Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Dec 20;207(13):2799-807.
doi: 10.1084/jem.20101326. Epub 2010 Nov 29.

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

Jose M González-Navajas  1 , Jason Law, Kim Phung Nguyen, Meha Bhargava, Mary Patricia Corr, Nissi Varki, Lars Eckmann, Hal M Hoffman, Jongdae Lee, Eyal Raz
Affiliations

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

Jose M González-Navajas et al. J Exp Med. .

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.

PubMed Disclaimer

Figures

Figure 1.
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.
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.
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.
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.
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abe K., Nguyen K.P., Fine S.D., Mo J.H., Shen C., Shenouda S., Corr M., Jung S., Lee J., Eckmann L., Raz E. 2007. Conventional dendritic cells regulate the outcome of colonic inflammation independently of T cells. Proc. Natl. Acad. Sci. USA. 104:17022–17027 10.1073/pnas.0708469104 - DOI - PMC - PubMed
    1. Arai Y., Takanashi H., Kitagawa H., Okayasu I. 1998. Involvement of interleukin-1 in the development of ulcerative colitis induced by dextran sulfate sodium in mice. Cytokine. 10:890–896 10.1006/cyto.1998.0355 - DOI - PubMed
    1. Bauer C., Duewell P., Mayer C., Lehr H.A., Fitzgerald K.A., Dauer M., Tschopp J., Endres S., Latz E., Schnurr M. 2010. Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome. Gut. 59:1192–1199 10.1136/gut.2009.197822 - DOI - PubMed
    1. Bresnihan B., Alvaro-Gracia J.M., Cobby M., Doherty M., Domljan Z., Emery P., Nuki G., Pavelka K., Rau R., Rozman B., et al. 1998. Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist. Arthritis Rheum. 41:2196–2204 10.1002/1529-0131(199812)41:12<2196::AID-ART15>3.0.CO;2-2 - DOI - PubMed
    1. Cominelli F., Nast C.C., Llerena R., Dinarello C.A., Zipser R.D. 1990. Interleukin 1 suppresses inflammation in rabbit colitis. Mediation by endogenous prostaglandins. J. Clin. Invest. 85:582–586 10.1172/JCI114476 - DOI - PMC - PubMed

Publication types

MeSH terms