ONX-0914, a selective inhibitor of immunoproteasome, ameliorates experimental autoimmune myasthenia gravis by modulating humoral response

Accumulating evidence shows that the immunoproteasome participates in the immune response, beyond its initial role in the protein degradation. Here, we tested the effects of the selective immunoproteasome inhibitor, ONX-0914, on experimental autoimmune myasthenia gravis (EAMG). We found that ONX-0914 ameliorated the severity of ongoing EAMG by reducing the autoantibody affinity, accompanied with decreased Tfh cells and antigen presenting cells. Also it reduced the percentage of Th17 cells and inhibited the secretion of IL-17. Our data indicated ONX-0914 may bring benefit for MG therapy.

Myasthenia gravis (MG) is an antibody-mediated, T-cell-dependent and complement-involved autoimmune disease (Zuckerman et al., 2010). The production of function-blocking antibody against the acet- ylcholine receptor (AChR) at the neuromuscular junction (NMJ) leads to neuromuscular transmission failure, thereby triggering excessive muscle weakness and fatigue. EXperimental autoimmune myasthenia gravis (EAMG) of rat can be induced by immunization with torpedo acetylcholine receptor (TAChR) or a synthetic peptide corresponding tothe rat AChRα97-116 (AChR R97-116) region, which is a reliable model for investigating novel therapeutic strategies.Proteasomes, multisubunit proteases, are responsible for the de- gradation of many abnormal/misfolded proteins in every eukaryotic cell. Multiple cellular processes, such as cell adhesion (Read et al., 1995), transcription (Desterro et al., 2000), antigen processing for presentation on MHC-I (Kloetzel, 2001) and cell cycle control are as- sociated with the proteasome. In vertebrates, each catalytic subunit of the proteasome is encoded by two genes. Most cell types express the constitutive subunits (β1c, β2c, β5c), however, in dendritic cells (DCs),lymphocytes and monocyte-derived cells, the constitutive subunits are replaced by the immunosubunits(β1i,β2i,β5i) to form immunoproteasomes (Tanaka, 1994). Additionally, immunoprotea- somes can also be induced in most cell types after exposure to in- flammatory cytokines, such as interferon (IFN)-γ and tumour necrosisfactor (TNF)-α (Griffin et al., 1998). Accumulating evidence revealsthat apart from its role in antigen processing, immunoproteasomes contribute to the regulation of cytokine production (Muchamuel et al., 2009), the expansion and survival of T cells (Moebius et al., 2010) and the differentiation of T helper cell (Kalim et al., 2012).

Therefore, the ONX-0914, a newly developed selective inhibitor of the immunosubunitβ5i (also known as low–molecular mass polypeptide-7 (LMP7)), hasshown inspiring therapeutic effects in animal models of rheumatoid arthritis (Muchamuel et al., 2009), systemic lupus erythematosus (Ichikawa et al., 2012), multiple sclerosis (Basler et al., 2014), Ha- shimoto’s thyroiditis (Nagayama et al., 2012) and inflammatory bowel disease (Basler et al., 2010). According to these researches, selective inhibition of immunoproteasome subunit LMP7 could efficiently sup-press production of proinflammatory cytokines like IL-6, IFN-γ, TNF-α, GM-CSF and IL-23 (Basler et al., 2014; Muchamuel et al., 2009), sup-press T helper-1(Th1) and T helper-17 (Th17) but enhances regulatory T cell differentiation in vivo and in vitro by inhibiting the phosphor- ylation of STAT3 (Kalim et al., 2012), suppressing function of Toll-like receptor (TLR)-activated dendritic cells and reducing autoantibody- secreting cells (Ichikawa et al., 2012). However, whether ONX-0914 could bring benefit for MG is still unknown.In this study, we investigated the effects of ONX-0914 on the ex- perimental myasthenia gravis model of rat, focusing on its effects on the humoral immunity. We found that ONX-0914 inhibited antibody affi- nity maturation and mildly abated autoantibody production, amelio- rated the severity of ongoing EAMG accompanied with the decline of antigen presenting cells, follicular helper T cells (Tfh) and germinal center B cells. Our data indicate that ONX-0914 could be a choice for MG therapy.

2.Materials and methods
ONX-0914 was dissolved in 10% (w/v) sulfobutylether-b-cyclodex- trin and 10 mM sodium citrate (pH 6) and administered to rats as an i.v. bolus dose of 3.5 mg/kg (in a volume of 300 μl) every 3 days throughout the course of the experiment, starting from 5 days post immunization.For mononuclear cell (MNC) preparation, the spleen and bilateral inguinal lymph nodes of individual rat were harvested and grinded through the cell strainer (70 μm, biologiX, USA) in PBS on day 50 post immunization. Osmotic lysis method was used to deplete the ery- throcytes to get spleen MNC.Tfh cells were detected by surface staining of PE-anti-CD4 Formaldehyde-fiXed and paraffin embedded MG patient’s and (Biolegend, USA), PE-Cy7-anti-ICOS (Biolegend, USA), rabbit-anti- thymic cyst patient’s thymuse were retrieved from the Tai’an central hospital. Sections, 4 μm in thickness, were baked for 2 h, and then deparaffinized in xylepes and rehydrated in ethanol. Antigen retrieval was performed by boiling slides in citrate buffer (pH = 6.0) using the presser cooker for 2 min. Endogenous peroXidase activity was blockedby applying hydrogen peroXide 3%. 5% bovine serum albumin was used to deplete unspecific protein-protein interactions. After blocking, sec- tions were stained by monoclonal rabbit anti-rat/human Proteasome 20S LMP7 IgG (abcam, USA) and followed by Horseradish PeroXidase- labeled anti-rabbit IgG secondary antibody (ZSGB-BIO, Beijing, China). DAB PeroXidase substrate kit (ZSGB-BIO, Beijing, China) was used as CXCR5 and second antibody Alexa Fluor 488-goat-anti-rabbit IgG (both from Abcam, USA). Treg cells were identified by first surface staining with FITC-anti-CD4 (eBioscience, USA) and then after fiXation and permeabilization, Alexa Fluor 647-anti-foXp3 was applied for staining according to the manufacturer recommended. Spleen DC was char- acterized by extracellular staining of Alexa Fluor 647-anti-OX62 (Biolegend, USA).

And PE-anti-MHC II (eBioscience, USA), FITC-anti- MHC II (Biolegend, USA), PE-anti-CD80 (eBioscience, USA), FITC-anti- CD86 (eBioscience, USA) were used to detect its maturation phenotype. Fluorescein labeled Peanut Agglutinin (PNA) (Vector laboratory, USA), PE-Cyanine7-anti-B220 (eBioscience, USA), PE-anti-MHC II were used chromogen. Counterstaining was performed with hematoXylin and in lymph node MNC to gate GC B cells and MHC II+ cells. eosin. Olympus BX51 microscope was used to evaluate the slides.Female Lewis rats weighing 160–180 g (SiX to eight-week-old) were For Th-cell subtypes analysis, lymph node MNCs were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (BI, Israel), 1% penicillin–streptomycin (Hycolone, Logan, UT, USA) and cell stimula- tion cocktail plus protein transport inhibitors (eBioscience, USA) for 5 hin the incubator according to the product instruction. Then, MNCs were harvested and washed with PBS and stained by FITC-anti-CD4 antibody purchased from Vital River Laboratories (Beijing, China) and kept at the (eBioscience, USA). After that, cells were fiXed with 2% paraf- local animal house under specific pathogen-free conditions, with a 12/ 12-h light/dark cycle and standard rat chow and water ad libitum. All the experimental protocols met the guidelines of the Animal Ethics Committee of Shandong University. To achieve the scientific objectives, minimum number of rats necessary was used. R97-116 peptide (DGDFAIVKFTKVLLDYTGHI) was synthesized by ChinaPeptides Corporation (Shanghai, China). ONX-0914 was purchased in ApexBio (Houston, USA).EAMG rats were induced following the protocol as Baggi F et al. described (Baggi et al., 2004) with some modifications. Lewis rats were anesthetized by isoflurane and immunized subcutaneously at the base of tail with 200 μl inoculum containing 75 μg R97-116 peptide, 1 mg Mycobacterium tuberculosis (strain H37RA; Difco, Detroit, MI, USA) inincomplete Freund’s adjuvant (Sigma-Aldrich) on day 0 followed by a boost on day 30 only with the same peptide in incomplete Freund’s adjuvant.

Clinical scoring was based on the presence of tremor, hun- ched posture, muscle strength and fatigability evaluated as Baggi F described (Baggi et al., 2004) in a double-blind fashion. Fatigability was assessed after repetitive paw grips on the cage grid for 30s. Disease severity was expressed as follows: 0, normal; 1, mildly decreased ac- tivity and weak grip or cry, more evident after exercise; 2, more evident clinical signs (tremor, hunched posture, head down, weak grip) present before exercise; 3, severe generalized weakness, marked decrease in body weight, moribund; 4, dead. Intermediate signs were graded as 0.5, 1.5, 2.5 or 3.5. Results were expressed as the mean score for each group at each time point. ormaldehyde and permeabilized with permeabilization wash buffer (Biolegend, USA) and stained with APC-anti-IL-17A (eBioscience, USA), APC-anti-IFN-γ (eBioscience, USA), PE-IL-4 (Biolegend, USA). Finally,cells were analyzed with a flow cytometer.To measure the level of anti-R97-116 IgG, flat-bottomed polystyrene 96-well plates (Corning, USA) were coated with 100 μl R97-116 peptide (5 μg/ml) overnight at 4 °C. Then, the plates were blocked with 200 μl of PBS containing 0.05% Tween 20 and 10% FBS at room temperaturefor 2 h. After that, diluted serum (1:100) was added and incubated for 2 h at 37 °C, followed by biotin-labeled anti-rat IgG (1:2000; Biolegend) for 1 h at 37 °C and streptavidin–horseradish peroXidase (1:1000; Bios,Beijing, China) at 37 °C for 30 min. For washing every time, 300 μl PBSsupplemented with 0.05% Tween 20 was added.

Finally, added the tetramethylbenzidine (TMB) substrate (Tiangen Biotechnology, Beijing, China) and read the OD value at 450 nm using a microplate ELISA reader. Results expressed as optical density (OD values) ± standard deviation (SD).The relative affinity of serum anti-R97-116 antibodies was de- termined by ELISA using thiocyanate elution (Pullen et al., 1986). Briefly, 96-well plates were coated and blocked following the protocol described as above. Then, diluted serum with a predetermined amountof anti-97-116 antibody was added for 2 h at 37 °C. Then, 200 μl ofappropriate quantities of potassium thiocyanate (KSCN) were added and incubated at room temperature for 30 min. The amount of antibody left was measured as described above. The relative affinity is expressed as affinity index, equal to the molarity of KSCN resulting in 50% of the absorbance obtained in the absence of KSCN. Spleen MNC from either EAMG or ONX-0914 group rats (10^6/ml) were cultured in the presence of AchR R97-116 (10 μg/ml). After a 72 h incubation, supernatants were collected and evaluated by anti-rat IFN-γ ELISA kit (DAKEWE, China) and anti-rat IL-17 ELISA kit (eBioscience,USA) according to the manufacturer’s instructions. The analyses were performed in duplicate and the results are expressed as OD values ± SD.Statistical analysis was performed with GraphPad Prism 6. Differences between two groups were tested by two-tailed Student t- test. Results were displayed as means ± SD. The significance level was set at p < 0.05. 3.Results Compared with healthy humans, MG patients' thymuses especially young patients' are often associated with follicular hyperplasia and ectopic germinal centers (Truffault et al., 2017). Consistent with the previous reports that LMP7 is abundant in monocyte-derived cells, higher expression of LMP7 in the ectopic germinal center was demon- strated by immunohistochemistry in the thymus of a MG patient (Fig. 1D) compared to a normal thymus, although normal medullary thymic epithelial cells (mTECs) can also express immunoproteasome (Nil et al., 2004).ONX-0914 is a potent selective inhibitor of LMP7. According to a previous report, sharp decline of LMP7 activity occurred at doses ran- ging from 1 to 10 mg/kg without apparent disturbance in other im- munosubunits' activity in mice (the half-maximal inhibitory concentration < 1 mg/kg) (Muchamuel et al., 2009). And the effective administration interval ranged from one day to about one week (Basler et al., 2014). To investigate the effects of LMP7 inhibition on EAMG rats, after dose conversion, ONX-0914 was administered to rats as ani.v. bolus at dose of 3.5 mg/kg every three days from day 5 to day 50 post-immunization. Rats began to exhibit clinical signs on about day 32, and by day 40, most of the immunized rats exhibited EAMG manifes- tations at different degree. At the peak phase of the disease (on day 38, p.i.), ONX-0914 treated group exhibited a lower average clinical score (0.4 ± 0.49), compared to EAMG group (clinical score 1.4 ± 0.85) (p < 0.05) (Fig. 2).To investigate the effect of ONX-0914 treatment on antibody re- sponse, we measured the quantity and affinity of anti-R97-116 IgG. We found that ONX-0914 significantly reduced antibody on day 25 (Fig. 3A) p.i. However, after the second immunization at day 30, anti- R97-116 IgG rapidly increased and even returned to the same level as EAMG group on day 35 and persisted until day 50. Since antibody avidity was another functional property of autoantibody, we then checked it. Interestingly, we found the affinity of antibody against R97- 116 was significantly brought down on day 50, as shown in Fig. 3B (p < 0.05).Since follicular helper T (Tfh) cells are specialized effector T cells that stimulate B cells proliferation and differentiation in germinal center (GC) to produce high-affinity antibody (Crotty, 2014), we as- sessed the effect of LMP7 inhibitor treatment on them. As shown in Fig. 4, the percentage of Tfh cells among CD4+ cells in ONX-0914 group was lower than that in EAMG group (p < 0.05). Meanwhile, the percentage of GC-B cells in the ONX-0914 group, characterized by po- sitive peanut agglutinin (PNA) staining and B cell marker B220 staining (Shinall et al., 2000), was also reduced (p < 0.05 vs EAMG group) (Fig. 4B). To further investigate the mechanism underlying the effects of ONX- 0914 on EAMG, we assessed the antigen presenting cells (APCs) both in spleen and lymph nodes. Our data showed that ONX-0914 treatment significantly decreased the percentage of OX62+DC in the spleens (p < 0.01, Fig. 5A). However, the phenotypic analysis showed no difference in the expression of MHC class II, CD80 and CD86 on spleen DC (when gated on OX62) between these two groups (Fig. 5A), which meant ONX-0914 only reduced the number of dendritic cells while leaving their immunogenicity unchanged. As for lymph node, less MHC class II positive cells were found in ONX-0914 group than EAMG group (p < 0.001, Fig. 5B).We also examined the Th subset distribution following ONX-0914 treatment. The results showed no difference in the percentage of Th2 cells (CD4+IL-4+) and Treg cells (CD4+FoXp3+) between ONX-0914 group and EAMG group (data not shown). However, compared to EAMG group, the percentage of Th1 cells (CD4+IFN-γ+) in the lymphnodes was increased in ONX-0914 group (p < 0.05) (Fig. 6A). Meanwhile, the percentage of Th17 cells (CD4+IL-17+) in the spleen were reduced in ONX-0914 group (p < 0.05), consistent with the level of IL-17 secreted by spleen MNC after R97-116 stimulation for 3 days (p < 0.01) (Fig. 6B and C). 4.Discussion Proteasome inhibitor was originally used to eliminate multiple myeloma cells (Meister et al., 2007; Obeng et al., 2006). Then, re- searchers reported that pan-proteasome (both constitutive subunits and immunosubunits) inhibition could eliminate normal plasma cells in several humoral immunity-related diseases, including myasthenia gravis (Gomez et al., 2011; Neubert et al., 2008; Taddeo et al., 2015). Nevertheless, the fact that as high as 91.7% patients receiving pan- proteasome inhibitor bortezomib experienced adverse events like polyneuropathy, headache and fever, impedes its long-term usage (Alexander et al., 2015; Rampen et al., 2013). Selective inhibition of immunoproteasome subunits which are predominantly expressed in the immune effector cells, becomes a promising strategy. Although the role of immunoproteasome in the cell is not well known, it but not the constitutive proteasome regulates cytokine secretion (Muchamuel et al., 2009). And the positive effects on the differentiation of Th cells makes the immunoproteasomes an alternative choice in autoimmune diseases treatment (Kalim et al., 2012; Liu et al., 2017). The abundant LMP7expression in the germinal center as we show in Fig. 1 indicates that immunoproteasome may participate in the antibody response to some extent. The aim of this study was to identify the effect of ONX- 0914 (selective inhibitor of LMP7) on EAMG, a model of human hu- moral disease, myasthenia gravis. Results showed administration of ONX-0914 inhibited the development of ongoing EAMG, together with the decline of the relative affinity of anti-R97-116 IgG. Tfh cells and GC B cells in the draining lymph nodes were also found decreased. Furthermore, we found ONX-0914 efficiently eliminated MHC II+ cells in the lymph node and DCs in the spleen. As far as the Th cells, ONX-0914 was found to reduce the percentage of Th17 and the IL-17 secretion by the spleen MNC. Our data indicate that ONX-0914 treatment could modulate humoral immunity and may provide benefit for MG therapy. MG is a typical organ specific autoimmune disease mediated by anti-AchR IgG antibodies. High affinity and high titer of Anti-AchR antibody is essential for the degradation of the neuromuscular junction. Thus, we examined the levels of anti-R97-116 antibodies but also the relative affinity which determined the intensity of antibody-antigen complex. Data here showed that the level of anti-R97-116 IgG was only reduced on day 25 post immunization. After the booster immunization on day 30 p.i., it quickly rises to the same level as the control group on day 35 p.i. and persisted until day 50 p.i. This may suggest that ONX- 0914 cannot efficiently affect the memory B and T cells. Once resti- mulated by the same antigen, the memory B cells may rapidly differ- entiate into plasma cells and secrete lots of antibody. This's consistent with the observation that although pan-proteasome inhibitor efficiently depleted antibody secreting cells, it didn't affect their precursors, and once withdrawn, plasma cells were quickly regenerated and antibody increased (Alexander et al., 2015). However, in spite of the still high autoantibody level, the relative affinity of anti-R97-116 antibody was decreased significantly. As we known, the defective neuromuscular transmission in MG resulted from antibody-induced modulation, steric interference with acetylcholine binding or ionophoric function, or by directing the activities the complement cascade (Brown and Krolick, 1988). Any of these antibody-mediated disturbances might be influ- enced the quantity or quality of the anti-AChR antibody (Brown et al., 1988). The amount of anti-AChR autoantibody in the circulation was not the sole determining factor in MG (Lindstrom et al., 1976). As an important functional property of antibody, antibody avidity determines the intensity of autoantibody-AchR complex which leads to AchR blockade or receptor degradation (Vincent, 2002). In MG patients, the affinity of autoantibody was reported extremely high (Bray et al., 1982). It's also associated with EAMG clinical symptom as reported previously (Wang et al., 2015). We here attributed the therapeutic ef- fects mainly to the reduction of antibody avidity, which may reduce the stability of antigen-antibody complex and the disturbances of the post- synapse neuromuscular junction. Considering the help from Tfh cells is the limiting factor in affinity selection in germinal center (DeFranco, 2016), we further investigate the drug's effects on Tfh cells. Tfh cells, characterized by the expression of the chemokine receptor CXCR5 and the transcription factor B-cell lymphoma 6 (Bcl6), inducible T-cell costimulator (ICOS) were newly defined in CD4+ T cell subset (Breitfeld et al., 2000; Ma et al., 2012a). It's believed that only GC B cell with high affinity for antigen can ac- quire and present enough antigen on MHC class II molecules for re- cognition by Tfh cells (Natkanski et al., 2013). Once contacting, signals like interleukin-21 and CD40L from Tfh cell will support the high af- finity GC B cells to survive and proliferate (Gitlin et al., 2014; Victora et al., 2010). Our result showed the ONX-0914 administration reduced Tfh cells in the draining lymph node, and we believed the lack of Tfh cells intervened in the process of antibody affinity maturation. More- over, Tfh cells have been confirmed contributing to many antibody- mediated autoimmune disease (Ma et al., 2012b; Simpson et al., 2010). In MG patients, the CXCR5+CD4+T cells correlated with MG disease presentation via undefined mechanisms (Saito et al., 2005). In our study, with the improvement of symptoms in the ONX-0914 group, the percentages of Tfh cells declined, which is consistent with previous reports (Saito et al., 2005; Xie et al., 2013). However, an inevitable shortcoming here is no further investigation on the interleukin-21 se- cretion, for the lack of appropriate antibody.Data presented in this study demonstrated that following ONX-0914 treatment, DC in the spleen, GC B cells and MHC II+ cells in the lymph nodes were all decreased. However, a previous report did not observe any change in the number of DC, B cells and macrophages (Muchamuel et al., 2009). One explanation for this contradiction is the duration time of drug administration. The rats have received ONX-0914 over 45 days in our study, while only 5 days in their study. Another report that knockout of another immunoproteasome subunit LMP2 also found the decrease of splenic B cells (Hensley et al., 2010), may provides evidence for our results. Besides, DCs had also been shown to be sensitive to bortezomib, an inhibitor of constitutive and immunoproteasome (Hirai et al., 2011; Nencioni et al., 2006). It's believed that these APCs were sensitive to the selective LMP7 inhibitor in the long period of drug administration, for the constitutively expression of immunoprotea- somes within them. As we known, APCs play a crucial role in the humoral immunity. MHC II-dependent antigen presentation by both DCs and B cells is ne- cessary for optimal differentiation of Tfh cells and germinal center (Barnett et al., 2014). Tfh cell differentiation could be dived into priming and maintenance stages. At the priming stage, DC is sufficient to induce Bcl6 expression and initiate Tfh cell differentiation (Kerfoot et al., 2011). At the latter stage, B cells are generally required for maintaining functional Tfh cells. Deficiency of not only B cells but also MHC class II molecules on B cell, could result in the decreased number of Tfh cells (Shin et al., 2015). Although our data showed that ONX- 0914 only reduced the number of DC while leaving their im- munogenicity or tolerance unchanged, however, the reduced total MHC II+ expression following ONX-0914 treatment may account for the lack of Tfh cells for optimal germinal center response.Apart from Tfh cells, conventional CD4+ T cells have an important role in EAMG development and progression. Th1 cells and Th17 cells have been shown to play a critical role in EAMG development (Balasa et al., 1997; Mu et al., 2009), by the secretion of IFN-γ and IL-17 respectively. Treg cells are protective in EAMG (Aricha et al., 2008), while T helper 2 (Th2) cells are controversial (Milani et al., 2003; Wang et al., 2007). To explore the effects of ONX-0914 on the differentiation of T cells, we analyzed the percentages of IFN-γ+ and IL-17+ cells among CD4+ cells both in lymph nodes and spleens. Our data showed that in the spleen, ONX-0914 decreased the percentage of Th17 cells and the secretion of IL-17, but, in the lymph nodes, increased the percentage of Th1 cells, without influencing Th2 and Treg cells. It seemed in conflict with the previous report that LMP7 inhibition not only suppressed Th1 and Th17 but also enhanced Treg cell differ- entiation (Kalim et al., 2012) in vitro and in colitis model. In line with our data, Liu H et al. recently also reported no significant effects on the differentiation of Th1,Th2 and Treg cells following ONX-0914 (pre- viously called PR-957) treatment on experimental autoimmune neuritis, but Th17 cells were significantly decreased (Liu et al., 2017). It's re- ported that ONX-0914 down-regulates the phosphorylation of STAT3 and directly suppresses Th17 cell differentiation (Liu et al., 2017). Since the differentiation of Th1 and Th17 were antagonistic (Weaver et al., 2007), IFN-γ inhibits Th17 differentiation and IL-17A suppresses the expansion of Th1 cells (Pan et al., 2014; Zhou et al., 2009), the increased Th1 cells may root in the direct decline of IL-17 secretion. Meanwhile, according to the greater role of IL-17 than IFN-γ in assisting B cells to prolife and form germinal center (Subbarayal et al., 2016), the integrated effects of ONX-0914 may be positive for the humoral im- munity. In conclusion, our data confirmed that the immunoproteasome in- hibitor ONX-0914 can ameliorate the severity of ongoing EAMG by inhibiting antibody affinity maturation, decreasing Tfh cells, antigen presenting cells and GC B cells. ONX-0914 also reduced the percentage of Th17 cells and the secretion of IL-17. These data suggest that the immunoproteasome inhibitor ONX-0914 may be a choice for the MG therapy, but more studies would be PR-957 needed.