Łukasz Krych

A maternal gluten-free diet reduces inflammation and diabetes incidence in the offspring of NOD mice

Early-life interventions in the intestinal environment have previously been shown to influence diabetes incidence. We therefore hypothesized that a gluten-free (GF) diet, known to decrease the incidence of type 1 diabetes, would protect against the development of diabetes when fed only during the pregnancy and lactation period. Pregnant nonobese diabetic (NOD) mice were fed a GF or standard diet until all pups were weaned to a standard diet. The early-life GF environment dramatically decreased the incidence of diabetes and insulitis. Gut microbiota analysis by 16S rRNA gene sequencing revealed a pronounced difference between both mothers and their offspring on different diets, characterized by increased numbers of Akkermansia, Proteobacteria, and TM7 in the GF diet group. In addition, pancreatic forkhead box P3 regulatory T cells were increased in GF-fed offspring, as were M2 macrophage gene markers and tight junction-related genes in the gut, while intestinal gene expression of proinflammatory cytokines was reduced. An increased proportion of T cells in the pancreas expressing the mucosal integrin α4β7 suggests that the mechanism involves increased trafficking of gut-primed immune cells to the pancreas. In conclusion, a GF diet during fetal and early postnatal life reduces the incidence of diabetes. The mechanism may involve changes in gut microbiota and shifts to a less proinflammatory immunological milieu in the gut and pancreas.

Mode of Delivery Shapes Gut Colonization Pattern and Modulates Regulatory Immunity in Mice

Delivery mode has been associated with long-term changes in gut microbiota composition and more recently also with changes in the immune system. This has further been suggested to link Cesarean section (C-section) with an increased risk for development of immune-mediated diseases such as type 1 diabetes. In this study, we demonstrate that both C-section and cross-fostering with a genetically distinct strain influence the gut microbiota composition and immune key markers in mice. Gut microbiota profiling by denaturing gradient gel electrophoresis and 454/FLX-based 16S rRNA gene amplicon sequencing revealed that mice born by C-section had a distinct bacterial profile at weaning characterized by higher abundance of Bacteroides and Lachnospiraceae, and less Rikenellaceae and Ruminococcus. No clustering according to delivery method as determined by principal component analysis of denaturing gradient gel electrophoresis profiles was evident in adult mice. However, the adult C-section–born mice had lower proportions of Foxp3+ regulatory T cells, tolerogenic CD103+ dendritic cells, and less Il10 gene expression in mesenteric lymph nodes and spleens. This demonstrates long-term systemic effect on the regulatory immune system that was also evident in NOD mice, a model of type 1 diabetes, born by C-section. However, no effect of delivery mode was seen on diabetes incidence or insulitis development. In conclusion, the first exposure to microorganisms seems to be crucial for the early life gut microbiota and priming of regulatory immune system in mice, and mode of delivery strongly influences this.

Beyond genetics. Influence of dietary factors and gut microbiota on type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease ultimately leading to destruction of insulin secreting β‐cells in the pancreas. Genetic susceptibility plays an important role in T1D etiology, but even mono‐zygotic twins only have a concordance rate of around 50%, underlining that other factors than purely genetic are involved in disease development. Here we review the influence of dietary and environmental factors on T1D development in humans as well as animal models. Even though data are still inconclusive, there are strong indications that gut microbiota dysbiosis plays an important role in T1D development and evidence from animal models suggests that gut microbiota manipulation might prove valuable in future prevention of T1D in genetically susceptible individuals.

Gut microbial markers are associated with diabetes onset, regulatory imbalance, and IFN-γ level in NOD Mice

Gut microbiota regulated imbalances in the hosts immune profile seem to be an important factor in the etiology of type 1 diabetes (T1D), and identifying bacterial markers for T1D may therefore be useful in diagnosis and prevention of T1D. The aim of the present study was to investigate the link between the early gut microbiota and immune parameters of non-obese diabetic (NOD) mice in order to select alleged bacterial markers of T1D. Gut microbial composition in feces was analyzed with 454/FLX Titanium (Roche) pyro-sequencing and correlated with diabetes onset age and immune cell populations measured in diabetic and non-diabetic mice at 30 weeks of age. The early gut microbiota composition was found to be different between NOD mice that later in life were classified as diabetic or non-diabetic. Those differences were further associated with changes in FoxP3+ regulatory T cells, CD11b+ dendritic cells, and IFN-γ production. The model proposed in this work suggests that operational taxonomic units classified to S24–7, Prevotella, and an unknown Bacteriodales (all Bacteroidetes) act in favor of diabetes protection whereas members of Lachnospiraceae, Ruminococcus, and Oscillospira (all Firmicutes) promote pathogenesis.

Gut microbiota regulates NKG2D ligand expression on intestinal epithelial cells

Intestinal epithelial cells (IECs) are one of a few cell types in the body with constitutive surface expression of natural killer group 2 member D (NKG2D) ligands, although the magnitude of ligand expression by IECs varies. Here, we investigated whether the gut microbiota regulates the NKG2D ligand expression on small IECs. Germ‐free and ampicillin‐treated mice were shown to have a significant increase in NKG2D ligand expression. Interestingly, vancomycin treatment, which propagated the bacterium Akkermansia muciniphila and reduced the level of IFN‐γ and IL‐15 in the intestine, decreased the NKG2D ligand expression on IECs. In addition, a similar increase in A. muciniphila and a decreased NKG2D ligand expression was seen after feeding with dietary xylooligosaccharides. A pronounced increase in NKG2D ligand expression was furthermore observed in IL‐10‐deficient mice. In summary, our results suggest that the constitutive levels of NKG2D ligand expression on IECs are regulated by microbial signaling in the gut and further disfavor the intuitive notion that IEC NKG2D ligand expression is caused by low‐grade immune reaction against commensal bacteria. It is more likely that constitutively high IEC NKG2D ligand expression is kept in check by an intestinal regulatory immune milieu induced by members of the gut microbiota, for example A. muciniphila.

Early gradual feeding with bovine colostrum improves gut function and NEC resistance relative to infant formula in preterm pigs

It is unclear when and how to start enteral feeding for preterm infants when mothers milk is not available. We hypothesized that early and slow advancement with either formula or bovine colostrum stimulates gut maturation and prevents necrotizing enterocolitis (NEC) in preterm pigs, used as models for preterm infants. Pigs were given either total parenteral nutrition (TPN, n = 14) or slowly advancing volumes (16-64 ml·kg(-1)·day(-1)) of preterm infant formula (IF, n = 15) or bovine colostrum (BC, n = 13), both given as adjunct to parenteral nutrition. On day 5, both enteral diets increased intestinal mass (27 ± 1 vs. 22 ± 1 g/kg) and glucagon-like peptide 2 release, relative to TPN (P < 0.05). The incidence of mild NEC lesions was higher in IF than BC and TPN pigs (60 vs. 0 and 15%, respectively, P < 0.05). Only the IF pigs showed reduced gastric emptying and gastric inhibitory polypeptide release, and increased tissue proinflammatory cytokine levels (IL-1β and IL-8, P < 0.05) and expression of immune-related genes (AOAH, LBP, CXCL10, TLR2), relative to TPN. The IF pigs also showed reduced intestinal villus-to-crypt ratio, lactose digestion, and some plasma amino acids (Arg, Cit, Gln, Tyr, Val), and higher intestinal permeability, compared with BC pigs (all P < 0.05). Colonic microbiota analyses showed limited differences among groups. Early feeding with formula induces intestinal dysfunction whereas bovine colostrum supports gut maturation when mothers milk is absent during the first week after preterm birth. A diet-dependent feeding guideline may be required for newborn preterm infants.

Provision of Amniotic Fluid During Parenteral Nutrition Increases Weight Gain With Limited Effects on Gut Structure, Function, Immunity, and Microbiology in Newborn Preterm Pigs

BACKGROUND Small enteral boluses with human milk may reduce the risk of subsequent feeding intolerance and necrotizing enterocolitis in preterm infants receiving parenteral nutrition (PN). We hypothesized that feeding amniotic fluid, the natural enteral diet of the mammalian fetus, will have similar effects and improve growth and gastrointestinal (GI) maturation in preterm neonates receiving PN, prior to the transition to milk feeding. MATERIALS AND METHODS Twenty-seven pigs, delivered by cesarean section at ~90% of gestation, were provided with PN and also fed boluses with amniotic fluid (AF; n = 13, 24-72 mL/kg/d) or no oral supplements (nil per os [NPO]; n = 14) until day 5 when blood, tissue, and fecal samples were collected for analyses. RESULTS Body weight gain was 2.7-fold higher in AF vs NPO pigs. AF pigs showed slower gastric emptying, reduced meal-induced release of gastric inhibitory peptide and glucagon-like peptide 2, changed gut microbiota, and reduced intestinal permeability. There were no effects on GI weight, percentage mucosa, villus height, plasma citrulline, hexose absorptive capacity, and digestive enzymes. Intestinal interleukin (IL)-1β levels and expression of IL1B and IL8 were increased in AF pigs, while blood biochemistry and amino acid levels were minimally affected. CONCLUSION Enteral boluses of AF were well tolerated in the first 5 days of life in preterm pigs receiving PN. Enteral provision of AF before the initiation of milk feeding may stimulate body growth and improve hydration in preterm infants receiving PN. Furthermore, it may improve GI motility and integrity, although most markers of GI maturation remain unchanged.

Phytase-producing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of Pichia kudriavzevii strains

Phytate is known as a strong chelate of minerals causing their reduced uptake by the human intestine. Ninety-three yeast isolates from traditional African fermented food products, belonging to nine species (Pichia kudriavzevii, Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces marxianus, Millerozyma farinosa, Candida glabrata, Wickerhamomyces anomalus, Hanseniaspora guilliermondii and Debaryomyces nepalensis) were screened for phytase production on solid and liquid media. 95% were able to grow in the presence of phytate as sole phosphate source, P. kudriavzevii being the best growing species. A phytase coding gene of P. kudriavzevii (PHYPk) was identified and its expression was studied during growth by RT-qPCR. The expression level of PHYPk was significantly higher in phytate-medium, compared to phosphate-medium. In phytate-medium expression was seen in the lag phase. Significant differences in gene expression were detected among the strains as well as between the media. A correlation was found between the PHYPk expression and phytase extracellular activity.

Have you tried spermine? A rapid and cost-effective method to eliminate dextran sodium sulfate inhibition of PCR and RT-PCR

The Dextran Sulfate Sodium (DSS) induced colitis mouse model is commonly used to investigate human inflammatory bowel disease (IBD). Nucleic acid extracts originating from these animals are often contaminated with DSS, which is a strong inhibitor of many enzymatic based molecular biology reactions including PCR and reverse-transcription (RT). Methods for removing DSS from nucleic acids extracts exist for RNA, but no effective protocol for DNA or cDNA is currently available. However, spermine has previously been shown to be an effective agent for counteracting DSS inhibition of polynucleotide kinase, which led to the hypothesis, that spermine could be used to counteract DSS inhibition of PCR and RT. We investigated the means of adding spermine in an adequate concentration to PCR based protocols (including qPCR, two-step RT-qPCR, and amplicon sequencing library preparation) to remove DSS inhibition. Within the range up to 0.01g/L, spermine can be added to PCR/qPCR or RT prophylactically without a significant reduction of reaction efficiency. Addition of spermine at the concentration of 0.08g/L can be used to recover qualitative PCR signal inhibited by DSS in concentrations up to 0.32g/L. For optimal quantitative analysis, the concentration of spermine requires fine adjustment. Hence, we present here a simple fluorometric based method for adjusting the concentration of spermine ensuring an optimal efficiency of the reaction exposed to an unknown concentration of DSS. In conclusion, we demonstrate a cost effective and easy method to counteract DSS inhibition in PCR and two-step RT-qPCR. Fixed or fine-tuned concentrations of spermine can be administered depending on the qualitative or quantitative character of the analysis.

Immunological effects of reduced mucosal integrity in the early life of BALB/c mice

Certain stimuli at the gut barrier may be necessary in early life to establish a proper balance of immune tolerance. We evaluated a compromised barrier in juvenile mice in relation to microbiota and local and systemic immunity. BALB/c mice were treated with a low dose of dextran sulfate sodium (DSS) with or without ampicillin and lipopolysaccharide (LPS) to clarify the importance of microbial antigens and interaction between microbial-associated patterns and toll-like receptors. The barrier breach resulted in increased plasma LPS, which was highest in mice treated simultaneously with ampicillin. Adding LPS in the food reduced its levels in plasma. Regulatory T cells were acutely increased in mesenteric lymph nodes (MLN) and spleen during DSS treatment regardless of simultaneous ampicillin treatment. In contrast, NK T and NK cells decreased in MLN and in spleen. This acute DSS effect was reflected in fold changes of haptoglobin and Il1a in colon, and this was also more pronounced in mice simultaneously treated with ampicillin. On day 1 post-treatment, major upregulations of Ifng, Foxp3, Il1b, Il2, and Il6 genes in colon were only observed in the mice simultaneously treated with ampicillin. A two-fold upregulation of colonic Foxp3 and Il1a was evident 25 days post-treatment. DSS skewed the microbiota in favor of Gram negative phyla. Therefore, increased permeability induced tolerogenic immunity independent of microbiota, and this was enhanced by LPS stimulation.