Acquisition of adult-like TLR4 and TLR9 responses during the first year of life

Abstract

Background: Characteristics of the human neonatal immune system are thought to be responsible for heightened susceptibility to infectious pathogens and poor responses to vaccine antigens. Using cord blood as a source of immune cells, many reports indicate that the response of neonatal monocytes and dendritic cells (DC) to Toll-like receptor (TLR) agonists differs significantly from that of adult cells. Herein, we analyzed the evolution of these responses within the first year of life.

Methodology/principal findings: Blood samples from children (0, 3, 6, 9, 12 month old) and healthy adults were stimulated ex vivo with bacterial lipopolysaccharide (LPS, TLR4 agonist) or CpG oligonucleotides (TLR9 agonist). We determined phenotypic maturation of monocytes, myeloid (m) and plasmacytoid (p) DC and production of cytokines in the culture supernatants. We observed that surface expression of CD80 and HLA-DR reaches adult levels within the first 3 months of life for mDCs and 6-9 months of life for monocytes and pDCs. In response to LPS, production of TNF-alpha, IP-10 and IL-12p70 reached adult levels between 6-9 months of life. In response to CpG stimulation, production of type I IFN-dependent chemokines (IP-10 and CXCL9) gradually increased with age but was still limited in 1-year old infants as compared to adult controls. Finally, cord blood samples stimulated with CpG ODN produced large amounts of IL-6, IL-8, IL-1beta and IL-10, a situation that was not observed for 3 month-old infants.

Conclusions: The first year of life represents a critical period during which adult-like levels of TLR responses are reached for most but not all cytokine responses.

Figure 1. HLA DR expression on the surface of circulating monocytes, myeloid and plasmacytoid DCs.

Blood samples were incubated with PBS or the indicated stimulant. The different subpopulations were gated as described in the Material and Methods section. Expression was compared in samples from the different age groups: Cord blood (CB, n = 10), 3-month (3 m, n = 10), 6&9-month (6–9 m pooled data from n = 4 and 8, respectively), 12-month (12 m, n = 9) old infants and healthy adults (n = 16). Data are represented as median+interquartile range. *p<0.05, **p<0.01, ***p<0.001 as compared to stimulated adult samples.

Figure 2. CD80 expression on the surface of circulating monocytes, myeloid and plasmacytoid DCs.

The different subpopulations were gated as described in the Material and Methods section. Expression was compared in samples from the different age groups: Cord blood (CB, n = 10), 3-month (3 m, n = 10), 6 and 9-month (6–9 m, pooled data from n = 4 and 8, respectively), 12-month (12 m, n = 9) old infants and healthy adults (n = 16). Data are represented as median+interquartile range. *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Age-dependent upregulation of LPS-induced production of TNF-α, IP-10, IL-12p70 and IFN-γ.

Whole blood samples were stimulated with LPS and culture supernatants were collected after 16–18 h. Production in the different infant groups was compared to adult values. Cord blood (CB, n = 13), 3-month (3 m, n = 15), 6-month (6 m, n = 8), 9-month (9 m, n = 9), 12-month (12 m, n = 9) old infants and healthy adults (n = 10). Data are represented as median+interquartile range. *p<0.05, **p<0.01, ***p<0.001 as compared to stimulated adult samples.

Figure 4. Age-dependent upregulation of CpG-induced production of IP-10 and MIG.

Whole blood samples were stimulated with CpG A+B combination and culture supernatants were collected after 16–18 h. Production in the different infant groups was compared to adult values. Cord blood (CB, n = 13), 3-month (3 m, n = 10), 6&9-month (6 m, n = 3, 9 m, n = 5), 12-month (12 m, n = 11) old infants and healthy adults (n = 10). Data are represented as median+interquartile range. *p<0.05, **p<0.01, ***p<0.001 as compared to stimulated adult samples.

Figure 5. Hyperproduction of specific cytokines in early life.

Whole blood samples were stimulated with the indicated TLR ligand and culture supernatants were collected after 16–18 h. Production in the different infant groups was compared to adult values. Cord blood (CB, n = 13), 3-month (3 m, n = 10), 6&9-month (6 m, n = 3, 9 m, n = 5), 12-month (12 m, n = 11) old infants and healthy adults (n = 10). Data are represented as median+interquartile range. *p<0.05, **p<0.01, ***p<0.001 as compared to stimulated adult samples.