Arthritis & Rheumatism
Novel Proteasome Inhibitors Have a Beneficial Effect in
Murine Lupus via the dual inhibition of Type I Interferon
and autoantibody secreting cells
Arthritis and Rheumatism
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Journal:
Manuscript ID:
Wiley - Manuscript type:
Complete List of Authors:
Full Length
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Date Submitted by the
Author:
ar-11-0412.R3
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Ichikawa, H. Travis; University of Rochester Medical Center, Department
of Medicine- Division of Allergy, Immunology & Rheumatology
Conley, Thomas; University of Rochester Medical Center, Department of
Medicine- Division of Allergy, Immunology & Rheumatology
Muchamuel, Tony; Onyx Pharmaceuticals
Jiang, Jing; Onyx Pharmaceuticals
Lee, Susan; Onyx Pharmaceuticals
Owen, Teresa; University of Rochester Medical Center, Department of
Medicine- Division of Allergy, Immunology & Rheumatology
Barnard, Jennifer; University of Rochester Medical Center, Department of
Medicine- Division of Allergy, Immunology & Rheumatology
Nevarez, Sarah; University of Rochester Medical Center, Department of
Medicine- Division of Allergy, Immunology & Rheumatology
Goldman, Bruce; University of Rochester Medical Center, Pathology &
Laboratory Medicine
Kirk, Christopher; Onyx Pharmaceuticals
Looney, Richard J.; University of Rochester Medical Center, Department
of Medicine- Division of Allergy, Immunology & Rheumatology
Anolik, Jennifer; University of Rochester Medical Center, Department of
Medicine- Division of Allergy, Immunology & Rheumatology
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Keywords:
Interferon, Dendritic Cells, Systemic lupus erythematosus (SLE),
Autoimmune Diseases
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Arthritis & Rheumatism
Novel Proteasome Inhibitors Have a Beneficial Effect in Murine Lupus via the dual
inhibition of Type I Interferon and autoantibody secreting cells
Running Title: Novel Proteasome Inhibitors inhibit interferon activation in Lupus
H. Travis Ichikawa*, Thomas Conley*, Tony Muchamuel†, Jing Jiang†, Susan Lee†,
Teresa Owen*, Jennifer Barnard*, Sarah Nevarez*, Bruce I. Goldman‡, Christopher J.
Kirk†, R. John Looney*, and Jennifer H. Anolik*
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*
Department of Medicine- Division of Allergy, Immunology & Rheumatology,
University of Rochester Medical Center, Rochester, New York; †Onyx Pharmaceuticals,
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Inc., South San Francisco, CA, ‡Pathology & Laboratory Medicine, University of
Rochester Medical Center, Rochester, New York
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Erythematosus, autoimmunity
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Key words: interferon, plasmacytoid dendritic cells, plasma cells, Systemic Lupus
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Dr. Anolik has been supported by the Lupus Research Institute and National Institutes of
Health Grants R01AI077674-01A and a grant from Onyx Pharmaceuticals, Inc. Tony
Muchamuel, Jing Jiang, Susan Lee, and Christopher Kirk are employed by Onyx
Pharmaceuticals, Inc.
Correspondence and reprint requests: Jennifer Anolik, MD, PhD, University of
Rochester School of Medicine, Box 695, 601 Elmwood Avenue, Rochester, NY 14642
Phone: 585-275-1632; Fax: 585-442-3214; e-mail: jennifer_anolik@urmc.rochester.edu
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ABSTRACT
Objective: We postulated that proteasome inhibition (PI) may be useful in the treatment
of SLE by targeting plasmacytoid dendritic cells (pDCs) and plasma cells (PCs), both
critical to disease pathogenesis.
Methods: Lupus prone mice were treated with the non-selective PIs carfilzomib and
bortezomib, the LMP7-selective immunoproteasome inhibitor ONX 0914, or vehicle
control. Tissues were harvested and analyzed by flow cytometry using standard markers.
Nephritis was monitored by proteinuria and kidney harvest. Serum anti-dsDNA levels
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were measured by ELISA and total IgG and dsDNA antibody secreting cells (ASC) by
ELIspot. Human PBMCs or mouse bone marrow cells were incubated with TLR agonists
and PIs and interferon α measured by ELISA and flow cytometry.
Results: Early treatment of lupus prone mice with the dual targeting PIs carfilzomib or
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bortezomib or the immunoproteasome specific inhibitor ONX 0914 prevented disease
progression, and treatment of mice with established disease dramatically abrogated
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nephritis. Treatment had profound effects on plasma cells with greater reductions in
autoreactive than total IgG ASCs, an effect that became more pronounced with prolonged
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treatment, and was reflected in decreasing serum autoantibodies. Remarkably,
proteasome inhibition efficiently suppressed production of interferon α by toll-like
receptor activated pDCs in vitro and in vivo, an effect mediated by both an inhibition of
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pDC survival and function.
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Conclusions: Inhibition of the immunoproteasome is equally efficacious to dual targeting
agents in preventing lupus disease progression by targeting two critical pathways in
disease pathogenesis, type I interferon activation and autoantibody production by plasma
cells.
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Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by
dysregulation in multiple arms of the immune system and the production of hallmark
autoantibodies and α interferon. Because this disease continues to be associated with
significant morbidity and mortality, there is great interest in the development of new and
targeted treatment approaches and correspondingly a better understanding of disease
pathogenesis (1). Although several open-label studies of B cell depletion (BCD) as a
targeted treatment have demonstrated clinical benefit (2), only a minority of SLE patients
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have lasting clinical responses (3, 4). Moreover, the recent failure of two large
randomized trials of BCD in SLE (5) highlights the need for other therapeutic strategies.
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In particular, anti-CD20 has variable impact on autoantibodies that are produced by
CD20 negative plasma cells. Decreasing autoantibodies may be particularly critical in
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SLE where they are often directly pathogenic, e.g. antibody induction of cytopenias and
stimulate α interferon.
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antibodies to DNA or DNA- and RNA-binding proteins forming immune complexes that
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Bortezomib is a proteasome inhibitor (PI) that effectively kills plasma cells and has
demonstrated success in the treatment of multiple myeloma (6-8). Recent promising
results suggest that PIs are an effective therapy in a murine model of lupus (9), with
relatively selective induction of the unfolded protein response in plasma cells, killing of
both long lived and short lived plasma cells, and elimination of autoantibodies. Despite
the success of bortezomib in the clinic and in this pre-clinical lupus model, its use in
human autoimmune disease is limited because of the development of painful neuropathy
in >30% of patients (10).
Thus, there is great interest in developing alternative
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proteasome inhibitors that are less toxic. Carfilzomib is an irreversible proteasome
inhibitor that, like bortezomib, primarily inhibits the chymotrypsin-like active sites of the
ubiquitously expressed constitutive proteasome and the immunoproteasome, which is
found predominantly in immune effector cells (11, 12).
In contrast to bortezomib,
clinical trials of carfilzomib have shown a low rate of peripheral neuropathy (13).
Recently, it has also been shown that the immunoproteasome and not the constitutive
proteasome regulates cytokine production in human peripheral blood mononuclear cells
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(PBMCs) and that selective inhibition of the immunoproteasome with the irreversible
inhibitor ONX 0914 (formerly PR-957) is efficacious in both anti-collagen antibody-
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induced arthritis and collagen-induced arthritis models in mice (14).
Given that bortezomib and other PIs have been shown to block activation of myeloid
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dendritic cells (mDCs) by Toll-like receptor 4 (TLR) in vitro (15) we postulated that pDC
activation via TLR signaling, a pathway that is known to be triggered via immune
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complex (IC) binding and lead to the production of large quantities of IFN-α, may be
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similarly blocked by the proteasome inhibitors (16-19). Here, we demonstrate for the first
time that proteasome inhibitors target both plasmacytoid dendritic cells producing α
interferon and plasma cells producing pathogenic antibodies, creating a powerful
synergistic effect in the treatment of SLE.
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MATERIALS AND METHODS
Mice and experimental design
NZB/NZWF1, MRL/lpr, and C57BL/6 mice were supplied by Jackson
Laboratories. All mice were housed in the animal facility in the University of Rochester
Medical Center or Onyx Pharmaceuticals. All experiment protocols were reviewed and
approved by the University of Rochester or Onyx Committee on Animal Resources.
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NZB/NZWF1 mice with established nephritis (24-30 weeks with durable proteinuria ≥2+
proteinuria or 100 mg/dl) (female) and female MRL/lpr mice (10 weeks) were treated
with the indicated proteasome inhibitors intravenously via tail vein route, bortezomib
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(0.50-0.75 mg/kg D1D3), carfilzomib (3-5 mg/kg D1D2), ONX 0914 (10 mg/kg QOD x
3 or 20 mg/kg QW) or vehicle solution. Proteinuria was monitored weekly once or twice
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with urine dipstick (Uristix by Bayer). Spleen, peripheral blood and bone marrow
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lymphocytes were collected after sacrifice for flow cytometry and ELISPOT analysis.
Kidneys and a portion of spleen were collected for histological analysis.
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ELISA assay
Sera from PI treated mice blood samples were collected and frozen prior to quantifying
total IgG antibody levels as described (20). Serum was also used for anti-dsDNA analysis
following the manufacturer’s instructions (Alpha Diagnostic International). Animal
identification and treatment information were blinded for all ELISA samples. A
proteasome active site ELISA was utilized for proteasome analysis and to monitor
inhibition of constitutive and immunoproteasome active sites in PBMC, pDCs and tissue
samples from treated animals, and was performed as described previously (21).
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Flow cytometry analysis
FITC-CD21, biotin-CD23, PE-CD23, PE-CD1d, APC-B220, FITC-B220, APCAA4.1,biotin-IgM, FITC-IgM, biotin-CD5 (BD Biosciences) and PE-CD11b (BD
Biosciences) were used for B cell subset identification. APC-CD4 (BD Biosciences) and
biotin-CD69 (BD Biosciences) were used for T cell analysis. PE-CD11b (BD
Biosciences) and FITC-CD11c (BD Biosciences) were used as dendritic cell markers.
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Plasma cells were identified as k (kappa) light chain+ and CD138+ among the
lymphocyte gated cells. Samples were run on a FACSCanto. All antibodies were
Histological analysis
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purchased from eBioscience except where indicated.
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Kidneys of mice from different treatment groups and control mice were fixed in 10%
formalin and paraffin embedded or frozen. Kidney sections (4 mm) were stained with
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hematoxylin and eosin. Pathology was analyzed and scored in a blinded fashion (B.I.G.).
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Briefly, the severity of glomerular, interstitial, and vascular lesions was determined on a
scale of 0 to 4+. Multiple sections at a minimum of two different levels were examined,
with each section typically containing >50 glomeruli and >25 blood vessels as described.
Spleen sections were frozen and cut into 4 mm sections. Immunohistochemistry slides
were stained for FDC-M1 (BD Bioscience), PE-B220, and Biotin-GL7 using a Dako
LSAB2 kit. 3-color fluorescent slides were stained for FITC-MOMA (ABD serotec),
AMCA-IgM (Vector labs), ALEXA647-IgD or -FITC-B220, BIOTIN-GL7 followed by
SA-PE, and APC-CD4 (BD Bioscience) and sections quantitated as described (20).
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Detection of antibody-secreting cells by enzyme-linked immunosorbent spot assay
Anti-dsDNA and total IgG secreting cells were detected as previously described (20).
Briefly, Millipore were coated with poly–L-lysine (Sigma) and calf thymus DNA
(Sigma), blocked with 2% fetal calf serum in PBS, and spleen or BM cell suspensions
incubated as serial dilutions starting with 5E5 cells/well overnight at 37 oC. After
incubation, plates were washed and incubated with alkaline phosphatase–conjugated goat
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antibody to mouse IgG (Jackson) for 1 h at RT and detected with Vector Blue Alkaline
Phosphatase Substrate Kit III (Burlingame, CA). For assessment of IgG-secreting cells,
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serial dilutions starting with 1E5 cells/well were incubated in goat anti-mouse IgG coated
plates (Southern Biotech). The developed spots were measured by ImmunoSpot 5.0
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(Cellular Technology Ltd, Shaker Heights, OH).
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Inhibition of IFN-α by proteasome inhibitors and pDC counts
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Mouse bone marrow cells were cultured in RPMI complete medium at 0.25 million per
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200 ul in 96 flat bottom culture plate over night in the presence of 500 ng/ml CpG (2216,
Invitrogen) with or without PIs. Human PMBCs were cultured in serum free media
(invitrogen) containing 1ng/mL GM-CSF (Sigma) and 50 U/mL IFN-α (Sigma) at 2.5 x
106 cells/ml with DMSO control or proteasome inhibitor. After 1 h incubation, cells were
washed twice with media and cultured in media containing CpG2216. Cells were
harvested 24 h after initial treatment. IFN-α production in the culture supernatants was
measured by commercial ELISA (Mouse Interferon Alpha ELISA Kit or human ELISA
kit PBL interferon source, Piscataway, NJ) following the manufacturer’s instructions. In
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separate experiments mouse cells were harvested and stained for B220, CD11b, CD11c
and Ly6C to count pDC numbers by flow cytometry analysis. In some experiments
intracellular cytokine staining for IFN-α was performed (Fix & Perm Medium A and B;
Invitrogen) with GolgiPlug added during the final 3 h of the culture (BD Bioscience).
RNA analysis
Total RNA was extracted from mouse splenocytes using the Qiagen RNeasy Mini kit
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(Qiagen, Valenica, CA) and reverse-transcribed into cDNA using the iScript cDNA
synthesis kit (Bio-Rad, Hercules, CA). Mx1 expression was defined in duplicate or
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triplicate real-time quantitative reverse transcriptase-polymerase chain reactions using the
Rotor-Gene 3000 thermal cycler (Corbett Life Science, Sydney, New South Wales,
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Australia) with SYBR Green I (Applied Biosystems, Foster City, CA) for detection of
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DNA synthesis. Primers were as described (22) with normalization to the β2microglobulin housekeeping gene and the following cycling conditions: 95°C for 4
Statistical analysis
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60 seconds, and 72°C for 15 seconds.
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minutes followed by 50 cycles of 95°C for 15 seconds, 60°C (annealing temperature) for
Student’s t-test or non-parametric Mann-Whitney U was used for comparison between
treatment groups. Chi-squared test was performed on protein survival data. Significance
is based on a value of p<0.05.
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RESULTS
Novel proteasome inhibitors prevent nephritis progression in Lupus prone mice
To evaluate the ability of carfilzomib and ONX 0914 to prevent lupus nephritis, 10 weekold female MRL/lpr mice were treated for 13 weeks. Both carfilzomib and ONX 0914
inhibited progression of nephritis to a similar level as bortezomib (Fig. 1a left panel and
supplemental data). High levels of proteinuria (100 mg/dl) were observed in all the
vehicle treated mice by the end of the treatment, whereas less than 20% of treated mice
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reached this level of proteinuria (Fig. 1a right panel). Similarly, NZB/NZW F1 mice with
established nephritis (2+ proteinuria) showed a halt in disease progression (Fig. 1a, right).
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There was also a significant decrease in the severity of glomerulonephritis (GN) and
interstitial inflammation after treatment with ONX 0914 (p=0.03 and 0.003, respectively)
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or bortezomib (p=0.001 and 0.002, respectively). The impact of carfilzomib was less
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marked achieving significance only for GN (p=0.05) (Fig 1b). In contrast, the control
group displayed severe GN with crescents, necrosis, and mesangial hypercellularity and
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massive interstitial nephritis (Fig. 1b, left).
Serum anti-dsDNA IgG levels were lowered by carfilzomib and ONX 0914 treatments to
a level comparable to that of bortizomib treated mice (Fig 1c). The total IgG levels were
also significantly reduced by bortezomib and ONX 0914. Although carfilzomib had
effects on total IgG levels early in treatment, this effect became less pronounced over
time. This may be because the maximally tolerated dose for carfilzomib in the mouse
results in less inhibition of LMP7 (50 – 60%) relative to ONX 0914 and bortezomib
(≥80%) (data not shown). Taken together, these data support the hypotheses that
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proteasome inhibition, including selective inhibition of the immunoproteasome, results in
therapeutic improvement in mouse models of SLE.
Elimination of plasma cells and germinal center cells in Lupus prone mice by proteasome
inhibition
It has been previously demonstrated that bortezomib decreases plasma cell numbers in
the spleen and bone marrow of lupus prone mice (9). Furthermore, we have
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demonstrated that carfilzomib and ONX 0914 reduce both anti-dsDNA and total IgG
levels in the sera of treated animals. Therefore, we measured the impact of proteasome
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inhibition on plasma cell numbers in spleen and bone marrow of 23-week old MRL/lpr
mice after treatment with bortezomib (0.5 mg/kg), carfilzomib (3 mg/kg), ONX 0914 (10
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mg/kg), or vehicle solution for 13 weeks. Plasma cells decreased in bortezomib treated
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animals by 90% and 95% in both spleen and bone marrow, respectively. Carfilzomib and
ONX 0914 treatment also decreased plasma cell numbers although not as markedly, by
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50% and 65% in spleen and bone marrow, respectively (Fig. 2a). In the NZB/W F1 mice
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model, splenic plasma cell numbers also decreased approximately 80% after 8 weektreatment of established nephritis with ONX 0914 (20 mg/kg) (Fig. 2d).
Although flow cytometric analysis is sensitive for assessing numbers of plasma cells, it
does not allow analysis of sub-populations of antigen specific antibody secreting cells
(ASC). Thus, total IgG and anti-dsDNA IgG ASC numbers were measured by ELISPOT
assay in 24 week-old MRL/lpr mice that were treated with bortezomib (0.5 mg/kg),
carfilzomib (3 mg/kg), ONX 0914 (10 mg/kg) and vehicle solution for 13 weeks (Fig.
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2b). In spleen, the level of decrease in total IgG ASC numbers was highest with
bortezomib (74%) followed by carfilzomib (17%) and ONX 0914 (15%). The level of
decrease in anti-dsDNA IgG ASC numbers in spleen was even more pronounced:
bortezomib (87%), ONX 0914 (44%), and carfilzomib (39%) (Fig. 2b). In bone marrow,
the level of decrease in total IgG ASC number was highest with bortezomib (63%)
followed by ONX 0914 (44%) and carfilzomib (43%), again with more pronounced
declines in anti-dsDNA IgG ASC number [bortezomib (91%), ONX 0914 (79%) and
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carfilzomib (64%)] (Fig. 2b). Thus, it appears that proteasome inhibition preferentially
affects autoantibody secreting plasma cells. The impact on the plasma cell compartment
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was confirmed by histologic analysis of spleen, where decreases in both numbers and size
of IgM and IgG bright plasma cells were observed with proteasome inhibitor treatment
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(Fig. 2c). Therefore, proteasome inhibition not only eliminates plasma cells but also may
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impact their antibody secreting capacity or preferentially inhibit high antibody secretors.
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Given that lupus prone NZB/W F1 mice develop germinal centers (GC) spontaneously as
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they age, GC cells (B220+, GL-7+) were enumerated in mouse spleen after 8 weeks of
treatment with ONX 0914 (20 mg/kg) and compared to vehicle treated control mice. The
GC cells in the spleen of treated mice were 78% lower in numbers compared to the
control, and no GC formation was observed histologically (Fig. 2d). Therefore, one
explanation for the lower numbers of plasma cells found in spleen and bone marrow after
long-term treatment with proteasome inhibition is a reduction in the de novo synthesis of
plasma cells in GC reactions.
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Another pathway for reduction of the plasma cell compartment is direct killing of preexisting plasma cells. As an initial assessment of the in vivo plasma cell killing by
proteasome inhibition, we treated mice for a short-term (one week) during which the de
novo synthesis of GC experienced plasma cells should be negligible. Plasma cell
numbers and ASCs were enumerated by flow cytometry and ELISPOT analysis as
previously described and were reduced (supplemental Fig. 2). As with long-term
treatment, the effects of proteasome inhibition on autoreactive ASCs were more marked
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than for total IgG ASCs (supplemental Fig. 2). Although GC cells from spleen were
reduced modestly with ONX 0914 treatment there was no apparent reduction with
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carfilzomib treatment (data not shown).
Proteasome inhibitors preferentially target active autoantibody secreting cells
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To more firmly establish that PIs directly and preferentially kill anti-dsDNA ASCs, we
examined their effects on antibody secretion in vitro. When PIs were added in titrating
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amount to ELISPOT wells, the spot numbers of total IgG ASC did not decrease (Fig. 3a
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and b). However, the size distribution of spots changed from random to small size spots
(Fig. 3a and b), suggesting that more active IgG ASCs are more sensitive to proteasome
inhibiton (Fig. 3d, left shift from vehicle). In contrast, both the numbers and size of antidsDNA ASC spots decreased as proteasome inhibitor concentrations increased (Fig. 3a
and c). The most plausible explanation for this result is that the active anti-dsDNA ASCs
are more sensitive to proteasome inhibition, with either preferential killing and/or
inhibition of antibody secretion activity.
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Proteasome inhibitors abrogate IFN-α production in vitro
One of the signature pathogenic cytokines found in lupus patients and mice is IFN-α
produced in large quantities by pDCs. Given that PIs have been demonstrated to have
effects on the production of other pro-inflammatory cytokines by a variety of immune
cells, and pDCs effectively become factories for production of IFN-α, we postulated that
one of the important effects of proteasome inhibition in lupus may be abrogation of the
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IFN-α signature. First, we tested if PIs prevent the production of IFN-α by cells
stimulated ex vivo via TLR (CpG) activation. Remarkably, all three PIs significantly
suppressed the production of IFN-α by mouse BM cells in a concentration dependent
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manner (Fig. 4a). To evaluate the impact of selective immunoproteasome inhibition, we
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compared the IFN-α production in CpG stimulated bone marrow cells exposed to ONX
0914 or PR-893 at concentrations resulting in selective inhibition of LMP7 or β5,
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respectively (Fig. 4b). LMP7 inhibition blocked production of IFN-α by over 90%
whereas selective inhibition of β5 did not alter cytokine release. Bortezomib and
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carfilzomib, which have dual inhibition of β5 and LMP7, also abrogated IFN-α
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production.
Next, we examined the impact of proteasome inhibition on IFN-α production by human
PBMCs. Similar to the mouse studies, LMP7 inhibition blocked cytokine production
(Fig. 5a). Of note, this occurred with multiple TLR ligands, including TLR9 (CpG) and
TLR3 agonists, at multiple agonist concentrations (Fig. 5a and supplemental data). A
similar result was observed with purified pDCs, the cell responsible for the largest
production of IFN-α (data not shown). Moreover, analysis of purified pDCs revealed
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that over 95% of their proteasome activity is mediated by the immunoproteasome (Fig.
5b).
Proteasome inhibitors block IFN-α production in vivo
The in vivo effects of proteasome inhibition were assessed using bone marrow cells from
treated NZB/W F1 mice activated ex vivo by CpG. Both bortezomib and ONX 0914
reduced IFN-α production by approximately 75%, whereas carfilzomib caused a 40%
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reduction (Fig. 4c). These results demonstrate that proteasome inhibition can suppress
the production of IFN-α in lupus. As further demonstration of the in vivo relevance of
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this inhibition to the disease process, we observed a significant decrease in the expression
of the interferon-inducible gene Mx1 in spleen cells from NZB/W F1 mice treated with
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the proteasome inhibitors (Fig. 4d).
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To further define the mechanisms of this effect, we determined whether pDCs were
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altered in numbers after in vivo or in vitro treatment with PIs. Notably, the fractions of
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pDCs in bone marrow were similar after in vivo treatment (Fig. 6a), as were absolute
numbers (vehicle treated mice 97.3 x 103 +21.5 vs. PI treated mice 98.8 x 103+29.2).
However, upon in vitro stimulation there were moderate decreases in pDC numbers in a
PI concentration dependent fashion. Notably, IFN producing pDCs appeared to be
particularly susceptible to proteasome inhibitor induced cell death (Fig. 6c). Given the
presence of residual pDCs at PI concentrations that completely abrogate IFN production,
it is likely that proteasome inhibition suppresses pDC function as well via inhibition of
IFN production and/or secretion.
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DISCUSSION
In this report we present evidence that novel proteasome inhibitors, including selective
targeting of the immunoproteasome, are remarkably efficacious in the treatment of
murine lupus via a dual inhibition pathogenic IFN-α production and autoreactive plasma
cells. Thus, the levels of serum total IgG and anti-dsDNA IgG antibody declined during
treatment and correlated with a decrease in plasma cell numbers in spleen and bone
marrow even after short-term treatment. Of note, autoreactive plasma cells were more
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sensitive to immunoproteasome inhibition both in vivo and in vitro. The data presented
also show for the first time a unique role for the immunoproteasome in TLR driven
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interferon α production given that LMP7 (ONX 0914) but not β5 inhibition blocked
cytokine production in TLR stimulated murine bone marrow cells and human PBMCs
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and decreased IFN inducible gene expression in vivo. Overall, these results demonstrate
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the synergistic effects of proteasome inhibition in lupus, with unique targeting of
pathogenic IFN-α production, and provide strong rationale for the clinical development
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of these agents.
The elimination of plasma cells producing pathogenic autoantibodies observed here may
be mediated by multiple pathways, including direct elimination, altered survival, or
impaired generation. Given the rapid in vivo effects of proteasome inhibition on the
plasma cell compartment and demonstration of in vitro elimination of anti-dsDNA
secreting plasma cells, we favor a direct action on PCs as part of the effect. This is in
accord with prior data in the literature demonstrating that proteasome inhibition induces
apoptosis of multiple myeloma cells (12) and other antibody secreting PCs (9).
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Terminally differentiated PCs may be particularly sensitive to the effects of proteasome
inhibition since they produce antibodies rapidly with accumulation of misfolded peptides
and induction of an unfolded protein response (UPR) (23), which leads to activation of
proapoptotic proteins and caspases and ultimately apoptosis (24). The proteasome is the
key catalytic machinery that degrades misfolded peptides, and indeed modulation of
proteasome expression and activity within the PC compartment may be one mechanism
regulating PC survival (25). Our observations that autoreactive PCs were preferentially
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targeted by proteasome inhibition, as evidenced by the more pronounced reductions of
anti-dsDNA antibody secreting cells in vivo in spleen and bone marrow as well as their
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direct elimination in vitro, may be explained by a higher rate of antibody synthesis and
secretion (23). Bortezomib was previously demonstrated to strongly and relatively
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specifically activate the UPR in PCs in a murine lupus model, with depletion of both long
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and short-lived PCs (9). Our data extends these observations to novel PIs, including
immunoproteasome targeting, an important advance given that the toxicity profile of
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bortezomib may limit its development in autoimmune diseases.
The observed pronounced decrease in autoreactive PCs with in vivo treatment may in
part be because a higher fraction of anti-dsDNA PCs are short-lived and continually
generated. One of the key events in early PC differentiation is activation of a
transcription factor XBP-1 (26). Proteasome inhibition has been reported to inhibit the
auto-phosphorylation of IRE1alpha, leading to inhibition of XBP-1 activation and
induction of apoptosis without activation of UPR in myeloma cell lines (27). Therefore,
it is tempting to speculate that autoreactive plasmablasts may be highly sensitive to PIs
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due to the dependency on XBP-1 activity. Proteasome inhibition may also impair PC
survival by altering the micro-environmental milieu. It is clear that PCs can have variable
life-spans with long lived PCs maintaining protective serum antibodies for years
(reviewed in (28)). Some of the factors important for the long life of PCs include IL-6,
BAFF, APRIL, and TNF as well as chemokines such as CXCL12 found in bone marrow
and inflamed tissues. This milieu may be disrupted in lupus, contributing to the longevity
of autoreactive PCs (29). Interestingly, at least two key cytokines contributing to the
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plasma cell niche, TNF and IL-6, have been demonstrated to be dependent upon LMP7
(21).
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A particularly novel aspect of our manuscript is the demonstration that TLR induced
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IFN-α production is completely abrogated by proteasome inhibition. Evidence
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supporting a prominent role for Type I interferon activation in SLE includes
demonstration of serum elevations among patients with active SLE (30), the more recent
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demonstration that high IFN levels are a heritable risk factor for SLE (31), induction of
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autoimmunity with IFN-α treatment of malignancy and hepatitis C (32, 33), and the
presence of an IFN-α gene expression signature in human SLE (17, 34). In murine SLE,
the demonstration of a Type I IFN signature has been more variable. Thus, in some
studies type I IFN has actually been found to exert a protective effect in MRL/lpr mice
but a detrimental effect in NZB/W mice (35). Moreover, a prominent Type I IFN
signature is found in NZB/W, but is more variable in MRL/lpr mice (36, 37). Although
the mechanistic importance of PI targeting of the IFN pathway to the efficacy of these
drugs in lupus is strongly supported by our demonstration of a decrease in IFN inducible
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gene expression in NZB/W mice in vivo, the contribution of this inhibition to the
beneficial clinical effects in the MRL/lpr mouse remains unclear.
Plasmacytoid dendritic cells (pDCs) are typically the major source of IFN-α production.
In SLE activation occurs via immune complex binding and costimulation of TLRs (TLR7, -8, or-9) and FcRs on pDCs (38). In our ex vivo bone marrow culture, we confirmed
that the majority of IFN-α was indeed produced by pDCs. A transcriptional activator for
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the IFN-α gene, IRF-7, is induced by constitutively expressed NF-kB. The expression
level of IRF-7 also can be upregulated by the NF-kB/p38 MAPK pathway via TLR-9
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signaling. IRF-7 activation also requires chloroquine-sensitive machinery upstream of
NF-kB/p38 MAPK (39). Therefore, activated NF-kB plays a central role in IFN-α
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production via IRF-7. It is thus possible that proteasome inhibition blocks IFN-α
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production by NF-kB inactivation due to inefficient proteasome-dependent degradation
of its inhibitor IkB (40). Additionally, as recently suggested PIs may suppress the
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function of pDCs by disrupting the intracellular trafficking of TLRs and subsequently the
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nuclear localization of IRF-7 and NF-kB (41). Finally, another mechanism for decreased
IFN-α is direct apoptosis of pDCs due to proteasome inhibition. Indeed, pDCs may be
particularly sensitive to proteasome inhibition in a similar fashion to plasma cells given
the large amounts of IFN-α produced and secreted. Although we observed a moderate
decrease in pDCs after in vitro treatment with PIs, this decreased survival does not
account for the complete inhibition of IFN-α secretion. Moreover, absolute numbers of
pDCs were not altered in vivo, again indicating suppression of both survival and
function.
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Inhibition of IFN-α likely contributes to the plasma cell effects of proteasome inhibition.
Thus, CXCL12 expressed in bone marrow and inflamed tissue is an important factor for
plasma cell niches and is enhanced by IFN-α (42). Other important roles for pDCderived Type I interferon include T-cell independent activation of human B-cell
differentiation to plasma cell (43) (44) and activation of IgG secretion in synergy with IL-
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6 (45). Therefore, it is our speculation that the abrogation of IFN-α production by
proteasome inhibition causes more global and indirect benefits on autoantibody
regulation.
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In conclusion, proteasome inhibition has a beneficial effect in murine lupus with
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synergistic effects on plasma cells and unique targeting of Type I interferon pathways.
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The increased therapeutic margin of a selective immunoproteasome inhibitor as well as
the LMP7 dependence of IFN-α production provides strong rationale for clinical
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development in an autoantibody and immune complex driven autoimmune disease such
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as lupus.
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Figure 1. Carfilzomib and ONX 0914 prevent nephritis progression in Lupus prone mice.
(a) 10 week-old MRL/lpr mice (n = 10 each group) were treated with bortezomib 0.75
mg/kg D1D3 (closed squares), carfilzomib 3 mg/kg D1D2 (closed triangles), ONX 0914
10 mg/kg QOD (closed circles) or vehicle solution (open circles) for 13 weeks.
Significant differences in proteinuria from vehicle treated animals (p<0.05) were
observed beginning at 3 weeks for bortezomib, 4 weeks for CFZ, and 2 weeks for ONX
0914. NZB/W mice (proteinuria grade 2+) were treated with carfilzomib (n = 2), ONX
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0914 (n = 4) or vehicle solution (n = 6) for 8 weeks (significant differences beginning at
4 weeks for ONX 0914 and 7 weeks for CFZ). (b) Representative kidney sections of
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NZB/W mice after treatment with 20 mg/ml of ONX 0914 or vehicle solution for 8
weeks. Kidneys were scored from 0 to 4 for glomerulonephritis (GN), interstitial
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nephritis (IN), and perivascular infiltration (VI) (mean for MRL/lpr mice in A). (c)
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Serum anti-dsDNA IgG antibody levels and total IgG levels of MRL/lpr mice (significant
differences beginning at 7 weeks). Data are shown as mean + s.e.m and are
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representative of 3 independent experiments and cohorts of treated mice.
Figure 2. Carfilzomib and ONX 0914 reduce numbers of plasma cells and germinal
center cells in Lupus prone mice. (a) 10 week-old MRL/lpr mice were treated for 13
weeks with PIs as in Figure 1 and PC (CD138+, k light chain+) counts defined by flow
cytometry in spleen (left) and bone marrow (right). *p=0.0003, **p<0.0001, ***p=0.01,
****p=0.005 (b) The numbers of total IgG ASC (top panels) and anti-dsDNA IgG ASC
(bottom panels) from spleen and bone marrow were measured by ELISPOT assays.
*p=0.008, **p=0.005, ***p=0.03, ****p=0.01, #p=0.05). (c) Representative spleen
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sections of normal mice, NZB/W mice treated with vehicle, and NZB/W mice treated
with ONX 0914 for 8 weeks (2+protein at start). (d) GC cell (B220+, GL-7+) and PC
numbers in spleen of NZB/W mice after 8 weeks of ONX 0914 (n = 3, 20 mg/kg) vs.
vehicle solution (n = 3). Representative spleen sections stained with GL7. All data is
expressed as mean + s.e.m..
Figure 3. PIs abrogate antibody secretion from ASCs in vitro. Spleen cells of NZB/W
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mice (4+ protein) were incubated overnight with titrating amounts of the indicated PIs
during ELISPOT assays. (a) Representative wells of ELISPOT results for IgG (7.8 x 103
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cells/well) and anti-dsDNA (250 x 103 cells/well) ASC with bortezomib (31.25 nM),
carfilzomib (1 µM), ONX 0914 (2 µM) and control (no PIs) added in vitro. The spot
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numbers or spot size of IgG (a) or anti-dsDNA IgG secreting cells (b) per 0.1 million
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spleen cells were normalized to the control (no PI, n=9) and plotted against the titrated
amounts of PIs (mean (n=3) + s.e.m). (d). The average spot sizes of IgG ASC ELISPOT
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without PIs.
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were plotted against the average occurrence relative to the highest occurrence with or
Figure 4. PIs block IFN-α production in the mouse. (a) C57B6 mouse BM cells were
incubated in vitro with titrating amounts of the indicated PIs in the presence of CpG2216
(500 ng/ml) overnight. IFN-α levels in the cell culture supernatants were measured by
ELISA. (b) C57B6 mouse BM cells were incubated as above with the indicated PIs (125
nM ONX 0914, 125 nM PR-893, 40 nM bortezomib, or 40 nM carfilzomib) and CpG and
IFN-α production assessed. (c) NZB/W mice (proteinuria 3+) were injected in vivo with
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PIs (bortezomib 0.75 mg/ml D1D3, carfilzomib 5 mg/ml on D1D2, ONX 0914 20 mg/ml
on D1D3D5 or control vehicle) for 1 week and again 1 hour before sacrifice and analysis.
Extracted BM cells were incubated in vitro overnight in the presence of CpG2216 and
IFN-α levels in supernatants measured (mean (n = 3) + s.e.m.). *p=0.01, **p=0.03,
***p=0.06. (d) NZB/W mice were injected in vivo with PIs as above, RNA extracted
from spleen cells, and Mx1 expression quantitated by real-time PCR. Data is normalized
relative to the housekeeping gene β2-microglobulin (mean n=3 mice per group in
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duplicate or triplicate + s.e.m.). #p=0.05. Data are representative of 3 independent
experiments.
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Figure 5. Immunoproteasome inhibition abrogates IFN-α production by human PBMCs.
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(a) Human PBMCs were incubated with titrating amounts of the indicated PIs in the
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presence of CpG2216 (250 ng/ml) or Poly- IC (100 mg/ml) overnight. IFN-α levels in the
cell culture supernatants were measured by ELISA. Data are shown from a single donor
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and are representative of 5 independent experiments with different donors. (b) pDCs were
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purified from two different human donors, a healthy control and a hemachromatosis
patient. Purity of >95% was confirmed by flow cytometry. Proteasome activity was
measured as described using both an enzymatic activity assay and an active site ELISA.
β5 and LMP7 containing 20S subunit was quantitated in ng/mg protein and expressed as
a %total 20S, mean +/- SEM.
Figure 6. PIs suppress both the survival and function of IFN-α producing pDCs. (a)
Flow cytometry analysis of mouse BM cells 1 hour after in vivo injection of PIs as
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described in the Figure 4c. The mean (n = 2) + s.e.m of %pDCs in total lymphocytes
from BM of mice treated with different PIs is shown. (b) Representative flow cytometry
analysis of pDCs after 16 hrs incubation in vitro as described in Figure 4 (a). Line graph
depicts BM lymphocyte pDC % from in vitro culture incubated with different PIs at
titrating concentrations (representative of 3 experiments). (c) C57B6 mouse BM cells
were incubated with titrating amounts of the indicated PIs in vitro in the presence of
CpG2216 (500 ng/ml) for 5 hours and an additional 3 hours with GolgiPlug.
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Intracellularly stained IFN-α accumulating pDC and total pDC levels were normalized to
the vehicle treated control and plotted against the PI concentrations. Data presented are
single decay.
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representative of 3 experiments, and the curves were fitted to the non-linear regression
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Nonstandard abbreviations: ASC, antibody secreting cell; APRIL, A proliferationinducing ligand; BAFF, B cell activation factor; BCD, B cell depletion; BCR, B cell
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receptor; IFN, interferon; PBMC, peripheral blood mononuclear cells; pDC,
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plasmacytoid dendritic cell; PI, proteasome inhibition; TLR, Toll like receptor; UPR,
unfolded protein response
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A
bortezomib
carfilzomib
ONX‐0914
B
C
**
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*
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D
***
#
#
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Figure 4. PIs block IFN-α production in the mouse. (a) C57B6
mouse BM cells were incubated in vitro with titrating amounts of
the indicated PIs in the presence of CpG2216 (500 ng/ml)
overnight. IFN-α levels in the cell culture supernatants were
measured by ELISA. (b) C57B6 mouse BM cells were incubated as
above with the indicated PIs (125 nM ONX 0914, 125 nM PR-893,
40 nM bortezomib, or 40 nM carfilzomib) and CpG and IFN-α
production assessed. (c) NZB/W mice (proteinuria 3+) were
injected in vivo with PIs (bortezomib 0.75 mg/ml D1D3,
carfilzomib 5 mg/ml on D1D2, ONX 0914 20 mg/ml on D1D3D5
or control vehicle) for 1 week and again 1 hour before sacrifice and
analysis. Extracted BM cells were incubated in vitro overnight in
the presence of CpG2216 and IFN-α levels in supernatants
measured (mean (n = 3) + s.e.m.). *p=0.01, **p=0.03, ***p=0.06.
(d) NZB/W mice were injected in vivo with PIs as above, RNA
extracted from spleen cells, and Mx1 expression quantitated by
real-time PCR. Data is normalized relative to the housekeeping
gene β2-microglobulin (mean n=3 mice per group in duplicate or
triplicate + s.e.m.). #p=0.05. Data are representative of 3
independent experiments.
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A
CpG
bortezomib
carfilzomib
ONX‐0914
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Poly‐
IC
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B
Figure 5. Immunoproteasome inhibition abrogates IFN-α
production by human PBMCs.
(a) Human PBMCs were incubated with titrating amounts of the
indicated PIs in the presence of CpG2216 (250 ng/ml) or Poly- IC
(100 mg/ml) overnight. IFN-α levels in the cell culture supernatants
were measured by ELISA. Data are shown from a single donor and
are representative of 5 independent experiments with different
donors. (b) pDCs were purified from two different human donors, a
healthy control and a hemachromatosis patient. Purity of >95%
was confirmed by flow cytometry. Proteasome activity was
measured as described using both an enzymatic activity assay and
an active site ELISA. β5 and LMP7 containing 20S subunit was
quantitated in ng/mg protein and expressed as a %total 20S, mean
+/- SEM.
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carfilzomib
ONX-0914
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Supplemental Figure 1. Effects of alternative proteasome inhibitor dosing strategies on murine lupus
nephritis. Ten week-old MRL/lpr mice (n = 10) were treated with carfilzomib 3 mg/kg D1D2 or escalation to 5
mg/kg D1D2 and ONX-0914 10 mg/kg QOD or 20 mg/kg and compared to vehicle solution (open circles) for
13 weeks. Weekly proteinuria levels were measured and plotted as mean proteinuria grades + s.e.m. (left).
Significant differences from vehicle treated animals (p<0.05) were observed beginning at 4 weeks of
treatment for CFZ and 2 weeks of treatment for ONX 0914.
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A
**
*
*
B
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***
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Supplemental Figure 2. Carfilzomib and ONX-0914 reduce numbers of plasma cells in NZB/W mice after
short term treatment. NZB/W mice with proteinuria grade 3+ were treated with carfilzomib (3 mg /kg on
D1D2), ONX-0914 (20 mg/kg on D1D3D5) or vehicle solution with analysis after 1 week of treatment. (a)
Plasma cell (CD138+, k light chain+) numbers in spleen (control; n=8, carfilzomib; n=7 and ONX-0914; n=8)
(left) and bone marrow (control; n=8, carfilzomib; n=7 and ONX-0914; n=8) (right). *p=0.01, **p=0.03. (b)
The numbers of total IgG ASC in spleen (top left; control; n=8, carfilzomib; n=10 and ONX-0914; n=8), total
IgG ASC in bone marrow (top right; control; n=8, carfilzomib; n=10 and ONX-0914; n=8), anti-dsDNA IgG
ASC in spleen (bottom left; control; n=7, carfilzomib; n=10 and ONX-0914; n=8) and anti-dsDNA ASC in bone
marrow (bottom right; control; n=5, carfilzomib; n=7 and ONX-0914; n=6) were measured by ELISPOT
assays. Data are presented as the mean spot counts per indicated cell numbers + s.e.m. *p=0.01, **p=0.03,
***p=0.05.
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Supplemental Figure 3. Immunoproteasome inhibition abrogates IFN-α production by human PBMCs
regardless of CpG2216 concentration. Human PBMCs were incubated with titrating amounts of the indicated
proteasome inhibitors in the presence of CpG2216 A) 31.5 nM, B) 62.5 NM, or C) 125 nM overnight. IFN-α
levels in the cell culture supernatants were measured by ELISA. PR893 is a selective β5 inhibitor at
concentrations of 170 nM.
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