IFN alpha-induced gene expression in human NK cells
ABSTRACT: NK cells are believed to contribute to the control of hepatitis C virus infection and pathogenesis of liver disease. Standard treatment of both acute and chronic hepatitis C is based on the administration of interferon alpha, however, the effects of type I interferons on human NK cells have not been studied in the context of hepatitis C. We therefore first performed a microarray screen for genes differentially regulated in human NK cells after stimulation of PBMC with recombinant interferon alpha-2b. One of the genes upregulated was TRAIL which was confirmed in vitro on the protein level. Keywords: Interferon alpha; human NK cells; stimulation for 6 hours; cells sorted after stimulation of whole PBMC Overall design: NK cells of PBMC of five healthy controls have been either isolated directly or after culturing for 6h in media containing 10% human AB serum supplemented with 1 and 100 ng/ml recombinant IFNa-2b. RNA of NK cells of the healthy controls was isolated and RNA was pooled for the hybridization.
INSTRUMENT(S): [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
Project description:NK cells are believed to contribute to the control of hepatitis C virus infection and pathogenesis of liver disease. Standard treatment of both acute and chronic hepatitis C is based on the administration of interferon alpha, however, the effects of type I interferons on human NK cells have not been studied in the context of hepatitis C. We therefore first performed a microarray screen for genes differentially regulated in human NK cells after stimulation of PBMC with recombinant interferon alpha-2b. One of the genes upregulated was TRAIL which was confirmed in vitro on the protein level. Keywords: Interferon alpha; human NK cells; stimulation for 6 hours; cells sorted after stimulation of whole PBMC NK cells of PBMC of five healthy controls have been either isolated directly or after culturing for 6h in media containing 10% human AB serum supplemented with 1 and 100 ng/ml recombinant IFNa-2b. RNA of NK cells of the healthy controls was isolated and RNA was pooled for the hybridization.
Project description:Approximately 50% of patients with chronic hepatitis C (CHC) have a sustained virologic response (SVR) to treatment with pegylated interferon (pegINF)-α and ribavirin. Non-response to treatment is associated with constitutively increased expression of IFN-stimulated genes (ISGs) in the liver. Treatment of patients with acute hepatitis C (AHC) is more effective, with SVR rates >90%. We investigated mechanisms of the different responses of patients with CHC and AHC to pegIFN-α therapy. We analyzed IFN signaling and ISG expression in liver samples from patients with acute hepatitis C (AHC), patients with chronic hepatitis (CHC), and individuals without hepatitis C (controls) using microarray, immunohistochemical, and protein analyses. Findings were compared with those from primary human hepatocytes stimulated with IFN-α or IFN-γ, as reference sets. Expression levels of 100s of genes, primarily those regulated by IFN-γ, were altered in liver samples from patients with AHC compared with controls. Expression of IFN-γ–stimulated genes was induced in liver samples from patients with AHC, whereas expression of IFN-α–stimulated genes was induced in samples from patients with CHC. In an expression analysis of negative regulators of IFN-α signaling, we did not observe differences in expression of SOCS1 or SOCS3 between liver samples from patients with AHC and those with CHC. However, USP18 (another negative regulator of IFN-α signaling), was upregulated in liver samples of patients with CHC that did not respond to therapy, but not in AHC. In conclusion, differences in expression of ISGs might account for the greater response of patients with AHC, compared to those with CHC, to treatment with pegINF-α and ribavirin. Specifically, USP18 is upregulated in liver samples of patients with CHC that do not respond to therapy, but not in patients with AHC. (Interferon-γ Stimulated Genes, but not USP18, are Expressed in Livers of Patients with Acute Hepatitis C; Dill MT, Makowska Z et al, Gastroenterology 2012 (in press)) Primary human hepatocytes from 2 donors were analyzed. From each donor there are 5 samples: untreated cells, cells treated with interferon alpha (1000 IU/ml) for 6 and 24 hours and cells treated with interferon gamma (1000 IU/ml) for 6 and 24 hours.
Project description:This dataset is part of the paper: Pegylated interferon-α regulates hepatic gene expression by transient activation of the Jak-STAT pathway; Dill MT et al; Journal of Clinical Investigation; in review Pegylated interferon-α (pegIFN-α) has replaced un-modified recombinant IFN-α for the treatment of chronic viral hepatitis because of its superior anti-viral efficacy that is generally attributed to improved pharmacokinetic properties. However, the pharmacodynamic effects of pegIFN-α in the liver have not been studied. We analyzed pegIFN-α induced signaling and gene regulation in paired liver biopsies obtained before treatment and during the first week after injection of pegIFN-α in 18 patients. Despite sustained high serum concentrations of pegIFN-α over the entire one-week dosing interval, IFN-α signaling through the Jak-STAT pathway occurs only during the first day. PegIFN-α induces hundreds of genes that can be classified into 4 clusters based on different temporal expression profiles. In all clusters, gene transcription is mainly driven by IFN stimulated gene factor 3 (ISGF3). IFN induced secondary transcription factors do not cause additional waves of gene expression. We could not confirm a role of un-phosphorylated STAT1 in prolonging IFN-α induced gene transcription. Collectively, our results reveal that the major effects of pegIFN-α in the liver are caused by an early and transient activation of ISGF3. Prolonging the serum half-life of IFN-α does not necessarily improve its pharmacodynamic properties. Paired liver biopsy samples were collected before and during the first week of pegylated interferon alpha treatment of 21 chronic hepatitis C patients. Total: 21 patients and 42 samples.
Project description:Interferon (IFN)-alpha causes high rates of depression and fatigue, and is used to investigate the impact of innate immune cytokines on brain and behavior. However, little is known about transcriptional profiles of circulating immune cells during chronic IFN-alpha administration. Accordingly, genome-wide transcriptional profiling was performed on peripheral blood mononuclear cells from 21 patients with chronic hepatitis C virus either awaiting IFN-alpha therapy (n=10) or after 12 weeks of IFN-alpha treatment (n=11). Significance analysis of microarray data identified 252 up-regulated gene transcripts, the majority of which were related to IFN-alpha/antiviral or innate-immune/inflammatory signaling. Of these upregulated genes, 2'-5'-oligoadenylate synthetase 2 (OAS2) was the only gene that was differentially expressed in patients that developed IFN-alpha-induced depression/fatigue, and correlated with depression and fatigue scores at 12 weeks of IFN-alpha administration. Promoter-based bioinformatic and cellular origin analyses revealed IFN-alpha-induced increases in genes bearing transcription factor binding motifs (TFBMs) related to myeloid differentiation, IFN-alpha signaling, API and CREB/ATF family of transcription pathways, with changes derived primarily from monocytes and plasmacytoid dendritic cells. Patients with high depression/fatigue scores demonstrated up-regulation of genes bearing TFBMs for myeloid differentiation, IFN-alpha and AP1 signaling, and down regulation of TFBMs for CREB/ATF-related transcription factors. Cellular origin analyses indicated a shift toward genes derived from CD8+T and NK cells in subjects with high depression/fatigue scores. These results reveal an antiviral and inflammatory transcriptional profile after 12 weeks IFN-alpha, accompanied by increased OAS2 expression, decreased CREB/ATF transcriptional control, and a shift from monocyte-derived genes to those of cytotoxic lymphocytes in IFN-alpha-induced depression/fatigue. Total RNA was isolated from the peripheral blood mononuclear cells (PBMC) obtained at 12 weeks from HCV patients treated with IFN-alpha plus ribavirin (n=11) and untreated HCV patients awaiting IFN-alpha/ribavirin therapy (control subjects, n=10).
Project description:IFN alpha mediated gene expression pattern. The effect of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. This analysis examined the effects of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. Magnetically sorted untouched CD8+CD45R0- T cells from three different donors were unstimulated or stimulated with IFNa2b or with anti-CD3/CD28 Beads alone or along with IFNa2b or IFNa5 for 48 hours. Individual mRNA samples were analyzed using HG-U133A 2.0 array gene chips. Keywords: Gene expression analysis after different stimulation Magnetically sorted untouched CD8+CD45R0-T cells from three different donors (named A, B and C) unstimulated or stimulated with IFN alpha 2b or with anti-CD3/CD28 beads alone or along with IFN alpha 2b or IFN alpha 5 for 48 hours.
Project description:Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFN-alpha) and ribavirin. It achieves a sustained viral clearance in only 50–60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFN-alpha. In patients with a rapid virological response to treatment, pegIFN-alpha induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFN-alpha did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway. Keywords: Comparison of human patient biopses before and after interferon treatment Overall design: Total of 78 Samples (before and after interferon treatement) are analyzed using Affymetrix Human U133 Plus 2.0 Array.
Project description:Targeting the expansion and survival of pathogenic memory immune cells is a promising therapy to prevent chronic autoimmune attacks. Interleukin-7 receptor α chain (IL-7Rα) genetic variation were repeatedly associated with susceptibility of several chronic autoimmune and inflammatory diseases. Interleukin-7 (IL-7) is as a critical survival factor for the maintenance of mature T lymphocytes and numerous studies in rodent suggest targeting the IL-7/IL-7Rα pathway holds promise for the treatment of autoimmune diseases or transplant rejection. In this study, we evaluated agonist and antagonist properties of different anti-IL7Rα mAbs recognizing different type of epitope. To determine if agonist/antagonist anti-IL7Rα mAbs could deliver effective agonist signals capable of modifying human T cells, we analyzed by RNA-based-next-generation sequencing (RNA-SEQ) the transcriptome of human PBMC incubated during 3,5 hours with a site-1 (IgG4 #1 or IgG1 #2) or site-1/2b (IgG4) mAbs in the presence or not of exogenous human IL-7 (5 ng/ml). Altogether, transcriptional analysis confirmed that, while site-1 and site-1/2b anti-human IL-7Rα mAbs shared similar antagonist properties, the two site-1 mAbs induced significant human PBMC transcriptional modification compatible with T-cell activation and inflammatory responses associated by the MAPK/ERK pathway. Overall design: Human PBMC from seven independant healthy volunteers were incubated during 3,5 hours with a site-1 (IgG4 #1 or IgG1 #2) or site-1/2b (IgG4) mAbs in the presence or not of exogenous human IL-7 (5 ng/ml).
Project description:Abstract: Background Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles. Results: We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons a, b, w and g, IL12 and TNFa; and (2) characterise the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes) to IFNg stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNg and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFa stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNg, and emergent properties associated with IFN-mediated activation of mixed cell populations. Conclusions: This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the detection and definition of host immune responses in a variety of disease settings. This SuperSeries is composed of the following subset Series: GSE16251: PBMC Cytokine Comparison GSE16252: PBMC IFNg Dose Reponse GSE16253: PBMC Cell Subset Stimulation Refer to individual Series
Project description:Human rhinoviruses (HRV) are usually innocuous viruses; however, they can trigger serious consequences in certain individuals, especially in the setting of deficient interferon (IFN) synthesis. Plasmacytoid dendritic cells (pDC) are key IFN producing cells, though we know little about the mechanisms by which pDC regulate HRV-induced immune responses. Herein we used gene expression microarrays to examine HRV-induced mRNA in blood mononuclear cells from healthy people, in combination with pDC depletion to assess whether observed expression patterns were pDC dependent. As expected, pDC depletion led to a major reduction in HRV-induced IFN-α release, and this was associated with profound differences in gene expression between intact PBMC and pDC depleted PBMC. pDC depletion led to major changes in upstream regulators, with 70-80% of the HRV activated genes appearing to be pDC dependent. PCR validation experiments confirmed changes seen in the microarrays, specifically the extent to which the following differentially expressed genes were highly pDC dependent: the transcription factor IRF7, both IL-27 chains (IL-27 and EBI3), the alpha chain of the IL-15 receptor (IL-15RA) and the IFN stimulated gene IFI27. IL-6, IFN-γ and IL-27 protein synthesis were also highly pDC dependent. Supplementing pDC-depleted cultures with either recombinant IFN-γ, IL-15, IL-27 or IL-6 was able to restore the IFN-α response, thereby compensating for the absence of pDC. Though pDC comprise only a minority population of migratory leukocytes, our findings highlight the extent to which these cells are able to exert a profound effect on the immune response to HRV. Overall design: PBMC samples from healthy people (n=11) were cultured in the presence (RV16) or absence (CTRL) of rhinovirus type 16. To investigate the role of plasmacytoid dendritic cells (pDC) in the response, pDC were depleted from cultures of these PBMC samples. Hence, each individual has four samples (PBMC-CTRL, PBMC-RV16, pDCdepleted-CTRL, pDCdepleted-RV16) for a total of 44 samples.