Major Differences in the Responses of Primary Human Leukocyte Subsets to IFN-

Department of Molecular Genetics, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
The Journal of Immunology (Impact Factor: 4.92). 10/2010; 185(10):5888-99. DOI: 10.4049/jimmunol.0902314
Source: PubMed


Treatment of cell lines with type I IFNs activates the formation of IFN-stimulated gene factor 3 (STAT1/STAT2/IFN regulatory factor-9), which induces the expression of many genes. To study this response in primary cells, we treated fresh human blood with IFN-β and used flow cytometry to analyze phosphorylated STAT1, STAT3, and STAT5 in CD4(+) and CD8(+) T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes and CD4(+) and CD8(+) T cells, few B cells activated STAT1 in response to IFN-β, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of suppressor of cytokine signaling 1 or relevant protein tyrosine phosphatases in B cells. Microarray and real-time PCR analyses revealed the induction of STAT1-dependent proapoptotic mRNAs in monocytes but not in B cells. These data show that IFN-stimulated gene factor 3 or STAT1 homodimers are not the main activators of gene expression in primary B cells of healthy humans. Notably, in B cells and, especially in CD4(+) T cells, IFN-β activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-β increases the survival of primary human B cells and CD4(+) T cells but enhances the apoptosis of monocytes, as well as to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections and by type I IFN treatment of patients with multiple sclerosis, hepatitis, or cancer.

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Available from: Anette Van Boxel-Dezaire
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    • "Previous studies described a role for IFN-β in B cell maturation [18], and in modulation of the TLR7 signaling pathway, a pathway involved in viral response and autoimmunity [19]–[21]. IFN-β was reported to increase transcription of the pro-survival genes encoding the B cell activating factor (TNFSF13B/BAFF), STAT3, and STAT5 [15], [22], but also to suppress release of stimulatory cytokines such as IL-1β and IL23 [23]. In all, these studies focused on a few specific and known pathways, which suggest a complex manner in which IFN-β affects B cells. "
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    ABSTRACT: The effects of interferon-beta (IFN-β), one of the key immunotherapies used in multiple sclerosis (MS), on peripheral blood leukocytes and T cells have been extensively studied. B cells are a less abundant leukocyte type, and accordingly less is known about the B cell-specific response to IFN-β. To identify gene expression changes and pathways induced by IFN-β in B cells, we studied the in vitro response of human Epstein Barr-transformed B cells (lymphoblast cell lines-LCLs), and validated our results in primary B cells. LCLs were derived from an MS patient repository. Whole genome expression analysis identified 115 genes that were more than two-fold differentially up-regulated following IFN-β exposure, with over 50 previously unrecognized as IFN-β response genes. Pathways analysis demonstrated that IFN-β affected LCLs in a similar manner to other cell types by activating known IFN-β canonical pathways. Additionally, IFN-β increased the expression of innate immune response genes, while down-regulating many B cell receptor pathway genes and genes involved in adaptive immune responses. Novel response genes identified herein, NEXN, DDX60L, IGFBP4, and HAPLN3, B cell receptor pathway genes, CD79B and SYK, and lymphocyte activation genes, LAG3 and IL27RA, were validated as IFN-β response genes in primary B cells. In this study new IFN-β response genes were identified in B cells, with possible implications to B cell-specific functions. The study's results emphasize the applicability of LCLs for studies of human B cell drug response. The usage of LCLs from patient-based repositories may facilitate future studies of drug response in MS and other immune-mediated disorders with a B cell component.
    Full-text · Article · Jul 2014 · PLoS ONE
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    • "ISG15, Mx1, OAS1, IRF7). 2) The GAF complex activates genes containing a GAS binding site in their promoter, such as SOCS1 and IRF1 [3,4]. 3) A third class of STAT protein complexes activates other canonical pathways that exhibit crosstalk with the JAK/STAT pathway (such as PI3K, NFkB, MAPK) [5]. "
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    ABSTRACT: Interferon-beta (IFN-beta) activates the immune response through the type I IFN signaling pathway. IFN-beta is important in the response to pathogen infections and is used as a therapy for Multiple Sclerosis. The mechanisms of self-regulation and control of this pathway allow precise and environment-dependent response of the cells in different conditions. Here we analyzed type I IFN signaling in response to IFN-beta in the macrophage cell line RAW 264.7 by RT-PCR, ELISA and xMAP assays. The experimental results were interpreted by means of a theoretical model of the pathway. Phosphorylation of the STAT1 protein (pSTAT1) and mRNA levels of the pSTAT1 inhibitor SOCS1 displayed an attenuated oscillatory behavior after IFN-beta activation. In turn, mRNA levels of the interferon regulatory factor IRF1 grew rapidly in the first 50--90 minutes after stimulation until a maximum value, and started to decrease slowly around 200--250 min. The analysis of our kinetic model identified a significant role of the negative feedback from SOCS1 in driving the observed damped oscillatory dynamics, and of the positive feedback from IRF1 in increasing STAT1 basal levels. Our study shows that the system works as a biological damped relaxation oscillator based on a phosphorylation-dephosphorylation network centered on STAT1. Moreover, a bifurcation analysis identified translocation of pSTAT1 dimers to the nucleus as a critical step for regulating the dynamics of type I IFN pathway in the first steps, which may be important in defining the response to IFN-beta therapy CONCLUSIONS: The immunomodulatory effect of IFN-beta signaling in macrophages takes the form of transient oscillatory dynamics of the JAK-STAT pathway, whose specific relaxation properties determine the lifetime of the cellular response to the cytokine.
    Full-text · Article · Jul 2013 · BMC Systems Biology
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    • "IFNb has also been demonstrated to induce p53 signaling [22], [23]. Interestingly, a recent study by van Boxel-Dezaire et al. [24] pointed out to monocytes as the specific cell subset going into apoptosis after IFNb stimulation via activation of both STAT1 and STAT3. In our study, the transcription factors STAT1, p53 and DDIT3 (see Table S1) were all up-regulated in monocytes from non-responders, findings that, altogether, suggest increased apoptosis in monocytes from IFNb non-responders, most likely owing to mitochondrial depolarization. "
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    ABSTRACT: A relatively large proportion of relapsing-remitting multiple sclerosis (RRMS) patients do not respond to interferon-beta (IFNb) treatment. In previous studies with peripheral blood mononuclear cells (PBMC), we identified a subgroup of IFNb non-responders that was characterized by a baseline over-expression of type I IFN inducible genes. Additional mechanistic experiments carried out in IFNb non-responders suggested a selective alteration of the type I IFN signaling pathway in the population of blood monocytes. Here, we aimed (i) to investigate whether the type I IFN signaling pathway is up-regulated in isolated monocytes from IFNb non-responders at baseline; and (ii) to search for additional biological pathways in this cell population that may be implicated in the response to IFNb treatment. Twenty RRMS patients classified according to their clinical response to IFNb treatment and 10 healthy controls were included in the study. Monocytes were purified from PBMC obtained before treatment by cell sorting and the gene expression profiling was determined with oligonucleotide microarrays. Purified monocytes from IFNb non-responders were characterized by an over-expression of type I IFN responsive genes, which confirms the type I IFN signature in monocytes suggested from previous studies. Other relevant signaling pathways that were up-regulated in IFNb non-responders were related with the mitochondrial function and processes such as protein synthesis and antigen presentation, and together with the type I IFN signaling pathway, may also be playing roles in the response to IFNb.
    Full-text · Article · Apr 2013 · PLoS ONE
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