The regulation of mucosal immune function is critical to host protection from enteric pathogens but is incompletely understood. The nervous system and the neurotransmitter acetylcholine play an integral part in host defense against enteric bacterial pathogens. Here we report that acetylcholine producing-T-cells, as a non-neuronal source of ACh, were recruited to the colon during infection with the mouse pathogen Citrobacter rodentium . These ChAT + T-cells did not exclusively belong to one Th subset and were able to produce IFNγ, IL-17A and IL-22. To interrogate the possible protective effect of acetylcholine released from these cells during enteric infection, T-cells were rendered deficient in their ability to produce acetylcholine through a conditional gene knockout approach. Significantly increased C . rodentium burden was observed in the colon from conditional KO (cKO) compared to WT mice at 10 days post-infection. This increased bacterial burden in cKO mice was associated with increased expression of the cytokines IL-1β, IL-6, and TNFα, but without significant changes in T-cell and ILC associated IL-17A, IL-22, and IFNγ, or epithelial expression of antimicrobial peptides, compared to WT mice. Despite the increased expression of pro-inflammatory cytokines during C . rodentium infection, inducible nitric oxide synthase ( Nos2 ) expression was significantly reduced in intestinal epithelial cells of ChAT T-cell cKO mice 10 days post-infection. Additionally, a cholinergic agonist enhanced IFNγ-induced Nos2 expression in intestinal epithelial cell in vitro . These findings demonstrated that acetylcholine, produced by specialized T-cells that are recruited during C . rodentium infection, are a key mediator in host-microbe interactions and mucosal defenses.

Objectives Very little is known about dietary carbohydrate and intestinal microbe interactions during the introduction of solid foods in exclusively breastfed infants. The objective of the UC Davis IMiND study is to discover the relationships between plant-derived complementary foods commonly used in the early weaning period and the gut microbiome in a prospective feeding-trial in exclusively breast milk-fed infants. Methods In a randomized, crossover study, 6-month old, exclusively breastfed infants (n = 99) entered a 7-day lead-in period of exclusive breast milk, followed by 7 days of either study food (pear or sweet potato) plus breast milk. This was followed by a 4-day washout period of exclusive breast milk, then 7 days of the alternate study food, followed by a 4-day follow-up period of exclusive breast milk. The infant gut microbiome was measured by 16 s rRNA amplicon sequencing (n = 39). Fecal monosaccharides and short chain fatty acids were measured in a subset of mother-infant dyads (n = 20) by liquid chromatography-mass spectrometry. Results There was no significant difference in gut alpha diversity (Shannon index) but a significant difference in beta diversity (unweighted UniFrac, P = 0.03, R,2 = 0.02) between pre- and post- first food. Free fecal monosaccharide composition was similar across all feeding periods. Total bound fecal monosaccharides, including arabinose and xylose were 2-fold higher in response to pear consumption compared with the other feeding periods (P < 0.05). Infant fecal lactic acid was lower and succinic acid was higher by 2-fold during pear consumption compared with all other feeding periods (P < 0.05). Conclusions The change in gut microbiome beta diversity suggests a change in microbial composition with the introduction of solid foods despite the unchanged alpha diversity. The change in fecal short chain fatty acids in response to pear consumption suggests a change in microbial metabolism. These effects may be explained by the appearance of undigested, bound glycans in the colon during pear consumption. These data suggest a novel approach in using chemical analysis to document the diversity and complexity of dietary carbohydrates during weaning that influence gut microbial metabolism. Funding Sources Mongolia Mengniu Dairy (Group) Company Ltd. funded this research but had no part in the analysis or interpretations of the study findings.

IntroductionHuman milk oligosaccharides (HMOS) are indigestible carbohydrates that support infant development by establishing a healthy microbiota, preventing infectious diseases, and promoting immune and cognitive development. Individual HMOS have distinct functions based on their chemical structures. HMO profiles can vary largely among mothers, but the research on factors other than genetic background affecting HMO composition are limited.MethodsIn the present analysis, we examined the relationships between maternal characteristics and the HMO profiles of breastfeeding mothers (n = 392) in the STRONG kids 2 with the following demographic characteristics: average age: 30.8 y, 74.5% White, and 75.5% exclusively breastfeeding. Human milk samples were collected at 6 weeks postpartum and maternal information was obtained from self-reported surveys. Information on dietary intake changes since the participants have been breastfeeding was collected. HMO profiles were analyzed by high performance liquid chromatography coupled with mass spectrometry and secretor status was determined by the presence of four secretor markers [2′-fucosyllactose (2′-FL), LNFP I, LDFT, and TFLNH]. Spearmen correlation test was utilized to determine the relationships between individual HMOS and associations with maternal factors. Between-group differences in HMO relative abundances were examined with Kruskal-Wallis test.ResultsAmong all participants, 71.9% were secretors and 28.1% were non-secretors. The relative abundances of all HMOS differed (p < 0.05) by secretor status, with the exception for 6′-SL and 3′-SL. Positive correlations were observed among HMOS with similar structures, such as the 1,2-fucosylated HMOS. The abundances of selected HMOS were associated with maternal body weight, pregnancy complications, and dietary characteristics. Based on pre-pregnancy BMI, in all mothers, relative abundance of 3′-SL was significantly higher in overweight mothers than obese mothers (p = 0.013). In milk produced by non-secretor mothers, LNPF I + III abundances were greater in overweight than normal weight mothers (p = 0.020). Several HMO abundances were found to be associated with Gestational diabetes mellitus (GDM). Variations of HMO abundances were also observed with dietary food intake. In all mothers, egg consumption was positively correlated with LNT + LNnT (R = 0.13; p = 0.012) and cheese intake was positively associated with 2′-FL (R = 0.10; p = 0.046) and S-LNnH II (R = 0.11; p = 0.026) abundances.DiscussionHMO profiles were found to be associated with maternal characteristics and intake. Future research will investigate associations between HMOS and maternal and infant outcomes.

Background The stable isotope deuterium dose-to-mother (DTM) technique to estimate nonbreast milk water intake demonstrates that maternal self-report methods of infant feeding overestimate the true prevalence of exclusively breastfeeding practices. Objective We aimed to determine potential monosaccharide and oligosaccharide markers that distinguish between exclusively breastfed (EBF) versus nonexclusively breastfed (non-EBF) infants utilizing LC-MS-based methods. Methods Data for the analysis were collected as part of a larger, longitudinal study of 192 breastfed Indonesian infants aged 2 mo and followed up at 5 mo. Feces samples were collected from infants aged 2 mo (n = 188) and 5 mo (n = 184). EBF and non-EBF strata at each time point were determined via the DTM technique. Feces samples were analyzed to determine monosaccharide content using ultra-high-performance LC-triple quadrupole MS (UHPLC-QqQ MS). Relative abundances of fecal oligosaccharides were determined using nano-LC-Chip-quadrupole time-of-flight MS (nano-LC-Chip-Q-ToF MS). Results At age 2 mo, monosaccharide analysis showed the abundance of fructose and mannose were significantly higher (+377% and +388%, respectively) in non-EBF compared with EBF infants (P <0.0001). Fructose and mannose also showed good discrimination with areas under the curve (AUC) of 0.86 and 0.82, respectively. Oligosaccharide analysis showed that a 6-hexose (Hex6) isomer had good discrimination (AUC = 0.80) between EBF and non-EBF groups at 5 mo. Conclusion Carbohydrate products, particularly fecal mono- and oligosaccharides, differed between EBF and non-EBF infants aged under 6 mo and can be used as potential biomarkers to distinguish EBF versus non-EBF feeding practices.