Human neonates are highly susceptible to infections, which is generally ascribed to transient developmental deficiencies in the innate and adaptive immune system. Here, we demonstrate that despite similar or higher concentrations of innate immune cells, neonatal ability to produce Th1-type cytokines upon TLR stimulation is impaired compared to adults. The neonatal TLR system undergoes rapid and differential development during the first month of life. Whereas the ability to produce Th1-type cytokines in response to agonists for TLR3, TLR7 and TLR9 rapidly increases to adult levels during the first month of life, TLR4 mediated responses remain impaired at least up to one month of age. Age-dependent impairments in the TLR system may contribute to the high neonatal susceptibility to infection. Our results are consistent with previous studies, demonstrating that neonatal innate immune system is distinct and polarized towards Th-2-type responses6,7,16
. This polarization is thought to play an important role in the prevention of harmful maternal-fetal alloimmune reactions leading to preterm labour and delivery5
. However, the bias against Th-1-cell-polarizing cytokines leaves the newborn susceptible to infection. Insight into the kinetics and factors modulating neonatal TLR development may result in new strategies to prevent and/or treat infections and allergic diseases.
Studies investigating postpartum TLR development describe gradual maturation of Th-1 polarizing capacity from infancy to childhood12,17,18
. The importance of establishing the immune status in early life is underscored by several studies showing that variations in early immune development have long-term sequelae with regard to the prevalence of many diseases19-22
. To our knowledge, this is the first study to investigate postpartum TLR development during the first month of life in a large cohort of healthy newborns. The striking maturation of neonatal TLR responses identifies the first month of life as an essential period in the development of this part of the innate immune system.
Studies investigating the cause of impaired neonatal TLR responses have mainly focused on impaired production of Th1-type cytokines such as TNF-α and IL-124,6,7,13,23,24
. Different mechanisms have been suggested to explain decreased cord blood LPS-induced IL12p70 production. Firstly, plasma factors, such as LPS binding protein have been shown to modulate LPS-delivery to the TLR4 receptor complex25,26
. Adenosine, present in neonatal plasma, has been shown to increase intracellular levels of cyclic adenosine monophosphate (cAMP), thereby inhibiting TLR-mediated TNF-α production13
. Interestingly, increased levels of cAMP also decrease IL-12 production while increasing production of IL-10, exemplifying its immunopolarizing potential5
. In our assay, neonatal plasma showed a modest increase in LPS-induced release of IL-10 in two out of three adult PBMC donors, and had no effect on the production of IL-12p70. Because of limited volume, we were only able to use 10% plasma, whereas previous studies used 100% plasma. In addition, instead of cord blood mononuclear cells, we used adult PBMC, which may be less sensitive to the TLR-modifying effects of neonatal plasma. We hypothesize that TLR4-mediated cytokine production is modulated by a complex and dynamic interplay between soluble factors and other (cell intrinsic) factors. The concentration of soluble mediators and target cell sensitivity will be subject of further studies. Secondly, differences in LPS-induced cytokine production may be due to differences in expression of the TLR4 complex, consisting of TLR4, CD14 and MD2. The relative level of TLR4 expression in neonates compared to adults is still a matter of debate, which is further complicated by methodological differences between studies6,18,27
. However, while differences in TLR4 complex expression or soluble factors may explain a general increase or decrease in cytokine release, they are unlikely to explain the polarization observed in our study.
Thirdly, IL-10 has been shown to negatively modulate IL-12p70 production28,29
. Indeed, in our samples, there was a modest, but significant negative correlation between LPS+IFN-γ induced IL-10 and IL-12p70 release. This finding supports the notion that TLR4-mediated cytokine production is tightly regulated to maintain the balance between Th1 and Th2. From our results, we cannot conclude whether polarized neonatal TLR4 responses are primarily due to increased production of IL-10, decreased production of IL-12p70, or to a common factor influencing both cytokines.
Fourthly, signalling downstream of TLR4 also differs between newborns and adults. A microarray study comparing cord blood and adult LPS-activated monocytes showed significant differences in expression of several signal transduction factors and transcription factors, including JunB and STAT430
. LPS-induced mRNA levels of both subunits of the IL-12p70 heterodimer by cord blood monocytes has been shown to be decreased due to decreased half-life (IL-12p40) or defective nucleosome remodelling, resulting in impaired transcription (IL-12p35)31-34
. Our data indicate that, in a whole blood assay, defective IL-12p70 production at birth is due to impaired transcription of both IL-12A (encoding the IL-12p35 subunit) and IL-12B (IL-12p40), and that these impairments are maintained up until the age of one month. This study has potential limitations. Firstly, in vitro study of whole blood represents a minimally perturbed system, yet may not reflect patterns of response in vivo35
. Secondly, repeated sampling in the same children would have been the optimal strategy to address maturation of TLR responses during the neonatal age. This was not feasible for practical reasons. Thirdly, the whole blood stimulation assay limits conclusions on cell-specific mechanisms underlying impairments in neonatal TLR function. Similar limitations would have been encountered using isolated PBMC, because PBMC composition changed markedly during the neonatal age. We believe that the whole blood stimulation model is a relevant representation of the immunological status of newborns and infants that correlates with susceptibility to infection36-38
In conclusion, neonatal TLR responses are distinct from those of adults, and differentially mature during the first month of life. The propensity towards high IL-10 but low IL-12p70 production in response to TLR4 agonists during the first month of life might contribute to neonatal susceptibility to pathogens that are recognized by TLR4, such as Gram-negative bacteria and RSV39
. Finally, future studies aimed at identifying the factors influencing postpartum TLR development will further delineate the age-dependent maturation of this key aspect of innate immunity and may identify new strategies to modulate the maturation of the neonatal innate immune system, to prevent infections and/or allergy during this vulnerable age.