Diacylglycerol Kinase Zeta Positively Controls the Development of iNKT-17 Cells

Invariant natural killer T (iNKT) cells play important roles in bridging innate and adaptive immunity via rapidly producing a variety of cytokines. A small subset of iNKT cells produces IL-17 and is generated in the thymus during iNKT-cell ontogeny. The mechanisms that control the development of these IL-17-producing iNKT-17 cells (iNKT-17) are still not well defined. Diacylglycerol kinase ζ (DGKζ) belongs to a family of enzymes that catalyze the phosphorylation and conversion of diacylglycerol to phosphatidic acid, two important second messengers involved in signaling from numerous receptors. We report here that DGKζ plays an important role in iNKT-17 development. A deficiency of DGKζ in mice causes a significant reduction of iNKT-17 cells, which is correlated with decreased RORγt and IL-23 receptor expression. Interestingly, iNKT-17 defects caused by DGKζ deficiency can be corrected in chimeric mice reconstituted with mixed wild-type and DGKζ-deficient bone marrow cells. Taken together, our data identify DGKζ as an important regulator of iNKT-17 development through iNKT-cell extrinsic mechanisms.

Recently, iNKT cells capable of producing the IL-17 family of cytokines (iNKT-17), such as IL-17A, IL-17F, and IL-22, have been identified [15][16][17][18]. iNKT cell-derived IL-17-family cytokines are implicated in both inflammatory responses such as airway inflammation via recruiting neutrophils and protective roles such as suppression of liver inflammation [19,20]. iNKT-17 cells are generated in the thymus and are considered to be developmentally programmed [17,21]. iNKT-17 cells are mainly restricted to the NK1.1 -CD4 -population [15] and express the marker for recent thymic emigrant and nature-regulatory T cells neuropilin-1 [16]. Additionally, iNKT-17 cells express molecules that are usually characteristic of Th17 cells such as the orphan nuclear receptor RORγt, the IL-23 receptor (IL-23R), and the chemokine receptor CCR6 [17,22,23]. Although it has become clear that iNKT-17 represents a unique iNKT sublineage with important functions in the pathogenesis of diseases, the signal control for the generation/maintenance of this sublineage of iNKT cells is not well understood.
Diacyglcerol kinase ζ (DGKζ) belongs to a family of 10 enzymes that phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA), two important second messengers involved in signaling from numerous receptors [24][25][26]. DGKζ is expressed in many cell lineages in the immune system, such as T cells, macrophages, dendritic cells, and mast cells [27][28][29][30]. Recent studies have demonstrated that DGK activity plays important regulatory roles in these immune-cell lineages via terminating DAG and simultaneously generating PA [28,29,31]. In T cells, DGKζ negatively controls TCR-induced activation of the RasGRP1-Ras-Erk1/2 pathway, the PKCθ-NFκB pathway, and mTOR signaling [27,30,32,33], inhibits T cell activation in vitro and in vivo [27,30], inhibits primary anti-viral immune responses but promotes memory CD8 T-cell-mediated antiviral immune responses [34], contributes to T-cell anergy and tumor evasion [31], and, together with DGKα, promotes the positive selection of conventional αβ T (cαβT) cells [35]. DGKζ has also been demonstrated to regulate TLR signaling and the production of proinflammatory cytokines such as IL-12p40 and TNFα to control innate and adaptive immune responses to parasite infection [26] and to modulate mast-cell survival and activation [29]. Recently, we have demonstrated that deficiency of both DGKζ and α, another isoform expressed in T cells, causes severe decreases of iNKT cells in mice [33]. However, deficiency of either DGKα or DGKζ alone does not result in a noticeable abnormality of iNKT-cell numbers in mice. In this report, we demonstrate that germline deficiency of DGKζ leads to decreases of IL-17 producing iNKT cells without an obvious effect on IL-4-and IFNγ-producing iNKT (iNKT-4 and -1) cells. The decrease of iNKT-17 cells caused by DGKζ deficiency is correlated with a reduced expression of RORγt and IL-23R. Interestingly, in chimeric mice reconstituted with mixed WT and DGKζ bone marrow (BM) cells, an iNKT-17 defect caused by DGKζ deficiency can be corrected, suggesting that DGKζ controls iNKT-17 development via iNKT extrinsic mechanisms.

Mice and cells
DGKζ-deficient (DGKζKO) mice backcrossed to C57BL/6J background for at least nine generations were previously reported [27,31]. C57BL/6J and CD45.1 + congenic mice were generated by in-house breeding. TCRαKO mice were purchased from the Jackson Laboratory. All mice were housed in a pathogen-free facility. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mice were used according to protocols approved by the Institutional Animal Care and Use Committee of Duke University (Protocol Number: A132-10-5). Splenocytes, thymocytes, and liver MNCs were made according to previously published protocols [33,36].
Cell-surface staining was performed with 2% FBS-PBS. Intracellular staining for IFNγ, IL-17A, and IL-4 was performed using BD Biosciences Cytofix/Cytoperm ™ and perm/wash solutions following the manufacturer's protocol. All flow cytometry data were collected using FACS Canto-II (BD Biosciences) and analyzed with the FlowJo software. A solution of 0.5% Tween-20-PBS was used to dissolve α-GalCer (Enzo life science).

Purification of iNKT cells and real-time quantitative PCR
iNKT cells were enriched with PE-CD1dTet and anti-PE-MACS-beads according to a previously published protocol [33,36]. Enriched iNKT cells were stained with anti-TCRβ and 7-AAD and sorted for live CD1dTet + TCRβ + iNKT cells with greater than 98% purity using MoFlo. Sorted iNKT cells were immediately lysed in Trizol for RNA preparation. cDNA was made using the iScript Select cDNA Synthesis Kit (Biorad). Real-time quantitative PCR was conducted and analyzed as previously described [33,36]. Expressed levels of target mRNAs were normalized with β-actin and calculated using the 2 -ΔΔCT method. Primers were as follows: IL-23R, Forward: 5'-AGCAAAATCATCCCACGAAC-3', Reverse:

In vivo stimulation of iNKT cells
Mice were intraperitoneally injected with 150 µg Brefeldin A in 100 µl. Ninety minutes later, mice were intraperitoneally injected with 2 µg α-GalCer diluted in 200 µl PBS. Two hours after the α-GalCer injection, splenocytes and liver MNCs were DGKζ Controls iNKT-17 Development PLOS ONE | www.plosone.org intracellularly stained for IFNγ, IL-4, and IL-17A. Total RNA from splenocytes was also isolated from mice injected with α-GalCer without a Brefeldin A pretreatment.

Statistical analysis
Data are presented as mean ± SEM and statistical significance were determined by a Student's t-test.

DGKζ deficiency does not affect iNKT cell proliferation in vitro
DGKα, ζ, and δ are the dominant isoforms that expressed in T cells [25,31]. We compared the expression of these isoforms between cαβT cells and iNKT cells. As shown in Figure 1A, both DGKα and δ were expressed at reduced levels in iNKT cells compared with CD8 + cαβT cells. However, DGKζ was expressed at a higher level in iNKT cells than in CD8 + T cells. The reason for the differential expression of DGK isoforms between cαβT and iNKT cells remains to be defined.
Previously, studies have demonstrated that a deficiency of DGKζ does not affect iNKT-cell development. The total numbers and developmental stages of iNKT cells in DGKζKO mice are not obviously different from WT control mice [33]. To examine whether DGKζ regulates iNKT-cell activation in vitro, we labeled WT and DGKζ deficient thymocytes with CFSE and then stimulated the cells with α-GalCer in vitro for 72 hours. As shown in Figure 1B, DGKζKO and WT iNKT cells expanded and proliferated similarly, suggesting that DGKζ deficiency did not affect TCR-induced iNKT-cell proliferative response in vitro. It has been demonstrated that DGKζKO cαβT cells are hyperproliferative in response to TCR stimulation [27]. Thus, DGKζ differentially controls cαβT and iNKT-cell proliferation in vitro.

Decreased IL-17 but not IFNγ or IL-4 production by DGKζ deficient iNKT cells following in vitro stimulation of the iVα14TCR
iNKT cells produce multiple cytokines to regulate immune responses. To determine whether DGKζ regulates cytokine production by iNKT cells during in vitro activation, we stimulated WT and DGKζKO thymocytes with α-GalCer for 48 and 72 hours; IFNγ, IL-4, and IL-17 levels in culture supernatants were measured by ELISA. No obvious differences of IFNγ and IL-4 levels were observed between WT and DGKζKO iNKT cells. In contrast, IL-17A levels were considerably decreased in DGKζ iNKT cells (Figure 2A). Consistent with these ELISA data, intracellular staining of these cytokines in iNKT cells also showed decreased IL-17A but similar IFNγ-and IL-4-producing iNKT cells following α-GalCer stimulation ( Figure 2B and 2C). Taken together, these data indicate that DGKζ plays an important role for IL-17 production by iNKT cells in vitro.

Impaired iNKT-17 development in the absence of DGKζ
The impaired production of IL-17A by iNKT cells following α-GalCer stimulation can be caused by a developmental defect or impaired expansion of iNKT-17 cells. To determine whether DGKζ deficiency causes a developmental defect in generating iNKT-17 cells, we enriched iNKT cells from WT and DGKζKO thymocytes and stimulated enriched iNKT cells with PMA plus ionomycin in vitro for 5 hours in the presence of GolgiPlug. Intracellular staining of cytokines showed decreased IL-17A positive cells within DGKζKO iNKT cells than in WT controls ( Figure 3A and 3B). In contrast, the percentages of IFNγ-and IL-4-producing cells were similar in WT and DGKζKO iNKT cells. RORγt and IL-23R signaling is critical for the iNKT-17 differentiation [17,22]. We sorted iNKT cells from WT and DGK-ζKO thymocytes and measured RORγt and IL-23R mRNA levels by quantitative real-time PCR. Consistent with the iNKT-17 developmental defect, IL-23R and RORγt mRNA levels were obviously decreased in DGKζKO iNKT cells compared with WT iNKT cells ( Figure 3C). Consistent with these observations, DGKζKO thymic iNKT cells contained much less IL-17A + RORγ + double positive cells than WT controls ( Figure 3D). Together, these results suggest that DGKζ at least promotes iNKT-17 differentiation during development.

Impaired in vivo IL-17 induction in DGKζ deficiency mice following α-GalCer treatment
The data shown above reveal the important role of DGKζ of IL-17 production in vitro. We further examined how DGKζ deficiency may affect iNKT-cell cytokine production in vivo. As shown in Figure 4A and 4B, intracellular staining showed that the percentages of IL-4 or IFNγ positive iNKT cells were similar between WT and DGKζKO mice 2 hours after the α-GalCer injection. However, the percentage of IL-17-producing iNKT cells was obviously lower in DGKζKO mice than in WT mice. Moreover, the IL-17A mRNA level, although not IL-4 or IFNγ mRNA levels, was decreased in the DGKζKO spleen after the α-GalCer injection ( Figure 4C). Together, these observations suggest that DGKζ is important for optimal IL-17 expression in iNKT cells in vivo.

Promotion of iNKT-17 differentiation by DGKζ is not iNKT cell intrinsic
Because DGKζ was deficient in all cell lineages in DGKζKO mice, the aforementioned iNKT-17 defect in these mice could be caused by extrinsic or intrinsic mechanisms. To distinguish these possibilities, we generated mixed-bone-marrow chimeric mice by co-injecting CD45.1 + WT and CD45.2 + DGKζKO BM cells at a 1:2 ratio into sublethally irradiated TCRα -/-mice. Eight weeks after reconstitution, iNKT cells from thymocytes or splenocytes of the chimeric mice were enriched and stimulated with PMA plus ionomycin for 5 hours or stimulated with α-GalCer for 72 hours to induce IL-17 and IFNγ production. As shown in Figure 5, similar percentages of DGKζKO and WT iNKT cells produced IL-17A, suggesting that the impairment of iNKT-17 differentiation caused by DGKζ deficiency likely resulted from mechanisms extrinsic to iNKT cells.

Discussion
In this report, we demonstrated that DGKζ plays a selective role in promoting iNKT-17 development. We have shown that a deficiency of DGKζ resulted in impaired iNKT-17 correlated with decreased expression of RORγt and IL-23R. In contrast, IFNγ-producing iNKT-1 or IL-4-producing iNKT-4 cell development seemed not to be affected by DGKζ activity.
At least three DGK isoforms, α, δ, and ζ, are expressed in iNKT cells. While sharing common structural features such as the kinase domain and the cysteine-rich C1 domains, they also contain distinct structural domains/motifs and belong to different subtypes of the DGK family [37]. We have demonstrated that DGKα and ζ function synergistically to promote iNKT-cell development/homeostasis and cαβ T cell maturation [33,35]. Additionally, deficiency of either DGKα or ζ results in enhanced activation of cαβT-cell activation reflected by hyper-proliferation and elevated cytokine production [27,31]. However, DGKζ deficiency does not obviously impact iNKT cell activation. DGKζ-deficient iNKT cells proliferate and secrete IFNγ and IL-4 similarly to WT iNKT cells following TCR engagement. Thus, iNKT cells and cαβT cells display a differential requirement of DGKζ for modulating their activation. At present, we cannot rule out that DGKα or δ may function redundantly with DGKζ in the control of iNKT cell activation. The virtual absence of iNKT cells in DGKα and ζ doubledeficient mice prevents us from addressing this issue. Further generation and analysis of mice with conditional ablation of multiple DGK isoforms in mature iNKT cells should provide a solid conclusion regarding the role of DGK activity in iNKT cell activation.
Our data indicate that DGKζ promotes iNKT-17 differentiation via iNKT-extrinsic mechanisms. Important questions remain to be addressed about which cell lineage DGKζ controls iNKT-17 differentiation and how DGKζ exerts such functions in this cell lineage. iNKT-17 development is intrinsically dependent on RORγt but is negatively controlled by Th-POK, a transcript factor critical for CD4 lineage development [17,21,38,39]. Extracellular factors such as IL-23 and IL-1 are indispensable for iNKT-17 differentiation [22,40]. Interestingly, we have found that DGKζ is important for IL-12p40 expression in macrophages and dendritic cells [28]. A decrease of expression of IL-12p40, a subunit for both IL-12 and IL-23, could potentially lead to impaired iNKT-17 differentiation. Additionally, DGK activity inhibits mTOR activation in T cells [32]. mTOR activity can negatively control IL-12p40 transcription in dendritic cells and macrophages [41][42][43][44]. Thus, it is possible that a potential elevation of mTOR activity in dendritic cells may cause down-regulation of IL-23 expression by dendritic cells, leading to impaired iNKT-17 differentiation. Future studies using DGKζ conditional knockout mice should help to identify the lineage in which, and the mechanisms by which, DGKζ functions to promote iNKT-17 differentiation.