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. 2021 Jan 6;10(1):81.
doi: 10.3390/cells10010081.

VSports注册入口 - Extracellular DNA Correlates with Intestinal Inflammation in Chemically Induced Colitis in Mice

Martin Maronek  1 , Barbora Gromova  1 , Robert Liptak  2 , Barbora Konecna  1 , Michal Pastorek  1 , Barbora Cechova  3 , Maria Harsanyova  4   5 , Jaroslav Budis  5   6   7 , David Smolak  4   5 , Jan Radvanszky  5   6   8 , Tomas Szemes  4   5   6 , Jana Harsanyiova  9 , Alzbeta Kralova Trancikova  10 , Roman Gardlik  1
Affiliations

Extracellular DNA Correlates with Intestinal Inflammation in Chemically Induced Colitis in Mice

Martin Maronek et al. Cells. .

Abstract

Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption VSports手机版. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated. .

Keywords: PAD4; cell-free DNA; deoxyribonuclease activity; neutrophil extracellular traps; ulcerative colitis. V体育安卓版.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Total, nuclear and mitochondrial DNA in plasma and colon explants. (A) the concentration of total ecDNA in plasma showed a time-dependent trend and was highest in DSS 7 group. (B) total extracellular DNA (ecDNA) in explants rose on day 3, however was lower in other timepoints. (C) the concentration of nuclear (ncDNA) in plasma did not differ between the groups. (D) ncDNA in explants reached the highest amount on day 3 and subsequently decreased until the end of the experiment. (E) mitochondrial (mtDNA) in plasma did not differ between the groups. (F) mtDNA in explants showed a similar pattern compared to ncDNA in explants, where mtDNA concentration rose on day 3 and decreased in other timepoints. * = p < 0.05; ** = 0.05 > p > 0.01; *** = p < 0.001.
Figure 2
Figure 2
Neutrophil extracellular trap (NET) osis and deoxyribonuclease (DNase) activity. (A) percentage of cells undergoing NETosis was elevated in both DSS 3 and DSS 5 groups compared to control group, however the highest percentage was observed in DSS 7 group. (B) DNase activity in plasma did not differ between the groups. (C) DNase activity in explants showed a rising trend along with increasing disease severity. (D) DNase activity in the distal colon was the highest on day 5, however decreased on day 7. *** = p < 0.001.
Figure 3
Figure 3
Correlations of total ecDNA and disease markers. The concentration of total ecDNA in plasma correlated with (A) relative weight of mice (p = 0.001, r2 = 0.2), (B) colon length (p = 0.0025, r2 = 0.2) and (C) percentage of cells undergoing NETosis (p = 0.003, r2 = 0.36). The concentration of total ecDNA in explants correlated with (D) percentage of cells undergoing NETosis (p = 0.025, r2 = 0.29) and (E) colon length (p = 0.019, r2 = 0.28). (F) the concentration of TNF-α correlated with percentage of cells undergoing NETosis.
Figure 4
Figure 4
Distribution of ecDNA fragments. Size and relative abundance of plasma ecDNA fragments in (A) CTRL, (B) DSS3, (C) DSS5 and (D) DSS7. CTRL shows higher abundance of fragment size 120–160 bp compared with other groups. Intragroup variability in the number of specific fragment sizes rises from CTRL towards DSS7 group. DSS7 showed the highest variability of fragment sizes and relative abundance. (E) principal component analysis (PCA) comparison of fragment sizes shows that the groups do not form bordered clusters. However, a clear left-to-right trend on x-axis is obvious from CTRL to DSS7 group.
Figure 5
Figure 5
Origin of ecDNA. (A) Relative proportion of mapped nuclear reads shows an increasing trend from CTRL toward DSS7. Relative proportion of (B) mitochondrial, (C) bacterial and (D) viral mapped reads shows decreasing trend from CTRL towards DSS7 group. Graph shows proportion of reads relative to total number of mapped reads.
Figure 6
Figure 6
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.
Figure 7
Figure 7
Comparison of Cl-amidine and streptonigrin treatment. Administration of either Cl-amidine or streptonigrin did not lead to amelioration of weight loss (A,B), stool consistency score (C,D) or colon length (E,F) *** = p < 0.001.
Figure 8
Figure 8
Representative images of endoscopic visualization of the colonic mucosa. When compared to negative (A) and positive control (B), administration of Cl-amidine visibly alleviated intestinal inflammation (C). Application of strep (F) managed to partially suppress the inflammation as well compared to negative (D) and positive control (E). Endoscopy score confirmed the partial amelioration of both Cl-amidine (G) and strep (H) treatment. ** = 0.05 > p > 0.01.
Figure 9
Figure 9
Cl-amidine treatment led to decrease of the concentration of total ecDNA in plasma (A), however this effect was not observed upon strep treatment (B). The concentration of total ecDNA in explants was not lower compared to positive control in either Cl-amidine (C) or strep (D) treatment. * = p < 0.05; ** = 0.05 > p > 0.01.
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