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Reactive Oxygen Species Deficiency Due to Ncf1-Mutation Leads to Development of Adenocarcinoma and Metabolomic and Lipidomic Remodeling in a New Mouse Model of Dextran Sulfate Sodium-Induced Colitis

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Reactive Oxygen Species Deficiency Due to Ncf1-Mutation Leads to Development of Adenocarcinoma and Metabolomic and Lipidomic Remodeling in a New Mouse Model of Dextran Sulfate Sodium-Induced Colitis

Lina Carvalho et al. Front Immunol.

Abstract

Inflammatory bowel disease is characterized by chronic relapsing idiopathic inflammation of the gastrointestinal tract and persistent inflammation. Studies focusing on the immune-regulatory function of reactive oxygen species (ROS) are still largely missing. In this study, we analyzed an ROS-deficient mouse model leading to colon adenocarcinoma. Colitis was induced with dextran sulfate sodium (DSS) supplied via the drinking water in wild-type (WT) and Ncf1-mutant (Ncf1) B10.Q mice using two different protocols, one mimicking recovery after acute colitis and another simulating chronic colitis. Disease progression was monitored by evaluation of clinical parameters, histopathological analysis, and the blood serum metabolome using 1H nuclear magnetic resonance spectroscopy. At each experimental time point, colons and spleens from some mice were removed for histopathological analysis and internal clinical parameters. Clinical scores for weight variation, stool consistency, colorectal bleeding, colon length, and spleen weight were significantly worse for Ncf1 than for WT mice. Ncf1 mice with only a 7-day exposure to DSS followed by a 14-day resting period developed colonic distal high-grade dysplasia in contrast to the low-grade dysplasia found in the colon of WT mice. After a 21-day resting period, there was still β-catenin-rich inflammatory infiltration in the Ncf1 mice together with high-grade dysplasia and invasive well-differentiated adenocarcinoma, while in the WT mice, high-grade dysplasia was prominent without malignant invasion and only low inflammation. Although exposure to DSS generated less severe histopathological changes in the WT group, the blood serum metabolome revealed an increased fatty acid content with moderate-to-strong correlations to inflammation score, weight variation, colon length, and spleen weight. Ncf1 mice also displayed a similar pattern but with lower coefficients and showed consistently lower glucose and/or higher lactate levels which correlated with inflammation score, weight variation, and spleen weight. In our novel, DSS-induced colitis animal model, the lack of an oxidative burst ROS was sufficient to develop adenocarcinoma, and display altered blood plasma metabolic and lipid profiles. Thus, oxidative burst seems to be necessary to prevent evolution toward cancer and may confer a protective role in a ROS-mediated self-control mechanism.

Keywords: adenocarcinoma; colitis; dextran sulfate sodium; lipids; metabolism; nicotinamide adenine dinucleotide phosphate oxidase; nuclear magnetic resonance; reactive oxygen species.

Figures

Figure 1
Figure 1
Ncf1-mutant (Ncf1) mice presented more severe clinical scores than their wild-type (WT) counterparts. (A) Colon lengths in centimeters for days 0, 22, and 30 of control, recovery (r), and twice colitis-induced (i) WT and Ncf1 groups. (B) Change in body weights from the initial average baseline weight over the experimental period, average weight ± SE (baseline weights: WT = 29.8 ± 3.90 g, Ncf1 = 31.3 ± 3.9 g). Differences between groups and time points were calculated by two-way ANOVA. (C) Mice stool consistency score after colitis induction with dextran sulfate sodium (DSS). (D) Colorectal bleeding score after colitis induction. Weight, stool consistency, and colorectal bleeding scorings are detailed in Section “Materials and Methods.”. Asterisks indicate significant differences: *p < 0.05, **p < 0.001, ***p < 0.0001 between WT and Ncf1 mice receiving DSS; boxed asterisks indicate significant differences: *p < 0.05 between Ncf1 recovery (r) and induction (i) groups.
Figure 2
Figure 2
Colonic mucosa of control, recovery (r), and twice colitis-induced (i) wild-type (WT) and Ncf1-mutant (Ncf1) groups. Day 0: glands are smaller, fewer, and with small epithelial cell nuclei in Ncf1 mice colon compared with WT mice. Day 30 of recovery: WT mouse colon with basal small gland hyperplasia and villous epithelial adaptation; Ncf1 mouse colon with superficial villous adaptation, high-grade dysplasia, and intramucosal adenocarcinoma. Day 22 for both experiments: both WT and Ncf1 mice colons with superficial villous glandular hyperplasia, and inflammation-reactive atypia in WT mouse colon; low-grade dysplasia; and mucinous cells hyperplasia in tubular glands of WT mouse colon contrasting to basal high-grade dysplasia in Ncf1 mouse colon. Day 30 of the second colitis induction: Superficial villous mucosae and basal cell glandular persistence with high-grade dysplasia in WT mouse colon compared with the superficial villous atrophy and well-differentiated adenocarcinoma in Ncf1 mouse colon. hematoxylin/eosin staining with 40× and 400× magnifications.
Figure 3
Figure 3
Histological evaluation of inflammation (A), dysplasia (B), and cellular composition of the inflammatory infiltrate (C) at distal and proximal segments of the colon for Ncf1-mutant (Ncf1)* mice, and WT mice. Asterisks indicate p < 0.05, Mann–Whitney U test between Ncf1(r) and WT (r) and Ncf1(i) and WT(i). Inflammation and dysplasia scoring systems are detailed in Section “Materials and Methods.”
Figure 4
Figure 4
The extensive inflammatory infiltrates present in the recovery (r) and twice colitis-induced (i) Ncf1-mutant (Ncf1) mouse colons harbor lymphocytes expressing high levels of β-catenin. In wild-type (WT) mice, some inflammatory foci contain scattered β-catenin-expressing lymphocytes. (A) Hematoxylin/eosin staining of representative colon sections of each protocol at D30 with 100× magnification; and the corresponding (B) β-catenin staining (brown) with 100× magnification. The colon sections are from the same samples as Figure 2, thus corresponding to the description of the histological features in that legend.
Figure 5
Figure 5
Representative Carr–Purcell–Meiboom–Gill (CPMG) (A) and 1H (B) nuclear magnetic resonance spectra of blood sera from wild-type (WT) and Ncf1-mutant (Ncf1) mice at baseline, after recovery of the first period of dextran sulfate sodium (DSS)-induced colitis (r) and after the second period of DSS-induced colitis (i). The CPMG pulse sequence suppresses the broad signals from macromolecules allowing a better characterization of the signals arising from the small metabolites. Signal assignments: (1) isoleucine, (2) leucine, (3) valine, (4) β-hydroxybutyrate, (5) lactate, (6) threonine, (7) alanine, (8) acetate, (9) proline, (10) glutamate, (11) glutamine, (12) methionine, (13) malate, (14) creatine, (15) choline, (16) phosphorylcholine/glycerophosphocholine, (17) taurine, (18) glycine, (19) serine, (20) water, (21) α-glucose, (22) fumarate, (L1) lipid methyls, (L2) lipid aliphatic chain, (L3) lipid β-methylenes, (L4) lipid allylic methylenes, (L5) lipid α-methylenes, (L6) lipid polyunsaturated allylic methylenes, and (L7) lipid alkenes.
Figure 6
Figure 6
Control wild-type (WT) and Ncf1-mutant (Ncf1) mice display similar metabolomic profiles showing no difference in small molecule metabolites and lipid and macromolecular fractions. The scores plot (A) accounts for 66.3% of the total variance (PC1 = 51.8%, PC2 = 14.5%) and the loadings plot (B) shows the δ of each variable (bucket) for the principal component analysis (PCA) analysis of Carr–Purcell–Meiboom–Gill (CPMG) spectra of Ncf1 and WT control groups. The CPMG pulse sequence suppresses the broad signals from macromolecules allowing analyze small molecule metabolites. The scores plot (C) summarizes 67.3% of the total variance (PC1 = 36.2%, PC2 = 31.1%) and the loadings plot (D) shows the δ of each variable (bucket) of the PCA analysis of 1H spectra of Ncf1 vs WT control groups. The 1H spectra allow analysis of the aqueous lipid fraction and other macromolecules.
Figure 7
Figure 7
The presence of the Ncf1-mutant (Ncf1) mutation leads to distinct metabolomic profiles in mice either recovering from (r) or after a second period of DSS-induced colitis (i). This contrasts with wild-type (WT) mice which present overlapping metabolomic profiles between control, recovery, and second colitis-induced groups. Principal component analysis of 1H spectra of WT and Ncf1mice. The scores plot for the WT reaches 49.4% of the total variance (PC1 = 33.8%, PC2 = 15.6%) (A); for the Ncf1 achieves 49.6% of the total variance (PC1 = 35.1%, PC2 = 14.5%) (B). The loadings plot shows the δ of each variable (bucket) for the WT (C) and the Ncf1 (D) groups.
Figure 8
Figure 8
The systemic metabolic profile after a second period of dextran sulfate sodium-induced colitis is markedly distinct between mice bearing the Ncf1-mutation (Ncf1) and the wild-type (WT) group. However, the two recovery groups tend to diverge from the induced groupings and edge toward a common metabolic profile. Principal component analysis (PCA) and partial least squares–discriminant analysis (PLS–DA) analysis of 1H spectra of Ncf1 vs WT after the second colitis induction (i) and recovery (r) groups. The PCA scores plot explains 41.8% of the total variance (PC1 = 25.5%, PC2 = 16.3%) (A) and the loadings plot shows the δ of each variable (bucket) (B). The PLS–DA model (C) and variable importance in projection (VIP) variables (D) for the four groups (r2 = 0.70, q2 = 0.43).

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