Fig 2: DNase I administration reduces intestinal inflammation and restores intestinal barrier function in mice with 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced colitis. (A) Schematic overview of the experiment. Colitis was induced by the intra-rectal (i.r.) injection of TNBS into pre-sensitized C57BL/6 mice. PBS or 250 U/dose DNase I was i.v. administered to mice on day 0 and 2. (B) NET release, measured in colon homogenates of PBS- or DNase I-administered TNBS-induced colitis mice on day 4, by using MPO-DNA complex ELISA. n = 9 mice per group. (C) Representative NET release (white arrows) in the colon of PBS control or DNase I-treated mice 4 d after TNBS administration, assessed by immunofluorescence microscopy of myeloperoxidase (MPO; green), citrullinated histone H3 (citH3; red), and Hoechst 33342-stained DNA (blue). n = 5 mice per PBS group and n = 5 mice per DNase I group. (D) Daily weight of TNBS mice after PBS or DNase I treatment. (E) Colon lengths of TNBS-treated mice with or without DNase I treatment. (F) Fecal lipocalin-2 levels were analyzed from fecal samples obtained from PBS- or DNase I-administered TNBS mice at different time points by ELISA. (G) Protein level of Il-1ß, Tnf-a, and Il-17A were determined in the colon homogenates of PBS- or DNase I-administered TNBS mice on day 4 by ELISA. (H) Representative H&E staining of the colon and histopathology score of the TNBS mice with or without DNase I treatment. 400× images are the enlargement of the area outlined in the 100X images. (D-H) The results are pooled data from two separate experiments. n = 7 mice per PBS group and n = 9 mice per DNase I group. (I) Intestinal permeability was determined by quantifying the amount of FITC-dextran levels in the serum 4 h after its oral gavage. PBS- or DNase I-administered TNBS mice were tested on day 4. n = 5 mice per PBS group and n = 6 mice per DNase I group. (J) Bacterial counts in the colon and MLN of TNBS mice treated with PBS or DNase I were determined on day 4. (K) Quantitative PCR analysis of relative amount of 16S rDNA in the feces of TNBS mice treated with PBS or DNase I on day 4. h. J and K results are pooled data from two separate experiments. n = 7 mice per PBS group and n = 8 or 9 mice per DNase I group. (L) Representative immunofluorescence staining of occludin, ZO-1, E-Cadherin, and Hoechst 33342-stained DNA of the colon tissues isolated from PBS- or DNase I-administered TNBS mice on day 4. (M) Percentage of apoptotic cells per 200× high-power field (HPF) in the colon of TNBS mice with or without DNase I treatment. n = 5 mice per PBS group and n = 6 mice per DNase I group. The data represent the mean ± SEM. Statistical analyses were performed using unpaired two-tailed t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar, 20 µm in c, 100 µm in 100× pictures and 25 µm in 400× pictures of H, 10 µm in L.

Fig 3: MIA down-regulates microglial Gpr56 expression in fetal brains in an IL-17a–dependent manner.(A) A schematic timeline for MIA induction. (B) Serum concentration of IL-6 was significantly up-regulated at 3 hours after poly(I:C) injection and went back to baseline 48 hours later at E14.5 (n = 5). (C) Serum concentration of IL-17a was significantly up-regulated at E14.5 48 hours after poly(I:C) injection (n = 5). (D) Fluorescence-activated cell sorting (FACS) strategy to collect CD11b+CD45medium microglia and CD11b-CD45- cells as nonmicroglial cells. (E) Relative levels of Gpr56 mRNA in microglia isolated from E14.5 fetal brains whose mothers were treated with PBS (n = 6), poly(I:C) (n = 5), IgG2a isotype control antibodies and poly(I:C) (n = 5), and IL-17a antibodies and poly(I:C) (n = 6). (F) Relative levels of Gpr56 mRNA in nonmicroglia cells isolated from E14.5 fetal brains. PBS versus poly(I:C): P = 0.55; IgG2a + poly(I:C) versus IL-17a antibodies + poly(I:C): P = 0.69. Unpaired Student’s t test. n.s., not significant. *P < 0.05 and ***P < 0.001. Data presented as means ± SEM.

Fig 4: Immunohistochemical staining of cytokines deposited in the dorsal skin tissue of experimental animals. (A) Expression of IL-4. (B) Expression of IL-5. (C) Expression of IFN-?. (D) Expression of IL-17. Scale bar indicates 100 µm (200X). The small black square indicates the location of antibody staining, which is subsequently magnified in the corners of the image. (i- Control, ii- Compound 1 alone, iii- Compound 2 alone, iv- DNCB, v- DNCB+1, vi- DNCB+2).

Fig 5: Disruption of NETs reduces the colonic inflammation in mouse fed DSS-containing water. (A) Fecal lipocalin-2 (Lcn2) levels. Fecal samples obtained from PBS- or DNase I-administered wild-type C57BL/6 mice 8 d after the consumption of clean water or 2.5% DSS were analyzed at different time points by ELISA. The results are pooled data from two separate experiments. n = 9 mice per control groups and n = 12 mice per DSS groups. (B) Quantitative RT-PCR analysis of Tnfa, Il1b, and Il17a mRNA levels in the colon of control and DSS mice treated with PBS or DNase I. Values are normalized to the expression of Tbp. The results are pooled data from two separate experiments. n = 9 mice per control groups and n = 12 mice per DSS groups. (C) Levels of Tnf-a, Il-1ß, and Il-17a were determined in the colon homogenates of PBS- or DNase I-administered wild-type C57BL/6 mice 8 d after the consumption of 2.5% DSS. The results are pooled data from two separate experiments. n = 9 mice per PBS group and n = 10 mice per DNase I group. The data represent the mean ± SEM. Statistical analyses were performed using one-way ANOVA with Turkey’s multiple comparison. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.