From Alpha Diversity to Zzz: Interactions among sleep, the brain, and gut microbiota in the first year of life.

Sleep disorders have been linked to alterations of gut microbiota composition in adult humans and animal models, but it is unclear how this link develops. With longitudinal assessments in 162 healthy infants, we present a so far unrecognized sleep-brain-gut interrelationship. First, we report a link between sleep habits and gut microbiota: daytime sleep is associated with bacterial diversity, and nighttime sleep fragmentation and variability are linked with bacterial maturity and enterotype. Second, we demonstrate a sleep-brain-gut link: bacterial diversity and enterotype are associated with sleep neurophysiology. Third, we show that the sleep-brain-gut link is relevant in development: sleep habits and bacterial markers predict behavioral-developmental outcomes. Our results demonstrate the dynamic interplay between sleep, gut microbiota, and the maturation of brain and behavior during infancy, which aligns with the newly emerging concept of a sleep-brain-gut axis. Importantly, sleep and gut microbiota represent promising health targets since both can be modified non-invasively. As many adult diseases root in early childhood, leveraging protective factors of adequate sleep and age-appropriate gut microbiota in infancy could constitute a health promoting factor across the entire human lifespan.

Development of the equine gut microbiota.

Shortly after birth the mammalian gut is colonized, by a transient microbiota, highly susceptible to environment and diet, that eventually stabilizes and becomes the resident gut microbiota. In a window of opportunity during the colonization, oral tolerance is established towards resident bacteria. In this study, the development of the equine gut microbiota was investigated in ten foals from parturition until post weaning. We found great differences in the core species of the gut microbiota composition between time-matched samples on Day 7 and 20 post-partum. Between day 20 and Day 50 post-partum, we saw the gut microbiota became increasingly dominated by fiber fermenting species. After Day 50, no significant changes in species abundance were observed. Gene expression analysis of pro- and anti-inflammatory cytokines in the blood revealed no significant changes before and after weaning. In summary, relative stability of the gut microbiota was reached within 50 days post-partum and, weaning did not have a major impact on the microbial composition.

Expression of immune regulatory genes correlate with the abundance of specific Clostridiales and Verrucomicrobia species in the equine ileum and cecum.

Billions of bacteria inhabit the gastrointestinal tract. Immune-microbial cross talk is responsible for immunological homeostasis, and symbiotic microbial species induce regulatory immunity, which helps to control the inflammation levels. In this study we aimed to identify species within the equine intestinal microbiota with the potential to induce regulatory immunity. These could be future targets for preventing or treating low-grade chronic inflammation occurring as a result of intestinal microbial changes and disruption of the homeostasis. 16S rRNA gene amplicon sequencing was performed on samples of intestinal microbial content from ileum, cecum, and colon of 24 healthy horses obtained from an abattoir. Expression of genes coding for IL-6, IL-10, IL-12, IL-17, 18 s, TNFα, TGFß, and Foxp3 in the ileum and mesenteric lymph nodes was measured by qPCR. Intestinal microbiota composition was significantly different in the cecum and colon compared to the ileum, which contains large abundances of Proteobacteria. Especially members of the Clostridiales order correlated positively with the regulatory T-cell transcription factor Foxp3 and so did the phylum Verrucomicrobia. We conclude that Clostridiales and Verrucomicrobia have the potential to induce regulatory immunity and are possible targets for intestinal microbial interventions aiming at regulatory immunity improvement.

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

Gut microbiota regulated imbalances in the host's 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.

The effect of Lactobacillus paracasei subsp. paracasei L. casei W8® on blood levels of triacylglycerol is independent of colonisation.

Gut microbiota (GM) dysbiosis has been linked to obesity and its metabolic complications such as cardiovascular disease (CVD). The risk of developing CVD increases with elevated concentration of serum triacylglycerol (TAG). In a blinded, randomised two-arm parallel human intervention study we have previously found that four weeks of supplementation with Lactobacillus paracasei subsp. paracasei L. casei W8® (L. casei W8) compared to placebo reduced the concentration of TAG in 64 young healthy adults, an effect, likely mediated by a decreased stearoyl- CoA desaturase-1 (SCD1) activity. In the present study we analysed faecal samples obtained during the intervention study to investigate whether this effect was related to the ability of L. casei W8 to colonise the human gut after supplementation of L. casei W8 (1010 cfu daily) as determined by qPCR specific for L. paracasei and L. casei (L. casei group); whether L. casei W8 consumption affected GM composition as determined by 16S rRNA gene targeted 454/FLX amplicon sequencing; and whether these changes were associated with changes in TAG concentration and SCD1 activity. Faecal samples were collected at baseline, after four weeks supplementation and two weeks after the supplementation was ended, and fasting blood samples were collected at baseline and after 4 weeks. Four weeks supplementation with L. casei W8 did not affect the overall composition of the GM; however, an increase in the relative abundance of the L. casei group from 8.48×10-6% of the total GM compared to 2.83×10-3% at baseline (P<0.001) was observed. Two weeks after supplementation ended, the relative abundance of the L. casei group was still increased 14 times compared to before the intervention (P<0.01). However, neither the increase in the abundance of the L. casei group nor overall GM composition correlated with changes in blood lipids or SCD1 activity.

TL1A regulates TCRγδ+ intraepithelial lymphocytes and gut microbial composition.

TL1A is a proinflammatory cytokine, which is prevalent in the gut. High TL1A concentrations are present in patients with inflammatory bowel disease (IBD) and in IBD mouse models. However, the role of TL1A during steady-state conditions is relatively unknown. Here, we used TL1A knockout (KO) mice to analyse the impact of TL1A on the intestinal immune system and gut microbiota. The TL1A KO mice showed reduced amounts of small intestinal intraepithelial TCRγδ(+) and CD8(+) T cells, and reduced expression of the activating receptor NKG2D. Moreover, the TL1A KO mice had significantly reduced body weight and visceral adipose tissue deposits, as well as lower levels of leptin and CXCL1, compared with wild-type mice. Analysis of the gut microbial composition of TL1A KO mice revealed a reduction of caecal Clostridial cluster IV, a change in the Firmicutes/Bacteroidetes ratio in caecum and less Lactobacillus spp. in the mucosal ileum. Our results show that TL1A deficiency impacts on the gut microbial composition and the mucosal immune system, especially the intraepithelial TCRγδ(+) T-cell subset, and that TL1A is involved in the establishment of adipose tissue. This research contributes to a broader understanding of TL1A inhibition, which is increasingly considered for treatment of IBD.

Investigating the long-term effect of subchronic phencyclidine-treatment on novel object recognition and the association between the gut microbiota and behavior in the animal model of schizophrenia.

Subchronic phencyclidine (subPCP) treatment induces schizophrenic-like behavior in rodents, including cognitive deficits and increased locomotor sensitivity towards acute administration of PCP. Evidence is accumulating that the gut microbiota (GM) influences behavior through modulation of the microbiota-gut-brain axis, and hence, part of the variation within this animal model may derive from variation in the GM. The aims of this study was to investigate first, the duration of subPCP-induced cognitive impairment in the novel object recognition test, and second, the possible effect of subchronic PCP-treatment on the GM, and the association between the GM and the behavioral parameters. The association was further investigated by antibiotic reduction of the GM. Male Lister Hooded rats were dosed twice daily i.p. with either 5mg/kg PCP or sterile isotonic saline for seven days followed by a seven-day washout period. Rats were tested in the novel object recognition and the locomotor activity assays immediately after, three weeks after, or six weeks after washout, and the fecal GM was analyzed by high throughput sequencing. Antibiotic- and control-treated rats were tested in the same manner following washout. In conclusion, subPCP-treatment impaired novel object recognition up to three weeks after washout, whereas locomotor sensitivity was increased for at least six weeks after washout. Differences in the core gut microbiome immediately after washout suggested subPCP treatment to alter the GM. GM profiles correlated to memory performance. Administration of ampicillin abolished the subPCP-induced memory deficit. It thus seems reasonable to speculate that the GM influences memory performance, contributing to variation within the model.

Characterization of the gut microbiota in leptin deficient obese mice - Correlation to inflammatory and diabetic parameters.

Gut microbiota have been implicated as a relevant factor in the development of type 2 diabetes mellitus (T2DM), and its diversity might be a cause of variation in animal models of T2DM. In this study, we aimed to characterise the gut microbiota of a T2DM mouse model with a long term vision of being able to target the gut microbiota to reduce the number of animals used in experiments. Male B6.V-Lep(ob)/J mice were characterized according to a number of characteristics related to T2DM, inflammation and gut microbiota. All findings were thereafter correlated to one another in a linear regression model. The total gut microbiota profile correlated to glycated haemoglobin, and high proportions of Prevotellaceae and Lachnospiraceae correlated to impaired or improved glucose intolerance, respectively. In addition, Akkermansia muciniphila disappeared with age as glucose intolerance worsened. A high proportion of regulatory T cells correlated to the gut microbiota and improved glucose tolerance. Furthermore, high levels of IL-10, IL-12 and TNF-α correlated to impaired glucose tolerance, blood glucose or glycated haemoglobin. The findings indicate that gut microbiota may contribute to variation in various disease read-outs in the B6.V-Lep(ob)/J model and considering them in both quality assurance and data evaluation for the B6.V-Lep(ob)/J model may have a reducing impact on the inter-individual variation.

Early life treatment with vancomycin propagates Akkermansia muciniphila and reduces diabetes incidence in the NOD mouse.

AIMS/HYPOTHESIS: Increasing evidence suggests that environmental factors changing the normal colonisation pattern in the gut strongly influence the risk of developing autoimmune diabetes. The aim of this study was to investigate, both during infancy and adulthood, whether treatment with vancomycin, a glycopeptide antibiotic specifically directed against Gram-positive bacteria, could influence immune homeostasis and the development of diabetic symptoms in the NOD mouse model for diabetes. METHODS: Accordingly, one group of mice received vancomycin from birth until weaning (day 28), while another group received vancomycin from 8 weeks of age until onset of diabetes. Pyrosequencing of the gut microbiota and flow cytometry of intestinal immune cells was used to investigate the effect of vancomycin treatment. RESULTS: At the end of the study, the cumulative diabetes incidence was found to be significantly lower for the neonatally treated group compared with the untreated group, whereas the insulitis score and blood glucose levels were significantly lower for the mice treated as adults compared with the other groups. Mucosal inflammation was investigated by intracellular cytokine staining of the small intestinal lymphocytes, which displayed an increase in cluster of differentiation (CD)4(+) T cells producing pro-inflammatory cytokines in the neonatally treated mice. Furthermore, bacteriological examination of the gut microbiota composition by pyrosequencing revealed that vancomycin depleted many major genera of Gram-positive and Gram-negative microbes while, interestingly, one single species, Akkermansia muciniphila, became dominant. CONCLUSIONS/INTERPRETATION: The early postnatal period is a critical time for microbial protection from type 1 diabetes and it is suggested that the mucolytic bacterium A. muciniphila plays a protective role in autoimmune diabetes development, particularly during infancy.