Soluble CD83 Ameliorates Experimental Colitis in Mice
Figure S1: CD83+ cells in inflammed lamina propria. Flow cytometry analyses of LP
infiltrating cells in DNBS/ethanol-treated CD83eGFP reporter mice 72h after
treatment. Mononuclear cells from intestinal lamina propria were isolated using the
Lamina Propria Dissociation Kit mouse (Miltenyi Biotec). Gating strategies for (A)
CD83+ cells, (C) B220+ B cells, CD4+/CD8+ T cells, CD11c+ DC and CD11b+ cells
and (E) DC subpopulations, monocyte/macrophages, eosinophils and neutrophils are
shown. Average distribution of CD83eGFP+ cells in LP (B) together with CD83eGFP+
subpopulations (D, F) were monitored by flow cytometry (n=5).
Figure S2: sCD83-treatment regime of experimental colitis. The animals received 100
µg sCD83 on day -1, 0 and +1 i.p. in saline solution. Colitis was induced by intra
rectal administration of 4 mg DNBS in 50% ethanol on day 0. The mice have been
observed for clinical symptoms and reweighed daily. On day 3 the animals have been
euthanized and colon and mesenteric lymph nodes were prepared for further analysis
by histology, RNA analysis and flow cytometry.
Figure S3: sCD83 treatment inhibits leukocyte proliferation without impact on
migration and apoptosis. (A) additional experiment to Fig. 4: Foxp3-specific qRT-PCR
using colonic tissue after DNBS-induced colitis on day 3. mean ± SEM, n = 5-7
mice/group, data normalized to HPRT; (B) total splenocytes of Balb/c mice were
stimulated for 72h with anti-CD3/anti-CD28 Dynabeads (Lifetechnologies) adding
sCD83 (25µg/ml) or buffer control and their proliferation was monitored using H3Thymidine incorporation, n=6, ***p < 0.001, ANOVA Tukey´s multiple comparison
test; (C) qRT-PCR for CCR9 and CD49d expression on colon and MLN after DNBSinduced colitis on day 3 (mean ± SEM, n=5-10 mice/group, data normalized to HPRT;
control [open bar], DNBS [black bar], DNBS+sCD83 [grey bar]; *p < 0.05, ANOVA
Tukey´s multiple comparison test; (D) transwell migration assay of LPS-stimulated
BM-DC from Balb/c mice towards a CCL19 gradient. Migration was determined by
measuring the β-glucuronidase activity of migrated cells compared to the standard
curve; n=2 mice/group; (E) ex vivo apoptosis assay using flow cytometry: Balb/c mice
received 100 µg sCD83 on 3 days (i.p.) in saline solution. On day 4 spleens were
prepared for flow cytometry using fluorochrome-conjugated antibodies against CD3,
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Soluble CD83 Ameliorates Experimental Colitis in Mice
CD11b, CD11c, CD19 to distinguish major leukocyte populations, and 7-AAD
together with Annexin V to monitor cell survival as follows: 7-AAD-/Annexin V- =
healthy, 7-AAD-/Annexin V+ = apoptotic
AAD+/Annexin
V-
=
dead;
n=5
mice
and
/group;
7-AAD+/Annexin V+ plus 7(F)
Representative
colonic
photomicrographs of characteristic Ki67 (top) or caspase-3 (middle) and caspase-8
(bottom) are shown at day 4 post first sCD83 i.p. treatment (right) or buffer control
(left) (as described above). Ki67, caspase-3 and -8 immunofluorescence is depicted
in red and Hoechst 33342 nuclear staining is depicted in blue. n=5 for each condition.
Original magnification 20×.
Figure S4: IDO inhibitor 1-MT diminished protective effects of sCD83 and increases
weight loss in DNBS-treated mice. sCD83- or control- treated DNBS-induced colitis
mice were pre-implanted with 1-MT impregnated pellets or vehicle (placebo) and
monitored from day 0 to day 3 for (A, B) mortality, and (C) weight loss: total overview
of
all
groups
(upper
left),
only
ethanol,
DNBS+1-MT+sCD83
and
DNBS+Placebo+sCD83 group (upper right), and weight loss at day 3 of
DNBS+Placebo vs. DNBS+1-MT and ethanol control; n=10-17 mice/group; mean ±
SEM; *p < 0.05, ***p < 0.001, ANOVA Tukey´s multiple comparison test.
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