Fig 2: Minocycline ameliorates axonal degeneration and functional deficit in aquaporin-4 (AQP4)–immunoglobulin (IgG)-induced optic neuritis (ON) rats. A Experimental time course. B, D Left: Representative images of Iba1 staining(green). Scale bar: 50 µm. Right: Representative images of MPO staining (red). Scale bar: 50 µm. C, E Left: Quantification of Iba1-positive area (%) (AQP4-IgG + PBS: n = 6, AQP4-IgG + minocycline: n = 6, mean ± SEM ***p < 0.001, assessed by Student’s t test). Right: Quantification of MPO-positive area (%) (AQP4-IgG + PBS: n = 6, AQP4-IgG + minocycline: n = 6, mean ± SEM, ns means no significant difference, assessed by Student’s t test). F Experimental time course of the analysis on retinal ganglion cell (RGC) axon quantification. G Representative confocal images showing CTB-labeled axons in the longitudinal optic nerve section. White arrows indicate the injection site. Scale bar: 500 µm. H Representative images of CTB-labeled axons in transverse sections at 1 mm and 4 mm posterior to the IgG-injected site. Scale bar: 20 µm. I, J Quantification of CTB-labeled axons at 1 mm and 4 mm posterior to the IgG-injected site (AQP4-IgG+PBS: n = 5, AQP4-IgG+minocycline: n = 5, mean ± SEM, ***p < 0.001 assessed by Student’s t test). K Representative images of FluoroMyelin staining at 1 mm and 4 mm posterior to the IgG-injected site. L, M Quantification of FluoroMyelin-positive area at 1 mm and 4 mm posterior to the IgG-injected site (AQP4-IgG + PBS: n = 5, AQP4-IgG + minocycline: n = 5, mean ± SEM, **p < 0.01, assessed by Student’s t test). N Experimental time course of PLR experiment. O Quantification of pupillary constriction (%) (AQP4-IgG+PBS: n = 7, AQP4-IgG+minocycline: n = 7, mean ± SEM, **p < 0.001, ***p < 0.001, assessed by two-way ANOVA, followed by Bonferroni test). P Quantification of CTB-labeled axons at 4 mm posterior to the IgG-injected site. (AQP4-IgG+PBS: n = 7, AQP4-IgG+minocycline: n = 7, mean ± SEM, **p < 0.01 assessed by Student’s t test). Q Spearman's correlation coefficient test was used to assess the correlations between the number of CTB-positive axons (day 24) and constriction rate of Pupillary light reflex (day 21) (AQP4-IgG+PBS: n = 7, AQP4-IgG+minocycline: n = 7)

Fig 3: COVID-19 patients produces high concentrations of NETs. Plasma and neutrophils were isolated from healthy controls and COVID-19 patients. (A) NET quantification by MPO-DNA PicoGreen assay in plasma from healthy controls (H.Control; n = 21) or COVID-19 patients (n = 32). (B) Supernatants from cultures of blood isolated neutrophils from healthy controls (n = 10) or COVID-19 patients (n = 11). NET quantification was performed using MPO-DNA PicoGreen assay. (C) Representative confocal analysis of NETs release by neutrophils isolated from healthy controls (n = 10) or COVID-19 patients (n = 11), cultured for 4 h at 37°C. Cells were stained for nuclei (DAPI, blue), MPO (green), and H3Cit (red). Scale bar indicates 50 µm. (D) Colocalization of DAPI and MPO between healthy controls (n = 10) and COVID-19 (n = 10). The data depicts Pearson’s correlation coefficient assessed by Fiji/ImageJ software. (E) Percentage of NETosis in neutrophil from COVID-19 patients (n = 7). (F) NET length quantification. (G) NET quantification by MPO-DNA PicoGreen assay in the supernatants of blood-isolated neutrophils from COVID-19 patients (n = 3) preincubated, or not, with PAD-4 inhibitor (Cl-Amidine; 200 µM) for 4 h at 37°C. (H) Representative confocal images showing the presence of NETs in isolated neutrophils from COVID-19 patients, treated or not, with Cl-Amidine (200 µM). Cells were stained for nuclei (DAPI, blue), MPO (green), and H3Cit (red). Scale bar indicates 50 µm. Data are representative of at least two independent experiments and are shown as mean ± SEM. P value were determined by two-tailed unpaired (A, B, and D–F) or paired (G) Student t test.

Fig 4: Degradation of NETs using rhDNase improves neurological outcomes after TBI.(A) Administration of rhDNase (5 mg/kg i.v.) at 1 hour after TBI reduced the extracellular expression of MPO and NE on Ly6G+TLR4+ neutrophils in both blood and brain tissue after TBI. (B) Quantification of data from (A). Scatterplots are representative of n = 8 mice per group and depict the % TLR4+ neutrophils exhibiting extracellular expression of NE and MPO. (C) Administration of rhDNase (5 mg/kg i.v.) at 1 hour after TBI reduced cerebral edema by MRI (top) and improved cerebral perfusion by MRI (middle) and LSCI (bottom) at 24 hours after injury. Representative images are provided from n = 8 mice per group. (D) Quantification of edema, absolute CBF, and area of slow flow/reduced reflow from (C). Data were analyzed using a one-way ANOVA followed by Tukey’s post hoc test. (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not statistically significant). (E) Administration of rhDNase (5 mg/kg i.v.) at 1 hour after TBI improved chronic neurological function, as assessed at 2 months after TBI, as compared to placebo-treated mice. Top panels depict representative heat maps obtained in the open-field test, which demonstrated an increased time spent within the center zone (a measure of decreased anxiety) after rhDNase treatment following TBI (see middle panel, left). Representative heat maps obtained following the NOR test show that rhDNase increased the time spent exploring a novel object. The increased discrimination index (middle, right) suggests that rhDNase improves recognition memory at 8 weeks after TBI. Bottom panels demonstrate improvements in chronic, posttraumatic motor function following rhDNase treatment. Administration of rhDNase at 1 hour after TBI reduced the time required to traverse a narrow beam and decreased the number of slips, indicative of better motor function and coordination (bottom left and middle). Treatment with rhDNase also improved grip strength after TBI, as compared to placebo-treated mice, suggestive of better neuromuscular function. Data are means ± SEM from n = 9 to 13 mice per group and were analyzed by one-way ANOVA followed by Tukey’s post hoc test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig 5: IEC-specific loss of OGT disrupts intestinal proliferative/apoptotic homeostasis ARepresentative images of the H&E staining of ileum and colon of 10-week-old male wild-type and Vil-Ogt KO mice. Scale bars = 100 µm.BCombined scores of the mucosal injury in 10-week-old male wild-type and Vil-Ogt KO mice (n = 11, ileum: P < 0.0001, colon: P = 0.0012).CRepresentative images of MPO, F4/80, and CD4 immunohistochemistry in the ileum tissue of male wild-type and Vil-Ogt KO mice. Scale bars = 50 µm.DRT–qPCR of inflammatory markers in jejunum, ileum, and colon (n = 5, jejunum: Il6 P = 0.029, Ifng P = 0.0009; ileum: Tnfa P = 0.0094, Ifng P = 0.0066; colon: Tnfa P = 0.028).EImmunofluorescent staining of Ki-67 in ileal tissues (n = 5). Scale bars = 50 µm.F, GTUNEL staining (F) and immunostaining of Cleaved-CASPASE3 (G) in the ileum. Quantification of the numbers of positive cells is shown at the right. (TUNEL WT n = 9, KO n = 6, P = 0.0003; Cleaved Cas3 WT = 8, KO = 7, P < 0.0001).HNumbers of survived organoids per 1000 isolated ileal crypts (WT n = 4, KO n = 23, P < 0.0001).Data information: Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed t-test.