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Weber G IL 3 Sepsis Science 2015
Weber G IL 3 Sepsis Science 2015
1260
To characterize the host response more completely, we performed time-course tissue, cellular, and molecular experiments. At 1 day after CLP,
WT mice developed neutrophilia and inflammatory Ly-6Chigh monocytosis (Fig. 1E), whereas in
Il3/ mice, monocyte and neutrophil numbers
remained relatively unchanged. The increased cell
numbers in WT mice were associated with higher
serum levels of IL-1b, IL-6, and TNF-a (Fig. 1F).
Phagocytic leukocytes were major sources of IL-1b,
IL-6, and TN-Fa, because phagocyte depletion
with clodronate liposomes and antiLy-6G before CLP abolished the cytokine storm (fig. S3A).
However, IL-3mediated cytokine induction was
indirect: Both WT and Il3/ neutrophils and
monocytes contained similar intracellular reservoirs of the three cytokines (fig. S3B). Analyzing other leukocytes showed IL-3dependent
differences in T and B cell numbers after CLP
(fig. S4A), but no differences in basophils, mast
cells (1012) (fig. S4, B and C), or histamine (fig.
S4D), which suggests that IL-3 had little to no
effect on basophil and mast cell production
and function during the initial inflammationdominant phase. Consequently, WT but not
Il3/ mice accumulated monocytes and neutrophils in the lung (Fig. 1G) and liver (Fig. 1H);
developed lung pathology (fig. S5A) with increased protein in bronchoalveolar lavage (fig.
S5B); and evolved abnormal liver morphology
(fig. S5C) with increased markers of cytolysis in
serum (fig. S5D). These data show that IL-3
contributed to septic shock, the most severe form
of sepsis (13, 14).
IL-3 promotes hematopoiesis by acting on its
receptor, a heterodimer that consists of the IL-
R ES E A RC H | R E PO R TS
RE S EAR CH | R E P O R T S
(Fig. 3D), IL-3+ cells were CD19+ B cells. According to enzyme-linked immunosorbent assay, IL-3
levels increased in serum after CLP (Fig. 3E)
but to a lesser extent in splenectomized mice
(Fig. 3E).
Identifying B cells as sources of IL-3 prompted
testing of whether IL-3producing B cells resemble
innate response activator (IRA) B cells (fig. S8A),
100
Clinical
score
20
100
points
WT + Imip-cilast
Il3 / + Imip-cilast
0
1 2
3 4
5 6 7
105
24
12
Ly-6C high
monocytes
Neutrophils
20
WT
Il3/
1.6
WT
Il3/
105/ml
105
25
***
100
80
24
**
10
800
IL-1
(pg/ml)
WT
Il3 /
nd
Steady CLP
state
Serum
IL-6
(ng/ml)
60
200
30
100
TNF(pg/ml)
**
0.8
400
cells
0 6 12 24 120
24
24
50 m
106
1.5
50 m
0
**
*
0
Il3 /
WT
Il3 /
1.4
106
106
cells
WT
0
3
50 m
24
cells
50 m
24
***
cells
0 6 12 24 120
50 m
0.7
**
50 m
0
10
107
CFU
1010
***
Blood
Blood
1010
32
Bacterial titre
Peritoneum
12
120
39
WT
Il3 /
10
RR (systolic)
cells
WT
Il3 /
50
Body
Temperature
***
***
***
50
mmHg
Survival on
antibiotics
Survival
[C]
50 m
50 m
0
1261
R ES E A RC H | R E PO R TS
15
12
7.5
CD123
CMP/MEP
GMP/MDP
2.5
**
24
24
+IL-3
3.61 105
0.38 105
0.90 105
0.07 105
105
+LPS
0.48 105
0.03 105
Bone marrow
GMP
Steady
state CLP
CLP
Blood
Ly-6Chigh Monocytes
Steady
state CLP
CLP
0.2
Blood
Neutrophils
Steady
state CLP
CLP
2
***
**
**
**
**
0.4
*
***
**
Survival
100
0.5
0.1
0.2
0.8
**
1262
1.6
**
1
0.8
Il3/
Il3/ + rIL-3
WT
WT + anti-CD123
WT
WT + rIL-3
Il3/
Il3/ + rIL-3
WT + anti-CD123
WT
WT + rIL-3
WT
Il3/
Il3/ + rIL-3
WT + anti-CD123
WT
WT + rIL-3
WT
Il3/
Il3/ + rIL-3
WT
WT + anti-CD123
WT
WT + rIL-3
Il3/
Il3/ + rIL-3
WT
WT + anti-CD123
WT
WT + rIL-3
Il3/
Il3/ + rIL-3
WT
WT + anti-CD123
WT
WT + rIL-3
2
Time (d)
Survival
100
1.6
50
***
* 6
****
***
IL-6 ( 10 ng/ml)
Steady
state CLP
CLP
**
0
****
cells
106
CD11b-AlexaFluor700
1.5
IL-6
( 102 pg/ml)
**
12
10
24
*
****
24
4.24 105
0.22 105
CD115-APC
Medium
Il3/
GFP
IL-1
(pg/ml)
Post-culture
Pre-culture
WT
105
HSPC
WT
Il3/
cells
106
24
CD45.2-Pacific blue
WT
Il3/
cells
Events
HSPC
MEP
CMP
GMP
MDP
MFI ( 102)
50
***
0
2 3 4
Time (d)
Il3/ CLP
RE S EAR CH | R E P O R T S
Il3 expression
n.d.
SP
BM
TH
LN
n.d.
n.d.
n.d.
n.d.
LG
LV
PR
DD
Spleen
Thymus
Non-B
B
Non-B
B
cells cells cells cells
CLP
0.1
106
1
1.6
Spleen
Thymus
0.5
cells
CD19-APCCy7
fold induction
16
IL-3-producing
B cells
0
IL-3 (ic)-PE
01 4 01 4
days after CLP
IRA B cells
CD23-PECy5
white
pulp
20 m
CLP
20 m
CLP
(2 days)
white pulp
IgM (B cells)
CD43-PECy7
648
IL-3 (ic)-PE
GFP
CD11b
Blood
IL-1
Neutro
Ly-6Chigh
phils
Mono
( 102
6
( 10 /ml) pg/ml)
cytes
( 106/ml)
4
0.6
0.2
p=
0.055
*
Serum
IL-6
( 10
ng/ml)
Clinical
Score
IL-3
IL-3
20
IL-3
10
0.1
0.3
1.5
p=
0.055
TNF( 102
pg/ml)
2
CLP
points
Steady state
red
pulp
CD19-APCCy7
Adoptive transfer of
peritoneal B1 cells
VLA4-AlexFluor
20
SSC
10
pg/ml
IL-3 (ic)-PE
CD19-PacOrange
**
IgM-APC
SSC-A
Serum IL-3
SPx CLP
+
+ +
CD138-PacBlue
GAPDH
IL-3
Spleen (CLP)
Live singlets
IL-3
200 m
merge
merge
0
0
Il3/ WT
Donor cells: Il3/ WT
Recipients:
Fig. 3. IRA B cells are major sources of IL-3 in sepsis. (A) Il3 mRNA expression in the indicated organs during a steady state and 3, 6, 12, and
24 hours after CLP (n = 6 to 8). (B) Identification of IL-3producing cells in the
spleen 4 days after CLP. (C) Enumeration of IL-3producing B cells in spleen
and thymus in a steady state and 1 and 4 days after CLP (n = 5). (D) Western
blot showing IL-3 expression by B cells and non-B cells sorted from the spleen
and thymus 1 day after CLP. (E) IL-3 serum levels in a steady state and 1 day
after CLP with and without splenectomy (SPx) (n = 3 to 6). (F) Flow cytometric
plots show the phenotype of IL-3+ and IL-3 cells retrieved from the spleen
after CLP. A representative plot of n = 5 is shown. (G) Immunofluorescence
SCIENCE sciencemag.org
0
0
0
0
Il3/ WT Il3/ WT Il3/ WT Il3/ WT
Il3/
microscopy of spleen tissue in the steady state and 1 day after CLP. (H) Costaining of representative IL-3+ cells with IgM. (I) Adoptive transfer of 1.5 106
peritoneal B1 B cells from GFP+ mice into WT mice subjected to CLP at the
time of cell transfer. Representative plots from flow cytometric analysis of
n = 3 mice are shown. (J) Adoptive transfer of 3 106 peritoneal B1 B cells from
WT or Il3/ mice to the peritoneum of Il3/ recipients subjected to CLP.
Data show the clinical score, number of Ly-6Chigh monocytes, neutrophils,
and serum cytokines 1 day after CLP (n = 5). (*P < 0.05, **P < 0.01). Error
bars indicate means T SEM. Significance was assessed by a Kruskal-Wallis
test with Dunns multiple comparison test (E) and a Mann-Whitney test (J).
1263
R ES E A RC H | R E PO R TS
1/nl
****
**
50
Cells
25
14
28
50
25
800
CD14 +
r 2=0.51
p=0.02
400
Cells
Spleen
(CD45+ live singlets)
IL-3-PE
Cells
CD20-PECy7
60
0 1
Spleen
20 m
p=0.001
IgM (B cells)
89.4 pg/ml
>89.4 pg/ml
40
0
1/nl
10
20
30
100
IgM-APC
1264
54
40
80
IL-3-producing
leukocytes
HLADR-PerCPCy5.5
SSC
1.8
1/nl
CD16 +
r 2=0.85
p=0.001
r 2=0.73
p<0.001
0.8
IL-3
( 10 2 pg/ml)
TNF( 10 2 pg/ml)
1.6
****
CD19-FITC
16
***
IL-6
(ng/ml)
IL-1
(pg/ml)
Plasma IL-3
Total leukocyte
number
IL-3-producing
human T cells
CD11b-APCCy7
with IL-3 plasma levels >87.4 pg/ml at admission had a poor prognosis (fig. S12, A and B,
and table S2). We therefore decided to test, in a
new prospective cohort [SEPIL-3 cohort, n = 37
(table S3)], whether IL-3 and blood monocytes
correlate. In septic patients monitored over 28 days,
blood leukocyte numbers peaked at the onset
of sepsis and decreased slowly thereafter (Fig.
4A). The increase was associated with a sharp
spike of plasma cytokines (Fig. 4B). Compared to
healthy volunteers, mean IL-3 in septic patient
plasma did not differ (Fig. 4C). Nevertheless, the
detectable levels of IL-3 correlated with circu-
IL-3
74
CD3-BV421
merge
1 day after sepsis onset) versus the survival of patients with IL-3 89.4 pg/ml.
(F) Representative flow cytometry plot of n = 2 patients showing the identity
of IL-3producing human splenocytes. (G) Immunofluorescence of human
spleen showing IL-3producing B cells in high magnification (60). A representative immunofluorescence section of n = 6 spleens is shown (*P <
0.05, ****P < 0.0001). Error bars indicate means T SEM. Significance was
assessed by one-way ANOVA with Tukeys multiple comparison test [(A) and
(C)], a Pearson correlation test (D), and log rank (E). Data in (A) to (D) are
from the SEPIL-3 cohort; data in (E) are pooled from the RAMMSES and
SEPIL-3 cohorts.
sciencemag.org SCIENCE
RE S EAR CH | R E P O R T S
RE FE RENCES AND N OT ES
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/347/6227/1260/suppl/DC1
Materials and Methods
Figs. S1 to S15
Tables S1 to S6
References (3436)
4 December 2014; accepted 21 January 2015
10.1126/science.aaa4268
CIRCADIAN RHYTHMS
Time-restricted feeding
attenuates age-related cardiac
decline in Drosophila
Shubhroz Gill,1,2 Hiep D. Le,1 Girish C. Melkani,3* Satchidananda Panda1*
Circadian clocks orchestrate periods of rest or activity and feeding or fasting over
the course of a 24-hour day and maintain homeostasis. To assess whether a
consolidated 24-hour cycle of feeding and fasting can sustain health, we explored the
effect of time-restricted feeding (TRF; food access limited to daytime 12 hours every
day) on neural, peripheral, and cardiovascular physiology in Drosophila melanogaster.
We detected improved sleep, prevention of body weight gain, and deceleration of
cardiac aging under TRF, even when caloric intake and activity were unchanged. We
used temporal gene expression profiling and validation through classical genetics
to identify the TCP-1 ring complex (TRiC) chaperonin, the mitochondrial electron
transport chain complexes, and the circadian clock as pathways mediating the
benefits of TRF.
SCIENCE sciencemag.org
1265
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