Breastfeeding profoundly shapes the infant gut microbiota, which is critical for early life immune development, and the gut microbiota can impact host physiology in various ways, such as through the production of metabolites. However, few breastmilk-dependent microbial metabolites mediating host–microbiota interactions are currently known. Here, we demonstrate that breastmilk-promoted Bifidobacterium species convert aromatic amino acids (tryptophan, phenylalanine and tyrosine) into their respective aromatic lactic acids (indolelactic acid, phenyllactic acid and 4-hydroxyphenyllactic acid) via a previously unrecognized aromatic lactate dehydrogenase (ALDH). The ability of Bifidobacterium species to convert aromatic amino acids to their lactic acid derivatives was confirmed using monocolonized mice. Longitudinal profiling of the faecal microbiota composition and metabolome of Danish infants (n = 25), from birth until 6 months of age, showed that faecal concentrations of aromatic lactic acids are correlated positively with the abundance of human milk oligosaccharide-degrading Bifidobacterium species containing the ALDH, including Bifidobacterium longum, B. breve and B. bifidum. We further demonstrate that faecal concentrations of Bifidobacterium-derived indolelactic acid are associated with the capacity of these samples to activate in vitro the aryl hydrocarbon receptor (AhR), a receptor important for controlling intestinal homoeostasis and immune responses. Finally, we show that indolelactic acid modulates ex vivo immune responses of human CD4+ T cells and monocytes in a dose-dependent manner by acting as an agonist of both the AhR and hydroxycarboxylic acid receptor 3 (HCA3). Our findings reveal that breastmilk-promoted Bifidobacterium species produce aromatic lactic acids in the gut of infants and suggest that these microbial metabolites may impact immune function in early life.

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The intestinal microbiota has been linked to the development and prevalence of steatohepatitis in humans. Interestingly, steatohepatitis is significantly lower in individuals taking a plant‐based, low‐animal‐protein diet, which is thought to be mediated by gut microbiota. However, data on causality between these observations in humans is scarce. In this regard, fecal microbiota transplantation (FMT) using healthy donors is safe and is capable of changing microbial composition in human disease. We therefore performed a double‐blind randomized controlled proof‐of‐principle study in which individuals with hepatic steatosis on ultrasound were randomized to two study arms: lean vegan donor (allogenic n = 10) or own (autologous n = 11) FMT. Both were performed three times at 8‐week intervals. A liver biopsy was performed at baseline and after 24 weeks in every subject to determine histopathology (Nonalcoholic Steatohepatitis Clinical Research Network) classification and changes in hepatic gene expression based on RNA sequencing. Secondary outcome parameters were changes in intestinal microbiota composition and fasting plasma metabolomics. We observed a trend toward improved necro‐inflammatory histology, and found significant changes in expression of hepatic genes involved in inflammation and lipid metabolism following allogenic FMT. Intestinal microbial community structure changed following allogenic FMT, which was associated with changes in plasma metabolites as well as markers of . Conclusion: Allogenic FMT using lean vegan donors in individuals with hepatic steatosis shows an effect on intestinal microbiota composition, which is associated with beneficial changes in plasma metabolites and markers of steatohepatitis.

26Breastfeeding profoundly shapes the infant gut microbiota, which is critical for early life immune 27 development. However, few breastmilk-dependent microbial metabolites mediating host-microbiota 28 interactions are currently known. We here demonstrate that breastmilk-promoted Bifidobacterium 29 species convert aromatic amino acids (tryptophan, phenylalanine and tyrosine) into their respective 30 aromatic lactic acids (indolelactate, phenyllactate and 4-hydroxyphenyllactate) via a previously 31 unrecognised aromatic lactate dehydrogenase. By longitudinal profiling of the gut microbiota 32 composition and metabolome of stool samples of infants obtained from birth until 6 months of age, 33 we show that stool concentrations of aromatic lactic acids is determined by the abundance of human 34 milk oligosaccharide degrading Bifidobacterium species containing the aromatic lactate 35 dehydrogenase. Finally, we demonstrate that stool concentrations of Bifidobacterium-derived 36 indolelactate are associated with the capacity of infant stool samples to activate the aryl 37 hydrocarbon receptor, a receptor important for maintenance of intestinal homeostasis and immune 38 system development. These findings open up new directions towards understanding the role of 39 breastmilk-promoted Bifidobacterium in mediating host-microbiota interactions in early life. 40 INTRODUCTION 41Human breastmilk is a perfectly adapted nutritional supply for the infant 1 . Breastfeeding provides 42 children with important short-term protection against infections, and may also provide long-term 43 metabolic benefits 1,2 . These benefits may partly be mediated through the gut microbiota, since 44 breastfeeding is the strongest determinant of gut microbiota composition and function during 45 infancy 3-5 . Human breastmilk contains human milk oligosaccharides (HMOs), which are complex, 46 highly abundant sugars serving as substrates for specific microbes including certain species of 47 Bifidobacterium 6 . This co-evolution between bifidobacteria and the host, mediated by HMOs, to a 48 large extent directs the colonization of the gut in early life, which has critical impact on the immune 49 system 7 . Depletion of specific microbes, including Bifidobacterium, in early life has been associated 50 with increased risk of allergy and asthma development in childhood 8,9 , and is suggested to 51 compromise immune function and lead to increased susceptibility to infectious disease 10,11 . Despite 52 Bifidobacterium dominating the gut of breastfed infants and being widely acknowledged as 53 beneficial, mechanistic insights on the contribution of these bacteria and their metabolites to 54 immune development during infancy remain limited. Recent studies show that microbial aromatic 55 amino acid metabolites including tryptophan-derived indoles 12 , via activation of the aryl 56 hydrocarbon receptor (AhR), can fortify the intestinal barrier 13,14 , protect against pathogenic 57 infections 15,16 and influence host metabolism 13,17,18 , which makes this...

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Breastfeeding protects against diseases, with potential mechanisms driving this being human milk oligosaccharides (HMOs) and the seeding of milk-associated bacteria in the infant gut. In a cohort of 34 mother–infant dyads we analyzed the microbiota and HMO profiles in breast milk samples and infant’s feces. The microbiota in foremilk and hindmilk samples of breast milk was compositionally similar, however hindmilk had higher bacterial load and absolute abundance of oral-associated bacteria, but a lower absolute abundance of skin-associated Staphylococcus spp. The microbial communities within both milk and infant’s feces changed significantly over the lactation period. On average 33% and 23% of the bacterial taxa detected in infant’s feces were shared with the corresponding mother’s milk at 5 and 9 months of age, respectively, with Streptococcus, Veillonella and Bifidobacterium spp. among the most frequently shared. The predominant HMOs in feces associated with the infant’s fecal microbiota, and the dominating infant species B. longum ssp. infantis and B. bifidum correlated inversely with HMOs. Our results show that breast milk microbiota changes over time and within a feeding session, likely due to transfer of infant oral bacteria during breastfeeding and suggest that milk-associated bacteria and HMOs direct the assembly of the infant gut microbiota.

Objective: Insulin resistance develops prior to the onset of overt type 2 diabetes, making its early detection vital. Direct accurate evaluation is currently only possible with complex examinations like the stable isotope-based hyperinsulinemic euglycemic clamp (HIEC). Metabolomic profiling enables the detection of thousands of plasma metabolites, providing a tool to identify novel biomarkers in human obesity.Design: Liquid chromatography mass spectrometry-based untargeted plasma metabolomics was applied in 60 participants with obesity with a large range of peripheral insulin sensitivity as determined via a two-step HIEC with stable isotopes [6,6-2 H 2 ] glucose and [1,1,2,3,3-2 H 5 ]glycerol. This additionally enabled measuring insulinregulated lipolysis, which combined with metabolomics, to the knowledge of this research group, has not been reported on before.Results: Several plasma metabolites were identified that significantly correlated with glucose and lipid fluxes, led by plasma (gamma-glutamyl)citrulline, followed by betaine, beta-cryptoxanthin, fructosyllysine, octanylcarnitine, sphingomyelin (d18:0/18:0, d19:0/17:0) and thyroxine. Subsequent machine learning analysis showed that a panel of these metabolites derived from a number of metabolic pathways may be used to predict insulin resistance, dominated by non-essential amino acid citrulline and its metabolite gamma-glutamylcitrulline. Conclusion:This approach revealed a number of plasma metabolites that correlated reasonably well with glycemic and lipolytic flux parameters, measured using gold standard techniques. These metabolites may be used to predict the rate of glucose disposal in humans with obesity to a similar extend as HOMA, thus providing potential novel biomarkers for insulin resistance. K E Y W O R D Scitrulline, human insulin resistance, plasma metabolites, stable isotope hyperinsulinemic clamp

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