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. 2015 Nov 2;14(4):380–391. doi: 10.1038/cmi.2015.90

Interferon gamma boosts the nucleotide oligomerization domain 2-mediated signaling pathway in human dendritic cells in an X-linked inhibitor of apoptosis protein and mammalian target of rapamycin-dependent manner

Tünde Fekete 1, Gabor Koncz 1,2, Brigitta Szabo 1, Andrea Gregus 1, Eva Rajnavölgyi 1,2
PMCID: PMC5380943  PMID: 26521691

Abstract

The cytoplasmic nucleotide oligomerization domain 2 (NOD2) receptor recognizes the bacterial cell wall component muramyl dipeptide (MDP). NOD2 ligation initiates the nuclear factor kappa B and the mitogen-activated protein kinase cascades. However, administering MDP alone is insufficient to elicit strong cytokine responses in various immune cells, including dendritic cells (DCs). Because the simultaneous presence of various microbial products and cytokines in inflamed tissues modulates DC function, we initiated this study to examine how interferon gamma (IFNγ), a central modulator of inflammation, affects the NOD2-mediated signaling pathway in human conventional DCs (cDCs). Synergistic stimulation of DCs with MDP and IFNγ increased the expression of CD40, CD80, CD83, CD86, and human leukocyte antigen DQ proteins and significantly elevated the production of pro-inflammatory cytokines IL-1β, IL-6, IL-12, and tumour necrosis factor (TNF), as well as anti-inflammatory cytokine IL-10. Furthermore, the simultaneous presence of MDP and IFNγ was necessary to decrease IkBα protein levels. By investigating various mechanisms implicated in MDP- and IFNγ-mediated signaling pathways, we revealed that the increased production of pro-inflammatory cytokines is highly dependent on the X-linked inhibitor of apoptosis protein (XIAP) but not on cellular IAP1 and IAP2. We also found that the NOD2 signaling pathway is regulated by the mammalian target of rapamycin (mTOR) but is not affected by phosphatidylinositol-3 kinase or signal transducer and activator of transcription 1 inhibition. Our results demonstrate, for the first time, that IFNγ positively affects NOD2-mediated signaling in human cDCs, in a manner considerably dependent on XIAP and partially dependent on mTOR.

Keywords: dendritic cell, mTOR, NOD2, XIAP

Introduction

Dendritic cells (DCs) have the potential to sense a wide range of microbial structures via multiple pattern recognition receptors (PRRs). The intracellular NOD-like receptors (NLR) are crucial sensors of defined bacterial structures. Among them, nucleotide oligomerization domain 2 (NOD2) is specialized in the recognition of muramyl dipeptide (MDP), a cell wall component of both Gram-positive and Gram-negative bacteria.1 Signaling through NOD2 culminates in the stimulation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) cascades and the subsequent upregulation of inflammatory and host defense genes. It has recently been shown that single-stranded RNA can initiate the NOD2 signaling pathway, leading to the activation of the interferon regulatory factor 3 (IRF3) transcription factor that drives the production of type I interferons.2

Members of the inhibitor of apoptosis protein (IAP) family were initially identified as apoptosis regulators.3 Later, it was found that their E3 ubiquitin ligase activity enables them to control innate immune responses.3 The polyubiquitination of receptor-interacting serine-threonine kinase 2 (RIP2) is a pivotal subsequent step in NOD2 stimulation that is directed by IAPs including cellular IAP1 (cIAP1), cIAP2, and X-linked IAP (XIAP). Macrophages in mice deficient in the cIAP1 and cIAP2 genes show defects in the activation of the MAPK and NF-κB signaling pathways.4 The same report has also demonstrated that the loss of RIP2 or cIAP2 protects mice from dextran sulphate sodium-induced colitis, suggesting a key role for cIAPs in regulating NOD2-mediated signaling. Another report has also shown that XIAP-deficient mice are unable to produce tumour necrosis factor (TNF) and IL-6 cytokines upon MDP treatment.5 Moreover, XIAP has been found to be essential for the formation of the receptor-signaling complex by recruiting the linear ubiquitin chain assembly complex (LUBAC) required for NOD2-mediated NF-κB activation.5

The potency of MDP alone to activate immune cells is low. However, MDP can act in synergy with various toll-like receptor (TLR) ligands and inflammatory cytokines to induce strong immune responses. NOD receptors cooperate with TLR2 to regulate human DC maturation.6 MDP administration in combination with ligands specific to TLR3, TLR4, and TLR9 increases the production of IL-12 and interferon gamma (IFNγ) and also enhances the T helper type 1 (Th1)-polarizing capacity of human monocyte-derived DCs (moDCs).7 Furthermore, co-stimulation of human moDCs with TLR2 ligand lipoteichoic acid and MDP augments DC maturation and induces the concomitant secretion of pro-inflammatory cytokines.8 MDP-Lys, a stearoyl-MDP derivative in combination with IFNα or IFNβ induces human moDCs to produce IL-12 and stimulates the differentiation of IFNγ-producing T-cells. It has also been demonstrated that co-administration of MDP-Lys with IFNβ significantly suppresses melanoma growth in mice.9 IFNγ has also been implicated in increasing the amplitude of NOD2-mediated responses because intravitreal co-injection of MDP and IFNγ results in the exacerbation of ocular inflammation in mice.10 Mouse bone marrow-derived macrophages produce nitrogen-monoxide upon NOD2 stimulation, specifically when the cells are primed with IFNγ.11 It has also been demonstrated that IFNγ synergistically acts with MDP to activate the antitumor activity of human blood monocytes.12

Based on these findings, IFNγ appears to be an important and abundant mediator of inflammation. IFNγ dominantly signals through the Janus kinase and the signal transducer and activator of transcription (STAT) pathway. The activation of STAT1 by IFNγ leads to the formation of homodimers that translocate to the nucleus and initiate the transcription of IFN-stimulated genes.8 In addition to the well-known STAT1-dependent mechanisms, IFNγ can also activate the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is required for the phosphorylation of STAT1 and the activation of gene expression in response to IFNγ.13 The mammalian target of rapamycin (mTOR), which acts as a downstream mediator of PI3K, also plays an important role in IFNγ-driven signaling by controlling events involved in mRNA translation.14 Furthermore, IFNγ activates several members of the MAPK cascade, including p38, extracellular signal-regulated kinase 1/2, and c-Jun.15,16

In the current study, our aim was to characterize the role of IFNγ in the NOD2-mediated inflammatory responses of human DCs. We found that the co-administration of MDP and IFNγ augmented DC activation, which required the contribution of XIAP. Furthermore, we also identified the contribution of the mTOR pathway to the MDP- and IFNγ-induced production of IL-6.

Results

MDP in combination with IFNγ activates moDCs more efficiently than MDP alone

To analyze the role of IFNγ in the MDP-induced stimulation of moDCs, we first tested the expression levels of membrane-bound activation-dependent molecules, including the CD83 maturation marker, the CD80 and CD86 costimulatory molecules, the chemokine receptor CCR7 (which drives DC migration from the periphery to the draining lymph nodes), and the Major Histocompatibility class II human leukocyte antigen DQ (HLA-DQ). Five-day moDCs were left untreated or were cultured in the presence of MDP (10 µg ml−1), IFNγ (10 ng ml−1), or both MDP and IFNγ. After a 24-h stimulation, the cells were analyzed by flow cytometry. In line with previous reports,9,17 MDP and IFNγ alone had no significant effects on the protein expression levels of CD40, CD80, CD83, CD86, and HLA-DQ (Figure 1). However, the synergistic stimulation of moDCs with MDP and IFNγ significantly enhanced the fluorescence intensities of all tested molecules. Interestingly, CCR7 upregulation was more prominent when IFNγ was used as a single stimulus compared to stimulation by MDP and IFNγ, indicating the essential role of IFNγ in this process.

Figure 1.

Figure 1

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MDP and IFNγ used in combination upregulate the expression of various DC maturation markers. moDCs were treated with MDP, IFNγ, or both MDP and IFNγ or were left untreated. After 24 h, the cells were subjected to flow cytometry analysis. Bars represent the mean ± SD of median fluorescence intensity (MFI) from at least four independent experiments.

Next we analyzed the secreted cytokine profile of moDCs when stimulated as described above (Figure 2). The results confirmed that MDP alone was a weak activator of pro-inflammatory cytokines IL-6, IL-23, and TNF, as well as anti-inflammatory mediator IL-10. Additionally, IL-1β in the supernatant of MDP-treated moDCs did not reach detectable levels. IFNγ alone was a poor inducer of IL-6, IL-10, and IL-12 and could not elicit the secretion of TNF and IL-1β. However, when MDP and IFNγ were used in combination, the production of all cytokines tested significantly increased. To clarify whether the elevated levels of IL-1β are a consequence of increased gene transcription or are due to the release from pre-formed granules, we measured the gene expression of IL-1β with qualitative polymerase chain reactiong (qPCR) after 6 h of stimulation (Supplementary Figure S1). We found that the mixture of MDP and IFNγ increased the transcriptional levels of IL-1β, which is in line with the significantly elevated levels of bioactive IL-1β protein. Overall, these results indicate that IFNγ is an efficient inducer of NOD2-mediated activation and concomitant cytokine production by moDCs.

Figure 2.

Figure 2

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IFNγ augments the MDP-stimulated pro- and anti-inflammatory cytokine production of moDCs. moDCs were treated with MDP (10 µg ml−1), IFNγ (10 ng ml−1), or both MDP and IFNγ or were left untreated. After 24 h, cell culture supernatants were collected, and cytokine levels were determined by ELISA. Data represent the mean ± SD of at least four independent experiments.

IFNγ boosts the MDP-induced cytokine production of blood circulating CD1c+ DCs

moDCs, together with primary CD1c+ and CD141+ DCs, belong to the family of classical/conventional DCs (cDCs).18 To reveal how DCs derived from circulating DC precursors respond to combined stimulation with MDP and IFNγ, we repeated the experiments with blood circulating CD1c+ DCs (Figure 3). The cells were stimulated with both MDP and IFNγ directly after isolation, and 24 h later, the supernatants of the CD1c+ DCs were collected and subjected to ELISA analysis. The results showed that the production of IL-1β, IL-6, TNF, and IL-10 significantly increased upon combined stimulation with MDP plus IFNγ, but no significant changes were detected when MDP or IFNγ were used alone for moDC stimulation. In contrast to moDCs, IFNγ alone was able to induce strong TNF production that could be further increased by co-treatment with MDP. Interestingly, none of the stimuli elicited the secretion of IL-12 and IL-23 in CD1c+ DCs, indicating that these cytokines are not involved in the MDP-mediated response. To the best of our current knowledge, these results demonstrate for the first time, that CD1c+ DCs are able to respond to NOD2 stimulation, which can be enhanced by IFNγ.

Figure 3.

Figure 3

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IFNγ enhances the MDP-stimulated pro- and anti-inflammatory cytokine production of CD1c+ DCs similarly to moDCs. CD1c+ DCs were treated with MDP, IFNγ, or a mixture of MDP and IFNγ or were left untreated. After 24 h, cell culture supernatants were collected, and the cytokine levels were determined by ELISA. Data represent the mean ± SD of four independent experiments.

Synergistic stimulation of moDC with MDP and IFNγ affects both the NF-κB and the MAPK signaling pathways

NOD2 stimulation initiates the activation of the IκB kinase complex (IKK), which is followed by the degradation of inhibitory protein IκBα and the subsequent release of NF-κB protein.1 The IKK complex is composed of three subunits: IKKα, IKKβ, and IKKγ. It has previously been shown that IKKβ but not IKKα is essential for the activation of the NF-κB pathway.19 By measuring the phosphorylation status of IKKβ, we observed a significant increase upon stimulation with MDP alone and with MDP combined with IFNγ. IKKβ phosphorylation occurred earlier and was higher when moDCs were synergistically activated with MDP and IFNγ compared with stimulation with MDP alone. However, the difference did not reach statistical significance. The antibody we used also detected the phosphorylation of IKKα, which was undetectable upon NOD2 stimulation. Nevertheless, the time course analysis of IκBα degradation revealed that the treatment with MDP in combination with IFNγ significantly reduces the levels of IκBα (Figure 4a). Contrary to this finding, the stimulation of moDCs with the individual stimuli resulted in a minor decrease in IκBα protein levels.

Figure 4.

Figure 4

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MDP and IFNγ co-treatment affects both NF-κB and MAPK signaling pathways. moDCs were stimulated with MDP, IFNγ, or both MDP and IFNγ or were left untreated. (a) Kinetics of IKKβ phosphorylation and IκBα degradation were determined by western blotting. Bar graphs show the IkBα/β-actin and phospho-IKKβ/IKKβ ratios measured at 60 min of stimulation. Bars represent the mean ± SD of four independent experiments. A representative blot (data set) is shown. (b) The cells were lysed 20 min after activation, and the phosphorylation of MAPKs was detected and analyzed according to the manufacturer's protocol. The results of two independent experiments are shown.

Given that NOD2 stimulation can also culminate in the activation of the MAPK pathway,20 we also tested the activity of several MAPKs with the use of a phospho-protein array (Figure 4b). These data confirmed that the stimulation of moDCs with MDP alone or in combination with IFNγ led to the phosphorylation of a defined set of MAPK family members, including ribosomal protein S6 kinase (RSK1). Furthermore, the addition of MDP to moDCs resulted in the activation of Akt2, p53, and p38γ, which was counteracted by the addition of IFNγ. In contrast, the MDP-induced phosphorylation of mitogen-activated protein kinase kinase 6 (MKK6) and mitogen- and stress-activated protein kinase 2 (MSK2) further increased after co-administration of IFNγ, confirming that IFNγ has the potential to modulate the MDP-mediated activity of MAPK family members to different extents.

Expression of cIAP1, cIAP2, and XIAP are regulated differently by the NOD2 signaling pathway

IAP ubiquitin ligases have been shown to be involved in signaling processes of several receptors including cytokine receptors, TLRs, and NLRs.21 Thus, we sought to investigate the expression levels of cIAP1, cIAP2, and XIAP in moDCs at both the mRNA and protein levels (Figure 5). For this experiment, the control cells were left untreated, were stimulated with MDP alone, or were stimulated with MDP in combination with IFNγ in a time-dependent manner. The results of the kinetic studies revealed that XIAP was consistently expressed in moDCs at the mRNA level over time and was not affected by any of the applied stimuli (Figure 5a). The transcript levels of cIAP1 were not modulated when MDP or IFNγ was used separately. However, when MDP and IFNγ were simultaneously added to the moDC cultures, we observed a moderate but significant upregulation of cIAP1 expression. Interestingly, the expression of cIAP2 was significantly upregulated by both MDP alone and MDP in combination with IFNγ. To confirm these results at the protein level, the cell lysates were collected at 12 and 24 h after activation and were subjected to western blot analysis (Figure 5b). Similar to the results obtained with qPCR analysis, we did not observe changes in the protein level of XIAP. Although the levels of cIAP1 protein were slightly increased upon activation with MDP alone and MDP in combination with IFNγ, these changes did not reach statistical significance. More intriguingly, cIAP2 protein was scarcely detectable in resting moDCs, but its expression was dramatically upregulated upon stimulation with MDP alone or MDP in combination with IFNγ, showing an increased response to dual stimuli. These results suggest that IFNγ modulates the expression of IAP proteins in response to NOD2 stimulation.

Figure 5.

Figure 5

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The expression levels of cIAP1, cIAP2, and XIAP proteins are differentially regulated by MDP and IFNγ. moDCs were activated by MDP, IFNγ, or their combination or were left untreated. (a) The upper panel represents the kinetics of cIAP1, cIAP2, and XIAP mRNA expression. The lower panel shows fold changes in gene expression compared to the untreated control (taken as 1) measured at 12 h after moDC stimulation. (b) Protein levels of cIAP1, cIAP2, and XIAP were determined in a time-dependent manner by western blotting. Bar graphs represent the mean ± SD of five independent experiments. (c) Representative blots are shown.

The production of inflammatory cytokines TNF and IL-6 is dependent on XIAP but not cIAP1 or cIAP2

To identify the possible regulatory role of IAP proteins, moDCs were transfected by siRNAs specific for cIAP1, cIAP2, or XIAP or scrambled siRNAs (as a control) on day 3 of DC differentiation. The efficacy of gene silencing was verified by western blot analysis at 48 h post-transfection (Supplementary Figure S2a). Cells were left untreated or were stimulated with MDP, IFNγ, or both MDP and IFNγ. Because our previous results verified the robust production of TNF and IL-6 in response to MDP and IFNγ co-administration (Figure 2), we tested the production of these cytokines after 24 h by ELISA (Figure 6a). The silencing of cIAP1 and cIAP2 had no effect on the secretion of TNF and IL-6, suggesting that none of these proteins play a role in the regulation of MDP- and IFNγ-mediated signaling. In contrast, XIAP depletion resulted in a significant decrease in the production of TNF and IL-6, pointing to the crucial role of XIAP in regulating moDC functionality upon dual stimulation with MDP and IFNγ.

Figure 6.

Figure 6

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Pro-inflammatory cytokine production of moDCs is regulated by XIAP and mTOR, but not by STAT1 and PI3K. (a) Three-day moDCs were transfected with siRNAs specific for cIAP1, cIAP2, or XIAP or scrambled siRNAs for 48 h. On day 5, the cells were activated with MDP, IFNγ, or with a mixture of MDP and IFNγ for 24 h. (b) Cells were transfected with siRNAs specific for STAT1 or scrambled siRNAs, followed by treatments as indicated in (a). (c) Five-day moDCs were left alone or were pre-treated with LY294002 (10 µM) or rapamycin (100 nM) for 2 h. The cells were washed, and they were activated by MDP, IFNγ, or both MDP and IFNγ or were left untreated. After 24 h of stimulation, the supernatants were collected, and the levels of TNF and IL-6 were assessed by ELISA. The results represent the mean ± SD of at least three independent experiments.

Contribution of the STAT1 and the PI3K/mTOR signaling pathways to the MDP- and IFNγ-induced moDC activation

To identify the molecular mechanism behind increased DC activity in response to MDP and IFNγ stimulation, we investigated the involvement of a candidate signaling pathway targeted by IFNγ. STAT1, a major downstream modulator of IFNγ, was depleted with siRNA silencing, as previously described (Supplementary Figure S2b). Interestingly, STAT1 depletion did not affect the production of IL-6 and TNF upon costimulation with MDP and IFNγ, excluding the direct regulatory role of STAT1 in this signaling pathway (Figure 6b).

The PI3K/mTOR pathway represents a central regulator of many different cellular processes and innate immune responses.22 To dissect whether the PI3K/mTOR pathway was engaged with NOD2- and IFNγ-mediated signaling events, we initiated blocking experiments using two different strategies: treatment of moDCs with the synthetic inhibitor LY294002, which targets PI3K activity, and using rapamycin to inhibit mTOR function. In these experiments, the cells were pre-treated with the inhibitory drugs for 2 h followed by thorough washing procedures to eliminate the blocking agents. After this step, the cells were incubated in the presence of MDP, IFNγ, or MDP plus IFNγ or were left untreated for 24 h. As shown in Figure 6c, neither PI3K-mediated inhibition nor mTOR blockade affected the production of TNF induced by the co-administration of MDP and IFNγ. In contrast to this finding, the production of IL-6 was diminished as a result of rapamycin pre-treatment, suggesting a potential regulatory role of mTOR in the NOD2- and IFNγ-mediated signaling pathway. Overall, the results suggest the involvement of mTOR in MDP- and IFNγ-induced DC activation and exclude the role of STAT1 and PI3K in this signaling cascade.

Discussion

DCs are important players in the immune system, and their display of various PRRs allows them to sense a wide range of pathogen- and damage-associated molecules.23 In inflamed tissues, resting DCs encounter a diverse set of microbial structures and are affected by the cytokine milieu established by surrounding cells, such as activated lymphocytes, epithelial cells, and fibroblasts.24 In the current study, we set out to explore how IFNγ, an inflammatory cytokine produced abundantly in response to infections by natural killer, Th1, and cytotoxic T-lymphocytes, might affect the NOD2-mediated activation of human cDCs. We demonstrated for the first time that MDP synergistically acts with IFNγ in stimulating DC maturation. We also found that the priming capacity of IFNγ on the NOD2-mediated production of pro-inflammatory cytokines depended on XIAP and partially on mTOR.

NLRs can cooperate with different TLRs and cytokines to initiate the maturation of various immune cells, including DCs.9,11,25 In the present study, we show that MDP itself is a weak inducer of DC activation because it cannot induce remarkable upregulation of moDC maturation markers and co-stimulatory molecules. Our results are in line with those from previous publications, indicating that MDP fails to provoke full DC maturation.17,25,26 Nevertheless, MDP in combination with IFNγ had a strong potential to induce increased expression of CD83, CD40, CD80, CD86, and HLA-DQ. It has also been found that IFNγ upregulates CD40, CD80, and MHC class II molecules in immortalized murine DC2.4 cells27 and CD86 in human moDCs.28 However, this result was not observed in our experimental system. IFNγ efficiently increases the expression of the chemokine CCR7 in human moDCs29 and in the DC2.4 cell line.27 In line with these results, we also detected elevated levels of CCR7 in moDCs stimulated with IFNγ. Interestingly, a single stimulation by IFNγ induced a more robust upregulation of CCR7 expression than the mixed stimulation of MDP and IFNγ, a phenomenon that requires further clarification.

It has been known that IFNγ promotes the TLR-induced production of inflammatory mediators, such as nitric oxide and IL-12, in bone marrow-derived macrophages, pointing to its priming ability in innate immune responses.30 IFNγ synergizes with MDP in inducing enhanced nitric oxide production in macrophages11 and augmenting ocular inflammation in an in vivo mouse model.10 Our present study demonstrates that the administration of IFNγ to moDCs significantly increases the NOD2-mediated secretion of both pro- and anti-inflammatory cytokines. However, it is important to note that the cytokine profile of primary CD1c+ DCs differs from that of moDCs.31 In our hands, IFNγ alone was able to induce efficient TNF secretion in this unique DC subset, whereas none of the applied stimuli elicited the production of IL-12 and IL-23. Increased TNF production induced by IFNγ stimulation can be explained by the concurrent presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the CD1c+ DCs culture required for the maintenance of cell survival.32 GM-CSF synergistically enhances the IFNγ-induced production of TNF in human monocytes.33 Furthermore, our observations pertaining to the diverse cytokine responses of these cDC subsets are in line with published reports34,35,36 suggesting that CD1c+ DCs display different functional properties than those of their monocyte-derived counterparts.

Induction of cytokine production by moDCs requires the activation of the NF-κB pathway, which acts through RIP2 in response to NOD2 stimulation. RIP2 is responsible for the polyubiquitination of IKKγ, which leads to IKKβ phosphorylation and the subsequent degradation of IκBα.1 To determine how NOD2-mediated NF-κB activation could be affected by IFNγ, we followed the time-dependent phosphorylation of IKKβ in parallel with IκBα degradation. Compared to MDP alone, the simultaneous presence of MDP and IFNγ marginally increased the phosphorylation of IKKβ but induced the degradation of IκBα to a significant level an hour after stimulation. Furthermore, remarkable differences were detected in the activity of the MAPK pathway upon stimulation of moDCs with MDP alone or in combination with IFNγ. The MDP-induced phosphorylation of Akt2, p53, and p38γ was counteracted by the presence of IFNγ, whereas the activity of MKK6 and MSK2 was further increased. We speculate that decreased p38γ phosphorylation might be a consequence of decreased MKK3 activity (data not shown). However, the contribution of MKK3 and MKK6, the major upstream mediators of p38γ, depends on the cell type, the nature, and the magnitude of the given stimulus.37 MSK2 acts downstream of MKK6 and has been found to negatively regulate p53 activity.38 Published reports also suggest that MSK2, together with MSK1, promotes lipopolysaccharide(LPS)-induced IL-10 production. Based on these results, we hypothesize that enhanced MSK2 activity might contribute to increased IL-10 production in moDCs as a result of MDP and IFNγ stimulation.

Our results showed that the priming effect of IFNγ on NOD2-mediated signaling is an early event, and this led us to hypothesize that this phenomenon might be driven by regulatory factors already present in the cytoplasm. In an attempt to identify the link between the NOD2- and IFNγ-mediated signaling pathways, we initially assessed the possible regulatory role of IAP family members because they are critical regulators of inflammatory processes. Based on published studies, cIAP1 and cIAP2 proteins are weakly expressed, whereas XIAP mRNA is not detectable in moDCs.39 Another study has reported that 7-day moDCs express cIAP1, cIAP2, and XIAP proteins.40 Our results showed that cIAP1 and XIAP were constitutively expressed in moDCs both at the mRNA and protein levels, which could not be further modulated by MDP or IFNγ. In contrast to these findings, cIAP2 was scarcely detectable in resting moDC but was strongly upregulated with MDP alone or in combination with IFNγ. Silencing of cIAP1 and cIAP2 did not affect the secretion of pro-inflammatory cytokines IL-6 and TNF. In contrast, the depletion of XIAP resulted in significantly reduced production of IL-6 and TNF. It is important to note that in this experimental setting, the depletion of XIAP was incomplete (Supplementary Figure S2a), confirming an even more prominent role for XIAP in the regulation of NOD2-mediated signaling in moDCs. Further experiments revealed that the knockdown of cIAP1, cIAP2, and XIAP by a siRNA cocktail decreased the level of pro-inflammatory cytokines to a similar extent as the individual depletion of XIAP. This result indicates that no interplay exists between the investigated IAP molecules (data not shown). XIAP acts as an essential ubiquitin ligase in the NOD2 signaling pathway by ubiquitinating RIP2 at sites distinct from the K63- and K48-linked chains.5 XIAP recruits LUBAC, which provides additional ubiquitin chains needed to complete IKK activation.5 A published study reported that cIAP1 and cIAP2 also contribute to RIP2 ubiquitination. However, their effects are not significant in the presence of XIAP. In addition to the well-known IAP molecules, the Pellino3 protein also binds directly to RIP2 and catalyzes its ubiquitination.41 Furthermore, the authors suggested that XIAP and Pellino3 collaborate to enhance NOD2-mediated activation of NF-κB and MAPKs. In our experiments, moDCs stimulated with MDP alone or in combination with IFNγ exhibited similar levels of RIP2 ubiquitination (Supplementary Figure S3). Because co-treatment with MDP and IFNγ did not result in increased ubiquitination of RIP2, we speculate that the induction of cytokine production by XIAP could be involved in the formation of the NOD2 signalosome.

Next, we tested the possible regulatory role of STAT1, a transcription factor capable of upregulating IFNγ-responsive genes. A recent report demonstrated that STAT1 can mediate pro-inflammatory cytokine responses induced by multiple TLRs through direct interaction with TRAF6, which is a crucial mediator of TLR signaling.42 Interestingly, the depletion of STAT1 did not alter MDP- and IFNγ-induced production of pro-inflammatory cytokines.

In an attempt to further clarify the underlying molecular mechanism of IFNγ-mediated priming of NOD2 responses, we tested the possible contribution of the PI3K/mTOR signaling pathway. The results showed that neither PI3K blockade nor mTOR inhibition affects the levels of TNF production. Nevertheless, rapamycin pre-treatment of moDCs decreased IL-6 levels that were elicited by MDP and IFNγ co-stimulation. An interesting link between these signaling events is that mTOR controls the production of pro-inflammatory cytokines differentially because the secretion of IL-6 is upregulated and the production of TNF is not affected by rapamycin treatment. These results are contradictory to previous findings showing that the mTOR pathway contributes to NOD2-mediated tolerance induction by promoting anti-inflammatory and inhibiting pro-inflammatory cytokine secretion in human macrophages.43 It has also been revealed that rapamycin-resistant and rictor-independent mTOR complex 1 downregulates IL-10 production.44 mTOR differentially controls TLR-induced cytokine production by innate immune cells; this finding shows that rapamycin pre-treatment increases LPS-induced IL-6 production in CD1c+ DCs, whereas it exerts a decreasing effect in moDCs.35 Furthermore, mTOR inhibition upregulates IL-12p40 in bone marrow-derived macrophages, whereas it downregulates TNF and IFNβ in myeloid DCs stimulated with LPS or CpG oligonucleotide.45 The opposing experimental outcomes of our findings with previous observations43 could be explained by cell type-dependent differences and/or different culture conditions. We hypothesize that mTOR controls gene expression by histone modification because a link between mTOR and epigenetic events has already been proposed.46 It is known that post-translational modifications of histones, particularly acetylation and methylation, are associated with the regulation of inflammatory and cytokine gene expression.47,48 Moreover, Tsaprouni and colleagues have found that H4 acetylation is significantly upregulated in inflamed biopsies from patients with Crohǹs disease, a disease that is associated with NOD2 mutation. Thus, it would be relevant to study the epigenetic regulation in mTOR-silenced moDCs upon stimulation with various PRR ligands.

The enhancement of NOD2-mediated signaling events can also be a consequence of functional inactivation or an inhibitory feedback mechanism.49 One possibility is that IFNγ mediates the suppression of IL-10 production. Because the synergistic stimulation by MDP and IFNγ induced an immediate response and enhanced IL-10 secretion to a similar extent as pro-inflammatory cytokines, we excluded the regulatory role of this cytokine. Other mechanisms, such as the inhibition of transcriptional repressors, have also been suggested to be initiated by IFNγ, hence causing augmented TLR-activated signal transduction.49 Further experiments are needed to elucidate the full spectrum of interrelated regulatory events by which IFNγ can promote NOD2-mediated signal transduction.

In summary, our results demonstrate for the first time that IFNγ boosts NOD2-mediated inflammatory responses in human cDCs. The priming effect of IFNγ on NOD2-induced inflammatory cytokine production requires the expression of XIAP. Moreover, the underlying mechanism of IFNγ-mediated priming is independent of STAT1 and implicates a specific regulation by mTOR.

Several decades ago, MDP was identified as a component of Freund's complete adjuvant.50 Since then, its synthetic analogs have extensively been studied as vaccine components to promote immune responses for the treatment of tumors and elimination of pathogens.51 Our finding that the administration of MDP together with IFNγ exerts strong immune stimulatory effects on human cDCs is clinically relevant and provides a rationale for designing more potent vaccines against microbial pathogens and cancer cells.

Materials and methods

Reagents

IFNγ and IL-4 were purchased from PeproTech EC (London, UK). GM-CSF was purchased from Gentaur Molecular Products (Brussels, Belgium). MDP was obtained from InvivoGen (San Diego, CA, USA). Rapamycin was ordered from Merck Millipore (Darmstadt, Germany). The PI3K inhibitor LY294002 hydrochloride and dimethyl sulfoxide were purchased from Sigma-Aldrich (Schnelldorf, Germany). Rapamycin and LY294002 were used at a concentration of 100 nM and 10 µM, respectively.

DC generation and stimulation

Peripheral blood mononuclear cells (PBMCs) from healthy donors were separated from buffy coats by Ficoll gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). Monocytes were isolated by positive selection using magnetic cell separation with anti-CD14-conjugated microbeads (Miltenyi Biotech, Bergish Gladbach, Germany). The purified cells were seeded in 24-well cell culture plates at a density of 106 cells ml−1 in serum-free AIMV medium (Life Technologies Corporation, Carlsbad, CA, USA) supplemented with 80 ng ml−1 GM-CSF and 50 ng ml−1 IL-4 for 5 days. cDCs were separated from PBMCs using the CD1c isolation kit (Miltenyi Biotech) and cultured in 48-well cell culture plates at a density of 5 × 105 cells ml−1 in AIMV medium supplemented with 20 ng ml−1 GM-CSF. For activation, 5-day moDCs or freshly isolated CD1c+ DCs were cultured with MDP (10 µg ml−1), IFNγ (10 ng ml−1), or both stimuli for the indicated time period in all experiments.

Flow cytometry

Anti-CD80- fluorescein isothiocyanate (FITC), anti-CD86-PE, CCR7-FITC, and isotype-matched control antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Anti-CD40-FITC, anti-CD83-PE, anti-HLA-DQ-FITC, and isotype-matched control antibodies were ordered from BioLegend (San Diego, CA, USA). Samples were analyzed with FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software (Tree Star, Ashland, OR, USA).

Cytokine production

The levels of IL-23 were measured with the human IL-23 ELISA Ready-Set Go kit (eBioscience, San Diego, CA, USA). The concentrations of IL-1β, IL-6, IL-10, IL-12, and TNF in culture supernatants were analyzed by BD-OptEIA Human ELISA kits (BD Pharmingen, San Diego, CA, USA) according to the manufacturer's instructions.

Real-time qPCR

Total RNA was extracted using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and was reverse-transcribed using the High Capacity cDNA RT Kit of Applied Biosystems (Carlsbad, CA, USA). All gene expression assays were purchased from Applied Biosystems, except for the housekeeping gene cyclophilin (Integrated DNA Technologies, Coralville, IA, USA). qPCR was performed using the ABI StepOne Real-Time PCR System (Applied Biosystems). Cycle threshold values were determined using the StepOne v2.1 Software (Applied Biosystems). Gene expression levels were normalized to cyclophilin.

Western blotting

Protein extraction was performed by lysing the cells in Laemmli sample buffer. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 10% polyacrylamide gels and electrotransferred onto nitrocellulose membranes (Bio-Rad Laboratories GmbH, Munich, Germany). Nonspecific binding was blocked by TBS-Tween with 5% non-fat dry milk. Anti-STAT1, anti-cIAP1, anti-cIAP2, anti-XIAP, anti-IκBα, anti-phospho-IKKα/β (Ser176/177), and IKKβ were purchased from Cell Signaling (Danvers, MA, USA). Anti-β-actin was ordered from Sigma-Aldrich. Primary antibodies were used at a dilution of 1:1000, and secondary antibodies (GE Healthcare, Little Chalfont, Buckinghamshire, UK) were used at 1:5000. Proteins were visualized using the ECL system (SuperSignal West Pico/Femto Chemiluminescent Substrate; Thermo Scientific, Rockford, IL, USA). The protein bands were scanned, and the band density was determined by using the Kodak 1D Image Analysis Software version 3.6 (Kodak Digital Science Imaging, Eastman Kodak Company, New Haven, CT, USA).

Phospho-MAPK proteome profiler array

Human phospho-MAPK array kit was purchased from R&D Systems. Monocytes were seeded in 6-well plates at a density of 106 cells per ml. Five-day moDCs were activated by MDP, MDP plus IFNγ, or left untreated. After 20 min of activation, cell lysates were generated, mixed with a phospho-specific antibody cocktail, then incubated with the arrays overnight at 4°C. Following incubation with streptavidin-horseradish peroxidase, the arrays were exposed to chemiluminescent reagents. Phospho-MAPK array spot signals were developed on X-ray films and analyzed by using the image analysis software Kodak 1D 3.6. The relative density values were calculated by normalizing to positive control signal intensities. The assays were carried out in duplicate.

siRNA-mediated gene silencing

STAT1, cIAP1, cIAP2, and XIAP-specific Silencer Select Validated siRNAs and Silencer Select Negative Control siRNA were ordered from Life Technologies. Cells were transfected at day 3 of DC differentiation in Opti-MEM medium (Life Technologies) in 4-mm cuvettes (Bio-Rad Laboratories Inc) using GenePulser Xcell instrument (Bio-Rad Laboratories Inc). Following transfection, the cells were seeded in 48-well cell culture plates at a density of 5 × 105 cells ml−1 in AIMV medium supplemented with 80 ng ml−1 GM-CSF and 50 ng ml−1 IL-4.

Statistical analysis

Data are expressed as the mean ± SD. Statistical analysis was performed by one-way analysis of variance with Bonferroni post-hoc test using GraphPad Prism v.6. (GraphPad Software Inc., La Jolla, CA, USA). Differences were considered to be statistically significant if P-values were less than 0.05. Significance is indicated by asterisks: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant.

Acknowledgments

This work was supported by the Hungarian Scientific Research Grants OTKA NN114423 and the Romanian Ministry of Education, Executive Agency For Higher Education, Research, Development and Innovation Funding, PNCDI II, project no. 119/2014. Supplementary information of this article can be found on the Cellular & Molecular Immunology's website (http://www.nature.com/cmi)

Footnotes

Supplementary information of this article can be found on the Cellular & Molecular Immunology's website (http://www.nature.com/cmi).

Supplementary Information

Supplementary information
Click here for additional data file. (620.6KB, pdf)

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Supplementary information
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