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671
Figure legends
672
Fig. 1
673
KikGR mice
674
The mice were administered ad libitum 2% (w/v) DSS in drinking water to induce
675
colitis for 7 days (Day 0 to Day 7) and then given drinking water for 2 days (Day 8 and
676
Day 9). From 7 days before Day 0, the mice were daily administered 100 μl of lentinan
677
(200 µg/mouse) or sterilized water as a vehicle via an oral route for 14 days. On day 8,
678
the region of the intestine was exposed to violet light (405 nm, 54 mW/cm2) for 2
679
minutes from the front and behind following laparotomy. The mice were killed on day
680
10. The cells were incubated with Mouse BD Fc BlockTM (BD Pharmingen) to block Fc
681
binding, then stained with Allophycocyanin (APC) (for CD4) mAb. (A) The level of
682
KikGR-Red cells (%) in lymphocyte of the ileum and the colon in the mice. The level of
683
CD4+ cells in the ileum (B), and the colon of the mice (C). Moreover, the cells were
684
stained with FITC-labeled (for CD25) mAbs, and then with Brilliant Violet 421-
685
conjugated anti-FoxP3 antibody (D). Data are expressed as mean ± SE (n=3). For
686
statistical analysis, a one-way ANOVA followed by the Tukey multiple comparisons test
687
was used; there are significant differences between different letters (p < 0.05).
Migratory of CD4+ cells from ileum to colon in the DSS-induced colitis
688
41
689
Fig. 2
690
in mice
691
Mice (C57BL/6NCrSlc) were administered ad libitum 2% (w/v) DSS in drinking water
692
to induce colitis for 7 days (Day 0 to Day 7) and then given drinking water for 2 days
693
(Day 8 and Day 9). From 7 days before Day 0, the mice were daily administered 50 μl
694
of lentinan (100 µg/mouse) or sterilized water as a vehicle via an oral or rectal route.
695
The body weight of mice was measured daily during the experiment and expressed as a
696
percentage of that on Day 0. Data are expressed as mean ± SE (n=4). *, P < 0.05; **, P
697
< 0.01; ***, P < 0.001, DSS (+), lentinan by a rectal route group (
698
lentinan by an oral route group ( ) versus DSS (+), vehicle group ( ). #, P < 0.05
699
DSS (+), lentinan by a rectal route group (
700
group ( ).
Effects of oral or rectal administration of lentinan on DSS-induced colitis
) or DSS (+),
) versus DSS (+), lentinan by an oral route
701
702
Fig. 3
703
colon
704
Mice were sacrificed on Day 9. Total RNA was extracted from the colon, and Ifng, Il1b,
705
Tnf, Il6, Il4, and, Il10 expression levels were determined by real-time RT-PCR, as
706
described in the Materials and Methods. The values except for Il4 were calculated as
Effects of lentinan administration on cytokine expression levels in the
42
707
relative expression levels to untreated controls without DSS or lentinan. The values for
708
Il4 were calculated as the relative expression levels to mice treated with DSS but not
709
lentinan. Data are expressed as mean ± SE (n=4).
710
711
Fig. 4
712
ilium
713
The total RNA was extracted from the ilium of the same mice as analyzed in Fig. 3, and
714
Ifng, and Il4 expression levels were determined by real-time RT-PCR, as described in
715
the Materials and Methods. The values were calculated as relative expression levels to
716
those in untreated controls with DSS or lentinan. Data are expressed as mean ± SE
717
(n=4).
Effects of lentinan administration on cytokine expression levels in the
718
719
Fig. 5
720
expression in normal mice
721
Mice were daily administered lentinan (100 µg/mouse) for 16 days or sterilized water as
722
a vehicle without DSS treatment via oral or rectal routes. Total RNA was extracted from
723
the ilium (A) and colon (B) of mice, and Ifng expression levels were analyzed by real-
724
time RT-PCR, as described in the Materials and Methods. The values were calculated as
Different effects of oral and rectal administration of lentinan on Ifng
43
725
relative expression levels to the vehicle control. Data are expressed as mean ± SE (n=4).
726
*, P < 0.05; **, P < 0.01 versus vehicle control group.
727
728
Fig. 6
729
Tbx21 expression levels in the ilium of the same mice as used in Fig. 5 were analyzed
730
by semi-quantitative RT-PCR as described in the Materials and Methods. The values
731
were calculated as relative expression levels to the vehicle control. Data are expressed
732
as mean ± SE (n=4). *, P < 0.05 versus vehicle control group.
Effects of lentinan administration on Tbx21 expression in the ilium
733
734
Fig. 7
735
colon
736
Il12b expression levels in the ilium (A) and colon (B) of the same mice as used in Fig. 5
737
were analyzed by real-time RT-PCR, as described in the Materials and Methods. The
738
values were calculated as relative expression levels to the vehicle control. Data are
739
expressed as mean ± SE (n=4). *, P < 0.05 versus vehicle control group.
Effects of lentinan administration on Il12b expression in the ilium and
740
741
Fig. 8
742
Caco-2 cells in a co-culture system of Caco-2 cells and BMDCs were treated with 100
Effects of lentinan on Il-12b expression in BMDCs in vitro
44
743
µg lentinan, as described in the Materials and Methods. The expression levels of TNF
744
receptor1 in Caco-2 cells (A) and Il12b in BMDCs (B) were analyzed by real-time RT-
745
PCR 6 h after treatment. The values were calculated as relative expression levels to the
746
vehicle control. Data are expressed as mean ± SE (n=3). (C) BMDCs were cultured as a
747
monolayer and received by direct treatment with 100 µg lentinan. Il12b expression
748
levels in BMDCs were analyzed by real-time RT-PCR 6 h after treatments. The values
749
were calculated as relative expression levels to the vehicle control. Data are expressed
750
as mean ± SE (n=3). *, P < 0.05; **, P < 0.01 versus vehicle control.
751
752
Fig. 9. The oral administration of lentinan induces the Th1 immune responses
753
mainly in the ileum, and then exerts the suppressive effects on a colitis
754
In this study, it was ascertained that oral administration of lentinan enhanced Th1-type
755
immune responses mainly at ileum and then accelerate the migration of CD4+ cells in
756
lymphocyte from ileum into the colon in which colitis was induced by DSS treatment.
757
These findings suggested that alternation of the immunological environment in the
758
small intestine affects the colon, resulting in suppressive effects on colitis. The present
759
results indicate the possibility that colitis might be controlled from the small intestine by
760
food intake, such as lentinan.
45
761
762
Figure S1. Expression of KikGR-Green and KikGR-Red in the murine lymphocytes
763
before and after photoconversion.
764
Upon exposure violet light for 2 minutes, cells were converted from green (KikGR-
765
Green) to red (KikGR-Red).
766
46
FoxP3+ cell
(% in KikGR-red D4+ CD25+
lymphocyte)
CTRL
10
LNT
DSS
Ileum
10
25
+L
ileum
SS
+L
SS
B)
SS
15
LN
TR
15
SS
LN
TR
SS
+L
a,b
SS
LN
TR
20
KikGR-red cell
(% in Lymphocyte)
A)
Ileum
Colon
10
C)
colon
20
15
D)
Colon
DSS + LNT
Up
Fig. 1
1１0
Relative weight (%)
100
**
***
***
90
DSS(-), vehicle
DSS(-), lentinan by a rectal route
DSS(+), vehicle
DSS(+), lentinan by a rectal route
DSS(+), lentinan by an oral route
80
70
Days after treatment with DSS
Up
Fig. 2
600
Il1b expression
50
40
30
20
400
300
200
100
250
200
1.5
100
50
Il10 expression
50
1.0
0.5
150
Il4 expression
500
10
Il6 expression
Ifng expression
700
Tnf expression
60
N.D. N.D.
30
20
10
2% DSS
Lentinan
－ Rectal － Rectal Oral
40
2% DSS
Lentinan
－ Rectal － Rectal Oral
Up
Fig. 3
2.5
2.0
Il4 expression
Ifng expression
1.5
1.0
0.5
2% DSS
2% DSS
Lentinan
Rectal
Oral
Lentinan
Rectal
Oral
Up
Fig. 4
Ileum
Ifng expression
A)
**
1.5
1.0
Lentinan
0.5
Oral
Lentinan
Rectal
Rectal
Colon
Ifng expression
B)
1.5
1.0
0.5
Lentinan
Oral
Lentinan
Up
Fig. 5
Lentinan (－)
Orally administered
Lentinan
Tbx21
Hprt
Tbx21 expression
2.5
2.0
1.5
1.0
0.5
Lentinan
Oral
Up
Fig. 6
Ileum
Il12b expression
A)
1.5
1.0
0.5
Lentinan
Oral
Lentinan
Rectal
Rectal
Colon
1.5
2.0
Il12b expression
1.5
1.0
1.0
0.5
2.
B)
0.5
Lentinan
Oral
Lentinan
Up
Fig. 7
1.2
tnfr1 expression
A)
1.0
0.8
0.6
0.4
0.2
1.5
Il12b expression
B)
1.0
0.5
**
Il12b expression
C)
Vehicle
Lentinan
Up
Fig. 8
Th0
Differentiation
IL-12
Th1
Migration
Th1
Dendritic cell
Th2
TNFR1
Colitis
Dectin-1
Intestinal epithelial cells
Ileum
colon
Digestive tract lumen
Lentinan
Lentinan
Up
Fig. 9
...
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