Chemical and genetic control of IFN γ‐induced MHCII expression
2018; Springer Nature; Volume: 19; Issue: 9 Linguagem: Inglês
10.15252/embr.201745553
ISSN1469-3178
AutoresRuud H. Wijdeven, Marvin M. van Luijn, Annet F. Wierenga‐Wolf, Jimmy J.L.L. Akkermans, Peter J. van den Elsen, Rogier Q. Hintzen, Jacques Neefjes,
Tópico(s)T-cell and B-cell Immunology
ResumoArticle18 July 2018free access Source DataTransparent process Chemical and genetic control of IFNγ-induced MHCII expression Ruud H Wijdeven Department of Cell and Chemical Biology, LUMC, Leiden, The Netherlands Search for more papers by this author Marvin M van Luijn Department of Immunology, MS Center ErasMS, Erasmus MC, University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Annet F Wierenga-Wolf Department of Immunology, MS Center ErasMS, Erasmus MC, University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Jimmy J Akkermans Department of Cell and Chemical Biology, LUMC, Leiden, The Netherlands Search for more papers by this author Peter J van den Elsen Department of Immunohematology and Blood Transfusion, LUMC, Leiden, The Netherlands Search for more papers by this author Rogier Q Hintzen Department of Immunology, MS Center ErasMS, Erasmus MC, University Medical Center, Rotterdam, The Netherlands Department of Neurology, MS Center ErasMS, Erasmus MC, University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Jacques Neefjes Corresponding Author [email protected] orcid.org/0000-0001-6763-2211 Department of Cell and Chemical Biology, LUMC, Leiden, The Netherlands Search for more papers by this author Ruud H Wijdeven Department of Cell and Chemical Biology, LUMC, Leiden, The Netherlands Search for more papers by this author Marvin M van Luijn Department of Immunology, MS Center ErasMS, Erasmus MC, University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Annet F Wierenga-Wolf Department of Immunology, MS Center ErasMS, Erasmus MC, University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Jimmy J Akkermans Department of Cell and Chemical Biology, LUMC, Leiden, The Netherlands Search for more papers by this author Peter J van den Elsen Department of Immunohematology and Blood Transfusion, LUMC, Leiden, The Netherlands Search for more papers by this author Rogier Q Hintzen Department of Immunology, MS Center ErasMS, Erasmus MC, University Medical Center, Rotterdam, The Netherlands Department of Neurology, MS Center ErasMS, Erasmus MC, University Medical Center, Rotterdam, The Netherlands Search for more papers by this author Jacques Neefjes Corresponding Author [email protected] orcid.org/0000-0001-6763-2211 Department of Cell and Chemical Biology, LUMC, Leiden, The Netherlands Search for more papers by this author Author Information Ruud H Wijdeven1, Marvin M Luijn2, Annet F Wierenga-Wolf2, Jimmy J Akkermans1, Peter J Elsen3, Rogier Q Hintzen2,4 and Jacques Neefjes *,1 1Department of Cell and Chemical Biology, LUMC, Leiden, The Netherlands 2Department of Immunology, MS Center ErasMS, Erasmus MC, University Medical Center, Rotterdam, The Netherlands 3Department of Immunohematology and Blood Transfusion, LUMC, Leiden, The Netherlands 4Department of Neurology, MS Center ErasMS, Erasmus MC, University Medical Center, Rotterdam, The Netherlands *Corresponding author. Tel: +31715263800; E-mail: [email protected] EMBO Rep (2018)19:e45553https://doi.org/10.15252/embr.201745553 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 The cytokine interferon-γ (IFNγ) can induce expression of MHC class II (MHCII) on many different cell types, leading to antigen presentation to CD4+ T cells and immune activation. This has also been linked to anti-tumour immunity and graft-versus-host disease. The extent of MHCII upregulation by IFNγ is cell type-dependent and under extensive control of epigenetic regulators and signalling pathways. Here, we identify novel genetic and chemical factors that control this form of MHCII expression. Loss of the oxidative stress sensor Keap1, autophagy adaptor p62/SQSTM1, ubiquitin E3-ligase Cullin-3 and chromatin remodeller BPTF impair IFNγ-mediated MHCII expression. A similar phenotype is observed for arsenite, an oxidative stressor. Effects of the latter can be reversed by the inhibition of HDAC1/2, linking oxidative stress conditions to epigenetic control of MHCII expression. Furthermore, dimethyl fumarate, an antioxidant used for the treatment of several autoimmune diseases, impairs the IFNγ response by manipulating transcriptional control of MHCII. We describe novel pathways and drugs related to oxidative conditions in cells impacting on IFNγ-mediated MHCII expression, which provide a molecular basis for the understanding of MHCII-associated diseases. Synopsis IFNγ-induced expression of MHC class II molecules in non-immune tissue is critical for inflammation and autoimmune diseases. This study identifies novel genetic and chemical factors that control MHCII expression, and potentially autoimmune diseases. Oxidative stressors affect IFNγ-induced MHC class II expression in non-haematopoietic tissues. A Keap1-Cul3-MYST1-BPTF axis and HDAC1/2 control IFNγ-induced MHC class II expression. These pathways are targeted by arsenite but also by autoimmune suppressing drugs such as DMF. Introduction Antigen presentation by major histocompatibility complex class II (MHCII) molecules is critical for the initiation of an adaptive CD4+ helper T-cell response and for efficient CTL responses to infections and cancer 1, 2. Many MHCII alleles are correlated to specific autoimmune disorders, for example, HLA-DRB1*1501 to multiple sclerosis 3, 4, and it is anticipated that MHCII antigen presentation sensitizes to many autoimmune diseases. Expression of MHCII is limited to antigen-presenting immune cells such as dendritic cells and B cells. However, under inflammatory conditions normal tissue cells can also express and present peptides on MHCII following release of various cytokines, predominantly interferon gamma (IFNγ) 5. This is important in a series of pathologies, including the onset of graft-versus-host disease (GVHD) 6, 7, transplant rejection 8, 9, autoimmune diseases 10, as well as T-cell priming by tumour cells 11, 12. Transcription of MHCII, as well as the associated invariant chain (Ii), which aides MHCII trafficking and occupies the peptide binding groove before antigen loading, is governed by transcriptional master regulator CIITA 13, 14. CIITA does not bind the MHCII promoter directly, but rather assembles a complex of transcription factors at the MHCII promoter, which includes RFX5, CREB and NF-Y 15. Furthermore, CIITA alters the chromatin environment by recruiting remodelling factors such as BRG-1, histone acetyltransferases (HATs) and deacetylates (HDACs) 16-18, as well as by its intrinsic HAT activity 19. CIITA itself is transcribed from different promoters in different cell types, with its IFNγ-induced isoform being initiated by transcription factor IRF-1 20. Besides upregulating MHC class II antigen presentation, IFNγ induces a broad pro-inflammatory gene signature in both immune and non-immune cells and is important for clearance of viral and bacterial infections 21. For these reasons, cancer cells promote resistance to immunotherapy by altering their IFNγ signalling pathway 22, 23, illustrating the importance of an intact IFNγ response for immune recognition. Sustained IFNγ signalling can lead to uncontrolled activation of the immune system, causing MHCII-dependent transplant rejection 24 as well as autoimmunity, but its importance in the disease pathology for different autoimmune diseases is ambiguous 25-29. At the molecular level, engagement of the IFNγ receptor by IFNγ leads to the activation of JAK kinases, which phosphorylate and stimulate nuclear translocation of transcription factor STAT1 30. STAT1 subsequently induces transcription of IRF-1 that controls the expression of many pro-inflammatory genes including CIITA 31. While the central pathway leading to transcription of IRF-1, CIITA and MHCII is conserved in most cells, many IFNγ-induced genes are expressed in a cell type-specific manner 16, 32-34, suggesting additional regulation by epigenetic modifiers and signalling pathways to steer the response. This is illustrated by the observation that HDAC inhibitors increase the expression of IFNγ-target genes in different tumours, thereby sensitizing tumour cells to immune checkpoint inhibition 35, 36. Understanding the factors regulating the IFNγ response and MHCII expression could thus provide novel means of interfering with this important signalling pathway. Here, we identified several novel regulators of IFNγ-mediated MHCII expression, including oxidative stress sensor Keap1, autophagy adaptor p62, E3-ligase Cullin-3 and chromatin remodeller BPTF. We illustrate the role of oxidative stress on MHCII expression with the immunotoxic agent arsenite and the autoimmune suppressive drug dimethyl fumarate, providing additional mechanisms of action for these compounds. Our experiments show that the expression MHCII is controlled by complex pathways, allowing chemical intervention for controlling MHCII-based pathologies. Results Keap1 regulates IFNγ-induced expression of MHCII The highly variable induction of MHCII by IFNγ in different cell types implies that additional proteins are in charge of regulating this process. Previously, using an RNAi screen focused on de-ubiquitinating enzymes, we identified OTUD1 as a regulator of constitutive MHCII transcription in melanoma cells (A. Sapmaz, I. Berlin, E. Bos, R. H. Wijdeven, H. Janssen, R. Konietzny, A. E. Erson-Bensan, R. I. Koning, B. M. Kessler, J. Neefjes & H. Ovaa, submitted). A secondary siRNA screen for potential interactors of OTUD1 37 in different cell types yielded E3-ligase adaptor Keap1 as the most prominent hit. Keap1 is a multifunctional protein that is best known for inhibiting oxidative stress responses by facilitating Cullin-3-dependent ubiquitination and degradation of Nrf2, a transcription factor for antioxidant genes 38. In addition, Keap1 regulates NF-κB signalling 39, 40, autophagy 41, DNA repair 42, drug resistance 43 and cell migration 44, through binding to a variety of substrates, but it has not been linked to MHCII expression. Silencing Keap1 attenuated IFNγ-induced MHCII expression in HeLa and U118 cells, but not constitutive expression in MelJuSo melanoma cells, setting it apart from OTUD1 (Figs 1A and B, and EV1A). Similarly, no downregulation of constitutive MHCII expression in THP-1 cells and U937 cells was observed after Keap1 depletion (Fig EV1B). However, IFNγ-induced MHCII expression on both cells was also not (THP-1) or only marginally (U937) affected in these cells, suggesting that Keap1 is more important in non-hematopoietic cells or cells not expressing MHCII constitutively. Figure 1. Keap1 positively regulates IFNγ-mediated MHCII expression HeLa cells were transfected with siCtrl or siRNAs targeting Keap1 and stained 72 h later for peptide-loaded MHCII (L243-cy5), stimulated or not with 100 ng/ml IFNγ for the indicated time. Representative histogram and quantifications are shown. Right: Keap1 silencing was determined by Western blot analysis (bottom) and qRT–PCR (top, normalized to GAPDH). U118 cells were analysed for MHCII levels according to the same protocol as in (A). HeLa cells transfected with siCtrl or siKeap1 were either or not exposed to IFNγ for 24 h, and mRNA expression of the indicated genes was analysed using qRT–PCR. MHCII promoter activity was analysed using a luciferase under control of the MHCII promoter (DR300) in cells transfected with the indicated siRNAs and treated with IFNγ when indicated. siSTAT1 was used as a positive control, and signals were normalized to a Renilla control plasmid. Cells transfected with siCtrl or siKeap1 were treated with IFNγ for 24 h and lysed, or actinomycin D (2 μM) was added and cells were lysed 2, 4 or 8 h later. mRNA expression level of HLA-DRα and IRF1 was analysed using qRT–PCR, and IRF1 was used as a control for effectivity of actinomycin D. Individual data points are represented by dots, and the line is the average of the two experiments. Data information: Experiments shown represent mean + SD of at least three independent experiments (except E, n = 2). Statistical significance was calculated as compared to control cells using a Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are available online for this figure. Source Data for Figure 1 [embr201745553-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Keap1 regulates IFNγ-induced MHCII expression FM3, FM78 and MelJuSo melanoma cells were transfected with siCtrl or siRNAs targeting Keap1 and stained 72 h later for peptide-loaded MHCII (L243-cy5) before analysis by flow cytometry. THP-1 and U937 cells stably transfected with the indicated shRNAs were stimulated or not with IFNγ for 48 h, and surface expression of peptide-loaded MHCII was assessed using flow cytometry. For stimulated samples, the signal for the unstimulated cells was subtracted to allow analysis of the IFNγ-induced expression. Right: Keap1 silencing was determined by Western blot analysis. HeLa cells transfected with the indicated siRNAs were stimulated or not for 24 h with IFNγ and stained for MHCII, CD63 (late endosomal marker) and DAPI (DNA). For quantification, average signal intensity of at least six fields was quantified over two independent experiments. Individual experimental averages are represented by dots, and the line is the average of the two experiments. Data information: Experiments shown in (A and B) represent mean + SD of at least three independent experiments, for (C) n = 2. Statistical significance was calculated compared to control cells using a Student's t-test (*P < 0.05). Source data are available online for this figure. Download figure Download PowerPoint We then deciphered in which step of the MHCII pathway Keap1 controls MHCII surface expression. To analyse defects in biosynthesis and cell surface transport of MHCII, the subcellular localization of MHCII was visualized. Cells silenced for Keap1 contained significantly reduced total amounts of MHCII, but the intracellular distribution was not affected (Fig EV1C). qPCR analyses of different genes in HeLa cells stimulated with IFNγ for 24 h indicated that Keap1 silencing inhibited the transcription of HLA-DRα, as well as Ii, but not CIITA (Fig 1C). To assess whether Keap1 controls the activity of CIITA or the associated transcription factors, a luciferase construct under control of the MHCII promoter was utilized. In contrast to the control knockdown of STAT1, depletion of Keap1 had no effect on MHCII promoter activity (Fig 1D), indicating that Keap1 does not control any of the factors involved in promoter activation. Furthermore, Keap1 did not affect the stability of HLA-DRα transcripts, since inhibition of RNA polymerase II by actinomycin D followed by culturing did not yield any differences in degradation rates (Fig 1E). Thus, Keap1 is a novel regulator of MHCII transcription, independently of promoter activation or mRNA stability. HDAC1/2 inhibition negates effect of Keap1 on MHCII expression The discrepancy between the effect of Keap1 on endogenous MHCII transcription and the effect of Keap1 on exogenous MHCII promoter activity suggested a role for epigenetic regulation controlled by Keap1 to influence IFNγ-induced MHCII transcription. MHCII expression is reportedly controlled by various epigenetic markers, including H3K27me3, DNA methylation and histone deacetylation 18, 45, 46. Treatment of cells with inhibitors of EZH2, which prevent H3K27me3 modifications 47, 48, as well as HDAC inhibitors, induced MHCII expression in HeLa cells (Fig 2A), whereas inhibition of DNA methylation by decitabine had no effect (data not shown). Whereas the relative effect of Keap1 depletion remained intact upon treatment with EZH2-inhibitors, all three HDAC inhibitors corrected the inhibition of IFNγ-induced MHCII expression following Keap1 silencing. This suggested that Keap1 controls MHCII expression by manipulating histone acetylation/deacetylation activity. Figure 2. Keap1 regulates MHCII expression through HDACs HeLa cells transfected with the indicated siRNAs were treated for 48 h with IFNγ and indicated EZH2 or HDAC inhibitors; expression of MHCII was analysed by flow cytometry and normalized to MFI of control HeLa cells. Maximum non-toxic doses of the inhibitors were used: EPZ6438 (2 μM), GSK343 (10 μM), SAHA (5 μM), MS-275 (0.1 μM) and MGCD0103 (1 μM). Right: Western blot for H4ac, to test effectivity of the HDAC inhibitors. Cells transfected with siCtrl (75 nM), siHDAC1 (37.5 nM siHDAC1 + 37.5 nM siCtrl), siHDAC2 (37.5 nM siHDAC2 + 37.5 nM siCtrl) or siHDAC1 + 2 (37.5 nM siHDAC1 + 37.5 nM siHDAC2). Cells were exposed for 48 h to IFNγ, and expression of MHCII was analysed by flow cytometry and MFI normalized to siCtrl. Bottom: silencing efficiency was evaluated using Western blot. Cells transfected with the indicated siRNAs (37.5 nM HDAC1, 37.5 nM HDAC2 and 37.5 nM Keap1 or siCtrl) were stimulated for 48 h with IFNγ, and expression of MHCII was determined by flow cytometry. Left: representative histogram. Right: bar graph of the average of three independent experiments. Signal for siKeap1 was normalized to the respective siCtrl. HeLa cells transfected with indicated siRNAs were lysed after 3 days, and HDAC activity was determined using Fluor de Lys assay reagents. Data information: All experiments shown represent mean + SD of at least three independent experiments. Statistical significance was calculated compared to control cells using a Student's t-test (*P < 0.05, **P < 0.01). Source data are available online for this figure. Source Data for Figure 2 [embr201745553-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint SAHA is a pan-HDAC inhibitor, while MGCD0103 specifically targets HDAC1/2 and MS-275 inhibits HDAC1 and to a minor extent HDAC2 49, arguing that HDAC1 or HDAC2 is the primary regulator of MHCII expression, which is in line with data that overexpression of HDAC1 or HDAC2 represses MHCII expression 18. However, silencing of HDAC1 did not upregulate MHCII expression, while knockdown of HDAC2 had only a minor effect (Fig 2B and C). HDAC1 and HDAC2 have partly overlapping functions and can compensate for each other 50; therefore, we simultaneously silenced both HDACs. This strongly increased IFNγ-induced MHCII expression and decreased the sensitivity of cells to Keap1 depletion (Fig 2B and C). This suggests that both HDAC1 and HDAC2 are involved in the inhibition of IFNγ-induced MHCII expression in a pathway that intermingles with Keap1. However, Keap1 does not directly regulate the activity of HDAC1 and HDAC2, as concluded by determining HDAC activity in cells either or not depleted of Keap1 (Fig 2D). Keap1 interactors p62/SQSTM1, Cul3 and BPTF regulate MHCII expression How does Keap1 interfere with MHCII expression in non-professional antigen-presenting cells (APCs)? Keap1 mainly serves as a substrate adaptor for Cul3 and binds many proteins via its Kelch-domain, including Nrf2 and other substrates containing an ETGE motif, to properly position these for Cul3-mediated ubiquitination 51. Two point mutants of Keap1 were generated, one that renders Keap1 unable to bind ETGE motif-containing proteins (Y572A) 52 and one that eliminates ubiquitin transfer to substrate proteins (G186R) 53. Stable expression of wild-type RNAi resistant Keap1 allowed rescue of IFNγ-induced MHCII expression upon Keap1 silencing, confirming that the effect of the siRNAs relied on Keap1 depletion. Yet, MHCII expression could not be rescued by either point mutant of Keap1 (Fig 3A and B), indicating a role for both substrate binding and ubiquitination in IFNγ-induced MHCII expression by Keap1. This effect was independent of the canonical substrate Nrf2, as co-depletion of Nrf2 did not restore IFNγ-induced MHCII expression to normal levels (Fig 3C). Figure 3. Keap1 interaction partners regulate MHCII expression HeLa cells stably expressing GFP or RNAi resistant GFP-Keap1 with the indicated mutations were transfected with siRNAs and stimulated with IFNγ for 48 h before analysis by flow cytometry. Shown is MFI relative to siCtrl. Bottom panel: Western blot for expression of the indicated GFP-Keap1 constructs. HeLa cells as in (A) were stimulated for 24 h with IFNγ, and mRNA levels of HLA-DRα were measured using qRT–PCR and related to siCtrl. MHCII expression on HeLa cells transfected with the indicated siRNAs and stimulated with IFNγ for 48 h was measured using flow cytometry and related to siCtrl. Bottom: Western blot analyses for expression of the indicated proteins. Screen for effect of silencing Keap1 interacting proteins on MHCII surface levels. HeLa cells transfected with 106 different siRNAs targeting Keap1-interacting proteins were stimulated with IFNγ for 48 h and analysed by flow cytometry. Right: summary of screening data for the indicated proteins. U118 cells were transfected with the indicated siRNAs and the next day stimulated with IFNγ. 48 h later, MHCII expression was analysed by flow cytometry. HeLa cells transfected with the indicated siRNAs were stimulated for 24 h with IFNγ and mRNA transcript levels were quantified using qRT–PCR, signal was normalized to GAPDH and siCtrl + IFNγ for each sample. Right graph: knockdown efficiency of the different siRNAs. HeLa cells transfected with the indicated siRNAs were stimulated for 48 h with IFNγ and the indicated HDAC inhibitors, followed by MHCII expression by flow cytometry. Data information: All experiments shown represent mean + SD of at least three independent experiments. Statistical significance was calculated compared to control cells using a Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are available online for this figure. Source Data for Figure 3 [embr201745553-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint To identify proteins that cooperate with Keap1 in the control IFNγ-induced MHCII transcription, we performed an RNAi screen targeting all 106 described Keap1 interactors (gene search on www.ncbi.nlm.nih.gov) and measured the effect on IFNγ-induced MHCII surface expression by flow cytometry. Using this screen, several proteins were identified as regulating IFNγ-induced MHCII expression (Fig 3D). Of the top ten hits, only the three most significant hits—BPTF/FALZ, p62/SQSTM1 and Cullin-3—regulated MHCII at the transcriptional level. BPTF is a chromatin remodeller that binds histone modifications H3K4me3 and H4K16ac and unwinds local chromatin for transcription 54, 55, p62 is an adaptor protein involved in autophagy, perinuclear endosome positioning and cell signalling 56-58, and Cullin-3 (Cul3) the ubiquitin ligase that executes the ubiquitination reaction of proteins selected by adaptors such as Keap1 59. Like for Keap1, depletion of these genes also reduced IFNγ-induced MHCII expression in U118 cells (Fig 3E) and controlled Ii but not CIITA or IRF1 expression (Fig 3F). However, the depletion phenotype of none of these three genes could be restored using HDAC inhibitors (Fig 3G), suggesting that the function of these genes does not fully overlap with that of Keap1. Arsenite regulates IFNγ-induced MHCII expression Keap1 contains several cysteine residues that are modified during oxidative stress, rendering it inactive and facilitating NRF2-dependent expression of antioxidant genes. Oxidative stress could then also impair IFNγ-induced MHCII expression by inactivating Keap1. In vivo exposure to sodium arsenite (AS(III)), an oxidative stressor that activates NRF2, has already been reported to decrease the expression of different MHCII alleles and is linked to an impaired immune response 60, 61. To assess a direct role for AS(III) in IFNγ-induced MHCII expression, HeLa and U118 cells were exposed to different concentrations of AS(III) during stimulation with IFNγ. A dose-dependent decrease in MHCII expression was observed, indicating a role for AS(III) in the regulation of IFNγ-induced MHCII expression (Fig 4A). Arsenite indeed targeted Keap1, since Nrf2 target NQO1 was upregulated in a dose-dependent manner (Fig 4B). Similar to Keap1 depletion, this decrease was transcription-dependent and confined to MHCII and Ii, but not CIITA (Fig 4B). Furthermore, treatment with HDAC inhibitor MGCD0103 fully restored IFNγ-induced MHCII expression (Fig 4A). However, AS(III) can also target the H4K16-specific histone acetyltransferase MYST1 62, suggesting it could exert its effect also via MYST1. In support of this, MYST1 knockdown reduced IFNγ-induced MHCII expression (Fig 4C). When cells were depleted for either Keap1 or MYST1 and exposed to AS(III), a very minimal additional effect was observed (Fig 4D), substantiating the notion that AS(III) acts through these molecules. Thus, sodium arsenite impaired IFNγ-mediated MHCII expression, probably via Keap1 and MYST1, and this effect could be negated by HDAC inhibitors. Figure 4. Arsenite controls IFNγ and histone acetylation-dependent MHCII expression HeLa and U118 cells were stimulated with IFNγ for 48 h in combination with the indicated concentration of NaAs2O3 in the presence or absence of 1 μM MGCD0103 and analysed for MHCII expression by flow cytometry. MGCD0103-treated samples were normalized to corresponding measured in the absence of NaAs2O3. HeLa cells either or not exposed to NaAs2O3 were stimulated for 24 h with IFNγ, and mRNA levels were measured by qRT–PCR. Data normalized within each sample to condition lacking NaAs2O3. MHCII levels on HeLa cells transfected with the indicated siRNAs and stimulated with IFNγ for 48 h were determined by flow cytometry. Data were normalized to siCtrl condition. HeLa cells transfected with the indicated siRNAs and stimulated with IFNγ for 48 h in the presence or absence of 5 μM NaAs2O3 were analysed for MHCII expression by flow cytometry. Data were normalized to untreated siCtrl condition. Data information: All experiments shown represent mean + SD of at least three independent experiments. Statistical significance was calculated compared to control cells using a Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Download figure Download PowerPoint Antioxidants control IFNγ-induced MHCII expression Besides oxidative stress, Keap1 is also a primary target for antioxidants such as tert-butylhydroquinone (tBHQ) and dimethyl fumarate (DMF) 38, 63. Both of these drugs display immunomodulatory activity, with their mechanism of action not fully understood 64, 65. DMF has been approved by the FDA for the treatment of psoriasis and multiple sclerosis (MS) 66-68, both autoimmune diseases that have been linked to IFNγ expression and activation of CD4+ T cells 26, 69, 70, implying a possible role for IFNγ-induced MHCII expression in disease pathology. To test whether DMF, like Keap1 inhibition, reduces IFNγ-induced MHC class II expression, we exposed various cell lines to IFNγ in the absence or presence of DMF. DMF reduced IFNγ-induced MHCII expression in all cell lines, whereas constitutive MHCII expression by monocyte-like THP-1 cells was unaffected (Fig 5A). Similarly, tBHQ specifically reduced IFNγ-induced MHCII expression (Fig 5A). In multiple sclerosis (MS), as well as experimental autoimmune encephalomyelitis (EAE, a mouse model for MS), macrophages play an important role in the initiation of the inflammatory response 71. To assess whether DMF also affects MHCII expression by these cells, monocyte-derived macrophages (MDMs), as well as MDMs that were cultured in the presence of myelin to generate foamy macrophages, which are present in brain lesions of MS patients 72, were treated with different doses of DMF. In both types of macrophages, DMF caused a dose-dependent decrease in IFNγ-induced MHCII expression but not constitutive MHCII expression (Fig 5B and C). Constitutive MHCII expression by B cells was also not affected by these drugs (Fig 5D). Figure 5. Dimethyl fumarate inhibits IFNγ-induced MHCII and chemokine expression THP-1, HeLa and U118 cells either or not exposed to IFNγ for 48 h were cultured in the presence of DMF (left) or tBHQ (right) at indicated concentrations before analyses of MHCII expression by flow cytometry. Since THP1 express constitutive MHCII, MHCII expression is normalized by subtracting MFI of non-IFNγ exposed THP1. Data normalized to MFI measured in the absence of drugs. Shown is mean + SD, n = 3. Monocyte-derived macrophages were treated with the indicated concentration of DMF and either or not stimulated with IFNγ for 48 h. MHCII expression was determined by flow cytometry. Shown is mean + SD of four independent experiments. For IFNγ-treated samples, MFI of control unstimulated macrophages was subtracted. Foamy macrophages were treated with the indicated concentrations of DMF and either or not activated with IFNγ for 24 h when indicated before MHCII expression was determined by flow cytometry. Shown is mean + SD, n = 3. Primary human B cells were cultured for 24 h in different concentrations of DMF as indicated, and surface MHCII expression was analysed by flow cytometry. Shown is mean + SD, n = 4. Macrophages (left) or HeLa cells (right) were cultured for 24 h in the presence of IFNγ and DMF when indicated before mRNA expression analysis of the indicate
Referência(s)