Fig 2: NETs were excessively released after MHV-3 infection. (A) Neutrophil infiltration in liver was detected by immunochemistry staining with LY6G. Arrows indicate neutrophil infiltration. (B) Liver samples were detected for NETs by immunofluorescence staining with Cit-H3 (red), neutrophil elastase (green), and 4',6-diamidino-2-phenylindole (DAPI) (blue). Arrows indicate NETs areas. (C) The mean fluorescence intensity of Cit-H3, NE, and their colocalization were quantified using ImageJ (National Institutes of Health, Bethesda, MD) analysis software. Scale bar: 50 µm. Ten microscopic fields per liver section from 3 mice in each group were counted. (D) Serum MPO-DNA levels were detected at indicated time points after MHV-3 infection. n = 5 in each group. (E) The expression of hepatic Cit-H3 and PAD4 were measured by Western blot and analyzed by Image lab (Bio-Rad, Hercules, CA). *P < .05, **P < .01, ***P < .001, and ****P < .0001, determined by 1-way analysis of variance followed by the Tukey follow-up test.

Fig 3: Effects of FTB on CLP-induced pathological changes and tissue injury scores of the lung, liver and kidney in rats with sepsis after inhibiting the NETs formation and the activity of PAD4. Treatment with FTB, DNase1 (NETs inhibitor) and Cl-amidine (PAD4 inhibitor) improved the CLP-induced pathological changes in the lung (red arrows indicate the pulmonary interstitial congestion, yellow arrows indicate the thickened alveolar walls and black arrows indicate the infiltration of inflammatory cells), liver (yellow arrows indicate the necrotic hepatocytes and black arrows indicate the infiltration of inflammatory cells) and kidney (yellow arrows indicate the enlarged lumen of focal tubules and black arrows indicate the interstitial inflammatory cell infiltration), and decreased the tissue injury scores. However, no differences were observed in the pathological changes and tissue injury scores between the FTB and DNase1 groups, FTB and Cl-amidine groups, DNase1 and FTB + DNase1 groups, and Cl-amidine and FTB + Cl-amidine groups (magnification: ×100). Sham group (n = 15), CLP group (n = 8), FTB group (n = 10), DNase1 group (n = 9), FTB + DNase1 group (n = 10), Cl-amidine group (n = 10), FTB + Cl-amidine group (n = 10).

Fig 4: Assessment of PAD4 expression in the lungs during sepsis. Total protein was extracted from lung tissues and PAD4 expression at protein levels was measured by Western blotting. Each blot was quantified by densitometric analysis. PAD4 expression in each sample was normalized to ß-actin expression and the mean values of WT sham groups were standardized as one for comparison. Representative blots for PAD4 and ß-actin from three independent experiments are shown. The full-length gel from where the cropped bands are taken is displayed in Supplemental Fig. 4, in which the cropped bands are shown in yellow box. Data are expressed as means ± SE (n = WT sham 10, WT CLP 10, CIRP-/- sham 10, and CIRP-/- CLP 10 mice) and compared using one-way ANOVA and SNK method (*p < 0.05 vs WT sham, #p < 0.05 vs WT CLP).

Fig 5: TREM1-induced SYK activation triggered proinflammatory subtype microglia and the formation of NETs after SAH. (A, B) Quantification of neurological function (modified Garcia score and beam balance test) at 24 h after SAH. n = 11/group. (C–J) Representative western blotting images and quantitative analysis of TREM1, p-SYK, SYK, Card9, p-NF-?B, NF-?B, and PAD4 in cortex in different groups. Control images of ß-actin are re-used for illustrative purposes. n = 6/group. (K–M) Representative western blotting images and quantitative analysis of MPO and CitH3 in cortex in different groups. Control images of ß-actin are re-used for illustrative purposes. n = 6/group. (N, O) Relative mRNA levels of proinflammatory subtype microglia marker genes (CD16, CD86). n = 5/group. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs Sham group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs SAH+vehicle group.