In this study, we report that IFITM can significantly inhibit EMCV infectivity in HEK293 cells. Overexpression of IFITM proteins could promote IFN-β production. Meanwhile, IFITMs facilitated type I IFN signaling pathway adaptor MDA5 expression. We detected the binding of IFITM2 to MDA5 in a co-immunoprecipitation assay. It was also found that the ability of IFITM2 to activate IFN-β was significantly inhibited after interfering with MDA5 expression, suggesting that MDA5 may play an important role in the activation of the IFN-β signaling pathway by IFITM2. Moreover, the N-terminal domain plays an active role in the antiviral activity and the activation of IFN-β by IFITM2. These findings suggest that IFITM2 plays a vital role in antiviral signaling transduction. In addition, a positive feed-forward loop between IFITM2 and type I IFN establishes a key role for IFITM2 in enforcing innate immune responses.

Release of High-Mobility Group Box-1 after a Raynaud's Attack Leads to Fibroblast Activation and Interferon-γ Induced Protein-10 Production: Role in Systemic Sclerosis Pathogenesis.

Raynaud's Phenomenon (RP) leading to repetitive ischemia and reperfusion (IR) stress, is the first recognizable sign of systemic sclerosis (SSc) leading to increased oxidative stress. High-mobility group box-1 (HMGB1) is a nuclear factor released by apoptotic and necrotic cells after oxidative stress. Since HMGB1 can signal through the receptor for advanced glycation end products (RAGE), we investigated whether an RP attack promotes the release of HMGB1, leading to fibroblast activation and the upregulation of interferon (IFN)-inducible genes. A cold challenge was performed to simulate an RP attack in patients with SSc, primary RP (PRP), and healthy controls. We measured levels of HMGB1 and IFN gamma-induced Protein 10 (IP-10) at different time points in the serum. Digital perfusion was assessed by photoplethysmography. In vitro, HMGB1 or transforming growth factor (TGF-β1) (as control) was used to stimulate healthy human dermal fibroblasts. Inflammatory, profibrotic, and IFN-inducible genes, were measured by RT-qPCR. In an independent cohort, sera were obtained from 20 patients with SSc and 20 age- and sex-matched healthy controls to determine HMGB1 and IP-10 levels. We found that HMGB1 levels increased significantly 30 min after the cold challenge in SSc compared to healthy controls. In vitro stimulation with HMGB1 resulted in increased mRNA expression of IP-10, and interleukin-6 (IL-6) while TGF-β1 stimulation promoted IL-6 and Connective Tissue Growth Factor (CTGF). In serum, both HMGB1 and IP-10 levels were significantly higher in patients with SSc compared to healthy controls. We show that cold challenge leads to the release of HMGB1 in SSc patients. HMGB1 induces IP-10 expression in dermal fibroblasts partly through the soluble RAGE (sRAGE) axis suggesting a link between RP attacks, the release of HMGB1 and IFN-induced proteins as a putative early pathogenetic mechanism in SSc.

Longitudinal analysis of immunocyte responses and inflammatory cytokine profiles in SFTSV-infected rhesus macaques.

Severe fever with thrombocytopenia syndrome virus (SFTSV), an emerging bunyavirus, causes severe fever with thrombocytopenia syndrome (SFTS), with a high fatality rate of 20%-30%. At present, however, the pathogenesis of SFTSV remains largely unclear and no specific therapeutics or vaccines against its infection are currently available. Therefore, animal models that can faithfully recapitulate human disease are important to help understand and treat SFTSV infection. Here, we infected seven Chinese rhesus macaques (Macaca mulatta) with SFTSV. Virological and immunological changes were monitored over 28 days post-infection. Results showed that mild symptoms appeared in the macaques, including slight fever, thrombocytopenia, leukocytopenia, increased aspartate aminotransferase (AST) and creatine kinase (CK) in the blood. Viral replication was persistently detectable in lymphoid tissues and bone marrow even after viremia disappeared. Immunocyte detection showed that the number of T cells (mainly CD8+ T cells), B cells, natural killer (NK) cells, and monocytes decreased during infection. In detail, effector memory CD8+ T cells declined but showed increased activation, while both the number and activation of effector memory CD4+ T cells increased significantly. Furthermore, activated memory B cells decreased, while CD80+/CD86+ B cells and resting memory B cells (CD27+CD21+) increased significantly. Intermediate monocytes (CD14+CD16+) increased, while myeloid dendritic cells (mDCs) rather than plasmacytoid dendritic cells (pDCs) markedly declined during early infection. Cytokines, including interleukin-6 (IL-6), interferon-inducible protein-10 (IP-10), and macrophage inflammatory protein 1 (MCP-1), were substantially elevated in blood and were correlated with activated CD4+ T cells, B cells, CD16+CD56+ NK cells, CD14+CD16+ monocytes during infection. Thus, this study demonstrates that Chinese rhesus macaques infected with SFTSV resemble mild clinical symptoms of human SFTS and provides detailed virological and immunological parameters in macaques for understanding the pathogenesis of SFTSV infection.

The Antiviral Compound PSP Inhibits HIV-1 Entry via PKR-Dependent Activation in Monocytic Cells.

Actin depolymerization factor (ADF) cofilin-1 is a key cytoskeleton component that serves to lessen cortical actin. HIV-1 manipulates cofilin-1 regulation as a pre- and post-entry requisite. Disruption of ADF signaling is associated with denial of entry. The unfolded protein response (UPR) marker Inositol-Requiring Enzyme-1α (IRE1α) and interferon-induced protein (IFN-IP) double-stranded RNA- activated protein kinase (PKR) are reported to overlap with actin components. In our published findings, Coriolus versicolor bioactive extract polysaccharide peptide (PSP) has demonstrated anti-HIV replicative properties in THP1 monocytic cells. However, its involvement towards viral infectivity has not been elucidated before. In the present study, we examined the roles of PKR and IRE1α in cofilin-1 phosphorylation and its HIV-1 restrictive roles in THP1. HIV-1 p24 antigen was measured through infected supernatant to determine PSP's restrictive potential. Quantitative proteomics was performed to analyze cytoskeletal and UPR regulators. PKR, IRE1α, and cofilin-1 biomarkers were measured through immunoblots. Validation of key proteome markers was done through RT-qPCR. PKR/IRE1α inhibitors were used to validate viral entry and cofilin-1 phosphorylation through Western blots. Our findings show that PSP treatment before infection leads to an overall lower infectivity. Additionally, PKR and IRE1α show to be key regulators in cofilin-1 phosphorylation and viral restriction.