Добірка наукової літератури з теми "ISGs phenotype"

Objective To characterize the phenotype and function of fibroblasts derived from airway scar in idiopathic subglottic stenosis (iSGS) and to explore scar fibroblast response to interleukin 17A (IL-17A). Study Design Basic science. Setting Laboratory. Subjects and Methods Primary fibroblast cell lines from iSGS subjects, idiopathic pulmonary fibrosis subjects, and normal control airways were utilized for analysis. Protein, molecular, and flow cytometric techniques were applied in vitro to assess the phenotype and functional response of disease fibroblasts to IL-17A. Results Mechanistically, IL-17A drives iSGS scar fibroblast proliferation ( P < .01), synergizes with transforming growth factor ß1 to promote extracellular matrix production (collagen and fibronectin; P = .04), and directly stimulates scar fibroblasts to produce chemokines (chemokine ligand 2) and cytokines (IL-6 and granulocyte-macrophage colony-stimulating factor) critical to the recruitment and differentiation of myeloid cells ( P < .01). Glucocorticoids abrogated IL-17A-dependent iSGS scar fibroblast production of granulocyte-macrophage colony-stimulating factor ( P = .02). Conclusion IL-17A directly drives iSGS scar fibroblast proliferation, synergizes with transforming growth factor ß1 to promote extracellular matrix production, and amplifies local inflammatory signaling. Glucocorticoids appear to partially abrogate fibroblast-dependent inflammatory signaling. These results offer mechanistic support for future translational study of clinical reagents for manipulation of the IL-17A pathway in iSGS patients.

Abstract Placental immunity is dichotomous: tolerance of the semiallogenic fetus is balanced with limiting transmission of maternal pathogens. HCs are the major fetal immune cell at the placenta, but mechanisms responsible for maintaining immune homeostasis while preventing infection require elucidation. We determined the phenotype of human HCs throughout gestation and analyzed stimulation response. Activated HCs were present in early pregnancy and reduced in number by term while maintaining similar phenotypes. Tolerant HC numbers were highest in midgestation, after a relatively intolerant phenotype early in gestation. We saw a significant shift in macrophage polarization as gestation progressed. Transcription of ARG2 exceeded iNOS at all points, reaching 10-fold higher at term. Following treatment with IL4+IL13 or IFNγ+LPS midgestation HCs underwent significant phenotype change and activation. Basal expression of antiviral IFN stimulated genes (ISGs) was lowest at midgestation and was enhanced by IFN-α and IFN-λ1 with a 10-fold stronger response to IFN-α. RIG-I agonism induced HC activation, 10-fold iNOS upregulation and enhanced transcription of IFNs, MDA5, RIG-I, and ISGs. Response to stimulation by IFNγ+LPS, IL4+IL13, IL1β+HAGG was limited to loss of tolerance at term. IL-10 treatment increased numbers of CD163+ HCs, and IFNγ+LPS caused loss of discernible polarization patterns. Basal expression of RIG-I, MDA5 and ISGs was highest in term HCs, but IFN-α did not activate them. RIG-I agonism reduced markers of tolerance. HCs are variable macrophages, with phenotype and immune capacity strongly dependent on gestational age. Understanding placental immunobiology is fundamental to addressing key pregnancy morbidities.

Interferon (IFN) -stimulated genes (ISGs) are critical effectors of IFN response to viral infection, but whether ISG expression is a correlate of protection against HIV infection remains elusive. A well-characterized subcohort of Kenyan female sex workers, who, despite being repeatedly exposed to HIV-1 remain seronegative (HESN), exhibit reduced baseline systemic and mucosal immune activation. This study tested the hypothesis that regulation of ISGs in the cells of HESN potentiates a robust antiviral response against HIV. Transcriptional profile of a panel of ISGs with antiviral function in PBMC and isolated CD4+ T cells from HESN and non-HESN sex worker controls were defined following exogenous IFN-stimulation using relative RT-qPCR. This study identified a unique profile of proinflammatory and proapoptotic ISGs with robust but transient responses to exogenous IFN-γ and IFN-α2 in HESN cells. In contrast, the non-HESN cells had a strong and prolonged proinflammatory ISG profile at baseline and following IFN challenge. Potential mechanisms may include augmented bystander apoptosis due to increased TRAIL expression (16-fold), in non-HESN cells. The study also identified two negative regulators of ISG induction associated with the HESN phenotype. Robust upregulation of SOCS-1 and IRF-1, in addition to HDM2, could contribute to the strict regulation of proinflammatory and proapoptotic ISGs in HESN cells. As reducing IRF-1 in the non-HESN cells resulted in the identified HESN ISG profile, and decreased HIV susceptibility, the unique HESN ISG profile could be a correlate of protection against HIV infection.

Differences between female and male immunity may contribute to variations in response to infections and predisposition to autoimmunity. We previously reported that neutrophils from reproductive-age males are more immature and less activated than their female counterparts. To further characterize the mechanisms that drive differential neutrophil phenotypes, we performed RNA sequencing on circulating neutrophils from healthy adult females and males. Female neutrophils displayed significant up-regulation of type I IFN (IFN)-stimulated genes (ISGs). Single-cell RNA-sequencing analysis indicated that these differences are neutrophil specific, driven by a distinct neutrophil subset and related to maturation status. Neutrophil hyperresponsiveness to type I IFNs promoted enhanced responses to Toll-like receptor agonists. Neutrophils from young adult males had significantly increased mitochondrial metabolism compared to those from females and this was modulated by estradiol. Assessment of ISGs and neutrophil maturation genes in Klinefelter syndrome (47, XXY) males and in prepubescent children supported that differences in neutrophil phenotype between adult male and female neutrophils are hormonally driven and not explained by X chromosome gene dosage. Our results indicate that there are distinct sex differences in neutrophil biology related to responses to type I IFNs, immunometabolism, and maturation status that may have prominent functional and pathogenic implications.

Type I interferons (IFN-Is) are a group of potent inflammatory and antiviral cytokines. They induce IFN stimulated genes (ISGs), which act as proinflammatory mediators, antiviral effectors, and negative regulators of the IFN-I signaling cascade itself. One such regulator is interferon stimulated gene 15 (ISG15). Humans with complete ISG15 deficiency express persistently elevated levels of ISGs, and consequently, exhibit broad spectrum resistance to viral infection. Here, we demonstrate that IFN-I primed fibroblasts derived from ISG15-deficient individuals are more resistant to infection with single-cycle HIV-1 compared to healthy control fibroblasts. Complementation with both wild-type (WT) ISG15 and ISG15ΔGG (incapable of ISGylation while retaining negative regulation activity) was sufficient to reverse this phenotype, restoring susceptibility to infection to levels comparable to WT cells. Furthermore, CRISPR-edited ISG15ko primary CD4+ T cells were less susceptible to HIV-1 infection compared to cells treated with non-targeting controls. Transcriptome analysis of these CRISPR-edited ISG15ko primary CD4+ T cells recapitulated the ISG signatures of ISG15 deficient patients. Taken together, we document that the increased broad-spectrum viral resistance in ISG15-deficiency also extends to HIV-1 and is driven by a combination of T-cell-specific ISGs, with both known and unknown functions, predicted to target HIV-1 replication at multiple steps.

ABSTRACT Herpes simplex virus VP16 and ICP0 mutants replicate efficiently in U2OS osteosarcoma cells but are restricted in other cell types. We previously showed that the restrictive phenotype is dominant in a transient cell fusion assay, suggesting that U2OS cells lack an antiviral mechanism present in other cells. Recent data indicate that unscheduled membrane fusion events can activate the expression of interferon-stimulated genes (ISGs) in fibroblasts, raising the possibility that our earlier results were due to a fusion-induced antiviral state. However, we show here that the permissive phenotype is also extinguished following fusion with Vero cells in the absence of ISG induction.

Abstract The molecular mechanisms regulating the inflammatory response during malaria are still poorly defined. Inflammatory cytokines and type I IFNs induced when innate immune sensors recognize components of the malaria parasite can contribute to clearance of the parasite and in some circumstances these same effectors lead to experimental cerebral malaria (ECM). Infection of C57BL/6 mice with Plasmodium berghei ANKA (PbA) leads to ECM where animals succumb to infection and die. Upon infection with PbA infected red blood cells (iRBCs), C57/Bl6 mice succumb to death 6-12 days post-infection. Recently, we have found that mice lacking the type I IFN receptor, IFNAR-/-, are protected from ECM-mediated death, implicating an important role of type I IFNs in exacerbating the ECM phenotype. Alternatively, when mice are infected with liver-tropic PbA sporozoites, a type I IFN response is induced while the parasites develop inside hepatocytes. This host response is responsible for upregulating interferon stimulated genes (ISGs) and limit the parasite load in the liver. The expression of ISGs are abrogated in IFNAR-/- mice. This protective phenotype is dependent on IRF3/7 and the adaptor MAVS suggesting that parasite RNA is recognized by host cells. Collectively, these findings reveal a dual role of type I IFNs that contribute to ECM-mediated death during PbA blood stage infections, but are also responsible for reducing parasite load during a liver stage infection.

Abstract Background: IL28B gene polymorphisms associated with hepatitis C virus (HCV) clearance suggests a role for type III interferons (IFNs. However, the intrinsic innate immune response following HCV infection remains poorly understood. Methods: Immunofluorescence staining of innate immune molecules (IRF3, ISG15) were performed on HCV infected PHHs. PHHs were also studied for correlation of IL28 genotype with type III IFN and ISG expression. In addition, RNA was extracted from 60 pre-treatment archived liver biopsies and hepatic expression of IFNs was measured by qPCR. Rs12979860 genotype was determined in PHHs and patients. Results: Primary human hepatocytes infected with HCV, but not contaminating non-parenchymal cells, demonstrate activated nuclear IRF3, supporting a direct anti-viral response to HCV pathogen associated molecular pattern (PAMP). In the patient cohort, there was a significant correlation between the hepatic expression of type III IFNs (IL28B & IL29, r=0.60, P &lt;0.0001). ISGs expression was positively correlated with Type III IFNs (e.g. for ISG115 r=0.55, p&lt;0.001). Conclusions: Our study demonstrates that HCV infection results in a rapid and robust intrinsic innate response in the liver. Although non-parenchymal cells may subsequently participate in this response, it is the infected hepatocyte that signals the initial danger signal. Finally, the hepatic expression of ISGs in HCV patients is mainly associated with type III IFNs.

Most isolates of human rhinovirus, the common cold virus, replicate more robustly at the cool temperatures found in the nasal cavity (33–35 °C) than at core body temperature (37 °C). To gain insight into the mechanism of temperature-dependent growth, we compared the transcriptional response of primary mouse airway epithelial cells infected with rhinovirus at 33 °C vs. 37 °C. Mouse airway cells infected with mouse-adapted rhinovirus 1B exhibited a striking enrichment in expression of antiviral defense response genes at 37 °C relative to 33 °C, which correlated with significantly higher expression levels of type I and type III IFN genes and IFN-stimulated genes (ISGs) at 37 °C. Temperature-dependent IFN induction in response to rhinovirus was dependent on the MAVS protein, a key signaling adaptor of the RIG-I–like receptors (RLRs). Stimulation of primary airway cells with the synthetic RLR ligand poly I:C led to greater IFN induction at 37 °C relative to 33 °C at early time points poststimulation and to a sustained increase in the induction of ISGs at 37 °C relative to 33 °C. Recombinant type I IFN also stimulated more robust induction of ISGs at 37 °C than at 33 °C. Genetic deficiency of MAVS or the type I IFN receptor in infected airway cells permitted higher levels of viral replication, particularly at 37 °C, and partially rescued the temperature-dependent growth phenotype. These findings demonstrate that in mouse airway cells, rhinovirus replicates preferentially at nasal cavity temperature due, in part, to a less efficient antiviral defense response of infected cells at cool temperature.

Skeletal aging is associated with reduced proliferative potential of bone marrow (BM) multipotential stromal cells (MSCs). Recent data suggest the involvement of type 1 interferon (IFN1) signalling in hematopoietic stem cell (HSC) senescence. Considering that BM-HSCs and BM-MSCs share the same BM niche, we investigated IFN1 expression profile in human BM-MSCs in relation to donor age, culture-expansion and IFN1 (α and β) stimulation. Fluorescence-activated cell sorting was used to purify uncultured BM-MSCs from younger (19–41, n = 6) and older (59–89, n = 6) donors based on the CD45lowCD271+ phenotype, and hematopoietic-lineage cells (BM-HLCs, CD45+CD271−) were used as controls. Gene expression was analysed using integrated circuits arrays in sorted fractions as well as cultured/stimulated BM-MSCs and Y201/Y202 immortalised cell lines. IFN1 stimulation led to BM-MSC growth arrest and upregulation of many IFN1-stimulated genes (ISGs), with IFNβ demonstrating stronger effects. Uncultured MSCs were characterised by a moderate-level ISG expression similar to Y201 cells. Age-related changes in ISG expression were negligible in BM-MSCs compared to BM-HLCs, and intracellular reactive oxygen species (ROS) levels in BM-MSCs did not significantly correlate with donor age. Antiaging genes Klotho and SIRT6 correlated with more ISGs in BM-MSCs than in BM-HLCs. In patients with osteoarthritis (OA), BM-MSCs expressed considerably lower levels of several ISGs, indicating that their IFN1 signature is affected in a pathological condition. In summary, BM-MSCs possess homeostatic IFN1 gene expression signature in health, which is sensitive to in vitro culture and external IFN1 stimulation. IFN signalling may facilitate in vivo BM-MSC responses to DNA damage and combating senescence and aberrant immune activation.

Le terapie con virus oncolitici stanno diventando una possibile alternativa per tumori difficilmente trattabili con le terapie standard. Il nostro gruppo ha dimostrato l’esistenza in cellule neoplastiche di due possibili fenotipi distinguibili in base all’espressione di geni stimolati dagli interferoni (ISGs) e di geni indotti dallo stress virale (VSIG) che possono essere facilmente distinti utilizando la proteina MxA come marcatore. Abbiamo dimostrato che l’esistenza di questo fenotipo ISGs e’ capace di bloccare nelle cellule tumorali sia la lisi virale sia l’espressione genica di proteine inserite in vettori virali. Inoltre, abiamo pubblicato che questo fenotipo caratterizza cellule di tumore che costituiscono una recidiva di tumore ovarico che era stato inizialmente curato con terapie con virus oncolitici. Poiche’ questo fenomeno e’ condiviso da tumori solidi ed ematologici, capire il meccanismo che induce l’espressione di questa signature di geni associati agli interferoni e’ di importanza chiave nel cercare di ottimizzare terapie con virus oncolitici o trials di terapie geniche basate su vettori adenovirali. Per individuare le vie di segnalazione coinvolte nel sostenere questo fenotipo abbiamo utilizzato linee cellulari di adenocarcinoma di pancreas e di mieloma multiplo. L’adoncarcinoma di pancreas e’ uno dei tumori piu’ aggressivi per cui ancora ad oggi non esiste una cura. Invece il mieloma multiplo e’ fra i tumori piu’ diffusi e ci sono gia’ molti trials clinici che sperimentano possibili terapie alternative con virus oncolitici. La finalita’ del presente progetto e’ confermare l’esistenza del dualismo del fenotipo ISGs in tumori di diversa origine istologica, e di dissezionare i pathways che sono maggiormente coinvolti nella risposta agli interferoni, e che portano all’upregolaizone di geni ISGs e VSGs, andando di volta in volta a bloccare in modo selettivo. Abbiamo focalizzato la nostra attenzione al pathway di NFkB nell’adenocarcinoma di pancreas e in quello di STAT3 nel mieloma multiplo. Dai nostril risultati sembra che l’acido acetilsalicilico e la curcumina possono bloccare l’espressione endogena degli ISGs nei tumori di pancreas, ristabilendo la normale permissivita’ alle infezioni con adenovirus, mentre lo stesso effetto lo abbiamo ottenuto nelle line di mieloma multiplo utilizzando il resveratrolo. Questi risultati possono essere considerati come dati preliminari utilizabili nel disegno di terapie combinatorie cosi’ da aumentare l’efficacia di terapie oncolitiche o di terapie geniche mediate da vettori adenovirali che possono costituire il futuro terapeutico per molti tipi di tumori ancora incurabili.
Oncolytic viral therapy is becoming an interesting alternative to standard therapies for incurable diseases. Our group has demonstrated the existence of two distinguishable phenotypes based on the expression of interferon-stimulated genes (ISGs) and Virus Stress Induced Genes (VSIG) with Myxovirus-resistance-A (MxA) protein as a marker. The existence of this ISGs phenotype has been proved to block viral oncolysis and viral mediated gene expression in cancer cells. We demonstrated that this caused in vivo an acquisition of a viral resistant phenotype in recurrent tumors cured in the first place with adeno-mediated oncolytic therapies. This is a phenomenon shared in both, solid and hematological tumors as well. The understanding of the mechanism that induces the expression of this interferon-related signature became of a key importance in optimizing oncolytic and gene therapies. In order to discover the pathways involved in the tumor-acquired ISGs phenotype, we focused our attention on tumor cell lines, and in particular pancreatic cancer and multiple myeloma cancer cell lines in vitro. Pancreatic ductal adenocarcinoma represents one of the most vicious cancers, for which no effective cure is optimised. On the other hand, multiple myeloma is the most frequent hematological tumor in humans and was often used as a possible target tumor for virus based therapies. The main aim of the present work was to confirm the existence of the dualism of ISGs phenotype in different cancer models and dissect the most important interferon pathways by specifically silencing different steps that could be responsible for the downstream ISGs up-regulation. We focused our attention to NFkB pathway in PDAC cancer cells and STAT3 pathway in multiple myeloma. We conclude that ASA and curcumin treatement can effectively revert the resistance in PDAC cell lines, while only resveratrol treatment can affect the infectivity of multiple myeloma cell lines to adenoviral vectors. Those findings can be considered the rational for a combinatory therapy with the aim of increasing the efficacy of oncolytic or gene therapy adeno-based approaches that might constitute the future for curing incurable cancers as pancreatic ductal adenocarcinoma.

Drought stress is a major limitation to bread wheat (Triticumaestivum L.) productivity and its yield stability in arid and semi-arid regions of world including parts of Israel and the U.S. Currently, breeding for sustained yields under drought stress is totally dependent on the use of yield and several key physiological attributes as selection indices. The attempt to identify the optimal genotype by evaluating the phenotype is undermining progress in such breeding programs. Osmotic adjustment (OA) is an effective drought resistance mechanism in many crop plants. Evidence exists that there is a genetic variation for OA in wheat and that high OA capacity supports wheat yields under drought stress. The major objective of this research was to identify molecular markers (RFLPs, restriction fragment length polymorphisms; and AFLPs, amplified fragment length polymorph isms) linked to OA as a major attribute of drought resistance in wheat and thus to facilitate marker-assisted selection for drought resistance. We identified high and low OA lines of wheat and from their cross developed recombinant inbred lines (RILs) used in the molecular tagging of OA in relation to drought resistance in terms of plant production under stress. The significant positive co-segregation of OA, plant water status and yield under stress in this RIL population provided strong support for the important role of OA as a drought resistance mechanism sustaining wheat production under drought stress. This evidence was obtained in addition to the initial study of parental materials for constructing this RIL population, which also gave evidence for a strong correlation between OA and grain yield under stress. This research therefore provides conclusive evidence on the important role of OA in sustaining wheat yield under drought stress. The measurement of OA is difficult and the selection for drought resistance by the phenotypic expression of OA is practically impossible. This research provided information on the genetic basis of OA in wheat in relations to yield under stress. It provided the basic information to indicate that molecular marker assisted selection for OA in wheat is possible. The RIL population has been created by a cross between two agronomic spring wheat lines and the high OA recombinants in this population presented very high OA values, not commonly observed in wheat. These recombinants are therefore an immediate valuable genetic recourse for breeding well-adapted drought resistant wheat in Texas and Israel. We feel that this work taken as a whole eliminate the few previous speculated . doubts about the practical role of OA as an important mechanism of drought resistance in economic crop plants. As such it should open the way, in terms of both concept and the use of marker assisted selection, for improving drought resistance in wheat by deploying high osmotic adjustment.