Type I and III interferons differentially encode inflammatory gene activation through threshold control of ISGF3 dynamics

Type I and type III interferons (IFNs) activate the shared Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway but generate distinct inflammatory transcriptional responses in addition to their shared antiviral transcriptional induction. While some mechanisms including cytokine-receptor affinity and receptor abundance have been proposed for regulating type I vs type III IFN specificity, they account for differences in signal amplitude but do not fully explain IFN-specific changes in JAK-STAT activity over time (i.e. the signaling temporal dynamics) which has been shown to be predictive of inflammatory gene activation. Here, we investigated mechanisms that could explain how type I and III IFNs produce distinct temporal dynamics of the IFN-activated transcription factor IFN-stimulated gene factor 3 (ISGF3), leading to IFN-type specific activation of inflammatory genes in lung epithelial cells. We show that IFN-{beta}, a type I IFN, and IFN-{lambda}3, a type III IFN, induced comparable initial ISGF3 activation but subsequently generated distinct signaling trajectories, with IFN-{beta} undergoing potentiation and IFN-{lambda}3 limiting the duration of high ISGF3 activity, preventing robust inflammatory gene induction despite prolonged lower-level activity. Inflammatory gene induction was associated with the magnitude and duration of ISGF3 activity above an activation threshold during the intermediate phase of the signal response. The characteristic IFN-{lambda}3-induced ISGF3 signaling dynamics were maintained at saturating doses of IFN-{lambda}3 indicating the lack of signal activation above the threshold was independent of the dose. Additionally, differences in the abundance of the ISGF3 complex subunits STAT1, STAT2, and interferon regulatory factor 9 (IRF9), or in the induction of known negative regulators did not readily explain the response. Blocking protein synthesis caused rapid loss of IFN-{lambda}3-induced STAT phosphorylation and ISGF3 activity, supporting the presence of a pre-existing negative regulator limiting IFN-{lambda}3-mediated JAK-STAT pathway activation above the threshold required for activating inflammatory genes, while IFN-{beta} and IFN-{lambda}3 co-stimulation localized this regulation upstream of the shared ISGF3 complex. Increasing pathway competence through IFN-{beta} priming enhanced the IFN-{lambda}3-induced ISGF3 response above the inflammatory gene activation threshold leading to induction of inflammatory genes, albeit transiently. Together, these findings support a model in which the magnitude and duration of ISGF3 activity above an inflammatory gene activation threshold, rather than pathway activation alone, contribute to stimulus-specific transcriptional responses and suggest that a negative regulatory mechanism upstream of the ISGF3 complex constrains the temporal dynamics of IFN-{lambda}3 signaling.