Pyruvate dehydrogenase phosphatase catalytic subunit 2 limits Th17 differentiation Michihito Konoa,1,2, Nobuya Yoshidaa,1,2, Kayaho Maedaa, Nicole E. Skinnera, Wenliang Pana, Vasileios C. Kyttarisa, Maria G. Tsokosa, and George C. Tsokosa,2 aDepartment of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 Edited by Dennis A. Carson, University of California, San Diego, La Jolla, CA, and approved August 1, 2018 (received for review April 3, 2018) Th17 cells favor glycolytic metabolism, and pyruvate dehydrogenase conversion for rapid nonmitochondrial ATP generation, Th17 (PDH) is the key bifurcation enzyme, which in its active dephosphory- cells were shown to be uniquely regulated by this enzyme (11). lated form advances the oxidative phosphorylation from glycolytic Accordingly, the enzymatic activity of PDH is repressed in Th17 pathway. The transcriptional factor, inducible cAMP early repressor/ cells to promote conversion of pyruvate to lactate by enhancing cAMP response element modulator (ICER/CREM), has been shown to the activity of PDH kinase (PDHK) that phosphorylates PDH be induced in Th17 cells and to be overexpressed in CD4+ T cells from (active form) to phospho-PDH (inactive form) (11). However, the patients with systemic lupus erythematosus (SLE). We found that whether PDH phosphatase (PDP), the balancing counterpart of glycolysis and lactate production in in vitro Th17-polarized T cells was PDHK that dephosphorylates phospho-PDH (inactive form) to reduced and that the expression of pyruvate dehydrogenase phos- PDH (active form), is also affected during Th17 differentiation phatase catalytic subunit 2 (PDP2), an enzyme that converts the in- is not known. active PDH to its active form, and PDH enzyme activity were increased Since genome-wide analysis of cAMP-response element (CRE) in Th17 cells from ICER/CREM-deficient animals. ICER was found to binding protein occupancy has indicated the possibility that CRE- bind to the Pdp2 promoter and suppress its expression. Furthermore, binding proteins can regulate genes involved in cell metabolism forced expression of PDP2 in CD4+ cells reduced the in vitro Th17 (12), we considered that ICER/CREM may control the activity of differentiation, whereas shRNA-based suppression of PDP2 expression metabolic enzymes. We observed reduced glycolysis and lac- increased in vitro Th17 differentiation and augmented experimental tate production in in vitro Th17-polarized ICER/CREM-deficient autoimmune encephalomyelitis. At the translational level, PDP2 ex- T cells. We found that Pdp2, one of the subunits of PDP, is in- pression was decreased in memory Th17 cells from patients with creased in Th17 cells from ICER/CREM-deficient mice, and its + SLE and forced expression of PDP2 in CD4 T cells from lupus-prone expression is controlled directly by ICER/CREM at the tran- MRL/lpr mice and patients with SLE suppressed Th17 differentiation. scriptional level. Forced PDP2 expression reduced the in vitro These data demonstrate the direct control of energy production dur- Th17 differentiation, whereas shRNA-based suppression of PDP2 ing Th17 differentiation in health and disease by the transcription expression increased in vitro Th17 differentiation and augmented factor ICER/CREM at the PDH metabolism bifurcation level. experimental autoimmune encephalomyelitis (EAE) in mice. Importantly, PDP2 expression was decreased in memory Th17 cells from patients with SLE and forced expression of PDP2 to ICER | PDP2 | Th17 | SLE | glycolysis + naïve CD4 T cells from the patients with SLE reduced IL-17 production during Th17 differentiation. Our data demonstrate ystemic lupus erythematosus (SLE) is a multiorgan disorder that ICER accomplishes Th17 differentiation through direct Sof largely unknown etiology characterized by an autoreactive control of a metabolic enzyme, which diverts energy production cellular and humoral immune response. While most types of to the glycolytic pathway. immune cells have been reported to be involved in the develop- ment and progression of SLE, abnormalities of T cells including Significance Th17 cells appear to play a major role in the pathogenesis (1, 2). Patients with SLE have a higher frequency of Th17 cells, which contribute to the establishment of proinflammatory conditions by Th17 cells favor glycolytic metabolism. Pyruvate dehydroge- infiltrating multiple organs (3, 4). nase, which facilitates entry into the oxidative phosphorylation The cAMP response element modulator (CREM) controls the circle, is inhibited by pyruvate dehydrogenase phosphatase transcription of cAMP-responsible genes (5). Interestingly, ex- catalytic subunit 2 (PDP2). Our studies demonstrate that the pression of CREM splice variants that repress cAMP transcription, transcription factor ICER/CREM, which is known to promote such as CREMα and inducible cAMP early repressor (ICER), are Th17 differentiation and related pathology, suppresses the + increased in CD4 T cells from patients with SLE (6). Several lines expression of PDP2 and diverts energy production into the of evidence suggest a strong association between ICER/CREM glycolytic pathway. In lupus-prone mice and people with sys- and Th17 cell differentiation (6), including the facts that forced temic lupus erythematosus, PDP2 levels are decreased and its expression of CREMα in human T cells enhances IL-17A ex- replenishment suppresses Th17 differentiation. pression (7), ICER is most exclusively induced in Th17 cells and promotes Th17 cell differentiation, and ICER/CREM-deficient Author contributions: M.K., N.Y., and G.C.T. designed research; M.K., N.Y., K.M., N.E.S., mice display less Th17-related autoimmune pathology. This has W.P., V.C.K., and M.G.T. performed research; M.K. and N.Y. contributed new reagents/ analytic tools; M.K., N.Y., and K.M. analyzed data; and M.K., N.Y., and G.C.T. wrote also been underscored by the genome-wide analysis of the Th17 the paper. transcription regulatory network that has revealed that Crem was induced among other genes during Th17 differentiation and that The authors declare no conflict of interest. silencing of Crem led to reduced Th17 differentiation (8). This article is a PNAS Direct Submission. Activated T cells including Th17 cells use glucose metabolism Published under the PNAS license. to fulfill the metabolic requirements for rapid proliferation and 1M.K. and N.Y. contributed equally to this work. biosynthesis supporting cellular growth and differentiation (9, 2To whom correspondence may be addressed. Email: [email protected], nyoshida@ 10). A previous metabolic profiling study has revealed that py- bidmc.harvard.edu, or [email protected]. ruvate dehydrogenase (PDH), which converts pyruvate to acetyl- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CoA, is a key bifurcation enzyme between T cell glycolytic and 1073/pnas.1805717115/-/DCSupplemental. oxidative metabolism. Because Th17 cells favor pyruvate to lactate Published online August 27, 2018. 9288–9293 | PNAS | September 11, 2018 | vol. 115 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1805717115 Downloaded by guest on September 26, 2021 Results catalytic subunit 2 (PDP2) is expressed in the liver (14) and many ICER Promotes Glycolysis and Lactate Production in in Vitro Th17 other cells, including white blood cells. Cells. Previously, we had shown that ICER/CREM promotes To investigate how ICER promotes glycolysis and lactate Th17 cell differentiation (6). Th17 cells depend on glycolysis more production in Th17 cells, we assessed the expression levels of the than other T cell subsets (11). Glycolysis involves the conversion of Pdhk1, Pdhk3, Pdp2, and pyruvate dehydrogenase phosphatase Pdpr glucose to pyruvate, which is subsequently converted to lactate by regulatory subunits ( ) in in vitro polarized Th17 cells using qRT-PCR. As shown in Fig. 2B, Pdhk1 expression was decreased lactate dehydrogenase (LDH), and lactate itself has been shown to in ICER/CREM-deficient Th17 cells compared with ICER/ induce the generation of Th17 cells (13). We hypothesized that CREM-sufficient Th17 cells, whereas Pdp2 expression was in- ICER/CREM promotes Th17 differentiation through the control creased in ICER/CREM-deficient Th17 cells. There were no of enzymes that enable lactate production. To examine the function + significant differences in Pdhk3 or Pdpr between the two groups of ICER in glycolysis and lactate production, naïve CD4 T cells (Fig. 2B and SI Appendix, Fig. S1A). However, when empty or from ICER/CREM-sufficient or ICER/CREM-deficient mice were ICERγ vector was overexpressed in Th17-polarized ICER/ cultured under Th17 conditions in vitro and extracellular acidifi- CREM-deficient cells, Pdp2 expression was significantly de- cation rate (ECAR) was analyzed by an extracellular flux analyzer creased in ICERγ-overexpressed Th17 cells, while the expression (Fig. 1A). Glycolysis can be measured as a change in ECAR that of Pdhk1 was not affected (Fig. 2D). In agreement with the reflects lactate production (11). Th17 cells from ICER/CREM- changes in mRNA levels, PDP2 protein expression was increased sufficient mice had more glycolysisasreflectedbytheincreased in ICER/CREM-deficient Th17 cells (Fig. 2C) and was de- lactate production and more glycolytic capacity than cells from creased in ICERγ-overexpressing Th17 cells (Fig. 2E). Next, ICER/CREM-deficient mice (Fig. 1B). To confirm these observa- LDH and PDH enzyme activity are determined. LDH activity is + tions, we overexpressed