Comparison of the levels of expression of IFN mRNA in and levels of mRNA (Figure 4A)

Comparison of the levels of expression of IFN mRNA in and levels of mRNA (Figure 4A). in fresh media. Data are representative of three similar experiments.(0.65 MB TIF) pbio.1000361.s003.tif (639K) GUID:?2ECA23ED-7EDC-466E-9017-067CBFDAF510 Figure S4: Reconstitution of STAT1 in IFNAR1 knockout cells restores IFN-mediated upregulation of IFN response genes. MEFs, IFNAR1?/? MEFs, and C57/BL6 (B6) splenocytes. Histograms represent the mean and error bars the standard error of four independent experiments (* cells. However, expression of STAT1 was rescued by culturing fibroblasts suggesting they secreted a STAT1-inducing factor. The STAT1-inducing factor in fibroblast-conditioned media was IFN, as it was inhibited by antibodies to IFNAR1, or when IFN expression was knocked down in cells. IFNAR1?/? fibroblasts, which cannot respond to this priming, also expressed reduced levels of STAT1, which correlated with their poor responses to IFN. The lack of priming in IFNAR1?/? fibroblasts was compensated by over-expression of STAT1, which rescued molecular responses to IFN and restored the ability of IFN to induce protective anti-viral immunity. This study provides a comprehensive description of the molecular events involved in priming by type I IFN. Adding to the previous working model that proposed an interaction between type I and II IFN receptors, our work and that of others demonstrates that type I IFN primes AF-9 IFN-mediated immune responses by regulating expression of STAT1. This may also explain how type I IFN can additionally prime cells to respond to a range of other cytokines that use STAT1 (e.g., IL-6, M-CSF, IL-10) and suggests a potential mechanism for the changing levels of STAT1 expression observed during viral infection. Author Summary Cells of the immune system release interferons (IFNs) in response to pathogens or tumor cells; these proteins signal to other immune cells to initiate the body’s defense mechanisms. The two classes of IFNstypes I and IIhave different receptors and distinct effects on the cells; however, there is crosstalk between them. In particular, small quantities of type I IFN can prime cells to produce a robust response to type II IFN. In this paper, we provide evidence to explain the molecular basis of this crosstalk. We show that continuous expression of the transcriptional activator c-Jun is responsible for producing basal, priming levels of a type I IFN; this signals to immune cells with the type I IFN receptor (IFNAR1) to maintain expression of STAT1 inside these cells. STAT1 Azomycin (2-Nitroimidazole) is a key factor for immune cell responses to type II IFN. Thus, signaling by low levels of type I IFN primes the cells with sufficient STAT1 to respond robustly to a subsequent type II IFN signal. This work provides an alternative explanation of the priming phenomenon to a previous proposal that the ligand-bound type I receptor, IFNAR1, acts as a component of the type II IFN receptor. Introduction Although type I and type II interferons (IFNs) have distinct roles in immune responses, there is substantial overlap between the genes and cellular responses they regulate. It has been known for some time that many cells secrete small priming quantities of type I IFNs that facilitate more potent responses to Azomycin (2-Nitroimidazole) subsequent stimuli [1]C[3]. Moreover, cellular responses to CSF-1 or IFN can be affected by neutralizing type Azomycin (2-Nitroimidazole) I IFN antibodies or knockout of type I IFN-Receptors (IFNAR) [2],[4],[5]. Notably, the protective anti-viral effects of IFN were much less potent in fibroblasts which appeared to be caused by a lack of type I IFN priming [4],[5]. The Azomycin (2-Nitroimidazole) molecular events that underpin these priming events have not been fully characterized, although it has been proposed that type I and II IFNs shared receptor components [5]. However, as the majority of responses to type I and II IFNs require the expression of the STAT1 transcription factor [6], this is also a possible point of crosstalk between them. STAT1 is a key mediator of cytokine-induced gene expression as it is Azomycin (2-Nitroimidazole) activated either as homo- or heterodimer with other STATs by many cytokines including type I and type II IFNs, interleukin (IL)-6 and IL-10. STAT1 activity is of particular importance to the IFN system as STAT1?/? mice display many similar phenotypes to mice lacking IFNAR1 or the IFN Receptor (IFNGR)1. In particular, anti-viral, anti-mycobacterial, and anti-tumor responses are compromised [6]C[9]. Induction of STAT1 expression is a potential explanation for the priming activity of type I IFN because it is an IFN-stimulated gene (ISG) itself [10]C[12] and.