BackgroundMechanistic data to support health claims is often generated using rodent models, and the influence of prebiotic supplementation has largely been evaluated using male rodents. Given that sex-based differences in immune parameters are well recognized and recent evidence suggests differences in microbiota composition between sexes, validation of the effectiveness of prebiotics merits assessment in both males and females. Here, we have compared the effect of oligofructose (OF) supplementation on the fecal bacterial community, short chain fatty acid profiles, and gut mucosal and systemic immune parameters in male and female rats.MethodsMale and female rats were fed rodent chow or chow supplemented with OF (5 % w/w). Fecal community change was examined by analyzing 16S rRNA gene content. To compare effects of OF between sexes at the gut microbial and mucosal immune level, fecal short chain fatty acid and tissue cytokine profiles were measured. Serum lipopolysaccharide levels were also evaluated by the limulus amebocyte lysate assay as an indirect means of determining gut permeability between sexes.ResultsIn the fecal community of females, OF supplementation altered community structure by increasing abundance in the Phylum Bacteroidetes. In male rats, no changes in fecal community structure were observed, although fecal butyrate levels significantly increased. Liver Immunoglobulin A (IgA) levels were higher in males relative to females fed OF, and serum LPS concentrations were higher in males independent of diet. Females had higher basal levels of the regulatory cytokine interleukin-10 (IL-10) in the colon and liver, while males had higher basal levels of the pro-inflammatory cytokines IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) in the cecum and liver.ConclusionsWe have shown that male and female rat gut communities metabolize an OF-supplemented diet differently. Sex-specific responses in both the fecal community and systemic immune parameters suggest that this difference may result from an increase in the availability of gut peptidyl-nitrogen in the males. These findings demonstrate the importance of performing sex-comparative studies when investigating potential health effects of prebiotics using rodent models.

Aim: To examine change in the gut community of rats fed high amylose maize starch (HAMS). Methods and Results: Rats were fed AIN93G diets containing HAMS (5% resistant starch type 2) or alphacell (control). HAMS increased faecal shortchain fatty acid output, faecal propionate and total bacteria output but reduced gut pH and blood urea concentrations compared with rats ingesting the control diet. Feeding HAMS resulted in a gut community dominated by four phylotypes homologous with Ruminococcus bromii, Bacteroides uniformis and with yet to be cultivated organisms aligning into the Family Porphyromonadaceae. Enrichment of phylotypes aligning within the Bacteroidetes occurred primarily in the caecum, whereas those homologous with R. bromii were found primarily in the faeces. HAMS altered community structure such that the phylum Bacteroidetes represented the dominant gut lineage and progressively reduced faecal community phylotype richness over the duration of feeding. Conclusions: Feeding HAMS resulted in a caecal and faecal community dominated by organisms that require ammonia as a primary nitrogen source. Gut ammonia derived from endogenous urea represents an important factor contributing to caecal community composition in addition to the ability to utilize HAMS. Increases in faecal propionate, rather than butyrate as is often observed following resistant starch feeding, reflected a gut community dominated by the Bacteroidetes. Significance: Diet-mediated change is often viewed strictly in terms of available carbohydrate. Here, we have shown that ammonia derived from endogenous urea is an important factor contributing to gut community composition and structure in rats fed this substrate. IntroductionResistant starches (RS) are starches or products of starch degradation, which are not digested or absorbed in the small intestine and pass into the colon where they become available as substrate for the resident microbiota (Asp et al. 1992). Resistant starches are currently classified into four categories according to their physical and chemical properties (Topping and Clifton 2001). RS type 1 (RS 1 ) is physically inaccessible to the enzymes of the small intestine, RS type 2 (RS 2 ) is native granular starch that is difficult to hydrate, RS type 3 (RS 3 ) is retrograded amylose, and RS type 4 (RS 4 ) is resistant due to chemical modification. The fermentation of RS, as with other carbohydrates, has been associated with various physiological benefits including increased faecal short-chain fatty acid

Genome-wide transcriptional analysis in intestinal epithelial cells (IEC) can aid in elucidating the impact of single versus multi-strain probiotic combinations on immunological and cellular mechanisms of action. In this study we used human expression microarray chips in an in vitro intestinal epithelial cell model to investigate the impact of three probiotic bacteria, Lactobacillus helveticus R0052 (Lh-R0052), Bifidobacterium longum subsp. infantis R0033 (Bl-R0033) and Bifidobacterium bifidum R0071 (Bb-R0071) individually and in combination, and of a surface-layer protein (SLP) purified from Lh-R0052, on HT-29 cells’ transcriptional profile to poly(I:C)-induced inflammation. Hierarchical heat map clustering, Set Distiller and String analyses revealed that the effects of Lh-R0052 and Bb-R0071 diverged from those of Bl-R0033 and Lh-R0052-SLP. It was evident from the global analyses with respect to the immune, cellular and homeostasis related pathways that the co-challenge with probiotic combination (PC) vastly differed in its effect from the single strains and Lh-R0052-SLP treatments. The multi-strain PC resulted in a greater reduction of modulated genes, found through functional connections between immune and cellular pathways. Cytokine and chemokine analyses based on specific outcomes from the TNF-α and NF-κB signaling pathways revealed single, multi-strain and Lh-R0052-SLP specific attenuation of the majority of proteins measured (TNF-α, IL-8, CXCL1, CXCL2 and CXCL10), indicating potentially different mechanisms. These findings indicate a synergistic effect of the bacterial combinations relative to the single strain and Lh-R0052-SLP treatments in resolving toll-like receptor 3 (TLR3)-induced inflammation in IEC and maintaining cellular homeostasis, reinforcing the rationale for using multi-strain formulations as a probiotic.

Spaceflight is associated with several health issues including diminished immune efficiency. Effects of long-term spaceflight on selected immune parameters of wild type (Wt) and transgenic mice over-expressing pleiotrophin under the human bone-specific osteocalcin promoter (PTN-Tg) were examined using the novel Mouse Drawer System (MDS) aboard the International Space Station (ISS) over a 91 day period. Effects of this long duration flight on PTN-Tg and Wt mice were determined in comparison to ground controls and vivarium-housed PTN-Tg and Wt mice. Levels of interleukin-2 (IL-2) and transforming growth factor-beta1 (TGF-β1) were measured in mucosal and systemic tissues of Wt and PTN-Tg mice. Colonic contents were also analyzed to assess potential effects on the gut microbiota, although no firm conclusions could be made due to constraints imposed by the MDS payload and the time of sampling. Spaceflight-associated differences were observed in colonic tissue and systemic lymph node levels of IL-2 and TGF-β1 relative to ground controls. Total colonic TGF-β1 levels were lower in Wt and PTN-Tg flight mice in comparison to ground controls. The Wt flight mouse had lower levels of IL-2 and TGF-β1 compared to the Wt ground control in both the inguinal and brachial lymph nodes, however this pattern was not consistently observed in PTN-Tg mice. Vivarium-housed Wt controls had higher levels of active TGF-β1 and IL-2 in inguinal lymph nodes relative to PTN-Tg mice. The results of this study suggest compartmentalized effects of spaceflight and on immune parameters in mice.

A B S T R A C TImmune measures and the fecal bacterial community were examined in female Biobreeding rats housed in wire bottom cages (wire) or in solid bottom cages containing hardwood chips (bedding). Housing did not affect food intake, weight gain, fecal output or fibre content, serum liver enzymes, or spleen and mesenteric lymph node immune cell populations. Bedding-housed rat feces were enriched in phylotypes aligning within the phylum Firmicutes (families Lactobacillaceae and Erysipelotrichaceae) and had a 2-fold lower content of phylotypes aligning within the phylum Bacteroidetes. Feces from bedding-housed rats also contained significantly more acetic acid and less propionic, isobutyric, valeric and isovaleric acids than those housed on wire. Bedding-housed rats had significantly higher splenic concentrations of interleukin-4 (P < 0.001). These results demonstrate that bedding can indirectly influence systemic and mucosal immune measures, potentially adding additional complexities and confounding results to nutrition studies investigating the health effects of dietary fibres.

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Dietary fibre can mediate effects on mucosal immunity and the gut microbial community. The aim of this study was to examine the impact of dietary fibre intake on immunization efficacy, and to assess potential sex-based differences. Following a 6 week feeding trial, male and female rats fed purified diets containing single sources of fermentable material (cellulose; C, wheat bran; WB, oat bran; OB, resistant starch; RS or oligofructan; OF) were parenterally immunized with 0.3 mg/kg Keyhole Limpet Hemocyanin (KLH). Anti-KLH and total antibody concentrations were evaluated at intervals from tail vein blood, and splenic lymphocyte percentages and cytokine levels were measured at the end of the trial. Circulating anti-KLH IgG, IgG2a and IgG2b levels decreased significantly in females fed OB, while anti-KLH IgG2a levels decreased in males fed either RS or OF-supplemented diets compared to control-fed rats. In contrast to females, males fed OB had significantly lower splenic TGF-b1/IFN-g ratios and higher total IgG antibody levels. Additionally, females fed RS or OF had significantly reduced splenic CD3+ and CD3+4+ percentages while only CD3+ percentages were significantly reduced in males fed OF, relative to their control-fed counterparts. Collectively these results suggest dietary fibre can impact systemic immune responses to immunization, and that these effects can differ between sexes.

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