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. Author manuscript; available in PMC: 2019 Sep 13.
Published in final edited form as: Cell Rep. 2019 Apr 16;27(3):699–707.e4. doi: 10.1016/j.celrep.2019.03.052

Keap1-Nrf2 System Plays an Important Role in Invariant Natural Killer T Cell Development and Homeostasis

Kalyani Pyaram 1,4,*, Ajay Kumar 1, Yeung-Hyen Kim 1, Sanjeev Noel 2, Sekhar P Reddy 3, Hamid Rabb 2, Cheong-Hee Chang 1,4,5,*
PMCID: PMC6743333  NIHMSID: NIHMS1049435  PMID: 30995469

SUMMARY

Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) proteins work in concert to regulate the levels of reactive oxygen species (ROS). The Keap1-Nrf2 antioxidant system also participates in T cell differentiation and inflammation, but its role in innate T cell development and functions remains unclear. We report that T cell-specific deletion of Keap1 results in defective development and reduced numbers of invariant natural killer T (NKT) cells in the thymus and the peripheral organs in a cell-intrinsic manner. The frequency of NKT2 and NKT17 cells increases while NKT1 decreases in these mice. Keap1-deficient NKT cells show increased rates of proliferation and apoptosis, as well as increased glucose uptake and mitochondrial function, but reduced ROS, CD122, and Bcl2 expression. In NKT cells deficient in Nrf2 and Keap1, all these phenotypic and metabolic defects are corrected. Thus, the Keap1-Nrf2 system contributes to NKT cell development and homeostasis by regulating cell metabolism.

Graphical Abstract

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In Brief

Keap1 and Nrf2 proteins work in concert to regulate redox balance in the cells. Pyaram et al. report that Keap1 governs NKT cell development and peripheral homeostasis by regulating proliferation and apoptosis. In an Nrf2-dependent manner, Keap1 also controls NKT cell metabolism, including glucose uptake and ROS.

INTRODUCTION

Invariant natural killer T (NKT) cells are T cells of innate lineage, characterized by the expression of a Vα14-Jα18 invariant T cell receptor (TCR). They recognize glycolipids presented by major histocompatibility complex I (MHC I)-like CD1d molecules (Kohlgruber et al., 2016). Upon activation, NKT cells produce abundant amounts of a range of cytokines (Crosby and Kronenberg, 2018). Thus, NKT cells play a critical role in multiple types of immune responses against pathogens, cancer, and autoimmunity (Krijgsman et al., 2018; Wu and Van Kaer, 2011). Although NKT cells are found at the highest levels in spleen, liver, and adipose tissues, their development primarily occurs in the thymus, where they branch out from conventional T cells at the CD4CD8 double-positive (DP) stage (Benlagha et al., 2005; Egawa et al., 2005). Their development thereafter occurs through four stages: 0, 1, 2, and 3. As the newly committed stage 0 cells lose CD24 and move to stage 1, they undergo a proliferative burst. At stage 2, they acquire CD44, and the subsequent acquisition of NK1.1 results in NKT cells’ maturing to stage 3 with a full cytokine profile (Bennstein, 2018). NKT cell stages in the thymus can be divided into functional subsets NKT1, NKT2, and NKT17, which overlap in function with Th1, Th2, and Th17 cells, respectively (Wang and Hogquist, 2018).

Recently, we reported that peripheral NKT cells harbor much higher levels of reactive oxygen species (ROS) than CD4 T cells at steady state, and maintenance of higher ROS levels is important for bringing about the inflammatory functions of NKT cells (Kim et al., 2017). ROS include superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH), which are released as by-products of cellular oxygen metabolism (Turrens, 2003). ROS levels and in turn the redox balance of a cell are tightly regulated by Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) proteins, which work in concert (Itoh et al., 1999). Nrf2, a transcription factor that controls the expression of antioxidant pathway genes, is regulated by Keap1 under homeostatic conditions using the ubiquitin-proteasome pathway (Furukawa and Xiong, 2005). Under oxidative stress conditions, Nrf2 dissociates from Keap1 and translocates to the nucleus, where it activates transcription of cytoprotective genes (Itoh et al., 1997; Taguchi et al., 2011). In addition, Keap1 has been shown to play a role in autophagy (Dodson et al., 2015), apoptosis (Lo and Hannink, 2006), NF-κB signaling (Lee et al., 2009), and cell proliferation (Mulvaney et al., 2016). Activation of Nrf2 has been shown to negatively affect the maturation and effector functions in the related natural killer (NK) cells (Boss et al., 2018). Keap1 has been linked to T cell functions and thereby to inflammation (Ahmed et al., 2017; Noel et al., 2018; Tsai et al., 2018). T cell-specific deletion of Keap1 did not affect the conventional T cell numbers or percentages in the thymus, indicating that Keap1-Nrf2 is dispensable for T cell development (Noel et al., 2015). However, the role of Keap1-Nrf2 complex in the innate T cell development or functions is unknown.

Here, we report that Keap1 controls NKT cell development in a cell-intrinsic manner and affects the survival and proliferation of NKT cells in the thymus as well as peripheral organs. Furthermore, Keap1 is important for balanced effector functions of NKT cells. Our study demonstrates that Keap1 functions by regulating cell metabolism in an Nrf2-dependent manner.

RESULTS

Keap1 Is Important for NKT Cell Development and Homeostasis

To study the role of Keap1 in NKT cell development, we used mice with T cell-specific deletion of Keap1 (Keap1fl/fl-CD4Cre, referred to as Keap1−/−) that were reported previously (Noel et al., 2015). We first compared the thymic and splenic cellularity between Keap1−/− mice and their wild-type (WT) littermates and found little difference (Figure S1A). Consistent with the previous report (Noel et al., 2015), the percentages and numbers of single-positive CD4 (SPCD4), SPCD8 T cells, and DP thymocytes were comparable (Figure S1B). We next compared the NKT cell compartment between the Keap1−/− and WT mice. The percentages as well as cell numbers of CD1d tetramer-positive NKT cells were significantly reduced in the thymus, spleen, and liver of Keap1−/− mice (Figures 1A and 1B). When we analyzed NKT cell developmental stages and compared their frequencies, Keap1−/− mice showed higher percentages of stage 0 and stage 1 NKT cells than WT mice (Figures 1C and 1D). Little difference was observed in the percentages of stage 2 cells, but the percentage of mature stage 3 NKT cells was reduced in the Keap1−/− thymus. This reduction in the frequency of stage 3 Keap1−/− NKT cells was also reflected in the cell numbers (Figure 1D). Although the percentages of stages 0 and 1 were increased in the Keap1−/− thymus, the cell numbers were lower (Figure 1D) because the number of total NKT cells was greatly reduced in Keap1−/− thymus compared with the WT (Figure 1B). These data indicated a defect in NKT cell development across early as well as late stages in the Keap1−/− thymus. Thus, we calculated the efficiency of transition from the DP stage to stage 0 (S0/DP), stage 0 to 1 (S1/S0), stage 1 to 2 (S2/S1), and stage 2 to 3 (S3/S2) in the WT and Keap1−/− thymi by calculating the ratio of the cell number of the later stage to that of the preceding stage. The efficiency of transition was lower across all stages of the Keap1−/− NKT cells compared with the WT NKT cells except the stage 0-to-stage 1 transition, which was higher (Figure 1E).

Figure 1. Keap1 Is Important for NKT Cell Development and Homeostasis.

Figure 1.

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(A) NKT cells were identified as TCRβ+ and CD1d-PBS57 tetramer+ cells in the thymus (Thy), spleen (Spl), and liver (Liv) of Keap1−/− and wild-type (WT) littermate mice (n = 5).

(B) NKT cell percentages (top) and numbers (bottom) in indicated tissues from WT and Keap1−/− mice (n = 5).

(C) NKT cells from total thymocytes from WT and Keap1−/− mice were analyzed for expression of CD24, CD44, and NK1.1 to identify CD24+ stage 0 cells, stage 1 (CD44−NK1.1−), stage 2 (CD44+NK1.1−), and stage 3 (CD44+NK1.1+) cells (n = 7).

(D) Graphs show the percentages (top) and cell numbers (bottom) of thymic NKT cell developmental stages (Stg) in WT and Keap1−/− mice (n = 7).

(E) The progression efficiency at each stage was calculated by dividing the cell numbers of each stage by the numbers of the previous stage. S0, S1, S2, and S3 represent stage 0, stage 1, stage 2, and stage 3, respectively (n = 5).

All data are shown as mean ± SEM from four to six independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

To identify if the developmental defect observed in Keap1−/− NKT cells is cell intrinsic or extrinsic, we generated mixed bone marrow (BM) chimeric mice by transferring equal proportions of Keap1−/− (CD45.2) and WT (CD45.1) BM cells to irradiated Rag−/− hosts. The regeneration of total cells in the thymus (Figure S1C), and CD4 and CD8 T cells in thymus, spleen, and liver, was comparable between WT and Keap1−/− mice, as expected (Figure S1D). In the NKT cell compartment however, Keap1−/− cells were poorly reconstituted in all three organs (Figure S1E). The efficiency of NKT cell recovery, as calculated by the ratio of knockout (KO)/WT NKT cells, was very poor from Keap1−/− cells (Figure S1F). Furthermore, accumulation of NKT stages 0, 1, and 2 was seen within the Keap1−/− NKT cells at the cost of stage 3 cells (Figure S1G), as was seen earlier in the Keap1−/− mice (Figure 1D). We also tested the possibility that Keap1 deficiency might alter the level of expression or presentation capability of CD1d, both of which can result in a poor selection of Keap1−/− NKT cells. The expression of CD1d on WT and Keap1−/− DP thymocytes was comparable (Figure S1H). Second, we compared the ability of WT and Keap1−/− DP thymocytes to activate DN32.D3 NKT hybridomas in the presence of α-GalCer. Both WT and Keap1−/− thymocytes were able to activate the DN32.D3 cells to similar levels as measured by their IL-2 secretion (Figure S1I). Overall, these data demonstrate that Keap1 controls NKT development in the thymus and homeostasis in the peripheral organs in a cell-intrinsic manner.

Keap1 Controls NKT Cell Proliferation, Survival, and Effector Functions

The reduced number of thymic and peripheral NKT cells in Keap1-deficient mice could be due to increased apoptosis or decreased proliferation. We first assessed the rate of spontaneous apoptosis in the NKT cells from WT and Keap1−/− thymi by staining for annexin V. Keap1−/− NKT cells had more annexin V+ cells than WT NKT cells (Figure 2A). In particular, stage 0 Keap1−/− NKT cells showed a higher proportion of apoptotic cells compared with other stages (Figure 2B and Figure S2A). The higher rate of apoptosis in Keap1−/− NKT cells was cell intrinsic because Keap1−/− NKT cells from BM chimeric mice were more apoptotic than WT NKT cells (Figure S2B).

Figure 2. Keap1 Controls NKT Cell Proliferation, Survival, and Effector Functions.

Figure 2.

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(A) Representative histograms and graph show the frequencies of annexin V+ in the thymic NKT cells from Keap1−/− and WT mice (n = 6).

(B) Percentages of annexin V+ cells within each developmental stage of thymic NKT cells from WT and Keap1−/− mice were compared (n = 6).

(C) The expression of Ki-67 in the WT and Keap1−/− thymic NKT cells was measured (n = 4).

(D) Percentages of BrdU+ cells within thymic and splenic NKT cells from WT and Keap1−/− mice were compared. Percentages of BrdU+ thymic NKT cells at each stage were also compared (n = 3).

(E) Representative BrdU and 7-AAD profiles of NKT cells from thymus and spleen of BrdU-injected WT and Keap1−/− mice (n = 3).

(F) Representative plots comparing NKT1, NKT2, and NKT17 subsets gated using T-bet, PLZF, and RORgt expression in the thymus of WT and Keap1−/− mice (n = 5).

(G) Graphs showing the percentages of NKT subsets in thymi (n = 5) and spleens (Spl) (n = 3) of WT and Keap1−/− mice.

(H) Representative plots comparing the intracellular expression of cytokines between thymic NKT cells WT and Keap1−/− mice after PMA-ionomycin stimulation in the presence of Golgi-plug for 4 h (n = 5).

(I) NKT cells sorted from WT and Keap1−/− spleens were stimulated with α-GalCer. On day 3, intracellular cytokines were measured after PMA-ionomycin treatment. Representative plots from three independent experiments are shown (n = 3).

(J) WT and Keap1−/− mice were injected intraperitoneally (i.p.) with PBS or α-GalCer, and 4 h later, the spleens were harvested to measure intracellular cytokines from freshly isolated NKT cells. Representative plots from three independent experiments are shown (n = 3).

All data are represented as mean ± SEM from the indicated number of sets of experiments. *p < 0.05 and **p < 0.01.

We next compared the expression of Ki-67, a proliferation marker. Unexpectedly, more Ki-67+ cells were observed within the Keap1−/− NKT cells compared with WT cells (Figure 2C). To further confirm the enhanced proliferation potential, we performed in vivo BrdU incorporation assays. Consistently, higher frequencies of BrdU+ NKT cells were observed in the Keap1−/− thymi and spleens compared with the WT organs (Figure 2D). Stage-wise analysis revealed that stage 0 and stage 1 NKT cells in the Keap1−/− thymus undergo more proliferation than the WT NKT cells of same stages (Figure 2D and Figure S2C). Interestingly, most of the BrdU+ NKT cells were also positive for 7AAD, a dye indicative of cell death, and BrdU+7AAD+ NKT cells were significantly increased in Keap1−/− thymus and spleen (Figure 2E). Thus, absence of Keap1 results in increased rates of cell proliferation and apoptosis, particularly in the early stages of NKT cell development.

To then assess NKT cell functions, we analyzed the frequency of NKT1, NKT2 and NKT17 (Cohen et al., 2013; Lee et al., 2013). In the Keap1−/− thymus and spleen, the percentage of NKT1 subset was decreased, while the NKT2 and NKT17 subsets were increased (Figures 2F and 2G). The three effector subsets NKT1, NKT2, and NKT17 majorly express the cytokines IFN-γ, IL-4, and IL-17, respectively (Lee et al., 2013). Thus, we next assessed the expression of these cytokines after stimulating the freshly isolated thymic and splenic cells with PMA and ionomycin for 4 h. Consistent with the low-frequency NKT1 subset, a trend of low IFN-γ+ NKT cells was observed in the Keap1−/− mice (Figures 2H, S2D, and S2E). Higher percentages of IL-4+ and IL-17+ NKT cells were noted in the Keap1−/− mice, in parallel with the increased NKT2 and NKT17 cells compared with WT mice (Figures 2H, S2D, and S2E). We further explored the functional effects of Keap1 deficiency using α-galactosylceramide (α-GalCer), an NKT cell-specific ligand, in vitro and in vivo. Upon in vitro stimulation, splenic NKT cells from Keap1−/− mice had lower percentages of IFN-γ+ cells and a trend of higher IL-4+ cells but proliferated poorly compared with WT NKT cells (Figures 2I and S2F) indicating that although Keap1−/− NKT cells undergo higher proliferation at steady state, they expand at a slower rate upon stimulation. Upon in vivo stimulation with α-GalCer, Keap1−/− NKT cells showed a similar trend of lower IFN-γ+ cells (Figures 2J and S2G). Thus, Keap1 is important for the balanced cytokines and proliferation of NKT cells.

Ablation of Nrf2 Rescues NKT Cell Developmental Defect in Keap1−/− Mice

T cells from Keap1−/− mice show increased accumulation of Nrf2 as well as increased mRNA levels of Nrf2 target genes Nqo1, Ho1, and Gclc (Noel et al., 2015). We also observed increased mRNA levels of Nqo1 in the sorted Keap1−/− thymic NKT cells compared with the WT cells (Figure S3A). To analyze if the defects observed in the Keap1−/− NKT cells are mediated by increased Nrf2, we generated mice with T cell-specific deletion of both Keap1 and Nrf2 (Keap1fl/fl Nrf2fl/fl-CD4Cre, called Keap1−/− Nrf2−/− henceforth) and examined their NKT cell compartment.

In line with our supposition, the NKT cell percentages in the Keap1−/− Nrf2−/− thymus were restored to WT levels (Figures 3A and 3B). A similar trend was observed in the spleen and liver of these mice (Figure 3B). Interestingly, Nrf2 deficiency (Nrf2fl/fl-CD4Cre [Nrf2−/−]) alone did not affect the NKT cell frequencies in any of the organs. Furthermore, the percentages of all the NKT cell stages in the Keap1−/− Nrf2−/− thymus were comparable with that in WT thymus (Figure 3C and Figure S3B). In addition, the frequencies of NKT2 and NKT17 subsets, which were higher in Keap1−/− thymus, were reduced in the Keap1−/− Nrf2−/− thymus, while the NKT1 cells were increased and restored to the WT levels in the Keap1−/− Nrf2−/− thymus (Figure 3D and Figure S3C). Also, the rates of proliferation and apoptosis, which were increased in the thymic Keap1−/− NKT cells, were restored in the NKT cells of Keap1−/− Nrf2−/− mice to the levels of WT mice (Figures 3E and 3F and Figure S3D). In all, these data indicate that the defect in NKT cell development and homeostasis observed in the Keap1−/− NKT cells seems to be due to dysregulated Nrf2.

Figure 3. Nrf2 Deficiency in Keap1−/− Mice Restores NKT Cell Development.

Figure 3.

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(A) Representative dot plots showing the NKT cell percentages in the thymus of the indicated mice (n = 4).

(B) Summary of NKT cell percentages in the thymus (Thy), spleen (Spl), and liver (Liv) of the indicated mice (n = 4).

(C) Percentages of thymic NKT cells at each developmental stage (STG) of indicated mice were compared (n = 4).

(D) Graphs showing the percentages of NKT subsets in thymi of indicated mice.

(E and F) Percentages of Ki-67+ (E) and annexin V+ (F) NKT cells in the thymus were compared (n = 3).

Data are shown as mean ± SEM from more than three sets of experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

Keap1 Deficiency Alters Cellular Metabolism in NKT Cells

Considering that the Keap1-Nrf2-regulated antioxidant system regulates cellular ROS (Taguchi et al., 2011), we investigated if the defective NKT cell development in the Keap1−/− mice was due to reduced levels of cellular ROS. We found that Keap1−/− NKT cells in the thymus have lower ROS levels than the WT NKT cells (Figure 4A). Furthermore, in WT NKT cells, the ROS levels rose significantly from DP stage to the stage 0 NKT cells and were maintained throughout the remaining stages, but Keap1−/− NKT cells had significantly lower ROS levels than WT cells across stages 1, 2, and 3 (Figure 4B and Figure S4A). Keap1-Nrf2 system crosstalk with autophagy pathway (Dodson et al., 2015; Taguchi et al., 2012) and autophagy is essential for NKT cell development, survival, and homeostasis (Pei et al., 2015; Salio et al., 2014). However, autophagy, measured by CytoID staining, was not altered in Keap1−/− NKT cells compared with WT NKT cells (Figure S4B).

Figure 4. Keap1 Deficiency Alters Cellular Metabolism in NKT Cells.

Figure 4.

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(A) Histogram overlay comparing the levels of ROS (DCFDA) in the NKT cells from WT and Keap1−/− thymi (n = 5).

(B) ROS levels in each NKT cell stage were compared. Fold increase was calculated as (mean fluorescence intensity [MFI] of each stage of NKT cells/MFI of DP thymocytes) (n = 4).

(C) Glucose uptake by thymic NKT cells was assessed after incubating the thymocytes from WT and Keap1−/− mice with 2-NBDG (n = 6).

(D) Expression of Glut1 was compared between WT and Keap1−/− thymic NKT cells (n = 3).

(E and F) Mitochondrial mass (E) and mitochondrial potential (F) in the WT and Keap1−/− thymic NKT cells were measured after incubating thymocytes with MitoTracker green and TMRM, respectively (n = 3 or 4).

(G–I) Graphs comparing DCFDA (G), glucose up-take (H), and mitochondrial mass (I) of thymic NKT cells from the indicated mice (n = 3 or 4).

(J) The expression of CD122 in the NKT cells from the thymi of WT, Keap1−/−, and Keap1−/− Nrf2−/− mice was compared (n = 3).

(K) Representative histograms showing the expression of Bcl2 by NKT cells from the thymi of indicated mice (n = 3). The graph summarizes the data from three independent experiments.

Data are shown as mean ± SEM from indicated number of sets of mice. *p < 0.05, **p < 0.01, and ****p < 0.0001.

Nrf2 has also been reported to positively regulate glucose metabolism and mitochondrial metabolism (Dinkova-Kostova and Abramov, 2015; Mitsuishi et al., 2012; Wang et al., 2018). Interestingly, the frequency of Keap1−/− thymic NKT cells taking up a higher amount of glucose was significantly increased (Figure 4C), together with elevated levels of glucose transporter Glut1 compared with WT NKT cells (Figure 4D). Additionally, Keap1−/− NKT cells had increased mitochondrial mass (Figure 4E) as well as higher mitochondrial potential than WT cells (Figure 4F). Importantly, deficiency of Nrf2 in the Keap1−/− NKT cells not only restored glucose up-take (Figures 4G and S3D) and mitochondrial mass (Figures 4H and S3D) but also increased ROS (Figures 4I and S3D) to levels comparable with WT NKT cells. Thus, Keap1 regulates NKT cell metabolism via Nrf2.

Next, we measured and compared the expression of CD122 (IL-15R), which has been shown to be essential for NKT cell maturation (Gordy et al., 2011; Ohteki et al., 1997). The expression levels of CD122 were lower in the Keap1−/− NKT cells than the WT cells, but the levels on Keap1−/− Nrf2−/− NKT cells were comparable with WT, indicating that reduced CD122 expression in the Keap1−/− cells is mediated by Nrf2 (Figure 4J). Because IL-15 signaling induces the expression of anti-apoptotic Bcl-family members (Gordy et al., 2011), we measured the expression of Bcl-2 and found that Keap1−/− NKT cells but not Keap1−/− Nrf2−/− NKT cells had more Bcl2-low cells than WT NKT cells (Figure 4K). Furthermore, this difference in the expression of CD122 and Bcl-2 was more prominent in NK1.1 population of thymic Keap1−/− NKT cells (Figures S4C–S4E). Consistently, NK1.1−early stages of development in Keap1−/− NKT cells also exhibited the most defect in survival (Figure 2B and Figure S2A). These data suggest that the Keap-Nrf2 complex controls NKT cell survival in the thymus by regulating the expression of CD122 and, subsequently, Bcl2.

DISCUSSION

In the present study, we revealed a critical role of Keap1 and Nrf2 proteins in NKT cell development and peripheral homeostasis. The defects in Keap1−/− NKT cells correlate not only with high proliferation but also with elevated apoptosis in these cells. In most cases, a defect in NKT cell frequency or development in the thymus is accompanied by a reduction in the peripheral NKT cell numbers, which was observed in Keap1−/− mice. This could be a result of lower number of mature NKT cells egressing to the periphery from the thymus or due to defects in the NKT cell survival and homeostasis after reaching the peripheral organs. In Keap1−/− mice, higher frequencies of annexin V+ and BrdU+ NKT cells were observed in the spleens, indicating that peripheral homeostasis and survival of the NKT cells was also compromised in the absence of Keap1. It is possible that the apoptosis is a consequence of high proliferation rate. In line with this, in the thymus and spleen, Keap1−/− NKT cells that incorporated BrdU were also positive for the cell death marker 7AAD, indicating that these highly proliferating cells undergo cell death simultaneously.

Another possibility is that proliferation and apoptosis are two parallel but independent outcomes of Keap1 deficiency. It is well established that Nrf2 positively regulates mitochondrial biogenesis and mitochondrial function (Dinkova-Kostova and Abramov, 2015). NKT cells are reported to have lower mitochondrial content than conventional T cells (Salio et al., 2014). Thus, an increase in mitochondrial activity might lead to increased proliferation of NKT cells in Keap1−/− mice. In tumor cells, Nrf2 is reported to enhance proliferation (Wang et al., 2018). Our data also shows that Keap1−/− NKT cells express lower levels of CD122 and Bcl2, both of which are important for NKT cell survival. The mechanism by which Keap1-Nrf2 complex regulates CD122 expression in NKT cells is enigmatic.

In line with the increased proliferation, mitochondrial mass, and mitochondrial potential, Keap1−/− NKT cells also exhibited a significant increase in glucose uptake and GLUT1 expression. It is well known that dividing cells take up more glucose using glucose transporters, which are expressed at increased levels in proliferating T cells (Frauwirth et al., 2002; Maciver et al., 2008). Therefore, the increased glucose uptake and Glut1 expression of Keap1−/− NKT cells could be a result of their high metabolic needs. Alternatively, studies showed that Nrf2 controls glucose metabolism (Heiss et al., 2013; Mitsuishi et al., 2012). According to a recent report, Nrf2 transcriptionally upregulates the expression of O-GlcNAc transferase (OGT), an important enzyme in hexosamine biosynthesis pathway of glucose metabolism (Li et al., 2017). It remains to be determined if OGT-mediated regulation of STAT3 activity or glucose consumption has any role in the proliferation of NKT cells or in the expression of CD122.

Interestingly, in contrast to the increased proliferation of Keap1-deficient NKT cells at steady state, lower proliferation was observed compared to the WT NKT cells upon stimulation with the NKT cell-specific ligand α-GalCer. It is possible that NKT cells undergo metabolic reprogramming upon stimulation in order to accommodate the energy requirements for supporting increased proliferation, which is defective in Keap1−/− NKT cells. Thus, Keap1-deficient NKT cells respond differently upon stimulation. Unfortunately, little is known about NKT cell metabolism at steady state or after stimulation, leaving much to speculation. More extensive investigations are required in order to determine the role of Keap1 in NKT cell metabolism.

We reported previously that low levels of ROS in NKT cells skews them toward NKT2 subset while decreasing NKT1 and NKT17 subsets (Kim et al., 2017). But here, we observed that Keap1−/− NKT cells, which harbor lower ROS levels, had lower frequencies of NKT1 but higher frequencies of both NKT2 and NKT17 subsets. It is likely that Keap1-Nrf2 system induces NKT17 independent of ROS. In the absence of Keap1, Nrf2 levels are expected to increase in the cytoplasm, leading to an upregulation of antioxidant genes. Indeed, levels of total cellular ROS in the thymic Keap1−/− NKT cells across all the stages were lower compared with the Keap1-sufficient NKT cells. It is likely that an appropriate amount of ROS at each NKT cell stage is critical for their developmental progression, and this balance may be compromised in Keap1−/− mice. In parallel, introducing Nrf2 deficiency to Keap1−/− NKT cells increased the cell numbers, and the developmental progression of NKT cells in Keap1−/− Nrf2−/− mice was restored to levels similar to WT mice. Intriguingly, apart from ROS, the proliferation rate and cell death as well as all metabolic parameters, including glucose uptake and mitochondrial mass, were also restored in the Keap1−/− Nrf2−/− NKT cells. These data imply that Nrf2-driven metabolic state of Keap1−/− NKT cells mediate the proliferative and apoptotic defects observed in these cells. Further investigation of the role of Keap1 and Nrf2 in NKT cell susceptibility to oxidative stress and subsequent change in effector functions is warranted. However, it is beyond the scope of the present study.

It should be pointed out that the defective development and altered metabolic state, as well as low ROS levels, were specific to NKT cells but not to CD4 T cells in Keap1−/− mice. Multiple studies have shown that unlike CD4 T cells, NKT cells are sensitive to changes in signaling pathways mediated by mTOR (Prevot et al., 2015; Zhang et al., 2014), CD28 (Williams et al., 2008), PI3K (Kishimoto et al., 2007), SAP-Fyn (Nunez-Cruz et al., 2008), and Wnt/β-catenin (Pyaram et al., 2017), most of which are known to regulate the metabolic pathways. Our present study together with these reports suggests that metabolic needs and signaling pathways necessary for NKT cell development and that effector functions are distinct from CD4 T cells.

In summary, we uncover an important role of the Keap1-Nrf2 complex in regulating cell metabolism to control NKT cell development, homeostasis, and effector functions. Taking into account the important and varied functions of Keap1-Nrf2 system, its pharmacological manipulation may be explored as a therapeutic strategy for targeting T cells in pathological conditions including NKT cells. It remains to be seen if clinical reagents targeting Nrf2 or oxidative stress would alter NKT cell metabolism, frequency, or functions.

STAR★METHODS

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to, and will be fulfilled by, the Lead Contact, Cheong-Hee Chang (heechang@umich.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice

T cell-specific Keap1-deficient mice, Keap1 fl/fl-CD4Cre (referred to as Keap1KO or Keap1−/−) were generated by crossing Keap1fl/fl mice which have been previously described (Blake et al., 2010) with CD4-Cre mice (The Jackson Laboratory). Nrf2 fl/fl-CD4Cre mice were generated by crossing Nrf2 fl/fl mice described earlier (Reddy et al., 2011) with CD4-Cre mice (The Jackson Laboratory). Keap1fl/flNrf2 fl/fl-CD4Cre mice were generated by crossing Keap1fl/flCD4-Cre mice with Nrf2fl/fl mice. B6.SJL-Ptprca Pepcb/BoyJ (CD45.1 congenic), Rag1−/− and C57BL/6 mice were purchased from The Jackson Laboratory. Age matched (8 to 14 weeks of age) Keap1fl/fl mice without Cre transgene obtained as littermates were used as WT controls in all experiments. All the mice were bred and maintained under specific pathogen-free conditions at the University of Michigan animal facility. All animal experiments were performed under protocols approved by the University of Michigan Institutional Animal Care and Use Committee.

Bone Marrow Chimeras

Bone marrow chimeric mice were generated as described earlier (Prevot et al., 2015; Zeng et al., 2013) with few modifications. Rag1−/− mice, that served as recipients, were sub-lethally irradiated (950 rad) 24 hours before receiving bone marrow transfers. Total bone marrow (BM) cells were harvested from the femurs and tibias of donor mice (3–4 months of age) and depleted of T cells, B cells, and MHC class II-positive lymphocytes by using a cocktail of antibodies against Thy1.2 (30H12), CD19 (1D3), and MHC class II (M5/114), followed by complement-mediated lysis using 10% Guinea pig serum. BM cells from Keap1fl/fl-CD4Cre and B6.SJL-Ptprca Pepcb/BoyJ donor mice were mixed at a 1:1 ratio and total 5 3 106 cells in 200 μl of PBS were transferred to each recipient mouse via tail vein injection. BM recipients were then housed under specific pathogen-free conditions and received acid water (pH 3.0) for 6 weeks. Mice were analyzed 12 to 16 weeks after BM transfer.

METHOD DETAILS

Cell Preparation and Flow Cytometry

Primary cell suspensions were prepared from thymi and spleens as per standard protocol(Kim et al., 2017). Lymphocytes from livers were isolated after loading homogenized liver cells resuspended in 40% isotonic Percoll solution on top of a 70% Percoll solution (GE Healthcare), and then centrifuged at 970 g at room temperature for 30 minutes. Liver mononuclear cells were harvested at the interface of the two layers of Percoll, washed and resuspended.

Up to 3 3 106 cells per sample were resuspended in 100 μl of FACS buffer (1% FBS in 1x PBS) and stained with following fluorophore conjugated anti-mouse antibodies, details of which are provided in the key resources table-TCR-β-Pacific blue or TCR-β-APC, CD4-PerCP-Cy5.5, CD8a-V500, CD44-FITC or CD44- PerCP-Cy5.5, CD45.1-FITC or CD45.1-PE-Cy7, NK1.1-PerCP-Cy5.5 or NK1.1-PE-Cy7, CD122-PE, CD24-PE-CF594, CD45.2-FITC, IFN-γ-FITC, IL-4-PECy7, IL-17-APC-Cy7, Ki-67-PerCP-Cy5.5, T-bet-PerCP-Cy5.5, PLZF-FITC, Bcl2-FITC and RORγT-PE. PE- or APC-conjugated murine CD1d tetramers loaded with PBS-57 were provided by the National Institutes of Health Tetramer Facility. Dead cells were excluded by staining with 1 mg/ml propidium iodide (Sigma-Aldrich).

For Glut1 staining, total thymocytes were fixed with 4% paraformaldehyde after staining for surface markers. Glut1 was then stained using anti-Glut antibody (EPR3915). Transcription factor staining to identify committed cells was performed using the Foxp3/transcription factor staining kit (BD) and T-bet and PLZF antibodies. Up to 2–3 × 106 total thymic or splenic cells were first stained for surface markers followed by Annexin V staining as per the manufacturer’s instructions (BD) for identifying for apoptotic cells. For the quantification of autophagy levels, thymocytes Cyto ID Autophagy Detection kit (Enzo Life Sciences) was used as per manufacturer’s instructions.

NKT Cell Hybridoma Assay

Double positive (CD4+CD8+) thymocytes were sorted out from Keap1fl/fl-CD4Cre and Keap1fl/fl littermate mice using BD Aria II flow cytometer after staining the total thymocytes with surface antigens. Per well, 1 × 105 DN32.D3 NKT-cell hybridoma cells (Lantz and Bendelac, 1994) were incubated with 3 × 105 sorted thymocytes from WT or Keap1KO mice in the presence of 0, 0.1 or 1 mg/ml of a-galactosylceramide (α-GalCer) in a 96-well plate for 72 hours. On day3, the culture supernatants were collected and analyzed for secreted IL-2 by ELISA (Core facility at University of Michigan).

In Vivo BrdU Incorporation

Six-week old Keap1fl/fl-CD4Cre and Keap1fl/fl littermate mice were injected twice (every 6 hours) intraperitoneally with 0.5 mg of BrdU (Sigma-Aldrich) in 0.3ml PBS. Mice injected with PBS alone were included as controls. Around eighteen hours after the last injection, animals were euthanized and single cell suspensions were prepared from thymi, spleens, and livers as described earlier. Following surface staining, cells were stained for BrdU incorporation using BrdU flow kit (BD bioscience), as per the manufacturer’s protocol.

NKT Cell Functional Assays

Freshly isolated total thymic and splenic cells (5 × 106 cells) were stimulated for 4 hours with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) and 1.5 μM of ionomycin (Sigma-Aldrich) in the presence of GolgiPlug (BD Biosciences). To measure intracellular cytokines, the cells were permeabilized using Cytofix/Cytoperm Plus (BD), then stained with the appropriate antibodies.

For antigen-specific responses, NKT cells were sorted from WT and Keap1−/− mice using BD Aria II flow cytometer after staining the total splenic cells with surface antigens. To compare the responses of NKT cells after activation, sorted cells were activated with α-GalCer (100 ng/ml) in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine and penicillin/streptomycin at 37°C. On day 3 intracellular cytokine expression was measured after the cells were re-stimulated with PMA and ionomycin. To measure antigen-stimulated cell proliferation, sorted NKT cells were labeled with CellTrace™ Violet (CTV; 5 μM) (Invitrogen) in 1X PBS containing0.1× BSA for 20 min at 37°C and activated with α-GalCer (100 ng/ml). Cells were analyzed by flow cytometry on day 3 to assess dilution of CTV.

To measure antigen-specific responses in vivo, WT and Keap1−/− mice were injected with 5 μg of α-GalCer in 200 mL of PBS intraperitoneally. After 4 hours, organs were harvested, and intracellular expression of cytokines was measured from freshly isolated NKT cells.

Detection of Reactive Oxygen Species (ROS)

To measure total cellular ROS, 2–3 × 106 total thymic cells were incubated with 1 μM 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA), also called DCFDA (Invitrogen) in RPMI media containing 10% fetal bovine serum (FBS) for 30 minutes at 37°C. After washing once, the cells were stained with relevant surface antibodies for flow cytometry analysis.

Glucose Uptake Assay

Up to 3 3 106 total thymic cells were incubated with 20 mM 2-NBDG [2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2-Deoxyglucose; Invitrogen] in Glucose-free RPMI media for 1 hour at 37 C. After washing once, the cells were stained with relevant surface antibodies for flow cytometry analysis.

Detection of Mitochondrial Parameters

Up to 3 × 106 total thymic cells were incubated with 30 nM MitoTracker Green (Invitrogen) or 60 nM Tetramethylrhodamine, Methyl Ester (TMRM; Invitrogen) in RPMI media containing 10% fetal bovine serum (FBS) for 30 minutes at 37°C. After washing once, the cells were stained with relevant surface antibodies for flow cytometry analysis.

Quantitative Real-Time PCR

Total cellular RNA was isolated from sorted CD1d-PBS57 tetramer-positive thymic NKT cells using RNeasy kit (QIAGEN). 50 ng RNA was reverse transcribed to cDNA using the RT2 First Strand Kit (QIAGEN). qPCR was performed using specific primers (synthesized from IDT) for the target gene Nqo-1. Gene expression of the target genes was normalized to b-Actin expression levels. The data analysis was performed using the 2 ΔΔCT method.

QUANTIFICATION AND STATISTICAL ANALYSIS

All data were obtained from at least three or more independent experiments. The n values described in the figure legends correspond to the number of animals used for each comparable. Data for all experiments were analyzed with Prism software (GraphPad Prism ver. 6). An unpaired student’s t test was used for the comparison of two experimental groups, and one-way analysis of variances (ANOVA) was used for multiple comparisons. All graphical data are represented as mean ± SEM. P values < 0.05 were considered statistically significant and were represented as < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****).

Supplementary Material

1

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Anti-Mouse CD4 (clone RM4–5) eBioscience Cat# 45-0042-82
Anti-Mouse CD8a (clone 53-6.7) BD Biosciences Cat# 560778
Anti-Mouse TCRb (clone H57-597) eBioscience Cat# 48-5961-82, 17-5961-83
Anti-Mouse CD44 (clone IM7) eBioscience Cat# 11-0441-85, 45-0441-82
Anti-Mouse NK1.1 (clone PK136) eBioscience Cat# 45-5941-82, 25-5941-82
Anti-Mouse CD45.1 (clone A20) eBioscience Cat# 11-0453-85, 25-0453-82
Anti-Mouse CD45.2 (clone 104) BD Biosciences Cat# 553772
Anti-Mouse CD122 (clone 5H4) eBioscience Cat# 12-1221-81
Anti-Mouse CD24 (clone (M1/69) BD Biosciences Cat# 562477
Anti-Mouse IFN-g (clone XMG1.2) eBioscience Cat# 11-7311-82
Anti-Mouse IL-4 (clone 11B11) eBioscience Cat# 25-7041-82
Anti-Mouse IL-17 (clone TC11-18H10) BD Biosciences Cat# 560821
Anti-Human/Mouse T-bet (clone eBio4B10) eBioscience Cat# 45-5825-82
Anti-Human/Mouse PLZF (clone Mags-21F7) eBioscience Cat# 53-9320-82
Anti-Human/Mouse Bcl-2 (clone BCL/10C4). Biolegend Cat# 633505
Anti-Mouse Ki-67 (clone SolA15) eBioscience Cat# 46-5698-80
Anti-Mouse/Rat Thy1.2 (30H12) BioXCell Cat# BE0066
Anti-mouse Glut1 antibody (clone EPR3915) Abcam Cat# ab115730
Anti-Human/Mouse RORgT (clone AFKJS-9) eBioscience Cat# 12-6988-80
Chemicals, Peptides, and Recombinant Proteins
PBS57-loaded mouse CD1d tetramers NIH tetramer facility http://tetramer.yerkes.emory.edu/reagents/cd1
PMA Sigma-Aldrich Cat# P8139
Ionomycin Sigma-Aldrich Cat# I0634
BrdU Sigma-Aldrich Cat# B9285
α-galactosylceramide (α-GalCer) Diagnocine KRN7000
Propidium iodide Sigma-Aldrich Cat# P4170
Percoll GE Healthcare Cat# 45001-747
GolgiPlug BD Biosciences Cat# 555029
Guinea pig serum MP Biomedicals Cat# 642831
CellTrace™ Violet Invitrogen Cat# C34557
DCFDA Invitrogen Cat# C6827
2-NBDG Cayman Chemicals Cat# 11046
MitoTracker Green Invitrogen Cat# M7514
TMRM Invitrogen Cat# T668
SYBR green PCR mix Applied Biosystems Cat# 4309155
Critical Commercial Assays
BrdU flow kit BD Biosciences Cat# 559619
Foxp3/transcription factor staining kit BD Biosciences Cat# 560409
Annexin V staining kit BD Biosciences Cat# 559763
Cyto ID Autophagy Detection kit Enzo Life Sciences Cat# ENZ-51031-0050
Cytofix/Cytoperm Plus BD Biosciences Cat# 554714
RNeasy kit QIAGEN Cat# 74004
RT2 First Strand Kit QIAGEN Cat# 330404
Experimental Models: Cell Lines
Vα14+ NKT cell hybridoma cell line DN32.D3 DOI https://doi.org/10.1084/jem.180.3.1097. N/A
CD19 (1D3), ATCC HB-305
MHC class II (M5/114) ATCC TIB-120
Experimental Models: Organisms/Strains
Mouse: Keap1 fl/fl-CD4Cre Noel etal. (2015) N/A
Mouse: Nrf2fl/fl Reddy et al., 2011 N/A
Mouse: B6.SJL-Ptprca Pepcb/BoyJ The Jackson Laboratory Stock No: 002014
Mouse: C57BL/6J The Jackson Laboratory Stock No: 000664
Mouse: B6.129S7-Rag1tm1Mom/J The Jackson Laboratory Stock No: 002216
Mouse: B6.Cg-Tg(Cd4-cre)1Cwi/BfluJ The Jackson Laboratory Stock No: 022071
Oligonucleotides
β-Actin primers (5′−3′): For, AGCCATGTACGTAG CCATC; Rev, CTCTCAGCTGTGGTGGTG IDT This paper
Nqo-1 primers (5′−3′): For, TTCTCTGGCCGATT CAGAGT; Rev, GGCTGCTTGGAGCAAAATG IDT This paper
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Highlights.

  • Keap1 deficiency results in defective NKT cell development and homeostasis

  • Keap1-Nrf2 govern NKT cell proliferation, apoptosis, and effector functions

  • Nrf2 deletion restores low ROS and NKT cell defects inKeap1-deficient mice

  • The Keap1-Nrf2 system regulates NKT cell metabolism

ACKNOWLEDGMENTS

We would like to thank Ms. Chauna Black for diligent maintenance and genetic screening of the mice. We also thank the Chang lab members for their valuable input to the manuscript. We acknowledge the NIH Tetramer Facility for providing us with the mouse CD1d tetramers. This work was supported in part by NIH grants AI121156 (to C.-H.C.) and DK111209 (to H.R.).

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10.1016/j.celrep.2019.03.052.

DECLARATION OF INTERESTS

The authors declare no competing interests.

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Supplementary Materials

1
NIHMS1049435-supplement-1.pdf (1.1MB, pdf)

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