Early Probiotic Supplementation Enhances Infant Immune Maturation

Researchers from the PROOM-3 study have demonstrated that early pre- and postnatal supplementation with the probiotic Limosilactobacillus reuteri significantly modulates the development of immune responses in infants. Published in Allergy, Asthma & Clinical Immunology, the randomized, double-blind, placebo-controlled trial investigated how starting supplementation at gestational week 20 influences cytokine and chemokine production during the first two years of life. The study found that while probiotic supplementation promoted age-dependent changes indicative of immune maturation, supplementation with omega-3 polyunsaturated fatty acids (PUFAs) alone did not produce clear immunomodulatory effects on these specific markers.

The research addresses a critical gap in understanding how early-life interventions might prevent allergic diseases. Allergic conditions affect approximately 30% to 40% of the global population, often beginning in early childhood through a progressive pattern known as the atopic march. The study hypothesized that a longer prenatal supplementation period, beginning at week 20 of pregnancy rather than the third trimester, could more effectively guide immune maturation. This approach aims to help the infant immune system distinguish between harmful pathogens and harmless environmental stimuli, a process essential for establishing long-term immunological resilience.

The study included 117 mother-baby pairs from the Östergötland region of Sweden, who were randomized into four groups: placebo, L. reuteri, omega-3 PUFAs, or a combination of both. Pregnant women in the probiotic group received 10^9 colony-forming units of L. reuteri twice daily from week 20 until delivery, while their infants received 10^8 CFU once daily from birth until one year of age. The omega-3 group received capsules containing 640 mg of omega-3 PUFAs, with infants receiving the supplement indirectly via breast milk until three months of age. This design allowed for the assessment of both isolated and combined effects of these interventions on immune development.

To evaluate immune function, the team isolated peripheral blood mononuclear cells (PBMCs) from infants at birth and at 6, 12, and 24 months of age. These cells were stimulated ex vivo with environmental allergens such as birch pollen and cat dander, food allergens like ovalbumin, and ligands for Toll-like receptors (TLRs), including lipopolysaccharide, lipoteichoic acid, and CpG oligodeoxynucleotides. The researchers measured a comprehensive panel of cytokines and chemokines associated with Th1, Th2, Th17, and regulatory T-cell responses, using Meso Scale Discovery assays to quantify the net immune response to each stimulus.

The primary finding was that L. reuteri supplementation induced significant age-dependent changes in 56% of the tested conditions, compared to only 22% in the placebo group. This substantial difference, which was statistically significant, suggests that the probiotic enhances the natural maturation of the immune system. In contrast, the double supplementation group showed similar but less pronounced changes in 40% of the conditions. The results indicate that the probiotic component is the primary driver of these immunological shifts, as the addition of omega-3 PUFAs did not significantly amplify the effect beyond what was observed with the probiotic alone.

A specific area of interest was the response to CpG stimulation, which activates TLR9, a receptor involved in innate immune recognition. In the placebo group, there were no significant age-dependent changes in the secretion of IL-1β, IL-6, IL-10, or TNF following CpG stimulation. However, infants who received L. reuteri supplementation showed significantly greater IL-10 responses at 12 and 24 months compared to birth. Additionally, IL-1β, IL-6, and TNF secretion were significantly elevated at 24 months in the probiotic group. These changes suggest a long-term effect of the probiotic on the maturation of innate immune pathways, which are crucial for regulating allergic responses.

At six months of age, L. reuteri supplementation also led to a significant increase in Th1-associated CXCL10 levels in response to birch and cat allergens compared to the placebo group. CXCL10 is a chemokine that may counteract Th2-mediated allergic responses, and its lower plasma levels have been associated with allergen sensitization in children. The increase in CXCL10 secretion supports the hypothesis that earlier initiation of L. reuteri supplementation may more effectively promote Th1 responses, which are generally considered protective against the development of allergic diseases.

The lack of clear effects from omega-3 PUFA supplementation in this study contrasts with previous findings that suggested anti-inflammatory benefits. The authors suggest that this may reflect the distinct mechanisms through which these interventions act. While omega-3 PUFAs are well-documented for their anti-inflammatory properties, their role in early immune development remains less clear. The positive effects observed in the combined group were likely attributed to the immune-modulatory properties of L. reuteri, which may directly activate immune cells, whereas omega-3 PUFAs likely exert their effects by modulating inflammatory pathways over time.

The study highlights the importance of distinguishing between the immune-stimulatory properties of probiotics and the anti-inflammatory effects of omega-3 fatty acids. The microbiota plays a pivotal role in training and modulating the immune system during early life, and factors such as diet and environmental exposure influence the development of a healthy microbiota. The findings suggest that L. reuteri supplementation may positively influence immune maturation by selectively modulating innate immune pathways, including those mediated by TLR9, which has been shown to suppress IgE synthesis.

Limitations of the study include the fact that the observed immunological changes have not yet been directly correlated with clinical allergy outcomes. While the study provides compelling evidence for enhanced immune maturation, future research will focus on linking these immunological profiles to the actual prevention of allergic diseases in infancy. The variability in sample sizes across time points was due to randomization and sample availability, which required the use of non-parametric statistical tests to account for incomplete data contributions.

The results of the PROOM-3 study contribute to a growing body of evidence supporting the role of early-life microbiota modulation in allergy prevention. The study demonstrates that the timing and type of supplementation can significantly influence the trajectory of immune development. By starting supplementation at gestational week 20, the researchers were able to capture a longer window of prenatal exposure, which may be critical for shaping the infant immune system. The findings underscore the potential of probiotics as a tool for promoting immune homeostasis and reducing the risk of allergic sensitization.

In conclusion, the study provides a detailed characterization of how pre- and postnatal supplementation affects cytokine and chemokine responses during infancy. The significant age-dependent changes observed in the L. reuteri group suggest that the probiotic supports the natural maturation of the immune system. While omega-3 PUFAs did not show clear effects on these specific markers, the combined approach did not negate the benefits of the probiotic. These findings highlight the complex interplay between diet, microbiota, and immune development, and provide a foundation for future studies aimed at translating immunological insights into clinical allergy prevention strategies.

The trial implemented distinct vehicle controls and defined nutritional formulations across the study arms. Pregnant participants in the probiotic arm ingested Limosilactobacillus reuteri suspended in refined coconut and peanut oils, with infants receiving the identical liquid preparation, whereas the corresponding control cohort received unsupplemented vehicle oil drops. For the fatty acid intervention, mothers took three daily capsules providing 640 milligrams of omega-3 polyunsaturated fatty acids composed of 35 percent eicosapentaenoic acid and 25 percent docosahexaenoic acid, while the control group received olive oil capsules. Baseline demographic and clinical characteristics remained well balanced across the randomized cohorts, with the recorded presence of household pets at the twelve-month timepoint representing the only variable that differed significantly between the placebo group and the active supplementation arms.

The ex vivo laboratory protocols incorporated precise culturing conditions to evaluate cellular reactivity while conserving limited infant blood samples. Mononuclear cells were suspended at a standard density of one million viable cells per milliliter in culture medium containing beta-mercaptoethanol. When available cell numbers restricted the full battery of seventy experimental conditions, stimulations followed a strict hierarchy prioritizing ovalbumin, birch allergen, and phytohemagglutinin ahead of cat allergen and Toll-like receptor ligands. To reliably quantify T-helper-2 cytokine production during six-day cultures challenged with environmental allergens or tetanus toxoid, investigators supplemented the wells with blocking antibodies directed against the interleukin-4 receptor, preventing the cultured mononuclear cells from consuming released interleukin-4 before harvest. Innate responses were concurrently assessed after twenty-four hours using targeted Toll-like receptor agonists, including lipoteichoic acid for TLR2 and lipopolysaccharide for TLR4.

Temporal cytokine profiling in the dual-supplementation arm further demonstrated distinct kinetics following Toll-like receptor 9 activation by CpG oligodeoxynucleotides. Infants administered both Limosilactobacillus reuteri and omega-3 fatty acids showed significant increases in interleukin-6 and tumor necrosis factor secretion at twelve and twenty-four months compared to birth measurements, as well as significantly elevated interleukin-10 concentrations spanning six, twelve, and twenty-four months. All biochemical measurements, derived from customized multiplex electrochemiluminescence arrays after subtracting unstimulated baseline levels, were analyzed by a blinded researcher using non-parametric Kruskal-Wallis testing and Dunn’s post hoc evaluations. To control for multiple testing, investigators applied the Benjamini-Hochberg adjustment, establishing statistical significance at p less than 0.05 and adjusted discrimination at q less than 0.10, while assigning values below the lower detection limit to half the detection threshold.

Subject of Research: Effects of pre- and postnatal probiotic and ω-3 fatty acid supplementation on cytokine and chemokine responses to allergens and TLR ligands during infancy

Article Title: Effects of pre- and postnatal probiotic and ω-3 fatty acid supplementation on cytokine and chemokine responses to allergens and TLR ligands during infancy

Article References: Fontes-Oliveira, C. C., Nylén, A., Ljung, J., Welin, A., Arvidsson, L., Martí, M., Govindaraj, D., Martín, I. G., Janefjord, C., Tingö, L., Al-Kaabawi, A., Severin, E., Duchén, K., & Jenmalm, M. C. (2026). Effects of pre- and postnatal probiotic and ω-3 fatty acid supplementation on cytokine and chemokine responses to allergens and TLR ligands during infancy. Allergy, Asthma & Clinical Immunology, 22(1), Article 27. https://doi.org/10.1186/s13223-026-01036-y

Image Credits: AI Generated

DOI: 10.1186/s13223-026-01036-y

Keywords: Effects, pre-, postnatal, probiotic, fatty, acid, supplementation, cytokine, chemokine, responses, allergens, ligands