Disruption of the primocolonizing microbiota alters epithelial homeostasis and imprints stem cells in the colon of neonatal piglets.

The gut microbiota plays a key role in the postnatal development of the intestinal epithelium. However, the bacterial members of the primocolonizing microbiota driving these effects are not fully identified and the mechanisms underlying their long-term influence on epithelial homeostasis remain poorly described. Here, we used a model of newborn piglets treated during the first week of life with the antibiotic colistin in order to deplete specific gram-negative bacteria that are transiently dominant in the neonatal gut microbiota. Colistin depleted Proteobacteria and Fusobacteriota from the neonatal colon microbiota, reduced the bacterial predicted capacity to synthetize lipopolysaccharide (LPS), and increased the concentration of succinate in the colon. The colistin-induced disruption of the primocolonizing microbiota was associated with altered gene expression in the colon epithelium including a reduction of toll-like receptor 4 (TLR4) and lysozyme (LYZ). Our data obtained in porcine colonic organoid cell monolayers suggested that these effects were not driven by the variation of succinate or LPS levels nor by a direct effect of colistin on epithelial cells. The disruption of the primocolonizing microbiota imprinted colon epithelial stem cells since the expression of TLR4 and LYZ remained lower in organoids derived from colistin-treated piglet colonic crypts after several passages when compared to control piglets. Finally, the stable imprinting of LYZ in colon organoids was independent of the H3K4me3 level in its transcription start site. Altogether, our results show that disruption of the primocolonizing gut microbiota alters epithelial innate immunity in the colon and imprints stem cells, which could have long-term consequences for gut health.

The Early Life Microbiota Is Not a Major Factor Underlying the Susceptibility to Postweaning Diarrhea in Piglets.

Postweaning diarrhea (PWD) in piglets impair welfare, induce economic losses and lead to overuse of antibiotics. The early life gut microbiota was proposed to contribute to the susceptibility to PWD. The objective of our study was to evaluate in a large cohort of 116 piglets raised in 2 separate farms whether the gut microbiota composition and functions during the suckling period were associated with the later development of PWD. The fecal microbiota and metabolome were analyzed by 16S rRNA gene amplicon sequencing and nuclear magnetic based resonance at postnatal day 13 in male and female piglets. The later development of PWD was recorded for the same animals from weaning (day 21) to day 54. The gut microbiota structure and α-diversity during the suckling period were not associated with the later development of PWD. There was no significant difference in the relative abundances of bacterial taxa in suckling piglets that later developed PWD. The predicted functionality of the gut microbiota and the fecal metabolome signature during the suckling period were not linked to the later development of PWD. Trimethylamine was the bacterial metabolite which fecal concentration during the suckling period was the most strongly associated with the later development of PWD. However, experiments in piglet colon organoids showed that trimethylamine did not disrupt epithelial homeostasis and is thus not likely to predispose to PWD through this mechanism. In conclusion, our data suggest that the early life microbiota is not a major factor underlying the susceptibility to PWD in piglets. IMPORTANCE This study shows that the fecal microbiota composition and metabolic activity are similar in suckling piglets (13 days after birth) that either later develop post-weaning diarrhea (PWD) or not, which is a major threat for animal welfare that also causes important economic losses and antibiotic treatments in pig production. The aim of this work was to study a large cohort of piglets raised in separates environments, which is a major factor influencing the early life microbiota. One of the main findings is that, although the fecal concentration of trimethylamine in suckling piglets was associated with the later development of PWD, this gut microbiota-derived metabolite did not disrupt the epithelial homeostasis in organoids derived from the pig colon. Overall, this study suggests that the gut microbiota during the suckling period is not a major factor underlying the susceptibility of piglets to PWD.

The phenotype of the gut region is more stably retained than developmental stage in piglet intestinal organoids.

Intestinal organoids are innovative in vitro tools to study the digestive epithelium. The objective of this study was to generate jejunum and colon organoids from suckling and weaned piglets in order to determine the extent to which organoids retain a location-specific and a developmental stage-specific phenotype. Organoids were studied at three time points by gene expression profiling for comparison with the transcriptomic patterns observed in crypts in vivo. In addition, the gut microbiota and the metabolome were analyzed to characterize the luminal environment of epithelial cells at the origin of organoids. The location-specific expression of 60 genes differentially expressed between jejunum and colon crypts from suckling piglets was partially retained (48%) in the derived organoids at all time point. The regional expression of these genes was independent of luminal signals since the major differences in microbiota and metabolome observed in vivo between the jejunum and the colon were not reproduced in vitro. In contrast, the regional expression of other genes was erased in organoids. Moreover, the developmental stage-specific expression of 30 genes differentially expressed between the jejunum crypts of suckling and weaned piglets was not stably retained in the derived organoids. Differentiation of organoids was necessary to observe the regional expression of certain genes while it was not sufficient to reproduce developmental stage-specific expression patterns. In conclusion, piglet intestinal organoids retained a location-specific phenotype while the characteristics of developmental stage were erased in vitro. Reproducing more closely the luminal environment might help to increase the physiological relevance of intestinal organoids.

Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions.

In mammals, the introduction of solid food is pivotal for the establishment of the gut microbiota. However, the effects of the first food consumed on long-term microbiota trajectory and host response are still largely unknown. This study aimed to investigate the influences of (i) the timing of first solid food ingestion and (ii) the consumption of plant polysaccharides on bacterial community dynamics and host physiology using a rabbit model. To modulate the first exposure to solid nutrients, solid food was provided to suckling rabbits from two different time points (3 or 15 days of age). In parallel, food type was modulated with the provision of diets differing in carbohydrate content throughout life: the food either was formulated with a high proportion of rapidly fermentable fibers (RFF) or was starch-enriched. We found that access to solid food as of 3 days of age accelerated the gut microbiota maturation. Our data revealed differential effects according to the digestive segment: precocious solid food ingestion influenced to a greater extent the development of bacterial communities of the appendix vermiformis, whereas life course polysaccharides ingestion had marked effects on the cecal microbiota. Greater ingestion of RFF was assumed to promote pectin degradation as revealed by metabolomics analysis. However, transcriptomic and phenotypic host responses remained moderately affected by experimental treatments, suggesting little outcomes of the observed microbiome modulations on healthy subjects. In conclusion, our work highlighted the timing of solid food introduction and plant polysaccharides ingestion as two different tools to modulate microbiota implantation and functionality. IMPORTANCE Our study was designed to gain a better understanding of how different feeding patterns affect the dynamics of gut microbiomes and microbe-host interactions. This research showed that the timing of solid food introduction is a key component of the gut microbiota shaping in early developmental stages, though with lower impact on settled gut microbiota profiles in older individuals. This study also provided in-depth analysis of dietary polysaccharide effects on intestinal microbiota. The type of plant polysaccharides reaching the gut through the lifetime was described as an important modulator of the cecal microbiome and its activity. These findings will contribute to better define the interventions that can be employed for modulating the ecological succession of young mammal gut microbiota.

Microbiability and microbiome-wide association analyses of feed efficiency and performance traits in pigs.

BACKGROUND: The objective of the present study was to investigate how variation in the faecal microbial composition is associated with variation in average daily gain (ADG), backfat thickness (BFT), daily feed intake (DFI), feed conversion ratio (FCR), and residual feed intake (RFI), using data from two experimental pig lines that were divergent for feed efficiency. Estimates of microbiability were obtained by a Bayesian approach using animal mixed models. Microbiome-wide association analyses (MWAS) were conducted by single-operational taxonomic units (OTU) regression and by back-solving solutions of best linear unbiased prediction using a microbiome covariance matrix. In addition, accuracy of microbiome predictions of phenotypes using the microbiome covariance matrix was evaluated. RESULTS: Estimates of heritability ranged from 0.31 ± 0.13 for FCR to 0.51 ± 0.10 for BFT. Estimates of microbiability were lower than those of heritability for all traits and were 0.11 ± 0.09 for RFI, 0.20 ± 0.11 for FCR, 0.04 ± 0.03 for DFI, 0.03 ± 0.03 for ADG, and 0.02 ± 0.03 for BFT. Bivariate analyses showed a high microbial correlation of 0.70 ± 0.34 between RFI and FCR. The two approaches used for MWAS showed similar results. Overall, eight OTU with significant or suggestive effects on the five traits were identified. They belonged to the genera and families that are mainly involved in producing short-chain fatty acids and digestive enzymes. Prediction accuracy of phenotypes using a full model including the genetic and microbiota components ranged from 0.60 ± 0.19 to 0.78 ± 0.05. Similar accuracies of predictions of the microbial component were observed using models that did or did not include an additive animal effect, suggesting no interaction with the genetic effect. CONCLUSIONS: Our results showed substantial associations of the faecal microbiome with feed efficiency related traits but negligible effects with growth traits. Microbiome data incorporated as a covariance matrix can be used to predict phenotypes of animals that do not (yet) have phenotypic information. Connecting breeding environment between training sets and predicted populations could be necessary to obtain reliable microbiome predictions.

Developmental Stage, Solid Food Introduction, and Suckling Cessation Differentially Influence the Comaturation of the Gut Microbiota and Intestinal Epithelium in Rabbits.

BACKGROUND: In mammals, the establishment around weaning of a symbiotic relationship between the gut microbiota and its host determines long-term health. OBJECTIVES: The aim of this study was to identify the factors driving the comaturation of the gut microbiota and intestinal epithelium at the suckling-to-weaning transition. We hypothesized that the developmental stage, solid food ingestion, and suckling cessation contribute to this process. METHODS: From birth to day 18, Hyplus rabbits were exclusively suckling. From day 18 to day 25, rabbits were 1) exclusively suckling; 2) suckling and ingesting solid food; or 3) exclusively ingesting solid food. The microbiota (16S amplicon sequencing), metabolome (nuclear magnetic resonance), and epithelial gene expression (high-throughput qPCR) were analyzed in the cecum at days 18 and 25. RESULTS: The microbiota structure and metabolic activity were modified with age when rabbits remained exclusively suckling. The epithelial gene expression of nutrient transporters, proliferation markers, and innate immune factors were also regulated with age (e.g., 1.5-fold decrease of TLR5). Solid food ingestion by suckling rabbits had a major effect on the gut microbiota by increasing its α diversity, remodeling its structure (e.g., 6.3-fold increase of Ruminococcaceae), and metabolic activity (e.g., 4.6-fold increase of butyrate). Solid food introduction also regulated the gene expression of nutrient transporters, differentiation markers, and innate immune factors in the epithelium (e.g., 3-fold increase of nitric oxide synthase). Suckling cessation had no effect on the microbiota, while it regulated the expression of genes involved in epithelial differentiation and immunoglobulin transport (e.g., 2.5-increase of the polymeric immunoglobulin receptor). CONCLUSIONS: In rabbits, the maturation of the microbiota at the suckling-to-weaning transition is driven by the introduction of solid food and, to a lesser extent, by the developmental stage. In contrast, the maturation of the intestinal epithelium at the suckling-to-weaning transition is under the influence of the developmental stage, solid food introduction, and suckling cessation.

Genetic relationships between feed efficiency and gut microbiome in pig lines selected for residual feed intake.

This study aimed to evaluate the genetic relationship between faecal microbial composition and five feed efficiency (FE) and production traits, residual feed intake (RFI), feed conversion ratio (FCR), daily feed intake (DFI), average daily gain (ADG) and backfat thickness (BFT). A total of 588 samples from two experimental pig lines developed by divergent selection for RFI were sequenced for the 16 rRNA hypervariable V3-V4 region. The 75 genera with less than 20% zero values (97% of the counts) and two α-diversity indexes were analysed. Line comparison of the microbiota traits and estimations of heritability (h2 ) and genetic correlations (rg ) were analysed. A non-metric multidimensional scaling showed line differences between genera. The α-diversity indexes were higher in the LRFI line than in the HRFI line (p < .01), with h2 estimates of 0.19 ± 0.08 (Shannon) and 0.12 ± 0.06 (Simpson). Forty-eight genera had a significant h2 (>0.125). The rg of the α-diversities indexes with production traits were negative. Some rg of genera belonging to the Lachnospiraceae, Ruminococcaceae, Prevotellaceae, Lactobacillaceae, Streptococcaceae, Rikenellaceae and Desulfovibrionaceae families significantly differed from zero (p < .05) with FE traits, RFI (3), DFI (7) and BFT (11). These results suggest that a sizable part of the variability of the gut microbial community is under genetic control and has genetic relationships with FE, including diversity indicators. It offers promising perspectives for selection for feed efficiency using gut microbiome composition in pigs.

Gut Microbiota-Derived Metabolite Signature in Suckling and Weaned Piglets.

The gut microbiota plays a key role in intestinal development at the suckling-to-weaning transition. The objective of this study was to analyze the production of metabolites by the gut microbiota in suckling and weaned piglets. We studied piglets raised in two separate maternity farms and weaned at postnatal day 21 in the same farm. The fecal metabolome (1H nuclear magnetic resonance) and the microbiota composition (16S rRNA gene amplicon sequencing) and its predicted functions (PICRUSt2) were analyzed in the same piglets during the suckling period (postnatal day 13) and 2 days after weaning (postnatal day 23). The relative concentrations of the bacterial metabolites methylamine, dimethylamine, cadaverine, tyramine, putrescine, 5-aminovalerate, succinate, and 3-(4-hydroxyphenylpropionate) were higher during the suckling period than after weaning. In contrast, the relative concentrations of the short-chain fatty acids acetate and propionate were higher after weaning than during the suckling period. The maternity of origin of piglets also influenced the level of some bacterial metabolites (propionate and isobutyrate). The fecal metabolome signatures observed in suckling and weaned piglets were associated with specific microbiota-predicted functionalities, structure, and diversity. Gut microbiota-derived metabolites, which are differentially abundant between suckling and weaned piglets (e.g., short-chain fatty acids and biogenic amines), are known to regulate gut health. Thus, identification of metabolome signatures in suckling and weaned piglets paves the way for the development of health-promoting nutritional strategies, targeting the production of bacterial metabolites in early life.

Early Introduction of Solid Feeds: Ingestion Level Matters More Than Prebiotic Supplementation for Shaping Gut Microbiota.

Early introduction of a nutritional substrate is a promising biomimetic strategy for controlling the implantation of the microbiota and preserving the health of young animals. In this study, we provided experimental solid substrate in a gel form to stimulate suckling rabbits' intake and to investigate its effects on microbiota implantation and colonization. All the rabbits had access to solid feed outside the nest as of 15 days of age. Except for the control group, rabbits were offered starter feed gels inside the nests from 3 to 18 days of age. These gels were either free of additives (AF_GEL) or contained 4% of fructo-oligosaccharides (FOS_GEL) or 4% of mannan-oligosaccharides and ß-glucans mixtures (MOS_GEL). The cecal content of 160 rabbits was sampled at 18, 29, 38, and 57 days of age and analyzed using 16S rRNA gene sequencing. Pups consumed an average of 3.95 ± 1.07 g of starter feed gel with a higher intake when it was supplemented with fructo-oligosaccharides (+1.2 g; P < 0.05). Starter feed gel consumption increased the ensuing intake of pellets (+17 g from 15 to 21 days; P < 0.05). Alpha-diversity indexes were similar between groups and prebiotic supplementation did not induce a clear shift in microbiota pattern. Conversely, when considering rabbits that consumed more starter feed, the highest proportions of bacteria with plant-degrading abilities, such as species from the Lachnospiraceae and Ruminococcaceae families, were observed at 18 days of age. However, fermentative activities were not affected by starter feed intake at 29, 38, and 57 days of age. By providing comprehensive results on the regulation of microbial community structure at the onset of solid feed intake, this research paves the way for further studies on digestive ecosystem maturation.

Gut microbiota derived metabolites contribute to intestinal barrier maturation at the suckling-to-weaning transition.

In suckling mammals, the onset of solid food ingestion is coincident with the maturation of the gut barrier. This ontogenic process is driven by the colonization of the intestine by the microbiota. However, the mechanisms underlying the microbial regulation of the intestinal development in early life are not fully understood. Here, we studied the co-maturation of the microbiota (composition and metabolic activity) and of the gut barrier at the suckling-to-weaning transition by using a combination of experiments in vivo (suckling rabbit model), ex vivo (Ussing chambers) and in vitro (epithelial cell lines and organoids). The microbiota composition, its metabolic activity, para-cellular epithelial permeability and the gene expression of key components of the gut barrier shifted sharply at the onset of solid food ingestion in vivo, despite milk was still predominant in the diet at that time. We found that cecal content sterile supernatant (i.e. containing a mixture of metabolites) obtained after the onset of solid food ingestion accelerated the formation of the epithelial barrier in Caco-2 cells in vitro and our results suggested that these effects were driven by the bacterial metabolite butyrate. Moreover, the treatment of organoids with cecal content sterile supernatant partially replicated in vitro the effects of solid food ingestion on the epithelial barrier in vivo. Altogether, our results show that the metabolites produced by the microbiota at the onset of solid food ingestion contribute to the maturation of the gut barrier at the suckling-to-weaning transition. Targeting the gut microbiota metabolic activity during this key developmental window might therefore be a promising strategy to promote intestinal homeostasis.

Fumonisin-Exposure Impairs Age-Related Ecological Succession of Bacterial Species in Weaned Pig Gut Microbiota.

Pigs are highly affected by dietary mycotoxin contamination and particularly by fumonisin. The effects of fumonisin on pig intestinal health are well documented, but little is known regarding its impact on gut microbiota. We investigate the effects of the fumonisin (FB1, 12 mg/kg feed) on the fecal microbiota of piglets (n = 6) after 0, 8, 15, 22, and 29 days of exposure. A control group of six piglets received a diet free of FB1. Bacterial community diversity, structure and taxonomic composition were carried out by V3⁻V4 16S rRNA gene sequencing. Exposure to FB1 decreases the diversity index, and shifts and constrains the structure and the composition of the bacterial community. This takes place as early as after 15 days of exposure and is at a maximum after 22 days of exposure. Compared to control, FB1 alters the ecological succession of fecal microbiota species toward higher levels of Lactobacillus and lower levels of the Lachnospiraceae and Veillonellaceae families, and particularly OTUs (Operational Taxonomic Units) of the genera Mitsuokella, Faecalibacterium and Roseburia. In conclusion, FB1 shifts and constrains age-related evolution of microbiota. The direct or indirect contribution of FB1 microbiota alteration in the global host response to FB1 toxicity remains to be investigated.

PEGylation of antibody fragments greatly increases their local residence time following delivery to the respiratory tract.

Inhalation aerosols offer a targeted therapy for respiratory diseases. However, the therapeutic efficacy of inhaled biopharmaceuticals is limited by the rapid clearance of macromolecules in the lungs. The aim of this research was to study the effects of the PEGylation of antibody fragments on their local residence time after administration to the respiratory tract. We demonstrate that the conjugation of a two-armed 40-kDa polyethylene glycol (PEG) chain to anti-interleukin-17A (IL-17A) F(ab')2 and anti-IL-13 Fab' greatly prolonged the presence of these fragments within the lungs of mice. The content of PEGylated antibody fragments within the lungs plateaued up to 4h post-delivery, whereas the clearance of unconjugated proteins started immediately after administration. Forty-eight hours post-delivery, F(ab')2 and Fab' contents in the lungs had decreased to 10 and 14% of the dose initially deposited, respectively. However, this value was 40% for both PEG40-F(ab')2 and PEG40-Fab'. The prolonged pulmonary residency of the anti-IL-17A PEG40-F(ab')2 translated into an improved efficacy in reducing lung inflammation in a murine model of house dust mite-induced lung inflammation. We demonstrate that PEGylated proteins were principally retained within the lung lumen rather than the nasal cavities or lung parenchyma. In addition, we report that PEG increased pulmonary retention of antibody fragments through mucoadhesion and escape from alveolar macrophages rather than increased hydrodynamic size or improved enzymatic stability. The PEGylation of proteins might find broad application in the local delivery of therapeutic proteins to diseased airways.

Production, purification and biological characterization of mono-PEGylated anti-IL-17A antibody fragments.

The aim of this study was to maximize the yield of the production of mono-PEGylated anti-interleukin-17A (anti-IL-17A) antibody fragments using large (≥ 20 kDa) polyethylene glycol (PEG) chains. Particular attention was paid to selectively yield mono-PEGylated species to maintain the maximum possible functionality and to simplify the purification. Neutralization of IL-17A by antibody constructs might find application for the treatment of bronchial hyperreactivity. Amino-directed and sulfhydryl-directed PEGylation of the native antibody fragments were compared. The former was selected as it produced the most interesting construct in terms of yield and preservation of biological activity. In particular, the F(ab')2-PEG conjugate with one 40 kDa branched PEG prepared in this study was produced at a 42% yield. The conjugate presented only a slight decrease in its binding activity and in its in vitro inhibitory potency offering interesting perspectives for in vivo studies.