Interleukin-23 production in dendritic cells is negatively regulated by protein phosphatase 2A

Abstract

IL-12 and IL-23 are produced by activated antigen-presenting cells but the two induce distinct immune responses by promoting Th1 and Th17 cell differentiation, respectively. IL-23 is a heterodimeric cytokine consisting of two subunits: p40 that is shared with IL-12 and p19 unique to IL-23. In this study, we showed that the production of IL-23 but not IL-12 was negatively regulated by protein phosphatase 2A (PP2A) in dendritic cells (DC). PP2A inhibits IL-23 production by suppressing the expression of the IL-23p19 gene. Treating DC with okadaic acid that inhibits the PP2A activity or knocking down the catalytic subunit of PP2A with siRNA enhanced IL-23 but not IL-12 production. Unlike PP2A, MAP kinase phosphatase-1 or CYLD did not show an effect on IL-23 production supporting the specificity of PP2A. PP2A-mediated inhibition requires a newly made protein that is likely responsible for bringing PP2A and IKKbeta together upon LPS stimulation, which then results in the termination of IKK phosphorylation. Thus, our results uncovered an important role of the protein phosphatase in the regulation of IL-23 production and identified PP2A as a previously uncharacterized inhibitor of IL-23p19 expression in DC.

Fig. 1.

Distinct regulation of IL-23 expression in DC after LPS treatment. (A and B) DC prepared from C57BL/6 mice were stimulated with LPS (1 μg/mL) for the indicated time. IL-10, IL-6, IL-12p35, IL-12p40 (A), and IL-23p19 (B) mRNA levels were quantified by qRT-PCR (lines) and ELISA was performed to measure IL-10, IL-6, IL-12p70, IL-12p40 (A), and IL-23 (B) production in culture supernatants (bars). (C) DC from C57BL/6 and IL-10-deficient mice were stimulated with LPS for the indicated time. The amount of IL-23p19 mRNA was quantified by qRT-PCR. Values are presented as means ± SD.

Fig. 2.

Inhibition of IKK activity primarily accounts for IL-23p19 repression. (A and B) DC from C57BL/6 mice were pretreated with cycloheximide (10 μg/mL) or DMSO for 30 min and then stimulated with LPS (1 μg/mL) for the indicated time. IL-23p19, IL-12p35, and IL-12p40 mRNA levels were quantified by qRT-PCR (A) and P-TAK1, TAK1, P-Erk1/2, Erk1/2, P-p38, p38, P-JNK, JNK, P-IKKα/β, IKKβ, IκBα, and GAPDH were detected from the whole-cell lysates by immunoblot analysis (B). (C) Cells were pretreated with indicated inhibitors (10 μM) for 30 min, followed by LPS stimulation for 1 h. IL-23p19 mRNA level was determined by qRT-PCR.

Fig. 3.

IL-23p19 expression was not regulated by MKP-1 or CYLD. (A) DC from C57BL/6 and MKP-1-deficient mice were stimulated with LPS (1 μg/mL) for the indicated time. Whole cell lysates were used to detect P-p38, p38, P-IKKα/β, and IKKβ by immunoblotting. (B) Cells were stimulated for the indicated time and used to prepare mRNA and cell culture supernatants. qRT-PCR and ELISA were performed to measure levels of IL-23p19 and IL-12p35 mRNA and IL-23 and IL-12p70 proteins at 24 h, respectively. *, P < 0.05. (C) DC from C57BL/6 mice were stimulated as indicated in Fig. 2. Cells were lysed and IKKγ was immunoprecipitated. The ubiquitinated proteins were determined by immunoblot analysis with anti-ubiquitin (Ub). The amounts of immunoprecipitated proteins were determined by immunoblot analysis with anti-IKKγ. (D) DC from C57BL/6 and CYLD-deficient mice were stimulated with LPS (1 μg/mL) for the indicated time. mRNA levels of IL-23p19 and IL-12p35 were determined by qRT-PCR and IL-23 and IL-12p70 production was measured by ELISA.

Fig. 4.

Treatment of okadaic acid (OA), an inhibitor of PP2A, prevented the down-regulation of IL-23 but not IL-12 production. DC were pretreated with or without OA (100 nM) for 1 h and then stimulated with LPS (1 μg/mL) for the indicated time. (A) P-p38, p38, P-Erk1/2, Erk1/2, P-JNK, JNK, P-IKKα/β, and IKKβ were detected by immunoblotting. (B) IL-23p19, IL-12p35, and IL-12p40 mRNA levels were determined by qRT-PCR. (C) IL-23, IL-12p70, and IL-12p40 production at 24 h was measured by ELISA. (D) DC from BALB/c mice were treated with or without OA for 1 h and then stimulated with LPS for 24 h. DC were then pulsed with OVA peptide (5 μM) for 2 h, washed, and cocultured with CD4⁺ DO11.10 T cells. After 6 days, cells were restimulated with plate-bound anti-CD3 antibody for 48 h. ELISA was performed to detect IL-17A and IFN-γ production. *, P < 0.05; LPS versus LPS + OA treatment.

Fig. 5.

Knockdown of PP2A C with siRNA enhanced IL-23 production in DC. (A) siRNA targeted against PP2A C or control nontargeting siRNA was delivered into DC as described in Materials and Methods. Knockdown of PP2A C was determined by immunoblotting with anti-PP2A C antibody. Anti-GAPDH antibody was used as a control. (B) siRNA-delivered DC were stimulated with LPS (1 μg/mL) for 24 h. IL-23 and IL-12p70 production was measured by ELISA. *, P < 0.05. (C and D) PP1 C was knockdown as in A and the levels of IL-23 and IL-12p70 were measured as in B.

Fig. 6.

PP2A interaction with IKKβ requires new other protein synthesis. DC from C57BL/6 mice were pretreated with cycloheximide (10 μg/mL) or DMSO for 30 min and then stimulated with LPS (1 μg/mL) for the indicated time (A) or 1 h (B, C, and D). (A) PP2A A, PP2A B, PP2A C, GAPDH, and IκBα were detected from the whole-cell lysates by immunoblotting. (B) Cells were subjected to subcellular fractionation into nuclear and cytoplasmic fractions. The relative purity of each fraction was confirmed by immunoblotting with anti-CREB and anti-p38 antibodies. The subcellular localization of PP2A was determined by immunoblotting with anti-PP2A C antibody. (C) Phosphatase activity of PP2A was assessed as described in Materials and Methods (Left). PP2A subunits present in immunoprecipitation were detected by immunoblotting (Right). (D) Cells were treated as indicated and total cell lysates were subjected to immunoprecipitation with the anti-PP2A (Left) or the anti-IKKγ antibody (Right). The amounts of coprecipitated IKKβ were shown at top and the control immunoblots at bottom.

Fig. 7.

Down-regulation of IL-23p19 expression is due to IKK. (A) The diagram shows the experimental design. (B and C) DC from C57BL/6 mice were pretreated with or without cycloheximide (B) at 10 μg/mL for 30 min or OA (C) at 100 nM for 1 h. Cells were then stimulated with LPS (1 μg/mL). At 50 min after LPS stimulation, indicated inhibitor (10 μM) or DMSO was added to the cells (depicted by an arrow). IL-23p19 mRNA level was determined by qRT-PCR. Values are presented as means ± SD.