Activation of

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Activation of ß-Catenin in Dendritic Cells
Regulates Immunity Versus Tolerance in the
Intestine

• Santhakumar Manicassamy,1 Boris Reizis,2 Rajesh
  Ravindran,1 Helder Nakaya,1 Rosa Maria
  Salazar-Gonzalez,1,3 Yi-chong Wang,1 Bali
  Pulendran1,4

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Immune Cells
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Tolerance vs. Immunity
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Cytokine Expression
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(No Transcript)
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Pathogenesis of Inflammatory Bowel Disease
In normal conditions, a balance is established
between the generation of proinflammatory (TH1
TH2, TH17) and anti-inflammatory (TH3, Treg1) T
cells
Mediators indicated in blue (boldface)
down-regulate inflammation, and those indicated
in red promote inflammation.
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Pathogenesis of Inflammatory Bowel Disease
Induction of Inflammatory Cascade Bacterial
antigens inappropriately leak across the
intestinal epithelial cell barrier. Tissue
macrophages and dendritic cells (DCs) present
these antigens to resident CD4 T cells and
activate proinflammatory T cells relative to
regulatory T cells (Tr1, TH3, and NK-T cells)
leading to excess proinflammatory cytokine
release (IL-1, TNF-a) from the macrophages.
Activation of the immune and vascular systems is
regulated by nerve cells and their mediators
(substance P). NK-T cell indicates natural killer
T cell TNF, tumor necrosis factor.
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S1
Analysis of gene expression profiles using
microarray analysis of purified intestinal lamina
propria DCs (LP-DCs) and compared with those of
splenic DCs (spl-DCs) mice.

• Several Wnt-ligands were up-regulated in LP-DCs
  as compared with spl-DCs. Consistent with this,
  reverse transcriptase polymerase chain reaction
  (RT-PCR) analysis showed that LP-DCs
  constitutively expressed several Wnt-ligands as
  compared with that of spl-DCs in the steady state
  (fig. S1)

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Background

• ß-catenin, a central component in Wnt signaling,
  is widely expressed in hematopoietic stem cells,
  macrophages, DCs, and lymphocytes.
• The Wntß-catenin pathway has been implicated in
  the differentiation of DCs from hematopoietic
  stem cells.
• Activation of Wnt-Frizzled (fzd) receptor
  signaling causes translocation of ß-catenin from
  the cytoplasm to the nucleus, where it interacts
  with T cell factor/ lymphoid enhancer factor
  (TCF/LEF) family members and regulates
  transcription of several target genes

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S2
9. Mucosa10. Lamina propria11. Epithelium

• Assessment of whether the ß-catenin pathway was
  active in intestinal Lamina Propria-DCs using the
  T Cell Factor/Lymphoid Enhancer FactorlacZ
  reporter mice. In contrast to splenic-DCs, LP-DCs
  from TCF-reporter mice showed strong
  ß-galactosidase expression, suggesting that
  ß-catenin signaling pathway is constitutively
  active in intestinal DCs (fig. S2).

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s3A

• To directly assess the role of ß-catenin
  specifically
• in DC function, they crossed floxed ß-catenin
  allele mice (ß-catfl/fl) with transgenic mice
  (CD11c-cre) expressing the cre enzyme under the
  control of the CD11c promoter . This
  specifically abrogated ß-catenin expression in
  DCs (fig. S3A).

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Conditional targeting construct
In this cartoon, the loxP sites are represented
by arrowheads, the first and second exons by open
boxes, introns by lines, and the targeted gene by
the shaded box.
Cre enzyme is introduced by transient
transfection with a Cre-expressing plasmid CD11c
under the control of CD11c promoter PCR is then
used to find clones that are lacking the targeted
gene but still have the first and second loxP
sites, as illustrated by the next cartoon.
The ES cells resulting from the partial
recombination reaction illustrated above are said
to have a floxed gene, meaning the gene of
interest has one or more exons flanked by loxP
sites (flox flanked by loxP). When a mouse is
made from these ES cells, it has an almost
completely wildtype allele that can be knocked
out by the expression of Cre inside its cells
(assuming removal of the first exon results in a
knockout).
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S3B
Distinct subsets of intestinal DCs and
macrophages can be distinguished by the
expression patterns of CD11c and CD11b
(CD11cCD11b and CD11cCD11b DCs and
CD11cCD11b macrophages)

• ß-catenin was highly expressed and
  constitutively active in LP-DC subsets and LP
  macrophages in the small and large intestine
  (Fig. 1A and fig. S3B). In ß-catDC-/- mice,
  ß-catenin expression was abrogated in the
  CD11cCD11b and CD11cCD11b DCs but only
  partially abrogated in CD11cCD11b macrophages
  (fig S3B).

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Figure 1A
ß-catenin signaling is constitutively active in
intestinal APCs and regulates the induction of
Treg cells and effector T cells.
(A) Expression of ß-galactosidase
(representing ß-catenin activity) by intestinal
DCs and macrophages from SI-LP and LI-LP of
TCF-reporter mice.
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S4A

• Whether ß-catenin signaling in DCs is critical
  for intestinal homeostasis.
• We compared the frequencies of Treg cells and
  TH17/TH1 cells in the intestine of ß-catDC-/- and
  ß-catfl/fl mice because intestinal DCs and
  macrophages
• play an important role in the induction of these
  subsets. ß-catDC-/- mice had
• lower frequencies of Treg cells in the small
  intestine (SI)LP, the large intestine (LI)LP,
  and caecum, but not the spleen, as compared with
  that
• of ß-catfl/fl mice (Fig. 1B and fig S4A).

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Figure 1B
(1B and 1C) Percentages of CD4 T cells positive
for FoxP3 (Treg cells), IL-17 (TH17), and IFN-g
(TH1) isolated from Small Intestine-LP, Large
Intestine-LP, Ceacum, and Spleen of ß-catfl/fl
and ß-catDC-/- mice.
Fluorescence activated cell sorting (FACS) plots
Numbers in FACS plots represent of cells
positive for the indicated protein.
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S4B,C
To determine whether the reduced frequencies of
Treg cells observed in the LP of ß-catDC-/- mice
was due to increased frequencies of effector CD4
cells, we examined the frequencies of TH1 and
TH17 in the intestine and periphery.
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Figure 1C
They observed higher frequencies of TH17 and TH1
cells in the SI-LP and LI-LP but not in the
spleen of ß-catDC-/- mice as compared with that
of the ß-catfl/fl mice (Fig. 1C and fig. S4, B
and C).
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S5
ß-catenin signaling in intestinal DCs is critical
in maintaining the balance between Treg cells and
CD4 T effector populations
CD4 T cells isolated from SI-LP and LI-LP of
ß-catDC-/- mice showed elevated expression of the
TH17 cellassociated mRNAs interleukin (IL)17,
IL-21, Rorgt, and the TH1 cellassociated mRNA
interferon (IFN)g (23, 24) as compared with the
CD4 T cells isolated from ß-catfl/fl mice.
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Figure 1D
(D and E) Intracellular expression of FoxP3,
IL-17, and IFN-g in naïve CD4OT-II T cells
stimulated to differentiate in vitro by
intestinal APCs (CD11cCD11b and CD11cCD11b
DCs and CD11cCD11b macrophages) isolated from
ß-catfl/fl or ß-catDC-/- mice, in the presence of
TGF-ß (1 ng/ml
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Figure 1E
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Figure 2A
Fig. 2. ß-catenin signaling in intestinal DCs
promotes the expression of Raldh and suppresses
the expression of proinflammatory cytokines.
(A) Expression of Aldh1a1 and Aldh1a2 mRNA in
CD11cCD11b and CD11cCD11b DCs and
CD11cCD11b macrophages isolated from SI-LP of
ß-catfl/fl or ß-catDC-/- mice.
Retinal dehydrogenase (RALDH) catalyzes the
oxidation of retinal to all-trans and 9-cis
retinoic acid, which function as ligands
controlling RAR and RXR nuclear
receptor-signaling pathways.
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S11
Because ß-catDC-/- LP-DCs had reduced levels of
vitamin Ametabolizing enzymes, we assessed
whether addition of exogenous RA can rescue their
defect in Treg induction. Addition of exogenous
RA to ß-catDC-/- LP-DCs did indeed enhance their
capacity to induce Treg cells as well as
ß-catfl/fl DCs (fig S11). Thus,
ß-catenin mediated signaling promotes Treg
induction through the expression of
vitaminAmetabolizing enzymes in DCs.
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Figure 2B
(B)Expression of Raldh protein by
CD11cCD11b and CD11cCD11b DCs and CD11c
CD11b macrophages isolated from SILP of
b-catfl/fl or b-catDC-/- mice, as assessed by
intracellular staining and flow cytometry.
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Figure 2C
LP-DCs had higher mRNA levels of the
pro-inflammatory cytokines IL-23a and IL-6 and
lower mRNA levels of the anti-inflammatory
cytokines IL-10 and TGF-b, as compared with that
of b-catfl/fl LP-DCs, from the SI and LI
(C) Expression of Il-10, TGf-b1, Il-23a, and Il-6
mRNAs in CD11cCD11b and CD11cCD11b DCs and
CD11cCD11b macrophages isolated from SI-LP of
ß-catfl/fl or ß-catDC-/- mice.
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Figure 2D
ß-catenin signaling in LP-DCs is critical for the
induction of anti-inflammatory cytokines, which
are crucial for the induction of Treg cells and
suppression of TH1 and TH17 responses
(D) Cytokine concentrations in the supernatants
of CD11cCD11b and CD11cCD11b DCs and
CD11cCD11b macrophages isolated from SI-LP of
b-catfl/fl or b-catDC-/- mice after 24 hours. P
lt 0.05 P lt 0.005.
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Figure 3A
Fig. 3. ß-catenin activation in intestinal DCs is
independent of commensals and induced by
Wnt-signaling.
(A and B) FACS plots representing the percentage
of CD4 T cells that express (A) FoxP3, IL-17,
mice treated with or without Antibiotics
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Figure 3B
(B) IFN-g isolated from SI-LP of ß-catfl/fl or
ß-catDC-/- mice treated with (Atx) or without
(None) antibiotics.
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Figure 3C
(C) FACS plots showing the intracellular
expression levels of ß-galactosidase or ß-catenin
protein in CD11cCD11b and CD11cCD11b DCs and
CD11cCD11b macrophages isolated from SI-LP of
TCF-reporter mice treated with or without
antibiotics.
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Figure 3D 3E
(D and E) mRNA expression of (D) Fzd receptors
and (E) Wnt ligands in CD11cCD11b and
CD11cCD11b DCs and CD11cCD11b macrophages
isolated from SI-LP of ß-catfl/fl mice.
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Figure 3F

• (F) mRNA expression levels of Wntß-catenin
  target genes Wisp1, Wisp2, and Axin1 in
  CD11cCD11b and CD11cCD11b DCs and
  CD11cCD11b
• Macrophages isolated from SI-LP of ß-catfl/fl
  mice. Error bars indicate mean T SEM. Data are
  from one experiment representative of three.

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Figure 4A
Ablation of ß-catenin expression in DCs enhanced
inflammatory responses and disease in a mouse
model of inflammatory bowel disease.
ß-catenin signaling in LP-DCs regulates
intestinal homeostasis. A) Percent body weight
of ß-catfl/fl or ß-catDC-/- mice treated with 2
DSS (Dextran Sulfate Sodium) for 7 days induced
weight loss indicative of severe inflammation.
Error bars indicate mean T SD.
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Figure 4B
(B) FACS plot representing percentage of CD4
cells positive for IL-17 and IFN-? isolated on
day 8, from Ceacum and LI-LP of ß-catfl/fl or
ß-catDC-/- mice treated with DSS for 6 days.
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Figure 4C
(C) Histopathological changes in colon tissue
from ß-catfl/fl or ß-catDC-/- mice treated with
or without 2 DSS treatment for 7 days. Areas of
interest are infiltrations, edema (yellow
arrows), goblet cells (black arrows), and
basement membrane (green arrows).
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In Summary

• Demonstrated that a signaling pathway involving
  ß-catenin in DCs regulates the balance between
  inflammatory versus regulatory responses in the
  intestine.
• The present data demonstrate that ß-catenin
  signaling in DCs is critical for maintaining DCs
  in tolerogenic state, via induction of various
  anti-inflammatory factors such as RA, IL10, and
  TGFß.
• Strategies that can activate ß-catenin signaling
  specifically in DCs may be attractive means of
  controlling autoimmune diseases such as IBD via
  the induction of Treg cells and concomitant
  suppression of inflammatory effector T cells.