SUPPLEMENTAL
IL-10 down-regulates CXCR3 expression on Th1 cells and
interferes with their migration to intestinal inflammatory sites
Munisch Wadwa,1 Robert Klopfleisch,2 Alexandra Adamczyk,1 Annika Frede,1 Eva Pastille,1
Karsten Mahnke,3 Wiebke Hansen,1 Robert Geffers,4 Karl Sebastian Lang,5 Jan Buer,1 Jürgen
Büning,6,7 Astrid M. Westendorf1,7,*
1
Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
2
Institute of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany
3
Department of Dermatology, Ruprecht-Karls University Heidelberg, Heidelberg, Germany
4
Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig, Germany
5
Institute of Immunology, Medical Faculty, University of Duisburg-Essen, Germany
6
Department of Internal Medicine I, University Hospital of Schleswig-Holstein, Lübeck, Germany
7
contributed equally to this work
Keywords: DEC-205, colitis, Th1 cells, CXCR3, IL-10
Conflict of interest: The authors declare that no conflict of interest exists.
*
Correspondence to: Astrid M. Westendorf, PhD, Infection Immunology, Institute of Medical
Microbiology, University Hospital Essen, Hufelandstrasse 55, 45122 Essen, Germany. Phone:
+492017231826; Fax: +492017235602; e-mail: astrid.westendorf@uk-essen.de
Supplemental Methods
Immunofluorescence microscopy. Endoscopically obtained biopsy samples were snapfrozen in liquid nitrogen. Cryo-sections (7 µm thick) were air-dried and fixed in acetonemethanol (1:1). Double labeling of CD4 (clone MT310; DAKO, Glostrup, Denmark) and
CXCR3 (antibodies-online, Aachen, Germany) was performed with the Zenon labeling
technique (Zenon α-mouse AlexaFluor488 and Zenon α-mouse AlexaFluor647; Invitrogen,
Karlsruhe, Germany). Hoechst 33342 dye was used to stain nuclei (Sigma-Aldrich, Bonn,
Germany). Immunofluorescence staining was analyzed by scanning with a Zeiss LSM 510
Meta confocal microscope (Jena, Germany). Eight individual fields of view were applied for
three separate sections per mucosal sample for quantitative analysis with ImageJ v1.47
software. For quantification of CD4+CXCR3+ cells the number of blue pixels (cell nuclei) was
divided by the number of yellow pixels (CD4+CXCR3+) in each image and is depicted as
relative CD4/CXCR3 expression in the bar diagrams.
Antibodies and flow cytometry. Flow cytometric analysis was performed using
antibodies against α-mouse CD4 (RM4-5), α-mouse CD11c (N418), α-mouse CD40 (3/23), αmouse CD80 (16-10A1), α-mouse CD86 (GL1), α-human CD4 (RPA-T4) (all BD Bioscience,
Heidelberg, Germany), α-mouse CXCR3 (CXCR3-173), α-mouse Vβ5.1/5.2 (MR9-4), αmouse Foxp3 (FJK-16s), α-mouse Ki-67 (SolA15) and α-human CXCR3 (CEW33D) (all
eBioscience,
San
Diego,
CA,
USA),
as
fluorescin
isothiocyanat,
pacific
blue,
allophycocyanin, PeCy7, or phycoerythrin conjugates. The monoclonal antibody 6.5 (α-TCRHA) was purified from hybridoma supernatants and coupled to the fluorochrome
AlexaFluor647 using the AlexaFluor647 Protein Labeling Kit (Life Technologies, Darmstadt,
Germany) according to the manufacturer´s instructions. Intracellular staining for Foxp3 and
Ki-67 was performed using the Foxp3 staining buffer set (eBioscience, San Diego, CA, USA)
according to the manufacturer´s recommendation. IFN-γ expression was analyzed by
stimulating cells for 4 h with 10 ng/ml PMA and 100 µg/ml ionomycin in the presence of 5
µg/ml Brefeldin A (all from Sigma-Aldrich, Bonn, Germany), treating them with 2%
paraformaldehyde and 0.1% NP-40, and staining them with α-mouse IFN-γ (XMG1.2) (BD
Bioscience, Heidelberg, Germany). Dead cell exclusion was achieved by co-staining cells
with 7AAD (eBioscience) or fixable viability dye eFluor780 (both eBioscience, San Diego,
CA, USA). FACS analysis was performed on a LSRII flow cytometer using FACS DIVA
software (BD Bioscience, Heidelberg, Germany).
RNA isolation and quantitative real-time PCR. Isolation of RNA from biopsy samples
from the small intestine and the colons was performed with the RNeasy Fibrous Tissue Mini
Kit from Qiagen (Hilden, Germany). The NucleoSpin XS Kit (Macherey-Nagel, Düren,
Germany) was used for RNA isolation from single cell suspensions according to the
manufacturer’s instructions. After RNA isolation 1 µg of whole RNA was subjected to a
reverse transcription reaction using M-MLV reverse transcriptase (Promega, Mannheim,
Germany). Quantitative real-time PCR analysis was performed with an ABI PRISM cycler
(Applied Biosystems, Foster City, CA, USA) using the Fast SYBR Green Master Mix (Life
Technologies, Darmstadt, Germany) and specific primers for CXCR3 (5'-GGA GCC CCA
CCG CCC TAC TAA A-3' and 5'-AAC GCC TGG CCC TCT GTG CTA TT-3'), CXCL9 (5'AAG TTA GCC TGT GTG GG AGC-3' and 5'-TTA CCG AAG GGA GGT GGA CA-3'),
CXCL10 (5'-CTC TCC ATA CTC CCC TTT ACC C-3' and 5'-GCT TCG GCA GTT ACT
TTT GTC TCA-3'), CXCL13 (5'-ATT CAA GTT ACG CCC CCT GG-3' and 5'-TTG GCA
CGA GGA TTC ACA CA-3'), IL-10 (5'-CTG GAC AAC ATA CTG CTA ACC GAC TC-3'
and 5'-ATT TCT GGG CCA TGC TTC TCT GC-3'), IL-10R (5'-TCA TGG TGA CAT TCC
AGG GC-3' and 5'-TGG AGG CCA AGC CAA ATC AT-3'), TGF-β (5'-ACC TGG GTT
GGA AGT GGA T-3' and 5'-GAA GCG CCC GGG TTG TGT TGG TT-3'), IL-6 (5'-ACC
ACG GCC TTC CCT ACT TC-3' and 5'-GCC ATT GCA CAA CTC TTT TCT C-3'), IL-1β
(5'-AAG GAG AAG ACC AGC CCG TGT TG-3' and 5'-TGG ATA AGC AGC TGA TGT
GAA GTA-3'), TNF-α (5'-CAA TGC ACA GCC TTC CTC ACA G-3' and 5'-CCC GGC
CTT CCA AAT AAA TAC AT-3'), and RPS-9 (5'-CTG GAC GAG GGC AAG ATG AAG
C-3' and 5'-TGA CGT TGG CGG ATG AGC ACA-3'). Relative mRNA levels were
calculated with included standard curves for each individual gene and further normalization to
the housekeeping gene RPS-9.
DNA microarray hybridization and analysis. Total RNA from sorted HA-specific
CD4+ T cells was isolated with the RNeasy kit (Qiagen, Hilden, Germany). The quality and
integrity of the total RNA were controlled with an Agilent Technologies 2100 Bioanalyzer
(Agilent Technologies, Waldbronn, Germany). Next, 500 ng of total RNA was applied for a
Cy3-labelling reaction with the one-color Quick Amp Labeling Kit (Agilent Technologies,
Waldbronn, Germany). Labeled cRNA was hybridized to Agilent’s mouse 4x44k microarrays
for 16 h at 68°C and was scanned with the Agilent DNA Microarray Scanner. Expression
values were calculated with the Feature Extraction 10.5.1.1 software package (Agilent
Technologies, Waldbronn, Germany). Statistical analysis of the expression data was
performed with the Gene Spring software package (Agilent Technologies, Waldbronn,
Germany). Clustering analysis was performed with the Genesis platform.
Histopathological analysis. Small intestines and colons were immersion fixed with 4 %
buffered formalin, embedded in paraffin, sectioned at a thickness of 4 µm, and stained with
hematoxylin and eosin. Disease severity was determined by blinded scoring of inflammation
markers such as infiltration of the lamina propria by immune cells, atrophy and fusion of
intestinal villi, cell hyperplasia, and necrosis on a scale ranging from 0 (no signs of
inflammation) to 3 (severe signs of inflammation).
Suppl. Fig.-1 Westendorf
Fig. S1 Adoptive transfer of HA-specific Th1 cells into VILLIN-HA transgenic
mice leads to severe intestinal inflammation. HA-specific CD4+ T cells from
spleens of TCR-HA mice were sorted by flow cytometry and differentiated in vitro into
Th1 effector cells using α-CD3/α-CD28 antibodies in the presence of recombinant IL12 (rIL-12) and α-IL-4 antibody. On day 6, the cells were analyzed for IFN-γ
expression by flow cytometry (white histogram, unstained control; grey histogram, αIFN-γ antibody). Dot plots from one representative experiment are shown.
Suppl. Fig.-2 Westendorf
b
-log10 p-
-log10 p-value
a
log fold change (DEC-HA vs. PBS)
log fold change (DEC-HA vs. ISO-HA)
Fig. S2 Differential gene expression of ex vivo isolated Th1 cells from DEC-HA,
ISO-HA, and PBS treated mice. VILLIN-HA transgenic mice were injected i.p. with
DEC-HA, ISO-HA, or PBS before adoptive transfer of HA-specific Th1 cells. Five
days after cell transfer, HA-specific CD4+ T cells were re-isolated from the colonic
lamina propria by flow cytometry and subjected to gene expression profiling.
Differential gene expression and statistical significance thereof were determined by
limma package using an empirical Bayes method to moderate the standard errors of
the estimated log-fold changes. Statistical errors were corrected using BonferroniHochberg method. For each condition the number of biological replicates was n=2.
The entire datasets were visualized by volcano plots: The averaged log fold change
is plotted on the x-axis and the negative log10 p-value (error-corrected p-value) is
plotted on the y-axis. Red colored dots encode for significant expression changes at
corrected p-values below 0.05. Differential Expression for the transcripts of Cxcr3
und Cxcr5 is indicated. a) DEC-HA vs. PBS; b) DEC-HA vs. ISO-HA
Suppl. Fig.-3 Westendorf
Fig. S3. DEC-OVA Treatment leads to CXCR3 down-regulation on OVA-specific
Th1 cells. Wild-type C57BL/6 mice were injected i.p. on days -2 and -1 with DECOVA or ISO-OVA. On day 0 OVA-specific Th1 cells were adoptively transferred. On
day 6 after transfer Th1 cells from the spleens were analyzed for CXCR3 expression
by flow cytometry (a). Additionally, transferred Th1 cells were re-isolated by flow
cytometry from the spleens and subjected to in vitro migration assays using
recombinant CXCL10 as chemoattractant (b). Data from one representative out of
two independent experiments is shown. Statistical analysis was performed with
Student´s t-test (*p<0.05; ***p<0.001).
Suppl. Fig.-4 Westendorf
Fig. S4 Phenotype of intestinal and splenic DCs after DEC-HA treatment.
BALB/c mice were injected i.p. with PBS, ISO-HA or DEC-HA. On the following day
DCs from the colonic lamina propria (LP), the mesenteric lymph nodes (MLNs) and
the spleen were analyzed by quantitative real-time PCR (a) or flow cytometry (b) for
the expression of cytokines and activation markers. DCs were gated for
CD45+CD11c+F4/80-. Data from two independent experiments are show as mean
arbitrary units (a) or fluorescence intensity (MFI) or percentages (b) ± SEM.
Statistical analysis was performed using one-way ANOVA (*p<0.05; **p<0.01).
Suppl. Fig.-5 Westendorf
Fig. S5 α-DEC-205 mAb or α-DEC-205 mAb coupled to an irrelevant antigen do
not protect from Th1-cell-mediated inflammation. BALB/c mice were injected i.p.
with PBS, α-DEC-205 mAb (DEC-205 mAb), α-DEC-205 mAb coupled to the βgalactosidase protein (DEC-β-gal) or DEC-HA. 24 hours later, serum of the mice was
collected and analyzed for IL-10 with Luminex technology. Additionally, CD11c+ DCs
were isolated from spleens and stimulated in vitro with LPS overnight. Stimulated DC
supernatants were then analyzed with Luminex technology for secretion of IL-10 (a)
VILLIN-HA transgenic mice were injected i.p. with PBS, DEC-205 mAb, DEC-β-gal,
or DEC-HA on days -2 and -1 before adoptive transfer of HA-specific Th1 cells. Mice
were monitored daily for clinical signs of sickness. On day 5 after transfer, the body
weight of the mice was determined (b) and histopathological analysis (c) was
performed on small intestines and colons. Data from one representative out of two
independent experiments is shown. n.d., not detectable
Suppl. Fig.-6 Westendorf
Fig. S6 IL-10 does not directly act on Th1 cells. CD4+ T cells were differentiated
in vitro into IFN-γ-producing Th1 cells. Naive CD4+ T cells and CD4+CD25+ Tregs
were sorted from naïve mice. Expression of IL-10Rα in all T cell subsets was
determined by quantitative real-time PCR analysis (a). Differentiated Th1 cells were
re-stimulated with α-CD3/α-CD28 antibodies in the absence (medium) or presence of
recombinant mouse IL-10 (rm-IL-10, 100 ng/ml) or a neutralizing α-IL-10 antibody (5
µg/ml) in vitro for 3 days. CXCR3 expression was determined by quantitative realtime PCR (b). Data from one representative out of two independent experiments are
shown as mean ± SEM.
Suppl. Fig.-7 Westendorf
Fig. S7 Co-transfer of CD4+ T cells from DEC-HA treated mice does not protect
against inflammation. HA-specific Th1 cells were adoptively transferred into
ISO-HA or DEC-HA treated BALB/c mice. Five days after transfer, CD4+ T cells were
isolated from the spleens of these mice and adoptively co-transferred with freshly
differentiated HA-specific Th1 cells into VILLIN-HA transgenic mice (Th1 + ISO-HA
CD4+ and Th1 + DEC-HA CD4+, respectively). As control VILLIN-HA mice received
HA-specific Th1 cells only (Th1 only) (a). Mice were monitored daily for loss of body
weight (b) and clinical signs of sickness. On day 5 after transfer histopathological
analysis was performed on sections of the small intestine and the colon (c).