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Inhibitor of NF B Kinase Subunit 2 Blockade Hinders the Initiation but Aggravates the Progression of Crescentic GN

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The NFκB 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 NFκB inhibitors are promising anti-inflammatory drug candidates. Here, we investigated whether inhibiting the NFκB-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 during 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 responses in vivo, it may aggravate such responses in clinically relevant situations, because it also impairs regulatory T cells and thereby, unleashes preexisting nephritogenic responses. Our findings argue against using IKK2 inhibitors in chronic GN and perhaps, other immune-mediated diseases.
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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 inammatory disease, and
NFkB inhibitors are promising antiinammatory drug candidates. Here, we inves-
tigated whether inhibiting the NFkBinducing kinase IKK2 can attenuate crescentic
GN, a severe DCand Th celldependent kidney inammatory disease. Prophylactic
pharmacologic IKK2 inhibition reduced DC and Th cell activation and ameliorated
nephrotoxic seruminduced 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 ndings argue against using IKK2 inhibitors in chronic GN
and perhaps, other immunemediated diseases.
J Am Soc Nephrol 27: cccccc, 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
NFkBdependent 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 inamma-
tion, and various NFkB inhibitors are
currently being tested for the treatment
of immune-mediated and inammatory
diseases.
5
Some IKK2 inhibitors have
shown anti-inammatory 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
inammatory 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.
1113
They also require classic NFkB pathway
components, including IKK2, for their
development in the thymus.
4,1416
Furthermore, recent studies described
distinct NFkB components with anti-
inammatory properties.
17,18
Thus, there
are both pro- and anti-inammatory
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: cccccc, 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 inammatory kidney disease,
which is mediated by Th cells specicfor
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 specicformurineglo-
merular components into mice. In the
immune activation phase of this model,
which lasts until days 45afterserum
injection, DCs in lymphatic organs
capture sheep Ig and activate specic
Th cells. In the effector phase starting at
days 34, these Th cells enter the kidney,
where the antiserum is deposited be-
cause of its specicity, 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 (ow 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 antisheep 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 modied 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, BF), and neither
renal inammation (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,
wheremicearerst immunized with
sheep Ig to allow for the activation of spe-
cic 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, BD, com-
pare the rst two experimental groups),
lower creatinine clearance, more elevated
BUN, and higher proteinuria (Figure 3,
EG). 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). Antisheep 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 nonsignicant 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
2629
and because the thymic gener-
ation of T
reg
cells requires IKK2,
4,1416
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, BG, all
four groups). Likewise, the parameters for
intrarenal inammation (Figure 3, HJ)
and antisheep 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
NFkBdependent inammatory 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)weresignicantly changed, indi-
cating that KINK-1 acted by reducing ei-
ther the numbers of cytokineproducing
immune cells or hypothetic cytokine pro-
duction by nonimmune cells.
2Journal of the American Society of Nephrology J Am Soc Nephrol 27: cccccc,2015
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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 proinammatory.
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-inammatory only when initiated
before disease onset, which is impractical
for clinical use. Therapeutic IKK2 inhibi-
tion has potential proinammatory con-
sequences, because it compromises the
regulation of pathogenic immune re-
sponses through T
reg
cells, and this func-
tion prevails over the anti-inammatory
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 acidSchiffstained 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 IFNgproducing Th cells, (I) IgG titers of antisheep 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 MannWhitney test). cGN, crescentic glomerulonephritis; MFI, mean uorescence intensity; NTS,
nephrotoxic serum nephritis. *P,0.05; **P,0.01; ***P,0.001.
J Am Soc Nephrol 27: cccccc, 2015 IKK2 Inhibition in GN 3
www.jasn.org BRIEF COMMUNICATION
maturation. These ndings 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
ndings imply that great care is neces-
sary in clinical studies aiming to treat
chronic kidney inammation by NFkB
inhibition, at least when IKK2 inhibi-
tors are used, because these might ag-
gravate rather than ameliorate disease.
Our ndings 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 specic
pathogenfree conditions. Animal experiments
Figure 2. Shortterm therapeutic IKK2 inhibition fails to attenuate pNTN. (A) Experimental plan, (B) representative periodic acidSchiff
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
IFNgproducing Th cells, (I) IgG titers of antisheep 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; KruskalWallis test with post hoc analys is by MannWhitney test). cGN, crescent ic glomerulonephritis; MFI,
mean uorescence intensity; NTS, nephrotoxic serum nephritis.
4Journal of the American Society of Nephrology J Am Soc Nephrol 27: cccccc,2015
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Figure 3. IKK2 inhibition aggravates aNTN by reducing intrarenal T
reg
cells. (AK) 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 acidSchiffstained 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 eld
(hpf), (I) expression of the DC activation markers CD40 and CD80, (J) numbers of activated and percentages of IFNgproducing Th cells,
and (K) serum antisheep IgG titers. (LN) Number of intrarenal T
reg
cells in the experiments shown in Figures 1 and 2 and AK, 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 uorescence intensity; NTS, nephrotoxic serum. *P,0.05; **P,0.01; ***P,0.001.
J Am Soc Nephrol 27: cccccc, 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 ATPcompetitive 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 sexmatched male mice (2025 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 antimouse glomerular basement
membrane antiserum (nephrotoxic nephritis
serum) as described.
24
Controls received
an equal amount of nonspecic sheep IgG.
In aNTN, mice were injected on day 25
with 2.5 mg/kg body wt sheep IgG in incom-
plete Freunds 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%)xed parafn tissue
sections stained by periodic acidSchiff 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 inltration was then quanti-
ed 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
highpower elds 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 uorochrome-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
(536.7), B220 (RA36B2), CD25 (PC61.5),
forkhead/wingedhelix box P3 (FJK-16S),
CD11c (HL3), MHC-II (M5/114.15.2),
F4/80 (BM8), Gr1 (RB68C5), CD40 (3/23),
CD69 (1H.2F3), CD80 (1610A1), 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 singlecell 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 (EG) 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 ow
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 uorescence intensity. *P,0.05.
6Journal of the American Society of Nephrology J Am Soc Nephrol 27: cccccc,2015
BRIEF COMMUNICATION www.jasn.org
by ELISA or Luminex according to the man-
ufacturers 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 lms
were quantied 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 KruskalWallis test
with post hoc analysis usingthe MannWhitney
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 signicance was dened as P,0.05.
Experiments yielding insufcient 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/
purications per group, but in some cases, four
samples/purications 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 nancial interest.
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ASN.2015060699/-/DCSupplemental.
8Journal of the American Society of Nephrology J Am Soc Nephrol 27: cccccc,2015
BRIEF COMMUNICATION www.jasn.org
... A study published from our laboratory by Gotot et al. (20) showed that systemic IKK-2 inhibition after the induction of the disease increased renal injury in the NTN model and that activated cytotoxic T lymphocytes were resistant to IKK-2 inhibition because of other pathways-in particular, nuclear factor of activated T-cell signaling (21). NF-kB activation was demonstrated by our group after ischemia-reperfusion kidney injury, and more Th17 cells were found in ischemic CD4xNEMO D mice but not in CD4xIKK2 D mice (22). ...
... We and others have demonstrated NF-kB activation in glomerulonephritis, and in 2016 we demonstrated that IKK-2 inhibition before NTN induction attenuated the nephritogenic T h cell response (20). Therefore, genetically modified mice were used to examine the effects of IKK-2 or NEMO deletion, specifically in T lymphocytes, in the experimental NTN model. ...
... We have shown previously that total kidney NF-kB was activated after the induction of NTN (20), which was also confirmed in CD4xIKK2 D and CD4xNEMO D mice and may explain why no significant functional and morphologic changes were observed at 10 d after NTN induction. Our results do not exclude, however, that functional or morphologic changes may occur at later time points and influence the long-term outcome in these mice. ...
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Experimental nephrotoxic serum nephritis (NTN) is a model for T-cell-mediated human rapid progressive glomerulonephritis. T-cell receptor stimulation involves intracellular signaling events that ultimately lead to the activation of transcription factors, such as NF-κB. We explored the involvement of the NF-κB components IKK-2 and NEMO in NTN, by using cell-specific knockouts of IKK-2 and NEMO in CD4+ T lymphocytes. Our results demonstrate that although the course of disease was not grossly altered in CD4xIKK2Δ and CD4xNEMOΔ animals, renal regulatory T cells were significantly reduced and T helper (Th)1 and Th17 cells significantly increased in both knockout mouse groups. The expression of the renal cytokines and chemokines IL-1β, CCL-2, and CCL-20 was also significantly altered in both knockout mice. Lymphocyte transcriptome analysis confirmed the increased expression of Th17-related cytokines in spleen CD4+ T cells. Moreover, our array data demonstrate an interrupted canonical NF-κB pathway and an increased expression of noncanonical NF-κB pathway-related genes in nephritic CD4xNEMOΔ mice, highlighting different downstream effects of deletion of IKK-2 or NEMO in T lymphocytes. We propose that better understanding of the role of IKK-2 and NEMO in nephritis is essential for the clinical application of kinase inhibitors in patients with glomerulonephritis.-Guo, L., Huang, J., Chen, M., Piotrowski, E., Song, N., Zahner, G., Paust, H.-J., Alawi, M., Geffers, R., Thaiss, F. T-lymphocyte-specific knockout of IKK-2 or NEMO induces Th17 cells in an experimental nephrotoxic nephritis mouse model.
... We recently observed that prolonged treatment with the IKKb inhibitor KINK-1 (kinase inhibitor of NF-kB-1) surprisingly aggravated a T helper (Th) cell-mediated kidney disease model (Gotot et al., 2016). Given that NF-kB activation promotes FoxP3 expression (Ruan et al., 2009;Schuster et al., 2012;Zheng et al., 2010) and that CD25 signaling promotes Treg survival (Furtado et al., 2002), we hypothesized that mature Tregs may require IKKb for expansion, maintenance, and/or recruitment. ...
... We recently noted lower Treg numbers after prolonged IKKb inhibition in a kidney disease model (Gotot et al., 2016). To investigate whether Tregs require cell-intrinsic IKKb, we crossed IKKb fl/fl mice (Park et al., 2002) with FoxP3 Cre mice (Rubtsov et al., 2008) to generate mice whose FoxP3-expressing cells lacked IKKb (termed FoxP3 DIKKb mice). ...
... Flow cytometric analysis after 15 days showed almost 50% less splenic FoxP3 + Tregs, whereas Th cells, CTLs ( Figure 2F), and total numbers of circulating leukocytes (data not shown) remained normal, indicating that systemic IKKb inhibition acted preferentially on Tregs. DCs and macrophages showed elevated signs of activation ( Figure S2B), presumably because of the loss of Tregs, consistent with our previous study on the effects of KINK-1 in myeloid cells (Gotot et al., 2016). Scurfy symptoms were not observed under KINK-1 treatment, consistent with the reduction of Tregs by only 50%. ...
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Regulatory T cells (Tregs) prevent autoimmunity but limit antitumor immunity. The canonical NF-κB signaling pathway both activates immunity and promotes thymic Treg development. Here, we report that mature Tregs continue to require NF-κB signaling through IκB-kinase β (IKKβ) after thymic egress. Mice lacking IKKβ in mature Tregs developed scurfy-like immunopathology due to death of peripheral FoxP3⁺ Tregs. Also, pharmacological IKKβ inhibition reduced Treg numbers in the circulation by ∼50% and downregulated FoxP3 and CD25 expression and STAT5 phosphorylation. In contrast, activated cytotoxic T lymphocytes (CTLs) were resistant to IKKβ inhibition because other pathways, in particular nuclear factor of activated T cells (NFATc1) signaling, sustained their survival and expansion. In a melanoma mouse model, IKKβ inhibition after CTL cross-priming improved the antitumor response and delayed tumor growth. In conclusion, prolonged IKKβ inhibition decimates circulating Tregs and improves CTL responses when commenced after tumor vaccination, indicating that IKKβ represents a druggable checkpoint.
... Both pro-and anti-inflammatory NF-kB effects exist, and inhibiting the wrong subunit at the wrong time can be deleterious. 25,26 Thus, the exact NF-kB contributions need to be determined for each disease type. We tested the hypothesis that endothelial NF-kB is activated by ANCAstimulated neutrophils, mediates glomerular necrosis and crescent formation, and provides a potential treatment target. ...
... We previously described NF-kB activation by inflammatory mediators in human neutrophils and its role in apoptosis. 36 However, NF-kB activation has also been reported in renal diseases, 23 including experimental anti-GBM crescentic GN. 20,26,37 The starting point of our NF-kB study in AAV was to reanalyze kidneys from a previous study, where we had induced NCGN by anti-MPO antibodies and treated some of the mice with bortezomib. 27 This study was particularly informative because the proteasome inhibitor bortezomib reduces IkBa degradation and thereby NF-kB activation. ...
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Full-text available
ANCA-associated vasculitis (AAV) is a highly inflammatory condition in which ANCA-activated neutrophils interact with the endothelium, resulting in necrotizing vasculitis. We tested the hypothesis that endothelial NF-κB mediates necrotizing crescentic GN (NCGN) and provides a specific treatment target. Reanalysis of kidneys from previously examined murine NCGN disease models revealed NF-κB activation in affected kidneys, mostly as a p50/p65 heterodimer, and increased renal expression of NF-κB-dependent tumor necrosis factor α (TNF-α). NF-κB activation positively correlated with crescent formation, and nuclear phospho-p65 staining showed NF-κB activation within CD31-expressing endothelial cells (ECs) in affected glomeruli. Therefore, we studied the effect of ANCA on NF-κB activation in neutrophil/EC cocultures in vitro ANCA did not activate NF-κB in primed human neutrophils, but ANCA-stimulated primed neutrophils activated NF-κB in ECs, at least in part via TNF-α release. This effect increased endothelial gene transcription and protein production of NF-κB-regulated interleukin-8. Moreover, upregulation of endothelial NF-κB promoted neutrophil adhesion to EC monolayers, an effect that was inhibited by a specific IKKβ inhibitor. In a murine NCGN model, prophylactic application of E-selectin-targeted immunoliposomes packed with p65 siRNA to downregulate endothelial NF-κB significantly reduced urine abnormalities, renal myeloid cell influx, and NCGN. Increased glomerular endothelial phospho-p65 staining in patients with AAV indicated that NF-κB is activated in human NCGN also. We suggest that ANCA-stimulated neutrophils activate endothelial NF-κB, which contributes to NCGN and provides a potential therapeutic target in AAV.
... Therefore, it could be concluded that systemic IKK inhibition has more beneficial effects on tubular epithelial cells than on infiltrating lymphocytes. Dual nature of systemic use of IKK2 inhibition has also been confirmed in experimental model of rapidly progressive glomerulonephritis [118]. Therapeutic IKK2 inhibition even produced proinflammatory effects, because suppressed Treg cells' function prevailed over the inhibited DC maturation, arguing against using IKK2 inhibitors in glomerulonephritis and possibly other immune-mediated renal diseases [118]. ...
... Dual nature of systemic use of IKK2 inhibition has also been confirmed in experimental model of rapidly progressive glomerulonephritis [118]. Therapeutic IKK2 inhibition even produced proinflammatory effects, because suppressed Treg cells' function prevailed over the inhibited DC maturation, arguing against using IKK2 inhibitors in glomerulonephritis and possibly other immune-mediated renal diseases [118]. ...
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Acute kidney injury caused by ischemia and subsequent reperfusion is associated with a high rate of mortality and morbidity. Ischemia/reperfusion injury in kidney transplantation causes delayed graft function and is associated with more frequent episodes of acute rejection and progression to chronic allograft nephropathy. Alloantigen-independent inflammation is an important process, participating in pathogenesis of injurious response, caused by ischemia and reperfusion. This innate immune response is characterized by the activity of classical cells belonging to the immune system, such as neutrophils, macrophages, dendritic cells, lymphocytes, and also tubular epithelial cells and endothelial cells. These immune cells not only participate in inflammation after ischemia exerting detrimental influence but also play a protective role in the healing response from ischemia/reperfusion injury. Delineating of complex mechanisms of their actions could be fruitful in future prevention and treatment of ischemia/reperfusion injury. Among numerous so far conducted experiments, observed immunomodulatory role of adenosine and adenosine receptor agonists in complex interactions of dendritic cells, natural killer T cells, and T regulatory cells is emphasized as promising in the treatment of kidney ischemia/reperfusion injury. Potential pharmacological approaches which decrease NF- κ B activity and antagonize mechanisms downstream of activated Toll-like receptors are discussed.
... Another example can be found in the process of dendritic cell maturation during experimental cGN, which is generally mediated by the transcription factor nuclear factor-κB (NF-κB). In murine cGN, pharmacological inhibition of NF-κB diminished the maturation of DCs, but the subsequent loss of regulatory T cells exacerbated multiple features of crescentic disease (Gotot et al. 2016). ...
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Crescentic glomerulonephritis represents a group of kidney diseases characterized by rapid loss of kidney function and the formation of glomerular crescents. While the role of the immune system has been extensively studied in relation to the development of crescents, recent findings show that parietal epithelial cells play a key role in the pathophysiology of crescent formation, even in the absence of immune modulation. This review highlights our current understanding of parietal epithelial cell biology and the reported physiological and pathological roles that these cells play in glomerular lesion formation, especially in the context of crescentic glomerulonephritis.
... TANK-binding kinase 1 (TBK1) can suppress the production of IgA in B cells by accelerating the degradation of NIK, which plays an important role in the NFκB noncanonical pathway (68,69). Our data showed that in crescentic GN, an inhibitor of IKK2, unexpectedly accelerated the disease, which may have resulted from Treg cell impairment (70). In another experimental model of crescentic GN, deficiency in NFκB1 (p50) increased the severity of acute glomerular injury but NFκB1 did not influence the chronic, fibrotic phase (71). ...
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The global burden of chronic kidney disease will increase during the next century. As NFκB, first described more than 30 years ago, plays a major role in immune and non-immune-mediated diseases and in inflammatory and metabolic disorders, this review article summarizes current knowledge on the role of NFκB in in vivo kidney injury and describes the new and so far not completely understood crosstalk between canonical and non-canonical NFκB pathways in T-lymphocyte activation in renal disease.
... Subsequently, antibodies against foreign IgG and T cells accumulate in the kidney and cause crescentic GN within 1-2 wk. NTN is associated with renal Treg cell accumulation, and mice with depleted or functionally impaired Treg cell populations develop aggravated crescentic GN (6,9,17,21,23,26,27,29,36). On the other hand, mice with increased Treg cell populations are protected (38,40), which is also the case when Treg cells are expanded by anti-IL-2/IL-2 treatment (Fig. 7). ...
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Anti-glomerular basement membrane (anti-GBM) disease is characterized by antibodies and T cells directed against the Goodpasture antigen, the noncollagenous domain of the α3-chain of type IV collagen [α3(IV)NC1] of the GBM. Consequences are the deposition of autoantibodies along the GBM and the development of crescentic glomerulonephritis (GN) with rapid loss of renal function. Forkhead box protein P3 (Foxp3)+ regulatory T (Treg) cells are crucial for the maintenance of peripheral tolerance to self-antigens and the prevention of immunopathology. Here, we use the mouse model of experimental autoimmune GN to characterize the role of Treg cells in anti-GBM disease. Immunization of DBA/1 mice with α3(IV)NC1 induced the formation of α3(IV)NC1-specific T cells and antibodies and, after 8-10 wk, the development of crescentic GN. Immunization resulted in increased frequencies of peripheral Treg cells and renal accumulation of these cells in the stage of acute GN. Depletion of Treg cells during immunization led to enhanced generation of α3(IV)NC1-specific antibodies and T cells and to aggravated GN. In contrast, depletion or expansion of the Treg cell population in mice with established autoimmunity had only minor consequences for renal inflammation and did not alter the severity of GN. In conclusion, our results indicate that in anti-GBM disease, Treg cells restrict the induction of autoimmunity against α3(IV)NC1. However, Treg cells are inefficient in preventing crescentic GN after autoimmunity has been established.
... 99 However, in a mouse model of crescentic GN, inhibition of NF-kB prior to disease induction resulted in improved disease phenotype, but treatment after onset of disease resulted in marked worsening of disease with higher proteinuria, lower creatinine clearance, and greater severity of histology. 100 Further work investigating the role of this complex pathway is needed before translation to clinical studies. ...
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Regulatory T cells (Tregs) exert their immunosuppressive activity through several immunoregulatory mechanisms, including the production of anti-inflammatory cytokines such as IL-10. Although several studies suggest a role for Tregs in modulating crescentic GN, the underlying mechanisms are not well understood. Here, using IL-10 reporter mice, we detected IL-10-producing Foxp3(+) T cells in the kidney, blood, and secondary lymphoid tissue in a mouse model of crescentic GN. Specific inactivation of Il10 in Foxp3(+) Tregs eliminated the ability of these cells to suppress renal and systemic production of IFNγ and IL-17; these IL-10-deficient Tregs lost their capacity to attenuate renal tissue injury. These data highlight the suppressive functions of Tregs in crescentic GN and suggest the importance of Treg-derived IL-10 in ameliorating disease severity and in modulating both the Th1 and most notably Th17 immune response.
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Authors: A.B. and D.B. contributed equally to this work. Effector functions of inflammatory IL-17-producing Th (Th17) cells have been linked to autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). However, what determines Th17 cell encephalitogenicity is still unresolved. Here, we show that after EAE induction, mice deficient for the NF-κB regulator MALT1 (Malt1-/- mice) exhibit strong lymphocytic infiltration in the CNS, but do not develop any clinical signs of EAE. Loss of Malt1 interfered with expression of the Th17 effector cytokines IL-17 and GM-CSF both in vitro and in vivo. In line with their impaired GM-CSF secretion, Malt1-/- Th cells failed to recruit myeloid cells to the CNS to sustain neuroinflammation, whereas autoreactive WT Th cells successfully induced EAE in Malt1-/- hosts. In contrast, Malt1 deficiency did not affect Th1 cells. Despite their significantly decreased secretion of Th17 effector cytokines, Malt1-/- Th17 cells showed normal expression of lineage-specific transcription factors. Malt1-/- Th cells failed to cleave RelB, a suppressor of canonical NF-κB, and exhibited altered cellular localization of this protein. Our results indicate that MALT1 is a central, cell-intrinsic factor that determines the encephalitogenic potential of inflammatory Th17 cells in vivo.
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Inflammation conveys the development of glomerular injury and is a major cause of progressive kidney disease. NF-κB signaling is among the most important regulators of pro-inflammatory signaling. Its role in podocytes, the epithelial cells at the kidney filtration barrier, is poorly understood. Here, we inhibited NF-κB signaling in podocytes by specific ablation of the NF-κB essential modulator (NEMO, IKKγ). Podocyte-specific NEMO-deficient mice (NEMO(pko)) were viable and did not show proteinuria or overt changes in kidney morphology. After induction of glomerulonephritis both NEMO(pko) and control mice developed significant proteinuria. However, NEMO(pko) mice recovered much faster, showing rapid remission of proteinuria and restoration of podocyte morphology. Interestingly, quantification of infiltrating macrophages, T-lymphocytes and granulocytes at day 7 revealed no significant difference between wild-type and NEMO(pko). To further investigate the underlying mechanisms we created a stable NEMO knock-down mouse podocyte cell line. Again, no overt changes in morphology were observed. Translocation of NF-κB to the nucleus after stimulation with TNFα or IL-1was sufficiently inhibited. Moreover, secretion of proinflammatory chemokines from podocytes after stimulation with TNFα or IL-1 was significantly reduced in NEMO deficient podocytes and in glomerular samples obtained at day 7 after induction of nephrotoxic nephritis. Collectively, these results show that proinflammatory activity of NF-κB in podocytes aggravates proteinuria in experimental glomerulonephritis in mice. Based on these data it may be speculated that immunosuppressive drugs may not only target professional immune cells but also podocytes directly to convey their beneficial effects in various types of glomerulonephritis.
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Migratory non-lymphoid tissue dendritic cells (NLT-DCs) transport antigens to lymph nodes (LNs) and are required for protective immune responses in the context of inflammation and to promote tolerance to self-antigens in steady-state. However, the molecular mechanisms that elicit steady-state NLT-DC maturation and migration are unknown. By comparing the transcriptome of NLT-DCs in the skin with their migratory counterparts in draining LNs, we have identified a novel NF-κB-regulated gene network specific to migratory DCs. We show that targeted deletion of IKKβ in DCs, a major activator of NF-κB, prevents NLT-DC accumulation in LNs and compromises regulatory T cell conversion in vivo. This was associated with impaired tolerance and autoimmunity. NF-κB is generally considered the prototypical pro-inflammatory transcription factor, but this study describes a role for NF-κB signaling in DCs for immune homeostasis and tolerance that could have implications in autoimmune diseases and immunity. Copyright © 2015 Elsevier Inc. All rights reserved.
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Immune responses in the skin are important for host defence against pathogenic microorganisms. However, dysregulated immune reactions can cause chronic inflammatory skin diseases. Extensive crosstalk between the different cellular and microbial components of the skin regulates local immune responses to ensure efficient host defence, to maintain and restore homeostasis, and to prevent chronic disease. In this Review, we discuss recent findings that highlight the complex regulatory networks that control skin immunity, and we provide new paradigms for the mechanisms that regulate skin immune responses in host defence and in chronic inflammation.
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The NF-kappa B signal transduction pathway is best known as a major regulator of innate and adaptive immune responses, yet there is a growing appreciation of its importance in immune cell development, particularly of T lineage cells. In this Review, we discuss how the temporal regulation of NF-kappa B controls the stepwise differentiation and antigen-dependent selection of conventional and specialized subsets of T cells in response to T cell receptor and costimulatory, cytokine and growth factor signals.
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The nuclear factor-κB (NF-κB) family of transcription factors plays important roles in various biological processes including apoptosis, stress response, immunity, and inflammation. NF-κB signaling is involved in both immune cell development and function, and it is critical in modulation of the immune response through the transcriptional regulation of cytokine and chemokine expression. An area of great interest in T-cell-mediated adaptive immunity is the ability of naive CD4(+) T cells generated in the thymus to differentiate into various subsets including T-helper 1 (Th1), Th2, Th17, Th9, follicular helper T (Tfh), Th22, and regulatory T (Treg) cells, upon encountering different pathogens and microenvironments. In this review, we discuss the role of NF-κB pathway in the development and functional divergence of the different helper T-cell subsets as well as in regulatory T cells.