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• W2912267252 abstract "Chronic antibody-mediated rejection is the leading cause of allograft dysfunction and loss after kidney transplantation, and current immunosuppressive regimens fail to target the plasma cells that produce alloantibodies. We previously showed that treatment with the immunoproteasome inhibitor ONX 0914 prevented the expansion of plasma cells and prevented chronic allograft nephropathy and organ failure after kidney transplantation in rats, but the mechanism has remained elusive. In the current study, we confirmed a long-term reduction in alloantibody production and improvements in allograft histology in rats treated with ONX 0914 or with the broad-spectrum proteasome inhibitor bortezomib. Plasma cells from allotransplanted rats expressed immunoproteasomes at high levels. Immunoproteasome inhibition with ONX 0914 led to ubiquitin-conjugate accumulation, activation of the unfolded protein response, and induction of apoptosis in plasma cells. In addition, ONX 0914 suppressed the expression of adhesion molecules (VLA-4 and LFA-1), plasma cell survival factors (APRIL and IL-6), and IFN-γ−inducible chemokines in bone marrow, while the APRIL receptor BCMA, the IL-6 receptor, and the chemokine receptors CXCR4 and CXCR3 were down-regulated on plasma cells. Taken together, immunoproteasome inhibition blocked alloantibody production by inducing apoptosis of plasma cells through activating the unfolded protein response and suppressing plasma cell survival factors in the bone marrow. Chronic antibody-mediated rejection is the leading cause of allograft dysfunction and loss after kidney transplantation, and current immunosuppressive regimens fail to target the plasma cells that produce alloantibodies. We previously showed that treatment with the immunoproteasome inhibitor ONX 0914 prevented the expansion of plasma cells and prevented chronic allograft nephropathy and organ failure after kidney transplantation in rats, but the mechanism has remained elusive. In the current study, we confirmed a long-term reduction in alloantibody production and improvements in allograft histology in rats treated with ONX 0914 or with the broad-spectrum proteasome inhibitor bortezomib. Plasma cells from allotransplanted rats expressed immunoproteasomes at high levels. Immunoproteasome inhibition with ONX 0914 led to ubiquitin-conjugate accumulation, activation of the unfolded protein response, and induction of apoptosis in plasma cells. In addition, ONX 0914 suppressed the expression of adhesion molecules (VLA-4 and LFA-1), plasma cell survival factors (APRIL and IL-6), and IFN-γ−inducible chemokines in bone marrow, while the APRIL receptor BCMA, the IL-6 receptor, and the chemokine receptors CXCR4 and CXCR3 were down-regulated on plasma cells. Taken together, immunoproteasome inhibition blocked alloantibody production by inducing apoptosis of plasma cells through activating the unfolded protein response and suppressing plasma cell survival factors in the bone marrow. Kidney transplantation has become the gold-standard therapy for patients with end-stage renal function failure. In past decades, T cell–mediated acute rejection after kidney transplantation has been effectively inhibited by immunosuppressive agents such as immunophilin inhibitors and antilymphocyte/cytokine antibodies. Nevertheless, one-half of renal allotransplants are lost over the course of 10 years after transplantation,1Serrano O.K. Kandaswamy R. Gillingham K. et al.Rapid discontinuation of prednisone in kidney transplant recipients: 15-year outcomes from the University of Minnesota.Transplantation. 2017; 101: 2590-2598Crossref PubMed Scopus (24) Google Scholar mainly because of donor-specific alloantibody-mediated chronic rejection.2Colvin R.B. Smith R.N. Antibody-mediated organ-allograft rejection.Nat Rev Immunol. 2005; 5: 807-817Crossref PubMed Scopus (394) Google Scholar Currently no effective therapeutic approaches exist to prevent antibody-mediated chronic rejection because they fail to deplete plasma cells (PCs) that produce allo-antibodies.3Ejaz N.S. Alloway R.R. Halleck F. et al.Review of bortezomib treatment of antibody-mediated rejection in renal transplantation.Antioxidants Redox Signaling. 2014; 21: 2401-2418Crossref PubMed Scopus (35) Google Scholar Bortezomib, a broad-spectrum proteasome inhibitor applied in multiple myeloma therapy, can reduce alloantibody production via induction of PC depletion in patients with kidney transplantation3Ejaz N.S. Alloway R.R. Halleck F. et al.Review of bortezomib treatment of antibody-mediated rejection in renal transplantation.Antioxidants Redox Signaling. 2014; 21: 2401-2418Crossref PubMed Scopus (35) Google Scholar, 4Everly M.J. Everly J.J. Susskind B. et al.Bortezomib provides effective therapy for antibody- and cell-mediated acute rejection.Transplantation. 2008; 86: 1754-1761Crossref PubMed Scopus (330) Google Scholar, 5Perry D.K. Burns J.M. Pollinger H.S. et al.Proteasome inhibition causes apoptosis of normal human plasma cells preventing alloantibody production.Am J Transplantation. 2009; 9: 201-209Crossref PubMed Scopus (265) Google Scholar, 6Walsh R.C. Everly J.J. Brailey P. et al.Proteasome inhibitor-based primary therapy for antibody-mediated renal allograft rejection.Transplantation. 2010; 89: 277-284Crossref PubMed Scopus (153) Google Scholar and improve the pathologic status of transplanted kidneys in human patients and rats.7Vogelbacher R. Meister S. Guckel E. et al.Bortezomib and sirolimus inhibit the chronic active antibody-mediated rejection in experimental renal transplantation in the rat.Nephrol Dialysis Transplantation. 2010; 25: 3764-3773Crossref PubMed Scopus (43) Google Scholar However, bortezomib inhibits all types of proteasomes expressed in human tissues.8Huber E.M. Basler M. Schwab R. et al.Immuno- and constitutive proteasome crystal structures reveal differences in substrate and inhibitor specificity.Cell. 2012; 148: 727-738Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 9Livneh I. Cohen-Kaplan V. Cohen-Rosenzweig C. et al.The life cycle of the 26S proteasome: from birth, through regulation and function, and onto its death.Cell Res. 2016; 26: 869-885Crossref PubMed Scopus (195) Google Scholar Because the ubiquitin-proteasome system regulates virtually all pathways of cell biology via targeted protein degradation,10Hershko A. The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle.Angew Chem Int Ed. 2005; 44: 5932-5943Crossref PubMed Scopus (86) Google Scholar, 11Sledz P. Baumeister W. Structure-driven developments of 26S proteasome inhibitors.Ann Rev Pharmacol Toxicol. 2016; 56: 191-209Crossref PubMed Scopus (19) Google Scholar nonselective proteasome inhibition causes severe adverse effects, thus limiting its clinical applicability.12Schlafer D. Shah K.S. Panjic E.H. et al.Safety of proteasome inhibitors for treatment of multiple myeloma.Exp Opin Drug Safety. 2017; 16: 1-17Crossref PubMed Scopus (4) Google Scholar, 13Schmidt N. Alloway R.R. Walsh R.C. et al.Prospective evaluation of the toxicity profile of proteasome inhibitor-based therapy in renal transplant candidates and recipients.Transplantation. 2012; 94: 352-361Crossref PubMed Scopus (35) Google Scholar Consequently, selective inhibition of proteasomes in immune cells involved in chronic allograft rejection would be a more attractive alternative. Immunoproteasomes bearing the catalytic β-type subunits β1i (LMP2), β2i (MECL-1), and β5i (LMP7) are steadily expressed in many immune cell types and in inflamed tissues that are exposed to interferon (IFN)-γ or tumor necrosis factor (TNF).14Groettrup M. Kirk C.J. Basler M. Proteasomes in immune cells: more than peptide producers?.Nat Rev Immunol. 2010; 10: 72-77Crossref Scopus (240) Google Scholar Apart from its function in antigen presentation, the immunoproteasome plays a pivotal role in the pathogenesis of several autoimmune diseases.15Basler M. Dajee M. Moll C. et al.Prevention of experimental colitis by a selective inhibitor of the immunoproteasome.J Immunol. 2010; 185: 634-641Crossref PubMed Scopus (181) Google Scholar, 16Basler M. Mundt S. Muchamuel T. et al.Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis.EMBO Mol Med. 2014; 6: 226-238Crossref PubMed Scopus (123) Google Scholar, 17Ichikawa H.T. Conley T. Muchamuel T. et al.Beneficial effect of novel proteasome inhibitors in murine lupus via dual inhibition of type I interferon and autoantibody-secreting cells.Arthritis Rheumatism. 2012; 64: 493-503Crossref PubMed Scopus (198) Google Scholar, 18Liu R.T. Zhang P. Yang C.L. et al.ONX-0914, a selective inhibitor of immunoproteasome, ameliorates experimental autoimmune myasthenia gravis by modulating humoral response.J Neuroimmunol. 2017; 311: 71-78Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 19Muchamuel T. Basler M. Aujay M.A. et al.A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis.Nat Med. 2009; 15: 781-787Crossref PubMed Scopus (445) Google Scholar, 20Nagayama Y. Nakahara M. Shimamura M. et al.Prophylactic and therapeutic efficacies of a selective inhibitor of the immunoproteasome for Hashimoto's thyroiditis, but not for Graves’ hyperthyroidism, in mice.Clin Exp Immunol. 2012; 168: 268-273Crossref PubMed Scopus (41) Google Scholar The inhibition of the immunoproteasome subunit LMP7 with the peptide-epoxyketone inhibitor ONX 0914 (formerly named PR-957) interfered not only with antibody-mediated autoimmune diseases such as systemic lupus erythematosus17Ichikawa H.T. Conley T. Muchamuel T. et al.Beneficial effect of novel proteasome inhibitors in murine lupus via dual inhibition of type I interferon and autoantibody-secreting cells.Arthritis Rheumatism. 2012; 64: 493-503Crossref PubMed Scopus (198) Google Scholar and myasthenia gravis18Liu R.T. Zhang P. Yang C.L. et al.ONX-0914, a selective inhibitor of immunoproteasome, ameliorates experimental autoimmune myasthenia gravis by modulating humoral response.J Neuroimmunol. 2017; 311: 71-78Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar but also with chronic antibody-mediated rejection of allotransplanted kidneys in rats.21Li J. Basler M. Alvarez G. et al.Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation.Kidney Int. 2018; 93: 670-680Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar In a recent study, we showed that ONX 0914 treatment prevented the rejection of rat donor kidneys of the F344 strain by allogeneic Lewis recipient rats.21Li J. Basler M. Alvarez G. et al.Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation.Kidney Int. 2018; 93: 670-680Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar The mechanism of how ONX 0914 achieved this therapeutic success was not addressed in the previous study. However, it was found that ONX 0914 treatment lowered the plasma levels of donor-specific alloantibodies and IgG deposition in the kidney allografts, as well as the number of CD45R–CD138+ PCs in spleen and bone marrow of the recipients. Investigating the molecular pathways of how immunoproteasome inhibition affects PC survival is rendered difficult by the low numbers of PCs that can be recovered from the spleen (approximately 40,000) and bone marrow (approximately 15,000) of mice. In this study we exploited the high numbers of PCs that can be retrieved from allotransplanted rats (2 million per animal) in order to study the consequences of LMP7 inhibition. We found that immunoproteasome inhibition in vivo caused accumulation of polyubiquitin conjugates, activation of the unfolded protein response (UPR), and apoptosis in PCs. Moreover, crucial adhesion molecules, survival factors, and chemokines for PCs in bone marrow were downregulated by ONX 0914 treatment. Together, these findings reveal the mechanistic basis of how immunoproteasome inhibition leads to the depletion of PCs and thereby to the reduction of alloantibodies and the preservation of allotransplanted organs. We previously showed that ONX 0914 treatment reduced donor-specific alloantibody production in allogeneic recipient rats during the fourth and fifth week after kidney transplantation.21Li J. Basler M. Alvarez G. et al.Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation.Kidney Int. 2018; 93: 670-680Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar However, we were unable to trace the long-term production of alloantibodies because in the previous study the right kidney of allogeneic recipient rats was resected after transplantation. As a consequence, all vehicle-treated recipients died from renal allograft function failure in the fifth week after transplantation. Therefore, we now kept the right kidney in recipient rats and treated allogeneic recipient rats with ONX 0914 (5 mg/kg) or bortezomib (0.2 mg/kg) for 7 weeks from the third week until the tenth week after transplantation. We found that after 10 weeks of kidney transplantation, ONX 0914 and bortezomib treatment still maintained low donor-specific alloantibody levels for IgG1, IgG2a, IgG2b, and IgG3 in recipient rats in contrast to vehicle treatment (Figure 1). Because the donor-specific alloantibodies were reduced by ONX 0914 treatment, we also examined the antibody-mediated immune response in renal grafts. First, immunofluorescence staining showed that IgG was significantly deposited along tubular epithelia in renal allografts after 10 weeks of transplantation. Double staining for IgG and cell death with use of terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) staining revealed that deposition of IgG increased the death of glomerular and tubular cells in allograft kidney compared with isograft kidney (Figure 2a). Treatment of allorecipients with ONX 0914 (5 mg/kg) or bortezomib (0.2 mg/kg) for 7 weeks reduced IgG deposition and the number of TUNEL+ cells (Figure 2). In addition, immunohistochemistry showed that complement C4d was significantly deposited and CD11c+ dendritic cells and NK1.1+ natural killer cells had infiltrated into allografts to an enhanced extent (Figure 3). However, after a decrease in IgG deposition, treatment with ONX 0914 or bortezomib for 7 weeks also inhibited deposition of C4d and infiltration of dendritic cells and natural killer cells in renal allografts (Figure 3).Figure 3ONX 0914 treatment suppresses the inflammatory response in renal allografts. (a) Immunohistochemistry staining of C4d, CD11c, and NK1.1 in different renal grafts. Complement C4d was significantly deposited and CD11c+ dendritic cells and natural killer (NK) cells significantly infiltrated into renal allografts after 10 weeks of transplantation in contrast to renal isograft. Treatment of allogeneic recipients with ONX 0914 (5 mg/kg) or bortezomib (0.2 mg/kg) for 7 weeks from the third week after transplantation onward remarkably suppressed deposition of complement C4d and infiltration of dendritic cells and NK cells in renal allografts. Data are representative images of each group (n = 5) from 3 separate experiments. Brown color indicates positive staining. Bars = 50 μm. (b) Quantitation for relative C4d-positive area and percentage of CD11c+ dendritic cells and NK1.1 cells in different renal grafts according to immunohistochemistry staining. Data are shown as individual spots per animal with the mean of each group (n = 5) from 3 separate experiments. P values are indicated in each graph. Statistical analysis was performed by Student-Newman-Keuls test after 1-way analysis of variance. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We previously showed that chronic graft nephropathy, which is characterized by glomerular sclerosis, interstitial fibrosis, and interstitial arteriosclerosis, is alleviated by ONX 0914 treatment.21Li J. Basler M. Alvarez G. et al.Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation.Kidney Int. 2018; 93: 670-680Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Here, we also investigated 2 other characteristic pathologic changes of renal allografts, that is, glomerular basement membrane (GBM) thickening and peritubular capillary basement membrane (PTC-BM) multilayers by transmission electron microscopy. At 10 weeks after transplantation, GBM thickening, fusion of podocyte foot processes, and PTC-BM multilayers obviously occurred in renal allografts compared with isografts. Treatment with ONX 0914 or bortezomib for 7 weeks significantly inhibited GBM thickening, fusion of podocyte foot processes, and PTC-BM multilayers in kidney allografts (Figure 4a–c). Next we investigated proteinuria as an early indicator of renal dysfunction. We collected urine at 5 weeks after transplantation to avoid anuria caused by loss of renal function later on. Treatment of recipient rats with ONX 0914 or bortezomib for 2 weeks from the third week after transplantation onward significantly reduced albuminuria that appeared in vehicle-treated recipient rats (Figure 4d). LMP7 inhibition by ONX 0914 has reduced proinflammatory cytokine production from T cells in vitro and in mouse models of autoimmunity and fungal infection.16Basler M. Mundt S. Muchamuel T. et al.Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis.EMBO Mol Med. 2014; 6: 226-238Crossref PubMed Scopus (123) Google Scholar, 19Muchamuel T. Basler M. Aujay M.A. et al.A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis.Nat Med. 2009; 15: 781-787Crossref PubMed Scopus (445) Google Scholar, 22Basler M. Groettrup M. Immunoproteasome-specific inhibitors and their application.Meth Mol Biol. 2012; 832: 391-401Crossref PubMed Scopus (15) Google Scholar, 23Kalim K.W. Basler M. Kirk C.J. et al.Immunoproteasome subunit LMP7 deficiency and inhibition suppresses Th1 and Th17 but enhances regulatory T cell differentiation.J Immunol. 2012; 189: 4182-4193Crossref PubMed Scopus (103) Google Scholar, 24Mundt S. Basler M. Buerger S. et al.Inhibiting the immunoproteasome exacerbates the pathogenesis of systemic Candida albicans infection in mice.Sci Rep. 2016; 6: 19434Crossref PubMed Scopus (29) Google Scholar Here, we show that the concentrations of TNF and IFN-γ significantly increased in sera of allogeneic recipients after kidney transplantation. Remarkably, treatment with ONX 0914 or bortezomib significantly reduced the TNF and IFN-γ concentrations in sera of allogeneic recipients (Figure 5). To investigate how immunoproteasome inhibition prevents chronic antibody-mediated renal rejection, we studied how ONX 0914 reduces donor-specific alloantibodies in recipient rats after kidney transplantation. Previously, we demonstrated that ONX 0914 reduced PC numbers in allogeneic recipients.21Li J. Basler M. Alvarez G. et al.Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation.Kidney Int. 2018; 93: 670-680Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar To further explore the underlying mechanisms, we magnetically isolated CD45R–CD138+ PCs from allogeneic recipient rats treated with either vehicle or ONX 0914 to a purity of more than 90% (Figure 6a–c). Double staining of these PCs with annexin V and propidium iodide showed that treatment of recipients with ONX 0914 significantly induced apoptosis of PCs in contrast to treatment with vehicle (Figure 6d–e). To investigate how ONX 0914 induced apoptosis of PCs in allogeneic recipients, we first determined the expression levels of LMP2 (β1i) and LMP7 (β5i), as well as their constitutive counterparts β1 and β5, by Western blot analysis. Through comparison with the Epstein-Barr virus–transformed lymphoblastoid cell line LCL721.145,25Kavathas P. Bach F.H. DeMars R. Gamma ray-induced loss of expression of HLA and glyoxalase I alleles in lymphoblastoid cells.Proc Natl Acad Sci U S A. 1980; 77: 4251-4255Crossref PubMed Scopus (223) Google Scholar which almost exclusively expresses immunoproteasomes,26Schmidtke G, Schregle R, Alvarez G, et al. The 20S immunoproteasome and constitutive proteasome bind with the same affinity to PA28αβ and equally degrade FAT10 [e-pub ahead of print]. Mol Immunol. https://doi.org/10.1016/j.molimm.2017.11.030. Accessed November 30, 2018.Google Scholar and its LMP2/LMP7 doubly deficient mutant LCL721.174, we found that PCs from vehicle-treated recipients expressed very high levels of immunoproteasomes and low levels of constitutive proteasomes (Figure 7a). Interestingly, the immunoproteasome expression in PCs was significantly lowered by ONX 0914 treatment, consistent with suppressed levels of IFN-γ and TNF in the serum (Figure 5) and in kidney allografts.21Li J. Basler M. Alvarez G. et al.Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation.Kidney Int. 2018; 93: 670-680Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar To better quantitate the influence of ONX 0914 on immunoproteasome and constitutive proteasome contents in PCs, their expression levels were normalized to the 721.145 and 721.174 cells, respectively. In contrast to vehicle treatment, ONX 0914 treatment significantly reduced LMP2 expression from 71% to 48% and reduced LMP7 expression from 54% to 31% in PCs from allogeneic recipients (Figure 7b and 7c). Because the immunoproteasome was the predominant proteasome type in PCs, we tested by Western blotting whether ONX 0914 treatment would affect the degradation of polyubiquitin conjugates in PCs. Indeed, we found that in PCs from ONX 0914-treated allorecipients, polyubiquitin conjugates significantly accumulated compared with PCs from vehicle-treated recipients (Figure 7d). The accumulation of misfolded proteins triggers the UPR,27Walter P. Ron D. The unfolded protein response: from stress pathway to homeostatic regulation.Science. 2011; 334: 1081-1086Crossref PubMed Scopus (3914) Google Scholar and bortezomib has been reported to deplete PCs by activating the UPR in a mouse lupus nephritis model, at least according to mRNA analysis.28Neubert K. Meister S. Moser K. et al.The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis.Nat Med. 2008; 14: 748-755Crossref PubMed Scopus (481) Google Scholar Consistent with accumulation of polyubiquitin conjugates in PCs by immunoproteasome inhibition, we found that the expression of UPR signaling molecules, sliced X-box binding protein-1, activating transcription factor 4, and C/EBP-homologous protein were upregulated in PCs (Figure 7e). These data provide strong evidence that ONX 0914 treatment in allogeneic recipients activated the UPR in PCs in vivo. As a consequent downstream effect, cleaved caspase-3 expression was increased in PCs (Figure 7f), indicating the activation of apoptosis in PCs from allogeneic recipients. Survival of long-lived PCs depends on survival niches in the bone marrow.29Roth K. Oehme L. Zehentmeier S. et al.Tracking plasma cell differentiation and survival.Cytometry A. 2014; 85: 15-24Crossref PubMed Scopus (30) Google Scholar, 30Tellier J. Kallies A. Finding a home for plasma cells--a niche to survive.Eur J Immunol. 2014; 44: 2243-2246Crossref PubMed Scopus (6) Google Scholar Hence, we investigated whether immunoproteasome inhibition affected the PCs’ survival niche in the bone marrow. Western blotting showed that treatment of allogeneic recipients with ONX 0914 significantly inhibited expression of adhesion molecules required for PCs’ residence in the bone marrow niche: integrin α4/β1 (very late antigen-4 [VLA-4]) and integrin β2/CD11a (lymphocyte function–associated antigen-1 [LFA-1]) were strongly reduced in bone marrow cells from ONX 0914-treated recipients compared with vehicle-treated recipients (Figure 8a). In addition, ONX 0914 treatment suppressed expression of a proliferation-inducing ligand (APRIL) and interleukin (IL)-6, which are pivotal PC prosurvival factors expressed by nonplasma cells of the bone marrow (Figure 8b), corresponding to the finding that the percentage of eosinophils, one of the main APRIL and IL-6-producing cell types in bone marrow, was significantly reduced by ONX 0914 treatment (Figure 8c and d). Furthermore, because ONX 0914 treatment systemically diminished IFN-γ (Figure 5), the IFN-γ–inducible, PC-attracting chemokines CXCL9 (monokine induced by IFN-γ), CXCL10 (IFN-induced protein [IP]-10), and CXCL-11 (IFN-inducible T-cell α chemoattractant) in bone marrow also were reduced by ONX 0914 treatment in allorecipients (Figure 8e). Unfortunately, we failed to measure the chemokine CXCL12 in bone marrow cells. Nevertheless, we could show that the expression of CXCR3, CXCR4, B-cell maturation antigen (BCMA), and IL-6 receptor (IL-6R), which are the corresponding receptors of CXCL9-11, CXCL12, APRIL, and IL-6, was significantly downregulated in PCs from allogeneic recipients 7 weeks after the onset of ONX 0914 treatment (Figure 9a and b ). Consequently, the expression of the anti-apoptotic protein Mcl-1 was strongly downregulated in PCs from allogeneic recipients upon ONX 0914 treatment (Figure 9c). Taken together, ONX 0914 interfered with expression of survival cues in the PC niche, which probably contributed to the ONX 0914–dependent reduction in PC numbers.Figure 9ONX 0914 treatment reduces the cell surface expression of receptors for prosurvival factors on plasma cells from kidney allograft recipients. Treatment with ONX 0914 (5 mg/kg) of allogeneic recipient rats for 7 weeks from the third week after transplantation onward downregulated the expression of CXCR3, CXCR4, BCMA, and interleukin (IL)-6R on plasma cells. (a) Representative flow cytometry histograms of CXCR3, CXCR4, B-cell maturation antigen (BCMA), and IL-6R on plasma cells from different recipient rats. Data are representative histograms for each group (n = 4) from 3 separate experiments. (b) Quantitation of the mean fluorescence intensity (MFI) of CXCR3, CXCR4, BCMA, and IL-6R on plasma cells from different recipient rats according to flow cytometry. Data are shown as individual spots per animal with the mean of each group (n = 4) from 3 separate experiments. (c) Representative Western blots (left panel) and quantitation (right panel) for Mcl-1 expression in plasma cells from different renal allograft recipients. Treatment with ONX 0914 (5 mg/kg) in allogeneic recipient rats for 7 weeks starting at the third week after transplantation reduced the expression of the antiapoptotic protein Mcl-1 in plasma cells. Densitometric analysis (right panel) of Mcl-1 expression normalized to β-actin. Data are shown as individual spots per animal with mean of each group (n = 4) from 3 separate experiments. P values are indicated in the graphs. Statistical analysis was performed by 2-tailed unpaired Student t-test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study we investigated the mechanistic basis of why the immunoproteasome holds great potential as a drug target to interfere with chronic antibody-mediated rejection of allotransplants. Probably as a result of the specific expression of immunoproteasomes in immune cells and inflamed tissues,31Kremer M. Henn A. Kolb C. et al.Reduced immunoproteasome formation and accumulation of immunoproteasomal precursors in the brains of lymphocytic choriomeningitis virus-infected mice.J Immunol. 2010; 185: 5549-5560Crossref PubMed Scopus (52) Google Scholar, 32Stohwasser R. Standera S. Peters I. et al.Molecular cloning of the mouse proteasome subunits MC14 and MECL-1: reciprocally regulated tissue expression of interferon-γ- modulated proteasome subunits.Eur J Immunol. 1997; 27: 1182-1187Crossref PubMed Scopus (54) Google Scholar LMP7 inhibition did not cause overt adverse effects when it was used to prevent chronic antibody-mediated rejection in this study and a previous study.21Li J. Basler M. Alvarez G. et al.Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation.Kidney Int. 2018; 93: 670-680Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar This expression profile raises the question of which type of the immunoproteasome-rich leukocytes must be inhibited to suppress chronic renal allograft rejection. The residing of PCs in renal allografts results in acute rejection as a result of acute interstitial nephritis, probably through local antibody secretion, whereas the chronic antibody-mediated rejection is caused by late systemic production of antibodies by PCs in immune organs. Hence, as the main producers of alloantibodies after transplantation, PCs move into focus. Nevertheless, most likely because of limitations in available cell numbers, the immunoproteasome protein content of primary PCs from spleen or bone marrow of rats, mice, or humans previously has not been determined. Here we made use of the elevated PC numbers in allotransplanted rats to show that immunoproteasome subunits LMP2 and LMP7, both of which contribute to the chymotrypsin-like activity of immunoproteasomes, are very highly expres" @default.
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• W2912267252 title "Immunoproteasome inhibition induces plasma cell apoptosis and preserves kidney allografts by activating the unfolded protein response and suppressing plasma cell survival factors" @default.
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