Interleukin 1 receptor signaling regulates DUBA expression and facilitates Toll-like receptor 9-driven antiinflammatory cytokine production

Abstract

The interleukin 1 receptor (IL-1R) and the Toll-like receptors (TLRs) are highly homologous innate immune receptors that provide the first line of defense against infection. We show that IL-1R type I (IL-1RI) is essential for TLR9-dependent activation of tumor necrosis factor receptor-associated factor 3 (TRAF3) and for production of the antiinflammatory cytokines IL-10 and type I interferon (IFN). Noncanonical K63-linked ubiquitination of TRAF3, which is essential for type I IFN and IL-10 production, was impaired in Il1r1(-/-) CD11c(+) dendritic cells. In contrast, degradative ubiquitination of TRAF3 was not affected in the absence of IL-1R1 signaling. Deubiquitinating enzyme A (DUBA), which selectively cleaves K63-linked ubiquitin chains from TRAF3, was up-regulated in the absence of IL-1R1 signaling. DUBA short interference RNA augmented the TLR9-dependent type I IFN response. Mice deficient in IL-1RI signaling showed reduced expression of IL-10 and type I IFN and increased susceptibility to dextran sulphate sodium-induced colitis and failed to mount a protective type I IFN response after TLR9 ligand (CpG) administration. Our data identifies a new molecular pathway by which IL-1 signaling attenuates TLR9-mediated proinflammatory responses.

Figure 1.

Il1r1^−/− mice are more susceptible to DSS-induced colitis than WT mice. (A) DAI score in WT and Il1r1^−/− mice. Mice were given DSS (2%) in their drinking water for 7 d with or without pretreatment with CpG oligonucleotides (10 µg/mouse) 2 h before DSS administration. (B) Survival of WT and Il1r1^−/− mice treated as described in A. (C) Hematoxylin and eosin staining of colon sections from untreated mice or WT and Il1r1^−/− on day 7 of DSS treatment. Bar, 50 µm. (D) Quantitative PCR analysis of pro- and antiinflammatory mediators in colonic homogenates from WT and Il1r1^−/− mice on day 7 of DSS treatment. (A–D) Data are representative of four different experiments (n = 6). Error bars represent mean ± SEM. ns, not significant. *, P < 0.05; **, P < 0.01.

Figure 2.

Il1r1^−/− mice have impaired IL-10 and type I IFN response. (A) Cytokine levels in serum of WT and Il1r1^−/− mice 2 h after i.v. administration of 50 µg CpG oligonucleotides or vehicle (C). Data are representative of two independent experiments (n = 4). (B) Cytokine levels in supernatants from WT and Il1r1^−/− CD11c⁺ BMDCs 24 h after stimulation with 10 µg/ml CpG, 10 µg/ml Poly(I:C), or vehicle (C). (C) WT BMDCs were incubated with isotype control antibody, αIL-1RI blocking antibodies, or αIL-1β neutralizing antibody for 2 h. Cells were then stimulated with CpG for 24 h and cytokine levels were determined in the supernatants. (D) Effect of IL-1β prestimulation on cytokine production. WT BMDCs were cultured in the presence of 0, 1, or 10 ng/ml of recombinant IL-1β for 12 h. Cells were then washed and restimulated or not with 10 µg/ml CpG for 24 h, and the levels of cytokines were determined in the supernatants. (B–D) Data are representative of three different experiments. Error bars represent mean ± SEM. ns, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

Il1r1^−/− BMDCs display impaired IRF3 and IRF7 responses. (A) Electrophoretic mobility shift assay (EMSA) analysis of NF-κB activation in WT and Il1r1^−/− BMDCs after CpG stimulation (10 µg/ml) for the indicated time periods. (B) BMDCs from WT and Il1r^−/− mice were stimulated with 10 µg/ml CpG for 3 h, followed by RNA isolation and quantitative PCR analysis using GAPDH expression as internal reference. (C) Immunoblot analysis of MAPK activation in BMDCs from WT and Il1r1^−/− stimulated with 10 µg/ml CpG oligonucleotides for the indicated time periods. (D) Immunoblot analysis of IRF3 and IRF7 in nuclear extracts of BMDCs from WT and Il1r1^−/− mice stimulated as in C. (E) Baseline levels of IRF3 and IRF7 in nuclear extracts from freshly isolated unstimulated splenocytes from WT and Il1r1^−/− mice. (F) WT BMDCs were left unstimulated (0) or were stimulated with 10 ng/ml of recombinant IL-1β, 10 µg/ml CpG, or both for the indicated time periods. Nuclear extracts were then isolated and IRF7 translocation was analyzed by immunoblotting. (A–F) Data are representative of three independent experiments. Error bars represent mean ± SEM. ns, not significant.

Figure 4.

IL-1RI signaling modulates the ubiquitination profile of TRAF3. (A) Immunoblot analysis of TRAF3 in total cell lysates from WT and Il1r1^−/− BMDCs stimulated with 10 µg/ml CpG for the indicated time periods. Ratio expresses the densitometry analysis of the TRAF3 bands normalized to the expression of β-actin. (B) TRAF3 ubiquitination assay. WT and Il1r1^−/− BMDCs were treated with the proteasome inhibitor MG132 for 2 h and then stimulated with 10 µg/ml CpG for the indicated time periods. Overnight immunoprecipitation of TRAF3 was then followed by immunoblotting with anti–K48-linked ubiquitin (K48-Ub), anti–K63-linked ubiquitin (K63-Ub), or anti-TRAF3. (A and B) Data are representative of three different experiments.

Figure 5.

DUBA inhibits type I IFN production by Il1r1^−/− BMDCs. (A) Immunoblot analysis of DUBA in total cell lysates from WT and Il1r1^−/− BMDCs stimulated with 10 µg/ml CpG for the indicated time periods. (B) Immunoblot analysis of DUBA expression in total cell lysates from freshly isolated splenocytes from WT and Il1r1^−/− mice. (C) Quantitative PCR analysis of Duba mRNA expression in freshly isolated splenocytes and mesenteric LN-derived cells from WT and Il1r1^−/− mice. (D) Immunoblot analysis of DUBA expression in total cell lysates from Il1r^−/− BMDCs transfected with 0.5 µM of either control (Ctrl) or DUBA siRNAs. (E) Pro- and antiinflammatory cytokine production in Il1r1^−/− BMDCs after DUBA knockdown. Il1r1^−/− BMDCs were transfected as described in D. 24 h after transfection, cells were stimulated with 10 µg/ml CpG oligonucleotides for an additional 24 h and cytokine production was determined by ELISA. (A–E) Data are representative of three different experiments. Error bars represent mean ± SEM. *, P < 0.05; **, P < 0.01.