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. Author manuscript; available in PMC: 2013 Nov 23.
Published in final edited form as: Immunity. 2013 Apr 25;38(5):984–997. doi: 10.1016/j.immuni.2013.02.021

The microRNA miR-181 is a critical cellular metabolic rheostat essential for NKT cell ontogenesis and lymphocyte development and homeostasis

Jorge Henao-Mejia 1,*, Adam Williams 1,*, Loyal A Goff 2,3,4, Matthew Staron 1, Paula Licona-Limón 1, Susan M Kaech 1,6, Maki Nakayama 5, John L Rinn 2,3, Richard A Flavell 1,6,**
PMCID: PMC3738211  NIHMSID: NIHMS485597  PMID: 23623381

Abstract

Regulation of metabolic pathways in the immune system provides a mechanism to actively control cellular function, growth, proliferation and survival. Here, we report that miR-181 is a non-redundant determinant of cellular metabolism and is essential to support the biosynthetic demands of early NKT cell development. As a result, miR-181-deficient mice showed a complete absence of mature NKT cells in the thymus and periphery. Mechanistically, miR-181 modulated expression of the phosphatase PTEN to control PI3K signaling, which was a primary stimulus for anabolic metabolism in immune cells. Thus miR-181-deficient mice also showed severe defects in lymphoid development and T cell homeostasis associated with impaired PI3K signaling. These results uncover miR-181 as essential for NKT cell development, and establish this family of miRNAs as central regulators of PI3K signaling and global metabolic fitness during development and homeostasis.

Keywords: miR-181, PTEN, Natural killer T cells, Metabolism, PI3K

Introduction

Until recently metabolism was considered a ‘house-keeping’ utility that merely supported cellular processes. However, it is now clear that the regulation of metabolic pathways in the immune system provides a mechanism to actively control cellular function, growth, proliferation and survival. Signaling through the phosphatidylinositol 3-kinase (PI3K) pathway provides a potent stimulus for anabolic metabolism during lymphoid development and immune responses, but it must be tightly controlled to prevent cancer and autoimmunity (Alimonti et al., 2010; Pal et al., 2012). The phosphatase PTEN is the principal and non-redundant negative regulator of the PI3K pathway; as a result organisms are exquisitely sensitive to PTEN expression (Alimonti et al., 2010). Robust mechanisms must therefore exist to precisely coordinate PTEN expression with the transcriptional programs that promote lineage specification and differentiation in the immune system, and regulation by miRNAs provides one exciting possibility. We therefore hypothesized that miRNA families specialized in the coordination of anabolic metabolism during lymphoid development and homeostasis in vivo are likely to have evolved. Previous work has revealed miRNAs with the capacity to regulate the PI3K-PTEN axis; however, the lack of in vivo developmental phenotypes associated with impaired anabolic metabolism in miRNA-deficient mice indicates that miRNAs responsible for cellular metabolic regulation have yet to be identified (Ebert and Sharp, 2012; Huse et al., 2009; Inui et al., 2010; Ma et al., 2011; Mendell and Olson, 2012; Olive et al., 2009; Park et al., 2010; Patrick et al., 2010; Small et al., 2010; Ventura et al., 2008).

The development of T cells and Natural Killer T (NKT) cells in the thymus is a life-long process that requires high proliferation rates and therefore elevated biosynthetic demands; PI3K signaling is a critical anabolic determinant required to support these proliferative developmental stages (Fayard et al., 2010; Finlay et al., 2010). While much is known about the transcriptional programs and signaling pathways that regulate these essential metabolic adaptations during NKT cell and T cell development, the role of non-coding RNAs in controlling such processes is mostly unknown. Interestingly, thymic ablation of the miRNA-processing enzyme Dicer causes defects in thymocyte development as well as a complete loss of NKT cells in the thymus and periphery; however, the identity of the individual microRNAs and the mechanism through which they regulate NKT development remain largely undetermined(Cobb et al., 2005; Fedeli et al., 2009; Zheng et al., 2012).

We revealed that miR-181 was an essential regulator of PI3K signaling strength, through PTEN modulation, and therefore was a critical determinant of cellular metabolic adaptations required to support high proliferation rates during development. As a result, miR-181-deficient mice showed a complete absence of mature NKT cells in the thymus and periphery. In addition, we showed that miR-181-deficient mice displayed several hematopoietic and non-hematopoietic defects associated with reduced metabolic fitness driven by impaired PI3K signaling. Altogether these results provide important insights into the physiological function of this miRNA family; moreover, it places miR-181 as a central in vivo regulator of cellular metabolic fitness during development and homeostasis.

Results

miR-181 determines organism size

The miR-181 family is composed of six mature miRNAs: miR-181a-1, miR-181a-2, miR-181b-1, miR-181b-2, miR-181c, and miR-181d which are encoded in three independent paralog precursor transcripts on three separate chromosomes (Ji et al., 2009). The mature forms of miR-181a-1 and miR-181a-2, as well as miR-181b-1 and miR-181b-2 are identical in sequence. Furthermore, all family members contain the same 5’ “seed” sequence suggesting a significant degree of functional redundancy (Ji et al., 2009). To test the function of the miR-181 family in vivo, we generated mice in which each of the three miR-181 gene clusters were flanked by loxP sites to enable tissue specific deletion (Figures S1A–C). Mice deficient for the microRNA clusters miR-181a1b1 (containing Mir181α-1 and Mir181b-1), miR-181a2b2 (containing Mir181α-2 and Mir181b-2) and miR-181cd (containing Mir181c and Mir181d ) were obtained in predicted Mendelian ratios and none of these lines displayed any obvious gross phenotypic abnormalities in terms of growth, development or survival. In contrast, mice carrying compound deletions of the different miR-181 clusters demonstrated reduced survival and decreased body weight when compared to littermates, suggesting that this miRNA family regulates an essential pathway in vivo (Figures 1A, S1D and data not shown). Indeed, mice deficient for all three miR-181 clusters have yet to be obtained; providing evidence that complete deficiency of the miR-181 family may not be compatible with life.

Figure 1. miR-181 regulates survival, organism size and PTEN expression in thymocytes.

Figure 1

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(A) Survival rates of mice with compound deletions of the miR-181a1b1 (a1b1WT, a1b1HET, or a1b1KO) and the miR-181a2b2 (a2b2WT, a2b2HET, or a2b2KO) clusters (n=245). (B) (Panel 1) Scatter plot of gene-level expression estimates from RNA-Seq of WT (a1b1WT) vs miR-181a1b1 deficient (a1b1KO) DP thymocytes. (Panel 2) Volcano plot highlighting log2 ratios (a1b1WT/a1b1KO) of gene expression estimates vs differential expression significance values. (C) GSEA plot demonstrating enrichment of miR-181 target genes in miR-181a1b1 deficient DP thymocytes. The x-axis represents the rank ordering (a1b1WT/a1b1KO) of all genes. A running GSEA enrichment score for miR-181 target genes (red) is plotted along the rank order. miR-181 target genes are individually identified with a black tick mark at their rank positions. A density plot of miR-181 target genes is presented with the darker blue indicating a greater number of target genes. (D) Relative amounts of Pten expression from RNA-Seq data in DP thymocytes from WT and miR-181a1b1 deficient mice. (E) Protein blot analysis of PTEN protein in total thymocytes from WT and miR-181a1b1 deficient mice. Each lane represents thymocytes from a single mouse. (F) Protein blot analysis of PTEN protein in sorted DN1–3 and DN4 thymocytes from WT and miR-181a1b1 deficient mice. Each lane represents thymocytes from a single mouse. (G) Intracellular stain of phospho-AKT (Ser473) after stimulation with CXCL12 for indicated times. Representative of 5 independent experiments. (H) Abbreviated heatmap of FOXO target gene log2 fold-change values (fill color) with indications of membership in the differentially expressed gene set (black border). (I and J) Cell surface expression of CD62L on thymocytes from WT and miR-181a1b1 deficient mice. (n=5, data is representative of 4 independent experiments). All error bars indicate mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S1.

miR-181 regulates PI3K signaling by modulating PTEN expression

NKT cells and conventional T cells develop from a common progenitor cell in the thymus in an ordered multi-step program. As miR-181a and miR-181b are highly expressed in the thymus from the mir-181a1b1 cluster locus we focused on the characterization of the mir-181a1b1-deficient line (Figures S1A and S1E). As an initial approach we performed an unbiased RNA-Seq analysis of sorted CD4+CD8+ double positive (DP) thymocytes. DP thymocytes were chosen as they represent the point of divergence between NKT cell and conventional T cell development, and because Dicer-deficient mice have a complete block during the very early stages of NKT cell development (Fedeli et al., 2009). Triplicate RNAs were sequenced on an Illumina HiSeq 2000, and ≥ 64.0×106 76-bp paired-end reads per replicate were mapped to the mouse genome (mm9) using Tophat (Trapnell et al., 2009). Mouse UCSC transcripts were quantified and differentially expressed genes were identified using Cuffdiff2 (data not shown). Analysis of replicate FPKM (Fragments Per Kilobase of transcript model, per Million fragments mapped) distributions, appropriate hierarchical clustering of biological replicates, and a low squared coefficient of variation between replicate expression values confirmed that the samples were of good quality (Figure S1F–H). Differential gene analysis revealed 2612 differentially expressed genes between wild-type (WT) and miR-181a1b1-deficient DP thymocytes; more than 80% of these genes were downregulated in the miR-181a1b1 deficient cells (Figure 1B). The observation that deletion of a single miR-181 cluster was sufficient to cause deregulation of a vast number of genes suggests that this miRNA family is controlling a central regulatory pathway. To confirm a global derepression of miR-181 targets in miR-181a1b1 deficient mice, a FPKM-rank-ordered (WT/ miR-181a1b1-deficient) list of genes was prepared and used as input for a pre-ranked GSEA analysis (Subramanian et al., 2005) against the MSigDB microRNA target gene set collection (C3 miRNA v3.0; MSigDB). The greatest significant association (p<0.001; Kolmogorov-Smirnov test) was observed for genes in the miR-181 target gene set and confirms a transcriptome-wide increase in predicted miR-181 target genes in miR-181a1b1 deficient DP thymocytes (Figure 1C). Of the 146 predicted miR-181 targets that were identified as significantly upregulated in miR-181a1b1 deficient DP thymocytes, we were particularly intrigued by the gene Pten (phosphatase and tensin homolog), the principal negative regulator of the PI3K pathway. Pten was an attractive target as it is a non-redundant regulator of a central pathway common in all cells; moreover, small changes in PTEN transcript levels usually have large phenotypic consequences (Alimonti et al., 2010; Carracedo et al., 2011; Garcia-Cao et al., 2012; Ortega-Molina et al., 2012). RNA-Seq analysis showed that Pten mRNA was approximately 65% higher in miR-181a1b1 deficient DP thymocytes (Figure 1D). Upregulation of PTEN in miR-181a1b1 deficient thymocytes was confirmed by immunoblot analysis (Figures 1E, 1F and S1I). In support of Pten mRNA as a potential miR-181 target there are 4 predicted (Dianalab algorithm) miR-181 sites in the 3’ UTR of Pten (Figure SIJ).

We predicted that increased PTEN expression would result in reduced PI3K signaling in DP thymocytes. Thymocyte development requires PI3K activation through CXCR4, and ex vivo stimulation of thymocytes with the CXCR4 ligand CXCL12 represents a physiologically relevant system to interrogate PI3K signaling ex vivo (Janas et al., 2010). Using intracellular flow cytometry, we measured the amount of AKT phosphorylation on serine 473 (p-AKT) in freshly isolated thymocytes after in vitro stimulation with CXCL12 (Figure 1G). Although DP thymocytes from miR-181a1b1 deficient mice contained a similar amount of p-AKT at 1 minute post-stimulation, loss of p-AKT occurred at a faster rate, suggesting that the increased PTEN expression attenuated PI3K signaling downstream of CXCR4. Importantly, Cxcr4 transcription (as determined by RNA-Seq) was not significantly changed in the absence of miR-181a1b1 (data not shown). It has previously been suggested that miR-181 is an intrinsic modulator of TCR signaling strength, through the downregulation of different phosphatases that dampen ERK signaling(Ebert et al., 2009; Li et al., 2007). Moreover, it has been suggested to regulate NOTCH1 signaling indirectly through modulation of negative regulators of this pathway(Fragoso et al., 2012). However, we found no evidence to support this (Figures S2A–D). Moreover, we did not observe any gross defects in TCR signaling strength (Figure S2B and S2E-H). Downstream of PI3K, activated AKT phosphorylates and inhibits FOXO proteins; FOXO proteins are transcription factors, which can either activate or repress target genes to regulate numerous cellular responses. Analysis of gene expression changes of FOXO targets serves as a useful surrogate marker of PI3K-PTEN activity (Carracedo et al., 2011). Examination of the RNA-Seq data revealed that a significant number of predicted FOXO target genes (36/369; p=9.21×10−4; hypergeometric test) were differentially regulated in miR-181a1b1 deficient DP thymocytes (Figure 1H). Moreover, genes that are known to be activated by FOXO proteins were significantly upregulated in miR-181a1b1 deficient DP thymocytes whereas FOXO repressed genes were significantly upregulated in WT DP thymocytes. Of interest we found an upregulation of the confirmed FOXO target genes Ctla4, Cd28 and Sell (encoding CD62L) in miR-181a1b1 deficient DP thymocytes. We further validated these RNA-Seq findings by confirming that surface expression of CD62L was significantly elevated in DN4 (double negative 4), DP and CD4 SP (single positive) thymocytes of miR-181a1b1 deficient mice (Figures 1I and 1J). Thus, miR-181a1b1 ablation increased PTEN activity resulting in decreased signaling downstream of PI3K.

miR-181a1b1 deficiency alters cellular metabolism

PI3K–AKT signaling leads to increased MYC activity and activation of the mTORC1 complex, which together are responsible for driving multiple anabolic processes, including the transcriptional upregulation of metabolic enzymes required to meet the energetic and biosynthetic demands of growth and proliferation. We therefore examined the RNA-Seq data to determine if there was evidence of reduced signaling downstream of PI3K in the form of a disrupted metabolic gene signature in miR-181a1b1 deficient DP thymocytes. This analysis revealed that key components of the glycolytic pathway, pentose phosphate pathway and nucleotide biosynthetic pathways were significantly downregulated in miR-181a1b1 deficient DP thymocytes (Figures 2A and 2B). In contrast, expression of the genes involved in glutaminolysis and beta-oxidation were minimally dysregulated in miR-181a1b1 deficient thymocytes. Expression of genes involved in cell cycle regulation were also minimally changed in miR-181a1b1 deficient thymocytes, highlighting the specificity of the metabolic reprogramming gene signature (Figure 2B).

Figure 2. Thymocytes deficient in miR-181a1b1 display an altered metabolic gene signature and reduced rates of glycolysis.

Figure 2

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(A) Heatmap of log2 fold-change values for genes in specific metabolic pathways. Those genes identified as significantly differentially expressed are indicated with a black border. (B) Hypergeometric p-values for PI3K-associated metabolic pathways with a significant number of differentially expressed genes, analysis of various cell cycle pathways is also included. (C) Relative amount of Pkm2 mRNA from RNA-Seq data in DP thymocytes from WT and miR-181a1b1 deficient mice. (D) Flow cytometry analysis of 2-NBDG uptake by WT and miR-181a1b1 deficient DP thymocytes. (n=5–6, data is representative of two independent experiments). (E) Cell surface expression of CD98 on DP thymocytes from WT and miR-181a1b1 deficient mice, as revealed by flow cytometry. (n=5–6, data is representative of two independent experiments). (F) Cell surface expression of CD71 on DP thymocytes from WT and miR-181a1b1 deficient mice, as revealed by flow cytometry. (n=5–6, data is representative of two independent experiments). (G and H) Metabolic analysis using the Seahorse XF96 Extracellular Flux Analyzer. (G) Extracellular acidification rates (ECAR) and (H) oxygen consumption rates (OCR) of thymocytes from WT and miR-181a1b1 deficient mice. Each point represents a single mouse. (n=4, data is representative of three independent experiments). (I) Flow cytometry assessment of mitochondrial mass (using mitotracker green) and (J) mitochondrial reactive oxygen species (using mitosox red) in DP thymocytes from WT and miR-181a1b1 deficient mice. Each point represents a single mouse. (n=5–6, data are representative of two independent experiments). All error bars indicate mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S2.

The pyruvate kinase isoform PKM2 promotes both aerobic glycolysis and anabolic metabolism (Christofk et al., 2008). Interestingly, miR-181a1b1 deficient DP thymocytes cells had significantly reduced levels of PKM2 mRNA as compared to WT DP thymocytes, which could potentially impair anabolic metabolism and the ability of miR-181a1b1 deficient cells to proliferate (Figure 2C).

GLUT1 (also known as SLC2A1) is the primary glucose transporter expressed on developing thymocytes and it is required during the DN to DP transition to meet the metabolic requirements of proliferation (Juntilla et al., 2007). PI3K–AKT signaling and MYC positively regulate transcription of the Slc2a1 gene (Barthel et al., 1999; Osthus et al., 2000). PI3K signaling also initiates trafficking of GLUT1 to the plasma membrane (Wieman et al., 2007). GLUT1 mRNA was significantly downregulated in miR-181a1b1 deficient DP thymocytes (Figure 2A). As a consequence, thymocytes displayed a significantly reduced capacity for glucose uptake as revealed by decreased acquisition of the fluorescent deoxyglucose analog 2-NBDG (Figure 2D). Like GLUT1, PI3K signaling is also required for expression of the nutrient transporters CD98 (amino acid transporter, SLC3A2) and CD71 (transferrin receptor) (Kelly et al., 2007). Cell surface expression of both CD98 and CD71 was significantly reduced in the absence of the miR-181a1b1 cluster on multiple thymocyte populations, as determined by flow cytometry (Figure 2E, 2F and Figure S2I).

Activation of mTORC1 by AKT drives increased translation through multiple mechanisms, including activation of ribosomal proteins, as well as increased transcription of ribosomal RNA and ribosomal protein genes (Hay and Sonenberg, 2004). In agreement with this, analysis of the RNA-Seq data revealed a significant reduction in the expression of ribosomal proteins in miR-181a1b1 deficient DP thymocytes (Figure 2A). Altogether these data reveal that deletion of the single miR-181a1b1 cluster is sufficient to induce a major metabolic reprogramming at the genetic level.

To directly assess whether the altered metabolic gene signature of miR-181a1b1 deficient thymocytes translated to altered metabolism we measured rates of aerobic glycolysis and oxidative phosphorylation. The extracellular acidification rate (ECAR) is used as a measure of lactate produced during glycolysis. Thymocytes from miR-181a1b1 deficient mice displayed a 50% reduction in ECAR indicating that glycolysis is significantly decreased (Figure 2G). In contrast oxygen consumption rates (OCR), which report levels of oxidative phosphorylation, were not significantly altered between WT and miR-181a1b1 deficient thymocytes (Figure 2H). Flow cytometric assessment of mitochondrial mass and production of mitochondrial reactive oxygen species (ROS) also revealed no significant differences between WT and miR-181a1b1 deficient thymocytes (Figures 2I and 2J). The suboptimal glucose uptake and reduced glycolytic rates indicate that miR-181a1b1 deficient thymocytes have reduced metabolic fitness, which would result in an impaired capacity for cell growth and proliferation.

miR-181a1b1 deficiency perturbs T cell development

The results presented above show that deletion of the miR-181a1b1 cluster causes a significant metabolic reprogramming in DP thymocytes. As DP thymocytes represent the point of divergence for the development of NKT cells and conventional T cells (which are both PI3K dependant), we predicted that the generation of both lineages would be severely impacted in miR-181a1b1 deficient mice.

Mice with targeted deletion of individual components of the PI3K pathway display characteristic developmental phenotypes in the thymus (Fayard et al., 2007; Finlay et al., 2010; Hinton et al., 2004; Ji et al., 2007; Sasaki et al., 2000; Swat et al., 2006; Webb et al., 2005). These include thymic hypocellularity, decreased percentage of DN1 thymocytes and maintained expression of CD25 on thymocytes from the DN3 to DP stages. Interestingly, miR-181a1b1 deficient mice displayed evidence for each of these developmental defects, reflecting reduced capacity for PI3K signaling as a consequence of PTEN overexpression (Figures 3A, 3B and S3A–D).

Figure 3. Deletion of miR-181a1b1 perturbs thymocyte development.

Figure 3

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(A) Absolute thymocyte numbers and representative flow cytometry plots showing thymocyte populations from age matched WT and miR-181a1b1 deficient mice (n=8–9, data represents two independent experiments). (B) Cell surface expression of CD25 on indicated populations of thymocytes from WT and miR-181a1b1 deficient mice, as revealed by flow cytometry (n=5, data is representative of at least 4 independent experiments). (C) Absolute numbers, percentage and representative flow cytometry histogram of BrdU incorporation in double positive (DP) thymocytes from WT and miR-181a1b1 deficient mice after a single BrdU injection (n=6, data representative of three independent experiments). (D) Percentage of annexin V negative WT and miR-181a1b1 deficient DP thymocytes at indicated time points of in vitro culture. A representative flow cytometry histogram is shown (n=3, data is representative of three independent experiments). (E) Relative decreased in DP thymocyte numbers and representative flow cytometry plots in WT and miR-181a1b1 deficient mice injected with a single dose of dexamethasone. Numbers shown are relative to PBS injected control mice. (n=6–7, data represents two independent experiments) (F) Ratio of miR-181a1b1 deficient (KO) to WT Tcra J gene segment usage obtained from sequencing Tcra chain mRNA in sorted CD4+ thymocytes and plotted as a function of relative distance along the Tcra locus. Each point represents the ratio obtained from the average values of thymocytes independently sorted and sequenced from 3 WT and 6 miR-181a1b1 deficient mice. All error bars indicate mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S3.

Metabolic alterations associated with PI3K signaling deficiency drives thymic hypocellularity by altering the balance of proliferation and apoptosis in the thymus (Fayard et al., 2007; Finlay et al., 2010; Hinton et al., 2004; Sasaki et al., 2000; Swat et al., 2006; Webb et al., 2005). Therefore, to assess the proliferation of thymocytes in vivo, WT and miR-181a1b1 deficient mice were injected with a single dose of BrdU and thymocytes were harvested 24 hours later. The absolute number and percentage of BrdU+ DP thymocytes were reduced in miR- 181a1b1 deficient mice, revealing that proliferation during this developmental stage is indeed significantly impaired (Figure 3C). When cultured ex vivo, DP cells rapidly undergo apoptosis in the absence of survival signals. Thymocytes from miR-181a1b1 deficient mice showed increased apoptosis in culture over time, as revealed by flow cytometric quantification of annexin V staining (Figure 3D). Dexamethasone, a glucocorticoid steroid, induces thymocyte apoptosis in vitro and in vivo, and is thought to mimic a process termed “death by neglect” (Wang et al., 1999; Zilberman et al., 2004). miR-181a1b1 deficient DP thymocytes cultured in the presence of increasing concentrations of dexamethasone were more prone to apoptosis compared to WT cells (Figure S3E). To test survival in vivo, WT and miR-181a1b1 deficient mice were injected with a single dose of dexamethasone to induce thymocyte apoptosis. In agreement with in vitro observations, miR-181a1b1 deficient thymocytes were more susceptible to dexamethasone-induced apoptosis in vivo (Figure 3E).

Mutations that reduce thymocyte survival manifest as a decreased in T cell receptor frequency of distal Jα gene segment usage (D'Cruz et al., 2010; Guo et al., 2002). As a functional readout of thymocyte survival in vivo we assessed usage of each Vα and Jα gene segment in sorted CD4 SP thymocytes by global sequencing of Tcra transcripts (Figures 3F and S3F). From this analysis, it was evident that in miR-181a1b1 deficient mice Jα gene usage was significantly biased in favor of proximal Jα gene segments over more distal Jα gene segments, indicating that miR-181a1b1 deficient thymocytes have a decreased lifespan in vivo. Thus, in line with a reduced PI3K-driven metabolic fitness, miR-181a1b1 deficient thymocytes show decreased proliferative capacity and decreased survival in vivo.

Dysregulation of PTEN levels in miR-181a1b1 deficient mice blocks NKT cell development

NKT cells are a highly prevalent population of innate-like T cells that have been shown to be important in clearing tumors, preventing autoimmunity and fighting infections. Most NKT cells express the canonical TCRα-chain composed of α-chain variable region 14 and α-chain joining region 18 (Vα14-Jα18), together with the TCR Vβ8, Vβ7 or Vβ2 chain, which as a heterodimeric complex confers specificity to glycolipid ligands. Upon analysis of Tcra gene segment usage in CD4 SP thymocytes we noted a significant underrepresentation of the Vα14-Jα18 NKT TCR (Figure 4A) (designated Trav11.02-Traj18.01 using updated nomenclature).

Figure 4. miR-181a1b1 deficient mice lack NKT cells.

Figure 4

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(A) Relative frequency of the Vα14-Jα18 NKT TCR usage in sorted CD4+ thymocytes (designated Trav11.02-Traj18.01) from WT and miR-181a1b1 deficient mice, revealed by sequencing Tcra mRNA. Each point represents data from a single mouse. (B) Absolute numbers and percentages of αGalCer-CD1d tetramer+TCRβ+ NKT cells in the thymus, spleen and liver of WT and miR-181a1b1 deficient mice. (n=7–8, data represents two independent experiments). (C) Absolute numbers of αGalCer-CD1d tetramer+TCRβ+ NKT cells in the thymus, spleen and liver of WT (a1b1fl/fl) and miR-181a1b1fl/flCd4-Cre (a1b1fl/fl;Cd4-Cre) mice. (n=7–8, data represents two independent experiments). All error bars indicate mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S4.

Flow cytometry, using CD1d tetramers loaded with alpha-galactosylceramide (αGalCer), revealed an almost complete absence of NKT cells in the thymus, spleen and liver of miR-181a1b1 deficient mice (Figure 4B). As mice in which the miR-181a1b1 cluster had been deleted with Cd4-Cre also lacked NKT cells we reasoned that the defect in NKT development most likely occurred during or after the DP thymocyte stage (Figure 4C). NKT deficiency was specific to loss of the miR-181a1b1 cluster as miR-181a2b2 and miR-181cd deficient mice did not display reductions in NKT cell numbers, reflecting the expression profiles of each of the miR-181 members (Figures S4A and S4B)(Kuchen et al., 2010).

NKT cells are selected by glycolipids presented in the context of CD1d displayed on the surface of DP cortical thymocytes, and defects in CD1d expression preclude NKT development. However, we found no evidence for reduced CD1d expression or for a processing or presentation defect by miR-181a1b1 deficient cortical thymocytes, indicating that the deficiency in NKT cells was cell intrinsic (Figures 5A–C and S5C). We have also excluded defects in three molecules critical for two survival axes that have previously been shown to regulate NKT cell development (RORγt, Bcl-xL and BIM) (Figures 5D, 5E and S5A–D). Moreover, as the deficit in NKT cell development prevails in NKT TCR transgenic mice, it suggests that defects in Tcra rearrangement due to reduced thymocyte survival are not the singular cause of the NKT cell developmental block in miR-181a1b1 deficient mice (Figures 5F and S5E).

Figure 5. NKT cell developmental defects in miR-181a1b1 deficient mice are cell intrinsic and independent of BCL-XL, BIM, or Tcra rearrangement.

Figure 5

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(A) Cell surface expression of CD1d on DP thymocytes from WT and miR-181a1b1 deficient mice, as revealed by flow cytometry (n=4). Representative flow cytometry histograms are shown. (B) Briefly, 0.5×106 WT and 0.5×106 miR-181a1b1 deficient bone marrow cells were injected into lethally irradiated hosts. Six weeks later engraftment was assessed by percentages of CD45.1 and CD45.2 cells in the spleen. Absolute numbers of WT (CD45.1) and miR-181a1b1 deficient (CD45.2) αGalCer-CD1d tetramer+TCRβ+ NKT cells in the thymus, spleen and liver of transplanted mice. (n=8, data representative of 2 independent experiments). (C) Representative flow cytometry dot plots from the spleen of bone marrow chimeric mice. (D-F) Absolute numbers of αGalCer-CD1d tetramer+TCRβ+ NKT cells in the thymus, spleen and liver of (D) Tg-Bcl-xl (a1b1WT; Tg-Bcl- xl) and Tg-Bcl-xl-miR-181a1b1 (a1b1KO; Tg-Bcl-xl) deficient mice (n=7–8, data represents two independent experiments), (E) Bim−/− (a1b1WT; BimKO) and Bim−/−-miR-181a1b1 deficient (a1b1KO; BimKO) mice (n=4–5), and (F) Tg-Vα14-Jα18 (a1b1WT; Tg-Vα14-Jα18) and Tg-Vα14-Jα18-miR-181a1b1 deficient (a1b1KO; Tg-Vα14-Jα18) mice. (n=4–5, data is representative of two independent experiments). All error bars indicate mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S5.

Genetic reduction of PTEN levels restores NKT cell development in miR-181a1b1 deficient mice

NKT development can be divided into four (0–3) discrete stages based on the expression of the cell surface markers NK1.1, CD44, and CD24. Expression of miR-181a and miR-181b was highest in DP thymocytes and decreased as NKT development progressed to stage 3 (Figure 6A). In order to determine at which stage NKT development is blocked in miR-181a1b1 deficient mice we performed tetramer enrichment of NKT cells pooled from the thymus of 4–5 mice using αGalCer-loaded CD1d tetramers, followed by flow cytometry (Figures 6B and 6C). Immediately following positive selection, stage 0 developmental intermediaries, which are present in very low numbers, undergo a massive proliferative expansion in a PI3K dependent manner (Finlay et al., 2010). Similar to what has been shown in PDK1- and Dicer-deficient animals, deletion of the miR-181a1b1 cluster resulted in a developmental block during the metabolically demanding transition from stage 0–1, whereas later stages showed no obvious defects (Figures 6B and 6C). Due to the similarity to PDK1-deficient animals, we hypothesized that proliferation of early stage NKT cells would also be defective in the miR-181a1b1 deficient mice. Indeed BrdU incorporation experiments in Tg-Vα14-Jα18 and Tg-Vα14-Jα18 miR-181a1b1deficient mice showed that the absence of the miR-181a1b1 cluster leads to decreased proliferation of developing NKT cells (Figure 6D).

Figure 6. miR-181a1b1 is essential for early stages of NKT cell development.

Figure 6

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(A) Relative expression of miR-181a and miR-181b in sorted DP thymocytes and sorted NKT cells MACs purified from the thymus of 5 pooled mice after tetramer enrichment representing developmental stages 0–3, as determined by semi-quantitative RT-PCR. Data is representative of two independent experiments. Enriched αGalCer-CD1d tetramer+ NKT cells were subsequently sorted based on the following markers; Stage 0: NK1.1-;CD44-;CD24+; Stage 1: NK1.1−;CD44−;CD24−; Stage 2: NK1.1−;CD44+; Stage 3: NK1.1+;CD44+). (B) Absolute numbers of αGalCer-enriched CD1d tetramer+ cells/thymus, absolute numbers of Stage 0 tetramer+ NKT cells/Thymus and relative numbers of Stage 0 tetramer+ NKT cells / Total CD1d tetramer+ NKT cells obtained after tetramer enrichment from the thymus of WT and miR-181a1b1 deficient mice. Purifications were performed using thymi pooled from multiple mice; each point represents 4–5 mice, data is from three independent experiments. (C) Relative frequency of NKT stages 1–3 in αGalCer-CD1d tetramer+/TCRβ+ NKT cells (n=4, data representative of at least 4 independent experiments). Representative plots are shown. (D) Representative flow cytometry histograms showing BrdU incorporation in αGalCer-CD1d tetramer+/TCRβ+ NKT cells (Stage 0 to Stage 3) from Tg-Vα14-Jα18 (a1b1WT; Tg-Vα14-Jα18) and Tg-Vα14-Jα18-miR-181a1b1 deficient (a1b1KO; Tg-Vα14-Jα18) mice. after a single BrdU injection (data representative of two independent experiments). (E) Absolute number and relative frequency of αGalCer-CD1d tetramer+/TCRβ+ NKT cells in the thymus of miR-181a1b1fl/fl (a1b1fl/fl), miR-181a1b1fl/flCd4-Cre (a1b1fl/fl; Pten+/+; Cd4-Cre) and miR-181a1b1fl/flPtenfl/flCd4-Cre (a1b1fl/fl; Ptenfl/fl; Cd4-Cre) mice (data from two independent experiments). (F) Representative flow cytometry plots from (E). (G) Cell surface expression of CD62L and CD25 on developing thymocytes from miR-181a1b1fl/fl(a1b1fl/fl), miR-181a1b1fl/flCd4-Cre (a1b1fl/fl; Pten+/+; Cd4-Cre) and miR-181a1b1fl/flPtenfl/flCd4-Cre (a1b1fl/fl; Ptenfl/fl; Cd4-Cre) mice (data from two independent experiments). All error bars indicate mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001.

The paucity of NKT cells in miR-181a1b1 deficient mice reveals that development of this lineage is uniquely sensitive to PI3K-mediated metabolic dysregulation. We hypothesized that reducing PTEN levels would allow rescue of NKT cell numbers in the thymus of miR-181a1b1 deficient mice, however, as Pten transcripts were upregulated by 65% in DP thymocytes (and visibly more at other stages) we reasoned that deletion of only one Pten allele would be insufficient to rescue NKT development. It must be noted that although PTEN is not absolutely required for the generation of NKT cells, it is required for their maturation from stage 2 to stage 3 of NKT cell ontogenesis. Thus we performed experiments in which either one or both Pten alleles were ablated using Cd4-Cre. As predicted, deletion of one Pten allele resulted in a partial rescue of NKT development, PI3K signaling, and CD25 and CD62L expression (data not shown), whereas deletion of both Pten alleles caused a complete restoration of the number of developing NKT cells in the thymus and completely restored appropriate expression of CD25 and CD62L on developing thymocytes (Figures 6E–G).

miR-181a1b1 deficient mice display multiple hematopoietic defects

As metabolic regulation has global importance in biological processes requiring high rates of proliferation and because the miR-181 family is ubiquitously expressed, we hypothesized that miR-181 deletion would affect additional PI3K-dependent events in the hematopoietic system. We therefore assessed the effect of miR-181 cluster ablation on peripheral T cell homeostatic proliferation and early B cell development, both of which are known to be regulated by PI3K signaling. PDK1-deficient T cells, which lack PI3K signaling, are defective in their ability to undergo homeostatic proliferation in a lymphopenic host (Finlay et al., 2010). In concordance with this, miR-181a1b1 deficient conventional αβ T cells are significantly reduced (∼75%) in peripheral tissues of mixed bone marrow (BM) chimeras reconstituted with miR-181a1b1 deficient and WT BM cells. (Figures 7A–D). Identical results were obtained when mixed BM chimeras were reconstituted with miR-181a1b1fl/flCd4-Cre and WT BM cells, excluding any potential contribution of differences in thymic reconstitution (Figures S6A–C). In addition, to exclude any potential influence of thymic output, we injected a mixture (1:1) of CFSE labeled congenically marked WT and miR-181a1b1-miR-181a2b2 deficient naïve T cells into lymphopenic hosts (Rag1−/− mice) and determined the relative engraftment of T cells and their proliferation status after 10 days (Figures 7E–H). In agreement with our previous results, we noted reduced frequency, reduced total numbers and reduced proliferative capacity of miR-181a1b1-miR-181a2b2 deficient T cells compared to WT T cells in peripheral lymphoid organs.

Figure 7. Aberrant lymphopenia-induced T cell proliferation in the absence of miR-181a1/b1 and miR-181a2/b2.

Figure 7

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(A to D) Proliferation of WT and miR-181a1b1 deficient cells induced by lymphopenia. (A and B) Briefly, 0.5×106 WT and 0.5×106 miR-181a1b1 deficient bone marrow cells were injected into lethally irradiated hosts. Six weeks later engraftment was assessed by determining percentages of CD45.1 and CD45.2 cells in the thymus and spleen. (C and D) Absolute numbers and percentages of WT (CD45.1) and miR-181a1b1 deficient (CD45.2) B cells (B200+) and T cells (TCRβ+) in the spleen were determined by flow cytometry. (n=8, data is representative of two independent experiments). (E to H) Analysis of lymphopenia induced proliferation. Sorted naïve CD4+ T cells from WT (CD45.1) and miR-181a1b1-miR-181a2b2 deficient (CD45.2) mice were labeled with CFSE and injected into Rag−/− mice in equal proportions. The relative proportion of cells of each genotype in the injected mixture was assessed by flow cytometry. Ten days after injection, spleen and lymph nodes were harvested from transplanted mice and (E) the absolute number and (F) percentage of injected cells was quantified by flow cytometry. (G and H) Proliferation of injected cells was assessed by CFSE dilution. (n=6, data is representative of two independent experiments). All error bars indicate mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S6.

The mir-181a1b1 and mir-181a2b2 clusters are both highly expressed during the early stages of B cell development (pro-B cells and pre-B cells) (Kuchen et al., 2010). Recent evidence indicates that PI3K signaling is required for the development of pro-B cells and the propagation of pre-B cells (Baracho et al., 2011). Moreover, PI3K signaling promotes the retention of FOXO1 in the nucleus, which drives Il7ra, Cd62l, and Rag expression during early B cell development (Amin and Schlissel, 2008; Dengler et al., 2008). Therefore, we explored the potential for B cell developmental defects in miR-181a1b1-miR-181a2b2 deficient mice. The frequency and total numbers of B220+ cells in the bone marrow were decreased approximately 50% in miR-181a1b1-miR-181a2b2 deficient mice as compared to WT animals (Figure S6D). Furthermore, miR-181a1b1-miR-181a2b2 deficient mice showed a significant accumulation of pro-B and reduction of pre-B cells and immature B cells, suggesting a defect in the pro-B cell to pre-B cell transition (Figures S6E–G). Finally, as a surrogate marker of reduced PI3K signaling we noted aberrant expression of CD62L on pro-B and pre-B cells in miR-181a1b1-miR-181a2b2 deficient mice (Figure S6H). Thus, we conclude that miR-181 is also required for normal lymphopenia-induced T cell proliferation and for normal B cell development.

Discussion

Metabolic regulation is emerging as a principal mechanism to actively control cellular function, growth, proliferation and survival. In the present study, we reveal that miR-181 is a non-redundant rheostat of PTEN activity and cellular metabolism during lymphoid development and homeostasis, highlighting this as one of the most important miRNA families described to date.

Through the regulation of PI3K signaling the mir-181a1b1 cluster promotes a cellular anabolic reprogramming that is necessary to sustain the biosynthetic demands encountered during development. The physiological importance of this finding is highlighted by the observation that miR-181a1b1 cluster deletion results in a complete absence of NKT cells in the thymus and the periphery; indeed this is a unique example where deletion of a single miRNA cluster causes the loss of entire cell lineage. In addition, miR-181 deficient mice show severe defects in T cell development and homeostasis, defects during early B cell development, as well as reduced organism size, revealing that miR-181 mediated metabolic regulation has a global physiological importance.

Interestingly, NKT cell development appears to be particularly sensitive to alterations in metabolic fitness, which likely reflects a number of aspects unique to NKT cell ontogenesis. First, developing thymocytes must successfully express a functional NKT TCR; this necessitates use of one of the most distal Tcra J gene segments and therefore requires extended thymocyte survival(D'Cruz et al., 2010). Second, after diverging from the T cell lineage, developing NKT cells must undergo a rapid and vast burst of proliferation. Third, expression of the key metabolic regulator MYC is specifically required during early NKT cell development (Dose et al., 2009; Mycko et al., 2009). Each of these three independent biological events depends upon PI3K signaling to provide adequate metabolic fitness necessary to support prolonged thymocyte survival and foster early NKT cell proliferation. Therefore, consistent with the data presented herein, the paucity of NKT cells in miR-181a1b1 deficient mice most likely results from the cumulative effect of impaired PI3K signaling during multiple stages of NKT development.

Although miRNAs have previously been shown to target different components of the PI3K-PTEN axis, the physiological relevance of the majority of these interactions remains to be established, especially when considering the general lack of gross phenotypes in miRNA knockout mice (Ebert and Sharp, 2012; Huse et al., 2009; Inui et al., 2010; Ma et al., 2011; Mendell and Olson, 2012; Olive et al., 2009; Park et al., 2010; Patrick et al., 2010; Small et al., 2010; Ventura et al., 2008). In contrast, the robust metabolic phenotypes observed on deletion of miR-181 family members at the organism and cellular level, combined with the ability to rescue developmental abnormalities through genetic reduction of PTEN levels, confirms the function of this miRNA family in metabolic regulation. Thus, miR-181 represents an example of a miRNA family that functions as a bona fide cellular metabolic rheostat during development in vivo.

Finally, whereas PTEN downregulation is a hallmark of many cancers and obesity, overexpression of miR-181 is considered a signature of a wide variety of solid tumors and leukemias (Calin et al., 2005; Carracedo et al., 2011; Jones et al., 2012). We therefore propose that dysregulation of the miR-181-PTEN axis may drive development of cancer and metabolic syndrome.

Experimental procedures

NKT purification

Single cell suspensions from thymus were incubated with phycoerthryin-conjugated αGalCer-loaded CD1d tetramers. NKT cells were then isolated by positive selection using an LS column according to the manufactures instructions.

CXCL12 stimulation and Phosphoflow

Freshly isolated thymocytes were resuspended at 1×107 cells/ml in DMEM with 0.1% BSA. Cells were rested at 37°C for 20 minutes before the addition of 10 nM CXCL12 (Peprotech) for indicated time points. Cells were then fixed by the addition of and equal volume of 4% PFA and incubated at 37°C for an additional 15 minutes. Fixed cells were washed twice with FACS buffer and permeabilized in ice cold 90% methanol and stored at −20°C for 1 hour. Before staining, fixed cells were washed 3 times in FACS buffer. Staining was performed at room temperature for 1 hour. Stained cells were washed twice and subsequently stained with an Alexa Fluor 488 conjugated anti-rabbit antibody at room temperature for 30 minutes. After washing three times cells were analyzed by flow cytometry. The following phospho-specific antibodies from Cell Signaling were used: Phospho-AKT (Ser473) (193H12) and Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E).

TCR stimulation

Thymocytes were resuspended at 5×106 cells/ml in RPMI with 10% FBS and rested at 37°C for 20 minutes before spinning onto tissue culture plates pre-coated with anti-CD3 (10µg/ml) and anti-CD28 (50µg/ml). At indicated time points cells were removed with a cell scraper and immediately transferred onto ice, centrifuged and resuspended in ice cold lysis buffer (Cell signaling) containing a protease inhibitor cocktail (Roche) and a phosphatase inhibitor (Thermo Scientific). Protein lysates were analyzed by Western blot.

Thymocyte survival culture

Thymocytes were cultured at a concentration of 1–3×106 cells/ml in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin-L-glutamine (PSG). Cells were harvested at indicated time points and survival was assessed by Annexin V staining.

Dexamethasone culture

Thymocytes were cultured at a concentration of 3×106 cells/ml in DMEM supplemented with 10% FBS and 1% PSG Cells were incubated with indicated concentrations of dexamethasone for 24 hours before survival was assessed by annexin V staining.

Dexamethasone injection

Age, weight and sex matched mice were injected with a single intra-peritoneal dose of dexamethasone (150 µg/mouse) and 20 hours later mice were euthanized and thymocytes analyzed by annexin V and PI staining.

BrdU Incorporation

Mice were injected with a single intra-peritoneal dose of BrdU (1mg) and 24 hours later were sacrificed and single cells suspensions were obtained from thymus. BrdU flow kit (BDbiosciences) was used to detect BrdU as directed by the manufacturer’s instructions.

Seahorse metabolic studies

Analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of thymocytes was performed using a Seahorse XF96 Extracellular Flux Analyzer instrument. Briefly, WT or KO thymocytes were seeded at least in triplicate at a density of 1×106/well on poly-D-lysine coated plates, and the OCR and ECAR were measured in real-time.

Glucose uptake/Mitosox/Mitochondrial mass

For analysis of glucose uptake, mitochondrial mass and mitochondrial reactive oxygen species thymocytes were pulsed with either 2-NBDG, mitotracker green and mitosox red dyes (Invitrogen) for 10 minutes at 37°C in 1% FCS/RPMI or 1% FCS/glucose-free RPMI (glucose uptake assay, specifically). Cells were then washed with FACS buffer and stained as usual for flow cytometry.

Statistical analysis

Unless otherwise stated we performed statistical analysis using a one-way ANOVA with a Bonferroni multiple comparison post test and Student’s T test. We considered P<0.05 to be statistically significant. *P<0.05, **P<0.001, ***P<0.0001

Supplementary Material

01

Acknowledgments

We would like to thank Stephanie C. Eisenbarth, Jon Alderman, and Caroline Lieber for technical assistance and discussions. J.H.M. is supported by Leukemia and Lymphoma Society Postdoctoral Fellowship. M.S is supported by F32, 1F32AI096718-01A1. M.N is supported by R00 DK080885. P.L-L was supported by post-doctoral fellowship from PEW charitable trust: PEW Latin American Fellow Program in Biomedical Sciences. This work was supported in part by the Howard Hughes Medical Institute (G.I.S and R.A.F.).

Footnotes

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The authors report no conflict of interest.

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