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. 2009 Oct 27;106(43):18327-32.
doi: 10.1073/pnas.0905815106. Epub 2009 Oct 7.

Negative regulation of MyD88-dependent signaling by IL-10 in dendritic cells

Jihoon Chang  1 , Steven L Kunkel, Cheong-Hee Chang
Affiliations

Negative regulation of MyD88-dependent signaling by IL-10 in dendritic cells

Jihoon Chang et al. Proc Natl Acad Sci U S A. .

Abstract

IL-10 produced by dendritic cells (DC) can limit or terminate ongoing inflammatory responses by inhibiting the proinflammatory cytokine production. Currently, the molecular mechanism by which IL-10 suppresses cytokine production is still ill-defined. In this study, we showed that IL-10 produced by DC dampens myeloid differentiation factor (MyD)88-dependent, but not MyD88-independent signaling. At the molecular level, IL-10 induces ubiquitination and subsequent protein degradation of MyD88-dependent signaling molecules, including IL-1 receptor-associated kinase 4 and TNF-receptor associated factor 6. Protein degradation by IL-10 was associated with decreased phosphorylation of p38, JNK, and IKK. All of these events were prevented by either blocking IL-10 receptor signaling or inhibiting proteasome degradation. IL-10 induced LPS hyporesponsiveness using the same mechanisms, i.e., ubiquitination and protein degradation. Thus, a previously undescribed regulatory mechanism by which IL-10-mediated protein degradation contributes to the inhibition of inflammatory cytokine production and endotoxin tolerance in DC.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
IL-10 inhibits the cytokine production in DC on stimulation with LPS and CpG, but not curdlan or PolyI:C. (A and B) DC prepared from C57BL/6 or Il10−/− mice were treated with 1 μg/mL LPS (A) or 100 μg/mL curdlan (B) alone or together with α-IL-10R or rat IgG1 (both at 15 μg/mL) for 24 h or as indicated. IL-1β and IL-6 levels were quantified by ELISA in culture supernatants. (C) DC prepared from C57BL/6 mice were treated with 1 μg/mL CpG or 100 μg/mL PolyI:C alone or together with α-IL-10R for the indicated time. IL-6 level was quantified by ELISA. (D) DC from C57BL/6 mice were treated with 100 μg/mL PolyI:C alone or together with IL-10 for the indicated time. IL-6 was measured by ELISA and IFN-β was quantified by qRT-PCR. Values are presented as means ± SD.
Fig. 2.
Fig. 2.
IL-10 induces degradation of MyD88-dependent signaling molecules and down-regulation of phosphorylation of p38, JNK, and IKK. DC prepared from C57BL/6 mice were treated with 1 μg/mL LPS + 15 μg/mL α-IL-10R or 1 μg/mL LPS + 15 μg/mL rat IgG1. At the indicated time, whole-cell lysates were prepared. (A) IRAK4, IRAK1, TRAF6, p38, and β-actin expression was analyzed by immunoblotting. (B) P-p38, p38, P-JNK, JNK, P-Erk1/2, Erk1/2, P-IKKα/β, and IKKβ were detected by immunoblotting. (C) DC from Myd88−/− mice were treated the same way as in A. IRAK4, p38, IκBα, P-p38, and P-IKKα/β were detected by immunoblotting. (D) DC from Myd88−/− mice were treated with 1 μg/mL of LPS or 1 μg/mL of LPS together with 10 ng/mL of IL-10. At the indicated time, whole-cell lysates were prepared and used to examine IRAK4, IRAK1, TRAF6, p38, and β-actin by immunoblotting.
Fig. 3.
Fig. 3.
IL-10 signaling leads to ubiquitination of IRAK1, IRAK4, and TRAF6. (A and B) DC from C57BL/6 mice were treated with 1 μg/mL LPS or 10 ng/mL IL-10 as indicated. Cells were lysed and TRAF6, IRAK1, IRAK4, or TAK1 was immunoprecipitated as described in Materials and Methods. Ubiquitinated proteins and corresponding total proteins were determined by immunoblotting using the anti-ubiquitin (Ub) antibody (Upper) and the antibody recognizing TRAF6, IRAK1, IRAK4, or TAK1 (Lower), respectively. (C) DC prepared from C57BL/6 or Myd88−/− mice were treated with IL-10 and subjected to immunoprecipitation followed by blotting as described in A. The asterisk indicates heavy chain. Of note, the molecular weights shown are different due to use of a different percentage of gels (12 or 8%). (D) DC from C57BL/6 mice were stimulated as indicated in Fig. 2. Immunoprecipitates were immunoblotted using the anti-Ub or the anti-IRAK4 antibody (Left). Cells were treated with LPS (1 μg/mL) for the indicated time and the IL-10 level was quantified by ELISA (Right).
Fig. 4.
Fig. 4.
Cytokine production was partially rescued by preventing IL-10-mediated protein degradation. DC were treated with LPS (1 μg/mL) alone or together with α-IL-10R (15 μg/mL), MG132 (0.5 μM), chloroquine (10 μM), or left untreated for 48 h. The antibody and inhibitors were added 4.5 h after LPS treatment. IRAK4 and β-actin expression was analyzed at by immunoblotting (A); and IL-1β, IL-12p40, and IL-6 levels were quantified by ELISA (B). *, P < 0.05; **, P < 0.01, LPS versus LPS with inhibitor treatment.
Fig. 5.
Fig. 5.
IL-10-mediated protein degradation contributes to endotoxin tolerance. (A) DC prepared from C57BL/6 mice were treated with LPS (0.1 μg/mL) alone or together with α-IL-10R (15 μg/mL), MG132 (0.5 μM), or chloroquine (10 μM), or left untreated for 48 h. After extensive washing, cells were restimulated with 1 μg/mL LPS for 24 h, and cytokines in the culture supernatants were determined by ELISA. (B) DC were treated as in A, except that cells were harvested 6 h after restimulation. RNA was prepared, and qRT-PCR was performed to assess the amount of IL-23p19, IL-12p35 and IL-1β, and IFN-β mRNA. (C) DC were prepared and restimulated with LPS for the indicated time. Total cell lysates were prepared and subjected to immunoblot analysis to determine IRAK4, IRAK1, and p38 expression. (D) DC from Il10−/− mice were stimulated with LPS (0.1 μg/mL) alone or together with IL-10 (10 ng/mL), MG132 (0.5 μM), or left untreated for 48 h. After extensive washing, cells were restimulated with 1 μg/mL LPS for 24 h, and IL-12p40 and IL-6 levels were quantified by ELISA. (E) DC from C57BL/6 mice were treated with CpG (1 μg/mL), curdlan (100 μg/mL), or left untreated for 48 h. Cells were then restimulated for 24 h with CpG (1 μg/mL) or curdlan (100 μg/mL). IL-12p40 and IL-6 in the culture supernatants were assayed by ELISA. *, P < 0.05; **, P < 0.01.
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