Breastmilk-Saliva Interactions Boost Innate Immunity by Regulating the Oral Microbiome in Early Infancy

Abstract

Introduction: Xanthine oxidase (XO) is distributed in mammals largely in the liver and small intestine, but also is highly active in milk where it generates hydrogen peroxide (H2O2). Adult human saliva is low in hypoxanthine and xanthine, the substrates of XO, and high in the lactoperoxidase substrate thiocyanate, but saliva of neonates has not been examined.

Results: Median concentrations of hypoxanthine and xanthine in neonatal saliva (27 and 19 μM respectively) were ten-fold higher than in adult saliva (2.1 and 1.7 μM). Fresh breastmilk contained 27.3 ± 12.2 μM H2O2 but mixing baby saliva with breastmilk additionally generated >40 μM H2O2, sufficient to inhibit growth of the opportunistic pathogens Staphylococcus aureus and Salmonella spp. Oral peroxidase activity in neonatal saliva was variable but low (median 7 U/L, range 2-449) compared to adults (620 U/L, 48-1348), while peroxidase substrate thiocyanate in neonatal saliva was surprisingly high. Baby but not adult saliva also contained nucleosides and nucleobases that encouraged growth of the commensal bacteria Lactobacillus, but inhibited opportunistic pathogens; these nucleosides/bases may also promote growth of immature gut cells. Transition from neonatal to adult saliva pattern occurred during the weaning period. A survey of saliva from domesticated mammals revealed wide variation in nucleoside/base patterns.

Discussion and conclusion: During breast-feeding, baby saliva reacts with breastmilk to produce reactive oxygen species, while simultaneously providing growth-promoting nucleotide precursors. Milk thus plays more than a simply nutritional role in mammals, interacting with infant saliva to produce a potent combination of stimulatory and inhibitory metabolites that regulate early oral-and hence gut-microbiota. Consequently, milk-saliva mixing appears to represent unique biochemical synergism which boosts early innate immunity.

Fig 1. Concentration of nucleotide precursors in whole saliva samples of human adults, term neonates, and domesticated mammals.

(A) Healthy non-smoking adults (n = 77) and (B) healthy full-term vaginally-delivered neonates (n = 60), lines show median values. Non-parametric analyses were conducted to estimate the significance using a Mann-Whitney U test, (C) Longitudinal study of median concentrations of metabolites in saliva from full-term neonates aged 1–4 days (n = 60), 6 weeks (n = 20), 6 months (n = 19), and 12 months (n = 14), (D) Median metabolite concentrations in saliva from selected domesticated mammals (8 cows, 5 sheep, 4 goats, 5 horses, 1 camel, 7 dogs, 5 cats). Metabolites were divided into five functional groups: Pyrimidines (Pseudouridine to Orotate); Purine bases (Hypoxanthine, Xanthine); Purine nucleosides (Inosine, Guanosine); ATP precursors (Adenine, Adenosine); Deoxynucleosides (Deoxyadenosine to Deoxyuridine).

Fig 2. Breastmilk XO generates H₂O₂ when interacted with neonatal saliva.

(A) Distribution of XO activity in breastmilk. Samples of colostrum were collected 1–5 day postpartum (n = 24). Error bars show mean±SD. XO activity was 8.0 ± 5.3 U/L. (B) Dose dependent inhibition of breastmilk XO by oxypurinol. Pooled breastmilk samples containing 0–25 μM oxypurinol concentrations (n = 3) were incubated with the peroxidase reagent containing 400 uM hypoxanthine. (C) The mean concentration of H₂O₂ in fresh breastmilk samples (n = 22) was 27.3±12.2 μM. (D) Kinetics of H₂O₂ generation after mixing 33 μL of diluted breastmilk, 33 μL of 1/3 diluted neonatal saliva and 34 μL peroxidase assay working solution (green circles). The negative control was buffer and peroxidase reagents (blue squares), (n = 2). The original concentrations of hypoxanthine and xanthine in the neonatal saliva were 70 μM and 30 μM respectively. Calculation of [H₂O₂] was based on two-fold dilutions assuming that breastmilk and neonatal saliva is 1:1 during suckling (1 mole xanthine produces 1 mole H₂O₂, 1 mole hypoxanthine generates 2 moles H₂O₂).

Fig 3. Thiocyanate in saliva.

Median concentrations and interquartile ranges (mM) of thiocyanate (SCN^-) in saliva of neonates (n = 16), infants at 6 weeks (n = 16), 6 months (n = 18), and 12 months (n = 12), and adults (n = 20).

Fig 4. Effect of H₂O₂ on bacterial growth.

The effect of increasing H₂O₂ concentration (0–400 μM) on bacterial growth. Hydrogen peroxide was added to nutrient broth inoculated with bacteria and incubated for 24 h at 37°C, except for L. plantarum, which was incubated for 48 h. The concentration of cells was determined as turbidity by measuring absorbance at 600 nm. Values represent the mean±SD (n = 3).

Fig 5. Salivary nucleotide metabolites and XO regulate bacterial growth.

Bacteria were incubated with breast milk and simulated neonatal saliva for 24 h at 37°C and then enumerated by sub-culturing onto agar plates. Values represent the mean±SD (n = 3). (A) Bactericidal effects of H₂O₂ generated by breastmilk (XO) and increasing concentrations of salivary xanthine and hypoxanthine (0–50 μM). Figure displays the concentration-dependent effect of xanthine and hypoxanthine versus log CFU/mL. ANOVA was used to compare between multiple means. (B) The effect of breastmilk plus simulated neonatal saliva (with and without supplements) on bacterial counts (CFU/mL). Bacteria were incubated with breastmilk and saliva alone (Control), or supplemented with nucleotide metabolites omitting xanthine/hypoxanthine (Full supplement except X&HX), or the latter with xanthine/hypoxanthine (Full supplement), or the latter with oxypurinol (Full supplement + Oxypurinol) (see Table 2). Means were compared by t-test: * p<0.05; ** p<0.01; ns, not significant.