Type I IFN drives unconventional IL-1ß secretion in lupus monocytes.

Opsonization of red blood cells that retain mitochondria (Mito+ RBCs), a feature of systemic lupus erythematosus (SLE), triggers type I interferon (IFN) production in macrophages. We report that monocytes (Mos) co-produce IFN and mature interleukin-1ß (mIL-1ß) upon Mito+ RBC opsonization. IFN expression depended on cyclic GMP-AMP synthase (cGAS) and RIG-I-like receptors' (RLRs) sensing of Mito+ RBC-derived mitochondrial DNA (mtDNA) and mtRNA, respectively. Interleukin-1ß (IL-1ß) production was initiated by the RLR antiviral signaling adaptor (MAVS) pathway recognition of Mito+ RBC-derived mtRNA. This led to the cytosolic release of Mo mtDNA, which activated the inflammasome. Importantly, mIL-1ß secretion was independent of gasdermin D (GSDMD) and pyroptosis but relied on IFN-inducible myxovirus-resistant protein 1 (MxA), which facilitated the incorporation of mIL-1ß into a trans-Golgi network (TGN)-mediated secretory pathway. RBC internalization identified a subset of blood Mo expressing IFN-stimulated genes (ISGs) that released mIL-1ß and expanded in SLE patients with active disease.

Host factor MxA restricts Dabie bandavirus infection by targeting the viral NP protein to inhibit NP-RdRp interaction and ribonucleoprotein activity.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high case mortality rates, which is caused by Dabie bandavirus (DBV), a novel pathogen also termed as SFTS virus (SFTSV). Currently, no specific therapeutic drugs or vaccines are available for SFTS. Myxovirus resistance protein A (MxA) has been shown to inhibit multiple viral pathogens; however, the role of MxA in DBV infection is unknown. Here, we demonstrated that DBV stimulates MxA expression which, in turn, restricts DBV infection. Mechanistic target analysis revealed that MxA specifically interacts with the viral nucleocapsid protein (NP) in a manner independent of RNA. Minigenome reporter assay showed that in agreement with its targeting of NP, MxA inhibits DBV ribonucleoprotein (RNP) activity. In detail, MxA interacts with the NP N-terminal and disrupts the interaction of NP with the viral RNA-dependent RNA polymerase (RdRp) but not NP multimerization, the critical activities of NP for RNP formation and function. Furthermore, MxA N-terminal domain was identified as the functional domain inhibiting DBV infection, and, consistently, then was shown to interact with NP and obstruct the NP-RdRp interaction. Additionally, threonine 103 within the N-terminal domain is important for MxA inhibition to DBV, and its mutation (T103A) attenuates MxA binding to NP and obstruction of the NP-RdRp interaction. This study uncovers MxA inhibition of DBV with a series of functional and mechanistical analyses, providing insights into the virus-host interactions and probably helping inform the development of antiviral agents in the future.IMPORTANCEDBV/SFTSV is an emerging high-pathogenic virus. Since its first identification in China in 2009, cases of DBV infection have been reported in many other countries, posing a significant threat to public health. Uncovering the mechanisms of DBV-host interactions is necessary to understand the viral pathogenesis and host response and may advance the development of antiviral therapeutics. Here, we found that host factor MxA whose expression is induced by DBV restricts the virus infection. Mechanistically, MxA specifically interacts with the viral NP and blocks the NP-RdRp interaction, inhibiting the viral RNP activity. Further studies identified the key domain and amino acid residue required for MxA inhibition to DBV. Consistently, they were then shown to be important for MxA targeting of NP and obstruction of the NP-RdRp association. These findings unravel the restrictive role of MxA in DBV infection and the underlying mechanism, expanding our knowledge of the virus-host interactions.

Interleukin-8 in HepG2 cells: Enhancing antiviral proteins in uninfected cells but promoting HBV replication in infected cells.

In vitro studies have revealed that hepatitis B virus (HBV) infection upregulates interleukin-8 (IL-8), which enhances HBV replication. Clinically, elevated IL-8 levels in chronic HBV patients are associated with diminished therapeutic efficacy of interferon-α (IFN-α). Our study advances these findings by demonstrating that IL-8 promotes the expression of myxovirus resistance A (MxA) and protein kinase R (PKR) in HepG2 cells via the PI3K-AKT pathway. However, HBV-infected cells fail to exhibit IL-8-induced upregulation of MxA and PKR, likely due to HBV's upregulation of PP2A that inhibits the PI3K-AKT pathway. Notably, IL-8 targets the C/EBPα transcription factor, increasing HBV promoter activity and viral replication, which in turn partially suppresses the expression of MxA and PKR induced by IFN-α. Our findings uncover a mechanism by which HBV may evade immune responses, suggesting potential new strategies for immunotherapy against chronic HBV infection.

Constitutive type-1 interferons signaling activity in malignant gliomas.

PURPOSE: Recent studies revealed a pro-tumor effect of constitutive Type-1 interferons (IFN-I) production and the downstream signaling activity in several malignancies. In contrast, heterogeneity and clinical significance of the signaling activity in gliomas remain unknown. Thus, we aimed to depict the heterogeneity and clinical significance of constitutive Type-1 interferon (IFN-I) production and the downstream signaling activity in gliomas. METHODS: We utilized multiplex immunofluorescence (mIF) on a 364 gliomas tissue microarray from our cohort. Moreover, we conducted bioinformatic analyses on the Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) databases to investigate the heterogeneity and clinical significance of constitutive IFN-I signaling activity in gliomas. RESULTS: We observed high heterogeneity of the constitutive IFN-I signaling activity among glioma subtypes. Signaling increased with the WHO malignancy grade while decreasing in the gliomas with IDH mutations. Additionally, high IFN-I activity served as an independent predictor of unfavorable outcomes, and global DNA hypermethylation in IDH-mutant gliomas was associated with decreased IFN-I signaling activity. Positive correlations were observed between the IFN-I activity and glioma-associated inflammation, encompassing both anti-tumor and pro-tumor immune responses. Furthermore, the IFN-I activity varied significantly among tumor and immune cells in the glioma microenvironment (GME). Notably, a distinct pattern of IFN-I signaling activity distribution in GME cells was observed among glioma subtypes, and the pattern was independently associated with patient overall survival. CONCLUSIONS: Constitutive IFN-I signaling activity varies significantly among glioma subtypes and represents a potential indicator for increased glioma inflammation and unfavorable clinical outcomes.

VP24 matrix proteins of eight filoviruses downregulate innate immune response by inhibiting the interferon-induced pathway.

Filoviruses encode viral protein 24 (VP24) which effectively inhibit the innate immune responses in infected cells. Here we systematically analysed the effects of nine mammalian filovirus VP24 proteins on interferon (IFN)-induced immune response. We transiently expressed Ebola, Bombali, Bundibugyo, Reston, Sudan and Taï Forest ebolavirus (EBOV, BOMV, BDBV, RESTV, SUDV, TAFV, respectively), Lloviu virus (LLOV), Mengla dianlovirus (MLAV) and Marburgvirus (MARV) VP24 proteins and analysed their ability to inhibit IFN-α-induced activation of myxovirus resistance protein 1 (MxA) and interferon-induced transmembrane protein 3 (IFITM3) promoters. In addition, we analysed the expression of endogenous MxA protein in filovirus VP24-expressing cells. Eight filovirus VP24 proteins, including the VP24s of the recently discovered MLAV, BOMV and LLOV, inhibited IFN-induced MxA and IFITM3 promoter activation. MARV VP24 was the only protein with no inhibitory effect on the activation of either promoter. Endogenous MxA protein expression was impaired in cells transiently expressing VP24s with the exception of MARV VP24. We mutated nuclear localization signal (NLS) of two highly pathogenic filoviruses (EBOV and SUDV) and two putatively non-pathogenic filoviruses (BOMV and RESTV), and showed that the inhibitory effect on IFN-induced expression of MxA was dependent on functional cluster 3 of VP24 nuclear localization signal. Our findings suggest that filovirus VP24 proteins are both genetically and functionally conserved, and that VP24 proteins of most filovirus species are capable of inhibiting IFN-induced antiviral gene expression thereby efficiently downregulating the host innate immune responses.

Symptomatic myopathies in sarcoidosis: disease spectrum and myxovirus resistance protein A expression.

OBJECTIVES: Symptomatic myopathy in sarcoidosis patients is not always due to sarcoid myopathy (ScM). We investigated the clinical and pathological spectrum including myxovirus resistance protein A (MxA) expression among sarcoidosis patients. METHODS: We reviewed the Mayo Clinic database (May 1980-December 2020) to identify sarcoidosis patients with myopathic symptoms and pathological evidence of myopathy. RESULTS: Among 5885 sarcoidosis patients, 21 had symptomatic myopathy. Eight carried a diagnosis of sarcoidosis 5.5 years (median) prior to myopathy onset. Eleven patients had ScM. The remaining had non-sarcoid myopathies (five IBM, one immune-mediated necrotizing myopathy, one non-specific myositis, two non-specific myopathy and one steroid myopathy). Estimated frequency of IBM is 85 per 100 000 sarcoidosis patients. The following features were associated with non-sarcoid myopathies (P < 0.05): (i) predominant finger flexor and quadriceps weakness, (ii) modified Rankin scale (mRS) >2 at time of diagnosis, (iii) creatine kinase >500 U/l, and (iv) absence of intramuscular granulomas. Sarcoplasmic MxA expression was observed in scattered myofibres in three patients, two of whom were tested for DM-specific autoantibodies and were negative. Immunosuppressive therapy led to improvement in mRS ≥1 in 5/10 ScM, none of the five IBM, and 3/3 remaining patients with non-sarcoid myopathies. DISCUSSION: Symptomatic myopathy occurred in 0.36% of sarcoidosis. IBM was the second most common cause of myopathies after ScM. Frequency of IBM in sarcoidosis is higher than in the general population. Recognition of features suggestive of alternative aetiologies can guide proper treatment. Our findings of abnormal MxA expression warrant a larger study.

Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication.

The antiviral response against influenza A virus (IAV) infection includes the induction of the interferon (IFN) signaling pathway, including activation of the STATs protein family. Subsequently, antiviral myxovirus resistance (MxA) protein and other interferon-stimulated genes control virus replication; however, the molecular interaction of viral-mediated IFN signaling needs more investigation. Host microRNAs (miRNAs) are small non-coding molecules that posttranscriptionally regulate gene expression. Here, we sought to investigate the possible involvement of miR-141 in IAV-mediated IFN signaling. Accordingly, the microarray analysis of A549 cells transfected with precursor miR-141 (pre-miR-141) was used to capture the potentially regulated genes in response to miR-141 overexpression independent of IAV infection. The downregulation of targeted genes by miR-141, in addition to viral gene expression, was investigated by quantitative real-time PCR, western blot analysis, and flow cytometric assay. Our findings showed a significant upregulation of miR-141 in infected A549 cells with different strains of IAV. Notably, IAV replication was firmly interrupted in cells transfected with the miR-141 inhibitor. While its replication significantly increased in cells transfected with pre-miR-141 confirming the crucial role of miRNA-141 in supporting virus replication. Interestingly, the microarray data of miR-141 transduced A549 cells showed many downregulated genes, including MxA, STAT3, IFI27, and LAMP3. The expression profile of MxA and STAT3 was significantly depleted in infected cells transfected with the pre-miR-141, while their expression was restored in infected cells transfected with the miR-141 inhibitor. Unlike interleukin 6 (IL-6), the production of IFN-ß markedly decreased in infected cells that transfected with pre-miR-141, while it significantly elevated in infected cells transfected with miR-141 inhibitor. These data provide evidence for the crucial role of miR-141 in regulating the antiviral gene expression induced by IFN and IL-6 signaling during IAV infection to ensure virus replication.

MxA for differentiating viral and bacterial infections in adults: a prospective, exploratory study.

PURPOSE: Inappropriate antibiotic prescription in patients with viral infections contributes to the surge of antibiotic resistance. Viral infections induce the expression of the antiviral protein MxA in monocytes, which is a promising biomarker to differentiate between viral and bacterial diseases. In this prospective, exploratory study, we aimed to determine the diagnostic value of monocyte MxA expression in adults with viral, bacterial or co-infections. METHODS: We measured monocyte MxA expression using flow cytometry in a cohort of 61 adults with various viral, bacterial and co-infections including patients receiving immunosuppressive therapy. RESULTS: Monocyte MxA expression in virus-infected patients was significantly higher compared to bacterial infections (83.3 [66.8, 109.4] vs. 33.8 [29.3, 47.8] mean fluorescence intensity [MFI]; p < 0.0001) but not co-infections (53.1 [33.9, 88.9] MFI). At a threshold of 62.2 MFI, the area under the ROC curve (AUC) to differentiate between viral and bacterial infections was 0.9, with a sensitivity and specificity of 92.3% and 84.6%, respectively. Immunosuppressive therapy did not affect monocyte MxA expression in virus-infected patients. CONCLUSION: Our findings corroborate the diagnostic performance of MxA in differentiating viral and bacterial infections but also point to an important caveat of MxA in viral-bacterial co-infections. This study extends previous reports and indicates that MxA is also a useful biomarker in immunocompromised patients.

Potential of green tea EGCG in neutralizing SARS-CoV-2 Omicron variant with greater tropism toward the upper respiratory tract.

Background: COVID-19 due to SARS-CoV-2 infection has had an enormous adverse impact on global public health. As the COVID-19 pandemic evolves, the WHO declared several variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and Omicron. Compared with earlier variants, Omicron, now a dominant lineage, exhibits characteristics of enhanced transmissibility, tropism shift toward the upper respiratory tract, and attenuated disease severity. The robust transmission of Omicron despite attenuated disease severity still poses a great challenge for pandemic control. Under this circumstance, its tropism shift may be utilized for discovering effective preventive approaches. Scope and approach: This review aims to estimate the potential of green tea epigallocatechin gallate (EGCG), the most potent antiviral catechin, in neutralizing SARS-CoV-2 Omicron variant, based on current knowledge concerning EGCG distribution in tissues and Omicron tropism. Key findings and conclusions: EGCG has a low bioavailability. Plasma EGCG levels are in the range of submicromolar concentrations following green tea drinking, or reach at most low µM concentrations after pharmacological intervention. Nonetheless, its levels in the upper respiratory tract could reach concentrations as high as tens or even hundreds of µM following green tea consumption or pharmacological intervention. An approach for delivering sufficiently high concentrations of EGCG in the pharynx has been developed. Convincing data have demonstrated that EGCG at tens to hundreds of µM can dramatically neutralize SARS-CoV-2 and effectively eliminate SARS-CoV-2-induced cytopathic effects and plaque formation. Thus, EGCG, which exhibits hyperaccumulation in the upper respiratory tract, deserves closer investigation as an antiviral in the current global battle against COVID-19, given Omicron's greater tropism toward the upper respiratory tract.

The Effect of Interferon-Beta Therapy on T-Helper 17/miR-326 and T-Helper 1/miR-29b-3p Axis in Relapsing-Remitting Multiple Sclerosis Patients.

BACKGROUND: We aimed to evaluate the therapeutic effects of interferon-beta (IFN-ß) on hsa-miR29b-3p and hsa-miR326 in isolated T-helper (Th)1 and Th17 cells expressed by relapsing-remitting multiple sclerosis (RRMS) patients before and after 1 year of treatment with IFN-ß. METHODS: The study was done on 19 RRMS patients pre- and posttreatment with IFN-ß to evaluate the frequency of Th1 and Th17 cells by flow cytometry. The expression level of hsa-miR-29b-3p and hsa-miR-326 in isolated Th1 and Th17 cells was assessed by quantitative polymerase chain reaction. Enzyme-linked immunosorbent assay was also used to measure the plasma levels of I interferon -gamma and interleukin (IL)-17A. RESULTS: Th17 cells and plasma levels of IL-17A decreased in RRMS patients after IFN-ß therapy but hsa-miR-29b-3p and hsa-miR-326 expression had no significant change in treated RRMS patients versus baseline. MxA gene expression was significantly induced upon IFN-ß therapy in patients with RRMS. CONCLUSION: IFN-ß therapy is more effective on Th17 than Th1, but it does not reform altered expression of hsa-miR-326 and hsa-miR-29b-3p in Th17 and Th1, respectively.

Association of human myxovirus resistance protein A with severity of COVID-19.

BACKGROUND: In this retrospective cohort study, we explored the correlation of blood human myxovirus resistance protein A (MxA) level with severity of disease in hospitalized COVID-19 patients. METHODS: All 304 patients admitted for COVID-19 in our hospital until 30th of April 2021 were included in this study. MxA was measured from peripheral blood samples in 268 cases. Patients were divided into groups based on their level of MxA on admission. We studied baseline characteristics and severity of disease on admission based on clinical parameters and inflammatory biomarker levels in each group. Severity of disease during hospitalization was determined by the applied level of respiratory support, by the usage of corticosteroids and by the duration of hospitalization. RESULTS: Higher MxA levels on admission were associated with a shorter duration of symptoms before admission, and with more severe disease. Adjusted Odds Ratios for any respiratory support were 9.92 (95%CI 2.11-46.58; p = 0.004) in patients with MxA between 400 µg/L and 799 µg/L (p = 0.004) and 20.08 (95%CI 4.51-89.44; p < 0.001) in patients with MxA ≥ 800 µg/L in comparison with patients with initial MxA < 400 µg/L. The usage of corticosteroids was significantly higher in the high-MxA group (77%) in comparison with the intermediate-MxA group (62%, p = 0.013) and low-MxA group (47%, p < 0.001). CONCLUSIONS: Higher initial levels of MxA were associated with more severe COVID-19. MxA may be a helpful additional biomarker to predict the severity of the disease.

Detection of group A streptococcus in children with confirmed viral pharyngitis and antiviral host response.

Our aim was to study the detection of group A streptococcus (GAS) with different diagnostic methods in paediatric pharyngitis patients with and without a confirmed viral infection. In this prospective observational study, throat swabs and blood samples were collected from children (age 1-16 years) presenting to the emergency department with febrile pharyngitis. A confirmed viral infection was defined as a positive virus diagnostic test (nucleic acid amplification test [NAAT] and/or serology) together with an antiviral immune response of the host demonstrated by elevated (≥ 175 µg/L) myxovirus resistance protein A (MxA) blood concentration. Testing for GAS was performed by a throat culture, by 2 rapid antigen detection tests (StrepTop and mariPOC) and by 2 NAATs (Simplexa and Illumigene). Altogether, 83 children were recruited of whom 48 had samples available for GAS testing. Confirmed viral infection was diagnosed in 30/48 (63%) children with febrile pharyngitis. Enteroviruses 11/30 (37%), adenoviruses 9/30 (30%) and rhinoviruses 9/30 (30%) were the most common viruses detected. GAS was detected by throat culture in 5/30 (17%) with and in 6/18 (33%) patients without a confirmed viral infection. Respectively, GAS was detected in 4/30 (13%) and 6/18 (33%) by StrepTop, 13/30 (43%) and 10/18 (56%) by mariPOC, 6/30 (20%) and 9/18 (50%) by Simplexa, and 5/30 (17%) and 6/18 (30%) patients by Illumigene. CONCLUSION: GAS was frequently detected also in paediatric pharyngitis patients with a confirmed viral infection. The presence of antiviral host response and increased GAS detection by sensitive methods suggest incidental throat carriage of GAS in viral pharyngitis. WHAT IS KNOWN: •The frequency and significance of GAS-virus co-detection are poorly characterised in children with pharyngitis. •Detection of a virus and the antiviral host response likely indicates symptomatic infection. WHAT IS NEW: •Group A streptococcus (GAS) was detected in 17-43% of the children with confirmed viral pharyngitis depending on the GAS diagnostic method. •Our results emphasize the risk of detecting and treating incidental pharyngeal carriage of GAS in children with viral pharyngitis.

Pathological features of inflammatory myopathy as a manifestation of chronic graft-versus-host disease after allogeneic bone marrow transplantation.

Chronic graft-versus-host disease (cGVHD) is the most important complication resulting in the death of bone marrow transplantation (BMT) survivors. It is also a relatively rare cause of inflammatory myopathy (IM). We report the case of a 46-year-old woman who developed severe cGVHD-related IM after BMT for myelodysplastic syndrome. She presented with severe muscle pain and weakness with cGVHD-related symptoms in other organs. Myopathological analysis showed moderate cell infiltration with remarkable necrotic and regenerative fibers. Sarcoplasm and capillaries expressed C5b9 and myxovirus resistance protein 1. Non-necrotic fibers in perifascicular regions expressed MHC-II. Steroid therapy did not sufficiently control cGVHD-related IM, and the patient was concurrently treated with an immunosuppressant. Our findings show that IM is a key manifestation of cGVHD and that the expression of interferon-inducible proteins in muscle pathology is useful for identifying cGVHD-related IM.

Rapid Reversible Osmoregulation of Cytoplasmic Biomolecular Condensates of Human Interferon-α-Induced Antiviral MxA GTPase.

We previously discovered that exogenously expressed GFP-tagged cytoplasmic human myxovirus resistance protein (MxA), a major antiviral effector of Type I and III interferons (IFNs) against several RNA- and DNA-containing viruses, existed in the cytoplasm in phase-separated membraneless biomolecular condensates of varying sizes and shapes with osmotically regulated disassembly and reassembly. In this study we investigated whether cytoplasmic IFN-α-induced endogenous human MxA structures were also biomolecular condensates, displayed hypotonic osmoregulation and the mechanisms involved. Both IFN-α-induced endogenous MxA and exogenously expressed GFP-MxA formed cytoplasmic condensates in A549 lung and Huh7 hepatoma cells which rapidly disassembled within 1-2 min when cells were exposed to 1,6-hexanediol or to hypotonic buffer (~40-50 mOsm). Both reassembled into new structures within 1-2 min of shifting cells to isotonic culture medium (~330 mOsm). Strikingly, MxA condensates in cells continuously exposed to culture medium of moderate hypotonicity (in the range one-fourth, one-third or one-half isotonicity; range 90-175 mOsm) first rapidly disassembled within 1-3 min, and then, in most cells, spontaneously reassembled 7-15 min later into new structures. This spontaneous reassembly was inhibited by 2-deoxyglucose (thus, was ATP-dependent) and by dynasore (thus, required membrane internalization). Indeed, condensate reassembly was preceded by crowding of the cytosolic space by large vacuole-like dilations (VLDs) derived from internalized plasma membrane. Remarkably, the antiviral activity of GFP-MxA against vesicular stomatitis virus survived hypoosmolar disassembly and subsequent reassembly. The data highlight the exquisite osmosensitivity of MxA condensates, and the preservation of antiviral activity in the face of hypotonic stress.

Murine GFP-Mx1 forms nuclear condensates and associates with cytoplasmic intermediate filaments: Novel antiviral activity against VSV.

Type I and III interferons induce expression of the "myxovirus resistance proteins" MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles ("biomolecular condensates"). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40-50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20-30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.

Updates on the Immunopathology in Idiopathic Inflammatory Myopathies.

PURPOSE OF REVIEW: To review recent advances in immunopathology for idiopathic inflammatory myopathies, focusing on widely available immunohistochemical analyses. RECENT FINDINGS: Sarcoplasmic expression of myxovirus resistance protein A (MxA) is specifically observed in all types of dermatomyositis and informs that type I interferons are crucially involved in its pathogenesis. It is a more sensitive diagnostic marker than perifascicular atrophy. Diffuse tiny dots in the sarcoplasm highlighted by p62 immunostaining are characteristically seen in immune-mediated necrotizing myopathy. This feature is linked to a chaperone-assisted selective autophagy pathway. Myofiber invasion by highly differentiated T cells, a marker of which is KLRG1, is specific to inclusion body myositis and has a crucial role in its pathogenesis. The recent advances in immunopathology contribute to increased diagnostic accuracy and a better understanding of the underlying pathophysiology in different types of idiopathic inflammatory myopathies.

Increased MxA protein expression and dendritic cells in spongiotic dermatitis differentiates dermatomyositis from eczema in a single-center case-control study.

BACKGROUND: Dermatomyositis (DM) is conventionally characterized by interface dermatitis (ID) on skin histopathology. A subset of DM patients has skin biopsies showing spongiotic dermatitis (SD), a histopathology more commonly seen in eczema. In this study, we aimed to (a) identify the percentage of clinically diagnosed DM patients with SD skin biopsies, (b) identify cytokine and cell markers that can help determine if a SD skin biopsy is consistent with DM. METHODS: In this case-control study, biopsy specimens from ten DM patients with SD (DM-SD) were compared to specimens from ten healthy controls, ten patients with eczema, and 12 patients with DM with ID (DM-ID). Specimens were stained by immunohistochemistry for MxA, IFN-ß, CD11c, and BDCA2. One-way ANOVA with Bonferroni's multiple comparison test was used to compare protein expression between groups. RESULTS: Eleven of 164 (6.7%) patients with a clinical diagnosis of DM at our tertiary care center were identified as having SD. MxA, IFN-ß, CD11c, and BDCA2 protein expression was significantly higher in DM-SD compared to eczema and healthy controls. Expressions of MxA, IFN-ß, and BDCA2 were not significantly different between DM-SD and DM-ID. CONCLUSION: Increased MxA, IFN-ß, CD11c, and BDCA2 protein expression may aid in distinguishing between DM-SD and eczema and warrants further investigation.

Long Noncoding RNA Lnc-MxA Inhibits Beta Interferon Transcription by Forming RNA-DNA Triplexes at Its Promoter.

Previously, we identified a set of long noncoding RNAs (lncRNAs) that were differentially expressed in influenza A virus (IAV)-infected cells. In this study, we focused on lnc-MxA, which is upregulated during IAV infection. We found that the overexpression of lnc-MxA facilitates the replication of IAV, while the knockdown of lnc-MxA inhibits viral replication. Further studies demonstrated that lnc-MxA is an interferon-stimulated gene. However, lnc-MxA inhibits the Sendai virus (SeV)- and IAV-induced activation of beta interferon (IFN-ß). A luciferase assay indicated that lnc-MxA inhibits the activation of the IFN-ß reporter upon stimulation with RIG-I, MAVS, TBK1, or active IRF3 (IRF3-5D). These data indicated that lnc-MxA negatively regulates the RIG-I-mediated antiviral immune response. A chromatin immunoprecipitation (ChIP) assay showed that the enrichment of IRF3 and p65 at the IFN-ß promoter in lnc-MxA-overexpressing cells was significantly lower than that in control cells, indicating that lnc-MxA interfered with the binding of IRF3 and p65 to the IFN-ß promoter. Chromatin isolation by RNA purification (ChIRP), triplex pulldown, and biolayer interferometry assays indicated that lnc-MxA can bind to the IFN-ß promoter. Furthermore, an electrophoretic mobility shift assay (EMSA) showed that lnc-MxA can form complexes with the IFN-ß promoter fragment. These results demonstrated that lnc-MxA can form a triplex with the IFN-ß promoter to interfere with the activation of IFN-ß transcription. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that lnc-MxA can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we revealed that lnc-MxA is an interferon-stimulated gene (ISG) that negatively regulates the transcription of IFN-ß by forming an RNA-DNA triplex.IMPORTANCE IAV can be recognized as a nonself molecular pattern by host immune systems and can cause immune responses. However, the intense immune response induced by influenza virus, known as a "cytokine storm," can also cause widespread tissue damage (X. Z. J. Guo and P. G. Thomas, Semin Immunopathol 39:541-550, 2017, https://doi.org/10.1007/s00281-017-0636-y; S. Yokota, Nihon Rinsho 61:1953-1958, 2003; I. A. Clark, Immunol Cell Biol 85:271-273, 2007). Meanwhile, the detailed mechanisms involved in the balancing of immune responses in host cells are not well understood. Our studies reveal that, as an IFN-inducible gene, lnc-MxA functions as a negative regulator of the antiviral immune response. We uncovered the mechanism by which lnc-MxA inhibits the activation of IFN-ß transcription. Our findings demonstrate that, as an ISG, lnc-MxA plays an important role in the negative-feedback loop involved in maintaining immune homeostasis.

Human Antiviral Protein MxA Forms Novel Metastable Membraneless Cytoplasmic Condensates Exhibiting Rapid Reversible Tonicity-Driven Phase Transitions.

Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles (e.g., stress granules and P-bodies) in the mammalian cell cytoplasm and nucleus. In contrast to the long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA) associated with the endoplasmic reticulum (ER) and Golgi apparatus, we report that MxA formed membraneless metastable (shape-changing) condensates in the cytoplasm. In our studies, we used the same cell lines and methods as those used by previous investigators but concluded that wild-type MxA formed variably sized spherical or irregular bodies, filaments, and even a reticulum distinct from that of ER/Golgi membranes. Moreover, in Huh7 cells, MxA structures associated with a novel cytoplasmic reticular meshwork of intermediate filaments. In live-cell assays, 1,6-hexanediol treatment led to rapid disassembly of green fluorescent protein (GFP)-MxA structures; FRAP revealed a relative stiffness with a mobile fraction of 0.24 ± 0.02 within condensates, consistent with a higher-order MxA network structure. Remarkably, in intact cells, GFP-MxA condensates reversibly disassembled/reassembled within minutes of sequential decrease/increase, respectively, in tonicity of extracellular medium, even in low-salt buffers adjusted only with sucrose. Condensates formed from IFN-α-induced endogenous MxA also displayed tonicity-driven disassembly/reassembly. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with spherical GFP-MxA condensates in cells showing an antiviral effect. These observations prompt comparisons with the extensive literature on interactions between viruses and stress granules/P-bodies. Overall, the new data correct a long-standing misinterpretation in the MxA literature and provide evidence for membraneless MxA biomolecular condensates in the uninfected cell cytoplasm.IMPORTANCE There is a long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA), which displays antiviral activity against several RNA and DNA viruses, associates with the endoplasmic reticulum (ER) and Golgi apparatus. We provide data to correct this misinterpretation and further report that MxA forms membraneless metastable (shape-changing) condensates in the cytoplasm consisting of variably sized spherical or irregular bodies, filaments, and even a reticulum. Remarkably, MxA condensates showed the unique property of rapid (within 1 to 3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions. Moreover, GFP-MxA condensates included the VSV nucleocapsid (N) protein, a protein previously shown to form liquid-like condensates. Since intracellular edema and ionic changes are hallmarks of cytopathic effects of a viral infection, the tonicity-driven regulation of MxA condensates may reflect a mechanism for modulation of MxA function during viral infection.

Biomolecular condensates in cell biology and virology: Phase-separated membraneless organelles (MLOs).

Membraneless organelles (MLOs) in the cytoplasm and nucleus in the form of 2D and 3D phase-separated biomolecular condensates are increasingly viewed as critical in regulating diverse cellular functions. These functions include cell signaling, immune synapse function, nuclear transcription, RNA splicing and processing, mRNA storage and translation, virus replication and maturation, antiviral mechanisms, DNA sensing, synaptic transmission, protein turnover and mitosis. Components comprising MLOs often associate with low affinity; thus cell integrity can be critical to the maintenance of the full complement of respective MLO components. Phase-separated condensates are typically metastable (shape-changing) and can undergo dramatic, rapid and reversible assembly and disassembly in response to cell signaling events, cell stress, during mitosis, and after changes in cytoplasmic "crowding" (as observed with condensates of the human myxovirus resistance protein MxA). Increasing evidence suggests that neuron-specific aberrations in phase-separation properties of RNA-binding proteins (e.g. FUS and TDP-43) and others (such as the microtubule-binding protein tau) contribute to the development of degenerative neurological diseases (e.g. amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer's disease). Thus, studies of liquid-like phase separation (LLPS) and the formation, structure and function of MLOs are of considerable importance in understanding basic cell biology and the pathogenesis of human diseases.