Male and female homozygous Ncf1 (BQ.Ncf1^m1J/m1J, abbreviated to Ncf1, n = 30) and wild-type (WT, n = 30) B10.Q mice between 6 and 8 weeks old were obtained from breeding heterozygous mice followed by genotyping as previously described (28). Animals were bred and maintained under standard conditions at the specific pathogen-free animal facility of the Faculty of Pharmacy, University of Coimbra with food and water supplied ad libitum within a controlled temperature environment, and alternating 12-h light/dark cycles. All animal studies were approved by the internal FFUC Animal Facility Ethics Committee and were in accordance with EU legislation for experimental animal welfare.

Colitis was induced by oral administration of 3% w/v DSS (average mol. wt. 40,000 g/mol, AppliChem, Darmstadt, Germany) in the first induction period and 2.5% w/v DSS in the second induction period via drinking water supplied ad libitum. The DSS concentration was reduced for the second cycle to prevent premature death so that animals survived till the end of the experiment. DSS reduction also minimized weight loss and rectal bleeding, thus limiting excessive suffering.

Two different colitis-induction protocols were used: in protocol remission (r), mice were subjected to a 7-day DSS induction followed by 21 days of resting on normal water; in protocol induction (i), mice were subjected to 7 days of DSS induction, followed by 14 days of resting on normal water and then a second 7-day DSS-induction period.

Animals were monitored every third day for alterations in colitis-related clinical scores: weight variation, stool consistency, and colorectal bleeding. Pain was monitored on a daily basis through observation of animal activity for 5 min during the resting (light cycle) and active (dark cycle) periods. Blood and stool scorings were performed as previously described (25). In brief, blood scoring: 0- no blood; 1- visible blood; 2- rectal bleeding; stool consistency scoring: 0- normal; 1- soft but formed; 2- very soft; 3- diarrhea. Five mice from each group were sacrificed by cervical dislocation under anesthesia at three experimental time points: days 0 (baseline controls), 22 and 30. Tissues were collected for histopathological analysis (vide infra) and assessment of colon length and spleen weight.

Swiss rolls of the whole colon, rolled from the rectum to cecum, were formalin fixed and paraffin embedded. Sections were hematoxylin/eosin (HE) stained according to standard protocols.

Immunohistochemistry was performed on distal colon sections. In brief, endogenous peroxidase activity was quenched by 15 min incubation with 3% diluted hydrogen peroxide. Non-specific binding was blocked with Ultra V Block (Ultra Vision Kit; TP-125-UB; Lab Vision Corporation, Fremont, CA, USA). Incubation with the primary rabbit monoclonal antibody against β-catenin (clone D10A8, Cell Signaling Technology Europe, Frankfurt am Main, Germany) was followed by incubation with biotin-labeled secondary antibody (Ultra Vision Kit; TP-125-BN; Lab Vision Corporation, Fremont, CA, USA). Primary antibody binding was localized in tissues using peroxidase-conjugated streptavidin (Ultra Vision Kit; TP-125-HR; Lab Vision Corporation, Fremont, CA, USA) and 3,3-diaminobenzidine tetrahydrochloride (RE7190-K; Novocastra Laboratories Ltd., Newcastle, UK) was used as chromogen, according to manufacturer’s instructions. Hematoxylin was used to counterstain the slides.

Inflammation was scored in proximal and distal segments halves regarding the number of inflammatory foci: 0- no inflammatory focus; 1- one inflammatory focus; 2- two inflammatory foci; 3- three or more inflammatory foci. Lymphocytes, plasma cells, and neutrophils infiltration were scored based on cell morphology, with approximate percentage validation of each subset within the total observed inflammatory cells in inflammatory foci, as previously described (25).

Epithelial morphology was scored based on the presence of tubular and villous patterns: tubular-glandular hyperplasia above the muscularis mucosae and villous formation of superficial villous projections. Dysplasia was scored in the same segments using a semi-quantitative scale according to the World Health Organization (WHO) 2010 guidelines for colon adenocarcinoma classification (26): 0- no dysplasia; 1- hyperchromatic nuclear pluristratification and lamina propria separated glands; 2- epithelial low-grade dysplasia (complex ramified glands with cell hyperplasia and pluristratified hyperchromatic nuclei); 3- epithelial high-grade dysplasia (beyond low-grade dysplasia, nuclear atypia, mitosis and reduced lamina propria area).

Blood serum from mice was collected for metabolic profiling using¹ H NMR spectroscopy. Samples consisted of 70 µL of sera plus 70 µL of D₂O (99.9%) together with 35 µL of sodium fumarate (10 mM) and phosphate buffer dissolved in D₂O (99.9%) for use as an internal reference. Samples (total volume 175 µL) were placed into 2.5 mm NMR tubes and spectra acquired using a 600 MHz Bruker NMR spectrometer equipped with an inverse configuration probe. For all samples, regular ¹H acquisition with presaturation (sw = 10 kHz, ns = 32, td = 60 k, aq = 3 s) and Carr–Purcell–Meiboom–Gill (CPMG) (28) (sw = 10 kHz, ns = 350, spin-echo time = 200 168 ms, td = 60 k, aq = 3 s) spectra were acquired. Spectra were processed with TopSpin using 0.2 Hz of line broadening and manual phasing while AMIX was used for metabolite assignment and multivariate statistics. For metabolomics integration, spectra were subjected to bucketing by AMIX from 0.5–9 ppm (excluding the solvent region) with the spectral area normalized to the sum of all points. All metabolomics data have been deposited to the EMBL-EBI MetaboLights database (DOI: 10.1093/nar/gks1004. PubMed PMID: 23109552) with the identifier MTBLS593.

All data were tested for normal distribution with Levene’s test. Since data did not follow a normal distribution, the non-parametric Kruskal–Wallis test followed by a non-parametric Mann–Whitney U test were used to compare values between groups and time points using GraphPrism 6.01 (GraphPad Software Inc., CA, USA).

Statistical differences between curves were determined using a two-sided hypothesis permutation test with 10,000 permutations (http://bioinf.wehi.edu.au/software/compareCurves/index.html) (29). Bivariate correlation studies (Spearman, two-sided) were performed using SPSS 17. Differences were considered significant for p < 0.05. For NMR metabolomics analysis, data were analyzed using MetaboAnalyst software performing interquartile range filtering, log transformation, and pareto scaling prior to principal component analysis (PCA) or partial least squares–discriminant analysis (PLS–DA) modeling (30).

Colitis induction with DSS led to a reduction in the colon length in both the WT and the Ncf1 mice resulting in a significant shortening of the colon after a second induction period (Figure 1A).

Body weight was monitored throughout the study (Figure 1B). At baseline, the weights of Ncf1 and WT mice were comparable (WT = 29.8 ± 3.90 g, Ncf1 = 31.3 ± 3.9 g). Weight loss began on day 3 after DSS colitis induction with all DSS-treated animals reaching the minimum weight on day 13 (i.e., during the recovery period) and with Ncf1 mice presenting a greater weight loss than WT mice. In protocol (r), the WT mice recovered their baseline weight while Ncf1 only recovered, at most, up to 90% of their original weight. The mice which were subjected to a second cycle of DSS-induced colitis [protocol (i)], partially recovered their weight until day 25 when a new phase of weight loss set in. Ncf1 mice suffered greater weight loss (reduction to 75% of baseline weight) than the WT mice (reduction to only 90% of baseline weight). While WT mice on both protocols had comparable weights until the end of the experimental period, Ncf1 mice under the (i) protocol had a greater weight loss than those under the (r) protocol.

The presence of colorectal blood and the consistency of the feces are two further clinical signs of DSS-induced colitis. During the first induction period, both Ncf1 and WT groups had decreased stool consistency and increased anal bleeding. During the resting period, both groups started to recover with respect to these two clinical parameters. WT and Ncf1 mice submitted to protocol (i) both presented a new surge in anal bleeding together with softer stools, these clinical symptoms were significantly different to those animals undergoing protocol (r) (Figures 1C,D).

To evaluate epithelial morphology, inflammation, and dysplasia, the defined colon histopathology scores were applied using untreated WT and Ncf1 animals as controls and registered after colitis induction at day 22 for both experiments and day 30 for protocol r and protocol i.

At baseline, WT mice had well defined glands above muscularis mucosae, whereas Ncf1 mice presented a reduced number of glandular tubules (Figure 2 D0), both with the absence of dysplasia and inflammation.

On day 22, WT mice maintained preserved epithelial morphology and superficial villous projections with low-grade distal dysplasia (1.4 ± 0.2) and low-distal inflammation (1.8 ± 0.4). The Ncf1 group also presented villous projections, but these were formed by compacted glands with less interstitial vascularization in addition to glandular higher grade dysplasia (2.0 ± 0.3, p = 0.5) and foci of well-differentiated adenocarcinoma accompanied by higher distal inflammation (3.0 ± 0.0, p = 0.039) (Figure 2 D22; Figure 3). Even though Ncf1 inflammatory score was higher than the WT, the cellular composition of the inflammatory infiltrates had similar frequencies of grnaulocytes, lymphocytes, and plasmacytes in both groups. Proximal inflammation and dysplasia were low in both groups.

On day 30 for protocol r [Figure 2 D30(r); Figures 3 and 4 D30(r)], WT mice maintained villous projections corresponding to half of the mucosae length, supported by tubular-glandular hyperplasia above the muscularis mucosae, with different sizes and segments of very small glands with low-grade dysplasia foci. Scattered lymphocytes in small inflammatory infiltrates expressed β-catenin. In Ncf1 mice colons, there were a reduced number of glands under villous projections in the colon, although the morphology was similar to day 22. In general, Ncf1 mice had more high-grade dysplasia (with anisocariosis with persistent nucleoli and visible mitosis) and foci of well-differentiated adenocarcinoma with large inflammatory infiltrates rich in β-catenin-expressing lymphocytes (total mean scores for dysplasia WT = 2.20 ± 0.49, Ncf1 = 3.20 ± 0.74, p = 0.42; total mean scores for inflammation WT = 2.80 ± 0.66, Ncf1 = 4.00 ± 0.95, p = 0.31).

On day 30 for protocol i [Figure 2, Day 30(i); Figures 3 and 4 D30(i)], a persistent adaptation of the colonic sections was observed. WT colon presented superficial epithelial villous projections without extensive inflammation. In distal segments, high-grade dysplasia persisted in both groups. Ncf1 proximal and distal colonic segments maintained a glandular morphology with hyperchromatic nuclei and mucosa-associated lymphoid tissue hyperplasia. Additionally, Ncf1 colon developed invasive well-differentiated adenocarcinoma in segments where narrower, reserve microglands were visible above the mucularis mucosae, invading till the muscularis propria, accompanied by extensive inflammatory infiltrates harboring lymphocytes expressing high levels of β-catenin (mean scores for total dysplasia WT = 1.71 ± 0.52, Ncf1 = 4.38 ± 0.26, p = 0.001; mean scores for total inflammation WT = 4.14 ± 0.51, Ncf1 = 5.25 ± 0.31, p = 0.03).

Considering the dysplasia score in WT vs Ncf1 mice, we found a very strong positive correlation between dysplasia score and inflammation score (r_s = 0.835) in Ncf1 mice, while this correlation was only moderate for the WT mice (r_s = 0.591) (Table 1). Moderate-to-strong positive correlations could be observed for the infiltrating leukocytes in both groups (Table 1). The dysplasia score is also negatively correlated with the colon length in both groups, consistent with the previous results in Figure 1A.

Blood serum samples were analyzed using two types of ¹H NMR experiments, the basic ¹H spectrum (single pulse-acquire), and the CPMG pulse sequence. The basic ¹H spectrum exhibits all of the signals from both small metabolites and macromolecules resulting in an uneven baseline and the overlap of various signals from different compounds. The CPMG pulse sequence, on the other hand, suppresses the broad signals from macromolecules, namely lipids and proteins, resulting in clear peaks and a very well-defined baseline, thereby allowing better characterization and assignment of the signals arising from the small metabolites (Figure 5). A number of metabolites were able to be identified and quantified: (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, and (22) fumarate. Due to the breadth of the lipid signals, lipid signals were assessed and designated by moieties only: (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 (31). Figure 6A depicts the scores plot for the comparison of the CPMG spectra between control groups. Each sample is plotted according to the scores for PC1 and PC2, values that are calculated through the loadings plot (Figure 6B) where each dot represents a data point (a spectral bucket). Although the CPMG and the basic ¹H (Figures 6C,D) provide slightly different information, the score plots are similar and WT and Ncf1 groups still overlap. However, considering the WT and Ncf1 separately, on the basis of the PCA for the Ncf1 mice, the control group is separable from the DSS-induced groups with the recovery group brought closer to the control group (Figures 7B,D). This contrasts with WT mice which present overlapping metabolomics profiles between control, recovery, and second colitis-induced groups (Figures 7A,C). Given these differences, a plot of all DSS-induced groups was prepared to see if the metabolic profile is different between WT and Ncf1 mice. The unsupervised PCA (Figures 8A,B) still shows some group overlap, but for this data set we were able to calculate a valid PLS–DA model (r² = 0.70, q² = 0.43) (Figures 8C,D). While the recovery groups drifted toward a common metabolic profile, the Ncf1 and WT groups after the second DSS-induction colitis are distinct from each other with lower blood glucose [variable importance in projection (VIPs) at 3.23, 3.37, 3.41, 3.52, and 5.22] and higher lactate levels (VIPs at 1.31, 4.10, and 4.15) on the Ncf1 group.

The metabolites assigned by NMR were quantified from both ¹H and CPMG spectra and were correlated with clinical parameters. Due to the broad signals of the lipids in the ¹H spectra, lipid signals were assessed and designated by moieties relative to the lipid methyl group (L1). Regarding the dysplasia score and the metabolome, we could only find weak-to-moderate correlations in pooled samples (Table 2). Analyzing in more detail, these correlations are only statistically significant for isoleucine, lactate, and proline in the Ncf1 group.

As regards the inflammation score, the pooled data set only presents weak-to-moderate correlations (Table 3). However, the inflammation score seems to have a more profound effect on the WT mice blood serum metabolome, with seven moderate-to-strong statistically significant correlations: positive for L2/L1, L6/L1, and L7/L1 and negative for L3/L1, valine, acetate, and choline while the Ncf1 mice blood serum metabolome only depicts a moderate negative correlation with glucose.

Weight variation [defined as the percentage of weight loss (negative) or gain (positive) over the course of the experiment relative to the baseline] was only weakly to moderately correlated in the pooled group but when separated into WT and Ncf1 groups, significant correlations arise, mostly for the Ncf1 group (Table 4). With the exception of glucose which is positively correlated with weight gain, all other significant correlations are negative, namely L4/L1, L6/L1, L7/L1, leucine, lactate, creatine, and proline.

Colon length was diminished in the treated groups (Figure 1A) and this marker shows stronger correlations with blood serum metabolites for the WT group (positive for L3/L1, valine, and acetate and negative for L2/L1, L6/L1, L7/L1, and GPC/Cho) than for the Ncf1 group (negative for leucine and isoleucine) (Table 5).

For spleen weight, several weak-to-moderate correlations are present in the pooled data set (Table 6). When analyzing groups by genotype, the WT group shows strong positive correlations for the lipid moieties L2/L1, L3/L1, L6/L1, and L7/L1 and negative moderate correlations for valine, acetate, and choline. Conversely, spleen weight for the Ncf1 group only achieves moderate correlations, positive for L2/L1 and L7/L1 and negative for glucose, glutamine, phosphocholine, and glycine.