Impacts of diets fed after weaning on gut microbiota and susceptibility to DSS-induced colitis in mice

Background Living in a sanitary environment and taking Western-style diet in early life are both risk factors for inflammatory bowel disease and important factors for shaping host gut microbiota. Here, we aimed to establish whether different dietary composition fed during the early period after weaning would associate the susceptibility to DSS-induced colitis with different gut microbiota shifts. Methods Eighty weaned Balb/c mice were fed with high sugar, fat, protein, fiber, and standard diet for 8weeks respectively. Inflammation was induced by administration of 2.5% (wt/vol) dextran sulfate sodium (DSS) in drinking water for 7 days, and the microbiota characterized by 16s rRNA based pyrosequencing. Analyzed the inflammatory factors and toll-like receptors by Real-time PCR Results The high protein and high fiber+protein group exacerbated severity of DSS-induced colitis, the high fiber and high protein+fiber groups had the effect of reducing colitis, and the high sugar, fat and standard group show the similar disease phenotype of colitis. The diversity and richness of the microflora were significantly decreased in the high fiber group, while only decreased richness of flora was observed in the high protein group. The abundance of Firmicutes was decreased and the abundance of Bacteroides was increased in the high fat, high sugar, high protein and high fiber groups, especially in the high protein and high fiber group. The microbial community structure was slightly different at the species/genus level. The microbial community structure of high protein-fiber group and high fiber-protein group was still similar. Conclusions Mice were fed with different dietary compositions of high sugar, fat, protein and fiber diets since weaning, and similar gut microbiota of high-abundance Bacteroides and low-abundance Firmicutes are formed in adult mice. These microbiota do not cause colonic mucosal damage directly. Only high protein diet aggravated DSS-induced colitis, while high fiber diet alleviated the colitis.


Introduction 2
The pathogenesis of inflammatory bowel disease (IBD) is 3 considered to be intolerance of the genetically susceptible individuals 4 to gut microbiota, triggering a dysregulated immune response 5 resulting in inflammatory injuries of intestinal mucosa [1][2][3] . 6 Epidemiologic data show that the incidence of IBD among 7 first-generation immigrants to regions with high incidence of IBD is as 8 low as that among natives in the countries from which they emigrated, 9 yet second-generation immigrants have an increased risk of IBD 10 similar to that in locations to which they immigrated [4] . The variance in 11 the disease incidence cannot be solely attributed to genetic 12 susceptibility, suggesting environmental factors exposed in early life 13 play important roles in the development of IBD [4,5] . Current evidences 14 support risk factors associated with IBD include hygiene hypothesis, 15 Cesarean section, formula feeding, and antibiotic use in infants and 16 young children [6][7][8] . Differences in delivery mode, infant feeding type, 17 sanitary condition and antibiotic use have also been linked with 18 differences in the intestinal flora of babies and children [9] . Several 19 studies have demonstrated marked alterations in the gut microbiota of 20 patients with IBD [10][11][12] . Thus, the composition of gut microbiota is 5 1 associated with IBD. 2 Studies have found significant differences in fecal microflora 3 between people eating western diet and people from regions where 4 fiber content is high in the diet, revealing impacts of different diets in 5 shaping gut microbiota [13,14] . Recent reports indicate that the gut 6 microbiota and alterations thereof, due to a consumption of 7 a diet high in saturated fats and low in fibers, can trigger factors 8 regulating the development of IBD [15] . The incidence of IBD is steadily 9 rising in developed as well as in developing countries paralleling the 10 escalating consumption of western-style diets, characterized by high 11 protein and fat as well as excessive sugar intake, with less vegetables 12 and fiber [16] . Animal studies also find that "western-style" diets can 13 lead to intestinal flora shifts in mice and aggravate inflammatory 14 injuries of colonic mucosa induced by DSS [17] . All these findings 15 suggest that changes in dietary composition contribute to the 16 development of IBD by altering the structure of gut microbiota. 17 In the recent years, the incidence of pediatric IBD is rapidly on the 18 rise, indicating that gut microbiota shaped in early life is related to the 19 risk of IBD [18] . The development of intestinal flora consists of two 6 1 stages, one is lactation and the other is exposure to environment and 2 diet after weaning [19] . A comparative study has found similarities in 3 fecal microflora between European children of preweaning and 4 children of the same age from rural Africa, and significant differences 5 between European children aged 2 to 6 years and children of same 6 age from Africa [20] . A recent analysis of the intestinal flora composition 7 of one infant followed over 2.5 years showed a considerable alteration 8 in the microflora with the introduction of solid foods and a shift towards 9 a more stable, adult-like microbiota with weaning [21] . By 3 years of age, 10 microbiota composition approaches that of adults [21] . These results

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indicate that the development of intestinal flora after weaning is 12 influenced by diets. Thus, the development of gut microbiota during 13 the early period after weaning may be related to the development of 14 IBD.

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Taken together, we propose a hypothesis that different dietary 16 composition fed during the early period after weaning may affect the 17 risk of the development of IBD by different gut microbiota shifts.

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Therefore, the aim of this study is to investigate the developments of 19 gut microbiota compositions in mice fed on different diets after 20 weaning as well as the susceptibility to DSS-induced colitis. caged under standard conditions (20-22°C, 50-55% humidity, 12/12h 5 dark/light cycle), and randomly assigned to eight groups (10 mice in 6 each group): control group (Con), standard group (Sta), group with 7 high sugar (Sug), group with high fat (Fat), group with high protein 8 (Pro), group with high fiber (Fib), group with one-month high protein 9 and one-month high fiber (Pro+Fib), group with one-month high fiber 10 and one-month high protein (Fib+Pro) ( Table 1). All mice were 11 provided with water and diets, and the calories were balanced in all 12 the groups every day. The stool of mice was collected once after 13 8-week treatment and stored at -70°C for further analysis.
14 Susceptibility to colitis was tested by the administration of 2.5% (wt/vol) Disease activity index (DAI) was quantified using the parameters of 5 weight loss, stool consistency, and gross blood in the feces which 6 were recorded daily for each animal [22] . Colons were immediately excised, rinsed with ice-cold 10 phosphate-buffered saline. 2 sections were snap-frozen in liquid 11 nitrogen and stored at -70°C for RNA isolation and protein preparation.   The PCR amplification profiles consisted of hot-start activation at 95℃ 10 for 10 min, followed by cycles of denaturation at 95℃ for 15 s,  Genomic DNA was extracted from the stool of enrolled mice (3 mice 2 per group) using the QIAamp DNA Stool Mini kit ( Qiagen, Germany ).

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The extracted DNA was amplified using primers targeting the V1 to V3 4 hypervariable regions of bacterial 16S rRNA gene (27F:  to process the sequence data [23] . Sequencing reads from the different 13 samples were sorted by unique barcodes. Primers and barcodes were 14 trimmed from each read. To minimize the sequencing errors, chimeric 15 sequences were removed using UCHIME (http://drive5.com/uchime).

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Low-quality sequences were excluded if the sequences met one of the 17 following criteria: (1) sequences with more than two inexact match to 11 1 average quality < 25. The trimmed sequences were clustered into 2 operational taxonomic units (OTUs) by setting a 0.03 distance limit 3 (97% of similarity). The alpha diversity (Shannon index and Simpson 4 index) and richness (ACE and Chao1) were analyzed based on 5 identified OTUs using the Mothur program. Representative sequence 6 of each OTU was taxonomically classified using the Silva database [24] 7 at a 70% confidence threshold with Mothur. shrinking or DAI were observed in the Fib or Pro+Fib groups (Fig 1, 2). Simpson index and ACE did not differ among different groups (P > 4 0.05). OTU (97%) of the Fat group was more than that of Pro and Fib 5 group (P < 0.05), and OTU (97%) of the Fib group was less than that 6 of the Sta group (  Pro group, with no significant difference between them (P > 0.05).  IBD patients, which is seems to be related to pathogenesis of IBD [32] .

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In fact, the researches in both IBD patients and animal studies have 16 shown that high fiber diet protect colon mucosa, and high protein diet 17 aggravate colon mucosa damage [26,28,33] . Obviously, the bacteria Fusobacteriales, Coriobacteriales and Clostridiales [35] .
10 Fusobacteriales are implicated in many different inflammatory 11 processes, including colonic inflammation [36,37] . However, in this study 12 there were no significant difference in Fusobacteriales in each group, 13 which cannot explain why protein diet can aggravate the colitis.
14 Therefore, the early adaptation of bacteria to intestinal mucosa may 15 not damage the colonic mucosa. In our study, although we found that