MaruYama Takashi Gifu Univ School of Med Introduction Nuclear I κ B family proteins form a complex with NF κ B and control NF κ B target gene regulation I κ B ζ a member of this protein family can be induced in T cells in response to TGF ID: 908909
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Slide1
I
k
BNS controls Th17 differentiation and Experimental Autoimmune Encephalomyelitis
MaruYama
, Takashi (Gifu Univ., School of Med.)
Introduction
Nuclear I
κ
B family proteins form a complex with NF-
κB and control NF-κB target gene regulation. IκB-ζ, a member of this protein family, can be induced in T cells in response to TGF-β+IL-6 and positively regulates Th17 differentiation. Thus, IκB-ζ-deficient mice are able to resist some Th17-dependent autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE). However, these mice develop different types of autoimmune disorders such as Sjogren syndrome with advanced age, limiting the utility of IκB-ζ as a therapeutic target. In this study, we focus on another nuclear IκB family protein, IκBNS (IκBNS is a most similar homolog of IκB-ζ) and asked whether IκBNS have a potential therapeutic target of EAE without any immune dysregulations.
Ankyrin
-repeats
Ankyrin
-repeats
Transactivation
domain
Nuclear
Localize
S
ignal
I
k
B
-
z
I
k
B
NS
43% match
・
Less
Th17 differentiation
Nature
. 2010
29;464:1381-5
・
Sjogren
syndrome like diseases
Immunity
.
2013 21;38:450-60
.
・
Not observe immune dysregulation
with ages
Deficient mice
Materials & Methods
Plasmids:
Expression
vectors encoding FLAG-tagged mouse
I
B-
were constructed as described previously (
JBC
2005 280
:
1678-1687
)
.
Mouse I
B
NS
was inserted into a pcDNA3-FLAG vector at the
EcoR
I
and
BamH
I
sites.
M
ouse
IL-17A promoter (-6647 to +1) was inserted into a pGL4.12 vector at the
Nhe
I
&
Hin
dIII
sites.
EAE induction
:
M
ice
were injected subcutaneously (on the lower back) on day 0 with emulsions containing complete Freund’s adjuvant (
CFA),
100
μg
MOG
peptide,
and 0.5 mg
Mycobacterium tuberculosis
H37RA.
In addition, these mice received 500 ng pertussis
toxin
by
i.p
. injection to boost immunological responses on day 0 and
2.
In vitro
T cell
culture:
Purified
CD4
+
CD25
-
T cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf
serum
at
37°C
in 5%
CO
2
.
Th0 condition:
anti-CD3 (1
μg
/mL) + anti-CD28 (1
μg
/mL)
stimulation.
Th17 condition:
anti-CD3 (1
μg
/mL) + anti-CD28 (1
μg
/mL) with TGF-
1 (2 ng/mL) and IL-6 (50 ng/mL)
stimulation.
Data Analysis
: Data (Fig.1-3)
shown represent mean ± S.E. Paired data were evaluated using the Student’s t test. *
p
< 0.05, **
p
< 0.01.
Results
Fig.2 Less severity in
I
k
B
NS
deficient T cells
transfer model
(
A
) Diagram of methods of Passive EAE models.
(
B
) Disease progression of EAE
(
n =
3-4)
EAE
model in
I
k
B
NS
deficient (
Nfkbid
-/-
) mice
Fig.1
I
k
B
NS
deficient mice have resistant to EAE
(
A
) Disease progression of EAE in
Nfkbid
+/+
(n = 11–13) and
Nfkbid
-/-
mice (n = 9–11). (
B-C
)
Analysis of mice 12 days after immunization. (
B
) Measurement
of IL-17A
supernatant
concentrations by ELISAs (
Nfkbid
+/+
: n = 5;
Nfkbid
-/-
: n = 6), using cultured draining LNs incubated in the presence or absence of MOG peptide (10 ng/ml) for 72h.
(
C
)
Histology of spinal cord specimens in EAE models.
S
ections
were stained with
hematoxylin
and eosin (HE
) or
Klüver
-Barrera staining (
KB)
A
B
C
Passive EAE model using CD4
+
T cells
MOG+CFA
12 days
after immunization
Inguinal
draining LNs
Cultured
in the presence of
MOG
Purified CD4+ T cells
& transfer to WT mice
A
B
Th17 generation
In vitro
S
pleen
Naïve CD4
+
T
Cultured
With/without
TGF-
b
+IL-6
ELISA (A)
Supernatant
RT-PCR (B)
Cells
A
B
Fig.3
I
k
B
NS
deficient T cells fail to
generate Th17
(
A, B
)
Expression of IL-17A
protein (
A
)
or
Il-17a
mRNA
(
B
) in
CD4
+
T cells from
Nfkbid
+/+
and
Nfkbid
-/-
mice, cultured for 48 h under Th0 or Th17 conditions.
Transcriptional activity of
I
k
B
NS
to IL-17A gene
TCR
ROR
g
t
I
k
B
-
z
TGF-
b
IL-17 A
IL-6
I
k
B
NS
?
A
B
Fig.4
I
k
B
NS
did not have transcriptional activity to IL-17A
(
A
) Schematic of Th17 development controlled by nuclear
I
k
B
family
proteins.
(
B
)
Il-17a
reporter
activity presented as the fold-increase over that observed in HEK293 cells transfected with the empty vector.
Data shown are the mean ± S.D. of duplicate samples and are representative of 3 independent experiments.
Status of Acetyl Histone & Th17 master regulator ‘
ROR
g
t
’ expression
Fig.5
I
k
B
NS
deficient T cells were less acetylation of CNS1 &
ROR
g
t
expression
(
A
) Scheme of IL-17A gene conservation. (
B
)
ChIP
analysis of H3K27Ac. Cells were cultured under
Th17
conditions for 48 h.
(
C
)
RORγt
expression in CD4
+
T cells from
Nfkbid
+/+
and
Nfkbid
-/-
mice, cultured for 72 h under
Th17
conditions. Data are representative of three independent experiments.
S
pleen
Naïve CD4
+
T
Cultured (48h)
With
TGF-
b
+IL-6
ChIP
(B)
Cells
FACS (C)
ROR
g
t
&
I
k
B
-
z
binding element
A
B
C
Conclusions
O
ur
results indicate that
Nfkbid
-/-
T cells showed impaired Th17 cells differentiation because of a reduction in
RORγt
expression and histone H3 acetylation in the CNS 1 region. In conclusion, we show that I
BNS deficiency causes resistance to Th17-dependent autoimmune disease through the control of Th17 differentiation.PLoS One 2014 Oct 27;9(10):e110838.
Acknowledgements
I am
grateful to Dr.
Muta
,
Tatsushi
(Tohoku University) for supervising this research and for helpful discussions. We are also grateful to Mr.
Goto
,
Yasuyuki
(Tohoku University) and Ms. Takahashi, Kyoko (Gifu University) for providing technical assistance.