TLR-driven early glycolytic reprogramming via the kinases TBK1-IKKɛ supports the anabolic demands of dendritic cell activation (original) (raw)

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Acknowledgements

We thank members of the Pearce laboratory for discussions; H. Virgin for support of the 14C-glucose–tracing experiments; W. Beatty and the Molecular Microbiology Imaging Facility for technical assistance with electron microscopy; and G. Bridon and B. Faubert for help with the glucose-tracing experiments. Some observations reported here were made while B.E., E.A., T.C.F., G.J.W.v.d.W., E.L.P. and E.J.P. were at the Trudeau Institute; we thank the institute for its support during that time. Supported by the US National Institutes of Health (AI53825 and CA164062 to E.J.P.; AI091965 and CA158823 to E.L.P.; and AI049823 to E.A.), the Netherlands Organisation for Scientific Research (B.E. and G.J.W.v.d.W.) and The Arthritis Society of Canada (R.G.J.).

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Authors and Affiliations

  1. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA
    Bart Everts, Stanley Ching-Cheng Huang, Amber M Smith, Chih-Hao Chang, Wing Y Lam, Veronika Redmann, Gerritje J W van der Windt, Maxim N Artyomov, Erika L Pearce & Edward J Pearce
  2. Department of Medical Laboratory and Radiation Sciences, University of Vermont, Burlington, Vermont, USA
    Eyal Amiel
  3. Trudeau Institute, Saranac Lake, New York, USA
    Tori C Freitas & Julianna Blagih
  4. Department of Physiology, McGill University, Montreal, Quebec, Canada
    Russell G Jones

Authors

  1. Bart Everts
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  2. Eyal Amiel
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  3. Stanley Ching-Cheng Huang
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  4. Amber M Smith
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  5. Chih-Hao Chang
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  6. Wing Y Lam
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  7. Veronika Redmann
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  8. Tori C Freitas
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  9. Julianna Blagih
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  10. Gerritje J W van der Windt
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  11. Maxim N Artyomov
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  12. Russell G Jones
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  13. Erika L Pearce
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  14. Edward J Pearce
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Contributions

B.E., E.A., S.C.-C.H., C.-H.C., G.J.W.v.d.W., R.G.J., E.L.P. and E.J.P. designed experiments; B.E., E.A., S.C.-C.H., C.-H.C., A.M.S., W.Y.L., V.R., T.C.F. and J.B. did experiments. B.E., E.A., S.C.-C.H., J.B., R.G.J., M.N.A., E.L.P. and E.J.P. analyzed data; and B.E. and E.J.P. wrote the paper.

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Correspondence toEdward J Pearce.

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Integrated supplementary information

Supplementary Figure 1 GM-DCs display an early iNOS-independent and a late iNOS-dependent increase in glycolysis in response to LPS.

GM-DCs were seeded in a Seahorse XF-96 analyzer and ECAR was determined at indicated times after LPS stimulation in the presence or absence of NOS inhibitor SEITU. Data represent mean ±SEM of triplicate wells. One representative experiment out of 3 is shown.

Supplementary Figure 2 Effects of 2-DG on TLR-induced cytokine production, on binding of GAPDH to cytokine transcripts, on cell death and on activation of Flt3L-DCs.

(a,c) _Nos2_-/- GM-DCs were stimulated with LPS in the presence or absence of 2-DG and after 6 h (a) culture supernatants were analyzed for indicated cytokines (c) and cell death was determined by flow cytometry. (a) Bars represent mean ± SEM of triplicate wells. (b) GM-DCs were stimulated with LPS in the presence or absence of 2-DG and after 3 h GAPDH was immunoprecipitated and binding of mRNA of indicated genes to GAPDH was quantified by RT-qPCR. Bars represent mean ±SEM of 3 independent experiments. (d) SIRPα+ and CD24+ DCs were flow cytometry-sorted from Flt3L-BM cultures, stimulated with indicated TLR agonists in the presence or absence of 2-DG and 16 h later expression of indicated surface marker was determined by flow cytometry. Bars represent mean ±SEM of 3 independent experiments. (a,c) One representative experiment out of 2 is shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Supplementary Figure 3 Glycolytic ATP is not essential for DC activation.

(a) Relative ATP levels are shown in GM-DCs at indicated time points following LPS stimulation. Data represent mean ±SEM of duplicate wells. (b) GM-DCs were analyzed as in Fig. 1a in response to treatment with LPS following pretreatment with LDHA inhibitor oxamate. Data represent triplicate wells and are shown as mean ±SEM. (c,d) GM-DCs, pretreated with oxamate, were analyzed for expression of indicated markers (c) or cytokines (d) 6 h after LPS stimulation by flow cytometry, based on surface or intracellular staining, respectively. (c) Expression levels are shown relative to LPS-stimulated DCs. (c) Bars represent at least 3 independent experiments ±SEM. (d) Grey solid and black open histograms represent unstimulated and stimulated cells, respectively. (a,b,d) One representative experiment out of 3 is shown.

Supplementary Figure 4 Glycolysis-derived carbon, but not that of glutaminolysis or FA oxidation, is required for DC activation.

(a,b) GM-DCs were stimulated with LPS in the presence or absence pyruvate carrier inhibitor α-cyanocinnamate (αCC), CPT1a inhibitor etomoxir (eto) or glutaminase inhibitor 6-Diazo-5-oxo-L-norleucine (DON) and 6 h later expression of indicated surface markers (a) and indicated cytokines (b) was determined by flow cytometry. (a) Data are shown relative to LPS-stimulated DCs and bars represent mean ±SEM of 3 independent experiments. (b) Grey solid and black open histograms represent unstimulated and stimulated cells, respectively. (c) GM-DCs were transduced with retroviral vectors expressing hairpins (hp) or over-expressing constructs (oe) of PDK1 and LDHA or their respective controls. DCs expressing huCD8 and GFP, as reporters for transduction, were analyzed for surface expression of indicated markers after stimulation with medium or LPS for 24 h. (b,c) One representative experiment out of 3 is shown. *, P < 0.01

Supplementary Figure 5 Mitochondrial ATP is not required for DC activation.

(a,b) GM-DCs, pretreated with oligomycin, were analyzed for expression of indicated markers (a) or cytokines (b) 6 h after LPS stimulation by flow cytometry, based on surface or intracellular staining, respectively. (a) Expression levels are shown relative to LPS-stimulated DCs and bars represent mean ±SEM of 4 independent experiments. (b) Grey solid and black open histograms represent unstimulated and stimulated cells, respectively. (c) GM-DCs were stimulated with LPS in the presence or absence of C75 and after 6 h PGE2 levels were determined in culture supernatants. Bars represent mean ±SEM of duplicates (b,c) (d,e) GM-DCs, pretreated with C75, were stimulated with LPS in the presence or absence of rosiglitazone (Rgz) and 6 h later analyzed for expression of indicated markers (d) or cytokines (e) by flow cytometry, based on surface or intracellular staining respectively. (b,e) Grey solid and black open histograms represent unstimulated and stimulated cells, respectively. One representative experiment out of (b) 4 or (c,d,e) 2 is shown. *, P < 0.05

Supplementary Figure 6 TLR-driven Akt activation and ECAR burst is TBK1-IKKɛ dependent but mTOR and PI(3)K independent.

(a) GM-DCs were analyzed for real-time changes in ECAR in response to stimulation with LPS following pretreatment with Akt inhibitor Akt1/2 VIII, mTORC1 inhibitor rapamycin (rap) or PI(3)K inhibitor wortmannin (WM). ECAR is shown relative to basal levels prior to inhibitor incubation, which is set to 1. (b) GM-DCs were pre-incubated with indicated inhibitors and stimulated with or without LPS for 15 minutes after which Akt phosphorylation was assessed by flow cytometry. (c) GM-DCs were pre-incubated with Akt inhibitors triciribine (Tric) and Akt1/2 VIII, TBK1-IKKɛ inhibitor BX795 (BX), rapamycin or wortmannin, and stimulated with LPS for 15 minutes after which phosphorylation of indicated proteins were assessed by immunnoblot. (d) GM-DCs were stimulated with TLR2 agonist Pam3CSK4 and analyzed for TBK1 phosphorylation by immunoblot. (e) GM-DCs were stimulated as in (c) and analyzed for TBK1 phosphorylation. (f) WT or _Ikbke_-/-GM-DCs were transduced with retroviral vectors expressing a control or TBK1 hairpin, sorted based on huCD8 expression as a reporter for transduction and analyzed for Akt phosphorylation by flow cytometry 15 min after LPS stimulation. (g) GM-DCs were analyzed for real-time changes in ECAR in response to stimulation with LPS following pretreatment with Akt inhibitor triciribine or TBK1-IKKɛ inhibitor Amlexanox (Amx). ECAR is shown relative to basal levels prior to inhibitor incubation, which is set to 1. Data represent are shown as mean ±SEM (a,g) triplicate or (f) duplicate wells. One representative experiment out of (b) 5, (a,c,e) 3 or (d,f,g) 2 is shown.

Supplementary Figure 7 Chemical dissociation of HK from mitochondria impairs glycolytic flux and DC activation.

(a) Following pretreatment with Akt1/2 VIII, GM-DCs were stimulated with LPS for 1 h after which HK activity was determined. Bars represent mean ±SEM of duplicates. (b) GM-DCs were treated with clotrimazole (CLT), a drug that dissociates HK from mitochondria, and cytosolic and mitochondrial fractions were analyzed for the presence of HK-II. (c) Following pretreatment with CLT, GM-DCs were stimulated with LPS for 1 h after which HK activity was determined. Bars represent mean ±SEM of 3 independent. (d) GM-DCs were analyzed as in Fig. 1a following pretreatment with CLT. Data represent mean ±SEM of triplicate wells. (e,f) GM-DCs, pretreated with CLT, were stimulated with LPS for 6 h and subsequently analyzed as in Fig. 2b,c. (e) Bars represent mean ±SEM of 3 independent experiments. (f) Grey solid and black open histograms represent unstimulated and stimulated cells, respectively. One representative experiment out of (a,b) 2 or (d,f) 3 is shown. *, P < 0.05

Supplementary Figure 8 TLR-induced early glycolytic burst is not dependent on increased GLUT1 translocation to the cell surface, PFK-2 isoforms PFKFB2 and PFKFB3, mTORC1 or HIF-1α.

(a) Expression of glucose transporter genes in GM-DCs (derived from public database GSE17721). Relative expression is shown. (b) GLUT1 surface expression on GM-DCs was determined by flow cytometry following stimulation with LPS for 1 or 24 h. (c) mRNA expression levels of indicated genes were determined by RT-qPCR of GM-DCs transduced with retroviral vectors expressing hairpins (hp) to Pfkfb2 and Pfkfb3 or a control. Relative expression is shown. (d) Sorted transduced DCs were analyzed as in Fig. 1a. Data are representative of triplicate wells and are shown as mean ±SEM. (e,f) sorted CD11c+ GM-DCs derived from _Rpto_rfl/fl _Itgax_cre (CD11cΔraptor) or _Rpto_rfl/fl (CD11cWT) mice were analyzed (e) for indicated proteins by immunoblot and (f) analyzed as in Fig. 1a. (g) GM-DCs were stimulated with LPS for 1 h and analyzed for indicated proteins by immunoblot. (h) Global transcription was determined in GM-DCs by detection of incorporation of nucleotide homolog ethylene uridine (EU) by flow cytometry following a 3 h pulse in the presence or absence of Actinomycin-D (ActD). (i) GM-DCs were analyzed as in Fig. 1a following preincubation with ActD. (j) mRNA expression of Hif1a was determined as in (c). (k) Sorted transduced DCs were analyzed as in Fig. 1a. Data represent mean ±SEM from (d,f,I,k) triplicate wells or (a,c) 2 or (j) 3 experiments. One representative experiment out of (e,f,k) 2 or (b,d,g,h,i) 3 is shown. *, P < 0.05; **, P < 0.01.

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Everts, B., Amiel, E., Huang, SC. et al. TLR-driven early glycolytic reprogramming via the kinases TBK1-IKKɛ supports the anabolic demands of dendritic cell activation.Nat Immunol 15, 323–332 (2014). https://doi.org/10.1038/ni.2833

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