In the present study, we investigated the effects of age and environment on several innate and T cell immune parameters in children living in a tropical region of Ecuador. The findings show that immune development continues to occur until at least 5 years of age as indicated by the increasing percentages of memory CD4+ and CD8+ T-cells and decreasing percentages of naïve CD4+ T cells. Further, this maturation of the immune system was associated with a strong age-dependent down-regulation of pro-inflammatory innate immune responses during the first 5 years of life, an observation that was linked to a decline in the percentages of regulatory T cells (CD4+CD25+FoxP3+) and IL-10 production by SEB-stimulated peripheral blood leukocytes.
We recruited urban infants and children of the same ethnicity (Afro-Ecuadorian) of parents that had migrated from the same rural Districts as used for the rural sampling. ‘Matching’ by ethnicity likely will have reduced recall biases from parental questionnaires and confounding by factors associated with ethnicity. Because we conducted our stimulations using in vitro
stimulation of whole blood samples, the results we observed are likely a fairly accurate representation of what occurs during viremias or bacteremias of humans. However, because the individual cell types that make up the population of circulating blood cells likely differ as we age, we are not able to discern from our results whether changes in response to TLR agonists are due to intrinsic decreases in responsiveness to these molecules or whether this phenomenon occurs due to a changes in subsets of circulating blood cells. Another potential limitation was that we did not collect data earlier in infancy. Such data would have provided an important baseline with which to interpret age-dependent trends. The observation of significant levels of IFN-γ by 6–9 months and no age-dependent up-regulation in production of this cytokine, often referred to as a marker of immune maturation 
, may indicate that IFN- γ responses had matured markedly by 6–9 months. However, the finding that memory T-cells progressively increased with increasing age, along with a corresponding decrease in percentages of naïve T-cells, demonstrates that substantial immune system development continues through the first 5 years of life and occurred at similar rates in urban and rural populations.
To examine the effects of environment on immune responses we selected two distinct environments that we believed would differ fundamentally in terms of infectious and microbial exposures. We expected infectious exposures to be much greater in the rural environment because of poorer access to sanitation and clean drinking water compared to urban households (). Similarly we expected more intense environmental exposures to microbes (e.g. endotoxin) in the rural populations. Such exposures are considered to provide important signals for immune development in early life 
. In fact, environment appeared had only modest effects on the immune parameters we measured. It is possible that many of those environmental exposures that influence immune maturation occurred with a similar intensity and at similar ages in both populations. Matching by ethnicity may have made our urban and rural populations more similar with respect to important environmental exposures - recent rural migrants to the city of Esmeraldas tend to settle at the periphery of the city and bring with them “ruralised” lifestyles 
so several important environmental exposures may be shared by our urban and rural study populations.
The potential effects of environment on immunity were measured in infants aged 6 to 9 months, a time when we expected the immune system to be most plastic and subject to environmental influences. The effects observed by comparing data from urban with rural infants did show several interesting findings: 1) a stronger down-regulation of IL-10 responses but a delay in maximal SEB-induced IFN-γ responses in rural compared to urban infants; 2) a more rapid increase in memory CD8+
T cells in rural compared to urban infants; and 3) a more rapid down-regulation of IL-8 responses to TLR 3 and TLR6 in urban compared to rural children - similar but non-significant trends were observed for other TLR agonists. Few differences were observed for comparisons of urban and rural children for the older age groups. We observed only one significant association between a specific environmental exposure and immune parameter in multivariate analysis: an increase in SEB-induced IL-10 production with increasing levels of crowding. A study of children aged 4–11 years in urban Brazil showed an elevated frequency of IL-10 production (the spontaneous accumulation of IL-10 after 5 days of culture used as a marker for homeostatic immune regulation) by peripheral blood leukocytes (PBLs) from children without access to drinking water and sanitation in early life compared to children from more hygienic households 
. In the present study we did not observe an effect of bathroom or water source on SEB-induced IL-10 after controlling for other covariates. Possible explanations are that the two parameters measure different regulatory effects or the fact that there is no municipal sewage drainage system or infrastructure for the provision of potable water that can be considered to be truly clean in urban Esmeraldas and that would be comparable to the city of Salvador in Brazil. No significant associations were observed between the immune parameters measured and geohelminth infection. Our data, therefore, provides only limited support that the different environmental exposures evaluated in this study have specific effects on different immune parameters. It is important to note, however, that the present study had limited power to detect effects for individual environmental exposures—larger and appropriately powered studies will be required to fully explore interactions between individual environmental exposures and immune parameters.
Previous studies have documented that the capacity of peripheral blood leukocytes to secrete a wide spectrum of inflammatory cytokines (e.g. IL-6, IL-10, TNF-α, and IFN- γ) is downregulated after birth 
. LPS-induced production of IL-6 [15,16]
and IL-8 
is elevated in neonates compared to young children 
or adults 
, indicating an attenuation of inflammatory cytokine production postnatally 
. In fact, neonatal mice appear to be highly susceptible to the pro-inflammatory effects of TLR agonists such as LPS and poly(I:C), a deficit that is related to the low numbers of T cells in neonates, making neonates susceptible to uncontrolled pro-inflammatory innate responses 
. Our data showing an age-dependent decline in PBL responses to TLR agonists are consistent with these observations.
What then may be the consequences of enhanced innate immune responsiveness in early life? The ability of the innate immune system to respond to microbes may be critical in early life because of the immaturity of the adaptive immune response. However, an exaggerated innate immune response may contribute to the immunopathology of infection through excessive inflammatory cell recruitment and activation. For example, severe infections with respiratory syncytial virus in children have been linked to a higher capacity for LPS-induced production of IL-6 and IL-8 at birth 
. In the present study, declines in IL-8 responses were strongly associated with percentages of FoxP3+
T regulatory cells that declined in parallel. The role of FoxP3+
T regs in modulating innate immunity is unclear - FoxP3+
T regs are predominantly of thymic origin 
and can be considered ‘natural’ rather than ‘induced’. There is evidence that FoxP3+
T regs can directly suppress the activation of innate immune cells [20–22]
although the ligation of TLRs of innate immune cells can also modulate the suppressive activity of FoxP3+
T regs 
. IL-8 is a neutrophil activator and chemoattractant and, through its effects on neutrophils, likely provides a first line of defense against bacterial pathogens that are common causes of morbidity and mortality in early life. However, excessive neutrophil activation may carry severe immunopathological consequences, and it is likely that mechanisms exist that function to down-regulate such responses (e.g. FoxP3+
T regs) until the immune response matures sufficiently to respond to such infectious challenges in a more targeted and regulated manner.
The age-dependent decline in regulatory function observed in the present study (SEB-induced IL-10 and percentages of FoxP3+
T regs) was unexpected—we expected to observe age-dependent increases in both parameters. However, high levels of IL-10 production may have an important role early life in regulating the inflammatory response to infections. Greater mitogen-induced production of IL-I0 has been associated with a low risk of severe respiratory infections 
and deficient IL-10 was associated with greater viral load following in vivo
challenge with rhinovirus 
. The source of IL-10 in SEB-stimulated peripheral blood is most likely to be T cells including populations of T regs although other cellular sources of IL-10 such as monocytes and B cells cannot be excluded. A study of Swedish infants showed that the percentages of CD4+
T cells increase after birth reaching a peak during the first month of life, but thereafter a downward trend was observed 
. Age-dependent declines in SEB-induced IL-10 and FoxP3+ T regs, that may reflect non-specific regulatory responses, may be paralleled by a reciprocal increase in antigen-specific regulation (i.e. frequencies of IL-10 expressing Tr1 cells and antigen-specific production of IL-10 by T cells) reflecting an increasingly important role of antigen-specific regulation with age.