Interleukin-23 production in dendritic cells is negatively regulated by ...

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May 4, 2010 - IL-12 and IL-23 are produced by activated antigen-presenting cells but ... the maintenance of a T cell subset producing IL-17 and has a major.
Interleukin-23 production in dendritic cells is negatively regulated by protein phosphatase 2A JiHoon Changa, Timothy J. Voorheesa, Yusen Liub, Yongge Zhaoc, and Cheong-Hee Changa,1 a Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109; bCenter for Perinatal Research, Research Institute at Nationwide Children’s Hospital, Department of Pediatrics, Ohio State University College of Medicine, Columbus, OH 43205; and cLaboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20817

Edited by Laurie H. Glimcher, Harvard University, Boston, MA, and approved March 26, 2010 (received for review December 18, 2009)

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 IKKβ 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. cytokine

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L-23 belongs to the IL-6/IL-12 family of cytokines. By searching for IL-6-like cytokines, IL-23 was discovered using a computational screen in the late 1990s (1, 2). One of the sequences, named p19, was distantly related to IL-12p35. p19 protein had no biological activity by itself, but it combined with IL-12p40 forming another cytokine, named IL-23, with biological activities similar as well as distinct from IL-12 (2). IL-23 is produced mainly by activated antigen-presenting cells (APC) including dendritic cells (DC). The activation of DC plays a pivotal role in shaping the immune responses. Following the detection of microbial products, for example via TLRs, activated DC can provide signals to prime naïve T cells to mount appropriate adaptive immune responses. It has long been recognized that IL-12 has a central role in the generation of Th1 cells. IL-23 also regulates Th1 responses by stimulating IFN (IFN)-γ, but it is now widely accepted that IL-23 has a unique role in the maintenance of a T cell subset producing IL-17 and has a major role in pathogenesis of autoimmune diseases (3). Given that IL-12 and IL-23 produced by APC are predominantly responsible for promoting Th1 and Th17 cells, respectively, it is essential to understand the pathways that lead to IL-12 and IL-23 production. Several studies have reported regulators of IL-23 versus IL-12 production. Commensal Gram-negative bacteria but not Gram-positive bacteria prime DC for enhanced IL-23 expression, suggesting that the expression of the different IL-12 family members could be dictated by different priming conditions of DC (4). In addition, prostaglandin E2 induced both the expression of IL-23p19 and IL-12p40 without affecting IL-12p35 expression in bone marrow-derived DC (5). Bone marrow-derived DC generated in the presence of prostaglandin E2 also strongly favored IL-23 production and promoted differentiation of Th17 cells (6). Recently, Gerosa and colleagues (7) provided additional insights into the differential regulation of IL-23 and IL-12 production in human monocytederived DC. It was shown that the selective engagement of different 8340–8345 | PNAS | May 4, 2010 | vol. 107 | no. 18

combinations of innate immune receptors led to striking differences in the expression of IL-23 and IL-12 (7). TLR signaling pathways rapidly activate NF-κB transcription factor, which plays a key role in inflammation and immune responses by regulating the expression of numerous inflammatory cytokine genes (8). The activation of NF-κB requires phosphorylation and subsequent degradation of IκB and is primarily mediated by two protein kinases, IκB kinase (IKK) α and β. IKKα and IKKβ form a complex with a regulatory component called IKKγ [also known as NF-κB essential modulator (NEMO)] (9). Interestingly, IKKα and IKKβ have distinct functions although they have the extensive sequence similarity. IKKβ but not IKKα was shown to be involved in rapid NF-κB activation triggered by LPS or TNF-α (9, 10). However, IKK activation is transient and subjected to many negative feedback regulations, most notably by deubiquitinases including CYLD (11). Deubiquitinase activity of CYLD was necessary for the inhibitory effect. CYLD likely acts at the level of IKK as CYLD was unable to deubiquitinate IκB (12). CYLD acts as a negative regulator of NF-κB signaling through its interaction with IKKγ and TNF receptor associated factor 2 (12, 13). The regulation of NF-κB signaling is also controlled by a series of kinases and phosphatases. However, the roles of phosphatases involved in the NF-κB pathway are still poorly understood. The serinethreonine protein phosphatase 2A (PP2A) is a heterotrimer composed of two structural (A and B) subunits and a catalytic C subunit (14). Of particular interest is that PP2A associates with IKK and leads to the modulation of NF-κB transcriptional activity (15, 16). It was shown that PP2A C physically interacts with IKKβ (16). Because PP2A Cα null mutant mice were embryonic lethal (17), alternative approaches, such as use of knockdown or pharmacological inhibitors, have been taken to study the role of PP2A in the NF-κB regulation. Here, we studied the regulation of IL-23 production in DC and identified PP2A as a negative regulator of IL-23 but not IL-12 production. Following a quick induction by LPS, IL-23p19 gene expression was rapidly down-regulated by PP2A. Interestingly, PP2Amediated down-regulation requires newly synthesized protein(s) that may act as adaptor(s) to form a complex between PP2A and IKKβ. This association then prevents sustained IKK phosphorylation and leads to rapid suppression of IL-23p19 gene expression in DC. Results Distinct Regulation of IL-23p19 Gene Expression by LPS in DC. To understand how IL-23 expression in DC is regulated in depth, we analyzed the temporal expression of the IL-23p19 gene together with other cytokine genes. After cells were stimulated with LPS, most of the cytokine gene expression steadily increased up to 6 h and declined over time (Fig. 1A Top). Concurrently, each cytokine production showed a gradual accumulation in the culture super-

Author contributions: J.C. and C.-H.C. designed research; J.C. and T.J.V. performed research; Y.L. and Y.Z. contributed new reagents/analytic tools; J.C., T.J.V., and C.-H.C. analyzed data; and J.C., T.J.V., and C.-H.C. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

To whom correspondence should be addressed. E-mail: [email protected].

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natant (Fig. 1A Bottom). By contrast, when IL-23p19 gene expression was examined, it reached maximum level at 1 h after LPS treatment followed by a rapid decrease (Fig. 1B). IL-23 protein level in the supernatant followed the pattern of IL-23p19 mRNA expression (Fig. 1B). IL-10 negatively regulates cytokine production in DC upon LPS stimulation (18). We tested therefore whether IL-10 participates in the inhibition of IL-23p19 gene expression. DC from IL-10-deficient mice, however, showed a similar pattern of IL-23p19 gene expression compared with wild-type DC although the expression level was elevated (Fig. 1C). Protein Synthesis Is Necessary for IL-23p19 Repression. The rapid decline in IL-23p19 gene expression prompted us to investigate the underlying mechanisms responsible for the down-regulation. To determine whether the decrease is mediated by newly synthesized proteins, cells were pretreated with cycloheximide (CHX), followed by LPS stimulation. The induction of IL-23p19 gene expression was not affected in the presence of CHX (Fig. 2A Left). More importantly, the level of IL-23p19 mRNA was sustained when the protein synthesis was blocked (Fig. 2A Left). These data suggest that protein

synthesis is not necessary for the activation of IL-23p19 gene expression but is for the down-regulation. By contrast, IL-12p35 gene expression was completely abolished in the presence of CHX, implicating that the transcription of the IL-12p35 gene depends on newly made proteins (Fig. 2A Center). IL-12p40 gene expression was moderately inhibited when protein synthesis was blocked (Fig. 2A Right). LPS treatment activates MAPK and IKK that are known to play a role in the regulation of cytokine gene expression. Therefore, we asked whether the inhibition of protein synthesis affects MAPK or IKK activity. To do this, we treated DC with LPS in the presence or absence of CHX and assessed the phosphorylation status of key signaling molecules by immunoblot analysis. The phosphorylation status of TAK1 and Erk1/2 was comparable with or without CHX (Fig. 2B). The pretreatment of CHX, however, increased the basal level of phospho-p38. In addition, the amount of phosphorylation of JNK and IKK was greatly enhanced when cells were treated with CHX (Fig. 2B). IκBα showed a prolonged degradation with the CHX treatment, which suggests a continuous activation of the NF-κB signaling pathway. To further substantiate the role of MAPK and IKK

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. IL23p19 mRNA level was determined by qRT-PCR.

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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.

for IL-23p19 gene expression, we used pharmacological reagents. U0126, LY294002 and SP600125, which selectively inhibits MEK1/2, PI3K and JNK, respectively, had marginal effects on IL-23p19 gene expression. By contrast, IL-23p19 gene expression was severely suppressed when cells were treated with the p38 specific inhibitor SB202190 or with BMS-345541 that blocks IKKβ (Fig. 2C). Thus, these data suggest that p38 and IKK are key regulators of IL-23p19 gene expression. IL-23 Expression Is Not Regulated by MKP-1 or CYLD. MKP-1 is a well characterized phosphatase for p38 and MKP-1-deficient (Mkp-1−/−) cells exhibited prolonged p38 activation and resulted in enhanced TNF-α and IL-6 production upon LPS stimulation (19). Therefore, we tested whether MKP-1 is responsible for the down-regulation of IL-23p19 gene expression. As reported, p38 activation was extended in the absence of MKP-1 (Fig. 3A). But, IKK activation measured by phosphorylation was moderately reduced in Mkp-1−/− DC compared with the control (Fig. 3A). When we assessed the cytokine production in DC, levels of both IL-23p19 mRNA and IL-23 protein were still inhibited without MKP-1, implying that MKP-1 is not likely mediating the repression. In addition, IL-12p35 gene expression and IL-12p70 production were greatly diminished in Mkp-1−/− DC (Fig. 3B). We showed that IKK and IκBα, but not TAK1, were affected by the inhibition of protein synthesis (Fig. 2B). In addition, inhibiting IKKβ by a pharmacological reagent abolished IL-23p19 gene expression (Fig. 2C). Together, these data suggest that the effect of CHX could be at the level of IKK, which led us to test IKKγ,

a regulatory component of IKK. Ubiquitination of IKKγ is believed to be involved in IKK activation (11). Therefore, it is possible that preventing protein synthesis changes ubiquitination of IKKγ and, consequently, IL-23p19 expression. To explore this possibility, ubiquitination of IKKγ was examined. As shown in Fig. 3C, ubiquitination of IKKγ was sustained in the presence of CHX, suggesting a prolonged IKK activation. Deubiquitinase CYLD is known to act as a negative regulator of NF-κB signaling through its interaction with IKKγ (13). However, when CYLD-deficient (Cyld−/−) DC was treated with LPS, IL-23p19 gene expression was not affected (Fig. 3D). Together, these data indicate that MKP-1 and CYLD play a minor role, if any, in the regulation of IL-23p19 gene expression. Okadaic Acid Prevented Down-Regulation of IL-23 but Not IL-12 Expression. Purified IKK is inactivated by PP2A (20), presumably via

the physical interaction between both proteins (16). Therefore, we asked whether PP2A is involved in the inhibition of IL-23p19 gene expression in DC. To do this, we tested the effect of okadaic acid (OA), a potent inhibitor of PP2A. Similar to CHX treatment, the phosphorylation status of p38 and Erk1/2 was not altered but that of JNK and IKK was greatly enhanced in the presence of OA (Fig. 4A). Next, we examined cytokine expression. Expression of IL-23p19 was no longer inhibited when cells were treated with OA suggesting the essential role of protein phosphatase activity for IL-23p19 gene regulation (Fig. 4B). In contrast, OA had no effect on the expression of IL-12p35 and IL-12p40. Likewise, IL-23 protein level was substantially increased in the presence of OA, whereas neither IL12p70 nor IL-12p40 production was affected by OA (Fig. 4C). Consistent with the report that IL-23 supports survival and expansion of IL-17-producing T cells (21), DC treated with OA together with LPS were more potent to induce IL-17 production by CD4+ T cells than DC with LPS alone (Fig. 4D). However, IFN-γ production was comparable between the two (Fig. 4D). Thus, our data indicate that IL-23 but not IL-12 expression was selectively affected by OA, which is likely associated with the PP2A activity. Enhanced IL-23 Production by Down-Regulating PP2A. To further delineate the role of PP2A in the inhibition of IL-23p19 gene expression, PP2A C expression was knocked down by delivering siRNA into DC, which resulted in very efficient reduction of PP2A C protein (Fig. 5A). Upon LPS stimulation, the same cells showed a significant increase in IL-23 production but not in IL-12p70 (Fig. 5B). OA can effectively inhibit PP1 in addition to PP2A, albeit at a relatively high concentration (22, 23). Therefore, we tested whether PP1 also participates in IL-23 gene regulation by introducing siRNA specific to PP1 C. Unlike PP2A, modulating PP1 C did not change the amount of IL-23 (Fig. 5 C and D), demonstrating that PP2A is specifically responsible for suppressing IL-23 expression.

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 IL12p70 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.

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Association of PP2A with IKKβ Requires New Protein Synthesis. So far, we have demonstrated that PP2A seems to be a critical regulator of IL-23 down-regulation. In addition, the data indicated the necessity of de novo protein synthesis for the repression. Based on these observations, we hypothesized the level or the activity of PP2A depends on a newly synthesized protein. To test this hypothesis, we first compared the protein level of PP2A in the presence and absence of CHX. We found that the protein levels of all subunits of PP2A were not altered by CHX treatment (Fig. 6A). PP2A is known to exist in the cytosol of most cell types (14). Therefore, we wondered if its cellular localization in DC was dysregulated in the presence of CHX. To test this postulation, cell lysates were subjected to nuclear and cytoplasmic fractionations followed by immunoblotting. As shown in Fig. 6B, a majority of PP2A C was detected in the cytoplasmic fraction at comparable levels with or without CHX treatment. We next asked whether the function of PP2A has been influenced by the CHX treatment. PP2A possesses the phosphatase activity and also interacts with IKK to modulate NF-κB activation. Therefore, Chang et al.

we measured the both functions. To test whether the enzymatic activity of PP2A is regulated by CHX, DC were treated with LPS in the presence or absence of CHX. We then determined the phosphatase activity of PP2A by immunoprecipitating it from cell lysates. The phosphatase activity of PP2A seemed to be constitutively active in DC regardless of CHX (Fig. 6C, Left) and immunoprecipitated PP2A C was associated with its structural subunits in all conditions (Fig. 6C, Right). Therefore, PP2A synthesis and its enzymatic activity were not affected without new protein synthesis. However, because PP2A activity is exerted on IKK, we then sought to determine whether the ability of PP2A to associate with IKK was compromised by the CHX treatment. To test this, we used coimmunoprecipitation to measure their association in the presence or absence of CHX. As shown in Fig. 6D, the association of PP2A with IKKβ was enhanced after LPS stimulation. However, in the pres-

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. AntiGAPDH 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.

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ence of CHX, the association of PP2A with IKKβ was greatly reduced (Fig. 6D Left). The lack of interaction between PP2A and IKKβ was not stemmed from the inability of IKKβ forming the complex with IKKγ (Fig. 6D Right). Collectively, our data indicate that PP2A plays an essential role in regulating IL-23p19 gene expression in DC by modulating IKK activation. Down-Regulation of IL-23p19 Expression Is Due to IKK. We next investigated the role of IKK during the phase of IL-23p19 downregulation. To do this, we pretreated DC with CHX and then stimulated with LPS to have the IL-23p19 gene expressed and sustained. We then added an inhibitor to block IKK followed by harvesting cells at different time points to measure the level of IL23p19 transcripts (Fig. 7A). If IKK plays a role during down-regulation, adding an IKK inhibitor would reduce the amount of IL23p19 transcripts. Indeed, adding the IKKβ blocker BMS-345541 to CHX-treated cells rapidly decreased IL-23p19 gene expression (Fig. 7B). By contrast, IL-23p19 gene expression was still sustained when CHX-treated cells were treated with the p38 inhibitor SB202190. A similar inhibitory effect on IL-23p19 expression was observed with BMS-345541 when DC were pretreated with OA (Fig. 7C). Thus, these data indicate that that IKK plays a key role in the down-regulation of IL-23p19 gene expression.

Discussion The roles of protein phosphatases in the regulation of cytokine production remain unexplored. Our study showed that the expression of IL-23, but not IL-12, is negatively regulated by PP2A in DC. At the molecular level, upon LPS stimulation PP2A interacts with the IKK complex in a manner dependent on de novo protein synthesis, which then leads to deactivation of IKK and subsequent termination of the NF-κB cascade. In addition, we showed that deubiquitinase CYLD, a known regulator of NF-κB signaling, did not participate in IL-23 production. In this report, we demonstrated a unique pattern of IL-23p19 expression in DC. IL-23p19 induction does not require new protein synthesis and this may explain the rapid increase in mRNA by using existing transcription factors. However, the induction was quickly turned down by PP2A. This pattern of up- and downregulation of IL-23p19 expression is opposite from that of the IL-12p35 gene. The activation of the IL-12p35 gene requires newly PNAS | May 4, 2010 | vol. 107 | no. 18 | 8343

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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) IL23, 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. 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 antiPP2A 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.

synthesized proteins upon LPS stimulation and PP2A showed no effect on its expression. Because the expression of both IL-23p19 and IL-23p35 genes depend on IKK/NF-κB signaling, it suggests that the activation of default IKK/NF-κB signaling is sufficient for IL-23p19 but not IL-12p35 induction. Additional transcription factors that are made upon LPS stimulation are likely necessary for IL-12p35 gene activation. Given the importance of NF-κB signaling in the transcriptional regulation of IL-23p19 and IL-12p35 genes, it remains elusive how the blocking IKK by PP2A selectively affects IL-23p19 but not IL12p35 expression. Perhaps different protein phosphatases are involved in the termination of the IKK activity for IL-12p35. PP2Cβ was shown to associate with IKKβ and regulate NF-κB activity in HEK293 cells (24). Alternatively, IL-12p35 expression in DC could be mediated by IKK-independent NF-κB regulation. It was shown that IκBNS, a TLR-induced nuclear IκB protein, controls the expression of a subset of genes in an IKK-independent manner (25). We showed that MKP-1, a phosphatase of p38, is essential for IL-12 but not IL-23 production. Moreover, deficiency in the deubiquitinase CYLD, a well-known NF-κB regulator, did not affect the production of IL-12 or IL-23 in DC. Together, our study suggests that although the production of both IL-12 and IL-23 requires NFκB, the activation of NF-κB seems to be achieved by different signaling to produce two cytokines. This seems to reflect that the demand of these two cytokines could be dictated by the nature of the initial trigger of an immune response such as the type of pathogens, which then results in Th1- or Th17 cell-mediated immunity.

We demonstrated that PP2A is constitutively expressed in the cytoplasm. Moreover, PP2A isolated from DC without stimulation was enzymatically active. Yet functionally competent PP2A cannot remove phosphates from IKK, presumably due to the inability to form a complex between PP2A and IKK until new proteins with unknown identity are made upon LPS stimulation. Based on these observations, it is tempting to speculate that the interaction between PP2A to IKKβ requires an adaptor protein that is only made and thus available after stimulation. Recently, a similar case has been reported that AIP1 [apoptosis signal-regulating kinase 1(ASK1)interacting protein 1] recruits PP2A to ASK1 after TNF treatment (26). Alternatively, PP2A may undergo a modification that is necessary to interact with IKK. In fact, the phosphatase activity of PP2A was shown to be regulated by covalent modifications such as methylation and phosphorylation (27). Therefore, it is possible that new protein synthesis is necessary to produce a modifying enzyme for PP2A, which then allows PP2A to interact with IKKβ. The PP2A binding sites to IKKβ may not be exposed without the modifier. The identification of a putative adaptor or a modifier is needed to have better understanding of how PP2A interacts with IKK. Mice lacking IL-23p19 but not IL-12p35 were resistant to autoimmune inflammatory diseases, including experimental autoimmune encephalomyelitis and inflammatory bowel disease (28). Furthermore, emerging data support that IL-23 is closely linked to autoimmune inflammatory diseases in humans. IL-23p19 expression was increased in psoriatic lesions (29). Interestingly, IL-23p19 but not IL-12p35 mRNA expression was elevated in Crohn’s disease and DC from multiple sclerosis patients had an increased IL23 but equivalent amounts of IL-12 (30, 31). Because IL-23/Th17 is strongly associated with autoimmune inflammation in local tissues it is not surprising that IL-23 expression is tightly regulated. In conclusion, the present study discovered that protein phosphatase PP2A plays a critical role in the regulation of IL-23 expression by modulating LPS-induced NF-κB activation. Future studies are warranted to unveil many aspects of immune regulation mediated by protein phosphatases including PP2A. Materials and Methods

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.

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Mice and Cells. BALB/c, DO11.10 TCR-transgenic mice and IL-10–deficient mice on the BALB/c background were purchased from Jackson Laboratory. The bone marrow cells from CYLD-deficient mice were provided by Dr. A. Jain (National Institute of Health, Bethesda, MD) (32). MKP-1–deficient mice on a C57/129 background were kindly provided by Bristol-Myers Squibb, and these mice were back-crossed to C57BL/6 mice for 10 generations in the Research Institute at Nationwide Children’s Hospital. C57BL/6 mice and MKP-1– deficient mice were bred and maintained under specific pathogen-free conditions at the University of Michigan and Research Institute at Nationwide Children’s Hospital animal facility, respectively. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Uni-

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Reagents. LPS and cycloheximide were obtained from Sigma-Aldrich. Okadaic acid, SB202190, BMS-345541, LY294002, and SP600125 were obtained from Calbiochem. U0126 was from Promega. Antibodies used for immunoblot analysis included p38 MAPK, phospho-p38 MAPK (Tyr180/Tyr182), p44/42 MAPK, phospho-p44/42 MAPK (Thr202/Tyr204), IKKβ, phospho-IKKα (Ser180)/IKKβ (Ser181), SAPK/JNK, phospho-SAPK/JNK (Thr183/Tyr185), phospho-TAK1 (Thr184/187), CREB (48H2), IκBα (Cell Signaling Technology), TAK1, IKKγ, Ub (P4D1) (Santa Cruz Biotechnology), GAPDH (6C5) (Abcam), PP2A A, PP2A B (2G9), PP2A C (1D6), and PP1 C (Millipore). ELISA and Quantitative Real-Time RT-PCR (qRT-PCR). Cytokine concentrations in supernatants were detected by ELISA and mRNA accumulation of cytokines was analyzed by qRT-PCR as previously described (33). The primers used for GAPDH, IL-12p35, IL-12p40, IL-10, IL-6, and IL-23p19 were described previously (18). T Cell Differentiation. CD4+ T cells from DO11.10 TCR-transgenic mice were purified using anti-CD4 magnetic beads (Miltenyi Biotec). DC from BALB/c mice were pretreated with or without OA (100 nM) for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. DC (5 × 104) were then pulsed with 5 μM ovalbumin (OVA) peptide (residues 323–339) for 2 h, washed, and cocultured with CD4+ DO11.10 T cells (2.5 × 105). Cultures were split (1:2) on day 4 and were restimulated on day 6 with plate-bound anti-CD3 (145-2C11) antibody for 48 h. ELISA was performed to detect IL-17A and IFN-γ production. siRNA. Predesigned Accell siRNA targeted against mouse PP2A C (A-040657-13), PP1 C (A-040960-15), and control kits (K-005000-G1-02) were purchased from Dharmacon. siRNAs were delivered into DC according to the manufacturer’s instructions. Briefly, cells were resuspended in Accell siRNA delivery media containing 2.5% serum and plated into a 48-well plate to a concentration of 0.3 × 106 cells/well; 1 μM Accell siRNAs were added to each well in a plate and cells were incubated at 37 °C with 5% CO2 for 96 h to assess siRNA delivery efficiency or protein knockdown. Flow cytometric analysis was performed to assess delivery efficiency of FAM-labeled control Accell siRNA using FACSCanto

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(BD Biosciences) and analyzed using FlowJo software (Tree Star); 85.7% and 97.4% of DC at 72 h and 96 h, respectively, were detected as FITC-positives. The protein knockdown was confirmed by immunoblot analysis. Following 96-h incubation, cells were treated with 1 μg/mL LPS for an additional 24 h for ELISA. Protein Phosphatase Assay. Phosphatase activity of PP2A was determined by using PP2A immunoprecipitation phosphatase assay kit (Millipore). Briefly, PP2A was immunoprecipitated from cell lysates. Immunoprecipitated purified PP2A was diluted in pNPP Ser/Thr assay buffer and incubated with phosphopeptide (final 750 μM) for 10 min at 30 °C. Malachite green detection solution was added and absorbance at 650 nm was measured at different time points. The lysates were immunoprecipitated with a control mouse IgG antibody and used as a negative control (−). Subcellular Fractionation. DC were plated at 5 × 106 cells/well in six-well plates. After treating cells with LPS in the presence or absence of CHX, cells were collected, washed once with PBS, and treated with 180 μL of ice cold Buffer A [10 mM hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1% protease inhibitor mixture (Sigma-Aldrich)]. Cells were incubated on ice for 30 min; 20 μL of 10% Nonidet P-40 was added and tubes were vortexed for 1 min. Lysates were centrifuged for 10 min at 16,100 × g at 4 °C. Cytoplasmic supernatant was removed and collected in a fresh tube. Pelleted nuclei were treated with 75 μL of ice cold Buffer B [20 mM hepes (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF]. Nuclear pellets were incubated on ice for 1 h and vortexed every 10 min and then centrifuged for 20 min at 16,100 × g at 4 °C. Nuclear supernatants were collected in fresh tubes. Samples were stored at −20 °C. Statistical Analysis. Results are given as the mean ± SD. Data were analyzed by a two-tailed t test. Differences with a P value of 0.05 or less were regarded as statistically significant. ACKNOWLEDGMENTS. We thank Dr. Philip King (University of Michigan, Ann Arbor) and Dr. Mark Kaplan (Indiana University, Indianapolis) for critical reading of the manuscript. This work was partly supported by National Institutes of Health Grants AI070448 (to C.-H.C.) and AI057798 (to Y.L.).

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IMMUNOLOGY

versity of Michigan and Research Institute at Nationwide Children’s Hospital. Bone marrow-derived DC were prepared as previously described (33).