Critical Role of AZI2 in GM-CSF–Induced Dendritic Cell Differentiation

Masahiro Fukasaka, Daisuke Ori, Tatsukata Kawagoe, Satoshi Uematsu, Kenta Maruyama, Toshihiko Okazaki, Tatsuya Kozaki, Tomoko Imamura, Sarang Tartey, Takashi Mino, Takashi Satoh, Shizuo Akira and Osamu Takeuchi
J Immunol June 1, 2013, 190 (11) 5702-5711; DOI: https://doi.org/10.4049/jimmunol.1203155

Abstract

TNFR-associated factor family member–associated NF-κB activator (TANK)–binding kinase 1 (TBK1) is critical for the activation of IFN regulatory factor 3 and type I IFN production upon virus infection. A set of TBK1-binding proteins, 5-azacytidine–induced gene 2 (AZI2; also known as NAP1), TANK, and TBK1-binding protein 1 (TBKBP1), have also been implicated in the production of type I IFNs. Among them, TANK was found to be dispensable for the responses against virus infection. However, physiological roles of AZI2 and TBKBP1 have yet to be clarified. In this study, we found that none of these TBK1-binding proteins is critical for type I IFN production in mice. In contrast, AZI2, but not TBKBP1, is critical for the differentiation of conventional dendritic cells (cDCs) from bone marrow cells in response to GM-CSF. AZI2 controls GM-CSF–induced cell cycling of bone marrow cells via TBK1. GM-CSF–derived DCs from AZI2-deficient mice show severe defects in cytokine production and T cell activation both in vitro and in vivo. Reciprocally, overexpression of AZI2 results in efficient generation of cDCs, and the cells show enhanced T cell activation in response to Ag stimulation. Taken together, AZI2 expression is critical for the generation of cDCs by GM-CSF and can potentially be used to increase the efficiency of immunization by cDCs.

Introduction

Dendritic cells (DCs) play a central role in instructing T cells to activate acquired immunity upon infection (1–4). They phagocytose microbes such as bacteria and viruses, process them into Ags, and present the Ags on MHC molecules. Besides Ag presentation, innate immune cells such as DCs and macrophages sense the invasion of pathogens through a set of pattern-recognition receptors, including TLRs and retinoic acid inducible gene-I (RIG-I)–like receptors (RLRs) (5, 6). Whereas TLRs are transmembrane proteins that recognize microbial components on the cell surface or in endosomes, RLRs sense viral dsRNA in the cytoplasm. The signaling pathways triggered by the receptors lead to the production of proinflammatory cytokines, including TNF, IL-6, IL-12p40, and type I IFNs (7). In addition, the signaling pathways increase the surface expression of costimulatory molecules, such as CD40, CD80, and CD86, on DCs (8). It is well documented that costimulation of DCs is critical for mounting efficient T cell activation and vaccination (9).

DCs are classified into several different subsets, including CD8− and CD8+ conventional DCs (cDCs) and plasmacytoid DCs (pDCs) (10, 11). pDCs, which are characterized by surface expression of CD11c and B220, are known to produce vast amounts of IFN-α in response to virus infection via TLR7 and TLR9 (12). On the other hand, cDCs recognize pathogen infection via TLRs and RLRs for the production of cytokines. Among the cDCs, CD8+ DCs differentiate in a Batf3-dependent manner (13) and have a strong capacity for cross-presentation of foreign Ags to MHC class I molecules (11). In mice, both cDCs and pDCs can be generated from bone marrow (BM) cells by culture with distinct cytokines. FLT3 ligand (FLT3L) is essential for pDC and a set of cDC differentiation from BM precursor cells (14). In contrast, culture of mouse BM cells or human PBMCs in the presence of GM-CSF is widely used to induce cDCs (15). GM-CSF induces the survival, proliferation, and differentiation of hematopoietic cells including DCs (16). The GM-CSF receptor is composed of an α subunit and a common β subunit shared by IL-3Rs and IL-5Rs (17). After stimulation of GM-CSF receptors, signaling pathways are activated through the formation of a unique dodecameric receptor complex (18, 19). The transcription factors STAT5 and IFN regulatory factor 4 (IRF4) (20, 21) are essential for GM-CSF–mediated signaling pathways, and MAPK and AKT are also activated by GM-CSF (17).

The molecular mechanisms for how TLRs and RLRs transactivate type I IFN genes and proinflammatory cytokines have been extensively studied. With the exception of TLR3, TLRs trigger an MyD88-dependent signaling pathway that induces activation of the transcription factor NF-κB, thereby leading to the expression of proinflammatory cytokine genes. In contrast, TLR3 and TLR4 signal through another adaptor molecule, Toll/IL-1R domain–containing adaptor protein inducing IFN-β, which results in the activation of a set of kinases called IκB kinase-i (IKK-i, also known as IKKε) and TNFR-associated factor (TRAF) family member–associated NF-κB activator (TANK)–binding kinase 1 (TBK1). These kinases phosphorylate IRF3 and IRF7, which induce transactivation of type I IFN and IFN-inducible genes (6, 22). RLR signaling also activates IKK-i and TBK1 for the production of type I IFNs. IKK-i and TBK1 are known to associate with several adaptor molecules, including TANK, 5-azacytidine–induced gene 2 (AZI2; also known as NAP1), and TBK1-binding protein 1 (TBKBP1; also known as SINTBAD). These three proteins harbor a coiled-coil motif and a TBK1-binding motif (23), and were reported to be required for the downstream signaling of TLRs and RLRs to activate TBK1 and IKK-i (23–25). However, the generation of TANK-deficient mice revealed that TANK is dispensable for TLR- and RLR-mediated type I IFN production. In contrast, TANK acts as a negative regulator of MyD88-dependent signaling by suppressing the activation of TRAF6 (26). TANK-deficient mice experience development of fatal autoimmune glomerular nephritis and osteoporosis because of enhanced activation of immune cells and osteoclasts (26, 27).

In this study, we analyzed the functional roles of AZI2 and TBKBP1 in vivo by generating mice that lack these genes. Surprisingly, both AZI2 and TBKBP1 were dispensable for the signaling pathways emanating from TLRs and RLRs. We found that AZI2, but not TBKBP1, is essential for the differentiation of cDCs in response to culture with GM-CSF by activating TBK1, and at the same time, AZI2–/– GM-CSF–derived BM-derived DCs (BMDCs [GM-DCs]) showed severely impaired T cell activation both in vitro and in vivo. Taken together, these data demonstrate a novel function of AZI2 for the induction of cDCs and further suggest the potential of AZI2-targeting approaches for enhancement of DC vaccine potency.

Materials and Methods

Generation of AZI2−/− and TBKBP1−/− mice

The AZI2 and TBKBP1 genes were isolated from genomic DNA extracted from embryonic stem cells by PCR. The targeting vector was constructed by replacing fragments encoding the exon 3 (AZI2) or exons 4–6 (TBKBP1) with a neomycin-resistance gene cassette (neo), and an HSV thymidine kinase driven by PGK promoter was inserted into the genomic fragment for negative selection (Fig. 1A, 1B). After the targeting vector was transfected into embryonic stem cells, G418 and ganciclovir doubly resistant colonies were selected and screened by PCR, and further confirmed by Southern blotting. Homologous recombinants were microinjected into C57BL/6 female mice, and heterozygous F1 progenies were intercrossed to obtain knockout (KO) mice. Double KO (DKO) mice were generated by intercrossing AZI2 hetero and TBKBP1 hetero mice. KO and littermate control mice were used throughout the experiments. All animal experiments were carried out with the approval of the Animal Research Committee of the Research Institute for Microbial Diseases at Osaka University.

Cells, viruses, and reagents

At 3 d after injection of 2 ml of 4% (w/v) thioglycollate medium (Sigma) i.p., peritoneal exudate cells were isolated by washing with ice-cold HBSS as previously described (28). CD4+ T cells or CD90.2− cells were isolated from splenocyte single-cell suspensions by negative selection with CD4+ T cell Isolation kit (Miltenyi Biotec) or CD90.2 Microbeads (Miltenyi Biotec). Encephalomyocarditis virus (EMCV), HSV, Newcastle disease virus, and influenza virus (Flu; PR8) have been described previously (29, 30). Polyinosinic-polycytidylic acid [poly(I:C)] and CpG DNA (oligodeoxynucleotide 1668) were purchased from Invivogen. LPS from Escherichia coli was purchased from Sigma. GM-CSF and FLT3L were purchased from PeproTech.

Generation of GM-DCs and FLT3L-derived BMDCs

BM cells were harvested from the femurs and tibias of mice by flushing the marrow cavity with HBSS. GM-DC:BM cells were resuspended at the density of 1 × 106 cells/ml in complete RPMI 1640 (supplemented with 10% FCS, 50 mM 2-ME, 1% penicillin [100 U/ml] and 1% streptomycin [100 U/ml]) and 10 ng/ml mouse GM-CSF. The cell culture medium was replaced on days 2 and 4, and cells were grown for 6 d. FLT3L-derived BMDC (Flt3L-DC):BM cells were lysed with RBC lysing buffer (Sigma) and resuspended in complete RPMI 1640 containing 20 ng/ml Flt3L, and cells were grown for 8 d.

Quantitative real-time PCR and RT-PCR

Total RNA was extracted using TRIzol (Invitrogen), and cDNA was generated by using ReverTraAce (Toyobo) according to the manufacturer’s instructions. Quantitative real-time PCR was carried out with primers, purchased from Applied Biosystems, specific for IL-6, TNF, IFN-β, and 18S rRNA, as an internal control, using a 7700 Sequence Detector (Applied Biosystems) as described previously (31). RT-PCR analysis was conducted on cDNA samples with the following primers: human AZI2 (forward: 5′-ggatgcactggtagaagatgatatctg-3′, reverse: 5′-tcagctggaggagttctacttctttagat-3′) and GAPDH (forward: 5′-cttactccttggaggccatg-3′, reverse: 5′-ttagcccccctggccaagg-3′). The primer pairs and rTaq polymerase (Toyobo) were used for PCR.

ELISA

Cell culture supernatants were assessed for the concentration of IL-6, IL-12p40, TNF-α, IFN-β, and IFN-γ according to manufacturers’ instructions (R&D Systems or PBL Interferon Source).

Flow cytometry

All Abs used were purchased from BD Pharmingen or BioLegend. Cells were washed in flow cytometry buffer (2% [v/v] FCS and 2 mM EDTA in PBS, pH 7.5) and then, after treatment with each Ab, incubated for 20 min and washed twice with flow cytometry buffer. The cells were analyzed by flow cytometry on a FACSCalibur instrument (BD Biosciences), followed by data analysis using FlowJo v7.2.2 software (Tree Star).

DC maturation

Day 6 GM-DCs were harvested and stimulated at 1 × 106 cells/ml with LPS (1 μg/ml) or CpG DNA (1 μM) to induce DC maturation. After 24 h, the cells were subjected to flow cytometric analysis and the culture supernatants were frozen at −20°C for measurement of secreted cytokines by ELISA.

CD4+ T cell proliferation assays

Wild-type (WT) and AZI2−/− GM-DCs or FLT3L-DCs were stimulated with LPS (1 μg/ml) and CpG DNA (1 μM) for 24 h, and irradiated (3000 rad) to prevent cell division. For the MLR, CD4+ T cells were isolated from BALB/c mice splenocytes. A total of 1 × 106 CD4+ T cells was mixed with (1/3)2 × 105, (1/3)1 × 105, or 1 × 105 GM-DCs or FLT3L-DCs in each well. After 72 h of coculture, for proliferation assays, cells were pulsed with 2 μCi [3H]thymidine for the last 18 h. Levels of cytokines in the culture supernatant was measured by ELISA.

Proliferation and cell-cycle assay

BM cells were isolated from WT or AZI2−/− mice and stimulated with GM-CSF (10 ng/ml) for 48 h. For cell-cycle analysis, cells were stained with propidium iodide buffer (0.4% sodium citrate, 0.03% Nonidet P-40, 0.05 mg/ml propidium iodide [Sigma], and 0.02 mg/ml RNaseA) for 15 min on ice and assessed by flow cytometry. For cell proliferation assay, the BM cells were pulsed with BrdU for the last 24 h, and BrdU incorporation was analyzed using the BrdU Flow kit (BD Pharmingen) according to the manufacturer’s instructions or cells were pulsed with 2 μCi [3H]thymidine for the last 12 h.

Immunoblot analysis

BM cells were isolated from WT or AZI2−/− mice, and stimulated with GM-CSF for 0–20 min and lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1% [v/v] Nonidet P-40) containing compete mini protease inhibitor mixture (Roche). Cell lysates were separated by standard SDS-PAGE and analyzed by immunoblot. The following Abs were used: phospho-STAT5 (Cell Signaling Technology), phospho-ERK1/2 (T202/Y204; Cell Signaling Technology), phospho-AKT (S473; Cell Signaling Technology), and β-actin (Santa Cruz Biotechnology).

Retroviral transduction

BM cells were isolated from AZI2−/− mice and incubated with retroviral supernatant supplemented with 10 ng/ml GM-CSF and 10 ng/ml polybrene for 12 h on days 0 and 2. Virus was produced by PlatE packaging cells transfected with indicated plasmids by using Fugene6 Transfection Reagent (Roche) 3 d before infection. Postinfection, the cells were cultivated in RPMI 1640 medium supplemented with 10% FCS, 10 ng/ml GM-CSF until day 6, and 2 μg/ml puromycin was added for the last 2 d before harvesting floating cells.

Immunization

WT or AZI2−/− derived GM-DCs were pulsed with OVA (100 μg/ml; Sigma) in the presence of CpG DNA (1 μM) for 18 h. For in vivo transfer experiments, mice were injected i.p. with Ag-pulsed GM-DCs (1 × 105 cells). Two weeks after the injection, CD4+ T cells were purified from the spleen and stimulated with OVA (0–1000 μg/ml) in the presence of irradiated (3000 rad) CD90.2+ depleted spleen cells of WT littermates for 72 h. For proliferation assays, cells were pulsed with 2 μCi [3H]thymidine for the last 18 h. Levels of cytokines in the culture supernatant were measured by ELISA.

Statistics

Statistical significance was calculated with the two-tailed Student t test.

Results

Generation of mice that lack AZI2 and TBKBP1

To investigate the roles of AZI2 and TBKBP1 in vivo, we generated AZI2–/– and TBKBP1–/– mice (Fig. 1A, 1B). Homologous recombination of the AZI2 and TBKBP1 loci was confirmed by Southern blotting (Fig. 1C, 1D). RT-PCR analysis with total RNA isolated from spleens showed deletion of AZI2 or TBKBP1 expression in AZI2–/– and TBKBP1–/– mice (Fig. 1E, 1F). AZI2–/–, TBKBP1–/–, and AZI2–/–TBKBP1–/– doubly deficient mice were born in a typical Mendelian pattern, appeared to grow normally, and were fertile. In addition, the absence of AZI2 and TBKBP1 did not alter the differentiation of T and B cells, macrophages, CD11c+B220−CD8− cDCs, CD8+ cDCs, and CD11c+B220+ pDCs in the spleen and lymph nodes (data not shown).

FIGURE 1.
FIGURE 1.

Generation of AZI2- and TBKBP1-deficient mice. (A and B) Structure of the murine AZI2 (A) and TBKBP1 (B) gene, their targeting construct, and disrupted gene. (C and D) Southern blot analysis of genomic DNA from offspring from the heterozygote intercrosses in AZI2 (C)- or TBKBP1 (D)-deficient mice. (C) Genomic DNAs were extracted, digested with BamHI, separated by electrophoresis, and hybridized with the radiolabeled probe indicated in (A). (D) Genomic DNAs were digested with XhoI and EcoRV, and hybridized with probe indicated in (B). (E and F) RT-PCR analysis of cDNA in AZI2−/− (E) or TBKBP1−/− (F) mice and their controls (WT). The expression of GAPDH gene was analyzed with the same RNA.

AZI2 and TBKBP1 are dispensable for TLR- and RLR-induced cytokine and type I IFN production in macrophages

Because AZI2 and TBKBP1 are implicated in the activation of IRF3 by interacting with TBK1 and IKK-i (23, 24), we first examined the responses of peritoneal macrophages to infection with NDV, an RNA virus recognized by RIG-I in macrophages and cDCs. As shown in Fig. 2A and 2B, the IL-6 and IFN-β mRNA expression levels were comparable between WT, AZI2–/–, TBKBP1–/–, and DKO macrophages, indicating that AZI2 and TBKBP1 are dispensable for the IFN responses resulting from IRF3 activation in macrophages. Furthermore, the levels of IL-6 and TNF production in response to various TLR ligands, such as poly(I:C) (TLR3 ligand), LPS (TLR4 ligand), and CpG DNA (TLR9 ligand), were comparable between WT, AZI2–/–, TBKBP1–/–, and DKO macrophages (Fig. 2C, 2D). These findings indicate that AZI2 and TBKBP1 are dispensable for cytokine responses after RLR and TLR stimulation.

FIGURE 2.
FIGURE 2.

Production of IL-6, TNF, and type I IFN was not altered between WT, AZI2−/−, TBKBP1−/−, and DKO mouse macrophages. (A–D) Thioglycollate-elicited peritoneal macrophages were collected from WT, AZI2−/−, TBKBP1−/−, and DKO mice. (A and B) Quantitative PCR analysis showing expression of IL-6 (A) and IFN-β (B) mRNAs in peritoneal macrophages infected with NDV (multiplicity of infection [MOI] 1) for 0, 4, and 8 h. (C and D) Cells were stimulated for 24 h with poly(I:C) (10 or 100 μg/ml), LPS (10 or 100 ng/ml), or CpG DNA (0.1 or 1 μM), and IL-6 (C) or TNF (D) production of supernatants was measured by ELISA. Results are representative of two independent experiments (error bars indicate SD).

AZI2−/−, but not TBKBP1−/−, GM-DCs showed impaired production of cytokines

Next, we generated pDCs from mouse BM cells by culture in the presence of FLT3L. When FLT3L-DCs were infected with NDV, IFN-α production was not impaired in the absence of AZI2 and TBKBP1 (Fig. 3A). Furthermore, IL-6 production in response to CpG-DNA stimulation and Flu infection was comparable between AZI2–/–, TBKBP1–/–, and DKO FLT3L-DCs (Fig. 3B). These findings demonstrate that AZI2 and TBKBP1 are dispensable for the production of type I IFNs and proinflammatory cytokines activated via IRF3 and IRF7 signaling in macrophages and pDCs.

FIGURE 3.
FIGURE 3.

AZI2−/−, but not TBKBP1−/−, GM-DCs impaired the production of cytokines after stimulation of various TLR ligands and viruses. (A and B) BM cells from WT, AZI2−/−, TBKBP1−/−, and DKO mice were cultured in the presence of FLT3L for 8 d. (A) The cells were infected with NDV (MOI 1), and the production of IFN-α in the culture supernatant was measured by ELISA. (B) FLT3L-DCs were stimulated with CpG DNA (1 μM) or Flu (PR8) (MOI 1), and the IL-6 production was measured. (C–E) GM-DCs from WT, AZI2−/−, TBKBP1−/−, and DKO mice were stimulated with poly(I:C) (100 μg/ml), LPS (1 μg/ml), CpG DNA (1 μM), and infected with EMCV (MOI 1), HSV (MOI 1), NDV (MOI 1), and Flu (PR8) (MOI 1) for 24 h. Production of IFN-α (C), IL-6 (D), and TNF (E) in the culture supernatants was determined by ELISA. (F and G) GM-DCs from WT and AZI2−/− mice were stimulated with LPS (1 μg/ml) and CpG DNA (1 μM) for 2 and 4 h. The total RNAs were prepared, and the IL-6 and TNF mRNA levels were determined by quantitative PCR. Similar experiments were taken at least three times and one representative experiment is shown (error bars indicate SD). *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student t test.

Next, we generated GM-DCs by cultivating BM cells in the presence of GM-CSF. Interestingly, the production of IL-6 and TNF in response to stimulation with various TLR ligands, including poly(I:C), LPS, and CpG DNA, as well as infection with RNA viruses, including NDV, Flu, and EMCV, was severely impaired in the absence of AZI2, but was unaffected by TBKBP1 deficiency (Fig. 3D, 3E). In addition, IFN-α production in response to NDV infection was impaired in the absence of AZI2 in GM-DCs (Fig. 3C). The cytokine production levels in AZI2–/– and DKO GM-DCs were impaired to similar extents to WT cells, indicating that AZI2, but not TBKBP1, is involved in the production of cytokines in GM-DCs. GM-DCs are known to recognize RNA virus infection via RLRs for the production of type I IFNs and proinflammatory cytokines, indicating that AZI2 is required for the cytokine production in response to stimulation of TLRs and RLRs in this cell type. In addition to RNA viruses, IL-6 and TNF production in response to HSV infection was also significantly reduced in AZI2–/– and DKO, but not TBKBP1–/–, GM-DCs compared with WT cells (Fig. 3D, 3E). The expression of IL-6 and TNF mRNAs in response to LPS and CpG DNA stimulation was impaired in AZI2–/– GM-DCs (Fig. 3F, 3G), indicating that the impaired cytokine production was controlled at the gene expression levels. Collectively, these findings demonstrate that AZI2 controls the production of proinflammatory cytokines in a cell-type–specific manner and is critical for the production of cytokines in GM-DCs.

T cell–stimulating activity of GM-DCs is impaired in the absence of AZI2

GM-DCs harbor the ability to stimulate T cells, and the activity is increased in response to TLR ligand stimulation. To examine whether AZI2 is important for the T cell–stimulating activity of GM-DCs, we stimulated WT and AZI2–/– GM-DCs with LPS (1 μg/ml) or CpG DNA (1 μM) for 24 h and then examined them for their allogeneic T cell–stimulating activities. As shown in Fig. 4A, the proliferation of allogeneic CD4+ T cells after coculture with GM-DCs was severely impaired in the absence of AZI2, irrespective of LPS and CpG DNA stimulation. The ability of GM-DCs from AZI2–/– mice to support IFN-γ production from WT CD4+ T cells was severely impaired compared with cells from WT mice (Fig. 4B). Then we examined the T cell–stimulating activity of WT and AZI2–/– FLT3L-DCs. Allogenic CD4+ T cells proliferated comparably after coculture with FLT3L-DCs from WT and AZI2–/– mice with or without stimulation with LPS and CpG DNA (Fig. 4C). These findings demonstrate that AZI2 is important for GM-DCs, but not pDCs, to confer the ability to activate CD4+ T cells.

FIGURE 4.
FIGURE 4.

AZI2−/− GM-DCs show impaired activity to stimulate T cells. (A and B) WT and AZI2−/− GM-DCs were stimulated with LPS (1 μg/ml) and CpG DNA (1 μM) or cultivated without stimulation for 24 h. Then the cells were irradiated (3000 rad) and cocultured with splenic CD4+ T cells isolated from BALB/c mice. (A) Proliferation of CD4+ T cells was determined by [3H]thymidine incorporation 72 h after cultivation. (B) Levels of IFN-γ in the culture supernatants (1 × 105 cells/well GM-DCs) were measured by ELISA 72 h after coculture. (C) WT and AZI2−/− FLT3L-DCs were stimulated with LPS (1 μg/ml) and CpG DNA (1 μM) or cultivated without stimulation for 24 h. Then the cells were irradiated (3000 rad) and cocultured with splenic CD4+ T cells isolated from BALB/c mice. Proliferation of CD4+ T cells was determined by [3H]thymidine incorporation 72 h after cultivation. All results are representative of at least three (A and B) or two (C) independent experiments (error bars indicate SD). *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student t test.

AZI2 is critical for the differentiation of GM-DCs

Because macrophages and GM-DCs transactivate cytokine gene expression downstream of identical intracellular signaling pathways, it is unlikely that AZI2 directly controls RLR- and TLR-mediated signaling molecules. Thus, we examined the differentiation of BMDCs in response to culture in the presence of GM-CSF. As shown in Fig. 5A, culture of BM cells from WT mice with GM-CSF increased the population of CD11c+ cells in a time-dependent manner, and ∼70% of cells were CD11c+ after 6 d of culture. In contrast, CD11c expression was severely impaired in cells that lack AZI2 (Fig. 5A). Although WT cells increased during the course of GM-CSF culture, AZI2–/– cells failed to proliferate (Fig. 5B). In contrast, the population of CD11c+ cells in TBKBP1–/– BM cells cultured with GM-CSF was comparable with that of WT cells (data not shown). To investigate whether AZI2 controls GM-DC differentiation in a cell-intrinsic manner, we cocultured CD45.1+ WT and CD45.2+ AZI2–/– BM cells in the presence of GM-CSF (Fig. 5C). The proportion of CD11c+ DCs generated from AZI2–/– BM cells was severely decreased, even in the presence of WT cells, indicating that AZI2 is required for GM-DC differentiation in a cell-autonomous manner. Furthermore, the number of cells expressing the costimulatory markers CD40, CD80, and CD86 on the surface was severely decreased in AZI2–/– GM-DCs even without TLR ligand stimulation, and even after stimulation with TLR ligands, a severe reduction in the expression of these costimulatory molecules was observed in the absence of AZI2 (Fig. 5D). Next, we sorted CD11c+ and CD11c− cells from WT and AZI2–/– GM-DCs, and examined the production of IL-6 in response to LPS and CpG DNA stimulation. IL-6 levels were much more abundant in CD11c+ cells compared with CD11c− cells, and the production of IL-6 was comparable between CD11c+ cells sorted from WT and AZI2−/− GM-DCs (Fig. 5E). Thus, impaired cytokine production to TLR ligands in the absence of AZI2 is due to impaired generation of CD11c+ DCs in AZI2−/− GM-DCs. Collectively, AZI2 deficiency impairs GM-DC differentiation, causing defects in costimulatory molecule expression in response to various stimuli. Although AZI2 is required for proper development of DCs in response to GM-CSF, we failed to observe a defect in lung histology in AZI2−/− mice (data not shown).

FIGURE 5.
FIGURE 5.

Impaired differentiation of GM-DCs in AZI2−/− mice. (A and B) WT and AZI2−/− BM cells were cultured with GM-CSF. At days 2, 4, and 6 of culture, cells were harvested and examined by flow cytometry (A) and the total cell numbers were counted (B). (C) BM cells derived from WT CD45.1 mice and WT or AZI2−/− CD45.2 mice were harvested and were cocultured (1:1 ratio) with GM-CSF to generate GM-DCs. At day 6, cells were assessed by flow cytometry. (D) WT and AZI2−/− GM-DCs were stimulated with LPS (1 μg/ml) or CpG DNA (1 μM) for 24 h. The maturation of GM-DCs was assessed by measuring the expression levels of CD40, CD80, and CD86 by flow cytometry. (E) CD11c+ and CD11c− cells were sorted from WT and AZI2−/− GM-DCs, and stimulated with LPS (1 μg/ml) or CpG DNA (1 μM) for 24 h. Then the production of IL-6 in the culture supernatant was measured by ELISA. Results are representative of over 10 (A), 3 (B and D), or 2 (C and E) independent experiments (error bars indicate SD). **p < 0.01, two-tailed Student t test.

AZI2 is required for the proliferation of GM-DCs via TBK1

Next, we investigated the mechanisms underlying how AZI2 controls GM-CSF–induced DC differentiation. When we examined the contribution of AZI2 to cell-cycle progression in BM cells cultured with GM-CSF for 48 h, we found that cells in S and G2/M phases were severely decreased in the absence of AZI2 (Fig. 6A), In addition, incorporation of BrdU as well as [3H]thymidine was impaired in AZI2–/– BM cells cultured with GM-CSF (Fig. 6B, 6C). Collectively, AZI2 is critical for cell-cycle progression of BM cells in response to GM-CSF.

FIGURE 6.
FIGURE 6.

The AZI2-TBK1 pathway is crucial for the generation of GM-DCs by controlling cell-cycle progression. (A–C) BM cells derived from WT and AZI2−/− mice were cultured with GM-CSF (10 ng/ml) for 48 h. (A) The cells were then stained with propidium iodide and analyzed by flow cytometry. (B) The cells were pulsed with BrdU for last 24 h of culture, and BrdU+ proliferating cells were assessed by flow cytometry. (C) The cells were pulsed with [3H]thymidine for last 12 h of culture and [3H]thymidine incorporation to the cells was measured. (D) BM cells were incubated with GM-CSF for indicated periods, and cell lysates were subjected to immunoblot analysis using Abs against p-STAT5, p-ERK, and p-AKT, and β actin as a loading control. (E) Schematic representation of human AZI2 domain structure. (F–I) GM-DCs were generated from AZI2−/− BM cells infected with retroviruses expressing human AZI2 (hAZI2) or its deletion mutants (aa residues 1–158, 1–270, or 1–392). (F) Semiquantitative RT-PCR showing expression of hAZI2. GAPDH was amplified as a control. (G) Levels of CD11c expression in GM-DCs expressing indicating constructs were determined by flow cytometry. (H and I) Production of IL-6 (H) and IL-12p40 (I) in cells expressing hAZI2 and its mutants in response to LPS and CpG DNA stimulation was measured by ELISA. (J) GM-DCs were generated from AZI2−/− BM cells infected with retroviruses expressing hAZI2 or a mutant lacking the TBK1 binding domain (Δ158–270). (J) Levels of CD11c expression in GM-DCs expressing indicating constructs were determined by flow cytometry. (K) GM-DCs were generated from AZI2−/− BM cells infected with retroviruses expressing human TBK1, and the levels of CD11c expression on the cell surface were analyzed by flow cytometry. Results are representative of two (A, H–J), three (B–D, K), or four (F, G) independent experiments (error bars indicate SD). *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student t test.

The GM-CSF receptor consists of α and β subunits (18), and treatment with GM-CSF initiates the formation of a unique dodecameric receptor complex that leads to activation of the JAK2/STAT5, ERK1/2, and PI3K/AKT pathways (17). Thus, we assessed whether AZI2 deficiency had any effects on GM-CSF receptor signaling. STAT5, ERK, and AKT were phosphorylated to comparable levels in WT and AZI2–/– BM cells after 5–20 min of GM-CSF stimulation (Fig. 6D), indicating that AZI2 is not required for triggering of the initial GM-CSF receptor signaling. We then retrovirally expressed human AZI2 in AZI2–/– BM cells, followed by culture in the presence of GM-CSF (Fig. 6F). Expression of full-length AZI2 (1–392) greatly increased the CD11c+ cell population (Fig. 6G). AZI2 is composed of N-terminal coiled-coil domains and a following TBK1-binding domain (Fig. 6E) (32, 33). Although expression of the coiled-coil domains of AZI2 (1–158) failed to rescue the defect in DC differentiation in AZI2–/– BM cells, expression of AZI2 (1–270) containing the TBK1-binding domain greatly increased the proportion of CD11c+ cells (Fig. 6G). Furthermore, expression of AZI2 (1–270) and full-length AZI2 (1–392), but not AZI2 (1–158), in AZI2–/– BM cells cultured with GM-CSF increased the production of IL-6 and IL-12p40 in response to LPS and CpG DNA stimulation (Fig. 6H, 6I). In addition, an AZI2 mutant lacking the TBK1-binding domain (Δ158–270) also failed to rescue the generation of CD11c+ GM-DCs (Fig. 6J). These findings indicate that expression of AZI2 containing the TBK1-binding domain rescued the generation of GM-DCs and their ability to produce cytokines in response to TLR ligands. These observations suggest that AZI2 controls the generation of GM-DCs by binding to TBK1, IKK-i, or both.

Besides acting as IRF3/7 kinases, TBK1 and IKK-i have been implicated in cell proliferation and oncogenesis (34, 35). To investigate the contribution of TBK1 to GM-DC differentiation, we assessed the effect of human TBK1 overexpression in AZI2–/– BM cells on the differentiation of GM-DCs. Interestingly, overexpression of TBK1 in AZI2–/– BM cells increased the CD11c+ cell population (Fig. 6K), suggesting that TBK1 acts downstream of AZI2 to facilitate the differentiation of GM-DCs.

AZI2 is critical for the ability of GM-DCs to stimulate Ag-specific T cell activation in vivo

To investigate the role of AZI2 in inducing the T cell–stimulating activity of GM-DCs in vivo, we immunized WT mice with Ag-loaded GM-DCs from WT and AZI2–/– mice. GM-DCs were pulsed with OVA and CpG DNA for 18 h, and the Ag-loaded cells were i.p. injected into C57BL/6 mice. At 2 wk after the injection, CD4+ T cells were collected from splenocytes and cocultured with OVA and CD90.2− cells to assess the DC vaccination-induced Ag-specific responses. Mice immunized with AZI2–/– GM-DCs showed severely impaired T cell proliferation compared with WT cells (Fig. 7A). The IFN-γ production in response to OVA was also significantly reduced in T cells immunized with AZI2–/– DCs (Fig. 7B). These findings indicate that AZI2 is required for efficient immunization and T cell activation by GM-DCs in vivo.

FIGURE 7.
FIGURE 7.

AZI2 is crucial for T cell activation by GM-DCs in vivo. (A and B) Ag-pulsed GM-DCs require AZI2 to stimulate T cells in vivo. WT and AZI2−/− GM-DCs pulsed with OVA (100 μg/ml) and CpG DNA (1 μM) for 18 h were injected into the peritoneal cavities of C57BL/6 mice. Two weeks after the immunization, splenic CD4+ T cells were collected from the mice and stimulated with indicated concentrations of OVA in the presence of CD90.2− splenocytes for 72 h. (A) CD4+ T cell proliferation was measured by [3H]thymidine incorporation, and (B) levels of IFN-γ in supernatants (1000 μg/ml OVA) were determined by ELISA. (C–F) GM-DCs were generated from WT BM cells infected with a retrovirus expressing full-length hAZI2 (aa residues 1–392). (C) Cells expressing CD11c+ were determined by flow cytometry. (D and E) Production of IL-6 (D) and IL-12p40 (E) in AZI2 overexpressing cells in response to stimulation with LPS and CpG DNA was measured by ELISA. (F) C57BL/6 mice were treated with hAZI2-overexpressing GM-DCs pulsed with OVA and CpG DNA. Two weeks later, proliferation of Ag-specific CD4+ T cells in the spleen was determined by [3H]thymidine incorporation. Results are representative of three (A, B, D, E), four (C), or two (F) independent experiments (error bars indicate SD). *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student t test.

Overexpression of AZI2 enhances the T cell–stimulating activity of GM-DCs

The earlier observations prompted us to hypothesize that increased expression of AZI2 potentiates the efficiency of GM-DC generation and T cell activation. To evaluate the potential use of AZI2 in modified DC vaccines, we retrovirally overexpressed human AZI2 in WT BM cells, followed by DC generation in the presence of GM-CSF. Interestingly, overexpression of human AZI2 in BM cells significantly increased the efficiency of CD11c+ DC generation (Fig. 7C). Furthermore, the levels of IL-6 and IL-12p40 production in response to LPS and CpG DNA were significantly higher in cells overexpressing AZI2 (Fig. 7D, 7E). These data demonstrate that increased expression of AZI2 facilitates the generation of GM-DCs. Finally, we assessed the ability of AZI2-overexpressing DCs to stimulate T cells in vivo. AZI2-overexpressing GM-DCs were loaded with OVA and stimulated with CpG DNA before vaccination. The proliferation of CD4+ T cells in response to OVA was significantly enhanced in mice vaccinated with GM-DCs overexpressing AZI2 (Fig. 7F). Thus, AZI2 overexpression in GM-DCs increases the efficiency of immunization for activation of CD4+ T cells in vivo.

Discussion

In this study, we discovered a novel function of AZI2, but not TBKBP1, in controlling the differentiation of GM-DCs in mice. In contrast, AZI2 and TBKBP1 are dispensable for the TLR- or RLR-mediated signaling pathways leading to IRF3 activation and type I IFN production. Furthermore, the production of cytokines in response to TLR stimulation was unaltered in macrophages and pDCs that lack AZI2 and TBKBP1.

AZI2 (also known as NAP1) comprises an N-terminal coiled-coil domain and a central TBK1-binding domain (36). It forms a family with proteins harboring coiled-coil domains and a TBK1-binding domain, namely, TANK and TBKBP1 (23). AZI2 interacts with TBK1 and IKK-i, and was shown to be involved in IFN-β induction in response to TLR and RLR stimulation (32, 33). AZI2 was also found to be involved in the activation of IRF3 in response to poly(I:C) (25). Nevertheless, this study revealed that deficiencies in AZI2 and TBKBP1 do not alter the poly(I:C)- and RNA virus-mediated responses in macrophages and pDCs. Although GM-DCs lacking AZI2 showed impaired cytokine production in response to TLR ligands and virus infection, the defects appear to be caused by impaired development of DCs in response to GM-CSF. Therefore, it is likely that AZI2 and TBKBP1 are not directly involved in the signaling pathways that activate IRF3 and type I IFNs. We previously showed that TANK is also dispensable for the production of type I IFNs in response to RNA virus infection and TLR stimulation (26). Therefore, this study clearly demonstrates that a set of proteins possessing a TBK1-binding domain, namely, AZI2, TBKBP1, and TANK, are dispensable for the regulation of IRF3 downstream of TBK1. TANK deficiency in mice leads to overproduction of cytokines in response to TLR stimulation and the development of autoimmune glomerular nephritis (26). In contrast, deficiencies in AZI2 and TBKBP1 did not enhance the proinflammatory cytokine responses in macrophages or lead to the development of autoimmune diseases (M. Fukasaka and O. Takeuchi, unpublished observations), suggesting that the functions of AZI2 and TBKBP1 in vivo are distinct from that of TANK, despite the molecular similarity. Our data clearly show that this set of TBK1-binding proteins is not essential for the activation of IRF3 downstream of RLRs and TLRs. Given that TRAF3 has been shown to interact directly with TBK1 and IKK-i (37), it is possible that IFN-β promoter stimulator-1/TRAF3 proteins may directly activate TBK1 for phosphorylation of IRF3.

TBK1-deficient mice show embryonic lethality at embryonic day 15.5 because of liver apoptosis (38). Although IKK-i–deficient mice are viable, deficiency in both TBK1 and IKK-i leads to earlier death during embryonic development (39). In contrast, mice that lack both IRF3 and IRF7 are viable and do not show obvious developmental defects (40, 41). Therefore, it is apparent that the functions of TBK1 and IKK-i are not limited to the phosphorylation of IRF proteins and subsequent production of type I IFNs. It has been shown that TBK1 and IKK-i are critical for oncogenic transformation (35, 42). Although the mechanism is not yet fully understood, it was reported that this occurs through activation of AKT kinase (43, 44). In this study, we have shown that AZI2 is critical for the cell cycling of BM cells in response to GM-CSF stimulation. Ectopic expression of TBK1 in AZI2−/− BM cells rescued the defects in the generation of GM-DCs, suggesting that AZI2 controls the cell cycling of BM cells by activating TBK1 in response to GM-CSF.

Although GM-CSF has been widely used to generate cDCs from BM cells (15), GM-CSF deficiency does not affect the generation of tissue-resident cDCs in mice (45). In contrast, GM-CSF deficiency leads to the development of pulmonary alveolar proteinosis through macrophage dysfunction. Human pulmonary alveolar proteinosis can be explained by depletion of GM-CSF by anti–GM-CSF Abs, suggesting that the functional roles of GM-CSF in vivo are similar between humans and mice. We found that AZI2 is dispensable for preventing pulmonary alveolar proteinosis. Because the initial activation of signaling pathways in response to GM-CSF stimulation was not impaired, even in the absence of AZI2, the differentiation of alveolar macrophages seems to be controlled independently of AZI2.

Although GM-CSF is dispensable for the development of splenic DCs, this growth factor has been used to generate cDCs ex vivo in mice (46). In addition, GM-CSF is now widely used to generate cDCs for human DC therapies that target cancer. We found that AZI2 was required for the activation of T cell responses by transferring GM-DCs. Furthermore, overexpression of AZI2 led to efficient generation of cDCs in response to GM-CSF and enhanced the T cell–stimulating activity of GM-CSF–derived BM cells. Thus, it is possible that overexpression of AZI2 in human cells can reinforce the efficiency of DC generation required for DC therapies. Because AZI2 deficiency does not affect the development of mouse tissues, it is possible that manipulation of AZI2 can enhance cDC generation without affecting other important cellular processes. AZI2 has been identified as a gene induced in response to 5-azacytidine treatment. Therefore, AZI2 expression can be an efficient target of therapeutic strategies to enhance DC therapies against cancer and other diseases.

In contrast with AZI2, lack of TBKBP1 did not cause any defects in mouse development, type I IFN responses, or DC generation. We also found no differences in the differentiation and activation of immune cells between WT and TBKBP1–/– mice. It is possible that TBKBP1, AZI2, and TANK act redundantly in unknown biological processes, although AZI2 and TANK have distinct functions. Further studies are required to clarify the functional role of TBKBP1 in vivo.

A DC vaccine is defined as DCs loaded with Ags, especially on a tumor-associated Ag (47, 48). Recently, strategies have been developed to generate large-scale production of DCs, and many different protocols have been designed to load Ags onto DCs. The associated findings made it possible to start clinical studies as cancer immunotherapies, but the limited success indicated that it was necessary to improve and enhance the DC vaccine potency. For instance, nanoparticle–Ag complexes are efficiently taken up by DCs (49). Downregulation of SRA/CD204, a pattern recognition scavenger receptor, strongly enhances DC-mediated antitumor immunity (50). We have shown that increased AZI2 expression in GM-DCs significantly enhanced the efficacy to activate CD4+ T cells in vivo. Therefore, it may be possible to enhance the efficiency of DC vaccination by manipulating the levels of AZI2 expression, although further studies are required to control AZI2 expression.

In summary, this study has clearly demonstrated that AZI2 is critical for the development of DCs in response to GM-CSF stimulation. Given that GM-CSF is useful for developing DC therapies, future studies involving manipulation of AZI2 expression will be beneficial for improving the methods for developing DCs ex vivo.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Y. Asahira, E. Kamada and M. Kageyama for secretarial assistance and Y. Fujiwara, M. Kumagai, and N. Umano for technical assistance.

Footnotes

  • This work was supported by Special Coordination Funds of the Japanese Ministry of Education, Culture, Sports, Science and Technology and grants from the Ministry of Health, Labour and Welfare in Japan and the Japan Society for the Promotion of Science through the Funding Program for World-Leading Innovative Research and Development on Science and Technology (FIRST Program). This work was also supported in part by grants from the Takeda Science Foundation, the Naito Foundation, and the Mochida Foundation.

  • Abbreviations used in this article:

    AZI2
    5-azacytidine–induced gene 2
    BM
    bone marrow
    BMDC
    bone marrow–derived dendritic cell
    cDC
    conventional dendritic cell
    DC
    dendritic cell
    DKO
    double knockout
    EMCV
    encephalomyocarditis virus
    FLT3L
    FLT3 ligand
    FLT3L-DC
    FLT3L-derived BMDC
    Flu
    influenza virus
    GM-DC
    GM-CSF–derived BMDC
    IKK-i
    IκB kinase-i
    IRF
    IFN regulatory factor
    KO
    knockout
    MOI
    multiplicity of infection
    NDV
    Newcastle disease virus
    pDC
    plasmacytoid DC
    poly(I:C)
    polyinosinic-polycytidylic acid
    RLR
    retinoic acid–inducible gene-I–like receptor
    TANK
    TNFR-associated factor family member–associated NF-κB activator
    TBK1
    TANK-binding kinase 1
    TBKBP1
    TBK1-binding protein 1
    TRAF
    TNFR-associated factor
    WT
    wild-type.

  • Received November 14, 2012.
  • Accepted March 26, 2013.

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