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.