PKM2 e Diferenciação de Células Th17 - Mecanismos e Inflamação Autoimune

ARTICLE PKM2 promotes Th17 cell differentiation and autoimmune inflammation by finetuning STAT3 activation Luis Eduardo Alves Damasceno12 Douglas Silva Prado12 Flavio Protasio Veras12 Miriam M Fonseca12 Juliana E TollerKawahisa12 Marcos Henrique Rosa12 Gabriel Azevedo Publio12 Timna Varela Martins12 Fernando S Ramalho3 Ari Waisman4 Fernando Queiroz Cunha12 Thiago Mattar Cunha12 and Jose Carlos AlvesFilho12 Th17 cell differentiation and pathogenicity depend on metabolic reprogramming inducing shifts toward glycolysis Here we show that the pyruvate kinase M2 PKM2 a glycolytic enzyme required for cancer cell proliferation and tumor progression is a key factor mediating Th17 cell differentiation and autoimmune inflammation We found that PKM2 is highly expressed throughout the differentiation of Th17 cells in vitro and during experimental autoimmune encephalomyelitis EAE development Strikingly PKM2 is not required for the metabolic reprogramming and proliferative capacity of Th17 cells However T cellspecific PKM2 deletion impairs Th17 cell differentiation and ameliorates symptoms of EAE by decreasing Th17 cellmediated inflammation and demyelination Mechanistically PKM2 translocates into the nucleus and interacts with STAT3 enhancing its activation and thereby increasing Th17 cell differentiation Thus PKM2 acts as a critical nonmetabolic regulator that finetunes Th17 cell differentiation and function in autoimmunemediated inflammation Introduction Th17 cells are critical components of the adaptive immunity that contribute to the host defense against extracellular pathogens but they are also implicated in the pathogenesis of autoimmune mediated inflammatory diseases Korn et al 2009 Cosignaling of IL6 and TGFβ induces the differentiation of Th17 cells Veldhoen et al 2006 Bettelli et al 2006 Mangan et al 2006 IL6 drives the phosphorylation of STAT3 that translocates into the nucleus and induces the expression of the transcription factors Rorα and Rorγt Ivanov et al 2006 Yang et al 2007 2008 TGFβ inhibits IL6induced SOCS3 expression thus prolonging STAT3 activation Qin et al 2009 Chen et al 2006 In combination with other transcription factors STAT3 and retinoic acid orphan receptor gamma T synergize to regulate transcription of the T helper type 17 Th17 cellsignature genes IL17A IL17F IL22 and IL23R Korn et al 2009 Another cytokine IL23 mediates the final differentiation stabilization and induction of GMCSF production by Th17 cells making these cells pathogenic ElBehi et al 2011 Codarri et al 2011 McGeachy et al 2009 However much remains unclear about the regulatory signaling pathways that control the differentia tion and pathogenicity of Th17 cells Recent studies have shown that immune cells undergo a dynamic metabolic reprogramming to support the bioenergetic and biosynthetic requirements for proper activation prolifera tion and differentiation Mammalian target of rapamycin complex 1 mTORC1 and hypoxiainducible factor 1α HIF1α are critical regulators of cellular metabolism and also have a central role in controlling immune cell activation and func tions ONeill et al 2016 Buck et al 2015 Almeida et al 2016 Indeed the HIF1α and mTORC1dependent metabolic reprogram ming toward aerobic glycolysis a phenomenon that resembles the welldescribed Warburg effect in tumor cells is also especially important for Th17 cell development Shi et al 2011 Delgoffe et al 2011 Dang et al 2011 Kurebayashi et al 2012 Consis tent with this the blockade of glycolysis with 2deoxyglucose inhibits Th17 cell generation in vitro and ameliorates the devel opment of experimental autoimmune encephalomyelitis EAE Shi et al 2011 1Department of Pharmacology Ribeirao Preto Medical School University of Sao Paulo Ribeirao Preto Brazil 2Center for Research in Inflammatory Diseases Ribeirao Preto Medical School University of Sao Paulo Ribeirao Preto Brazil 3Department of Pathology Ribeirao Preto Medical School University of Sao Paulo Ribeirao Preto Brazil 4Institute for Molecular Medicine University Medical Center of the Johannes GutenbergUniversity Mainz Germany Correspondence to Jose C AlvesFilho jcafilhouspbr 2020 Damasceno et al This article is distributed under the terms of an AttributionNoncommercialShare AlikeNo Mirror Sites license for the first six months after the publication date see httpwwwrupressorgterms After six months it is available under a Creative Commons License AttributionNoncommercialShare Alike 40 International license as described at httpscreativecommonsorglicensesbyncsa40 Rockefeller University Press httpsdoiorg101084jem20190613 1 of 16 J Exp Med 2020 Vol 217 No 10 e20190613 Pyruvate kinase PK is a glycolytic enzyme that converts phosphoenolpyruvate to pyruvate Israelsen and Vander Heiden 2015 Gui et al 2013 Four isoforms of PK are present in mam mals and differentially distributed according to the cell type Particularly expressions of the PK isoforms M1 PKM1 and M2 PKM2 are derived through alternative splicing of the Pkm gene Noguchi et al 1986 PKM1 is constitutively expressed at a con stant level in most tissues while PKM2 is mainly expressed in proliferating and tumor cells Structurally PKM1 forms constitu tive and stable tetramers with high metabolic activity whereas the PKM2 tetrameric conformation requires allosteric modulation being mostly expressed as metabolically inactive monomeric and dimeric forms Israelsen and Vander Heiden 2015 Gui et al 2013 Although the dimeric PKM2 has low metabolic activity it gains the ability to translocate into the nucleus and act as a nuclear transcriptional coactivator regulating gene expression by inter action with some transcriptional factors including HIF1α Yang et al 2011 Luo et al 2011 Yang et al 2012a Pharmacological inhibition of PKM2 nuclear translocation or its silencing decreases aerobic glycolysis and the proliferation of tumor cells Christofk et al 2008 Anastasiou et al 2012 Moreover recent reports show that PKM2 regulates the production of inflammatory cyto kines in LPSactivated macrophages Shirai et al 2016 Yang et al 2014 PalssonMcDermott et al 2015 In this study we demonstrated that PKM2 mediates the differentiation of Th17 cells but not Th1 Th2 or regulatory T T reg cells through activation of STAT3 We found that the di meric PKM2 translocates into the nucleus and interacts with STAT3 enhancing its phosphorylated status throughout the differentiation of Th17 cells T cellspecific PKM2 deletion im pairs the development of Th17 cells and ameliorates symptoms of EAE by decreasing Th17 cellmediated inflammation and de myelination PKM2 therefore represents a potential therapeutic target for autoimmunemediated inflammation Results Th17 cells express PKM2 throughout differentiation To determine the role of PKM2 in the activation proliferation and differentiation of T cells we initially analyzed the expres sion of PKM splice isoforms in Th cell subtypes To this end we cultured naive CD4CD25 T cells from C57BL6 mice under Th1 Th2 Th17 and induced T reg iT reg cellpolarizing conditions in vitro to obtain T cells with selective expression of Ifng Il4 Il17a and Foxp3 respectively Fig S1 A As controls naive CD4CD25 T cells were activated with antiCD3εCD28 anti bodies without the addition of differentiating cytokines Th0 cells We found that Pkm1 mRNA is constitutively expressed in freshly isolated naive T cells and did not increase substantially in Th cell subtypes whereas Pkm2 mRNA expression was up regulated in all Th cell subtypes when compared with naive T cells at 48 h of culture However significantly more Pkm2 mRNA expression was observed in Th17 cells Fig 1 A Of note effectormemory CD62LloCD44hi CD4 T cells in homeostatic conditions show a slight increase in Pkm2 mRNA expression but not Pkm1 in comparison to naive cells but lower than that ob served in fully differentiated Th17 cells in vitro Fig S1 B In a kinetic analysis of Th17 cell differentiation Pkm2 mRNA expression was detectable at 24 h and it reached a peak at 48 h of culture whereas Pkm1 expression remained constant through out differentiation Fig 1 B Immunoblot analysis confirmed that PKM2 protein levels were very low or undetectable in naive T cells However it markedly increased throughout Th17 cell differentiation while PKM1 protein expression is constitu tively expressed in resting naive T cells showing a slight in crease in differentiated Th17 cells Fig 1 C According to our findings on Pkm2 gene expression PKM2 protein expression was higher in Th17 cells than Th1 cells Fig S1 C Additionally using the flow cytometry approach we observed that IL17A CD4 T cells exhibited higher intracellular staining for PKM2 than IL17A CD4 T cells from the same culture wells after Th17 differentiation Moreover the addition of IL23 to the cell cultures concomitantly increased both Th17 cell differentiation and PKM2 expression in IL17Aproducing T cells Fig 1 D mTORC1 signaling upregulates the expression of PKM2 in tumor cells Sun et al 2011 whereas T cellspecific deletion of mTORC1 activity impairs Th17 differentiation in vitro and in vivo Kurebayashi et al 2012 Delgoffe et al 2011 We therefore investigated whether mTORC1 signaling is involved in the expression of PKM2 during the differentiation of Th17 cells As expected inhibition of mTOR with rapamycin dramatically reduced IL17A and increased Foxp3 expression in CD4 T cells cultured under Th17 cellpolarizing conditions Fig S1 D and E Rapamycin did not affect Pkm1 expression but it significantly reduced PKM2 mRNA and protein expression Fig 1 E and F These observations led us to determine if the expression of PKM2 changes in a Th17 cellmediated inflammatory disease model Sie et al 2014 Its expression profile was evaluated throughout the course of EAE development This was done by immunizing mice with myelin oligodendrocyte glycoprotein MOG3555 peptide Fig 2 A and B We found that Pkm2 mRNA expression had increased in draining LNs DLNs before disease onset day 10 and in the spinal cord at the peak of the EAE symptoms day 15 following the expression profile observed with the transcription of Th17 cellrelated genes such as Il17a Csf2 and Il23r Fig 2 C Immunoblot analysis confirmed the increased PKM2 protein levels in the spinal cord of EAE mice Fig 2 D Moreover histopathological analysis with HE and immunofluorescent staining of spinal cord lesions in EAE mice showed that PKM2 expression was confined almost exclusively into the inflammatory cell infiltration region while absent in spinal cord sections of naive mice Fig 2 E Consistent with this CD4 T cells isolated from the spinal cords of EAE mice expressed significantly higher Pkm2 mRNA transcription levels along with the Th17 cellrelated genes Il17a Csf2 Il23r Rora and Rorc compared with CD45 cells from the spinal cord of naive mice Fig 2 F Taken together these results indicate that PKM2 ex pression is induced in Th17 cells in vitro and in vivo suggesting that it might affect their differentiation Th17 cells require PKM2 for the complete differentiation Activated T cells undergo a dynamic metabolic reprogramming to support the bioenergetic and biosynthetic demands for proper proliferation and differentiation Buck et al 2015 Almeida Damasceno et al Journal of Experimental Medicine 2 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 et al 2016 Cancer cells and macrophages require PKM2 ex pression for their metabolic reprogramming toward aerobic glycolysis PalssonMcDermott et al 2015 Christofk et al 2008 whereas defective glycolysis dramatically impairs Th17 cell differentiation and proliferation Shi et al 2011 We hy pothesized that similar to macrophages and tumor cells PKM2 upregulation would also be required for metabolic reprogram ming of T cells To test this hypothesis we crossed mice carrying the LoxPflanked Pkm2specific exon 10 Pkm2flfl Israelsen et al 2013 with CD4Cre mice Lee et al 2001 to generate T cellspecific PKM2 deficient mice CD4CrePkm2flfl Litter mates Pkm2flfl and CD4Cre mice were used as WT controls No significant difference in LN and spleen sizes were ob served between WT or CD4CrePkm2flfl mice Fig S2 A and CD4CrePkm2flfl pups did not display any apparent abnormalities presenting a grossly healthy development data not shown Figure 1 Th17 cell differentiation accompanies high PKM2 expression levels A Pkm1 and Pkm2 gene expression were evaluated by RTqPCR in freshly isolated CD4 T cells naive and polyclonally activated CD4 T cells Th0 and Th1 Th2 Th17 and iT reg cells at 48 h after culture n 3 B Naive CD4 T cells were differentiated into Th17 cells and gene expression of Pkm1 and Pkm2 was determined at different time points by RTqPCR n 3 C Protein expression levels of PKM1 and PKM2 during Th17 cell differentiation were detected by immunoblot βactin was used as a loading control D Th17 cells were differ entiated in the presence or absence of IL23 and PKM2 expression was determined by flow cytometry MFI mean fluorescence intensity E Rapamycin 01 µM an mTOR inhibitor was added to the Th17 cell cultures After 96 h cells were collected and the expression of Pkm1 and Pkm2 was determined by RTqPCR For gene expression analysis the cycle threshold values were normalized to Gapdh fold change was calculated relative to untreated cells n 3 F Rapamycintreated Th17 cells were also collected for immunoblot analysis of PKM2 protein levels n 3 βActin was used as a loading control Data are representative of two independent experiments Error bars show mean SEM P values were determined by oneway ANOVA followed by Tukeys post hoc test A and F twoway ANOVA followed by Tukeys post hoc test B and D or twotailed Students t test E P 005 ns not significant Damasceno et al Journal of Experimental Medicine 3 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 Figure 2 PKM2 expression increases during EAE development A and B EAE was induced in WT C57BL6 mice by subcutaneous immunization with MOG3555 the clinical score was evaluated throughout the days after immunization C DLNs top and spinal cord samples bottom were collected at the indicated time points depicted in B red arrows for analysis of Pkm2 Il17a Csf2 and Il23r gene expression by RTqPCR n 7 per time point Cycle threshold values were normalized to Gapdh D PKM2 total protein levels in the spinal cord of EAEbearing mice were determined by immunoblot βactin was used as a loading control E Inflammatory cell infiltration was observed in the spinal cord by using HE staining left black arrows Scale bar indicates 500 and 50 µm PKM2 protein expression in the spinal cord was analyzed by immunofluorescence red DAPI was used as a nuclear marker blue and myelin was stained with fluoromyelin stain probe green n 3 Scale bar represents 50 µm F Mononuclear cells were isolated from CNS of naive and EAE mice n 9 per group followed by magnetic separation of CD4 T cells Expression of Pkm2 Il17a Csf2 Il23r Rora and Rorc was analyzed by RTqPCR Each sample was a pool of three Damasceno et al Journal of Experimental Medicine 4 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 Moreover the proportion of CD4 or CD8 T cells in the thymus and the frequency of naive or memory CD4 T cell populations in LNs and spleen was not significantly different between WT and CD4CrePkm2flfl mice Fig S2 BD We then cultured naive PKM2deficient CD4 T cells under a Th17 cellpolarizing condition and examined the expression levels of key molecules required for glycolysis Fig 3 A The loss of Pkm2 in CD4 T cells was confirmed by immunoblot analysis of PKM2 protein expression in Th17 cells after 72 h in culture Fig 3 B Consistent with previous studies WT Th17 cells showed increased expression of Slc2a1 the gene encoding glu cose transporter GLUT1 Ldha lactate dehydrogenase LDH and Hif1a HIF1α compared with naive CD4 T cells Fig 3 C Strikingly PKM2 deficiency did not affect the expression of those proglycolytic genes Fig 3 C Correspondingly LDH and HIF1α proteins levels were also comparable between WT and PKM2deficient Th17 cells Fig 3 D Nevertheless deficiency of PKM2 led to compensatory upregulation of PKM1 expression Fig 3 B and C Thus we next examined whether PKM2 affects the glucose metabolism of Th17 cells This evaluation was per formed by monitoring the uptake of fluorescent glucose ana logue 2NBDG in concert with glucose consumption and lactate secretion in vitro WT Th17 cells were highly glycolytic showing increased glucose uptake when compared with naive T cells Consistent with the normal expression of the main proglycolytic molecules in the absence of PKM2 expression glucose uptake by PKM2deficient Th17 cells was not different from that of WT Th17 cells even in the presence of IL23 Fig 3 E and Fig S2 E mice Cycle threshold values were normalized to Gapdh and fold change was calculated relative to CNS CD45 cells from naive mice Data are representative of two C E and F or three D independent experiments Error bars indicate mean SEM P values were determined by twotailed Students t test C and F P 005 Figure 3 PKM2 deficiency does not alter Th17 cell metabolic reprogramming A Naive CD4 T cells were obtained from CD4CrePkm2flfl or control littermates WT and cultured under Th17 cellskewing conditions B PKM1 protein expression in PKM2deficient Th17 cells was determined by immunoblot PKM2 deficiency was also confirmed by immunoblot analysis C Th17 cells were harvested to evaluate the expression of glycolysisrelated genes Slc2a1 Ldha Hif1a and Pkm1 by RTqPCR data were normalized to Gapdh and fold change calculated relative to freshly isolated naive CD4 T cells n 3 D WT and PKM2 deficient Th17 cells were harvested at 96 h to determine protein levels of LDHA and HIF1α by immunoblot βActin was used and loading control E Th17 cells were incubated with a fluorescent glucose analogue 2NBDG 30 µM for glucose uptake evaluation by flow cytometry dotted lines indicate fluorescence minusone FMO control values n 34 MFI mean fluorescence intensity F Glucose consumption and lactate production were measured in Th17 cell culture supernatants n 45 Data are representative of two D E and F or three B and C independent experiments Error bars show mean SEM P values were determined by oneway ANOVA followed by Tukeys post hoc test C twoway ANOVA followed by Tukeys post hoc test E or twotailed Students t test F P 005 ns not significant Damasceno et al Journal of Experimental Medicine 5 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 Moreover glucose consumption and lactate production were not significantly different between WT and PKM2deficient Th17 cells Fig 3 F and Fig S2 F Collectively these results indicate that PKM2 deficiency did not impair the metabolic reprogram ming of Th17 cells toward aerobic glycolysis To assess whether loss of PKM2 function affects T cells pro liferative capacity we next performed flow cytometric analysis of fluorescent dye dilution in CD4 T cells stimulated with anti CD3ε and antiCD28 in the presence of Th17 cellpolarizing cy tokines PKM2deficient CD4 T cells showed normal proliferative capacity after 72 h in culture Fig 4 A Additionally we evalu ated the proliferation of PKM2deficient T cells cultured under a Th0 condition and again no significant differences were de tected even in the presence of IL2 Fig S2 G However pro liferating PKM2deficient Th17 cells showed significantly reduced expression of IL17A Fig 4 B suggesting that PKM2 is required for differentiation but not Th17 cell proliferation Consistent with this association expression of the Th17 cell related genes Il17a Csf2 Il22 Il23r Rora and Rorc were also markedly reduced in PKM2deficient Th17 cells Fig 4 C Fur ther analysis confirmed that PKM2 deficiency impaired rises in IL17A expression in Th17 cells even after they had been dif ferentiated in the presence of IL23 Fig 4 D and E Never theless reduced IL17A expression was not accompanied by alteration of Foxp3 expression in PKM2deficient Th17 cells Fig S3 A Moreover PKM2 deficiency did not affect Th1 Th2 or iT reg cell differentiation Fig S3 BD suggesting that Th17 dif ferentiation requires PKM2 whereas the differentiation of other Th cell subtypes is unaffected by the loss of PKM2 function in vitro Loss of PKM2 in T cells ameliorates autoimmunemediated neuroinflammation To determine the in vivo relevance of these findings we next examined whether the loss of PKM2 in T cells influenced the pathogenesis of EAE To this end CD4CrePkm2flfl mice were Figure 4 PKM2 deficiency impairs Th17 cell differentiation A Naive CD4 T cells from WT or conditional knockout CD4CrePkm2flfl mice were stained with CellTrace Violet proliferation dye CTV 5 µM Cells were then cultured under Th17 cellskewing conditions and after 96 h cell proliferation was evaluated by flow cytometry MFI mean fluorescence intensity n 5 B Cells were stained with 5 µM eFluor 670 proliferation dye and cultured under Th17 cell polarizing conditions for 96 h Cells were intracellularly stained for IL17A after 4 h of PMAionomycin stimulation n 3 C The expression of Th17 cell signature genes was evaluated by RTqPCR and displayed in a heatmap Gene expression correlates with color intensity data normalized by Zscore row cycle threshold values were normalized to Gapdh n 3 D Naive CD4 T cells from WT or CD4CrePkm2flfl were also differentiated in the presence of IL23 and frequency of IL17A CD4 T cell population determined by flow cytometry n 3 E Supernatants of Th17 cultures were collected and IL17A levels detected were by ELISA n 3 Data are representative of two AC or more than five D and E independent experiments Error bars indicate mean SEM P values were determined by twoway ANOVA followed by Tukeys post hoc test D and E or twotailed Students t test A and B P 005 Damasceno et al Journal of Experimental Medicine 6 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 immunized with MOG3555 peptide Notably the loss of PKM2 in T cells not only significantly reduced the clinical severity but also decreased the incidence of EAE Fig 5 AC Consistently histopathological analysis with HE and fluoromyelin staining of spinal cords showed that CD4CrePkm2flfl mice had lower in flammatory cell infiltration and decreased demyelination than WT mice Fig 5 D To evaluate the impact of PKM2 deficiency on Th17 differentiation during EAE we next analyzed the ex pression of Th17 cellassociated genes in DLNs Deficiency of PKM2 in T cells resulted in a significant reduction of the Th17 cellrelated genes Il17a Rora and Rorc in DLNs before disease onset day 6 Fig 5 E suggesting that T cellspecific deletion of PKM2 inhibits Th17 cell differentiation in the EAE model In deed loss of PKM2 in T cells significantly reduced the frequency of IL17A CD4 T cells in DLNs at the peak of the disease day 15 whereas the population of Foxp3 CD4 T cells remained unal tered Fig S4 A Moreover when we restimulated cells isolated from DLNs of EAE mice with MOG3555 ex vivo the frequency of IL17ARorγt CD4 T cells and the production of IL17A GMCSF and IFNγ by PKM2deficient T cells were significantly lower compared to WT cells Fig 5 F and Fig S4 B PKM2 deficiency also reduced the transcription of mature pathogenic Th17 cell effector genes Csf2 and Ifng in the total spinal cord tissue at the peak of EAE symptoms Fig S4 C Consistent with this mRNA transcription levels in Pkm2 and Th17 cellrelated genes in cluding Il17a Il21 Csf2 Ifng Il23r Rora and Rorc were lower in CD4 T cells isolated from the spinal cords of CD4CrePkm2flfl mice than in those obtained from WT mice Fig 5 G Accordingly mice lacking PKM2 in T cells had significantly decreased fre quencies of IL17A CD4 T cells coproducing the pathogenic Th17 cytokines GMCSF or IFNγ in their spinal cords Fig 5 H Of note we noticed that deficiency of PKM2 did not affect the frequency of IFNγGMCSF Th1 cells in vitro while the gen eration of IL17AGMCSF Th17 cells was impaired Fig S3 E To further confirm the role of PKM2 for the development of encephalitogenic Th17 cells we adoptively transferred enriched MOGspecific WT or PKM2deficient Th17 cells in vitro into Rag1 mice We found that the loss of PKM2 in Th17 cells sig nificantly reduced their ability to induce passive EAE when compared with WT cells Fig S4 D Collectively these data in dicate that PKM2 is required for Th17 cell differentiation in vitro and in vivo contributing to the pathogenesis of EAE PKM2 promotes STAT3 phosphorylation in Th17 cells In its tetrameric form PKM2 has high metabolic activity con verting phosphoenolpyruvate to pyruvate in the glycolytic pathway However its less enzymatically active dimeric form has the potential to translocate into the nucleus and act as a transcriptional coactivator Yang et al 2011 Gao et al 2012 In this context phosphorylation of PKM2 at tyrosine 105 Y105 prevents tetramer conformation favoring the dimeric state Hitosugi et al 2009 To investigate how PKM2 regulates Th17 cell differentiation we initially examined its phosphorylation status and conformational state We found that increases in total PKM2 expression preceded parallel increases in its phospho rylation at Y105 during Th17 cell differentiation peaking at 72 h of culture Fig 6 A Phosphorylated PKM2 at Y105 was also increased in the spinal cord tissue of EAE mice and was posi tively associated with the clinical score of the disease Fig S4 E Moreover immunoblot analysis of protein crosslinking assay revealed that all oligomeric forms of PKM2 were upregulated in Th17 cells when compared with Th0 cells after 72 h of culture However the dimeric PKM2 was the most prevalent confor mation detected mainly when Th17 cells were differentiated in the presence of IL23 Fig 6 B suggesting that PKM2 can be translocated into the nucleus Indeed confocal immunofluores cence microscopy analysis revealed a punctate staining pattern of PKM2 in Th17 cells with both cytoplasmic and nuclear lo calization whereas Th0 cells showed an evenly distributed pattern of PKM2 mainly in the cytoplasm Fig 6 C Immunoblot analysis of cytoplasmic and nuclear fractions further confirmed the nuclear translocation of PKM2 in Th17 cells Fig 6 D To investigate the functional importance of the PKM2 nu clear translocation in mediating Th17 cell differentiation we next used the small molecule TEPP46 which is a wellcharacterized PKM2specific allosteric activator that promotes tetramer for mation and inhibits nuclear translocation Anastasiou et al 2012 Notably treating CD4 T cells with TEPP46 significantly reduced Th17 cell differentiation to the same level as that observed in PKM2deficient T cells Fig 6 E and Fig S5 A suggesting that dimeric PKM2 nuclear translocation is required for the regula tion of Th17 cell differentiation Indeed immunoblot analysis of nuclear fractions of Th17 cells showed that TEPP46 completely abrogated the translocation of PKM2 into the nucleus Fig 6 F Of note we found that PKM2 also translocates into the nucleus of Th1 cells which is also inhibited by TEPP46 Fig S5 B Nev ertheless TEPP46 did not affect the differentiation of Th1 cells Fig S5 C IL6 and IL23 induce Th17 cell differentiation through acti vation of the STAT3 signaling Korn et al 2009 Dong 2008 Moreover the phosphorylation of STAT3 phosphoSTAT3 at Y705 residue is known to be required for Th17 cell differentia tion Guanizo et al 2018 Renner et al 2008 Interestingly the nuclear dimeric PKM2 form can act as a protein kinase and phosphorylate STAT3 at Y705 in the nucleus enhancing its transcriptional activity and promoting tumor growth Gao et al 2012 We then examined whether PKM2 and STAT3 proteins can interact during the differentiation of Th17 cells Confocal immunofluorescence images indicated that STAT3 and PKM2 colocalize in the nucleus of Th17 cells Fig 7 A Indeed immu noprecipitation coupled to immunoblot analysis showed that PKM2 coimmunoprecipitated with STAT3 in WT Th17 cells Fig 7 B The specific PKM2STAT3 interaction was supported by immunoprecipitating STAT3 in PKM2deficient Th17 cells and using a control IgG antibody for the immunoprecipitation assay To further confirm the direct interaction between PKM2 and STAT3 we also conducted a proximity ligation assay PLA in Th17 cells We found a robust fluorescent signal generated by PLA probes targeting STAT3 and PKM2 in WT but not PKM2 deficient Th17 cells indicating a nuclear PKM2STAT3 inter action Fig 7 C and Fig S5 D We then evaluated whether the absence of PKM2 could affect the phosphorylation status of STAT3 Immunoblot analysis demonstrated that acute phosphorylation of STAT3 at Y705 by Damasceno et al Journal of Experimental Medicine 7 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 Figure 5 T cellspecific PKM2 deletion ameliorates autoimmunemediated inflammation AC WT or CD4CrePkm2flfl mice were immunized with MOG3555 and monitored daily for clinical signs n 1824 per group A Cumulative EAE clinical scores B Representation by linear regression curves dashed lines indicate the 95 confidence intervals C Disease incidence by severity is represented on a bar chart as no EAE score 1 mild EAE score 12 and severe EAE score 25 D Inflammatory cell infiltration in the spinal cord top black arrowheads was observed by using HE staining the number of inflammatory cells in transverse spinal cord sections was determined in a blinded fashion right n 7 per group Scale bars represent 500 and 50 µm Fluoromyelin staining green was performed to detect demyelination sites bottom white arrowheads nuclei labeled with DAPI blue Scale bar indicates 50 µm E Analysis of Il17a Rora and Rorc gene expression in DLN cells collected 6 d after immunization Data were normalized to Gapdh foldchange is relative to naive controls n 5 per group F DLN cells were harvested 6 d after immunization and restimulated in vitro with MOG3555 the frequencies of IL17ARorγt CD4 T cells were then determined by flow cytometry n 3 G CNSinfiltrating CD4 T cells were isolated Each sample was a pool of cells from two mice and analyzed for expression of Th17 cellassociated genes n 6 per group Cycle threshold values were normalized to Gapdh fold change is relative to CNS CD45 cells from naive mice Data were normalized by Z score row and depicted in a heatmap H Spinal cordinfiltrating mononuclear cells were collected from WT Damasceno et al Journal of Experimental Medicine 8 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 IL6 stimulation was reduced in TCRactivated PKM2deficient CD4 T cells when compared with WT CD4 T cells Fig 7 D Additionally fully differentiated PKM2deficient Th17 cells 96 h culture showed significantly lower levels of phosphorylated STAT3 at Y705 while the total STAT3 protein expression was not altered Fig 7 E and Fig S5 E In accordance TEPP46 also substantially reduced the levels of nuclear Y705phosphorylated STAT3 in Th17 cells Fig 7 F Of note although the abundance of phosphorylated STAT3 in Th1 cells is markedly lower than that observed in Th17 cells PKM2deficient Th1 cells showed reduced levels of phosphorylated STAT3 when compared with WT cells Fig S5 E In vivo the deficiency of PKM2 in T cells resulted in a significant reduction of Y705phosphorylated STAT3 in the spinal cord tissue of EAE mice compared to and CD4CrePkm2flfl mice 15 d after immunization for flow cytometric analysis of IL17A CD4 T cell populations coproducing GMCSF or IFNγ n 58 per group Data are pooled from three AC or representative of two EH or three D independent experiments Error bars represent mean SEM P values were determined by oneway ANOVA followed by Tukeys post hoc test E twoway ANOVA followed by Tukeys post hoc test A and B or twotailed Students t test D F and H P 005 Figure 6 PKM2 translocates into the nucleus of Th17 cells A The degree of PKM2 phosphorylation at Y105 was determined by immunoblot at different time points of Th17 cell culture βactin was used as a loading control B Th0 or Th17 cells underwent protein crosslinking using disuccinimidyl suberate followed by immunoblot analysis to identify PKM2 oligomer states C Naive CD4 T cells were cultured under Th17 cellinducing conditions for 96 h and prepared for confocal immunofluorescence analysis Cells were stained with fluorophoreconjugated antiPKM2 red and nuclei labeled with DAPI blue Confocal images were acquired scale bar indicates 5 µm D Cytoplasmic and nuclear protein extracts from Th17 cell culture were obtained and analyzed by immunoblot to determine PKM2 levels in these compartments GAPDH and NPM were used as cytoplasm and nuclear loading controls respectively E WT or PKM2deficient CD4 T cells were cultured under Th17 cellskewing conditions in the presence of or absence of TEPP46 100 µM a PKM2 activator followed by flow cytometry analysis of IL17A CD4 T cells frequencies n 35 F Nuclear fractions from Th17 cells were obtained and analyzed by immunoblot to determine PKM2 protein expression GAPDH and NPM were used as cytoplasm and nuclear loading controls respectively Data are representative of two independent experiments Error bars are mean SEM P values were determined by twoway ANOVA followed by Tukeys post hoc test E P 005 Damasceno et al Journal of Experimental Medicine 9 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 WT mice Fig S5 F Finally we examined the importance of STAT3 activation by PKM2 in the differentiation of Th17 cells To this end we investigated the effect of a suboptimal con centration of Stattic a smallmolecule inhibitor of STAT3 activation Schust et al 2006 on the differentiation of Th17 cells As expected a suboptimal concentration of Stattic par tially reduced the differentiation of WT Th17 cells whereas it had no additive inhibition on the Th17 differentiation ob served in PKM2deficient T cells Fig 7 G Of note the in hibition of STAT3 activation did not affect Th1 cell differentiation in WT or PKM2deficient T cells Fig S5 G Taken together our results provide strong evidence for the nonmetabolic role of PKM2 in the Th17 cell differentiation program Figure 7 Nuclear PKM2 regulates STAT3 activation in Th17 cells A Immunofluorescence staining of intracellular PKM2 red and STAT3 green was performed in differentiated Th17 cells nuclei were labeled with DAPI blue Confocal analysis was used for image acquisition Scale bar represents 5 µm B The interaction between STAT3 and PKM2 was examined by immunoprecipitation IP Briefly Th17 cell lysates were subjected to IP with a mouse anti STAT3 or control IgG antibody followed by immunoblot analysis using a rabbit antiPKM2 and antiSTAT3 Protein extracts without immunoprecipitation input served as positive controls WB Western blot C PLA was performed to detect the interaction between PKM2 and STAT3 in red in differentiated Th17 cells The blue signal indicates DAPIstained nuclei Confocal images were acquired scale bar represents 5 µm D WT or PKM2deficient naive CD4 T cells were activated with antiCD3εCD28 for 48 h Cells were then acutely stimulated with recombinant mouse IL6 10 ngml and collected 15 or 30 min later for immunoblot analysis E Immunoblot was performed to identify total and phosphorylated Y705 levels of STAT3 in WT or PKM2deficient Th17 cells βactin was used as the loading control F Immunoblot analysis of nuclear fraction from Th17 cells to determine phosphorylated STAT3 Y705 protein expression GAPDH and NPM were used as cytoplasm and nuclear loading controls respectively G Stattic 2 µM an inhibitor of STAT3 activation was added to Th17 cell cultures for 96 h followed by flow cytometric analysis n 3 Data are representative of two AD F and G or four E independent experiments Error bars show mean SEM P values were determined by twoway ANOVA followed by Tukeys post hoc test G or twotailed Students t test E P 005 Damasceno et al Journal of Experimental Medicine 10 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 Discussion Recent studies connecting the fields of cellular metabolism and immunology have dramatically improved our understanding of how immune cells benefit from a metabolic reprogramming to support their activation proliferation and differentiation ONeill et al 2016 Buck et al 2015 Almeida et al 2016 Emerging ev idence proposes that metabolic enzymes rather than solely being components of biochemical pathways are also proteins that me diate many other biological functions including gene transcrip tion and cell cycle progression Yu and Li 2017 Seki and Gaultier 2017 The enzyme PK is a critical ratelimiting enzyme in the glycolytic pathway that catalyzes the formation of pyruvate from phosphoenolpyruvate Notably the PK isoform M2 is not only present in the cytoplasm as a metabolic enzyme but also can translocate into the nucleus indicating that it has additional noncanonical or nonmetabolic functions unrelated to glycolysis Israelsen and Vander Heiden 2015 Gui et al 2013 In the present study we have shown that PKM2 acts as a transcriptional coactivator during Th17 cell differentiation by finetuning STAT3 nuclear activation We have shown that PKM2 is hardly detectable in naive T cells whereas the TCR activation of T cells substantially increases its expression at least in part through mTORC1 signaling mTORC1 is a wellknown metabolic sensor that promotes aerobic glycolysis by inducing the expression of several glycolysisrelated genes Saxton and Sabatini 2017 Consistent with our results it has been previ ously reported that the mTORC1HIF1α signaling axis upregulates the expression of PKM2 in tumor cells Sun et al 2011 Iqbal et al 2013 Importantly T cellspecific deletion of mTORC1 activity or HIF1α impairs Th17 differentiation Delgoffe et al 2011 Kurebayashi et al 2012 Shi et al 2011 Dang et al 2011 In this context we found that PKM2 mRNA and protein ex pression are higher in differentiated Th17 cells than other Th cell subtypes Consistent with this we found that CD4 T cells isolated from the spinal cords of mice undergoing EAE a well characterized animal autoimmune disease model for the ef fector function of Th17 cells Sie et al 2014 express high levels of PKM2 in parallel with the upregulation of the Th17 cell related genes Il17a Csf2 Il23r Rora and Rorc This association implies a potential role of this glycolytic enzyme in supporting Th17 cell differentiation It is noteworthy that while we did not detect alteration in Pkm1 mRNA levels throughout Th17 cell differentiation we observed a particular increase in PKM1 protein levels in fully differentiated Th17 cells which might result from reduced proteasomal degradation Thus it will be important to determine if the later upregulation PKM1 ex pression is solely part of the differentiation process or a re sponse to the metabolic demand of mature Th17 cells PKM2 acts as a transcriptional coactivator for βcatenin and HIF1α in tumor cells promoting the expression of genes in volved in glycolysis and proliferation Yang et al 2011 Luo et al 2011 Moreover LPSinduced glycolytic reprogramming and IL1β production by macrophages require nuclear interac tion of PKM2 with HIF1α PalssonMcDermott et al 2015 We therefore hypothesized that PKM2 would also be required for the metabolic reprogramming and proliferation of Th17 cells Strikingly PKM2 deficiency neither impaired the metabolic reprogramming toward aerobic glycolysis nor affected the pro liferative capacity of Th17 cells Nonetheless the loss of PKM2 in T cells selectively inhibited Th17 differentiation without altering Th1 Th2 or iT reg cell differentiation in vitro As described above the neuroinflammation observed in mice undergoing EAE is mainly mediated by autoantigenspecific Th17 cells Sie et al 2014 Consistent with our in vitro re sults the specific loss of PKM2 in T cells not only significantly reduced the clinical severity but also decreased the incidence of EAE These declines were associated with a lower frequency of IL17A CD4 T cells and less Th17 cellrelated cytokine produc tion by T cells upon ex vivo stimulation with MOG3555 Nev ertheless while we did not find a role for PKM2 in Th1 cell polarization in vitro mice lacking PKM2 in T cells showed a reduced frequency of T cells expressing IFNγ during EAE A plausible explanation for these last findings is the potential conversion of Th17 cells into IL17AIFNγ CD4 T cells which is one of the signatures of pathogenic Th17 cells and the dominant T cell population found in the spinal cord of EAE mice Kurschus et al 2010 Hirota et al 2011 In support of this we also demonstrated that GMCSF another pathogenic Th17 cell sig nature cytokine Codarri et al 2011 ElBehi et al 2011 was similarly reduced in mice lacking PKM2 in T cells during EAE However cytokinedriven T cell polarization in vitro is different from pathophysiological differentiation in vivo where other mediators might be directly or indirectly involved Thus we cannot rule out the possibility that PKM2 might regulate Th1 cell differentiation in vivo Indeed it was reported that homocys teine stimulation of T cells increases glycolytic metabolism and IFNγ expression in a PKM2dependent manner Lü et al 2018 In agreement with our findings during the revision process of this article a study was published supporting the role of PKM2 in the generation of Th17 cells Kono et al 2019 How ever using shikonin a pharmacological inhibitor of PKM2 it was proposed that inhibition of PKM2 impairs Th17 cell differ entiation by reducing glycolysis which contrasts with our metabolic findings obtained with the genetic approach Cre LoxP The differences in the metabolic profile might be ex plained by offtarget effects of shikonin in other enzymes that regulate glycolysis including the inhibition of glycogen synthase kinase 3β GSK3β and cell division cycle 25 Cdc25 phospha tases Chen et al 2019 Zhang et al 2019 Liang et al 2016 Furthermore we found that the deficiency of PKM2 in Th17 cells led to a compensatory upregulation of PKM1 expression which might explain the normal glycolytic profile that we have ob served and also supports the hypothesis of a nonmetabolic mechanism of PKM2 in mediating Th17 cell differentiation The differential role of PKM1 in Th17 cell differentiation is currently unclear and merits further investigation PKM1 and PKM2 isoforms are products of alternative splicing of the same Pkm gene Noguchi et al 1986 PKM2 in its tet rameric form has high metabolic activity in the glycolysis pathway similar to PKM1 However the less enzymatically ac tive dimeric form of PKM2 can translocate into the nucleus and act as a transcriptional coactivator Yang et al 2011 Luo et al 2011 Yang et al 2012a Phosphorylation of PKM2 on Y105 is indicative of the dimeric form of PKM2 as it prevents the Damasceno et al Journal of Experimental Medicine 11 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 tetrameric conformation Hitosugi et al 2009 Herein we found that the expression of total PKM2 was followed by increased phosphorylation at Y105 during Th17 cell differentiation Consis tently the dimeric form of PKM2 was the most prevalent con formation detected in Th17 cells localized in both cytoplasmic and nuclear compartments Other posttranslational modifications of PKM2 including acetylation and succinylation have been re ported to affect PKM2 conformation favoring the dimeric form Lv et al 2013 Wang et al 2017 Therefore we cannot exclude the role of other posttranslational modifications affecting the translocation of PKM2 into the nucleus of Th17 cells However consistent with the wellcharacterized effect of small compound TEPP46 in promoting PKM2 tetramer formation and inhibiting its nuclear translocation Anastasiou et al 2012 we observed that TEPP46 reduces Th17 cell differentiation suggesting that nuclear translocation of PKM2 is required for the generation of Th17 cells Indeed this hypothesis was recently supported by Angiari et al 2020 Since the nuclear translocation of PKM2 requires the binding of its nuclear localization signal sequence to the importin α5 an adaptor protein that imports proteins into the nucleus Yang et al 2012b further analysis showing the inter action of the PKM2 nuclear localization signal with importin α5 in Th17 cells may help to confirm our conclusions Nuclear PKM2 has been shown to interact with and enhance STAT3 phosphorylation at Y705 contributing to increases in cancer cell proliferation Gao et al 2012 and inflammatory cytokine production by macrophages Shirai et al 2016 Moreover it was reported that the mutation of PKM2 at residue R399 locks it in di meric conformation enhancing its ability to phosphorylate STAT3 Gao et al 2012 In this context it is well known that IL6 and IL23 promote differentiation of Th17 cells through activation of the STAT3 signaling pathway Korn et al 2009 Dong 2008 Inter estingly integrative phosphoproteomics analysis of IL23activated T cells revealed predominant phosphorylation of preexisting STAT3 nuclear subsets in addition to the translocation of phosphorylated STAT3 Lochmatter et al 2016 In the current study we demon strated that PKM2 interacts with STAT3 in the nucleus of Th17 cells and deficiency or inhibition of nuclear translocation of PKM2 sig nificantly reduced phosphorylation of STAT3 levels in Th17 cells Whether the observed phosphorylation of STAT3 is due to direct phosphorylation catalyzed by the nuclear dimeric PKM2 or caused by an indirect mechanism via another protein kinase needs to be further investigated Of note the potential role of nuclear PKM2 as a protein kinase has been recently debated Hosios et al 2015 In conclusion we have demonstrated that PKM2 acts as a critical nonmetabolic regulator of Th17 cell differentiation by enhancing the activation of STAT3 Fig 8 Our study also highlights the role of PKM2 in the regulation of pathogenic Th17 cells during autoimmunemediated neuroinflammation PKM2 therefore may represent a potential therapeutic target for autoimmunemediated inflammation Materials and methods Mice C57BL6 CD4Cre TgCd4Cre1CwiBfluJ Lee et al 2001 Pkm2flox B6129SPkmtm11MgvhJ Israelsen et al 2013 and Rag1 mice were purchased from Jackson Laboratories Conditional knockout mice CD4CrePkm2flfl were generated by crossing CD4Cre to Pkm2flox mice which were maintained on a C57BL6 genetic background All animals were housed in a specific pathogenfree facility at the Ribeirao Preto Medical School under controlled temperature 2225C and 12h lightdark cycle and provided with water and food ad libitum Mice used in experiments were sex and age matched All experiments were performed in accordance with protocols approved by the Ethics Committee on Animal Use of Ribeirao Preto Medical School University of São Paulo In vitro T cell differentiation Naive CD4CD25 T cells were purified from LNs and spleen of WT C57BL6 CD4CrePkm2flfl or control littermate CD4Cre and Pkm2flfl mice with the untouched CD4 T cell isolation kit Miltenyi Biotec and a biotinylated CD25 monoclonal antibody eBioscience by using an AutoMACS magnetic cell sorter Miltenyi Biotec according to the manufacturers protocol Pu rified cells were activated with soluble antiCD3εCD28 both 1 µgml BD Biosciences on Ubottomed plates 105well Skewing conditions were as follows Th17 25 ngml rhTGFβ1 eBioscience plus 20 ngml rmIL6 RD Systems with or without 20 ngml rmIL23 RD Systems Th1 rmIL12 and rmIL2 both 20 ngml RD Systems Th2 antiIFNγ 10 µgml rmIL4 and rmIL 2 both 20 ngml RD Systems For iT reg cell polarization naive T cells were cultured with platebound CD3εCD28 both 1 µgml BD Biosciences in the presence of 1 ngml rhTGF β1 eBioscience When indicated 01 µM rapamycin Cayman Chemical 100 µM TEPP46 Millipore or 2 µM Stattic Tocris was used Induction and assessment of EAE EAE was induced by subcutaneously immunizing mice in the flanks with MOG3555 Proteimax The 300 µg of administered MOG3555 was composed of 100 µl PBS and 100 µl CFA SigmaAldrich supplemented with 5 mgml heatinactivated Mycobacterium tuber culosis H37Ra Difco Additionally mice received 200 ng pertussis toxin SigmaAldrich ip followed on the day of immunization as well as 2 d later For adoptive transfer experiments DLNs cells were harvested from WT or CD4CrePkm2flfl donor 8 d after immunization and cultured in vitro with MOG3555 under Th17 cellpolarizing conditions for 72 h CD4 T cells were isolated by magnetic separa tion Miltenyi Biotec and a total of 106 CD4 T cells were injected iv into naive Rag1 recipients 1 d later the recipient mice were immunized with MOG3555 plus pertussis toxin as previously de scribed Clinical signs of EAE were scored on a standard 05 scale according to previous recommendations Stromnes and Goverman 2006 as follows 0 unaffected 05 partial limp tail 1 para lyzed tail 15 loss of coordinated movements 2 hindlimb paresis 25 one hindlimb paralyzed 3 both hindlimbs paralyzed 35 hindlimbs paralyzed and weakness in forelimbs 4 one forelimb paralyzed 45 both forelimbs paralyzed and 5 moribunddeath RNA isolation and quantitative realtime PCR RTqPCR Total RNA from cultures or sorted CD4 T cells were isolated using the RNeasy Isolation Kit according to the manufacturers instructions Qiagen Total RNA from spinal cords was harvested Damasceno et al Journal of Experimental Medicine 12 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 following the TRIzol Reagent Invitrogen protocol The RNA was quantified and then converted to cDNA using the High Capacity cDNA Reverse Transcription Kit Applied Biosystems RTqPCR was performed with SYBR Green PCR Master Mix Applied Biosystems using a StepOnePlus RealTime PCR machine Applied Biosystems Gene expression was determined relative to Gapdh and fold change calculated by using the 2ΔΔCT threshold cycle method In some cases gene expression was represented as heatmaps generated by using the opensource software Morpheus httpssoftwarebroadinstituteorgmorpheus A list of primers is presented in Table S1 Flow cytometry For intracellular cytokine staining cells were stimulated in culture medium with PMA 50 ngml SigmaAldrich and ion omycin 500 ngml SigmaAldrich for 4 h in the presence of monensin GolgiStop 15 µgml BD Biosciences at 37C in a humidified 5 CO2 chamber The cells were then washed and stained for 10 min at room temperature with fixable viability dye Invitrogen for dead cells exclusion and fluorochromelabeled monoclonal antibodies against surface cell markers Afterward cells were fixed and permeabilized using CytofixCytoperm BD Biosciences and PermWash buffer BD Biosciences followed by intracellular staining with monoclonal antibodies for 20 min Intracellular staining of transcription factors was done without stimulation with the eBioscience Foxp3 FixationPermeabiliza tion Kit Data were acquired on FACSVerse or FACSCanto II machines BD Biosciences and analyzed using FlowJo software Tree Star Cell proliferation assay Naive CD4CD25 T cells were labeled with Cell Proliferation Dye eFluor 670 or CellTrace Violet both 5 µM Invitrogen following the manufacturers protocol Cells were then resuspended in culture medium and activated with antiCD3εCD28 both 1 µgml BD Bi osciences in the presence or absence of rmIL2 20 ngml RD Systems or cultured under Th17 cellskewing conditions for 3 d The stepwise dilution of the fluorescence in daughter cells as in dicative of cell proliferation was assessed by flow cytometry Cytokine measurement Supernatants from cell cultures were collected after centrifu gation and IFNγ IL17A GMCSF and IL13 levels were mea sured by ELISA according to the manufacturers instructions RD Systems Glucose uptake consumption and lactate production For flow cytometrybased glucose uptake assay naive CD4 T cells or differentiated Th17 cells were stimulated 1 µgml anti CD3εCD28 and incubated with 30 µM 2NBDG Invitrogen a fluorescent glucose analogue diluted in the glucosefree me dium for 30 min at 37C before measuring fluorescence by flow cytometry Lactate and glucose concentrations in the cell culture supernatants were measured with colorimetric kit assays ac cording to the manufacturers instructions Bioclin Immunoprecipitation and immunoblot analysis Wholecell lysates were prepared using radioimmunoprecip itation assay lysis buffer SigmaAldrich supplemented with Figure 8 Schematic representation describing how PKM2 induces Th17 cell differentiation The cooperation between TCR activation and costimulatory signals per se leads to a significant increase of Pkm2 expression 1 which is highly augmented by the presence of IL6 and IL23 important cytokines for controlling the Th17 cell phenotype program This cascade boosts the activity of the metabolic sensor mTOR that in turn contributes to Pkm2 transcription 2 IL6R and IL23R signaling cascade promote STAT3 phosphorylationactivation 3 concomitantly with an accumulation of PKM2 dimers in Th17 cells 4 The dimeric oligomer state facilitates PKM2 translocation into the nucleus 5 and its interaction with STAT3 increasing its transcriptional activity 6 This process culminates in enhanced transcription of Th17 cellassociated genes contributing to the development of autoimmune neuroinflammation Damasceno et al Journal of Experimental Medicine 13 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 protease and phosphatase inhibitor cocktail Cell Signaling Protein concentrations were determined with a bicinchoninic acid protein assay reagent kit SigmaAldrich For separation by electrophore sis 10 µg total protein was loaded onto SDSpolyacrylamide gels according to standard protocols SDSPAGE and then transferred to nitrocellulose membrane GE Healthcare Membranes were blocked with 5 wtvol nonfat milk Cell Signaling in Tris buffered saline with 01 Tween20 TBST for 1 h at room tem perature and then incubated overnight at 4C with 11000 dilutions of primary antibodies against PKM1 PKM2 phosphoPKM2 Y105 STAT3 phosphoSTAT3 Y705 LDHA or HIF1α all from Cell Signaling Subsequently membranes were repeatedly washed with TBST and incubated for 2 h with the appropriate HRPconjugated secondary antibody 15000 dilution SigmaAldrich Immunore activity was detected using the ECL prime reagent GE Healthcare and then the chemiluminescence signal was recorded on the ChemiDoc XRS Imager BioRad Laboratories Data were analyzed with Image Lab software BioRad Laboratories Total βactin levels were used as a loading control Immunoprecipitation was per formed using the Pierce coIP kit Thermo Scientific following the manufacturers protocol Briefly control IgG antibody and mouse antiSTAT3 Cell Signaling were immobilized using AminoLink Plus coupling resin Equal amounts of Th17 cell lysates were pre cleared and subsequently incubated with the antibodycoupled resin overnight at 4C Afterward the resin was washed and pro teins were eluted using elution buffer The immunoprecipitated samples were analyzed for PKM2 and STAT3 protein expression by immunoblot as described above Subcellular fractionation Nuclear and cytosolic fractionation was performed by using the NEPER Nuclear and Cytoplasmic Extraction Reagents kit according to the manufacturers recommendations Thermo Scientific Protein levels were quantified bicinchoninic acid and samples separated by SDSPAGE before immunoblot analy sis The housekeeping proteins GAPDH and nucleophosmin NPM were used as cytosolic and nuclear loading controls respectively Crosslinking reaction Th0 cells no cytokines or differentiated Th17 cells were cross linked with 500 µM disuccinimidyl suberate SigmaAldrich for 30 min and then cell lysates were prepared with radioim munoprecipitation assay buffer The subsequent steps were performed as described in the immunoblot analysis section Antigenspecific T cell response Cells from DLNs and spleen of EAEbearing mice were isolated and cultured in 96well roundbottom plates 3 105 cellswell with MOG3555 50 µgml in culture medium for 4 d at 37C The concentration of IL17A GMCSF and IFNγ in the culture su pernatants was measured using ELISA kits RD Systems Preparation of central nervous system CNS mononuclear cells EAE mice were deeply anesthetized and transcardially perfused with icecold PBS The spinal cord was collected and minced with a sharp razor blade following digestion for 30 min at 37C with collagenase D 25 mgml Roche Diagnostics Mononu clear cells were isolated by the passage of the tissue through a cell strainer 70 µm followed by centrifugation through a 37 70 Percoll gradient GE Healthcare For intracellular cytokine staining isolated cells were stimulated as previously described followed by flow cytometric analysis Conversely cell suspen sions were labeled with antiCD4 L3T4 microbeads Miltenyi Biotec and separated using an AutoMACS magnetic cell sorter Miltenyi Biotec The purity of cell preparations was 90 and total RNA was extracted for RTqPCR analysis Histology Mice were anesthetized and perfused with cold PBS followed by 4 paraformaldehyde PFA Spinal cords were collected post fixed in 4 PFA and then cryoprotected in 30 sucrose solution for 72 h Tissues were then embedded in OCT compound Tissue Tek Sakura Finetek and snapfrozen on dry ice Spinal cords were cryostatcut Leica into 20µmthick transverse sections mounted on glass slides and stained with HE A pathologist assessed transverse spinal cord tissue sections for inflammatory cell infiltration in a blinded fashion Immunofluorescence For immunofluorescence analysis spinal cord cryosections were permeabilized with 02 Triton X100 in PBS for 20 min blocked with 2 BSA in PBS for 30 min and then incubated overnight at 4C with primary antibodies Subsequently sec tions were incubated for 2 h at room temperature with species specific Alexa Fluorconjugated secondary antibodies Abcam CNS tissue sections were incubated for 1 h with the fluorescent myelin stain FluoroMyelin Green 1200 Invitrogen Slides were rinsed in PBS and coverslipped in ProLong Gold antifade reagent with DAPI Invitrogen For T cell immunofluorescence cells were incubated on polyLlysinecoated coverslips fixed 4 PFA and permeabilized After incubation with primary and secondary antibodies coverslips were washed and mounted onto microscope slides using a DAPIcontaining mounting me dium The following primary antibodies were used antiPKM2 1200 Abcam and antiSTAT3 1200 Cell Signaling The slides were visualized with a highresolution SP5 confocal mi croscope Leica and image analysis performed on Fiji software PLA PLA was performed using a Duolink In Situ Kit MouseRabbit according to the manufacturers instructions SigmaAldrich Briefly Naive CD4 T cells were cultured under Th17 cell skewing conditions for 96 h Cells were attached to polyL lysinecoated cover slides fixed with 2 PFA 10 min at room temperature washed with PBS and blocked with blocking buffer 30 min at room temperature Cells were permeabilized 01 Triton X100 and intracellularly stained with primary mouse antiSTAT3 and rabbit antiPKM2 overnight at 4C both from Cell Signaling followed by incubation with oligonucleotide labeled secondary antibodies Ligase was added for the hy bridization of PLA probes 30 min at 37C to form a circular ized DNA strand if in close proximity Samples were incubated Damasceno et al Journal of Experimental Medicine 14 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 with amplification solution containing fluorescently labeled oligonucleotides plus polymerase 100 min at 37C for the rollingcircle amplification reaction Fluorescent signals indi cating proximity was visualized by confocal microscopy Statistical analysis GraphPad Prism 70 software was used for statistical analysis Multiplegroup comparisons were performed with either one way ANOVA or twoway ANOVA followed by Tukeys post hoc test Unpaired twotailed Students t test was used for compar ison of two conditions Data are expressed as means SEM P value 005 was considered significant Online supplemental material Fig S1 validates the expression of signature genes of CD4 T cell subsets and exhibits the differential expression of Pkm1 and Pkm2 among naive effectormemory and Th17 cells and it also shows the PKM2 protein levels in Th1 and Th17 cells Fig S2 includes data showing that T cellspecific PKM2 deletion in mice does not cause gross defect and present data reinforcing that loss of PKM2 does not affect glucose uptake lactate production and prolifer ation of CD4 T cells Fig S3 demonstrates that loss of PKM2 in CD4 T cells does not impair Th1 Th2 or iT reg cell differentia tion Fig S4 displays additional data confirming that PKM2 boosts Th17 cellmediated EAE pathogenesis Fig S5 shows that both STAT3 activation and PKM2 are dispensable for Th1 dif ferentiation and provide data showing reduced levels of STAT3 activation in the spinal cord of EAE PKM2deficient mice Table S1 contains the sequences of mouse primer pairs used for RT qPCR analysis Table S2 lists the reagents used in the study Acknowledgments We thank all members of the Laboratory of Inflammation and Pain at Ribeirao Preto Medical School for technical support and discussions This work was supported by São Paulo Research Foundation grant 2013082162 Center for Research in Inflammatory Dis eases and National Council for Scientific and Technological Development grant 43082320185 This work was also sup ported by São Paulo Research Foundation fellowships for LEA Damasceno 2016102809 DS Prado 16053773 JE Toller Kawahisa 17017148 and MH Rosa 18239106 Author contributions LEA Damasceno DS Prado FP Veras FQ Cunha TM Cunha and JC AlvesFilho designed experiments and provided conceptual input LEA Damasceno DS Prado FP Veras MM Fonseca JE TollerKawahisa MH Rosa GA Publio and TV Martins performed experiments A 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targeting Cdc25s BMC Cancer 1920 httpsdoiorg101186s128850185220x Damasceno et al Journal of Experimental Medicine 16 of 16 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 Supplemental material Figure S1 Expression of signature genes of CD4 T cell subsets and differential expression of Pkm1 and Pkm2 among naive effectormemory and Th17 cells A Naive CD4 T cells were isolated and cultured under Th1 Th2 Th17 or iT reg cells polarizingconditions cells were collected and expression of Ifng Il4 Il17a and foxp3 was determined by RTqPCR B Naive CD4CD62LhiCD44lo or effectormemory CD4 T cells CD4CD62LloCD44hi were sorted from LNs and spleen of C57BL6 WT mice n 3 Naive cells were also cultured under Th17 cellpolarizing conditions 96 h Cells were collected and total mRNA extracted for RTqPCR analysis C WT or CD4CrePkm2flfl CD4 T cells were differentiated into Th1 or Th17 cells for 96 h and collected for immunoblot analysis of PKM2 protein expression βactin was used as a loading control D and E Rapamycin 01 µM an mTOR inhibitor was added to the Th17 cell cultures Cells were collected and intracellularly stained for IL17A and Foxp3 followed by flow cytometric analysis Il17a and Foxp3 gene expression levels were determined by RTqPCR n 3 For gene expression analysis the cycle threshold values were normalized to Gapdh fold change was calculated relative to naive cells in A and B or untreated cells medium in D and E Data are representative of two independent experiments Error bars show mean SEM P values were determined by twoway ANOVA followed by Tukeys post hoc test B or twotailed Students t test D and E P 005 ns not significant Damasceno et al Journal of Experimental Medicine S1 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 Figure S2 T cellspecific PKM2 deletion in mice does not cause gross defects or affect glucose uptake lactate production and proliferation of CD4 T cells A Photograph of spleen and LNs isolated from WT and CD4CrePkm2flfl mice n 3 B Flow cytometric analyses of thymic CD4 and CD8 fre quencies n 3 C and D Proportion of activated CD62LloCD44hi and naive CD62LhiCD44lo CD4 T cells in LNs and spleen n 3 SSC side scatter E WT or PKM2deficient Th17 cells differentiated in the presence or absence of IL23 were incubated with 2NBDG 30 µM for 30 min The glucose uptake ability of Th17 cells was evaluated by flow cytometry MFI mean fluorescence intensity F Levels of lactate produced by Th17 cells were determined in culture su pernatants n 3 G Naive CD4 T cells were labeled with 5 µM proliferation dye and then activated with antiCD3εCD28 and cultured in the presence or absence of IL2 for 72 h Flow cytometric analyses were performed to determine their proliferative capacity n 3 Data are representative of two E and F or three BD independent experiments Error bars show mean SEM P values were determined by twotailed Students t test Damasceno et al Journal of Experimental Medicine S2 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 Figure S3 Loss of PKM2 in CD4 T cells does not impair Th1 Th2 or iT reg differentiation A WT or PKM2deficient CD4 T were cultured under Th17 cellskewing conditions and stained for both IL17A and Foxp3 followed by flow cytometric analysis n 5 BD Naive CD4 T cells were also cultured under Th1 Th2 or iT regskewing conditions and analyzed for expression of IFNγ IL4 and Foxp3 respectively by flow cytometry n 3 In addition IFNγ and IL13 levels in supernatants of Th1 and Th2 cultures respectively were measured by ELISA n 3 E Naive CD4 T cells were cultured under Th1 or Th17 cell polarizing conditions for 96 h Intracellular staining for IFNγ and GMCSF in Th1 cells top and both IL17A and GMCSF in Th17 cells bottom was performed followed by flow cytometric analysis n 5 Data are representative of at least three independent experiments Error bars show mean SEM P values were determined by twotailed Students t test P 005 Damasceno et al Journal of Experimental Medicine S3 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 Figure S4 PKM2 boosts Th17 cellmediated EAE pathogenesis A EAE was induced in WT or CD4CrePkm2flfl mice and DLN cells collected on day 15 n 5 per group Cells were stimulated and intracellularly stained for IL17A or Foxp3 followed by flow cytometric analysis B DLN cells were harvested and restimulated with MOG3555 in vitro for 72 h The supernatants were collected and the levels of IL17A GMCSF and IFNγ were measured by ELISA n 5 C Lumbar spinal cord sections were collected from naive or EAE mice with PKM2 deficiency in CD4 T cells Homogenates were obtained and mRNA extracted followed by cDNA conversion RTqPCR was performed to analyze the expression of Il17a Csf2 and Ifng Gapdh was used for normalization n 5 D DLN cells were collected from WT or CD4CrePkm2flfl EAE mice day 8 and cultured in the presence of MOG3555 under Th17 cellskewing conditions for 72 h CD4 T cells were sorted and intravenously transferred 106 into Rag1 mice 1 d later EAE was induced in the recipient mice n 6 per group Mice were monitored for clinical signs of EAE and CNS inflammatory cell infiltrate analyzed by HE staining Scale bar represents 50 µm E PKM2 and phosphoPKM2 Y105 protein levels in the spinal cord of EAEbearing mice were determined by immunoblot βactin was used as a loading control Data are representative of two AD or three E independent experiments Error bars show mean SEM P values were determined by twoway ANOVA followed by Tukeys post hoc test BD and twotailed Students t test A P 005 Damasceno et al Journal of Experimental Medicine S4 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613 Tables S1 and S2 are provided online as separate Word documents Table S1 lists mouse primer pairs used for RTqPCR analysis Table S2 lists reagents used in this study Figure S5 STAT3 activation and PKM2 are dispensable for the generation of Th1 cells A Naive CD4 T cells were cultured under Th17 cellskewing conditions in the presence or absence of TEPP46 100 µM followed by flow cytometric analysis n 3 B and C Th1 cells were differentiated with or without TEPP46 and then cytoplasmic and nuclear fractions collected to determine PKM2 protein expression by immunoblot NPM was used as a nuclear loading control Flow cytometric analysis of Th1 cell differentiation was conducted n 3 D PLA assay was performed in WT or PKM2deficient Th17 cells followed by confocal microscopy analysis The close proximity of STAT3 and PKM2 is represented in green The blue signal indicates DAPIstained nuclei Scale bar indicates 10 µm E WT or PKM2deficient Th1 or Th17 cell lysates were subjected to immunoblot analysis of total and phosphoSTAT3 Y705 expression GAPDH was used as a loading control F STAT3 and phosphoSTAT3 Y705 levels were determined in spinal cords of WT or CD4CrePkm2flfl EAEbearing mice by immunoblot analysis βActin was used as a loading control G Naive WT and PKM2lacking Th1 cells were differentiated in the presence or absence of Stattic 2 µM flow cytometric analysis of IFNγproducing cells was performed n 3 Data are representative of two BG or three A independent experiments Error bars show mean SEM P values were determined by twoway ANOVA followed by Tukeys post hoc test G and twotailed Students t test A and C P 005 ns not significant Damasceno et al Journal of Experimental Medicine S5 Nonmetabolic role of PKM2 drives Th17 development httpsdoiorg101084jem20190613