in serum. Indomethacin (Sigma-Aldrich, I7378),
a nonsteroidal anti-inflammatory drug (NSAID)
that can induce intestinal damage in mice,
wasusedasapositivecontrol.
For the experimental group, wild-type and
Sprr2a−/−mice were treated with 190mlof7%
DMSO in PBS by oral gavage. For the positive
control group, mice were treated with 190ml
of indomethacin (1.5 mg/ml in 7% DMSO in
PBS) by oral gavage. After 1 hour, mice in both
groups were treated with 190ml of FITC-dextran
(80 mg/ml in PBS) by oral gavage. Mice were
sacrificed 4 hours later, and sera were collected.
The sera were then centrifuged for 20 min at 4°C
at 1740g, and the supernatants were collected.
Serum FITC-dextran levels were measured by
fluorescence microplate assay against a FITC-
dextran standard curve using a Spectramax
plate reader (Molecular Devices).
L. monocytogenesinfection
L. monocytogenesstrain EGDe (erythromycin-
resistant) was grown overnight in brain-heart
infusion (BHI) broth containing 20mg/ml eryth-
romycin at 37°C. Wild-type andSprr2a−/−mice
were treated with erythromycin (0.4 mg per
mice) by oral gavage the day before infection.
Mice were inoculated with 1.0 × 10^9 L. mono-
cytogenesfor tissue dissemination experiments
or 2.5 × 10^9 bacteria for survival experiments
by oral gavage. For tissue dissemination ex-
periments, mice were sacrificed 1 day after
infection, and the mesenteric lymph nodes
(MLN), liver, and spleen were collected and
weighed. Tissues were homogenized in ice-
cold PBS and the numbers ofL. monocyto-
geneswere counted by dilution plating on
BHI-erythromycin agar plates. We calculated
the limit of detection as [CFU (minimum)] ×
[dilution factor] / [tissue weight]. Data points
below the limit of detection indicate that no
L. monocytogenescolonies grew on any of the
three replicate plates.
Recombinant IL-13 and IL-22 treatment
Wild-typeBALB/cmicewereinjectedintra-
peritoneally with 1.5mg of carrier-free recom-
binant mouse IL-13 (BioLegend, 575906) or
IL-22 (BioLegend, 576206) every other day for
a total of four treatments. Mice were sacri-
ficedonthedayafterthelastinjection.
Heligmosomoides polygyrusinfection
Heligmosomoides polygyrus[H. polygyrus,
recently renamedH. bakeri( 44 )] stocks were
propagated as previously described ( 45 , 46 ),
with a few exceptions. Briefly, B6.129S2(C)-
Stat6tm1Gru(Jackson stock, 005977) ( 38 ) or wild-
type C57BL/6 mice housed in a BSL2 animal
facility were infected with 400H. polygyrus
L3 larvae by oral gavage. On day 13 of infec-
tion, grates were placed in the cages and feces
were collected for the next 3 days. Feces were
mashed and mixed with activated, washed
charcoal (Sigma-Aldrich, C2764-500G), then
plated on Whatman filter paper in petri dishes.
PBS was added to the dishes until a pool formed
around the filter paper. After 7 to 10 days of
incubation in a humid box, the L3 larvae were
collected with a modified Baermann appara-
tus filled with warm PBS. The tube of the
Baermann apparatus was plugged with a stop-
per. A metal grate was placed at the top of the
funnel, then one layer of muslin and one layer
of Kimwipe (Fisher Scientific) were placed on
top of that. The fecal mixture was spread on
top of both layers, and the larvae were allowed
to fall for 2 hours. The PBS from the apparatus
was collected into a beaker and the larvae
were concentrated by centrifugation at 300g
for 10 min with no brake. After the larvae
were concentrated, they were put through the
Baermann apparatus and then concentrated
by centrifugation a second time. The larvae
were washed three times with PBS and stored
at 4°C in PBS. A new stock was generated every
6 months.
ForH. polygyrusinfection experiments, wild-
type,Stat6−/−, andSprr2a−/−mice were inocu-
lated with 200 L3H. polygyruslarvae through
oral gavage in the BSL2 animal facility. After
2 weeks, mice were sacrificed, and the intesti-
nal tissues were collected for further studies.H. polygyrusworm and egg counting
To quantify worm burden, small intestines
and their luminal contents were collected and
placed in 10 ml of PBS in Petri dishes at room
temperature. The intestines were dissected lon-
gitudinally and worms associated with the in-
testinal tissue were collected by scraping with
forceps. After 1 hour of incubation at 37°C,
worms were counted manually using forceps.
For egg counting, 1 to 2 fecal pellets were
collected and weighed. Three milliliters of
water was added to the fecal pellets in 15-ml
conical tubes and vortexed periodically until
the pellets were completely dispersed. Just be-
fore each sample was added to the McMaster
egg counter (Electron Microscopy Sciences, 63512-
75), an equal volume of supersaturated NaCl
solution was added to the tube. Eggs were
counted under a microscope, and two inde-
pendent counts were averaged. Egg numbers
were normalized to the weight of fecal pellets.Statistics
We used two-tailed StudentÕsttests to deter-
mine the statistical significance of a difference
between two treatments when a parametric test
was appropriate. We used the Mann-Whitney
Utest for experiments requiring a nonpara-
metric statistical test (e.g., Fig. 3E). For data
having unequal variances among different ex-
perimental groups and following a lognormal
distribution, a logarithmic transform was per-
formed before the statistical analysis. The
log-rank test was used in survival experimentswhere the null hypothesis was that there is no
difference between experimental groups in
the probability of lethal morbidity at any time
point. Statistical details of experiments are
provided in the figure legends, including how
significance was defined and the statistical
methods used. Data represent means ± stan-
dard errors of the mean (SEM). All statistical
analyses were performed with GraphPad Prism
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