Mx Expression Research Articles

Red elemental selenium (Se0 ) improves the immunoactivities of EPC cells, crucian carp and zebrafish against spring viraemia of carp virus.

Selenium, as an essential trace element, interferes through selenoproteins in many physiological processes of plants and mammals. Its antiviral activity has recently attracted much attention because selenium improves the antiviral capacity of animal cells against a few viruses relevant to human diseases. In this study, the red elemental selenium was purified from the fermentative culture of Herbaspirillum camelliae WT00C and then used to culture epithelioma papulosum cyprinid (EPC) cells or feed crucian carp and zebrafish. Finally, its antiviral effects were investigated at the cell level and living fishes after spring viraemia of carp virus infection. At the cell level, 5, 10 and 20 μg ml-1 red elemental selenium significantly induced the expression of interferon (IFN) and ISG15 genes in EPC cells. The viral TCID50 (50% tissue culture infective dose) values in the EPC cells incubated with 5, 10 and 20 μg ml-1 red elemental selenium were significantly less than those of the control. More expression of IFN and ISG15 genes and less TCID50 values indicate that red elemental selenium indeed improves the antiviral capability of EPC cells. In the crucian carp fed with the food containing 5 and 10 μg g-1 red elemental selenium, IFN expressions showed 13- and 39-fold increases at the 16th day of post-injection, and its expression was dependent on selenium concentrations. Meanwhile, no fish death occurred in all the experimental groups. In the zebrafish fed with the red worm containing 5 μg g-1 red elemental selenium, IFN and Mx expressions and survival rate were significantly higher than those of the control. The results of this study show that red elemental selenium indeed improves the antiviral activity of fish. The antiviral effects of selenium mainly come from its immune regulation through its incorporation into selenoproteins. The optimum level of selenium contributes to improving fish immunity, whereas excess selenium causes excessive immune and inflammatory responses.

Alveolar Macrophages Are Key Players in the Modulation of the Respiratory Antiviral Immunity Induced by Orally Administered Lacticaseibacillus rhamnosus CRL1505.

The oral administration of Lacticaseibacillus rhamnosus CRL1505 differentially modulates the respiratory innate antiviral immune response triggered by Toll-like receptor 3 (TLR3) activation in infant mice, improving the resistance to Respiratory Syncytial Virus (RSV) infection. In this work, by using macrophages depletion experiments and a detailed study of their production of cytokines and antiviral factors we clearly demonstrated the key role of this immune cell population in the improvement of both viral elimination and the protection against lung tissue damage induced by the CRL1505 strain. Orally administered L. rhamnosus CRL1505 activated alveolar macrophages and enhanced their ability to produce type I interferons (IFNs) and IFN-γ in response to RSV infection. Moreover, an increased expression of IFNAR1, Mx2, OAS1, OAS2, RNAseL, and IFITM3 was observed in alveolar macrophages after the oral treatment with L. rhamnosus CRL1505, which was consistent with the enhanced RSV clearance. The depletion of alveolar macrophages by the time of L. rhamnosus CRL1505 administration abolished the ability of infant mice to produce increased levels of IL-10 in response to RSV infection. However, no improvement in IL-10 production was observed when primary cultures of alveolar macrophages obtained from CRL1505-treated mice were analyzed. Of note, alveolar macrophages from the CRL1505 group had an increased production of IL-6 and IL-27 suggesting that these cells may play an important role in limiting inflammation and protecting lung function during RSV infection, by increasing the maturation and activation of Treg cells and their subsequent production of IL-10. In addition, we provided evidence of the important role of CD4+ cells and IFN-γ in the activation of alveolar macrophages highlighting a putative pathway through which the intestinal and respiratory mucosa are communicated under the influence of L. rhamnosus CRL1505.

SARS-CoV-2 Infection Boosts MX1 Antiviral Effector in COVID-19 Patients.

SummaryIn a published case-control study (GSE152075) from SARS-CoV-2-positive (n = 403) and -negative patients (n = 50), we analyzed the response to infection assessing gene expression of host cell receptors and antiviral proteins. The expression analysis associated with reported risk factors for COVID-19 was also assessed. SARS-CoV-2 cases had higher ACE2, but lower TMPRSS2, BSG/CD147, and CTSB expression compared with negative cases. COVID-19 patients' age negatively affected ACE2 expression. MX1 and MX2 were higher in COVID-19 patients. A negative trend for MX1 and MX2 was observed as patients' age increased. Principal-component analysis determined that ACE2, MX1, MX2, and BSG/CD147 expression was able to cluster non-COVID-19 and COVID-19 individuals. Multivariable regression showed that MX1 expression significantly increased for each unit of viral load increment. Altogether, these findings support differences in ACE2, MX1, MX2, and BSG/CD147 expression between COVID-19 and non-COVID-19 patients and point out to MX1 as a critical responder in SARS-CoV-2 infection.

Abstract 2255: Enhancing immunogenicity in immunologically cold ER+ breast cancer using estrogen receptor blockade and radiation therapy

Abstract Metastatic, hormone therapy-resistant breast cancers expressing estrogen receptor (ER+) account for the majority of breast cancer deaths. ER+ breast cancers are immunologically “cold” with low tumor mutation burden and few tumor-infiltrating lymphocytes (TILs). Consequently, application of immune checkpoint inhibitors (ICIs) to ER+ breast cancers has not proven effective. Radiation therapy (RT) can increase immune susceptibility of “cold” tumors, but can also increase recruitment of immunosuppressive myeloid-derived suppressor cells (MDSCs) to the radiated tumor microenvironment (TME). Recent studies demonstrate that ER inhibition may antagonize the trafficking and activation of MDSCs in tumors. We hypothesized that combining RT and a selective ER degrader (SERD), fulvestrant, would cooperate to relieve immunosuppression, increase tumor immune susceptibility, and facilitate response to ICIs in a syngeneic murine model of ER+ breast cancer. Using a murine breast cancer model of ER+ breast cancer developed (TC11), we examined effects in vitro and in vivo. In vitro, TC11 cells were treated with 8Gy RT and/or fulvestrant (1uM) to evaluate cell autonomous effects. In vivo, 25,000 TC11 cells were transplanted to caudal mammary fat pads of female FVB/N mice. When tumors reached ~200mm3, mice were randomized and treated. We initially evaluated the time course of immune responses to 8Gy RT in the TC11 TME, euthanizing mice every 5 days up to 20 days after RT. We subsequently treated mice with vehicle, fulvestrant (250mg/kg sc, weekly), 8Gy RT, and fulvestrant+8Gy RT. Flow cytometry was performed on day 5 and day 10 after RT to examine the innate and adaptive immune cell infiltrates, respectively. A therapeutic intervention study was performed using combinations of fulvestrant, 8Gy RT, and/or anti-PDL1 (200µg, ip, at days 3, 6, and 9 after RT). 8Gy RT elicited a type-1 interferon response in TC11 cells in vitro and in vivo; as measured by upregulated expression of Mx1, Pdl1, Oas2, Oas3, Trex1, and IfnB mRNAs peaking at 3-5 days post RT and persisting to day 10 in TC11 tumors. RT increased MDSCs (Ly6C+Ly6G+) in the TC11 TME compared to vehicle or fulvestrant alone in tumors taken at day 5, but MDSCs were reduced in tumors treated with fulvestrant and RT. Together, fulvestrant and RT increased TILs (CD45+) and CD8+ T cells at day 10 compared to vehicle control and single treatments. Preliminary data indicate RT and fulvestrant together primed a greater response to anti-PDL1 ICI compared to single or dual combinations of these treatments. These results demonstrate a cooperative interaction between RT and fulvestrant in favorably modulating the TME and response to ICIs in an immunologically cold, hormone-therapy resistant, murine ER+ breast cancer model. Further study is warranted to determine whether this combination may elicit a more effective in situ vaccine effect compared to RT alone. Citation Format: Amber M. Bates, Kathleen A. O'Leary, Sarah Emma, Erin Nystuen, Elizabeth G. Sumiec, Linda A. Schuler, Zachary S. Morris. Enhancing immunogenicity in immunologically cold ER+ breast cancer using estrogen receptor blockade and radiation therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2255.

Microalgal extracts induce larval programming and modify growth and the immune response to bioactive treatments and LCDV in Senegalese sole post-larvae

Immunostimulants are key molecules in aquaculture since they heighten defensive responses and protection against pathogens. The present study investigated the treatment of Senegalese sole larvae with a whole-cell crude extract of the microalgae Nannochloropsis gaditana (Nanno) and programming of growth and the immune system. Larvae at hatch were treated with the Nanno extracts for 2 h and thereafter were cultivated for 32 days post-hatch (dph) in parallel with an untreated control group (CN). Dry weight and length at 21 days post-hatch (dph) were higher in post-larvae of the Nanno than CN group. These differences in weight were later confirmed at 32 dph. To evaluate changes in the immune response associated with Nanno-programming treatments, the Nanno and CN post-larvae were supplied with two bioactive compounds yeast β-glucan (Y) and a microalga extract from the diatom Phaeodactylum tricornutum (MAe). The bioactive treatments were administrated to the treatment groups through the live prey (artemia metanauplii, 200 artemia mL−1) enriched for 30 min with MAe or Y (at 2 mg mL−1 SW) or untreated prey in the case of the negative control (SW). The effect of the treatments was assessed by monitoring gene expression, enzyme activity and mortality over 48 h. The post-larvae sole supplied with the bioactive compounds Y and MAe had increased mortality at 48 h compared to the SW group. Moreover, mortality was higher in Nanno-programmed than CN post-larvae. Lysozyme and total anti-protease enzymatic activities at 6 and 24 h after the start of the trial were significantly higher in the Nanno and MAe supplied post-larvae compared to their corresponding control (CN and SW, respectively). Immune gene transcripts revealed that il1b, cxc10 and mx mRNAs were significantly different between Nanno and CN post-larvae at 6 and 24 h. Moreover, the expression of il1b, tnfa, cxc10, irf3, irf7 and mx was modified by bioactive treatments but with temporal differences. At 48 h after bioactive treatments, Y and SW post-larvae were challenged with the lymphocystis disease virus (LCDV). No difference existed in viral copy number between programming or bioactive treatment groups at 3, 6 and 24 h after LCDV challenge although the total number of copies reduced with time. Gene expression profiles in the LCDV-challenged group indicated that post-larvae triggered a wide defensive response compared to SWC 24 h after challenge, which was modulated by programming and bioactive compound treatments. Cluster analysis of expressed genes separated the SW and Y groups indicating long-lasting effects of yeast β-glucan treatment in larvae. A noteworthy interaction between Nanno-programming and Y-treatment on the regulation of antiviral genes was observed. Overall, the data demonstrate the capacity of microalgal crude extracts to modify sole larval plasticity with long-term effects on larval growth and the immune responses.

Lsh/HELLS is required for B lymphocyte development and immunoglobulin class switch recombination

Mutation of HELLS (Helicase, Lymphoid-Specific)/Lsh in human DNA causes a severe immunodeficiency syndrome, but the nature of the defect remains unknown. We assessed here the role of Lsh in hematopoiesis using conditional Lsh knockout mice with expression of Mx1 or Vav Cre-recombinase. Bone marrow transplantation studies revealed that Lsh depletion in hematopoietic stem cells severely reduced B cell numbers and impaired B cell development in a hematopoietic cell-autonomous manner. Lsh-deficient mice without bone marrow transplantation exhibited lower Ig levels in vivo compared to controls despite normal peripheral B cell numbers. Purified B lymphocytes proliferated normally but produced less immunoglobulins in response to in vitro stimulation, indicating a reduced capacity to undergo class switch recombination (CSR). Analysis of germline transcripts, examination of double-stranded breaks using biotin-labeling DNA break assay, and End-seq analysis indicated that the initiation of the recombination process was unscathed. In contrast, digestion-circularization PCR analysis and high-throughput sequencing analyses of CSR junctions and a chromosomal break repair assay indicated an impaired ability of the canonical end-joining pathway in Lsh-deficient B cells. Our data suggest a hematopoietic cell-intrinsic role of Lsh in B cell development and in CSR providing a potential target for immunodeficiency therapy.

Construction of a DNA vaccine and its protective effect on largemouth bass (Micropterus salmoides) challenged with largemouth bass virus (LMBV)

Largemouth bass virus (LMBV) is the causative agent of a disease causing high mortality rates in largemouth bass during summer. However, there is little information available about the development of vaccines for LMBV disease. Hence, a DNA vaccine, named pCDNA3.1(+)-MCP-Flag, was constructed by inserting the cloned LMBV major capsid protein (MCP) gene into the pCDNA3.1(+)-Flag plasmid. The expression of the recombinant plasmid was confirmed by Western blot (WB) and RT-PCR. The WB result revealed that the MCP protein produced a band of approximately 53 kDa, consistent with the expected result. The RT-PCR results also confirmed that MCP was transcribed in the EPC cells transfected with the recombinant plasmid. The largemouth bass in the DNA vaccine group were immunized with the pCDNA3.1(+)-MCP-Flag plasmid by pectoral fin base injection, and the relative percent survival (RPS) of fish challenged with LMBV was 63%. The relative immunological analyses were as follows. Compared with the PBS and pCDNA3.1(+) groups, the DNA vaccine group showed significantly upregulated expression of IL-1β, IL-8, TNF-α and Mx in the spleen, head kidney and liver. All largemouth bass immunized with the DNA vaccine produced a high titre of LMBV-specific neutralizing antibody during the immunization period. The titre was 1:375 ± 40 and peaked at 14 days post-vaccination. The expression of the recombinant plasmid was analysed in the tissues of the DNA vaccine group by RT-PCR. The recombinant plasmid was expressed in the spleen, head kidney and liver, and MCP protein was successfully expressed after vaccination. In conclusion, the recombinant plasmid expressing LMBV MCP induced significant immune responses in largemouth bass, and might represent a potential LMBV vaccine candidate for largemouth bass.

Increased antiviral response in circulating lymphocytes from hypogammaglobulinemia patients.

B cells play a crucial role during rhinovirus (RV) infections by production of virus-neutralizing antibodies. A main feature of common variable immunodeficiency (CVID) is hypogammaglobulinemia (HG). HG patients have severely reduced levels of antibody-producing B cells and suffer from prolonged virus infections. Here, we addressed whether antiviral response of peripheral blood lymphocytes differs between HG patients and healthy individuals during natural RV infection. Using fluorescence-activated cell sorting, B-cell subsets were analyzed. Simultaneously, CD19+B cells, CD14+monocytes, and CD3+T cells were sorted from frozen peripheral blood mononuclear cells from 11 RV-infected hypogammaglobulinemia patients, 7 RV-infected control subjects, and 14 noninfected control subjects. Real-time PCR was used to study expression of antiviral genes. A pan-RV PCR was used to detect RV genome in all samples. In HG patients, total B-cell numbers, as well as IgA+and IgG+switched memory B cells, were reduced while naïve B cells and T cells were increased. STAT1 expression was increased in HG patients compared to controls in all lymphocyte subsets analyzed. The expression of antiviral genes IFITM1 and MX1 correlated with STAT1 expression in B cells and monocytes. RV RNA was found in 88.9% of monocytes from infected HG patients, 85.7% of monocytes from infected controls, and 7.1% of monocytes from uninfected controls. We demonstrate an increased antiviral response in B cells and monocytes in HG patients and their correlation with STAT1 expression. Monocytes of infected HG patients and infected non-HG controls carry RV RNA.

Porcine enteric alphacoronavirus Inhibits IFN-α, IFN-β, OAS, Mx1, and PKR mRNA Expression in Infected Peyer's Patches in vivo.

Porcine enteric alphacoronavirus (PEAV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhea in neonatal piglets. The pathogenesis and host immune responses of PEAV infection are not fully characterized. The reason lies in the stomach environment, which would degrade cell-cultured live viruses via oral infection, making it difficult to establish an effective infection model to study the pathogenesis and host immune responses in pigs with a mature immune system. To solve this problem, in this study, coated PEAV-loaded microspheres were developed by centrifugal granulation-fluidized bed coating and demonstrated as an effective oral delivery system/animal infection model to protect PEAV virion against the complex gastrointestinal environment in vitro and to cause infection in weaned piglets in vivo. Weaned piglets orally inoculated with coated PEAV-loaded microspheres developed diarrhea and virus RNA was detected in rectal swabs from one to seven days post inoculation. In addition, microscopic lesions in the small intestine were observed, and viral antigens were also detected in the small intestines with PEAV immunohistochemical staining. Importantly, PEAV significantly inhibited mRNA expression of IFN-α, IFN-β, OAS, Mx1, and PKR, the genes involved in modulation of the host immune responses, in infected Peyer's patches, indicating that PEAV can overcome the antiviral response to cause damage when infection occurs. Collectively, our research successfully established a PEAV animal infection model in weaned piglets and suggested that the observed gene expression profile might help explain immunological changes associated with PEAV infection.

Molecular cloning, tissue distribution and function analysis of duck TLR7

Toll-like receptors (TLRs) play an important role in detecting pathogen-associated molecular patterns (PAMPs). Among the TLRs, TLR7 is involved in the recognition of antiviral compounds and single-stranded RNA. This study was designed to explore the structure and function of TLR7 in duck (Anas platyrhynchos), a natural host for avian influenza virus. Firstly, the full-length cDNA of Shaoxing egg-laying duck TLR7 (duTLR7) was obtained using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). It consisted of 38 base pairs (bp) 5′-untranslated region (UTR), 187 bp 3′-UTR, and 3270 bp open reading frame that encodes a single protein of 1089 amino acid residues. DuTLR7 shares high identity with TLR7 genes from other vertebrates. In healthy ducks, duTLR7 transcripts were broadly expressed in different tissues, with higher expression levels in the liver, kidney, and thymus. The highest relative transcript level of duTLR7 could be induced with R848 stimulation. In addition, overexpression of duTLR7 by stimulating with poly(I:C) significantly promoted IFN-β, NF-κB, IRF7, TRIF, Mx, STAT1 and STAT2 expressions. Taken together, these results suggest that TLR7 may play an important role in the innate immune response of ducks.

Pulmonary Involvement in a Mouse Model of Sjögren's Syndrome Induced by STING Activation.

Sjögren’s Syndrome (SS), a chronic autoimmune disorder affecting multiple organ systems, is characterized by an elevated type I interferon (IFN) response. Activation of Stimulator of Interferon Genes (STING) protein induces type I IFN and in mice, several features of SS, including anti-nuclear antibodies, sialadenitis, and salivary gland dysfunction. Since lung involvement occurs in one-fifth of SS patients, we investigated whether systemic activation of STING also leads to lung inflammation. Lungs from female C57BL/6 mice injected with the STING agonist 5, 6-Dimethylxanthenone-4-acetic acid (DMXAA), were evaluated for acute and chronic inflammatory responses. Within 4h of DMXAA injection, the expression of Ifnb1, Il6, Tnf, Ifng, and Mx1 was significantly upregulated. At 1 and 2 months post-treatment, lungs showed lymphocytic infiltration in the peri-bronchial regions. The lungs from DMXAA treated mice showed an increased expression of multiple chemokines and an increase in lymphatic endothelial cells. Despite STING expression in bronchial epithelium and cells lining the alveolar wall, bone marrow chimeras between STING knockout and wild type mice showed that STING expression in hematopoietic cells was critical for lung inflammation. Our results suggest that activation of the STING pathway might be involved in SS patients with concomitant salivary gland and lung disease.

SARS-CoV-2 infection risk assessment in the endometrium: viral infection-related gene expression across the menstrual cycle

ObjectiveTo determine the susceptibility of the endometrium to infection by—and thereby potential damage from—SARS-CoV-2.DesignAnalysis of SARS-Cov-2 infection-related gene expression from endometrial transcriptomic data sets.SettingInfertility research department affiliated with a public hospital.Patient(s)Gene expression data from five studies in 112 patients with normal endometrium collected throughout the menstrual cycle.Intervention(s)None.Main Outcome Measure(s)Gene expression and correlation between viral infectivity genes and age throughout the menstrual cycle.Result(s)Gene expression was high for TMPRSS4, CTSL, CTSB, FURIN, MX1, and BSG; medium for TMPRSS2; and low for ACE2. ACE2, TMPRSS4, CTSB, CTSL, and MX1 expression increased toward the window of implantation. TMPRSS4 expression was positively correlated with ACE2, CTSB, CTSL, MX1, and FURIN during several cycle phases; TMPRSS2 was not statistically significantly altered across the cycle. ACE2, TMPRSS4, CTSB, CTSL, BSG, and MX1 expression increased with age, especially in early phases of the cycle.Conclusion(s)Endometrial tissue is likely safe from SARS-CoV-2 cell entry based on ACE2 and TMPRSS2 expression, but susceptibility increases with age. Further, TMPRSS4, along with BSG-mediated viral entry into cells, could imply a susceptible environment for SARS-CoV-2 entry via different mechanisms. Additional studies are warranted to determine the true risk of endometrial infection by SARS-CoV-2 and implications for fertility treatments.

The Stronger Downregulation of in vitro and in vivo Innate Antiviral Responses by a Very Virulent Strain of Infectious Bursal Disease Virus (IBDV), Compared to a Classical Strain, Is Mediated, in Part, by the VP4 Protein.

IBDV is economically important to the poultry industry. Very virulent (vv) strains cause higher mortality rates than other strains for reasons that remain poorly understood. In order to provide more information on IBDV disease outcome, groups of chickens (n = 18) were inoculated with the vv strain, UK661, or the classical strain, F52/70. Birds infected with UK661 had a lower survival rate (50%) compared to F52/70 (80%). There was no difference in peak viral replication in the bursa of Fabricius (BF), but the expression of chicken IFNα, IFNβ, MX1, and IL-8 was significantly lower in the BF of birds infected with UK661 compared to F52/70 (p < 0.05) as quantified by RTqPCR, and this trend was also observed in DT40 cells infected with UK661 or F52/70 (p < 0.05). The induction of expression of type I IFN in DF-1 cells stimulated with polyI:C (measured by an IFN-β luciferase reporter assay) was significantly reduced in cells expressing ectopic VP4 from UK661 (p < 0.05), but was higher in cells expressing ectopic VP4 from F52/70. Cells infected with a chimeric recombinant IBDV carrying the UK661-VP4 gene in the background of PBG98, an attenuated vaccine strain that induces high levels of innate responses (PBG98-VP4UK661) also showed a reduced level of IFNα and IL-8 compared to cells infected with a chimeric virus carrying the F52/70-VP4 gene (PBG98-VP4F52/70) (p < 0.01), and birds infected with PBG98-VP4UK661 also had a reduced expression of IFNα in the BF compared to birds infected with PBG98-VP4F52/70 (p < 0.05). Taken together, these data demonstrate that UK661 induced the expression of lower levels of anti-viral type I IFN and proinflammatory genes than the classical strain in vitro and in vivo and this was, in part, due to strain-dependent differences in the VP4 protein.

MG132 Attenuates the Replication of Classical Swine Fever Virus in vitro.

The 26S proteasome, in charge of intracellular protein degradation, plays significant roles in the modulation of various cellular activities as well as in the interplay between virus and host. However, studies about the relationship between 26S proteasome and classical swine fever virus (CSFV) is limited up to now. MG132 is a proteasome inhibitor and has been extensively used in studies about replication of many viruses. Herein, we investigated the role of MG132 in CSFV replication and results showed that MG132 significantly decreased virus titers and viral RNA copies in CSFV-infected PK-15 cells. Further studies demonstrated that MG132 upregulated the expression of several interferon-stimulated genes (ISGs), in CSFV-infected cells. Since the activation of ISGs is controlled by the JAK-STAT signal pathway, we next examined the effect of MG132 on the expression and localization of key molecular STAT1 in the infected cells using Western blot and confocal laser scanning microscopy, respectively. Results showed that CSFV infection and viral NS4A protein decreased the protein level of STAT1, and MG132 promoted the accumulation of STAT1 in the nucleus of cells adjacent to the CSFV-infected cells. Besides, MG132 did not affect the expressions of IFN-α, STAT1, Mx1, OAS1, and PKR genes in cells without CSFV. In conclusion, we identify that MG132 significantly inhibits CSFV replication in vitro, in which the activation of the JAK-STAT pathway and the subsequent upregulation of expressions of ISGs might play significant roles, providing a potential preventive method for CSF.

TLR Stimulation Produces IFN-β as the Primary Driver of IFN Signaling in Nonlymphoid Primary Human Cells.

Several human autoimmune diseases are characterized by increased expression of type 1 IFN-stimulated genes in both the peripheral blood and tissue. The contributions of different type I IFNs to this gene signature are uncertain as the type I IFN family consists of 13 alphas and one each of β, ε, κ, and ω subtypes. We sought to investigate the contribution of various IFNs to IFN signaling in primary human cell types. We stimulated primary skin, muscle, kidney, and PBMCs from normal healthy human donors with various TLR ligands and measured the expression of type I IFN subtypes and activation of downstream signaling by quantitative PCR. We show that IFNB1 is the dominant type I IFN expressed upon TLR3 and TLR4 stimulation, and its expression profile is associated with subsequent MX1 transcription. Furthermore, using an IFN-β-specific neutralizing Ab, we show that MX1 expression is inhibited in a dose-dependent manner, suggesting that IFN-β is the primary driver of IFN-stimulated genes following TLR3 and TLR4 engagement. Stimulation with TLR7/8 and TLR9 ligands induced IFNB1 and IFNA subtypes and MX1 expression only in PBMCs and not in tissue resident cell types. Concordantly, IFN-β neutralization had no effect on MX1 expression in PBMCs potentially because of the combination of IFNB1 and IFNA expression. Combined, these data highlight the potential role for IFN-β in driving local inflammatory responses in clinically relevant human tissue types and opportunities to treat local inflammation by targeting IFN-β.

Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma

Genome-wide association studies (GWAS) have identified ~20 melanoma susceptibility loci, most of which are not functionally characterized. Here we report an approach integrating massively-parallel reporter assays (MPRA) with cell-type-specific epigenome and expression quantitative trait loci (eQTL) to identify susceptibility genes/variants from multiple GWAS loci. From 832 high-LD variants, we identify 39 candidate functional variants from 14 loci displaying allelic transcriptional activity, a subset of which corroborates four colocalizing melanocyte cis-eQTL genes. Among these, we further characterize the locus encompassing the HIV-1 restriction gene, MX2 (Chr21q22.3), and validate a functional intronic variant, rs398206. rs398206 mediates the binding of the transcription factor, YY1, to increase MX2 levels, consistent with the cis-eQTL of MX2 in primary human melanocytes. Melanocyte-specific expression of human MX2 in a zebrafish model demonstrates accelerated melanoma formation in a BRAFV600E background. Our integrative approach streamlines GWAS follow-up studies and highlights a pleiotropic function of MX2 in melanoma susceptibility.

OP0048 TYPE I INTERFERON EXPRESSION IDENTIFIES DIFFERENT SUBSETS OF ANTIPHOSPHOLIPID SYNDROME

Background: Type I Interferons (IFN) play a key role in the pathogenesis and development of various autoimmune conditions. Among them, a solid amount of data demonstrates that the expression of various IFN regulated genes (IRGs), the so-called “IFN signature”, has been linked to disease activity and disease progression in systemic lupus erythematosus (SLE) patients. Nevertheless, to date, a limited number of studies have analysed the IFN signature in antiphospholipid syndrome (APS) setting. Objectives: This study aims to describe the activation and structure of the type I IFN signature among different subsets of APS, including primary APS (PAPS) and when associated with other autoimmune conditions (secondary APS - SAPS), and antiphospholipid antibodies positive individuals (“aPL carriers”). Methods: A total of 116 patients were enrolled, including 19 PAPS patients, 13 SAPS, 75 SLE patients, and 9 aPL carriers [1,2]. Thirty-two subjects were also recruited as healthy controls (HCs). IFI44, IFI44L, IFI6, MX1 and IRF4 gene expression was determined in whole blood in the entire cohort. Expression levels were normalized to Z-scores and averaged into a global IFN signature. Differences were measured by Kruskal-Wallis tests and associations among genes were studied by cluster and correspondence analyses. Correlations were plotted by network analyses. Results: A global activation of the type I IFN signature was observed (HCs: -0.44±0.08, aPL carriers: -0.38±0.12, PAPS: -0.31±0.80, SAPS: -0.17±0.39, SLE: 0.09±0.80; p(Kruskal-Wallis) 0.050). No associations were observed with traditional cardiovascular risk factors or current treatments (all p >0.050). Conclusion: In line with other autoimmune conditions, APS is associated with a broad type I IFN activation, with distinct profiles of the IFN signature structure among different clinical subsets, suggesting that different pathogenic pathways are involved in the pathogenesis of these conditions.

Proteomics and transcriptomics study reveals the utility of ISGs as novel molecules for early pregnancy diagnosis in dairy cows

An early and precise diagnosis of pregnancy in cows is critical to short the calving interval and to improve their reproductive efficiency. Neutrophils are the first blood cells to sensitize the embryo in the uterus and participate in maternal recognition of pregnancy after getting induced by interferon tau (IFNτ). To study the protein abundance ratio, blood samples were collected on 0th, 10th, 18th and 36th day post-artificial insemination (AI) from crossbred Karan Fries cows. Neutrophils were isolated through density gradient centrifugation and studied for protein abundance by high-performance liquid chromatography coupled with mass spectrometry (LC–MS). Protein abundance ratios for Myxovirus resistance (MX1 and MX2) were found to be higher (P < 0.05) on day 10 and day 18 post-AI, whereas Oligoadenylate synthetase-1 (OAS1) and Interferon stimulated gene-15 ubiquitin-like modifier (ISG15) proteins were more abundant on day 18 post-AI. The relative mRNA expressions of these molecules were also studied by qPCR. The gene expression of ISG15, MX1, MX2 and OAS1 was found to be higher (P < 0.05) on day 10th, 18th and 36th post-AI compared to day 0. The study indicates that ISGs on blood neutrophils are essential for the establishment of pregnancy and may be targeted as potential biomarkers for pregnancy diagnosis in cows.

Efficient Selection of New Immunobiotic Strains With Antiviral Effects in Local and Distal Mucosal Sites by Using Porcine Intestinal Epitheliocytes

Previously, we evaluated the effect of the immunobiotic strain Lactobacillus rhamnosus CRL1505 on the transcriptomic response of porcine intestinal epithelial (PIE) cells triggered by the challenge with the Toll-like receptor 3 (TLR-3) agonist poly(I:C) and successfully identified a group of genes that can be used as prospective biomarkers for the screening of new antiviral immunobiotics. In this work, several strains of lactobacilli were evaluated according to their ability to modulate the expression of IFNα, IFNβ, RIG1, TLR3, OAS1, RNASEL, MX2, A20, CXCL5, CCL4, IL-15, SELL, SELE, EPCAM, PTGS2, PTEGES, and PTGER4 in PIE cells after the stimulation with poly(I:C). Comparative analysis of transcripts variations revealed that one of the studied bacteria, Lactobacillus plantarum MPL16, clustered together with the CRL1505 strain, indicating a similar immunomodulatory potential. Two sets of in vivo experiments in Balb/c mice were performed to evaluate L. plantarum MPL16 immunomodulatory activities. Orally administered MPL16 prior intraperitoneal injection of poly(I:C) significantly reduced the levels of the proinflammatory mediators tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-15 in the intestinal mucosa. In addition, orally administered L. plantarum MPL16 prior nasal stimulation with poly(I:C) or respiratory syncytial virus infection significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced levels of the proinflammatory mediators TNF-α, IL-6, and IL-8 were found in MPL16-treated mice. Improved levels of IFN-β and IFN-γ in the respiratory mucosa were observed in mice treated with L. plantarum MPL16 when compared to control mice. The immunological changes induced by L. plantarum MPL16 were not different from those previously reported for the CRL1505 strain in in vitro and in vivo studies. The results of this work confirm that new immunobiotic strains with the ability of stimulating both local and distal antiviral immune responses can be efficiently selected by evaluating the expression of biomarkers in PIE cells.

Transcriptomic Analysis of the Chicken MDA5 Response Genes.

RIG-I and MDA5 are two key pattern recognition receptors that sense RNA virus invasion, but RIG-I is absent in chickens. Although chickens have intact MDA5, the genes downstream of chicken MDA5 (chMDA5) that may mediate antiviral response are not well studied. We compared the transcriptional profile of chicken embryonic fibroblasts (DF1) transfected with chMDA5, and poly(I:C), using RNA-seq. Transfected chMDA5 and poly(I:C) in DF1 cells were associated with the marked induction of many antiviral innate immune genes compared with control. Interestingly, nine interferon-stimulated genes (ISGs) were listed in the top 15 upregulated genes by chMDA5 and poly(I:C) transfection. We used real-time PCR to confirm the upregulation of the nine ISGs, namely, MX1, IFI6, IFIT5, RSAD2, OASL, CMPK2, HELZ2, EPSTI1, and OLFML1, by chMDA5 and poly(I:C) transfection in DF1 cells. However, avian influenza virus H5N6 infection only increased MX1, IFI6, IFIT5, RSAD2, and OASL expression levels. Further study showed that the overexpression of these five genes could significantly inhibit H5N6 virus replication. These results provide some insights into the gene expression pattern induced by chMDA5, which would be beneficial for understanding and identifying innate immune genes of chicken that may lead to new antiviral therapies.