Skewed pattern of Toll-like receptor 4-mediated cytokine production in human neonatal blood: low LPS-induced IL-12p70 and high IL-10 persist throughout the first month of life

Abstract

Newborns are highly susceptible to infectious diseases, which may be due to impaired immune responses. This study aims to characterize the ontogeny of neonatal TLR-based innate immunity during the first month of life. Cellularity and Toll-like receptor (TLR) agonist-induced cytokine production were compared between cord blood obtained from healthy neonates born after uncomplicated gestation and delivery (n=18), neonatal venous blood obtained at the age of one month (n=96), and adult venous blood (n=17). Cord blood TLR agonist-induced production of the Th1-polarizing cytokines IL-12p70 and IFN-alpha was generally impaired, but for TLR3, 7 and 9 agonists, rapidly increased to adult levels during the first month of life. In contrast, TLR4 demonstrated a slower maturation, with low LPS-induced IL-12p70 production and high IL-10 production up until the age of one month. Polarization in neonatal cytokine responses to LPS could contribute to neonatal susceptibility to severe bacterial infection.

Figure 1. Whole blood concentrations of innate immune cells

Whole blood leukocyte differential was performed in cord blood (n=18), neonatal venous blood obtained at the age of one month (n=96) and adult venous blood (n=17). Cell percentages were determined by FACS and multiplied by the total concentration of lymphocytes and monocytes from the complete blood count. Data are represented as geometric mean + 95% CI. *; p<0.05.

Figure 2. Rapid maturation of TLR responses in healthy newborns

Cytokine production in cord blood (n=18), neonatal venous blood at age one month (n=96) and adult venous blood (n=17) was measured after a 24h-incubation with poly I:C (TLR3, panel A+B), Loxoribine (TLR7, C+D) and ODN CpG (TLR9, E+F). Neonatal ability to produce Th1-type cytokines upon TLR-agonist stimulation was impaired at birth, but rapidly increased to adult levels. Data are represented as geometric mean + 95% CI. *; p<0.05.

Figure 3. Distinct TLR4 responses up until the age of one month

Cytokine production in cord blood (n=18), neonatal venous blood at age one month (n=96) and adult venous blood (n=17) was measured after a 24h-incubation with LPS+IFN-γ. Cord blood TLR4 responses were characterized by high levels of IL-10 (A) and low levels of IL-12p70 (B). Although LPS+IFN- γ-induced production of IL-10 and IL-12p70 gradually decreased or increased respectively, TLR4-mediated cytokine responses at the age of one month remained significantly impaired compared to adult responses. *, p<0.05.

Figure 4. Human neonatal cord blood demonstrates reduced LPS-induced cytokine mRNA responses compared to adults

Cytokine mRNA concentrations in cord blood (n=7), neonatal venous blood at age one month (n=7) and adult venous blood (n=7) were measured after a 5h-incubation with medium, LPS or LPS+IFN-γ. TLR4-mediated transcription of IL-12A and IL-12B and TNF-α was impaired both in cord blood and in venous blood at age one month, compared to adult venous blood. In contrast, despite higher medium-induced levels in cord blood, TLR4 agonist-induced production of IL-6 and IL-10 was similar in all age groups. *; p<0.05.

Figure 5. Donor- and cytokine specific effect of neonatal plasma on LPS-induced cytokine production

Adult PBMC derived from heparinized blood from three separate donors were stimulated with LPS and IFN-γ in the presence of 10% heterologous adult or neonatal plasma. Each dot represents a different plasma donor. After 24h incubation, the extracellular medium was collected for IL-10 and IL-12p70 measurements. Bars represent median values.