Fig 2: Characterization of NET formation in a mouse model of ischemic stroke.Mice were subjected to 1 hour of tMCAO or sham surgery. Plasma was isolated 6 or 24 hours after stroke onset, and brains were collected and processed for histology. (A) MPO-DNA complexes were measured using an in-house MPO-DNA ELISA. n = 4 per group. (B) Plasma HMGB1 levels were measured by ELISA. n = 4 per group. (C) Brain tissue was stained for the presence of NETs by a combination of Ly6G (white), MPO (red), H3cit (green), and DNA (DAPI, blue). Scale bar: 50 µm. (D–F) Both neutrophils not undergoing NET formation (H3cit–, highlighted in D) and neutrophils undergoing NET formation (H3cit+, highlighted in E and F) were observed. Scale bars: 25 µm. (G–I) Brain tissue was stained for the colocalization of NETs with platelets by a combination of MPO (red), H3cit (green), platelets (CD41, white), and DNA (DAPI, blue). Scale bars: 15 µm. Images are representative of 4 mice. Groups were compared by ordinary 1-way ANOVA. *P < 0.05, **P < 0.01.

Fig 3: tACPA inhibits NET formation in vivo and in vitro. To induce inflammation in the peritoneum, pristane was injected, followed by cIgG or dc-tACPA. a The composition of peritoneal cell infiltrates was analyzed after 24 h (n = 7–8). b Representative images showing NET formation in the peritoneum of mice treated with cIgG or dc-tACPA. NETs were stained with Hoechst (blue) and anti-citH3 antibody (red). Scale bars: 50 µm. c Quantification of NETs (colocalization of citH3 and Hoechst; n = 10). d Representative images showing in vitro human NET release induced by activated PLTs or gout SF without antibody (No Ab) or in the presence of cIgG or hz-tACPA. Scale bars: 20 µm, bottom right image: 10 µm. NETs were stained with DAPI (blue), anti-citH3 antibody (red), and anti-NE antibody (green). MPO/DNA complexes were measured in harvested NETs upon stimulation with activated e PLTs or f gout SF (n = 5). g The activity of MPO present in NETs of neutrophils from HVs was measured after A23187 stimulation in the presence of different concentrations of hz-tACPA (n = 4). h The percentage of NETs at t = 240 min in A23187-stimulated neutrophils from HVs that were treated with either full-length dc-tACPA or dc-tACPA F(ab')2 antibody fragments was quantified (n = 8). MPO activity in NETs or NET-DNA was quantified in A23187-stimulated neutrophils from i RA and j SLE patients (n = 5 and n = 3, respectively) in the absence or presence of cIgG or hz-tACPA. k MPO activity in human A23187-induced NETs without antibodies or with cIgG, m-tACPA, antibodies against acetylated H2A (anti-acH2A), methylated H2A (anti-metH2A), or phosphorylated H2A (anti-phosH2A) was measured (n = 4). To determine the percentage of DNA and MPO activity in NETs, the mean of the No Ab group was set at 100%, and individual percentages were calculated (g and k). In (i) and (j), the mean of the cIgG group was set at 100%. The results are presented as the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 using two-tailed Mann–Whitney statistical test

Fig 4: HBV-mediated S100A9 facilitates NETs generation. (A) Procedure for NETs analysis by co-culture of neutrophils with QSG-7701, HepG2 or HepG2.2.15 cells for 48 h. (B) The representative images of IF staining for DNA/NE/MPO in neutrophils co-cultured with QSG-7701, HepG2 or HepG2.2.15 cells for 48 h. White scale bars, 50 µm. (C) Western blot analysis for CitH3 in neutrophils co-cultured with QSG-7701, HepG2 or HepG2.2.15 cells for 48 h. The fold change of densitometric ratios was normalized to the ß-actin and then compared to the control and was shown on the right panel. (D) ELISA analysis for MPO-DNA in cultured supernatant of co-culture system. (E) Western blot analysis for endogenic expression of S100A9 in QSG-7701, HepG2 and HepG2.2.15 cells. The fold change of densitometric ratios was normalized to the ß-actin and then compared to the control and was shown on the right panel. (F) ELISA analysis for S100A9 levels in cultured supernatant of QSG-7701, HepG2 and HepG2.2.15 cells. (G) ELISA analysis for S100A9 levels in cultured supernatant of HepG2.2.15 cells transfected with siNC or siS100A9 for 48 h. (H) The representative images of IF staining for DNA/NE/MPO in neutrophils treated with CM (siNC or siS100A9-transfected HepG2.2.15) for 48 h. White scale bars, 50 µm. (I) Western blot analysis for CitH3 in neutrophils treated with CM (siNC or siS100A9-transfected HepG2.2.15) for 48h. The fold change of densitometric ratios was normalized to the ß-actin and then compared to the control and was shown on the right panel. (J) ELISA analysis for MPO-DNA in cultured supernatant of neutrophils treated with CM (siNC or siS100A9-transfected HepG2.2.15) for 48 h. (K) The representative images of IF staining for DNA/NE/MPO in neutrophils treated with and without GST, GST-S100A9 or PMA for 12 h. White scale bars, 50 µm. (L) Western blot analysis for CitH3 in neutrophils treated with and without GST, GST-S100A9 or PMA for 12 h. The fold change of densitometric ratios was normalized to the ß-actin and then compared to the control and was shown on the right panel. (M) ELISA analysis for MPO-DNA in cultured supernatant of neutrophils treated with and without GST, GST-S100A9 or PMA for 12 h. None, no treatment. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: siS100A9, the siRNA to interfere with S100A9; siNC, negative control siRNA; GST, glutathione-s-transferase.

Fig 5: NETs levels are elevated in HCC, especially in HBV-positive HCC. (A-B) The representative images of IF staining for CD66b (A) in tissue sections from HCC patients, HBV-HCC patients and their matched para-carcinoma tissues. The IOD of CD66b (B) IF-stained materials were analyzed utilizing the Image Pro Plus software. Data from histomorphometric analysis are presented as mean ± standard deviation calculated from 8 HCC patients, 8 HBV-HCC patients and their matched para-carcinoma tissues. (C-D) The representative images of IF staining for CitH3 (C) in tissue sections from HCC patients, HBV-HCC patients and their matched para-carcinoma tissues. The IOD of CitH3 (D) IF-stained materials were analyzed utilizing the Image Pro Plus software. Data from histomorphometric analysis are presented as mean ± standard deviation calculated from 8 HCC patients, 8 HBV-HCC patients and their matched para-carcinoma tissues. (E) Western blot analysis for CitH3 expression in tissue sections from 8 HCC patients, 8 HBV-HCC patients and their matched para-carcinoma tissues. The fold change of densitometric ratios was normalized to the ß-actin and then compared to the control and was shown on the below panel. (F) Western blot analysis for CitH3 expression in peripheral neutrophils from 6 healthy controls, 6 HCC patients and 6 HBV-HCC patients. The fold change of densitometric ratios was normalized to the ß-actin and then compared to the control and was shown on the right panel. (G) The representative images of IF staining for CitH3 in human neutrophils from healthy controls, HCC patients and HBV-HCC patients. (H) The IOD of CitH3 IF-stained materials were analyzed utilizing the Image Pro Plus software. Data from histomorphometric analysis are presented as mean ± standard deviation calculated from 6 healthy controls, 6 HCC patients and 6 HBV-HCC patients. (I) The representative images of IF staining for DNA/NE/MPO in human neutrophils from healthy controls, HCC patients and HBV-HCC patients. (J) ELISA analysis for serum levels of MPO-DNA (serum marker for NETs) in healthy controls, HCC patients and HBV-HCC patients. White scale bars: 50 µm. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: HCC, HBV-negative hepatocellular carcinoma; HBV-HCC, HBV-positive hepatocellular carcinoma; IOD, integrated optical density; DAPI, 4’, 6-diamidino-2-phenylindole; CitH3, citrullinated modification of histone 3; NE, neutrophil elastase; MPO, myeloperoxidase; IF, immunofluorescence; N, normal; C, cancer; HC, healthy controls.