Targeting prohibitins at the cell surface prevents Th17‐mediated autoimmunity
2018; Springer Nature; Volume: 37; Issue: 16 Linguagem: Inglês
10.15252/embj.201899429
ISSN1460-2075
AutoresUlrike Buehler, Katharina Schulenburg, Hajime Yurugi, Maja Šolman, Daniel Abankwa, Alexander Ulges, Stefan Tenzer, Tobias Bopp, Bernd Thiede, Frauke Zipp, Krishnaraj Rajalingam,
Tópico(s)Cancer Immunotherapy and Biomarkers
ResumoArticle26 July 2018free access Source DataTransparent process Targeting prohibitins at the cell surface prevents Th17-mediated autoimmunity Ulrike Buehler Department of Neurology, Focus Program Translational Neurosciences (FTN) and Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Katharina Schulenburg Cell Biology Unit, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Hajime Yurugi Cell Biology Unit, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Maja Šolman Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland Search for more papers by this author Daniel Abankwa Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland Cancer Cell Biology and Drug Discovery Group, Life Sciences Research Unit, University of Luxembourg, Esch-sur-Alzette, Luxembourg Search for more papers by this author Alexander Ulges Institute of Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Stefan Tenzer orcid.org/0000-0003-3034-0017 Institute of Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Tobias Bopp Institute of Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Bernd Thiede Department of Biosciences, University of Oslo, Oslo, Norway Search for more papers by this author Frauke Zipp Corresponding Author [email protected] orcid.org/0000-0002-1231-1928 Department of Neurology, Focus Program Translational Neurosciences (FTN) and Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Krishnaraj Rajalingam Corresponding Author [email protected] orcid.org/0000-0002-4175-9633 Cell Biology Unit, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Ulrike Buehler Department of Neurology, Focus Program Translational Neurosciences (FTN) and Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Katharina Schulenburg Cell Biology Unit, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Hajime Yurugi Cell Biology Unit, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Maja Šolman Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland Search for more papers by this author Daniel Abankwa Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland Cancer Cell Biology and Drug Discovery Group, Life Sciences Research Unit, University of Luxembourg, Esch-sur-Alzette, Luxembourg Search for more papers by this author Alexander Ulges Institute of Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Stefan Tenzer orcid.org/0000-0003-3034-0017 Institute of Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Tobias Bopp Institute of Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Bernd Thiede Department of Biosciences, University of Oslo, Oslo, Norway Search for more papers by this author Frauke Zipp Corresponding Author [email protected] orcid.org/0000-0002-1231-1928 Department of Neurology, Focus Program Translational Neurosciences (FTN) and Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Krishnaraj Rajalingam Corresponding Author [email protected] orcid.org/0000-0002-4175-9633 Cell Biology Unit, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Author Information Ulrike Buehler1,‡, Katharina Schulenburg2,‡, Hajime Yurugi2,‡, Maja Šolman3, Daniel Abankwa3,4, Alexander Ulges5, Stefan Tenzer5, Tobias Bopp5, Bernd Thiede6, Frauke Zipp *,1,‡ and Krishnaraj Rajalingam *,2,‡ 1Department of Neurology, Focus Program Translational Neurosciences (FTN) and Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany 2Cell Biology Unit, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany 3Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland 4Cancer Cell Biology and Drug Discovery Group, Life Sciences Research Unit, University of Luxembourg, Esch-sur-Alzette, Luxembourg 5Institute of Immunology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany 6Department of Biosciences, University of Oslo, Oslo, Norway ‡These authors contributed equally to this work ‡These authors contributed equally to this work *Corresponding author. Tel: +49 6131 172224; E-mail: [email protected] *Corresponding author. Tel: +49 6131 178051; E-mail: [email protected] EMBO J (2018)37:e99429https://doi.org/10.15252/embj.201899429 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract T helper (Th)17 cells represent a unique subset of CD4+ T cells and are vital for clearance of extracellular pathogens including bacteria and fungi. However, Th17 cells are also involved in orchestrating autoimmunity. By employing quantitative surface proteomics, we found that the evolutionarily conserved prohibitins (PHB1/2) are highly expressed on the surface of both murine and human Th17 cells. Increased expression of PHBs at the cell surface contributed to enhanced CRAF/MAPK activation in Th17 cells. Targeting surface-expressed PHBs on Th17 cells with ligands such as Vi polysaccharide (Typhim vaccine) inhibited CRAF-MAPK pathway, reduced interleukin (IL)-17 expression and ameliorated disease pathology with an increase in FOXP3+-expressing Tregs in an animal model for multiple sclerosis (MS). Interestingly, we detected a CD4+ T cell population with high PHB1 surface expression in blood samples from MS patients in comparison with age- and sex-matched healthy subjects. Our observations suggest a pivotal role for the PHB-CRAF-MAPK signalling axis in regulating the polarization and pathogenicity of Th17 cells and unveil druggable targets in autoimmune disorders such as MS. Synopsis Prohibitins are highly expressed on the surface of Th17 cells contributing to high CRAF-MAPK activation and polarization. Employment of PHB ligands inhibits CRAF activation and impairs Th17 mediated pathogenicity. Surface expressed prohibitins (PHBs) regulate Th17 differentiation. Prohibitins (PHBs) are highly expressed on the surface of Th17 cells contributing to enhanced CRAF kinase activation. Targeting surface-expressed PHBs inhibited the CRAF–MAPK pathway and Th17 differentiation and promoted expression of FOXP3. PHBs are highly surface expressed in a subset of CD4+ T cells from patients suffering from multiple sclerosis. Introduction Among the different subsets of CD4+ T cells, T helper (Th)17 cells have gained enormous attention recently due to their established role in the aetiology of autoimmune disorders (Singh et al, 2014; Yang et al, 2014; Patel & Kuchroo, 2015). Th17 cells are primarily recognized by their ability to produce interleukin (IL)-17, mostly IL-17A and IL-17F (Harrington et al, 2005). In addition to IL-17, Th17 cells coproduce IL-21, IL-22, granulocyte–macrophage colony-stimulating factor (GM-CSF) and chemokine receptor CCR6, which coordinates Th17-mediated tissue inflammation. Other cytokines such as IL-6, IL-1β, IL-21, IL-23 and transforming growth factor (TGF)-β contribute to the differentiation of Th17 cells from naïve CD4+ T cells (Bettelli et al, 2006; Mangan et al, 2006; Veldhoen et al, 2006). While naïve CD4+ T cells lack IL-23 receptor (IL-23R), the IL-6-STAT3 signalling axis leads to the expression of IL-23R; IL-23 is required for Th17-mediated autoimmune responses (Ciofani et al, 2012). The repertoire of Th17-associated factors is primarily controlled through the activation of transcription factors RORα, RORγt and STAT3 (Yang et al, 2007, 2008). While the contribution of Th17 cells towards the manifestation of autoimmune disorders is well established, the molecular signalling pathways that control the polarization and pathogenicity of Th17 cells have been intensively studied (Gaffen et al, 2014). Multiple sclerosis (MS) is a neuroinflammatory disease which affects more than two million people worldwide, women more than twice as often as men, and may lead to devastating disability in young adults. Experimental autoimmune encephalomyelitis (EAE) is a well-established essential animal model for understanding the pathology of MS (Luchtman et al, 2014). While the critical role of IL-17-producing Th17 cells in the pathology of EAE animal models is established, their role in the aetiology of MS remains controversial. Nevertheless, it could be shown recently that IL-17-producing T cells in the periphery correlate with T1 hypointense lesions in the brain of highly active MS patients (Buehler et al, 2016). Moreover, antibodies against IL-17 or IL-17 receptors showed promising results in EAE animal models, and furthermore, these effects could also be detected in a human phase II clinical trial (Luchtman et al, 2014). IL-17 antibodies are already approved for treatment of autoimmune disorders such as psoriasis (Waisman, 2012). Apart from protein-based biologics targeting inflammatory cytokines or their cognate receptors, drugs targeting the “druggable genome” in autoimmune disorders are rare. Thus far, the FDA has approved tofacitinib, an inhibitor of Janus kinases (JAKs), for treatment of rheumatoid arthritis; several other small molecule inhibitors targeting JAKs are in clinical development (van Vollenhoven, 2013). Here, we demonstrate that targeting the PHB-CRAF axis and the CRAF-MAPK cascade could possibly be a strategy to combat autoimmune disorders such as MS. Prohibitins (PHB1/2) are flagship members of the SPFH superfamily of proteins (named after stomatin, prohibitin, flotillin and Hflk/C) that carry an evolutionarily conserved PHB domain (Browman et al, 2007). PHB1/2 function as stable heteromers and a large fraction of the PHB1/2 proteins are present in the mitochondrial inner membrane where they form large multimeric ring-like complexes which are critical for the functional and structural integrity of the mitochondria (Merkwirth & Langer, 2009). Loss of PHBs leads to abnormal cristae morphology due to accelerated processing of the dynamin-like GTPase OPA1 (Merkwirth et al, 2008). Recent studies revealed an emerging role for the plasma membrane (PM)-associated fraction of PHB1 in mediating signal transduction (Mishra et al, 2010). More than a decade ago, we showed that the PM-associated fraction of PHB1 is required for the activation of CRAF kinase by oncogenic RAS (Rajalingam et al, 2005). CRAF is the central member of the RAS-RAF-MAPK cascade that controls fundamental cellular processes. RAF kinases are serine/threonine kinases and comprise three isoforms (ARAF, BRAF and CRAF) (Matallanas et al, 2011). RAS are small GTPases comprising three isoforms HRAS, NRAS and KRAS. Upon activation, RAS binds to CRAF and recruits CRAF to the PM, a critical event in the activation of the CRAF kinase. At the PM, association with PHB1 is required for the full activation of this kinase. Recent studies have identified PHB1/2 as a direct target of rocaglamides (RocA) which are natural anti-tumour drugs. Further, binding of rocaglamides to the PHB1/2 complex directly disrupts the interaction between CRAF and PHB1, leading to inactivation of CRAF kinase (Polier et al, 2012). Vi polysaccharide of Salmonella Typhi has been shown to target the PHB1/2 complex at the cell surface to modulate MAPK and IL-8 signalling in human intestinal epithelial cells (Sharma & Qadri, 2004). Furthermore, Vi polysaccharide is a WHO-recommended vaccine (Typhim) that can be administered to healthy individuals to protect them from Salmonella enterica (serovar Typhi) infections. In T cells, where PHBs are found to be surface-expressed upon activation, Siglec-9 expressed on antigen-presenting cells (APC) was identified to be a natural, physiological ligand of surface-exposed PHB1 (Yurugi et al, 2013). Siglec-9 binding to PHBs led to inhibition of MAPK signalling and IL-2 production. Notably, in all these examples, the ligands target surface-exposed PHB1. Indeed, the PM-associated fraction of PHB1 has been associated with several distinct functions. For example, translocation of PHB1 to the PM is required for the activation of mast cells (Kim et al, 2013). Moreover, the PM-associated fraction of PHB1 has been shown to contribute to paclitaxel resistance in tumour cells, as well as enhanced metastases in cervical carcinomas due to high CRAF-MAPK activation (Patel et al, 2010; Chiu et al, 2013). In this study, we found that PHB1 is highly expressed on the surface of Th17 cells, contributing to their pathogenicity. Targeting surface-expressed PHB1/2 led to inhibition of the CRAF-MAPK cascade that controls the plasticity of Th17 cells. Results With an aim to provide novel insights into mechanisms driving the differentiation of Th17 cells, we performed label-free quantitative surface proteomics of Th2 versus Th17 cells as described in the methods section. In short, cells were biotinylated and surface-expressed proteins were isolated by streptavidin beads post-lysis and subjected to liquid chromatography–mass spectrometry (LC-MS) analysis. After confirming the purity of the fractions, label-free quantitative analysis of the mass spectrometry data led to identification of several factors which were specifically expressed in the different subsets of CD4+ T cells (Table EV1). More than 100 proteins were found to be differentially regulated in Th17 cells in comparison with Th2 cells which were identified in all three biological replicates from three healthy human donors (Fig 1A and Table EV1). We then focussed primarily on the signalling factors that were found to be surface-expressed in Th17 cells (Table EV1). Prohibitins (PHB1/2) were consistently upregulated on the surface of Th17 cells in all the screens with an average fold-change of 3.20 ± 1.34 for PHB1 and 2.57 ± 0.79 for PHB2 (Fig 1B and Table EV1). To validate the mass spectrometry-based results, we repeated the surface biotinylation of Th17 and Th2 cells and confirmed by Western blotting that PHBs are indeed highly surface expressed in Th17 cells (Fig 1C). We then tested whether PHBs were specifically surface expressed in Th17 cells derived from murine CD4+ T cells. As expected, we detected an increase in PHB1 exposure in Th17 cells in comparison with Th1, Th2 and Tregs from mice (Fig 1D). These results were further confirmed by employing a specific PHB1 binding peptide (CKGGRAKDC) coupled to rhodamine (Kolonin et al, 2004; (Fig EV1A). Immunofluorescence analysis also confirmed that PHB1 is surface expressed in plasma membrane microdomains as revealed by a dotted, granular staining pattern exhibited throughout the surface of Th17 cells (Fig 1E). PHBs were also highly expressed at both the mRNA and protein levels in Th17 cells (Figs 1F–H and EV1B–D). Finally, we detected high PHB1 levels on the cell surface of Th17 but not Th1 or Th2 cells in ex vivo cultures both on day 1 and on day 7 of differentiation (Fig EV1E). Taken together, these data suggest that PHBs are highly expressed at the cellular level as well as on the surface of Th17 cells. Figure 1. Prohibitins are surface exposed and highly expressed in Th17 cells Venn diagram showing overlap between proteins identified in the surface biotinylation experiments. Shown are short listed factors that are consistently detected in three biological replicates (n = 3 healthy human donors). The 366 proteins identified in both cell types are split into proteins showing at least twofold higher abundance in Th17 cells, proteins with a log2 ratio between −1 and 1 (considered as non-cell type specific) and proteins with at least twofold higher abundance in Th2 cells. Graphical presentation of the calculated fold-change values for prohibitin-1 (PHB1) and prohibitin-2 (PHB2) based on label-free quantification results from three biological replicates, shown as mean (± SEM). Surface biotinylation of murine Th2 and Th17 cells was performed, and the surface expression of PHB1 was checked by immunoblots. Th1, Th2, Th17 and Treg cells were stained for PHB1 expression. Isotype antibody was used as a control (filled histogram). MFI (mean fluorescence intensity) is shown as mean (± SEM); n = 3 animals per group. Cells were treated and stained with Alexa594-conjugated secondary antibody. Red signal indicates surface expression of PHB1 in Th17, Th1 and Th2 cells; the nucleus was stained (blue) with Hoechst. Scale bar = 5 μm. The expression levels of PHB1 and PHB2 in the murine whole-cell lysates were analysed by immunoblots. PHB1 and PHB2 are highly expressed in Th17 cells. The expression analysis of PHB1 mRNA levels reveals a steady increase during the course of Th17 cell differentiation. Shown are the data from a single representative experiment. CD4+ T cells subjected to Th17 differentiation were lysed on indicated days, and the levels of PHB1 and PHB2 total protein were monitored by immunoblots. Data Information: In (F) and (H), Ponceau staining of the entire membrane was performed to monitor loading of proteins. Source data are available online for this figure. Source Data for Figure 1 [embj201899429-sup-0004-SDataFig1.tif] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. PHB1 is highly surface expressed in Th17 cells The surface expression of PHBs was monitored by FACS analysis by employing a PHB-binding peptide and a scrambled control peptide coupled to rhodamine. Shown are data from a representative experiment. The activation of MEK1 (a downstream substrate of CRAF kinase) was monitored by immunoblots in Th0 and Th17 cells isolated from a healthy human donor. Shown are the data from a representative experiment. Kinetics of total PHB1 and PHB2 protein levels during the differentiation Th17 and Th2 cells was analysed by Western blots. The protein levels of PHB1/2 shown in (C) were quantified by ImageJ. Ponceau staining of the entire membrane (in B and C) was employed for total protein levels. Naïve T cells were treated with corresponding cytokines to obtain Th1, Th2 or Th17 cells. The surface expression of PHB1 was measured by FACS analysis on Day 1 and Day 7. Shown are data from three independent experiments. Error bars represent the standard error of the mean (± SEM). P-values were calculated using a two-way ANOVA test with a SIDAK multiple comparisons test; **P < 0.01. HeLa cells were transfected with empty vector (EV), CMV-PHB1 or pDISPLY-PHB1. The cells were lysed, and the proteins were separated in an SDS–PAGE. Western blot analysis was performed to measure the activation of CRAF, MEK1/2 and ERK1/2. The quantification of the bands was performed by ImageJ and presented as a table. Source data are available online for this figure. Download figure Download PowerPoint As the PM-associated fraction of PHB1 is shown to contribute to CRAF activation in other cell types, we hypothesized that enhanced PHB1 expression at the cell surface might contribute to increased CRAF-MAPK activation in Th17 cells. In fact, artificial targeting of PHB1 to the cell surface enhanced CRAF-MEK1 activation in HeLa cells (Fig EV1F). As expected, we detected high levels of active CRAF serine 338 phosphorylation in the steady-state Th17 cells isolated from healthy human blood samples (Fig 2A). In turn, we also detected an increase in the activation of MEK1/2 and ERK1/2 kinases in Th17 cells (Figs 2A and EV1B and C). As PHB1 is required for the activation of CRAF, we tested whether interfering with the PHB-CRAF interface using established ligands of PHBs could influence the activation dynamics of MAPK activation in Th17 cells. Figure 2. CRAF-MAPK is activated in Th17 cells; targeting of PHB1/2 complex with specific ligands leads to the inhibition of this pathway Th17 cells were isolated from the blood of healthy donors and protein lysates were prepared. Immunoblot analysis was performed to monitor the activation status of CRAF and ERK1/2 kinases. PHB1 levels were also monitored. Ponceau staining of the entire membrane was performed; quantification of each band was normalized to the total protein levels. Representative of n = 3 healthy donors. Murine Th17 cells were differentiated in vitro in the presence of Vi polysaccharide and/or RocA; the activation dynamics of CRAF, MEK1/2 and ERK1/2 kinases were monitored with phospho-specific antibodies. HeLa cells were incubated with PHB-binding peptide (CKGGRAKDC coupled to rhodamine) for various time periods and monitored under a confocal microscope for localization studies. Scale bar = 10 μm. Acceptor in-growth FLIM-FRET measurements in HeLa cells expressing EGFP-C-RAF and incubated with CKGGRAKDC-rhodamine B peptide (20 μM) for 0, 10, 20, 30 or 60 min to labelled plasma membrane PHB. The effect of the 100 μg/ml Vi polysaccharide treatment was examined. Numbers in bars indicate the number of analysed cells from two biological replicates. Analysis of variance (ANOVA) complemented by Tukey's honestly significant difference test (Tukey's HSD) performed in the software R version 2.15.2 was used to determine the statistical differences. Statistical significance levels are annotated as NS = non-significant P > 0.05, *P < 0.05, **P > 0.01, ***P < 0.001. Error bars represent ± SEM. The surface proteins on Th17 cells bound to immobilized-Vi polysaccharide were isolated; the presence of PHB1/2 complex was monitored by immunoblots. Source data are available online for this figure. Source Data for Figure 2 [embj201899429-sup-0005-SDataFig2.tif] Download figure Download PowerPoint We employed Vi polysaccharide which has been shown to interact directly with surface-expressed PHB1/2 complex in epithelial cells (Sharma & Qadri, 2004). As expected, Vi polysaccharide treatment of Th17 cell cultures differentiated in vitro from mice led to a striking reduction in the activation of the CRAF and MEK1 kinases (Fig 2B). To further corroborate these observations, we treated Th17 cell culture with rocaglamide (RocA), a natural anti-tumour drug that has been shown to disrupt CRAF-PHB interaction in tumour cells (Polier et al, 2012). Interestingly, treatment with RocA led to a strong reduction in the activation of CRAF–MAPK kinase cascade in Th17 cells (Fig 2B). While treatment with Vi polysaccharide did not affect cell viability in CD4+ T cells, RocA treatment led to a reduction in the proliferation of CD4+ T cells (Fig EV2A–C). Furthermore, RocA has also been shown to influence protein translational machinery by directly targeting eIF4a (Iwasaki et al, 2016). As most PHBs are localized to the mitochondrial inner membrane, we tested whether binding of Vi polysaccharide or RocA under these settings altered mitochondrial viability and function in Th17 cells. Neither treatment disturbed the mitochondrial membrane potential nor Opa 1 processing, a frequent event observed during PHB1/2-depleted and/or knockout conditions (Fig EV2D and E). To further confirm that Vi polysaccharide treatment led to disruption of PHB1-CRAF at the plasma membrane, we performed FLIM-FRET experiments in the acceptor in-growth mode (Harpur et al, 2001). We employed a fluorescently labelled PHB-binding peptide and EGFP-tagged CRAF in HeLa cells. Time-lapse imaging experiments confirmed that PHB-specific peptide decorated the plasma membrane of epithelial cells (Fig 2C) as described earlier (Chiu et al, 2013). Furthermore, we could determine that Vi polysaccharide treatment successfully led to the disruption of PHB1-CRAF interaction at the plasma membrane (Fig 2D). To verify that Vi polysaccharide directly bound to PHB1/2 complex at the surface of Th17 cells, we employed a Vi-trap approach, where surface biotinylated proteins bound to immobilized Vi polysaccharide beads were analysed as described in the methods section. These results indeed confirmed that Vi polysaccharide bound to PHB1/2 complex exposed in the cell surface of Th17 cells (Fig 2E). We then tested whether intervention of PHB-CRAF-MAPK signalling has any influence on the generation and/or pathogenicity of Th17 cells. Click here to expand this figure. Figure EV2. Effects of PHB ligands on cell viability Th17 cells from mice were treated with various concentrations of either Vi polysaccharide or RocA for 3 days, and the viability of the cells was measured by MTT assays. Shown are data from three independent experiments (n = 3). Error bars represent standard error of the mean (± SEM). The proliferation of Vi polysaccharide-treated CD4+ T cells was monitored by incorporation of Alexa421-labelled BrdU. The CD4+ T cells were treated with various concentrations of Vi polysaccharide for 48 h. Shown are FACS analysis data from a single representative experiment. CFSE staining of Th17 cells incubated for 72 h with Vi polysaccharide or RocA was performed, and the samples were analysed by FACS. Shown are data from a single representative experiment. The processing of Opa1 in RocA-treated Th17 cells was monitored by immunoblot analysis. Ponceau staining of the entire membrane was performed to control the total protein levels. The mitochondrial membrane potential of Th17 cells incubated with Vi polysaccharide for 72 h was monitored by FACS analysis. Shown are histograms from a representative experiment. Source data are available online for this figure. Download figure Download PowerPoint Recent studies employing single-cell genomics identified a unique set of pathogenicity factors that contribute to disease pathology in Th17 cells (Gaublomme et al, 2015). We checked whether any of the key factors are altered in Th17-polarized cells treated with PHB ligands. Interestingly, pathogenic factors including IL-17A, IL-17F and RORγt were downregulated at the mRNA level in Th17 cells treated with PHB ligands (Vi polysaccharide and RocA) (Fig 3A). Surprisingly, we detected a strong upregulation of FOXP3 both on the mRNA and on protein levels in the same population, suggesting that interfering with CRAF activation led to a switch towards a more regulatory Treg population in these cultures (Fig 3A and B). Figure 3. Interfering with PHB-CRAF signalling axis ameliorates Th17-mediated pathogenicity Murine Th17 cells were differentiated in the presence of either Vi polysaccharide or RocA, and the mRNA levels of IL-17A, IL-17F, RORγt and FOXP3 were monitored by real-time PCR analysis. Shown are data from three independent experiments (n = 3). Murine Th17 cells were treated with either solvent (PBS) or Vi polysaccharide or RocA or both; the cell lysates were subjected to immunoblot analysis for the protein levels of RORγt, FOXP3 and actin. Ponceau staining (PS) of the entire membrane was performed to monitor total protein levels. The effects of RocA and Vi polysaccharide on IL-17 during polarization of CD4+ T cells to Th17 cells were monitored by FACS (n = 5). The relative secretion of IL-17A during culture and treatment with either RocA or Vi polysaccharide was monitored by ELISA (control, n = 8 and Vi polysaccharide treatment n = 7 each; RocA treatment n = 5). The effects of RocA and Vi polysaccharide on FOXP3 during the polarization of CD4+ T cells to Th17 cells were monitored by FACS (n = 5). Mice undergoing active EAE were treated with either placebo PBS control (n = 15) or Vi polysaccharide (n = 12) and monitored for disease pathology. Data Information: In (A, C–F), error bars represent ± SEM (NS, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001). Source data are available online for this figure. Source Data for Figure 3 [embj201899429-sup-0006-SDataFig3.tif] Download figure Download PowerPoint Consistent with these observations, we detected a strong decrease in the protein levels of RORγt in Th17 cells treated with Vi polysaccharide (Fig 3B). Vi polysaccharide or RocA treatment led to a significant reduction both in the intracellular and in secreted IL-17 levels as revealed by both FACS analysis and ELISA (Fig 3C and D); however, the treatment significantly induced FOXP3 expression (Fig 3E). Similar results were obtained with IL-17, RORγt and FOXP3 when the Th17 cultures were treated with MEK1 inhibitor U0126 or with RAF/multikinase inhibitor sorafenib (Fi
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