Pyruvate kinase M2 is requisite for Th1 and Th17 differentiation
Michihito Kono, … , Nobuya Yoshida, George C. Tsokos
Michihito Kono, … , Nobuya Yoshida, George C. Tsokos
Published June 20, 2019
Citation Information: JCI Insight. 2019;4(12):e127395. https://doi.org/10.1172/jci.insight.127395.
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Research Article Immunology

Pyruvate kinase M2 is requisite for Th1 and Th17 differentiation

Abstract

Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin–dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4+ T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4+ T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4+ T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell–dependent autoimmune diseases.

Authors

Michihito Kono, Kayaho Maeda, Irina Stocton-Gavanescu, Wenliang Pan, Masataka Umeda, Eri Katsuyama, Catalina Burbano, Seo Yeon K. Orite, Milena Vukelic, Maria G. Tsokos, Nobuya Yoshida, George C. Tsokos

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Figure 3

PKM2 promotes in vitro Th1 and Th17 differentiation.

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PKM2 promotes in vitro Th1 and Th17 differentiation. (A–F) Naive CD4+ T ...
(A–F) Naive CD4+ T cells were cultured for 3 days under Th1- and Th17-polarizing conditions and various concentration of shikonin were added on day 0. (A and B) Extracellular acidification rate (ECAR) in Th1 (A) and Th17 cells (B) with or without shikonin was measured on day 3. Cumulative data are shown (mean ± SEM); n = 3. (C and E) Representative flow plots of IFN-γ–producing (C) and IL-17A–producing (E) CD4+ T cells are shown. (D and F) Cumulative data of IFN-γ–producing (D) and IL-17A–producing (F) CD4+ T cells are shown (mean ± SEM); n = 5. (G–I) Naive CD4+ T cells were cultured for 4 days under Th17-polarizing conditions and transfected with control shRNA or Pkm2-specific shRNA on day 1. (G) PKM2 and actin protein expression on day 3 was assessed by Western blotting. Representative blots are shown. Data are representative of 4 experiments. (H) Cumulative data of the Western blotting densitometric ratio (PKM2/actin) are shown (mean ± SEM); n = 4. (I) Cumulative data of flow plots of IL-17A–producing CD4+ T cells are shown (mean ± SEM); n = 4. (J) Naive CD4+ T cells were cultured for 3 days under Th17-polarizing conditions and transfected with empty vector or mouse PKM2–expressing vector on day 1. Cumulative data of IL-17A–producing CD4+ T cells are shown (mean ± SEM); n = 4. *P < 0.05; **P < 0.01 by 2-tailed t test (A, B, and J) or 1-way ANOVA with Bonferroni’s multiple-comparisons test (D, F, H, and I).