Addition of soluble fiber to standard purified diets is important for gut morphology in mice

Purified diets (PD) increase standardization and repeatability in rodent studies but lead to differences in the phenotype of animals compared to grain-based “chow” diets. PD contain less fiber and are often devoid of soluble fiber, which can impact gut health. Thus, the aim of the present study was to modify the PD AIN93G by addition of soluble fiber, to promote more natural gut development as seen with chow diets. One hundred twenty male C57BL/6J mice were fed over 12 weeks either a chow diet, AIN93G or one of three modified AIN93G with increased fiber content and different ratios of soluble fiber to cellulose. Gut health was assessed through histological and immunohistochemical parameters and gut barrier gene expression. Gut microbiota composition was analyzed and its activity characterized through short chain fatty acid (SCFA) quantification. Feeding AIN93G led to tissue atrophy, a less diverse microbiota and a lower production of SCFA compared to chow diet. The addition of soluble fiber mitigated these effects, leading to intermediate colon and caecum crypt lengths and microbiota composition compared to both control diets. In conclusion, the addition of soluble fibers in PDs seems essential for gut morphology as well as a diverse and functional gut microbiome.

Mice were fed either chow diet, AIN93G or one of three AIN-based diets with varying ratios of soluble fiber to cellulose for 12 weeks.Boxplots display median and quartiles of the data points.Goblet cells were counted in 14 -23 samples per diet group.70S: experimental diet with fiber fraction consisting of 70% soluble fiber and 30% cellulose; 50S: experimental diet with fiber fraction consisting of 50% soluble fiber and 50% cellulose; 30S: experimental diet with fiber fraction consisting of 30% soluble fiber and 70% cellulose.Boxplots display median and quartiles of the data points.Crypt lengths were measured in 16 -22 samples and villus lengths in 14 -19 samples per diet group.
70S: experimental diet with fiber fraction consisting of 70% soluble fiber and 30% cellulose; 50S: experimental diet with fiber fraction consisting of 50% soluble fiber and 50% cellulose; 30S: experimental diet with fiber fraction consisting of 30% soluble fiber and 70% cellulose.Boxplots display median and quartiles of the data points (n = 24 per group).Values below the limit of quantification are displayed as 0.
70S: experimental diet with fiber fraction consisting of 70% soluble fiber and 30% cellulose; 50S: experimental diet with fiber fraction consisting of 50% soluble fiber and 50% cellulose; 30S: experimental diet with fiber fraction consisting of 30% soluble fiber and 70% cellulose.70S: experimental diet with fiber fraction consisting of 70% soluble fiber and 30% cellulose; 50S: experimental diet with fiber fraction consisting of 50% soluble fiber and 50% cellulose; 30S: experimental diet with fiber fraction consisting of 30% soluble fiber and 70% cellulose.Histology Crypt length was measured in colon, ileum and caecum from crypt base to gut lumen using the segmented line tool in Image J (Supplementary Figure 5).For measurements in colon and ileum two cross-sections from within 1 cm of gut tissue were used.In colon 20 measurements were made, in ileum 10 -20 and in cecum 8 -20.For villus length in ileum cross-sections 8 -20 villi were measured accordingly.In AB-stained sections of colon, ileum and caecum goblet cells and epithelial cells lining the crypt were counted and percentage of goblet cells calculated.

Immunohistochemistry
Formalin-fixed paraffin embedded tissues were sectioned (3 µm), placed on coverslips and deparaffinized.Next, slides were blocked with 3% H2O2 for 10 min at room temperature (RT).Then, slides were unmasked in the microwave (180° for 15 min) with sodium citrate (pH 6.0) and afterwards blocked with 10% normal goat serum (NGS).The primary antibody (KI-67; AB9260, Merck; dilution 1:300) was diluted in blocking solution (NSG) and incubated overnight at 4°C.Next day, slides were washed with PBS, incubated with secondary antibody (goat Anti-Rabbit IgG H&L (HRP) (ab205718, Abcam; dilution: 1:1000) at RT for 1 hour, and washed again with PBS.KI67+ cells were visualized with a DAB stain.Micrographs (40x magnification) of proliferating cells were taken and KI67+ cells were counted.For assessment, we calculated the proliferation index: [all KI67 positive cells / all epithelial cells] x 100.Sections with less than 1000 epithelial cells were excluded.Only crypts that were cut perpendicularly along their entire axis and had a clear opening towards lumen were included in the counting.

Gene expression experiments
For lysation 30 mg of each tissue were added to 30 mg dia Zirconia Silica beads (0.1 mm) and 600 µL RLT Buffer (Qiagen #79216) in 2 mL scMirco tubes.Next, tissues were lysed using FastPrep (6 m/sec for 40 sec) and samples were centrifugated at 13000 rpm for 5 minutes at 4 °C.
For DNase digestion, 1 µg RNA was mixed with 1 µL DNase, 1 µL 10x Reaction Buffer containing MgCl2 and DEPC water to 10 µL.Samples were placed in RNase free tubes for 30 minutes at 37 °C, followed by addition of 1 µL of 50 mM EDTA at 65 °C for 10 minutes.
Details for PCR reaction were as follows: Each PCR amplification reaction (20 µL total volume) containing 50 ng of synthesized cDNA template and 0.4 to 0.8 µM of forward and reverse primers (Supplementary Table 4).The thermal cycle started with hot start polymerase activation at 95 °C (2 min), followed by 40 cycles of denaturation at 95 °C (15 sec) and annealing at 60 °C (1 min) for the most primer pairs.For some primers, the annealing temperature and time were adjusted, or elongation was performed (72 °C, 30 sec).Adjustments to the cycling program are listed in Supplementary Table 4.
Intestinal microbiome analysis: -Detailed protocol for 16S rRNA gene sequencing

DNA extraction
Mouse faeces was collected for microbiome analysis one day after feeding with the experimental diets started (= week 0) and after 36 days (= week 5).For one mouse of diet group 30S, no faeces could be collected at week 0. Intestinal contents were collected during the dissection of the mice (= week 12) from the ileum, caecum and colon.All samples were flash frozen in liquid N2 and stored at -80 °C.DNA was extracted from all samples using the NucleoSpin DNA Stool kit (Macherey-Nagel).Up to 200 mg of faeces were transferred to the bead tubes, incubated with 850 µL lysis buffer at 70 °C for 5 min and then subjected to 40 s treatment on a FastPrep 24 machine (MP biomedicals) at 6.0 m/s, followed by washing and elution of DNA according to the kit manufacturer's protocol.Purity and concentration of the extracted DNA were checked on a microvolume photometric device.

16S rRNA gene sequencing
The variable region V4 of the prokaryotic 16S rRNA gene was amplified in a single PCR step with dual indexing primers as described by Kozich et al. [1] with modifications by Parada et al. [2] and Apprill et al. [3] (Supplementary Table 5).NEBNext® Ultra™ II Q5® Master Mix (New England Biolabs) was used in a 25 µL reaction mix with 1 ng template DNA, 0.5 µM of each PCR primer (forward and reverse), 0.2 mM dNTP mix, 12.5 µL Q5 Master Mix, filled up with HyPure water (Cytiva Europe GmbH).The PCR program included initial denaturation for 30 sec at 98 °C, 25 cycles of 10 s at 98 °C, 30 s at 55 °C and 20 s at 72 °C, followed by terminal elongation for 2 min at 72 °C.PCR products were purified using magnetic beads (Mag-Bind® RxnPure Plus, Omega) and their concentration was measured using a Quantus Fluorometer (Promega) with the QuantiFluor One dsDNA System (Promega), according to the manufacturer's recommendation.Negative control samples included amplification without template and using DNA purifications without sample as template (= blank control).The PCR products were pooled at equimolar concentration to 5 libraries that were sequenced on individual runs (some including libraries of other projects) using the Illumina MiSeq v3 kit following the manufacturer's instruction with modifications.To enable sequencing of the dual indexing primers, 3.4 µL of 100 µM custom sequencing primers (Supplementary Table 5) were added to the cartridge as described in the MiSeq WetLab SOP by Kozich et al. [4].Detailed information on conditions of the 5 sequencing runs are listed in Supplementary Table 6.

Bioinformatic analysis
Bioinformatic analysis was performed in R version 4.1.0[5].Raw sequence data were trimmed to 200 (fwd) and 150 (rev) bases and reads with ambiguous base calls or an expected error rate > 2 after trimming were discarded.Remaining reads were error corrected, forward and reverse reads merged, and chimeric sequences removed using dada2 version 1.22 [6] with the 'pseudo-pooling' option yielding 1,249 amplicon sequence variants (ASVs) of the V4 region.ASVs were classified using the RDP Naïve Bayesian Classifier algorithm as implemented in dada2 against the Silva database v138.1 [7].
Detailed protocol for GC-Analysis of SCFA and BCFA in caecum content and feces About 100 mg of caecum or faeces were freeze-dried using an Alpha 1-2 freeze-drying manifold (Martin Christ), resulting in about 25 mg (range 5 to 70 mg) of dry material.880 µL deionized water, 20 µL internal standard (ISTD, 4-ethylbutyric acid, 1 g/L) and 100 µL HCl (25 %) were successively added, leading to an end volume of 1000 µL.Samples were placed in an overhead mixer for 10 min, followed by vortexing for 10 min, and then centrifugation for 10 min at 16,100 rpm (23,100 g).The supernatant was transferred to another tube and 200 µL were transferred to conic GC vials.Samples were analyzed using a 6890 GC (Agilent), equipped with a split/splitless injector.A 0.5 µL sample was injected using a split ratio of 1:10 at 200 °C.Water/HCl and methanol were used as wash solvents.SCFAs were separated on a ZB-WAXplus column with the dimensions 30 m × 0.25 mm × 0.25 µm (Phenomenex).Hydrogen was used as carrier gas with a flow rate of 42 cm/sec.The following temperature program was used: 70 °C (0.3 min)/70 to 230 °C (21° C/min; 8.4 min)/230 (0.5 min).Individual SCFAs were detected by FID at 250 °C using helium as makeup gas.Compounds were quantified by internal calibration (7 levels).A volatile acid mix (10 mM) was complemented by additional acetic acid spike and those single FA which were not included in the commercial mixture (all standards from Sigma-Aldrich).Appropriate volumes of these stock solutions were mixed and further diluted to desired calibration levels by adding 20 µL ISTD, 100 µL HCl (25 %) and filling up to 1000 µL with deionized water.
The calibration range was up to 1000 µM, and 6-fold higher for acetic acid (6250 µM).Freeze-dried matrix controls (2 levels, with and without spike) were used to monitor method performance.

Comparison between microbiota and SCFA/BCFA profiles
Associations between the caecal microbiota and SCFA/BCFA contents were calculated as correlations between relative abundances of taxa and concentrations (μmol/g) of metabolites.Taxa abundances were filtered to remove rare taxa with a maximum relative abundance of 1 % within the caecum samples.Correlations were calculated as Pearson's linear correlation coefficient using the corr.testfunction of R package psych [13].Correlations were visualized as a heatmap with heatmap.2,package gplots [16].Clusters of taxa that showed similar correlations with SCFA/BCFA profiles were calculated using hierarchical clustering (hclust, UPGMA method) of the euclidean distances, and dividing the resulting dendrogram into three clusters using cutree (k = 3).Relative abundances of the three clusters were calculated merging all ASVs of the same cluster using the merge_taxa command of the phyloseq package.

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and # indicate significant difference to AIN93G and Chow, respectively (p < 0.05, Dunnett's Test).Supplementary Figure S2: Ileum crypt length (a) and villus length (b) of mice fed either chow diet, AIN93G or one of three AIN-based diets with varying ratios of soluble fiber to cellulose.
Supplementary FigureS3: Average microbiome composition in caecum samples and faeces/colon samples.Bargraphs display the average relative abundance of the most abundant genera for each diet group in in caecum samples (a, c) and faeces and colon samples at different timepoints (b, d).Taxa were grouped at the phylum (a, b) and genus level (c, d), respectively.Taxa that could not be classified on the genus level were assigned the name of the next highest taxonomic level that could be classified with a '_g' suffix indicating the genus level.SupplementaryFigure S4: Concentrations of the short chain fatty acids (SCFA) in faeces of mice fed either chow diet, AIN93G or one of three AIN-based diets with varying ratios of soluble fiber to cellulose.

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and # indicate significant difference to AIN93G and Chow, respectively (p < 0.05, Dunnett's Test).Supplementary Figure S5: Scatter plots of colon crypt length (a), colon proliferation index (b), caecum crypt length (c), caecum proliferation index (d) and the caecal concentrations of the three main SCFA acetate, propionate and butyrate.