Blood First Edition Paper, prepublished online April 27, 2004; DOI 10.1182/blood-2003-12-4302
Immunoregulation of dendritic cells by IL-10 is mediated through suppression of the PI3K/Akt pathway and IκB kinase activity. Running Title: Regulation of NF-κB activation in DC. Sandip Bhattacharyya*, Pradip Sen*, Mark Wallet*, Brian Long*, Albert S. Baldwin, Jr.+#, and Roland Tisch*+# *Department of Microbiology and Immunology, School of Medicine, +Lineberger Comprehensive Cancer Center, and # Curriculum in Oral Biology, University of North Carolina at Chapel Hill, NC 27599. Corresponding Author: Roland Tisch, Ph.D. Department of Microbiology and Immunology Mary Ellen Jones Bldg., Rm 804 Campus Box# 7290 University of North Carolina at Chapel Hill Chapel Hill, NC 27599-7290 Tel#: 919-966-7020, FAX#: 919-962-8103, email:
[email protected] This work was supported Grant 1-P60-DE 13079 from the National Institute of Dental and Craniofacial Research. Scientific Heading: Immunobiology Total word count: 4,948 Abstract word count: 190
Copyright (c) 2004 American Society of Hematology
ABSTRACT IL-10 has potent immunoregulatory effects on the maturation and antigen presenting cell (APC) function of dendritic cells (DC). The molecular basis underlying these effects in DC, however, is ill defined. It is well established that the transcription factor NF-κB has a key role regulating DC development, maturation and APC function. This study was initiated to determine the effects of IL-10 on the NF-κB signaling pathway in immature DC. IL-10 pretreatment of myeloid DC cultured from bone marrow resulted in reduced DNA binding and nuclear translocation of NF-κB following anti-CD40 Ab or LPS stimulation. Furthermore, inhibition of NF-κB activation was characterized by reduced: i) degradation and/or phosphorylation of IκBα and IκBε but not IκBβ, and ii) phosphorylation of serine 536 located in the trans-activation domain of p65. Notably, IL10 mediated inhibition of NF-κB coincided with suppressed IκB kinase (IKK) activity in vitro. Furthermore, inducible Akt phosphorylation was blocked by IL-10, and inhibitors of phosphatidylinositol 3-kinase (PI3K) effectively suppressed activation of Akt, IKK and NF-κB. These findings demonstrate that IL-10 targets IKK activation in immature DC, and that blockade of the pathway is mediated in part through suppression of the PI3K pathway.
INTRODUCTION Dendritic cells (DC) play a key role in regulating immune responses to foreign and self antigens.1,2 Upon uptake of antigen, DC process and present peptides to naïve T cells, and secrete pro- or anti-inflammatory cytokines that influence the nature of the response. The capacity of DC to promote immunity versus tolerance within a responding population of T cells is believed to be a function of the subset, maturation and/or activation state of the DC.1,2 For example, T cell tolerance is induced upon antigen presentation by inactivated, immature DC characterized by low expression of the costimulatory molecules CD80 and CD86. IL-10 has also been reported to be a potent regulator of DC maturation and effector function. Upregulation of co-stimulatory molecule expression, secretion of pro-inflammatory cytokines such as IL-12 and TNFα, and the capacity of DC to stimulate T cells are effectively suppressed by IL-10.3-9 Furthermore, APC function can be influenced by IL-10 secreted by DC in an autocrine manner.10,11
The NF-κB family of transcription factors has a major role in mediating inflammatory responses,12-14 which includes regulation of DC development, maturation and function.1523
Notably, NF-κB activity controls the expression of many genes involved in the APC
function of DC such as IL-12, TNFα, MHC II, and costimulatory molecules. The NF-κB complex consists of homo- or hetero-dimers of the structurally related proteins p50, p52, p65 (RelA), c-Rel and RelB. In most resting cells, NF-κB is sequestered in the cytoplasm complexed with the inhibitory molecules IκBα, IκBβ, and IκBε.12-14 In response to a variety of stimuli such as LPS, TNFα, IL-12 and CD40 engagement, IκB proteins are
phosphorylated, poly-ubiquitinated and then degraded by the 26S proteosome.
The
release of NF-κB from IκB proteins facilitates nuclear translocation and subsequent binding to consensus sequences and induction of gene transcription. Reduced numbers of myeloid DC in mice in which the relB gene has been disrupted provides evidence that NF-κB has a role in DC development.16-18 Furthermore, we and others have shown that hyperactivation or inhibition of NF-κB activity can enhance or block, respectively, the capacity of DC to secrete pro-inflammatory cytokines and stimulate naïve CD4+ and CD8+ T cells.21-24
Phosphorylation of the IκB inhibitory molecules is catalyzed by the IκB kinase (IKK), a multisubunit complex consisting of IKK1/IKKα, IKK2/IKKβ, and IKKγ/NEMO.14,25 Kinase activity is contained in the IKK1 and IKK2 subunits, whereas IKKγ serves a scaffold/regulatory function. Biochemical analyses and targeted gene deletions have demonstrated that IKK2 is essential for NF-κB activation in response to proinflammatory mediators such as IL-1, TNFα or LPS.26-28 Activation of the complex is achieved in part, by the phosphorylation of IKK catalyzed by diverse kinases namely, NF-κB inducing kinase (NIK), MEKK1, MEKK3, Cot, NAK/TBK, and PKCζ.14,25 Included in this group is the serine-threonine kinase Akt found in the phosphatidylinositol 3-kinase (PI3K) pathway.29-31 We and others have demonstrated that the PI3K/Akt pathway can regulate NF-κB activation, although different mechanisms have been described.31-39
Studies have demonstrated that IL-10 inhibits the activity of NF-κB in different cell types such as human and mouse macrophages, and T cells.40-42 Furthermore, we previously demonstrated that IL-10 blocked NF-κB activity through the inhibition of IKK in a human monocyte cell line.43 Nevertheless, the mechanism by which IL-10 inhibits NF-κB transcriptional activity varies depending on the cell type. Moreover, IL-10 mediated suppression has been reported to have no effect on NF-κB activity in human macrophages.44 The current study was initiated to determine the mechanism by which IL10 regulates the APC function of DC. Evidence is provided that activation of NF-κB is blocked in myeloid DC following pre-treatment with IL-10, and that this inhibition correlates with suppressed Akt and downstream IKK activity.
MATERIALS AND METHODS Mice NOD/LtJ, BALB/cJ and NOR/Lt mice were maintained and bred under specific-pathogen free conditions. The NOD.CL4 mouse line expresses transgenes encoding the CL4 clonotypic T cell receptor (TCR).23 The CL4-clonotypic TCR is H2Kd restricted and specific for an influenza hemagglutinin (HA) peptide spanning amino acid residues 512520.
Preparation of primary DC Femurs of male or female mice 8-12 wk of age were used to isolate bone marrow-derived DC as previously described.22,23 Bone marrow was depleted of CD4 (GK1.5), CD8 (HO2.2), MHC class II (M1/42.3.9.8 HLK), and B220 (RA3-3A1/6.1) expressing cells via complement-mediated lysis. DC precursors were plated on 6-well ultra low-cluster tissue culture plates (Corning Incorporated, Corning, NY) in RPMI 1640 medium containing 10% FBS and penicillin / streptomycin (base medium), 10 ng/ml murine GM-CSF (PeproTech, Rocky Hill, NJ), and 10 ng/ml murine IL-4 (PeproTech). On day two, nonadherent cells were harvested and cultured as above for 8 days. Culture medium was added on days 4 and 7. For all experiments DC were harvested on day 10 of the cultures. 7-amino-actinomycin D (7-AAD) (BD PharMingen, San Diego, CA) staining was used to access the viability of DC.
DC were isolated from the spleen by preparing a single cell suspension depleted of RBC that was incubated with 0.5 mg/ml collagenase A (Roche, Indianapolis, IN) and 5 µg/ml DNase I (Roche) for 20 min at 370C. Cells were washed and DC purified using an
EasySep Murine CD11c Positive Selection Kit (Stem Cell Technologies, Vancouver, Canada). Flow cytometric analysis demonstrated >90% purity based on CD11c expression.
EMSA and Western blotting DC cultured in base medium at 5x105 cells / well in 6 well low cluster plates (Corning Incorporated) were stimulated with either 500 ng/ml LPS, 10 µg/ml anti-CD40 Ab (HM40-3, BD PharMingen, San Diego, CA) or 10 µg/ml purified hamster IgM isotype control (clone G235-11, BD PharMingen) for specified times. Nuclear and cytoplasmic extracts were prepared as described.22 EMSA was performed as described using NF-κB binding
32
P-labelled double stranded DNA probes from the MHC class I H2K promoter:
5′-CAGGCTGGGGATTCCCATCTCCACAGTTTCACTTC-3′,22 the murine IL-12(p40) promoter : 5′-CTTCTTAAAATTCCCCCAGA-3.45 A double-stranded OCT-1 DNA probe: 5′-TGTCGAATGCAAATCACTAGAA-3′
(Santa Cruz Biotechnology, Santa
Cruz, CA) was used as control. Bands were visualized using a phosphoimager (Molecular Dynamics, Sunnyvale, CA). For supershift experiments, 2 µg of anti-p50 (sc-114X), antip52 (sc-298), anti-p65 (sc-8008X), anti-cRel (sc-70X), anti-RelB (sc-226X) (Santa Cruz Biotechnology) were added to lysates and incubated for 1hr at room temperature prior to the addition of 32P-labelled probe.
For Western blotting, 80µg of cytoplasmic extract was resolved via SDS-PAGE using a 810% separating gel. Proteins were transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ) using a semi-dry transfer system and
blocked with 5% dried milk in PBS and 0.1% Tween-20. Blots were probed with antiIκBα (sc-371), anti-IκBβ (sc-969), anti-IκBε (sc-7156), anti-IKK1 (sc-7182) (Santa Cruz Biotechnology); anti-IKK2 (2684), anti-phospho-IκBα (9246), anti-phospho-p65 (3031), anti-phospho-IKK1/IKK2 (2681) anti-phospho Akt (9271), anti-Akt (9272)
(Cell
Signaling TechnologyTM (Beverly, MA)), and anti-β actin (A2066, Sigma) Abs. Binding of HRP-labeled goat anti-rabbit Ab (sc-2004) or goat anti-mouse Ab (sc-2005) (Santa Cruz Biotechnology) was determined using SuperSignalR West Pico Chemiluminescent Substrate (Pierce, Rockland, IL).
IKK Assay DC (5x106) were stimulated and whole cell lysates prepared. IKK signalosome was immunoprecipitated using the Catch and ReleaseTM kit (Upstate, Lake Placid, NY) and rabbit polyclonal anti-IKK1 and anti-IKK2. In vitro kinase experiments were performed as described22 by incubating 0.25µg of immunoprecipitated protein with 0.5 µg GST-IκBα substrate. The reaction mixtures were incubated for 60 min at 300 C with 5 µCi [γ-32P] ATP in kinase buffer. Kinase reactions were terminated upon addition of SDS-PAGE sample buffer and were analyzed by SDS-PAGE. Bands were visualized by autoradiography.
Akt Assay DC (4x106) were stimulated as described, and whole cell lysates prepared. Akt kinase activity was determined using an Akt Kinase Assay Kit (Cell Signaling TechnologyTM). Briefly, Akt was immunoprecipitated as per the manufacturer’s instructions and kinase activity determined by
measuring phosphorylation of a GSK-3 substrate via an immunoblot probed with a phosphoGSK-3α/β (Ser21/9) specific Ab.
Measurement of DC secretion of IL-12(p70) and TNFα. DC were harvested on day 10 of culture and plated at 106 cells/ml in a 24 well plate with 1 ml of base medium and treated accordingly. Cells were washed and stimulated with LPS (500 ng/ml), anti-CD40 (10 µg/ml) or isotype-matched control Ab (10 µg/ml) for 48 hr. Supernatants were collected and assayed via ELISA for IL-12(p70), and TNFα as per the manufacturer’s instructions (BD PharMingen).
DC:T cell assays CD8+ T cells were purified from the spleens of NOD.CL4 mice using anti-CD8 magnetic beads (Miltenyi Biotech, Auburn, CA). T cells were CD62hiCD44loCD69lo and >90% CD8+. DC were pre-treated with murine IL-10 for 24 hr, washed 3 times and incubated with 5 ug/ml of HA peptide in 1.0 ml of base medium supplemented with 50 µM βmercaptoethanol, 1 mM sodium pyruvate, 1x nonessential amino acids, and 1 mM glutamine (complete medium) in a 24-well plate. After 30 min, DC were washed 4 times, and 3.5x105 T cells added to each well in 1.0 ml of complete medium. At 48 hrs, culture supernatants were harvested and levels of IL-2 determined via ELISA as per the manufacturer’s instructions (BD Pharmingen).
RESULTS IL-10 pretreatment suppresses the APC function of DC. To confirm the immunoregulatory effects of IL-10, immature DC were pretreated with varying concentrations of IL-10 and examined for the capacity to: i) secrete proinflammatory cytokines following anti-CD40 Ab or LPS stimulation, and ii) stimulate T cells. As reported,22,23 DC cultured from the bone marrow of NOD mice in GM-CSF and IL-4 for 10 days were >98% CD11c+CD11b+CD8α- and exhibited an immature phenotype (Table 1). Cultures of DC pretreated with increasing concentrations of IL-10 exhibited in a dose dependent manner, reduced levels of IL-12p70 and TNFα secretion after stimulation with anti-CD40 Ab (Figure 1A) or LPS (Figure 1B). IL-10 concentrations of 50 ng/ml or greater resulted in maximum cytokine suppression. DC exhibited >96% viability in the above treatment groups based on flow cytometry (data not shown). No significant effect on IL-12p70 or TNFα secretion was detected in cultures, however, when DC were co-treated with 50 ng/ml of IL-10 and LPS (Figure 1C) or anti-CD40 Ab (data not shown). Pretreatment with IL-10 for 24 hr also inhibited the capacity of peptide pulsed DC to stimulate naïve CD8+ T cells. IL-2 secretion by CD8+ T cells prepared from NOD.CL4 mice transgenic for a H2Kd-restricted TCR specific for HA was reduced in a dose dependent manner after pretreatment of DC with increasing concentrations of IL-10 (Figure 1D). Finally, IL-10 pretreatment inhibited LPS induced upregulation of CD40, CD86 and IAg7 cell surface expression (Table 1). In agreement with work reported by others,3-9 these results demonstrate that pretreatment but not costimulation with IL-10 inhibits the APC function of DC.
IL-10 pretreatment inhibits nuclear DNA binding activity of NF-κB. Since NF-κB plays a key role in regulating the APC function of DC, the effect of IL-10 pretreatment on NF-κB activation was investigated. An EMSA was performed on nuclear extracts prepared from DC pretreated with 50 ng/ml of IL-10 for varying periods of time, and then stimulated with either anti-CD40 Ab or LPS for 30 min. Because maximum suppression of cytokine secretion and T cell stimulation was detected in DC pretreated with 50 ng/ml of IL-10 (Figure 1), this concentration was used here and in subsequent experiments. IL-10 pretreatment inhibited nuclear DNA binding activity of NF-κB in a time dependent manner (Figure 2). Significant inhibition of DNA binding by nuclear NFκB, represented by two sets of complexes, was detected between 12 and 24 hr of IL-10
pretreatment for either LPS or anti-CD40 Ab stimulation (Figure 2A, 2B). For example, LPS-induced NF-κB DNA binding was reduced 16- and 22-fold upon IL-10 pretreatment for 12 and 24 hr, respectively, relative to LPS stimulation alone (Figure 2A). Similar levels of OCT-1 DNA binding were observed regardless of the duration of IL-10 pretreatment (Figure 2) demonstrating that suppression of nuclear DNA binding was NFκB-specific.
Using Abs specific for each Rel family member, supershift analysis demonstrated that complex 1 contained heterodimeric complexes of p50, p65, c-Rel and RelB, whereas complex 2 was comprised of p50 homodimers (Figure 2C). Interestingly, LPS induced DNA binding of p50/p50 homodimer complexes (complex 2) was significantly inhibited after only 6 hr of IL-10 pretreatment relative to the heterodimeric complexes (Figure 2A).
At this time point, a 5.7-fold reduction of p50/p50 DNA binding was detected compared to extracts prepared from DC stimulated with LPS alone.
Further analysis of nuclear extracts prepared from DC pretreated with IL-10 for 24 hr demonstrated early and persistent inhibition of NF-κB DNA binding after anti-CD40 Ab or LPS stimulation (Figure 2D). Reduced DNA binding was detected with either H2K(Figure 2D) or IL-12p40-specific oligonucleotide probes (data not shown). For example, IL-10 pretreatment reduced NF-κB DNA binding to the H2K probe between 7.3- to 15.8fold within 30 min to 2 hr of LPS stimulation, relative to extracts prepared from DC treated with LPS-only (Figure 2D). Supershift analysis indicated that binding of the different NF-κB complexes to the H2K-probe was uniformly suppressed after 24 hr of IL10 pretreatment (Figure 2C). In contrast to the suppression observed by IL-10 pretreatment, co-incubation with IL-10 and LPS had no significant effect on the level or kinetics of NF-κB DNA binding compared to LPS treatment alone (Figure 2E). Similarly, NF-κB DNA binding induced via anti-CD40 Ab was unaltered by IL-10 co-treatment (data not shown).
To ensure that the effects of IL-10 pretreatment were not intrinsic to bone marrowderived DC, DC were directly isolated from the spleen and NF-κB DNA binding assessed following LPS stimulation. Splenic DC exhibited a typical myeloid-like phenotype characterized by a CD11b+CD11c+CD8α- profile. IL-10 pretreatment of splenic DC reduced the nuclear DNA binding activity of NF-κB by 7-fold, comparable to that observed for bone marrow-derived DC (Figure 2F).
These results demonstrate that IL-10 pretreatment of cultured or ex vivo DC suppresses nuclear DNA binding of NF-κB independent of the type of stimulus or DNA probe. However, NF-κB DNA binding is unaltered when DC are co-treated with IL-10 and antiCD40 Ab or LPS. This latter result is consistent with the inability of IL-10 to suppress cytokine secretion upon co-stimulation with anti-CD40 Ab or LPS (Figure 1C).
IL-10 pretreatment inhibits IκB degradation and p65 phosphorylation. The lack of nuclear DNA binding by NF-κB suggested that IL-10 pretreatment was blocking degradation of the IκB proteins. Accordingly, the status of IκBα, IκBβ, and IκBε in cytoplasmic extracts was determined by Western blot using Abs specific for the respective inhibitory proteins. Degradation of the three IκB proteins was readily detected in cytoplasmic extracts prepared from DC stimulated with LPS (Figure 3A). However, pretreatment with IL-10 for 24 hr significantly inhibited degradation of IκBα, and to a lesser extent IκBε following LPS stimulation. This was most evident during the early kinetics of LPS stimulation (Figure 3A). After 30 min of LPS treatment at which maximum IκBα and IκBε degradation was observed, IL-10 pretreatment reduced degradation by 7- and 3-fold, respectively. IL-10 pretreatment also inhibited phosphorylation of IκBα approximately 3-fold following either LPS or anti-CD40 Ab stimulation (Figure 3B). Interestingly, LPS induced degradation of IκBβ was not significantly effected by IL-10 pretreatment (Figure 3A). Furthermore, IκB protein degradation was unaffected when DC were co-treated with IL-10 and LPS (Figure 3C). These results demonstrate that IL-10 pretreatment of DC suppresses degradation and/or phosphorylation of IκBα and IκBε, but not IκBβ.
LPS has been reported to induce phosphorylation of the p65 Rel homology and transactivation domains thereby increasing p65 transcription activity.46-49 To determine whether IL-10 pretreatment also inhibited p65 phosphorylation, nuclear extracts were prepared from LPS stimulated DC and analyzed via Western blot. Ab was employed that is specific for phosphorylated serine 536 found in the trans-activation domain of p65. LPS treatment induced increased phosphorylation of nuclear p65 in a time dependent manner (Figure 3D). In contrast, IL-10 pretreatment for 24 hr reduced nuclear p65 phosphorylation up to a 3.5-fold (Figure 3D). Importantly, reduced phospho-p65 correlated with up to a 3.0-fold decrease in nuclear p65 protein compared to DC stimulated with LPS-only (Figure 3D). These results indicate that IL-10 pretreatment inhibits phosphorylation and nuclear translocation of p65 in DC.
IL-10 pretreatment inhibits IKK activity. Phosphorylation and subsequent degradation of the IκB proteins is initiated by the IKK complex upon appropriate cellular stimulation.12-14,25 Therefore the possibility that IL-10 pretreatment inhibited IKK activity was investigated. Initially, the activation status of IKK was assessed by measuring phosphorylation of the complex. The IKK complex is activated in part by phosphorylation of serine residues located in the activation loops of IKK1 (Ser176 and Ser180)50,51 and IKK2 (Ser177 and Ser181)26. Cytoplasmic extracts prepared from DC pretreated with IL-10 and stimulated with LPS or anti-CD40 Ab were examined via Western blot using Ab specific for phosphorylated IKK1 Ser180 and IKK2 Ser181. Treatment with either LPS (Figure 4A) or anti-CD40 Ab (Figure 4B) alone induced IKK1 and IKK2 phosphorylation. In contrast, IL-10 pretreatment reduced IKK1
and IKK2 phosphorylation following stimulation with LPS (Figure 4A) or anti-CD40 Ab (Figure 4B). A comparative study with DC isolated from NOD, NOR and BALB/C demonstrated comparable inhibition of IKK1 and IKK2 phosphorylation indicating that suppression of IKK activity in DC via IL-10 pretreatment was not mouse strain specific (data not shown).
Whether reduced IKK activation following IL-10 pretreatment also reflected diminished kinase activity was determined. Cultured DC were incubated with IL-10 or left untreated, stimulated with anti-CD40 Ab or LPS for varying times, and IKK1 or IKK2 immunoprecipitated from cytoplasmic extracts using corresponding Abs. IKK activity was assessed by measuring phosphorylation of a GST-IκBα substrate in vitro. IL-10 pretreatment suppressed IKK activity in a time dependent manner (Figure 5A). For example, IKK activity induced by LPS or anti-CD40 Ab stimulation was inhibited approximately 4- to 5-fold by 12 and 24 hr of IL-10 pretreatment (Figure 5A). Furthermore, IL-10 pretreatment for 24 hr inhibited IKK1 and IKK2 activity (3- to 6fold) at all times examined following stimulation with LPS (Figure 5B) or anti-CD40 Ab (Figure 5C). In contrast, co-treatment of DC with IL-10 and LPS had no significant effect on IKK activity relative to treatment with LPS-only (Figure 5D). Together, these results demonstrate that IL-10 pretreatment influence the NF-κB signaling pathway in DC by reducing phosphorylation and subsequent activation of IKK1 and IKK2 activity.
IL-10 pretreatment inhibits Akt activation.
Akt has been reported to regulate NF-κB in an inducer and cell specific manner.33-37 The effect of IL-10 pretreatment on Akt activation was assessed by measuring phosphorylation of the C-terminus Ser473. Western blot analysis using Ab specific for phospho-Ser473 demonstrated that LPS stimulation induced Akt phosphorylation (Figure 6A). In contrast, IL-10 pretreatment markedly inhibited LPS-induced phosphorylation 3to 5-fold, although phosphorylated Akt was detected after 12 hr of LPS stimulation (Figure 6A). Notably, Akt protein expression was comparable between the two treatment groups (Figure 6A).
Next, Akt kinase activity was measured in DC lysates. LPS stimulation alone induced persistent Akt kinase activity based on in vitro phosphorylation of a GSK-3 substrate (Figure 6B). In contrast, IL-10 pretreatment significantly reduced Akt kinase activity up to 6 hrs post-LPS stimulation (Figure 6B), consistent with Akt phosphorylation (Figure 6A). Densitometric analysis indicated 6- to 8- fold inhibition of Akt kinase activity in IL10 pretreated DC, whereas Akt protein expression was comparable between the two treatment groups (Figure 6B). Notably, IL-10 pretreatment of isolated, splenic DC also resulted in an 8-fold reduction of LPS induced Akt kinase activity (Figure 6C). These results indicate that Akt activation and kinase activity are suppressed at early time points in both cultured and isolated DC following IL-10 pretreatment.
PI3K inhibition suppresses activation of Akt, IKK and NF-κB, and inhibits the APC function of DC.
Since upstream PI3K activation is required for Akt phosphorylation, the effect of PI3K inhibitors on Akt, and subsequent IKK and NF-κB activation was investigated. DC were treated 1 hr prior to LPS stimulation with PI3K inhibitors LY294002 (50 mM) or wortmannin (200 nM). Incubation with the PI3K inhibitors resulted in marked inhibition of Akt phosphorylation (3- to 9-fold) at all time points examined following LPS stimulation (Figure 7A). No significant difference in Akt protein expression, however, was observed between the two treatment groups (Figure 7A). Importantly, at 200 nM wortmannin and 50 mM LY294002, maximum inhibition of Akt phosphorylation was obtained with >94% viability of DC as determined by flow cytometry.
The effect of the PI3K inhibitors on IKK activity and NF-κB activation was investigated. In vitro IKK1 and IKK2 activity was reduced 3- to 7-fold and 4- to 8-fold, respectively, by treatment with wortmannin or LY294002 prior to LPS stimulation (Figure 7B&C). No significant difference was observed in the level of IKK1 or IKK2 protein in extracts used to measure kinase activity (Figure 7B&C). Kinetic analysis of LPS stimulation indicated reduced NF-κB DNA binding activity at all time points tested when DC were pretreated with either wortmannin or LY294002 (Figure 7D). DNA binding of NF-κB was reduced 2- to 6-fold under the respective conditions (Figure 7D). Similar levels of OCT-1 DNA binding were observed demonstrating that suppression of nuclear DNA binding was NFκB-specific (Figure 7D). Collectively, these results indicate that inhibition of PI3K
activity suppresses downstream activation of Akt, IKK and NF-κB.
Finally, the effects of PI3K inhibition on the capacity of cultured DC to secrete proinflammatory cytokines following LPS stimulation, and stimulate T cells was determined. Pretreatment with 50 mM LY294002 reduced levels of LPS-induced IL-12p70 and TNFα secretion by 6- and 4-fold, respectively (Table 2). Similarly, pretreatment of DC with 200 nM wortmannin inhibited the secretion of IL-12p70 and TNFα by 4-fold subsequent to LPS stimulation (Table 2). Pretreatment with LY294002 or wortmannin also significantly suppressed the stimulatory capacity of peptide pulsed DC to activate NOD.CL4 CD8+ T cells as determined by IL-2 secretion (Table 3). These results demonstrate that pretreatment with PI3K inhibitors significantly suppresses the APC function of DC.
DISCUSSION IL-10 is a potent inhibitor effecting the expression of pro-inflammatory molecules and the function of a number of cell types such as T cells, monocytes, macrophages and a variety of non-immune cells.52 IL-10 has also been shown to effectively block the maturation and APC function of myeloid-derived DC.3-11 In agreement with these studies, pretreatment of immature DC with IL-10 effectively suppressed: i) IL-12p70 and TNFα secretion following stimulation with LPS or antiCD40 Ab (Figure 1A&B), and ii) the capacity to stimulate CD8+ T cells (Figure 1D). The latter correlated with a limited increase of surface CD40 and CD86 (and IAg7) expression by IL-10 pretreated DC stimulated with LPS (Table 1).
The key molecular events involved in the immunoregulation of DC by IL-10 have yet to be delineated. Different mechanisms associated with IL-10-mediated immunoregulation have been reported which are dependent on the cell type and/or the signaling pathway engaged. For example, IL-10 suppresses TNFα gene expression in LPS stimulated human macrophages through transcriptional and post-transcriptional events independent of NF44
κB.
Furthermore, IL-10 inhibits LPS-induced activation of macrophages by eliciting
expression of suppressor of cytokine signaling 3.53 On the other hand, IL-10 mediated suppression of NF-κB activation has been observed in a variety of cell types, and different mechanisms modulating the pathway have been reported.40-43 For instance, IL10 has been shown to selectively upregulate expression of IκBα in brain astrocytes to block NF-κB dependent expression of the nitric oxide synthase-2 gene.54 Evidence that the NF-κB pathway is also targeted in DC comes from two studies showing that IL-10
prevents nuclear translocation and DNA binding of RelB in human DC precursors or immature DC, respectively.9,10 We and others have reported that blockade of NF-κB activation in general, significantly effects the maturation and APC function of DC.19,21-23 NOD DC transfected with a vector encoding a modified IκBα molecule that inhibits NFκB activation exhibit a phenotype analogous to NOD DC treated with IL-10, namely
reduced levels of IL-12p70 and TNFα secretion upon stimulation, and an impaired ability to stimulate naïve T cells.22,23 The current study provides insight into the molecular basis for IL-10-mediated immunoregulation of immature DC by demonstrating that the NF-κB pathway is inhibited through suppression of IKK activity.
Consistent with the suppression of IKK activity, various downstream events associated with NF-κB activation were inhibited by IL-10 pretreatment. DNA binding of nuclear NF-κB was blocked independent of the stimulus or the oligonucleotide probe used for EMSA via IL-10 pretreatment (Figures 2). Notably, the kinetics of IL-10 mediated suppression of NF-κB DNA binding (Figure 2A&B) and IKK activity (Figure 5A) coincided. For example, maximum inhibition of each event was detected following 12 to 24 hr of IL-10 pretreatment. Supershift analysis also demonstrated that under optimal conditions of IL-10 pretreatment, consisting of 24 hr incubation with 50 ng/ml of IL-10, DNA binding of the different NF-κB components was uniformly suppressed (Figure 2C). However, DNA binding of p50/p50 homodimers compared to heterodimeric complexes was markedly reduced after 6 hr of IL-10 pretreatment (Figure 2A), a pretreatment time found to be suboptimal for all other parameters examined. This latter observation indicates that sensitivity to IL-10 and its effects on DNA binding can vary among the NF-
κB complexes. Wang et al. demonstrated that IL-10 may inhibit proteasomal processing
of the p100 and p105 subunits of NF-κB, thereby blocking synthesis of p52 and p50, respectively.40 Accordingly, IL-10 may also effect NF-κB signaling by blocking p105 phosphorylation and subsequent processing.
The lack of DNA binding by nuclear NF-κB correlated with diminished nuclear translocation of p65 (Figure 3D). This result is compatible with reduced IKK mediated phosphorylation of the IκB proteins resulting in persistent retention of NF-κB complexes in the cytoplasm. Indeed, IL-10 pretreatment reduced phosphorylation and/or degradation of IκBα and IκBε following anti-CD40 Ab or LPS stimulation (Figure 3A&B). Surprisingly, IL-10 pretreatment failed to prevent degradation of IκBβ following LPS (or anti-CD40 Ab) treatment (Figure 3A) suggesting that IL-10 differentially effects IκB protein degradation. Furthermore, LPS induced phosphorylation of p65 Ser536 was effectively suppressed by IL-10 pretreatment (Figure 3D). Recently, Yang et al. reported that phosphorylation of Ser536 located in the trans-activation domain of p65 was dependent on IKK2 activity.49 Therefore, IL-10 mediated suppression of IKK may directly reduce the transcription function of p65 containing NF-κB complexes.
The finding that IL-10 pretreatment inhibited IKK phosphorylation and activity agrees with earlier work by our group.43 IL-10 treatment was found to suppress TNFα-induced IKK activity in vitro in the human monocytic cell line THP-1. Interestingly, the suppressive effect was observed in THP-1 cells with either no or a brief i.e. 5 min pretreatment period with IL-10. In contrast, IKK activity (Figure 5A) and subsequent NF-
κB activation (Figure 2A&B) were effectively suppressed in immature DC only after a 12
to 24 hr pretreatment with IL-10. This result is consistent with reports demonstrating that the effects of IL-10 on immature DC maturation and APC function are typically observed after 24 hr of pretreatment.3-11 These observations indicate that in immature DC, inhibition of the NF-κB pathway by IL-10 requires de novo protein synthesis. Moreover, the difference between THP-1 cells and DC regarding the need for IL-10 pretreatment suggests that cell-specific mechanisms are involved in the suppression of IKK activity, even for the same signaling pathway. In view of its dominant role activating NF-κB in response to pro-inflammatory signals,26-28 a block in IKK2 activity likely accounts for the suppressive effects of IL-10. Although a recent study reported that NF-κB activation in DC via CD40 signaling is IKK2 dependent, whereas LPS stimulation is not.24
The effects of IL-10 on IKK activity are likely to be mediated via multiple mechanisms. The transient effect of IL-10 on Akt activation (Figure 6A&B), supports this hypothesis. IL-10 pretreatment for example, may interfere with assembly and/or maintenance of an active IKK complex. Oligomerization of IKKγ has been shown to be essential for proper assembly and autophosphorylation of the IKK complex.55,56 IL-10 may inhibit the function or expression of proteins such as chaperones that are involved in assembling the heteromeric complex and/or maintaining the conformation between IKK2 and the IKKγ scaffold necessary for efficient IKK2 autophosphorylation.
As an initial effort to define the mechanism(s) by which IL-10 inhibits IKK activity in immature DC, a role for the PI3K signaling pathway was investigated. We and others
have demonstrated that NF-κB activation can be mediated through the PI3K/Akt pathway.31-39 Furthermore, a recent study reported that the survival of LPS-stimulated monocyte derived human DC is markedly reduced by inhibition of PI3K and downstream phosphorylation of Akt.57 Accordingly, LPS-mediated phosphorylation (Figure 6A) and in vitro kinase activity of Akt (Figure 6B) were significantly inhibited by IL-10 pretreatment, suggesting that the PI3K pathway in DC is indeed a target of IL-10. Consistent with this conclusion are findings that pretreatment with wortmannin or LY294002 inhibited Akt phosphorylation (Figure 7A), in vitro IKK1 and IKK2 activity (Figure 7B&C), and NF-κB DNA binding induced by LPS (Figure 7D). Moreover, the PI3K inhibitors significantly suppressed LPS-induced secretion of IL-12p70 and TNFα (Table 2), and the capacity of DC to stimulate naïve CD8+ T cells (Table 3). Collectively, these results demonstrate that the PI3K signaling pathway has a key role in regulating the activation and APC function of DC. Interestingly, the effects of IL-10 on the PI3K pathway, and in turn the role of PI3K in NF-κB activation appear to be dependent on the cell type. In contrast to our results, IL-10 stimulates PI3K and Akt activation in murine progenitor myeloid cells.58 Furthermore, suppression of PI3K enhances LPS induced NFκB activation in human monocytes.
39
Currently the focus of ongoing efforts is to define
the mechanism(s) by which the PI3K pathway is suppressed by IL-10 in DC.
Importantly, the effects of IL-10 on bone marrow-derived DC were also evident for myeloid-like DC isolated directly from the spleen. IL-10 pretreatment of splenic DC ex vivo effectively inhibited NF-κB DNA binding (Figure 2F) and Akt kinase activity (Figure 6C) induced by LPS stimulation to a similar extent observed in cultured DC.
These findings indicate that the inhibitory effects of IL-10 are applicable to myeloid-like DC in general.
In summary, this work demonstrates that IL-10 effectively immunoregulates the APC function of DC through suppression of IKK activity and subsequent NF-κB activation. Inhibition of NF-κB activation is in part the consequence of IL-10 targeting the PI3K pathway.
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Table 1. Phenotypic analysis of bone marrow-derived DCa
Untreated
IL-10b
LPSb
IL-10+LPSc
Unstained
14
13
13
13
CD11b
104
110
113
86
CD11c
63
79
70
64
CD40
22
22
100
40
CD80
35
35
161
153
CD86
40
44
210
77
H2Db
64
62
91
83
IAg7
67
59
324
112
a
Bone marrow DC were cultured for 10 days as indicated in Materials and Methods. The
data are presented as mean fluorescence intensities. b
Treatment for 24 hrs.
c
Pre-treatment with IL-10 for 24 hrs followed by LPS stimulation for an additional 24
hrs .
Table 2. PI3K inhibitors suppress secretion of pro-inflammatory cytokines of DC
TNF-α (pg/ml)
IL-12 p70 (pg/ml)
Untreated
24±6
49±3
LPSa
1036±161
1831±134
IL-10b+LPS
156±21d
263±43d
LYc+LPS
310±28e
402±131e
Woc+LPS
284±40e
501±16d
a
Treatment for 48 hrs.
b
Pre-treatment for 24 hrs.
c
Pre-treatment for 1 hr.
d
p < 0.01 vs LPS (Student t test).
e
p < 0.02 vs LPS (Student t test).
Table 3. PI3K inhibitors suppress APC function of DC
IL-2 (pg/ml) Untreated HAa
22±4 1546±124
IL-10b+HA
348±18d
LYc+HA
571±48e
Woc+HA
647±28e
a
Treatment for 1 hr.
b
Pre-treatment for 24 hrs.
c
Pre-treatment for 1 hr.
d
p < 0.01 vs HA (Student t test).
e
p < 0.02 vs HA (Student t test).
FIGURE LEGENDS
Figure 1. IL-10 pretreatment suppresses secretion of pro-inflammatory cytokines and APC function of DC. Bone marrow derived DC were pretreated with varying doses of IL-10 for 24 hr, stimulated with (A) 10 µg/ml of anti-CD40 Ab or (B) 500 ng/ml of LPS for 48 hr, and levels of IL-12p70 (open circle) and TNF-α (closed circle) measured in culture supernatants by ELISA. Levels of pIL-12p40 and TNFα were below 25 pg/ml in the absence of (A) anti-CD40 Ab or (B) LPS stimulation. (C) DC were stimulated with varying concentrations of LPS alone (solid circle or triangle) or co-treated with 50 ng/ml of IL-10 for 48 hr (open circle or triangle), and the amount of IL-12p70 (circle) and TNFα (triangle) measured in culture supernatants. (D) DC were pretreated with varying concentrations of IL-10 for 24 hr, pulsed with 5µg/ml of HA peptide, and cultured with 3.5x105 NOD.CL4 T cells for 48 hr. Levels of IL-2 secretion in culture supernatants were measured via ELISA. Less than 20 pg/ml of IL-2 was detected in cultures lacking HA peptide. Data represent mean ±SD of four individual cultures and are representative of three independent experiments. *P
