The prevalence of allergic diseases has increased in the recent decades and is forecasted to continue to increase worldwide (1). Current evidence suggests that a perturbation of gut microbiota influences the development of allergic diseases (2). The human gut microbiota is involved in various metabolic processes that collectively regulate immunity and gut health. Infancy is an important stage for the establishment and maturation of the gut microbiome. Factors such as gestational age, mode of delivery, antibiotics exposure, feeding modes, environmental exposures and host genetics are known to influence this maturation process (2).

Short chain fatty acids (SCFAs) are derived from gut microbial fermentation of dietary fibers and polyphenols. These metabolites play a key role in maintaining gut and immune homeostasis (3, 4). They are absorbed in the colon and exert systemic effects by regulating various physiological processes and acting as signaling molecules, maintaining balance between pro- and anti-inflammatory properties (3). Two main anti-inflammatory signaling mechanisms of SCFAs have been identified: inhibition of histone deacetylases (HDACs), and activation of G-protein-coupled receptors (GPCRs) (4, 5). In addition, SCFAs have also been found to potentiate de novo extrathymic Treg cell generation (4). In support, seven prospective birth cohort studies have demonstrated that infants with higher stool SCFAs, particularly butyric acid have less atopic outcomes in childhood (6–12).

This study leveraged data from a sub-cohort from the Growing Up in Singapore Toward Healthy Outcomes (GUSTO) birth cohort to evaluate the biological implication of infant stool SCFA profiles on clinical outcomes of atopic eczema, wheezing and allergen sensitization up to the age of 8 years. To investigate the protective role of SCFAs against the development of atopic disorders, serial infant stool SCFA measurements at 4 time points (3 weeks, 3, 6, and 12 months) were longitudinally analyzed in order to determine their potential influence on atopic outcomes in childhood.

This study investigated the longitudinal association between stool SCFA profiles across 4 time points in first year of life with the risk of developing allergy-related outcomes up to 8 years. Our results showed that subjects with lower longitudinal stool SCFA levels specifically butyric and propionic acids, especially during the weaning period, have a higher propensity of developing allergy-related outcomes. We also observed similar trends in association with eczema when single time points were analyzed, though statistical significance was only seen at the 3- and 6-month time points. Recently, few studies indicated that a compromised microbiome in the first 100 days of life prevents the establishment of butyrate producers at the time of weaning (13, 17). Those observations reinforce the notion that the infant gut microbiome follows a successive pattern of microbial colonization in the first year of life. First, establishment of breast milk sugars consumers in the first months of life, followed by the establishment of butyrate producers or fibers/polyphenols consumers at the time of weaning (13, 18).

Interestingly, lower butyric acid levels in the first 6 months and before 12 months were associated with eczema, and statistical significance was lost when the 12-month time point was included. This could be explained by the recommended introduction of solid food around 6 months which could influence the SCFA levels afterwards (19). Our selected cohort also showed a mean weaning age at 5.5 months (Supplementary Table 4). This suggests that the strong influence of stool SCFA levels on eczema risk occurs mainly early in infancy before the introduction of solid food.

Our findings add to the body of evidence that lower stool butyric acid levels before 6 months is associated with increased risk of developing wheezing, eczema, food sensitization and combined outcomes of both wheezing and eczema up to age 8 years, compared to those with higher levels. Other studies have also demonstrated the association between lower levels of stool butyric acid in infancy and increased likelihood of eczema by 18 months (10), food sensitization, food allergy, inhalant allergen and allergic rhinitis by 6 years (11). Additionally, we observed that higher odds of developing recurrent wheezing till age 8 years was associated with lower longitudinal levels of propionic acid, which has not been observed in previous studies. However, lower levels of stool propionic acid have been associated with inhalant allergen sensitization by 6 years (11).

Although the associations between lower levels of acetic acid with atopic wheeze at 12 months of age (12), with food sensitization and food allergy by 6 years (11) have been reported, we did not observe any significant association in terms of acetic acid levels with any of the allergy-related outcome. Taken together, these studies provide robust evidence for the protective effect of higher stool levels of SCFAs, particularly butyric acid between 3 and 6 months of age, against allergy-related outcomes. A preclinical study defined the “weaning reaction” as a sensitive period allowing the establishment of butyrate producers which have a paramount role in programming the immune system. Thus, early life provides a potential intervention window through a modulation of the infant gut microbiome (20). Table 1 summarizes the studies to date that have made similar observations to our study.

This notion is further substantiated by preclinical studies in mice, demonstrating that SCFAs such as acetic, butyric and propionic acids, produced in the gut can regulate homeostasis of Tregs in the colon (23, 24). These SCFAs have varying influence on Treg differentiation and accumulation due to varying degrees of interaction with different GPCRs that would then have different impact on immune cells (T cells and dendritic cells), hence conferring different immunological protection (24). These differential effects on the immune system may partly explain the different associations of each SCFA with different allergic outcomes. Evidence on protective effects and their mechanism of action for minor SCFAs is still lacking and thus deserves further study.

Current studies suggest that early life nutritional intervention could be a solution to modulate SCFA concentrations in the gut and reduce susceptibility to allergic diseases. Increased consumption of foods with SCFAs such as butyric acid in yogurt could be beneficial to directly increase gut SCFA concentration and hence increase levels of SCFAs in the body. This strategy has shown to be beneficial in the prevention of inflammation-related diseases such as chronic inflammatory bowel disease (25, 26). Evidence for the direct supplementation of SCFA intake in relation to the development of allergy-related outcomes remains sparse but deserves further attention.

Prebiotics and probiotics supplementation have been reported to promote the growth of SCFA-producers, which were shown to be associated with a beneficial effect against eczema (27–29). SCFA supplementation, particularly butyric acid, via oral supplements or consumption of food high in butyric acid, to treat colonic diseases has shown promising results in animal studies, with some of these effects being observed in human trials (25, 26). In relation to allergic disorders, preclinical studies in mice have also shown that fructo-oligosaccharide- (FOS-) supplemented diet and butyric acid supplementation improved efficacy of oral immunotherapy (OIT) for cow's milk allergy through the effective reduction of mast cell and basophil activation and enhanced suppressive activity of Tregs (30). These dietary interventions promote the growth of beneficial commensals, increase SCFA production, thus conferring immunological protective effects on the host (31). A translation of our findings into additional supportive clinical evidence could be alluded to epidemiological studies indicating that increased food diversity and higher intake of fruits and vegetables in the first years of life were associated with a robust immunity (less risk of allergy) (32–34).

The strength of this study is the serial assessment of stool SCFAs obtained in early life among children from a large birth cohort subjected to adjustment for various key confounding factors which have been known to affect the propensity of developing allergic outcomes. The long follow-up period of this cohort allowed for better monitoring of the trajectory of allergy manifestation as certain allergy-related disorders present later in childhood. The limitation of this study, however, is the relatively small sample size, and might be the reason for the few associations found with single time points of SCFAs, and wide confidence intervals with adjORs.

In conclusion, low levels of gut SCFAs, particularly butyric acid between 3 and 6 months of life and propionic acid up to 12 months are shown to be significantly associated with an increased susceptibility to allergic outcomes in childhood.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: http://www.metabolomicsworkbench.org, PR000983.

The studies involving human participants were reviewed and approved by Domain Specific Review Board of Singapore, National Healthcare Group and the Centralized Institutional Review Board of SingHealth (DSRB D/09/021 and CIRB 2009/280/D). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

HC performed the statistical analysis and manuscript writing. JC and DK carried out the experiment. GY carried out the data analysis. ET, EL, LS, NK, AG, HV, and OT were involved in the subject recruitment and sample collection. CH-H, YC, CL, and JK contributed to the interpretation of the results. FY, KT, Y-SC, and KG were involved in project administration. EC, BL, and LT were involved in planning and supervising the project. All authors contributed to the article and approved the submitted version.

This work was supported by the Singapore Ministry of Health's National Medical Research Council [NMRC/CIRG/1414/2014].

CL and JK are employees of Danone Nutricia Research. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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