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Inhibitor of NFkB Kinase Subunit 2 Blockade Hinders the
Initiation but Aggravates the Progression of Crescentic
GN
Janine Gotot,* Eveline Piotrowski,
†
Martin S. Otte,*
‡
André P. Tittel,* Guo Linlin,
†
Chen Yao,
†
Karl Ziegelbauer,
§
Ulf Panzer,
†
Natalio Garbi,* Christian Kurts,* and Friedrich Thaiss
†
*Institute of Experimental Immunology, Rheinische Friedrich Wilhelms University, Bonn, Germany;
†
Third Medical
Department of Clinical Medicine, University Hospital Hamburg Eppendorf, Hamburg, Germany;
‡
Department of
Otorhinolaryngology, Head and Neck Surgery, University of Cologne Germany; and
§
Global Drug Discovery,
TRG Oncology/GT, Bayer Pharma AG, Berlin, Germany
ABSTRACT
The NFkB transcription factor family facilitates the activation of dendritic cells (DCs)
and CD4
+
T helper (Th) cells, which are important for protective adaptive immunity.
Inappropriate activation of these immune cells may cause inflammatory disease, and
NFkB inhibitors are promising anti–inflammatory drug candidates. Here, we inves-
tigated whether inhibiting the NFkB–inducing kinase IKK2 can attenuate crescentic
GN, a severe DC–and Th cell–dependent kidney inflammatory disease. Prophylactic
pharmacologic IKK2 inhibition reduced DC and Th cell activation and ameliorated
nephrotoxic serum–induced GN in mice. However, therapeutic IKK2 inhibition dur-
ing ongoing disease aggravated the nephritogenic immune response and disease
symptoms. This effect resulted from the renal loss of regulatory T cells, which have
been shown to protect against crescentic GN and which require IKK2. In conclusion,
although IKK2 inhibition can suppress the induction of nephritogenic immune re-
sponses in vivo, it may aggravate such responses in clinically relevant situations,
because it also impairs regulatory T cells and thereby, unleashes preexisting neph-
ritogenic responses. Our findings argue against using IKK2 inhibitors in chronic GN
and perhaps, other immune–mediated diseases.
J Am Soc Nephrol 27: ccc–ccc, 2015. doi: 10.1681/ASN.2015060699
Inhibitor of NFkB kinase subunit 2
(IKK2; also known as IKKb)triggers
the classic NFkB activation pathway,
which is critical for the activation of den-
dritic cells (DCs) and CD4
+
Thelper
(Th) cells during adaptive immune re-
sponses.
1,2
NFkB–dependent DC ac-
tivation (for example, in response to
microbial molecular patterns) results in
the upregulation of costimulatory mol-
ecules, such as CD80, CD86, or CD40,
which promote immunogenic Th cell ac-
tivation.
3
IKK2 deletion in T cells pre-
vented their activation and effector func-
tion.
1,4
Thus, the NFkBpathwayis
widely considered to promote inflamma-
tion, and various NFkB inhibitors are
currently being tested for the treatment
of immune-mediated and inflammatory
diseases.
5
Some IKK2 inhibitors have
shown anti-inflammatory effects in pre-
clinical studies on arthritis and pulmo-
nary disease.
6,7
By contrast, the targeted genetic de-
letion of IKK2 in nonimmune cells, such
as keratinocytes or hepatocytes, caused
inflammatory disease of skin and liver,
respectively.
8,9
Targeted IKK2 deletion
in DCs suppressed not only their acti-
vation but also, their migration into
draining lymph nodes and their ability
to induce differentiation of regulatory
T(T
reg
) cells.
10
Such T
reg
cells are im-
portant to maintain immunologic self-
tolerance by inhibiting autoreactive T and
B cells and require the forkhead/winged–
helix box P3 transcription factor.
11–13
They also require classic NFkB pathway
components, including IKK2, for their
development in the thymus.
4,14–16
Furthermore, recent studies described
distinct NFkB components with anti-
inflammatory properties.
17,18
Thus, there
are both pro- and anti-inflammatory
functions of the NFkBpathway,buttheir
interplay and regulation in the in vivo sit-
uation are unclear.
4,19
Received June 25, 2015. Accepted October 8,
2015.
J.G. and E.P. contributed equally to this work.
Present address: Dr. Chen Yao, Organ Transplant
Institute, Chinese PLA 309th Hospital, Beijing, China.
Published online ahead of print. Publication date
available at www.jasn.org.
Correspondence: Prof. Christian Kurts, Institute of
Experimental Immunology, Rheinische Friedrich
Wilhelms University, Sigmund Freud Street 25,
53105 Bonn, Germany, or Prof. Friedrich Thaiss,
Third Medical Department of Clinical Medicine,
University Hospital Hamburg Eppendorf, Martini
Street 52, Hamburg 20246, Germany. Email:
ckurts@web.de or thaiss@uke.de
Copyright © 2015 by the American Society of
Nephrology
J Am Soc Nephrol 27: ccc–ccc, 2015 ISSN : 1046-6673/2707-ccc 1
NFkB activation has also been ob-
served in patients
20
and experimental
models of GN.
21
Crescentic GN (cGN)
is a severe inflammatory kidney disease,
which is mediated by Th cells specificfor
glomerular antigens and may rapidly
progress to terminal kidney failure.
22,23
It can be mimicked by the passive neph-
rotoxic nephritis (pNTN) model, which
is induced by injecting a nephrotoxic
sheep antiserum specificformurineglo-
merular components into mice. In the
immune activation phase of this model,
which lasts until days 4–5afterserum
injection, DCs in lymphatic organs
capture sheep Ig and activate specific
Th cells. In the effector phase starting at
days 3–4, these Th cells enter the kidney,
where the antiserum is deposited be-
cause of its specificity, and produce
effector cytokines, like IFNg,thatacti-
vate macrophages.
22,23
During their ef-
fector phase, Th cells can be regulated
by kidney-resident DCs, whose activa-
tion state determines whether they stim-
ulate or inhibit the Th cells and thus,
whether nephritis progresses or heals.
24
Here, we hypothesized that inhibiting
DC and Th cell activation with an NFkB
inhibitor should attenuate GN. We tested
this hypothesis by treating mice every
other day with the IKK2 inhibitor kinase
inhibitorof NFkB-1 (KINK-1)
7,25
starting
1 day before administration of the neph-
rotoxic serum (experimental plan is in
Figure 1A). This drug reduced NFkBnu-
clear translocation in vivo (Supplemental
Figure 1) and attenuated pNTN, which
was evidenced by fewer crescents in his-
tologic sections (Figure 1B), lower tubu-
lointerstitial injury (Figure 1C), higher
creatinine clearance (Figure 1D), lower
BUN (Figure 1E), and lower proteinuria
(Figure 1F). Renal DCs (flow cytometric
gating strategy is in Supplemental Figure
2) showed a less activated phenotype
(Figure 1G), and intrarenal activated Th
cells producing IFNgwere less frequent
(Figure 1H). Thus, IKK2 inhibition start-
ing before pNTN induction attenuated
the nephritogenic Th cell response and
disease symptoms.
We noted that anti–sheep Ig titers,
which can be considered a parameter
for the nephritogenic Th cell response,
were systemically reduced after IKK2 in-
hibition (Figure 1I). Furthermore, DCs
in the spleen appeared less mature (Fig-
ure 1J). This indicated that the induction
of the pNTN model had been compro-
mised; in other words, prophylactic
IKK2 inhibition before pNTN induction
(Figure 1A) had suppressed the induc-
tion of this disease model rather than
preventing its progression. Because pro-
phylactic IKK2 inhibition does not
mimic the clinically relevant situation
of a patient presenting with ongoing dis-
ease, we modified our protocol and ap-
plied the IKK2 inhibitor on days 4, 6, and
8 after disease induction (experimental
plan is in Figure 2A). However, under
these conditions, pNTN was no longer
attenuated (Figure 2, B–F), and neither
renal inflammation (Figure 2, G and H)
nor systemic antirenal immune response
(Figure 2, I and J) were decreased. This
might indicate that IKK2 inhibition can-
not attenuate ongoing pNTN. Alterna-
tively, the duration of IKK2 inhibition
(on days 4, 6, and 8 compared with six
times treatment in Figure 1) may have
been too short to affect disease.
To distinguish between these possibil-
ities, we decided to use a protocol that
allows prolonged IKK2 inhibition selec-
tively in the Th cell effector phase. Because
these phases overlap between days 3 and 5
in pNTN,
23
we switched to the accelerated
nephrotoxic nephritis (aNTN) model,
wheremicearefirst immunized with
sheep Ig to allow for the activation of spe-
cific Th cells without a nephritogenic ef-
fector phase; 5 days later, we injected a
lower dose of nephrotoxic sheep serum
to target these Th cells to the kidney and
applied KINK-1 selectively in the effector
phase (experimental plan is in Figure 3A).
Surprisingly, in this setting, aNTN was
markedly and consistently aggravated,
which was evident by more severe histo-
logic kidney damage (Figure 3, B–D, com-
pare the first two experimental groups),
lower creatinine clearance, more elevated
BUN, and higher proteinuria (Figure 3,
E–G). There were more F4/80
+
immune
cells (Figure 3H), intrarenal DCs were
more activated (Figure 3I), and more ac-
tivated IFNg
+
Th cells were detected (Fig-
ure 3J). Anti–sheep Ig titers as a parameter
for the nephritogenic Th1 cell response
were increased as well (Figure 3K).
Importantly, we noted that intrarenal
T
reg
cells (gating strategy is in Supplemen-
tal Figure 2) were less frequent in both
pNTN and aNTN when the KINK-1 was
given six times (Figure 3, L and N) but
that only a slight and nonsignificant re-
duction was seen in the therapeutic
pNTN setting with only three applications
(Figures 2 and 3M). Because T
reg
cells are
well documented to suppress pNTN and
aNTN
26–29
and because the thymic gener-
ation of T
reg
cells requires IKK2,
4,14–16
we
hypothesized that KINK-1 might have tar-
geted these cells.
We tested this hypothesis by treating
DEREG mice, in which T
reg
cells can be
conditionally depleted, with KINK-1 (ex-
perimental setting is in Figure 3A, all four
groups). Both T
reg
depletion and IKK2 in-
hibition aggravated aNTN, but no additive
aggravation was seen when these proce-
dures were combined (Figure 3, B–G, all
four groups). Likewise, the parameters for
intrarenal inflammation (Figure 3, H–J)
and anti–sheep Ig titers (Figure 3K) were
increased by T
reg
depletion and IKK2
inhibition, but no synergy between these
maneuvers was evident. Thus, therapeutic
IKK2 inhibition aggravated aNTN but was
unable to further increase damage when
T
reg
cells were absent, indicating that
KINK-1 aggravated aNTN by reducing
T
reg
numbers.
Finally, we examined the mechanisms
by which T
reg
cells can suppress immune
responses. After KINK-1 application and
after T
reg
depletion, less TGF-band less
IL-10 was detectable in kidney digests, but
no additive effect of these measures was
noted (Figure 4, A and C, Supplemental
Figure 3). As a control, we measured the
NFkB–dependent inflammatory cytokine
TNFaand found that it was increased af-
ter both maneuvers but again, not syner-
gistically (Figure 4E, Supplemental Figure
3). Intracellular levels of neither TGF-bor
IL-10 in T
reg
cells (Figure 4, B and D) nor
TNFain DCs or macrophages (Figure 4, F
andG)weresignificantly changed, indi-
cating that KINK-1 acted by reducing ei-
ther the numbers of cytokine–producing
immune cells or hypothetic cytokine pro-
duction by nonimmune cells.
2Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc,2015
BRIEF COMMUNICATION www.jasn.org
In summary, our study shows diamet-
rical consequences of prophylactic and
therapeutic in vivo inhibition of IKK2:
prophylactic inhibition prevented DC
and Th cell activation, consistent with the
widely accepted view that the NFkBpath-
way is proinflammatory.
2,3,19
Prophylactic
inhibition over at least 1 week suppressed
T
reg
cellsaswell,butthiswasinconsequen-
tial; there was no nephritogenic immune
response that T
reg
cells would have to regu-
late, because the Th effector cells had not
been properly activated. However, when we
applied the IKK2 inhibitor in a realistic dis-
ease situation (i.e., after Th cell activation),
which mimics the situation of patients pre-
senting with ongoing crescentic GN, the
suppression of T
reg
cells was able to unleash
the preexisting nephritogenic Th cell re-
sponse, and disease was aggravated.
We conclude that IKK2 inhibition is
anti-inflammatory only when initiated
before disease onset, which is impractical
for clinical use. Therapeutic IKK2 inhibi-
tion has potential proinflammatory con-
sequences, because it compromises the
regulation of pathogenic immune re-
sponses through T
reg
cells, and this func-
tion prevails over the anti-inflammatory
effect resulting from inhibiting DC
Figure 1. Prophylactic IKK2 inhibition blocks the induction of pNTN. Prophylactic IKK2 inhibition in pNTN. (A) Experimental plan, (B)
representative periodic acid–Schiff–stained glomeruli and percentage of crescentic glomeruli, (C) tubulointerstitial injury, (D) creatinine
clearance, (E) BUN, (F) albumin-to-creatinine ratio, (G) expression of the DC activation markers CD40 and CD80, (H) numbers of activated
and percentages of IFNg–producing Th cells, (I) IgG titers of anti–sheep Ig antibodies, and (J) DC activation markers on splenic cells in
nephritic mice prophylactically treated with KINK-1 or vehicle. Data are representative of three independent experiments (n=5; Kruskal–
Wallis test with post hoc analysis by Mann–Whitney test). cGN, crescentic glomerulonephritis; MFI, mean fluorescence intensity; NTS,
nephrotoxic serum nephritis. *P,0.05; **P,0.01; ***P,0.001.
J Am Soc Nephrol 27: ccc–ccc, 2015 IKK2 Inhibition in GN 3
www.jasn.org BRIEF COMMUNICATION
maturation. These findings highlight the
need for in vivo studies in which a com-
plex pathway, such as NFkB, can exert
antagonistic functions in different cell
types or at different time points. Our
findings imply that great care is neces-
sary in clinical studies aiming to treat
chronic kidney inflammation by NFkB
inhibition, at least when IKK2 inhibi-
tors are used, because these might ag-
gravate rather than ameliorate disease.
Our findings may apply to immune-
mediated diseases affecting other or-
gans as well.
CONCISE METHODS
Mice and Reagents
Mice were bred at the animal facilities of the
University Hospital Bonn and University
Hospital Hamburg Eppendorf under specific
pathogen–free conditions. Animal experiments
Figure 2. Short–term therapeutic IKK2 inhibition fails to attenuate pNTN. (A) Experimental plan, (B) representative periodic acid–Schiff–
stained glomeruli and percentage of crescentic glomeruli, (C) tubulointerstitial injury, (D) creatinine clearance, (E) BUN, (F) albumin-
to-creatinine ratio, (G) expression of the DC activation markers CD40 and CD80, (H) numbers of activated and percentages of
IFNg–producing Th cells, (I) IgG titers of anti–sheep Ig antibodies, and (J) DC activation markers on splenic cells in nephritic mice treated
therapeutically (starting day +4 after the induction of nephrotoxic nephritis) with KINK-1 or vehicle. Data are representative of three in-
dependent experiments (n=4; Kruskal–Wallis test with post hoc analys is by Mann–Whitney test). cGN, crescent ic glomerulonephritis; MFI,
mean fluorescence intensity; NTS, nephrotoxic serum nephritis.
4Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc,2015
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Figure 3. IKK2 inhibition aggravates aNTN by reducing intrarenal T
reg
cells. (A–K) Therapeutic IKK2 inhibition was performed in the
effector phase of accelerated cGN. (A) When indicated, T
reg
cells were depleted using DT in DEREG mice as shown in the experimental
plan. (B) Representative periodic acid–Schiff–stained glomeruli, (C) percentage of crescentic glomeruli, (D) tubulointerstitial injury, (E)
creatinine cleara nce, (F) BUN, (G) albumin-to-cr eatinine ratio, (H) number of F4/8 0
+
immune cells counte d on sections per high-power field
(hpf), (I) expression of the DC activation markers CD40 and CD80, (J) numbers of activated and percentages of IFNg–producing Th cells,
and (K) serum anti–sheep IgG titers. (L–N) Number of intrarenal T
reg
cells in the experiments shown in Figures 1 and 2 and A–K, respectively.
Data are representative of four independent experiments (n=5; post hoc analysis by ANOVA with Bonferroni post-test). cGN, crescentic
glomerulonephritis; DT, diphtheria toxin; MFI, mean fluorescence intensity; NTS, nephrotoxic serum. *P,0.05; **P,0.01; ***P,0.001.
J Am Soc Nephrol 27: ccc–ccc, 2015 IKK2 Inhibition in GN 5
www.jasn.org BRIEF COMMUNICATION
were performed according to national and insti-
tutional animal care and ethical guidelines and
had been approved by government committees
(Behörde für Gesundheit und Verbraucherschutz
BGV der Freien und Hansestadt Hamburg
and Landesamt für Natur and Umwelt und
Verbraucherschutz Nordrhein-Westfalen).
T
reg
cells were depleted in nephritic DEREG
micebyinjecting15ng/gbodywtdiphtheria
toxin.
27,28
KINK-1 is an ATP–competitive selec-
tive IKK2 kinase inhibitor.
7
KINK-1 was dis-
solved in 10% cremophor (Sigma-Aldrich,
St. Louis, MO) diluted with PBS. A KINK-1
dose of 5 mg/kg body wt subcutaneously every
second day inhibited IKK2 in mice.
pNTN and aNTN Models
pNTN was induced in 8- to 10-week-old age–
and sex–matched male mice (20–25 g body
wt; Charles River Laboratories, Wilmington,
MA) on the C57BL/6J background by intra-
peritoneal injection of 2.5 mg/kg body wt
sheep anti–mouse glomerular basement
membrane antiserum (nephrotoxic nephritis
serum) as described.
24
Controls received
an equal amount of nonspecific sheep IgG.
In aNTN, mice were injected on day 25
with 2.5 mg/kg body wt sheep IgG in incom-
plete Freund’s adjuvant subcutaneously and
day 0 with 2 mg/kg body wt nephrotoxic ne-
phritis serum intraperitoneally. This dose
caused intermediate disease severity, and doses
$2.5 mg/kg body wt caused complete renal fail-
ure after 2 weeks. Urine was collected in metabolic
cages for 24 hours, and serum was derived from
whole blood after cardiac puncture. Urinary cre-
atinine, serum creatinine, and BUN were mea-
sured using standard methods in the central
laboratory of Bonn University Hospital.
24
Albuminuria (Mice Albumin Kit; Bethyl) and se-
rum IgG (Dianova) were determined by ELISA.
Histology
Renal tissue injury was assessed in 2-mm
paraformaldehyde (4%)–fixed paraffin tissue
sections stained by periodic acid–Schiff reac-
tion. A semiquantitative score for acute glo-
merular injury was assessed in 30 glomeruli
per mouse by a double-blinded observer as
described before.
28
Kidney damage was his-
tologically scored by an observer blinded to
the identity of samples. For determining
the proportion of crescentic glomeruli,
$80 glomeruli per section were examined.
F4/80
+
cells were stained using rabbit anti-
body to F4/80 (MCA497B; Serotec) diluted
1:50. F4/80
+
cell infiltration was then quanti-
fied using ImageJ software (National Institutes
of Health). Light microscopic evaluation was
performed under an Axioskop (Carl Zeiss
GmbH, Jena, Germany) and photographed
with an Axiocam HRc (Carl Zeiss GmbH)
using the Axiostar software (Carl Zeiss
GmbH). F4/80
+
cells in 30 tubulointerstitial
high–power fields per kidney were counted
by light microscopy.
Flow Cytometry
Complete kidneys and spleens were digested
with collagenase (Roche Diagnostics, Indian-
apolis, IN) and DNAse-I as previously de-
scribed.
30
Single-cell suspensions were
stained with fluorochrome-conjugated anti-
bodies in PBS containing 10% FCS. The fol-
lowing antibodies from BD Pharmingen or
eBioscience (San Diego, CA) were used:
CD4 (GK1.5), anti-CD45 (30F11), CD8
(53–6.7), B220 (RA3–6B2), CD25 (PC61.5),
forkhead/winged–helix box P3 (FJK-16S),
CD11c (HL3), MHC-II (M5/114.15.2),
F4/80 (BM8), Gr1 (RB6–8C5), CD40 (3/23),
CD69 (1H.2F3), CD80 (16–10A1), and anti-
IFNg(XMG1.2). Dead cells were excluded
by staining with the LIVE/DEAD Fixable
Violet Dead Cell Stain Kit. Viable CD11c
+
MHC II
+
cells were considered DCs. Intra-
cellular staining was performed as recently
described.
13
Cells were analyzed with a BD
Biosciences (San Jose, CA) LSRII using Diva
and FlowJo software. Kidney single–cell sus-
pensions were overnight restimulated with
25 mg/ml sheep Ig, and then, concentrations
of TGF-b, IL-10, and TNFawere measured
Figure 4. Intrarenal cytokine milieu after IKK2 inhibition in aNTN. (A and B) TGF-b, (C and D) IL-10, and (E–G) TNFalevels in (A, C, and E)
whole-kidney digests measured by ELISA or Luminex and (B and D) T
reg
cells, (F) DCs, and (G) macrop hages measured by intracellular flow
cytometry. Data are representative of two independent experiments (n=4; post hoc analysis by ANOVA with Bonferroni post-test). cGN,
crescentic glomerulonephritis; DT, diphtheria toxin; MFI, mean fluorescence intensity. *P,0.05.
6Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc,2015
BRIEF COMMUNICATION www.jasn.org
by ELISA or Luminex according to the man-
ufacturer’s instructions (eBioscience).
Nuclear Fractionation and Gel Shift
Experiment
Kidneys were perfused with 50 ml sterile PBS
per animal before harvesting. Nuclear mini-
ature extracts were prepared, and gel shift
assays were carried out using an NFkB oligo-
nucleotide probe (Promega, Heidelberg,
Germany) end labeled with
32
P-g-ATP
(3000 Ci/mmol; GE Healthcare, Waukesha,
WI). Afterward, 30 mg nuclear protein was
incubated for 30 minutes at room tempera-
ture with 100,000 cpm probe in 20 mM
HEPES (pH 7.9), 0.3 mM EDTA, 0.2 mM
EGTA, 80 mM NaCl, and 2 mg poly(dI-dC)
poly(Di-dC) (Amersham Pharmacia Biotech)
in a total volume of 20 ml. Where indicated,
competition experiments were performed by
adding unlabeled consensus oligonucleotides
in a 100-fold molar excess to the binding re-
action. The DNA-protein complexes were
separated by electrophoresis and autoradio-
graphed at 280°C for 1 week. Exposed films
were quantified using a phosphoimager Bio-
Rad GS-363 (multianalyst software; Hercules,
CA) and corrected to the density of the probe.
Statistical Analyses
Results are expressed as me ans6SEM. Differ-
ences between experimental groups were
compared by either the Kruskal–Wallis test
with post hoc analysis usingthe Mann–Whitney
test or one-way ANOVA with post hoc Bonferroni
test of selected groups (GraphPad Prism
Software; GraphPad Software, La Jolla, CA).
Paired ttest was used to compare mean values
within one experimental series. No random-
ization or exclusion of data points was used.
Statistical significance was defined as P,0.05.
Experiments yielding insufficient data for sta-
tistical analysis because of the experimental
setup were repeated at least three times. Tests
were reported only where data met assumptions
of tests. On the basis of preliminary experimental
data, a power analysis of 0.8 with P,0.05
indicates a minimum number of three samples/
purifications per group, but in some cases, four
samples/purifications per group were used.
ACKNOWLEDGMENTS
We thank Chrystel Flores and Anna Kaffke
for excellent technical assistance and Tim
Sparwasser for DEREG mice. C.K. is a member
of the Excellence Cluster ImmunoSensation.
We acknowledge technicalsupport from the
Central Animal Facilities and the Flow Cy-
tometry Core Facilitiesof the Medical Faculties
both in Bonn and in Hamburg. This work was
funded by Deutsche Forschungsgemeinschaft
Grants KFO228 and SFBTR57, Gottfried
Wilhelm Leibniz Price (to C.K.), and the
European Union Consortia INTRICATE and
RELENT.
DISCLOSURES
K.Z. is a full-time employee and stockholder of
Bayer AG (Berlin, Germany). The other authors
declare no competing financial interest.
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ASN.2015060699/-/DCSupplemental.
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