Increase of aerobic glycolysis mediated by activated T helper cells drives synovial fibroblasts towards an inflammatory phenotype: new targets for therapy?

RESEARCH ARTICLE Open Access Increase of aerobic glycolysis mediated by activated T helper cells drives synovial fibroblasts towards an inflammatory phenotype: new targets for therapy? Peter Kvacskay 1 , Nina Yao 1 , Jürgen-Heinz Schnotz 1 , Roberta Scarpone 1 , Rui de Albuquerque Carvalho 2,3 , Karel D. Klika 4 , Wolfgang Merkt 1 , Theresa Tretter 1 , Hanns-Martin Lorenz 1 and Lars-Oliver Tykocinski 1* Abstract Background: A dysregulated glucose metabolism in synovial fibroblasts (SF) has been associated with their aggressive phenotype in rheumatoid arthritis (RA). Even though T helper (Th) cells are key effector cells in the propagation and exacerbation of synovitis in RA, little is known about their influence on the metabolism of SF. Thus, this study investigates the effect of Th cells on the glucose metabolism and phenotype of SF and how this is influenced by the blockade of cytokines, janus kinases (JAKs) and glycolysis. Methods: SF from patients with RA or osteoarthritis (OA) were cultured in the presence of a stable glucose isotopomer ([U13 C]-glucose) and stimulated with the conditioned media of activated Th cells (ThCM). Glucose consumption and lactate production were measured by proton nuclear magnetic resonance ( 1 H NMR) spectroscopy. Cytokine secretion was quantified by ELISA. The expression of glycolytic enzymes was analysed by PCR, western blot and immunofluorescence. JAKs were blocked using either baricitinib or tofacitinib and glycolysis by using either 3-bromopyruvate or FX11. Results: Quiescent RASF produced significantly higher levels of lactate, interleukin (IL)-6 and matrix metalloproteinase (MMP) 3 than OASF. Stimulation by ThCM clearly changed the metabolic profile of both RASF and OASF by inducing a shift towards aerobic glycolysis with strongly increased lactate production together with a rise in IL-6 and MMP3 secretion. Interestingly, chronic stimulation of OASF by ThCM triggered an inflammatory phenotype with significantly increased glycolytic activity compared to unstimulated, singly stimulated or restimulated OASF. Finally, in contrast to cytokine-neutralizing biologics, inhibition of JAKs or glycolytic enzymes both significantly reduced lactate production and cytokine secretion by Th cell-stimulated SF. (Continued on next page) © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: [email protected] 1 Department of Medicine V, Division of Rheumatology, University of Heidelberg, INF 410, 69120 Heidelberg, Germany Full list of author information is available at the end of the article Kvacskay et al. Arthritis Research & Therapy (2021) 23:56 https://doi.org/10.1186/s13075-021-02437-7 (Continued from previous page) Conclusions: Soluble mediators released by Th cells drive SF towards a glycolytic and pro-inflammatory phenotype. Targeting of JAKs or glycolytic enzymes both potently modulate SF’s glucose metabolism and decrease the release of IL-6 and MMP3. Thus, manipulation of glycolytic pathways could represent a new therapeutic strategy to decrease the pro-inflammatory phenotype of SF. Keywords: Rheumatoid arthritis, Synovial fibroblasts, Fibroblast-like synoviocytes, T lymphocytes, T helper cells, Metabolism, Glycolysis, Janus kinases, Inflammation, Translational research Background Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation, recurring synovitis and destruction of the cartilage and bone [1,2]. The characteristic transformation of the synovial membrane into a destructive tumour-likepannusisaccompanied by a persistent intra-articular invasion of immune cells. Synovial fibroblasts (SF), also known as fibroblast-like synoviocytes, are the major cell type within the hyperplastic pannus and the main effector cells of joint destruction. SF of RA patients (RASF) present an aggressive phenotype with abnormally increased proliferation, secretion of proinflammatory cytokines and tissue-invasive properties [3– 5]. The aberrant fibroblast phenotype and the hypoxic and nutrient-deprived micro-environment of the pannus are characteristics which are also found in solid tumours. For tumour cells, it is well known that they adapt their glucose metabolism to meet the increased bioenergetic and biosynthetic demands in a tumour micro-environment [6,7]. Already in 1924, Warburg showed that malignant cells produce significantly higher amounts of lactate than normal cells under normoxic conditions [8,9]. He stated that upregulation of aerobic glycolysis allowed malignant cells to survive the hypoxic conditions prevailing in highly proliferative tumour tissues. There is growing evidence lately that this well-described Warburgeffect,i.e.themetabolic switch from oxidative phosphorylation towards glycolysis, is also a characteristic feature of inflamed joints of RA patients [10,11]. Several studies have demonstrated that lactate levels were significantly increased while glucose levels were decreased in synovial fluid or in synovial tissue of RA patients compared to those of osteoarthritis (OA) patients or healthy individuals [12–15]. In the serum of RA patients, an increase of both glucose and lactate levels has been described [16]. Using fluorodeoxyglucose-positron emission tomography, the increased glucose uptake by cells of the pannus was able to be imaged, suggesting an enhanced glycolytic activity of RASF and invading immune cells [17,18]. Moreover, a significant correlation between mitochondrial dysfunction, enhanced aerobic glycolysis and the inflammatory, destructive properties of RASF has been described [19–23]. Although a dysregulated glucose metabolism in RASF has been suggested to play a critical role in the pathogenesis of RA, very little is known about the impact of activated immune cells on the regulation of the glucose metabolism of SF. The association of RA, especially in the most erosive form, with specific HLA-DR alleles provides strong evidence for a key role of T helper (Th) cells in RA pathogenesis and disease severity. T lymphocytes infiltrate the joints of RA patients and constitute about 30–50% of all cell types in the sub-lining region of synovial tissues. In this way, T cells and SF are in close contact and stimulate each other by direct cellcell contact or by the release of soluble factors [24]. The interaction between Th17 cells and SF has been described as a key mechanism for the development of synovial tissue inflammation [25]. Interaction and reciprocal activation of Th cells and RASF have recently been shown to induce and amplify inflammatory responses and to result in a metabolic shift towards glycolysis in SF to meet the increased metabolic demand [26]. On the other hand, interaction with Th cells induces immunosuppressive functions of SF, a capacity that has been shown to be reduced in RASF compared to OASF [27, 28]. Altogether, cross-talk with Th cells clearly affects SF’s phenotype and function. In this study, we investigated the impact of Th cells on the glucose metabolism and phenotype of SF. Further, we compared the effect of chronic stimulation with single stimulation and re-stimulation by Th cells on SF. Finally, we analysed the potency of cytokine-neutralizing biologics and janus kinase inhibitors (JAKi), used in the treatment of inflammatory rheumatic diseases like RA, as well as of inhibitors targeting glycolytic enzymes in limiting the T cell-mediated induction of a glycolytic and inflammatory phenotype in SF. OASF have been described to differ from RASF in many ways, e.g. OASF are less resistant to apoptosis, have a different epigenetic signature and do not possess an intrinsically activated, aggressive and invasive phenotype like RASF [29–31]. In the present study, OASF were used as a degenerative disease control to RASF. Methods Cell isolation and culture SF from patients with either OA or RA were isolated from synovial tissues collected during diagnostic Kvacskay et al. Arthritis Research & Therapy (2021) 23:56 Page 2 of 15 arthroscopy or therapeutic joint surgery as described previously [28]. CD4 + Th cells were isolated from heparinized venous blood of RA patients or normal healthy donors (NHD) by density gradient centrifugation and separation using the MojoSort human CD4 T cell isolation kit (BioLegend) according to the manufacturer’sinstructions (purity ≥98%). All RA patients fulfilled the American College of Rheumatology/European League Against Rheumatism criteria for the classification of RA [32]. SF were cultured in DMEM-F12 medium (Merck) supplied with 10% heat-inactivated foetal calf serum (Thermo Fisher Scientific). Passages 4–10 were used for experiments. Th cells were stimulated with anti-CD3 and anti-CD28 (both 1 μg/ml, Thermo Fisher Scientific) in glucose-free RPMI-1640 medium (Biological Industries) supplemented with 10% foetal calf serum and the stable glucose isotope tracer [U13 C]-glucose (2 g/l, Tracer tec). Th cell-conditioned media (ThCM) were collected on day 4. For most experiments, SF were stimulated with ThCM diluted 1:5 in RPMI-1640 medium. Hypoxic culture conditions were maintained using a glove box (Coylab) and an incubator (Heracell 150i, Thermo Fisher Scientific) with oxygen level control. In some experiments, SF were stimulated with different concentrations of recombinant human interleukin (IL)- 1β, IL-17A (both BioLegend), interferon (IFN)γ(R&D Systems) or tumour necrosis factor (TNF)α(Peprotech). Cytokines or cytokine receptors were neutralized using canakinumab (Novartis), etanercept (Pfizer), secukinumab (Novartis) or tocilizumab (Roche). Janus kinases (JAKs) were targeted by baricitinib (TargetMol) or tofacitinib (Pfizer). Hexokinase (HK) 2 was inhibited by 3bromopyruvate (3-BrPa) (Gentaur) and lactate dehydrogenase (LDH)-A by FX11 (Merck). Cell survival was determined by Annexin V and propidium iodide staining and measured by flow cytometry using a BD FACSCanto II analyser (BD Biosciences). Staurosporin-treated cells (2 μM, 24 h) served as a positive control. Analysis of lactate and glucose levels by 1 H NMR spectroscopy SF were cultured in the presence of [U13 C]-glucose either with or without stimulation by ThCM. Culture supernatants were harvested on day 4 and analysed using a Bruker Avance II spectrometer equipped with a 5-mm indirect detection probe (Bruker BioSpin) operating at 600 MHz for 1 H. The 1 H NMR spectra with water suppression using presaturation were acquired with 43k points defining a spectral width of 7.2 kHz using a 30° radiofrequency pulse and a total repetition time of 10 s to ensure full relaxation of the 1 H nuclei. Spectral analyses were performed using NUTSpro™NMR software (Acorn NMR Inc.). Each free induction decay was multiplied by a decaying exponential with a decay constant of 0.2 Hz prior to Fourier transformation. For quantification, sodium fumarate (10 mM) dissolved in a 0.2-M phosphate buffer solution prepared with D 2 O (99.9%) was used as an internal standard. Quantification of cytokine secretion The concentrations of IL-6, IL-8 and matrix metalloproteinase (MMP) 3 in culture supernatants were quantified by enzyme-linked immunosorbent assay (ELISA) using the Duo Set ELISA kits (R&D Systems) for all cytokines according to the manufacturer’s instructions. Cytokines in the ThCM were quantified using the LEGENDplex Human Th Cytokine Panel 12-plex assay kit (BioLegend). In vitro scratch migration assay To assess the migration of SF in the presence or absence of stimulation by soluble mediators released by activated Th cells or recombinant human TNFα, IL-17A or IFNγ (10 ng/ml each, all PeproTech), we performed an in vitro scratch assay as described [33]. RNA isolation, cDNA transcription and quantitative RTPCR The High Pure RNA isolation kit (Roche) was used to isolate total RNA. Total RNA was reverse transcribed into cDNA with the QuantiTect reverse transcription kit (Qiagen). RT-PCRs were performed using the PowerUp SYBR Green master mix and a StepOnePlus system (both Applied Biosystems). The following primers were utilized: HK2—Fwd-AAGGCTTCAAGGCATCTG, revCCACAGGTCATCATAGTTCC; PFKp—Fwd-AGAT CCATAAGGAGGCCGTG, rev-AGAACGAAGGTCCT CTGGTG; PKM2—Fwd-ATTATTTGAGGAACTCCG CCGCCT, rev-ATTCCGGGTCACAGCAATGATGG; and LDH-A—Fwd-ACCCAGTTTCCACCATGATT, revCCCAAAATGCAAGGAACACT. Western blot analysis Isolation of total cell extracts from SF was performed by lysing cells in RIPA buffer (ccpro) containing a protease inhibitor cocktail (completeMini, Boehringer) for 20 min on ice. After dilution in a loading buffer, protein samples were resolved using standard SDS-PAGE and transferred onto nitrocellulose membranes. HK2, phosphofructokinase (PFK)p, pyruvate kinase M2 (PKM2) and LDH-A were detected by corresponding antibodies (CellSignaling). β-Actin was used as a loading control. Quantification was performed by densiometric analysis using ImageJ2. Fluorescence microscopy SF were left to adhere on glass coverslips and were either stimulated with ThCM for 4 days or left Kvacskay et al. Arthritis Research & Therapy (2021) 23:56 Page 3 of 15 unstimulated. Cells were fixed by paraformaldehyde and permeabilized with ethanol. Fluorescent antibody staining was performed using antibodies against HK2 (eBioscience), PKM2 (CellSignaling) and Cy3-labelled anti-rabbit-IgG. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The analysis was performed at the Imaging Facility of the Center for Molecular Biology Heidelberg (ZMBH) using an Olympus IX81 microscope. Statistics Results are presented as the mean ± SEM. The MannWhitney Utest and the Wilcoxon signed-rank test were applied to unpaired and paired, respectively, sample sets using GraphPad Prism for statistical analysis. Values of p less than 0.05 were considered statistically significant. Results Activated Th cells release mediators that induce a metabolic shift towards aerobic glycolysis in SF In order to investigate the influence of Th cells on the glucose metabolism of both OASF and RASF, SF were cultured in the presence of a stable glucose isotope ([U13 C]-glucose) either under resting conditions or stimulated by ThCM. The amount of [U-13C]-glucose remaining and the glucose-derived [U13 C]-lactate produced by the SF was measured using 1 H NMR spectroscopy. This method allowed us to quantify the concentration of residual, non-metabolized glucose in the culture supernatants as well as the concentration of generated and secreted lactate as the product of glycolysis. By calculating the amount of glucose that was consumed by SF, but not metabolized into lactate, it was possible to indirectly quantify the level of oxidative glucose metabolism. Figure 1a shows representative 1 H NMR spectra of culture supernatants from unstimulated SF and from SF stimulated by ThCM. Stimulation with ThCM resulted in a decrease of glucose and a clear increase of lactate in SF culture supernatants. Comparing OASF and RASF under resting conditions, RASF showed significantly more lactate production and a significantly higher ratio of glucose metabolized by aerobic glycolysis versus that metabolized by oxidative phosphorylation than OASF (Fig. 1b). Stimulation by conditioned culture medium from Th cells of RA patients resulted in a significant increase in lactate production and a significant shift towards glycolytic glucose metabolism by both RASF and OASF (Fig. 1b). However, in contrast to resting conditions, no significant difference in lactate production was detected between RASF and OASF under stimulation with ThCM, although a trend towards still higher lactate levels and glycolytic rates in RASF could be seen (Fig. 1b). Similar to the stimulation by RA patients’ThCM, stimulation of OASF and RASF by conditioned culture medium from Th cells of healthy donors resulted in a strong increase of lactate production by SF (Additional file 1: Fig. S1). Since there were no significant detectable differences between the induction of glycolysis in SF by ThCM from the T cells of healthy individuals or RA patients, all of the experiments in this study were carried out with stimulation of SF by RA patients’ThCM. As shown in Additional file 2: Fig. S2, the stimulatory effect of ThCM on SF’s lactate production rates was dose-dependent. Analysis of the cytokines present in the ThCM used to stimulate the SF revealed that IFNγrepresented the highest content of all the cytokines tested (Additional file 3: Fig. S3). Additionally, TNFα, IL-6, IL-9, IL22, IL-5, IL-13 and to a lesser extent IL-17A and IL-10 were also detected. However, at 151.07 ± 36.42 ng/ml, the concentration of IFNγwas more than 25-fold higher than any of the other cytokines (Additional file 3: Fig. S3). Since hypoxia has been described as a characteristic micro-environmental feature of inflamed joints of RA patients with a mean ambient oxygen tension of only 3.2% O 2 [34], we therefore next investigated the influence of hypoxic conditions on the glucose metabolism of SF. As expected, culture under hypoxic conditions (3% O 2 ) resulted in an increased lactate production by both OASF and RASF. Similar to what had been observed under normoxic conditions, RASF showed significantly higher lactate production than OASF under hypoxia in the absence of further stimulation (Additional file 4: Fig. S4). Stimulation by ThCM under hypoxic conditions equally induced a significant and dose-dependent upregulation of lactate production in both RASF and OASF. However, a further shift from oxidative towards glycolytic glucose metabolism could not be detected in stimulated versus un-stimulated SF under hypoxia (Additional file 4: Fig. S4). Together, these results demonstrate that RASF displayed a higher basic rate of glycolysis under resting conditions compared to OASF; but under stimulation by Th cells, OASF acquired a similar high glycolytic activity as RASF. Moreover, the data indicate that particularly RASF use glycolysis as a means of energy production under hypoxic conditions. Soluble mediators released by activated Th cells induce a pro-inflammatory phenotype in SF To determine whether activated Th cells also stimulated the development of a pro-inflammatory profile in SF, we analysed the secretion of IL-6, IL-8 and MMP3 by OASF and RASF cultured under resting conditions or under stimulation by ThCM. In the absence of stimulation, RASF showed significantly higher levels of IL-6 and MMP3 secretion compared to OASF (Fig. 2a). Stimulation with ThCM strongly augmented the secretion of ILKvacskay et al. Arthritis Research & Therapy (2021) 23:56 Page 4 of 15 6, IL-8 and MMP3 in both groups. Cytokines IL-6 and IL-8 can also be secreted by activated Th cells, however only in much lower amounts compared to Th cellstimulated SF as reported previously [27,28] and shown in Additional file 3: Fig. S3. The ThCM used here to stimulate SF only contained approximately 6 ng/ml IL-6 (6.04 ± 2.95 ng/ml). Therefore, the proportion of Th cellderived IL-6 and IL-8 in the supernatants of ThCMstimulated SF can be neglected. To investigate the impact of stimulation by Th cells on the migration of SF, we performed an in vitro scratch assay. No difference in the migratory capacities could be detected between OASF and RASF under resting conditions. Interestingly, under stimulation by ThCM, both RASF and OASF displayed significantly decreased migration rates compared to unstimulated conditions (Fig. 2b and Additional file 5: Fig. S5). Similarly, stimulation of SF with a combination of IFNγ,TNFαand IL-17A also strongly reduced their migration. This effect was less pronounced when the SF were stimulated by individual cytokines alone (Additional file 5: Fig. S5). Thus, mediators released by activated Th cells induced a strong metabolic shift towards glycolysis in SF in tandem with an augmented secretion of inflammatory mediators and reduced migratory properties. Chronic stimulation by Th cells triggers a highly glycolytic and inflammatory phenotype in OASF Fibroblasts have been described to play a critical role in the induction of a chronic persistent inflammation in RA, and stimulation seems to prime even SF from noninflamed joints for a pro-inflammatory memory response [35]. Here, we wanted to investigate whether a single stimulation or a chronic stimulation by Th cells primes Fig. 1 Th cells induce a metabolic shift towards aerobic glycolysis in SF. OASF (n= 12) and RASF (n= 12) were cultured for 4 days in the presence of [U13 C]-glucose either under resting conditions or stimulated by conditioned culture media of activated Th cells (ThCM). The amount of [U13 C]-glucose and [U13 C]-lactate produced by the SF was measured using 1 H NMR spectroscopy. aRepresentative examples of 1 H NMR spectra of SF stimulated by ThCM (red) and unstimulated SF (blue). bThe amount of secreted [U13 C]-lactate and the ratio of [U13 C]-glucose metabolized by glycolysis (Gly) and those metabolized by oxidative phosphorylation (OXPHOS) was determined for SF culture supernatants. Results are presented as the mean ± SEM. *p< 0.05, **p< 0.01, Mann-Whitney Utest and Wilcoxon signed-rank test. w/o stim, without stimulation Kvacskay et al. Arthritis Research & Therapy (2021) 23:56 Page 5 of 15 the phenotype and glucose metabolism of OASF. Therefore, OASF were cultured under four different conditions: The first group was cultured without stimulation for a total period of 18 days (unstimulated). The second group of OASF was stimulated by ThCM on day 14 of culture (primary stimulation (1st stim)). The third group was first stimulated between days 1 and 4 of culture, then washed and remained unstimulated until a second stimulation on day 14 (re-stimulation (2nd stim)). And finally, the fourth group was repeatedly stimulated between days 1 and 12 and re-stimulated on day 14 by ThCM (chronic stimulation). On day 18, we quantified the concentrations of lactate, IL-6 and MMP3 in the culture supernatants of all four groups. As presented in Fig. 3, unstimulated SF showed very low lactate production and nearly no IL-6 or MMP3 secretion. Single stimulation on day 14 resulted in a sharp increase of lactate and IL-6 production and a mild upregulation of MMP3 secretion. Remarkably, pre-stimulation and later re-stimulation of SF did not induce a memory response with higher lactate, IL-6 or MMP3 production compared to the singly stimulated group. However, chronic stimulation of the SF resulted in a significantly increased secretion of lactate, as well as of IL-6 and MMP3 (Fig. 3). Hence, a repeated stimulation of OASF by Th cells, but not a single re-stimulation, triggered an aggressive phenotype with significantly higher production of lactate and inflammatory cytokines compared to once only stimulated OASF. Stimulation of SF by Th cells enhance the expression of glycolytic enzymes Since RASF displayed increased baseline levels of glycolysis under resting conditions when compared to OASF and stimulation by ThCM boosted the glycolytic activity of both OASF and RASF, to further elucidate the role of glycolytic key regulators in the enhanced glycolytic activity of RASF and the shift in glucose metabolism towards glycolysis upon stimulation by Th cells, we quantified the expression of HKII, PFKp, PKM2 and Fig. 2 Mediators released by Th cells induce pro-inflammatory cytokine expression by SF but diminish their migration. Secretion of proinflammatory cytokines correlates with enhanced glycolytic activity in SF. OASF and RASF were cultured in the presence or absence of ThCM. a After 4 days, the concentration of interleukin (IL)-6, IL-8, and matrix metalloprotease (MMP)3 within the culture supernatants was quantified by ELISA (n= 10). bAn in vitro scratch assay was performed to investigate the cell migration of SF cultured with or without ThCM (n= 6). Data are presented as the mean ± SEM. *p< 0.05, **p< 0.01, Mann-Whitney Utest and Wilcoxon signed-rank test Kvacskay et al. Arthritis Research & Therapy (2021) 23:56 Page 6 of 15 LDH-A in OASF and RASF under resting conditions and under stimulation by ThCM. Even though there was more aerobic glycolysis in resting RASF than in OASF, we did not find any differences in the mRNA and protein expression of the analysed glycolytic enzymes between resting OASF and RASF (Fig. 4a, b). Stimulation with ThCM resulted in a significant increase in the expression of HK2 mRNA by both RASF and OASF compared to unstimulated conditions. For PFKp, PKM2 and LDH-A, a tendency towards higher mRNA expression upon stimulation by ThCM could be observed (Fig. 4a). Comparable results were obtained on the protein level by western blot and immunofluorescence analysis (Fig. 4b and Additional file 6Fig. S6). Targeting particular cytokines by biologics is not sufficient to reduce the Th cell-stimulated glycolysis in SF Our data presented above showed that both the production of pro-inflammatory cytokines and a metabolic shift towards aerobic glycolysis were induced in SF by ThCM by soluble factors in a cell contact-independent way. We next investigated whether cytokines known to play key roles in the pathogenesis of RA and to have the capacity to activate SF, namely TNFα, IL-1βand IL-17A, and IFNγ, which we found in highest concentrations in ThCM, are known to play a role in the pathogenesis of RA or other rheumatic diseases, alone or in combination, are able to induce the production of lactate by SF. Therefore, OASF and RASF were incubated with different concentrations of recombinant TNFα,IL-1β, IL-17A and IFNγ, either individually or with all four cytokines. On day 4, the lactate production by the stimulated SF was quantified. As presented in Fig. 5a, all individual cytokines induced a trend towards an enhanced lactate production by both OASF and RASF, at least at the highest concentrations tested. However, only IL1-βhad a significant and dose-dependent glycolysis-promoting effect on OASF and RASF. In addition, simultaneous stimulation by all four cytokines together revealed a synergistic effect resulting in a higher increase in lactate Fig. 3 Chronic stimulation by Th cells triggers a glycolytic and proinflammatory phenotype in OASF. OASF were cultured for a total period of 18 days under four different conditions: The first group was cultured without stimulation (w/o stim). The second group was stimulated on d14 of culture (1st stim). The third group was first stimulated between d1 and d4, then remained unstimulated until a second stimulation on d14 (2nd stim). The fourth group was repeatedly stimulated between d1 and d12 and re-stimulated on d14 (chronic stim). On d18, the concentrations of lactate, IL-6 and MMP3 were quantified for all four groups (n= 7). Results are presented as the mean ± SEM. Statistical significances between the chronically stimulated group and the unstimulated, the singly stimulated and the re-stimulated group are shown. *p< 0.05, Wilcoxon signed-rank test Kvacskay et al. Arthritis Research & Therapy (2021) 23:56 Page 7 of 15 production by both OASF and RASF than the one observed with the individual cytokines (Fig. 5a). Neutralizing cytokines or cytokine receptors by specific antibodies have proven to be greatly effective in the treatment of rheumatic diseases. In order to determine whether such biologics could abrogate the upregulation of aerobic glycolysis in Th cell-stimulated SF, OASF were incubated with ThCM in the presence of different concentrations of etanercept (anti-TNFα), tocilizumab (anti-IL-6-receptor), secukinumab (anti-IL-17A) or canakinumab (anti-IL-1β), and the production of lactate was analysed on day 4. As depicted in Fig. 5b, none of the biologics caused a significant reduction in lactate production by OASF stimulated with ThCM. Thus, proinflammatory cytokines, especially IL-1β, can induce aerobic glycolysis in SF. However, targeting only one cytokine or cytokine receptor by the biologics was not sufficient to significantly affect the Th cell-mediated metabolic switch in SF. Inhibition of JAKs as well as glycolytic enzymes both efficiently block the Th cell-mediated switch towards a glycolytic and inflammatory phenotype in SF Since the biologics targeting single cytokines were inefficient in lowering the stimulatory effect of ThCM on SF’s glycolytic metabolism, we next tested whether blocking intracellular cytokine signalling through the JAK pathway could more efficiently block Th cell-mediated SF Fig. 4 Stimulation of SF by Th cells results in an enhanced expression of glycolytic enzymes. OASF and RASF were cultured under resting conditions or under stimulation by ThCM and cells were harvested on d4. aThe mRNA expression of HK2, LDH-A, PKM2 and PFKp were determined by RT-PCR. Each bar indicates the mRNA expression of the corresponding gene as 2 −deltaCT using β-actin mRNA expression as reference (n= 6). bProtein levels of HK2, LDH-A, PKM2 and PFKp were quantified by western blot. β-Actin was used as a loading control. The depictions are representative examples from one of three independent experiments. HK2, hexokinase 2; LDH-A, lactate dehydrogenase A; PKM2, pyruvate kinase M2; PFKp, phosphofructokinase p. Data are presented as the mean ± SEM. *p< 0.05, Mann-Whitney Utest and Wilcoxon signed-rank test Kvacskay et al. Arthritis Research & Therapy (2021) 23:56 Page 8 of 15 activation. Most of the cytokines which we detected in ThCM are known to induce cytokine receptor signalling via the JAK-STAT pathway, and stimulation by soluble mediators released by activated Th cells strongly induced phosphorylation of STAT in OASF and RASF [28]. For this reason, SF were stimulated with ThCM in the presence or absence of the JAKi baricitinib or tofacitinib. Remarkably, both baricitinib and tofacitinib significantly diminished the production of lactate by SF stimulated by ThCM in a dose-dependent manner (Fig. 6a). Additionally, baricitinib significantly abrogated the shift from oxidative to glycolytic glucose metabolism in SF. Importantly, in parallel to their effect on the glycolytic rate, both JAKi significantly reduced the secretion of IL6 by Th cell-stimulated SF (Fig. 6b). Secretion of MMP3 was less affected by JAKi, though baricitinib significantly reduced MMP3 expression by SF at the highest concentration tested (500 nM) (Fig. 6b). Fig. 5 Effects of stimulation with cytokines and blocking of cytokines on the glucose metabolism of SF. aOASF and RASF were stimulated with different concentrations of IL-1β, IL-17A, TNFαand IFNγ, either individually or with all four cytokines. After 4 days of culture, the supernatants were harvested, and lactate concentrations were measured by 1 H NMR spectroscopy (n= 6). bOASF were cultured under resting conditions or with stimulation by ThCM in the presence or absence of anti-TNFα(etanercept), anti-IL-6 receptor (tocilizumab), anti-IL-17A (secukinumab) and anti-IL-1β(canakinumab) at the three given concentrations. Lactate concentrations were quantified on d4 (n= 4). Data are shown as the mean ± SEM. *p< 0.05, Mann-Whitney U test and Wilcoxon signed-rank test Kvacskay et al. Arthritis Research & Therapy (2021) 23:56 Page 9 of 15