Post on 18-Jan-2018
description
FSC-A FSC-A ICOS
Life
/Dea
d
SSC-
A
IL-1
3 eG
FP
21 dpi
Figure S1
Figure S1 Identification of IL-13-competent cells in lungs of infected IL-13eGFP/+ miceIL-13eGFP reporter mice were infected intranasally with C. neoformans 1841 and analysed at indicated time points (n = 6 mice/experiment). Gating strategy for identification of IL-13-competent pulmonary cells is shown. One of two experiments is shown.
il17rb/st2-
/-
naïve 21dpi
wt
IL-33R
GATA
3
Figure S2
Figure S2 Gating scheme for ILC2 from wt and il17rb/st2-/- mice based on GATA3 and IL-33R Wt and il17rb/st2-/- mice were infected intranasally with C. neoformans 1841 and analysed at indicated time points (n = 7 mice/experiment). Gating of Lineage negative pulmonary cells and subsequent GATA3 analysis, i.e. ILC2, is shown. One of two experiments is shown.
Figure S3
Figure S3 Comparison of cytokine production by pulmonary cells from st2-/- and il17rb/st2-/- miceMice were infected intranasally with C. neoformans 1841 and analysed 21 dpi (n = 5-7 mice/experiment). ELISA of supernatants of pulmonary leukocytes restimulated with ionomycin and phorbol 12-myristate 13-acetate for 4 h. Ratios are calculated as x-fold amount of each cytokine produced by individual st2-/- or il17rb/st2-/- mouse compared to corresponding wt control mice used in the same experiment as experiments with st2-/- and il17rb/st2-/- mice were independently performed. Two-tailed unpaired t-test was performed with * = P ≤ 0.05 for IFN-γ. Mean with range is shown for IFN-γ and median with range for IL-13, IL-5, and IL-17A. One of two experiments is shown for each ratio.
IL-5
0.0
0.5
1.0
1.5
2.0
x-fo
ld (o
f wt c
ontro
l)IL-17A
0
5
10
15x-
fold
(of w
t con
trol
)IFN-
0.0
0.5
1.0
1.5
2.0 *
x-fo
ld (o
f wt c
ontr
ol)
IL-13
0.0
0.5
1.0
1.5
2.0st2-/-il17rb/st2-/-
x-fo
ld (o
f wt c
ontr
ol)
Figure S4
Figure S4 Pulmonary PAS-score and gob5 expression in il17rb/st2-/- miceil17rb/st2-/- mice were infected intranasally with C. neoformans 1841 and analysed at indicated time points (n = 7 mice/experiment). PAS score was calculated from counting PAS+ epithelial cells of ten bronchi of each mouse and gob5 expression was analysed by qPCR (as described in materials and methods section). One of two experiments is shown.
7 14 21
0.00.1
12345 il17rb/st2-/-
dpi
% P
AS+
cells
of b
ronc
hial
epi
thel
ium
gob5
naïve 7 14 21
0
10
20
30il17rb/st2-/-
dpi
x-fo
ldhp
rt
wt 21dpi
wt naïve
il17rb/st2-/-
21dpi
Foxp3
IL-3
3R
Gated on living CD4+ T singlet cells
Figure S5
GATA3
wt 21dpi
Figure S5 Th cells subsets from wt mice show IL-33R-dependent GATA3 expressionWt and il17rb/st2-/- mice were infected intranasally with C. neoformans 1841 and analysed at indicated time points (n = 7 mice/experiment). Gating strategy for analysis of GATA3 in pulmonary Th cell subsets based on IL-33R and Foxp3 expression is shown. GATA3 MFI (APC) is shown for 21dpi wt mouse.
102
103
104
naïve 21dpi
*** ******wtil17rb/st2-/-
Ym1
grey
val
ue (m
ean)
il17rb/st2-/- wt21 dpinaïve
il17rb/st2-/- wt
Ym1
Hoec
hst
Figure S6
Figure S6 Ym1 immunofluorescence with sorted alveolar macrophages from wt and il17rb/st2-/- miceYm1 (protein product of chi3l3) staining intensity expressed as grey values (see materials and methods section) is shown for alveolar macrophages from naïve and 21 dpi mice (il17rb/st2-/- mice white boxes, wt mice grey boxes) with representative staining for Ym1 (top row) and corresponding nucleus stain with Hoechst 33342 (bottom row). Kruskal-Wallis test with Dunn's Multiple Comparison Test was performed with *** = P ≤ 0.001. One of two experiments is shown.
naïve 14 dpi0.0
0.5
1.0
1.5rag2-/-rag2/c-/-
notdetected
notdetected
notdetected
notdetected
IL-1
3 [n
g/m
L]
naïve 14 dpi0.0
0.5
1.0
1.5rag2-/-rag2/c-/-
notdetected
notdetected
IL-5
[ng/
mL]
Figure S7
Figure S7 Supernatant analysis of pulmonary cells from rag2-/- and rag2/γc-/- mice
il2 (Th cells)
naïve 21dpi0.0
0.1
0.2
0.3
0.4
0.5
wtil17rb/st2-/-
x-fo
ldhp
rt
Figure S8
Figure S8 Quantitative real time PCR analysis of sorted Th cells from wt and il17rb/st2-/- mice