A controlled piglet study (Merrifield et al., Gut 2013) showed that a four-week egg- versus soya-based weaning diet induces a sustained shift of the urinary metabolic phenotype and alters gut mucosal immunoglobulin production that persists for at least 28 days after all animals are switched to a common washout diet, with probiotic Bifidobacterium lactis NCC2818 modulating both. The study left open five linked questions: whether the diet-induced metabolic and immunological imprint converges or persists over longer follow-up; whether probiotic intervention can accelerate reversion of the imprint and at what dose and timing; which host regulatory mechanisms (e.g., epigenetic) mediate the sustained reprogramming; what the normal developmental trajectories and ranges of urinary metabolic and mucosal immune parameters are (needed for diagnostic thresholds and windows of opportunity); and whether diet- or probiotic-responsive microbes causally drive the observed immunoglobulin modulation rather than merely correlating with it. Same-group follow-up studies and adjacent work in pigs, mice, and human infants characterize parts of these threads but resolve none of them; the problem remains open.

Background

Weaning — the transition from milk to solid food — causes a major ecological shift in the gut microbiota and is a critical window for the development of mucosal immunity in young mammals. Because the pig's gastrointestinal physiology, microbiology, and genome are closer to humans than rodents are, piglets are used as a translational model of the human infant.

Metabonomics is the systematic profiling of small molecules in biofluids by, for example, proton nuclear magnetic resonance (¹H NMR) spectroscopy of urine. Many urinary metabolites are co-metabolites produced jointly by host tissues and gut bacteria (e.g., hippurate, phenylacetylglycine, and indoleacetylglycine, which arise from microbial transformation of dietary phenolics and tryptophan followed by host conjugation), so the urinary metabolic phenotype (metabotype) reports on both host metabolism and microbial activity. Mucosal immunity can be quantified ex vivo by culturing intestinal tissue fragments and measuring the immunoglobulins IgA and IgM that they produce; secretory IgA is the dominant antibody class at mucosal surfaces, where it shapes the dialogue with the commensal microbiota.

Merrifield et al. (Gut 2013) combined these platforms in a 2×2 factorial piglet experiment: piglets were weaned at three weeks onto either an egg-based or a soya-based diet for four weeks, after which all animals received a common fish-based washout diet for another four weeks; half of each diet group also received the human probiotic strain Bifidobacterium lactis NCC2818 from weaning onwards. At post-mortem (11 weeks), urinary ¹H NMR profiles still separated strongly by the initial weaning diet, showing that a transient early diet leaves a sustained metabolic imprint that survives four weeks of dietary standardization. Both the weaning diet and the probiotic independently shifted mucosal IgA and IgM production in a tissue-specific manner, and urinary co-metabolites correlated with local immunoglobulin output, suggesting urine could serve as a non-invasive readout of mucosal immune status. The design carries statistical limitations (a small cohort of 14 animals, a single terminal time point, and only correlative evidence linking the urinary metabolic profile to mucosal immunoglobulin output).

Five unresolved directions for future dedicated experiments follow from the study: (1) temporal durability — do the diet-induced metabolic and immunological differences observed 28 days after dietary standardization converge or remain stable over longer follow-up; (2) reversibility and intervention efficacy — can B. lactis NCC2818 or other probiotic strains accelerate reversion from the early-life diet-induced metabolic imprint, and with what dosing and timing; (3) mechanistic basis — which host regulatory mechanisms, such as DNA methylation, histone modification, or chromatin remodelling, mediate the sustained reprogramming; (4) normal ranges and windows of opportunity — the normal developmental trajectories and ranges of urinary metabolic and mucosal immune parameters in piglets (and human infants) are not established, blocking definition of diagnostic thresholds and optimal intervention timing; and (5) causality within the microbiota axis — whether the microbial taxa or microbial metabolic activities that shift with diet or probiotic are causal drivers of the immunoglobulin modulation, or only correlating with it. These five strands jointly serve one objective: to determine whether, how, and to what extent early weaning diet durably programs host metabolism and mucosal immunity in a human-relevant model, so that the imprint can be monitored non-invasively and redirected by timely intervention.

Problem Statement

Determine the fate, reversibility, and mechanistic basis of the sustained metabolic and mucosal-immunological imprint induced by early weaning diet in the piglet model of Merrifield et al. (Gut 2013). This one experimental objective comprises five components, each an independent sub-question: resolving one component does not require resolving the others:

  1. Temporal durability: establish whether the metabolic and mucosal immunological differences induced by a brief egg- versus soya-based weaning diet, still present 28 days after switching to a common washout diet, converge back toward a common phenotype or remain stable (or diverge further) over longer follow-up — i.e., characterize the imprint's trajectory into later juvenile and adult life.

  2. Reversibility and intervention: determine whether Bifidobacterium lactis NCC2818 or other probiotic strains can accelerate reversion — here defined as recovery of the egg- versus soya-diet groups toward a common, diet-group-independent metabolic and immunological phenotype (i.e., loss of the between-diet separation) — from an established early-life diet-induced metabolic and immunological imprint, and identify dosing and timing regimens that achieve this.

  3. Mechanistic basis: identify the host regulatory mechanisms — for example DNA methylation, histone modification, or chromatin remodelling — that mediate the sustained metabolic and immunological reprogramming induced by early diet and/or probiotics.

  4. Normal ranges and windows of opportunity: establish the normal developmental trajectories and ranges of urinary metabolic and mucosal immune parameters in piglets, with human infants as a translational extension, sufficient to define diagnostic thresholds for aberrant imprinting — a sustained profile that deviates outside the normal developmental range — and optimal timing (windows of opportunity) for intervention.

  5. Causality within the microbiota axis: establish whether the microbial taxa or microbial metabolic activities that change in response to weaning diet or probiotic are causal drivers of the observed mucosal immunoglobulin modulation, as opposed to merely correlating with it.

References

  1. Merrifield CA, Lewis MC, Claus SP, Pearce JTM, Cloarec O, Duncker S, Heinzmann SS, Dumas M-E, Kochhar S, Rezzi S, Mercenier A, Nicholson JK, Bailey M, Holmes E. Weaning diet induces sustained metabolic phenotype shift in the pig and influences host response to Bifidobacterium lactis NCC2818. Gut 2013;62(6):842–851. doi:10.1136/gutjnl-2011-301656. https://doi.org/10.1136/gutjnl-2011-301656
  2. Lewis MC, Patel DV, Fowler J, Duncker S, Zuercher AW, Mercenier A, Bailey M. Dietary supplementation with Bifidobacterium lactis NCC2818 from weaning reduces local immunoglobulin production in lymphoid-associated tissues but increases systemic antibodies in healthy neonates. British Journal of Nutrition 2013;110(7):1243–1252. doi:10.1017/S0007114513000251. https://doi.org/10.1017/S0007114513000251
  3. Merrifield CA, Lewis MC, Berger B, Cloarec O, Heinzmann SS, Charton F, Krause L, Levin NS, Duncker S, Mercenier A, Holmes E, Bailey M, Nicholson JK. Neonatal environment exerts a sustained influence on the development of the intestinal microbiota and metabolic phenotype. The ISME Journal 2016;10(1):145–157. doi:10.1038/ismej.2015.90. https://doi.org/10.1038/ismej.2015.90
  4. Lewis MC, Merrifield CA, Berger B, Cloarec O, Duncker S, Mercenier A, Nicholson JK, Holmes E, Bailey M. Early intervention with Bifidobacterium lactis NCC2818 modulates the host–microbe interface independent of the sustained changes induced by the neonatal environment. Scientific Reports 2017;7:5310. doi:10.1038/s41598-017-05689-z. https://doi.org/10.1038/s41598-017-05689-z
  5. Christoforidou Z, Mora Ortiz M, Poveda C, Abbas M, Walton G, Bailey M, Lewis MC. Sexual dimorphism in immune development and in response to nutritional intervention in neonatal piglets. Frontiers in Immunology 2019;10:2705. doi:10.3389/fimmu.2019.02705. https://doi.org/10.3389/fimmu.2019.02705
  6. Li Y, Thelen KM, Matos Fernández K, Nelli R, Fardisi M, Rajput M, Trottier NL, Contreras GA, Moeser AJ. Developmental alterations of intestinal SGLT1 and GLUT2 induced by early weaning coincides with persistent low-grade metabolic inflammation in female pigs. American Journal of Physiology-Gastrointestinal and Liver Physiology 2022;322(3):G346–G359. doi:10.1152/ajpgi.00207.2021. https://doi.org/10.1152/ajpgi.00207.2021
  7. Metzler-Zebeli BU, Lerch F, Yosi F, Vötterl JC, Koger S, Aigensberger M, Rennhofer PM, Berthiller F, Schwartz-Zimmermann HE. Creep feeding and weaning influence the postnatal evolution of the plasma metabolome in neonatal piglets. Metabolites 2023;13(2):214. doi:10.3390/metabo13020214. https://doi.org/10.3390/metabo13020214
  8. Marincola FC, Corbu S, Lussu M, Noto A, Dessì A, Longo S, Civardi E, Garofoli F, Grenci B, Mongini E, Budelli A, Grinzato A, Fasano F, Fanos V, Stronati M. Impact of early postnatal nutrition on the NMR urinary metabolic profile of infant. Journal of Proteome Research 2016;15(10):3712–3723. doi:10.1021/acs.jproteome.6b00537. https://doi.org/10.1021/acs.jproteome.6b00537
  9. Corbett RJ, Luttman AM, Wurtz KE, Siegford JM, Raney NE, Ford LM, Ernst CW. Weaning induces stress-dependent DNA methylation and transcriptional changes in piglet PBMCs. Frontiers in Genetics 2021;12:633564. doi:10.3389/fgene.2021.633564. https://doi.org/10.3389/fgene.2021.633564
  10. Planer JD, Peng Y, Kau AL, Blanton LV, Ndao IM, Tarr PI, Warner BB, Gordon JI. Development of the gut microbiota and mucosal IgA responses in twins and gnotobiotic mice. Nature 2016;534(7606):263–266. doi:10.1038/nature17940. https://doi.org/10.1038/nature17940
  11. Thaiss CA, Itav S, Rothschild D, Meijer MT, Levy M, Moresi C, Dohnalová L, Braverman S, Rozin S, Malitsky S, Dori-Bachash M, Kuperman Y, Biton I, Gertler A, Harmelin A, Shapiro H, Halpern Z, Aharoni A, Segal E, Elinav E. Persistent microbiome alterations modulate the rate of post-dieting weight regain. Nature 2016;540(7634):544–551. doi:10.1038/nature20796. https://doi.org/10.1038/nature20796
  12. Meng Q, Luo Z, Cao C, Sun S, Ma Q, Li Z, Shi B, Shan A. Weaning alters intestinal gene expression involved in nutrient metabolism by shaping gut microbiota in pigs. Frontiers in Microbiology 2020;11:694. doi:10.3389/fmicb.2020.00694. https://doi.org/10.3389/fmicb.2020.00694