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 5

PKM2 inhibitor–treated CD4+ T cells ameliorate disease activity in an adoptive cell transfer EAE model.

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PKM2 inhibitor–treated CD4+ T cells ameliorate disease activity in an ad...
(A–D) CD4+ T cells from 2D2 mice were cultured with 20 μg/ml MOG35-55, mitomycin-treated splenocytes, and 0.5 ng/ml rIL-12 for 48 hours and DMSO or shikonin was added on day 0. On day 2, these cells were transferred into Rag1-deficient mice intravenously and EAE was induced. (A and B) Clinical scores (A) and body weight changes (B) of recipient mice are shown. Cumulative results are shown (mean ± SEM); n = 5. (C and D) Spinal cords were harvested on day 14 and stained with H&E to evaluate inflammation. (C) Representative images show H&E staining of spine from the mice that received DMSO- or shikonin-treated cells. Scale bars: 500 μm or 100 μm (higher-magnification images on right). (D) Quantitative cumulative data are shown (mean ± SEM); n = 5. (E and F) Absolute cell numbers of spinal cord–infiltrating IFN-γ–producing (E) and IL-17A–producing CD4+ T cells (F) from the mice that received DMSO- or shikonin-treated cells were evaluated by flow cytometry on day 14. Cumulative data are shown (mean ± SEM); n = 8. *P < 0.05. **P < 0.01 by 2-way ANOVA (A and B) or 2-tailed t test (D–F).