Fig 2: PAO induces calcium- and ER stress–dependent NETosis.(A) NETs’ formation is measured by dsDNA in the control, DPI-, PAO-, and DPI+PAO-cotreated neutrophil supernatants. Each dot represents neutrophils from an individual subject. Here, “ns” means not significant, as compared with PAO- vs. DPI+PAO-cotreated human neutrophils. Intracellular calcium flux measurements in the neutrophils isolated from human subjects loaded with calcium-sensitive dye Fluo-4 and stimulated with PAO for indicated time points in the absence or presence of (B) calcium chelator, BAPTA, and (C) PAD4 inhibitor, GSK484. All the intracellular calcium levels were measured in 3–5 individual subjects. Data are shown as the mean ± SEM. (D) Transmission electron microscopy images (n = 3). A total of 8 images were taken from each group. The endoplasmic reticulum is delineated with red lines in the lower panel. Scale bar: 2 nm. (E) Immunoblots of PAO-exposed neutrophils (3 hours) for ER stress marker proteins ATF4 and eIF2-a and (F and G) densitometry measurements. Individual lanes in immunoblots and symbol in graphs represent neutrophils from individual subjects. ß-Actin was used as loading control. Measurements of intracellular calcium flux in Fluo-4–loaded human neutrophils stimulated with PAO in the absence or presence of (H) ISRIB, (I) 4-phenylbutyric acid (4-PBA), and (J) 2-APB for indicated time points. All the intracellular calcium levels were measured in 3–5 individual subjects. (K) Immunoblot for Cit-H3 levels in the NETs. Each lane represents lysate from an individual human subject. (L) Densitometry for Cit-H3 from the immunoblot. All data are shown as the mean ± SEM. *P < 0.05, **P < 0.01. Statistics: 1-way ANOVA followed by Tukey’s multiple comparisons test (A and L), 2-way ANOVA for repeated measures over period of time (B, C, and H–J), and Wilcoxon’s test (F and G).

Fig 3: Mice exposed to lewisite and PAO on skin display NETosis in the lung.(A) dsDNA in the BALF of control and lewisite groups (n = 5/group). (B) Immunofluorescence staining of lung sections and (C) measurement of fluorescence intensity for Cit-H3 (red) and NE (green) from control (n = 5) and lewisite groups (n = 5). Scale bar: 100 µm. (D) IHC for PAD4 in lung sections from control and lewisite groups (n = 5/group). Scale bar: 100 µm. Inset: original magnification, ×200. (E) Measurements of DAB intensity for PAD4 from IHC images. An average measurement from 5 mice was calculated from the average of 8 images. Mice were subjected to skin exposure to PAO and sacrificed after 6 hours (F–L). (F) BALF cell pellets stained with hematoxylin (nuclear stain). Scale bar: 5 µm. (G) Percentage of neutrophils counted in BAL cell pellets (n = 5/group). (H) Measurement of dsDNA in BALF (n = 5/group). (I) IHC images for PAD4 staining (n = 5/group). Scale bar: 80 µm. The inset demonstrates an enlarged area of the image: original magnification, ×200. (J) Measurements of DAB intensity from IHC for PAD4. An average measurement from 5 mice was calculated from the average of 8 images. (K) IHC of the lung sections for Cit-H3. Scale bar: 100 µm. (L) Mean intensity of DAB for PAD4 from IHC images. An average measurement from 5 mice was calculated from the average of 8 images. All data are representations of one experiment out of 3 independent experiments. Each dot represents an individual mouse in the graphs. Data are shown as the mean ± SEM. **P < 0.01. Statistics: 2-tailed t test.

Fig 4: Immunostaining of colonic mucosa. Crypts in (A) CTRL and (B) DSS3 group show no sign of damage and no expression of PAD4. (C) colonic mucosa in DSS5 group is damaged and with peptidyl arginine deiminase (PAD)4-specific signal at the crypt borders. (D) colonic mucosa crypts in DSS7 group are disorganized and with strong PAD4-specific signal. Arrows show locations of PAD4-specific signal.

Fig 5: PAD4–/– mice have diminished ALI following cutaneous exposure to PAO.WT and PAD4–/– mice were exposed to PAO on skin and sacrificed after 6 hours. (A) H&E staining of lung sections (n = 5/group). Scale bar: 100 µm. (B) Lung injury score (n = 5/group). (C) IHC staining of lung sections for Cit-H3 (n = 5/group). Scale bar: 100 µm. Lower panel original magnification, ×200. (D) Measurements of DAB intensity from IHC for Cit-H3. An average measurement from 5 mice was calculated from the average of 8 images. (E) Percentage of neutrophils in BAL fluid (n = 5–6 /group). (F) Measurement of dsDNA levels in BAL fluid. WT and PAD4–/– mice were treated with Cit-H3 peptides (25 µg/mouse, intratracheally) and sacrificed after 12 hours. (G) H&E staining of lung sections (n = 5/group). Scale bar: 100 µm; inset original magnification, ×100. (H) Lung injury score (n = 5/group) and (I) protein in BAL fluid (n = 5). Statistics: (B, F, H, and I) box-and-whiskers plots display median, and whiskers show maximum to minimum value. Each symbol represents an individual mouse. *P < 0.05, **P < 0.01. Data were analyzed by using 1-way ANOVA followed by Tukey’s multiple comparisons test. In graphs (D and E) data are shown as the mean ± SEM. **P < 0.01.