Figure legends to supplemental material
FIGURE S1. Increased fractalkine / CX3CL1 expression in C. rodentium infected animals.
Total RNA were isolated from the colonic tissues of non-infected and C. rodentium infected B6,
(age- and sex-matched) and CX3CR1GFP/+ and CX3CR1GFP/GFP animals, reverse transcribed to
cDNA and fractalkine/CX3CL1 expression was analyzed by qRT-PCR. In the non-parametric
student´s t test p< 0.05 was considered statistically significant.
FIGURE S2. Serum anti-C. rodentium titers of infected mice. Sera were taken from every
individual mouse at sacrifice (per group 6 individual mice were analyzed). Individual sera were
assayed for antigen-specific total IgG by ELISA. In the non-parametric student´s t test p< 0.05
was considered statistically significant.
FIGURE S3. CX3CR1+ macrophages accumulate in the colonic lamina propria of C.
rodentium infected CX3CR1GFP/+ animals. A. Blood was taken from littermate controls,
heterozygous and homozygous CX3CR1-GFP animals, red blood cells lysed and stained for
CD11b, DX-5, CD3 and CD19. Histograms were gated on the indicated cell population. Filled
histograms represent blood samples from wt mice, grey open histograms from heterozygous
and black open histograms from homozygous CX3CR1-GFP animals. B. DCs and macrophages
were isolated from the colonic lamina propria of non-infected and infected homozygous
CX3CR1-GFP animals and stained for CD11c, F4/80 and CD103 and analyzed by flow
cytometry. Dot blots were gated on CD11c+ cells. Numbers indicate the percentage in the
respective histograms. C. CX3CR1-GFP expression was analyzed by gating on F4/80+CD103CD11c+, F4/80-CD103-CD11c+ and on F4/80-CD103+CD11c+ cells and presented as histograms.
Open curves indicate CX3CR1-GFP expression; filled histograms were obtained by gating on
the indicated cell population from wildtype animals. D. Confocal images and of CX3CR1GFP/+ and
CX3CR1GFP/GFP animals infected with C. rodentium at day 0, 12 and 27 post infection. E. At the
indicated time points after infection colonic lamina propria cell isolates were obtained, stained
for CD11c and analyzed by flow cytometry. Numbers indicated the percentages of cells in the
respective quadrants of the dot blots. F. Mean (± SEM) total CX3CR1+ cell numbers isolated
from the colonic lamina propria of infected of CX3CR1GFP/+ and CX3CR1GFP/GFP animals.
FIGURE S4. Generation of a red fluorescent C. rodentium ICC169 derivative. A. Plasmid
map of p16S_PT5mRuby with relevant restriction sites and arrangement of the 730 bp fragment
of the 16S rDNA sequence of E. coli K12 (blue), the T5 promoter (green), the gene encoding the
bright monomeric red fluorescent protein mRuby and ermAM, the gene conferring erythromycin
resistance (red arrows), and the thermosensitive origin of replication of plasmid pGh9::ISS1
(blue)
45
.B. Bright field and fluorescent microscopic images of C. rodentium ICC169 wt and
ICC169::p16S_mRuby.
FIGURE S5. Fractalkine / CX3CL1 induce the expression of IL1ß and IL-12p40 in the colon
of C. rodentium infected animals. The colon was removed from non-infected and C.
rodentium infected B6, (age- and sex-matched) and CX3CR1GFP/+ and CX3CR1GFP/GFP animals at
peak infection (12 days post infection), RNA isolated, reverse transcribed to cDNA and IL-1ß,
IL-12p40, IL-23p19, mucine 1, mucine 3 and mucine 4 expression was analyzed by qRT-PCR.
In the non-parametric student´s t test p< 0.05 was considered statistically significant.
FIGURE S6. CX3CR1+ macrophages facilitate IL-22 production by lymphoid tissue inducer
cells. CX3CR1+ macrophages phagocytose C. rodentium and facilitate IL-22 production by
lymphoid tissue inducer cells (LTi). LTi derived IL-22 promotes RegIII and RegIIIß expression
by intestinal epithelial cells facilitating the clearance of C. rodentium.