Co‐inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity
2018; Springer Nature; Volume: 19; Issue: 12 Linguagem: Inglês
10.15252/embr.201846512
ISSN1469-3178
AutoresMichael Basler, Michelle Lindström, Jacob LaStant, J. Michael Bradshaw, Timothy D. Owens, Christian Schmidt, Elmer Maurits, Christopher Tsu, Herman S. Overkleeft, Christopher J. Kirk, Claire L. Langrish, Marcus Groettrup,
Tópico(s)Immunodeficiency and Autoimmune Disorders
ResumoArticle2 October 2018free access Source DataTransparent process Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity Michael Basler Corresponding Author [email protected]i-konstanz.de orcid.org/0000-0002-9428-2349 Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, Switzerland Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany Search for more papers by this author Michelle M Lindstrom Principia Biopharma, South San Francisco, CA, USA Search for more papers by this author Jacob J LaStant Principia Biopharma, South San Francisco, CA, USA Search for more papers by this author J Michael Bradshaw Principia Biopharma, South San Francisco, CA, USA Search for more papers by this author Timothy D Owens Principia Biopharma, South San Francisco, CA, USA Search for more papers by this author Christian Schmidt orcid.org/0000-0001-9568-895X Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Konstanz, Germany Search for more papers by this author Elmer Maurits Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands Search for more papers by this author Christopher Tsu Takeda Pharmaceuticals International Co., Cambridge, MA, USA Search for more papers by this author Herman S Overkleeft Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands Search for more papers by this author Christopher J Kirk Kezar Life Sciences, South San Francisco, CA, USA Search for more papers by this author Claire L Langrish Principia Biopharma, South San Francisco, CA, USA Search for more papers by this author Marcus Groettrup Corresponding Author [email protected] orcid.org/0000-0002-5423-6399 Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, Switzerland Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany Search for more papers by this author Michael Basler Corresponding Author [email protected] orcid.org/0000-0002-9428-2349 Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, Switzerland Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany Search for more papers by this author Michelle M Lindstrom Principia Biopharma, South San Francisco, CA, USA Search for more papers by this author Jacob J LaStant Principia Biopharma, South San Francisco, CA, USA Search for more papers by this author J Michael Bradshaw Principia Biopharma, South San Francisco, CA, USA Search for more papers by this author Timothy D Owens Principia Biopharma, South San Francisco, CA, USA Search for more papers by this author Christian Schmidt orcid.org/0000-0001-9568-895X Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Konstanz, Germany Search for more papers by this author Elmer Maurits Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands Search for more papers by this author Christopher Tsu Takeda Pharmaceuticals International Co., Cambridge, MA, USA Search for more papers by this author Herman S Overkleeft Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands Search for more papers by this author Christopher J Kirk Kezar Life Sciences, South San Francisco, CA, USA Search for more papers by this author Claire L Langrish Principia Biopharma, South San Francisco, CA, USA Search for more papers by this author Marcus Groettrup Corresponding Author [email protected] orcid.org/0000-0002-5423-6399 Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, Switzerland Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany Search for more papers by this author Author Information Michael Basler *,1,2, Michelle M Lindstrom3, Jacob J LaStant3, J Michael Bradshaw3, Timothy D Owens3, Christian Schmidt2,4, Elmer Maurits5, Christopher Tsu6, Herman S Overkleeft5, Christopher J Kirk7, Claire L Langrish3 and Marcus Groettrup *,1,2 1Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, Switzerland 2Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany 3Principia Biopharma, South San Francisco, CA, USA 4Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Konstanz, Germany 5Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands 6Takeda Pharmaceuticals International Co., Cambridge, MA, USA 7Kezar Life Sciences, South San Francisco, CA, USA *Corresponding author. Tel: +49 7531 882258; Fax: +49 7531 883102; E-mail: [email protected] *Corresponding author. Tel: +49 7531 882130; Fax: +49 7531 883102, E-mail: [email protected] EMBO Rep (2018)19:e46512https://doi.org/10.15252/embr.201846512 This manuscript is dedicated to the late Jens Oliver Funk, to acknowledge his contributions to the initial design of studies and supervision of the collaboration. 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 Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible proteasome variant comprising the proteolytic subunits LMP2 (β1i), MECL-1 (β2i), and LMP7 (β5i). Targeting the immunoproteasome in pre-clinical models of autoimmune diseases with the epoxyketone inhibitor ONX 0914 has proven to be effective. ONX 0914 was previously described as a selective LMP7 inhibitor. Here, we show that PRN1126, developed as an exclusively LMP7-specific inhibitor, has limited effects on IL-6 secretion, experimental colitis, and experimental autoimmune encephalomyelitis (EAE). We demonstrate that prolonged exposure of cells with ONX 0914 leads to inhibition of both LMP7 and LMP2. Co-inhibition of LMP7 and LMP2 with PRN1126 and LMP2 inhibitors LU-001i or ML604440 impairs MHC class I cell surface expression, IL-6 secretion, and differentiation of naïve T helper cells to T helper 17 cells, and strongly ameliorates disease in experimental colitis and EAE. Hence, co-inhibition of LMP2 and LMP7 appears to be synergistic and advantageous for the treatment of autoimmune diseases. Synopsis Simultaneous targeting of the immunoproteasome subunits LMP2 and LMP7 is required for optimal therapeutic efficacy in the treatment of autoimmunity. These findings will promote the design of novel immunoproteasome inhibitors with optimal therapeutic efficacy. Simultaneously targeting the immunoproteasome subunits LMP2 and LMP7 blocks autoimmunity. Co-inhibition of LMP2 and LMP7 is required to reduce MHC-I surface expression, IL- 6 production, and Th17 differentiation. Co-inhibition strongly ameliorates disease phenotypes of experimental colitis and EAE. Introduction The ubiquitin–proteasome system is expressed in all eukaryotic cells and exerts numerous essential regulatory functions in nearly all cell biological pathways. The 26S proteasome degrades polyubiquitylated protein substrates and consists of a 19S regulator bearing ubiquitin receptors and an ATPase ring in charge of protein unfolding as well as a 20S proteolytic core complex. The 20S proteasome has a barrel-shaped structure consisting of four rings with seven subunits each. In the inner two rings, made of β-subunits, the catalytically active β1c, β2c, and β5c subunits are located 1. In hematopoietic cells and in cells stimulated with interferon (IFN)-γ or tumor necrosis factor (TNF)-α, these proteolytically active subunits are replaced by β1i [low molecular mass polypeptide (LMP)2], β2i [multicatalytic endopeptidase complex-like (MECL)-1], and β5i (LMP7) forming the immunoproteasome. Proteasome inhibitors are used in anti-cancer therapy to treat multiple myeloma and relapsed mantle cell lymphoma 2. To improve pharmacological and toxicological profiles, new proteasome inhibitors with immunoproteasome selectivity have been developed. In 2009, the epoxyketone inhibitor PR-957 (later renamed ONX 0914) was described and characterized to be selective for LMP7 in a concentration-dependent manner 3. The molecular basis for this selectivity was explained after solving crystal structures of ONX 0914-soaked immunoproteasomes, showing that the LMP2 substrate pocket could theoretically favor ONX 0914 binding, but steric hindrance by Phe31 of LMP2 counteracts this 1. Hence, ONX 0914 was used as the prototype LMP7-selective inhibitor in many studies. LMP7 inhibition with ONX 0914, for example, protected from immunopathological damage in the brain after virus infection 4, exacerbated the pathogenesis of experimental systemic Candida albicans infection 5, protected from colitis-associated cancer formation 6, 7, and prevented several autoimmune diseases in pre-clinical mouse models 3, 8-14 (summarized in Ref. 13). At the cellular level, these effects were shown to involve two major pathways of disease development, namely cytokine secretion and T helper cell differentiation 15. The secretion of different proinflammatory cytokines from LPS-stimulated human PBMCs or mouse splenocytes as well as TCR-activated T cells was strongly suppressed by LMP7 inhibition with ONX 0914 3, 8, 10, 12, 16, 17. Additionally, ONX 0914 treatment prevented the differentiation of naïve T helper cells to polarized Th17 cells in vitro 3, 7, 14, 18. Hence, these findings propose the immunoproteasome to be an interesting therapeutic target for the treatment of inflammatory diseases. Therefore, the novel highly selective LMP7-specific inhibitor PRN1126 was developed. However, PRN1126, in contrast to ONX 0914, had no effect on cytokine secretion, Th17 differentiation, and autoimmune diseases. We demonstrated that prolonged exposure of cells with ONX 0914 led to inhibition of both LMP7 and LMP2. Combining the strictly LMP7-selective inhibitor PRN1126 with LMP2-specific inhibitors, we found that co-inhibition of both LMP2 and LMP7 is required to reduce IL-6 secretion and to impair Th17 differentiation. In vivo, only combined LMP2 and LMP7 inhibition could significantly ameliorate experimental colitis and inhibit EAE disease induction under dosing conditions where PRN1126 treatment alone was not efficacious. Results PRN1126 is an LMP7-specific inhibitor PRN1126 was discovered at Principia Biopharma during a medicinal chemistry effort focused on the discovery of inhibitors with selectivity for the immunoproteasome LMP7 subunit. PRN1126 is a reversible covalent inhibitor of LMP7, covalently anchoring via reversible interactions to Cys48 on LMP7 to enhance potency, selectivity, and prolonged target inhibition in immune cells (Figs 1A and EV1A). Figure 1. PRN1126 is selective for the chymotrypsin-like activity of the immunoproteasome A. Structure of PRN1126, a reversible covalent selective LMP7 inhibitor. B, C. Hydrolysis of fluorogenic substrates (Suc-LLVY-AMC) for the chymotrypsin-like activity of human (B) or mouse (C) 20S constitutive proteasome (CP) or 20S immunoproteasome (IP) at various PRN1126 concentrations. Data are presented as the means ± s.d. from quadruplicate assays. The highest fluorescence value was set to 100%. The experiments were repeated twice with similar results. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Extended LMP7 inhibition in human PBMC A. PBMCs were treated with PRN1126 at 1 μM for 1 h, washed to remove compound, and incubated in media for the time indicated. LMP7 inhibition was determined by hydrolysis of fluorogenic substrate (Suc-LLVY-AMC). Data points represent the mean ± s.d. (n = 3). B–G. Hydrolysis of fluorogenic substrates Bz-VGR-AMC for trypsin-like activity (B–E) or z-LLE-βNA for caspase-like activity (F, G) of human (B, C, F) or mouse (D, E, G) 20S constitutive proteasome (B, D, F, G) or immunoproteasome (C, E) at various concentrations of PRN1126 and MG132. Data are presented as the means of fluorescence ± s.d. from quadruplicate assays. The experiments were repeated three times with similar results. Download figure Download PowerPoint To further characterize the selectivity of PRN1126, fluorogenic peptide assays with purified proteasomes were performed. To this aim, human constitutive proteasomes (CPs) and human immunoproteasomes (IPs) were respectively purified from LCL721.174 (source of CP) or LCL721.145 (source of IP) lymphoblastoid cell lines 19. CP and IP were incubated with different concentrations of PRN1126 and assayed with a fluorogenic substrate specific for the chymotrypsin-like activity of the proteasome (Suc-LLVY-AMC; Fig 1B). PRN1126 showed IC50 values for the IP of 7.2 ± 1.3 × 10−9 M and 2.1 ± 0.8 × 10−7 M for the CP, and is therefore approximately 30 times more selective for the IP than for the CP. A similar experiment was performed with mouse proteasome. Immunoproteasomes were isolated from livers of BALB/c mice 8 days after infection with lymphocytic choriomeningitis virus (LCMV), and constitutive proteasomes were purified from livers of uninfected LMP7−/−/MECL-1−/− gene-targeted mice 1, 20. PRN1126 showed IC50 values for the mouse IP of 1.6 ± 0.3 × 10−8 M and 2.1 ± 0.3 × 10−7 M for the mouse CP, and is therefore at least 13 times more selective for the mouse IP than for the mouse CP (Fig 1C). Next, mouse and human immunoproteasome were incubated with PRN1126, and the trypsin-like activity was assessed with the fluorogenic substrate Bz-VGR-AMC (Fig EV1B–E). PRN1126 did not affect the trypsin-like activity below 10−6 M, indicating that PRN1126 does not inhibit β2c in the CP and MECL-1 in the IP, the proteolytically active subunits in the proteasome responsible for the trypsin-like activity. To investigate the impact of PRN1126 on the caspase-like activity, which is evoked by β1c, human and mouse CPs were incubated with different inhibitor concentrations and assayed with the fluorogenic substrate z-LLE-βNA (Fig EV1F and G). PRN1126 did not reduce the caspase-like activity. Taken together, PRN1126 is a potent and LMP7-selective inhibitor of the mouse and human immunoproteasome. Next, we tested the cell permeability of PRN1126. LCL721.174 (contain CP) or LCL721.145 (contain IP) cells were incubated with different concentrations of PRN1126 and assayed with the cell-permeable substrate MeO-Suc-GLF-AMC 21 specific for the chymotrypsin-like activity (Fig EV2A). PRN1126 inhibited the cleavage of this substrate in cells containing immunoproteasomes, demonstrating that PRN1126 is cell-permeable. Click here to expand this figure. Figure EV2. PRN1126 cell permeability and target occupancy of LMP7 or LMP2 in mice LCL721.174 cells expressing constitutive proteasomes (CP) or LCL721.145 cells expressing immunoproteasomes (IP) were treated with the indicated concentrations of the LMP7 inhibitor PRN1126. The chymotrypsin-like activity in the cells was determined by the hydrolysis of the cell-permeable fluorogenic proteasome substrate Meo-Suc-GLF-AMC. Depicted is the mean ± s.d. % of maximal hydrolytic activity of triplicate cultures. The highest fluorescence value was set to 100%. Mice were treated with a single dose of PRN1126 (40 mg/kg, s.c.) or vehicle. Spleens were harvested at the time points indicated, and the drug occupancy of LMP7 or LMP2 subunits was assessed in splenocytes with the ProCISE assay. Data points represent the means ± s.d. of three mice. Download figure Download PowerPoint PRN1126 affects the presentation of an LMP7-dependent epitope Since LMP7 plays an important role in major histocompatibility complex (MHC) class I-restricted antigen presentation, we investigated whether PRN1126 can alter the presentation of an LMP7-dependent MHC-I T-cell epitope. Therefore, the CTL epitope UTY246–254 (ubiquitously transcribed tetratricopeptide repeat gene, Y-linked), derived from the endogenously expressed Y chromosome-encoded HY-Ag, was chosen. It has been shown that the generation of the UTY246–254 epitope is strictly LMP7-dependent 22, 23. Furthermore, the presentation of the UTY246–254 epitope can be reduced by an LMP7-selective inhibitor (ONX 0914), but not an LMP2-selective inhibitor 3, 23. Male derived mouse splenocytes were incubated with PRN1126, and the UTY246–254 presentation on the H-2Db class I molecule was determined with an UTY246–254-specific T-cell hybridoma in lacZ assays (Fig 2A). The presentation of UTY246–254 was reduced in a dose-dependent manner by PRN1126, confirming previous results obtained with different inhibitors targeting LMP7 3, 19 and corroborating that PRN1126 is an LMP7-specific inhibitor. Figure 2. PRN1126 does not affect dextran sulfate sodium (DSS)-induced colitis or experimental autoimmune encephalomyelitis (EAE) A. Presentation of UTY246–254 on splenocytes after exposure to indicated concentrations of PRN1126. Data are presented as the mean absorbance ± s.d. of three replicate cultures. The experiment has been performed twice, yielding similar results. B. Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 mice treated with the indicated concentrations of PRN1126 overnight. Pooled data from three independent experiments (n = 9) are shown as the means of median fluorescence intensity ± s.e.m. All data were statistically compared to the DMSO-treated group. ***P < 0.001. One-way ANOVA. C, D. Splenocytes from C57BL/6 mice (C) or human PBMCs (D) were exposed (continuous treatment) to 300 nM PRN1126, or vehicle (DMSO), or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. (C) IL-6 concentrations are presented as mean and individual data points from triplicate wells. The experiment has been performed twice, yielding similar results. (D) Data are presented as single dots from three independent donors. The highest cytokine concentration was set to 100%. *P < 0.05. Unpaired Student's t-test. E. Colitis was induced by oral administration of 3% DSS for 5 days. Mice were treated daily (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. Data points represent mean ± s.e.m. of 15 mice pooled from three independent experiments. All data were statistically compared to the vehicle-treated group. *P < 0.05, ***P < 0.001. Two-way ANOVA. F. Mice were immunized with MOG35–55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated three times a week (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. All data were statistically compared to the vehicle-treated group. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA. Shown are the means of the clinical scores ± s.e.m. (n = 6 per group). The experiments have been performed twice, yielding similar results. Download figure Download PowerPoint PRN1126 does not reduce MHC class I cell surface expression and cytokine secretion LMP7-deficient mice display reduced levels of MHC-I surface expression 24, and MHC-I surface expression on splenocytes can be reduced with LMP7-selective inhibitors 3, 19. Therefore, splenocytes from wild-type C57BL/6 mice were incubated with 300 nM PRN1126 or with the previously described LMP7-selective inhibitor ONX 0914 3. Whereas the expression of the MHC-I molecule H-2Kb on splenocytes from wild-type mice was reduced with ONX 0914, no effect could be observed with PRN1126 (Fig 2B). Next, we investigated whether PRN1126 can alter cytokine secretion as previously reported for different LMP7-selective inhibitors 3, 12, 16, 19. Mouse splenocytes or human PBMCs were exposed to PRN1126 or ONX 0914, stimulated with LPS and IL-6 secretion into the supernatant was determined (Fig 2C and D). In contrast to ONX 0914, no influence on IL-6 secretion could be observed with PRN1126 up to a concentration of 300 nM. PRN1126 does not ameliorate DSS-induced colitis and EAE Targeting the immunoproteasome has been shown to protect mice from DSS-induced colitis 8 and to ameliorate experimental autoimmune encephalomyelitis (EAE) 12. DSS-induced colitis was elicited by oral administration of 3% DSS in the drinking water for 5 days. Mice were treated daily with 40 mg/kg PRN1126, a dose which inhibits LMP7 in vivo (as shown in naïve mice achieving 76, 48, and 0% LMP7 occupancy at 1, 6, and 14 h, respectively post-dose; Fig EV2B). Compared to vehicle-treated mice, a minor reduction in colitis-associated weight loss could be observed in mice treated with PRN1126 as expected based on intermittent target inhibition (Fig 2E). In contrast, ONX 0914 protected mice from colitis as previously observed 8. Similar to colitis, in EAE, a mouse model for multiples sclerosis, PRN1126 had no significant impact on the clinical manifestation of EAE when dosed intermittently (3×/week), while ONX 0914 delayed the onset and reduced the severity of disease symptoms (Fig 2F) with the same dosing schedule. In contrast to ONX 0914, PRN1126 is selective for LMP7 and does not inhibit LMP2 It has been reported previously that LMP7 inhibition alters MHC-I surface expression 3, reduces cytokine secretion 3, 12, 16, protects from experimental colitis 8, and ameliorates EAE 12 using the inhibitor ONX 0914, and this was further confirmed in this study (Fig 2). Nevertheless, PRN1126 alone had limited effect in these experiments, although PRN1126 was LMP7-selective as shown in Fig 1. These discrepant results posed the question how two LMP7-selective inhibitors can have such different effects in biological assays in vitro and in vivo? Therefore, we had a closer look at the activity profile of the two immunoproteasome inhibitors PRN1126 and ONX 0914. Ac-PAL-AMC is a fluorogenic peptide substrate exclusively cleaved by LMP2 23, 25. Mouse and human immunoproteasomes were incubated with different concentrations of PRN1126 and assayed with the fluorogenic substrate specific for LMP2 activity (Fig 3A). Whereas PRN1126 did not alter Ac-PAL-AMC cleavage up to 10 μM, ONX 0914 partially inhibited LMP2 starting at concentrations above 10 nM [human: IC50 1.1 ± 0.5 × 10−7 M (Fig 3A upper panel); mouse IC50 6.5 ± 0.5 × 10−8 M (Fig 3A lower panel)]. We have observed that binding of ONX 0914 to LMP7 and LMP2 leads to a shift of LMP7 and LMP2 bands to higher apparent molecular weights in Western blot. Incubation of enriched lymphocytes from mouse spleens with 300 nM ONX 0914 for 2 h led to an irreversible modification of almost all LMP2 molecules with ONX 0914 (Fig 3B). These results indicate that ONX 0914 is inhibiting both LMP7 and LMP2 at 300 nM in cells if they are incubated with ONX 0914 for extended periods of time. Therefore, we hypothesized that inhibition of both LMP7 and LMP2 might be required to induce broad anti-inflammatory effects in vitro and in vivo. To investigate this hypothesis, the LMP7-specific inhibitor PRN1126 was combined with the well-characterized LMP2-specific inhibitors LU-001i 19, 26 or ML604440 23, and inhibition of IL-6, T-cell differentiation, and therapeutic efficacy in mouse models of inflammatory bowel disease and multiple sclerosis was assessed. LU-001i is a pure LMP2-specific inhibitor not affecting other subunits of the CP or the IP 19, 26 and leads to a shift of LMP2 but not LMP7 bands to higher apparent molecular weights in Western blot (Fig EV3A). In contrast to the epoxyketone LU-001i, the dipeptide boronate ML604440 does not lead to a shift of immunoproteasome subunits bands to higher apparent molecular weights in Western blot (Fig EV3A). However, it has been demonstrated that ML604440 does not affect the chymotrypsin-like 23 and the trypsin-like activity (Fig EV3B–E) and is therefore an LMP2-specific inhibitor. Figure 3. ONX 0914 but not PRN1126 inhibits LMP2 Hydrolysis of fluorogenic substrates (Ac-PAL-AMC) for LMP2 activity of human (upper panel) or mouse (lower panel) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914. Data are presented as the means of fluorescence ± s.d. from quadruplicate assays. The experiments were repeated three times with similar results. Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914. Ficoll-enriched lymphocytes from C57BL/6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro. SDS–PAGE and immunoblotting against indicated proteins were performed. Shown are representative Western blots out of three independent experiments with similar outcome. Source data are available online for this figure. Source Data for Figure 3 [embr201846512-sup-0002-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Electrophoretic mobility shifts of IP subunits with different inhibitors A. Ficoll-enriched lymphocytes from C57BL/6 mice were treated with DMSO, 300 nM ONX 0914, 300 nM PRN1126, 300 nM LU-001i, or 300 nM ML604440 for 2 h in vitro. SDS–PAGE and immunoblotting against indicated proteins were performed. Shown are representative Western blots out of two independent experiments with similar outcome. B–E. Hydrolysis of the fluorogenic substrate Bz-VGR-AMC for trypsin-like activity of human (B, C) or mouse (D, E) 20S constitutive proteasome (B, D) or immunoproteasome (C, E) at various concentrations of ML604440 and MG132. Data are presented as the means of fluorescence ± s.d. from quadruplicate assays. The experiments were repeated three times with similar results. Source data are available online for this figure. Download figure Download PowerPoint A reduction of MHC class I surface expression requires the co-inhibition of LMP7 and LMP2 Splenocytes derived from wild-type or LMP7-deficient mice were incubated with 300 nM ONX 0914, 300 nM PRN1126, 300 nM ML604440, 300 nM LU-001i, 300 nM PRN1126 + 300 nM ML604440, or 300 nM PRN1126 + 300 nM LU-001i overnight. On the next day, the H-2Kb surface expression on wild-type or LMP7-deficient splenocytes was analyzed by flow cytometry (Fig 4). Whereas LMP2 inhibition with ML604440 or LU-001i alone or LMP7 inhibition with PRN1126 alone had no influence on the surface expression of H-2Kb, combined LMP2 and LMP7 inhibition (PRN1126 + ML604440 or PRN1126 + LU-001i) reduced H-2Kb expression to an extent, which was similar to that achieved with ONX 0914. Basal H-2Kb expression on LMP7−/− splenocytes, which is approximately 50% lower relative to wild-type levels, was not affected when LMP2 and LMP7 inhibitors were combined, thus confirming the specificity of the inhibitors. Figure 4. Co-inhibition of LMP7 and LMP2 reduces H-2Kb surface expression on splenocytesFlow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 wild-type (left side) or LMP7−/− (right side) mice treated with vehicle (DMSO), ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126 + ML604440 (300 nM each), or PRN1126 + LU-001i (300 nM each) overnight. Unstained splenocytes were used as negative control. Pooled data from three independent experiments (n = 9) are shown as the means of median fluorescence intensity ± s.e.m. All data were statistically compared to the DMSO-treated group. **P < 0.01, ***P < 0.001. One-way ANOVA. Download figure Download PowerPoint The reduction in IL-6 production depends on combined inhibition of LMP7 and LMP2 Next, we tested whether co-inhibition of LMP2 and LMP7 is also needed to alter cytokine secretion. Mouse splenocytes or human PBMCs from different healthy volunteers were incubated with 300 nM ONX 0914, 300 nM PRN1126, 300 nM ML604440, 300 nM LU-001i, 300 nM PRN1126 + 300 nM ML604440, or 300 nM PRN1126 + 300 nM LU-001i. After stimulation with LPS overnight, the concentration of IL-6 in the supernatant was determined by ELISA (Fig 5A and B). No significant inhibition of IL-6 secretion by mouse splenocytes (Fig 5A) or human PBMCs (Fig 5B) was observed when LMP7 (PRN1126) or LMP2 (ML604440, LU-001i) alone were targeted. In contrast, co-inhibition of LMP2 and LMP7 reduced IL-6 secretion to a similar degree as ONX 0914. This reduction in IL-6 secretion is not due to increased cell death, since splenocytes incubated overnight with LMP7 (PRN1126) and LMP2 (ML604440, LU-001i) inhibitors did not result in increased propidium iodide-positive cells (Fig EV4). Hence, these data indicate that the previously reported reduction in cytokine secretion of cells exposed to ONX 0914 was due to the co-inhibition of LMP2 and LMP7. Figure 5. The reduction in IL-6 production and the suppression of Th17 differentiation rely on the co-inhibition of LMP7 and LMP2 A, B. Splenocytes from C57BL/6 mice (A) or human PBMCs (B) were incubated (continuous treatment) with DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126 + ML604440 (300 nM each), or PRN1126 + LU-001i (300 nM each) and stimulated with LPS overnight. The IL-6 concentration in the supernatant was analyzed by ELISA. The highest IL-6 concentration from each experiment was set to 100%. (A) Data are presented as the mean ± s.e.m. of four independent experiments each measured in triplicates. (B) Data are presented as the mean ± s.e.m. of six different healthy donors. All data were statistically compared to the DMSO-treated group. ***P < 0.001. One-way ANOVA. C. The differentiation of CD4+ T cells isolated from the spleens of C57BL/6 mice that were exposed (continuous treatment) to DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (3
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