FIGURE LEGENDS
Figure 1. The effects of antibiotics on ROS generation and NETs
formation. Human peripheral neutrophils were pre-incubated with
antibiotics or PBS for 2 hours and stimulated to form NETs with or
without PMA (100nM) for 3 hours. A. Kinetics of NETs releasing
in PMA-stimulated neutrophils was recorded
(n=3).B . NETs release in activated (with PMA) or resting (antibiotics
alone) neutrophils was
measured
using Sytox Green
fluorescence
plate reader assay at 3 hour and expression as ratio of percentage of
total DNA (n=3). Both antibiotics group showed effects on the NETs
formation only in activated neutrophils. C. To further confirm
the existence of NETs, neutrophils were pre-incubated with antibiotics
or PBS for 2 hours and stimulated to form NETs with PMA (100nM) for 3
hours. DNA (blue) stained with DAPI, MPO (green) stained with anti-MPO
antibody and cit-H3 (red) stained with anti-cit-H3 were
counterstained
in
neutrophils
for immunofluorescence. DNA stained with
Sytox
Green alone in neutrophils for immunofluorescence to show the morphology
of NETs. D . Neutrophils were pre-incubated with β-Lactams (2
mM) for 2 hour and then treated with or without PMA. Intracellular ROS
were measured with DHR123 at 45 min (n=3). E . Neutrophils
pre-cultivated with DPI, a NADPH oxidase inhibitor, were incubated with
β-Lactams for 2 hour and then activated with PMA. The ROS production was
measured using DHR123 fluorescence plate reader assay at time of 45min
(n=3). F . To confirm the role of ROS, exogenous
H2 O2(30μM) was added after 2-hour incubating with β-Lactams. NETosis was
measured using Sytox Green fluorescence plate reader assay at time of 3
hour and expression as relative fluorescence unit (n=3). Images are
representative of three independent experiments. Bars were shown in the
figure; data were analyzed by Student’s t test. *P<0.05, **
P<0.01, ***P<0.001, ns, no significance.
Figure 2. De-escalation strategy exerted a diverse effect on the
serum MPO-DNA at different stages of sepsis. A . At the early stage of
sepsis (Day 3), blood was drawn from different groups (n=25), and serum
MPO-DNA levels were determined with mouse MPO ELISA kit. DE group showed
higher serum MPO-DNA levels than ES group, and both were lower that
control group (p<0.001). Higher MPO-DNA levels were diminished
with the administration of
DNaseI
(p<0.001). B . At the late stage of sepsis (Day 6),
serum MPO-DNA levels were determined the same way (n=20). ES group
showed higher levels of serum MPO-DNA than DE group (p<0.01),
and both were higher than sham group (p<0.001).
Figure 3. De-escalation strategy caused less organ injuries and
inflammatory response attenuated by DNaseI in early sepsis stage. A-B .
The serum AST and Creatine were determined by ELISA (n=25). Reduced
levels of AST and Creatine were shown in DE compared with ES group
(p<0.01) , however, this effect was reversed by NETs
scavenger, DNaseI (p<0.001). C-F . The serum levels of
IL-6, IFN-γ, IL-10 and MCP-1 were determined using Luminex ® xMAP
(n=25). The inflammatory cytokines levels were lower in DE group than
that in ES
group
(p<0.01), except for the level of MCP-1 (p<0.001).
When DNaseI was administrated, all were elevated compared with DE
group
(p<0.05).
Figure 4. De-escalation strategy alleviates organ injuriesin
the late sepsis stage. A-D. The serum levels of AST, creatine,
ALT and LDH were measured with ELISA kit
in
all groups (n=17). The levels blood chemistries were lower in DE group
than those in ES group (p<0.01).
However,
those were extremely higher in control group though there was 2 of 10
left at day 6.
Figure 5. De-escalation strategy allays inflammatory response in
the late sepsis stage. A-F. The serum levels of MIP-2, MCP-1, TNF-α,
IL-10, IL-6 and IFN-γ were measured using Luminex ® xMAP (n=17). The
inflammatory cytokines levels were lower in DE group than that in ES
group (p<0.01), which was coincident with that in early sepsis
stage. And in control group (n=5), those biomarkers were extremely
higher than those in antibiotic groups.
Figure 6. Morphologic changes in liver, intestine and lung
tissues in late sepsis stage and survival rates in different groups (day
#6). The image A-C are representative of liver, intestine and lungs
from sham, DE and ES group. Mice in control group did not shown due to
severe necrosis. A. The same liver tissues in the same mice
were exanimated by HE and ICH in each group. Mice in sepsis groups
showed more leukocyte infiltration and hydropic degeneration in liver
tissues. And DE treatment presented alleviated pathological changes than
ES treatment. And poor pathological morphologic changes were consistent
with more positive citH3 and apoptotic cells in liver tissues. Stars
indicate leukocyte cells infiltration. Solid triangles indicate
hydropic
degeneration. Arrows indicate positive cells in ICH. B.Intestine tissues collected in each group were treated with HE or ICH
examination. Mice suffered from sepsis showed severe inflammatory cells
infiltration and
microvilli
damage. And severer intestine injuries were correlative to more positive
citH3 and apoptotic cells in intestine tissues. Stars indicate leukocyte
cells infiltration. Triangles indicate microvilli damage. Arrows
indicate positive cells in ICH. C. CLP procedure induced
inflammatory
cells infiltration comparing normal tissue, and DE showed less
pathological changes than ES group. Stars indicate inflammatory cells
infiltration. D . Survival rates of CLP sepsis mice treated with
DE or ES comparing with control group at day 6 after CLP operation.
Although there was no significant difference between DE and ES group
(p=0.051), DE has the greatest survival benefits compared with the
control group. DE, de-escalation group. ES, escalation group. N=10 per
experimental group and N=5 in sham group.