Tyrosine kinase 2 controls IL-1ß production at the translational level

Abstract

IL-1beta is an important proinflammatory cytokine with a major role in several inflammatory diseases. Expression of IL-1beta is tightly regulated at the level of transcription, mRNA stability, and proteolytic processing. In this study, we report that IL-1beta expression in response to LPS is also regulated at the translational level. LPS-induced IL-1beta protein levels in macrophages derived from murine bone marrow are markedly increased in the absence of tyrosine kinase 2 (Tyk2). Increased IL-1beta is found intra- and extracellularly, irrespective of the efficiency of IL-1beta processing. We show that the absence of Tyk2 results both in higher translational rates and in enhanced association of IL-1beta mRNA with polysomes. Induction and stability of IL-1beta mRNA are not affected by the lack of Tyk2. We show further that the Tyk2-dependent translational inhibition is mediated by autocrine/paracrine type I IFN signaling and requires signal transducer and activator of transcription 1. Enhanced IL-1beta production in Tyk2- and IFN receptor 1-deficient macrophages is also observed following Listeria monocytogenes infection. Taken together, the data describe a novel mechanism for the control of IL-1beta synthesis.

FIGURE 1

Pro–IL-1β protein expression is elevated in the absence of Tyk2. Macrophages were treated with LPS for the indicated times and whole cell lysates were subjected to 2D-DIGE (A, B) or Western blot analysis (C). A, Selected regions from Cy3 and Cy5 images converted to gray scale showing pro–IL-1β spot positions (indicated by arrows). B, Protein expression levels are given as fold ratios relative to untreated WT cells. Mean values ± SD of three biological replicates are shown. **p ≤ 0.01. C, Five microgram protein per lane were separated by 14%T SDS-PAGE and subjected to Western blot analysis. Membranes were probed with an anti–IL-1β Ab; protein loading was controlled by reprobing with an anti-panERK Ab.

FIGURE 2

IL-1β mRNA expression and stability is unaltered in Tyk2^–/– cells. Macrophages were treated with LPS for the times indicated and total RNA was subjected to RT-qPCR for IL-1β (A) or TNF-α (B). mRNA expression levels were calculated relative to untreated WT cells, with Ube2d2 as endogenous control. Mean values ± SE from at least three independent experiments are shown. No significant differences were found. Macrophages were treated with LPS for 4 h and act D was added for the indicated times (C–F). Total RNA was isolated and subjected to RT-qPCR analysis for IL-1β (C), TNF-α (D), NFκBia (E), and Ube2d2 (F). Mean values ± SD from three replicates derived from two independent experiments are depicted.

FIGURE 3

Extracellular protein levels of IL-1β are elevated in Tyk2^–/– cells. Macrophages were treated with LPS for the times indicated and cell culture supernatants collected. Extracellular protein concentrations of IL-1β (A) and TNF-α (B) were measured by ELISA. Mean values ± SD from three independent experiments are shown; **p ≤ 0.01. nd, not detectable.

FIGURE 4

Pro- and mature IL-1β, and intra- and extracellular protein levels of IL-1β are higher in the absence of Tyk2, independent of the presence of ATP. Macrophages were treated with LPS for 4 h with or without subsequent addition of ATP at the concentrations indicated for 30 min. A, Extracellular IL-1β concentrations were measured by ELISA. Mean values ± SD from three independent experiments are shown, **p ≤ 0.01. B, Proteins were precipitated from supernatants and 15 μl per lane were subjected to Western blot analysis by 15%T SDS-PAGE. C, Five microgram protein from whole cell lysates per lane were separated by 15%T SDS-PAGE. B and C, Membranes were probed with an anti–IL-1β Ab. Data are representative for three independent experiments. T, Tyk2^–/–; W,WT.

FIGURE 5

Effect of IFNAR1 and Stat1 deficiency on IL-1β production induced by LPS and Listeria monocytogenes infection. A and B, Macrophages were treated with LPS for the times indicated with or without subsequent addition of ATP for 30 min. C–E, Macrophages were infected with L. monocytogenes at an MOI of 10 for the indicated times. A, Proteins were precipitated from supernatants and 15 μl per lane separated by 15%T SDS-PAGE. B, Five microgram or (C) 10 μg protein from whole cell lysates per lane were separated by 15%T SDS-PAGE. A–C, Membranes were probed with an anti–IL-1β Ab. B and C, Protein loading of whole cell extracts was controlled by reprobing membranes with an anti-panERK Ab. D and E, Extracellular IL-1β and TNF-α concentrations were measured by ELISA. Mean values ± SD from three independent experiments are depicted. nd, not detectable; p* ≤ 0.05, **p ≤ 0.01 as compared with WT cells. Western blot results (A–C) are representative of at least two independent experiments. I, IFNAR1^–/–; MOI, multiplicity of infection; S, Stat1^–/–; T, Tyk2^–/–; W,WT.

FIGURE 6

Effect of exogenous IFN-β on IL-1β protein levels. Macrophages were pretreated with IFN-β for 1 h or left untreated and subsequently treated with LPS or left untreated for 24 h. A, 5 μg protein from whole cell lysates per lane were separated by 15%T SDS-PAGE. Membranes were probed with an anti–IL-1β Ab and protein loading controlled by reprobing membranes with an anti-panERK Ab. W, WT; T, Tyk2^–/–. B, Mean values of pro–IL-1β signal intensities from three independent experiments. C, IL-1β and (D) TNF-α were measured in cell supernatants by ELISA, mean values ± SD from three independent experiments are depicted.

FIGURE 7

Pro–IL-1β protein stability and synthesis in the presence or absence of Tyk2. A, Macrophages were treated with LPS for 4 h, 10 μg/ml CHX was added and cells were further cultivated for the times indicated. Five microgram protein from whole cell lysates per lane were separated by 15%T SDS-PAGE. Membranes were probed with an anti–IL-1β Ab, protein loading was controlled by reprobing with an anti-panERK Ab. B, Macrophages were pulse labeled with [³⁵S] methionine/cysteine and chased for the indicated times. IL-1β was immunoprecipitated from 400 μg whole cell extracts and detected by autoradiography. C and D, Quantifications of signal intensities from B for (C) the three different pulse-periods and (D) 1 h pulse and the different chase periods. Results are representative of two independent experiments. T, Tyk2^–/–; W, WT.

FIGURE 8

Polysome profile of IL-1β, TNF-α, TBP, and PAI2 mRNAs. Macrophages were treated with LPS for 4 h and cytoplasmic extracts separated in a continuous 15–40% sucrose gradient by ultracentrifugation. Fractions were manually collected from top to bottom and deproteinized and RNAwas extracted. A, Polysomal fractions were separated on 0.8% agarose gels. Representative results are shown for WT and Tyk2^–/– macrophages. B–E, Two fractions each were pooled and mRNA levels determined by RT-qPCR. Amounts of the mRNA per fraction for IL-1β (B), TNF-α (C), TBP (D), and PAI2 (E) are given as percentage of mRNA present in all fractions of each genotype. Mean values ± SD of three independent experiments are shown. *p ≤ 0.05; **p ≤ 0.01.

FIGURE 9

Effect of Tyk2 deficiency on IL-1β production in vivo. WT and Tyk2^–/– mice were injected with LPS (1 mg/20 g body weight) i.p. IL-1β was measured 4 h after challenge in (A) peritoneal lavages and (B) sera by ELISA. Mean values ± SE for (A) 16 or (B) 15 mice from three independent experiments are depicted. *p ≤ 0.05.