Fermentable fibres condition colon microbiota and promote diabetogenesis in NOD mice
Gut microbiota (GM) and diet both appear to be important in the pathogenesis of type 1 diabetes. Fermentable fibres (FFs), of which there is an ample supply in natural, diabetes-promoting diets, are used by GM as a source of energy. Our aim was to determine whether FFs modify GM and diabetes incidence in the NOD mouse.
Female NOD mice were weaned to a semisynthetic diet and the effects of FF supplementation on diabetes incidence and insulitis were evaluated. Real-time quantitative PCR was employed to determine the effects imposed to gene transcripts in the colon and lymph nodes. Changes to GM were analysed by next-generation sequencing.
NOD mice fed semisynthetic diets free from FFs were largely protected from diabetes while semisynthetic diets supplemented with the FFs pectin and xylan (PX) resulted in higher diabetes incidence. Semisynthetic diet free from FFs altered GM composition significantly; addition of PX changed the composition of the GM towards that found in natural-diet-fed mice and increased production of FF-derived short-chain fatty acid metabolites in the colon. The highly diabetogenic natural diet was associated with expression of proinflammatory and stress-related genes in the colon, while the semisynthetic diet free from FFs promoted Il4, Il22, Tgfβ and Foxp3 transcripts in the colon and/or pancreatic lymph node. PX in the same diet counteracted these effects and promoted stress-related IL-18 activation in gut epithelial cells. 16S RNA sequencing revealed each diet to give rise to its particular GM composition, with different Firmicutes to Bacteroidetes ratios, and enrichment of mucin-degrading Ruminococcaceae following diabetes-protective FF-free diet.
FFs condition microbiota, affect colon homeostasis and are important components of natural, diabetes-promoting diets in NOD mice.
KeywordsDietary intervention Gut immune system Microbiota Type 1 diabetes
Binding immunoglobulin protein
Cellulose containing diet
C/EBP homologous protein
Mesenteric lymph node
Normal rodent chow
Pancreatic lymph node
Pectin- and xylan-containing diet
Short-chain fatty acid
Regulatory T cell
The NOD mouse spontaneously develops autoimmune diabetes, which closely resembles human type 1 diabetes (reviewed in [1, 2]). Current evidence suggests a close relationship between development of type 1 diabetes and the gut immune system [3, 4, 5]. Inflammatory cells and markers in intestinal biopsies, increased levels of antibodies to food antigens in children who develop type 1 diabetes, shared determinants of leucocyte traffic between gut and pancreas [6, 7] and increased permeability of the gut [8, 9] all provide evidence supporting such a relationship. The importance of dietary factors in type 1 diabetes development is directly proven in animal models of autoimmune diabetes [10, 11, 12].
Studies in rodent models of autoimmune diabetes, as well as in humans, also suggest that the composition of gut microbiota (GM) is an important determinant of islet autoimmunity and type 1 diabetes . Disruption of immune homeostasis in the colon by chemical irritation or Citrobacter rodentium infection promotes activation of islet-reactive T cells in the pancreatic lymph node (PaLN) [14, 15]. In NOD mice, anti-diabetogenic infant formula diet both alleviates inflammatory phenomena in the gut and affects microbiota composition in the large intestine [12, 16], suggesting that the immune mechanisms evoked by commensal microbes in the large intestine associate with autoimmune diabetes.
Studies in the BB rat and NOD mouse indicate that natural cereal-based rodent diets are the most diabetogenic, while hypoallergenic infant formulae consisting of hydrolysed peptides or soy protein are associated with protection against diabetes. While most dietary factors, including proteins, are digested and absorbed in the small intestine by host-dependent mechanisms, dietary fibres pass to the large intestine intact and form an important part of luminal gut contents there. Certain gut bacteria are capable of hydrolysing these complex polysaccharides for their energy supply. While cellulose is hydrolysed inefficiently, pectin and xylan are hydrolysed more readily and thus may be called fermentable fibres (FFs). Hydrolysis of FFs results in the production of short-chain fatty acids (SCFAs), such as butyrate, which support epithelial cells and the integrity of immune homeostasis in the large intestine [17, 18]. Interestingly, some complex polysaccharides also irritate the large intestine [19, 20] and can disturb gut homeostasis with potential relevance to islet autoimmunity .
Due to the proposed roles of GM in type 1 diabetes and their effects on the gut immune system, we set forth to investigate whether the FFs pectin and xylan, present in natural diabetes-promoting diets and used by GM as energy source, have a role in diabetogenesis in the NOD mouse. Our data suggest that the lack of these compounds in semisynthetic diets protects NOD mice effectively from diabetes. This protection is associated with several potentially important phenomena, including a change in the composition of GM, reduction in stress-related transcripts C/EBP homologous protein (CHOP) and binding immunoglobulin protein (BiP) and promotion of anti-inflammatory transcripts in the gut and PaLN and reduced IL-18 production in gut epithelial cells.
Female NOD mice (NOD/ShiLtJ; Jackson Laboratories, Bar Harbor, ME, USA) were bred and housed in Turku University animal facility under specific-pathogen-free conditions and caged under filter covers. For a brief description of conditions see ESM Methods. All animal work was carried out by trained scientists with permission from the Finnish Laboratory Animal Board (license number: ESAVI/3210/04.10.03/2011). Mice from several litters were allocated into groups fed different diets from weaning. For complete semisynthetic diets, Basal mix 80% (Harlan Laboratories, Madison, WI, USA) was used for base and was supplemented with 20% whey and casein and fibres (pectin and xylan or cellulose alone) as indicated (ESM Table 1). The blood glucose level was monitored weekly from 12 to 34 weeks of age by puncturing the tail vein. Blood glucose exceeding 14 mmol/l for 2 consecutive days was deemed diabetic. Samples were collected for colon epithelial cell isolation and cytokine profiling (colon, lymph nodes) after diets had been consumed for 4 weeks (n = 7). Insulitis was quantified by evaluating the degree of infiltration and categorised as follows: 0, no insulitis; 1, peri-insulitis with or without minimal infiltration in islets; 2, insulitis with <50% infiltration of islets; 3, invasive insulitis with >50% infiltration of islets. Insulitis was determined from non-consecutive cryosections of pancreases after hematoxylin and eosin (H&E) staining. At least 60 individual islets were graded for the level of infiltrating cells. Colon samples were fixed in paraformaldehyde and embedded in paraffin and sections were stained with H&E staining for evaluation of colon mucosa thickness.
RNA isolation and quantitative PCR
To analyse diet-derived changes in cytokines and transcription factors, colon and lymph nodes were collected into RNAlater (Qiagen, Germantown, MD, USA). Total RNA was extracted with bead-based PowerLyzer RNA Isolation Kit (MoBio, Carlsbad, CA, USA). cDNA was synthesised with Maxima reverse transcriptase and oligo (dT) primers (Thermo Fisher, Waltham, MA, USA). For all primer and probe details, see ESM Table 2. Relative cytokine expression was determined by real-time quantitative PCR (LightCycler 480; Roche, Basel, Switzerland). All cytokine signals were normalised to β-actin expression. Data are shown in relation to 4-week-old normal chow signal. The p values are calculated from ΔCt values [Ct(target) − Ct(reference)].
Western blot analysis of epithelial IL-18 expression
For epithelial cell IL-18 immunoblot analysis the colon was emptied, rinsed with PBS and cut open longitudinally. The colon was agitated in 1 mmol/l EDTA–Hanks’ balanced salt solution at 37°C for 30 min. The solution was collected and centrifuged to pellet extracted cells. Cells were resuspended in 1 ml RIPA buffer and lysed for 30 min at 4°C. The lysate was centrifuged for 30 min at 14,000 g (4°C) to remove cell debris. Protein concentration was measured using the Bradford assay (Bio-Rad, Hercules, CA, USA). Western blot was performed on 10% SDS-PAGE gels and proteins were transferred onto PVDF membrane (Millipore, Darmstadt, Germany). Proteins IL-18 and actin were probed with monoclonal antibodies (anti-mouse IL-18, clone 39-3F from MBL, Nagoya, Japan; anti-actin, AC-40 from Sigma-Aldrich, St Louis, MO, USA) followed by second-step peroxidase-labelled anti-rat IgG (Cell Signaling Technology, Beverly, MA, USA). Immunoreactive bands were visualised on x-ray films after reaction with ECL substrate (Thermo Fisher). The density of the IL-18 band was compared with that of the actin band from the same membrane. Band densities were measured using Multiskan EX photometer (Thermo Fisher).
Analysis of SCFAs and bacterial DNA in the large intestine
Colon and caecum contents were pooled from three 7-week-old mice (n = 15). Concentrations of SCFAs were measured by gas chromatography. Total bacterial DNA was isolated and stratified on the basis of guanosine and cytidine (G+C) nucleotide content. The total amount of bacteria was calculated as copy numbers per weight of content (Alimetrics, Espoo, Finland; www.alimetrics.com/, ).
DNA extraction for the next-generation sequencing analysis
Total DNA was extracted from 100 mg frozen, pooled (three mice) colon and caecum samples with GenoXtract Stool Extraction Kit VER 2.0 (Hain Lifescience, Nehren, Germany). The purity and concentration of the DNA samples were measured using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher) at 230, 260 and 280 nm.
Next-generation sequencing analysis for microbiota samples
Next-generation sequencing was performed on an Illumina MiSeq system (Illumina, San Diego, CA, USA). For each genomic DNA sample, the V4 region of 16S rRNA gene (250 bp) was amplified by using Kapa HiFi PCR kit (Kapa Biosystems, Wilmington, MA, USA) and Veriti Thermal cycler (Thermo Fisher). The quality of libraries was confirmed by using Bioanalyzer 2100 (Agilent, Santa Clara, MA, USA) and the dataset was analysed by QIIME pipeline . Data filtering was performed as described previously .
Statistical analyses were carried out using GraphPad Prism 5 software (GraphPad, La Jolla, CA, USA). Statistical significance was determined using a two-tailed Student’s t test or one-way ANOVA with Dunnett’s post hoc test. Test for survival was made with Gehan–Breslow–Wilcoxon test and test for trend with more than two groups with Mantel–Cox logrank test. A p value of <0.05 was considered statistically significant.
Supplementation of semisynthetic diets with FF promotes diabetes and insulitis
In addition, the severity of insulitis in the various diet groups was evaluated by grading islets according to lymphocyte infiltration (Fig. 1b, c). The CE diet significantly reduced insulitis when compared with NC (p = 0.023), whereas the PX diet did not. There was also a significant difference between PX and CE diets with PX resulting in 1.8 times higher insulitis score than CE (p = 0.034). At 7 weeks of age, destruction of insulin-producing cells was not visible in islets of the CE group, but had begun in islets of both the PX and NC groups (Fig. 1d). These results suggest that pectin and xylan, but not cellulose, contribute to the development of autoimmune diabetes in NOD mice.
Expression of cytokines and epithelial stress-response genes in the colon depends on diet
Il22 was expressed in the colons of 4-week-old mice fed the CE and PX diets, whereas colons of the NC group showed very low expression levels. The CE diet induced a 20-fold (p = 0.039) increase and the PX diet induced a tenfold (p = 0.008) increase in Il22 expression when compared with the NC diet (Fig. 2c). Expression of Foxp3 was greatly upregulated in colons of the CE group at 4 weeks (5.5-fold, p = 0.033) when compared with NC (Fig. 2d). The PX diet did not induce Foxp3 expression in the colon at all. Expression of epithelial stress-response genes Chop (also known as Ddit3) and Bip (also known as Hspa5) was significantly higher in mice receiving the NC diet at 4 weeks of age, as compared with mice receiving the CE or PX diets (Fig. 2e, f).
Further, mRNA expression of transcription factor Rorγt (also known as Rorc) showed a significant decrease with semisynthetic diets when compared with NC. At 4 weeks this effect was not yet statistically significant, but reached significant difference by 7 weeks (Fig. 2g). At 7 weeks, the colons of mice fed the CE diet showed a sevenfold (p = 0.004) decrease in Rorγt expression when compared with NC colons (Fig. 2g) and those of the mice fed the PX diet showed a threefold (p = 0.002) decrease. Importantly the CE diet was significantly more effective than PX in reducing the expression of Rorγt. PX diet-induced Rorγt expression remained 2.3-fold higher than CE diet-induced Rorγt expression (p = 0.033). Also, we observed significantly increased Il4 expression in colons of the CE group (17.5-fold) at 4 weeks when compared with the NC group (p = 0.036, Fig. 2h).
FF-free diet promotes Foxp3 and Tgfβ transcripts in both mesenteric and pancreatic lymph nodes
FF-free diet reduces crypt length and epithelial IL-18 in the colon
Effect of FF on GM composition
Summary of p values describing differences in microbiota between diets
CE vs PX
CE vs NC
PX vs NC
Effect of diets on metabolic products in the large intestine
In this study, we show evidence for the first time that FFs in the diet associate with development of autoimmune diabetes. Our data indicate that the addition of two such fibres, pectin and xylan, to semisynthetic FF-free diet, significantly counteracts its diabetes-preventing effect in NOD mice. It is generally known that semisynthetic diets are diabetes-retardant both in NOD mice and BB rats and, apart from their usefulness in modifying protein composition in the diet, they are free from FF .
Our whole microbiome data demonstrated that inclusion of pectin and xylan in the rodent diet significantly increases the presence of Bacteroides and at the phylum level reverses the Bacteroidetes to Firmicutes ratio from 0.2 to 1.5, indicating a 7.5-fold increase in Bacteroidetes over Firmicutes. As previous studies in rodents and humans have suggested that predominance of Bacteroides associates with type 1 diabetes development [24, 28, 32, 33], the observed increase in Bacteroides induced by pectin and xylan further suggests a microbiota-mediated effect on diabetogenesis. Our 16S RNA gene sequencing results show that the NOD mice used in this study are devoid of known mucin-degrading bacteria such as Verrucomicrobiales and Prevotellaceae. These genera have been associated with protection from diabetes . Another mucin-degrading genus occupying the same environmental niche as Verrucomicrobiales (reviewed in ), namely the Ruminococcaceae, was enriched by the diabetes-protective CE diet. These results support the notion that mucin-degrading bacteria may counteract the development of autoimmune diabetes.
FFs are numerous and many of them are either constituents of roots, berries and vegetables or alimentary products of the modern food industry, ingested daily as part of the normal diet. In this study, we found that two such fibres, pectin and xylan, impact strongly on GM and condition the colon towards creating the environment observed in NOD mice fed standard, diabetogenic, laboratory chow. In light of the present study, it appears that previously observed associations between microbiota changes and type 1 diabetes-related autoimmunity may in part be explained by individual differences in ingestion of these fibres and their effects on GM.
The authors thank M. Jaakkola and L. Lauren (Medical Microbiology and Immunology, University of Turku) for their technical contributions to experiments, as well as A. Demasör and R. Mankonen for animal care at the Turku University central animal core.
This study was supported by Päivikki and Sakari Sohlberg Foundation, Diabetes Research Foundation Finland, Jalmari and Rauha Ahokas Foundation, Emil Aaltonen Foundation, Turku University Foundation and the Academy of Finland.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
All authors made substantial contributions to the conception and design of the study, acquisition of data or analysis and interpretation of data and drafted the article or revised it critically for important intellectual content. All authors gave final approval of the version to be published. RT and AH are responsible for the integrity of the work as a whole.