Circadian Variation of Immune Cell Populations in Preterm Human Milk: A Preliminary Study

Introduction

An estimated 15 million premature births occur annually, claiming nearly a million lives and making it the leading cause of death in children under five.^1,2 The immune system of premature babies is immature, which places them at greater risk of suffering serious complications; this prematurity is positively correlated with morbidity and mortality from infections.^3–5

The intrauterine environment and especially the nutritional conditions of the fetus affect the immune maturity of the newborn ⁶ ; the more premature the baby, the higher the risk of infection. Premature babies, exhibiting poor immunovigilance and a deregulated inflammatory response compared to full-term infants, are at heightened risk of infection due to a lower capacity of innate lymphoid cells and phagocytes, accompanied by a depleted repertoire of antibodies and complement proteins, along with diminished cytokine production.^7,8

Human milk boasts a diverse array of nutritional and nonnutritional components that contribute to the immunological well-being of premature infants. Interestingly, some nutritional components display remarkable rhythmicity, characterized by markedly different concentrations across the day–night cycle.^9,10 The objective of this work was to analyze the rhythmicity of immune cells and proinflammatory cytokines present in preterm intermediate milk.

Materials and Methods

Results

The presence of immune cells was monitored in all premature milk samples (day/night). A significant difference was observed in the percentage of CD34⁺ cells present in the day samples versus the night samples (41.3%, IQR; 30.2–52.4 versus 25.1%, IQR; 19.1–31.1) (p = <0.0001). On the other hand, 59.18% (IQR; 55.9–62.4) of CD45⁺ cells were observed in diurnal milk samples versus 57.7% (IQR; 51.7–63.7) in nocturnal milk samples, corresponding to a small increase in diurnal samples (p = 0.02). In addition, different subpopulations of immune cells were identified between day and night samples. There was a diurnal increase in the percentage of CD23⁺ populations (13.0%, IQR; 12.4–13.6 versus 12.3%, IQR; 11.6–12.8) (p = 0.004), CD32⁺ (36.5%, IQR; 31.4–41.5 versus 29.1%, IQR; 25.4–32.8) (p < 0.0001), CD36⁺ (27.9%, IQR; 26.4–29.3 versus 14.3%, IQR; 8.8–19.4) (p < 0.0001), CD2⁺ (18.5%, IQR; 15.8–21.2 versus 10.11%, IQR; 2.2–18.0) (p < 0.0001), and Tγδ cells (11.1%, IQR; 7.1–15.1 versus 6.6%, IQR; 0.7–12.5) (p = 0.002). On the other hand, a nocturnal increase was observed in the percentage of CD16⁺ populations (8.6%, IQR; 4.3–13.0 versus 15.0%, IQR; 12.8–17.2) (p = 0.002), CD19⁺ (6.4%, IQR; 3.8–9.0 versus 10.7%, IQR; 8.7–12.7) (p = 0.002), and CD4⁺ (29.9%, IQR; 24.7–35.0 versus 32.6%, IQR; 28.3–36.9) (p = 0.002). No variation was observed in the CD3⁺ and CD8⁺ populations (Fig. 1).

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FIG. 1.

Day/night cell subpopulation estimation graph in preterm transition milk samples. A circadian oscillation is observed in the percentage of stem cells and immune cells analyzed. The differences between means were analyzed by unpaired Student's t-test. *p = <0.05, **p = 0.001, ****p = 0.00001.

Analysis of proinflammatory cytokines in milk samples (day/night) revealed a diurnal increase in IL-1β concentrations (23.9 pg/mL; 19.3–28.5 versus 20.7 pg/mL; 18.6–22.9) (p = 0.03), IL-6 (187.5 pg/mL; 97.6–277.4 versus 12.3 pg/mL, −116.3 to 140.9) (p = 0.007), and TNF-α (193.2 pg/mL; 47.1–339.2 versus 127.4 pg/mL; 82.5–172.3) (p = 0.04) in comparison with nocturnal milk samples (Fig. 2). A positive correlation was observed between the age of the mothers with the daytime percentage of Tγδ cells (p = 0.04) and the daytime concentration of TNF-α (p = 0.03). No association was observed between the percentage of cells or the concentration of cytokines with factors such as the weight of the mother, the weight of the newborn, and gestational age, nor with the presence of sepsis or respiratory deficiencies in newborns.

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FIG. 2.

Differential concentration of proinflammatory cytokines in daytime and nighttime samples of milk from premature transition. The difference between means were analyzed by unpaired Student's t-test. *p = <0.05, **p = 0.001.

Discussion

Circadian rhythms modulate the migration of immune cells in the body, along with their ability to proliferate and secrete cytokines. These events typically occur during the individual's active behavioral period and respond to both endogenous and extrinsic rhythmic cues.^12,13 Our work demonstrates the rhythmic fluctuations of immune cell populations in the transitional breast milk of premature mothers. Previous studies have shown that rhythmic cell mobilization depends on specific factors associated with both the cell type and the originating tissue. ¹⁴

Microbial oscillations directly and indirectly influence both host metabolism and immune function. ¹⁵ Within the mother–child dyad, a neonate's immune response may initially mirror the mother's due to shared microbial exposures and immunological transfer, before establishing its own unique profile. ¹⁶ Notably, the presence of sepsis in premature infants could further modulate the rhythmicity of immune populations in their mothers' breast milk. This rhythmic pattern might have evolved to provide an effective defense against the diurnal fluctuations in bacterial load associated with sepsis. Additionally, as sepsis progresses, levels of inflammatory molecules, including cytokines, demonstrably increase.^17,18

While a key function of the circadian clock in immune response is anticipating pathogen exposure and orchestrating effective defense, our analysis of most immune cell and cytokine populations reveals a bias toward elevated defense components during the daytime. This trend could be attributable to the newborns' active period or the differential exposure of these cells to endotoxins. ¹⁹

Upon birth, newborns rely on rhythmic signals conveyed through the composition of breast milk. These signals mirror the mother's own rhythmic patterns and reflect a unique molecular connection within the mother–child dyad. Beyond its established benefits, our study reveals differential rhythmic behavior in immune cell subpopulations and proinflammatory cytokines within the transitional milk of mothers with premature infants. This suggests that milk can dynamically adjust its cellular content to meet the immunological needs of the infant, potentially through a rhythmic mechanism that enhances immune response effectiveness. However, the specific oscillator driving this rhythmicity in breast milk remains to be elucidated in future research.