RNAseq_IRF1: An IRF1-dependent IFNβ relay propagates IFNγ priming across macrophages

Abstract

Macrophages are central regulators of inflammation, and their functional state is shaped by exposure to interferons (IFNs). IFNγ-priming, the pre-exposure of macrophages to IFNγ before a secondary stimulus, reinforces subsequent inflammatory and IFN responses, but whether its consequences extend beyond the directly exposed macrophage has remained unclear. Here, we show that IFNγ-priming boosts IFNβ production in response to subsequent Toll-like receptor 4 (TLR4) stimulation. This response is associated with IFNγ-induced interferon regulatory factor 1 (IRF1) expression and chromatin poising at type-I IFN and IFN-stimulated gene loci, accompanied by remodeling of active enhancers and promoters. TLR4 stimulation then converts this poised state into productive IRF1 binding. Using an IFN-stimulated gene factor 3 (ISGF3) reporter system, co-cultures, conditioned-medium transfer and blockade of the type-I IFN receptor, we demonstrate that IFNγ-primed macrophages secrete IRF1-dependent IFNβ capable of priming neighbouring, previously unexposed macrophages. IFNγ-priming therefore does not merely sensitize macrophages to type-I IFNs but drives their production and intercellular spread, reframing the functional unit of IFNγ-priming from the individual macrophage to the population, with implications for chronic inflammatory diseases featuring elevated IFNγ and type-I IFN signatures.

Description

This repository contains raw and processed RNAseq data generated to characterize transcriptional differences IFNγ-primed BLaER1 macrophages in presence of lipopolysaccharid, for wild-type and IRF1-/-.

Culture

Human B cell Leukemia C/EBPα Estrogen Receptor clone 1 (BLaER1) eGFP^-/- cells were a gift from the lab of Holger Heine (Research Center Borstel) and cultured in RPMI 1640 (Thermo Fisher) supplemented with 100 U/mL penicillin (Gibco), 100 µg/mL streptomycin (Gibco), 2 mM L-glutamine (Gibco), 1 mM sodium pyruvate (Thermo Fisher) and 10% fetal calf serum (FCS) (Gibco). Cells were cultured at 37°C, 5% CO2. To induce macrophage trans-differentiation, BLaER1 cells were cultured for six days in trans-differentiation medium, consisting of complete RPMI 1640 with 10 ng/mL IL-3 (PeproTech), 10 ng/mL M-CSF (Miltenyi) and 200 nM β-estradiol (Sigma Aldrich), as previously described(9). Fresh trans-differentiation medium was added on day three of the trans-differentiation. On day five, cells were lifted from the culture flask using TrypLE Express (Thermo Fisher), density normalized, and replated for experiment specific purposes on day six and onwards. Stimulations were done with 10  ng/mL LPS (InvivoGen). For IFNγ priming, trans-differen