Increased Interferon Signaling in Vaginal Tissue of Patients With Primary Sjögren Syndrome.

OBJECTIVE: Vaginal dryness is an important factor influencing sexual function in women with primary Sjögren syndrome (pSS). Previous studies showed a higher degree of inflammation in vaginal biopsies from patients with pSS compared to non-pSS controls. However, the molecular pathways that drive this inflammation remain unclear. Therefore, the aim of this study was to investigate inflammatory pathway activity in the vaginal tissue of patients with pSS. METHODS: Vaginal biopsies of 8 premenopausal patients with pSS experiencing vaginal dryness and 7 age-matched non-pSS controls were included. Expression of genes involved in inflammation and tissue homeostasis was measured using NanoString technology and validated using TaqMan Real-Time PCR. Vaginal tissue sections were stained by immunohistochemistry for myxovirus resistance protein 1 (MxA) and CD123 (plasmacytoid dendritic cells [pDCs]). RESULTS: The most enriched pathway in vaginal biopsies from patients with pSS compared to non-pSS controls was the interferon (IFN) signaling pathway (P < 0.01). Pathway scores for Janus kinase and signal transducer and activator of transcription (JAK-STAT) and Notch signaling were also higher (P < 0.01 for both pathways). Conversely, transforming growth factor-ß signaling and angiogenesis pathway scores were lower in pSS (P = 0.02 and P = 0.04, respectively). Differences in IFN signaling between patients with pSS and non-pSS controls were confirmed by PCR and MxA tissue staining. No CD123+ pDCs were detected in vaginal biopsies. IFN-stimulated gene expression levels correlated positively with CD45+ cell numbers in vaginal biopsies and serum anti-SSA/Ro positivity. CONCLUSION: Upregulation of IFN signaling in vaginal tissue of women with pSS, along with its association with tissue pathology, suggests that IFNs contribute to inflammation of the vaginal wall and potentially also to clinical symptomatology (ie, vaginal dryness).

Oral Antiviral Defense: Saliva- and Beverage-like Hypotonicity Dynamically Regulate Formation of Membraneless Biomolecular Condensates of Antiviral Human MxA in Oral Epithelial Cells.

The oral mucosa represents a defensive barrier between the external environment and the rest of the body. Oral mucosal cells are constantly bathed in hypotonic saliva (normally one-third tonicity compared to plasma) and are repeatedly exposed to environmental stresses of tonicity, temperature, and pH by the drinks we imbibe (e.g., hypotonic: water, tea, and coffee; hypertonic: assorted fruit juices, and red wines). In the mouth, the broad-spectrum antiviral mediator MxA (a dynamin-family large GTPase) is constitutively expressed in healthy periodontal tissues and induced by Type III interferons (e.g., IFN-λ1/IL-29). Endogenously induced human MxA and exogenously expressed human GFP-MxA formed membraneless biomolecular condensates in the cytoplasm of oral carcinoma cells (OECM1 cell line). These condensates likely represent storage granules in equilibrium with antivirally active dispersed MxA. Remarkably, cytoplasmic MxA condensates were exquisitely sensitive sensors of hypotonicity-the condensates in oral epithelium disassembled within 1-2 min of exposure of cells to saliva-like one-third hypotonicity, and spontaneously reassembled in the next 4-7 min. Water, tea, and coffee enhanced this disassembly. Fluorescence changes in OECM1 cells preloaded with calcein-AM (a reporter of cytosolic "macromolecular crowding") confirmed that this process involved macromolecular uncrowding and subsequent recrowding secondary to changes in cell volume. However, hypertonicity had little effect on MxA condensates. The spontaneous reassembly of GFP-MxA condensates in oral epithelial cells, even under continuous saliva-like hypotonicity, was slowed by the protein-phosphatase-inhibitor cyclosporin A (CsA) and by the K-channel-blocker tetraethylammonium chloride (TEA); this is suggestive of the involvement of the volume-sensitive WNK kinase-protein phosphatase (PTP)-K-Cl cotransporter (KCC) pathway in the regulated volume decrease (RVD) during condensate reassembly in oral cells. The present study identifies a novel subcellular consequence of hypotonic stress in oral epithelial cells, in terms of the rapid and dynamic changes in the structure of one class of phase-separated biomolecular condensates in the cytoplasm-the antiviral MxA condensates. More generally, the data raise the possibility that hypotonicity-driven stresses likely affect other intracellular functions involving liquid-liquid phase separation (LLPS) in cells of the oral mucosa.

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.

Salmonid alphavirus non-structural protein 2 is a key protein that activates the NF-κB signaling pathway to mediate inflammatory responses.

Salmonid alphavirus (SAV) infection of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) causes pancreas disease (PD) with typical inflammatory responses, such as necrosis of the exocrine pancreas, cardiomyopathy and skeletal myopathy. However, the pathogenic mechanism underlying SAV infection is still unclear. Inflammation may cause damage to the body, but it is a defense response against infection by pathogenic microorganisms, of which nuclear factor-kappa B (NF-κB) is the main regulator. This study revealed that SAV can activate NF-κB, of which the viral nonstructural protein Nsp2 is the major activating protein. SAV activates the NF-κB signaling pathway by simultaneously up-regulating TLR3, 7, 8 and then the expression of the signaling molecule myeloid differentiation factor 88 (Myd88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). We found that Nsp2 can induce IκB degradation and p65 phosphorylation and transnucleation, and activate NF-κB downstream inflammatory cytokines. Nsp2 may simultaneously activate NF-κB through TLR3,7,8-dependent signaling pathways. Overexpression of Nsp2 can up-regulate mitochondrial antiviral signaling protein (MAVS) and then promote the expression of IFNa1 and antiviral protein Mx, which inhibits viral replication. This study shows that Nsp2 acts as a key activator protein for the NF-κB signaling pathway, which induces inflammation post-SAV infection. This study systematically analyzes the molecular mechanism of SAV activation of the NF-κB signaling pathway, and provides a theoretical basis for revealing the mechanism of innate immune response and inflammatory injury caused by SAV.

Antitumour Effects of Selected Pyridinium Salts on Sensitive Leukaemia HL60 Cells and Their Multidrug Resistant Topoisomerase II-Defective HL60/MX2 Counterparts.

Multidrug resistance (MDR), having a multifactorial nature, is one of the major clinical problems causing the failure of anticancer therapy. The aim of this study was to examine the antitumour effects of selected pyridinium salts, 1-methyl-3-nitropyridine chloride (MNP) and 3,3,6,6,10-pentamethyl-3,4,6,7-tetrahydro-[1,8(2H,5H)-dion]acridine chloride (MDION), on sensitive leukaemia HL60 cells and resistant topoisomerase II-defective HL60/MX2 cells. Cell growth was determined by the MTT test. Intracellular ROS level was measured with the aid of 2',7'-DCF-DA. The cell cycle distribution was investigated by performing PI staining. DSB formation was examined using the γ-H2AX histone phosphorylation assay. The activity of caspase-3 and caspase-8 was measured with the use of the FLICA test. The assays for examining the lysosome membrane permeabilization were carried out with the aid of LysoTracker Green DND-26. Both studied compounds exerted very similar cytotoxic activities towards sensitive HL60 cells and their MDR counterparts. They modulated the cellular ROS level in a dose-dependent and time-dependent manner and significantly increased the percentage of sensitive HL60 and resistant HL60/MX2 cells with sub-diploid DNA (sub-G1 fraction). However, the induction of DSB formation was not a significant mechanism of action of these pyridinium salts in studied cells. Both examined compounds triggered caspase-3/caspase-8-dependent apoptosis of sensitive HL60 cells and their MDR counterparts. Additionally, the findings of the study indicate that lysosomes may also participate in the programmed death of HL60 as well as HL60/MX2 cells induced by MDION. The data obtained in this work showed that both examined pyridinium salts, MNP and MDION, are able to retain high antileukaemic effects against multidrug resistant topoisomerase II-defective HL60/MX2 cells.

MX2 Viral Substrate Breadth and Inhibitory Activity Are Regulated by Protein Phosphorylation.

Human immunodeficiency virus type-1 (HIV-1) infection is potently inhibited by human myxovirus resistance 2 (MX2/MxB), which binds to the viral capsid and blocks the nuclear import of viral DNA. We have recently shown that phosphorylation is a key regulator of MX2 antiviral activity, with phosphorylation of serine residues at positions 14, 17, and 18 repressing MX2 function. Here, we extend the study of MX2 posttranslational modifications and identify serine and threonine phosphorylation in all domains of MX2. By substituting these residues with aspartic acid or alanine, hence mimicking the presence or absence of a phosphate group, respectively, we identified key positions that control MX2 antiviral activity. Aspartic acid substitutions of residues Ser306 or Thr334 and alanine substitutions of Thr343 yielded proteins with substantially reduced antiviral activity, whereas the presence of aspartic acid at positions Ser28, Thr151, or Thr343 resulted in enhanced activity: referred to as hypermorphic mutants. In some cases, these hypermorphic mutations, particularly when paired with other MX2 mutations (e.g., S28D/T151D or T151D/T343A) acquired the capacity to inhibit HIV-1 capsid mutants known to be insensitive to wild-type MX2, such as P90A or T210K, as well as MX2-resistant retroviruses such as equine infectious anemia virus (EIAV) and murine leukemia virus (MLV). This work highlights the complexity and importance of MX2 phosphorylation in the regulation of antiviral activity and in the selection of susceptible viral substrates. IMPORTANCE Productive infection by human immunodeficiency virus type-1 (HIV-1) requires the import of viral replication complexes into the nuclei of infected cells. Myxovirus resistance 2 (MX2/MxB) blocks this step, halting nuclear accumulation of viral DNA and virus replication. We recently demonstrated how phosphorylation of a stretch of three serines in the amino-terminal domain of MX2 inhibits the antiviral activity. Here, we identify additional positions in MX2 whose phosphorylation status reduces or enhances antiviral function (hypomorphic and hypermorphic variants, respectively). Importantly, hypermorphic mutant proteins not only increased inhibitory activity against wild-type HIV-1 but can also exhibit antiviral capabilities against HIV-1 capsid mutant viruses that are resistant to wild-type MX2. Furthermore, some of these proteins were also able to inhibit retroviruses that are insensitive to MX2. Therefore, we propose that phosphorylation comprises a major element of MX2 regulation and substrate determination.

Fifty Shades of Erns: Innate Immune Evasion by the Viral Endonucleases of All Pestivirus Species.

The genus Pestivirus, family Flaviviridae, includes four historically accepted species, i.e., bovine viral diarrhea virus (BVDV)-1 and -2, classical swine fever virus (CSFV), and border disease virus (BDV). A large number of new pestivirus species were identified in recent years. A common feature of most members is the presence of two unique proteins, Npro and Erns, that pestiviruses evolved to regulate the host's innate immune response. In addition to its function as a structural envelope glycoprotein, Erns is also released in the extracellular space, where it is endocytosed by neighboring cells. As an endoribonuclease, Erns is able to cleave viral ss- and dsRNAs, thus preventing the stimulation of the host's interferon (IFN) response. Here, we characterize the basic features of soluble Erns of a large variety of classified and unassigned pestiviruses that have not yet been described. Its ability to form homodimers, its RNase activity, and the ability to inhibit dsRNA-induced IFN synthesis were investigated. Overall, we found large differences between the various Erns proteins that cannot be predicted solely based on their primary amino acid sequences, and that might be the consequence of different virus-host co-evolution histories. This provides valuable information to delineate the structure-function relationship of pestiviral endoribonucleases.

Dietary Supplementation with Biobran/MGN-3 Increases Innate Resistance and Reduces the Incidence of Influenza-like Illnesses in Elderly Subjects: A Randomized, Double-Blind, Placebo-Controlled Pilot Clinical Trial.

Influenza-like illness (ILI) remains a major cause of severe mortality and morbidity in the elderly. Aging is associated with a decreased ability to sense pathogens and mount effective innate and adaptive immune responses, thus mandating the development of protective nutraceuticals. Biobran/MGN-3, an arabinoxylan from rice bran, has potent anti-aging and immunomodulatory effects, suggesting that it may be effective against ILI. The objective of the current study was to investigate the effect of Biobran/MGN-3 on ILI incidence, natural killer (NK) cell activity, and the expressions of RIG-1 (retinoic acid-inducible gene 1), MDA5 (melanoma differentiation-associated protein 5), and their downstream signaling genes ISG-15 (interferon-stimulated genes 15) and MX1 (myxovirus (influenza) resistance 1, interferon-inducible). A double-blind, placebo-controlled clinical trial included eighty healthy older adults over 55 years old, 40 males and 40 females, who received either a placebo or Biobran/MGN-3 (500 mg/day) for 3 months during known ILI seasonality (peak incidence) in Egypt. The incidence of ILI was confirmed clinically according to the WHO case definition criteria. Hematological, hepatic, and renal parameters were assessed in all subjects, while the activity of NK and NKT (natural killer T) cells was assessed in six randomly chosen subjects in each group by the degranulation assay. The effect of Biobran/MGN-3 on RIG-1 and MDA5, as well as downstream ISG15 and MX1, was assessed in BEAS-2B pulmonary epithelial cells using flow cytometry. The incidence rate and incidence density of ILI in the Biobran/MGN-3 group were 5.0% and 0.57 cases per 1000 person-days, respectively, compared to 22.5% and 2.95 cases per 1000 person-days in the placebo group. Furthermore, Biobran/MGN-3 ingestion significantly enhanced NK activity compared to the basal levels and to the placebo group. In addition, Biobran/MGN-3 significantly upregulated the expression levels of RIG-1, MDA5, ISG15, and MX1 in the human pulmonary epithelial BEAS-2B cell lines. No side effects were observed. Taken together, Biobran/MGN-3 supplementation enhanced the innate immune response of elderly subjects by upregulating the NK activity associated with reduction of ILI incidence. It also upregulated the intracellular RIG-1, MDA5, ISG15, and MX1 expression in pulmonary epithelial tissue cultures. Biobran/MGN-3 could be a novel agent with prophylactic effects against a wide spectrum of respiratory viral infections that warrants further investigation.

High accumulation of Mx2 renders limited multiplication of oncolytic herpes simplex virus-1 in human tumor cells.

Increasing studies demonstrated that oncolytic activities of oHSV-1 are limited to the capacity of virus replicating in tumors. In order to potentiate the oHSV-1 oncolytic activity and expand the application of oHSV-1 treatment in multiple types of tumors, it is critical to explore the potential factors or mechanisms mediating tumor resistance to oHSV-1 infection. Here we evaluated the levels of oHSV-1 multiplication in various tumor cell lines and showed that glioblastoma cell line A172 had the lowest virus yields but intrinsically accumulated the highest levels of Mx2 protein. Subsequently we demonstrated that genetic depletion of Mx2 specifically enhanced oHSV-1 productive replication in A172 cells through promoting the nuclear translocation of uncoated viral genomic DNA and down-regulating innate antiviral response. In the further investigation, we found that Mx2 knockdown could alter the intrinsic mRNA accumulation of diverse sets innate immune genes in A172 cells, in particular DHX36 and MyD88. Mx2 depletion led to a decrease in mRNA levels of MyD88 and DHX36 in A172 cells and MyD88/DHX36 knockdown increased virus yield in A172 cells and decreased the production of IFNα, activation of IRF3 activity and NF-κB signaling in A172 cells. This shed new lights on understanding the roles of some intrinsic antiviral genes in oHSV-1 resistance, facilitating to offer potential targets to improve oHSV-1 oncolytic efficacy and develop candidates of biomarkers to predict the efficiency of oHSV-1 multiplication in tumors.

Heat stress modulates polymorphonuclear cell response in early pregnancy cows: I. interferon pathway and oxidative stress.

One of the major causes of early pregnancy loss is heat stress. In ruminants, interferon tau (IFNT) is the embryo signal to the mother. Once the interferon signaling pathway is activated, it drives gene expression for interferon-stimulated genes (ISGs) and alters neutrophils responses. The aim of the present study was to evaluate interferon (IFN) pathway, ISGs and gene expression in polymorphonuclear leukocytes (PMN) and oxidative stress in dairy cows under heat stress. Pregnant cows had their estrous cycle synchronized and randomly assigned to a comfort or heat stress group. Blood samples were collected at artificial insemination (AI) and on Days 10, 14 and 18 following AI. Pregnant cows were pregnancy checked by ultrasound on Day 30 and confirmed on Day 60 post-AI. Results are presented as mean ± SEM. The corpus luteum (CL) diameter was not different between groups of pregnant cows; concentration of progesterone of pregnant cows on Day 18 following AI was greater in comfort group compared to heat stressed group. Comfort pregnant cows had higher expression of all analyzed genes from interferon pathway, except for IFNAR1, on both Days 14 and 18. Conversely, heat stressed cows did not show altered expression of IFNT pathway genes and ISGs between Days 10, 14, and 18 after AI. The oxidative stress, determined as malondialdehyde (MDA) levels, was greater in heat stress group on Days 10, 14 and 18, independent of pregnancy status. Heat stress negatively influences expression of ISGs, IFN pathway gene expression in neutrophils, and oxidative stress. Our data suggest that lower conception rates in cows under heat stress are multifactorial, with the association of interferon pathway activation and the unbalanced oxidative stress being main contributing factors.

Vitamin C Enhances Antiviral Functions of Lung Epithelial Cells.

Vitamin C is well documented to have antiviral functions; however, there is limited information about its effect on airway epithelial cells-the first cells to encounter infections. Here, we examined the effect of vitamin C on human bronchial epithelium transformed with Ad12-SV40 2B (BEAS-2B) cells, and observed that sodium-dependent vitamin C transporter 2 (SVCT2) was the primary vitamin C transporter. Transcriptomic analysis revealed that treating BEAS-2B cells with vitamin C led to a significant upregulation of several metabolic pathways and interferon-stimulated genes (ISGs) along with a downregulation of pathways involved in lung injury and inflammation. Remarkably, vitamin C also enhanced the expression of the viral-sensing receptors retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated protein 5 (MDA-5), which was confirmed at the protein and functional levels. In addition, the lungs of l-gulono-γ-lactone oxidase knockout (GULO-KO) mice also displayed a marked decrease in these genes compared to wild-type controls. Collectively, our findings indicate that vitamin C acts at multiple levels to exert its antiviral and protective functions in the lungs.

Human cytomegalovirus-induced host protein citrullination is crucial for viral replication.

Citrullination is the conversion of arginine-to-citrulline by protein arginine deiminases (PADs), whose dysregulation is implicated in the pathogenesis of various types of cancers and autoimmune diseases. Consistent with the ability of human cytomegalovirus (HCMV) to induce post-translational modifications of cellular proteins to gain a survival advantage, we show that HCMV infection of primary human fibroblasts triggers PAD-mediated citrullination of several host proteins, and that this activity promotes viral fitness. Citrullinome analysis reveals significant changes in deimination levels of both cellular and viral proteins, with interferon (IFN)-inducible protein IFIT1 being among the most heavily deiminated one. As genetic depletion of IFIT1 strongly enhances HCMV growth, and in vitro IFIT1 citrullination impairs its ability to bind to 5'-ppp-RNA, we propose that viral-induced IFIT1 citrullination is a mechanism of HCMV evasion from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection.

Induction of IFN-ß through TLR-3- and RIG-I-Mediated Signaling Pathways in Canine Respiratory Epithelial Cells Infected with H3N2 Canine Influenza Virus.

Canine influenza virus (CIV) induces acute respiratory disease in dogs. In this study, we aimed to determine the signaling pathways leading to the induction of IFN-ß in a canine respiratory epithelial cell line (KU-CBE) infected with the H3N2 subtype of CIV. Small interfering RNAs (siRNAs) specific to pattern recognition receptors (PRRs) and transcription factors were used to block the IFN-ß induction signals in H3N2 CIV-infected KU-CBE cells. Among the PRRs, only the TLR3 and RIG-I expression levels significantly (p < 0.001) increased in CIV-infected cells. Following transfection with siRNA specific to TLR3 (siTLR3) or RIG-I (siRIG-I), the mRNA expression levels of IFN-ß significantly (p < 0.001) decreased, and the protein expression of IFN-ß also decreased in infected cells. In addition, co-transfection with both siTLR3 and siRIG-I significantly reduced IRF3 (p < 0.001) and IFN-ß (p < 0.001) mRNA levels. Moreover, the protein concentration of IFN-ß was significantly (p < 0.01) lower in cells co-transfected with both siTLR3 and siRIG-I than in cells transfected with either siTLR3 or siRIGI alone. Also, the antiviral protein MX1 was only expressed in KU-CBE cells infected with CIV or treated with IFN-ß or IFN-α. Thus, we speculate that IFN-ß further induces MX1 expression, which might suppress CIV replication. Taken together, these data indicate that TLR3 and RIG-I synergistically induce IFN-ß expression via the activation of IRF3, and the produced IFN-ß further induces the production of MX1, which would suppress CIV replication in CIV-infected cells.

[A case of dermatomyositis positive for anti-nuclear matrix protein 2 antibody without dermatologic symptoms].

We report a 47-year-old woman who presented with progressive myalgia, weakness in the proximal limbs, and dysphagia for a month and a half. No skin rash was observed on admission. Examination of MRI data suggested inflammatory changes in the proximal limbs and trunk muscles. Biopsy specimens from the left biceps muscle showed no perifascicular atrophy, but immunohistochemical staining revealed the presence of myxovirus resistance protein A (MxA) in myofibers, strongly suggesting dermatomyositis (DM). In addition, her serum was positive for anti-nuclear matrix protein 2 (anti-NXP-2) antibody, which is reportedly useful as a marker of DM without skin lesions. Her symptoms gradually improved upon intravenous methylprednisolone pulse therapy in conjunction with oral prednisolone, oral tacrolimus, and intravenous immunoglobulin therapy. Our findings suggest that in cases where inflammatory muscle disease is suspected, anti-NXP-2 antibody analyses should be considered for precise diagnosis, even if there are no dermatological symptoms.

MX2 mediates establishment of interferon response profile, regulates XAF1, and can sensitize melanoma cells to targeted therapy.

MX2 is an interferon inducible gene that is mostly known for its antiviral activity. We have previously demonstrated that MX2 is also associated with the tumorigenesis process in melanoma. However, it remains unknown which molecular mechanisms are regulated by MX2 in response to interferon signaling in this disease. Here, we report that MX2 is necessary for the establishment of an interferon-induced transcriptional profile partially through regulation of STAT1 phosphorylation and other interferon-related downstream factors, including proapoptotic tumor suppressor XAF1. MX2 and XAF1 expression tightly correlate in both cultured melanoma cell lines and in patient-derived primary and metastatic tumors, where they also are significantly related with survival. MX2 mediates IFN growth-inhibitory signals in both XAF1 dependent and independent ways and in a cell type and context-dependent manner. Higher MX2 expression renders melanoma cells more sensitive to targeted therapy drugs such as vemurafenib and trametinib; however, this effect is XAF1 independent. In summary, we uncovered a new mechanism in the complex regulation of interferon signaling in melanoma that can influence both survival and response to therapy.

Inhibition of interferon-signalling halts cancer-associated fibroblast-dependent protection of breast cancer cells from chemotherapy.

BACKGROUND: Triple negative breast cancers (TNBC) have poor prognoses despite aggressive treatment with cytotoxic chemotherapy. Cancer-associated fibroblasts (CAFs) are prominent in tumour stroma. Our hypothesis was that CAFs modulate chemotherapy sensitivity. METHODS: TNBC cells and breast fibroblasts were cultured; survival after chemotherapeutics was assessed using luciferase or clonogenic assays. Signalling was investigated using transcriptomics, reporters, recombinant proteins and blocking antibodies. Clinical relevance was investigated using immunohistochemistry. RESULTS: Breast CAFs dose-dependently protected TNBC cell lines MDA-MB-231 and MDA-MB-157, but not MDA-MB-468s, from chemotherapy. CAF-induced protection was associated with interferon (IFN) activation. CAFs were induced to express IFNß1 by chemotherapy and TNBC co-culture, leading to paracrine activation in cancer cells. Recombinant IFNs were sufficient to protect MDA-MB-231 and MDA-MB-157 but not MDA-MB-468 cells. In TNBC patients, IFNß1 expression in CAFs correlated with cancer cell expression of MX1, a marker of activated IFN signalling. High expression of IFNß1 (CAFs) or MX1 (tumour cells) correlated with reduced survival after chemotherapy, especially in claudin-low tumours (which MDA-MB-231 and MDA-MB-157 cells represent). Antibodies that block IFN receptors reduced CAF-dependent chemoprotection. CONCLUSIONS: CAF-induced activation of IFN signalling in claudin-low TNBCs results in chemoresistance. Inhibition of this pathway represents a novel method to improve breast cancer outcomes.

TRIM26 Facilitates HSV-2 Infection by Downregulating Antiviral Responses through the IRF3 Pathway.

Herpes simplex virus type 2 (HSV-2) is the primary cause of genital herpes which results in significant morbidity and mortality, especially in women, worldwide. HSV-2 is transmitted primarily through infection of epithelial cells at skin and mucosal surfaces. Our earlier work to examine interactions between HSV-2 and vaginal epithelial cells demonstrated that infection of the human vaginal epithelial cell line (VK2) with HSV-2 resulted in increased expression of TRIM26, a negative regulator of the Type I interferon pathway. Given that upregulation of TRIM26 could negatively affect anti-viral pathways, we decided to further study the role of TRIM26 in HSV-2 infection and replication. To do this, we designed and generated two cell lines derived from VK2s with TRIM26 overexpressed (OE) and knocked out (KO). Both, along with wildtype (WT) VK2, were infected with HSV-2 and viral titres were measured in supernatants 24 h later. Our results showed significantly enhanced virus production by TRIM26 OE cells, but very little replication in TRIM26 KO cells. We next examined interferon-ß production and expression of two distinct interferon stimulated genes (ISGs), MX1 and ISG15, in all three cell lines, prior to and following HSV-2 infection. The absence of TRIM26 (KO) significantly upregulated interferon-ß production at baseline and even further after HSV-2 infection. TRIM26 KO cells also showed significant increase in the expression of MX1 and ISG15 before and after HSV-2 infection. Immunofluorescent staining indicated that overexpression of TRIM26 substantially decreased the nuclear localization of IRF3, the primary mediator of ISG activation, before and after HSV-2 infection. Taken together, our data indicate that HSV-2 utilizes host factor TRIM26 to evade anti-viral response and thereby increase its replication in vaginal epithelial cells.

Myxovirus resistance 1 (Mx1) gene: Molecular characterization of complete coding sequence and expression profile in the endometrium of goat (Capra hircus).

Myxovirus resistance 1 (Mx1) gene plays an important role in uterine receptivity and conceptus development by creating a strong defense mechanism in the uterine environment. However, the specific role of Mx1 gene is not yet documented in the goat. Therefore, in the present study, full-length coding sequence (CDS) of the Mx1 gene was amplified, sequenced and characterized through various Bioinformatic tools. Temporal expression profile of Mx1 mRNA and protein was also examined by quantitative real-time PCR (qPCR) and western blot, respectively, in the endometrium of cyclic stage (non-pregnant), pregnancy stage I (16-24 days of gestation) and pregnancy stage II (25-40 days of gestation) of caprine (cp). A fragment of the cpMx1 gene, 2144 bp in length, was amplified from complementary DNA (cDNA) with a 1965 bp open reading frame. Coding and deduced amino acid sequences of the cpMx1 were aligned with other species and it exhibited 98.8-81.5 % identities with different species. On phylogenetic analysis, sheep and goat were found belonging to the same clade but differing from large ruminants. The cpMx1 protein possess the conserved signature motif (LPRGTGIVTR) of dynamin superfamily and the tripartite guanosine-5'-triphosphate (GTP) binding motif (GDQSSGKS, DLPG, TKPD) at the N-terminal end, and the leucine zipper motifs at the C-terminal end. Both cpMx1 mRNA and protein were found to be expressed maximally (P < 0.05) in the pregnancy stage I as compared to cyclic stage. It was concluded that the cpMx1 gene shares major structural and probably functional similarities with other species.

The differing pathophysiologies that underlie COVID-19-associated perniosis and thrombotic retiform purpura: a case series.

BACKGROUND: There are two distinctive acral manifestations of COVID-19 embodying disparate clinical phenotypes. One is perniosis occurring in mildly symptomatic patients, typically children and young adults; the second is the thrombotic retiform purpura of critically ill adults with COVID-19. OBJECTIVES: To compare the clinical and pathological profiles of these two different cutaneous manifestations of COVID-19. METHODS: We compared the light microscopic, phenotypic, cytokine and SARS-CoV-2 protein and RNA profiles of COVID-19-associated perniosis with that of thrombotic retiform purpura in critical patients with COVID-19. RESULTS: Biopsies of COVID-19-associated perniosis exhibited vasocentric and eccrinotropic T-cell- and monocyte-derived CD11c+ , CD14+ and CD123+ dendritic cell infiltrates. Both COVID-associated and idiopathic perniosis showed striking expression of the type I interferon-inducible myxovirus resistance protein A (MXA), an established marker for type I interferon signalling in tissue. SARS-CoV-2 RNA, interleukin-6 and caspase 3 were minimally expressed and confined to mononuclear inflammatory cells. The biopsies from livedo/retiform purpura showed pauci-inflammatory vascular thrombosis without any MXA decoration. Blood vessels exhibited extensive complement deposition with endothelial cell localization of SARS-CoV-2 protein, interleukin-6 and caspase 3; SARS-CoV-2 RNA was not seen. CONCLUSIONS: COVID-19-associated perniosis represents a virally triggered exaggerated immune reaction with significant type I interferon signaling. This is important to SARS-CoV-2 eradication and has implications in regards to a more generalized highly inflammatory response. We hypothesize that in the thrombotic retiform purpura of critically ill patients with COVID-19, the vascular thrombosis in the skin and other organ systems is associated with a minimal interferon response. This allows excessive viral replication with release of viral proteins that localize to extrapulmonary endothelium and trigger extensive complement activation.

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.