Skip to main page content (V体育平台登录)
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. VSports app下载.

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2020 Aug 5;8(8):275.
doi: 10.3390/biomedicines8080275.

Neutrophil Extracellular Traps Impair Intestinal Barrier Function during Experimental Colitis

Elliot Yi-Hsin Lin  1 , Hsuan-Ju Lai  1 , Yuan-Kai Cheng  1 , Kai-Quan Leong  1 , Li-Chieh Cheng  1 , Yi-Chun Chou  2 , Yu-Chun Peng  1 , Yi-Hsuan Hsu  1 , Hao-Sen Chiang  1   2
Affiliations

Neutrophil Extracellular Traps Impair Intestinal Barrier Function during Experimental Colitis

Elliot Yi-Hsin Lin (VSports在线直播) et al. Biomedicines. .

Abstract

Aberrant neutrophil extracellular trap (NET) formation and the loss of barrier integrity in inflamed intestinal tissues have long been associated with inflammatory bowel disease (IBD). However, whether NETs alter intestinal epithelium permeability during colitis remains elusive. Here, we demonstrated that NETs promote the breakdown in intestinal barrier function for the pathogenesis of intestinal inflammation in mouse models of colitis. NETs were abundant in the colon of mice with colitis experimentally induced by dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS) VSports手机版. Analysis of the intestinal barrier integrity revealed that NETs impaired gut permeability, enabling the initiation of luminal bacterial translocation and inflammation. Furthermore, NETs induced the apoptosis of epithelial cells and disrupted the integrity of tight junctions and adherens junctions. Intravenous administration of DNase I, an enzyme that dissolves the web-like DNA filaments of NETs, during colitis restored the mucosal barrier integrity which reduced the dissemination of luminal bacteria and attenuated intestinal inflammation in both DSS and TNBS models. We conclude that NETs serve a detrimental factor in the gut epithelial barrier function leading to the pathogenesis of mucosal inflammation during acute colitis. .

Keywords: DNase I; DSS/TNBS-induced colitis; intestinal barrier integrity; neutrophil extracellular traps (NETs) V体育安卓版. .

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results V体育ios版.

Figures

Figure 1
Figure 1
Neutrophil extracellular traps (NETs) are induced in the colon of dextran sulfate sodium (DSS)-treated mice. (A) NET release was measured in colon homogenates of wild-type C57BL/6 mice 8 d after the consumption of clean water or 2.5% DSS, by using myeloperoxidase (MPO)-DNA ELISA. (The results are pooled data from two separate experiments. n = 9 mice per group.) (B,C) NET formation counts per 200X high-power field (HPF) and representative NET release (white arrows) in the colon of wild-type C57BL/6 mice 8 d after the consumption of clean water or 2.5% DSS, assessed by immunofluorescence microscopy of MPO (green), citrullinated histone H3 (citH3; red), and Hoechst 33342-stained DNA (blue). Bottom row, enlargement of the area outlined in the second-row images. (The results are pooled data from two separate experiments. n = 9 mice per group.) (D) Representative NET release (gray arrows) in the colon of wild-type C57BL/6 mice 8 d after the consumption of 2.5% DSS, assessed by immunofluorescence microscopy of MPO (green), neutrophil elastase (NE; red), and Hoechst 33342-stained DNA (blue). Bottom row, enlargement of the area outlined in the second-row images (n = 9 mice per group). s: NETs in spread form, d: NETs in diffuse form, a: aggregate NET. The data represent the mean ± SEM. Statistical analyses were performed using unpaired, two-tailed t-test. ** p < 0.01, **** p < 0.0001. Scale bars, 50 µm. 20 µm in zoom in pictures.
Figure 2
Figure 2
DNase I treatment attenuates DSS-induced colitis in mice. (A) Schematic overview of the experiment. Colitis was induced by providing 2.5% DSS in the drinking water for 8 d. Clean water was fed to the controls. Phosphate-buffered saline (PBS) or 250 U/dose DNase I was intravenously (i.v.) administered to wild-type C57BL/6 mice every other day, on days 0, 2, 4, and 6. (B,C) Daily weight and total clinical scores of the control or DSS-treated mice after PBS or DNase I treatment. (D) Colon lengths and representative colon images of the control or DSS-treated mice with or without DNase I treatment. (B–D) The results are pooled data from four separate experiments. n = 15 mice per control groups and n = 18 mice per DSS groups. (E) Representative hematoxylin and eosin (H&E) staining of the colon, 400X high-power field (HPF) images are the enlargement of the area outlined in the 100X HPF images. The black arrow indicates neutrophils in the lumen. (F) Histopathology score of the control or DSS-treated mice with or without DNase I treatment. (E,F) The results are pooled data from two separate experiments. n = 9 mice per control groups and n = 12 mice per DSS groups. (G) Percentage and absolute numbers of neutrophils (CD11b + Ly6G+F4/80-) in the intestinal epithelium (IE) and lamina propria (LP) of the colon (Total). n = 5 mice per group. The data represent the mean ± SEM. Statistical analyses were performed using (B–D,F), one-way ANOVA with Turkey’s multiple comparison or (G), an unpaired two-tailed t-test. ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar, 1 cm in (D), 100 µm in 100× pictures and 25 µm in 400× pictures of (E).
Figure 3
Figure 3
DNase I treatment significantly degrades NET structure in the colon of mice fed DSS. (A) Representative NET release (white arrows) in the colon of PBS control or DNase I-treated mice 8 d after the consumption of clean water or 2.5% DSS, assessed by the immunofluorescence microscopy of myeloperoxidase (MPO; green), citrullinated histone H3 (citH3; red), and Hoechst 33342-stained DNA (blue). (B) Enlargement of the area outlined in (A). White arrows indicate NETs. s: NETs in spread form, d: NETs in diffuse form, a: aggregate NET. (C) NET formation counts per 200X high-power field (HPF) in each group. (A–C) The results are pooled data from two separate experiments. n = 9 mice in water control groups and n = 12 mice in DSS-treated groups, at least 3 images/mouse. (D) NET release, measured in colon homogenates of PBS- or DNase I-administered wild-type C57BL/6 mice 8 d after the consumption of clean water or 2.5% DSS, by using MPO-DNA complex ELISA. The results are pooled data from three separate experiments. n = 9 mice per control groups and n = 15 mice per DSS groups. The data represent the mean ± SEM. Statistical analyses were performed using one-way ANOVA with Turkey’s multiple comparison. **** p < 0.0001. Scale bars, 50 µm in (A). 20 µm in (B).
Figure 4
Figure 4
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-α, Il-1β, 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.
Figure 5
Figure 5
NETs alter intestinal barrier function and cause the apoptosis of intestinal cells in the colon of DSS-treated mice. (A) Intestinal permeability was determined by quantifying the amount of fluorescein isothiocyanate (FITC)-dextran levels in the serum 4 h after its oral gavage. PBS- or DNase I-administered wild-type C57BL/6 mice fed clean water or 2.5% DSS were tested on day 8 from the beginning of the DSS treatment. (B,C) Bacterial counts in the colon and mesenteric lymph nodes (MLN) of control or DSS mice treated with PBS or DNase I were determined on day 8. (D) Quantitative PCR analysis of relative amount of 16S rDNA in the feces of control and DSS mice treated with PBS or DNase I on day 8. (A-D) The results are pooled data from two separate experiments. n = 9 mice per control groups and n = 12 mice per DSS groups. (E,F) Representative immunofluorescence staining of occludin, ZO-1, E-Cadherin, and Hoechst 33342-stained DNA and representative fluorescent images of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL, green) staining of the colon tissues isolated from PBS- or DNase I-administered wild-type C57BL/6 mice on day 8 after the consumption of clean water or 2.5% DSS. Slides were counterstained with Hoechst 33,342 (blue). (G) Percentage of apoptotic cells per 200X high-power field (HPF) in each group listed in f. The results are pooled data from two separate experiments. n = 8 mice per control groups and n = 9 mice per DSS groups. The data represent the mean ± SEM. Statistical analyses were performed using A-D, G one-way ANOVA with Turkey’s multiple comparison or E, unpaired two-tailed t-test. * p < 0.05, *** p < 0.001, **** p < 0.0001. Scale bar, 10 µm in E; 50 µm in the left panel and 20 µm in the zoom of F.
Figure 6
Figure 6
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-1β, Tnf-α, 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.
Figure 7
Figure 7
Neutrophil extracellular traps (NETs) exacerbate mouse experimental colitis by impairing intestinal barrier function. (Left) NETs are abundant in the colon of mouse in DSS-induced or TNBS-induced colitis models. In the colon of non-treated mouse, aberrant NET formation promotes the apoptosis of intestinal cells during colitis. NETs also alter intestinal epithelial permeability leading to luminal bacterial translocation into the colon and MLN as well as gut inflammation in vivo. Mechanistically, histones are the major protein components of NET structure that decrease the intestinal barrier integrity and function, as well as promotes the cytotoxicity of intestinal epithelial cells. (Right) Disruption of NET structure with DNase I in mice with DSS-induced or TNBS-induced colitis protects the host from intestinal inflammation and injury by restoring the intestinal barrier integrity and function that prevent luminal bacterial translocation into the colon and MLN, suggesting that NETs play a distinct role that is required for the development and pathogenesis of colitis.
See this image and copyright information in PMC

References

    1. Ng S.C., Shi H.Y., Hamidi N., Underwood F.E., Tang W., Benchimol E.I., Panaccione R., Ghosh S., Wu J.C.Y., Chan F.K.L., et al. Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: A systematic review of population-based studies. Lancet. 2018;390:2769–2778. doi: 10.1016/S0140-6736(17)32448-0. - DOI - PubMed
    1. Khor B., Gardet A., Xavier R.J. Genetics and pathogenesis of inflammatory bowel disease. Nature. 2011;474:307–317. doi: 10.1038/nature10209. - VSports手机版 - DOI - PMC - PubMed
    1. Okumura R., Takeda K. Roles of intestinal epithelial cells in the maintenance of gut homeostasis. Exp. Mol. Med. 2017;49:e338. doi: 10.1038/emm.2017.20. - DOI - PMC - PubMed
    1. Knights D., Lassen K.G., Xavier R.J. Advances in inflammatory bowel disease pathogenesis: Linking host genetics and the microbiome. Gut. 2013;62:1505–1510. doi: 10.1136/gutjnl-2012-303954. - DOI (VSports最新版本) - PMC - PubMed
    1. Wera O., Lancellotti P., Oury C. The Dual Role of Neutrophils in Inflammatory Bowel Diseases. J. Clin. Med. 2016;5:118. doi: 10.3390/jcm5120118. - DOI (V体育ios版) - PMC - PubMed